<Emphasis Type="SmallCaps">l</Emphasis>-Arginine Uptake in Normal and Diabetic Rat Retina and Retinal Pigment Epithelium

Diabetes-induced increase in oxidative stress is postulated as playing a significant role in the development of retinopathy. The retinal pigment epithelium (RPE) which forms part of the retinal blood barrier has been reported to be affected in diabetes. Besides functioning as a neurotransmitter, the radical nitric oxide (NO) can act as a cytotoxic agent. NO is synthesized by nitric oxide synthase (NOS) that oxidizes arginine to citrulline producing NO. Given that intracellular concentration of arginine depends mainly on its transport, we studied arginine transport in RPE and retina from normal and streptozotocin-induced diabetic rats. Retina and RPE take up arginine by a saturable system with an apparent K_M of 70–80 μM. Tissue incubation in the presence of insulin or high glucose concentrations significantly increased arginine transport in RPE but not in retina from control rats. Similarly, arginine uptake was enhanced in RPE, but not in the retina from streptozotocin-induced diabetic rats. However, NO content was two-fold higher in diabetic retina and RPE compared to that in the control rats. Such findings may suggest that diabetes induced an increase in NO levels in RPE, which may have brought about alterations in its functioning and in turn manifestations of diabetic retinopathy. Special issue article in honor of Dr. Ricardo Tapia.     

Diabetic retinopathy: recent advances towards understanding neurodegeneration and vision loss

Diabetic retinopathy(DR) is one of the most common retinal diseases world-wide. It has a complex pathology that involves the vasculature of the inner retina and breakdown of the blood-retinal barrier. Extensive research has determined that DR is not only a vascular disease but also has a neurodegenerative component and that essentially all types of cells in the retina are affected, leading to chronic loss of visual function. A great deal of work using animal models of DR has established the loss of neurons and pathology of other cell types, including supporting glial cells. There has also been an increased emphasis on measuring retinal function in the models, as well as further validation and extension of the animal studies by clinical and translational research. This article will attempt to summarize the more recent developments in research towards understanding the complexities of retinal neurodegeneration and functional vision loss in DR.     

Hyperactivation of retina by light in mice leads to photoreceptor cell death mediated by VEGF and retinal pigment epithelium permeability

Light toxicity is suspected to enhance certain retinal degenerative processes such as age-related macular degeneration. Death of photoreceptors can be induced by their exposure to the visible light, and although cellular processes within photoreceptors have been characterized extensively, the role of the retinal pigment epithelium (RPE) in this model is less well understood. We demonstrate that exposition to intense light causes the immediate breakdown of the outer blood–retinal barrier (BRB). In a molecular level, we observed the slackening of adherens junctions tying up the RPE and massive leakage of albumin into the neural retina. Retinal pigment epithelial cells normally secrete vascular endothelial growth factor (VEGF) at their basolateral side; light damage in contrast leads to VEGF increase on the apical side – that is, in the neuroretina. Blocking VEGF, by means of lentiviral gene transfer to express an anti-VEGF antibody in RPE cells, inhibits outer BRB breakdown and retinal degeneration, as illustrated by functional, behavioral and morphometric analysis. Our data show that exposure to high levels of visible light induces hyperpermeability of the RPE, likely involving VEGF signaling. The resulting retinal edema contributes to irreversible damage to photoreceptors. These data suggest that anti-VEGF compounds are of therapeutic interest when the outer BRB is altered by retinal stresses.     

Ultrastructural localization of blood-retinal barrier breakdown in diabetic and galactosemic rats

