Blood-retinal Barrier Leakage Research Articles

Pharmacological Modulation of β-Catenin Preserves Endothelial Barrier Integrity and Mitigates Retinal Vascular Permeability and Inflammation.

Compromised blood-retinal barrier (BRB) integrity is a significant factor in ocular diseases like uveitis and retinopathies, leading to pathological vascular permeability and retinal edema. Adherens and tight junction (AJ and TJ) dysregulation due to retinal inflammation plays a pivotal role in BRB disruption. We investigated the potential of ICG001, which inhibits β-catenin-mediated transcription, in stabilizing cell junctions and preventing BRB leakage. In vitro studies using human retinal endothelial cells (HRECs) showed that ICG001 treatment improved β-Catenin distribution within AJs post lipopolysaccharide (LPS) treatment and enhanced monolayer barrier resistance. The in vivo experiments involved a mouse model of LPS-induced ocular inflammation. LPS treatment resulted in increased albumin leakage from retinal vessels, elevated vascular endothelial growth factor (VEGF) and Plasmalemmal Vesicle-Associated Protein (PLVAP) expression, as well as microglia and macroglia activation. ICG001 treatment (i.p.) effectively mitigated albumin leakage, reduced VEGF and PLVAP expression, and reduced the number of activated microglia/macrophages. Furthermore, ICG001 treatment suppressed the surge in inflammatory cytokine synthesis induced by LPS. These findings highlight the potential of interventions targeting β-Catenin to enhance cell junction stability and improve compromised barrier integrity in various ocular inflammatory diseases, offering hope for better management and treatment options.

Retinal vascular abnormalities and blood-retinal barrier breakdown in Alzheimer's disease.

Vascular changes including cerebral amyloid angiopathy (CAA), reduced blood flow, pericyte loss and vascular platelet-derived growth factor receptor-β (PDGFRβ) deficiency have emerged as prominent features of Alzheimer's disease (AD). We previously identified the existence of the pathological hallmarks of AD, amyloid β-protein (Aβ) plaques, along with intracellular and vascular Aβ deposits in the neurosensory retina of AD patients. Data suggest that certain retinal signs manifest early in disease progression and appear similar to those observed in the brain. Yet, retinal vascular abnormalities in AD and their ability to predict cerebral pathology and cognitive decline are poorly understood. Here, we assessed vascular changes including capillary degeneration, PDGFRβ expression, vascular amyloidosis, pericyte loss, and blood-retinal barrier (BRB) permeability in postmortem retinas from a cohort of mild cognitively impaired (MCI) and AD patients. We evaluated retinal cross-sections from pre-defined topographical regions and isolated retinal vascular networks labeled by periodic acid-Schiff or immunofluorescence. We further quantified capillary degeneration, PDGFRβ expression, vascular amyloidosis, and inner BRB tight-junction molecule expression and BRB leakage in transgenic APPSWE /PS1ΔE9 mouse models of AD (ADtg mice) at different disease stages. Early and progressive PDGFRβ deficiency and pericyte loss were found in retinas from AD patients. PDGFRβ deficiency associated with vascular Aβ deposits in postmortem retinas from a cohort of MCI and AD patients. In ADtg mice as compared to age- and sex-matched wild-type mice, we found significant degeneration of retinal capillaries (P<0.0001). ADtg retinas also displayed a substantial vascular PDGFRβ deficiency (∼50% reduction, P=0.0017) and vascular Aβ deposition, which inversely correlated with the extent of degenerated capillaries (Pearson's r=-0.8, P=0.0016). Tight-junction alterations including claudin-1 downregulation and increased BRB permeability were found in ADtg mice. In postmortem retinas from the human cohort, retinal PDGFRβ loss significantly associated with increased retinal vascular Aβ40 and Aβ42 burdens. Decreased vascular LRP-1 and early apoptosis of pericytes in AD retina were further revealed. Correlations were identified between retinal vascular abnormalities and cerebral Aβ burden, CAA, and clinical status. Overall, the identification of age- and Alzheimer's-dependent retinal capillary degeneration and compromised BRB integrity starting at early disease stages provide novel targets for AD diagnosis and therapy.

The Herbal Combination CPA4-1 Inhibits Changes in Retinal Capillaries and Reduction of Retinal Occludin in db/db Mice.

