The 67-kd laminin receptor is preferentially expressed by proliferating retinal vessels in a murine model of ischemic retinopathy.

Endothelial cell association with vascular basement membranes is complex and plays a critical role in regulation of cell adhesion and proliferation. The interaction between the membrane-associated 67-kd receptor (67LR) and the basement membrane protein laminin has been studied in several cell systems where it was shown to be crucial for adhesion and attachment during angiogenesis. As angiogenesis in the pathological setting of proliferative retinopathy is a major cause of blindness in the Western world we examined the expression of 67LR in a murine model of hyperoxia-induced retinopathy that exhibits retinal neovascularization. Mice exposed to hyperoxia for 5 days starting at postnatal day 7 (P7) and returned to room air (at P12) showed closure of the central retinal vasculature. In response to the ensuing retinal ischemia, there was consistent preretinal neovascularization starting around P17, which persisted until P21, after which the new vessels regressed. Immunohistochemistry was performed on these retinas using an antibody specific for 67LR. At P12, immunoreactivity for 67LR was absent in the retina, but by P17 it was observed in preretinal proliferating vessels and also within the adjacent intraretinal vasculature. Intraretinal 67LR immunoreactivity diminished beyond P17 until by P21 immunoreactivity was almost completely absent, although it persisted in the preretinal vasculature. Control P17 mice (not exposed to hyperoxia) failed to demonstrate any 67LR immunoreactivity in their retinas. Parallel in situ hybridization studies demonstrated 67LR gene expression in the retinal ganglion cells of control and hyperoxia-exposed mice. In addition, the neovascular intra- and preretinal vessels of hyperoxia-treated P17 and P21 mice labeled strongly for 67LR mRNA. This study has characterized 67LR immunolocalization and gene expression in a murine model of ischemic retinopathy. Results suggest that, although the 67LR gene is expressed at high levels in the retinal ganglion cells, the mature receptor protein is preferentially localized to the proliferating retinal vasculature and is almost completely absent from quiescent vessels. The differential expression of 67LR between proliferating and quiescent retinal vessels suggests that this laminin receptor is an important and novel target for future chemotherapeutic intervention during proliferative vasculopathies.

Retinopathy is reduced during experimental diabetes in a mouse model of outer retinal degeneration

PURPOSE. Diabetic patients who also have retinitis pigmentosa (RP) appear to have fewer and less severe retinal microvascular lesions. Diabetic retinopathy may be linked to increased inner retinal hypoxia, with the possibility that this is exacerbated by oxygen usage during the dark-adaptation response. Therefore, patients with RP with depleted rod photoreceptors may encounter proportionately less retinal hypoxia, and, when diabetes is also present, there may be fewer retinopathic lesions. This hypothesis was tested in rhodopsin knockout mice (Rho(-/-)) as an RP model in which the diabetic milieu is superimposed. The study was designed to investigate whether degeneration of the outer retina has any impact on hypoxia, to examine diabetes-related retinal gene expression responses, and to assess lesions of diabetic retinopathy.

Characterisation of a C1qtnf5 Ser163Arg Knock-In Mouse Model of Late-Onset Retinal Macular Degeneration

A single founder mutation resulting in a Ser163Arg substitution in the C1QTNF5 gene product causes autosomal dominant late-onset retinal macular degeneration (L-ORMD) in humans, which has clinical and pathological features resembling age-related macular degeneration. We generated and characterised a mouse

Expression of the 67 kDa laminin receptor (67LR) during retinal development: correlations with angiogenesis

