Temporal phosphoproteomic analysis of VEGF-A signaling in HUVECs: an insight into early signaling events associated with angiogenesis

Abstract

Vascular endothelial growth factor-A (VEGF-A) is one of the primary factors promoting angiogenesis in endothelial cells. Although defects in VEGF-A signaling are linked to diverse pathophysiological conditions, the early phosphorylationdependent signaling events pertinent to VEGF-A signaling remain poorly defined. Hence, a temporal quantitative phosphoproteomic analysis was performed in human umbilical vein endothelial cells (HUVECs) treated with VEGF-A-165 for 1, 5 and 10 min. This led to the identification and quantification of 1971 unique phosphopeptides corresponding to 961 phosphoproteins and 2771 phosphorylation sites in total. Specifically, 69, 153, and 133 phosphopeptides corresponding to 62, 125, and 110 phosphoproteins respectively, were temporally phosphorylated at 1, 5, and 10 min upon addition of VEGF-A. These phosphopeptides included 14 kinases, among others. This study also captured the phosphosignaling events directed through RAC, FAK, PI3K-AKT-MTOR, ERK, and P38 MAPK modules with reference to our previously assembled VEGF-A/VEGFR2 signaling pathway map in HUVECs. Apart from a significant enrichment of biological processes such as cytoskeleton organization and actin filament binding, our results also suggest a role of AAK1-AP2M1 in the regulation of VEGFR endocytosis. Taken together, the temporal quantitative phosphoproteomics analysis of VEGF signaling in HUVECs revealed early signaling events and we believe that this analysis will serve as a starting point for the analysis of differential signaling across VEGF members toward the full elucidation of their role in the angiogenesis processes.

Isolation and characterization of ZK002, a novel dual function snake venom protein from Deinagkistrodon acutus with anti-angiogenic and anti-inflammatory properties

“…During angiogenesis, activation of VEGFR-2 promotes cell proliferation, migration, and vascular permeabilization (Apte et al, 2019). In our study, we found that ZK002 treatment inhibited activation of VEGFR2, an event located at the proximal point of VEGF-mediated signaling (Abhinand et al, 2016;Abhinand et al, 2023).…”

Roles and inhibitors of FAK in cancer: current advances and future directions

“…Temporal quantitative phosphoproteomic analysis of human umbilical vein endothelial cells revealed that FAK phosphorylation activation is an early phosphorylation-dependent signaling event in the VEGFA/VEGFR2 pathway, emphasizing the crucial role of FAK in initiating angiogenesis ( Abhinand et al, 2023 ). Notably, distinct phosphorylation sites on EC-FAK have divergent effects on tumor angiogenesis in vivo .…”