The Function And Mechanism Of The Signal Transduction Regulating Angiogenesis By G?i Mediated MYDGF

Intraocular pathological angiogenesis is an important pathogenetic mechanism for many ocular diseases,and inhibition of pathological angiogenesis is an effective treatment.Cell growth factors closely related to angiogenesis such as vascular endothelial growth factor(VEGF)? epidermal growth factor(EGF)can promote the proliferation of vascular endothelial cells and thus promote the progression of pathological angiogenesis.Finding a way to inhibit these growth factors can effectively slow down the development of pathological neovascularization to some extent.According to our previous research experience,G?i plays an important role in the activation of downstream AKT-mTOR and MAPK/ERK signaling pathway.MYDGF is a bone marrow-derived angiogenic factor,which has been confirmed to be expressed in various tissues and organs of the body by previous studies.MYDGF can also promote the proliferation and survival of vascular endothelial cells.We aim to study whether G?i plays an important role in MYDGF-mediated angiogenesis so as to provide a new direction for the treatment of pathological neovascular disease in the eyes.Purposes ? Analyze the molecular mechanism of MYDGF signal transduction mediated by G?i;?Clarify the effect of MYDGF and G?i expression on angiogenesis.Methods ? Cultivation of wild type mouse embryonic fibroblasts(WT-MEFs)?G?i1/3 double knockout mouse embryonic fibroblasts(DKO-MEFs),G?i1 single knockout mouse embryonic fibroblasts(G?i1KO-MEFs),G?i2 single knockout mouse embryonic fibroblasts(G?i2KO-MEFs),G?i3 single knockout mouse embryonic fibroblasts(G?i3KO-MEFs)and Gab1 knockout mouse embryonicfibroblasts(Gab1KO-MEFs);? Targeted and knocked down G?i1 and/or G?i3 in MEFs with the method of shRNA;?Transferred G?i1 and G?i3 expression plasmid to DKO-MEFs cells to obtain Rescue1-MEFs and Rescue3-MEFs;? The above cells were stimulated with different concentrations of MYDGF for different time to detect the activation levels of the downstream AKT-mTOR signaling pathway(AKT,S6,S6 K,and GSK3?/3?)and the MAPK/ERK signaling pathway(ERK);?Lentivirus vectors targeting to knock down G?i1 and G?i3 genes were transfected into HUVECs to obtain G?i1/3-shRNA HUVEC cells.Transferred negative vectors to obtain scr-shRNA HUVEC cells;?The modal of retinopathy of prematurity(ROP)in mice was established by high oxygen concentration.The content of MYDGF in retina of mouse was measured;?The content of MYDGF in proliferative membrance of proliperative diabetic retinopathy(PDR)was measured.Result ?In MEFs,G?i1 and G?i3 double knock blocked the activation of the MYDGF-induced AKT-mTOR and MAPK/ERK signaling pathway;? After the treatment of MYDGF,the G?i1KO-MEFs,G?i3KO-MEFs and shRNA-MEFs were weakened to varing degrees and the G?i2KO-MEFs after the stimulation of MYDGF,the activation of downstream signal did not have significant changes;?With the stimulation of MYDGF,downstream signal activation of Rescue1-MEFs and Rescue3-MEFs had obvious recovery;?Teated with MYDGF,Gab1 knockout MEFs showed significant reduction or even loss of activation level of downstream AKT-mTOR and MAPK/ERK signaling pathway;? MYDGF could promote the proliferation,migration and tube formation of HUVECs,but cell activity of HUVECs transfected with G?i1/3-shRNA lentivirus was significantly inhibited;?The content of MYDGF in retina of ROP mouse was higer than that of control mouse;? The content of MYDGF in retina of proliferative diabetic retinopathy was higher than that in normal retina.Conclusion ? Results in vitro suggested that G?i1 and G?i3 may mediate the downstream signal transduction induced by MYDGF;? Results of cell experiment showed that G?i1 and G?i3 paly an important role in the proliferation of HUVEC induced by MYDGF.? Results in vivo suggested that G?i1 and G?i3 may mediate the downstream signal transduction induced by MYDGF.