Increased formation of advanced glycation end products (AGEs) plays an important role in the development of diabetic retinopathy (DR) via blood-retinal barrier (BRB) dysfunction, and reduction of AGEs has been suggested as a therapeutic target for DR. In this study, we examined whether CPA4-1, a herbal combination of Cinnamomi Ramulus and Paeoniae Radix, inhibits AGE formation. CPA4-1 and fenofibrate were tested to ameliorate changes in retinal capillaries and retinal occludin expression in db/db mice, a mouse model of obesity-induced type 2 diabetes. CPA4-1 (100 mg/kg) or fenofibrate (100 mg/kg) were orally administered once a day for 12 weeks. CPA4-1 (the half maximal inhibitory concentration, IC50 = 6.84 ± 0.08 μg/mL) showed approximately 11.44-fold higher inhibitory effect on AGE formation than that of aminoguanidine (AG, the inhibitor of AGEs, IC50 = 78.28 ± 4.24 μg/mL), as well as breaking effect on AGE-bovine serum albumin crosslinking with collagen (IC50 = 1.30 ± 0.37 μg/mL). CPA4-1 treatment ameliorated BRB leakage and tended to increase retinal occludin expression in db/db mice. CPA4-1 or fenofibrate treatment significantly reduced retinal acellular capillary formation in db/db mice. These findings suggested the potential of CPA4-1 as a therapeutic supplement for protection against retinal vascular permeability diseases.

Role of intravitreal ranibizumab for rapid recovery of central serous chorioretinopathy

Background: Central serous chorioretinopathy (CSCR) is an idiopathic disorder in which there is leakage of fluid from hyper permeable choriocapillaris and the collection of fluid between neurosensory and neuropigmentry retina in the macular area that is responsible for decrease visual acuity. CSCR may be acute or chronic. Various treatment options include simple observation, argon laser photocoagulation of the leaking spot, photodynamic therapy (PDT), oral ketoconazole and oral rifampicin are available. Ranibizumab is a monoclonal antibody fragment that act as vascular endothelial growth factor inhibitor, stabilize blood retinal barrier and decrease leakage from choriocapillaris. This study aims to evaluate the role of intravitreal ranibizumab for rapid recovery in central serous chorioretinopathy.&#x0D; Patients and methods: This descriptive case series was carried out at Department of Ophthalmology, Nishtar Medical University Multan, Pakistan from 01-10-2019 till 30-04-2020. The study included twelve eyes of twelve patients suffering from acute CSCR. All patients were given a single injection of intravitreal ranibizumab (0.5mg/0.05ml) as a primary treatment and followed for two months after injection at one week, one month and two months interval to document efficacy of intravitreal ranibizumab. At each baseline and follow up visits, dilated fundus examination was carried out, ending up with patients’ best corrected visual acuity. Central retinal thickness (CRT) was also recorded and results were compared with prior visit results of patients. Major outcomes were the improvement in visual acuity and decrease in CRT. Baseline CRT values were also compared with post injection CRT values at one week, one month and two months intervals using paired sample t-test and best corrected visual acuity (BCVA) was compared using chi–square test.&#x0D; Results: Mean age of the patients was 39.6 years with a male to female ratio of 9:1. Best corrected visual acuity was 6/60 on Snellen chart at baseline. All patients exhibited mean improvement of best corrected visual acuity of three Snellen lines after one week. Eleven patients were back to best corrected visual acuity of 6/6 after one month. Remaining one patient gained best corrected visual acuity of 6/6 after two months of post injection. The mean CRT at presentation was 500 ± 80U (range; 386–580) which reduced significantly to 272 ± 52 U (range 220–338) from baseline after one month showing significant reduction (p&lt;0.001). At the last follow-up visit, the CRT was measured 230 ± 20 U (range 220—250) which shows complete resolution of sub-retinal fluid.&#x0D; Conclusion: Intravitreal ranibizumab can be used for rapid absorption of sub-retinal fluid in acute CSCR and significant reduction in CRT along with improvement in BCVA indicate that it may be safely employed in CSCR to achieve better clinical outcomes.

