Deletion of smooth muscle alpha-actin alters blood-retina barrier permeability and retinal function.

Purpose: Vascular smooth muscle (SM) cells and pericytes are essential for normal vascular development. SM alpha-actin null mice were used to determine whether vascular SM and pericyte contractile functions, and not merely their presence, are necessary for vascular development, normal blood-retina barrier (BRB) permeability, and retinal function.

Methods: Age-matched SM alpha-actin null and wild-type mice were analyzed.

RPE65 gene delivery restores isomerohydrolase activity and prevents early cone loss in Rpe65-/- mice.

Purpose: Recent in vitro evidence has shown that RPE65 is the isomerohydrolase that converts all-trans retinyl ester to 11-cis retinal, the chromophore for visual pigments in vertebrates. Homozygous RPE65 knockout (Rpe65-/-) mice lack 11-cis retinoids and have early cone degeneration. The purpose of this study is to determine whether RPE65 gene delivery restores the isomerohydrolase activity and normal profile of endogenous retinoids in Rpe65-/- mice.

Inhibition of ocular angiogenesis by diced small interfering RNAs (siRNAs) specific to vascular endothelial growth factor (VEGF).

The effects of diced small interfering RNAs (siRNAs) designed for vascular endothelial growth factor (VEGF) on the expression of VEGF in human retinal pigment epithelial cell line ARPE-19 cells in vitro and on corneal angiogenesis in vivo were examined. The exposure to diced siRNAs significantly reduced the VEGF mRNA expression in ARPE-19 cells with minimal toxicity. In suture-induced corneal angiogenesis models, diced siRNAs minimized the severity of angiogenesis.

Kringle 5 of human plasminogen suppresses hepatocellular carcinoma growth both in grafted and xenografted mice by anti-angiogenic activity.

Plasminogen kringle 5 (K5), a proteolytic fragment of plasminogen, is an endogenous angiogenic inhibitor. We have previously shown that K5 inhibits ischemia-induced retinal neovascularization in a rat model. However, its anti-angiogenic potential and application in the treatment of neoplastic diseases have not been well investigated.

Therapeutic potential of angiostatin in diabetic nephropathy.

Angiostatin is a proteolytic fragment of plasminogen and a potent angiogenic inhibitor. Previous studies have shown that angiostatin inhibits retinal neovascularization and reduces retinal vascular permeability in diabetic retinopathy. Here, it is reported for the first time that angiostatin is also implicated in diabetic nephropathy (DN).

Pigment epithelium-derived factor (PEDF) is an endogenous antiinflammatory factor.

Pigment epithelium-derived factor (PEDF) is a potent angiogenic inhibitor. Reduced PEDF levels are associated with diabetic retinopathy. However, the mechanism for the protective effects of PEDF against diabetic retinopathy (DR) is presently unclear.

RPE65 is an iron(II)-dependent isomerohydrolase in the retinoid visual cycle.

The isomerization of all-trans-retinyl ester to 11-cis-retinol in the retinal pigment epithelium (RPE) is a critical step in the visual cycle and is essential for normal vision. Recently, we have established that protein RPE65 is the isomerohydrolase catalyzing this reaction. The present study investigated if metal ions are required for the isomerohydrolase activity of RPE65.

Plasminogen kringle 5 inhibits alkali-burn-induced corneal neovascularization.

Purpose: Plasminogen kringle 5 (K5) is a potent angiogenic inhibitor. The purpose of the present study was to evaluate the therapeutic effect of K5 on alkali-burn-induced corneal neovascularization (NV) and to investigate its mechanism of action.

Methods: Corneal NV was induced in rabbits by NaOH.

Identification of conserved histidines and glutamic acid as key residues for isomerohydrolase activity of RPE65, an enzyme of the visual cycle in the retinal pigment epithelium.

