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

Semaphorin 3F forms an anti-angiogenic barrier in outer retina

Semaphorins are known modulators of axonal sprouting and angiogenesis. In the retina, we identified a distinct and almost exclusive expression of Semaphorin 3F in the outer layers. Interestingly, these outer retinal layers are physiologically avascular. Using functional in vitro models, we report potent anti-angiogenic effects of Semaphorin 3F on both retinal and choroidal vessels. In addition, human retinal pigment epithelium isolates from patients with pathologic neovascularization of the outer retina displayed reduced Semaphorin 3F expression in 10 out of 15 patients. Combined, these results elucidate a functional role for Semaphorin 3F in the outer retina where it acts as a vasorepulsive cue to maintain physiologic avascularity

Propranolol Inhibition of β-Adrenergic Receptor Does Not Suppress Pathologic Neovascularization in Oxygen-Induced Retinopathy

Purpose.: Retinopathy of prematurity (ROP) is a leading cause of blindness in children and is, in its most severe form, characterized by uncontrolled growth of vision-threatening pathologic vessels. Propranolol, a nonselective β-adrenergic receptor blocker, was reported to protect against pathologic retinal neovascularization in a mouse model of oxygen-induced retinopathy (OIR). Based on this single animal study using nonstandard evaluation of retinopathy, clinical trials are currently ongoing to evaluate propranolol treatment in stage 2 ROP patients who tend to experience spontaneous disease regression and are at low risk of blindness. Because these ROP patients are vulnerable premature infants who are still in a fragile state of incomplete development, the efficacy of propranolol treatment in retinopathy needs to be evaluated thoroughly in preclinical animal models of retinopathy and potential benefits weighed against potential adverse effects. Methods.: Retinopathy was induced by exposing neonatal mice to 75% oxygen from postnatal day (P) 7 to P12. Three routes of propranolol treatment were assessed from P12 to P16: oral gavage, intraperitoneal injection, or subcutaneous injection, with doses varying between 2 and 60 mg/kg/day. At P17, retinal flatmounts were stained with isolectin and quantified with a standard protocol to measure vasoobliteration and pathologic neovascularization. Retinal gene expression was analyzed with qRT-PCR using RNA isolated from retinas of control and propranolol-treated pups. Results.: None of the treatment approaches at any dose of propranolol (up to 60 mg/kg/day) were effective in preventing the development of retinopathy in a mouse model of OIR, evaluated using standard techniques. Propranolol treatment also did not change retinal expression of angiogenic factors including vascular endothelial growth factor. Conclusions.: Propranolol treatment via three routes and up to 30 times the standard human dose failed to suppress retinopathy development in mice. These data bring into question whether propranolol through inhibition of β-adrenergic receptors is an appropriate therapeutic approach for treating ROP

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

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

Confirmation of gene expression differentially regulated in Lrp5 null retina with RT-qPCR.

<p>Quantification of mRNA (A) <i>Cln5</i> and <i>Slc38a5</i>, (B) <i>Gja1</i>, <i>Msfd2</i>, <i>Sox18</i>, and <i>vWF</i>, and (C) <i>Plvap</i> and <i>EMP1</i> in WT and <i>Lrp5</i> null retina with RT-qPCR at P8. Expression levels were normalized against housekeeping gene <i>Cyclophilin A</i>. *<i>p</i><0.05, **<i>p</i><0.01.</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