Fibulin-7 C-terminal fragment and its active synthetic peptide suppress choroidal and retinal neovascularization.

Wet age-related macular degeneration (AMD) and diabetic retinopathy are the leading causes of blindness through increased angiogenesis. Although VEGF-neutralizing proteins provide benefit, inconsistent responses indicate a need for new therapies. We previously identified the Fibulin-7 C-terminal fragment (Fbln7-C) as an angiogenesis inhibitor in vitro. Here we show that Fbln7-C inhibits neovascularization in vivo, in both a model of wet AMD involving choroidal neovascularization (CNV) and diabetic retinopathy involving oxygen-induced ischemic retinopathy. Furthermore, a short peptide sequence from Fbln7-C is responsible for the anti-angiogenic properties of Fbln7-C. Our work suggests Fbln7-C as a therapeutic candidate for wet AMD and ischemic retinopathy.

Metipranolol promotes structure and function of retinal photoreceptors in the rd10 mouse model of human retinitis pigmentosa.

Metipranolol is a ß-adrenergic receptor antagonist that is given orally for the treatment of hypertension and also applied topically to the cornea for treating glaucoma. It also inhibits nitrosative stress which has previously been shown to be the cause of cone photoreceptor death in retinitis pigmentosa. In this study, we tested the hypothesis that metipranolol protects photoreceptor structure and function in the mouse model rd10. At P35, compared with vehicle-treated rd10 mice in which rod degeneration was nearly complete, rd10 mice given daily subcutaneous injections of 40 mg/kg of metipranolol had reduction in markers of nitrosative stress, fewer TUNEL-positive cells, increased outer nuclear layer thickness, and substantially more staining for rhodopsin. This was accompanied by significantly higher mean scotopic and photopic electroretinogram b-wave amplitudes indicating improved photoreceptor function. At P50, metipranolol-treated rd10 mice had decreased 3-nitrotyrosine staining in the retina, increased immunostaining for cone arrestin, a marker for cone photoreceptors, and significantly higher scotopic and photopic b-wave amplitudes at the highest stimulus intensity compared with vehicle-treated mice. At P65, cone density was significantly higher in metipranolol-treated versus vehicle-injected rd10 mice. Metipranolol applied as eye drops promoted cone photoreceptor function in retinas of rd10 mice greater than subcutaneously injected metipranolol. The reduced nitrosative damage and rescue of functional loss of photoreceptors in rd10 mice suggests that metipranolol, a drug with established ocular safety and tolerability, may have potential for treating patients with retinitis pigmentosa.

Identification of Tyrosine O Sulfated Proteins in Cow Retina and the 661W Cell Line.

Lack of tyrosine O Sulfation compromises both rod and cone electroretinographic responses emphasizing the importance of this post-translational modification for vision. To identify tyrosine sulfated proteins in retina, cow retinal lysates were subjected to immunoaffinity purification using an anti-sulfotyrosine antibody. The tyrosine sulfated proteins were eluted from the column using a sulfotyrosine pentapeptide and identified using mass spectrometry. Similarly, tyrosine sulfated proteins secreted by the 661W cell line were identified. Proteins identified were vitronectin, fibronectin, fibulin 2, nidogen, collagen V alpha 2, complement component 3 and C4 and fibrinogen beta. All proteins were subjected to analysis by 'Sulfinator' to determine potential sulfated tyrosines.

Photoreceptor Neuroprotection: Regulation of Akt Activation Through Serine/Threonine Phosphatases, PHLPP and PHLPPL.

Serine/threonine kinase Akt is a downstream effector of insulin receptor/PI3K pathway that is involved in many processes, including providing neuroprotection to stressed rod photoreceptor cells. Akt signaling is known to be regulated by the serine/threonine phosphatases, PHLPP (PH domain and leucine rich repeat protein phosphatase) and PHLPPL (PH domain and leucine rich repeat protein phosphatase-like). We previously reported that both phosphatases are expressed in the retina, as well as in photoreceptor cells. In this study, we examined the PHLPP and PHLPPL phosphatase activities towards non-physiological and physiological substrates. Our results suggest that PHLPP was more active than PHLPPL towards non-physiological substrates, whereas both PHLPP and PHLPP dephosphorylated the physiological substrates of Akt1 and Akt3 with similar efficiencies. Our results also suggest that knockdown of PHLPPL alone does not increase Akt phosphorylation, due to a compensatory increase of PHLPP, which results in the dephosphorylation of Akt. Therefore, PHLPP and PHLPPL regulate Akt activation together when both phosphatases are expressed.

