C-reactive Protein On The Function Of Endothelial Progenitor Cells And Its Mechanism

Part OneCulture of endothelial progenitor cells in human peripheral blood and their characteristicsObjective: To examine whether endothelial progenitor cells ( EPCs ) could be isolated from peripheral blood and could be cultured in vitro.Methods: Total mononuclear cells were isolated from peripheral blood of human male volunteers and were plated on human fibronectin-coated culture dishes and cultured in EndoCult^TM medium. EPCs were characterized and adherent cells which were positive for Dil-acLDL up take and lectin binding by direct fluorescent staining were observed under fluorescence microscope, consistent with endothelial lineage cells. EPCs were further identified by demonstrating the expression of AC133, CD34 and VEGFR-2 with flow cytometry. The expression of endothelial nitric oxide synthase (eNOS) mRNA was examined by RT-PCR.Results: Culture cells had the ability to endocytose acLDL and bind UEA-1 lectin. They also could be shown to have EPCs specific markers such VEGFR-2, CD34 and AC133. The EPCs also expressed eNOS mRNA.Conclusions: EPCs could be isolated and cultured from human peripheral blood, the expression of eNOS mRNA may provide a better way to indentify EPCs. The expression of AC133, CD34 and VEGFR-2 were positive. The expression of eNOS may be a better way to define what EPCs is. Culture of endothelial progenitor cells in human peripheral blood would provide a cell source for the further investigation of EPCs. Part TwoEffects of C-reactive protein on interleukin-8 expression in endothelial Progenitor cells from peripheral blood and itsmolecular mechanismsObjective: Vascular endothelial progenitor cells (EPCs) are the precursor of endothelial cells. Increased evidence suggests that circulating progenitor cells contribute to postnatal revascularization. These cells home to sites of ischemia, adopt an endothelial phenotype, and contribute to new blood vessel formation, the identity of the circulating cells that contribute to revascularization is not entirely clear. C-reactive protein (CRP) is an inflammatory modulator which also predicts cardiovascular disease. Interleukin-8 (IL-8) is an important mediator of the paracrine mitogenic effect of EPCs, which has direct angiogenic effects on mature endothelial cells. We, herein, investigated the direct effect of CRP on IL-8 production and gene expression in cultured human EPCs.Methods: EPCs were isolated from the peripheral venous blood of healthy male volunteers. Cells were cultured in EndoCult^TM liquid medium in the absence and presence of CRP at clinically relevant concentrations (5 to 25μg/ml) for different durations (3 to 48 hours). Furthermore, EPCs were pretreated with SB203580, a mitogen-activated protein kinase (p38 MAPK) inhibitors, anti CD32 and anti CD16 antibody before CRP treatment. IL-8 protein and mRNA of cultured EPCs were evaluated using ELISA and real-time PCR.Results: EPCs could be isolated and cultured from human peripheral blood. The results showed that CRP at concentrations 10μg/ml significantly reduced IL-8 secretion of cultured EPCs with a peak at 25μg/ml, and also decreased mRNA expression of EPCs with a peak at 12 hours. In addition, preincubation of EPCs with SB203580 (an inhibitor of p38 mitogen-activated protein kinase, MAPK) decreased CRP-inhibited IL-8 mRNA expression at 12 hours in EPCs. Anti-CD32 antibody effectively blocked CRP-induced IL-8 expression in addition.Conclusions: Our study, for the first time, demonstrated that CRP directly inhibited EPCs IL-8 secretion, key player of angiogenesis induced by EPCs, which occurred in part via an effect of CRP to active EPCs p38 MAPK signal transduction pathway. CD32 may be the receptor of CRP on EPCs. The ability of CRP to inhibit EPCs IL-8 secretion may represent an important mechanism that further links inflammation to cardiovascular disease.