EphB1 causes retinal damage through inflammatory pathways in the retina and retinal Müller cells.

Purpose: To examine whether increased ephrin type-B receptor 1 (EphB1) leads to inflammatory mediators in retinal Müller cells. Methods: Diabetic human and mouse retinal samples were examined for EphB1 protein levels. Rat Müller cells (rMC-1) were grown in culture and treated with EphB1 siRNA or ephrin B1-Fc to explore inflammatory mediators in cells grown in high glucose. An EphB1 overexpression adeno-associated virus (AAV) was used to increase EphB1 in Müller cells in vivo. Ischemia/reperfusion (I/R) was performed on mice treated with the EphB1 overexpression AAV to explore the actions of EphB1 on retinal neuronal changes in vivo. Results: EphB1 protein levels were increased in diabetic human and mouse retinal samples. Knockdown of EphB1 reduced inflammatory mediator levels in Müller cells grown in high glucose. Ephrin B1-Fc increased inflammatory proteins in rMC-1 cells grown in normal and high glucose. Treatment of mice with I/R caused retinal thinning and loss of cell numbers in the ganglion cell layer. This was increased in mice exposed to I/R and treated with the EphB1 overexpressing AAVs. Conclusions: EphB1 is increased in the retinas of diabetic humans and mice and in high glucose-treated Müller cells. This increase leads to inflammatory proteins. EphB1 also enhanced retinal damage in response to I/R. Taken together, inhibition of EphB1 may offer a new therapeutic option for diabetic retinopathy.

Bone Morphogenetic Protein-4 Impairs Retinal Endothelial Cell Barrier, a Potential Role in Diabetic Retinopathy.

Bone Morphogenetic Protein 4 (BMP4) is a secreted growth factor of the Transforming Growth Factor beta (TGFß) superfamily. The goal of this study was to test whether BMP4 contributes to the pathogenesis of diabetic retinopathy (DR). Immunofluorescence of BMP4 and the vascular marker isolectin-B4 was conducted on retinal sections of diabetic and non-diabetic human and experimental mice. We used Akita mice as a model for type-1 diabetes. Proteins were extracted from the retina of postmortem human eyes and 6-month diabetic Akita mice and age-matched control. BMP4 levels were measured by Western blot (WB). Human retinal endothelial cells (HRECs) were used as an in vitro model. HRECs were treated with BMP4 (50 ng/mL) for 48 h. The levels of phospho-smad 1/5/9 and phospho-p38 were measured by WB. BMP4-treated and control HRECs were also immunostained with anti-Zo-1. We also used electric cell-substrate impedance sensing (ECIS) to calculate the transcellular electrical resistance (TER) under BMP4 treatment in the presence and absence of noggin (200 ng/mL), LDN193189 (200 nM), LDN212854 (200 nM) or inhibitors of vascular endothelial growth factor receptor 2 (VEGFR2; SU5416, 10 µM), p38 (SB202190, 10 µM), ERK (U0126, 10 µM) and ER stress (Phenylbutyric acid or PBA, 30 µmol/L). The impact of BMP4 on matrix metalloproteinases (MMP2 and MMP9) was also evaluated using specific ELISA kits. Immunofluorescence of human and mouse eyes showed increased BMP4 immunoreactivity, mainly localized in the retinal vessels of diabetic humans and mice compared to the control. Western blots of retinal proteins showed a significant increase in BMP4 expression in diabetic humans and mice compared to the control groups (p < 0.05). HRECs treated with BMP4 showed a marked increase in phospho-smad 1/5/9 (p = 0.039) and phospho-p38 (p = 0.013). Immunofluorescence of Zo-1 showed that BMP4-treated cells exhibited significant barrier disruption. ECIS also showed a marked decrease in TER of HRECs by BMP4 treatment compared to vehicle-treated HRECs (p < 0.001). Noggin, LDN193189, LDN212854, and inhibitors of p38 and VEGFR2 significantly mitigated the effects of BMP4 on the TER of HRECs. Our finding provides important insights regarding the role of BMP4 as a potential player in retinal endothelial cell dysfunction in diabetic retinopathy and could be a novel target to preserve the blood-retinal barrier during diabetes.

Hyperhomocysteinemia dysregulates plasma levels of polyunsaturated fatty acids-derived eicosanoids.

