NG2 inhibition decreases endothelial cell sprouting along venules: A novel in situ angiogenesis assay to investigate multicellular interactions

Abstract

Elucidating the multicellular dynamics associated with angiogenesis requires the ability to examine mechanistic responses over the hierarchy of a microvascular network within a controlled environment. The objective of this study was to establish a simple in situ rat mesentery culture model to investigate pericyte‐endothelial interactions during angiogenesis in intact networks. Adult Wistar rat mesenteric windows were aseptically harvested and cultured in minimum essential media for 3 days. Media was left unstimulated or supplemented with bFGF, VEGF or bFGF + NG2 antibody. In supplement‐free media, viability/cytotoxicity analysis revealed that cells were alive after 3 days of culture. Immunohistochemical labeling with antibodies to PECAM, NG2 and LYVE‐1 identified endothelial cells, pericytes and lymphatic cells, respectively. VEGF and bFGF treatments stimulated approximately a 2‐fold increase in capillary sprouting, quantified as endothelial cell sprouts per venule length. NG2 function‐blocking antibodies inhibited the bFGF‐induced sprouting along venules. The results establish primary culture of the adult rat mesentery as a novel in situ angiogenesis model for investigating multicellular angiogenic mechanisms across intact microvascular networks, and implicate pericyte NG2 expression as a functional regulator of capillary sprouting. Supported by the Board of Regents of the State of Louisiana LEQSF(2009‐12)‐RD‐A‐19.Grant Funding Source : Supported by the Board of Regents of the State of Louisiana LEQSF(2009‐12)‐RD‐A‐19.