SHAPE Society

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. ENDOTHELIAL CELL DYSFUNCTIONENDOTHELIAL CELL DYSFUNCTION
Cardiovascular Disease
StartsStarts and EndsEnds with
Endothelial Cell Dysfunction

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. 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. 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. 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. 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. 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. 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. 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. 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. Time dependent activation of MAPK
pathway in C. pneumoniae-infected HAEC

13. ICAM-1 expression by C.pneumoniae-
infected HAEC is not mediated by MAPK
Activation

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. 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. PKC isozymes depletion in
C. pneumoniae-infected HAEC
Cy: Cytosol
M: Membrane
NI: Non-infected cells
PMA: PMA-treated cells

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. 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. 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. 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. 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