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

Immunology of AAV-mediated gene transfer in the eye

The eye has been at the forefront of translational gene therapy largely owing to suitable disease targets, anatomic accessibility and well-studied immunologic privilege. These advantages have fostered research culminating in several clinical trials and AAV has emerged as the vector of choice for many ocular therapies. Pre-clinical and clinical investigations have assessed the humoral and cellular immune responses to a variety of naturally occurring and engineered AAV serotypes as well as their delivered transgenes and these data have been compared to potential clinical sequelae. Encouragingly, AAV appears safe and effective with clinical follow up surpassing 5 years in some studies. As disease targets continue to expand for AAV in the eye, thorough and deliberate assessment of immunologic safety is critical. With careful study, the development of these technologies should concurrently inform the biology of the immune response in the eye

Neuroprotection mediated by ST266 requires full complement of proteins secreted by amnion-derived multipotent progenitor cells.

ST266 is the biological secretome of cultured Amnion-derived Multipotent Progenitor cells containing multiple growth factors and cytokines. While intranasally-administered ST266 improves the phenotype in experimental optic neuritis, specific ST266 components mediating these effects are not known. We compared the effects of ST266 with and without removal of large molecular weight proteins both in vitro and in the multiple sclerosis model experimental autoimmune encephalomyelitis (EAE) in C57BL/6J mice. Mice were treated daily with intranasal vehicle, ST266 or lower molecular weight fraction of ST266. Retinal ganglion cells were counted in isolated retinas, and optic nerves were assessed for inflammation and demyelination. ST266 treatment significantly improved retinal ganglion cell survival and reduced optic nerve demyelination in EAE mice. The lower molecular weight ST266 fraction significantly improved optic nerve demyelination, but only showed a trend towards improved retinal ganglion cell survival. ST266 fractions below 50kDa increased Schwann cell proliferation in vitro, but were less effective than non-fractionated ST266. Demyelination attenuation was partially associated with the lower molecular weight ST266 fraction, but removal of higher molecular weight biomolecules from ST266 diminishes its neuroprotective effects, suggesting at least some high molecular weight proteins play a role in ST266-mediated neuroprotection

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

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

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

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

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

Wnt ligands and receptor regulated in P8 <i>Lrp5</i> null retina.

<p>Wnt ligand <i>Norrin</i>, Wnt5a, Wnt7b, and Wnt10b mRNA expression in WT and <i>Lrp5^−/−</i> whole retinas were quantified with RT-qPCR and normalized to <i>Cyclophilin A</i> expression. *<i>p</i><0.05, **<i>p</i><0.01.</p