on August 24, 2006
Published online before print August 24, 2006, doi: 10.1161/01.ATV.0000242794.65541.02
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Submitted on January 7, 2006
Accepted on August 11, 2006
C-Reactive Protein Alters Antioxidant Defenses and Promotes Apoptosis in Endothelial Progenitor Cells
Hiroko Fujii ;
From the Toronto General Research Institute (H.F., S.-H.L.), Department of General Internal Medicine (P.E.S.), Division of Cardiac Surgery (P.W.M.F., S.V.), St. Michael’s Hospital, University of Toronto, Canada.
* To whom correspondence should be addressed. E-mail: subodh.verma{at}sympatico.ca.
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.
Methods and 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 µg/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.
Key words: antioxidative enzymes • apoptosis • C-reactive protein • endothelial progenitor cells • reactive oxygen species
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