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  1. 1. 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 pneumoniaeChlamydophila pneumoniae InducesInduces ICAM-1 Expression In Human AorticICAM-1 Expression In Human Aortic Endothelial CellsEndothelial Cells Via Protein Kinase C - DependentVia Protein Kinase C - Dependent Activation of Nuclear Factor-Activation of Nuclear Factor-κκBB
  2. 2. ENDOTHELIAL CELL DYSFUNCTIONENDOTHELIAL CELL DYSFUNCTION Cardiovascular Disease StartsStarts and EndsEnds with Endothelial Cell Dysfunction
  3. 3. Factors Leading to EndothelialFactors 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
  4. 4. 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
  5. 5. C. pneumoniae Infects and Activates Endothelial Cells Monocyte V-CAM-1 E-Selectin ICAM-1 Adhesion molecules Induction of Chemokines IL-8, MCP-1 C. pneumoniae Elementary bodies Endothelial cells
  6. 6. 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
  7. 7. Sequential steps of leukocyte adhesion Capture/Tethering Rolling Firm Adhesion Transmigration L-selectin P-selectin E-selectin Integrins, ICAM,VCAM ICAM PECAM Endothelial cells Price et. al. 1999
  8. 8. RATIONALE and GOALS It is known that C. pneumoniae activates p42/p44 (ERK1/2) and NF-κB 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 transductionTo determine which signaling transduction pathways are involved in the regulation of ICAM-1pathways are involved in the regulation of ICAM-1 byby C. pneumoniaeC. pneumoniae in human aortic endothelial cellsin human aortic endothelial cells
  9. 9. Structure and regulation of ICAM-1 promoter ARE ICAM-1ICAM-1 TATAIRE TATA H2O2 IL-1β TNF AP-1 AP-1/ETS AP-1/ETS NF-kB C/EBP-AP-3 TFIID AP-1 C/EBP NF-kB Ets-1 STAT Sp1 AP-2 TFIID IFN-γ • Major intracellular signal transduction pathways – NF-κB pathway – Mitogen-Activated Protein (MAP) kinase (ERK, JNK, and p38) pathway – Protein kinase C (PKC) pathway
  10. 10. 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 37°C Incubation, 37°C, 1h MOI: 5-10 EB/cell HAEC Differential centrifugation
  11. 11. Time Course Expression of ICAM-1 in C. pneumoniae-Infected Human Aortic Endothelial Cells NI: Non-infected TNF: TNF-treated cells Cp: C.pneumoniae infected cells UV: Cells infected with UV-treated Cp H: Cells infected with Heat-inactivated Cp M: Mock cells
  12. 12. Time dependent activation of MAPK pathway in C. pneumoniae-infected HAEC
  13. 13. ICAM-1 expression by C.pneumoniae- infected HAEC is not mediated by MAPK Activation
  14. 14. NF-κB activation mediates C. pneumoniae- induced ICAM-1 expression NI: Non-infected cells TNF: TNF-treated cells CAPE: Caffeic acid phenethyl ester
  15. 15. ICAM-1 expression induced by C. pneumoniae is PKC-dependent Cy:Cytosol M: Membrane NI:Non-infected cells Cal C: Calphostin C Bis I: Bisindolyl- maleimide I
  16. 16. PKC isozymes depletion in C. pneumoniae-infected HAEC Cy: Cytosol M: Membrane NI: Non-infected cells PMA: PMA-treated cells
  17. 17. ICAM-1 up-regulation in C. pneumoniae- infected cells is PKC and NF-κB dependent NI:Non-infected cells TNF:TNF-treated cells Cal C: Calphostin C Bay: Bay 117085
  18. 18. 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-κB 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-κB activation and that, in turn, leads to increased transcription of the ICAM-1 gene
  19. 19. C. pneumoniae’s EB HAEC Toll-like receptors Leucine-like receptors? ICAM-1 NIK IKKγ IKKα IKKβ IkB- p and ubiquination NF-kB translocation P65(RelA) cSrc PKCCalphostinC CAPE BAY117085 U0126 PD98059 c-Fos, Ets-1, Sap1, STAT c-Raf-1 MEK1/2 ERK1/2 c-Jun JNK SAPK MEKK1 MKK4 IkB NF-kB Signal transduction pathways that mediate ICAM-1Signal transduction pathways that mediate ICAM-1 up-regulation inup-regulation in C. pneumoniae-C. pneumoniae-infected HAECinfected HAEC
  20. 20. CONCLUSIONSCONCLUSIONS 1. 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
  21. 21. Questions?Questions? Vielma SA, Kreggs G, Lopes-Virella MF:Vielma SA, Kreggs G, Lopes-Virella MF: Circulation ResearchCirculation Research 92: 1130-7, 200392: 1130-7, 2003