Hyperhomocysteinemia (HHcy) contributes to the incidence of many cardiovascular diseases (CVD). Our group have previously established crucial roles of eicosanoids and homocysteine in the incidence of vascular injury in diabetic retinopathy and renal injury. Using cystathionine-ß-synthase heterozygous mice (cßs+/-) as a model of HHcy, the current study was designed to determine the impact of homocysteine on circulating levels of lipid mediators derived from polyunsaturated fatty acids (PUFA). Plasma samples were isolated from wild-type (WT) and cßs+/- mice for the assessment of eicosanoids levels using LC/MS. Plasma 12/15-lipoxygenase (12/15-LOX) activity significantly decreased in cßs+/- vs. WT control mice. LOX-derived metabolites from both omega-3 and omega-6 PUFA were also reduced in cßs+/- mice compared to WT control (P < 0.05). Contrary to LOX metabolites, cytochrome P450 (CYP) metabolites from omega-3 and omega-6 PUFA were significantly elevated in cßs+/- mice compared to WT control. Epoxyeicosatrienoic acids (EETs) are epoxides derived from arachidonic acid (AA) metabolism by CYP with anti-inflammatory properties and are known to limit vascular injury, however their physiological role is limited by their rapid degradation by soluble epoxide hydrolase (sEH) to their corresponding diols (DiHETrEs). In cßs+/- mice, a significant decrease in the plasma EETs bioavailability was obvious as evident by the decrease in EETs/ DiHETrEs ratio relative to WT control mice. Cyclooxygenase (COX) metabolites were also significantly decreased in cßs+/- vs. WT control mice. These data suggest that HHcy impacts eicosanoids metabolism through decreasing LOX and COX metabolic activities while increasing CYP metabolic activity. The increase in AA metabolism by CYP was also associated with increase in sEH activity and decrease in EETs bioavailability. Dysregulation of eicosanoids metabolism could be a contributing factor to the incidence and progression of HHcy-induced CVD.

Implication of N-Methyl-d-Aspartate Receptor in Homocysteine-Induced Age-Related Macular Degeneration.

Age-related macular degeneration (AMD) is a leading cause of vision loss. Elevated homocysteine (Hcy) (Hyperhomocysteinemia) (HHcy) has been reported in AMD. We previously reported that HHcy induces AMD-like features. This study suggests that N-Methyl-d-aspartate receptor (NMDAR) activation in the retinal pigment epithelium (RPE) is a mechanism for HHcy-induced AMD. Serum Hcy and cystathionine-ß-synthase (CBS) were assessed by ELISA. The involvement of NMDAR in Hcy-induced AMD features was evaluated (1) in vitro using ARPE-19 cells, primary RPE isolated from HHcy mice (CBS), and mouse choroidal endothelial cells (MCEC); (2) in vivo using wild-type mice and mice deficient in RPE NMDAR (NMDARR-/-) with/without Hcy injection. Isolectin-B4, Ki67, HIF-1α, VEGF, NMDAR1, and albumin were assessed by immunofluorescence (IF), Western blot (WB), Optical coherence tomography (OCT), and fluorescein angiography (FA) to evaluate retinal structure, fluorescein leakage, and choroidal neovascularization (CNV). A neovascular AMD patient's serum showed a significant increase in Hcy and a decrease in CBS. Hcy significantly increased HIF-1α, VEGF, and NMDAR in RPE cells, and Ki67 in MCEC. Hcy-injected WT mice showed disrupted retina and CNV. Knocking down RPE NMDAR improved retinal structure and CNV. Our findings underscore the role of RPE NMDAR in Hcy-induced AMD features; thus, NMDAR inhibition could serve as a promising therapeutic target for AMD.

N-Methyl-D-aspartate receptor activation, novel mechanism of homocysteine-induced blood-retinal barrier dysfunction.

Elevated levels of amino acid homocysteine (Hcy) recognized as hyperhomocysteinemia (HHcy) was reported in several human visual disorders, such as diabetic retinopathy (DR) and age-related macular degeneration (AMD). Breakdown of blood-retinal barrier (BRB) is concomitant with vision loss in DR and AMD. We previously reported that HHcy alters BRB. Here, we tested the hypothesis that HHcy alters BRB via activation of N-methyl-D-aspartate receptor (NMDAR). Human retinal endothelial cells subjected to high level of Hcy and mouse model of HHcy were used. We injected Hcy intravitreal and used a mouse model of HHcy that lacks cystathionine-ß-synthase (CBS). RT-PCR, western blot, and immunofluorescence showed that retinal endothelial cells (RECs) express NMDAR at the gene and protein levels both in vitro and in vivo and this was increased by HHcy. We assessed BRB function and retinal morphology using fluorescein angiogram and optical coherence tomography (OCT) under HHcy with and without pharmacological inhibition of NMDAR by (MK801) or in mice lacking endothelial NMDAR (NMDARE-/- mouse). Additionally, retinal albumin leakage and tight junction proteins ZO-1 and occludin were assessed by western blotting analysis. Inhibition or elimination of NMDAR was able to improve the altered retinal hyperpermeability and morphology under HHcy as indicated by significant decrease in retinal albumin leakage and restoration of tight junction proteins ZO-1 and occludin. Our findings underscore a potential role for endothelial NMDAR in mediating Hcy-induced breakdown of BRB and subsequently as a potential therapeutic target in retinal diseases associated with HHcy such as DR and AMD. KEY MESSAGES: • Elevated levels of homocysteine (Hcy) are defined as hyperhomocysteinemia (HHcy). • HHcy is implicated in diabetic retinopathy and age-related macular degeneration. • HHcy alters BRB via activation of N-methyl-D-aspartate receptor.

