Experimental inhibition of proliferative retinopathy

Abstract: Retinal ischemic diseases like retinopathy of prematurity (ROP), diabetic retinopathy (DRP) and retinal vein occlusion, with preretinal neovascularization (angiogenesis) that may lead to vitreous hemorrhages and retinal detachment with subsequent vision loss and blindness is a global health problem. By analyzing the potential role of molecules acting at different levels of the angiogenic cascade the presented studies aimed to widen our understanding of retinal ischemic diseases on a molecular, cellular and morphologic level. All studies were based on an experimental model of oxygen-induced retinopathy (OIR) where neonatal mice exposed to a hyperoxic environment develop pathologic retinal neovascularization. Genetically modified mice lacking functional genes (so called knock-out mice (KO)) for matrix metalloproteinase-2 (MMP-2), the adenosine A2A receptor (A2AR) or interleukin-10 (IL-10) were studied. In addition, purinergic P2 receptors were investigated in normal mice. Retinal neovascularization was quantified by counting neovascular nuclei in retinal cross-sections. Retinal vessel development and capillary free areas were investigated by fluorescein staining of retinal flatmounts. P2 receptor expression and studies of the inflammatory reaction were performed with immunohistochemistry. In situ hybridization and real time-Polymerase Chain Reaction (rt-PCR) were used for identification of mRNA of relevant target molecules. In the first study MMP-2, a member of an enzyme family essential for degradation of extracellular matrix (ECM) and the vascular basement membrane was not found to interfere with normal retinal vessel development. In OIR there was increased retinal neovascularization in the MMP-2 KO mice pointing at MMP-2 having an anti-angiogenic effect in this model. In the second study, the expression of P2X2 and P2Y2 receptors were found to be up-regulated by oxygen exposure. Retinal neovascularization was strongly inhibited by the P2 antagonists suramin and PPADS and this was accompanied with a down-regulation of P2X2 receptor expression suggesting a role for P2 receptors in OIR. In the third study, hyperoxia was seen to reduce A2AR mRNA expression but the decline in receptor expression between postnatal day 12 (P12) and P17 seen in control animals was absent in retinas exposed to hyperoxia. The area of vascular regression was smaller in A2AR KO mice and there was also reduced retinal neovascularization. Gene microarray expression analyses of retinas of hyperoxia-treated mice showed that the P12 wild-type (WT) mice differed considerably from the P12 A2AR KO mice and at all other time-points. The results thus show that the A2AR exacerbates the initial vasoobliterative phase in OIR. In the fourth study, the anti-inflammatory cytokine IL-10 was found to act as a pro-angiogenic factor in the OIR model and occasional macrophages/microglial cells were seen in WT mice. In conclusion, the presented work has, in different ways, tried to enlighten the complex process of retinal angiogenesis and has identified some potential strategies for treatment of neovascular retinopathies including inhibition of P2 receptor and A2AR signaling