International Society of Hypertension (ISH) New ...ish-world.com/data/uploads/ISH_symposium_2014_programme_2... · of Hypertension (ISH) New Investigator Symposium on Hypertension

International Societyof Hypertension (ISH)New Investigator Symposium on Hypertension and Cardiovascular Disease

Monday 8th September 2014Hilton San Francisco Union Square

www.ish-world.com/NIN

New Investigator CommitteeThe New Investigator Committee (NIC) is charged with encouraging and supporting New Investigator Network initiatives to attract and retain younger members of the ISH. Core members include: M. Tomaszewski (Chair), D. Burger, F. Charchar, A. Schutte and P. Veerabhadrappa.

Four NIC Working Groups (1) Awards & Finance, (2) Media, (3) Networking and Mentorship and (4) Recruitment were established at the start of 2013 to strengthen and further develop network activities and ensure regional representation where possible. Each group is lead by an NIC member. Current Working Group members are listed below.

ISH New Investigator Network (ISHNIN)The ISH New Investigator Network was established by the NIC to serve as a platform for interaction between students and new investigators and allow new avenues for communication, collaboration and education.

The New Investigator Network provides opportunities for all new scientists within the ISH to interact and build relationships, as well as to gain experience and exposure within the society as they start their scientific careers. Numerous initiatives are aimed at fostering relationships between researchers across the globe and include web and video casts, a quarterly newsletter, and a mentorship scheme. Moreover, annual New Investigator Symposia are held (similar to this year’s event in San Francisco). The San Francisco symposium has been organized, executed, adjudicated, and moderated by new scientists within the ISH. The network maintains a social media presence on both Facebook and Twitter.

1. Awards & Finance Working Group

LeAD:Alta SchuttePotchefstroom,South Africa

MeMBeRS: Matilde Alique Madrid, SpainSofie Brouwers Brussels, Belgiumevi Christofidou Leicester, UKKarla Haack Nebraska, USATomoyuki Honjo Kobe, JapanRichard Wainford Boston, USA

4. Recruitment

LeAD:Praveen VeerabhadrappaPhiladelphia, USA

MeMBeRS: Seema Bhanji Karachi, PakistanKeith Diaz Philadelphia, USARana el Bikai Montreal, CanadaGodsent Isiguzo Jos, NigeriaFrancine Marques Ballarat, AustraliaCesar Romero Córdoba, Argentina

2. Media Working Group

LeAD:Dylan BurgerOttawa,Canada

MeMBeRS: Ruan Kruger Potchefstroom, South AfricaRama Guggilla Andhra Pradesh, IndiaAndre Pascal Kengne Capetown, South AfricaMuhammad Oneeb Rehman Mian Montreal, CanadaStefan Naydenov Sofia, BulgariaAugustine Odili Nonso Abuja, NigeriaRicardo Pena Silva Bogota, Columbia

3. Networking and Mentorship

LeAD:Fadi CharcharBallarat,Australia

M. TomaszewskiChair – ISH NIC

MeMBeRS: Katrina Binger Berlin, GermanyFady Hannah-Shmouni New Haven, USAAugusto Montezano Glasgow, UKKrupa Savalia Nebraska, USAFouad Zouein Jackson, USA

3 ISH New Investigator Symposium, San Francisco, USA. 8 September 2014

Programme Overview 12.00-‐12.45 REGISTRATION and COFFEE Imperial Ballroom 12.50-‐13.00 Welcome and Introduction Maciej Tomaszewski, University of Leicester, UK / Chair -‐ ISH New Investigator Committee (NIC) 13.00-‐14.10 ORAL PRESENTATIONS -‐ SESSION 1: Imperial Ballroom Chairs: Fadi Charchar and Aaron Trask 13.00-‐13.10 1.1 Adenosine A3 Receptors Regulate Oxidative Stress and Inflammatory Responses in a Model of

Hypertension and Renal Disease T Yang, M Hezel, M Peleli, N Terrando, B Fredholm, M Carlström

Karolinska Institutet 13.10-‐13.20 1.2 Anti-‐inflammatory Effect of Ang-‐(1-‐7) non-‐peptide mimetic (AVE 0991), on T Cell Infiltration in

Perivascular Adipose Tissue in ApoE-‐/-‐ Mice D Skiba1, R Nosalski2, T Mikolajczyk2, R Olszanecki3, J Jawien3, F Rios1, AC Montezano1, R Touyz1, TJ Guzik1 1Institute of Cardiovascular and Medical Sciences, University of Glasgow, 2Translational Medicine

Laboratory, Department of Internal Medicine, Jagiellonian University School of Medicine, 3Department of Pharmacology, Jagiellonian University School of Medicine

13.20-‐13.30 1.3 Toll-‐like Receptor 2 Signaling Contributes To Cerebrovascular Dysfunction And Decreased Cerebral

Blood Flow In Type-‐1 And Type-‐2 Diabetes T Hardigan1, N Hoda2, M Abdelsaid3, A Ergul4 1Georgia Regents University, 2Georgia Regents University, 3Charlie Norwood Veterans Affairs Medical

Center, 4Charlie Norwood Veterans Affairs Medical Center 13.30-‐13.40 1.4 Angiotensin 1-‐7 Regulation of Endothelin-‐1 System In Pulmonary Hypertension K Hood1, H Yusuf2,J Findlay1, RA Santos3 CH Castro4, GS Baillie1, AC Montezano1, MR MacLean1, R Touyz1 1University of Glasgow, 2University of Ottawa, 3Institute of Biological Sciences, 4Institute of Biological

Sciences 13.40-‐13.50 1.5 Inflammasome activity is essential for deoxycorticosterone acetate/salt-‐induced hypertension in mice S Murali Krishnan, C Sobey, B Kemp-‐Harper, C Chan, H Diep, J Dowling, A Pinar, A Mansell, G Drummond

Monash University 13.50-‐14.00 1.6 Chloroquine, an inhibitor of endosomal Toll-‐like receptors, improves blood pressure and endothelial

function in spontaneously hypertensive rats C McCarthy1, C Wenceslau1, S Goulopoulou1, S Ogbi1, T Matsumoto2, C Webb1 1Georgia Regents University, 2Hoshi University 14.00-‐14.10 1.7 Vascular aging in aldosterone associated hypertension: role of NADPH oxidase 1 A Harvey1, AC Montezano1, D Graham1, Y He2, G Ceravolo2, C Yabe-‐Nishimura3, K Griendling4, R Touyz1 1University of Glasgow, 2University of Ottawa, 3Kyoto Prefectural University of Medicine, 4Emory

University 14.10-‐15.30 Posters viewing, judging and refreshments Grand Ballroom A The first 30 minutes will be for networking only Poster Session 1 Adipose tissue, nervous system and the kidney Chairs: Fadi Charchar and L. Gabriel Navar Poster Session 2 Blood vessel, endothelium Chair: Praveen Veerabhadrappa Poster Session 3 Oxidative stress, inflammation and immunity Chair: Dylan Burger


Poster Session 4 Clinical Studies Chairs: Maciej Tomaszewski and Augusto Montezano Poster Session 5 Miscellaneous Chairs: Alta Schutte and Richard Wainford 15.30-‐16.40 ORAL PRESENTATIONS -‐ SESSION 2 Imperial Ballroom Chairs: Praveen Veerabhadrappa and Theodora Szasz 15.30-‐15.40 2.1 Use of mass spectrometry for the development of new biomarkers for diabetic nephropathy N Grobe1, S Gutta1, H Osman2, M Saklayen2, K Elased1 1Wright State University, 2Dayton Veterans Affairs Medical Center 15.40-‐15.50 2.2 Effects of Carotid Body Tumor Resection on the Blood Pressure of Essential Hypertensive Patients M Fudim, K Groom, C Laffer, J Netterville, D Robertson, F Elijovich

Vanderbilt University 15.50-‐16.00 2.3 Activation of AT1 receptors on dendritic cells restrains angiotensin II-‐dependent hypertension through

a CCR7-‐mediated mechanism JD Zhang, SD Crowley

Duke University 16.00-‐16.10 2.4 The effects of positive allosteric modulation of GABAA receptors upon stress and hypertension in

Schlager hypertensive mice E Stevenson1, E Johns1, P Davern1, J-‐L Moretti1, K Jackson1, K Evans2, K Marques3, K Head1

1Baker IDI Heart and Diabetes Institute, 2Monash University, 3Federation University

16.10-‐16.20 2.5 Lymphocyte-‐Specific Adaptor Protein, LNK (SH2B3), Regulates Angiotensin-‐II Induced Hypertension and Renal/Vascular Inflammation M. Saleh, D. Harrison, M. Madhur Vanderbilt University, USA

16.20-‐16.30 2.6 Impaired PVN neuronal activity in rats lacking central Gαi2 proteins: Implications for the pathophysiology of salt-‐sensitive hypertension

C Carmichael, A Carmichael, P Vartanyan, R Wainford

Boston University School of Medicine 16.30-‐16.40 2.7 Reduced uterine perfusion pressure (RUPP) elicits increased sFlt-‐1 levels not only in the placenta but

also adipose tissue F Spradley, A Palei, J Granger

University of Mississippi Medical Center 16.40-‐17.20 SESSION 3: KEYNOTE PRESENTATION Imperial Ballroom Chair: Maciej Tomaszewski From Mentee to Mentor: Pathway to Emerging Independence L. Gabriel Navar, PhD, Chair, Department of Physiology and Director, Center of Biomedical Research,

Excellence in Hypertension and Renal Biology Tulane University Health Sciences Center 17.20-‐17.45 Closing Remark and Presentation of Awards Maciej Tomaszewski


List of Poster Presentations Poster Session 1 Adipose tissue, nervous system and the kidney Chairs: Fadi Charchar and L. Gabriel Navar P1. Beta3 adrenergic receptor activation increases anti-‐contractile effects of perivascular adipose tissue via cystathionine

gamma lyase and cyclooxygenase T Szasz1, T Matsumoto2, RC Webb1

1Georgia Regents University, 2Hoshi University P2 AT2 receptor signaling participates in the conversion of white-‐to-‐beige fat K Tsukuda, M Mogi, H Hirotomo, H Kan-‐no, T Chisaka, T Wang, T Kukida, T Bai, T Shan, T Iwanami Department of Molecular Cardiovascular Biology and Pharmacology, Ehime University, Graduate School of Medicine P3 CETP inhibitors Torcetrapib, Dalcetrapib, and Anacetrapib induce adipocyte-‐derived Aldosterone production through

Nox and STAT3 activation FJ Rios1, K Neves2, A Nguyen Dinh Cat1, S Even1, R Palacios3, R Jenkins1, R Montezano1, R Touyz1 1University of Glasgow, 2University of Sao Paulo, 3Universidad Rey Juan Carlos P4 Activation of central angiotensin II type 2 receptors attenuates neurogenic hypertension S Brouwers1, RD Wainford1, I Smolders2, AG Dupont2 1Boston University School of Medicine, 2Vrije Universiteit Brussel P5 Impaired brain-‐derived neurotrophic factor-‐TrkB signaling in nucleus tractus solitarius during chronic heart failure

blunts baroreflex sensitivity B Becker, H Wang, I Zucker 1University of Nebraska Medical Center P6 Contribution of orexin to neurogenic hypertension in BPH/2J mice K Jackson1, T Nguyen-‐Huu1, E Stevenson1, P Davern1, P Carrive2, P Head1

1BakerIDI Heart and Diabetes Institute, 2University of New South Wales P7 Overexpression of calcium/calmodulin-‐dependent protein kinase II (CaMKII) potentiates angiotensin II (AngII)-‐

mediated signaling in neurons U Basu, AJ Case, J Liu, Y Li, MC Zimmerman 1University of Nebraska Medical Center P8 Capsaicin mediated afferent renal denervation: Pharmacological confirmation and implications for the

pathophysiology of hypertension A Carmichael, R Wainford

Boston University School of Medicine P9 Testosterone propionate in Ischemia-‐reperfusion (I/R) induced Acute Kidney Injury (AKI) CN Patil, R Maranon, C Dalmasso, H Zhang, LA Juncos, JF Reckelhoff

University of Mississippi Medical Center P10 Telmisartan attenuates renal dysfunction in high-‐salt-‐loading RAS activated mice J Iwanami, M Mogi, K Tsukuda, XL Wang, M Kukida, H Nakaoka, T Chisaka, H Bai, B Shan, M Horiuchi Ehime University, Graduate School of Medicine P11 Soluble guanylate cyclase (sGC) stimulator reduces the cardiac and renal damage in angiotensin II (Ang II)-‐induced

diastolic heart failure N Haase1, N Wilck1, L Marko1, A Heuser2, D Brockschnieder3, D Kretschmer3, D Stasch3, D Dechend1, D Mueller1 1Experimental and Clinical Research Center (ECRC), 2Max-‐Delbrueck Center for Molecular Medicine, 3Bayer HealthCare,

Global Drug Discovery P12 Redox-‐regulated suppression of resting splenic T-‐lymphocytes during sympathoexcitation-‐associated hypertension AJ Case, MC Zimmerman

University of Nebraska Medical Center


Poster Session 2 Blood vessel, endothelium Chair: Praveen Veerabhadrappa P13 Castration impairs rat internal pudendal artery reactivity and structure R Lopes, K Neves, M Silva, V Olivon, S Ruginsk, S Ramalho, S Rodrigues, S Tostes, S Carneiro

University of Sao Paulo P14 17beta-‐Estradiol and 16alpha-‐Hydroxyestrone Increase Oxidative Stress Through Nrf2 Dysfunction In Human

Pulmonary Artery Smooth Muscle Cells – Implications in Pulmonary Hypertension K Hood1, RA Lopes1, A Johansen1, AC Montezano1, C Szyndralewiez2, P Page2, MR MacLean1, R Touyz1 1University of Glasgow, 2Genkyotex P15 Fetal growth restriction is a risk of vascular remodeling T Chisaka, M Mogi, H Nakaoka, M Kukida, H Kan-‐no, H Tsukuda, H Wang, H Bai, H Iwanami, H Higaki

Ehime University Graduate School of Medicine P16 G protein-‐coupled estrogen receptor contributes to the vascular effects of aldosterone and type two diabetes-‐

associated vascular dysfunction. N Ferreira1, S B A Cau2, C P Manzato1, M A B Silva1, F S Carneiro1, F C A Tostes1 1University of Sao Paulo, 2University of Juiz de Fora

P17 Increased adrenergic contractions, but preserved relaxations to insulin in renal arteries of obese mice O Baretella, A Xu, P Vanhoutte

Department of Pharmacology & Pharmacy and State Key Laboratory of Pharmaceutical Biotechnology, The University of Hong Kong

P18 Chemerin Decreases Vascular Insulin Signaling K Neves1, N Lobato2, R Lopes1, F Mestriner3, A Oliveira3, A Tostes3 1University of Glasgow, 2University of Goias, 3University of Sao Paulo P19 Role of vascular smooth muscle cell PPARgamma in aldosterone-‐induced vascular injury S Ouerd1, M Trindade1, N Idris-‐Khodja1, T Barhoumi1, S Offermanns2, S Gonzalez3, S Paradis1, EL Schiffrin4

1Lady Davis Institute for Medical Research, 2Max-‐Planck-‐Institute for Heart and Lung Research, 3National Cancer Institute, 4McGill University

P20 has been withdrawn by the authors. P21 Endothelin-‐1 overexpression preserves endothelial function in mice with vascular smooth muscle cell-‐specific

deletion of ppar-‐gamma N Idris Khodja1, S Ouerd1, T Barhoumi1, M Trindade2, J Gornitsky1, A Rehman1, S Offermanns3, F Gonzalez4, P Paradis1,

EL Schiffrin5

1Hypertension and Vascular Research Unit, Lady Davis Institute for Medical Research, 2Department of Clinical Medicine, State University of Rio de Janeiro, 3Department of Pharmacology, Max-‐Planck-‐Institute for Heart and Lung Research, 4Laboratory of Metabolism, Division of Basic Sciences, National Cancer Institute, National Institutes of Health, 5McGill University

P22 Chronic treatment with fluoxetine increases relaxation of rat resistance mesenteric arteries via ATP-‐sensitive

potassium channels activation C A Pereira, N S Ferreira, F L A C Mestriner, L B Resstel, F S Carneiro, F C A Tostes University of Sao Paulo P23 Endothelin-‐1 overexpression in endothelial cells increases blood pressure in an endothelin type A receptor-‐

dependent manner S Coelho1, Y Rautureau1, A Rehman1, S Offermanns2, P Paradis1, EL Schiffrin3 1Lady Davis Institute for Medical Research, 2Goethe University Frankfurt, 3Magill University. P25 Adipocyte-‐derived aldosterone and cortisol are Nox1/4 dependent: implications in obesity-‐associated vascular

dysfunction S Even

University of Glasgow


Poster Session 3 Oxidative stress, inflammation and immunity Chairs: Dylan Burger and Matthew C. Zimmerman P26 Nox isoforms, oxidative stress, inflammation and fibrosis in adipose tissue in obese mice: differential regulation in

epididymal, perirenal and perivascular depots S Even

University of Glasgow P27 AMP-‐activated Protein Kinase Activator AICAR Attenuates TNF-‐alpha-‐induced Inflammation In Murine Adipocytes A White, A Nguyen Dinh Cat, C Jenkins, S Mancini, A Montezano, A Salt, R Touyz University of Glasgow, P28 FOXP3+ T regulatory lymphocytes counteract angiotensin ii-‐induced vascular injury MOR Mian1, T Barhoumi1, M Briet2, AC Ene1, P Paradis1, EL Schiffrin1

1Lady Davis Institute for Medical Research, McGill University, 2Centre Hospitalo Universitaire d'Angers, Universite d'Angers

P29 High salt (NaCl) reduces the activation and function of alternatively activated (M2) macrophages via epigenetic

modification K Binger1, M Gebhardt1, C Rintisch1, M Henig1, A Schroeder2, W Neuhofer3, K Hilgers3, A Manzel8, C Schwartz9, M

Kleinewietfeld4

1Experimental and Clinical Research Ctr and Max-‐Delbrueck Ctr for Molecular Med, 2Friedrich-‐Alexander Univ of Erlangen-‐Nuremberg, 3University of Munich, 4Yale School of Medicine

P30 Chemerin, a novel adipokine, regulates human vascular cell function through redox-‐sensitive processes involving

Nox1/4 and eNOS K Neves1, R Lopes1, N Lobato2, A Cat Nguyen1, A Oliveira3, A Montezano1, A Tostes1, R Touyz1 1University of Glasgow, 2University of Goias, 3University of Sao Paulo; P31 Acute increase in O-‐GlcNAcylation reduces the release of cytokines and attenuates hypotension in mice with LPS-‐

induced sepsis V Olivon, R Ferreira, F Mestriner, F Cunha, J Alves-‐Filho, J Tostes University of Sao Paulo -‐ USP P32 The Role of Immunological CD8 Effector Memory T Cells In Hypertension H Itani, J Wu, L Xiao, F Zhang, D Michell, W Chen, D Harrison

Vanderbilt University P33 Downregulation of vascular Nrf2 and associated antioxidant enzymes in hypertension R Lopes1, A Montezano1, K Neves1, R Tostes2, R Touyz1 1University of Glasgow, 2University of Sao Paulo P34 Angiotensin II induces oxidative stress through ADP-‐ribose-‐TRPM2 dependent mechanisms in human microvascular

endothelial cells – Role of AT2 receptor M. Dulak-‐Lis, A. Nguyen, C. Jenkins, A. Montezano, R. Touyz Institute of Cardiovascular and Medical Sciences P35 A high-‐salt diet alters the composition of intestinal microbiota in mice N Wilck1, S Olesen2, M Matus2, A Balogh3, R Dechend3, R Alm2, R Müller1 1ECRC -‐a joint cooperation between Max Delbrück Center for Molecular Medicine Berlin and the Charite Medical

Faculty, 2Department of Biological Engineering, Massachusetts Institute of Technology, 3Helios Clinic Berlin-‐Buch, P36 Protein Tyrosine Phosphatase Oxidation and Redox Proteomics in Hypertension S Tsiropoulou, A Montezano, A Scott, R Burchmore, R Touyz University of Glasgow P37 Immune dysfunction in a vasopressin-‐induced mouse model of preeclampsia S Scroggins, D Santillan, N Pearson, J Grobe, M Santillan

1University of Iowa Carver College of Medicine


P38 Animal models of salt-‐sensitive hypertension show impaired renal sodium chloride cotransporter (NCC) function K Walsh, R Wainford

Boston University School of Medicine Poster Session 4 Clinical Studies Chairs: Maciej Tomaszewski and Augusto Montezano P39 Arterial blood pressure in the Congolese youths: the interaction of drinking alcohol and overweight. G Ngoyi, BM Bayauli, JR M'buyamba-‐Kabangu

University Hospital of Kinshasa P40 The role of traditional medicine in the fight against hypertension in Africa Albert ZE Health and Development Research Institute P41 This poster has been withdrawn by the author. P42 Family history of premature cardiovascular events and total plaque area H Perez Vascularis P43 has been withdrawn by the authors P44 Blood Pressure Variability during High Salt Intake, But Not Low Salt, Is Associated with Future Cardiovascular Events

in Patients with Essential Hypertension T Kisaka1, R Ozono1, TA Cox2, Y Kihara1 1Hiroshima University Graduate School of Biomedical Sciences, 2Los Angeles Biomedical Research Institute at Harbor-‐

UCLA Medical Center P45 Subcutaneous Resistance Artery Remodeling And Function In Chronic Kidney Disease Patients JC Fraulob Aquino1, M Briet2, T Barhoumi1, C Savoia3, P Paradis1, EL Schiffrin1 1Vascular and Hypertension Res Unit, Lady Davis Inst for Medical Res, SMBD-‐Jewhish General Hosp, Mcgill Univ,

2INSERM U1083, CNRS UMR 6214, Ctr Hospo-‐Univire d’Angers, Univ d'Angers, 3Cardiology Unit, Second Faculty of Medicine, Sant’Andrea Hospital, Sapienza University of Rome, and Research Center, Fatebenefratelli San Pietro Hospital

P46 The determinants of acute coronary syndrom in young adults at Jacques Monod Hospital I Iubenga Cliniques Universitaires de Kinshasa P47 has been withdrawn by the authors P48 has been withdrawn by the authors P49 Preliminary candidate gene analysis in the Women’s Interagency HIV Study shows pleiotropic effect of APOB and TNF

genes and novel association of SERPINE1 with Metabolic Syndrome traits Y Natanzon

Case Western Reserve University P50 Effect of nutritional transition on the incidence of hypertension in Africa ZE Albert Health and Development Research Institute P51 Novel neurohumoral responses to head upright tilt testing in children with chronic nausea and orthostatic

intolerance A Wagoner1, H Shaltout2, D Diz2, J Fortunato2

1Wake Forest School of Medicine, Hypertension and Vascular Research Center, 2Children's Hospital of Colorado


Poster Session 5 Miscellaneous Chairs: Alta Schutte and Richard Wainford P52 Renal endoplasmic reticulum (ER) stress is induced via Endothelin A (ETA) receptor activation C De Miguel, J Hobbs, D Pollock, J Pollock

University of Alabama at Birmingham P53 Biochemical and Biophysical validation of new inhibitors identified through rational structure based design against

Dopamine-‐beta-‐hydroxylase to combat hypertension SK Dey1, T Joseph2, S Kumar3, A Kamaladevi4, N Sarkar2, N Sarkar2, N Balamurugan4, N Thelma2, N Kundu1

1Department of Biochemistry, University of Delhi South Campus, 2Department of Genetics, University of Delhi South Campus, 3Institute of Genomics and Integrative Biology, 4Department of Biotechnology, Alagappa University,

P54 Uncovering the Molecular Mechanisms Underlying the Hypertension in Snx1 Knockout Mice J Yang, L Asico, J Feranil, J Jones, I Armando, I Weinman, I Jose, I Villar

University of Maryland School of Medicine P55 Evidence for the Expression of Renin Angiotensin System (RAS) and a Disintegrin and Metalloproteinase (ADAM) 17-‐

mediated shedding of ACE2 in COS-‐7 cells J Grobe, N Kashkari, H Chodavarapu, H Somineni, M Di Fulvio, K Elased Wright State University P56 Cardiac-‐specific deletion of ERBB4 in the adult mouse increases cardiac cell prolifertion and causes physiological

cardiac hypertrophy Z Wang, W Thomas, T Paravicini University of Queensland P57 Role of Cytochrome P450 4A2 in mediating the elevated blood pressure in a rat model of polycystic ovary syndrome R Maranon, C Patil, C Dalmasso, R Roman, J Reckelhoff University of Mississippi Medical Center P58 Role of melanocortin-‐4-‐receptor in the blood pressure regulation in female hyperandrogenemic rats, a model of

polycystic ovary syndrome R Maranon, R Lima, J do Carmo, A da Silva, J Hall, J Reckelhoff University of Mississppi Medical Center,

P59 has been withdrawn by the authors P60 Gender-‐specific angiogenesis locus on rat chromosome 13 identified with congenic strains that differ by one gene T Stodola, D Didier, M Flister, J Lazar, A Greene Medical College of Wisconsin P61 The effects of a high-‐fat diet (HFD) on blood pressure in pregnant rats A Palei, F Spradley, J Granger

University of Mississippi Medical Center P62 Angiotensin II-‐dependent aortic aneurysm and associated pathogenesis is facilitated by CYP1B1 via oxidative stress

and platelet aggregation S Thirunavukkarasu, N Khan, U Ghafoor, B Jennings, K Mukherjee, A Estes, K Malik The University of Tennessee Health Science Center P63 Binge Eating Disorder is Improved by Stimulation of Angiotensin II Type2 Receptor in Diabetic Mice H Nakaoka, M Mogi, H Kan-‐no, K Tsukuda, T Chisaka, T Kukida, T Wang, T Bai, T Shan, T Iwanami Ehime University Graduate School of Medicine


Abstracts of Oral Presentations 1.1 Adenosine A3 Receptors Regulate Oxidative Stress and Inflammatory Responses in a Model of Hypertension and

Renal Disease T Yang, M Hezel, M Peleli, N Terrando, B Fredholm, M Carlström

Karolinska Institutet

Objectives: Adenosine A1 and A2 receptors contribute to renal autoregulation and blood pressure control, but the role of A3 signaling is unknown. We previously demonstrated that early life reduction in nephron number (UNX) combined with high salt diet (HS) induced renal oxidative stress and hypertension in rats. This study aimed at investigating the role of A3 receptor in modulating renal and cardiovascular function in this disease model.

Methods: Wild-‐type (WT) and A3 knockout (A3KO) mice underwent UNX or sham-‐operation at 3-‐week-‐age followed by HS treatment. Blood pressure and renal function were measured in conscious aged mice and oxidative stress as well as inflammatory properties was characterized.