Homocysteine Induces Inflammation in Retina and Brain.

Homocysteine (Hcy) is an amino acid that requires vitamins B12 and folic acid for its metabolism. Vitamins B12 and folic acid deficiencies lead to hyperhomocysteinemia (HHcy, elevated Hcy), which is linked to the development of diabetic retinopathy (DR), age-related macular degeneration (AMD), and Alzheimer's disease (AD). The goal of the current study was to explore inflammation as an underlying mechanism of HHcy-induced pathology in age related diseases such as AMD, DR, and AD. Mice with HHcy due to a lack of the enzyme cystathionine-ß-synthase (CBS) and wild-type mice were evaluated for microglia activation and inflammatory markers using immuno-fluorescence (IF). Tissue lysates isolated from the brain hippocampal area from mice with HHcy were evaluated for inflammatory cytokines using the multiplex assay. Human retinal endothelial cells, retinal pigment epithelial cells, and monocyte cell lines treated with/without Hcy were evaluated for inflammatory cytokines and NFκB activation using the multiplex assay, western blot analysis, and IF. HHcy induced inflammatory responses in mouse brain, retina, cultured retinal, and microglial cells. NFκB was activated and cytokine array analysis showed marked increase in pro-inflammatory cytokines and downregulation of anti-inflammatory cytokines. Therefore, elimination of excess Hcy or reduction of inflammation is a promising intervention for mitigating damage associated with HHcy in aging diseases such as DR, AMD, and AD.

Role of Endothelial ADAM17 in Early Vascular Changes Associated with Diabetic Retinopathy.

ADAM17, a disintegrin and metalloproteinase 17, is a transmembrane metalloproteinase that regulates bioavailability of multiple membrane-bound proteins via ectodomain shedding. ADAM17 activity was shown to contribute to a number of vascular pathologies, but its role in the context of diabetic retinopathy (DR) is not determined. We found that expression and enzymatic activity of ADAM17 are upregulated in human diabetic postmortem retinas and a mouse model of streptozotocin-induced diabetes. To further investigate the contribution of ADAM17 to vascular alterations associated with DR, we used human retinal endothelial cells (HREC) treated with ADAM17 neutralizing antibodies and exposed to glucidic stress and streptozotocin-induced endothelial ADAM17 knockout mice. Evaluation of vascular permeability, vascular inflammation, and oxidative stress was performed. Loss of ADAM17 in endothelial cells markedly reduced oxidative stress evidenced by decreased levels of superoxide, 3-nitrotyrosine, and 4-hydroxynonenal and decreased leukocyte-endothelium adhesive interactions in vivo and in vitro. Reduced leukostasis was associated with decreased vascular permeability and was accompanied by downregulation of intercellular adhesion molecule-1 expression. Reduction in oxidative stress in HREC was associated with downregulation of NAD(P)H oxidase 4 (Nox4) expression. Our data suggest a role for endothelial ADAM17 in DR pathogenesis and identify ADAM17 as a potential new therapeutic target for DR.

A dual role of 12/15-lipoxygenase in LPS-induced acute renal inflammation and injury.

