Expression Of VEGF And FLK-1 In HBMEC Under The State Of UPR

ObjectiveThe blood - brain barrier ( BBB) is formed by the complex tight junctions between the endothelial cells of the brain capillaries. This structure results in the capillary wall that behaves as a continuous lipid bilayer controlling the access of solutes and toxic substances to the central nervous system (CNS) so as to maintain brain homeostasis. So, study of human brain microvascular endothelial cell ( HBVEM ) will lead us to understand the regulative mechanism of BBB homeostasis , and pathogenesis of some CNS disorders.Vascular endothelial growth factor ( VEGF) is a highly specific mitogen for vascular endothelial cells and induces neovascularization in development, trauma and tumor. Through its receptors, VEGFR2 (FLK - 1) VEGFR1 (FLt -1), VEGF induces endothelial cell proliferation and differentiation, promotes cell migration, and inhibits ap-optosis. In addition of its paracrine function, VEGF is an autocrine factor. VEGF can promotes endothelial cell survival and resist apopto-sis, which is mediated through the VEGFR-2 receptor and the phos-phatidylinositol 3'- kinase (PI3 - K)/Akt and MEK/ERK signaling pathway. These indicate that VEGF can promote endothelial cell homeostasis under stress situation, such as hypoxia and ischemia. And that these stresses also result in disturbance of homeostasis unavoidably , and launch some stress response, including unfolding protein re-sponse.UPR is a signal passway from endoplasmic reticulum to nucleus and results in up-regulation of expression of molecular chaperon, such as GRP78, GRP94 and PDI. These molecular chaperon help protein fold accurately, and reduce the overloading of unfolded and wrongly - folded protein resulted from stress.Since UPR and VEGF - FLK - 1 passway can help cell maintain homeostasis under stress, we can assume some relationship between them. So, taking the main part of BBB - HBMEC as the object, we tested Expression of VEGF and FLK - 1 in HBMEC under the state of UPR and accumulate more data for exploring the function of BBB thereby.Methods1. HBMEC Cell culture and induction by tunicamycin of different concentration(2,5 ,10,25g/ml)2. detection of GRP78,GRP94,VEGF mRNA by RT - PCR3. detection of GRP78,GRP94,VEGF protein by western blot4. detection of GRP78,GRP94,VEGF protein by immunohisto-chemistryResults1. Basal expressions of GRP78, GRP94,VEGF and FLK - 1 are detected in HBMEC.2. Levels of both mRNA and protein of GRP78 of HBMEC in pre - induction and post-induction are of evident difference. Levels inpost - induction are far higher than in pre - induction, and with the increase o f the concentration o f tunicamycin, Levels of mRNA andprotein also increase.3. Levels of both mRNA and protein of GRP94 of HBMEC in pre- induction and post - induction are of evident difference. Levels in post - induction are far higher than in pre - induction, and with the increase o f the concentration o f tunicamycin, Levels of mRNA and protein also increase.4. Levels of both mRNA and protein of VEGF of HBMEC in pre- induction and post - induction are of evident difference. Levels in post - induction are far higher than in pre - induction, and with the increase o f the concentration o f tunicamycin, Levels of mRNA and protein also increase.5. Levels of protein of FLk-1 of HBMEC in pre - induction and post - induction are of difference. Levels in post - induction are higher than in pre - induction, and with the increase of the concentration o f tunicamycin, Levels of protein also increase.Conclusion1. In HBVEM, there are basal expression of GRP78,GRP94 VEGF and VEGF may function as a autocrine survival factor.2. Expression of VEGF and FLk -1 correlates to inductive level of UPR in HBVEM.3. VEGF and its receptor-FLK - 1 may be the target gene of UPR, suggesting a mechanism of homeostasis of HBMEC under stress.