Abstract
It is now well established that inflammatory responses are pivotal in the development of atherosclerosis and that the prototypic marker of inflammatory status in humans is C-reactive protein (CRP). Recently, moderately raised levels of serum CRP have been claimed to be a risk factor for the development of cardiovascular disease. The role of CRP in atherogenesis has therefore; come under intensive investigation and several reports have claimed that CRP can directly affect endothelial cells, thereby affecting the development of atherosclerosis. Controversy, however, has arisen over these claims, as some of the observed effects on endothelial cell lines have been attributed to the presence of contaminating sodium azide in commercial preparations or endotoxin in recombinant preparations of CRP. The aim of this study was to examine the effect of a range of physiologically/pathologically relevant concentrations of highly purified CRP on endothelial cells by measuring the expression of a range of adhesion molecules, the secretion of inflammatory and haemostatic factors and to clarify whether FcyRI and FcyRII (the main receptors for CRP) can modulate CRP effects on endothelial-like cell lines and primary human endothelial cells. Moderate to high levels of CRP were observed to decrease the expression of the adhesion molecules VCAM-1 and PECAM-1 and the secretion of the chemotactic cytokine interleukin-8 and the nitric oxide product nitrite by endothelial cells. Whereas low, moderate and high concentrations of CRP had no effect on the level of surface expression of the adhesion molecules ICAM-1, or on the secretion of TNFa by any of the endothelial cells tested. Incubation of endothelial cells with moderately raised levels of CRP resulted in a significant increase in the prothrombotic mediator Von Willebrand Factor (vWF) with a concomitant decrease in secreted tissue plasminogen activator (tPA-1), which was confirmed at the transcriptional level using RT-PCR. Incubation of primary endothelial cells with high concentrations of CRP increased the expression of Fc gamma receptors (FcyRI and FcyRII) at 8 or 12 hours by HCAECs, while low-moderately raised levels of CRP (1-10ug/ml) decreased the expression of FcyRI and FcyRII at 24 hours suggesting interaction of CRP with these receptors. This was supported by the observation that CRP co-localized with both FcyRI and FcyRII on HCAECs using confocal fluorescence microscopy and that incubation with CRP resulted in the up-regulation of FcyR mRNA expression by these cells using RT-PCR. Knocking down FcyR genes by small interference RNA in HCAECs resulted in the abrogation of CRP-enhanced vWF mRNA expression (as detected by RT-PCR). Furthermore, this effect appeared to be CRP concentration dependent, with lower levels of CRP having greater effect through FcyRIIB and moderate levels through FcyRIIA and combination of FcyRI, FcyRIIA and FcyRIIB. These data suggest that CRP might exert direct angiogenic effects at low and anti-angiogenic effects at moderate and high levels of CRP which is reflected by the inhibition of PECAM-1, VCAM-1, NO and interleukin-8 production and may induce direct pro-coagulant effects on endothelial cells by promoting vWF secretion and attenuating tPA-1 production. These effects may occur mostly through expression and internalization of FcyRIIB and FcyRIIA at low to moderate and through FcyRI and FcyRIIB at high concentrations of CRP by endothelial cells. These data support the hypothesis that CRP is an effector molecule able at different concentrations, to provoke an angiogenic or anti-angiogenic and pro-atherothrombotic effect on endothelial cells through FcyR interaction in vitro.