The Angiogenic Properties of the Corneal Epithelial Metabolite 12(R)-Hydroxyeicosatrienoic Acid (12(R)-HETrE) in Human Microvessel Endothelial Cells

Abstract

Abstract: : Purpose: We have shown a strong correlation between the inflammatory/neovascular response in injured cornea and an enhanced production of the arachidonic acid metabolite 12(R)-HETrE which possesses biological activities indicative of a pro-inflammatory factor (vasodilation, increased capillary permeability, neutrophil chemotaxis and angiogenesis).It is readily released from the injured epithelium and its levels increased dramatically in tears from inflamed human eyes suggesting a paracrine role for 12(R)-HETrE. We studied whether human microvessel endothelial cells respond to 12(R)-HETrE. Methods: Endothelial cell cultures from human microvessel endothelial cells (HMVE) were grown until 70% confluent and then quiesced for 24-36 hours. The cells were treated with 12(R)-HETrE (1nM to 10 nM) for 24-72 h. Cell proliferation and capillary like tube formation in cells grown on Matrigel were measured using standard methods. Intracellular Ca was measured using Fura2AM. Results: 12(R)-HETrE (1 nM) caused a rapid and marked increase in intracellular calcium that was inhibited by the PLC inhibitor U73122. 12(R)-HETrE (1 nM) increased HMVE cell number by 3 fold; this increase was also abolished by U73122. Addition of VEGF antibody to HMVE grown in Matrigel-coated plates inhibited 12(R)-HETrE-induced capillary formation. Conclusions: 12(R)-HETrE is mitogenic and angiogenic in human endothelial cells; these effects include the activation of intracellular Ca coupled to PLC-IP3 signaling and are mediated, at least in part, by activation of VEGF. The findings that 12(R)-HETrE is present in inflamed human tears and that it potently activates human endothelial cells to form capillaries implicate its clinical relevance in human disease.

Keywords: eicosanoids • neovascularization • inflammation