Differential Expression and Localization of ADAMTS Proteinases in Proliferative Diabetic Retinopathy.

We analyzed the expression of ADAMTS proteinases ADAMTS-1, -2, -4, -5 and -13; their activating enzyme MMP-15; and the degradation products of proteoglycan substrates versican and biglycan in an ocular microenvironment of proliferative diabetic retinopathy (PDR) patients. Vitreous samples from PDR and nondiabetic patients, epiretinal fibrovascular membranes from PDR patients, rat retinas, retinal Müller glial cells and human retinal microvascular endothelial cells (HRMECs) were studied. The levels of ADAMTS proteinases and MMP-15 were increased in the vitreous from PDR patients. Both full-length and cleaved activation/degradation fragments of ADAMTS proteinases were identified. The amounts of versican and biglycan cleavage products were increased in vitreous from PDR patients. ADAMTS proteinases and MMP-15 were localized in endothelial cells, monocytes/macrophages and myofibroblasts in PDR membranes, and ADAMTS-4 was expressed in the highest number of stromal cells. The angiogenic activity of PDR membranes correlated significantly with levels of ADAMTS-1 and -4 cellular expression. ADAMTS proteinases and MMP-15 were expressed in rat retinas. ADAMTS-1 and -5 and MMP-15 levels were increased in diabetic rat retinas. HRMECs and Müller cells constitutively expressed ADAMTS proteinases but not MMP-15. The inhibition of NF-κB significantly attenuated the TNF-α-and-VEGF-induced upregulation of ADAMTS-1 and -4 in a culture medium of HRMECs and Müller cells. In conclusion, ADAMTS proteinases, MMP-15 and versican and biglycan cleavage products were increased in the ocular microenvironment of patients with PDR.

Apocynin ameliorates NADPH oxidase 4 (NOX4) induced oxidative damage in the hypoxic human retinal Müller cells and diabetic rat retina.

NADPH oxidase (NOX) is a main producers of reactive oxygen species (ROS) that may contribute to the early pathogenesis of diabetic retinopathy (DR). ROS has harmful effects on endogenous neuro-survival factors brain-derived neurotrophic factor (BDNF) and sirtuin 1 (SIRT1) are necessary for the growth and survival of the retina. The role of NOX isoforms NOX4 in triggering ROS in DR is not clear. Here we determine the protective effects of a plant-derived NOX inhibitor apocynin (APO) on NOX4-induced ROS production which may contribute to the depletion of survival factors BDNF/SIRT1 or cell death in the diabetic retinas. Human retinal Müller glial cells (MGCs) were treated with hypoxia mimetic agent cobalt chloride (CoCl2) in the absence or presence of APO. Molecular analysis demonstrates that NOX4 is upregulated in CoCl2-treated MGCs and in the diabetic retinas. Increased NOX4 was accompanied by the downregulation of BDNF/SIRT1 expression or in the activation of apoptotic marker caspase-3. Whereas, APO treatment downregulates NOX4 and subsequently upregulates BDNF/SIRT1 or alleviate caspase-3 expression. Accordingly, in the diabetic retina we found a positive correlation in NOX4 vs ROS (p = 0.025; R2 = 0.488) and caspase-3 vs ROS (p = 0.04; R2 = 0.428); whereas a negative correlation in BDNF vs ROS (p = 0.009; R2 = 0.596) and SIRT1 vs ROS (p = 0.0003; R2 = 0.817) respectively. Taken together, NOX4-derived ROS could be a main contributor in downregulating BDNF/SIRT1 expression or in the activation of caspase-3. Whereas, APO treatment may minimize the deleterious effects occurring due to hyperglycemia and/or diabetic mimic hypoxic condition in early pathogenesis of DR.

Matrix metalloproteinase-14 is a biomarker of angiogenic activity in proliferative diabetic retinopathy.

Purpose: Matrix metalloproteinase-14 (MMP-14) is a transmembrane MMP that plays a critical role in promoting angiogenesis. We investigated the expression levels of MMP-14 and correlated the levels with clinical disease activity and with the levels of the angiogenic factors vascular endothelial growth factor (VEGF) and MMP-9 in proliferative diabetic retinopathy (PDR). To reinforce the findings at the functional level, we examined the expression of MMP-14 in the retinas of diabetic rats. Methods: Vitreous samples from 34 patients with PDR and 18 nondiabetic patients and epiretinal membranes from 13 patients with PDR and the retinas of rats were studied with enzyme-linked immunosorbent assay, immunohistochemistry, western blotting, and real-time reverse transcription PCR (RT-PCR). Results: The MMP-14, VEGF, and MMP-9 levels were statistically significantly higher in the vitreous samples from patients with PDR than in the samples from the nondiabetic controls (p<0.001 for all comparisons). The MMP-14 levels in patients with PDR with active neovascularization were statistically significantly higher than those in patients with inactive PDR (p<0.001). There were statistically significant positive correlations between levels of MMP-14 and levels of VEGF (r = 0.3; p = 0.032) and MMP-9 (r = 0.54; p<0.001). In the epiretinal membranes, MMP-14 was expressed in vascular endothelial cells, leukocytes, and myofibroblasts. Statistically significant positive correlations were detected between the numbers of blood vessels expressing CD31 and the numbers of blood vessels (r = 0.74; p = 0.004) and stromal cells (r = 0.72; p = 0.005) expressing MMP-14. Statistically significant increases of MMP-14 mRNA and protein were detected in rat retinas after induction of diabetes. Conclusions: These results suggest that MMP-14 is involved in PDR angiogenesis.