Recent studies suggest a potential role of bioactive lipids in acute kidney injury induced by lipopolysaccharide (LPS). The current study was designed to determine the profiling activities of various polyunsaturated fatty acid (PUFA) metabolizing enzymes, including lipoxygenases (LO), cyclooxygenase, and cytochrome P450 in the plasma of LPS-injected mice using LC-MS. Heat map analysis revealed that out of 126 bioactive lipids screened, only the 12/15-LO metabolite, 12-HETE, had a significant (2.24 ±â€¯0.4) fold increase relative to control (P = 0.0001) after Bonferroni Correction (BCF α = 0.003). We then determined the role of the 12/15-LO in LPS-induced acute kidney injury using genetic and pharmacological approaches. Treatment of LPS injected mice with the 12/15-LO inhibitor, baicalein, significantly reduced levels of renal injury and inflammation markers including urinary thiobarbituric acid reactive substance (TBARs), urinary monocyte chemoattractant protein-1 (MCP-1), renal interleukin-6 (IL-6), and tumor necrosis factor-α (TNF-α). Similarly, knocking-out of 12/15-LO reduced levels of renal inflammation and injury markers elicited by LPS injection. Next, we tested whether exogenous supplementation with docosahexaenoic acid (DHA) as a substrate would divert the role of 12/15-LO from being pro-inflammatory to anti-inflammatory via increased production of the anti-inflammatory metabolite. DHA treatment restored the decreased in plasma level of resolvin D2 (RvD2) and reduced renal injury in LPS-injected mice whereas DHA treatment failed to provide any synergistic effects in reducing renal injury in LPS injected 12/15-LO knock-out mice. The ability of RvD2 to protect kidney against LPS-induced renal injury was further confirmed by exogenous RvD2 which significantly reduced the elevation in renal injury in LPS injected mice. These data suggest a double-edged sword role of 12/15-LO in LPS-induced acute renal inflammation and injury, depending on the type of substrate available for its activity.

Inhibition of interleukin-6 trans-signaling prevents inflammation and endothelial barrier disruption in retinal endothelial cells.

Vascular inflammation plays a critical role in the pathogenesis of diabetic retinopathy. Recently, Interleukin-6 (IL-6) trans-signaling via soluble IL-6 receptor (sIL-6R) has emerged as a prominent regulator of inflammation in endothelial cells. This study was designed to test the hypothesis that selective inhibition of the IL-6 trans-signaling pathway will attenuate inflammation and subsequent barrier disruption in retinal endothelial cells. Human retinal endothelial cells (HRECs) were exposed to IL-6 and sIL-6R to induce IL-6 trans-signaling and the commercially available compound sgp130Fc (soluble gp-130 fused chimera) was used to selectively inhibit IL-6 trans-signaling. IL-6 trans-signaling activation caused a significant increase in STAT3 phosphorylation, expression of adhesion molecules, ROS production and apoptosis in HRECs whereas a significant decrease in mitochondrial membrane potential and NO production was observed in IL-6 trans-signaling activated cells. These changes were not observed in cells pre-treated with sgp130Fc. IL-6 trans-signaling activation was sufficient to cause barrier disruption in endothelial monolayers and pre-treatment of HRECs with sgp130Fc, maintained endothelial barrier function similar to that of untreated cells. Thus, in conclusion, these results indicate that IL-6 trans-signaling is an important mediator of inflammation, apoptosis and barrier disruptive effects in the retinal endothelial cells and inhibition of the IL-6 trans-signaling pathway using sgp130-Fc attenuates vascular inflammation and endothelial barrier disruption.

Epigenetic modifications in hyperhomocysteinemia: potential role in diabetic retinopathy and age-related macular degeneration.

To study Hyperhomocysteinemia (HHcy)-induced epigenetic modifications as potential mechanisms of blood retinal barrier (BRB) dysfunction, retinas isolated from three- week-old mice with elevated level of Homocysteine (Hcy) due to lack of the enzyme cystathionine ß-synthase (cbs-/- , cbs+/- and cbs+/+ ), human retinal endothelial cells (HRECs), and human retinal pigmented epithelial cells (ARPE-19) treated with or without Hcy were evaluated for (1) histone deacetylases (HDAC), (2) DNA methylation (DNMT), and (3) miRNA analysis. Differentially expressed miRNAs in mice with HHcy were further compared with miRNA analysis of diabetic mice retinas (STZ) and miRNAs within the exosomes released from Hcy-treated RPEs. Differentially expressed miRNAs were further evaluated for predicted target genes and associated pathways using Ingenuity Pathway Analysis. HHcy significantly increased HDAC and DNMT activity in HRECs, ARPE-19, and cbs mice retinas, whereas inhibition of HDAC and DNMT decreased Hcy-induced BRB dysfunction. MiRNA profiling detected 127 miRNAs in cbs+/- and 39 miRNAs in cbs-/- mice retinas, which were significantly differentially expressed compared to cbs+/+ . MiRNA pathway analysis showed their involvement in HDAC and DNMT activation, endoplasmic reticulum (ER), and oxidative stresses, inflammation, hypoxia, and angiogenesis pathways. Hcy-induced epigenetic modifications may be involved in retinopathies associated with HHcy, such as age-related macular degeneration and diabetic retinopathy.

Hyperhomocysteinemia Alters Retinal Endothelial Cells Barrier Function and Angiogenic Potential via Activation of Oxidative Stress.