Breakdown of the blood-retinal barrier (BRB) is an early event in diabetic and galactosemic rats, but the location and nature of the specific defect(s) are controversial. Using an electron microscopic immunocytochemical technique, the retinas of normal, diabetic, and galactosemic rats were immunostained for endogenous albumin. Normal rats showed little evidence of BRB breakdown at either the inner barrier (retinal vasculature) or the outer barrier (retinal pigment epithelium) (RPE). In diabetic and galactosemic rats, as was true in human diabetics, BRB breakdown occurred predominantly at the inner BRB, but in some cases at the outer barrier as well. Treatment with the aldose reductase inhibitor sorbinil largely prevented BRB failure in galactosemic rats. In the inner retina of diabetic and galactosemic rats, albumin was frequently demonstrated on the abluminal side of the retinal capillary endothelium (RCE) in intercellular spaces, basal laminae, pericytes, ganglion cells, astrocytes, and the perinuclear cytoplasm of cells in the inner nuclear layer. Albumin did not appear to cross RCE cell junctions; however, it was occasionally seen in RCE cytoplasm of galactosemic rats. In the outer retina, albumin was frequently detected in the subretinal space, in the intercellular space between photoreceptors, and in the perinuclear cytoplasm of photoreceptor cells, but was only infrequently found in the RPE cells constituting the barrier. Albumin derived from the choroidal vasculature did not appear to cross the tight junctions of the RPE. These findings suggest that specific sites of BRB compromise are infrequent but that once albumin has crossed the RCE or RPE it freely permeates the retinal tissue by filling intercellular spaces and permeating the membranes of cells not implicated in BRB formation. The diffuse cytoplasmic staining of some RCE and RPE cells suggests that the predominant means of BRB breakdown in diabetes and galactosemia involves increased focal permeability of the surface membranes of the RCE and RPE cells rather than defective tight junctions or vesicular transport.     

Role of lysophosphatidic acid in the retinal pigment epithelium and photoreceptors

The human retina is a complex structure of organised layers of specialised cells that support the transmission of light signals to the visual cortex. The outermost layer of the retina, the retinal pigment epithelium (RPE), forms part of the blood retina barrier and is implicated in many retinal diseases. Lysophosphatidic acid (LPA) is a bioactive lipid exerting pleiotropic effects in various cell types, during development, normal physiology and disease. Its producing enzyme, AUTOTAXIN (ATX), is highly expressed by the pigmented epithelia of the human eye, including the RPE. Using human pluripotent stem cell (hPSC)-derived retinal cells, we interrogated the role of LPA in the human RPE and photoreceptors. hPSC-derived RPE cells express and synthesize functional ATX, which is predominantly secreted apically of the RPE, suggesting it acts in a paracrine manner to regulate photoreceptor function. In RPE cells, LPA regulates tight junctions, in a receptor-dependent mechanism, with an increase in OCCLUDIN and ZONULA OCCLUDENS (ZO)-1 expression at the cell membrane, accompanied by an increase in the transepithelial resistance of the epithelium. High concentration of LPA decreases phagocytosis of photoreceptor outer segments by the RPE. In hPSC-derived photoreceptors, LPA induces morphological rearrangements by modulating the actin myosin cytoskeleton, as evidenced by Myosin Light Chain l membrane relocation. Collectively, our data suggests an important role of LPA in the integrity and functionality of the healthy retina and blood retina barrier.     

骨钙素减轻糖尿病大鼠血-视网膜屏障的损伤

目的：研究骨钙素（Osteocalcin）对链脲佐菌素诱导的糖尿病大鼠血-视网膜屏障的影响。方法：取健康SD大鼠24只,随机分为正常对照组、糖尿病1月（DM1）组、糖尿病骨钙素干预（DM1＋OCGY）组。尾静脉注射STZ建立DM模型,成模后DM1＋OCGY组腹腔注射骨钙素（2．57mg·kg·1·d^-1）,DMl组腹腔注射等量生理盐水,1个月后处死动物。用伊文思蓝方法检测大鼠血-视网膜屏障的改变。结果：造模1月后大鼠视网膜血管渗透性显著增加,共聚焦显微镜显示红色荧光斑点主要分布在视网膜血管周围,给予骨钙素后,红色荧光斑点明显减少,进一步定量显示1M糖尿病大鼠视网膜伊文思蓝含量为57．4±8．7μg·g^-1,骨钙素能够改变这种变化,DM1＋OCGY组伊文思蓝含量为26．1±3．8μg·g^-1。结论：骨钙素能抑制糖尿病视网膜病的血管渗漏,对糖尿病引起的血一视网膜屏障破坏有保护作用。     

