Effects of multikinase inhibitors on pressure overload-induced right ventricular remodeling

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International Journal of Cardiology 167 (2013) 2630–2637

Contents lists available at ScienceDirect

International Journal of Cardiology

j ourna l homepage: www.e lsev ie r .com/ locate / i j ca rd

Effects of multikinase inhibitors on pressure overload-induced rightventricular remodeling☆

Baktybek Kojonazarov a, Akylbek Sydykov a, Soni Savai Pullamsetti a,b, Himal Luitel a, Bhola K. Dahal a,Djuro Kosanovic a, Xia Tian a, Matthaeus Majewski a, Christin Baumann a, Steve Evans c, Peter Phillips c,David Fairman c, Neil Davie c, Chris Wayman c, Iain Kilty c, Norbert Weissmann a, Friedrich Grimminger a,Werner Seeger a,b, Hossein Ardeschir Ghofrani a, Ralph Theo Schermuly a,⁎a Department of Internal Medicine, Justus-Liebig-University Giessen, Universities of Giessen and Marburg Lung Center (UGMLC), Member of the German Center for Lung Research, Germanyb Department of Lung Development and Remodelling, Max-Planck Institute for Heart and Lung Research, Bad Nauheim, Germanyc Pfizer Global Research and Development, Sandwich, United Kingdom

☆ This work was supported by the Deutsche Forschun5-1, Excellence Cluster Cardio-Pulmonary System andfrom Pfizer.⁎ Corresponding author at: Universities of Giessen

Aulweg 130, 35392 Giessen, Germany. Tel.: +49 641 99E-mail address: [email protected]

0167-5273/$ – see front matter © 2012 Elsevier Irelanddoi:10.1016/j.ijcard.2012.06.129

a b s t r a c t

a r t i c l e i n f o

Article history:

Received 14 February 2012Received in revised form 16 April 2012Accepted 24 June 2012Available online 31 July 2012

Keywords:Right ventricular remodelingPulmonary hypertensionSunitinibSorafenib

Background: Little is known about the effects of current PAH therapies and receptor tyrosine kinase inhibitorson heart remodeling. We sought to investigate the effects of the multikinase inhibitors sunitinib (PDGFR-,VEGFR- and KIT-inhibitor) and sorafenib (raf1/b-, VEGFR-, PDGFR-inhibitor) on pressure overload inducedright ventricular (RV) remodeling.Methods: We investigated the effects of the kinase inhibitors on hemodynamics and remodeling in ratssubjected either to monocrotaline (MCT)-induced PH or to surgical pulmonary artery banding (PAB). MCTrats were treated from days 21 to 35 with either vehicle, sunitinib (1 mg/kg, 5 mg/kg and 10 mg/kg/day)or sorafenib (10 mg/kg/day). PAB rats were treated with vehicle, sunitinib (10 mg/kg/day) or sorafenib(10 mg/kg/day) from days 7 to 21. RV function and remodeling were determined using echocardiography,invasive hemodynamic measurement and histomorphometry.

Results: Treatment with both sorafenib and sunitinib decreased right ventricular systolic pressure, pulmonaryvascular remodeling, RV hypertrophy and fibrosis in MCT rats. This was associated with an improvement ofRV function. Importantly, after PAB, both compounds reversed RV chamber and cellular hypertrophy,reduced RV interstitial and perivascular fibrosis, and improved RV function.Conclusion: We demonstrated that sunitinib and sorafenib reversed RV remodeling and significantlyimproved RV function measured via a range of invasive and non-invasive cardiopulmonary endpoints inexperimental models of RV hypertrophy.

© 2012 Elsevier Ireland Ltd. All rights reserved.

1. Introduction

Pulmonary arterial hypertension (PAH) is a disease of the smallpulmonary arteries, characterized by progressive increase in pulmo-nary vascular resistance (PVR) leading to right ventricular hypertro-phy (RVH), failure and finally death [1]. Pathological changes in thepulmonary arteries of patients with PAH include the formation ofplexiform lesions, as well as smooth muscle and fibroblast prolifera-tion [2]. While pulmonary vascular disease is the obvious primarypathologic focus, RVH and RV dysfunction are major determinantsof prognosis in PAH [3–6].

gsgemeinschaft (DFG), GH-9/an unrestricted research grant

and Marburg Lung Centre,42420; fax: +49 641 42419.

essen.de (R.T. Schermuly).