Association of HMGB1 with oxidative stress markers and regulators in PDR.

Purpose: We investigated the link among the proinflammatory cytokine high-mobility group box 1 (HMGB1) and 8-hydroxy-2'-deoxyguanosine (8-OHdG) as a marker of oxidative DNA damage, the endothelial adhesion molecule and oxidase enzyme vascular adhesion protein-1 (VAP-1), and the inducible cytoprotective molecule heme oxygenase-1 (HO-1) in proliferative diabetic retinopathy (PDR). We correlated the levels of these molecules with clinical disease activity and studied the proinflammatory activities of HMGB1 on rat retinas and human retinal microvascular endothelial cells (HRMECs). Methods: Vitreous samples from 47 PDR and 19 non-diabetic patients, epiretinal membranes from 11 patients with PDR, human retinas (16 from diabetic patients and 16 from non-diabetic subjects), rat retinas, and HRMECs were studied by enzyme-linked immunosorbent assay, immunohistochemistry, western blot immunofluorescence, and RT-PCR analyses. In addition, we assessed the adherence of leukocytes to HMGB1-stimulated HRMECs. Results: HMGB1, 8-OHdG, and soluble VAP-1 (sVAP-1) levels were significantly higher in vitreous samples from PDR patients than in those from non-diabetics (p = 0.001, <0.0001, <0.0001, respectively). The HMGB1, 8-OHdG, sVAP-1, and HO-1 levels in PDR with active neovascularization were significantly higher than those in inactive PDR (p = 0.025, <0.0001, <0.0001, 0.012, respectively). Significant positive correlations were observed between the levels of HMGB1 and the levels of 8-OHdG (r = 0.422; p = 0.001) and sVAP-1 (r = 0.354; p = 0.004) and between the levels of 8-OHdG and the levels of sVAP-1 (r = 0.598; p<0.0001). In epiretinal membranes, VAP-1 and 8-OHdG were expressed in vascular endothelial cells and stromal cells. Significant increases in the VAP-1 mRNA and protein levels were detected in the RPE, but not in the neuroretina of diabetic patients. Treatment of HRMEC with HMGB1, diabetes induction, and an intravitreal injection of HMGB1 in normal rats induced a significant upregulation of the adhesion molecule intercellular adhesion molecule-1 (ICAM-1) in HRMECs and retinas. On the other hand, the expressions of vascular cell adhesion molecule-1 and VAP-1 were not affected. Oral administration of the HMGB1 inhibitor glycyrrhizin in rats attenuated the diabetes-induced upregulation of the retinal ICAM-1 expression. Treatment of HRMECs with HMGB1 increased leukocyte adhesion and induced the upregulation of 8-OHdG and HO-1 and the membranous translocation of VAP-1. Conclusions: Our results suggest a potential link among the proinflammatory cytokine HMGB1, VAP-1, oxidative stress, and HO-1 in the pathogenesis of PDR.

High-Mobility Group Box-1 Protein Mediates the Regulation of Signal Transducer and Activator of Transcription-3 in the Diabetic Retina and in Human Retinal Müller Cells.

PURPOSE: The expression of high-mobility group box-1 (HMGB1) and signal transducer and activator of transcription-3 (STAT-3) is upregulated in the diabetic retina. We hypothesized that the activation of STAT-3 is under the control of HMGB1. METHODS: Retinas from 1-month-old diabetic rats and from normal rats intravitreally injected with HMGB1 and human retinal Müller glial cells (MIO-M1) stimulated with HMGB1 or high glucose were studied by Western blot analysis and immunofluorescence. We also studied the effect of the HMGB1 inhibitor glycyrrhizin (GA) on high-glucose-induced pSTAT-3 nuclear translocation and upregulation in Müller cells and on pSTAT-3 expression in the retinas of diabetic rats (n = 7-10 in each group). In addition, we studied the effect of STAT-3 inhibitor on the HMGB1-induced induction of vascular endothelial growth factor (VEGF) by Müller cells and human retinal microvascular endothelial cell (HRMEC) migration. RESULTS: Treatment of retinal Müller cells with recombinant HMGB1 induced nuclear translocation of pSTAT-3 but did not alter pSTAT-3 expression. High glucose induced a significant upregulation of HMGB1 and pSTAT-3 upregulation and nuclear translocation in retinal Müller cells. GA co-treatment normalized the high-glucose-induced upregulation of HMGB1 and pSTAT-3 upregulation and nuclear translocation in Müller cells. Intravitreal administration of HMGB1 in normal and diabetic rats upregulated pSTAT-3 expression in the retina. GA attenuated the diabetes-induced upregulation of pSTAT-3 in the retina. The STAT-3 inhibitor attenuated HMGB1-induced VEGF upregulation by Müller cells and HRMEC migration. CONCLUSIONS: The results suggest a role for HMGB1 in the modulation of STAT-3 expression in the diabetic retina.