Induction of necrotic cell death by oxidative stress in retinal pigment epithelial cells

Age-related macular degeneration (AMD) is a degenerative disease of the retina and the leading cause of blindness in the elderly. Retinal pigment epithelial (RPE) cell death and the resultant photoreceptor apoptosis are characteristic of late-stage dry AMD, especially geographic atrophy (GA). Although oxidative stress and inflammation have been associated with GA, the nature and underlying mechanism for RPE cell death remains controversial, which hinders the development of targeted therapy for dry AMD. The purpose of this study is to systematically dissect the mechanism of RPE cell death induced by oxidative stress. Our results show that characteristic features of apoptosis, including DNA fragmentation, caspase 3 activation, chromatin condensation and apoptotic body formation, were not observed during RPE cell death induced by either hydrogen peroxide or tert-Butyl hydroperoxide. Instead, this kind of cell death can be prevented by RIP kinase inhibitors necrostatins but not caspase inhibitor z-VAD, suggesting necrotic feature of RPE cell death. Moreover, ATP depletion, receptor interacting protein kinase 3 (RIPK3) aggregation, nuclear and plasma membrane leakage and breakdown, which are the cardinal features of necrosis, were observed in RPE cells upon oxidative stress. Silencing of RIPK3, a key protein in necrosis, largely prevented oxidative stress-induced RPE death. The necrotic nature of RPE death is consistent with the release of nuclear protein high mobility group protein B1 into the cytoplasm and cell medium, which induces the expression of inflammatory gene TNFα in healthy RPE and THP-1 cells. Interestingly, features of pyroptosis or autophagy were not observed in oxidative stress-treated RPE cells. Our results unequivocally show that necrosis, but not apoptosis, is a major type of cell death in RPE cells in response to oxidative stress. This suggests that preventing oxidative stress-induced necrotic RPE death may be a viable approach for late-stage dry AMD.     

Breaking down the blood-brain barrier: signaling a path to cerebral malaria?

Cerebral malaria is a major killer in the developing world, but we still know very little about the causes of this disease. How does Plasmodium falciparum cause such a devastating neurological disease while it is in the brain vasculature? Why do some patients die, whereas others survive? What processes contribute to disease in the brain, and can we reverse them? Here, the latest evidence from post-mortem, in vitro and animal studies is reviewed to highlight the role of blood-brain barrier breakdown in cerebral malaria. Blood-brain barrier integrity is disturbed during severe malaria, causing leakage of cerebral vessels. Understanding how this happens and how it contributes to the pathogenesis of coma may provide new opportunities for the treatment of cerebral malaria.     

Glucose Metabolism in Rat Retinal Pigment Epithelium

The retinal pigment epithelium (RPE) is the major transport pathway for exchange of metabolites and ions between choroidal blood supply and the neural retina. To gain insight into the mechanisms controlling glucose metabolism in RPE and its possible relationship to retinopathy, we studied the influence of different glucose concentrations on glycogen and lactate levels and CO₂ production in RPE from normal and streptozotocin-treated diabetic rats. Incubation of normal RPE in the absence of glucose caused a decrease in lactate production and glycogen content. In normal RPE, increasing glucose concentrations from 5.6 mM to 30 mM caused a four-fold increase in glucose accumulation and CO₂ yield, as well as reduction in lactate and glycogen production. In RPE from diabetic rats glucose accumulation did not increase in the presence of high glucose substrate, but it showed a four- and a seven-fold increase in CO₂ production through the mitochondrial and pentose phosphate pathways, respectively. We found high glycogen levels in RPE which can be used as an energy reserve for RPE itself and/or neural retina. Findings further show that the RPE possesses a high oxidative capacity. The large increase in glucose shunting to the pentose phosphate pathway in diabetic retina exposed to high glucose suggests a need for reducing capacity, consistent with increased oxidative stress.     

