Mural Cell Associated VEGF Is Required for Organotypic Vessel Formation

Abstract

BackgroundBlood vessels comprise endothelial cells, mural cells (pericytes/vascular smooth muscle cells) and basement membrane. During angiogenesis, mural cells are recruited to sprouting endothelial cells and define a stabilizing context, comprising cell-cell contacts, secreted growth factors and extracellular matrix components, that drives vessel maturation and resistance to anti-angiogenic therapeutics.Methods and FindingsTo better understand the basis for mural cell regulation of angiogenesis, we conducted high content imaging analysis on a microtiter plate format in vitro organotypic blood vessel system comprising primary human endothelial cells co-cultured with primary human mural cells. We show that endothelial cells co-cultured with mural cells undergo an extensive series of phenotypic changes reflective of several facets of blood vessel formation and maturation: Loss of cell proliferation, pathfinding-like cell migration, branching morphogenesis, basement membrane extracellular matrix protein deposition, lumen formation, anastamosis and development of a stabilized capillary-like network. This phenotypic sequence required endothelial-mural cell-cell contact, mural cell-derived VEGF and endothelial VEGFR2 signaling. Inhibiting formation of adherens junctions or basement membrane structures abrogated network formation. Notably, inhibition of mural cell VEGF expression could not be rescued by exogenous VEGF.ConclusionsThese results suggest a unique role for mural cell-associated VEGF in driving vessel formation and maturation.

A novel imaging‐based high‐throughput screening approach to anti‐angiogenic drug discovery

“…Images were acquired for every third day as 53 5 montages with a 310 objective and quantified with the parameter tube total length (BD Attovision software). Once the network is formed at day 3, this parameter reflects the stability of the network (day [3][4][5][6][7][8][9][10][11][12], and inhibition of network formation due to drug treatment can be measured. (b) Images from a developing network acquired every third day during a 12-day period showing fluorescent EC (gray).…”

“…The co-culture assay entails live image analysis of fluorescent protein-expressing primary human EC that form tubular networks within 72 h postseeding (6). Unless otherwise specified, the EC used in this study were transduced with a tdTomato retroviral expression construct to ensure stable expression of the fluorescent marker.…”

“…As they begin to integrate into a proper network (Day 3, Fig. 1b), the EC interdigitate to generate tubes with uniform diameter (6). The EC do not proliferate when co-cultured with vSMC, so the networks do not grow in size.…”

“…During network formation, EC are completely dependent on vSMC-derived VEGF. Inhibition of VEGF-signaling [by treatment with anti-VEGF antibody, (bevacizumab), VEGF receptor-targeting small molecule tyrosine kinase inhibitors, or RNA interference of VEGF expression in vSMC] potently blocks capillary-like network formation (6)(7)(8)(9). However, once formed, EC networks acquire resistance to anti-VEGF therapeutics reflecting a phenotypic VEGF-switch characteristic of blood vessel maturation.…”

Targeting Endothelium-Pericyte Cross Talk by Inhibiting VEGF Receptor Signaling Attenuates Kidney Microvascular Rarefaction and Fibrosis

“…[12][13][14][15] PDGF over expression in endothelial cells, however, paradoxically promotes vascular rarefaction, suggesting that pro-apoptotic factors are released from, or survival factors are down-regulated in, expanded pericytes/fibroblasts. 16 Although vascular endothelial growth factor (VEGF) signaling from pericytes to endothelial cells has been implicated in developmental angiogenesis, [17][18][19] and more recently VEGF signaling from the specialized pericytes of the kidney glomerulus known as podocytes has been shown to be crucial for vascular stabilization, 20 the presence of VEGF receptor 1 (VEGFR1) FMS-like tyrosine kinase 1 (Flt1) and receptor 2 (VEGFR2) VEGF receptor 3 and Neuropilin 1 on endothelial cells, the regulated expression of four distinct VEGF genes, and the regulated expression of multiple transcription splice variants render VEGF signaling more complicated than simple binary ligand-receptor interactions.…”