Retinal Expression of Wnt-Pathway Mediated Genes in Low-Density Lipoprotein Receptor-Related Protein 5 (Lrp5) Knockout Mice

Mutations in low-density lipoprotein receptor-related protein 5 (Lrp5) impair retinal angiogenesis in patients with familial exudative vitreoretinopathy (FEVR), a rare type of blinding vascular eye disease. The defective retinal vasculature phenotype in human FEVR patients is recapitulated in Lrp5 knockout $(Lrp5^{-/-})$ mouse with delayed and incomplete development of retinal vessels. In this study we examined gene expression changes in the developing $Lrp5^{−/−}$ mouse retina to gain insight into the molecular mechanisms that underlie the pathology of FEVR in humans. Gene expression levels were assessed with an Illumina microarray on total RNA from $Lrp5^{−/−}$ and WT retinas isolated on postnatal day (P) 8. Regulated genes were confirmed using RT-qPCR analysis. Consistent with a role in vascular development, we identified expression changes in genes involved in cell-cell adhesion, blood vessel morphogenesis and membrane transport in $Lrp5^{−/−}$ retina compared to WT retina. In particular, tight junction protein claudin5 and amino acid transporter slc38a5 are both highly down-regulated in $Lrp5^{−/−}$ retina. Similarly, several Wnt ligands including Wnt7b show decreased expression levels. Plasmalemma vesicle associated protein (plvap), an endothelial permeability marker, in contrast, is up-regulated consistent with increased permeability in $Lrp5^{−/−}$ retinas. Together these data suggest that Lrp5 regulates multiple groups of genes that influence retinal angiogenesis and may contribute to the pathogenesis of FEVR

Resveratrol Inhibits Pathologic Retinal Neovascularization in Vldlr -/- Mice

PURPOSE: Macular telangiectasia (MacTel) is a vision-threatening retinal disease with unknown pathogenesis and no approved treatment. Very low-density lipoprotein receptor mutant mice (Vldlr(-/-)) exhibit critical features of MacTel such as retinal neovascularization and photoreceptor degeneration. In this study, the authors evaluate the therapeutic potential of resveratrol, a plant polyphenol, in Vldlr(-/-) mice as a model for MacTel. METHODS: Vldlr(-/-) and wild-type mice at postnatal day (P) 21 to P60 or P10 to P30 were treated orally with resveratrol. The number of neovascular lesions was evaluated on retinal flatmounts, and resveratrol effects on endothelial cells were assessed by Western blot for phosphorylated ERK1/2, aortic ring, and migration assays. Vegf and Gfap expression was evaluated in laser-capture microdissected retinal layers of angiogenic lesions and nonlesion areas from Vldlr(-/-) and wild-type retinas. RESULTS: From P15 onward, Vldlr(-/-) retinas develop vascular lesions associated with the local upregulation of Vegf in photoreceptors and Gfap in the inner retina. Oral resveratrol reduces lesion formation when administered either before or after disease onset. The reduction of vascular lesions in resveratrol-treated Vldlr(-/-) mice is associated with the suppression of retinal Vegf transcription. Resveratrol also reduces endothelial ERK1/2 signaling as well as the migration and proliferation of endothelial cells. Furthermore, a trend toward increased rhodopsin mRNA in Vldlr(-/-) retinas is observed. CONCLUSIONS: Oral administration of resveratrol is protective against retinal neovascular lesions in Vldlr(-/-) mice by inhibiting Vegf expression and angiogenic activation of retinal endothelial cells. These results suggest that resveratrol might be a safe and effective intervention for treating patients with MacTel

Wnt Signaling Mediates Pathological Vascular Growth in Proliferative Retinopathy Clinical Perspective

BACKGROUND: Ischemic proliferative retinopathy, characterized by pathological retinal neovascularization, is a major cause of blindness in working-age adults and children. Defining the molecular pathways distinguishing pathological neovascularization from normal vessels is critical to controlling these blinding diseases with targeted therapy. Because mutations in Wnt signaling cause defective retinal vasculature in humans with some characteristics of the pathological vessels in retinopathy, we investigated the potential role of Wnt signaling in pathological retinal vascular growth in proliferative retinopathy. METHODS AND RESULTS: In this study, we show that Wnt receptors (Frizzled4 and low-density lipoprotein receptor-related protein5 [Lrp5]) and activity are significantly increased in pathological neovascularization in a mouse model of oxygen-induced proliferative retinopathy. Loss of Wnt coreceptor Lrp5 and downstream signaling molecule dishevelled2 significantly decreases the formation of pathological retinal neovascularization in retinopathy. Loss of Lrp5 also affects retinal angiogenesis during development and formation of the blood-retinal barrier, which is linked to significant downregulation of tight junction protein claudin5 in Lrp5(-/-) vessels. Blocking claudin5 significantly suppresses Wnt pathway-driven endothelial cell sprouting in vitro and developmental and pathological vascular growth in retinopathy in vivo. CONCLUSIONS: These results demonstrate an important role of Wnt signaling in pathological vascular development in retinopathy and show a novel function of Cln5 in promoting angiogenesis