Electron microscopic immunocytochemical demonstration of blood-retinal barrier breakdown in human diabetics and its association with aldose reductase in retinal vascular endothelium and retinal pigment epithelium

Summary Light-microscopic immunohistochemical staining for albumin has been used to localize sites of blood-retinal barrier (BRB) breakdown in ocular disorders, but the mechanism for BRB compromise cannot be resolved at this level. Using eyes up to 2 days post-mortem from normal patients or from patients with diabetic retinopathy, or other disorders known to cause BRB failure, electron-microscopic immunocytochemistry reveals focal breakdown of the inner BRB, comprised of the retinal vascular endothelium (RVE), which appears to be mediated by diffuse permeation of the RVE cells and by vesicular transport. Permeation of the retinal pigmented epithelial (RPE) cells that comprise the outer BRB also occurs, but there is no evidence of opening of tight junctions between RVE or RPE in any of the disorders evaluated. Increased aldose reductase (AR) expression in the RVE and RPE cells of diabetics as well as in the perivascular retinal astrocytes, which interact with RVE cells to establish the inner BRB, suggests that AR activity and the subsequent intracellular accumulation of sorbitol in these cell types may impair the function of the BRB in diabetes.     

Endoplasmic reticulum stress and inflammation: mechanisms and implications in diabetic retinopathy

The endoplasmic reticulum (ER) is the primary cellular compartment where proteins are synthesized and modified before they can be transported to their destination. Dysfunction of the ER impairs protein homeostasis and leads to the accumulation of misfolded/unfolded proteins in the ER, or ER stress. While it has long been recognized that ER stress is a major cause of conformational disorders, such as Alzheimer's disease, Huntington's disease, certain types of cancer, and type 2 diabetes, recent evidence suggests that ER stress is also implicated in many chronic inflammatory diseases. These diseases include irritable bowel syndrome, atherosclerosis, diabetic complications, and many others. Diabetic retinopathy is a common microvascular complication of diabetes, characterized by chronic inflammation, progressive damage to retinal vascular and neuronal cells, vascular leakage, and abnormal blood vessel growth (neovascularization). In this review, we discuss the role and mechanisms of ER stress in retinal inflammation and vascular damage in diabetic retinopathy.     

Characterization of a Spontaneous Retinal Neovascular Mouse Model

Background

Vision loss due to vascular disease of the retina is a leading cause of blindness in the world. Retinal angiomatous proliferation (RAP) is a subgroup of neovascular age-related macular degeneration (AMD), whereby abnormal blood vessels develop in the retina leading to debilitating vision loss and eventual blindness. The novel mouse strain, neoretinal vascularization 2 (NRV2), shows spontaneous fundus changes associated with abnormal neovascularization. The purpose of this study is to characterize the induction of pathologic angiogenesis in this mouse model.

Methods

The NRV2 mice were examined from postnatal day 12 (p12) to 3 months. The phenotypic changes within the retina were evaluated by fundus photography, fluorescein angiography, optical coherence tomography, and immunohistochemical and electron microscopic analysis. The pathological neovascularization was imaged by confocal microscopy and reconstructed using three-dimensional image analysis software.

Results

We found that NRV2 mice develop multifocal retinal depigmentation in the posterior fundus. Depigmented lesions developed vascular leakage observed by fluorescein angiography. The spontaneous angiogenesis arose from the retinal vascular plexus at postnatal day (p)15 and extended toward retinal pigment epithelium (RPE). By three months of age, histological analysis revealed encapsulation of the neovascular lesion by the RPE in the photoreceptor cell layer and subretinal space.

Conclusions

The NRV2 mouse strain develops early neovascular lesions within the retina, which grow downward towards the RPE beginning at p15. This retinal neovascularization model mimics early stages of human retinal angiomatous proliferation (RAP) and will likely be a useful in elucidating targeted therapeutics for patients with ocular neovascular disease.     