Ltd. All rights reserved.

Currently available therapies for PAH are considered to act princi-pally via vasodilation, and although offering symptomatic benefit andsome improvement in long term outcomes, their true impact ondisease progression is considered limited [1,7–11]. The developmentof novel therapies that directly target the fundamental processesinvolved in chronic pulmonary vascular remodeling and maladaptiveRV structure/function represents a key aim in this disease.

In this context, recent interest has focused on the potential thera-peutic application in PAH of receptor tyrosine kinase (RTK) inhibitors,agents previously developed for neoplastic indications. Activation ofRTK via growth factors leads tomultiple downstream cellular processes,including proliferation, resistance to apoptosis and migration. Inexperimental PH, the involvement of platelet-derived growth factor(PDGF) [12,13], basic fibroblast growth factor (bFGF) [14,15], vascularendothelial growth factor (VEGF) [16,17] and epidermal growth factor(EGF) [18,19] has been clearly demonstrated. However, the role ofgrowth factors in RV adaptation to increased afterload is currently poorlyunderstood.

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We have previously shown that inhibition of receptor tyrosinekinases PDGFR, BCR-ABL, and c-kit by imatinib reverses PH and RVHand improves survival in monocrotaline (MCT)-induced and hypoxicPH [13]. In addition, imatinib reversed PH in Tie2 PPARγ(−/−) micethat spontaneously develop PH [20]. The results of the pilot Phase IIstudy demonstrated that imatinib appeared safe and well toleratedover a 6 month period, the primary efficacy parameter (6MWD) didnot improve in patients randomized to imatinib compared withplacebo, despite significant improvement in secondary endpoints[21]. Experimental evidence of potential therapeutic use of sorafeniban inhibitor of the serine/threonine kinase Raf as well as the tyrosinekinases PDGFR, VEGFR, c-kit, and fms-like tyrosine kinase-3 (Flt-3),has been provided [22,23]. The Phase Ib trial suggests that, for PAHpatients receiving parenteral prostacyclins, sorafenib can be addedsafely at a starting dosage of 200 mg twice daily [24]. However,these studies did not answer the question whether the effects of TKinhibition were secondary to reduced PVR or due to a direct impacton the RV.

Sunitinib malate (SU11248 L-malate salt, hereafter referred to assunitinib) is a RTK inhibitor approved multi-nationally for the treat-ment of gastrointestinal stromal tumor after disease progression onor intolerance to imatinib and for the treatment of advanced renalcell carcinoma [25–27]. Sunitinib is a potent and selective inhibitorof VEGF receptors 1, 2 and 3, PDGF receptors alpha and beta (PDGFRαand PDGFRβ), as well as stem cell factor receptor (c-kit), Flt-3, colonystimulating factor receptor (CSF-1R), and the tyrosine kinase receptorencoded by the ret proto-oncogene (RET) [28,29].

Thus, the aim of this study was to investigate the effects of sun-itinib on RV remodeling in two different animal models of RVH; inthese studies, sorafenib was used as a reference compound. In orderto differentiate the indirect from direct effects of both compoundson RVH we used MCT-induced PAH model and surgical pulmonaryartery banding (PAB) model, where RVH and RV dysfunction areinduced independent of pulmonary vascular disease.

2. Materials and methods

Please see the online data supplement for additional details.All experiments were performed according to the institutional guidelines that

comply with National and international regulations. The local authorities for animalresearch approved the study protocol. The authors of this manuscript have certifiedthat they comply with the Principles of Ethical Publishing in the International Journalof Cardiology.

2.1. Monocrotaline-induced PH

Sprague–Dawley rats, 300–350 g body weight (BW) were obtained from CharlesRiver Laboratories. As described [13], rats were given a single subcutaneous injectionof 60 mg/kg monocrotaline (MCT, Sigma-Aldrich Chemie GmbH, Munich, Germany)or vehicle under isoflurane anesthesia. Twenty one days thereafter the animals wererandomized to receive either sunitinib twice daily (10 mg/kg, n=9; 5 mg/kg, n=9;1 mg/kg, n=8), sorafenib once daily (10 mg/kg/day, n=7) or placebo (n=12), byoral gavage, for 14 days until the day of the terminal hemodynamic measurementson day 35.

