Effects Of Erythropoietin On Vascular Endothelial Cells Apoptosis Induced By Tumor Necrosis Factor-alpha

Background:The endothelial cell layer is the "guardian" of molecular traffic between the blood and surrounding tissue. The endothelial integrity plays a pivotal role in many aspects of vascular function:e.g., regulates vascular tone, tissue perfusion, vascular permeability, myocardial function, blood fluidity, anticoagulant activity and inflammatory responses. The circulatory inflammatory cytokine tumor necrosis factor-alpha (TNF-α) is increased in pathological conditions, such as chronic kidney disease (CKD), especially in end stage renal disease, which initiates or exacerbates vascular endothelial injury. Ongoing endothelial dysfunction even disintegration are thought to be central processes toward cardiovascular disease (CVD) and progressive kidney damage. Thus, protecting endothelial cell could be potentially beneficial in clinical trials aimed at reducing the incidence of CVD and also the loss of kidney function in CKD patients. A molecule coming into the focus of cardiovascular medicine is erythropoietin (EPO), best known for its ability to increase red blood cell mass. However, beside its effect on hematopoietic cells, EPO protects endothelial cell function and integrity. A recent experimental studie has shown that EPO protect cardiomyocytes from apoptotic cell death induced by direct cytotoxicity of TNF-α. The role of endothelial nitric oxide synthase (eNOS) in the anti-apoptotic effects of EPO in cardiomyocytes has already been proved. Because its cytoprotective properties having been brought to light, we have reason to believe that EPO could prevent even reverse CVD and CKD progression due to endothelial cell lose.Objective:We investigated the effects of recombinant human erythropoietin (rhEPO) on human umbilical vein endothelial cells (HUVEC) apoptosis induced by TNF-α and whether the positive role of EPO on eNOS also occur in the vascular endothelial cells. This study was also designed to assess the role of EPO in regulating the expression of the cell apoptosis-associated intracellular signaling component Caspase-3in the endothelial cell stimulated with the proinflammatory cytokine TNF-a. The ultimate goal of this study was to provide a theoretical basis for application of EPO replacement therapeutic strategy approach to reduce CVD incidence and delay CKD progression.Methods:HUVEC were maintained at37℃in5%CO2in Dulbecco’s Modified Eagle’s Medium (DMEM) containing10%fetal bovine serum (FBS),100U/ml penicillin and100μg/ml streptomycin. The medium was changed every two days and the cells were passaged with trypsin-EDTA. Cultured endothelial cells were pre-incubated for24h in DMEM containing no serum before experiments. Cells were then randomly divided into five study groups:either not treated (control), or treated with TNF-a (50ng/ml) alone or rhEPO at different doses (12.5,25,50IU/ml) pretreated before TNF-a, respectively, for24h. Morphology of endothelial cells was observed by inverted fluorescence microscope. Cell viability was measured by methyl thiazolyl tetrazolium (MTT) assay. Cell apoptosis was evaluated using single cell gel electrophoresis (SCGE) assay. The concentration of eNOS was assessed using enzyme-linked immunosorbent assay (ELISA). The expression of Caspase-3protein was examined by immunocytochemistry assay.Results:TNF-a-treated HUVEC displayed morphological features of apoptosis, such as shrunken body, sparkling membrane, dense nucleu and so on. TNF-a-induced endothelial cell apoptosis was associated with reduced eNOS concentration but increased Caspase-3protein expression (all P<0.01). Compared to untreated cells, rhEPO-pretreated HUVEC exhibited better morphology. Pretreatment with rhEPO at25and50IU/ml restored eNOS activation as compared to TNF-a alone (all P<0.05). EPO at12.5IU/ml, however, did not have significant effect on TNF-a-induced eNOS decrease (P>0.05). This phenomenon was accompanied by reduced the relative content of Caspase-3protein, as resulting in improved cell viability and reduced apoptosis in a dose-dependent manner (all P<0.05). Such differences between rhEPO-pretreated groups was obvious (all P<0.05).Conclusion:EPO is a promising cell protector against cytotoxic effects of TNF-a, the anti-apoptotic effects on vascular endothelial cells are mediated by restoring eNOS activation and inhibiting Caspase-3expression.