Results: WT but not A3KO with UNX+HS developed hypertension (WT: 106;4 vs 83;1; A3KO: 82;2 vs 82;2 mmHg) and cardiac hypertrophy (WT: 4.5;0.1 vs 4.0;0.1; A3KO: 4.7;0.2 vs 4.6;0.1 mg/gBW), characterized by increased plasma creatinine, impaired renal plasma flow and increased renal NADPH oxidase activity. UNX+HS increased plasma IL-‐6 (36.1;5.7 vs 16.3;2.4 pg/ml) and IL-‐10 (37.8;3.9 vs 25.4;2.4 pg/ml) levels in WT, but not in A3KO (39.7;3.9 vs 38.5;5.9 pg/ml and 46.5;3.7 vs 43.9;6.0 pg/ml respectively). However, A3KO displayed higher baseline cytokine levels. Furthermore, bone marrow-‐derived macrophages from A3KO mice showed higher constitutive NF-‐kB p65 nuclear translocation and expressed higher M1 (PDL-‐1 and CD86) and M2 (PDL2 and CD206) markers under LPS stimulation compared with WT. These suggested an enhanced innate immune response in A3KO.

Conclusion: UNX followed by HS intake lead to renal and cardiovascular dysfunction, which importantly depends on A3 receptor-‐mediated regulation of oxidative stress and immune system.

1.2 Anti-‐inflammatory Effect of Ang-‐(1-‐7) non-‐peptide mimetic (AVE 0991), on T Cell Infiltration in Perivascular Adipose Tissue in ApoE-‐/-‐ Mice

D Skiba1, R Nosalski2, T Mikolajczyk2, R Olszanecki3, J Jawien3, F Rios1, AC Montezano1, R Touyz1, TJ Guzik1 1Institute of Cardiovascular and Medical Sciences, University of Glasgow, 2Translational Medicine Laboratory, Department of Internal Medicine, Jagiellonian University School of Medicine, 3Department of Pharmacology, Jagiellonian University School of Medicine

Angiotensin 1-‐7 (Ang-‐1-‐7) has anti-‐atherosclerotic effects, possibly through its anti-‐inflammatory properties. As perivascular inflammation is an important component of early atherosclerosis, we aimed to determine whether angiotensin-‐(1-‐7) non peptide mimetic (AVE0991) can modulate these inflammatory mechanisms in the early development of atherosclerosis. Twelve-‐week old ApoE-‐/-‐ mice and controls (C57BL/6J) on chow diet were treated with placebo or AVE0991 (0.58nM/g/day, per os). Mice were analysed at 16, 20 and 24 weeks of age. The level of perivascular leukocyte and T cell infiltration was measured by flow cytometry. Atherosclerotic plaque area was assessed by Oil Red O staining of en-‐face aortic preparations. Endothelial dysfunction was measured by wire myography and ROS production in the aorta by lucigenin-‐enhanced chemiluminescence (5µM). RESULTS: Levels of endogenous Ang-‐1-‐7, measured by mass spectrometry in the perivascular adipose tissue were low in both WT and ApoE-‐/-‐ (32.9;1.2 vs 31.1;1.1 pg/mg; p=0.1). AVE0991 significantly reduced plaque area in ApoE-‐/-‐ mice at all time points (14.2;1.9% vs 7.25;1.3% at 24 weeks; p<0.05). During this early stage of atherosclerosis there was no evidence of endothelial dysfunction and no significant increase in ROS generation in ApoE-‐/-‐ mice. However, there was significant perivascular inflammation in 16 week old ApoE-‐/-‐ mice: CD45+ leukocytes were 2 fold higher in ApoE-‐/-‐ mice vs controls (3200;850 vs 1700;267 cells/mg; p<0.05) and the number of CD3+ T in ApoE-‐/-‐ mice was higher than in C57Bl/6J (638;88 vs 310;64 cells/mg; p<0.05). With aging, the number of leukocytes and T cells further increased in ApoE-‐/-‐ but not in C57BL/6J mice, an effect abrogated by AVE0991. Moreover in cell culture of SW872 preadipocytes, AVE 0991 preincubation abolished TNF-‐a induced increase of chemokine RANTES and IL-‐6 mRNA expression. CONCLUSION: AVE0991 effectively inhibits early perivascular inflammation during the development of atherosclerotic plaque. This occurs independently of changes in redox status or endothelial dysfunction. 1.3 Toll-‐like Receptor 2 Signaling Contributes To Cerebrovascular Dysfunction And Decreased Cerebral Blood Flow In

Type-‐1 And Type-‐2 Diabetes T Hardigan1, N Hoda1, M Abdelsaid2, A Ergul2 1Georgia Regents University, 2Charlie Norwood Veterans Affairs Medical Center


It is recognized that vascular cognitive impairment may be a new complication in both type 1 and type 2 diabetes. Toll-‐like receptor-‐2 (TLR2) plays a role in cardiovascular complications of diabetes but its involvement in diabetic cerebrovascular disease is unknown. Since brain function heavily depends on constant perfusion, and decreased CBF precedes development of inflammation and cognitive deficits, we hypothesized that enhanced TLR2 signaling in both type 1 and 2 diabetes would contribute to cerebrovascular dysfunction and decreased CBF. Endothelium-‐dependent relaxation was assessed by measuring acetylcholine (ACh, 10-‐9 -‐10-‐4 M) induced dilatory response in basilar arteriesfrom GK rats in the presence and absence of an anti-‐TLR2 (1μg) antibody. Vascular contractility to serotonin (10-‐9 -‐10-‐5 M) stimulation was also assessed. Area under the curve (AUC) and maximal effective concentration (Emax as % of max KCl response) were calculated as indices of total relaxation and total contraction, respectively. Basilar artery relaxation was significantly improved in the vessels preincubated (30’) with antiTLR2 (184.8;24.0 vs. 87.7; 4, p =0.007). The EMax in response to serotonin stimulation in the anti-‐TLR2 treated vessels was not significantly different than the vessels from the untreated diabetic GK rats (110.6; 4.9% vs 99.4.;4.5%). To further assess the in vivo functional effects of TLR2 signaling, CBF (relative intensity) was measured using laser speckle imaging in wild type and TLR2-‐knockout (KO) mice using an STZ induced diabetes model. Six weeks after induction of diabetes, wild-‐type diabetic mice exhibited a significant decrease in CBF vs. control (210;22.5 vs. 300.3;18.4, p<0.05). This decrease in cerebral perfusion was attenuated in the TLR2-‐KO diabetic mice compared to TLR2 KO control (322.6;10.1 vs. 344. 5;11. 04). These findings suggest that TLR2 signaling leads to vascular dysfunction through decreased endothelium dependent relaxation, and could contribute to decreased CBF in diabetes predisposing to vascular cognitive impairment.

1.4 Angiotensin 1-‐7 Regulation of Endothelin-‐1 System In Pulmonary Hypertension K Hood1, H Yusuf2,J Findlay1, RA Santos3 CH Castro3, GS Baillie1, AC Montezano1, MR MacLean1, R Touyz1 1University of Glasgow, 2University of Ottawa, 3Institute of Biological Sciences

Studies suggest that activation of ACE2-‐Ang-‐(1-‐7)-‐Mas ameliorates vascular, cardiac and lung damage observed in pulmonary hypertension (PAH); a condition where endothelin-‐1 (ET-‐1) is important. We assessed whether Ang 1-‐7 regulates ET-‐1 system in pulmonary hypertension and putative mechanisms. Hypobaric hypoxia was used to induce PAH in mice, which were divided in 4 groups: normoxic controls (NC), hypoxic PAH (HP), normoxic (NA) and hypoxic PAH (HA) treated with Ang 1-‐7 (hydroxypropyl-‐βcyclodextrin-‐Ang(1-‐7) 30µg/kg/day given by oral gavage for 14 days after established hypoxia-‐induced PAH. In HP mice, RVSP (18.7 NC vs. 47.6mmHg HP, p<0.001), RVH by Fulton Index (0.198 NC vs. 0.279 HP, p<0.01) and urinary ET-‐1 levels (0.88 NC vs 2.48pg/ml HP, p<0.05 vs NC) were increased, an effect blocked by Ang1-‐7 treatment. In NA, Ang 1-‐7 increased urinary levels of ET-‐1 (2.24pg/ml, p<0.05 vs NC). Human microvascular endothelial cells (HMEC) were also stimulated with ET-‐1 in the absence/presence of Ang 1-‐7. Ang 1-‐7 stimulation increased preproET-‐1 mRNA levels (250%), ET-‐1 release (125%), and ETBR protein levels (50%), p<0.05 vs vehicle. To examine whether the regulation of ET-‐1 system by Ang 1-‐7 is beneficial, HMEC were stimulated with ET-‐1 in the absence/presence of Ang 1-‐7. ET-‐1 increased e-‐selectin mRNA (400%) and VCAM-‐1 protein (38%) levels, as well as TNFα production (30%); all effects blocked by Ang 1-‐7 (p<0.05). Ang 1-‐7 inhibition of ET-‐1 pro-‐inflammatory effects was dependent on NO production, since it was blocked by L-‐NAME and LY294002 (inhibitors of NO pathway). Interestingly, Ang 1-‐7 increased NO production (257%) through Mas and ETBR-‐dependent manner. An interaction between Mas and ETbR was observed in HMEC by immunoprecipitation and peptide array protocols. In conclusion, Ang 1-‐7 modulates ET-‐1 system in pulmonary hypertension and HMEC. Our findings indicate that Ang 1-‐7 negatively modulates proinflammatory signalling in human endothelial cells. The protective effect may involve crosstalk with ETBR. These data identify the ET-‐1 system as a novel molecular mechanism involved in the putative protective action of Ang 1-‐7 in pulmonary hypertension.

1.5 Inflammasome activity is essential for deoxycorticosterone acetate/salt-‐induced hypertension in mice S Murali Krishnan, C Sobey, B Kemp-‐Harper, C Chan, H Diep, J Dowling, A Pinar, A Mansell, G Drummond

Monash University

Background: Inflammasomes are signalling complexes comprised of a NOD-‐like receptor protein (NLRP), an adapter protein (ASC) and caspase-‐1. Inflammasomes detect host-‐derived danger signals, causing activation of caspase-‐1, which in turn cleaves the cytokines pro-‐interleukin(IL)-‐1β and pro-‐IL-‐18 into their active, pro-‐inflammatory forms. Hypertension is associated with chronic renal inflammation, but the role of the inflammasome in this process is not known.

Objectives: Investigate whether deoxycorticosterone acetate (DOCA)/salt-‐induced hypertension in mice is associated with increased expression and/or activation of the inflammasome in the kidney, and assess the impact of inhibition of inflammasome activity on blood pressure (BP) and markers of renal inflammation and fibrosis.

Methods and Results: Male C57BL6/J (wild type) and ASC-‐/-‐ mice were uninephrectomised, implanted with a DOCA pellet (2.4 mg/d, 21 d, s.c.) and had their drinking water replaced with 1% saline (1K/DOCA/salt). Control mice had a kidney removed but received a placebo pellet and normal drinking water. 1K/DOCA/salt-‐treated mice had elevated systolic BP (146;4 mmHg) compared to control mice (115;2 mmHg; n=13-‐16; P<0.05). 1K/DOCA/salt-‐induced hypertension was associated with increased renal mRNA expression (fold-‐change vs control; n=7-‐9; P<0.05) of inflammasome subunits NLRP3 (2.3;0.2), ASC (2.8;0.6) and pro-‐


caspase-‐1 (2.6;0.5), and the cytokine, pro-‐IL-‐1β (4.0;0.8). Moreover, protein levels of cleaved (active) caspase-‐1 and IL-‐1β were increased by 1.6;0.2-‐ and 2.1;0.3-‐fold, respectively in kidneys of 1K/DOCA/salt vs control mice (n=6; P<0.05). ASC-‐/-‐ mice, which lack an active inflammasome complex, displayed blunted hypertensive responses to 1K/DOCA/salt-‐treatment (140;3 mmHg) compared to wild types (155;8 mmHg; n=8-‐9; P<0.05). ASC-‐/-‐ mice were also protected from 1K/DOCA/salt-‐induced increases in renal expression of the inflammatory genes IL-‐6, IL-‐17a, CCL2, ICAM-‐1 and VCAM-‐1, and accumulation of collagen.

Conclusion: Renal inflammation, fibrosis and elevated BP in response to 1K/DOCA/salt-‐treatment are critically dependent on inflammasome activity, highlighting this signalling complex and its cytokine products as potential therapeutic targets to treat hypertension.

1.6 Chloroquine, an inhibitor of endosomal Toll-‐like receptors, improves blood pressure and endothelial function in spontaneously hypertensive rats

C McCarthy1, C Wenceslau1, S Goulopoulou1, S Ogbi1, T Matsumoto2, C Webb1

1Georgia Regents University, 2Hoshi University

Objectives: Circulating mitochondrial DNA (mtDNA) is elevated in spontaneously hypertensive rats (SHR) and the enzyme responsible for its degradation, DNase II, has decreased activity in SHR tissues. Moreover, a synthetic mtDNA mimetic, specific for TLR 9, causes endothelial dysfunction and elevated blood pressure. Therefore, we sought to inhibit the contribution of mtDNA on innate immune system activation, via inhibition of Toll-‐like receptor (TLR)9, in SHR.

Methods: We treated 12-‐15 week old SHR and Wistar-‐Kyoto rats (WKY) with chloroquine (CQ; 40mg/kg/day) or vehicle (veh; saline) for 21 days via intraperitoneal injection (i.p.). We hypothesized that CQ treatment would improve endothelial function and decrease blood pressure in SHR. Blood pressure was measured pre-‐ and post-‐treatment via tail cuff and endothelial function was measured via mesenteric resistance artery (MRA) relaxation to acetylcholine (ACh; 10-‐9-‐10-‐5 M) using a wire myograph.

Results: Treatment with CQ lowered post-‐systolic blood pressure in SHR (mmHg, Veh: 201;2 vs. CQ: 185;5 mmHg; p<0.05). No effects of treatment were observed on blood pressure in WKY (p>0.05). CQ abolished the contractile effect to high concentrations of ACh [AUC (%NE pre-‐contraction), Veh: 160;25 vs. CQ: 277;20; p<0.05] in MRA from SHR. However, no differences were observed in endothelium-‐independent relaxation to NO-‐donor sodium nitroprusside [Emax (%NE pre-‐contraction), Veh: 97;12 vs. CQ: 102;26; p<0.05] in MRAs from SHR. Again, no effects of treatment were observed for endothelium-‐dependent or -‐independent relaxation in WKY (p>0.05).

Conclusions: These data demonstrate the inhibition of endosomal TLRs, including TLR9, improves blood pressure and endothelial function in SHR. Inhibition of TLR9 abrogates the potentially deleterious contribution of increased mtDNA and impaired DNase II activity on innate immune system activation in hypertension.

1.7 Vascular aging in aldosterone associated hypertension: role of NADPH oxidase 1 A Harvey1, AC Montezano1, D Graham1, Y He2, G Ceravolo2, C Yabe-‐Nishimura3, K Griendling4, R Touyz1 1University of Glasgow, 2University of Ottawa, 3Kyoto Prefectural University of Medicine, 4Emory University In hypertension, vascular aging is accelerated leading to heart failure, stroke and renal dysfunction. Cellular and molecular mechanisms of age-‐associated vascular changes are unclear, but enhanced ROS generation by NADPH oxidases (Nox) and aldosterone (aldo) have been suggested. Nox1 is increased in hypertension therefore, we postulated that aldo plays an important role in vascular aging through Nox1-‐dependent mechanisms. We examined signalling molecules associated with aging in arteries from 2 experimental models: stroke-‐prone spontaneously hypertensive rats (SHRSP) and Nox1 knockout mice infused with aldo (300 ug/Kg/day). Gene expression was assessed by qPCR and protein levels by immunoblotting. Aldo was measured by ELISA. In SHRSP rats, aging-‐associated mRNA inflammatory markers, RANTES (5-‐ fold), MCP-‐1 (6-‐fold) and IL-‐6 (2-‐fold); as well as aldo levels (6-‐fold) and H2AX (marker of aging-‐associated DNA damage – 1.5-‐fold) were increased compared with control rats, p<0.05. In mice treated with aldo, JNK (pro-‐inflammatory – 69%) and p66SHC (pro-‐senescence – 92%) activation were increased in mesenteric arteries (p<0.05); an effect blunted in vessels from Nox1 KO mice. In parallel studies, vascular smooth muscle cells (VSMCs) from adult and aged control mice, as well as, from adult Nox1 transgenic mice (Nox1 overexpression in VSMCs) were extracted and stimulated with H2O2 and aldo. Basal levels of p66SHC (39%) and OGG-‐1 (48%), markers of senescence, were increased in VSMCs from aged animals, p<0.05. H2O2 (64%) and aldo (84%) increased p66SHC activation in VSMCs from adult mice to similar levels observed in cells from aged animals (p<0.05). However, aldo effects on p66SHC activation were exacerbated in VSMCs from adult Nox1 transgenic mice. In conclusion, aldosterone may play an important role in the aging-‐like phenotype in vascular injury associated with hypertension. Such processes may involve Nox1 and redox-‐sensitive p66Shc signalling.


2.1 Use of mass spectrometry for the development of new biomarkers for diabetic nephropathy N Grobe1, S Gutta1, H Osman2, M Saklayen2, K Elased1

1Wright State University, 2Dayton Veterans Affairs Medical Center Diabetes and its associated chronic kidney disease (CKD) is a major health burden and there is an urgent need for new sensitive biomarkers to detect and monitor the progression of CKD. Albuminuria is still the gold standard for the evaluation of kidney function. However, its sensitivity and reliability have recently been questioned. Angiotensin converting enzyme 2 (ACE2) and neprilysin (NEP) are highly expressed in renal tubules and responsible for generation of renoprotective angiotensin (Ang) 1-‐7. The aim of the study was to investigate whether urinary ACE2 and NEP are increased in diabetic patients with CKD indicating early renal damage before the onset of microalbuminuria. Participants were recruited from the Dayton VA Medical Center. Baseline urinary albumin creatinine ratio (UACR) and estimated glomerular filtration rate (eGFR) were determined three months before initiation of the study in non-‐diabetic patients (UACR <30 mg/g, eGFR=97.40;16 ml/min/1.73 m2), and in diabetic patients with normoalbuminuria (UACR <30 mg/g, eGFR=83.08;17 ml/min/1.73 m2), microalbuminuria (UACR = 30-‐300 mg/g, eGFR=47.13;23 ml/min/1.73 m2), and macroalbuminuria (UACR >300 mg/g, eGFR=39.68;20 ml/min/1.73 m2). Mass spectrometry-‐based enzyme assays were used to detect the ACE2 and NEP product Ang-‐(1-‐7) (m/z 899) in incubations of urine samples with the natural ACE2 substrate Ang II (m/z 1046) or in incubations of urine samples with the natural NEP substrate Ang I (m/z 1296). Urinary ACE2 and NEP were significantly increased in diabetic patients compared with non-‐diabetic controls (p<0.01). Western blot analysis, ELISA and fluorogenic substrate assays confirmed these findings. Ang II or Ang I enzyme activities were blocked by specific ACE2 inhibitor MLN-‐4760 or NEP inhibitor thiorphan, respectively. NEP, but not ACE2, was detected in plasma of patients. In conclusion, urinary ACE2 and NEP are increased in diabetic patients with CKD before the onset of microalbuminuria which suggests their use as early, noninvasive biomarkers for diabetic nephropathy. 2.2 Effects of Carotid Body Tumor Resection on the Blood Pressure of Essential Hypertensive Patients M Fudim, K Groom, C Laffer, J Netterville, D Robertson, F Elijovich

Vanderbilt University

Objectives: Removal of carotid body (CB) improves rodent models of hypertension (HTN) and heart failure (CHF), presumably via withdrawal of chemoreflex-‐induced sympathetic activation. CB tumor (CBT) resection in humans abolishes the chemoreflex but is associated with opposing effects on sympathetic regulation by associated baroreflex damage. The net effect of CBT resection on blood pressure (BP) in subjects with pre-‐existent HTN is unknown.

Methods: We retrospectively identified 20 subjects with pre-‐existent HTN (BP≥140/90 mmHg or use of antihypertensive drugs) from among a series of 134 patients who underwent uni-‐ or bilateral CBT resection by the same surgeon (JLN) between 1990-‐2012. Two subjects were excluded from analyses because of inadequate follow-‐up data. Changes in systolic BP (SBP) were assessed: a) an acute ∆SBP from baseline (average of all readings for the 3 months preceding surgery) to the first reading after 30 days from the procedure, and b) a chronic ∆SBP, i.e., the slope of the regression of SBP on time over the entire follow up, expressed as ∆SBP/year. Acute ∆SBP was adjusted (covariate analyses) by the interval between pre and post BP readings and the change in therapy during this interval (using a medication score based on equipotency). Analogously, chronic ∆SBP was adjusted by the total duration of follow-‐up, the total number of SBP readings and the change in therapy during the entire period of each subject's follow-‐up.

Results: Age and duration of HTN were 56;4 and 9;5 years, respectively. There was a significant relationship between the adjusted acute ∆SBP and the adjusted chronic slopes (r=0.47, p<0.05) indicating that initial BP responses to CBT resection tend to be sustained. Regression analysis showed that for every change of 0.4 mmHg in acute ∆SBP, there was a concomitant 1 mmHg/yr change in the chronic slope. Twelve subjects (67%) had concordant reductions of ∆SBP and slope of any magnitude. In 6 of them (33%), acute ∆SBP was ≥ -‐10 mmHg (corresponding to a concomitant slope ≥ -‐5.9 mmHg/yr).

Conclusions: This is the first study to show that CBT resection is associated with a sustained reduction of BP in a subset of patients with comorbid HTN. Because concomitant baroreceptor damage by CBT surgery most likely leads to underestimation of the depressor effect of chemoreflex disruption, development of a targeted removal of the CB chemoreflex may conceivably have a role in the therapy of human hypertension.

2.3 Activation of AT1 receptors on dendritic cells restrains angiotensin II-‐dependent hypertension through a CCR7-‐mediated mechanism

JD Zhang, SD Crowley

Duke University Dendritic cells (DCs) trigger an adaptive immune response by presenting processed antigens to T lymphocytes. Given the emerging importance of adaptive immunity in hypertension, we posited that DCs may sample neoantigens in the kidney in order to prime pro-‐hypertensive T cells. Using CD11c-‐Cre mT/mG (DC GFP) mice in which DCs fluoresce green, we detected robust


green signals within the kidney after 2 wks of angiotensin (Ang) II-‐induced hypertension, confirming infiltration of DCs into the hypertensive kidney. As activation of type 1 angiotensin (AT1) receptors on other immune cell populations has been shown to modulate inflammatory responses, we explored the role of the DC AT1 receptor in blood pressure regulation by generating mice with DC-‐specific deletion of the AT1A receptor (CD11c Cre

+Agtr1aflox/flox= DC KO) and littermate (Agtr1aflox/flox = WT) controls. Compared to WTs, the DC KOs had an ≈87% reduction in AT1A receptor mRNA expression in DCs (p<0.0001) but had preserved AT1A expression in B and T lymphocytes, kidney, and heart (p=NS). Next, we infused uni-‐nephrectomized DC KO mice and wild-‐type (WT) controls with Ang II (500ng/kg/min) for 4 wks. At baseline, the mean arterial blood pressure (MAP) was similar in WT and DC KO mice (123;2 vs. 126;2 mm Hg; p=NS). By contrast, after the 1st week of Ang II infusion, DC KO mice had markedly exaggerated elevations in blood pressure (158;5 vs.174;4mm Hg; p=0.026). Expression of chemokine receptor CCR7 on the mature DC and the T cell directs both cell lineages to the lymph node where the DC can display a processed antigen and activate the T cell. . We detected higher levels of CCR7 mRNA in the kidneys from the Ang II-‐infused DC KOs compared to WTs (2.98;0.90 vs. 1.00;0.16 au; p=0.04), and posited that enhanced DC priming of T cells in the DC KOs contributed to their exaggerated hypertension. Consistent with this possibility, uni-‐nephrectomized CCR7-‐deficient (KO) mice had a significantly blunted chronic hypertensive response to Ang II (165;4 vs. 178;2 mm Hg; p=0.03) and ameliorated albuminuria (2.1;0.2 vs. 3.7;0.8 mg/24hrs; p=0.02). We conclude from these data that stimulation of AT1 receptors on DCs constrains Ang II-‐induced blood pressure elevation, possibly by limiting the CCR7-‐dependent activation of T lymphocytes by DCs. 2.4 The effects of positive allosteric modulation of GABAA receptors upon stress and hypertension in Schlager

hypertensive mice E Stevenson1, E Johns1, P Davern1, J-‐L Moretti1, K Jackson1, K Evans2, K Marques3, K Head1

1Baker IDI Heart and Diabetes Institute, 2Monash University, 3Federation University

An exaggerated pressor response to stress has been linked to the subsequent development of hypertension. Hypertensive Schlager mice (BPH/2J) have neurogenic hypertension associated with abnormal reactivity of neurons in the forebrain integrating the response to aversive stress. Recent studies suggest they also have functional and molecular differences in GABAA

receptors compared with their normotensive counterparts (BPN/3J, Marques et al, 2011). Allopregnanolone is an endogenous neurosteroid reduced by chronic stress and when administered, decreases anxiety by positive allosteric modulation of GABAAreceptors.

Objectives: To determine if allopregnanolone reduces the pressor effects of stress and basal mean arterial pressure (MAP) in BPH/2J mice.

Methods: Male BPN/3J (n=9) and BPH/2J (n=5-‐7) mice received vehicle or allopregnanolone (5mg/kg/day) via subcutaneous minipumps for two weeks. Prior implantation of telemetric probes enabled recording of MAP, heart rate (HR) and activity before and following minipump implantation. The cardiovascular response to aversive (dirty cage switch and restraint) and non-‐aversive (feeding) stress tests as well as ganglionic blockade with 5mg/kg pentolinium were recorded before and following minipump implantation. Changes in mRNA expression of GABAA were assessed by qRT-‐PCR.

Results: Allopregnanolone reduced systolic arterial pressure (-‐7.4mmHg,P<0.01) and diastolic arterial pressure (-‐4.46mmHg, P=0.02) and attenuated the depressor response to pentolinium in BPH/2J mice; whereas no effect upon MAP was observed in BPN/3J mice. Allopregnanolone produced marked reductions in the pressor response to both cage switch and feeding stress (-‐20%,P<0.01) in BPH/2J mice; whilst increasing the pressor response to aversive stress in BPN/3J mice (+33-‐48%,P<0.001). In addition, allopregnanolone selectively increased the expression of α4 and δ subunits of GABAA receptors which mediate tonic inhibition in the hypothalamus of BPH/2J mice (1.7-‐4.8 fold, P<0.05).

Conclusions: The selective antihypertensive and stress inhibitory effects of allopregnanolonein BPH/2J hypertensive mice suggests that allosteric modulation of GABAA receptors in the hypothalamus may be a major cause of hypertension in this model.

