Tyrosine nitration of prostacyclin synthase is associated with enhanced retinal cell apoptosis in diabetes

The risk of diabetic retinopathy is associated with the presence of both oxidative stress and toxic eicosanoids. Whether oxidative stress actually causes diabetic retinopathy via the generation of toxic eicosanoids, however, remains unknown. The aim of the present study was to determine whether tyrosine nitration of prostacyclin synthase (PGIS) contributes to retinal cell death in vitro and in vivo. Exposure of human retinal pericytes to heavily oxidized and glycated LDL (HOG-LDL), but not native forms of LDL (N-LDL), for 24 hours significantly increased pericyte apoptosis, accompanied by increased tyrosine nitration of PGIS and decreased PGIS activity. Inhibition of the thromboxane receptor or cyclooxygenase-2 dramatically attenuated HOG-LDL-induced apoptosis without restoring PGIS activity. Administration of superoxide dismutase (to scavenge superoxide anions) or L-N(G)-nitroarginine methyl ester (L-NAME, a nonselective nitric oxide synthase inhibitor) restored PGIS activity and attenuated pericyte apoptosis. In Akita mouse retinas, diabetes increased intraretinal levels of oxidized LDL and glycated LDL, induced PGIS nitration, enhanced apoptotic cell death, and impaired blood-retinal barrier function. Chronic administration of tempol, a superoxide scavenger, reduced intraretinal oxidized LDL and glycated LDL levels, PGIS nitration, and retina cell apoptosis, thereby preserving the integrity of blood-retinal barriers. In conclusion, oxidized LDL-mediated PGIS nitration and associated thromboxane receptor stimulation might be important in the initiation and progression of diabetic retinopathy.

Abnormal Glycogen Storage by Retinal Neurons in Diabetes

PURPOSE: It is widely held that neurons of the central nervous system do not store glycogen and that accumulation of the polysaccharide may cause neurodegeneration. Since primary neural injury occurs in diabetic retinopathy, we examined neuronal glycogen status in the retina of streptozotocin-induced diabetic and control rats.

METHODS: Glycogen was localized in eyes of streptozotocin-induced diabetic and control rats using light microscopic histochemistry and electron microscopy, and correlated with immunohistochemical staining for glycogen phosphorylase and phosphorylated glycogen synthase (pGS).

RESULTS: Electron microscopy of 2-month-old diabetic rats (n = 6) showed massive accumulations of glycogen in the perinuclear cytoplasm of many amacrine neurons. In 4-month-old diabetic rats (n = 11), quantification of glycogen-engorged amacrine cells showed a mean of 26 cells/mm of central retina (SD ± 5), compared to 0.5 (SD ± 0.2) in controls (n = 8). Immunohistochemical staining for glycogen phosphorylase revealed strong expression in amacrine and ganglion cells of control retina, and increased staining in cell processes of the inner plexiform layer in diabetic retina. In control retina, the inactive pGS was consistently sequestered within the cell nuclei of all retinal neurons and the retinal pigment epithelium (RPE), but in diabetics nuclear pGS was reduced or lost in all classes of retinal cell except the ganglion cells and cone photoreceptors.

CONCLUSIONS: The present study identifies a large population of retinal neurons that normally utilize glycogen metabolism but show pathologic storage of the polysaccharide during uncontrolled diabetes.

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.

Reduced Nitro-oxidative Stress and Neural Cell Death Suggests a Protective Role for Microglial Cells in TNFα−/− Mice in Ischemic Retinopathy

Purpose. Neovascularization occurs in response to tissue ischemia and growth factor stimulation. In ischemic retinopathies, however, new vessels fail to restore the hypoxic tissue; instead, they infiltrate the transparent vitreous. In a model of oxygen-induced retinopathy (OIR), TNFa and iNOS, upregulated in response to tissue ischemia, are cytotoxic and inhibit vascular repair. The aim of this study was to investigate the mechanism for this effect.

Methods. Wild-type C57/BL6 (WT) and TNFa-/- mice were subjected to OIR by exposure to 75% oxygen (postnatal days 7–12). The retinas were removed during the hypoxic phase of the model. Retinal cell death was determined by TUNEL staining, and the microglial cells were quantified after Z-series capture with a confocal microscope. In situ peroxynitrite and superoxide were measured by using the fluorescent dyes DCF and DHE. iNOS, nitrotyrosine, and arginase were analyzed by real-time PCR, Western blot analysis, and activity determined by radiolabeled arginine conversion. Astrocyte coverage was examined after GFAP immunostaining.