Pathophysiology of leakage and the special role of vascular abnormalities in DME

AbstractBreakdown of the inner endothelial blood‐retinal barrier (BRB) in diabetic retinopathy and retinal vein occlusions results in vasogenic edema and neural tissue damage, causing loss of vision. The understanding of the complex cellular and molecular processes involved in BRB leakage has grown rapidly in recent years. Altered BRB function is caused by a change in the permeability characteristics of retinal endothelial cells induced by growth factors such as vascular endothelial growth factor (VEGF) and cytokines. This leads to increased paracellular and transcellular transport across the retinal vascular wall via opening of endothelial intercellular junctions and qualitative and quantitative changes in endothelial caveolar transcellular transport, respectively. Functional changes in pericytes and astrocytes, as well as structural changes in the composition of the endothelial glycocalyx and the basal lamina around BRB endothelium further facilitate BRB leakage. As Starling's rules apply, active transcellular transport of plasma proteins by the BRB endothelial cells causing increased interstitial osmotic pressure is probably the main factor in the formation of macular edema. In addition to these active, regulated and reversible mechanisms of leakage, long standing retinal vascular disease such as diabetic macular edema and retinal vein occlusion may also lead to the formation of vascular abnormalities in the form of telangiectatic capillaries (TelCaps). These are characterized by chronic uncontrolled leakage associated with formation of hard exudates, which may be independent of VEGF and require other forms of treatment such as laser photocoagulation instead of VEGF inhibitors or corticosteroids.

Increased serum proteins in non-exudative AMD retinas

The blood retinal barrier (BRB) closely regulates the retinal microenvironment. Its compromise leads to the accumulation of retinal fluid containing potentially harmful plasma components. While eyes with non-exudative age-related macular degeneration (AMD) were previously felt to have an intact BRB, we propose that the BRB in non-exudative AMD eyes may be subclinically compromised, allowing entry of retina-toxic plasma proteins. We test this hypothesis by measuring retinal levels of abundant plasma proteins that should not cross the intact BRB. Two cohorts of frozen, post mortem neurosensory retinas were studied by Western analysis. One cohort from Alabama had 4 normal controls and 4 eyes with various forms of AMD. Another cohort from Minnesota had 5 intermediate AMD eyes and 5 normals. Both cohorts were age/post mortem interval (PMI) matched. The non-exudative AMD retinas in the Alabama cohort had significantly higher levels of albumin and complement component 9 (C9) than normal controls. The positive control exudative AMD donor retina had higher levels of all but one serum protein. In both macular and peripheral neurosensory retina samples, intermediate AMD retinas in the Minnesota cohort had significantly higher levels of albumin, fibrinogen, IgG, and C9 than controls. Our results suggest that there may be moderate subclinical BRB leakage in non-exudative AMD. Potentially harmful plasma components including complement or iron could enter the neurosensory retina in AMD patients prior to advanced disease. Thus, therapies aiming to stabilize the BRB might have a role in the management of non-exudative AMD.

C1q/TNF-related protein-9 attenuates retinal inflammation and protects blood–retinal barrier in db/db mice

C1q/TNF-related protein-9(CTRP9) is an adipose cytokine, a closest adiponectin paralog, which has anti-inflammatory, antioxidant, vasodilation and anti-atherosclerosis effects. In addition, it can increase insulin sensitivity, decrease blood glucose level and inhibit the apoptosis of endothelial cells. However, it remains unclear whether CTRP9 has beneficial effects on diabetic retinopathy (DR). An adenoviral vector expressing CTRP9 was intravenously injected into db/db mice, aged 12 weeks, at day 15 post injection, and the process was repeated. The transfection efficiency of CTRP9 was assessed by enzyme linked immunosorbent assay. We used RT-PCR, immunofluorescence, and Western blot to determine proinflammatory cytokines, adhesion molecules and tight-junction proteins. The breakdown of blood–retinal barrier (BRB) was evaluated using Evans blue and retinal staining. CTRP9 suppresses the expression of interleukin-1 beta, tumor necrosis factor-alpha, monocyte chemotactic protein-1 and adhesion molecules in the retina of db/db mice. CTRP9 can balance the expression of pigment epithelium-derived factor and vascular endothelial growth factor. CTRP9 can also inhibit the activation of nuclear factor Kappa B in the retina of db/db mouse. In addition, CTRP9 can prevent the breakdown of BRB and downregulation of tight-junction proteins in the retina of db/db mice. Evans blue assay revealed the breakdown of BRB and vascular leakage in the retinas of diabetic mice. CTRP9 can both qualitatively and quantitatively alleviate the vascular leakage in the early stage of diabetic retinas. CTRP9 can inhibit the inflammation of diabetic retinopathy and protect blood–retinal barrier via decreasing proinflammatory cytokines and preventing the downregulation of tight-junction proteins.