Hyperhomocysteinemia (HHcy) is associated with several human visual disorders, such as diabetic retinopathy (DR) and age-related macular degeneration (AMD). Breakdown of the blood-retinal barrier (BRB) is linked to vision loss in DR and AMD. Our previous work revealed that HHcy altered BRB in retinal endothelial cells in vivo. Here we hypothesize that homocysteine (Hcy) alters retinal endothelial cell barrier function and angiogenic potential via activation of oxidative stress. Human retinal endothelial cells (HRECs) treated with and without different concentrations of Hcy showed a reduction of tight junction protein expression, increased FITC dextran leakage, decreased transcellular electrical resistance and increased angiogenic potential. In addition, HRECs treated with Hcy showed increased production of reactive oxygen species (ROS). The anti-oxidant N-acetyl-cysteine (NAC) reduced ROS formation and decreased FITC-dextran leakage in Hcy treated HRECs. A mouse model of HHcy, in which cystathionine-ß-synthase is deficient (cbs -/-), was evaluated for oxidative stress by dichlolorofluorescein (DCF), dihydroethidium (DHE) staining. There was a marked increase in ROS production and augmented GSH reductase and antioxidant regulator NRF2 activity, but decreased antioxidant gene expression in retinas of hyperhomocysteinemic mice. Our results suggest activation of oxidative stress as a possible mechanism of HHcy induced retinal endothelial cell dysfunction.

Endothelial adenosine A2a receptor-mediated glycolysis is essential for pathological retinal angiogenesis.

Adenosine/adenosine receptor-mediated signaling has been implicated in the development of various ischemic diseases, including ischemic retinopathies. Here, we show that the adenosine A2a receptor (ADORA2A) promotes hypoxia-inducible transcription factor-1 (HIF-1)-dependent endothelial cell glycolysis, which is crucial for pathological angiogenesis in proliferative retinopathies. Adora2a expression is markedly increased in the retina of mice with oxygen-induced retinopathy (OIR). Endothelial cell-specific, but not macrophage-specific Adora2a deletion decreases key glycolytic enzymes and reduces pathological neovascularization in the OIR mice. In human primary retinal microvascular endothelial cells, hypoxia induces the expression of ADORA2A by activating HIF-2α. ADORA2A knockdown decreases hypoxia-induced glycolytic enzyme expression, glycolytic flux, and endothelial cell proliferation, sprouting and tubule formation. Mechanistically, ADORA2A activation promotes the transcriptional induction of glycolytic enzymes via ERK- and Akt-dependent translational activation of HIF-1α protein. Taken together, these findings advance translation of ADORA2A as a therapeutic target in the treatment of proliferative retinopathies and other diseases dependent on pathological angiogenesis.Pathological angiogenesis in the retina is a major cause of blindness. Here the authors show that adenosine receptor A2A drives pathological angiogenesis in the oxygen-induced retinopathy mouse model by promoting glycolysis in endothelial cells via the ERK/Akt/HIF-1α pathway, thereby suggesting new therapeutic targets for disease treatment.

Targeting of 12/15-Lipoxygenase in retinal endothelial cells, but not in monocytes/macrophages, attenuates high glucose-induced retinal leukostasis.

AIMS: Our previous studies have established a role for 12/15-lipoxygenase (LO) in mediating the inflammatory response in diabetic retinopathy (DR). However, the extent at which the local or systemic induction of 12/15-LO activity involved is unclear. Thus, the current study aimed to characterize the relative contribution of retinal endothelial versus monocytic/macrophagic 12/15-LO to inflammatory responses in DR. MATERIALS & METHODS: We first generated a clustered heat map for circulating bioactive lipid metabolites in the plasma of streptozotocin (STZ)-induced diabetic mice using liquid chromatography coupled with mass-spectrometry (LC-MS) to evaluate changes in circulating 12/15-LO activity. This was followed by comparing the in vitro mouse endothelium-leukocytes interaction between leukocytes isolated from 12/15-LO knockout (KO) versus those isolated from wild type (WT) mice using the myeloperoxidase (MPO) assay. Finally, we examined the effects of knocking down or inhibiting endothelial 12/15-LO on diabetes-induced endothelial cell activation and ICAM-1 expression. RESULTS: Analysis of plasma bioactive lipids' heat map revealed that the activity of circulating 12/15-LO was not altered by diabetes as evident by no significant changes in the plasma levels of major metabolites derived from 12/15-lipoxygenation of different PUFAs, including linoleic acid (13-HODE), arachidonic acid (12- and 15- HETEs), eicosapentaenoic acid (12- and 15- HEPEs), or docosahexaenoic acid (17-HDoHE). Moreover, leukocytes from 12/15-LO KO mice displayed a similar increase in adhesion to high glucose (HG)-activated endothelial cells as do leukocytes from WT mice. Furthermore, abundant proteins of 12-LO and 15-LO were detected in human retinal endothelial cells (HRECs), while it was undetected (15-LO) or hardly detectable (12-LO) in human monocyte-like U937 cells. Inhibition or knock down of endothelial 12/15-LO in HRECs blocked HG-induced expression of ICAM-1, a well-known identified important molecule for leukocyte adhesion in DR. CONCLUSION: Our data support that endothelial, rather than monocytic/macrophagic, 12/15-LO has a critical role in hyperglycemia-induced ICAM-1 expression, leukocyte adhesion, and subsequent local retinal barrier dysfunction. This may facilitate the development of more precisely targeted treatment strategies for DR.