Coexpression of heparanase activity, cathepsin L, tissue factor, tissue factor pathway inhibitor, and MMP-9 in proliferative diabetic retinopathy.

PURPOSE: Heparanase cleaves heparan sulfate side chains of heparan sulfate proteoglycans, activity that is implicated in angiogenesis. Proteolytic cleavage of proheparanase by cathepsin L leads to the formation of catalytically active heparanase. We investigated the expression levels of heparanase enzymatic activity and correlated these with the levels of cathepsin L, the angiogenic factors tissue factor (TF) and matrix metalloproteinase-9 (MMP-9), and the angiostatic factor tissue factor pathway inhibitor (TFPI) in proliferative diabetic retinopathy (PDR). METHODS: Vitreous samples from 25 patients with PDR and 20 nondiabetic patients and epiretinal membranes from 12 patients with PDR were studied with enzyme-linked immunosorbent assay, western blot analysis, and immunohistochemistry. RESULTS: We observed a significant increase in the expression of heparanase activity in vitreous samples from patients with PDR compared to the nondiabetic controls (p=0.027). Significant positive correlations were found between the levels of heparanase activity and the levels of cathepsin L (r=0.51; p=0.001), TF (r=0.6; p<0.0001), and TFPI (r=0.49; p=0.001). The expression levels of cathepsin L (p=0.019), TF (p<0.0001), TFPI (p<0.0001), and MMP-9 (p=0.029) were significantly higher in the vitreous samples with detected heparanase activity compared to the vitreous samples with undetected heparanase activity. Western blot analysis demonstrated proteolytic cleavage of TFPI in the vitreous samples from patients with PDR. In the epiretinal membranes, cathepsin L, TF, and TFPI were expressed in vascular endothelial cells and CD45-expressing leukocytes. Significant positive correlations were detected between the number of blood vessels that expressed CD31 and the number of blood vessels that expressed TF (r=0.9; p<0.0001) and TFPI (r=0.81; p=0.001). CONCLUSIONS: The coexpression of these angiogenesis regulatory factors suggests cross-talk between these factors and pathogenesis of PDR angiogenesis.

The tumor necrosis factor superfamily members TWEAK, TNFSF15 and fibroblast growth factor-inducible protein 14 are upregulated in proliferative diabetic retinopathy.

PURPOSE: Tumor necrosis factor-like weak inducer of apoptosis (TWEAK) and tumor necrosis factor superfamily member 15 (TNFSF15), members of the TNF superfamily, play important roles in the modulation of inflammation and neovascularization. TWEAK activity is mediated via binding to fibroblast growth factor-inducible molecule 14 (Fn14). We investigated the expression of TWEAK, Fn14 and TNFSF15 and the correlation between TWEAK levels and the levels of the inflammatory biomarker soluble intercellular adhesion molecule-1 (sICAM-1) in proliferative diabetic retinopathy (PDR). In addition, we examined the expression of FN14 and TNFSF15 in retinas of diabetic rats. METHODS: Vitreous samples from 34 PDR and 23 nondiabetic patients were studied by enzyme-linked immunosorbent assay and Western blot analysis. Epiretinal membranes from 14 patients with PDR were studied by immunohistochemistry. The retinas of rats were examined by Western blot analysis. RESULTS: We identified a significant increase in the expression of TWEAK, Fn14, TNFSF15 and sICAM-1 in vitreous samples from PDR patients compared to controls. A significant positive correlation was found between levels of TWEAK and levels of sICAM-1 (r = 0.3, p = 0.02). In epiretinal membranes, TWEAK and TNFSF15 protein expression was confined to vascular endothelial cells, monocytes/macrophages and myofibroblasts. Significant positive correlations were observed between the number of blood vessels expressing CD34 and the number of blood vessels expressing TWEAK (r = 0.670; p = 0.017) and TNFSF15 (r = 0.784; p = 0.001). The expression level of TNFSF15 was upregulated in the retinas of diabetic rats, whereas Fn14 was not upregulated. CONCLUSIONS: Our findings suggest that TNFSF15 and the TWEAK/Fn14 pathway are novel mediators involved in persistent inflammation and modulation of pathological neovascularization associated with PDR.

S100A4 is upregulated in proliferative diabetic retinopathy and correlates with markers of angiogenesis and fibrogenesis.