Ginseng Extract Modified by Pectin Lyase Inhibits Retinal Vascular Injury and Blood-Retinal Barrier Breakage in a Rat Model of Diabetes

GS-E3D is an enzymatically modified ginseng extract by pectin lyase. In this study, we evaluated the preventive effects of GS-E3D on blood-retinal barrier (BRB) leakage in a rat model of diabetes. To produce diabetes, rats were injected with streptozotocin. GS-E3D was orally gavaged at 25, 50, and 100 mg/kg body weight for 6 weeks. We then compared the effect of GS-E3D with that of an unmodified ginseng extract (UGE) on retinal vascular leakage. The administration of GS-E3D significantly blocked diabetes-induced BRB breakdown. Immunofluorescence staining showed that GS-E3D reduced the loss of occludin in diabetic rats. In TUNEL staining, the number of apoptotic retinal microvascular cells was dose dependently decreased by GS-E3D treatment. GS-E3D decreased the accumulations of advanced glycation end products in the retinal vessels. In addition, the inhibition potential of GS-E3D on BRB breakage was stronger compared with UGE. These results indicate that GS-E3D could be a beneficial treatment option for preventing diabetes-induced retinal vascular injury.

Ethanol Extract of Chinese Propolis Attenuates Early Diabetic Retinopathy by Protecting the Blood-Retinal Barrier in Streptozotocin-Induced Diabetic Rats.

Propolis has been shown to reduce the level of blood glucose and suppress the histopathological changes in diabetics. However, it still remains unknown if propolis has a similar effect on diabetic retinopathy (DR). Our study aimed to evaluate the effect of the ethanol extract of Chinese propolis (EECP) on early DR in streptozotocin (STZ)-induced diabetic rats. EECP was given to diabetic rats by oral intubation for 12 weeks. The concentrations of fasting blood glucose (FBG), glycated hemoglobin (HbA1c), malondialdehyde (MDA), reactive oxygen species (ROS), and reactive nitrogen species (RNS) were measured. Pathological examinations, including hematoxylin and eosin (HE) staining, transmission electron microscopy (TEM), and immunofluorescence, were also conducted to provide further evidence of EECP's effect on early DR. EECP was able to attenuate diabetes via directly decreasing the levels of FBG and HbA1c, which also resulted in the reduction of MDA, ROS, and RNS. Furthermore, EECP could protect against the damages of photoreceptor cells, as well as retinal thickening. And the inhibition of blood-retinal barrier (BRB) leakage was also observed in EECP-treated diabetic rats, along with the inhibition the loss of tight junction proteins (occludin, ZO-1). These results suggest that EECP has an ameliorating effect on early DR by inhibition of blood-retinal barrier breakdown. PRACTICAL APPLICATION: This study sheds light on the protective effect of the ethanol extract of Chinese propolis on early diabetic retinopathy and the molecular actions underlying the inhibition of blood-retinal barrier breakdown. Our study suggests that ethanol extract of Chinese propolis can be considered as a potential therapeutic agent in the treatment of early diabetic retinopathy.

Occipital blood-brain barrier permeability is an independent predictor of visual outcome in type 2 diabetes, irrespective of the retinal barrier: A longitudinal study.

