C-reactive protein alters antioxidant defenses and promotes apoptosis in endothelial progenitor cells.
OBJECTIVE
C-reactive protein (CRP) has been suggested to participate in the development of atherosclerosis, in part, by promoting endothelial dysfunction and impairing endothelial progenitor cell (EPC) survival and differentiation. In the present study, we evaluated the effects of CRP on antioxidative enzymes, reactive oxygen species production, telomerase activity, and apoptosis in human circulating EPCs.


RESULTS
EPCs, isolated from peripheral venous blood, were cultured in the absence or presence of native pentameric azide and lipopolysaccharide (LPS)-free CRP (0, 5, 15, and 20 microg/mL), N-acetylcysteine (NAC), hydrogen peroxide (H2O2) or monoclonal anti-CRP antibodies. Fluorescence-activated cell sorter (FACS) analysis was used for the measurement of intracellular H2O2 and superoxide (O2(-)) by loading cells with 2',7'-dichlorodihydrofluorescein diacetate (H2DCF-DA). Apoptosis was evaluated with Annexin V immunostaining and cytosolic cytochrome c expression. Western blot analysis was used for the determination of manganese superoxide dismutase (MnSOD) and glutathione peroxidase expression, and polymerase chain reaction enzyme-linked immunosorbent assay was used to assess telomerase activity. Incubation of EPCs with CRP caused a concentration dependent increase in reactive oxygen species (ROS) production and apoptosis, with an effect quantitatively similar to H2O2. This effect was attenuated during coincubation with NAC or anti-CRP antibodies. Furthermore, CRP altered EPC antioxidative enzyme levels, demonstrating a reduced expression of glutathione peroxidase and a significant increase in MnSOD expression. Transfection of EPCs with MnSOD-RNAi resulted in a reduction in CRP-induced ROS production, apoptosis, and telomerase inactivation.


CONCLUSIONS
CRP, at concentrations known to predict cardiovascular events, may serve to impair EPC antioxidant defenses, and promote EPC sensitivity toward oxidant-mediated apoptosis and telomerase inactivation. These data further support a direct role of CRP in the development and/or progression of atherothrombosis.
Fujii
Hiroko
H
Toronto General Research Institute, St. Michael's Hospital, University of Toronto, Canada.
Li
Shu-Hong
SH
Szmitko
Paul E
PE
Fedak
Paul W M
PW
Verma
Subodh
S
D000111:Acetylcysteine / Q000494:pharmacology; D000906:Antibodies / Q000494:pharmacology; D017209:Apoptosis / Q000187:drug effects*; D002097:C-Reactive Protein / Q000008:administration & dosage / Q000276:immunology / Q000494:pharmacology*; D002478:Cells, Cultured; D019610:Cytoprotection; D004305:Dose-Response Relationship, Drug; D042783:Endothelial Cells / Q000187:drug effects / Q000378:metabolism / Q000473:pathology / Q000502:physiology*; D004789:Enzyme Activation / Q000187:drug effects; D005979:Glutathione Peroxidase / Q000037:antagonists & inhibitors; D006801:Humans; D009336:Necrosis; D009994:Osmolar Concentration; D010088:Oxidoreductases / Q000378:metabolism*; D034622:RNA Interference / Q000502:physiology; D017382:Reactive Oxygen Species / Q000378:metabolism; D013234:Stem Cells / Q000187:drug effects / Q000378:metabolism / Q000502:physiology*; D013482:Superoxide Dismutase / Q000235:genetics / Q000378:metabolism; D019098:Telomerase / Q000378:metabolism; D014162:Transfection
D000906:Antibodies; D017382:Reactive Oxygen Species; D002097:C-Reactive Protein; D010088:Oxidoreductases; D005979:Glutathione Peroxidase; D013482:Superoxide Dismutase; D019098:Telomerase; D000111:Acetylcysteine