ABSTRACT
Pericytes play a key role in the process of vascular maturation and stabilization however, the current methods for quantifying pericyte coverage of the neovasculature are laborious and subjective in nature. In this study, we have developed an objective, sensitive, and high-throughput method for quantifying pericyte coverage of angiogenic vessels by analyzing the expression of the pericyte-specific gene, the regulator of G-protein signaling 5 (RGS5). We determined that RGS5 expression was up-regulated during a defined developmental time period in which nascent vessel sprouts acquired a pericyte covering. Furthermore, RGS5 expression was dramatically reduced in vessels with poor pericyte coverage compared to normal angiogenic vasculature. Finally, we determined that the susceptibility of nascent vessels to regression by vascular endothelial growth factor (VEGF) inhibition was significantly reduced following RGS5 up-regulation, further implicating RGS5 in pericyte-endothelial cell interactions and the vascular maturation process. These studies establish the use of RGS5 gene expression as a quantitative and robust measure of pericyte coverage of neovasculature. This method provides a tool for vascular biologists studying pericyte-endothelial cell interactions and vascular maturation in both normal and pathological conditions, such as diabetic retinopathy and cancer.
Subject(s)
Pericytes/metabolism , RGS Proteins/metabolism , Animals , Aptamers, Nucleotide/pharmacology , Cornea/blood supply , Cornea/drug effects , Cornea/pathology , Gene Expression Regulation/drug effects , Male , Mice , Mice, Inbred C57BL , Neovascularization, Physiologic/genetics , Pericytes/drug effects , Platelet Endothelial Cell Adhesion Molecule-1/metabolism , Platelet-Derived Growth Factor/antagonists & inhibitors , RGS Proteins/genetics , Retina/drug effects , Retina/embryology , Retina/pathology , Vascular Endothelial Growth Factor A/pharmacologyABSTRACT
Formation of a functional vasculature during mammalian development is essential for embryonic survival. In addition, imbalance in blood vessel growth contributes to the pathogenesis of numerous disorders. Most of our understanding of vascular development and blood vessel growth comes from investigating the Vegf signaling pathway as well as the recent observation that molecules involved in axon guidance also regulate vascular patterning. In order to take an unbiased, yet focused, approach to identify novel genes regulating vascular development, we performed a three-step ENU mutagenesis screen in zebrafish. We first screened live embryos visually, evaluating blood flow in the main trunk vessels, which form by vasculogenesis, and the intersomitic vessels, which form by angiogenesis. Embryos that displayed reduced or absent circulation were fixed and stained for endogenous alkaline phosphatase activity to reveal blood vessel morphology. All putative mutants were then crossed into the Tg(flk1:EGFP)(s843) transgenic background to facilitate detailed examination of endothelial cells in live and fixed embryos. We screened 4015 genomes and identified 30 mutations affecting various aspects of vascular development. Specifically, we identified 3 genes (or loci) that regulate the specification and/or differentiation of endothelial cells, 8 genes that regulate vascular tube and lumen formation, 8 genes that regulate vascular patterning, and 11 genes that regulate vascular remodeling, integrity and maintenance. Only 4 of these genes had previously been associated with vascular development in zebrafish illustrating the value of this focused screen. The analysis of the newly defined loci should lead to a greater understanding of vascular development and possibly provide new drug targets to treat the numerous pathologies associated with dysregulated blood vessel growth.