Blood-brain barrier (BBB) permeability in type 2 diabetic patients has been previously shown to be altered in certain brain regions such as the basal ganglia and the hippocampus. Because of the histological and functional similarities between the BBB) and the blood-retinal barrier (BRB), we aimed to investigate how the permeability of both barriers predicts visual outcome. We included 2 control groups (acute unilateral stroke patients, n=9; type 2 diabetics without BRB leakage n=10) and a case study group of type 2 diabetics with established BRB leakage (n=17). We evaluated sex, age, disease duration, metabolic impairment, retinopathy grade and BBB permeability as predictors of visual acuity at baseline, 12 and 24months in the type 2 diabetics without BRB leakage group and the case study group. We have also explored differences in BBB permeability in the occipital lobe and frontal lobe in the 3 different groups. Ktrans (volume transfer coefficient) and Vp (fractional plasma volume) were estimated. The BBB permeability parameter Vp was higher in the case study group compared to the unaffected hemisphere of the stroke patient control group, suggesting vascular dynamics were changed in the occipital lobe of type 2 diabetics with established BRB leakage. These patients showed a significant correlation between glycated hemoglobin (HbA1C) levels and occipital and frontal Ktrans . We report for the first time that occipital BBB permeability is an independent predictor of visual acuity at baseline, as well as at 12and 24months, in type 2 diabetics with established BRB leakage. Our results suggest that occipital BBB permeability might be an independent biomarker for visual impairment in patients with established BRB leakage.

Conditional Müller Cell Ablation Leads to Retinal Iron Accumulation.

PurposeRetinal iron accumulation is observed in a wide range of retinal degenerative diseases, including AMD. Previous work suggests that Müller glial cells may be important mediators of retinal iron transport, distribution, and regulation. A transgenic model of Müller cell loss recently demonstrated that primary Müller cell ablation leads to blood–retinal barrier leakage and photoreceptor degeneration, and it recapitulates clinical features observed in macular telangiectasia type 2 (MacTel2), a rare human disease that features Müller cell loss. We used this mouse model to determine the effect of Müller cell loss on retinal iron homeostasis.MethodsChanges in total retinal iron levels after Müller cell ablation were measured using inductively coupled plasma mass spectrometry. Corresponding changes in the expression of iron flux and iron storage proteins were determined using quantitative PCR, Western analysis, and immunohistochemistry.ResultsMüller cell loss led to blood–retinal barrier breakdown and increased iron levels throughout the neurosensory retina. There were corresponding changes in mRNA and/or protein levels of ferritin, transferrin receptor, ferroportin, Zip8, and Zip14. There were also increased iron levels within the RPE of retinal sections from a patient with MacTel2 and both RPE and neurosensory retina of a patient with diabetic retinopathy, which, like MacTel2, causes retinal vascular leakage.ConclusionThis study shows that Müller cells and the blood–retinal barrier play pivotal roles in the regulation of retinal iron homeostasis. The retinal iron accumulation resulting from blood–retinal barrier dysfunction may contribute to retinal degeneration in this model and in diseases such as MacTel2 and diabetic retinopathy.

The Urokinase Receptor-Derived Peptide UPARANT Recovers Dysfunctional Electroretinogram and Blood-Retinal Barrier Leakage in a Rat Model of Diabetes.

The activation of the urokinase-type plasminogen activator and its receptor system is associated with retinal diseases. Among peptide inhibitors of this system, UPARANT acts by preventing the onset of pathologic signs of neovascular ocular diseases. We investigated whether systemic UPARANT may act in a therapeutic regimen by suppressing the retinal damage that characterizes diabetic retinopathy using a rat model of streptozotocin-induced diabetes. In healthy rats, plasma, eye, and retina concentrations of UPARANT were evaluated by mass spectrometry. In rat models of streptozotocin-induced diabetes, the appearance of diabetic retinopathy was assessed by electroretinogram (ERG). UPARANT was then administered at different dosages and daily regimens. ERG recording, Evans blue perfusion, and real-time PCR were used to evaluate UPARANT efficacy. UPARANT safety was also determined. UPARANT was found in plasma, eye, and retina soon after its administration and remained detectable after 24 hours. Between the 4th and the 5th week after diabetes onset, UPARANT at 8 mg/kg (daily for 5 days) was effective in recovering dysfunctional ERG. Three-day treatments at 8 mg/kg or a half dose for 5 days were ineffective. ERG recovery lasted approximately 2 weeks. ERG recovery was accompanied by restored blood-retinal barrier integrity and inhibition of inflammatory and angiogenic responses. UPARANT showed a safety profile. These data suggest that targeting the urokinase-type plasminogen activator and its receptor system by systemic UPARANT is a potential therapeutic approach for the treatment of early diabetic retinopathy, thus providing a potential alternative approach to delay disease progression in humans.