Results. The TNFa-/- animals displayed a significant reduction in TUNEL-positive apoptotic cells in the inner nuclear layer of the avascular retina compared with that in the WT control mice. The reduction coincided with enhanced astrocytic survival and an increase in microglial cells actively engaged in phagocytosing apoptotic debris that displayed low ROS, RNS, and NO production and high arginase activity.

Conclusions. Collectively, the results suggest that improved vascular recovery in the absence of TNFa is associated with enhanced astrocyte survival and that both phenomena are dependent on preservation of microglial cells that display an anti-inflammatory phenotype during the early ischemic phase of OIR.

Dysregulation in retinal para-inflammation and age-related retinal degeneration in CCL2 or CCR2 deficient mice

We have shown previously that a para-inflammatory response exists at the retinal/choroidal interface in the aging eye; and this response plays an important role in maintaining retinal homeostasis under chronic stress conditions. We hypothesized that dysregulation of the para-inflammatory response may result in an overt pro-inflammatory response inducing retinal degeneration. In this study, we examined this hypothesis in mice deficient in chemokine CCL2 or its cognate receptor CCR2. CCL2- or CCR2-deficient mice developed retinal degenerative changes with age, characterized as retinal pigment epithelial (RPE) cell and photoreceptor cell death. Retinal cell death was associated with significantly more subretinal microglial accumulation and increased complement activation. In addition, monocytes from CCL2- or CCR2-deficient mice had reduced capacity for phagocytosis and chemotaxis, expressed less IL-10 but more iNOS, IL-12 and TNF-a when compared to monocytes from WT mice. Complement activation at the site of RPE cell death resulted in C3b/C3d but not C5b-9 deposition, indicating only partial activation of the complement pathway. Our results suggest that altered monocyte functions may convert the protective para-inflammatory response into an overtly harmful inflammation at the retina/choroidal interface in CCL2- or CCR2-deficient mice, leading to RPE and photoreceptor degeneration. These data support a concept whereby a protective para-inflammatory response relies upon a normally functioning innate immune system. If the innate immune system is deficient chronic stress may tip the balance towards an overt inflammatory response causing cell/tissue damage.

Preparation of planar retinal specimens:verification by histology, mRNA profiling, and proteome analysis

Elucidation of the transcriptome and proteome of the normal retina will be difficult since it is comprised of at least 55 different cell types. However the characteristic layered cellular anatomy of the retina makes it amenable to planar sectioning, enabling the generation of enriched retinal cell populations. The aim of this study was to validate a reproducible method for preparing enriched retinal layers from porcine retina.

Immunohistochemical study of pig retinal development

PURPOSE: The pig eye is similar to the human eye in terms of anatomy, vasculature, and photoreceptor distribution, and therefore provides an attractive animal model for research into retinal disease. The purpose of this study was to characterize retinal histology in the developing and mature pig retina using antibodies to well established retinal cell markers commonly used in rodents.

METHODS: Eyes were enucleated from fetuses in the 9th week of gestation, 1 week old piglets and 6 months old adult animals. Eyeglobes were fixed and cryosectioned. A panel of antibodies to well established retinal markers was employed for immunohistochemistry. Fluorescently labeled secondary antibodies were used for signal detection, and images were acquired by confocal microscopy. Mouse retina at postnatal day (P) 5 was used as a reference for this study to compare progression of histogenesis. Most of the primary antibodies have previously been used on mouse tissue.

RESULTS: Most of the studied markers were detected in midgestation pig retina, and the majority had a similar distribution in pig as in P5 mouse retina. However, rhodopsin immunolabeling was detected in pig retina at midgestation but not in P5 mouse retina. Contrary to findings in all rodents, horizontal cells were Islet1-positive and cones were calbindin-immunoreactive in pig retina, as has also been shown for the primate retina. Recoverin and rhodopsin immunolabeling revealed an increase in the length of photoreceptor segments in 6 months, compared to 1 week old animals.

CONCLUSIONS: Comparison with the published data on human retina revealed similar marker distribution and histogenesis progression in the pig and human retina, supporting the pig as a valuable animal model for studies on retinal disease and repair. Furthermore, this study provides information about the dynamics of retinal histogenesis in the pig and validates a panel of antibodies that reliably detects developing and mature retinal cell phenotypes in the pig retina.