Interaction of vascular cells with the laminin component of basement membranes is important for normal cell function. Likewise, abnormal interactions may have a critical role in vascular pathology. It has been previously demonstrated that the 67 kDa laminin receptor (67LR) is expressed at high levels during proliferative retinopathy in a mouse model and in the current study we have examined 67LR in the neonatal mouse to determine if this receptor plays a role in aspects of developmental angiogenesis in the developing murine retina. Groups of C57/BL6 mice were killed at postnatal day P1, P3, P5, P7, P9 and P11 to assess the retinal vasculature. A number of mice were perfused with FITC-dextran and the eyes removed, fixed in 4% paraformaldehyde (PFA) and flat-mounted for confocal scanning laser microscopy. The eyes from the remaining mice were either placed in 4% PFA and embedded in paraffin-wax, or had the neural retina dissected off and total RNA or protein extracted. Immunofluorescence, in situ hybridization, quantitative reverse transcriptase polymerase chain reaction and Western blotting analysis were employed to locate and determine expression levels of 67LR. Both 67LR mRNA and protein expression showed a characteristic bi-phasic expression pattern which correlated with key stages of retinal vascular development in the murine retina. 67LR showed high expression levels at P1 (P < 0.05) (correlating with superficial vascular plexus formation) and at P7 (P < 0.05) (correlating with deep vascular plexus formation). Conversely, 67LR expression was decreased when active angiogenic activity was lowest. Significantly, optical sectioning of retinal flat-mounts revealed high levels of 67LR expression in developing segments of both superficial and deep capillary plexi, a pattern which co-localized strongly with laminin. 67LR is regulated during post-natal development of the retinal vasculature. High levels of 67LR during the two well-defined phases of retinal capillary plexus formation suggests that this receptor may play an important role in retinal angiogenesis.

Endostatin modulates VEGF-mediated barrier dysfunction in the retinal microvascular endothelium

Recent evidence indicates that the anti-angiogenic peptide endostatin may modulate some of the vasomodulatory effects of vascular endothelial growth factor (VEGF) in the retina, including reduction of blood retinal barrier function although it remains uncertain how endostatin promotes endothelial barrier properties. The current study has sought to examine how physiological levels of endostatin alters VEGF-induced inner BRB function using an in vitro model system and evaluation of occludin and ZO-1 regulatory responses. In addition, the ability of exogenous endostatin to regulate VEGF-mediated retinal vascular permeability in vivo was investigated.

Retinal microvascular endothelial cells (RMEC's) were exposed to various concentrations of endostatin. In parallel studies, RMEC monolayers were treated with vascular endothelial growth factor (VEGF165). Vasopermeability of RMEC monolayers and occludin expression were determined.

Blood retinal barrier integrity was quantified in mouse retina using Evans Blue assay following intravitreal delivery of VEGF165, endostatin or a VEGF/endostatin combination.

Endostatin increased the levels of expression of occludin whilst causing no significant change in FITC-dextran flux across the RMEC monolayer. Endostatin reversed the effects of VEGF165-enhanced permeability between microvascular endothelial cells and induced phosphorylation of occludin. Evans Blue leakage from retinas treated with VEGF was 2.0 fold higher than that of contra-lateral untreated eyes (P<0.05) while leakage of eyes from endostatin treated animals was unchanged. When eyes were injected with a combination of VEGF165 and endostatin there was a significant reduction in retinal vasopermeability when compared to VEGF-injected eyes (P<0.05).

We conclude that endostatin can promote integrity of the retinal endothelial barrier, possibly by preventing VEGF-mediated alteration of tight junction integrity. This suggests that endostatin may be of clinical benefit in ocular disorders where significant retinal vasopermeability changes are present.

Synthetic peptides interacting with the 67-kd laminin receptor can reduce retinal ischemia and inhibit hypoxia-induced retinal neovascularization.

The high-affinity 67-kd laminin receptor (67LR) is expressed by proliferating endothelial cells during retinal neovascularization. The role of 67LR has been further examined experimentally by administration of selective 67LR agonists and antagonists in a murine model of proliferative retinopathy. These synthetic 67LR ligands have been previously shown to stimulate or inhibit endothelial cell motility in vitro without any direct effect on proliferation. In the present study, a fluorescently labeled 67LR antagonist (EGF33–42) was injected intraperitoneally into mice and its distribution in the retina was assessed by confocal scanning laser microscopy. Within 2 hours this peptide was localized to the retinal vasculature, including preretinal neovascular complexes, and a significant amount had crossed the blood retinal barrier. For up to 24 hours postinjection, the peptide was still present in the retinal vascular walls and, to a lesser extent, in the neural retina. Non-labeled EGF33–42 significantly inhibited pre-retinal neovascularization in comparison to controls treated with phosphate-buffered saline or scrambled peptide (P <0.0001). The agonist peptide (Lamß1925–933) also significantly inhibited proliferative retinopathy; however, it caused a concomitant reduction in retinal ischemia in this model by promoting significant revascularization of the central retina (P <0.001). Thus, 67LR appears to be an important target receptor for the modulation of retinal neovascularization. Agonism of this receptor may be valuable in reducing the hypoxia-stimulated release of angiogenic growth factors which drives retinal angiogenesis.