We have recently reported that RPE65 from the retinal pigment epithelium is the isomerohydrolase, a critical enzyme in the visual cycle for regeneration of 11-cis retinal, the chromophore for visual pigments. Here, we demonstrated that mutation of any one of the absolutely conserved four histidine and one glutamic acid residues to alanine in RPE65 abolished its isomerohydrolase activity. Substitution of the conserved glutamic acid with glutamine also resulted in loss of the activity.

RPE65 is the isomerohydrolase in the retinoid visual cycle.

RPE65 is an abundant protein in the retinal pigment epithelium. Mutations in RPE65 are associated with inherited retinal dystrophies. Although it is known that RPE65 is critical for regeneration of 11-cis retinol in the visual cycle, the function of RPE65 is elusive.

Systemic and periocular deliveries of plasminogen kringle 5 reduce vascular leakage in rat models of oxygen-induced retinopathy and diabetes.

Purpose: Increased retinal vascular permeability is a common complication of diabetes and a major cause of vision loss in diabetic patients. The current study is to determine the effect of plasminogen kringle 5 (K5) on vascular leakage via systemic and periocular deliveries.

Methods: Oxygen-induced retinopathy (OIR) was generated by exposing newborn rats to 75% oxygen.

Bone marrow lacks a transplantable progenitor for smooth muscle type alpha-actin-expressing cells.

While some studies have suggested that hematopoietic stem cells might give rise to other tissue types, others indicate that transdifferentiation would have to be an extremely rare event. We have now exploited smooth muscle type alpha-actin (alphaSMA) promoter-driven green fluorescent protein (GFP) transgenic mice (alphaSMA-GFP mice) for bone marrow transplantation to evaluate their potential to generate donor-type tissues in irradiation chimeras. There was a highly restricted pattern of GFP expression in the transgenic mice, marking bone marrow stromal cells and mesangial cells in the kidney.

Downregulation of cone-specific gene expression and degeneration of cone photoreceptors in the Rpe65-/- mouse at early ages.

Purpose: RPE65 is essential for the generation of 11-cis retinal. Rod photoreceptors in the RPE65-knockout (Rpe65(-/-)) mouse are known to degenerate slowly with age. This study was designed to examine cone photoreceptors and the expression of cone-specific genes in the Rpe65(-/-) mouse.

Hsp27 upregulation by HIF-1 signaling offers protection against retinal ischemia in rats.

Purpose: Previous work from the authors' laboratory has shown that Hsp27 is specifically upregulated after retinal ischemic preconditioning (IPC), and this upregulation acts as a key cytoprotective factor in preventing retinal ischemic damage. The regulatory mechanisms involved in the upregulation of Hsp27 after IPC are unknown. The purpose of this study was to explore the transcriptional events responsible for the upregulation of Hsp27 after IPC.

Heat shock protein 27 delays Ca2+-induced cell death in a caspase-dependent and -independent manner in rat retinal ganglion cells.

Purpose: Hsp27 is a well-characterized and studied antiapoptotic protein. A recent study reported that Hsp27 is upregulated in the retina after retinal ischemic preconditioning. The timing of this upregulation of Hsp27 correlates with the protective effects of the treatment.

Targeted expression of Cre recombinase to cone photoreceptors in transgenic mice.

Purpose: Disruption of widely expressed essential genes in mice often leads to embryonic or neonatal lethality. To circumvent this problem and dissect gene functions in the cone photoreceptors, we elected to generate cone photoreceptor specific cre transgenic mice.

Methods: Transgenic mice expressing Cre recombinase directed by the human red/green pigment (HRGP) gene promoter were generated.

Genetic difference in susceptibility to the blood-retina barrier breakdown in diabetes and oxygen-induced retinopathy.

The breakdown of the blood-retina barrier (BRB) is a common feature of diabetic retinopathy. The purpose of the present study is to determine whether there are genetic differences in susceptibility to the breakdown of the BRB in diabetic retinopathy using two rat models. In streptozotocin (STZ)-induced diabetes, Brown Norway (BN) rats developed sustained vascular hyperpermeability in the retina during the entire experimental period (16 weeks of diabetes), while diabetic Sprague Dawley (SD) rats only showed retinal hyperpermeability from 3 to 10 days after the onset of diabetes.