2.2. Pulmonary artery banding

Pressure overload was produced by PAB in Sprague–Dawley rats, 180–200 g BW.Control rats were subjected to sham operations (n=9). Seven days after surgery, therats were randomized to receive either oral sunitinib twice daily (10 mg/kg/day, n=11) or sorafenib once daily (10 mg/kg/day, n=11) or placebo (n=11) for the following14 days until the day of the terminal hemodynamic measurements on day 21.

2.3. Echocardiography

Transthoracic echocardiography was performed and analyzed with a Vevo770high-resolution imaging system equipped with a 25-MHz transducer (VisualSonics,Toronto, Canada). The right ventricular wall thickness (RVWT), right ventricular internaldiameter (RVID), tricuspid annular plane systolic excursion (TAPSE), pulmonary arteryacceleration time (PAAT) and myocardial performance index (MPI) [30] were measured.Additionally peak pressure gradient (PPG) across pulmonary artery constriction in PABrats was measured.

2.4. Hemodynamic measurements

Invasive hemodynamic measurements were performed as described previously[13]. A right heart catheter (PE 50 tubing) was inserted through the right jugularvein for measurement of right ventricular pressure, and the left arteria carotis wascannulated for arterial pressure monitoring. Cardiac output was measured by thermo-dilution technique (Cardiotherm 500-X; Hugo-Sachs Electronic–Harvard ApparatusGmbH, March-Hugstetten, Germany). Briefly, a thermistor catheter (1.5 F) was for-warded into the ascending thoracic aorta via the right carotid artery for the measure-ment of transpulmonary thermodilution cardiac output. A 0.15-ml bolus of cold salinewas injected into the right ventricle as an indicator, and cardiac output was averagedfrom three consecutive determinations and indexed to the weight of the animal toobtain the cardiac index.

2.5. Assessment of RV hypertrophy

After excision of the heart, its RV wall was separated from the left ventricular (LV)wall and the interventricular septum (S). The ratio of the RV to LV and S weight (RV/LV+S) was calculated as an index of RV hypertrophy.

2.6. Pulmonary vascular morphology

Lung tissue preparation, sectioning, staining and vascular morphometry wereperformed as previously described [13]. All analyses were done in blind fashion.

2.7. RV cardiomyocyte size, fibrosis and vascularization assay

Myocardium was fixed in 4% formalin and stained using germ agglutinin to deter-mine myocyte cross-sectional area or picrosirius red to determine perivascular andinterstitial collagen fractions. For quantification of capillary density, transverse heartcryosections were stained with TRITC conjugated isolectin B4. Capillary density wasexpressed as the number of capillaries per cardiomyocyte per cardiomyocyte cross-sectional area. All analyses were done in blind fashion.

2.8. Sunitinib plasma concentration measurements

Plasma concentrations of sunitinib and its primary metabolite (SU12662) weremeasured as described [29].

2.9. Statistical analysis

All data are given as mean±SD. Differences between groups were assessed byone-way ANOVA and Student–Newman–Keuls post-hoc test for multiple comparisonswith a pb0.05 regarded as significant. The software packages GraphPad Prism 5.01were used for data analysis.

3. Results

3.1. Right ventricular function in MCT-induced PAH and PAB rats

In our study we have used two animal models of RVH. We havefound that both MCT and PAB rats developed the similar degree ofRVSP (71.8±14.5 mm Hg and 73.4±10.3 mm Hg, respectively) andRVH (ratio of the RV to LV+S was 0.60±0.14 and 0.64±0.08,respectively). Interestingly, that despite the similar degree of pressureload and RVH, the signs of the RV failure were more severe inMCT-injected rats than in PAB animals. Accordingly, the RV in MCTrats was more dilated compared to PAB rats (4.52±0.52 mm vs.3.43±0.24 mm, respectively, pb0.001). The dilatation of the RV asso-ciated with marked reduction of CI to 14.61±2.92 in MCT rats vs.17.65±2.33 in PAB animals (pb0.05) and TAPSE to 1.53±0.22 mmand 2.06±0.28 mm, respectively (pb0.001). Additionally, the RVMPI, which combines systolic and diastolic parameters of RV functionand is used as a marker of global RV function was more impaired inMCT rats vs. PAB (1.65±0.18 and 1.15±0.20 respectively, pb0.001).