Regenerative Therapeutic Potential of Adipose Stromal Cells in Early Stage Diabetic Retinopathy

Diabetic retinopathy (DR) is the leading cause of blindness in working-age adults. Early stage DR involves inflammation, vascular leakage, apoptosis of vascular cells and neurodegeneration. In this study, we hypothesized that cells derived from the stromal fraction of adipose tissue (ASC) could therapeutically rescue early stage DR features. Streptozotocin (STZ) induced diabetic athymic nude rats received single intravitreal injection of human ASC into one eye and saline into the other eye. Two months post onset of diabetes, administration of ASC significantly improved “b” wave amplitude (as measured by electroretinogram) within 1–3 weeks of injection compared to saline treated diabetic eyes. Subsequently, retinal histopathological evaluation revealed a significant decrease in vascular leakage and apoptotic cells around the retinal vessels in the diabetic eyes that received ASC compared to the eyes that received saline injection. In addition, molecular analyses have shown down-regulation in inflammatory gene expression in diabetic retina that received ASC compared to eyes that received saline. Interestingly, ASC were found to be localized near retinal vessels at higher densities than seen in age matched non-diabetic retina that received ASC. In vitro, ASC displayed sustained proliferation and decreased apoptosis under hyperglycemic stress. In addition, ASC in co-culture with retinal endothelial cells enhance endothelial survival and collaborate to form vascular networks. Taken together, our findings suggest that ASC are able to rescue the neural retina from hyperglycemia-induced degeneration, resulting in importantly improved visual function. Our pre-clinical studies support the translational development of adipose stem cell-based therapy for DR to address both retinal capillary and neurodegeneration.     

Inhibition of protein kinase C decreases prostaglandin-induced breakdown of the blood-retinal barrier

Breakdown of the blood-retinal barrier (BRB) occurs in several retinal diseases and is a major cause of visual loss. Vascular endothelial growth factor (VEGF) has been implicated as a cause of BRB breakdown in diabetic retinopathy and other ischemic retinopathies, and there is evidence to suggest that other vasopermeability factors may act indirectly through VEGF. In this study, we investigated the effect of several receptor kinase inhibitors on BRB breakdown resulting from VEGF, tumor necrosis factor-alpha (TNF-alpha), interleukin-1beta (IL-1beta), insulin-like growth factor-1 (IGF-1), prostaglandin E1 (PGE(1)), or PGE(2). Inhibitors of VEGF receptor kinase, including PKC412, PTK787, and SU1498, decreased VEGF-induced breakdown of the BRB. None of the inhibitors blocked leakage caused by TNF-alpha, IL-1beta, or IGF-1 and only PKC412, an inhibitor of protein kinase C (PKC) as well as VEGF and platelet-derived growth factor (PDGF) receptor kinases, decreased leakage caused by prostaglandins. Since the other inhibitors of VEGF and/or PDGF receptor kinases that do not also inhibit PKC had no effect on prostaglandin-induced breakdown of the BRB, these data implicate PKC in retinal vascular leakage caused by prostaglandins. PKC412 may be useful for treatment of post-operative and inflammatory macular edema, in which prostaglandins play a role, as well as macular edema associated with ischemic retinopathies.     

Immunohistochemical localization of blood-retinal barrier breakdown sites associated with post-surgical macular oedema

Summary Post-surgical macular oedema results from blood-retinal barrier breakdown, but it is not accompanied by structural abnormalities in the retinal vessels or retinal pigmented epithelium. Previous studies, using horseradish peroxidase in a primate model, suggested that leakage occurs primarily through this epithelium. This study was conducted to localize sites of the barrier breakdown in humans following different types of intra-ocular surgery and to compare them with eyes affected with ocular inflammatory disease, ocular infection, and choroidal melanoma. Paraffin sections of eyes were immunohistochemically stained for albumin to localize extravascular albumin, which was graded in a masked study. With aphakia/pseudophakia, penetrating keratoplasty, ocular inflammatory disease, ocular infection, and choroidal melanoma, barrier breakdown occurred primarily at the inner blood-retinal barrier (retinal vasculature), but leakage also occurred at the outer barrier (retinal pigmented epithelium). After retinal re-attachment surgery, the inner and outer blood-retinal barriers were equally compromised. Vascular leakage in the optic nerve head coincided with barrier failure in these disorders. The widespread pattern of blood-retinal barrier compromise with leakage at multiple sites suggests that soluble mediators are likely to play a role in postsurgical macular oedema, ocular inflammatory disease, and choroidal melanoma.     