Fibulin 2, a tyrosine O-sulfated protein, is up-regulated following retinal detachment.

Retinal detachment is the physical separation of the retina from the retinal pigment epithelium. It occurs during aging, trauma, or during a variety of retinal disorders such as age-related macular degeneration, diabetic retinopathy, retinopathy of prematurity, or as a complication following cataract surgery. This report investigates the role of fibulin 2, an extracellular component, in retinal detachment. A major mechanism for detachment resolution is enhancement of cellular adhesion between the retina and the retinal pigment epithelium and prevention of its cellular migration. This report shows that fibulin 2 is mainly present in the retinal pigment epithelium, Bruch membrane, choriocapillary, and to a lesser degree in the retina. In vitro studies revealed the presence of two isoforms for fibulin 2. The small isoform is located inside the cell, and the large isoform is present inside and outside the cells. Furthermore, fibulin 2 is post-translationally modified by tyrosine sulfation, and the sulfated isoform is present outside the cell, whereas the unsulfated pool is internally located. Interestingly, sulfated fibulin 2 significantly reduced the rate of cellular growth and migration. Finally, levels of fibulin 2 dramatically increased in the retinal pigment epithelium following retinal detachment, suggesting a direct role for fibulin 2 in the re-attachment of the retina to the retinal pigment epithelium. Understanding the role of fibulin 2 in enhancing retinal attachment is likely to help improve the current therapies or allow the development of new strategies for the treatment of this sight-threatening condition.

WITHDRAWN: Fibulin 2, a tyrosine o-sulfated protein, is up-regulated following retinal detachment.

The manuscript has been withdrawn by the author.

Reduced inspired oxygen decreases retinal superoxide radicals and promotes cone function and survival in a model of retinitis pigmentosa.

Retinitis pigmentosa (RP) is caused by many different mutations that promote the degeneration of rod photoreceptors and have no direct effect on cones. After the majority of rods have died cone photoreceptors begin to slowly degenerate. Oxidative damage contributes to cone cell death and it has been hypothesized that tissue hyperoxia due to reduced oxygen consumption from the loss of rods is what initiates oxidative stress. Herein, we demonstrate in animal models of RP that reduction of retinal hyperoxia by reducing inspired oxygen to continuous breathing of 11% O2 reduced the generation of superoxide radicals in the retina and preserved cone structure and function. These data indicate that retinal hyperoxia is the initiating event that promotes oxidative damage, loss of cone function, and cone degeneration in the RP retina.

Oxidative stress-induced alterations in retinal glucose metabolism in Retinitis Pigmentosa.

Retinitis pigmentosa occurs due to mutations that cause rod photoreceptor degeneration. Once most rods are lost, gradual degeneration of cone photoreceptors occurs. Oxidative damage and abnormal glucose metabolism have been implicated as contributors to cone photoreceptor death. Herein, we show increased phosphorylation of key enzymes of glucose metabolism in the retinas of rd10 mice, a model of RP, and retinas of wild type mice with paraquat-induced oxidative stress, thereby inhibiting these key enzymes. Dietary supplementation with glucose and pyruvate failed to overcome the inhibition, but increased reducing equivalents in the retina and improved cone function and survival. Dichloroacetate reversed the increased phosphorylation of pyruvate dehydrogenase in rd10 retina and increased histone acetylation and levels of TP53-induced glycolysis and apoptosis regulator (TIGAR), which redirected glucose metabolism toward the pentose phosphate pathway. These data indicate that oxidative stress induced damage can be reversed by shifting glycolytic intermediates toward the pentose phosphate pathway which increases reducing equivalents and provides photoreceptor protection.

Serine/threonine kinase akt activation regulates the activity of retinal serine/threonine phosphatases, PHLPP and PHLPPL.

