A novel in vivo model of puncture-induced iris neovascularization

Abstract

PurposeTo assess iris neovascularization by uveal puncture of the mouse eye and determine the role of angiogenic factors during iris neovascularization.MethodsUveal punctures were performed on BalbC mouse eyes to induce iris angiogenesis. VEGF-blockage was used as an anti-angiogenic treatment, while normoxia- and hypoxia-conditioned media from retinal pigment epithelium (RPE) cells was used as an angiogenic-inducer in this model. Iris vasculature was determined in vivo by noninvasive methods. Iris blood vessels were stained for platelet endothelial cell adhesion molecule-1 and vascular sprouts were counted as markers of angiogenesis. Expression of angiogenic and inflammatory factors in the puncture-induced model were determined by qPCR and western blot.ResultsPunctures led to increased neovascularization and sprouting of the iris. qPCR and protein analysis showed an increase of angiogenic factors, particularly in the plasminogen-activating receptor and inflammatory systems. VEGF-blockage partly reduced iris neovascularization, and treatment with hypoxia-conditioned RPE medium led to a statistically significant increase in iris neovascularization.ConclusionsThis study presents the first evidence of a puncture-induced iris angiogenesis model in the mouse. In a broader context, this novel in vivo model of neovascularization has the potential for noninvasive evaluation of angiogenesis modulating substances.

Gaining insight on mitigation of rubeosis iridis by UPARANT in a mouse model associated with proliferative retinopathy

“…An increase of similar angiogenic factors has been identified in hypoxia-exposed RPE cell media [ 19 , 23 ]. We have established a murine model of puncture-induced RI in association with PR by co-injection of pro-angiogenic factors derived from hypoxia-exposed RPE culture media [ 19 ], with increased neovascularization when compared with the puncture-induced RI mouse model (Suppl. Fig.…”

“…Patients with RI commonly express high VEGF levels as a consequence of the underlying PR [ 4 , 9 , 10 ]. Nonetheless, the puncture-induced RI mouse model previously established by us has been demonstrated to be independent of VEGF signaling [ 19 , 21 ]. Hypoxia-conditioned medium from ARPE-19 cells contains increased levels of VEGF and multiple other pro-angiogenic factors and cytokines [ 19 , 23 ].…”

“…Nonetheless, the puncture-induced RI mouse model previously established by us has been demonstrated to be independent of VEGF signaling [ 19 , 21 ]. Hypoxia-conditioned medium from ARPE-19 cells contains increased levels of VEGF and multiple other pro-angiogenic factors and cytokines [ 19 , 23 ]. Combination of the puncture-induced RI with co-injection of hypoxia-conditioned medium enhances the RI-PR mouse model to more closely parallel patients with iris neovascularization as a consequence of associated eye diseases.…”

“…Despite a considerable slower effect, anti-VEGF treatment produced similar outcomes within the study protocol. The mouse iris neovasculature develops through anastomosis [ 19 ], characterized by formation of vascular sprouts and branching. Analysis of irises microvasculature with PECAM-1 demonstrates that UPARANT-treated RI-PR eyes are indistinguishable from controls regarding number of blood vessels, sprouting, and branching, with a stronger reduction when compared with anti-VEGF.…”

“…To mimic PR (RI-PR model), three groups of six mice each, for a total of eighteen mice, were subjected to uveal punctures as described previously [ 19 , 20 ] and co-injected in both eyes with 1 μL of hypoxia-conditioned medium from ARPE-19 cells (ATCC, Manassas, VA, USA). Six mice were kept as untreated controls.…”