Diabetic Retinopathy in the Spontaneously Diabetic Torii Rat: Pathogenetic Mechanisms and Preventive Efficacy of Inhibiting the Urokinase-Type Plasminogen Activator Receptor System.

The spontaneously diabetic Torii (SDT) rat is of increasing preclinical interest because of its similarities to human type 2 diabetic retinopathy (DR). The system formed by urokinase-type plasminogen activator (uPA) and its receptor (uPAR) is a player in blood-retinal barrier (BRB) breakdown in DR. Here, we investigated whether in SDT rats, preventive administration of UPARANT, an inhibitor of the uPAR pathway, counteracts the retinal impairment in response to chronic hyperglycemia. Electroretinogram (ERG) monitoring was followed over time. Fluorescein-dextran microscopy, CD31 immunohistochemistry, quantitative PCR, ELISA, Evans blue perfusion, and Western blot were also used. UPARANT prevented ERG dysfunction, upregulation of vascular endothelial growth factor and fibroblast growth factor-2, BRB leakage, gliosis, and retinal cell death. The mechanisms underlying UPARANT benefits were studied comparing them with the acute streptozotocin (STZ) model in which UPARANT is known to inhibit DR signs. In SDT rats, but not in the STZ model, UPARANT downregulated the expression of uPAR and its membrane partners. In both models, UPARANT reduced the levels of transcription factors coupled to inflammation or inflammatory factors themselves. These findings may help to establish the uPAR system as putative target for the development of novel drugs that may prevent type 2 DR.

Alpha-mangostin attenuation of hyperglycemia-induced ocular hypoperfusion and blood retinal barrier leakage in the early stage of type 2 diabetes rats.

The present study examined effects of alpha-mangostin (α-MG) supplementation on the retinal microvasculature, including ocular blood flow (OBF) and blood-retinal barrier (BRB) permeability in a type 2 diabetic animal model. Male Sprague-Dawley rats were divided into four groups: normal control and diabetes with or without α-MG supplementation. Alpha-mangostin (200 mg/Kg/day) was administered by gavage feeding for 8 weeks. The effects of α-MG on biochemical and physiological parameters including mean arterial pressure (MAP), OBF, and BRB leakage were investigated. Additionally, levels of retinal malondialdehyde (MDA), advance glycation end products (AGEs), receptor of advance glycation end products (RAGE), tumour necrosis factor alpha (TNF-α), and vascular endothelial growth factor (VEGF) were evaluated. The elevated blood glucose, HbA1c, cholesterol, triglyceride, serum insulin, and HOMA-IR were observed in DM2 rats. Moreover, DM2 rats had significantly decreased OBF but statistically increased MAP and leakage of the BRB. The α-MG-treated DM2 rats showed significantly lower levels of retinal MDA, AGEs, RAGE, TNF-α, and VEGF than the untreated group. Interestingly, α-MG supplementation significantly increased OBF while it decreased MAP and leakage of BRB. In conclusion, α-MG supplementation could restore OBF and improve the BRB integrity, indicating its properties closely associated with antihyperglycemic, antioxidant, anti-inflammatory, and antiglycation activities.

Molecular basis of the inner blood-retinal barrier and its breakdown in diabetic macular edema and other pathological conditions

Breakdown of the inner endothelial blood-retinal barrier (BRB), as occurs in diabetic retinopathy, age-related macular degeneration, retinal vein occlusions, uveitis and other chronic retinal diseases, results in vasogenic edema and neural tissue damage, causing loss of vision. The central mechanism of altered BRB function is a change in the permeability characteristics of retinal endothelial cells caused by elevated levels of growth factors, cytokines, advanced glycation end products, inflammation, hyperglycemia and loss of pericytes. Subsequently, paracellular but also transcellular transport across the retinal vascular wall increases via opening of endothelial intercellular junctions and qualitative and quantitative changes in endothelial caveolar transcellular transport, respectively. Functional changes in pericytes and astrocytes, as well as structural changes in the composition of the endothelial glycocalyx and the basal lamina around BRB endothelium further facilitate BRB leakage. As Starling's rules apply, active transcellular transport of plasma proteins by the BRB endothelial cells causing increased interstitial osmotic pressure is probably the main factor in the formation of macular edema. The understanding of the complex cellular and molecular processes involved in BRB leakage has grown rapidly in recent years. Although appropriate animal models for human conditions like diabetic macular edema are lacking, these insights have provided tools for rational design of drugs aimed at restoring the BRB as well as for design of effective transport of drugs across the BRB, to treat the chronic retinal diseases such as diabetic macular edema that affect the quality-of-life of millions of patients.