References Marques FZ, Campain AE, Davern PJ, Yang, YHJ, Head, GA and Morris BJ. Global identification of the genes and pathways differentially expressed in hypothalamus in early and established neurogenic hypertension. Physiol Genom. 2011;43:766-‐771.

2.5 Lymphocyte-‐Specific Adaptor Protein, LNK (SH2B3), Regulates Angiotensin-‐II Induced Hypertension and Renal/Vascular Inflammation M. Saleh, D. Harrison, M. Madhur Vanderbilt University, USA

LNK, an adaptor protein primarily expressed in hematopoietic cells and endothelial cells, is a negative regulator of cytokine signaling and cell proliferation. A single nucleotide polymorphism in LNK is associated with hypertension, but the mechanism is


unknown. Our objective was to determine the effect of LNK on hypertension and inflammation using LNK-‐/-‐ mice. In response to angiotensin II (Ang II) infusion, LNK-‐/-‐ mice exhibit elevated systolic BP (SBP) compared to wild type C57Bl/6J mice (WT) as measured by telemetry. At baseline, kidneys from LNK-‐/-‐ mice exhibit greater levels of total leukocyte and T lymphocyte infiltration, superoxide production, and albuminuria compared to WT mice, and these parameters are further exacerbated by AngII infusion. Aortas from LNK-‐/-‐ mice exhibit enhanced inflammation, reduced nitric oxide production, and impaired endothelial-‐dependent relaxation. Bone marrow transplantation studies showed that loss of LNK in hematopoietic cells (not somatic cells) reproduced the phenotype of whole body deletion of LNK with SBP reaching 180 mmHg in response to a subpressor dose of Ang II. Splenic T cells from LNK-‐/-‐ mice produce elevated levels of interferon-‐gamma (IFNg) compared to WT mice. Furthermore, IFNg-‐/-‐ mice exhibit blunted hypertension in response to AngII infusion, suggesting that enhanced IFNg production is at least partly responsible for the aggravated hypertension in LNK-‐/-‐ mice. Data are summarized in the table (expressed as mean;SEM, n=5-‐10,*P<0.05 vs WT/Sham, #P<0.05 vs WT/AngII and†P<0.05 vs LNK-‐/-‐/Sham). In conclusion, LNK may serve as a novel therapeutic target for hypertension and its associated renovascular disorders.

Methodology WT/Sham WT/AngII LNK-‐/-‐/Sham LNK-‐/-‐/AngII

SBP in response to AngII (490 ng/kg/min) (mmHg). Telemetry

131.83;3.92

(baseline)

167.35;4.8*

(Day 14)

135.47;4.83

(baseline)

196.02;5.83*#†

(Day 14) SBP in response to subpressor dose of AngII (140 ng/kg/min)

Telemetry 134.04;1.64

(baseline)

136.54;1.98

(Day 14)

128.29;2.65

(baseline)

174.47;6.04*#†(Day14)

Renal infiltrating CD45+ cells

(x103)/2 kidneys

Flow cytometry 32.92;3.11 74.82;17.05* 70.24;9.88* 123.41;8.57*#†

Renal infiltrating T cells (x103)/2 kidneys

Flow cytometry 5.41;0.45 15.58;4.60* 13.12;3.08 29.30;3.51*#†

Renal cortical ROS production

(2-‐OH-‐ethidium pmol/mg protein)

HPLC 86.44;12.43 160.11;23.24* 159.00;16.40* 222.56;35.76*

Renal medullary ROS production

(2-‐OH-‐ethidium pmol/mg protein)

HPLC 106.11;13.05 202.11;21.62 244.56;43.08* 285.56;50.90*

Albuminuria (µg/day) ELISA 10.20;1.20 94.51;5.76* 27.21;5.27* 305.01;15.32*#† Aortic infiltrating CD45+ cells (x103)/thoracic aorta

Flow cytometry 29.63;2.57 63.19;11.91 63.11;18.76 171.77;32.94*#†

Aortic infiltrating T cells

(x103)/thoracic aorta Flow cytometry 9.13;1.04 20.01;5.07 21.42;7.92 67.14;13.22*#†

Acetylcholine induced vascular relaxation (log EC50)

Vascular reactivity 6.77;0.12 6.24; 0.13* 6.60;0.10* 5.60; 0.08*#†

Aortic NO production (AU)

Electron Paramagnetic Resonance

0.015;0.002 0.008;0.0009 0.010;0.002* 0.009;0.0017*

Splenic CD4+ IFNg production (ng/ml)

Luminex Assay 14.47;2.10 202.42;12.92* 88.50;10.17* 377.91;10.37*#†

Splenic CD8+ IFNg production (ng/ml)

Luminex Assay 176.83;79.73 568.88;36.91* 725.01;109.10* 1014.49;64.37*#†


2.6 Impaired PVN neuronal activity in rats lacking central Gαi2 proteins: Implications for the pathophysiology of salt-‐sensitive hypertension

C Carmichael, A Carmichael, P Vartanyan, R Wainford

Boston University School of Medicine Objectives: We have demonstrated downregulation of central Gαi2 proteins produces global sympathoexcitation and elevated MAP in response to acute high-‐salt challenge. The current study sought to define whether alterations in PVN neuronal activity in rats lacking brain Gαi2 proteins underlie the sympathoexcitatory and pressor responses observed upon acute high-‐salt challenge. Methods: Twenty-‐four hour ICV scrambled (SCR) or Gαi2 oligodeoxynucleotide (ODN; 25μg/5μl)-‐pretreated (PT) conscious Sprague-‐Dawley rats were monitored for changes in MAP in response to IV bolus NaCl (3M; 0.14 ml/100g). Rats were sacrificed at control, 40-‐min, or 100-‐min post NaCl administration time points and cFos IHC was performed. PVN Fos-‐positive nuclei were quantified for each subregion based on the respective rostral-‐caudal level of each slice to determine the phenotype of activated cells. Results: In response to IV sodium, no difference was observed in sodium-‐evoked peak change in MAP between groups. In SCR PT rats, MAP returned to control levels by 100-‐min whereas Gαi2 PT rats remained significantly elevated (MAP [mmHg] 100-‐min post NaCl SCR: 134;2 vs. Gαi2: 146;3, P<0.05). A significantly greater number of Fos+ventrolateral parvocellular, lateral parvocellular, and medial parvocellular neurons were observed in SCR as compared to Gαi2 PT rats at both 40-‐ and 100-‐min. No difference between groups was observed in magnocellular neurons. Conclusion: These data highlight Gαi2 protein signal transduction as a novel CNS mechanism acting to influence PVN parvocellular neuronal activity with projections to the intermediolateral spinal cord and rostral ventrolateral medulla upon an acute sodium challenge. Sodium-‐induced increases in Fos+PVN parvocellular cells in SCR PT rats likely reflect activation of autonomic neuronal activity to facilitate sympathoinhibition and sodium excretion to maintain physiological blood pressure control. In Gαi2 PT rats, failure to activate parvocellular neuronal activity represents a mechanism by which impairment of Gαi2 signal-‐transduction may contribute to the pathophysiology of salt-‐sensitive hypertension. 2.7 Reduced uterine perfusion pressure (RUPP) elicits increased sFlt-‐1 levels not only in the placenta but also adipose

tissue F Spradley, A Palei, J Granger

University of Mississippi Medical Center

Preeclampsia is defined as new-‐onset hypertension during pregnancy. Placental ischemia is causative with the release of antiangiogenic factors such as sFlt-‐1 into the maternal circulation promoting endothelial dysfunction and hypertension. Obesity is a major risk factor for preeclampsia, but the mechanisms are unclear. Adipose tissue is a potential source of sFlt-‐1. We tested the hypothesis that placental ischemia produced by RUPP elicits an increase in sFlt-‐1 in both placental and adipose tissue from pregnant rats. Pregnant Wistar-‐hannover rats (20 wks old) on NIH31 standard chow were subjected to RUPP (N=15) on gestational day (GD)14 or remained normal pregnant (NP, N=14). Rats were implanted with carotid catheters on GD18 and fasted overnight. On GD19, mean arterial blood pressure (MAP) was assessed in conscious, restrained rats. Statistical significance was P<0.05. MAP was greater in RUPP (114;2 v 101;1mmHg) with reduced fetal weight (1.73;0.02 v 1.88;0.01g) but similar placental weight (RUPP: 0.45;0.03 v NP: 0.47;0.03g). Although RUPP reduced maternal body weight (297;7 v 343;6g), visceral adipose tissue weight was not altered (RUPP: 11.5;1 v NP: 13.2;1g). Plasma total cholesterol was increased (RUPP: 223;35 v NP: 156;6mg/dL) but there was no difference in free fatty acids (RUPP: 8;2 v NP: 8;2mg/L). RUPP reduced leptin (3.2;0.2 v 4.2;0.4ng/mL) and adiponectin (2.8;0.2 v 3.3;0.2ug/mL) with increased glucose levels (191;6 v 163;8mg/dL). Interestingly, RUPP increased sFlt-‐1 levels in placenta (4702;375 v 3903;309pg/g tissue) and retroperitoneal adipose tissue (179;28 v 76;22pg/g tissue). These data indicate that RUPP-‐induced hypertension promotes metabolic disturbances along with increases in sFlt-‐1 not only in the placenta but in the adipose tissue. In conclusion, we propose that placental ischemia-‐induced hypertension is exaggerated in states linked to increased accumulation of adipose tissue, such as in diet-‐induced or genetic obesity, due to amplified metabolic derangements and increases in sFlt-‐1 levels from both placental and adipose sources.


Abstracts of Poster Presentations P1. Beta3 adrenergic receptor activation increases anti-‐contractile effects of perivascular adipose tissue via cystathionine

gamma lyase and cyclooxygenase T Szasz1, T Matsumoto2, RC Webb1

1Georgia Regents University, 2Hoshi University Objectives: Perivascular adipose tissue (PVAT) influences vascular contraction and may play an important role in vascular dysfunction in hypertension, obesity and atherosclerosis. Activation of beta 3 adrenergic receptors (β3AR), expressed on adipocytes, leads to adipose tissue remodeling with increased brown adipocyte mitosis, metabolic activity and mitochondrial biogenesis. It is however unclear what would be the functional result of β3AR activation in PVAT. We hypothesized that in vivo treatment with CL316243, a β3AR agonist, would increase the anti-‐contractile effect of PVAT. Methods and results: We used male Sprague-‐Dawley rats (12 weeks) infused with 1 mg/kg/day CL316243 for 7 days via osmotic minipump to evaluate vascular function in PVAT-‐intact mesenteric arteries. We observed the decrease in size and visible browning of fat depots following β3AR agonist treatment. Expression of uncoupling protein 1 (UCP-‐1) was increased in mesenteric PVAT from CL316243-‐treated compared to control rats. PVAT induced a decrease in the contractile responses to phenylephrine (PE) and endothelin-‐1 (ET-‐1) in mesenteric arteries from both groups. and this anti-‐contractile effect of PVAT was augmented in segments from CL316243-‐treated rats. The acetylcholine (ACh)-‐induced relaxation response was increased in the presence of PVAT in mesenteric arteries from CL316243-‐treated rats and this increase was annulled in the presence of inhibitors for cyclooxygenase (COX) or of CSE substrate L-‐cysteine. CSE expression was increased and COX-‐1 expression was decreased in mesenteric PVAT from CL316243-‐treated rats compared to control rats. Conclusion: We conclude that β3AR activation in PVAT leads to increased anticontractile effects of PVAT, probably via increased hydrogen sulfide and decreased prostanoid synthesis. P2 AT2 receptor signaling participates in the conversion of white-‐to-‐beige fat K Tsukuda, M Mogi, H Hirotomo, H Kan-‐no, T Chisaka, T Wang, T Kukida, T Bai, T Shan, T Iwanami Department of Molecular Cardiovascular Biology and Pharmacology, Ehime University, Graduate School of Medicine

Objectives: We have previously reported that treatment with angiotensin II type-‐2 receptor (AT2R) agonist (compound 21; C21) ameliorated insulin resistance with the improvement of adipocyte dysfunction in type 2 diabetic mouse model, KKAy. Until quite recently, adipose tissue has been divided into two groups; white adipose tissue is known for its role in energy store, while brown adipose tissue function is known for energy expenditure (thermogenesis). In addition, new clusters of adipocyte with thermogenic capability have been discovered in white adipose tissue. These adipocytes have been named ‘beige’. However, the role of AT2R signaling in the conversion from white to beige fat has not been explored.

Methods: KKAy mice were subjected to i.p. injection of C21 for 2 weeks. Expression of thermogenic genes such as UCP1, Cidea, PGC-‐1a and PRDM16 in adipose tissue was determined with real-‐time RT-‐PCR. We also examined the difference in white-‐to-‐beige conversion between wild-‐type (WT) mice and AT2R knockout (AT2KO) mice. Mice were fed with a high-‐cholesterol diet (HCD) from 8 weeks of age for 2 weeks. Furthermore, 9-‐week-‐old mice were forced to swim test for investigating the effect of exercise on fat conversion.

Results: In KKAy mice, treatment with C21 increased the mRNA expression of thermogenic genes in subcutaneous fat, but not in visceral fat. However, adipose tissue weight was not changed by treatment of C21 and immunohistochemical analysis of UCP1 did not show the beige-‐like adipocyte. In AT2KO mice, baseline mRNA levels of thermogenic genes were lower than WT mice. In WT mice, swim exercise increased the mRNA expression level of UCP1 in subcutaneous fat; however the up-‐regulation in AT2KO mice did not reach the level of that in WT mice.

Conclusion: These results suggest that AT2R signaling partly participates in the conversion of white-‐to-‐beige fat; however, the details are under investigation.

P3 CETP inhibitors Torcetrapib, Dalcetrapib, and Anacetrapib induce adipocyte-‐derived Aldosterone production through Nox and STAT3 activation

FJ Rios1, K Neves2, A Nguyen Dinh Cat1, S Even1, R Palacios3, R Jenkins1, R Montezano1, R Touyz1 1University of Glasgow, 2University of Sao Paulo, 3Universidad Rey Juan Carlos Large clinical trials indicated that CETP inhibitors increase HDL levels, but had unexpected side effects, such as hyperaldosteronism and hypertension. Some CETP inhibitors accumulate in adipocytes, which are a source of aldosterone (Aldo). Objectives:We questioned whether CETP inhibitors influence Aldo production in adipocytes and assessed the role of reactive oxygen species (ROS) and STAT3 in this process. Methods: Human adipocytes-‐SW872, expressing CETP, were studied and compared to mouse adipocytes -‐3T3-‐L1, lacking CETP. Cells were treated with torcetrapib (TOR), dalcetrapib (DAL), or


anacetrapib (ANA). To evaluate the role of ROS, cells were pre-‐treated with N-‐acetylcystein (NAC–ROS scavenger), ML171 and GKT136901 (Nox1/4 inhibitor, respectively), and Rotenone (Rot, mitochondrial ROS inhibitor). ROS were measured by lucigenin and amplex-‐red. Results: Aldo production, measured by ELISA, was induced by TOR (668pg/mL), DAL (348pg/mL), and ANA (434pg/mL) (p<0.05); an effect blocked by NAC. TOR, DAL, and ANA increased superoxide (2-‐fold) and H2O2 (1.5-‐fold), and STAT3-‐phosphorylation (p<0.05). TOR increased Nox1, 4, and 5 (2-‐fold); DAL increased Nox4 (2-‐fold) and ANA increased Nox1 and 4 (3-‐fold). In TOR-‐treated cells, Aldo was inhibited by GKT (62%), ML171 (62%), Rot (45%), and S3I-‐201 (STAT3-‐inhibitor, 66%). DAL-‐induced Aldo production was blocked by ML171(62%) and S3I-‐201(74%). In ANA treated cells, Aldo production was inhibited by GKT (94%), ML171 (83%), and Rot (66%). We also assessed the chemerin production, an adipokine associated with hypertension. Only TOR stimulated chemerin production (331pg/mL vscontrol 154pg/mL p<0.05); an effect blocked by GKT (95%) and S3I-‐201 (100%). In mouse adipocytes, TOR, DAL, and ANA induced Aldo (453pg/mL, 375pg/mL, and 445pg/mL, p<0.05) and ROS (1.7-‐fold, p<0.05). Conclusions: CETP inhibitors induced aldosterone in human and mouse adipocytes through redox-‐related mechanisms involving STAT3, Noxs and mitochondria. These findings have important clinical significance and may explain, in part, the hyperaldosteronism and hypertension reported in CETP clinical trials. P4 Activation of central angiotensin II type 2 receptors attenuates neurogenic hypertension S Brouwers1, RD Wainford1, I Smolders2, AG Dupont2 1Boston University School of Medicine, 2Vrije Universiteit Brussel Aim: The role of the angiotensin II type-‐2 receptor (AT2R) in hypertension is under debate and the expression can be modulated in different pathological states. In hypertension AT2R are upregulated, therefore we hypothesize that central AT2R-‐stimulation will lower blood pressure in conscious spontaneously hypertensive rats (SHR).

Methods: SHR were implanted with a radio-‐telemetry device and an icv cannula connected to a miniosmotic pump delivering saline vehicle, AT2R agonist Compound 21 (C21) (0.002μg/μl/hr) alone or in combination with AT2R antagonist PD123319. MAP was assessed for 21 days: 7 days baseline (saline), 14 days treatment (e.g. C21). To assess the role of the angiotensin II type-‐1 receptor (AT1R) and NO in these responses, AT1R-‐blocker losartan or NO-‐synthase-‐inhibitor L-‐NAME were administered centrally with C21 (n=5-‐7/group).

Results: Icv C21-‐infusion blocked the increase in MAP seen in the vehicle group (MAP (mmHg): baseline: vehicle 153;5 vs C21 157;4; day 21 (D21): vehicle 164;5 vs C21 155;5;p<0.05). PD123319 abolished this hypotensive effect (MAP (mmHg): baseline 154;5; D21 163;5;p<0.05). Co-‐infusion of C21 and losartan did not reinforce the MAP lowering effect seen with C21 alone (MAP (mmHg): baseline 157;2; D21 153;1;p<0.05). Simultaneous L-‐NAME administration abolished the C21 effects (MAP (mmHg): baseline 156;4; D21 190;5;p<0.05).

Mechanistically, an improved parasympathetic control of HR was seen in the C21-‐treated group (change in HR after i.p. propranolol (bpm): vehicle -‐68.2;2.4 vs C21 -‐36.3;4.1;p<0.05). Impaired baroreflex sensitivity (BRS) also improved under C21-‐infusion (BRS (ms/mmHg) D21: vehicle 1.81;0.16 vs C21 3.15;0.07;p<0.05)

Conclusion: Selective central AT2R-‐stimulation with C21 attenuates hypertension and corrects autonomic dysfunction in SHR. These effects are mediated through a NO-‐dependent mechanism. In contrast to an enhanced peripheral AT2R-‐effect following AT1R-‐blockade, concomitant central AT1R-‐blockade did not enhance these responses to central AT2R-‐stimulation. These findings suggest activation of the central AT2R represent a possible new therapeutic target for the treatment of neurogenic hypertension.

P5 Impaired brain-‐derived neurotrophic factor-‐TrkB signaling in nucleus tractus solitarius during chronic heart failure blunts baroreflex sensitivity

B Becker, H Wang, I Zucker 1University of Nebraska Medical Center

Objective: Blunted baroreflex function is a negative prognostic marker of autonomic imbalance in chronic heart failure (CHF), yet the mechanisms underlying baroreflex blunting in CHF are not fully understood. The nucleus tractus solitarius (NTS) is the primary central target of baroreceptor afferents, and plays a critical role in regulating central baroreflex sensitivity. Brain-‐derived neurotrophic factor (BDNF) and its receptor, TrkB, are highly expressed in the NTS, but little is known about the role of BDNF signaling in the NTS. We hypothesized that in the NTS, BDNF enhances baroreflex sensitivity, and impaired BDNF-‐TrkB signaling contributes to the blunted baroreflex function in CHF.

Methods: To test this hypothesis, 50 nl of BDNF or the selective TrkB antagonist ANA-‐12 were microinjected bilaterally into the dorso-‐medial NTS of sham-‐operated rats and myocardial infarct-‐induced CHF rats.


Results: BDNF evoked a greater decrease in blood pressure (MAP), heart rate (HR), and renal sympathetic nerve activity (RSNA) in sham vs. CHF rats. Injection of ANA-‐12 increased MAP, HR, and RSNA to a greater extent in sham vs. CHF rats (Table).

Sham

CHF

ΔMAP

(mmHg)

ΔHR

(bpm)

ΔRSNA

(%baseline)

ΔMAP

(mmHg)

ΔHR

(bpm)

ΔRSNA

(%baseline) BDNF

(100 pg) -‐31.5;3.2 -‐52.0;8.3 -‐70.0;4.1 -‐12.0;2.8* -‐20.5;4.1* -‐29.5;4.4*

ANA12 (125 µM)

32.0;2.8 37.5;3.8 61.0;5.0 8.4;1.1* 11.4;2.5* 21.0;3.0*

Changes are relative to baseline; *p<0.05 vs. sham; n=4/group

Following 125 µM ANA-‐12, baroreflex sensitivity was blunted in sham rats (3.7;0.3 vs. 1.9;0.1 bpm/mmHg max gain, p<0.05 before vs. after, n=4). CHF rats had blunted baroreflex sensitivity vs. sham (2.3;0.1 bpm/mmHg, p<0.05, n=4), and ANA-‐12 had little effect on baroreflex sensitivity in CHF (2.0;0.1 bpm/mmHg, n=4).

Conclusions: These data suggest that endogenous BDNF plays an important role in maintaining baroreflex sensitivity in the NTS, and that BDNF-‐TrkB signaling is impaired in CHF, which may contribute to blunted baroreflex sensitivity and autonomic imbalance.

P6 Contribution of orexin to neurogenic hypertension in BPH/2J mice K Jackson1, T Nguyen-‐Huu1, E Stevenson1, P Davern1, P Carrive2, P Head1

1BakerIDI Heart and Diabetes Institute, 2University of New South Wales

Objectives: Schlager BPH/2J mice are a genetic model of hypertension associated with an overactive sympathetic nervous system (SNS). Orexin is a neuropeptide which can regulate sympathetic activity, blood pressure and stress and its expression is greater in the hypothalamus of BPH/2J mice compared with normotensive BPN/3J control mice (Marques et al, 2011). The aim of the present study was to determine whether enhanced orexinergic signalling contributes to hypertension in BPH/2J mice.

Methods: BPH/2J and BPN/3J mice (n=3-‐4) were pre-‐implanted with radiotelemetry probes (DSI) to measure mean arterial pressure (MAP). During the dark (active) period the dual orexin receptor 1 and 2 antagonist, Almorexant (Actelion) was administered via intraperitoneal injection (i.p. 30 and 100mg/kg) and gavage (p.o. 100 and 300mg/kg). MAP was recorded for 6 hours and compared with baseline values 1 hour before treatment. Mid frequency (0.3–0.5 Hz) MAP power was used as an indicator of SNS activity in vehicle and Almorexant (i.p. 100mg/kg) treated mice.

Results: Baseline MAP was greater in BPH/2J compared with BPN/3J mice (135;2 vs 111;2mmHg respectively, P<0.001). Administration of Almorexant at 100mg/kg (i.p.) and 300mg/kg (p.o.) caused sustained hypotensive responses over the 6 hour period in BPH/2J mice (-‐18.1;2.0 and -‐9.2;1.6mmHg respectively) which were markedly greater than the effect of vehicle administration (~0.1;1.1mmHg, P<0.002). By contrast the responses to Almorexant in BPN/3J mice at all doses and routes were comparable with vehicle (P>0.09). Mid frequency MAP power was lower in BPH/2J mice following Almorexant treatment compared with vehicle (P<0.001), whilst there was no difference in BPN/3J mice (P=0.65).

Conclusion: The present results demonstrate that inhibition of orexin 1 and 2 receptors with Almorexant delivered either orally or i.p. can reduce BP and SNS activity in BPH/2J mice, suggesting that enhanced orexinergic signalling contributes to overactivation of the SNS and hypertension in BPH/2J mice.

References Francine Z. Marques, Global identification of the genes and pathways differentially expressed in hypothalamus in early and established neurogenic hypertension, Physiological Genomics, 43: 766–771, April 2011.

