Involvement of purinergic P2 receptors in experimental retinal neovascularization.

PURPOSE: The present study was aimed to investigate the expression of purinergic P2 receptors in oxygen-induced retinal neovascularization. METHODS: Immunohistochemistry was used to study the expression of purinergic P2Y2 and P2X2 receptors in the neonatal mouse retina during normal vascular development and after oxygen-induced retinopathy (OIR). The effect of the P2 antagonists, suramin and PPADS, on the extent of oxygen-induced retinal neovascularization was analyzed. RESULTS: In normal mice, the expression of P2Y2 receptors was weak throughout the retina, whereas P2X2 receptor expression was detected in the outer plexiform layer. In mice treated with oxygen, P2Y2 expression was detected in the ganglion and in the nerve fiber layers, whereas P2X2 expression was found in the inner and outer plexiform layers. Oxygen-induced preretinal neovascularization was strongly inhibited by the P2 antagonists, suramin (p<0.05) and PPADS (p<0.05), and this was accompanied by a down-regulation of P2X2 receptor expression in the inner plexiform layer in suramin-treated mice. CONCLUSIONS: The data suggest that purinergic P2 receptors are involved in neovascularization associated with OIR.

Retinal vascular development and pathologic retinal angiogenesis are not impaired in matrix metalloproteinase-2 deficient mice.

PURPOSE: Earlier studies have suggested a role for metalloproteinase-2 (MMP-2) in retinal angiogenesis. To investigate this further, we have studied retinal vascular development and pathologic ischemia-induced retinal angiogenesis in MMP-2-deficient and wild-type mice. METHODS: Vascular development of the retina was studied in retinal flatmounts, whereas pathologic retinal angiogenesis was analyzed in retinal flatmounts and on histologic sections using a model of ischemia-induced retinopathy. The time course of MMP-2 mRNA expression was determined by in situ hybridization and real-time polymerase chain reaction (PCR). RESULTS: Formation of the retinal vascular plexus was not significantly different in MMP-2-deficient mice as compared to wild-type mice. In ischemia-induced retinopathy, there was an increased formation of extraretinal neovascular tufts in the MMP-2-deficient mice (p < 0.05). MMP-2 mRNA expression did not correlate to either retinal vascular development or to ischemia-induced formation of extraretinal vascular tufts. CONCLUSIONS: The current data suggest that MMP-2 is not essential for either retinal vascular development or pathologic retinal neovascularization in the mouse.

Reduced choroidal neovascular membrane formation in matrix metalloproteinase-2-deficient mice.

PURPOSE: Findings in studies have suggested a role for matrix metalloproteinase (MMP)-2 in angiogenesis, including choroidal neovascularization (CNV). To investigate further, the current study was conducted to observe the formation of experimental CNV in MMP-2-deficient mice. METHODS: CNV was induced in wild-type and MMP-2-deficient mice by krypton laser photocoagulation of the fundus. The time-course of expression of MMP-2 mRNA after laser treatment was determined by in situ hybridization with anti-sense and sense cRNA probes. MMP-2 protein distribution was determined by immunohistochemistry. Ten days after treatment, the extent of CNV was evaluated on hematoxylin-eosin stained serial sections. The maximum height of the CNV lesions was calculated by image analysis of digitized histologic images. RESULTS: Expression of MMP-2 mRNA was detected in the CNV lesions at day 3 after laser treatment and peaked at day 5, after which it slowly declined. MMP-2 mRNA expression appeared to be highest at the margins of the membrane. Immunostaining for MMP-2 confirmed the presence of MMP-2 protein in the CNV lesions. The CNV lesions of MMP-2-deficient mice showed that relative thickness was reduced by 31% compared with wild-type mice (P = 0.006). CONCLUSIONS: The present study demonstrated that MMP-2 mRNA and protein are upregulated during experimental CNV in the mouse. The marked difference in thickness of the CNV membrane between wild-type and MMP-2-deficient mice shows that MMP-2 is involved in the formation of experimental CNV in the mouse. These results suggest that pharmacologic targeting of MMPs, including MMP-2, may reduce formation of CNV in conditions such as age-related macular degeneration.