Thioredoxin Interacting Protein (TXNIP) and Pathogenesis of Diabetic Retinopathy

Chronic hyperglycemia (HG)-associated reactive oxygen/nitrogen species (ROS/RNS) stress and low grade inflammation are considered to play critical roles in the development of diabetic retinopathy (DR). Excess glucose metabolic flux through the aldose reductase/polyol pathway, advanced glycation end product (AGE) formation, elevated hexosamine biosynthesis pathway (HBP), diacyl glycerol/PKC activation, and mitochondrial ROS generation are all implicated in DR. In addition, endoplasmic reticulum stress/unfolded protein response (er-UPR) and deregulation of mitochondrial quality control by autophagy/mitophagy are observed causing cellular bioenergetic deficiency and injury. Recently, a pro-oxidant and pro-apoptotic thioredoxin interacting protein (TXNIP) was shown to be highly upregulated in DR and by HG in retinal cells in culture. TXNIP binds to thioredoxin (Trx) inhibiting its oxidant scavenging and thiolreducing capacity. Hence, prolonged overexpression of TXNIP causes ROS/RNS stress, mitochondrial dysfunction, inflammation and premature cell death in DR. Initially, DR was considered as microvascular complications of endothelial dysfunction and pericyte loss characterized by capillary basement membrane thickening, pericyte ghost, blood retinal barrier leakage, acellular capillary and neovascularization. However, it is currently acknowledged that neuro-glia are also affected by HG in diabetes and that neuronal injury, glial activation, innate immunity/sterile inflammation, and ganglion apoptosis occur early in DR. In addition, retinal pigment epithelium (RPE) becomes dysfunctional in DR. Since TXNIP is induced by HG in most cells, its effects are not restricted to a particular cell type in DR. However, depending on the metabolic activity and anti-oxidant capacity, some cells may be affected earlier by TXNIP than others. Identification of TXNIP sensitive cells and elucidating the underlying mechanism(s) will be critical for preventing pre-mature cell death and progression of DR.

Reply: microcystic macular degeneration from optic neuropathy and Reply: microcystic macular oedema confirmed, but not specific for multiple sclerosis

Sir, we appreciate the thoughtful comments submitted in response to our recent article describing a new retinal phenotype (microcystic macular oedema) in a subset of patients with multiple sclerosis (Gelfand et al. , 2012). We thank Balk et al. and Abegg et al. for sharing similar observations of microcystic abnormalities of the inner nuclear layer in patients with optic nerve injury in the absence of multiple sclerosis. Clarification of the specificity of microcystic macular oedema will contribute towards understanding its aetiology, its relationship to optic neuropathy and its significance in multiple sclerosis. As discussed in our article, we considered two separate, but not mutually exclusive, mechanisms that could contribute to the development of microcystic macular oedema in multiple sclerosis: retinal inflammation with associated blood-retinal barrier leakage and/or trans-synaptic degeneration. We appreciate the proposition by Balk et al. that distortion or loss of Mueller cells in the inner nuclear layer could be an additional contributory mechanism. This warrants more detailed investigation in future studies. We agree that microcystic changes may occur strictly as a consequence of trans-synaptic degeneration; however, the individual cases highlighted by Balk et al. and Abegg et al. should be interpreted with caution. Balk et al. note an example of microcystic inner nuclear layer changes …

Methylglyoxal induces hyperpermeability of the blood–retinal barrier via the loss of tight junction proteins and the activation of matrix metalloproteinases