Adenosine Deaminase-2-Induced Hyperpermeability in Human Retinal Vascular Endothelial Cells Is Suppressed by MicroRNA-146b-3p.

Purpose: We recently demonstrated that adenosine deaminase-2 (ADA2) contributes to diabetic retinopathy (DR) via up-regulating the production of inflammatory cytokines in macrophages. Also, microRNA (miR)-146b-3p has the ability to inhibit ADA2. The goal of this study was to investigate the potential role of ADA2 and therapeutic benefit of miR-146b-3p in retinal inflammation and endothelial barrier dysfunction during diabetes. Methods: Adenosine deaminase-2 activity was determined by colorimetric method in diabetic human vitreous. Human monocyte cell line U937 was differentiated into macrophages and then treated with amadori glycated albumin (AGA), and conditioned medium (CM) was used to assess the changes in ADA2 activity and TNF-α and IL-6 levels by ELISA. Also, macrophages were transfected with miR-146b-3p before treatment with AGA. Permeability of human retinal endothelial cells (hRECs) was assessed by electric cell-substrate impedance sensing (ECIS) after treatment with macrophage CM. Zonula occludens (ZO)-1 was examined by immuno-fluorescence in hRECs. Leukocyte adhesion was assessed in hRECs by measuring myeloperoxidase (MPO) activity and intercellular adhesion molecule-1 (ICAM-1) expression. Results: Adenosine deaminase-2 activity was significantly increased in diabetic human vitreous. ADA2 activity and TNF-α and IL-6 levels were significantly increased in human macrophages by AGA treatment. Amadori glycated albumin-treated macrophage CM significantly increased hREC permeability, disrupted ZO-1 pattern, and increased leukocyte adhesion to hRECs through up-regulating ICAM-1. All these changes were reversed by miR-146b-3p. Conclusions: Adenosine deaminase-2 is implicated in breakdown of the blood-retinal barrier (BRB) in DR through macrophages-derived cytokines. Therefore, inhibition of ADA2 by miR-146b-3p might be a useful tool to preserve BRB function in DR.

Hyperhomocysteinemia disrupts retinal pigment epithelial structure and function with features of age-related macular degeneration.

The disruption of retinal pigment epithelial (RPE) function and the degeneration of photoreceptors are cardinal features of age related macular degeneration (AMD); however there are still gaps in our understanding of underlying biological processes. Excess homocysteine (Hcy) has been reported to be elevated in plasma of patients with AMD. This study aimed to evaluate the direct effect of hyperhomocysteinemia (HHcy) on structure and function of RPE. Initial studies in a mouse model of HHcy, in which cystathionine-ß-synthase (cbs) was deficient, revealed abnormal RPE cell morphology with features similar to that of AMD upon optical coherence tomography (OCT), fluorescein angiography (FA), histological, and electron microscopic examinations. These features include atrophy, vacuolization, hypopigmentation, thickened basal laminar membrane, hyporeflective lucency, choroidal neovascularization (CNV), and disturbed RPE-photoreceptor relationship. Furthermore, intravitreal injection of Hcy per se in normal wild type (WT) mice resulted in diffuse hyper-fluorescence, albumin leakage, and CNV in the area of RPE. In vitro experiments on ARPE-19 showed that Hcy dose-dependently reduced tight junction protein expression, increased FITC dextran leakage, decreased transcellular electrical resistance, and impaired phagocytic activity. Collectively, our results demonstrated unreported effects of excess Hcy levels on RPE structure and function that lead to the development of AMD-like features.

The Chemokine Platelet Factor-4 Variant (PF-4var)/CXCL4L1 Inhibits Diabetes-Induced Blood-Retinal Barrier Breakdown.