PURPOSE: The calcium-binding protein S100A4 is implicated in cancer cell invasion and metastasis, the stimulation of angiogenesis, the progression of fibrosis, and inflammatory disorders. We investigated the expression of S100A4 and correlated it with clinical disease activity as well as with the levels of osteopontin (OPN), soluble syndecan-1, and vascular endothelial growth factor (VEGF) in proliferative diabetic retinopathy (PDR). To reinforce the findings at the functional level, we examined the expressions of S100A4 and OPN in the retinas of diabetic rats and in human retinal microvascular endothelial cells (HRMECs) following exposure to VEGF and the proinflammatory cytokine tumor necrosis factor-α (TNF-α). METHODS: Vitreous samples from 30 PDR and 30 nondiabetic patients, epiretinal membranes from 14 patients with PDR, the retinas of rats, and HRMECs were studied by enzyme-linked immunosorbent assay (ELISA), immunohistochemistry, western blot analysis, and co-immunoprecipitation. RESULTS: ELISA revealed a significant increase in the expressions of S100A4, OPN, soluble syndecan-1, and VEGF in vitreous samples from PDR patients compared to nondiabetic controls (p = 0.001; <0.001; <0.001; <0.001, respectively). Significant positive correlations were found between the levels of S100A4, OPN (r = 0.52, p = <0.001), soluble syndecan-1 (r = 0.37, p = 0.012), and VEGF (r = 0.29, p = 0.044). In epiretinal membranes, S100A4 was expressed in the vascular endothelial cells and stromal CD45-expressing leukocytes. A significant positive correlation was detected between the number of blood vessels expressing CD31 and the number of stromal cells expressing S100A4 (r = 0.77, p = 0.001). Western blot analysis revealed a significant increase in the expressions of S100A4 and both intact and cleaved OPN in vitreous samples from PDR patients compared to nondiabetic controls, as well as in the retinas of diabetic rats. Co-immunoprecipitation studies revealed a positive interaction between S100A4 and the receptor for advanced glycation end products (RAGE) in the retinas of diabetic rats. TNF-α-but not VEGF-induced the upregulations of S100A4 and both intact and cleaved OPN in HRMECs. CONCLUSIONS: S100A4 represents a valuable vitreous marker molecule in the pathogenesis of PDR and might become a new target for the treatment of PDR.

Expression of bioactive lysophospholipids and processing enzymes in the vitreous from patients with proliferative diabetic retinopathy.

BACKGROUND: The bioactive lysophospholipids phosphatidic acid (PA), lysophosphatidic acid (LPA) and sphingosine-1-phosphate (S1P) have been implicated in mediating cell migration, proliferation and apoptosis, inflammation, angiogenesis and fibrosis. This study was conducted to measure the levels of PA, LPA, LPA-producing enzymes phospholipase A1/A2 (PLA1A/PLA2, respectively) and acylgylycerol kinase (AGK), the S1P receptor S1PR1, the S1P catabolising enzyme S1P lyase (SPL) and 5-lipoxygenase in the vitreous fluid from patients with proliferative diabetic retinopathy (PDR). In addition, we investigated the correlations between the levels of PA and LPA and the levels of the inflammatory and endothelial dysfunction biomarker soluble vascular cell adhesion molecule-1 (sVCAM-1). METHODS: Vitreous samples from 34 PDR and 29 nondiabetic patients were studied by biochemical and enzyme-linked immunosorbent assays and Western blot analysis. RESULTS: PA, LPA and sVCAM-1 levels in vitreous samples from PDR patients were significantly higher than those in nondiabetic patients. Significant correlations were observed between levels of LPA and levels of PA and sVCAM-1. Western blot analysis revealed a significant increase in the expression of PLA1A, AGK, S1PR1 and SPL in vitreous samples from PDR patients compared to nondiabetic controls, whereas PLA2 and 5-lipoxygenase were not detected. CONCLUSIONS: Our findings suggest that the enzymatic activities of PLA1A and AGK might be responsible for increased synthesis of LPA in PDR and that PLA1A, but not PLA2 is responsible for deacylation of PA to generate LPA. S1PR1 and SPL might regulate inflammatory, angiogenic and fibrogenic responses in PDR.

The proinflammatory cytokine high-mobility group box-1 mediates retinal neuropathy induced by diabetes.

To test the hypothesis that increased expression of proinflammatory cytokine high-mobility group box-1 (HMGB1) in epiretinal membranes and vitreous fluid from patients with proliferative diabetic retinopathy and in retinas of diabetic rats plays a pathogenetic role in mediating diabetes-induced retinal neuropathy. Retinas of 1-month diabetic rats and HMGB1 intravitreally injected normal rats were studied using Western blot analysis, RT-PCR and glutamate assay. In addition, we studied the effect of the HMGB1 inhibitor glycyrrhizin on diabetes-induced biochemical changes in the retina. Diabetes and intravitreal injection of HMGB1 in normal rats induced significant upregulation of HMGB1 protein and mRNA, activated extracellular signal-regulated kinase 1 and 2 (ERK1/2), cleaved caspase-3 and glutamate; and significant downregulation of synaptophysin, tyrosine hydroxylase, glutamine synthetase, and glyoxalase 1. Constant glycyrrhizin intake from the onset of diabetes did not affect the metabolic status of the diabetic rats, but it significantly attenuated diabetes-induced upregulation of HMGB1 protein and mRNA, activated ERK1/2, cleaved caspase-3, and glutamate. In the glycyrrhizin-fed diabetic rats, the decrease in synaptophysin, tyrosine hydroxylase, and glyoxalase 1 caused by diabetes was significantly attenuated. These findings suggest that early retinal neuropathy of diabetes involves upregulated expression of HMGB1 and can be ameliorated by inhibition of HMGB1.