In our previous studies, we have shown that insulin receptor (IR) activation leads to the activation of phosphoinositide 3-kinase (PI3K) and Akt activation in rod photoreceptors. This pathway is functionally important for photoreceptor survival as deletion of IR and one of the isoforms of Akt (Akt2) resulted in stress-induced photoreceptor degeneration. However, the molecular mechanism of this degeneration is not known. Akt signaling is known to be regulated by the serine/threonine phosphatases, PH domain and leucine-rich repeat protein phosphatases (PHLPP) and PHLPP-like (PHLPPL). In this study, we characterized these two phosphatases in the retina and examined the role of IR, PI3K, and Akt signaling on the activity of PHLPP and PHLPPL. Most of the studies published on PHLPP and PHLPPL are directed toward Akt dephosphorylation; however, there are no studies available to date on how the enzyme activities of these phosphatases are regulated. We made a novel finding in this study that both PHLPP and PHLPPL activities were significantly decreased in the presence of insulin ex vivo. The insulin-induced decrease of phosphatase activities were PI3K-dependent as pre-treatment of ex vivo retinal cultures with LY294002 significantly reversed the insulin-induced inhibition. It has been shown previously that PHLPP and PHLPPL regulate the dephosphorylation of Akt isoforms, and our results demonstrate for the first time that retinal PHLPP and PHLPPL activities are under the control of the IR-activated PI3K/Akt pathway.

Lack of protein-tyrosine sulfation disrupts photoreceptor outer segment morphogenesis, retinal function and retinal anatomy.

To investigate the role(s) of protein-tyrosine sulfation in the retina, we examined retinal function and structure in mice lacking tyrosylprotein sulfotransferases (TPST) 1 and 2. Tpst double knockout (DKO; Tpst1(-/-) /Tpst2 (-/-) ) retinas had drastically reduced electroretinographic responses, although their photoreceptors exhibited normal responses in single cell recordings. These retinas appeared normal histologically; however, the rod photoreceptors had ultrastructurally abnormal outer segments, with membrane evulsions into the extracellular space, irregular disc membrane spacing and expanded intradiscal space. Photoreceptor synaptic terminals were disorganized in Tpst DKO retinas, but established ultrastructurally normal synapses, as did bipolar and amacrine cells; however, the morphology and organization of neuronal processes in the inner retina were abnormal. These results indicate that protein-tyrosine sulfation is essential for proper outer segment morphogenesis and synaptic function, but is not critical for overall retinal structure or synapse formation, and may serve broader functions in neuronal development and maintenance.

Analysis of genes differentially expressed during retinal degeneration in three mouse models.

An estimated 100,000 people in the US alone have retinitis pigmentosa. This disease, caused by the loss of rods and cones, results in blindness. With the intention of identifying common cell death pathways that result in RP, the pattern of global gene expression in three different mouse models of retinal degeneration was analyzed using DNA arrays. The models used were opsin ( Delta255-256 ), a transgenic mouse line that expresses a mutant form of opsin with a deletion of an isoleucine at either position 255 or 256; the Bouse C mouse, whereby normal opsin is over-expressed by over 2 folds; MOT1, a model that expresses SV-40 T antigen downstream of opsin promoter and leads to retinal degeneration. We found that, at least in the 2 models of retinal degeneration that are characterized by rhodopsin abnormalities, death is due to the TNF pathway. In addition, there are a number of unknown genes not yet annotated in each of the models that could be promising in revealing novel functions in photoreceptors.

Sustained treatment of retinal vascular diseases with self-aggregating sunitinib microparticles.

Neovascular age-related macular degeneration and diabetic retinopathy are prevalent causes of vision loss requiring frequent intravitreous injections of VEGF-neutralizing proteins, and under-treatment is common and problematic. Here we report incorporation of sunitinib, a tyrosine kinase inhibitor that blocks VEGF receptors, into a non-inflammatory biodegradable polymer to generate sunitinib microparticles specially formulated to self-aggregate into a depot. A single intravitreous injection of sunitinib microparticles potently suppresses choroidal neovascularization in mice for six months and in another model, blocks VEGF-induced leukostasis and retinal nonperfusion, which are associated with diabetic retinopathy progression. After intravitreous injection in rabbits, sunitinib microparticles self-aggregate into a depot that remains localized and maintains therapeutic levels of sunitinib in retinal pigmented epithelium/choroid and retina for more than six months. There is no intraocular inflammation or retinal toxicity. Intravitreous injection of sunitinib microparticles provides a promising approach to achieve sustained suppression of VEGF signaling and improve outcomes in patients with retinal vascular diseases.