P7 Overexpression of calcium/calmodulin-‐dependent protein kinase II (CaMKII) potentiates angiotensin II (AngII)-‐mediated signaling in neurons

U Basu, AJ Case, J Liu, Y Li, MC Zimmerman 1University of Nebraska Medical Center AngII modulates neuronal ion channel activity and neuronal firing via reactive oxygen species (ROS) and redox-‐sensitive proteins. Our recent studies indicate that in neurons AngII increases activity (i.e. phosphorylation) of CaMKII, a signaling intermediate which inhibits potassium (K+) channel current. However, the potential cross-‐talk between AngII, ROS, and CaMKII in modulating


neuronal K+ channel activity and in vivo central AngII-‐induced pressor response remains unclear. Here, we tested the hypothesis that CaMKII overexpression exacerbates the AngII-‐dependent inhibition of K+ channel current and the acute central AngII-‐induced increase in blood pressure. Neuron specific CaMKIIα was cloned, and CaMKIIα adenovirus was generated (AdCaMKIIα). Adenovirus-‐mediated overexpression of total and active CaMKII protein were assessed in differentiated mouse catecholaminergic (CATH.a) neurons by measuring total and phosphorylated CaMKII protein levels, respectively, via Western blot analysis. To investigate the effect of CaMKIIα overexpression on K+ channel activity, voltage-‐gated K+current (IKV) was measured by the whole cell patch-‐clamp technique in CATH.a neurons transduced with AdCaMKIIα or control adenovirus, AdEmpty. Baseline IKV was measured followed by 5 mins of AngII (100 nM) superfusion. AngII lowered steady-‐state current (ISS) and peak current (Ipeak) in AdEmpty-‐transduced neurons (ISS by 20, 4%; Ipeak by 19, 3%), which was further significantly potentiated in AdCaMKIIα-‐transduced neurons (ISS by 39, 5%; Ipeak by 40, 4%; p< 0.05). In vivo studies suggest that the central AngII-‐induced pressor response is exacerbated in mice intracerebroventricularly injected with AdCaMKIIα as compared to control virus, AdGFP (10 mmHg change in mean blood pressure in AdGFP-‐injected mice vs 19 mmHg in AdCaMKIIα-‐injected mice; p< 0.05). These data indicate that CaMKIIα overexpression potentiates AngII-‐mediated inhibition of IKv and the central AngII-‐induced pressor response. Future studies will investigate ROS-‐mediated post-‐translational modifications of CaMKIIα in AngII-‐stimulated neurons and the contribution of these modifications to AngII signaling in neurons. P8 Capsaicin mediated afferent renal denervation: Pharmacological confirmation and implications for the

pathophysiology of hypertension A Carmichael, R Wainford

Boston University School of Medicine Aim: We assessed the efficacy of capsaicin-‐mediated denervation in preventing the physiological responses to activation of the afferent but not efferent renal nerves and in the development of salt-‐sensitive hypertension. Methods: Conscious Sprague-‐Dawley (SD) rats implanted with an ICV cannula, having undergone sham (S) or renal afferent nerve denervation (ADNX) (capsaicin 33mM), received intra-‐renal (IR) infusions of bradykinin (BK) (5-‐40 μg/kg/min) and adenosine (A) (2-‐12 μg/min). All animals then received ICV guanabenz (50ug) (N=5/gp). HR, MAP and sodium excretion were continuously monitored. Dahl salt-‐sensitive (DSS) rats underwent sham, ADNX or bilateral renal denervation (RNDX) surgery (N=5/group) – all animals were maintained on an 8% NaCl diet and MAP was monitored by radiotelemetry. Results: IR BK dose-‐dependently increased MAP and HR in sham, but not ADNX rats (BK 40 μg/kg/min; peak ΔHR [bpm] S +61;8 vs ADNX -‐5;3, P<0.05; peak ΔMAP [mmHg] S +19;3 vs ADNX +0.6;2, P<0.0.5). IR adenosine evoked a dose-‐dependent increase in natriuresis in sham animals, a response abolished by ADNX (A 12 μg/min; peak ΔUNaV [ueq/min] S +18.7;3 vs ADNX +2;0.4, P<0.05). In both sham and ADNX rats, ICV guanabenz evoked profound natriuresis (guanabenz 50 μg; peak ΔUNaV [μeq/min] S +13.4;1 vs ADNX +12.8;1). In DSS rats a high salt diet evoked hypertension in sham rats, hypertension was attenuated in RDNX rats but exacerbated in ADNX rats (DSS High-‐salt diet day 21 ΔMAP [mmHg]; S +30;3, RDNX +20;2*, ADNX +45;6*, P<0.05). Conclusion: Capsaicin-‐mediated afferent renal denervation prevents the physiological responses to activation of the renal afferent nerves without affecting the function of the renal efferent nerves, indicated by the natriuresis to central α2-‐adrenoceptor stimulation. Afferent renal denervation exacerbated DSS hypertension, suggesting a critical role of the renal afferent nerves as a negative feedback mechanism to evoke efferent renal nerve (and global) sympathoinhibition and the attenuation of hypertension. P9 Testosterone propionate in Ischemia-‐reperfusion (I/R) induced Acute Kidney Injury (AKI) CN Patil, R Maranon, C Dalmasso, H Zhang, LA Juncos, JF Reckelhoff


Acute kidney injury (AKI) is a leading cause of morbidity and mortality, and men are more prone to AKI than women implicating androgens as a causative factor for AKI susceptibility. We showed recently that infusion of testosterone 3 hrs post reperfusion protected rats from renal injury following ischemia/reperfusion (I/R). In the present study, we tested the hypothesis that testosterone works as a renal vasodilator perhaps by increasing nitric oxide. Male SD rats (8-‐14 wks; n=3-‐5/grp) were subjected to sham surgery or I/R induced AKI with bilateral clamping of renal vessels for 30 min. Three hrs after reperfusion, rats were given testosterone propionate (20 μg/kg i.v. over 10 min) or vehicle (0.75%EtOH). Rats were placed in metabolism cages for 24hrs for nitrate/nitrite excretion (UNOxV), and euthanized to collect blood for creatinine (PCr). I/R increased PCr and decreased UNOxV, compared to shams (0.58;0.05 vs. 3.99;0.36 mg/dL, p<0.0001; UNOxV: 8.54;1.7 vs 1.7;0.78 µmol/day/kg BW, p<0.001). Testosterone attenuated the increase in PCr (2.03;0.23 mg/dL, p< 0.01) but had no effect on UNOxV (3.79;0.91 µmol/day/kg). Pretreatment of rats with L-‐NAME (1mg/kg/day) for 48 hours prior to I/R abolished the improvement in PCr with testosterone (PCr: 3.73;0.82 mg/dL, p<0.05 vs no LNAME). Infusion of renal vasodilator PGE2 (30µg/kg iv over 10 min) attenuated PCr compared to I/R alone (2.95;0.21mg/dL, p<0.01) but was independent of increased NO (2.61;0.63 µmol/day/kg, p=NS vs I/R alone), and was not as effective as testosterone (p<0.05). These data show that low dose testosterone infusion 3 hrs post reperfusion improves renal function in AKI perhaps by causing renal vasodilation. The known renal vasodilator PGE2 also improves PCr in I/R, but not as effectively as testosterone. These result support the notion that testosterone infusion has therapeutic potential for the treatment of I/R induced AKI in humans. Supported by NIH R01HL66072, P01HL05971 and AHA 14POST18640015.


P10 Telmisartan attenuates renal dysfunction in high-‐salt-‐loading RAS activated mice J Iwanami, M Mogi, K Tsukuda, XL Wang, M Kukida, H Nakaoka, T Chisaka, H Bai, B Shan, M Horiuchi Ehime University, Graduate School of Medicine Objective: It is well known that high-‐salt intake and activation of renin angiotensin system (RAS) induces hypertension in part via an increase in renal injury and sympathetic nerve activity. We investigated the effect of telmisartan on salt-‐loading RAS activated mice.

Method: Ten weeks old male transgenic mice carrying human renin and angiotensinogen genes (hRN/hANG-‐Tg) were administrated control chow or the chow containing 8% NaCl with or without telmisartan (1 mg/kg/day) and PPAR-‐gamma antagonist, GW9662, in drinking water for 8 weeks. Blood pressure was measured by radio telemetry method. Concentration of urinary sodium was assessed before and after salt-‐loading. Urinary excretion of adrenaline and noradrenaline were measured by ELISA. Expression of ENaC in kidney was measured by real-‐time RT-‐PCR.

Results: Body weight did not differ in all groups. Survival rate 8 weeks after treatment was decreased in salt-‐loading hRN/hANG-‐Tg mice. This decrease was improved by treatment with telmisartan. Systolic blood pressure in salt-‐loading hRN/hANG-‐Tg mice at night was higher compared with control hRN/hANG-‐Tg mice and administration of telmisartan decreased blood pressure. Treatment with GW9662 did not influence these effects of telmisartan. Concentration of urinary sodium was increased by salt-‐loading, whereas this increase was decreased age-‐dependently. Expression of ENaC-‐alpha mRNA did not differ between control and salt-‐loading hRN/hANG-‐Tg mice. Treatment with telmisartan decreased expression of ENaC-‐alpha mRNA. ENaC-‐beta and gamma expression was increased by salt-‐loading. These increases were reduced by treatment with telmisartan. However, this reduction by telmisartan was not observed with co-‐administration of GW9662. Urinary excretion of adrenaline and noradrenaline were higher in salt-‐loading hRN/hANG-‐Tg mice. These increases were reduced by treatment with telmisartan. Co-‐treatment with GW9662 did not influence these effects of telmisartan.

Conclusion: These results suggested that salt-‐loading enhanced an increase in blood pressure and sympathetic activity and decrease renal function in concert with activated RAS. P11 Soluble guanylate cyclase (sGC) stimulator reduces the cardiac and renal damage in angiotensin II (Ang II)-‐induced

diastolic heart failure N Haase1, N Wilck1, L Marko1, A Heuser2, D Brockschnieder3, D Kretschmer3, D Stasch3, D Dechend1, D Mueller1 1Experimental and Clinical Research Center (ECRC), 2Max-‐Delbrueck Center for Molecular Medicine, 3Bayer HealthCare,

Global Drug Discovery Diastolic heart failure (DHF) is estimated to account for approximately 40% of heart failure cases. Due to the high prevalence and high mortality rates, diastolic heart failure represents a major challenge in cardiovascular medicine. The novel therapeutic alternative with NO-‐independent direct stimulators of sGC has recently been approved for the treatment of pulmonary hypertension.

We analyzed the role of the sGC stimulator BAY 41-‐8543 in a transgenic rat model of hypertension-‐induced diastolic heart failure. We used 4 week-‐old male double transgenic rats harboring both human renin and angiotensinogen genes (dTGRs). At age 7 weeks, dTGR show striking cardiac hypertrophy with fibrosis, severe diastolic dysfunction but preserved systolic function, albuminuria, and renal failure. We compared vehicle-‐treated dTGR receiving 10% transcutol, 20% cremophor, 70% water, dTGR receiving 3mg/kg/d BAY 41-‐8543, and vehicle-‐treated SD control rats (single oral dose per day for 3 weeks).

Systolic blood pressure increased progressively in vehicle-‐treated dTGRs compared with SD rats. BAY 41-‐8543 significantly reduced the blood pressure (197 +/-‐ 11 mmHg vehicle vs 133 +/-‐ 4 mmHg BAY 41-‐8543). Treatment with sGC stimulator ameliorated albuminuria (0.3 +/-‐ 0.06 mg/8h at week 7) compared with vehicle-‐treated dTGRs (12.4 +/-‐ 2.6 mg/8h at week 7). In addition BAY 41-‐8543 prevented fibrosis and inflammation in the kidney and heart. Cardiac hypertrophy or myocyte size were not reduced by BAY 41-‐8543 treatment. Cardiac echocardiography and hemodynamic investigations showed that BAY 41-‐8543 increased ejection fraction and improved diastolic function including strain rate and tissue doppler imaging. Programmed electrical stimulation showed a high non-‐sustained and sustained ventricular tachycardia induction rate in vehicle-‐treated dTGRs (46%), which was significantly reduced in BAY 41-‐8543-‐treated dTGR (11%). Finally, BAY 41-‐8543 treatment resulted in 100% survival at week 7, whereas 76% of vehicle-‐treated dTGRs died.

Our data demonstrate that sGC stimulators ameliorate cardiac and renal end-‐organ damage including electrical remodeling in a transgenic rat model of hypertension-‐induced DHF. Treatment of DHF with sGC stimulators offers a novel therapeutic potential.

P12 Redox-‐regulated suppression of resting splenic T-‐lymphocytes during sympathoexcitation-‐associated hypertension AJ Case, MC Zimmerman


University of Nebraska Medical Center

Over-‐activation of the sympathetic nervous system (i.e.sympathoexcitation) elevates circulating levels of norepinephrine (NE), which significantly contributes to hypertension and end organ damage. However, it is not clearly understood how NE affects various components of the immune system, such as resting T-‐lymphocytes. Herein, we tested the hypothesis that splenic T-‐lymphocyte activation is altered in NE-‐infused hypertensive mice. First, we observed subcutaneous infusion of NE (3.8 μg/kg/min) significantly increased circulating levels of NE (5.8 fold) and mean arterial pressure (20.6 +/-‐ 0.6 mmHg), which validated our model of NE-‐driven hypertension (p<0.05 vs. saline infused). Furthermore, splenic T-‐lymphocytes from NE-‐infused mice showed an approximate 20% +/-‐ 5% decrease in proliferation accompanied by a 50% +/-‐ 17% and 85% +/-‐ 6% reduction in interferon gamma (INFγ) and tumor necrosis factor alpha (TNFα) production respectively as compared to T-‐lymphocytes from saline-‐infused mice (p<0.05). Additionally, NE directly inhibited naïve T-‐lymphocyte proliferation and cytokine production ex vivo in a dose dependent manner. While NE did not demonstrate any pro-‐apoptotic effects on T-‐lymphocytes, a 21% +/-‐ 2% increase in G1 arrest was observed in NE-‐treated T-‐lymphocytes, and this was accompanied by a 60% +/-‐ 4% decrease in pro-‐growth cyclin D3, E1, and E2 mRNA expression (p<0.05 vs. saline). Interestingly, NE caused a 71% +/-‐ 17% (p<0.05 vs. saline) increase in cellular superoxide (O2

●-‐) production as evidenced by dihydroethidium (DHE) oxidation, which was shown to be partially-‐causal to the inhibitory effects of NE as the addition of Tempol, a O2

●-‐scavenger, to the drinking water of NE-‐infused mice was able to moderately restore T-‐lymphocyte growth and pro-‐inflammatory cytokine production while decreasing intracellular O2

●-‐ levels. Overall, our data indicates that increased NE has profound inhibitory effects on the resting population of T-‐lymphocytes, and may predispose hypertensive individuals to improper inflammatory responses during immune challenges.

P13 Castration impairs rat internal pudendal artery reactivity and structure R Lopes, K Neves, M Silva, V Olivon, S Ruginsk, S Ramalho, S Rodrigues, S Tostes, S Carneiro

University of Sao Paulo Testosterone deficiency is strongly associated with erectile dysfunction (ED). Inadequate penile arterial flow is one of the major causes of ED. Considering that blood flow to the corpus cavernosum is derived from the internal pudendal arteries (IPAs), we hypothesized that castration induces impairment of IPAs reactivity and structure, contributing to ED. Male Wistar rats were studied 30 days after castration (Cast). Functional and structural properties of rat IPA were determined in wire and pressure myograph systems, respectively. Castrated rats exhibited impaired erectile function, represented by decreased intracavernosal pressure/mean arterial pressure ratio [ICP/MAP (sham = 0.5, 0.1; castrated = 0.2, 0.1; 20 Hz. p <0.05). Castrated rats exhibited decreased phenylephrine (Phe)-‐ [Control: 175.4, 4.6vs Cast: 134,9, 11; Emax] and electrical field stimulation (EFS)-‐induced contraction (in mN) [Control: 213,0, 7.1 vs Cast: 137, 6.1; 12Hz] and decreased acetylcholine (ACh)-‐ [Control: 79.7, 2.2 vs Cast: 49.8, 2.8; Emax] and EFS-‐induced vasodilatation [Control: 54.6, 2.1vs Cast: 35.5, 1.6; 12hz]. IPAs from Cast rats exhibited decreased internal diameter [Control (µm): 499,9, 35.9vs Cast: 413.0, 14.8; 60 mmHg], external diameter [Control (µm): 669.4, 40.1 vs Cast: 552.0, 30.0; 60 mmHg], thickness of the arterial wall [Control (µm): 81.2, 6.4 vsCast: 55.8, 7.5; 80 mmHg] and cross-‐sectional area [Control (µm2): 157217, 12533 vsCast: 116383, 12424; 30 mmHg]. Castration decreased nNOS expression (60%) and increased p38 (Thr180/Tyr182)phosphorylation (450%), as well as cleavage of caspase 3 (270%). In conclusion, testosterone deficiency is associated with impairment of IPA reactivity and structure and increased apoptosis signalling markers. Our findings suggest that hypogonadism may contribute to IPA dysfunction, which can leads to ED. Financial support: FAPESP and CNPq. P14 17beta-‐Estradiol and 16alpha-‐Hydroxyestrone Increase Oxidative Stress Through Nrf2 Dysfunction In Human

Pulmonary Artery Smooth Muscle Cells – Implications in Pulmonary Hypertension K Hood1, RA Lopes1, A Johansen1, AC Montezano1, C Szyndralewiez2, P Page2, MR MacLean1, R Touyz1 1University of Glasgow, 2Genkyotex

Gender differences in pulmonary arterial hypertension (PAH) may be, in part, due to increased formation of the deleterious estrogen metabolite, 16α-‐hydroxyestrone (16αOHE1). Oxidative stress and Noxs have been implicated in the pathogenesis of PAH. We hypothesised that 17β-‐estradiol (E2) and 16αOHE1, specifically in human pulmonary artery smooth muscle cells (PASMCs), leads to Nox-‐induced oxidative stress, which promotes PASMC damage. Cultured human PASMCs were stimulated with either E2 (1nM) or 16αOHE1 (1nM). ROS production was assessed by chemiluminescence (O2-‐) and Amplex Red (H2O2); antioxidants, regulators of Nrf2, and PCNA (marker of growth) expression by immunoblotting; and Nrf2 activity by ELISA. E2 increased superoxide (219%) and H2O2 (52%) in PASMCs (p<0.05 vs vehicle). E2 induced ROS was blocked by PHTPP (ERβ antagonist), tempol (SOD mimetic), ML171 (Nox1 inhibitor) and GKT137831 (Nox1/4 inhibitor). Thioredoxin (71%), NQ01 (78%) and peroxiredoxin1 (69%) protein levels were decreased by E2, even though Nrf2 activity was increased (38%), p<0.05 vs vehicle. 16αOHE1 exhibited a rapid (5 min) and exaggerated increase in superoxide (355%) and a decrease in H2O2 (65%) production, p<0.05. 16αOHE1-‐induced ROS was blocked by MPP (ERα antagonist), G15 (GPR30 antagonist), tempol and ML171. 16αOHE1 increased BACH1 (129%; p<0.05), a competitor of Nrf2, which was decreased (92%). E2 stimulation resulted in decreased PCNA expression (30%), while 16αOHE1 increased PCNA levels (150%); p<0.05. E2 and 16αOHE1 induced a rapid and sustained ROS generation in PASMCs derived from PAH subjects. E2 and 16αOHE1 did not increase superoxide production in VSMCs from resistance arteries of healthy subjects. In conclusion, E2 induces ROS production through ERβ-‐Nox-‐dependent mechanisms,


while 16αOHE1 increases superoxide through an ERα/GPR30-‐Nox-‐dependent manner. The effects of E2 and 16αOHE1 on oxidative stress is present in PASMCs but not in VSMCs from peripheral arteries. and seems to be related to a dysregulation of the Nrf2 pathway. These processes may impact on molecular processes contributing to vascular remodeling in PAH.

P15 Fetal growth restriction is a risk of vascular remodeling T Chisaka, M Mogi, H Nakaoka, M Kukida, H Kan-‐no, H Tsukuda, H Wang, H Bai, H Iwanami, H Higaki

Ehime University Graduate School of Medicine Objective: We investigated the effect of FGR on inflammatory vascular remodeling using a cuff-‐ induced vascular injury mouse model.

Methods: Dams (C57BL/6J strain mice) were fed an isocaloric diet containing 20% protein (normal protein; NP) or 8% protein (low protein; LP) from 10 weeks of ages. On the day of delivery, all dams were returned to the NP diet. After weaning, offspring were fed the NP diet. Vascular injury was induced by polyethylene cuff-‐placement around the femoral artery in offspring at 10 weeks of age. Neointima formation was evaluated by Elastica van Gieson staining 2 weeks after cuff-‐placement. We assessed the following parameters in the femoral arteries prepared one week after cuff-‐placement. Inflammatory cytokine and NADPH oxidase subunit were assessed by RT-‐PCR. Superoxide anion production, cell proliferation were evaluated by dihydroethidium staining, proliferating cell nuclear antigen (PCNA) staining respectively. HIF-‐1 expression was evaluated by RT-‐PCR and immunoblot analysis.

Results: Birth weight in offspring from dams fed LP until delivery (LPO) was significantly lower at birth, but the same at 2 weeks after birth compared with that in NP offspring (NPO). Arterial blood pressure at 12 weeks of age did not differ between LPO and NPO. Neointima formation was more exaggerated in LPO compared with NPO associated with an increase in cell proliferation assed by proliferating cell nuclear antigen staining index. Moreover, LPO showed enhanced expression of monocyte chemotactic protein-‐1, interleukin (IL)-‐6, IL-‐1b, tumor necrosis factor-‐a, and production of superoxide anion in the injured artery. Moreover, mRNA expressions of isoforms of NAD(P)H oxidase subunits such as p22phox, p40phox, p47phox, p67phox, gp91phpx, and Rac1 in the injured arteries were enhanced in LPO. HIF-‐1 expression was increased in LPO more than that in NPO.

Conclusion: These results suggest that FGR is a risk for vascular remodeling in later life after birth.

P16 G protein-‐coupled estrogen receptor contributes to the vascular effects of aldosterone and type two diabetes-‐associated vascular dysfunction.

N Ferreira1, S B A Cau2, C P Manzato1, M A B Silva1, F S Carneiro1, F C A Tostes1 1University of Sao Paulo, 2University of Juiz de Fora

Aldosterone (Aldo) excess aggravates endothelial dysfunction in diabetes. Aldo exerts its effects via activation of both mineralocorticoid receptors (MR) and G protein-‐coupled estrogen receptors (GPER). Considering that GPER activation has beneficial effects in the vasculature, we hypothesized that GPER-‐mediated vascular effects of aldosterone are decreased/abrogated in diabetes. Second-‐order mesenteric arteries from control (B6BKS-‐Leprdb/+) and db/db female mice were incubated with 10nM Aldo or vehicle (veh), the MR antagonist eplerenone (Eple, 10µM) or the GPER antagonist G15 (1μM), and the effects on phenylephrine (Phe) vascular reactivity were determined. Aldo increased Phe maximal response (Emax, % KCl contraction) in arteries from control (veh:112.5;3.2 vs. Aldo:129.1;2.8 p<0.05), but not in arteries from db/db (veh:143.8;4.9vs. Aldo:136.0;5.2 p>0.05). In control vessels, Eple did not alter Phe Emax either in the presence of Aldo or veh (p>0.05), whereas G15 abrogated Aldo-‐induced increase in Phe Emax (Aldo:129.1;2.8vs. G15+Aldo:110.3;3.6 p<0.05). In db/db arteries, the Eple and G15 decreased Phe Emax both in the presence of veh and Aldo (veh:143.8;4.9 vs Aldo:136.0;5.2vs. Eple+veh:105.4;4.8 vs. Eple+Aldo:94.6;4.4 vs. G15+veh:96.4;4.2 vs. G15+Aldo:104.3;4.1 p<0.05). Arteries from db/db exhibited increased ERK1/2 activation vs. control [arbitrary units (Au), 229.4;3 vs. 100.0;11.8, respectively, p<0.05]. Aldo increased ERK1/2 activation in control (Au, 99.8;0.4 vs. 272.2;58.0 p<0.05) and db/db (Au,99.7;4.0 vs. 162.4;28.2 p<0.05). Eple and G15 reduced Aldo-‐induced ERK1/2 activation in control arteries (p<0.05). In db/db arteries, G15 decreased ERK1/2 activation both in the presence of veh and Aldo (Au, Aldo:162.4;28.2 vs. G15+veh:81.1;9.1 vs. G15+aldo:82.7;7.5 p<0.05). In summary, Aldo acutely increases contractile responses to PhE in mesenteric arteries from control animals by GPER-‐dependent mechanisms. In db/db, both MR and GPER seem to contribute to Aldo effects in the vasculature. Contrary to our hypothesis, GPER contributes to the vascular effects of Aldo in db/db as well as to diabetes-‐associated vascular dysfunction. Financial Support: CNPq, CAPES, FAPESP.

P17 Increased adrenergic contractions, but preserved relaxations to insulin in renal arteries of obese mice O Baretella, A Xu, P Vanhoutte

Department of Pharmacology & Pharmacy and State Key Laboratory of Pharmaceutical Biotechnology, The University of Hong Kong


Objectives: Obesity promotes hypertension. However, mice on a high fat diet do not exhibit an elevated arterial blood pressure, despite an increased heart rate. The renal circulation contributes to the regulation of blood pressure because of its sensitivity to vasoconstrictor stimuli. Less is known about relaxations in renal arteries from obese mice. The present experiments aimed to compare responsiveness in renal arteries from lean and obese mice.

Methods: Systolic arterial pressure and heart rate were measured [tail-‐cuff method] in obese [high fat diet over 30 weeks, 49.7;0.9 g] and lean [32.5;1.4 g] male C57BL/6N mice. Isometric tension was recorded in main and segmental renal artery rings in Halpern-‐Mulvany myographs. Increasing cumulative concentrations of contracting and relaxing agents were added.

Results: Systolic arterial pressure was similar between lean and obese mice, but heart rate was significantly increased in the latter (679;10 vs 632;16 bpm, n=10, P<0.05). Contraction-‐response curves to phenylephrine were shifted significantly to the left and the α1-‐adrenergic agonist was more effective in main renal arteries from obese mice (pD2 6.47;0.06 vs 6.12;0.06 in lean, Emax 145;3% vs 126;3% high K+, n=6, P<0.001). These differences were reduced in the presence of 3x10-‐4 mol/L L-‐NAME [inhibitor of nitric oxide synthases preventing basal NO release] with a remaining leftward shift in rings from obese animals (pD26.92;0.05 vs 6.68;0.07 in lean, n=6, P<0.01). Relaxations [in the presence of 10-‐6 mol/L meclofenamate] to acetylcholine (Emax 97;2% vs 90;3% phenylephrine, n=6) and also insulin were preserved in contracted segmental rings from obese animals

Conclusions: Renal arterial vascular smooth muscle of obese mice appears more responsive to α1-‐adrenergic vasoconstrictor stimulation, which may be due to an increased intrinsic responsiveness and/or reduced basal release of NO. However, endothelium-‐dependent relaxations to acetylcholine and insulin are sustained, which could help to prevent these hyperinsulinemic animals from becoming hypertensive.

P18 Chemerin Decreases Vascular Insulin Signaling K Neves1, N Lobato2, R Lopes1, F Mestriner3, A Oliveira3, A Tostes3 1University of Glasgow, 2University of Goias, 3University of Sao Paulo

Proinflammatory adipokines are key mediators of insulin resistance. The adipokine chemerin influences major aspects of the metabolic syndrome, including adipogenesis and glucose homeostasis in adipocytes and skeletal muscle cells. Amongst its many actions insulin also influences vascular function. Considering that chemerin impairs vascular reactivity, and that its effects in the vascular actions of insulin have not been investigated, we postulate that chemerin decreases vascular responses to insulin by reducing PI3K/Akt signaling. Isometric force was recorded in mesenteric arteries from C57Bl6 mice, incubated with chemerin (0.5 ng/mL, 1 h) or vehicle (veh). Chemerin decreased relaxation responses to insulin (0.1 -‐ 3000 ng/mL, pD2: chemerin: 94.5;0.1 vs. veh: 23.6;0,1) and to ACh (pD2: 5.2;0.1 vs. veh 6.6;0.1). Chemerin effects on insulin-‐induced vasodilatation were reverted by the PI3K activator YS-‐49 (pD2: veh= 23.0;0.1; chem= 108.5;0.1, YS-‐49+chem= 28.5;0.1) and by inhibitors of p38 (pD2: veh= 23.0;0.1; chem= 108.5;0.1, SB203580+chem= 27.1;0.1) and ERK1/2 (pD2: veh= 24.6;0.1; chem= 112.0;0.1, PD98059+chem= 25.4;0.1). Chemerin also decreased PI3K [arbitrary units (a.u.): 0.8;0.1 vs. veh 1.1;0.1) and Akt (a.u.: 0.8;0.1 vs. veh 1.2;0.1) phosphorylation in cultured vascular smooth muscle cells (VSMCs). Furthermore, chemerin inhibited insulin-‐stimulated glucose uptake by VSMCs ([3H]2-‐deoxy-‐glucose (2DG) uptake, counts per minute: 5668;729.0 vs. veh 9923;662.7). In conclusion, chemerin decreases insulin vascular responses by reducing PI3K/Akt signaling and by activating the MAPK pathway. These results suggest that chemerin may be involved in the pathogenesis of vascular insulin resistance. Our study may contribute to a better understanding of the role of factors released by the adipose tissue on insulin vascular responsiveness and, consequently, on the vascular dysfunction in obesity and obesity-‐associated diseases. Financial Support: FAPESP.

