Alpha2m the largest non-immunoglobulin protein present in blood plasma of mammals is an acute phase protein with its serum level elevated upon tissue damage and inflammation. In this study alpha2m administration in mEBs during early differentiation processes resulted in an enhancement of vascularization as assessed with PECAM1 and VE-Cadherin immunofluorescence studies. alpha2m stimulated a dose-dependent increase in protein expression of the endothelial specific markers PECAM1, FLK1 and VE-Cadherin along with up-regulation of the angiogenic growth factors FGF2, VEGF-165 and PDGF-BB. Alpha2m modulates it activity upon binding to and activating its receptors LRP1 and GRP78. Upon blocking with specific antibodies alpha2m mediated vascularization was abolished. Moreover the LRP1 antagonist RAP and inhibitors of the angiogenic growth factors blunted the expression of the growth factors FGF2, VEGF-165 and PDGF-BB along with abolishment of vascularization, suggesting the role of LRP1 receptor in triggering endothelial differentiation signaling. Alpha2m treatment also resulted in the activation of ERK1/2, PI3K, AKT and STAT3 which are all implicated in angiogenic signaling cascades. Their subsequent blocking with their respective inhibitors resulted in the abolishment of vascularization. Investigating the functional characteristic of alpha2m under systematic in-vitro analysis of sprout formation revealed that alpha2m stimulates endothelial cell proliferation and migration inducing vessel formation with longer and branched sprouts.Induction of inflammation in mEBs upon treatment with the inflammatory cytokine TNFa stimulated upregulation of alpha2m and its receptor LRP1 along with increase in vascularization. The vascularization induced by TNFa was abolished upon antibody blocking of the receptor LRP1 suggesting its activation in triggering increased vascularization. In contrast alpha2m treatment resulted in a dose-dependent decrease in the expression of intrinsic TNFalpha along with increase in vascularization, suggesting alpha2m mediates TNFalpha removal by activating LRP1 signaling which could also be responsible for enhanced vascularization in mEBs.In summary this study reports that alpha2m stimulates vasculogenesis in mESCs with upregulation of growth factors and activation of angiogenic signaling pathways. Additionally alpha2m exerts anti-inflammatory effects in mESCs through inhibition of TNFalpha expression.
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