High-mobility group box-1 protein activates inflammatory signaling pathway components and disrupts retinal vascular-barrier in the diabetic retina.

Extracellular high-mobility group box-1 (HMGB-1) functions as a pro-inflammatory cytokine and exhibits angiogenic effects. The purpose of this study was to investigate the expression of HMGB-1 signaling pathway components in the retinas of diabetic rats and to examine the effect of intravitreal administration of HMGB-1 on the retinas of rats. The retinas of diabetic and intravitreally injected HMGB-1 rats were studied using immunohistochemistry, Western blotting, co-immunoprecipitation and enzyme-linked immunosorbent assay. The effect of HMGB-1 on retinal endothelial cell barrier function was evaluated using electrical cell-substrate impedance sensing system (ECIS). Diabetes induced significant upregulation of the expression of HMGB-1, receptor for advanced glycation end products (RAGE), ERK(1/2) and nuclear transcription factor Kappa B (NF-κB), whereas the expression of toll-like receptor 2 (TLR2) and occludin was significantly downregulated. Co-immunoprecipitation studies revealed significant increase in interaction between HMGB-1 and RAGE. HMGB-1 reduced transendothelial electrical resistance of bovine retinal endothelial cells. Intravitreal administration of HMGB-1 to normal rats induced significant upregulation of intercellular adhesion molecule-1 (ICAM-1), soluble intercellular adhesion molecule-1 (sICAM-1), HMGB-1, RAGE, ERK(1/2), and NF-κB, and significantly increased retinal vascular permeability, whereas the expression of TLR2 and occludin was downregulated. Oral administration of glycyrrhizin, a specific inhibitor of HMGB-1, attenuated diabetes-induced upregulation of HMGB-1 expression, NF-κB activation and downregulation of occludin expression. Our findings provide evidence that in the diabetic retina, HMGB-1 possibly interacts with RAGE and activates ERK(1/2) and NF-κB to generate an inflammatory response and disrupt retinal vascular barrier.

Poly (ADP-ribose) polymerase mediates diabetes-induced retinal neuropathy.

Retinal neuropathy is an early event in the development of diabetic retinopathy. One of the potential enzymes that are activated by oxidative stress in the diabetic retina is poly (ADP-ribose) polymerase (PARP). We investigated the effect of the PARP inhibitor 1,5-isoquinolinediol on the expression of the neurodegeneration mediators and markers in the retinas of diabetic rats. After two weeks of streptozotocin-induced diabetes, rats were treated with 1,5-isoquinolinediol (3 mg/kg/day). After 4 weeks of diabetes, the retinas were harvested and the levels of reactive oxygen species (ROS) were determined fluorometrically and the expressions of PARP, phosporylated-ERK1/2, BDNF, synaptophysin, glutamine synthetase (GS), and caspase-3 were determined by Western blot analysis. Retinal levels of ROS, PARP-1/2, phosphorylated ERK1/2, and cleaved caspase-3 were significantly increased, whereas the expressions of BDNF synaptophysin and GS were significantly decreased in the retinas of diabetic rats, compared to nondiabetic rats. Administration of 1,5-isoquinolinediol did not affect the metabolic status of the diabetic rats, but it significantly attenuated diabetes-induced upregulation of PARP, ROS, ERK1/2 phosphorylation, and cleaved caspase-3 and downregulation of BDNF, synaptophysin, and GS. These findings suggest a beneficial effect of the PARP inhibitor in increasing neurotrophic support and ameliorating early retinal neuropathy induced by diabetes.

High-mobility group box-1 induces decreased brain-derived neurotrophic factor-mediated neuroprotection in the diabetic retina.

To test the hypothesis that brain-derived neurotrophic factor-(BDNF-) mediated neuroprotection is reduced by high-mobility group box-1 (HMGB1) in diabetic retina, paired vitreous and serum samples from 46 proliferative diabetic retinopathy and 34 nondiabetic patients were assayed for BDNF, HMGB1, soluble receptor for advanced glycation end products (sRAGE), soluble intercellular adhesion molecule-1 (sICAM-1), monocyte chemoattractant protein-1 (MCP-1), and TBARS. We also examined retinas of diabetic and HMGB1 intravitreally injected rats. The effect of the HMGB1 inhibitor glycyrrhizin on diabetes-induced changes in retinal BDNF expressions was studied. Western blot, ELISA, and TBARS assays were used. BDNF was not detected in vitreous samples. BDNF levels were significantly lower in serum samples from diabetic patients compared with nondiabetics, whereas HMGB1, sRAGE, sICAM-1, and TBARS levels were significantly higher in diabetic serum samples. MCP-1 levels did not differ significantly. There was significant inverse correlation between serum levels of BDNF and HMGB1. Diabetes and intravitreal administration of HMGB1 induced significant upregulation of the expression of HMGB1, TBARS, and cleaved caspase-3, whereas the expression of BDNF and synaptophysin was significantly downregulated in rat retinas. Glycyrrhizin significantly attenuated diabetes-induced downregulation of BDNF. Our results suggest that HMGB1-induced downregulation of BDNF might be involved in pathogenesis of diabetic retinal neurodegeneration.