P19 Role of vascular smooth muscle cell PPARgamma in aldosterone-‐induced vascular injury S Ouerd1, M Trindade1, N Idris-‐Khodja1, T Barhoumi1, S Offermanns2, S Gonzalez3, S Paradis1, EL Schiffrin4

1Lady Davis Institute for Medical Research, 2Max-‐Planck-‐Institute for Heart and Lung Research, 3National Cancer Institute, 4McGill University

Background: Peroxisome proliferator activated receptor γ (PPARγ) agonists improve vascular remodeling and endothelial dysfunction in hypertensive rodents and humans. PPARγ activation in vascular smooth muscle cells (VSMC) may be responsible for the vascular protective effects of PPARγ agonists. We previously observed a protective role of VSMC PPARγ in angiotensin II-‐induced endothelial dysfunction and vascular remodeling (Marchesi et al 2013). However, it is unknown whether VSMC PPARγ plays a similar protective role in adverse vascular effects of aldosterone. We hypothesized that inactivation of the Ppar gene in VSMC (smPparγ-‐/-‐) would exaggerate aldosterone-‐induced vascular injury.

Methods: Using a tamoxifen-‐inducible Cre/loxP system, Pparγ was ablated in VSMC of adult mice. Thirteen week-‐old control and smPparγ-‐/-‐ mice were infused or not with aldosterone (400 μg/kg/d, SC) for 14 days. Endothelial function and vascular remodeling were assessed in mesenteric arteries (MA) by pressurized myography.


Results: Endothelium-‐dependent relaxation (EDR) responses to acetylcholine were reduced to a similar extent in smPparγ-‐/-‐ and aldosterone-‐treated control and smPparγ-‐/-‐ mice compared to control mice (Emax: 62.5;8.7%, 50.8;8.6% and 56.8;7.9%, respectively, vs 86.4;3.2%, P<0.05). L-‐NAME, an inhibitor of nitric oxide (NO) synthase, completely blocked EDR in the four groups. Endothelium-‐independent relaxation response to the NO donor sodium nitroprusside and contractile responses to norepinephrine were similar in the four groups. Preliminary data indicated that aldosterone tended to increase MA stiffness in control mice, as shown by a leftward shift of the stress/strain relationship curve (strain at 140 mmHg, 0.79;0.07 vs 0.89;0.03). Furthermore, Pparγ deletion induced an increase in MA stiffness compared to control, which was not worsened by aldosterone (strain at 140 mmHg, 0.67;0.01, 0.65;0.03, vs 0.89;0.03).

Conclusion: These results indicate that either VMSC Pparγ inactivation or aldosterone treatment induce vascular remodeling and endothelial dysfunction, which are not mutually exaggerated. This suggests that PPARγ and aldosterone signal intracellularly through different pathways.

References: Marchesi, C., A. Rehman, Y. Rautureau, D. A. Kasal, M. Briet, A. Leibowitz, S. M. Simeone, T. Ebrahimian, M. F. Neves, S. Offermanns, F. J. Gonzalez, P. Paradis and E. L. Schiffrin (2013). Protective role of vascular smooth muscle cell PPARgamma in angiotensin II-‐induced vascular disease. Cardiovasc Res 97(3): 562-‐570.

P20 has been withdrawn by the authors. P21 Endothelin-‐1 overexpression preserves endothelial function in mice with vascular smooth muscle cell-‐specific

deletion of ppar-‐gamma N Idris Khodja1, S Ouerd1, T Barhoumi1, M Trindade2, J Gornitsky1, A Rehman1, S Offermanns3, F Gonzalez4, P Paradis1,

EL Schiffrin5

1Hypertension and Vascular Research Unit, Lady Davis Institute for Medical Research, 2Department of Clinical Medicine, State University of Rio de Janeiro, 3Department of Pharmacology, Max-‐Planck-‐Institute for Heart and Lung Research, 4Laboratory of Metabolism, Division of Basic Sciences, National Cancer Institute, National Institutes of Health, 5McGill University

Background: Peroxisome proliferator-‐activated receptor γ (PPARγ) agonists reduce blood pressure (BP) and vascular injury in hypertensive rodents and humans. Pparγ inactivation in vascular smooth muscle cells (VSMC) using a tamoxifen inducible Cre-‐Lox system enhanced angiotensin II-‐induced vascular remodeling and endothelial dysfunction. Transgenic mice overexpressing endothelin (ET)-‐1 selectively in the endothelium (eET-‐1) exhibit endothelial dysfunction, increased oxidative stress and inflammation. We hypothesized that inactivation of the Ppar gene in VSMC (smPparγ-‐/-‐) will exaggerate ET-‐1-‐induced vascular damage.

Methods/Results: Eleven week-‐old male control, eET-‐1, smPparγ-‐/-‐and eET-‐1/smPparγ-‐/-‐ mice were used. BP was determined by telemetry, mesenteric artery (MA) reactivity and structure by pressurized myography, reactive oxygen species (ROS) by dihydroethidium staining and expression of inflammatory markers by immunofluorescence. Systolic BP was 10 to 20 mmHg higher in eET-‐1 and eET-‐1/smPparγ-‐/-‐ compared to control and smPparγ-‐/-‐ (P<0.05). Endothelium-‐dependent relaxation (EDR) responses to acetylcholine were impaired 37% in smPparγ-‐/-‐ (P<0.05) but not in eET-‐1 and eET-‐1/smPparγ-‐/-‐ compared with control. Endothelium-‐independent relaxation responses to the nitric oxide donor, sodium nitroprusside, were similar in all groups. Media/lumen at 45 mmHg was increased 20% in eET-‐1/smPparγ-‐/-‐ compared with control (P<0.05). A similar increase in MA stiffness was observed in eET-‐1, smPparγ-‐/-‐ and eET-‐1/smPparγ-‐/-‐ compared to control, as indicated by a leftward displacement of the stress-‐strain curves (P<0.05). ROS levels were 1.7-‐fold greater in eET-‐1, 2.2-‐fold in smPparγ-‐/-‐ and 2.8-‐fold in eET-‐1/smPparγ-‐/-‐compared with control (P<0.05). Monocyte chemoattractant protein-‐1 levels were 1.7-‐fold higher in smPparγ-‐/-‐ compared with control (P<0.05), which was not exaggerated by ET-‐1 overexpression. Monocyte/macrophage specific antigen-‐2-‐positive cells in perivascular fat were ~2-‐fold higher in eET-‐1 and in smPparγ-‐/-‐ compared with control (P<0.05), which was further increased 2.0-‐fold in eET-‐1/smPparγ-‐/-‐ (P<0.05).

Conclusion: These results suggest that increased ET-‐1 paradoxically preserves endothelial function in mice with inactivated VSMC Pparγ,

P22 Chronic treatment with fluoxetine increases relaxation of rat resistance mesenteric arteries via ATP-‐sensitive potassium channels activation

C A Pereira, N S Ferreira, F L A C Mestriner, L B Resstel, F S Carneiro, F C A Tostes University of Sao Paulo

Fluoxetine, a selective serotonin reuptake inhibitor (SSRI) has properties that go beyond its antidepressant effects and alters mechanisms involved in the regulation of vasomotor tone. While there are many studies demonstrating the acute effects of fluoxetine in the vasculature, studies on the chronic effects of this SSRI are still limited. Here we postulated that chronic


treatment with fluoxetine enhances vascular reactivity to vasodilator stimuli by increasing nitric oxide (NO) signaling. The effects of chronic treatment with fluoxetine on vascular reactivity were determined in resistance mesenteric arteries from Wistarrats, which were treated with (I) vehicle (water for 21 days) or (II) fluoxetine (10 mg/kg/day for 21 days in the drinking water). Fluoxetine treatment increased endothelium-‐dependent (pEC50, Veh = -‐7.08;0.07; Fluox = -‐7.4;0.11, p<0.05) and -‐independent relaxant response (pEC50, Veh = -‐7.75;0.08; Fluox = -‐8.5, 0.11, p<0.05). Fluoxetine also increased vascular NOx (NO metabolites) levels (Veh = 1.2;0.13 µM/µg; Fluox = 2.0, 0.13 µM/µg, p <0.05), nitric oxide sintase (NOS) activity (Fluox = 76%) and phosphorylation of endothelial NOS (eNOS) at serine1177 [arbitrary units (a.u.), Veh = 0.36;0.10; Fluox = 1.2, 0.08, p <0.05]. Fluoxetine treatment did not change neuronal NOS (nNOS) or soluble guanylate cyclase (sGC) expression neither vascular responses to cyclic guanosine monophosphate (cGMP) or sGC activators. However, pinacidil-‐ (KATPchannels activator)-‐induced relaxation was increased by fluoxetine treatment(pEC50, Veh = -‐5.9;0.12; Fluox = -‐6.5;0.17, p>0.05). In conclusion, chronic treatment with fluoxetine increases endothelium-‐dependent and -‐independent relaxation response in resistance mesenteric arteries by mechanisms that involve increased NOS activity, NO generation and KATP channels activation. These effects may contribute to the cardiovascular side effects associated with fluoxetine treatment.

Financial Support: CNPq, CAPES.

P23 Endothelin-‐1 overexpression in endothelial cells increases blood pressure in an endothelin type A receptor-‐dependent manner

S Coelho1, Y Rautureau1, A Rehman1, S Offermanns2, P Paradis1, EL Schiffrin3

1Lady Davis Institute for Medical Research, 2Goethe University Frankfurt, 3Magill University. Background: The mechanisms of blood pressure (BP) regulation by endothelin (ET)-‐1 produced by endothelial cells (EC) are complex and remain unclear. Transgenic mice with constitutive EC-‐specific human ET-‐1 (EDN1) overexpression presented vascular injury but no change in BP, which could be due to adaptation to life-‐long high ET-‐1 exposure (Amiri et al. 2004) . We have now generated an inducible EC-‐restricted EDN1overexpressing mouse (ieET-‐1) in order to demonstrate the effects of ET-‐1 on BP regulation independent of developmental effects.

Method/Results: Two transgenic mouse lines (C134 and C170) expressing chloramphenicol acetyltransferase and EDN1 before and after Cre-‐mediated excision, respectively, were crossed withmice expressing tamoxifen-‐inducible CreERT2under the control of Tie2 promoter (ieCre) to generate ieET-‐1 mice. Mice were treated with tamoxifen (1 mg/kg/day, SC) or vehicle for 5 days and sacrificed 16 days later.Additional mice were treated with 5 or 10 mg/kg/day PO of ET type A receptor blocker, atrasentan, from day 9. BP was determined by telemetry, plasma ET-‐1 levels by ELISA and ET type A (Ednra) and B (Ednrb) receptors expression in the kidney by quantitative RT-‐qPCR.

Tamoxifen induced a 10-‐fold increase of plasma ET-‐1 in ieET-‐1-‐C134 and 13-‐fold in ieET-‐1-‐C170 (P<0.01), compared to control ieET-‐1 and tamoxifen-‐treated ieCre. ET-‐1 overexpression increased night systolic BP by ~20 mmHg in ieET-‐1-‐C134 and ieET-‐1-‐C170 compared to tamoxifen-‐treated ieCre, which was reversed partially or completely by 5 or 10 mg/kg/day of atrasentan, respectively (P<0.01). Tamoxifen-‐treated ieET-‐1-‐C134 presented a 17-‐fold increase in Ednrbexpression in renal cortex (P<0.05)and no change in renal medulla compared to control ieET-‐1, whereas renal Ednraexpression was unchanged.

Conclusion: This new inducible EC-‐restrictedEDN1 overexpressing mouse exhibits ET-‐1-‐dependent elevated BP mediated by ET type A receptors. Increased ET type B receptor expression in renal cortex could play a role in ET-‐1-‐induced BP rise.

References: Amiri, F., A. Virdis, M. F. Neves, M. Iglarz, N. G. Seidah, R. M. Touyz, T. L. Reudelhuber and E. L. Schiffrin (2004). Endothelium-‐restricted overexpression of human endothelin-‐1 causes vascular remodeling and endothelial dysfunction. Circulation 110(15): 2233-‐2240.

P25 Adipocyte-‐derived aldosterone and cortisol are Nox1/4 dependent: implications in obesity-‐associated vascular dysfunction

S Even

University of Glasgow We previously demonstrated that aldosterone (aldo) is produced by adipocytes, an effect associated with reactive oxygen species (ROS) and adipokine production. These processes are exaggerated in obesity. Whether ROS themselves play a role in adipocyte-‐derived aldo is unclear. Studies were performed in db/m (lean) and db/db (obese) mice, treated with low (20mg/kg/day) or high dose (60mg/kg/day) GKT137831 (GKT, Nox1/4 inhibitor, 16 weeks). Epididymal (EVAT) and perivascular (PVAT) fat were collected. Human adipocytes (SW872) were also studied. Aldo and corticosterone levels were measured by ELISA. Gene expression was assessed by qPCR. ROS generation was assessed by chemiluminescence and amplex red. Plasma aldo levels in db/db (pg/mL: 518 vs. 272g) and aldo levels in culture media from db/db adipocytes were increased (pg/mL/µg RNA: 1964 vs. 388), p<0.05. All effects were decreased by high dose GKT. In PVAT, CYP11B2 gene expression was increased in


db/db (2.6;0.8 vs control 1.1;0.1, p<0.05), an effect blocked by Nox1/4 inhibition. Corticosterone levels in culture media from db/db adipocytes were also increased. Gene expression of adipocyte differentiation marker, AP2, was increased (3.5;1.1 vs control 1.4;0.4) in obese mice. GKT decreased AP2 levels. In human adipocytes, AngII stimulation increased aldo (6 fold) and cortisol (4 fold) production, as well as superoxide (1 fold) and H2O2 (2 fold) levels (p<0.05 vs vehicle). Increased levels of superoxide by Ang II were blocked by GKT and ML171 (Nox1 inhibitor); while Ang II-‐induced H2O2 production was inhibited only by GKT. Ang II-‐induced aldo production was blocked by tempol (SOD mimetic), GKT and ML171. In contrast, cortisol was only blocked by tempol and GKT. In conclusion, aldo production in adipocytes is dependent on ROS formation and involves Nox1 and Nox4. These data suggest that Nox1/4 may play a role in adipocyte-‐derived aldosterone and cortisol production. Our findings suggest that adipocyte Nox1/4 may be a putative therapeutic target in obesity-‐associated hyperaldosteronism and cardiovascular damage. P26 Nox isoforms, oxidative stress, inflammation and fibrosis in adipose tissue in obese mice: differential regulation in

epididymal, perirenal and perivascular depots S Even

University of Glasgow

Adipose tissue inflammation and fibrosis contribute to cardiovascular complications observed in obese patients. Mechanisms related to these deleterious effects are still elusive. Here, we evaluated the oxidative, fibrotic and inflammatory status of obese mice and the putative role of NADPH oxidases. Epididymal (EPF), perivascular (PVF) and peri-‐renal (PRF) fat were collected from lean (db/m) and obese (db/db) mice. Gene expression was assessed by qPCR, fibrosis by picro Sirius red staining and polarized light microscopy, oxidative stress by Amplex Red (H2O2) and TBARS. In EPF, increased levels of TBARS (56%), CD206 mRNA (macrophage marker – 106%) and fibrosis (300%) were observed in obese mice (p<0.05 vs lean). Similar findings were found in PRF, where H2O2 (65%), TBARS (50%), and macrophage infiltration (F4/80 mRNA – 578%) levels were increased (p<0.05 vs lean). In PVF, H2O2(42%, p<0.05) levels, but not TBARS, were increased. mRNA levels of inflammatory marker such as, CD206 (120%), F4/80 (550%), TNFα (206%), iNOS (46%), as well as collagen 6a (marker of fibrosis), were increased in PVF from obese animals (p<0.05 vs lean). Treatment of obese mice with GKT137831 (GKT, Nox1/4 inhibitor, 16 weeks) did not decrease inflammation/fibrosis in EVF and increased mRNA levels of inflammatory markers in PRF. GKT decreased mRNA levels of pro-‐inflammatory markers, followed by an increase in mRNA levels of anti-‐inflammatory markers, in PVF. Levels of Nox2 (EPF: 1936%, PRF: 336%, PVF: 358%, p<0.05 vs lean), but neither Nox1 nor Nox4, were increased in any fat depots. In conclusion, oxidative stress is increased in fat from obese animals and may play a role in obesity-‐associated adipose tissue inflammation and fibrosis. Our data also suggests that Nox1/4 influence adipose inflammation and fibrosis in an adipose tissue-‐specific manner, with effects being pro-‐inflammatory in PRF and anti-‐inflammatory in PVF. Our findings highlight an important role for Nox isoforms in perivascular adipocyte tissue inflammation/fibrosis in obesity.

P27 AMP-‐activated Protein Kinase Activator AICAR Attenuates TNF-‐alpha-‐induced Inflammation In Murine Adipocytes A White, A Nguyen Dinh Cat, C Jenkins, S Mancini, A Montezano, A Salt, R Touyz University of Glasgow, Objectives: Adipose tissue, a metabolically active tissue and important regulator of vascular function, contributes to obesity-‐related cardiovascular diseases such as hypertension. Molecular mechanisms are still elusive, but inflammation and activation of a local renin-‐angiotensin-‐aldosterone system (RAAS) seem to play an important role in adipocyte dysfunction leading to vascular damage. AMP-‐activated protein kinase (AMPK), a key regulator of cell metabolism, may play an important role in adipose tissue biology/inflammation. In this study, we evaluated whether AMPK activation regulates TNFα-‐induced inflammation and RAAS status in adipocytes.

Methods: Murine adipocytes (3T3-‐L1) were used and treated with 10ng/ml TNFα in the absence/presence of AICAR (1mM – AMPK activator). Pro-‐inflammatory markers were assessed by qPCR or ELISA and protein expression by immunoblotting.

Results: TNFα stimulation increased IL-‐6 (113.4 fold) and MCP-‐1 (449.6 fold) mRNA levels, as well as, IL-‐6 release (17.67 fold) into culture medium in 3T3L1 adipocytes (p<0.05, vs vehicle); an effect abrogated by AICAR (75% reduction MCP-‐1 mRNA, 60% reduction IL-‐6 mRNA and 30% reduction IL-‐6 release). Adiponectin mRNA levels were decreased by TNFα (60%, p<0.05 vs vehicle), yet preincubation with AICAR had no effect. The effects of TNFα were also evaluated on components of the RAAS. TNFα induced a decrease in angiotensinogen (60%), mineralocorticoid receptor (70%) and AT2R (90%) mRNA levels in adipocytes (p<0.05), which was unaffected by AICAR. Activation of AMPK also suppressed TNFα-‐stimulated phosphorylation of MAP kinases and NFκB assessed by immunoblotting.

Conclusions: Our data highlights AMPK as an important anti-‐inflammatory signaling protein in adipose tissue and therefore an interesting potential target for the treatment of obesity-‐related vascular injury leading to hypertension. This is especially significant given that the commonly used drug metformin is an AMPK activator.


P28 FOXP3+ T regulatory lymphocytes counteract angiotensin ii-‐induced vascular injury MOR Mian1, T Barhoumi1, M Briet2, AC Ene1, P Paradis1, EL Schiffrin1

1Lady Davis Institute for Medical Research, McGill University, 2Centre Hospitalo Universitaire d'Angers, Universite d'Angers

Objectives: T effector lymphocytes contribute to vascular injury in angiotensin II-‐induced hypertension, but the role of T regulatory cells (Treg) is unclear. Angiotensin II-‐induced hypertension is blunted in T and B cell-‐deficient (Rag1-‐/-‐) mice, and restored with reconstitution of T cells. [1] We hypothesized that adoptive transfer of FOXP3-‐deficient (Scurfy) vs. wild-‐type T cells will exacerbate angiotensin II-‐induced vascular damage in Rag1-‐/-‐mice.

Methods and Results: Eleven-‐week old male Rag1-‐/-‐ mice were injected IV with vehicle, 10 million wild-‐type or Scurfy T cells, 1 million CD4+CD25+ Treg alone or with Scurfy T cells, and 2 weeks later were infused or not with angiotensin II (490 ng/kg/min, SC) for 14 days (n=3-‐8). Telemetric systolic and diastolic blood pressure (SBP and DBP), and mesenteric arteries (MA) function and structure, reactive oxygen species (ROS) production and fibronectin expression were assessed.

Angiotensin II induced a 40 mmHg SBP rise in vehicle, wild-‐type and Scurfy T cells groups, but DBP rise was ~10 mmHg greater in wild-‐type and Scurfy T cell-‐injected mice than in vehicles (P<0.01). Wild-‐type Treg injection alone tended to reduce SBP rise but did not affect DBP compared to control. Adoptive transfer of wild-‐type T cells restored angiotensin II induced-‐endothelial dysfunction (P<0.05), which was exaggerated in Scurfy T cell-‐injected mice (P<0.01) but not in mice receiving Scurfy T cells + wild-‐type Treg (P<0.05). Angiotensin II increased ROS production in MA wall and perivascular fat in Scurfy T cell-‐injected mice (P<0.01), but not when co-‐transferred with wild-‐type Tregs. Angiotensin II induced vascular stiffness (P<0.01) and hypertrophic remodeling (P<0.05) in vehicle and Scurfy T cell-‐injected mice, but not in other groups. Angiotensin II increased MA fibronectin expression (P<0.05) only in vehicle and Scurfy T cell-‐injected mice.

Conclusion: These results demonstrate that Foxp3+ T regulatory lymphocytes have a protective role against angiotensin II-‐induced vascular damage.

References: [1] Guzik TJ, Hoch NE, Brown KA, McCann LA, Rahman A, Dikalov S, Goronzy J, Weyand C, Harrison DG. Role of the t cell in the genesis of angiotensin ii induced hypertension and vascular dysfunction. The Journal of experimental medicine. 2007;204:2449-‐2460.

P29 High salt (NaCl) reduces the activation and function of alternatively activated (M2) macrophages via epigenetic modification

K Binger1, M Gebhardt1, C Rintisch1, M Henig1, A Schroeder2, W Neuhofer3, K Hilgers3, A Manzel8, C Schwartz9, M Kleinewietfeld4

1Experimental and Clinical Research Ctr and Max-‐Delbrueck Ctr for Molecular Med, 2Friedrich-‐Alexander Univ of Erlangen-‐Nuremberg, 3University of Munich, 4Yale School of Medicine

High intake of dietary salt (sodium chloride; NaCl) is attributed to the development of hypertension and cardiovascular disease, and has been proposed to be a cause for the rapid increase in autoimmune diseases in western civilisations. We have recently shown that NaCl has a pro-‐inflammatory effect and boosts the activation of Th17 cells in vitro, where mice fed a high salt diet have an accelerated and more severe experimental autoimmune encephalomyelitis. In this study, we examine how the activation of alternatively activated (M2) macrophages is affected by NaCl. In stark contrast to our study with Th17 cells, we find that high salt dose-‐dependently decreased M2 activation in IL-‐4+IL-‐13 stimulated BM-‐derived mouse macrophages. Genes important for M2 activation, including Mrc1, Arg1, Ym1, Fizz1 and PD-‐L2, all had a blunted expression in the presence of NaCl; an effect which was not observed in tonicity controls (mannitol or urea), implying a specific action of NaCl. In contrast to our previous findings in Th17 cells, the effect of salt on M2 activation was not mediated via Sgk-‐1. To explore the mechanism for the effect of NaCl on M2 activation we performed gene expression analysis simultaneously with genome wide chromatin modification analysis (H3K4me3 and H4ac). The results of this revealed that NaCl modulated epigenetic marks at genes important for M2 activation, and additionally identified new genes which were affected. Finally, we asked if these salt-‐mediated changes in M2 genes, translated into effects on their function. Using an in vitro assay, we found that NaCl-‐treated M2 macrophages have a reduced ability to suppress the activation of effector T cells. Our study reveals a novel effect of NaCl on M2 activation and function, and gives support to the notion that the modulation of immune cell function by high dietary salt is relevant to hypertension and autoimmune diseases.

P30 Chemerin, a novel adipokine, regulates human vascular cell function through redox-‐sensitive processes involving Nox1/4 and eNOS

K Neves1, R Lopes1, N Lobato2, A Cat Nguyen1, A Oliveira3, A Montezano1, A Tostes1, R Touyz1 1University of Glasgow, 2University of Goias, 3University of Sao Paulo;


Adipose tissue produces many adipokines, including chemerin, a chemoattractant for inflammatory cells that mediates its effects through chemokine-‐like receptor 1 (CMKLR1). Chemerin has been implicated in endothelial dysfunction and vascular inflammation, hallmarks of vascular injury in hypertension. These pathological processes are induced by reactive oxygen species (ROS). How chemerin regulates ROS is unknown. We postulated that chemerin, through redox-‐sensitive mechanisms, influences signalling in vascular cells. Human vascular smooth muscle cells (HVSMCs) and microvascular endothelial cells (HMECs) were studied. HVSMCs and HMECs were stimulated with recombinant chemerin (50ng/ml). eNOS and MAPK activation were assessed by immmunoblotting, ROS generation by chemiluminescence and nitric oxide (NO) levels by DAF-‐FM fluorescence. mRNA expression of Nox 1, Nox 4 and inflammatory markers was assessed by real time PCR. Chemerin stimulated ROS generation in HVSMCs (1 h -‐ 21%; 8 h – 41% increase) and HMECs (1 h -‐ 54% increase; 24 h -‐ 76% increase, p<0.05 vs vehicle), effects blocked by Nox1/4 inhibitor (GKT137831) and Nox1 inhibitor (ML171). Chemerin also increased phosphorylation of ERK1/2 (43% and 62%), p38MAPK (18% and 25%) and JNK (59% and 73%) in HVSMCs and HMECs respectively. eNOS phosphorylation (Ser1177, activation site) was decreased by chemerin in HMECs (45% decrease), while phosphorylation at nhibitory site, Thr495, was increased (72%). In parallel, chemerin decreased NO levels in HMECs by 33%. Finally, chemerin increased IL-‐6 (290%), MCP-‐1 (64%), VCAM-‐1 (210%) and ICAM-‐1 (130%) mRNA levels in HVSMCs. Nox 1 (49%) and Nox 4 (67%) mRNA levels were increased in HVSMCs stimulated with chemerin. In conclusion in human vascular cells, chemerin stimulates ROS generation and reduces NO formation, through Nox1/4 activation and eNOS inhibition respectively. These processes were associated with increased redox-‐sensitive MAPK signaling and inflammation. Our study identifies chemerin as a new vasoactive factor, which plays an important role in vascular injury and dysfunction.