The latency-associated nuclear antigen of Kaposi sarcoma-associated herpesvirus induces B cell hyperplasia and lymphoma.

Kaposi sarcoma-associated herpesvirus (KSHV) is a human lymphotropic herpesvirus. It is implicated in B cell neoplasias such as primary effusion lymphoma and multicentric Castleman disease in AIDS patients. The KSHV latency-associated nuclear antigen (LANA) is consistently expressed in all KSHV-associated tumor cells and was shown to bind the tumor suppressor proteins p53 and pRb. To test LANA's contribution to lymphomagenesis in vivo we generated transgenic mice expressing LANA under the control of its own promoter, which is B cell specific. All of the transgenic mice developed splenic follicular hyperplasia due to an expansion of IgM+ IgD+ B cells and showed increased germinal center formation. We also observed lymphomas, implying that LANA can activate B cells and provide the first step toward lymphomagenesis.

Protein tyrosine-O-sulfation in the retina.

Tyrosine-O-sulfation, a post-translational modification, is catalyzed by two independent tyrosylprotein sulfotransferases (TPSTs). As an initial step towards understanding the role of TPSTs in retinal function, this study was undertaken to determine the extent to which tyrosine-O-sulfation of proteins is utilized in the retina. A previously characterized anti-sulfotyrosine antibody was used to determine the presence and localization of tyrosine-O-sulfated proteins (TOSPs) in the retina. Using Western blot, RT-PCR and immunohistochemical analyses, we detected TOSPs in the retinas from diverse species, including frog, fish, mouse and human. Some of the variability in the observed sizes of retinal TOSPs in the mouse, at least, may result from differential patterns of glycosylation; however, there seem to be species-specific sulfated retinal proteins as well. TOSPs were detected in most of the retinal layers as well as in the retinal pigment epithelium from human and mouse. Several retinal TOSPs were detected in the inter-photoreceptor matrix, which is consistent with the secreted nature of some sulfated proteins. Transcripts for both TPST-1 and TPST-2 were expressed in both the human and mouse retinas. These data show that retinal protein tyrosine-O-sulfation is highly conserved which suggest a functional significance of these proteins to retinal function and structure.

Retinol dehydrogenases RDH11 and RDH12 in the mouse retina: expression levels during development and regulation by oxidative stress.

PURPOSE: RDH11 and RDH12 are closely related retinol dehydrogenases expressed in the retina. RDH12 has been linked to the early-onset retinal dystrophy Leber congenital amaurosis, whereas RDH11 has not been associated with human disease. To understand their physiological roles, the authors investigated their expression during development and their regulation by light-induced oxidative stress in mouse retina. METHODS: Quantitative RT-PCR and immunoblot analysis were used for quantification of RDH11 and RDH12 during development and oxidative stress. Expression during development was measured between embryonic day (E) 12 and postnatal day (P) 210 (7 months) in C57BL/6 mouse eyes. Expression during light-induced oxidative stress was measured between 2 and 24 hours of exposure to light in BALB/c mouse retina. RESULTS: The RDH11 level was low and remarkably constant during development and oxidative stress. RDH12 expression started at P7 and increased until P30 to approximately sevenfold higher than RDH11. Oxidative stress induced by exposure to constant bright light led to a rapid and significant decrease of RDH12 protein. CONCLUSIONS: The low and constant expression of RDH11 suggested a housekeeping function for this enzyme. The onset of RDH12 expression during the maturation of photoreceptor cells suggested a function related to the visual process. The light-induced rapid decrease of RDH12 protein, preceding the decrease of the mRNA, suggested a specific degradation of the protein rather than a regulation of gene expression.

VEGF/VEGFR2 blockade does not cause retinal atrophy in AMD-relevant models.