Thymoquinone Attenuates Retinal Expression of Mediators and Markers of Neurodegeneration in a Diabetic Animal Model.

BACKGROUND: Diabetic retinopathy (DR) is a slow eye disease that affects the retina due to a long-standing uncontrolled diabetes mellitus. Hyperglycemia-induced oxidative stress can lead to neuronal damage leading to DR. OBJECTIVE: The aim of the current investigation is to assess the protective effects of thymoquinone (TQ) as a potential compound for the treatment and/or prevention of neurovascular complications of diabetes, including DR. METHODS: Diabetes was induced in rats by the administration of streptozotocin (55 mg/kg intraperitoneally, i.p.). Subsequently, diabetic rats were treated with either TQ (2 mg/kg i.p.) or vehicle on alternate days for three weeks. A healthy control group was also run in parallel. At the end of the treatment period, animals were euthanized, and the retinas were collected and analyzed for the expression levels of brain-derived neurotrophic factor (BDNF), tyrosine hydroxylase (TH), nerve growth factor receptor (NGFR), and caspase-3 using Western blotting techniques in the retina of diabetic rats and compared with the normal control rats. In addition, dichlorofluorescein (DCF) levels in the retina were assessed as a marker of reactive oxygen species (ROS), and blood-retinal barrier breakdown (BRB) was examined for vascular permeability. The systemic effects of TQ treatments on glycemic control, kidney and liver functions were also assessed in all groups. RESULTS: Diabetic animals treated with TQ showed improvements in the liver and kidney functions compared with control diabetic rats. Normalization in the levels of neuroprotective factors, including BDNF, TH, and NGFR, was observed in the retina of diabetic rats treated with TQ. In addition, TQ ameliorated the levels of apoptosis regulatory protein caspase-3 in the retina of diabetic rats and reduced disruption of the blood-retinal barrier, possibly through a reduction in reactive oxygen species (ROS) generation. CONCLUSION: These findings suggest that TQ harbors a significant potential to limit the neurodegeneration and retinal damage that can be provoked by hyperglycemia in vivo.

Tissue Inhibitor of Metalloproteinase-3 Ameliorates Diabetes-Induced Retinal Inflammation.

Purpose: Endogenous tissue inhibitor of matrix metalloproteinase-3 (TIMP-3) has powerful regulatory effects on inflammation and angiogenesis. In this study, we investigated the role of TIMP-3 in regulating inflammation in the diabetic retina. Methods: Vitreous samples from patients with proliferative diabetic retinopathy (PDR) and non-diabetic patients were subjected to Western blot analysis. Streptozotocin-treated rats were used as a preclinical diabetic retinopathy (DR) model. Blood-retinal barrier (BRB) breakdown was assessed with fluorescein isothiocyanate (FITC)-conjugated dextran. Rat retinas, human retinal microvascular endothelial cells (HRMECs) and human retinal Müller glial cells were studied by Western blot analysis and ELISA. Adherence of human monocytes to HRMECs was assessed and in vitro angiogenesis assays were performed. Results: Tissue inhibitor of matrix metalloproteinase-3 in vitreous samples was largely glycosylated. Intravitreal injection of TIMP-3 attenuated diabetes-induced BRB breakdown. This effect was associated with downregulation of diabetes-induced upregulation of the p65 subunit of NF-κB, intercellular adhesion molecule-1 (ICAM-1), and vascular endothelial growth factor (VEGF), whereas phospho-ERK1/2 levels were not altered. In Müller cell cultures, TIMP-3 significantly attenuated VEGF upregulation induced by high-glucose (HG), the hypoxia mimetic agent cobalt chloride (CoCl2) and TNF-α and attenuated MCP-1 upregulation induced by CoCl2 and TNF-α, but not by HG. TIMP-3 attenuated HG-induced upregulation of phospho-ERK1/2, caspase-3 and the mature form of ADAM17, but not the levels of the p65 subunit of NF-κB and the proform of ADAM17 in Müller cells. TIMP-3 significantly downregulated TNF-α-induced upregulation of ICAM-1 and VCAM-1 in HRMECs. Accordingly, TIMP-3 significantly decreased spontaneous and TNF-α- and VEGF-induced adherence of monocytes to HRMECs. Finally, TIMP-3 significantly attenuated VEGF-induced migration, chemotaxis and proliferation of HRMECs. Conclusion: In vitro and in vivo data point to anti-inflammatory and anti-angiogenic effects of TIMP-3 and support further studies for its applications in the treatment of DR.