Decreased expression of pigment epithelium-derived factor is involved in the pathogenesis of diabetic nephropathy.

Pigment epithelium-derived factor (PEDF) is a potent angiogenic inhibitor. Previous studies have shown that decreased ocular levels of PEDF are associated with diabetic retinopathy. However, the implication of PEDF expression in diabetic nephropathy has not been revealed.

Identification of RDH10, an All-trans Retinol Dehydrogenase, in Retinal Muller Cells.

Purpose: To investigate the expression of RDH10, an all-trans retinol dehydrogenase identified in the retinal pigment epithelium (RPE), in retinal Muller cells.

Methods: The RDH10 protein levels in mouse eyecups and bovine tissues were examined by Western blot analysis using a polyclonal antibody against RDH10. The cellular localization in the retina was determined by immunohistochemistry.

Does constitutive phosphorylation protect against photoreceptor degeneration in Rpe65-/- mice?

Despite the presence of this virgin opsin, Rpe65-/- rods are behaving like dark-adapted rods. These results argue that opsin which has not been exposed to 11-cis retinal and is constitutively phosphorylated, does not generate the activity generally associated with the bleached apoprotein. However, increased light-independent activation of transducin (due to bleached opsin) could be demonstrated after the addition of exogenous 11-cis retinal.

Suppression of corneal neovascularization by PEDF release from human amniotic membranes.

Purpose: Human amniotic membrane (HAM) transplantation is commonly used in corneal surface reconstruction and is known to inhibit neovascularization of this tissue. The purpose of the present study is to reveal the molecular basis underlying antiangiogenic activity of HAM.

Methods: The effects of HAM protein on proliferation of vascular endothelial cells and corneal epithelial cells were determined by quantifying viable cells using the MTT assay.

Role of the 9-methyl group of retinal in cone visual pigments.

In rhodopsin, the 9-methyl group of retinal has previously been identified as being critical in linking the ligand isomerization with the subsequent protein conformational changes that result in the activation of its G protein, transducin. Here, we report studies on the role of this methyl group in the salamander rod and cone pigments. Pigments were generated by combining proteins expressed in COS cells with 11-cis 9-demethyl retinal, where the 9-methyl group on the polyene chain has been deleted.

The effect of angiostatin on vascular leakage and VEGF expression in rat retina.

Angiostatin is a potent angiogenic inhibitor. The present study identified a new activity of angiostatin: reducing vascular leakage, which is associated with diabetic macular edema, tumor growth and inflammation. An intravitreal injection of angiostatin significantly reduced retinal vascular permeability in rats with oxygen-induced retinopathy and in those with streptozotocin-induced diabetes, but not in normal rats.

Visual cycle retinoid processing proteins are present in HEK293S cells.

In HEK293S cells expressing opsin, rhodopsin regenerates on addition of all-trans retinol. This study was to determine if key proteins in the retinal pigment epithelium (RPE) are present in these cells. Cellular retinoid binding protein, cellular retinoic-acid binding protein, RPE65, caveolin-1-alpha- and -beta-isoforms, interphotoreceptor retinoid binding protein, and 11-cis retinol dehydrogenase, but not lecithin:retinol acyltransferase (LRAT), were identified by Western blot analysis.

Isorhodopsin rather than rhodopsin mediates rod function in RPE65 knock-out mice.

The chromophore of visual pigments is 11-cis-retinal and, thus, in its absence, opsin is not photosensitive and no visual function exists. However, in the RPE65 knockout (Rpe65-/-) mouse, where synthesis of 11-cis-retinal does not occur, a minimal visual response from rod photoreceptors is obtained. We have examined if an alternative pathway exists for cis-retinoid generation in the absence of RPE65.