Neuroprotective effects of quercetin in diabetic rat retina

Diabetic retinopathy (DR) is a severe complication of diabetes and the leading cause of blindness among working adults worldwide. DR is being widely recognized as a neurodegenerative disease of the retina, since, retinal neurons are damaged soon after diabetes onset. Diabetes-induced oxidative stress is considered as central factor that dysregulates neurotrophic factors and activates apoptosis, thereby damages neurons in the diabetic retina. Flavonoids being a powerful antioxidant have been considered to protect neurons in diabetic retina. The purpose of this study was to analyze the beneficial effects of flavonoid, quercetin to protect neurons in the diabetic rat retina. We quantitated the expression levels of BDNF, NGF, TrkB, synaptophysin, Akt, Bcl-2, cytochrome c and caspase-3 using Western blotting techniques in the diabetic retina with and without quercetin treatments and compared with non-diabetic rats. In addition, we employed ELISA techniques to determine the level of BDNF. Caspase-3 activity and the level of glutathione were analyzed by biochemical methods. Our results indicate that quercetin treatment to diabetic rats caused a significant increase in the level of neurotrophic factors and inhibited the level of cytochrome c and caspase-3 activity in the diabetic retina. Furthermore, the level of an anti-apoptotic protein Bcl-2 was augmented in quercetin treated diabetic retina. Thus, quercetin, may protect the neuronal damage in diabetic retina by ameliorating the levels of neurotrophic factors and also by inhibiting the apoptosis of neurons. Therefore, this study suggests that quercetin can be a suitable therapeutic agent to prevent neurodegeneration in diabetic retinopathy.     

视网膜微血管内皮细胞在糖尿病视网膜病变中的研究现状

糖尿病视网膜疾病是导致成年人失明的主要因素,是糖尿病的一种令人恐惧的并发症,高血糖被认为是促进其发展的主要原因。高血糖不断地破坏视网膜的微血管系统最终导致视网膜的许多代谢,结构和功能的紊乱。视网膜微血管内皮细胞在微脉管系统中形成树枝状供应视网膜神经,这些内皮细胞的解剖和生理符合重要视觉保护的营养需求[1]。一方面,内皮组织务必确保氧的供应和代谢活跃的视网膜营养供应;另一方面,内皮细胞有助于血-视网膜屏障将循环产生的毒素分子,白细胞促炎性物质排出体外来保护视网膜,这种特性也可能会引起疾病,比如:视网膜血管的渗漏和新生血管,炎性物质转移,因此,视网膜内皮细胞在视网膜缺血性病变,血管炎中起到重要作用,包括糖尿病视网膜病变和视网膜炎症或感染尤其是后葡萄膜炎。使用基因表达和蛋白质组学分析等研究方法,有助于了解这些疾病的发病机制。为了进一步开展对糖尿病视网膜疾病的研究,有必要就目前有关糖尿病视网膜病变患者微血管内皮细胞的研究进展予以综述,旨在为糖尿病视网膜病变的深入研究提供参考依据。     

Directional protein secretion by the retinal pigment epithelium: roles in retinal health and the development of age‐related macular degeneration

The structural and functional integrity of the retinal pigment epithelium (RPE) is fundamental for maintaining the function of the neuroretina. These specialized cells form a polarized monolayer that acts as the retinal–blood barrier, separating two distinct environments with highly specialized functions: photoreceptors of the neuroretina at the apical side and Bruch's membrane/highly vascularized choriocapillaris at the basal side. The polarized nature of the RPE is essential for the health of these two regions, not only in nutrient and waste transport but also in the synthesis and directional secretion of proteins required in maintaining retinal homoeostasis and function. Although multiple malfunctions within the RPE cells have been associated with development of age‐related macular degeneration (AMD), the leading cause of legal blindness, clear causative processes have not yet been conclusively characterized at the molecular and cellular level. This article focuses on the involvement of directionally secreted RPE proteins in normal functioning of the retina and on the potential association of incorrect RPE protein secretion with development of AMD. Understanding the importance of RPE polarity and the correct secretion of essential structural and regulatory components emerge as critical factors for the development of novel therapeutic strategies targeting AMD.