CD146/Soluble CD146 Pathway Is a Novel Biomarker of Angiogenesis and Inflammation in Proliferative Diabetic Retinopathy.

Purpose: Inflammation, angiogenesis and fibrosis are pathological hallmarks of proliferative diabetic retinopathy (PDR). The CD146/sCD146 pathway displays proinflammatory and proangiogenic properties. We investigated the role of this pathway in the pathophysiology of PDR. Methods: Vitreous samples from 41 PDR and 27 nondiabetic patients, epiretinal fibrovascular membranes from 18 PDR patients, rat retinas, human retinal microvascular endothelial cells (HRMECs) and human retinal Müller glial cells were studied by ELISA, Western blot analysis, immunohistochemistry and immunofluorescence microscopy analysis. Blood-retinal barrier breakdown was assessed with fluorescein isothiocyanate-conjugated dextran. Results: sCD146 and VEGF levels were significantly higher in vitreous samples from PDR patients than in nondiabetic patients. In epiretinal membranes, immunohistochemical analysis revealed CD146 expression in leukocytes, vascular endothelial cells and myofibroblasts. Significant positive correlations were detected between numbers of blood vessels expressing CD31, reflecting angiogenic activity of PDR, and numbers of blood vessels and stromal cells expressing CD146. Western blot analysis showed significant increase of CD146 in diabetic rat retinas. sCD146 induced upregulation of phospho-ERK1/2, NF-κB , VEGF and MMP-9 in Müller cells. The hypoxia mimetic agent cobalt chloride, VEGF and TNF-α induced upregulation of sCD146 in HRMECs. The MMP inhibitor ONO-4817 attenuated TNF-α-induced upregulation of sCD146 in HRMECs. Intravitreal administration of sCD146 in normal rats significantly increased retinal vascular permeability and induced significant upregulation of phospho-ERK1/2, intercellular adhesion molecule-1 and VEGF in the retina. sCD146 induced migration of HRMECs. Conclusions: These results suggest that the CD146/sCD146 pathway is involved in the initiation and progression of PDR.

The Chemokine-Based Peptide, CXCL9(74-103), Inhibits Angiogenesis by Blocking Heparan Sulfate Proteoglycan-Mediated Signaling of Multiple Endothelial Growth Factors.

Growth factors such as vascular endothelial growth factor (VEGF), fibroblast growth factor (FGF) and epidermal growth factor (EGF) are important angiogenesis-mediating factors. They exert their effects not only through their respective receptor tyrosine kinases (RTKs), but they also require molecular pairing with heparan sulfate proteoglycans (HSPGs). Angiogenic growth factors and their signaling pathways are commonly targeted in current anti-angiogenic cancer therapies but have unfortunately insufficient impact on patient survival. Considering their obvious role in pathological angiogenesis, HS-targeting drugs have become an appealing new strategy. Therefore, we aimed to reduce angiogenesis through interference with growth factor-HS binding and downstream signaling using a CXCL9-derived peptide with a high affinity for glycosaminoglycans (GAGs), CXCL9(74-103). We showed that CXCL9(74-103) reduced EGF-, VEGF165- and FGF-2-mediated angiogenic processes in vitro, such as endothelial cell proliferation, chemotaxis, adhesion and sprouting, without exerting cell toxicity. CXCL9(74-103) interfered with growth factor signaling in diverse ways, e.g., by diminishing VEGF165 binding to HS and by direct association with FGF-2. The dependency of CXCL9(74-103) on HS for binding to HMVECs and for exerting its anti-angiogenic activity was also demonstrated. In vivo, CXCL9(74-103) attenuated neovascularization in the Matrigel plug assay, the corneal cauterization assay and in MDA-MB-231 breast cancer xenografts. Additionally, CXCL9(74-103) reduced vascular leakage in the retina of diabetic rats. In contrast, CXCL9(86-103), a peptide with low GAG affinity, showed no overall anti-angiogenic activity. Altogether, our results indicate that CXCL9(74-103) reduces angiogenesis by interfering with multiple HS-dependent growth factor signaling pathways.

Interleukin-11 Overexpression and M2 Macrophage Density are Associated with Angiogenic Activity in Proliferative Diabetic Retinopathy.

PURPOSE: To investigate the expression of IL-11 and its receptor IL-11Rα and to quantify density of CD163+ M2 macrophages in proliferative diabetic retinopathy (PDR). METHODS: Vitreous samples from 29 PDR and 19 nondiabetic patients, epiretinal fibrovascular membranes from 15 patients with PDR and Müller cells were studied by enzyme-linked immunosorbent assay, immunohistochemistry and Western blot analysis. RESULTS: We showed a significant increase in expression of IL-11, soluble(s) IL-11Rα, sCD163 and VEGF in vitreous samples from PDR patients compared to nondiabetic controls. Significant positive correlations were found between levels of VEGF and levels of IL-11 and sCD163. Significant positive correlations were found between microvessel density and number of blood vessels and stromal cells expressing IL-11, IL-11Rα and CD163 in PDR epiretinal membranes. The hypoxia mimetic agent cobalt chloride induced upregulation of IL-11 and IL-11Ra in cultured Müller cells. CONCLUSIONS: IL-11/IL-11Rα signaling and CD163+ M2 macrophages might be involved in PDR angiogenesis.

