Silvana Vielma,MD Gregor Krings,BS
Maria Lopes-Virella, MD, PhD
Medical University of South Carolina and Ralph Johnson VA Medical Center, Charleston, SC
Chlamydophila pneumoniae Chlamydophila pneumoniae Induces Induces ICAM-1 Expression In Human AorticICAM-1 Expression In Human Aortic
Endothelial Cells Endothelial Cells Via Protein Kinase C - Dependent Via Protein Kinase C - Dependent
Activation of Nuclear Factor-Activation of Nuclear Factor-BB
ENDOTHELIAL CELL DYSFUNCTIONENDOTHELIAL CELL DYSFUNCTION
Cardiovascular Disease StartsStarts and EndsEnds with Endothelial Cell Dysfunction
Factors Leading to Endothelial Factors Leading to Endothelial DysfunctionDysfunction
• ConventionalConventional: Dyslipidemia, Hyperglycemia, Smoking• Non-ConventionalNon-Conventional:
– Increased Homocysteine, Angiotensin II, Pro-thrombotic Factors, Pro-Inflammatory Factors, Oxidative Stress
– Infectious Processes: Cytomegalovirus and other viral infections , C. pneumoniae
C. pneumoniae and Arteriosclerosis• C. pneumoniae has an epidemiological link with
arteriosclerosis and acute cardiovascular events
• C. pneumoniae has been detected in carotid, abdominal aorta, coronary , femoral, pulmonary and popliteal arteries
• C. pneumoniae is able to replicate in macrophages, endothelial and smooth muscle cells
C. pneumoniae Infects and Activates Endothelial Cells
MonocyteV-CAM-1E-SelectinICAM-1
Adhesion molecules
Induction of ChemokinesIL-8, MCP-1
C. pneumoniaeElementary bodies
Endothelial cells
ADHESION MOLECULESADHESION MOLECULES
• Activation of Endothelial Cells by C. pneumoniae leads to increased expression of adhesion molecules and, as a consequence, increased adherence of monocytes to the endothelium, an early hallmark of atherogenesis
• Adhesion molecules:– serve as mediators of cell-cell and cell-matrix
interactions– participate in cell migration and signaling functions
Sequential steps of leukocyte adhesion
Capture/Tethering Rolling Firm Adhesion Transmigration
L-selectin
P-selectin
E-selectin
Integrins, ICAM,VCAM
ICAMPECAM
Endothelial cells
Price et. al. 1999
RATIONALE and GOALS It is known that C. pneumoniae activates p42/p44 (ERK1/2) and NF-B in endothelial cells. Nothing is known, however, about regulation of ICAM-1 expression in chlamydia-infected HAEC. Thus the GOALGOAL of this study is:
To determine which signaling transduction To determine which signaling transduction pathways are involved in the regulation of ICAM-1 pathways are involved in the regulation of ICAM-1 by by C. pneumoniaeC. pneumoniae in human aortic endothelial cells in human aortic endothelial cells
Structure and regulation of ICAM-1 promoter
ARE
ICAM-1ICAM-1
TATAIRETATA
H2O2IL-1TNF
AP-1AP-1/ETS
AP-1/ETS
NF-kB
C/EBP-AP-3
TFIID AP
-1C/EBP
NF-kB Ets-1 STAT
Sp1AP-2
TFIID
IFN-
• Major intracellular signal transduction pathways– NF-B pathway – Mitogen-Activated Protein (MAP) kinase (ERK,
JNK, and p38) pathway– Protein kinase C (PKC) pathway
Protocol to infect HAEC with C. pneumoniae AR39 (ATCC)
Cycloheximide Tx Hep-2 cells
Chamber-slide system
Mechanical dysruption and sonication
IF staining
Rocker platform x 2h at 37CIncubation, 37°C, 1h
MOI: 5-10 EB/cell
HAEC
Differential centrifugation
Time Course Expression of ICAM-1 in C. pneumoniae-Infected
Human Aortic Endothelial Cells
NI: Non-infectedTNF: TNF-treated cellsCp: C.pneumoniae infected cellsUV: Cells infected with UV-treated CpH: Cells infected with Heat-inactivated CpM: Mock cells
Time dependent activation of MAPK pathway in C. pneumoniae-infected HAEC
ICAM-1 expression by C.pneumoniae- infected HAEC is not mediated by MAPK
Activation
NF-B activation mediates C. pneumoniae-induced ICAM-1 expression
NI: Non-infected cellsTNF: TNF-treated cellsCAPE: Caffeic acid phenethyl ester
ICAM-1 expression induced by C. pneumoniae is PKC-dependent
Cy:CytosolM: Membrane
NI:Non-infected cellsCal C: Calphostin CBis I: Bisindolyl- maleimide I
PKC isozymes depletion in C. pneumoniae-infected HAEC
Cy: CytosolM: Membrane NI: Non-infected cellsPMA: PMA-treated cells
ICAM-1 up-regulation in C. pneumoniae-infected cells is PKC and NF-B dependent
NI:Non-infected cellsTNF:TNF-treated cellsCal C: Calphostin CBay: Bay 117085
Summary of ResultsSummary of Results• The up-regulation of ICAM-1 expression in C. pneumoniae-infected
HAEC is time-dependent. Heat and UV inactivation of C. pneumoniae elementary bodies completely abolished the upregulation of ICAM-1
• Up-regulation of ICAM-1 expression in C. pneumoniae-infected HAEC is not mediated by MAPK activation
• Inhibition of NF-B activation completely abolishes C. pneumoniae-induced ICAM-1 expression by HAEC
• ICAM-1 upregulation in C. pneumoniae-stimulated HAEC is PKC dependent
• Activation of PKC leads to NF-B activation and that, in turn, leads to increased transcription of the ICAM-1 gene
C. pneumoniae’s EB
HAEC Toll-like receptorsLeucine-like receptors?
ICAM-1
NIKIKK
IKKIKK
IkB- p and ubiquination
NF-kB translocation
P65(RelA)
cSrc
PKCCalphostinC
CAPE
BAY117085
U0126PD98059
c-Fos, Ets-1, Sap1, STAT
c-Raf-1MEK1/2ERK1/2
c-Jun
JNKSAPK
MEKK1MKK4 IkB
NF-kB
Signal transduction pathways that mediate ICAM-1 Signal transduction pathways that mediate ICAM-1 up-regulation in up-regulation in C. pneumoniae-C. pneumoniae-infected HAECinfected HAEC
CONCLUSIONSCONCLUSIONS1. We have shown for the first time that PKC-mediated
activation of NF-kB by C. pneumoniae leads to a specific up-regulation of ICAM-1 in human aortic endothelial cells
2. Up-regulation of ICAM-1 by C. pneumoniae contributes to the chronic inflammatory events associated with atherosclerosis
Questions?Questions?
Vielma SA, Kreggs G, Lopes-Virella MF: Vielma SA, Kreggs G, Lopes-Virella MF: Circulation ResearchCirculation Research 92: 1130-7, 2003 92: 1130-7, 2003