The Effects And Mechanism Of Cellular Repressor Of E1A-stimulated Genes On Human Vascular Endothelial Cell Monolayer Permeability

Objective:The explore of angiogenesis approach brings a breakthrough in the treatment of ischemic heart disease.The purpose of angiogenesis approach is to restore the blood supply of ischemic myocardium and to improve symptoms and prognosis by increasing coronary artery branches or collateral circulation. The increase of vascular permeability is one of the sequence of events in angiogenesis. Most of cytokines and inflammatory mediators lead to ECs monolayer permeability increase by changing actin cytoskeleton which results in ECs retraction, the cell gap increases and damages VE-cadherin.The cellular repressor of E1A stimulated genes (CREG) was a transcriptional regulator found by Gill in 1998, which expressed at high level in the differentiated and mature tissues and cells in contrast to at low level in the undifferentiated and immature ones. Moreover, our lab has identified that CREG could promote differentiation of human vascular smooth muscle cells (VSMCs) and inhibit their proliferation and apoptosis in vitro and promote the migration and proliferation of ECs in vitro.These findings suggested that CREG is an important endogenous factor in regulating vascular homeostasis.But there is no reported research exploring roles of CREG in regulating arterial ECs monolayer permeability.In this study, human arterial ECs were tansfected with retroviral eukaryotic vectors and their expression of CREG were up-or down-regulated. The aim of this study was to investigate the effects and mechanism of CREG on human vascular ECs monolayer permeabilityMethods:1.The effects of CREG on the endothelial permeability were investigated by measuring paracellular flux of Biotin-BSA.Medium was then replaced with fresh serum free DMEM in the presence or absence of anti-VEGF neutralizing antibody and the incubation was continued for 30 minutes.The Biotin-BSA content of the samples was evaluated by ELISA reader. Enzyme linked immunosorbent assay (ELISA) were performed to test the concentration of VEGF in the medium.2.F-actin was stained by fluorescene isothiocyanate-conjugated phalloidin in the presence or absence of anti-VEGF neutralizing antibody and examined using OlympusⅨ-70 fluorescent microscope.3.VE-cadherin and claudin-5 were stained by fluorescene immunocyto-chemistry in the presence or absence of anti-VEGF neutralizing antibody and examined using OlympusⅨ-70 fluorescent microscope.Results:1. CREG overexpression ECs(group EO) monolayer permeability (0.0490±0.0016) was significantly increased compared with the normal control ECs (group EN) (0.0376±0.0061) (p<0.05). CREG suppression ECs (group ES) monolayer permeability(0.0324±0.0114) was decreased compared with group EN(0.0376±0.0061) (p<0.05). Results of ELISA with supernatant of cultivation suggested that the secretion of VEGF increased in group EO(1392.00±5.83) pg/ml but decreased in group ES (391.40±4.04) pg/ml compared with group EN(577.20±10.43)pg/ml (p<0.05).ECs permeability induced by CREG could be impeded by addition of anti-VEGF neutralizing antibody.2.Under basal conditions, F-actin in artery endothelial cells was arranged into a fine cortical network with a dense peripheral band at the cell boundaries;occasionally, stress fibers were observed. That F-actin cytoskeleton disorganized, polymerized and bundled obviously to form large amount of stress fiber in the central portion of cells was observed in group EO, whereas that F-actin mainly formed in the peripheral portion of cells and small amounts in the central portion of cells was observed in group ES. ECs cytoskeleton reorganization by CREG could be impeded by addition of anti-VEGF neutralizing antibody.3. A widespread gap formation and a loss of VE-cadherin staining at the periphery were found in group EO, inversely that VE-cadherin localized at the cell-cell contacts tightly and formed the zipper-like structures were found in group ES and EN. Expression of claudin-5 decreased in group EO but increased in group ES compared with group EN.The decrease of claudin-5 expression in group EO and the loss of VE-cadherin of ECs induced by CREG could be impeded by addition of anti-VEGF neutralizing antibody.Conclusion:As a kind of secreted protein, CREG over-expression can increase monolayer permeability of human vascular endothelial cells cultured in vitro through cytoskeleton reorganization and decreased expression of VE-cadherin and claudin-5 by the mechanism of enhancing the secretion of VEGF.