3.2. MCT-induced PH

3.2.1. Effects of sunitinib on hemodynamicsSunitinib administration dose-dependently decreased RVSP as

compared with placebo and the effect of sorafenib on RVSP was com-parable with that of sunitinib at a dose of 10 mg/kg (Fig. 1A). Addi-tionally both multikinase inhibitors dose-dependently improvedPAAT (Fig. 1A, Supplementary). Systemic arterial pressure (SAP) did

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Fig. 1. Effects of multikinase inhibitors on hemodynamics and RVH in MCT rats. (A) Right ventricular systolic pressure (RVSP), (B) Cardiac index (CI), (C) Total pulmonary resistance(TPR), (D) Ratio of RV to LV plus Septum (RV/LV+S). ***pb0.001, **pb0.01, *pb0.05 versus MCT placebo, #pb0.001 — MCT placebo versus healthy.

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not change in placebo-treated animals. There was a moderate butstatistically significant increase in SAP after sorafenib treatment. Nochanges in SAP were noted after any dose of sunitinib (Fig. 1B,Supplementary). Cardiac index (CI) significantly decreased and inparallel total pulmonary resistance (TPR) significantly increased inplacebo-treated animals. Sunitinib dose-dependently increased CI(Fig. 1B) and reduced TPR (Fig. 1C). Similarly, sorafenib significantlyincreased CI and reduced TPR. Left ventricular ejection fraction(LVEF) did not change significantly in response to either sunitinib orsorafenib treatments (data not shown). The hemodynamic benefitsin response to multikinase inhibitors were accompanied by improve-ment of pulmonary vascular remodeling (Fig. 2, Supplementary).

3.2.2. Sunitinib attenuates MCT mediated RV hypertrophyThe RV/LV+S ratiowas significantly increased inMCT rats compared

with healthy control (pb0.001). Sunitinib caused a dose-dependentreduction of RV/LV+S ratio as compared to placebo (Fig. 1D). TheRVWT measured by echocardiography increased from 0.50±0.01 mmin healthy rats to 1.51±0.16 mm in placebo rats (pb0.001, Fig. 1C,Supplementary). Treatment with sunitinib led to a dose- and time-dependent prevention of RV wall thickening. Similarly, sorafenibdecreased the RVWT and RV/LV+S.

3.2.3. Effects of sunitinib on RV function, fibrosis and gene expressionThe RV dilatation and RV function (TAPSE and RV MPI) were

significantly improved after treatment with either multikinase inhibi-tor (Fig. 2A–C). Importantly, improvement in RV function wasassociated with the reversal of RV interstitial (Fig. 2D) and perivascularfibrosis (Fig. 1D, Supplementary). RV expression of ANP, BNP, βMHC,αSMA, and COL3α1 was significantly elevated in MCT placebo rats.Both sunitinib and sorafenib significantly suppressed the expressionof ANP, BNP, αSMA, and COL3α1 (Fig. 2A–E, Supplementary).

3.2.4. Sunitinib plasma concentrationsPharmacokinetic/pharmacodynamic (PKPD) modeling of the

effect of sunitinib on MCT-induced increases in RVWT, measured byechocardiography over days 21–35 was performed to establish theconcentration effect relationship. Plasma concentrations of sunitiniband its primary metabolite in MCT rats increased with increasingdoses (Table 1, Supplementary). The placebo response was character-ized by a linear increase of the RVWT from day 21 until experimentalcompletion (Fig. 1C, Supplementary). The combined free plasma con-centrations of SU11248 and SU12662 throughout this period weretaken and applied to inhibiting this linear increase via an Emax model.The results were consistent with sunitinib demonstrating completeinhibition of the RVWT increase with an EC50 of 2.5 nM (95%CI0.314 to 4.67) for the combined unbound sunitinib and metaboliteconcentration (Table 2, Supplementary).