Resveratrol Inhibits Pathologic Retinal Neovascularization in Vldlr−/− Mice

Macular telangiectasia (MacTel) is a vision-threatening retinal disease with unknown pathogenesis and no approved treatment. Very low-density lipoprotein receptor mutant mice (Vldlr−/−) exhibit critical features of MacTel, such as retinal neovascularization and photoreceptor degeneration. In this study, the authors evaluated the therapeutic potential of resveratrol, a plant polyphenol, in Vldlr−/− mice as a model for MacTel

Selected groups of genes regulated in <i>Lrp5^−/−</i> retina.

<p>Note: Retinas were isolated from P8 <i>Lrp5^−/−</i> mice and age matched WT control mice. RNA was isolated and assessed with Illumina gene expression microarray. (−) indicates decreased expression in <i>Lrp5^−/−</i> retina and (+) indicates increased expression in <i>Lrp5^−/−</i> retina compared to WT control.</p

Regulation of tight junction, membrane transport, angiogenic, and cell adhesion genes in the <i>Lrp5</i> null retina.

<p>Heat maps illustrate the results of a gene array run from whole retinal total mRNA. The most regulated families of genes were (A) claudin family genes, (B) membrane transport genes, (C) angiogenic regulatory genes, and (D) cell adhesion/cell-cell junction genes. Each sample is represented by a block: either wild-type (WT) samples 1 through 3, and Lrp5 null samples 1–3. Relative down-regulation of expression in <i>Lrp5</i> null retina compared to WT retina is represented by green, while relative up-regulation is in red. No relative regulation is black (See scale on Figure).</p

Common genes regulated in <i>Lrp5^−/−</i> and <i>Norrin^−/−</i> retinas.

<p>Note: Retinas were isolated from P8 <i>Lrp5^−/−</i> mice and age matched WT control mice. RNA was isolated and assessed with Illumina gene expression microarray. (−) indicates decreased expression in <i>Lrp5^−/−</i> retina compared to WT retina, and (+) indicates increased expression in <i>Lrp5^−/−</i> retina.</p><p>*: Genes regulated in P7 <i>Norrin^−/−</i> retina were adapted from Schafer et. al. IOVS, 2009 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030203#pone.0030203-Schafer1" target="_blank">[33]</a>.</p

Abnormal vascular development in the inner and outer retina and forebrain of <i>Lrp5</i> null mice.

<p>(A) Retinal whole mounts of WT and <i>Lrp5</i> null mice stained for endothelial cells with isolectin B₄-594 at P12 and 17. Enlarged images highlight the abnormal vessel growth in the <i>Lrp5</i> null retina. (B) Retinal cross sections from P30 WT and <i>Lrp5</i> null mice stained for endothelial cells with isolectin B₄-594 (red) and cell nuclei with DAPI (blue). Arrows indicate deep layers of retinal vasculature which is present in WT retina but absent in <i>Lrp5</i> null retina. (C) Cross sections of the forebrain from P8 WT and <i>Lrp5</i> null mice with endothelial cells stained with isolectin B₄-594. Scale bar: 100 µm.</p

Delayed development of the superficial retinal vasculature and persistent hyaloid vessels in <i>Lrp5</i> null mice.

<p>(A) Left: retinal whole mounts stained with isolectin B₄-594 from WT and <i>Lrp5</i> null mice at post-natal day (P) 8. Right: quantification of vascularized retinal area in WT and <i>Lrp5</i> null mice at P8. n = 5–10 per group, ***<i>p</i><0.001. (B) Retinal cross sections of WT mice and <i>Lrp5</i> null mice at P8 stained for endothelial cells with isolectin B₄-594 (red) and cell nuclei (DAPI, blue). Arrows indicate persistent hyaloid vessels in <i>Lrp5</i> null retina. Scale bars: 500 µm.</p

Expression levels of <i>Lrp5</i>, <i>Norrin</i>, <i>Frizzled4</i> and <i>Dvl</i> mRNA during retinal development in wild type mice.

<p>Quantification of mRNA (A) <i>Lrp5</i>, (B) <i>Norrin</i>, (C) <i>Frizzled4</i>, (D) <i>Dvl1</i>, (E) <i>Dvl2</i>, and (F) <i>Dlv3</i> was performed with RT-qPCR during normal retinal development from P1-P17. Expression levels were normalized against house keeping gene <i>Cyclophilin A</i>. Trend lines were fitted with polynomial, linear or power regression.</p