In a recent study, we showed that transgenic βB1-TGF-β1 mice
35 with ocular overexpression of active TGF-β1 completely lack intraretinal capillaries and the layer of the choriocapillaris that is required for oxygen supply and nutrition of photoreceptors.
24 We now crossed transgenic βB1-TGF-β1 mice with βB1-Norrin animals that overexpress Norrin in the eye
8,35 to generate double transgenic βB1-TGF-β1/βB1-Norrin mice. After perfusion with FITC-coupled dextran, well-structured superficial and deep vascular plexuses were observed in retinae of wild type and transgenic βB1-Norrin mice at P16 (
Figs. 1A,
1B). In addition, in the inner plexiform layer, a few vessels were seen that began to form the intermediate vascular plexus (
Figs. 1A,
1B). In contrast, as expected, in transgenic βB1-TGF-β1 mice no intraretinal capillaries were observed (
Fig. 1C). However, in retinae of double transgenic βB1-TGF-β1/βB1-Norrin mice, well established superficial as well as deep vascular plexuses were detected (
Fig. 1D). Moreover, in meridional sections of retinae from βB1-Norrin mice and wild type littermates, vessels of the choriocapillaris were observed adjacent to the RPE (
Figs. 1E,
1F), suggesting an intact and dense choroidal capillary network, which was not detectable in transgenic βB1-TGF-β1 mice (
Fig. 1G). The loss of the choriocapillaris was rescued in double transgenic mice, in which vessels that lie close to the RPE were readily detected (
Fig. 1H). To confirm our observations in an independent assay, immunostaining for collagen type IV was performed to visualize the basal lamina of microvascular endothelial cells in the retina.
22 In the central inner retina of wild type mice, a dense capillary network was observed below the inner limiting membrane, which also was labeled (
Fig. 1I). As expected, at P8 the vessels of the superficial vascular plexus spread from the retinal center to its periphery (
Fig. 1I). In contrast, retinae of βB1-TGF-β1 transgenic mice completely lacked the retinal vasculature (
Fig. 1K). Only a thickened inner limiting membrane was seen covering the retinal surface (
Fig. 1K). The absence of retinal vessels in βB1-TGF-β1 mice was rescued in double transgenic littermates in which numerous vessels were detected in all regions of the inner retina (
Fig. 1L). Overall, our observations strongly indicated that Norrin inhibits the anti-angiogenic effects of TGF-β on retinal capillaries and choriocapillaris.