Pericyte Rho GTPase Mediates Both Pericyte Contractile Phenotype and Capillary Endothelial Growth State

Abstract

Pericytes regulate microvascular development and maturation through the control of endothelial cell motility, proliferation, and differentiation. The Rho GTPases have recently been described as key regulators of pericyte shape and contractile phenotype by signaling through the actin cytoskeleton in an isoactin-specific manner. In this report, we reveal that Rho GTPase-dependent signal transduction not only influences pericyte shape and contractile potential but also modulates capillary endothelial proliferative status and pericyte-endothelial interactions in vitro. We provide evidence that overexpression of mutant Rho GTPases, but not other Ras-related small GTPases, significantly alters pericyte shape, contractility, and endothelial growth state in microvascular cell co-cultures. In particular, we describe the use of a silicon substrate deformation assay to demonstrate that pericyte contractility is Rho GTP-and Rho kinase-dependent; further, we describe a novel in vitro system for examining pericyte-mediated endothelial growth arrest and show that control pericytes are capable of growtharresting capillary endothelial cells in a cell contactdependent manner, whereas pericytes overexpressing dominant-active and -negative Rho GTPase are comparably incompetent. These data strongly suggest that signaling through the pericyte Rho GTPase pathway may provide critical cues to the processes of microvascular stabilization, maturation, and contractility during development and disease.

Pericyte contractility controls endothelial cell cycle progression and sprouting: insights into angiogenic switch mechanics

“…Pericyte contractility was assayed as previously described (36,39,41), with the following modifications (22). Briefly, silicone (DMPS-12M, Sigma) was spread on coverslips at 42°C for 1 h. Siliconecoated coverslips were passed briefly over a flame to polymerize the substrate and then coated with plasma (36,39,41).…”

“…Briefly, silicone (DMPS-12M, Sigma) was spread on coverslips at 42°C for 1 h. Siliconecoated coverslips were passed briefly over a flame to polymerize the substrate and then coated with plasma (36,39,41). Rat tail collagen type I (Becton-Dickinson), diluted to 3 g/ml in PBS, was added to the coverslip and sterilized under UV light for 5 min.…”

“…The notion has been widely held that, early in DR, vascular cell death manifests, first by pericyte dropout and then by EC apoptosis and an excessive retinal neovascular response (2,13,40). However, recent work strongly suggests that pericyte dysfunction, not death or dropout, precedes pathological angiogenic induction and misregulation of microvascular growth (36,39,41).…”

“…Overexpression and selective inhibition studies have revealed that RhoA GTPase activity affects specifically the smooth muscle actin (SMA)-based cytoskeletal networks (35). Furthermore, alteration of pericyte Rho GTPase status via viral transduction of dominantactive or dominant-negative RhoA constructs leads to altered force generation on deformable substrates and hyper-or hypocontractile cells, respectively (39). Interestingly, alteration of normal pericyte tonus in either direction was shown to equally culminate in a loss of contact-dependent EC quiescence (39).…”

“…Furthermore, alteration of pericyte Rho GTPase status via viral transduction of dominantactive or dominant-negative RhoA constructs leads to altered force generation on deformable substrates and hyper-or hypocontractile cells, respectively (39). Interestingly, alteration of normal pericyte tonus in either direction was shown to equally culminate in a loss of contact-dependent EC quiescence (39). Thus a more complete understanding of the RhoA/ROCK pathway will lend insights into the physiological/pathological regulation of contractile phenotype and how pericyte-EC interactions govern angiogenesis.…”