3.3. Pulmonary artery banding

3.3.1. Effects of multikinase inhibitors on hemodynamics and RVHTo investigate whether the effect in MCT rats was due to a direct

impact of pharmacological interventions on RV hypertrophy or anindirect impact on RVH due to reduced pulmonary vascular resistance,we have investigated the effects of sunitinib and sorafenib in anestablished model of RVH by PAB, which induces RVH and RV dysfunc-tion independent of pulmonary vascular disease. We have found thatthe treatment with multikinase inhibitors did not significantly changeRVSP, SAP and LVEF (data not shown) compared to placebo. Likewise,PPGmeasured by echocardiography did not show any significant differ-ences between placebo and sunitinib or sorafenib treated animals(46.6±6.1, 44.7±3.4 and 45.9±4.7 mm Hg, respectively). The CIsignificantly decreased in the PAB placebo group as compared withsham-operated rats (pb0.05) and significantly increased in responseto sunitinib treatment as compared to PAB placebo (pb0.05, Fig. 3A).

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Fig. 2. Effects of multikinase inhibitors on RV function and fibrosis in MCT rats. (A) RV internal diameter (RVID), (B) Tricuspid annular plane systolic excursion (TAPSE), (C) RVmyocardial performance (MPI), (D) RV interstitial collagen area (%). ***pb0.001, **pb0.01 versus MCT placebo, #pb0.001 MCT placebo versus healthy.

Fig. 3. Effect of multikinase inhibitors on hemodynamics and RVH in PAB rats. (A) Cardiac index (CI), (B) Ratio of RV to LV plus Septum (RV/LV+S), (C) RV internal diameter (RVID)and representative echocardiographic images. ***pb0.001, **pb0.01,*pb0.05 versus PAB placebo, #pb0.001, ‡pb0.05 PAB placebo versus sham.

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RV/LV+S (Fig. 3B) and RVWT (Fig. 4A, Supplementary) were signifi-cantly reduced by sunitinib and sorafenib as compared to placebo.Sunitinib and sorafenib also reduced the PAB-induced hypertrophy ofindividual RV myocytes (Fig. 4B, Supplementary).

3.3.2. Effects of multikinase inhibitors on RV functionBoth kinase inhibitors significantly reduced RVID compared to

placebo in PAB rats (Fig. 3C). TAPSE was significantly reduced in PABplacebo versus sham-operated rats (pb0.001) and markedly improvedby sunitinib and sorafenib (Fig. 4A). The RV MPI was significantlyincreased in the PAB placebo group (pb0.001) and markedly improvedin response to both multikinase inhibitors (Fig. 4B).

3.3.3. Effects of multikinase inhibitors on RV fibrosis, vascularization andgenes expression

RV interstitial and perivascular fibrosis were significantly reducedby sunitinib and sorafenib treatments (Fig. 5A, B). The number ofcapillaries per cardiomyocyte per cardiomyocyte cross-sectional areawas reduced in the PAB placebo group as compared to sham(pb0.05). Neither sunitinib nor sorafenib changed this value (Fig. 6A,B). In addition, the expression of ANP, BNP, βMHC, αSMA, and collagen3α1 in RVwas significantly elevated in PAB placebo rats comparedwithsham. Both sunitinib and sorafenib reduced the expression of thesegenes (Fig. 5A–E, Supplementary).

4. Discussion

Our study demonstrates a potent effect of TK inhibition on RVHand remodeling including improved cardiac function in MCT- andPAB-rats. It has been demonstrated that application of some anticanceragents reversed pulmonary vascular remodeling in experimental PH[13,21–23,31]. Also the specific PDGFR/Abl/c-kit inhibitor imatinib re-verses pulmonary vascular remodeling in rodent models of PH [7,18]and therapeutic efficacies have been investigated in recently publishedcase report and Phase II study in PAH patients [21,31].

On the other hand, multikinase inhibitors like sorafenib are alsoefficacious in experimental PH [18,19], which raises the question of

Fig. 4. Effect of multikinase inhibitors on RV function in PAB rats. (A) Tricuspid annularrepresentative pictures. ***pb0.001, **pb0.01 versus PAB placebo, #pb0.001 PAB placebo v

advantages and disadvantages of multi-targeted versus singletargeted TK inhibitors [32]. While classical pharmacological develop-ment aims to modify molecules as selective as possible in order tominimize possible side effects, multi-pathway inhibitors may targetredundant pathways and more disease relevant targets. For example,activation of several growth factor pathways has been shown inexperimental and human PAH [12–19], suggesting that multikinaseinhibitors may be superior to single-targeted inhibitors. However,based on individual pharmacokinetics and RTK expression profilingone or two single-targeted inhibitors might be suitable for onepatient (-population) but multikinase inhibitors are preferable forothers.

