Differential Expression of Claudins in Retinas during Normal Development and the Angiogenesis of Oxygen-Induced Retinopathy
Investigative Ophthalmology and Visual Science
PURPOSE. Angiogenesis accompanies several retinal pathologies that impair the inner blood-retinal barrier. Claudins are key structural and functional proteins of the barrier. This study compared the expression of claudins during the normal angiogenesis of development with that of oxygen-induced retinopathy. METHODS. Real-time PCR was used to monitor mRNA from postnatal day 8 (P8) to P21 in normal mice and oxygen-induced retinopathy (OIR) mice. Protein expression was monitored by immunoblotting
... by immunoblotting and immunofluorescence. Isolectin B4 was used to identify blood vessels and occludin was used to identify tight junctions. Neovascularization and permeability were monitored using FITC-dextran and Evans blue. RESULTS. The mRNA of claudin-1, -2, -3, -4, -5, -12, -22, and -23 was developmentally regulated, but only claudin-1, -2, and -5 were found in the tight junctions of retinal vessels. OIR induced the formation of leaky neovascular vessels. The mRNA and protein of claudin-2 and -5 were overexpressed, whereas claudin-1 and occludin were unaffected. Despite their overexpression, each claudin was distributed throughout the cell, especially in the neovascular tufts. Occludin was retained at the lateral membranes but exhibited a punctate distribution. CONCLUSIONS. Claudin-1, -2, and -5 are the most prominent claudins of the inner blood-retinal barrier. The pathologic angiogenesis induced by oxygen formed a leaky barrier due to the mislocalization of these claudins. Studies of the mechanisms that regulate the intracellular distribution of claudins may lead to new therapeutic approaches for retinal vascular disease. (Invest Ophthalmol Vis Sci. 2011;52:7556 -7564) The number of pups present in each litter was about the same (n ϭ 8 -12); 23 litters were analyzed in this study. RNA Isolation and cDNA Preparation After mice were anesthetized by an intraperitoneal (IP) injection of 10% chloral hydrate (2.5 mL/kg), retinas of postnatal day 8 (P8), P11, P13, P15, P18, and P21 mice, together with kidney and brain of 6-week-old mice, were collected. Total RNA was extracted using a commercial reagent (TRIzol; Invitrogen, Carlsbad, CA) according to the manufacturer's instructions. Potential contamination by genomic DNA was prevented using a commercial kit (DNase I Kit; Sigma-Aldrich, St. Louis, MO). DNase I-treated RNA (1 g) was then converted to cDNA using reverse transcriptase (Takara, Siga, Japan). cDNA samples were aliquoted and stored at Ϫ80°C.