One of the early signs of diabetic retinopathy is the alteration of the blood-retinal barrier (BRB), which may involve the breakdown of endothelial cell tight junctions. Methylglyoxal (MGO) is a cytotoxic metabolite that is produced from glycolysis in vivo. Elevated levels of MGO are observed in a number of pathological conditions, including neurodegenerative disorders and diabetic complications. Herein, we hypothesize that increased levels of MGO disrupt the tight junction protein known as occludin protein by matrix metalloproteinases (MMPs), leading to breakage of the BRB. MGO was intravitreally injected into eyes of rats. BRB leakage, MMPs activity, and occludin were investigated in intravitreally MGO-injected eyes. When normoglycemic rats were intravitreally injected with 400μM MGO, there was widespread leakage of fluorescein isothiocyanate-bovine serum albumin (FITC-BSA) from the retinal vasculature when compared to control retinas. In addition, MGO-injected retinas demonstrated increases of both activity and expression of MMP-2 and MMP-9, and the degradation of occludin was found in the MGO-injected retinas. The results suggest that the activation of MMPs by elevated levels of MGO in the retina may facilitate an increase in vascular permeability by a mechanism involving proteolytic degradation of occludin. These findings may have implications for the role of MGO in the pathogenesis of diabetic retinopathy.

GSK-3β/CREB axis mediates IGF-1-induced ECM/adhesion molecule expression, cell cycle progression and monolayer permeability in retinal capillary endothelial cells: Implications for diabetic retinopathy

Various growth factors and cytokines are implicated in endothelial dysfunction and blood–retinal barrier (BRB) breakdown in early diabetic retinopathy (DR). However, cellular and molecular mechanisms that may underlie the pathology of DR are not fully understood yet. We therefore examined the effect of insulin-like growth factor (IGF)-1 on ECM/adhesion molecule expression, cell cycle regulation and monolayer permeability in an endothelial cell line (TR-iBRB2). We investigate whether the action of IGF-1 (1) involves glycogen synthase kinase 3beta (GSK-3β) and cAMP responsive transcription factor (CREB) and (2) alters ECM/adhesion molecule gene expression. Treatment of TR-iBRB2 cell with IGF-1 (100 ng/ml for 0–24 h) increases phosphorylation of (i) Akt Thr308, and its substrates including GSK-3β at Ser9, which inactivates its kinase function, and (ii) CREB at Ser133 (activation). These phosphorylations correlate positively with enhanced expression of CREB targets such as ECM protein fibronectin and cell cycle progression factor cyclin D1. However, stable transfection of a mutant GSK3β(S9A) or a dominant negative K-CREB in TR-iBRB2 prevents IGF-1-induced fibronectin and cyclin D1 expression. Furthermore, IGF-1 reduces the level of intercellular adherence molecule VE-cadherin and increases monolayer permeability in TR-iBRB2 cells when measured by FITC-dextran leakage. The effect of IGF-1 on VE-cadherin and membrane permeability is absent in TR-iBRB2 cells expressing the GSK-3β(S9A). Similarly, K-CREB reverses IGF-1 down-regulation of VE-cadherin and up-regulation of fibronectin. These results indicate that GSK-3β/CREB axis alters ECM/adhesion molecule expression and cell cycle progression in retinal endothelial cells, and may potentially contribute to endothelial dysfunction and BRB leakage in DR.

Photoreceptor degeneration changes magnetic resonance imaging features in a mouse model of retinitis pigmentosa

Retinal degeneration-1 (rd1) mice are animal models of retinitis pigmentosa, a blinding disease caused by photoreceptor cell degeneration. This study aims to determine magnetic resonance imaging (MRI) changes in retinas of 1- and 3-month-old rd1 mice. Apparent diffusion coefficient in retina was measured using diffusion MRI. The blood-retinal barrier leakage was evaluated using gadolinium-diethylenetriaminepentaacetic acid-enhanced T(1)-weighted MRI before and after systemic gadolinium-diethylenetriaminepentaacetic acid injection. Photoreceptor degeneration in rd1 retina was apparent by decreased retinal thickness and loss of water diffusion anisotropy in both 1- and 3-month-old rd1 mice. Furthermore, statistically significant increase of mean retinal apparent diffusion coefficient and gadolinium-diethylenetriaminepentaacetic acid-enhanced T(1)-weighted MRI signals were observed in 3-month-old rd1 mice comparing with age-matched wild-type mice. Together, these data suggest that MRI parameter changes can signature common pathological changes in photoreceptor-degenerated eyes, particularly blood-retinal barrier leakage-induced retinal edema.