Evaluation of Proteoforms of the Transmembrane Chemokines CXCL16 and CX3CL1, Their Receptors, and Their Processing Metalloproteinases ADAM10 and ADAM17 in Proliferative Diabetic Retinopathy.

The transmembrane chemokine pathways CXCL16/CXCR6 and CX3CL1/CX3CR1 are strongly implicated in inflammation and angiogenesis. We investigated the involvement of these chemokine pathways and their processing metalloproteinases ADAM10 and ADAM17 in the pathophysiology of proliferative diabetic retinopathy (PDR). Vitreous samples from 32 PDR and 24 non-diabetic patients, epiretinal membranes from 18 patients with PDR, rat retinas, human retinal Müller glial cells and human retinal microvascular endothelial cells (HRMECs) were studied by enzyme-linked immunosorbent assay, immunohistochemistry and Western blot analysis. In vitro angiogenesis assays were performed and the adherence of leukocytes to CXCL16-stimulated HRMECs was assessed. CXCL16, CX3CL1, ADAM10, ADAM17 and vascular endothelial growth factor (VEGF) levels were significantly increased in vitreous samples from PDR patients. The levels of CXCL16 were 417-fold higher than those of CX3CL1 in PDR vitreous samples. Significant positive correlations were found between the levels of VEGF and the levels of CXCL16, CX3CL1, ADAM10 and ADAM17. Significant positive correlations were detected between the numbers of blood vessels expressing CD31, reflecting the angiogenic activity of PDR epiretinal membranes, and the numbers of blood vessels and stromal cells expressing CXCL16, CXCR6, ADAM10 and ADAM17. CXCL16 induced upregulation of phospho-ERK1/2, p65 subunit of NF-κB and VEGF in cultured Müller cells and tumor necrosis factor-α induced upregulation of soluble CXCL16 and ADAM17 in Müller cells. Treatment of HRMECs with CXCL16 resulted in increased expression of intercellular adhesion molecule-1 (ICAM-1) and increased leukocyte adhesion to HRMECs. CXCL16 induced HRMEC proliferation, formation of sprouts from HRMEC spheroids and phosphorylation of ERK1/2. Intravitreal administration of CXCL16 in normal rats induced significant upregulation of the p65 subunit of NF-κB, VEGF and ICAM-1 in the retina. Our findings suggest that the chemokine axis CXCL16/CXCR6 and the processing metalloproteinases ADAM10 and ADAM17 might serve a role in the initiation and progression of PDR.

Galectin-1 studies in proliferative diabetic retinopathy.

PURPOSE: Galectin-1 regulates endothelial cell function and promotes angiogenesis. We investigated the hypothesis that galectin-1 may be involved in the pathogenesis of proliferative diabetic retinopathy (PDR). METHODS: Vitreous samples from 36 PDR and 20 nondiabetic patients, epiretinal fibrovascular membranes from 13 patients with PDR, rat retinas and human retinal Müller glial cells were studied by enzyme-linked immunosorbent assay (ELISA), immunohistochemistry and Western blot analysis. In vitro angiogenesis assays were performed and the adherence of leukocytes to galectin-1-stimulated human retinal microvascular endothelial cells (HRMECs) was assessed. RESULTS: The ELISA analysis revealed that galectin-1 and vascular endothelial growth factor (VEGF) levels were significantly higher in vitreous samples from PDR patients than in those from nondiabetics (p < 0.001 for both comparisons). A significant positive correlation was found between the levels of galectin-1 and VEGF (r = 0.354; p = 0.022). In epiretinal membranes, immunohistochemical analysis showed that galectin-1 was expressed in vascular endothelial cells expressing CD31, myofibroblasts expressing α-smooth muscle actin and leukocytes expressing CD45. The galectin-1 receptor neuropilin-1 was expressed on vascular endothelial cells. CD31 staining was used as a marker to assess microvessel density (MVD). Significant positive correlation was detected between MVD in epiretinal membranes and the number of blood vessels expressing galectin-1 (r = 0.848; p < 0.001). Western blot analysis demonstrated significant increase of galectin-1 protein in rat retinas after induction of diabetes. ELISA analysis revealed that hydrogen peroxide and cobalt chloride (CoCl2 ) induced upregulation of galectin-1 in Müller cells. Treatment with galectin-1 induced upregulation of VEGF in Müller cells and increased leukocyte adhesion to HRMECs. The galectin-1 inhibitor OTX008 attenuated VEGF-induced HRMECs migration and CoCl2 -induced upregulation of NF-κB, galectin-1 and VEGF in Müller cells. CONCLUSIONS: These results suggest that galectin-1is involved in the pathogenesis of PDR.