AGEs and Diabetic Retinopathy

The pathogenesis of diabetic retinopathy is multifactorial, and a range of hyperglycemia-linked pathways have been implicated in the initiation and progression of this condition. All cells in the retina are affected by the diabetic milieu, and in view of such disease and tissue complexity, it is unlikely that any single process is solely responsible for retinal pathophysiology. Nevertheless, establishing causal mechanisms remains an important research goal. This review concentrates on the formation of advanced glycation end products (AGEs) and the role they play in diabetic retinopathy. Perspective is provided on advanced glycation in the retina, the impact that this process has on retinal cell function, and how it relates to other pathogenic pathways. Emphasis is also placed the modulatory role of the receptor for AGEs (RAGE) and how its activation could evoke retinal inflammatory disease. Further research is needed to achieve a clear understanding of the cellular and molecular processes that underpin diabetic retinopathy's initiation and progression. Such advances in basic mechanisms may lead to effective treatments that can prevent progression of retinopathy from the point of the diagnosis of diabetes to sight-threatening proliferative diabetic retinopathy (PDR) and diabetic macular edema (DME).

Diabetes-related adduct formation and retinopathy

The pathogenesis of diabetic retinopathy is complex, reflecting the array of systemic and tissue-specific metabolic abnormalities. A range of pathogenic pathways are directly linked to hyperglycaemia and dyslipidaemia, and the retina appears to be exquisitely sensitive to damage. Establishing the biochemical and molecular basis for this pathology remains an important research focus. This review concentrates on the formation of a range of protein adducts that form after exposure to modifying intermediates known to be elevated during diabetes. These so-called advanced glycation end products (AGEs) and advanced lipoxidation end products (ALEs) are thought to play an important role in the initiation and progression of diabetic retinopathy, and mechanisms leading to dysfunction and death of various retinal cells are becoming understood. Perspective is provided on AGE/ALE formation in the retina and the impact that such adducts have on retinal cell function. There will be emphasis placed on the role of the receptor for AGEs and how this may modulate retinal pathology, especially in relation to oxidative stress and inflammation. The review will conclude by discussion of strategies to inhibit AGE/ALE formation or harmful receptor interactions in order to prevent disease progression from the point of diabetes diagnosis to sight-threatening proliferative diabetic retinopathy and diabetic macular oedema.

Advances in our understanding of diabetic retinopathy

Diabetic retinopathy remains the most common complication of diabetes mellitus and is a leading cause of visual loss in industrialized nations. The clinicopathology of the diabetic retina has been extensively studied, although the precise pathogenesis and cellular and molecular defects that lead to retinal vascular, neural and glial cell dysfunction remain somewhat elusive. This lack of understanding has seriously limited the therapeutic options available for the ophthalmologist and there is a need to identify the definitive pathways that initiate retinal cell damage and drive progression to overt retinopathy. The present review begins by outlining the natural history of diabetic retinopathy, the clinical features and risk factors. Reviewing the histopathological data from clinical specimens and animal models, the recent paradigm that neuroretinal dysfunction may play an important role in the early development of the disease is discussed. The review then focuses on the molecular pathogenesis of diabetic retinopathy with perspective provided on new advances that have furthered our understanding of the key mechanisms underlying early changes in the diabetic retina. Studies have also emerged in the past year suggesting that defective repair of injured retinal vessels by endothelial progenitor cells may contribute to the pathogenesis of diabetic retinopathy. We assess these findings and discuss how they could eventually lead to new therapeutic options for diabetic retinopathy.

Advanced glycation end products and diabetic retinopathy

Diabetic retinopathy (DR) has a complex pathogenesis which is impacted by a raft of systemic abnormalities and tissue-specific alterations occurring in response to the diabetes milieu. Many pathogenic processes play key roles in retinal damage in diabetic patients. One such pathway is the formation and accumulation of advanced glycation endproducts (AGEs) and advanced lipoxidation end products (ALEs) which are relevant modifications with roles in the initiation and progression of pathology. In this review, AGE/ALE formation in the diabetic retina is discussed alongside their impact on retinal cell function. In addition, various inhibitors of the AGE-RAGE system and their therapeutic utility for DR will also be evaluated.

Retinal Pigment Epithelial Cell DNA is Damaged by Exposure to Benzo[a]pyrene, a Constituent of Cigarette Smoke.

This study examined the effect of exogenous benzo[ a ]pyrene (BaP), an important constituent of cigarette smoke, on cultured bovine retinal pigment epithelial (RPE) cells. Evidence is presented for its metabolic conversion into benzo[ a ]pyrene diol epoxide (BPDE) and the consequent formation of potentially cytotoxic nucleobase adducts in DNA. Cultured RPE cells were treated with BaP at concentrations in the range of 0–100 µm. The presence of BaP was found to cause inhibition of cell growth and replication. BaP induced the expression of a phase I drug metabolizing enzyme which was identified as cytochrome P450 1A1 (CYP 1A1) by RT–PCR and by Western blotting. Coincident with the increased expression of CYP 1A1, covalent adducts between the mutagenic metabolite BPDE and DNA could be detected within RPE cells by immunocytochemical staining. Additional support for their formation was afforded by nuclease P1 enhanced 32P-postlabelling assays on cellular DNA. Single-cell gel electrophoresis (comet) assays showed that exposure of RPE cells to BaP rendered them markedly more susceptible to DNA damage induced by broad band UVB or blue light laser irradiation. In the case of UVB, this is consistent with the photosensitization of DNA cleavage by nucleobase adducts of BPDE. Collectively, these findings imply that BaP has a significant impact on RPE cell pathophysiology and suggest mechanisms whereby exposure to cigarette smoke might cause RPE dysfunction and cell death, thus possibly contributing to degenerative disorders of the retina.

