Abstract
Glycation of vessel wall proteins is thought to have an important role in the pathogenesis of vascular complications in diabetes mellitus. However, no previous study has implicated glycated vitronectin (VN) in the control of vascular endothelial growth factor (VEGF) signaling. To explore whether the glycation of VN affects angiogenic signaling and to understand the molecular mechanisms involved, we synthesized glycated VN by incubating VN with methylglyoxal (MGO) in vitro and identified the formation of glycated VN by an LC-ESI-MS/MS-based method. We tested the hypothesis that glycation of VN downregulates VEGF receptor-2 (VEGFR-2) activation by uncoupling the interaction between VEGFR-2 and αvβ3. Unmodified and MGO-glycated VN were used as substrates for human umbilical vein endothelial cells (HUVECs). The effects of glycated VN on VEGF signaling in HUVECs were investigated. The glycation of VN inhibited VEGF-induced phosphorylation of VEGFR-2 and the intracellular signaling pathway downstream of VEGFR-2. Glycated VN inhibited the binding of VEGFR-2 to β3 integrin and inhibited the phosphorylation of β3 integrin. Furthermore, glycation of VN significantly decreased VEGF-induced migration of HUVECs in vitro and vessel outgrowth in an ex vivo angiogenesis model. Collectively, these data indicate that the glycation of VN inhibits VEGF-induced VEGFR-2 activation by uncoupling VEGFR-2-αvβ3 integrin cross-talk. The glycation of VN causes a reduction in the migration of endothelial cells and vessel outgrowth. This may provide a mechanism for the failure of collateral sprouting in diabetic microangiopathy.