P31 Acute increase in O-‐GlcNAcylation reduces the release of cytokines and attenuates hypotension in mice with LPS-‐induced sepsis

V Olivon, R Ferreira, F Mestriner, F Cunha, J Alves-‐Filho, J Tostes University of Sao Paulo -‐ USP

The attachment of β-‐O-‐linked N-‐Acetylglucosamine (O-‐GlcNAc) is a common post-‐translational modification controlled by two enzymes: O-‐GlcNAc transferase (OGT) and β-‐N-‐acetylglucosaminidase (OGA). Acute increases in O-‐GlcNAc reduce release of pro-‐inflammatory mediators. We postulated that acute increases of O-‐GlcNAc reduce sepsis-‐associated mortality, release of pro-‐inflammatory mediators and changes in blood pressure and vascular reactivity. C57BL/6 mice received lipopolysaccharide (LPS) injections to produce mild (LPS M, 10mg/Kg, i.p.) or severe (LPS S, 20mg/Kg, i.p.) sepsis. Animals received glucosamine (GlcN), 300mg/Kg, i.v.) or vehicle 30 min. before the LPS administration and were euthanized 6 h later. GlcN treatment increased survival in mice with LPS-‐induced sepsis (LPS M=20%*; LPS S=50%*). GlcN treatment reduces serum levels expression of IL-‐1β (N and N+ GlcN = no detected in all cytokynes; LPS M = 286.3;14.5*, LPS M+GlcN = 212.9;3.1*#; LPS S= 343.9;29.1*, LPS S+GlcN = 128.4;11*# pg/mL), IL-‐6 (LPS M = 448.0;11.5*, LPS M+GlcN = 241.2;11.6*#; LPS S = 508.6;21.7*, LPS S+GlcN = 451.6;8.9* pg/mL) and TNF-‐α (LPS M = 311.3;15.7*, LPS M+GlcN = 76.5;5.5*#; LPS S = 354.2;32.1*, LPS S+GlcN = 136.2;10.2*# pg/mL)cytokines and in aorta mRNA (2-‐ΔΔCT) IL-‐1β (LPS M = 20.6;1.2*, LPS M+GlcN = 14.0;1.4*#; LPS S = 21.46;1.5*, LPS S+GlcN = 15.87;0.9*#), and TNF-‐α (LPS M = 0.07;0.004*, LPS M+GlcN = 0.02;0.004*#; LPS S = 0.13;0.01*, LPS S+GlcN = 0.02;0.006*#)expression. GlcN treatment attenuate, but did not normalize, LPS-‐induced decrease in blood pressure (N = 108;9.0, N + G = 112;10.0; LPS M = 72;6.0*, LPS M + G = 92;8.5*#; LPS S = 59;4.5*, LPS S+G = 88;7.5*# mmHg). No changes in vascular reactivity (thoracic aorta) to phenylephrine or acetylcholine were detected 6 h after LPS administration; (*p<0.05 vs. N; #p<0.05 vs. LPS). In conclusion, O-‐GlcNAc reduces sepsis-‐associated inflammatory and cardiovascular events, making this pathway a potential target for

P32 The Role of Immunological CD8 Effector Memory T Cells In Hypertension H Itani, J Wu, L Xiao, F Zhang, D Michell, W Chen, D Harrison

Vanderbilt University

Immunological memory is an important component of adaptive immunity. We have previously shown that T cells are important in hypertension. We therefore hypothesized that memory T cells contribute to long-‐term and repeated hypertensive challenges. To test this, mice received 2 weeks of angiotensin II, were allowed to recover for 3 weeks and then were received a low dose of ang II (140 ng/kg/min) that minimally raises blood pressure in naïve mice.

This low-‐dose of ang II increased blood pressure to 137;6 mmHg in mice that had previously received a vehicle infusion, but to 172;12 mmHg in mice that had received ang II

In another model we treated mice with L-‐NAME (0.5mg/ml) in drinking water for two weeks, and then fed a high salt diet (4% NaCl) two weeks after stopping L-‐NAME. The high salt diet had no effect on blood pressure in naïve mice, but increased blood pressure to 150;6 mmHg in mice that had received L-‐NAME. In keeping with a memory T cell response, we found that either the ang II or L-‐NAME and high salt protocols increased CD8 effector memory cells in the kidney by 4-‐fold while reducing these effector memory CD8. T cells in the spleen by 3-‐fold. CD8 memory T cells require co-‐stimulatory signaling between CD70 on macrophages and CD27 on T cells. We found that ang II infusion increased CD70 mRNA in isolated kidney vessels by 5 to 10-‐fold. Similarly, flow cytometry revealed that ang II increased macrophages expressing CD70 and CD8 T cells expressing CD27


expression in the kidney. Preliminary results show that anti-‐CD70 partially blocked memory formation by reducing the hypertensive response to the second low-‐dose of ang II by about 20 mmHg. Thus, these studies indicate that repeated exposures to ang II promote an immune memory response of CD8 T cells and markedly enhance the hypertensive response. The role of CD27 and CD70 signaling requires additional study but might serve as a therapeutic target

P33 Downregulation of vascular Nrf2 and associated antioxidant enzymes in hypertension R Lopes1, A Montezano1, K Neves1, R Tostes2, R Touyz1 1University of Glasgow, 2University of Sao Paulo

Oxidative stress plays an important role in vascular dysfunction in hypertension. While mechanisms regulating vascular pro-‐oxidants are emerging, there is a paucity of information on anti-‐oxidant systems. Nrf2 is a master regulator of antioxidants and its role in hypertension remains elusive. Weassessed vascular Nrf2 in hypertension by studying mesenteric vessels and VSMCs from WKY and SHRSP rats. Cells were stimulated with Ang II (10-‐7M) in the absence/presence of Nrf2 activators (bardoxolone or L-‐sulforaphane). ROS generation was assessed by chemiluminescence and amplex red. mRNA expression of anti-‐oxidant enzymes was assessed by qPCR. Nrf2 activity was analyzed by ELISA. Nrf2 activity was decreased inarteries(18%) andVSMCs (48%) in SHRSP (p<0.05 vs WKY). mRNA levels of antioxidant enzymes were reduced in SHRSP (SOD 1 (64%), catalase (60%), peroxiredoxin 1 (75%) and glutathione peroxidase (54%) Ang II increased Nrf2 activity in VSMCs from WKY (197%, 4h) and SHRSP (44%, 4h) (p<0.05, vs. vehicle). This was associated with increased antioxidant mRNA expression in WKY rats (SOD1-‐32%, catalase-‐42%, thioredoxin-‐71%, peroxiredoxin 1-‐90%, quinone oxidoreductase-‐84%; p<0.05 vs. vehicle) but not in SHRSP. ROS production and glucose-‐6-‐phosphate dehydrogenase (source of NADPH) mRNA levels were increased in SHRSP. Ang II-‐induced ROS generation in VSMCs from WKY and SHRSP was blocked by Nrf2activators. Vascular function assessment, by wire myography, demonstrated that increased contractility (Emax Phe: WKY113.4;5,67vs.SHRSP 159.0;8.29) and decreased endothelial-‐dependent relaxation (Emax ACh: WKY88.7;3.13 vs. SHRSP 74.7;3.25, p<0.05) in SHRSP were corrected by bardoxolone and L-‐sulforaphane. In conclusion, vascular dysfunction in SHRSP is associated with oxidative stress, decreased Nrf2 activity and reduced Nrf2-‐regulated antioxidant enzymes. A similar molecular phenotype was observed in Ang II-‐stimulated VSMCs. Nrf-‐2 agonists ameliorated vascular dysfunction in SHRSP. Our findings suggest that Nrf-‐2 downregulation may contribute to redox-‐sensitive vascular dysfunction and could be a therapeutic target in hypertension.

P34 Angiotensin II induces oxidative stress through ADP-‐ribose-‐TRPM2 dependent mechanisms in human microvascular endothelial cells – Role of AT2 receptor

M. Dulak-‐Lis, A. Nguyen, C. Jenkins, A. Montezano, R. Touyz Institute of Cardiovascular and Medical Sciences

Objectives: Endothelial dysfunction is associated with oxidative stress and dysregulation of calcium signalling. We previously demonstrated that calcium-‐dependent Nox5 is regulated by Ang II and may be implicated in vascular damage in hypertension. Elevation of intracellular free calcium concentration ([Ca2+]i) influences oxidative stress, whichmay be related to activation of redox sensitive calcium channel TRPM2. Here, we postulated that in human microvascular endothelial cells (HMEC), Ang II induces ROS generation through TRPM2-‐dependent processes.

Methods: HMECs were stimulated with either Ang II (100 nM) or H2O2 (1 mM). ROS production was measured by lucigenin (superoxide) and amplex red (H2O2); redox-‐sensitive MAPKs/eNOS activation by immunoblotting; and AT1R/AT2R mRNA by Q-‐PCR. In some experiments, the following inhibitors were used: aminoethoxydiphenyl borate (APB, TRPM2/SERCA/IP3R inhibitor), N-‐(p-‐amylcinnamoyl)anthranilic acid (ACA, TRPM2/PLA2 inhibitor), 8-‐bromo-‐cADP-‐ribose (8-‐Br-‐cADPR, ADPR antagonist), 3,4-‐Dihydro-‐5[4-‐(1-‐piperindinyl)butoxy]-‐1(2H)-‐isoquinoline (DPQ, poly-‐ADPR polymerase inhibitor) and PD123319 (AT2R antagonist).

Results: In hmECs, Ang II increased superoxide levels (1.9;0.2 fold, p<0.05 vs. vehicle), an effect blocked by APB, ACA, 8-‐Br-‐cADPR, DPQ and PD123319. In addition, Ang II-‐induced increase in H2O2production (1.6;0.21 fold, p<0.05 vs. vehicle) was inhibited by AT2R antagonism. Ang II stimulation increased ERK1/2 activation (185%), but not p38, through AT2R-‐dependent mechanisms (p<0.05). AT2R, but not AT1R was expressed in HMECs. H2O2, an activator of TRPM2, decreased superoxide production (25%) and increased MAPKs (p38: 82%; ERK1/2: 40%; JNK: 36%), as well as, eNOS (24%) activation, p<0.05. In addition, ROS formation by Ang II was measured in the presence of L-‐type calcium channel blocker diltiazem, which also blocked Ang II effects on superoxide production.

Conclusions: Our data demonstrate that Ang II-‐induced oxidative stress and redox-‐sensitive MAPK activation in hmEC cells may involve TRPM2 and calcium-‐dependent signalling, which are mediated via AT2R. Our findings identify a novel signalling pathways whereby Ang II/AT2R regulates endothelial cell ROS generation through TRPM2.

P35 A high-‐salt diet alters the composition of intestinal microbiota in mice N Wilck1, S Olesen2, M Matus2, A Balogh3, R Dechend3, R Alm2, R Müller1


1ECRC -‐a joint cooperation between Max Delbrück Center for Molecular Medicine Berlin and the Charite Medical Faculty, 2Department of Biological Engineering, Massachusetts Institute of Technology, 3Helios Clinic Berlin-‐Buch,

Objective: High-‐salt intake is associated with cardiovascular disease and hypertension. A substantial number of patients are sensitive to salt, but environmental factors influencing this phenomenon are poorly understood. The gut microbiome has not been considered in the context of salt and hypertension research to date. It is known to respond to fluctuations in lifestyle and diet, and is increasingly recognized as an important factor which influences host health and disease. Using next generation sequencing methods we aimed to determine the effect of high-‐salt diet on gut microbiome composition.

Methods and Results: Male C57BL6/J mice, 10 weeks of age, were individually housed and either subjected to a purified normal salt (0.5% sodium) or high-‐salt diet (4% sodium + 1% in drinking water) ad libitum for 14 days. Feces was collected on days 0 and 14 of the diets. DNA was extracted from feces, the V4 region of the 16S rRNA gene amplified with PCR. Samples were multiplexed and sequenced with MiSeq. Reads were quality controlled and operational taxonomic units (OTUs) were called by comparison to the Greengenes database.

The high-‐salt diet created a distinct gut composition compared to the normal-‐salt diet (analysis of Jensen-‐Shannon divergence). Whereas normal diet's gut microbiome is composed mostly of OTUs from the Bacteroidetes phylum (70% of reads), high-‐salt diet causes a shift in relative abundance to other phyla (increased Firmicutes:Bacteroidetes ratio). Moreover, the genus Allobaculum significantly spikes after high-‐salt feeding. Both phenomena have recently been implicated in obesity studies.

Conclusion: An increase in dietary sodium chloride alters the gut microbiome composition. Our findings indicate new perspectives on how

P36 Protein Tyrosine Phosphatase Oxidation and Redox Proteomics in Hypertension S Tsiropoulou, A Montezano, A Scott, R Burchmore, R Touyz University of Glasgow,

Aberrant signalling and vascular dysfunction in hypertension (HTN) have been associated with ROS-‐induced oxidation of redox-‐sensitive molecules. Protein tyrosine phosphatases (PTP) are susceptible to thiol-‐oxidation at their active site, which leads to PTP inactivation. We hypothesise that oxidative stress is associated with reversible PTP oxidation and consequent reduced activation, in HTN. VSMCs from normotensive and hypertensive rats (WKY and SHRSP) were stimulated with AngII (10-‐7 M) and ET-‐1 (10-‐7 M). Protein oxidation was assessed by oxyblot. PTP oxidation, TC-‐PTP and PTP1B expression were assessed by immunoblotting. Differential gel electrophoresis (DiGE) of CyDye-‐labelled cell lysates was employed for oxidised proteome screening. Global-‐protein and PTP-‐specific oxidation was increased in SHRSP versus WKY (fold change (FC)=1.29 and FC=1.53, p<0.05, respectively). AngII stimulation did not affect global oxidation in either WKY or SHRSP, but increased PTP-‐oxidation in WKY (FC=1.47 at 5min, p<0.05) to levels similar to SHRSP. Stimulation with ET-‐1 increased total oxidation in WKY (FC=1.52 at 15min, p<0.05) but not SHRSP, and had no effects on PTP oxidation. Moreover, AngII and ET-‐1 reduced protein expression of TC-‐PTP (FCAngII=0.8 at 2h, WKY; FCAngII=0.9 at 1h, SHRSP; FCET-‐1=0.8 at 2h for WKY and 1h for SHRSP) and PTPB1 (FCAngII=0.87 at 1h, WKY and SHRSP; FCET-‐1=0.9 at 2h, WKY and SHRSP), (p<0.05). DiGE proteomic data, filtered for FC>2, detected 1777 spots of which 78.1% were equally oxidised across SHRSP and WKY, and 14.9% exhibited increased oxidation levels in SHRSP. Our findings demonstrate increased PTP oxidation in VSMCs from hypertensive rats and differential regulation of PTP oxidation and expression by vasoactive agents, in HTN. Furthermore, the oxidised state of 21.9% of global proteome is altered in disease. Ongoing studies, in both rats and humans, are focusing on the characterisation of a PTP-‐oxidation signature in HTN, towards elucidation of aberrant redox signalling and identification of novel therapeutic targets.

P37 Immune dysfunction in a vasopressin-‐induced mouse model of preeclampsia S Scroggins, D Santillan, N Pearson, J Grobe, M Santillan

1University of Iowa Carver College of Medicine Objective: Preeclampsia is a potentially deadly hypertensive disease of pregnancy. We recently published that elevated secretion of arginine vasopressin (AVP) in humans is detectable early in women that subsequently develop preeclampsia. Our novel chronic AVP infusion mouse model closely mimics human preeclampsia. According central dogma, during a healthy pregnancy, a shift occurs from a Th1 to a Th2 response, which is required for maternal-‐fetal tolerance. Many pro-‐inflammatory cytokines are elevated and an aberrant immune response is clearly involved in preeclampsia. AVP secretion is both stimulated by and stimulates pro-‐inflammatory cytokine secretion and activation of lymphocytes. The objective of this study was to investigate the maternal-‐fetal immune alterations involved in preeclampsia associated with AVP. Methods:Wild-‐type female C57BL/6 mice were chronically infused with AVP or saline throughout pregnancy. Maternal and fetal tissues were obtained on gestational day 18. Cellular responses were determined using flow cytometry and cytokine concentrations via commercially available ELISAs. A Student’s t-‐test was used to determine differences with alpha=0.05.


Results: We observed an increase in the frequency of IL-‐12, IL-‐17, and TNF-‐alpha expressing splenic lymphocytes isolated from AVP-‐infused dams. While the frequency of IFN-‐gamma expressing lymphocytes trended higher it did not reach significance. The frequency of IL-‐6, IL-‐10, and LAP expressing lymphocytes were similar between groups. Interestingly, the maternal plasma and placenta revealed a significant reduction in IL-‐17 expression, yet IL-‐17 was elevated in the amniotic fluid isolated from AVP-‐infused dams. IL-‐4 and IFN-‐gamma were undetectable in the maternal plasma. Additionally, IL-‐4 and IFN-‐gamma in both the placenta and amniotic fluid of AVP-‐infused dams were not significantly different between groups. Conclusions: These data from our mouse model support our hypothesis that AVP hypersecretion results in maternal-‐fetal immune alterations associated with the development of preeclampsia. Future experiments will investigate local versus systemic immune mechanisms involved in AVP-‐induced preeclampsia. P38 Animal models of salt-‐sensitive hypertension show impaired renal sodium chloride cotransporter (NCC) function K Walsh, R Wainford

Boston University School of Medicine Aim: These studies test the hypothesis that norepinephrine (NE) impairs renal NCC regulation to evoke salt-‐sensitive hypertension in Sprague-‐Dawley (SD) and Dahl Salt-‐Sensitive (DSS) rats.

Methods: SD rats receiving a s.c. saline vehicle or NE (600ng/min) infusion were fed a 0.4% (NS) or high 8% NaCl (HS) diet for 14 days. Naïve DSS rats were placed on a NS or HS diet for 21 days. On day 14 or 21, MAP and peak natriuresis to i.v. hydrochlorothiazide (HCTZ; 2mg/kg) infusion was assessed. Plasma NE was measured and NCC immunoblotting was performed on kidney cortex tissue (N=5/6).

Results: Excess NE significantly increased MAP and plasma NE levels in NE infused SD rats. A HS diet further increased plasma NE levels and MAP in both NE infused SD and DSS rats. HS intake failed to suppress NCC activity in NE infused SD rats and naïve DSS rats. Impaired NCC expression was shown in NE and HS treated SD rats.

s.c. saline vehicle s.c. NE DSS NS HS NS HS NS HS

MAP (mmHg) 124;2 124;1 151;3*t 171;4* tf 138;5.8≠ 166;6* tf

Plasma NE (nmol/L) n.d. n.d. 99;3 125;20 47;6≠ 76;4 Peak ΔUNaV to HCTZ (μeq/min)

8.7;0.3

7.2;0.7

11.1;1.1

10.8;0.4

6.43;1.3

11.5;2f NCC expression

(ODU/mm2 normalized to b-‐

actin)

1.26;0.49

0.36;0.06

1.86;0.32

2.86;0.92

τ

TBD

TBD

ϕp<0.05 vs Respective NS gp; *p<0.05 vs Saline + NS gp; τp<0.05 vs Saline + HS gp; ≠p<0.05 vs NE + HS gp; n=5-‐6/gp.

Conclusion: These findings reveal that NE infusion combined with a high salt diet in a salt-‐resistant phenotype evokes the physiological responses found in a genetic model of salt-‐sensitive hypertension. These data from both animal models support a direct role of NE on NCC function and the pathophysiology of salt-‐sensitivity.

P39 Arterial blood pressure in the Congolese youths: the interaction of drinking alcohol and overweight. G Ngoyi, BM Bayauli, JR M'buyamba-‐Kabangu

University Hospital of Kinshasa Arterial blood pressure in the Congolese youths: the interaction of drinking alcohol and overweight. Aims: to evaluate the impact on Congolese youth blood pressure of the interaction between alcohol consumption and overweight.

Methods: lifestyle habits, blood pressure (by Omron M6, HEM 7001E), body mass index, plasma glucose and lipids were obtained in 532 youths (268 girls; 50.4%) aged 10 to 19 years from Adoula Quarter, (Kinshasa, DRC) in the framework of the


VITARAA study. Overweight according to age and sex was derived from international tables. We assessed the impact of alcohol and overweight on blood pressure using a generalized linear model procedure with age as a covariate. Determinants of BP were obtained by stepwise linear regression analysis.

Results: BP averaged 110;13/69;11mmHg and increased with age. Overweight/obesity (12.7%) predominated among girls (17.9% vs 7.5%). Alcohol consumption was reported by 23.11% of youths (20.7% of girls and 26.3% of boys, ns). The proportion of alcohol drinkers was similar among overweight subjects (17.6%) and those with normal weight (19.1%). Age-‐adjusted systolic (113. 5 [111.0 -‐ 115.9] vs 109.3[108.0 -‐ 110.7] mmHg; P=0.004) and diastolic (70.1 [67.9-‐72.2] vs 68.7[67.5-‐69.9]mmHg;P=0.271) BP were higher in alcohol drinkers for the whole study population and for girls and boys taken separately. BP was also higher in overweight youths (72.6;10.2 vs 68.9;11.1mmHg; P=0.028). Compared to abstinent, age-‐adjusted systolic BP was 3.6[0.8–6.5] mmHg (P=0.013) and 6.3[-‐4.1–16.7] mmHg, ( P=0.229) higher in alcohol drinkers, respectively in the absence and the presence of overweight. The respective differences in age-‐adjusted. diastolic BP were 1.4[-‐1.2–4.1]mmHg;(P=0.013) and 0.1[-‐6.8–7.1] mmHg (P=0.229).

Conclusion: Our results suggest that prevention of cardiovascular disease in the Africans should encompass the strategies to address overweight and alcohol intake at younger age.

P40 The role of traditional medicine in the fight against hypertension in Africa Albert ZE Health and Development Research Institute

The use of the traditional therapies in Africa is justified by the weakness of financial accessibility to medicines and cultural context. For several years, the majority of African countries recommend the valuation of traditional medicine. Indeed, they recommend, her association with modern medicine. It is widely known, nowadays, that traditional medicine plays an important role in health care and in re-‐forms of health sector in a global way. For that purpose, this research has for objective to show the importance of traditional medicine in treatment of hypertension in Africa.

Our analysis presents a global review of ethnobotanical survey allowing to list the various herbal medicinal products used in treatment of hypertension in sub-‐Saharan Africa. This work was made with the active participation of several traditional practitioners through countries.

In South Africa, the results show that 8 plants used for this study demonstrate an activity of Angiotensin ConvertingEnzyme (ACE), and a single plant showed more than 50 % of inhibition in the preparations (Ramesar et al, 2008). In the Central African Republic, there are 34 medicinal species which were distributed in 34 kinds and 27 families. Among these families, we have 4 families of Liliopsida and 23 families of Magnoliopsida (Apema et al, 2011). In Ivory Coast, 27 plants were identified as antihypertensives (Fézan et al, 2008). These plants belong to 25 kinds and 18 families. The majority of these plants are also identifying in Cameroon.

In spite of the insufficient knowledge of hypertension by the actors of traditional medicine as well in its naming as its diagnosis, these actors are able to find an important number of plants to fight this disease. In several other countries, we find the use of others plants in treatment of hypertension.

References: Zohoun, T et Flenon, J. (1997). La médecine traditionnelle et la pharmacopée africaines peuvent-‐elles constituer une alternative de soins face aux coûts prohibitifs actuels de la médecine moderne?, Pharm. Méd. Trad. Afr, 9, pp.3-‐16.

Akinkugbe, 0.0. (1979). Hypertension in developing counuies. in Arterial hypertension, Gross FH et Robert-‐sonJ IS Ed., pitman Press,K ent, 51-‐59.

Koate, P. (1978). L’hypertension artérielle en Afrique Noire. Bull. OMS, 56 : 841, 848.

Seedat, Y.K., Seedat, M.A., Hackland, D.B.T. (1982). Prevalence of hypertension in the urban and rural Zulu. J. Epidemiol. Community Hlth, 36 : 256-‐261.

Fézan, H. T.B., Irié, G.M., Kohué, C.C.N et Clejesson, H.B.M. (2008). Études de quelques plantes thérapeutiques utilisées dans le traitement de l’hypertension artérielle et du diabète : deux maladies émergentes en Côte d’Ivoire, Sciences & Nature, 5, 1, 39 – 48.

Ramesar, S., Baijnath, H., Govender, T., Mackraj, I. (2008). Angiotensin I-‐converting enzyme inhibitor activity of nutritive plants in KwaZulu-‐Natal, J Med Food, 11(2), 331-‐6.


Apema, R., Mozouloua, D., Kosh-‐Komba, E., et Ngoule, Y. (2011). les plantes médicinales utilisées dans le traitement de l’hypertension artérielle par les tradipraticiens à Bangui

P41 This poster has been withdrawn by the author. P42 Family history of premature cardiovascular events and total plaque area H Perez Vascularis

Introduction and Objective: Although the positive family history for premature cardiovascular events (FHPCVE) has been considered a putative risk factor for decades, it has not been incorporated along with other established risk factors such as hyperlipidemia, hypertension, and cigarette smoking in the daily clinical practice when physicians evaluates the cardiovascular risk of a patient. The objective of this research was to investigate if patients with a FHPCVE have higher total plaque area than patients without it, in the absence of other classical risk factors. Design and method Patients (2947) from the primary prevention database of Blossom DMO program were identified to have positive FHPCVE. After apply excluding criteria (absence of any classical risk factor) 23 patients qualified. A control group was generated reaching same data in variables (blood pressure, age, body weight, LDL cholesterol), except carotid total plaque area which was reported at the end of groups formation, once both groups were formed. We used Framingham study definitions (family history of <55 years in men and <65 years in women, first degree relatives) to consider a patient with FHPCVE. Results The group with FHPCVE was similar to the control group. Age 59;2 sv 60;1 yo, blood pressure 126;2/75;1 mmHg vs 127;1/74;1 mmHg, body mass index, 23;1 vs 24;1 Kg/cm² and LDL cholesterol 116;15 vs 139;20 mg/dl. Total plaque área (TPA) in patients with FHPCVE had 49;8 mm² while control group 33;4 mm² (p<0.05). Framingham risk score were similar, while the post test for acute myocardial infarction was lower in control group (16;1% vs 20;3%, p<0.05). Conclusions Our data indicates that patients with positive FHPCVE have larger TPA than patients without it, indicating enhanced risk to develop a cardiovascular event, even in the absence of other classical risk factor. P43 Withdrawn by the authors P44 Blood Pressure Variability during High Salt Intake, But Not Low Salt, Is Associated with Future Cardiovascular Events

in Patients with Essential Hypertension T Kisaka1, R Ozono1, TA Cox2, Y Kihara1 1Hiroshima University Graduate School of Biomedical Sciences, 2Los Angeles Biomedical Research Institute at Harbor-‐

UCLA Medical Center

Introduction: Accumulating evidence suggests that blood pressure variability (BPV) is a strong predictor of future cardiovascular events in patients with hypertension. However, it is unknown whether high salt intake may affect BPV and its predictive value.

Method: We analyzed the prevalence of cardiovascular events in 47 patients with essential hypertension in whom systolic BPV was determined with three different 7 day salt intake diets: regular (10g/day NaCl diet), low (3g), and high (20g). These BPV evaluations were performed after admission to our institute between 1988 and 1993. Details were reported elsewhere 1-‐2. Standard deviations (SD) in supine systolic blood pressures (SBP) measured every hour from 6:00 through 23:00 were determined as BPV under the three conditions. The patients were followed up for 22 +/-‐ 6 yrs.