Recombinant alpha 2(IV)NC1 domain of type IV collagen is an effective regulator of retinal capillary endothelial cell proliferation and inhibits pre-retinal neovascularisation

Background A recombinant form of the alpha 2(IV)NC1 domain of type IV collagen has been shown to have potent anti-angiogenic activity although this peptide has not been studied in the context of proliferative retinopathies. In the current investigation we examined the potential for alpha 2(IV) NC1 to regulate retinal microvascular endothelial cell function using a range of in vitro and in vivo assay systems.

Characterization of a spontaneous mouse retinal pigment epithelial cell line B6-RPE07.

PURPOSE. A spontaneously arising retinal pigment epithelial (RPE) cell line (B6-RPE07) was cloned from a primary culture of mouse RPE cells and maintained in culture for more than 18 months. Morphologic and functional properties of this cell line have been characterized.

METHODS. The morphology of the B6-RPE07 cells was examined by phase-contrast light microscopy, electron microscopy, and confocal microscopy. Barrier properties were measured by the flux of fluorescence from the apical to the basolateral compartment of culture chambers. The abilities of the cells to bind/phagocytose photoreceptor outer segments (POS) were determined by confocal microscopy, electron microscopy, and flow cytometry. Cytokine/chemokine secretion was measured by cytometric bead array. The expression of visual cycle proteins was determined by RT-PCR and Western blotting.

RESULTS. In standard culture conditions, B6-RPE07 cells display cobblestone morphology. When cultured on three-dimensional (3D) collagen gel–coated membranes, B6-RPE07 cells exhibit a monolayer epithelial polarization with apical surface microvilli. Immunohistochemistry of B6-RPE07 cultures revealed a high expression of pan-cytokeratin. B6-RPE07 cells also expressed the retinal pigment epithelium-specific marker CRALBP, but not RPE65. Cell junction proteins ZO-1 and ß-catenin, but not claudin-1/3 or occludin-1, were observed in B6-RPE07 cells. B6-RPE07 cells are able to bind, phagocytose, and digest POS. Finally, B6-RPE07 cells produce high levels of IL-6 and CCL2.

CONCLUSIONS. This is the first report of a mouse RPE cell line with morphology, phenotype, and function similar to those of in vivo mouse RPE cells. This cell line will be a valuable resource for future RPE studies, in particular for in vivo gene modification and transplantation studies.

Retinal Endothelial Cell Apoptosis Stimulates Recruitment of Endothelial Progenitor Cells

PURPOSE. Bone marrow–derived endothelial progenitor cells (EPCs) contribute to vascular repair although it is uncertain how local endothelial cell apoptosis influences their reparative function. This study was conducted to determine how the presence of apoptotic bodies at sites of endothelial damage may influence participation of EPCs in retinal microvascular repair.

METHODS. Microlesions of apoptotic cell death were created in monolayers of retinal microvascular endothelial cells (RMECs) by using the photodynamic drug verteporfin. The adhesion of early-EPCs to these lesions was studied before detachment of the apoptotic cells or after their removal from the wound site. Apoptotic bodies were fed to normal RMECs and mRNA levels for adhesion molecules were analyzed.

RESULTS. Endothelial lesions where apoptotic bodies were left attached at the wound site showed a fivefold enhancement in EPC recruitment (P < 0.05) compared with lesions where the apoptotic cells had been removed. In intact RMEC monolayers exposed to apoptotic bodies, expression of ICAM, VCAM, and E-selectin was upregulated by 5- to 15-fold (P < 0.05–0.001). EPCs showed a characteristic chemotactic response (P < 0.05) to conditioned medium obtained from apoptotic bodies, whereas analysis of the medium showed significantly increased levels of VEGF, IL-8, IL-6, and TNF-a when compared to control medium; SDF-1 remained unchanged.

CONCLUSIONS. The data indicate that apoptotic bodies derived from retinal capillary endothelium mediate release of proangiogenic cytokines and chemokines and induce adhesion molecule expression in a manner that facilitates EPC recruitment.