PURPOSE: To investigate the expression of platelet factor-4 variant (PF-4var/CXCL4L1) in epiretinal membranes from patients with proliferative diabetic retinopathy (PDR) and the role of PF-4var/CXCL4L1 in the regulation of blood-retinal barrier (BRB) breakdown in diabetic rat retinas and human retinal microvascular endothelial cells (HRMEC). METHODS: Rats were treated intravitreally with PF-4var/CXCL4L1 or the anti-vascular endothelial growth factor (VEGF) agent bevacizumab on the first day after diabetes induction. Blood-retinal barrier breakdown was assessed in vivo with fluorescein isothiocyanate (FITC)-conjugated dextran and in vitro in HRMEC by transendothelial electrical resistance and FITC-conjugated dextran cell permeability assay. Occludin, vascular endothelial (VE)-cadherin, hypoxia-inducible factor (HIF)-1α, VEGF, tumor necrosis factor (TNF)-α, receptor for advanced glycation end products (RAGE), caspase-3 levels, and generation of reactive oxygen species (ROS) were assessed by Western blot, enzyme-linked immunosorbent assays, or spectrophotometry. RESULTS: In epiretinal membranes, vascular endothelial cells and stromal cells expressed PF-4var/CXCL4L1. In vitro, HRMEC produced PF-4var/CXCL4L1 after stimulation with a combination of interleukin (IL)-1ß and TNF-α, and PF-4var/CXCL4L1 inhibited VEGF-mediated hyperpermeability in HRMEC. In rats, PF-4var/CXCL4L1 was as potent as bevacizumab in attenuating diabetes-induced BRB breakdown. This effect was associated with upregulation of occludin and VE-cadherin and downregulation of HIF-1α, VEGF, TNF-α, RAGE, and caspase-3, whereas ROS generation was not altered. CONCLUSIONS: Our findings suggest that increasing the intraocular PF-4var/CXCL4L1 levels early after the onset of diabetes protects against diabetes-induced BRB breakdown.

Pigment epithelium-derived factor inhibits retinal microvascular dysfunction induced by 12/15-lipoxygenase-derived eicosanoids.

We recently demonstrated that 12/15-lipoxygenase (LOX) derived metabolites, hydroxyeicosatetraenoic acids (HETEs), contribute to diabetic retinopathy (DR) via NADPH oxidase (NOX) and disruption of the balance in retinal levels of the vascular endothelial growth factor (VEGF) and pigment epithelium-derived factor (PEDF). Here, we test whether PEDF ameliorates retinal vascular injury induced by HETEs and the underlying mechanisms. Furthermore, we pursue the causal relationship between LOX-NOX system and regulation of PEDF expression during DR. For these purposes, we used an experimental eye model in which normal mice were injected intravitreally with 12-HETE with/without PEDF. Thereafter, fluorescein angiography (FA) was used to evaluate the vascular leakage, followed by optical coherence tomography (OCT) to assess the presence of angiogenesis. FA and OCT reported an increased vascular leakage and pre-retinal neovascularization, respectively, in response to 12-HETE that were not observed in the PEDF-treated group. Moreover, PEDF significantly attenuated the increased levels of vascular cell and intercellular adhesion molecules, VCAM-1 and ICAM-1, elicited by 12-HETE injection. Accordingly, the direct relationship between HETEs and PEDF has been explored through in-vitro studies using Müller cells (rMCs) and human retinal endothelial cells (HRECs). The results showed that 12- and 15-HETEs triggered the secretion of TNF-α and IL-6, as well as activation of NFκB in rMCs and significantly increased permeability and reduced zonula occludens protein-1 (ZO-1) immunoreactivity in HRECs. All these effects were prevented in PEDF-treated cells. Furthermore, interest in PEDF regulation during DR has been expanded to include NOX system. Retinal PEDF was significantly restored in diabetic mice treated with NOX inhibitor, apocynin, or lacking NOX2 up to 80% of the control level. Collectively, our findings suggest that interfering with LOX-NOX signaling opens up a new direction for treating DR by restoring endogenous PEDF that carries out multilevel vascular protective functions.

A lipidomic screen of hyperglycemia-treated HRECs links 12/15-Lipoxygenase to microvascular dysfunction during diabetic retinopathy via NADPH oxidase.