Intraocular injections of VEGF-neutralizing proteins provide tremendous benefits in patients with choroidal neovascularization (NV) due to age-related macular degeneration (AMD), but during treatment some patients develop retinal atrophy. Suggesting that VEGF is a survival factor for retinal neurons, a clinical trial group attributed retinal atrophy to VEGF suppression and cautioned against frequent anti-VEGF injections. This recommendation may contribute to poor outcomes in clinical practice from insufficient treatment. Patients with type 3 choroidal NV have particularly high risk of retinal atrophy, an unexplained observation. Herein we show in mouse models that VEGF signaling does not contribute to photoreceptor survival and functioning: (a) neutralization of VEGFR2 strongly suppresses choroidal NV without compromising photoreceptor function or survival; (b) VEGF does not slow loss of photoreceptor function or death in mice with inherited retinal degeneration, and there is no exacerbation by VEGF suppression; and (c) mice with type 3 choroidal NV develop retinal atrophy due to oxidative damage with no contribution from VEGF suppression. Intraocular injections of VEGF-neutralizing proteins, a highly effective treatment in patients with neovascular AMD, should not be withheld or reduced due to concern that they may contribute to long-term visual loss from retinal atrophy.

Light induces programmed cell death by activating multiple independent proteases in a cone photoreceptor cell line.

PURPOSE: Although the apoptotic death of photoreceptor cells in retinal degenerative disorders is well documented, the molecular mechanism is not understood. The objective of this study was to determine the molecular events leading to the death of photoreceptor cells. METHODS: An assay was developed wherein 661W cells, a cone photoreceptor cell line, were stressed with light and percentage of surviving cells was determined. The degree of cell death was established using the MTT assay. Western blot analysis was used to confirm the activation of multiple proteases. Amounts of retinaldehydes were determined by extraction and HPLC. RESULTS: 661W cells were more susceptible to light stress only in the presence of the chromophore 9-cis retinal for 4 hours. On exposure to light, 9-cis retinal was converted to all-trans retinal, which was found to be toxic to cells in the presence of light. However, all-trans retinol, which is the product of action by the enzyme retinol dehydrogenase on all-trans retinal, was not toxic. The sensitivity to light increased with serum deprivation. Light stress activated caspases, calpain 2, and cathepsin D independently and led to the demise of the cell. The mitochondria-dependent apoptotic pathway was also activated after the truncation of Bid, the pre-proapoptotic protein. Truncation of Bid led to the release of cytochrome c from the mitochondria and the activation of caspase 9. CONCLUSIONS: The activation of multiple proteases by light-induced stress is a relevant finding for studies conducted to investigate the use of pharmaceutical agents to retard or cure the loss of cone photoreceptors observed in age-related macular degeneration and other degenerative retinal diseases.

Tyrosine kinase blocking collagen IV-derived peptide suppresses ocular neovascularization and vascular leakage.

Vascular endothelial growth factor (VEGF)-neutralizing proteins provide benefit in several retinal and choroidal vascular diseases, but some patients still experience suboptimal outcomes, and the need for frequent intraocular injections is a barrier to good outcomes. A mimetic peptide derived from collagen IV, AXT107, suppressed subretinal neovascularization (NV) in two mouse models predictive of effects in neovascular age-related macular degeneration (NVAMD) and inhibited retinal NV in a model predictive of effects in ischemic retinopathies. A combination of AXT107 and the current treatment aflibercept suppressed subretinal NV better than either agent alone. Furthermore, AXT107 caused regression of choroidal NV. AXT107 reduced the VEGF-induced vascular leakage that underlies macular edema in ischemic retinopathies and NVAMD. In rabbit eyes, which are closer to the size of human eyes, intraocular injection of AXT107 significantly reduced VEGF-induced vascular leakage by 86% at 1 month and 70% at 2 months; aflibercept significantly reduced leakage by 69% at 1 month and did not reduce leakage at 2 months, demonstrating the longer effectiveness of AXT107. AXT107 reduced ligand-induced phosphorylation of multiple receptors: VEGFR2, c-Met, and PDGFRß. Optimal signaling through these receptors requires complex formation with ß3 integrin, which was reduced by AXT107 binding to αvß3 AXT107 also reduced total VEGFR2 levels by increasing internalization, ubiquitination, and degradation. This biomimetic peptide is a sustained, multitargeted therapy that may provide advantages over intraocular injections of specific VEGF-neutralizing proteins.