Role of a Rho GTPase guanidine exchange factor DOCK9 in cell signalling and angiogenesis

Abstract

Abstract Formation of the vascular network mainly occurs through two critical mechanisms, vasculogenesis and angiogenesis. Both vasculogenesis, the de novo formation of blood vessels during embryogenesis, and angiogenesis, the formation of new blood vessels from pre-existing ones by sprouting, are sophisticated and tightly regulated. To form functional vasculature, critical cellular mechanisms such as cell migration, adhesion, and proliferation are required, which are controlled by the activity of small Rho GTPases such as RhoA, Rac, and Cdc42. Small Rho GTPases are regulated in part by the action of the dedicator of cytokinesis (DOCKs) protein family. To date, 11 members of DOCKs protein have been discovered in the mammalian system. The subfamily member DOCK 9 (also known as ZIZIMIN1) is involved in vascular development and highly expressed in the heart. Thus, we hypothesised that DOCK 9 acts as GEF for Cdc42 in the heart; this is supported by the actions of several angiogenic signalling pathways, including CXCL12 and its receptor. Recently, a few studies have revealed that CXCL12 acts on endothelial cells to regulate cardiac development and angiogenesis. Yet, the downstream mechanism of CXCL12 and its receptor (CXCR4) need to be determined. In this study, the CXCL12 signalling mediatedby DOCK9 was elucidated. To this aim, human umbilical vein endothelial cells (HUVECs) were subjected to DOCK9 knockdown and stimulated by CXCL12. Through immunoblotting, we found a trend that with DOCK9 knockdown, HUVECs had less phosphorylated response and a decrease in ERK activation. However, the knockdownoftheDOCK9 was not efficient enough, so to further investigate the role of the DOCK9in angiogenesis, different studies were carried out in wild-type and knockout mice. First, analysis of postnatal mice retinasfrom WT and DOCK9 knockout by immunofluorescence staining revealed that retinal blood vessel length is affected by DOCK9 deletion, leading to a significant decrease in the length of angiogenic sprouts.In addition, gene expressions of marker genes in response to pressure overload was explored using a transverse aortic constriction (TAC) model in mice. Contrary to expectations, DOCK9 deficiency did not significantly reduced marker genes of cardiachypertrophy. Finally, aortic rings were treated with cxcl12; instead of stimulation inhibitory effect was observed.