Retinal hyperpermeability and subsequent macular edema is a cardinal feature of early diabetic retinopathy (DR). Here, we investigated the role of bioactive lipid metabolites, in particular 12/15-lipoxygenase (LOX)-derived metabolites, in this process. LC/MS lipidomic screen of human retinal endothelial cells (HRECs) demonstrated that 15-HETE was the only significantly increased metabolite (2.4 ± 0.4-fold, P = 0.0004) by high glucose (30 mM) treatment. In the presence of arachidonic acid, additional eicosanoids generated by 12/15-LOX, including 12- and 11-HETEs, were significantly increased. Fluorescein angiography and retinal albumin leakage showed a significant decrease in retinal hyperpermeability in streptozotocin-induced diabetic mice lacking 12/15-LOX compared with diabetic WT mice. Our previous studies demonstrated the potential role of NADPH oxidase in mediating the permeability effect of 12- and 15-HETEs, therefore we tested the impact of intraocular injection of 12-HETE in mice lacking the catalytic subunit of NADPH oxidase (NOX2). The permeability effect of 12-HETE was significantly reduced in NOX2(-/-) mice compared with the WT mice. In vitro experiments also showed that 15-HETE induced HREC migration and tube formation in a NOX-dependent manner. Taken together our data suggest that 12/15-LOX is implicated in DR via a NOX-dependent mechanism.

Caspase-14 expression impairs retinal pigment epithelium barrier function: potential role in diabetic macular edema.

We recently showed that caspase-14 is a novel molecule in retina with potential role in accelerated vascular cell death during diabetic retinopathy (DR). Here, we evaluated whether caspase-14 is implicated in retinal pigment epithelial cells (RPE) dysfunction under hyperglycemia. The impact of high glucose (HG, 30 mM D-glucose) on caspase-14 expression in human RPE (ARPE-19) cells was tested, which showed significant increase in caspase-14 expression compared with normal glucose (5 mM D-glucose + 25 mM L-glucose). We also evaluated the impact of modulating caspase-14 expression on RPE cells barrier function, phagocytosis, and activation of other caspases using ARPE-19 cells transfected with caspase-14 plasmid or caspase-14 siRNA. We used FITC-dextran flux assay and electric cell substrate impedance sensing (ECIS) to test the changes in RPE cell barrier function. Similar to HG, caspase-14 expression in ARPE-19 cells increased FITC-dextran leakage through the confluent monolayer and decreased the transcellular electrical resistance (TER). These effects of HG were prevented by caspase-14 knockdown. Furthermore, caspase-14 knockdown prevented the HG-induced activation of caspase-1 and caspase-9, the only activated caspases by HG. Phagocytic activity was unaffected by caspase-14 expression. Our results suggest that caspase-14 contributes to RPE cell barrier disruption under hyperglycemic conditions and thus plays a role in the development of diabetic macular edema.

Alterations of retinal vasculature in cystathionine-ß-synthase heterozygous mice: a model of mild to moderate hyperhomocysteinemia.

Mild to moderate hyperhomocysteinemia is prevalent in humans and is implicated in neurovascular diseases, including recently in certain retinal diseases. Herein, we used hyperhomocysteinemic mice deficient in the Cbs gene encoding cystathionine-ß-synthase (Cbs(+/-)) to evaluate retinal vascular integrity. The Cbs(+/+) (wild type) and Cbs(+/-) (heterozygous) mice (aged 16 to 52 weeks) were subjected to fluorescein angiography and optical coherence tomography to assess vasculature in vivo. Retinas harvested for cryosectioning or flat mount preparations were subjected to immunofluorescence microscopy to detect blood vessels (isolectin-B4), angiogenesis [anti-vascular endothelial growth factor (VEGF) and anti-CD105], gliosis [anti-glial fibrillary acidic protein (GFAP)], pericytes (anti-neural/glial antigen 2), blood-retinal barrier [anti-zonula occludens protein 1 (ZO-1) and anti-occludin], and hypoxia [anti-pimonidazole hydrochloride (Hypoxyprobe-1)]. Levels of VEGF, GFAP, ZO-1, and occludin were determined by immunoblotting. Results of these analyses showed a mild vascular phenotype in young mice, which progressed with age. Fluorescein angiography revealed progressive neovascularization and vascular leakage in Cbs(+/-) mice; optical coherence tomography confirmed new vessels in the vitreous by 1 year. Immunofluorescence microscopy demonstrated vascular patterns consistent with ischemia, including a capillary-free zone centrally and new vessels with capillary tufts midperipherally in older mice. This was associated with increased VEGF, CD105, and GFAP and decreased ZO-1/occludin levels in the Cbs(+/-) retinas. Retinal vein occlusion was observed in some Cbs(+/-) mouse retinas. We conclude that mild to moderate elevation of homocysteine in Cbs(+/-) mice is accompanied by progressive alterations in retinal vasculature characterized by ischemia, neovascularization, incompetent blood-retinal barrier, and vascular occlusion.