Antagonism of PDGFRβ Inhibits Pericyte Recruitment in a Mouse Model of Corneal Neovascularization

Abstract

Purpose: To explore the pathobiology of pericytes in ocular diseases such as diabetic retinopathy, in which pericyte loss is a hallmark. Pericytes are abluminally associated with the capillary endothelium and contribute to vessel formation, stabilization, and maturation. To examine the role of PDGF in these functions, we inhibited the PDGF pathway in a mouse model of corneal neovascularization (CoNV).

Methods: CoNV was induced in anesthetized C57BL/6 mice by mechanical abrasion of corneas. Animals were treated every other day, with control antibodies or with PDGFRβ and VEGF neutralizing antibodies administered IP. To quantify pericytes and neovascularization (NV), corneal flat mounts were stained with α-NG2 and α-PECAM1 antibodies, to detect pericytes and endothelial cells respectively, and imaged by fluorescence microscopy. Pixel area was measured using a program written in MatLab, and pericyte coverage (%) was calculated as NG2-labeled pixels/PECAM1-labeled pixels*100. NV area was measured using AxioVision software. Vessel morphology was assessed using AngioTool.

Results: In unabraded mice, pericytes were associated only with perilimbal vessels as there were no corneal vessels. In the new vessels observed in the abraded mice there was an increase in pericyte area (6.2-fold), vessel area (3.6-fold), and pericyte coverage (1.6-fold) relative to normal limbal vessels in unabraded controls. Neutralization of PDGFRβ dramatically reduced pericyte coverage but had no effect on vessel area or morphology. α-VEGF treatment resulted in reduced vessel area and altered vessel morphology. Neutralizing both PDGFRβ and VEGF did not elicit any additional effects on pericyte area, vessel area or morphology compared to neutralizing VEGF alone.

Conclusions: Corneal abrasion resulted in NV which could be inhibited with an antibody against VEGF. The new vessels were accompanied by an increase in pericyte area and coverage, which represents recruitment of new pericytes and can be blocked with systemic α-PDGFRβ treatment. This model allows visualization of both existing and newly recruited pericytes and appears to be a suitable model for studying pericytes.

Keywords: 609 neovascularization • 499 diabetic retinopathy • 481 cornea: endothelium