Results: SDs in SBP during the regular salt (12 +/-‐ 5 mm Hg) and high salt (13 +/-‐ 5 mm Hg) diets were significantly increased compared with the low salt (10 +/-‐ 4 mm Hg). Coefficients of variation (CV) in SBP for the regular, low, and high salt diets were 8%, 7% and 8%, respectively. Salt loading significantly increased both SD and CV (p < 0.05). During a 22-‐year follow-‐up period, 25 cardiovascular events were observed including 9 fatal events. ROC analyses revealed SD of SBP during regular salt diet had an AUC = 0.66 and high salt diet of AUC = 0.72, but that low salt diet was not significantly associated with events. CV of SBP during high salt diet, AUC = 0.72, was also significantly associated with events. Kaplan Meier analysis also showed that patients with CV of SBP larger than 8% had significantly greater number of cardiovascular events.

Conclusion: These results suggest that high and regular salt diet increase BPV, thereby leading to endogen damage and increased incidence of cardiovascular diseases.


References: 1. Oshima T, Matsuura H, Kido K, Matsumoto K, Fujii H, Masaoka S, et al. Intralymphocytic sodium and free calcium and plasma renin in essential hypertension. Hypertension. 1988;12(1): 26-‐31

2. Kisaka T, Ozono R, Ishida T, Higashi Y, Oshima T, Kihara Y. Association of elevated plasma aldosterone-‐to-‐renin ratio with future cardiovascular events in patients with essential hypertension. J Hypertens. 2012; 30(12): 2322-‐30

P45 Subcutaneous Resistance Artery Remodeling And Function In Chronic Kidney Disease Patients JC Fraulob Aquino1, M Briet2, T Barhoumi1, C Savoia3, P Paradis1, EL Schiffrin1

1Vascular and Hypertension Res Unit, Lady Davis Inst for Medical Res, SMBD-‐Jewhish General Hosp, Mcgill Univ, 2INSERM U1083, CNRS UMR 6214, Ctr Hospo-‐Univire d’Angers, Univ d'Angers, 3Cardiology Unit, Second Faculty of Medicine, Sant’Andrea Hospital, Sapienza University of Rome, and Research Center, Fatebenefratelli San Pietro Hospital

Background: Chronic kidney disease (CKD) is associated with cardiovascular (CV) complications. However, interventional trials targeting classical CV risks factors have been often unsuccessful in advanced stage CKD, which emphasizes the need to better understand CKD-‐associated vascular disorders. Resistance arteries are a key determinant of blood pressure (BP) and their changes in different CV conditions contribute to target organ damage. The aim of the present study was to characterize resistance artery remodeling and function in CKD patients, compared to vessels from hypertensive (HTN) subjects.

Method: Twenty-‐two stage 4 CKD patients (aged 63.6;3.1 years) and 16 HTN subjects (45.6;16.1 years) were included in the present study. They all underwent a subcutaneous biopsy under local anaesthesia. Small artery remodeling and function were studied on a pressurized myograph, and subcutaneous fat CD3 infiltration and media fibronectin expression by immunostaining. Vascular smooth muscle cells (VSMCs) were counted after hematoxilin-‐eosin staining.

Results: CKD systolic BP was similar to HTN (133;18vs.143;10 mmHg, respectively). Vasodilatory responses to acetylcholine were lower in CKD compared to HTN (maximal relaxation (%), 74.3;3.4vs87.5;2.7, P<0.05). Media/lumen at 60 mmHg was lower in CKD than in HTN (6.7;0.5vs8.8;0.7, P<0.05). Resistance artery stiffness was lower in CKD compared to HTN (strain at 120 mmHg, 0.845;0.126vs0.585;0.099,P<0.05). Fibronectin staining in resistance arteries was lower in CKD than HTN (8.2;0.8vs23.3;1.7 RFU/µm2,P<0.001). Less VSMCs were present in the arterial wall of CKD compared to HTN (5.4;0.4 vs 7.2;0.5 cells/µm2,P<0.05). Subcutaneous fat presented fewer CD3+ cells in CKD than HTN (12.8;4.1vs23.7;12.8 cells/mm2,P<0.05).

Conclusion: Despite higher levels of BP, resistance arteries isolated from CKD patients exhibited no vascular remodeling and lower arterial stiffness compared with HTN patients. These results are in line with the maladaptive hypotrophic remodeling observed in large vessels in CKD, suggesting a generalized vascular defect in mechanotransduction in CKD.

P46 The determinants of acute coronary syndrom in young adults at Jacques Monod Hospital 1Cliniques Universitaires de Kinshasa

Background: AMI has long been regarded as pathology of the elderly.

Objective: To assess frequency, profile, prognosis and determinants of MI in young adults in order to improve the management and prevention of cardiovascular disease.

Method: We obtained socio-‐demographic, clinical, biological and angiographic data and diagnosis in 1163 patients admitted during 2012 to the cardiac intensive care unit of GHH Jacques Monod. Complications during the hospital stay and the vital issue at discharge were recorded. We modelled the odds of IDM in the young (≤ 55years) and senior patients (>55 years) and the probability of death in logistic regression. Kaplan-‐meyer curves were used for the analysis of survival.

Results: Observed in 30.4% of cases, AMI predominated in males (sex ratio 1.46), with similar prevalence among young (32.4%) and senior patients (29.5%: P=0.33), but higher lethality compared to non-‐AMI patients (6% vs 2.7%; P=0.007). Family history of AMI (34.6% vs20.7%; P=0.006), smoking (88.8% vs 27.6%; P<0.001) and epigastric pain (18.7% vs 9.4%; P=0.014) predominated among young people whose total cholesterol (P<0.001), LDL-‐cholesterol (P<0.001) and triglycerides (P<0.001) were higher. Most patients had single coronary vessel lesions (57.8%) that predominated among young than old patients (79.4% vs 48.4%). Hemodynamic complications (killip 2-‐4) predominated among seniors, LV thrombosis among younger patients (4.7% vs 0.8%; P=0.029). Male gender and family history of AMI increased the risk of MI in all patients; smoking [5.69 (2.80-‐11.53); P<0.001] in young and hyperglycemia [1.87 (1.27-‐2.74); P=0.001] in seniors only. The probability of AMI death was higher in those with history of heart disease [4.12 (1.23-‐13.80); P=0.022] and cardiogenic shock [75.81 (22.98-‐250.16); P<0.001]. The probability of death was higher in patients with MI an cardiogenic shock (Log-‐rank P<0.001).

Conclusion: These results suggest the prevention of MI should encompass strategies to control smoking and dyslipidemia in the young.


References:

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J Am Coll Cardiol 2003; 41: 15S-‐22S. 8. Fuster V, Lewis A. Mechanisms leading to myocardial infarction: insight from studies of vascular biology. Circulation 1994; 90: 2126-‐46 9.Shah PK, et al. Mechanisms of plaque vulnerability and rupture. J Am Coll Cardiol 2003; 41:15S-‐22S. 10. Maresca G, Di Blasio A, Marchioli R, et al. Measuring plasma fibrinogen to predict stroke and myocardial infarction: an update. Arterioscler Thromb vasc Biol 1999; 19:1368-‐77. 11.Hoffman C, Miller R, Lawson W, et al. Elevation of factor VII activity and mass in young adults at risk of ischemic heart disease. J Am Coll Cardio 1989; 14:941-‐6. 12.Jansson J, Nilsson T, Olofsson B et al. Tissue plasminogen activator and other risk factors of cardiovascular events in patients with severeangina pectoris. Eur Heart Journal 1991; 12:157-‐61. 13. Gray R, Panahloo A, Mohamed-‐Ali V, et al. Proinsulin like molecules and plasminogen activator inhibitor type 1 PAI-‐1 activity in diabetic and non-‐diabetic subjects with or without myocardial infarction. Atherosclerosis1997; 130:171-‐6. 14. Cannon CP, McGabe CH, Stone PH, et al. Circadian variation in the incidence and prognosis of unstable angina and non Q-‐wave acute myocardial infarction the TIMI III registry and TIMI IIIb. Am J Cardiol1997; 7:253-‐258. 15.Selwyn A, Kinlay S, Libby P, Ganz P. Atherogenic lipids, vascular dysfonction, and clinical signs of ischemic heart disease. Circulation 1997; 95:5-‐7. 16.Brieger D, Eagle KA, Goodman SG, et al. Acute coronary syndromes without chest pain, an underdiagnosed and undertreated high-‐risk group: insights from the Global Registry of Acute Coronary Events. Chest2004;126:461–469. 17. Sgarbossa EB, Pinski SL, Barbagelata A, et al.Electrocardiographic diagnosis of evolving Acute Myocardial Infarction in the presence of Left Bundle-‐Branch Block. GUSTO-‐1 investigators.N Engl J Med 1996; 334:481-‐487. 18. Thygesen K, Alpert JS, White HD, et al. Universal definition of myocardial infarction. Circulation 2007; 116: 2634–2653. 19. Indications for fibrinolytic therapy in suspected acute myocardial infarction: collaborative overview of early mortality and major morbidity results from all randomised trials of more than 1000 patients. Fibrinolytic Therapy Trialists’ Collaborative Group. Lancet 1994; 343:311–322. 20. Boersma E, Maas AC, Deckers JW, et al. Early thrombolytic treatment in acute myocardial infarction: reappraisal of the golden hour. Lancet1996; 348:771–775. 21. White HD. Thrombolytic therapy in the elderly. Lancet 2000; 356:2028–2030. 22. Kastrati A, Dibra A, Spaulding C, et al. Meta-‐analysis of randomized trials on drug-‐eluting stents vs baremetalstents in patients with acute myocardial infarction. Eur Heart Journal2007; 28:2706 –2713. 23. Hoit BD, Gilpin AE,Henning H, et al. 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P47 has been withdrawn by the authors P48 has been withdrawn by the authors P49 Preliminary candidate gene analysis in the Women’s Interagency HIV Study shows pleiotropic effect of APOB and TNF

genes and novel association of SERPINE1 with Metabolic Syndrome traits Y Natanzon

Case Western Reserve University Purpose: To evaluate associations between candidate genes that promote pro-‐vasoconstriction, pro-‐coagulating, and pro-‐inflammatory vascular environment which lead to the constellation of phenotypes known as Metabolic Syndrome. Materials and


Methods: 3788 women from the Women’s Interagency HIV Study were genotyped using and Illumina 5M array. Thirty two candidate genes were selected from a comprehensive literature search that promoted pro-‐vasoconstrictive, pro-‐coagulative and pro-‐inflammatory vascular environment. Also three genes (APOB, CETP, LPL) were selected to serve as positive control due to their established association with Metabolic Syndrome and combination of Metabolic Syndrome components. Single SNP regression analysis was performed using the additive model, adjusting for age at baseline and BMI where appropriate. Nine principal components obtained from genome wide SNPs were added to the model to control for population structure. Phenotypes were derived using ATP III definition as a guideline. The baseline values of the phenotype were analyzed. 2000 SNPs were assayed within and 10kb upstream and downstream of each of the 32 candidate genes, which show an experiment-‐wide significance level of 2.5e-‐05. Results: Several previously reported SNPs in the APOB gene were marginally significant for HDL cholesterol and total cholesterol traits. Previously unreported SNP rs11687484 reached experiment-‐wide significance for both hemoglobin A1C and fasting glucose. Eight SNPs in CETP reached experiment-‐wide significance with HDL cholesterol, and nonHDL cholesterol/HDL cholesterol ratio phenotypes. A SNP rs72680532 in BMP4 gene is also significantly associated with total cholesterol/HDL cholesterol ratio. BMP4 gene has not been reported to be associated with any Metabolic Syndrome traits or cardiovascular disease. Previously unreported association between SNP rs3918229 in NOS3 gene and HDL cholesterol reached experimental wide significance. Conclusion: Multiple genes are associated with traits contributing to Metabolic Syndrome, which are suggestive of pleotropic effects. P50 Effect of nutritional transition on the incidence of hypertension in Africa ZE Albert Health and Development Research Institute

Food consumed by the populations, in their variety, determines largely their health, their growth and their development (OMS, 2003). But, however, the behaviour at risk as the tobacco consumption and lack of physical exercise, also play an important role. All this takes place in a social, cultural, political and economic environment which can damage the health of the populations (Zoyem et al, 2008). Thus, the objective of this study is to evaluate the relationship between westernization of behavior and hypertension.

For this work, we use the principalcomponent analysis (Saporta, 1990). Our analysis concerns 26 countries of sub-‐Saharan zone of Africa. These countries were chosen according to the criterion of availability of statistical data. The Variables are divided into two groups. The first one is constituted by variables concerning the phenomenon of westernization of behavior whereas the second concerns extra load metabolic diseases.

The results show that the affections such as hypertension, obesity and cardiovascular diseases are correlated in following behavioral factors: the urbanization, the offer of dietary energy and the addiction to smoking. Indeed, an increase of the rate of urbanization and offer of dietary energy cause an increase of obesity and hypertension. Furthermore, cardiovascular diseases also grow when the rate of urbanization and the addiction to smoking increase.

The food of societies in the world is marked by successive evolutions (Cambrezy, et Janin, 2003). So, several emerging countries and developing country are freeing gradually to problems of food insecurity and are confronted with the problems caused by chronic diseases connected to food (IRD, 2012). Associated to important changes in the food systems, this transition is made in a very fast way as a result of a steady urbanization and of the globalization of exchanges.

References: IRD. (2012). L’insécurité alimentaire en Afrique. Available at www.ird.fr

Cambrezy, L., & Janin, P. (2003). Le risque alimentaire en Afrique. Dossiers des images économiques du monde (DIEM), SEDES, 225p.

Organisation Mondiale de la Santé. (2003). Obésité: Prévention et prise en charge de l’épidémie mondiale. Organisation Mondiale de la Santé. Genève, pp 284.

Saporta, G. (1990). Probabilités, Analyse des données et statistique, Edition Tech-‐nip.

Zoyem, J.P., Diang’a, E., & Wodon, Q. (2008). Mesures et déterminants de l’insécurité alimentaire au Burundi selon l’approche de l’apport calorifique. The African statistical journal, 6, 35-‐66.


P51 Novel neurohumoral responses to head upright tilt testing in children with chronic nausea and orthostatic intolerance

A Wagoner1, H Shaltout2, D Diz2, J Fortunato2

1Wake Forest School of Medicine, Hypertension and Vascular Research Center, 2Children's Hospital of Colorado Background: Children with unexplained nausea have a high incidence of orthostatic intolerance (OI). The renin-‐angiotensin system (RAS) is known to play a role in disorders related to OI likely as a compensatory mechanism for CV regulation.

Objective: The objective of this study was to characterize the neurohumoral response to head upright tilt testing (HUT) in children with chronic nausea and OI. Methods: Forty-‐eight patients (mean age of 15 years) completed HUT. GI symptoms including nausea were reported during HUT. Subjects were supine for 15 min before 45 min 70° HUT. Plasma hormones catecholamines (Cat), epinephrine, norepinephrine (NE), vasopressin (AVP), aldosterone, renin, and angiotensins [Ang-‐(1-‐7) and AngII] were measured in blood sampled immediately before and after 15 min of HUT. Results: Thirty-‐five subjects demonstrated OI. There were no differences in supine blood pressures (BP) or humoral measures between groups. OI subjects had lower systolic (p=0.001), diastolic (p<0.0001), and mean arterial (p=0.0002) BPs during HUT compared to non-‐OI subjects. They also had higher Cat (p=0.02), AngII (p=0.04), and AVP (p=0.01) during HUT compared to non-‐OI subjects. There was a negative correlation between Ang-‐(1-‐7) and NE at baseline (r=-‐0.6, p<0.05) and HUT (r=-‐0.6, p=0.03) in non-‐OI subjects that was not seen in OI subjects. Nausea was reproduced by HUT in 42% of this cohort. Nausea subjects had significantly higher AVP during HUT compared to subjects who did not experience nausea (p=0.001). Conclusions: Children with chronic nausea testing positive for OI have elevated Cat, AngII, and AVP levels upon HUT. Elevated AVP may be a key trigger to nausea with orthostatic challenge independent of OI on HUT. In addition to this humoral response, the absence of change in DBP upon standing suggests a failure in the sympathetic nervous system and RAS compensatory mechanisms necessary to sustain HUT in children with chronic nausea and OI. P52 Renal endoplasmic reticulum (ER) stress is induced via Endothelin A (ETA) receptor activation C De Miguel, J Hobbs, D Pollock, J Pollock

University of Alabama at Birmingham

The endothelin (ET-‐1) system has been recently implicated in renal endoplasmic reticulum (ER) stress development, a type of cellular stress caused by misfolded protein accumulation within this organelle. To clarify the role of the ETA receptor in renal ER stress, we hypothesized that pharmacological blockade of this receptor via ABT-‐627 leads to reduced renal response to the ER stress inducer tunicamycin (TM). WT and ETBdeficient (sl/sl) rats (n=3-‐4/group) were pre-‐treated with ABT-‐627 (5mg/kg/day; drinking water) for 1 week and given a single injection of TM (2mg/g bwt; i.p.). Kidneys, plasma and urine were collected 24h later. ER stress markers in renal cortical and medullary regions were determined by qRT-‐PCR. Treatment of WT animals with ABT-‐627 significantly reduced the ER stress response to TM in cortex and outer medulla (fold change/WT+H2O; GRP78 (0.12;0.02 and 0.04; 0.01, respectively), ATF-‐4 (0.20; 0.02 and 0.15; 0.02), ATF-‐6 (0.28; 0.08 and 0.18; 0.04), spliced XBP-‐1 (0.03+0.01 and 0.05; 0.01) and CHOP (0.02; 0.01 and 0.04; 0.01). On the contrary, ABT-‐627 pre-‐treatment failed to decrease cortical and medullary ER stress development in sl/sl rats. These results indicate that ETAreceptor activation leads to induction of renal ER stress genes, while ETBreceptor activation has protective effects. Modulation of the ET system may have therapeutic value in protecting against ER stress-‐induced renal injury. P53 Biochemical and Biophysical validation of new inhibitors identified through rational structure based design against

Dopamine-‐beta-‐hydroxylase to combat hypertension SK Dey1, T Joseph2, S Kumar3, A Kamaladevi4, N Sarkar2, N Sarkar2, N Balamurugan4, N Thelma2, N Kundu1

1Department of Biochemistry, University of Delhi South Campus, 2Department of Genetics, University of Delhi South Campus, 3Institute of Genomics and Integrative Biology, 4Department of Biotechnology, Alagappa University,

Background: Human dopamine β-‐hydroxylase (hDBH), expressed in noradrenergic nerve terminals of nervous system and in chromaffin cells of adrenal medulla, is a key constituent of catecholamine biosynthetic pathway. DBH inhibition has been shown to help the treatment of hypertension and cardiac heart failure, which are major causes of mortality and morbidity worldwide. Existing hDBH inhibitors are too few, often result in side effects and are frequently non-‐responsive to specific population. Since no three-‐dimensional structure existed for full-‐length hDBH, structure based rational drug design has been elusive till date, an issue to which we provided solution lately by building an experimentally validated in silico model for hDBH (Kapoor et al 2011).

Objectives: Structure based peripherally active drug design against DBH and their validation to treat cardio-‐vascular diseases with special relevance to hypertension.


Methods and Results: The hDBH model was used for structure based virtual screening against small molecule database of NCI, USA; revealing 69 compounds as prospective inhibitors of DBH. These hits were then tested in vitro against human serum DBH and its nearly identical homologue, bovine DBH, with known inhibitors nepicastat and disulfiram as positive controls. NSC637578, NSC99657 and NSC379555 were discovered in the process as potent inhibitors of DBH with IC50s in micro-‐molar range. The binding of the inhibitors to the enzyme was validated using fluorescence and CD spectroscopy as well as ITC, revealing KDvalues in the range of 100nm to 1µM. In silico pharmacokinetic analysis indicated the molecules to be latest generation of DBH inhibitors having high cell permeability and inability to cross the BBB. Considerable doses (up to 50µM) of the lead compounds showed acceptable cellular tolerance against HEK 293 cell line and insignificant hemo-‐toxicities against human RBCs. In vivo evaluation of the lead molecules were attempted in model organisms like C. elegans and D. melanogaster. These studies reconfirmed their nontoxic properties up to 15µM doses. The lead compounds have been optimized computationally to improve their pharmacokinetic properties.

Conclusion: Three small molecules were identified with potential anti-‐hypertensive property with multi-‐fold advantages: (1) peripherally active inhibition with no risk of abnormal influence on CNS, (2) high cell permeability and tolerability, (3) no adverse effects with high in vivo doses, (4) low IC50 ensuring low dose requirement, (5) no structural deformities to DBH due to inhibitor binding and high KD value -‐ two most essential properties of lead molecules to be potent drug candidates. The lead molecules are ready for evaluation in rat models for hypertension.

References Kapoor, A., Shandilya, M., and Kundu, S.(2011) Structural Insight of Dopamine b-‐Hydroxylase, a Drug Target for Complex Traits, and Functional Significance of Exonic Single Nucleotide Polymorphisms. PLoS One 6(10): e26509. doi:10.1371/journal.pone.0026509

P54 Uncovering the Molecular Mechanisms Underlying the Hypertension in Snx1 Knockout Mice J yang, L Asico, J Feranil, J Jones, I Armando, I Weinman, I Jose, I Villar

University of Maryland School of Medicine Objectives: Sorting nexin 1 (SNX1) is crucial for dopamine D5 receptor (D5R) trafficking and function in human and mouse renal epithelial cells. C57Bl/6J and BALB/cJ mice have impaired sodium excretion and hypertension following renal-‐selective SNX1 depletion (Villar et al 2013). Here, we elucidated the renal molecular mechanisms that may be responsible for the hypertension in Snx1 knockout (Snx1-‐/-‐) mice.

Methods: We measured blood pressure (BP), sodium excretion, and renal expression of genes involved in reactive oxygen species (ROS) production by immunoblotting and measurement of ROS and NADPH oxidase (NOX) activity.

Results: Snx1-‐/-‐ mice had increased BP (systolic BP 131.3;6.4 mmHg vs. 105.5;6.4 in wild-‐type (WT) littermates, P<0.05, Student's t-‐test, n=5-‐6/group), and impaired natriuretic response to the D1-‐like receptor agonist fenoldopam (∆UNaV from baseline 83.0;11% vs. 201.5;4% in response to agonist treatment). Snx1-‐/-‐ mice had increased renal expression of AT1R (123.8;2.1 vs. 100;2.0%) and increased expression of NOX components, including NOX1 (153.4;12.2% vs. 100;4.1%), NOX2 (129.9;5.5% vs. 100;7.7%), and p47phox (118.2;2.7% vs. 100;5.0%). The decreased function of D1-‐like receptors in Snx1

-‐/-‐ mice was associated with increased renal expression of D5R (142.9;4.7% vs. 100;6.8%) and PON2 which have antioxidant activities. A renal-‐selective infusion of apocynin, which blocks NOX assembly by preventing p47phox translocation to NOX2, ameliorated the increased systolic BP in Snx1-‐/-‐ mice (131.3;4.8 mmHg to 105.7;1); vehicle treatment in both strains or apocynin treatment in WT mice had no effect. Basal renal NOX activity, which was higher in the Snx1-‐/-‐ than in WT mice (169;12.8 units/mg protein/min vs. 100;13.3), was normalized by apocynin (99.4;16.5), while basal ROS levels, which were 2-‐fold higher in the Snx1-‐/-‐ than in WT mice (218.6;7.7 units/mg protein vs. 100;17.9), were also ameliorated by apocynin (125.8;20.4).

Conclusion: Snx1-‐/-‐ mice have increased BP due to impaired D1-‐like receptor activity, increased renal NOX expression and activity, and AT1R expression.

References 1. Villar VA, Jones JE, Armando I, Asico LD, Escano CS Jr, Lee H, Wang X, Yang Y, Pascua-‐Crusan AM, Palmes-‐Saloma CP, Felder RA, Jose PA. Sorting nexin 1 loss results in D5 dopamine receptor dysfunction in human renal proximal tubule cells and hypertension in mice. J Biol Chem. 2013;288(1):152-‐63.

P55 Evidence for the Expression of Renin Angiotensin System (RAS) and a Disintegrin and Metalloproteinase (ADAM) 17-‐mediated shedding of ACE2 in COS-‐7 cells

J Grobe, N Kashkari, H Chodavarapu, H Somineni, M Di Fulvio, K Elased Wright State University The renin angiotensin system (RAS) plays a vital role in the regulation of the cardiovascular and renal system. COS-‐7 is a robust and easily transfectable cell line derived from the kidney of the African green monkey, Cercopithecus aethiops. The aims of this


study were to 1) demonstrate the presence of an endogenous and functional RAS in COS7 and 2) to investigate the role of ADAM17 in the ectodomain shedding of ACE2. COS7 cells were grown to 90% confluency followed by incubation in serum-‐free media for 24 hours. Western blot, immunohistochemistry and mass spectrometry (MS)-‐based enzyme assays were used to study RAS protein expression and activity. Western blot and immunostaining confirmed endogenous expression of ACE (195 kDa), ACE2 (75-‐70 kDa), AT1R (43 kDa), renin (41 kDa) and ADAM17 (130 kDa) in COS7 cells. A sensitive and selective MS approach determined endogenous RAS enzymatic activity using incubations of COS7 lysate and media with the natural RAS substrate Ang II (m/z 1046). MS analysis detected Ang-‐(1-‐7) formation (m/z899) in lysate and media of COS7 cells. While Ang-‐(1-‐7) formation in media was completely blocked by ACE2-‐specific inhibitor MLN-‐4760, Ang-‐(1-‐7) formation in lysate was significantly inhibited by prolyl carboxypeptidase-‐specific inhibitor Z-‐pro-‐prolinal. Using short-‐hairpin (sh) RNA-‐mediated technology, ADAM17 protein expression and activity was significantly reduced in silenced cells. Western blot analysis showed a significant increase of ACE2 protein expression in lysate and reduction of ACE2 shedding into the media in silenced cells compared to normal cell. This is the first study to demonstrate endogenous expression of the RAS and ADAM17 in COS7 and support the utility of COS7 for the study of ectodomain shedding of ACE2 in vitro. The transfectable nature of this cell line makes it an attractive in vitro cell model for studying the molecular, functional and pharmacological properties of the renal RAS. P56 Cardiac-‐specific deletion of ERBB4 in the adult mouse increases cardiac cell prolifertion and causes physiological

cardiac hypertrophy Z Wang, W Thomas, T Paravicini University of Queensland Objectives: The epidermal growth factor receptor type 4 (ErbB4) is a receptor tyrosine kinase essential for cardiac development. In this study we have investigated the physiological importance of ErbB4 in the adult heart.