The RV function is the most important determinant of prognosis inPAH patients, although the main pathologic abnormality is in thepulmonary vasculature [3–6]. However, the previous preclinical studieson the effects of TK inhibitors did not distinctly elucidate if the thera-peutic benefits in PH were due only to RV unloading secondary toimprovement in pulmonary vascular remodeling or a potential directmyocardial effects were also involved. In this context, the model ofPAB serves as a useful model of RVH, allowing investigation of themechanisms and any direct effects of therapeutic intervention on RVremodeling and functions. Therefore, we investigated the effects ofthe clinically approved multikinase inhibitor sunitinib on RV remodel-ing in two complementary animal models of RVH. Sorafenib was usedas a reference compound because it has recently been shown to suc-cessfully reduce MCT-induced PH [22] and hypoxia/SU5416 inducedPH [23].

First, we have investigated the RV function in two complementarymodels of RVH. In line with recently published study [33], we havedemonstrated that despite similar degree of the RVSP and RVH, theMCT-injected rats developed more severe RV dysfunction and failurecompared to PAB rats.

In earlier works, it was shown that progressive pulmonary steno-sis induced by PAB in rats is not associated with RV failure [34,35].Bogaard and co-authors demonstrated that RV pressure overload inrats does not lead to severe RV dysfunction, myocardial fibrosis andcapillary rarefaction [35]. In contrast, in our hands PAB model was

plane systolic excursion (TAPSE), (B) RV myocardial performance index (MPI) andersus sham.

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Fig. 5. Effects of multikinase inhibitors on RV fibrosis in PAB rats. (A) RV interstitial collagen area (%) with representative pictures, (B) RV perivascular collagen area (%) withrepresentative pictures. ***pb0.001, **pb0.01, *pb0.05 versus PAB placebo, #pb0.001 PAB placebo versus sham.

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associated with RV dilatation, impaired RV function, increased fibrosisin the RV and capillary rarefaction. The discrepancy between thosestudies and ours may be explained by different degrees of stenosisapplied to the pulmonary artery. Indeed, it was reported in congenitalpulmonary valve stenosis, that the degree of ventricular hypertrophydepends on the severity of obstruction and eventually, long-standinguntreated severe obstruction may lead to RV failure [36]. In line withour data, Schafer and co-authors showed that PAB in rats was associ-ated with progressive RV remodeling, decreased RV stroke volumeand upregulation of myocardial gene markers of hypertrophy andfibrosis [37]. Recently, an impaired RV function and a significantreduction in cardiac output were demonstrated in PAB rats [33].

TheMCT-induced PAHhas remained a frequently investigatedmodel.It has been shown that RVH strongly time- and dose-dependently

Fig. 6. Effects of multikinase inhibitors on RV vascularization in PAB rats. (A) Represen-tative pictures of the RV vascularization assay, (B) RV vascularization presented ascapillaries/RV CM cross sectional area. #pb0.001 PAB placebo versus sham.

correlates with MCT-induced pressure-overload [38], but the possibledirect effects ofMCT on the heart structure/function can not be excluded.Still it is not so clear whether RV failures are associated with heartinflammation/myocarditis [39,40] or secondary due to pulmonary vascu-lar injury. Unfortunately, we can only speculate that pulmonary vascularinjury due toMCT releasesmediatorswhich can increase stress on the RVand lead to maladaptive RVH. Therefore, further investigations arewarranted.

In our study we have demonstrated that multikinase inhibitorsmarkedly improved RV function in both MCT and PAB models ofRVH and failure. These improvements were associated with anti-remodeling effects on myocardium as evident from decreased expres-sion of hypertrophy and fibrosis markers in the RV. Further analysisshowed that the effects of sunitinib and sorafenib on RV remodelingand function were markedly superior in MCT rats compared to PABanimals. The therapeutic benefits of multikinase inhibitors in MCTrats can be attributable to the improvement of pulmonary vascularremodeling and RV function as well, whereas in PAB animals, im-provement of RV function was independent from pulmonary vascularremodeling.