Impaired retinal angiogenesis in diabetes: role of advanced glycation end products and galectin-3

Suppression of angiogenesis during diabetes is a recognized phenomenon but is less appreciated within the context of diabetic retinopathy. The current study has investigated regulation of retinal angiogenesis by diabetic serum and determined if advanced glycation end products (AGEs) could modulate this response, possibly via AGE-receptor interactions. A novel in vitro model of retinal angiogenesis was developed and the ability of diabetic sera to regulate this process was quantified. AGE-modified serum albumin was prepared according to a range of protocols, and these were also analyzed along with neutralization of the AGE receptors galectin-3 and RAGE. Retinal ischemia and neovascularization were also studied in a murine model of oxygen-induced proliferative retinopathy (OIR) in wild-type and galectin-3 knockout mice (gal3(-/-)) after perfusion of preformed AGEs. Serum from nondiabetic patients showed significantly more angiogenic potential than diabetic serum (P <0.0001) and within the diabetic group, poor glycemic control resulted in more AGEs but less angiogenic potential than tight control (P <0.01). AGE-modified albumin caused a dose-dependent inhibition of angiogenesis (P <0.001), and AGE receptor neutralization significantly reversed the AGE-mediated suppression of angiogenesis (P <0.01). AGE-treated wild-type mice showed a significant increase in inner retinal ischemia and a reduction in neovascularization compared with non-AGE controls (P <0.001). However, ablation of galectin-3 abolished the AGE-mediated increase in retinal ischemia and restored the neovascular response to that seen in controls. The data suggest a significant suppression of angiogenesis by the retinal microvasculature during diabetes and implicate AGEs and AGE-receptor interactions in its causation.

Isolation of retinal progenitor and stem cells from the porcine eye

Purpose: Retinal progenitor cells (RPCs) and retinal stem cells (RSCs) from rodents and humans have been isolated and characterized in vitro. Transplantation experiments have confirmed their potential as tools for cell replacement in retinal degenerative diseases. The pig represents an ideal pre-clinical animal model to study the impact of transplantation because of the similarity of its eye to the human eye. However, little is known about porcine RPCs and RSCs. We aimed to identify and characterize in vitro RPCs and RSCs from porcine ocular tissues. Methods: Cells from different subregions of embryonic, postnatal and adult porcine eyes were grown in suspension sphere culture in serum-free medium containing basic fibroblast growth factor (bFGF) and epidermal growth factor (EGF). Growth curves and BrdU incorporation assays were performed to establish the proliferative capacity of isolated porcine retina-derived RPCs and ciliary epithelium (CE)-derived RSCs. Self-renewal potential was investigated by subsphere formation assays. Changes in gene expression were assayed by reverse transcription polymerase chain reaction (RT-PCR) at different passages in culture. Finally, differentiation was induced by addition of serum to the cultures and expression of markers for retinal cell types was detected by immunohistochemical staining with specific antibodies. Results: Dissociated cells from embryonic retina and CE at different postnatal ages generated primary nestin- and Pax6-immunoreactive neurosphere colonies in vitro in numbers that decreased with age. Embryonic and postnatal retina-derived RPCs and young CE-derived RSCs displayed self-renewal capacity, generating secondary neurosphere colonies. However, their self-renewal and proliferation capacity gradually decreased and they became more committed to differentiated states with subsequent passages. The expansion capacity of RPCs and RSCs was higher when they were maintained in monolayer culture. Porcine RPCs and RSCs could be induced to differentiate in vitro to express markers of retinal neurons and glia. Conclusions: Porcine retina and CE contain RPCs and RSCs which are undifferentiated, self-renewing and multipotent and which show characteristics similar to their human counterparts. Therefore, the pig could be a useful source of cells to further investigate the cell biology of RPCs and RSCs and it could be used as a non-primate large animal model for pre-clinical studies on stem cell-based approaches to regenerative medicine in the retina.