Methods: We generated a mouse model with conditional deletion of ErbB4 in cardiomyocytes using a tamoxifen-‐inducible Cre recombinase (MHC-‐MerCreMer). Adult MHC-‐MerCreMer/ErbB4fl/fl (ErbB4 conditional knockout, cKO, n=6) and MHC-‐MerCreMer/ErbB4WT/WT (control, n=8) animals were injected with tamoxifen (20 mg/kg/day ip for 10 days) and cardiac specific deletion of ErbB4 confirmed by qPCR. At 3 months after ErbB4 deletion, cardiac function, structure and gene expression were measured using echocardiography (fractional shortening), histological staining (Masson's Trichrome for fibrosis and wheat germ agglutinin for cardiomyocyte cross-‐sectional area) and qPCR.

Results: Echocardiography revealed no differences in cardiac contractility between cKO and control animals. However, the heart weight:tibia length ratio was significantly increased in the cKO animals (5.1;0.2 vs 7.4;0.6 mg/mm, P<0.05). Cardiac-‐specific ErbB4 deletion did not increase expression of genes associated with pathological hypertrophy (BNP, α-‐MHC:β-‐MHC ratio), suggesting that the hypertrophy seen in the cKO animals may be physiological rather than pathological. Similarly, cardiac-‐specific ErbB4 deletion did not cause cardiac fibrosis or alter fibrotic gene expression (Col1A1, Col3A1 and PAI1). Cardiomyocyte cross sectional area was also unaffected by ErbB4 deletion. Interestingly, cardiac mRNA levels of both isoforms of the endogenous ErbB4 agonist NRG1 were increased by 4-‐5 fold compared to controls, and this increase correlated with a significant increase in the total number of cells positive for the proliferation marker phosphorylated-‐histone H3 (1.8;0.4 vs 8.2;2.8 cells/section, P<0.05).

Conclusion: Cardiac-‐specific deletion of ErbB4 in the adult increases heart weight without altering cardiac function, cardiomyocyte size or fibrosis. ErbB4 deletion also increases NRG1 expression, which may contribute to the maintenance of cardiac function and the development of cardiac hypertrophy via cell proliferation.

P57 Role of Cytochrome P450 4A2 in mediating the elevated blood pressure in a rat model of polycystic ovary syndrome R Maranon, C Patil, C Dalmasso, R Roman, J Reckelhoff University of Mississippi Medical Center

Women with polycystic ovary syndrome (PCOS) often have elevated blood pressure (BP). PCOS is characterized in part by increases in androgens, and androgens can increase cytochrome P450 (CYP) 4A isoforms and 20-‐HETE synthesis. We have found that CYP4A2 expression is increased in renal vasculature of hyperandrogenemic female rats, a model of PCOS. In the present study we tested the hypothesis that androgen increase would not cause elevated BP in CYP4A2-‐/-‐ rats compared with wild type SS.Bn5 rats. CYP4A2-‐/-‐ and SS.Bn5 rats (n=6-‐8/grp) were treated from 4 wks of age with dihydrotestosterone pellets (DHT 7.5 mg/90 d) or placebo pellets until 14 wks, and then telemetry transmitters were implanted. After 2 wks, mean arterial pressure (MAP) was measured for 10 days. DHT increased MAP and decreased HR in SS.Bn5 compared with placebo controls (placebo: 104;2 vs. DHT: 126;6 mmHg, p<0.001). In contrast, while placebo-‐treated CYP4A2-‐/-‐ rats had higher MAP than WT, DHT did not increase BP in CYP4A2-‐/-‐ rats (Placebo: 120;1 vs. DHT: 118;1 mmHg, p=NS). These data suggest that CYP4A2 may be necessary for DHT to increase BP in our model of PCOS. However, by what mechanism(s) CYP4A2-‐/-‐ rats have higher MAP than SS.Bn5 WT remains to be determined. Supported by NIH R01HL66072, P01HL05971 and AHA 14POST18640015.


P58 Role of melanocortin-‐4-‐receptor in the blood pressure regulation in female hyperandrogenemic rats, a model of polycystic ovary syndrome

R Maranon, R Lima, J do Carmo, A da Silva, J Hall, J Reckelhoff University of Mississppi Medical Center,

Obesity and hypertension are found in women with PCOS, although the mechanisms responsible for hypertension are unclear. We tested the hypothesis that the melanocortin-‐4 receptor (MC4R) contributes to the elevated blood pressure (BP) in hyperandrogenemic female rats. Female Sprague Dawley rats (4wks) were implanted with dihydrotestosterone (DHT; 7.5mg/90 days sc) or placebo pellets (PL) (n=5/grp) and aged to 12 wks. Body weight and food intake (whether rats were pair fed or had ad libitum access) were measured daily. Two wks following implantation of radiotelemetry transmitters and intracerebroventricular cannulae, baseline mean arterial pressure (MAP) was measured for 5 days; then rats received MC3/4R antagonist, SHU-‐9119 (SHU; 1 nmol/h ICV) or vehicle for 7 days and MAP was recorded. DHT-‐treated rats had higher body weight and MAP than PL rats (BW: PL: 266.0;8.7; DHT: 348.5;10.4 g, p<0.01; MAP: PL rats: 102;5; DHT: 114;5 mmHg, p<0.05). SHU significantly increased food intake and body weights in both placebo (PL) and DHT-‐treated rats fed ad libitum (PL: 379.2;28.5; DHT: 451/3;7.3 g, p<0.01 DHT vs PL; 0.01 SHU vs control), but had no effect on MAP compared to controls (PL: 104;5; HAF rats: 114;5 mmHg; p<0.05, HAF vs PL; p=NS, SHU vs controls). However, in other rats, when pair fed with little increase in body weight (PL: 253.7;2.0, SHU: 261.0;0.6, p<0.05; DHT: 306.7;2.6, SHU: 316.7;1.5 g, p<0.05), SHU decreased MAP in DHT treated rats but not placebo controls (PL rats: 102;1, SHU: 103;2 mmHg; p=NS; DHT rats: 110;1 vs. SHU: 97;1 mmHg; p<0.001). Thus MC4R antagonist reduces MAP in DHT-‐treated rats only when food intake and body weight are controlled. These data suggest that activation of MC4R contributes to elevated BP in our model of PCOS and may also contribute to the elevated BP in women with PCOS. NIH-‐R01HL66072, P01HL05971 and AHA 14POST18640015. P59 has been withdrawn by the authors P60 Gender-‐specific angiogenesis locus on rat chromosome 13 identified with congenic strains that differ by one gene T Stodola, D Didier, M Flister, J Lazar, A Greene Medical College of Wisconsin

Angiogenesis is the formation of new microvessels from existing vascular beds. AngII, a downstream product of renin, has been shown to be essential mediator of skeletal muscle angiogenesis in Dahl Salt Sensitive (SS) and Sprague Dawley rats, C57BL/6 mice, and human endothelial cells in vitro. Using partial chromosome introgression from the Brown Norway (BN) rat into the SS rat we have created two congenic lines with small (<300 Kbp) BN substitutions that differ by 23 Kbp. The congenic regions define an angiogenesis locus that contains one gene, Btg2. Sanger sequencing revealed 18 intergenic variants but no sequence variants in exons of Btg2between the BN and SS strains. Angiogenesis was measured using an in vivo electrical stimulation model of one hindlimb, with the contralateral leg acting as the control, in males and females of parental strains and both new congenic strains. Neither SS males nor females have angiogenesis after 7 days of stimulation. Males from Btg2BN have angiogenesis (TA=20.0;4.5% increase in vessel density in stimulated leg relative to unstimulated leg, EDL=15.8;5.8%,) while Btg2SSmales did not have angiogenesis (TA=5.1;2.2%, EDL=4.4;2.7%). Females of both strains had angiogenesis: Btg2BN (TA=15.8;4.3%, EDL=3.4%), Btg2SS(TA=14.5;2.0%, EDL=14.6;2.5%). Stimulation significantly increased Btg2 expression in both males and females in all strains except SS males. We cloned the renin proximal promoter into a vector to drive luciferase, co-‐transfected HEK-‐293 cells with this and vector(s) expressing Btg2 or Hoxb9 and Btg2, and found both Btg2and Hoxb9 affected renin promoter activity. However, no significant differences in renin expression were found in stimulated muscle between angiogenic and non-‐angiogenic strains. These data suggested there is a gender-‐specific angiogenesis locus containing one gene, Btg2, that is responding to the angiogenic stimulus and may be participating in a novel angiogenic pathway.

P61 The effects of a high-‐fat diet (HFD) on blood pressure in pregnant rats A Palei, F Spradley, J Granger


Objectives: While obesity is a major risk factor for preeclampsia (PE), the mechanisms whereby obesity increases this risk are unclear. Since blood pressure regulation during gestation relies greatly on the nitric oxide (NO) system, we aimed to determine the effects of a HFD on blood pressure and fetal outcomes in pregnant rats treated with NG-‐nitro-‐L-‐arginine methyl ester (L-‐NAME), a non-‐specific inhibitor of NO synthases.

Methods: Twelve-‐week-‐old Sprague-‐Dawley female rats were fed a normal diet (ND, 13% fat kcal) or a HFD (40% fat kcal) ad libitum. Body weight (BW) and total fat mass (FM) were recorded weekly. After 9 weeks of diet treatment, rats were allowed to breed. On gestation day (GD)14, L-‐NAME at the concentration 100 mg/L was added to the drinking water of a sub-‐group of animals (ND, n=5; ND + L-‐NAME, n=6; HFD, n=18; HFD + L-‐NAME, n=16). On GD19, mean arterial pressure (MAP) was assessed and tissues were harvested.


Results: Initial BW and FM were similar in ND, ND + L-‐NAME, HFD, and HFD + L-‐NAME rats. By the end of the study, HFD had no major effects on BW and FM (BW: 472.3;5.2 vs. 468.0;10.1 vs. 461.6;12.2 vs. 462.4;8.9g and FM: 97.5;2.4 vs. 87.3;9.3 vs. 103.3;7.3 vs. 97.5;6.0g, respectively). Although MAP was elevated in L-‐NAME treated pregnant rats, HFD did not alter significantly MAP or exacerbate the effect of L-‐NAME on MAP (106.6;5.0 vs. 131.3;4.9 vs. 116.0;2.7 vs. 130.2;2.8mmHg, respectively). Neither HFD nor L-‐NAME affected litter size (13.0;0.8 vs. 13.2;1.2 vs. 12.0;1.0 vs. 12.1;0.8 number of fetuses per rat, respectively). However, HFD reduced fetal weight equally in water and L-‐NAME treated pregnant rats (2.2;0.0 vs. 2.2;0.1 vs. 2.1;0.0 vs. 2.0;0.0g, respectively; P<0.05).

Conclusions: In summary, while HFD had a detrimental effect on fetal weight, it had no effect on blood pressure regulation in pregnant rats.

P62 Angiotensin II-‐dependent aortic aneurysm and associated pathogenesis is facilitated by CYP1B1 via oxidative stress and platelet aggregation

S Thirunavukkarasu, N Khan, U Ghafoor, B Jennings, K Mukherjee, A Estes, K Malik The University of Tennessee Health Science Center Objective: Previously we showed that development of hypertension is dependent on cytochrome P450 (CYP)1B1. This study addressed the role of CYP1B1 in Ang II-‐induced aortic aneurysms. Methods: Male ApoE-‐/-‐/Cyp1b1+/+and ApoE-‐/-‐/Cyp1b1-‐/-‐mice were administered Ang II (700 ng/min/kg) or vehicle for one month. Some Ang II treated mice were administered CYP1B1 inhibitor 2, 3’, 4, 5’-‐tetramethoxystilbene (TMS) (300 mg/kg/i.p) or its vehicle DMSO. Results: Ultrasound studies showed that Ang II infusion produced aortic aneurysms in ApoE-‐/-‐/Cyp1b1+/+mice that were prevented by TMS or Cyp1b1 gene deletion. Aneurysms were characterized by increased infiltration of platelets, macrophages and T lymphocytes. Increased degradation of collagen, elastin, actin; increased MMP2, 9, PDGFD; absence of PAI-‐1, increase in COX-‐2 and reactive oxygen species production observed in Ang II treated aneurysms in ApoE-‐/-‐/Cyp1b1+/+ mice were minimized by treatment with TMS or Cyp1b1 gene deletion. Microarray analysis indicated downregulation of markers of inflammation and oxidative stress including Angiopoeitin 2, P-‐selectin, platelet derived growth factor receptor, MMP 2, 9 and 12, CD276 and Hmox-‐1 in aortas of ApoE-‐/-‐/Cyp1b1-‐/-‐mice receiving Ang II. Conclusion: Data suggests that Ang II-‐induced aortic aneurysms and associated pathophysiological changes are mediated by CYP1B1 via increased oxidative stress and platelet aggregation. Parameters ApoE-‐/-‐/Cyp1b1+/+ ApoE-‐/-‐/Cyp1b1-‐/-‐

Veh Ang II DMSO AngII+TMS Veh Ang II

Aorta area ( mm2) 1.6;.4 3.2;.3* 1.1;.3 1.4;.2† 1.5;.3 1.3;.4

Aortic wall Collagen (Intensity x104 pixel2) 125;3 76;3* 130;7 148;9† 141;7 148;17

Elastin (No. of breaks/fiber) 1;0.13 4.;18* 1;0.17 2;.17† 1;0 1;0.1

Actin (Intensity of staining x104 pixel2) 345;23 949;* 438;66 412;30† 372;32 380;24

Aortic wall and perivascular area (A.U.)

CD62p+ 0 22;3* 0 1;.2† 0 0

F4/80+ 1;.3 9;.5* 1;.3 2;.3† 1;.2 1;.2

CD3+ 1;.2 6;.3* 1;.2 1;.3† 1;.2 1;.2

ROS 13;1 58;6* 19;1 21;2† 14;1 13;1

*ApoE-‐/-‐/Cyp1b1+/+ Ang II Vs veh; † ApoE-‐/-‐/Cyp1b1+/+Ang II+TMS vs. Ang II

P63 Binge Eating Disorder is Improved by Stimulation of Angiotensin II Type2 Receptor in Diabetic Mice H Nakaoka, M Mogi, H Kan-‐no, K Tsukuda, T Chisaka, T Kukida, T Wang, T Bai, T Shan, T Iwanami Ehime University Graduate School of Medicine

Objective: Binge eating disorder (BED) is associated with dopaminergic activation in food reward and contributes to pathogenesis of metabolism-‐related disorders. Recent studies have demonstrated that stimulation of angiotensin II type 2 receptor (AT2R) inhibits dopamine (DA) synthesis. Here, we investigated the relationship between DA signaling and BED after fasting condition and the effect of AT2R stimulation on BED and body weight.


Methods: Male wild-‐type mice (WT: C57BL6 strain), type 2 diabetic mice (KKAy) and AT2R-‐null mice (AT2KO) at 8 week-‐age were treated with an intraperitoneal injection of AT2R agonist, compound 21 (C21), at the dose of 10 mg/kg/day for 2 weeks. Two days after fasting, food and water intake and rebound weight gain were measured under re-‐feeding condition for 7 days. DA level in the striatum was measured by microdialysis. The expressions of DA receptor D1 (DRD1), DA receptor D2 (DRD2) and DA transporter (DAT) in the substantia nigra were evaluated by immunohistochemical staining.

Results: Food and water intake, and DA level in the striatum were significantly increased 48 hours after fasting compared with non-‐fasting KKAy and AT2KO. Administration of C21 significantly attenuated these increases in KKAy, but not in AT2KO. Moreover, C21 treatment significantly inhibited rebound weight gain after refeeding compared with vehicle-‐treated group, but not in AT2KO. In KKAy, the expressions of DRD1, DRD2 and DAT in the substantia nigra were markedly decreased compared with WT, whereas these reductions were blunted by administration of C21. Interestingly, rebound weight gain after re-‐feeding in AT2KO was significantly increased compared with WT; however, this increase was not inhibited by C21.

Conclusion: Activation of AT2R could contribute to inhibition of BED and rebound weight gain with a decrease in dopamine levels.These results indicate that stimulation of AT2R could be a new therapeutic approach to eating disorder with improving dopamine resistance.


Author Index Abdelsaid M 1.3 Alm E P.35 Alves-‐Filho J P.31 Armando I P.54 Asico L.D. P.54 Bai H P.2, P.10, P.15, P.63 Baillie G.S. 1.4 Balamurugan K P.53 Balogh A P.35 Baretella O P.17 Barhoumi T P.19, P.21, P.28, P.45 Basu U P.7 Bayauli P P.39 Becker B P.5 Binger K P.29 Briet M P.28, P.45 Brockschnieder D P.11 Brouwers S P.4 Burchmore R P.36 Carlström M 1.1 Carmichael A 2.6, P.8 Carmichael C 2.6 Carneiro F.S. P.13, P.16, P.22 Carrive P P.6 Case A.J. P.7, P.12 Castro C.H. 1.4 Cau S.B.A. P.16 Ceravolo G 1.7 Chan C 1.5 Chen W P.32 Chisaka T P.2, P.10, P.15, P.63 Chodavarapu H P.55 Coelho S P.23 Cox T P.44 Crowley S 2.3 Cunha F P.31 da Silva A P.58 Dalmasso C P.9, P.57 Davern P 2.4, P.6 De Miguel C P.52 Dechend R P.11, P.29, P.35 Dey S.K. P.53 Di Fulvio M P.55 Didier D P.60 Diep H 1.5 Diz D P.51 do Carmo J P.58 Dowling J 1.5 Drummond G 1.5 Dulak-‐Lis M P.34 Dupont A P.4 Elased K 2.1, P.55 Elijovich F 2.2 Ene A.C. P.28 Ergul A 1.3 Estes A P.62 Evans R 2.4 Even S P.3, P.25, P.26

Feranil J.B. P.54 Ferreira N P.16, P.22 Ferreira R P.31 Findlay J 1.4 Flister M P.60 Fortunato J P.51 Fraulob J.C. P.45 Fredholm B 1.1 Fudim M 2.2 Gebhardt M P.29 Ghafoor U P.62 Gonzalez F P.19, P.21 Gornitsky J P.21 Goulopoulou S P.24 Graham D 1.7 Granger J 2.7, P.61 Greene A P.60 Griendling K 1.7 Grobe J P.37 Grobe N 2.1, P.55 Groom K 2.2 Gutta S 2.1 Guzik T.J. 1.2 Haase N P.11 Hall J P.58 Hardigan T 1.3 Harrison D 2.5, P.32 Harvey A 1.7 He Y 1.7 Head G 2.4, P.6 Henig M P.29 Heuser A P.11 Hezel M 1.1 Higaki T P.15 Hilgers K P.29 Hirotomo N P.2 Hobbs J P.52 Hoda N 1.3 Hood K 1.4, P.14 Horiuchi M P.2, P.10, P.15, P.63 Huebner N P.29 Idris-‐Khodja N P.19, P.21 Ishii E P.15 Itani H P.32 Iubenga Y P.46 Iwanami J P.2, P.10, P.15, P.63 Jackson K 2.4, P.6 Jantsch J P.29 Jawien J 1.2 Jenkins C P.3, P.27, P.34 Jennings B P.62 Johansen A P.14 Johns E 2.4 Jones J.E. P.54 Jose P.A. P.54 Joseph T P.53 Juncos L.A. P.9 Kamaladevi A P.53


Kan-‐no H P.2, P.15, P.63, Kashkari N P.55 Kemp-‐Harper B 1.5 Khan N P.62 Kihara Y P.44 Kisaka T P.44 Kleinewietfeld M P.29 Kretschmer A P.11 Krishnan S.M. 1.5 Kukida M P.2, P.10, P.15, P.63 Kumar S P.53 Kundu S P.53 Laffer C 2.2 Lang F P.29 Lazar J P.60 Li Y P.7 Lima R P.58 Linker R P.29 Liu J P.7 Lobato N P.18, P.30 Lopes R.A. P.13, P.14, P.18, P.30, P.33 Lubenga Y P.46 MacLean M.R. 1.4, P.14 Madhur M 2.5 Malik K P.62 Mancini S P.27 Mansell A 1.5 Manzato C.P. P.16 Manzel A P.29 Maranon R P.9, P.57, P.58 Marko L P.11 Marques F 2.4 Matsumoto T P.1, P.24 Matus M P.35 M'buyamba-‐Kabangu J P.39 McCarthy C P.24 Mestriner F P.18, P.22, P.31 Mian MOR P28 Michell D P.32 Mikolajczyk T 1.2 Mogi M P.2, P.10, P.15, P.63 Montezano A.C. 1.2, 1.4, 1.7, P.3, P.14, P.27, P.30,

P.33, P.34, P.36 Moretti J 2.4 Müller D P.11, P.29, P.35 Mukherjee K P.62 Nakaoka H P.10, P.15, P.63 Natanzon Y P.49 Netterville J 2.2 Neuhofer W P.29 Neves K P.3, P.13, P.18, P.30, P.33 Ngoyi G P.39 Nguyen A P.3, P.27, P.30, P.34 Nguyen-‐Huu T P.6 Nosalski R 1.2 Offermanns S P.19, P.21, P.23 Ogbi S P.24 Olesen S P.35 Oliveira A P.30, P.18 Olivon V P.13, P.31

Olszanecki R 1.2 Oneeb Rehman Mian M P.28 Osman H 2.1 Ouerd S P.19, P.21 Ozono R P.44 Page P P.14 Palacios R P.3 Palei A 2.7, P.61 Paradis P P.19, P.21, P.23, P.28, P.45 Paravicini T P.56 Patil C.N. P.9, P.57 Pearson N P.37 Peleli M 1.1 Pereira C.A. P.22 Perez H P.42 Pinar A 1.5 Pollock D P.52 Pollock J P.52 Ramalho L P.13 Rautureau Y P.23 Reckelhoff J.F. P.9, P.57, P.58 Rehman A P.21, P.23 Resstel L.B. P.22 Rintisch C P.29 Rios F 1.2, P.3 Robertson D 2.2 Rodrigues J P.13 Roman R P.57 Ruginsk S P.13 Saklayen M 2.1 Saleh M 2.5 Salt I.P. P.27 Santillan D P.37 Santillan M P.37 Santos R.A. 1.4 Sarkar N P.53 Sarkar S P.53 Savoia C P.45 Schiffrin E.L. P.19, P.21, P.23, P.28, P.45 Schroeder A P.29 Schwartz C P.29 Scott A P.36 Scroggins S P.37 Shaltout H P.51 Shan B P.2, P.10, P.63 Silva M.A.B. P.13, P.16 Skiba D 1.2 Smolders I P.4 Sobey C 1.5 Somineni H P.55 Spradley F 2.7, P.61 Stasch J P.11 Stevenson E 2.4, P.6 Stodola T P.60 Szasz T P.1 Szyndralewiez C P.14 Terrando N 1.1 Thelma B P.53 Thirunavukkarasu S P.62 Thomas W P.56


Titze J P.29 Tostes R.C. P.13, P.16, P.18, P.22, P.30, P.31, P.33 Touyz R.M. 1.2, 1.4, 1.7, P.3, P.14, P.27, P.30,

P.33, P.34, P.36 Trindade M P.19, P.21 Tsiropoulou S P.36 Tsukuda K P.2, P.10, P.15, P.63 Vanhoutte P.M. P.17 Vartanyan P 2.6 Villar V.A.M. P.54 Voehringer D P.29 Wagoner A P.51 Wainford R 2.6, P.4, P.8, P.36, P.38 Walsh K P.38 Wang H P.5 Wang X.L. P.2, P.10, P.15, P.63 Wang Z P.56 Webb C P.24 Webb R.C. P.1 Weinman E.J. P.54 Wenceslau C P.24 White A P.27 Wilck N P.11, P.35 Wu J P.32 Xiao L P.32 Xu A p.17 Yabe-‐Nishimura C 1.7 Yang J P.54 Yang T 1.1 Yates Hood K P.14 Yusuf H 1.4 Ze A P.40, P.50 Zhang F P.32 Zhang H P.9 Zhang J 2.3 Zimmerman M.C. P.7, P.12 Zucker I P.5


The ISH NIC would like to thank the following people and organisations for their invaluable support of the ISH New Investigator Symposium:

Sponsors of the ISH NIC in 2014

ISH Corporate Members

American Heart Association Council on Hypertension AtCorBoehringer IngelheimDaiichi-SankyoDSI Omron europe

Clinical ScienceHypertension ResearchJournal of Human Hypertension

Abstract Reviewers

ALIQUe, Matilde Madrid, SpainCHATeRJee, Piyali Temple, USACHRISTOFIDIOU, Paraskevi Leicester, UKDIAZ, Keith Philadelphia, USA eL BIKAI, Rana Montreal, CanadaGeORGe, eric Jackson, USAHAACK, Karla Omaha, USAHANNAH-SHMOUNI, Fady New Haven, USAIVKOVIC, Vanja Zagreb, CroatiaKeNGNe, Andre Pascal Capetown, South AfricaKOROSTOVTSeVA, Lyudmila St. Petersburg, RussiaKRUGeR, Ruan Potchefstroom, South AfricaLORIA, Analia Augusta, USALORTHIOIR, Aurélien Paris, FranceMARQUeS, Francine Ballarat, Australia MeLS, Carina Potchefstroom, South AfricaMIAN, Muhammad Oneeb Rehman Montreal, Canada

MIRABITO, Katrina Melbourne, AustraliaMONTeZANO, Augusto Glasgow, UKMURPHY, Sydney Jackson, USANGUYeN DINH CAT, Aurelie Glasgow, UK PeNA SILVA, Ricardo Bogota, ColumbiaPeTTeY, Christina Little Rock, USAPOLICHNOWSKI, Aaron Milwaukee, USASeVA PeSSOA, Bruno Rotterdam, NetherlandsSMITH, Wayne Potchefstroom, South AfricaTRASK, Aaron Columbus, USAVeRHeYeN, Nicolas Graz, AustriaWAINFORD, Richard Boston, USA XAPLANTeRIS, Panagiotis Athens, GreeceZHANG, Yi Shanghai, ChinaZIMMeRMAN, Matt Omaha, USAZOUeIN, Fouad Jackson, USA

Join the ISHIf you are not already a member, why not become a part of the premier international society dedicated to research, education and clinical excellence in blood pressure and related cardiovascular disease?

Research Fellowships of the ISH are designed for graduate students and are entirely free. This is a special opportunity for any new research or clinical scientist undertaking a higher degree to enhance their CV. Tenure of this category is limited to three years and Research Fellows are required to confirm their status annually. Once they have completed their PhD (or other qualifying research degree), and wish to become a Regular Member, they are required to inform the ISH and provide and up-to-date CV.

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For further information on membership of the Society or the New Investigators Network please contact the NIC via the ISH Secretariat. Email: [email protected]

We also encourage you to speak to the ISH New Investigator Committee and Working Group members at the Symposium.

ContactInternational Society of Hypertension Secretariatc/o The Conference Collective, UKSuite 2, Churcham House, 1 Bridgeman Road, Teddington, Middlesex, TW11 9AJ

Tel: +44 (0)20 8977 7997Email: [email protected]: www.ish-world.com

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