VEGF and its receptors are targets for several antitumor drugs,including the approved compounds sorafenib and sunitinib. The roleof VEGF and its receptors in cardiomyocyte and RVH is still poorlyunderstood. It has been shown that conditional overexpression ofVEGF in cardiomyocytes leads to the growth of an abnormal vascularnetwork and edema, but an effect on cardiac hypertrophy was notreported [41]. In a pressure overload‐induced left ventricular hyper-trophy, VEGFR blockade resulted in a reduction inmyocardial capillarydensity and promoted the transition from compensatory hypertrophyto failure [42]. However, in our study we clearly demonstrated thatboth multikinase inhibitors not only effectively reduced RVH butalso improved RV function as compared to PAB placebo. This can beexplained by the fact that besides VEGFR both compounds block vari-ous other kinases like c-kit or PDGFR. The receptor tyrosine kinasec-kit is expressed on the surface of mast cells that have recentlybeen shown to be involved in left heart hypertrophy in spontaneoushypertensive rats [43] and can promote cell proliferation and collagenexpression in cardiac fibroblasts via the release of PDGF-A [44]. Othertyrosine kinase inhibitors like masitinib block via c-kit inhibition, cy-tokine production and migration of mast cells [45]. Thus, althoughnot addressed in detail, the c-kit blocking effects may well contributeto the anti-fibrotic effects of sunitinib and sorafenib in the models ofRVH. In addition, the inhibition of PDGFR by these compounds mayexert beneficial effects. It has been shown that adenoviral transfectionof the heart with PDGF-A, PDGF-C or PDGF-D significantly upregulated

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the profibrotic mediator TGFβ1 and accelerated cardiac fibrosis [46].In atrial fibrillation, characterized by atrial fibrosis, the injection of aneutralizing PDGF receptor‐specific antibody attenuated atrial fibrosis[44].

It has previously been shown that pressure overload of the RVmaylead to RV ischemia and reduction in capillary density [47,48], whichmay further aggravate ventricular dysfunction. Our study demon-strated that capillary density was reduced in PAB rats as comparedwith sham rats, but did not restore in response to treatment, despitereduced cardiomyocyte size. The inhibition of the VEGF receptors bysunitinib and sorafenib can not be excluded as one of the underlyingmechansims. In our study, treatment with either multikinase inhibi-tor was not associated with a further decline in capillary density. Tak-ing into account the reduction in cardiomyocyte hypertrophy and nochange in the numbers of capillaries per cardiomyocyte, it is conceiv-able that the capillary supply to individual cardiomyocytes was actu-ally increased compared to placebo PAB rats.

Most of our knowledge on the cardiac effects of anticancer therapiescomes from oncology efficacy trials that examine overall safety [25,26].It has been shown that, sunitinib and sorafenib have been associatedwith left ventricular dysfunction and systemic hypertension in cancerpatients [49,50]. In our study both compounds were well toleratedand we did not see serious adverse effects in the studying groups, ex-cept moderate BP elevation in MCT rats treated by sorafenib. However,SAP in sorafenib treated rats was in the range of normal values and wasnot significantly different compared to healthy control. Additionally, inPAB treated animals SAP was not significantly changed compared toplacebo and sham.

The PK/PD analysis showed that, sunitinib displayed completeinhibition of MCT-induced increases in RVWT at plasma exposures thatwere substantially lower than those required for efficacy in oncologymodels (50–100 ng/ml) [29] and also below clinical sunitinib exposuresin GIST and RCC patients (45.0–62.8 ng/ml and Cmin 84.3 ng/ml, respec-tively) [51,52]. This suggests that the sunitinib exposure required for ef-ficacy in PAH could be lower than in oncology. Probably it can explainthe absence of any cardiac side effects of sunitinib in the current study.

In summary, our study demonstrated that multikinase inhibitorswere beneficial not only in MCT-induced PH but also in the PABmodel, suggesting that they could improve RV function and reduceRVH by directly acting on the RV, independent of any reduction ofpulmonary artery resistance. Our findings imply that multikinaseinhibitors are active against the pathogenesis of RV and pulmonaryvascular remodeling simultaneously. Thus, our study is the first toour knowledge that describes the successful therapeutic use of themultikinase inhibitors in two well-accepted animal models of RVH.We believe that our encouraging data lay an adequate rational toembark on further preclinical/cellular/molecular studies the findingsof which would pave the way toward potential clinical application.

Supplementary data to this article can be found online at http://dx.doi.org/10.1016/j.ijcard.2012.06.129.

Acknowledgments

Weacknowledge the excellent technical support offered by ChristinaVroom and Stephanie Viehmann for this study.

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