Kaiser Alam

Myofibroblasts in proliferative diabetic retinopathy can originate from infiltrating fibrocytes and through endothelial-to-mesenchymal transition (EndoMT)

Myofibroblasts expressing α-smooth muscle actin (α-SMA) are the key cellular mediator of fibrosis. Fibrovascular epiretinal membranes from patients with proliferative diabetic retinopathy (PDR) are characterized by the accumulation of a large number of myofibroblasts. We explored the hypothesis that proliferating endothelial cells via endothelial-to-mesenchymal transition (EndoMT) and/or bone marrow-derived circulating fibrocytes contribute to the myofibroblast population present in PDR epiretinal membranes. Epiretinal membranes from 14 patients with PDR were studied by immunohistochemistry. All membranes contained neovessels expressing the endothelial cell marker CD31. CD31(+) endothelial cells co-expressed the fibroblast/myofibroblast markers fibroblast-specific protein-1 (FSP-1) and α-SMA, indicative for the occurrence of endoMT. In the stroma, cells expressing FSP-1, α-SMA, the leukocyte common antigen CD45, and the myelomonocytic marker CD11b were detected. Double labeling showed co-localization of CD45 with FSP-1 and α-SMA and co-localization of CD11b with α-SMA and matrix metalloproteinase-9, demonstrating the presence of infiltrating fibrocytes. In addition, we investigated the phenotypic changes that take place in human retinal microvascular endothelial cells following exposure to transforming growth factor-β1 (TGF-β1), connective tissue growth factor (CTGF) and the proinflammatory cytokines interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α). Retinal microvascular endothelial cells changed morphology upon cytokine exposure, lost the expression of endothelial cell markers (endothelial nitric oxide synthase and vascular endothelial-cadherin) and started to express mesenchymal markers (calponin, snail, transgelin and FSP-1). These results suggest that endothelial cells as well as circulating fibrocytes may differentiate into myofibroblasts in the diabetic eye and contribute to pathologic fibrosis in PDR.

Mutual enhancement between high-mobility group box-1 and NADPH oxidase-derived reactive oxygen species mediates diabetes-induced upregulation of retinal apoptotic markers

The expression of the proinflammatory cytokine high-mobility group box-1 (HMGB1) is upregulated in epiretinal membranes and vitreous fluid from patients with proliferative diabetic retinopathy (PDR) and in the diabetic retina. We hypothesized that a novel mechanism exists where HMGB1 and NADPH oxidase (Nox)-derived reactive oxygen species (ROS) are mutually enhanced in the diabetic retina, which may be a novel mechanism for promoting upregulation of retinal apoptotic markers induced by diabetes. Vitreous samples from 48 PDR and 34 nondiabetic patients, retinas from 1-month diabetic rats and from normal rats intravitreally injected with HMGB1 and human retinal microvascular endothelial cells (HRMEC) stimulated with HMGB1 were studied by enzyme-linked immunosorbent and spectrophotometric assays, Western blot analysis, RT-PCR, and immunofluorescence. We also studied the effect of the HMGB1 inhibitor glycyrrhizin and apocynin on diabetes-induced biochemical changes in the retinas of rats (n = 5–7 in each groups). HMGB1 and the oxidative stress marker protein carbonyl content levels in the vitreous fluid from PDR patients were significantly higher than in controls (p = 0.021; p = 0.005, respectively). There was a significant positive correlation between vitreous fluid levels of HMGB1 and the levels of protein carbonyl content (r = 0.62, p = 0.001). HMGB1 enhanced interleukin-1β, ROS, Nox2, poly (ADP-ribose) polymerase (PARP)-1, and cleaved caspase-3 production by HRMEC. Diabetes and intravitreal injection of HMGB1 in normal rats induced significant upregulation of ROS, Nox2, PARP-1, and cleaved caspase-3 in the retina. Constant glycyrrhizin and apocynin intake from onset of diabetes did not affect the metabolic status of the diabetic rats, but restored these increased mediators to control values. The results of this study suggest that there is a mutual enhancement between HMGB1 and Nox-derived ROS in the diabetic retina, which may promote diabetes-induced upregulation of retinal apoptotic markers.

Upregulated Expression of Heparanase in the Vitreous of Patients With Proliferative Diabetic Retinopathy Originates From Activated Endothelial Cells and Leukocytes.

PURPOSE To determine and interrelate the levels of heparanase, syndecan-1, and VEGF in proliferative diabetic retinopathy (PDR), and to study the production of heparanase by human retinal microvascular endothelial cells (HRMEC) and its effect on HRMEC barrier function. METHODS Vitreous samples from 33 PDR and 27 nondiabetic patients, epiretinal membranes from 16 patients with PDR and HRMEC were studied by enzyme-linked immunosorbent assay, immunohistochemistry, and Western blot analysis. The effect of heparanase on HRMEC barrier function was evaluated by transendothelial electrical resistance. RESULTS We showed a significant increase in the expression of heparanase, syndecan-1, and VEGF in vitreous samples from PDR patients compared with nondiabetic controls (P < 0.0001 for all comparisons). Significant positive correlations were found between the levels of heparanase and the levels of syndecan-1 (r = 0.75, P < 0.0001) and VEGF (r = 0.91, P < 0.0001) and between the levels of syndecan-1 and the levels of VEGF (r = 0.78, P < 0.0001). In epiretinal membranes, heparanase was expressed in vascular endothelial cells and CD45-expressing leukocytes. High-glucose, tumor necrosis factor alpha (TNF-α), and the combination of TNF-α and interleukin (IL)-1β, but not cobalt chloride induced upregulation of heparanase in HRMEC. Heparanase-reduced transendothelial electrical resistance of HRMEC. CONCLUSIONS Our findings suggest a link between heparanase, syndecan-1, and VEGF in the progression of PDR and that heparanase is a potential target for therapy of diabetic retinopathy.

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

BackgroundThe 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).MethodsVitreous samples from 34 PDR and 29 nondiabetic patients were studied by biochemical and enzyme-linked immunosorbent assays and Western blot analysis.ResultsPA, 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.ConclusionsOur 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 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)-1β 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.

Upregulation of Thrombin/Matrix Metalloproteinase-1/Protease-Activated Receptor-1 Chain in Proliferative Diabetic Retinopathy

ABSTRACT Purpose: Selective proteolytic activation of protease-activated receptor-1 (PAR1) by thrombin and matrix metalloproteinase-1 (MMP-1) plays a central role in enhancing angiogenesis. We investigated the expression levels of thrombin, MMP-1, and PAR1 and correlated these levels with vascular endothelial growth factor (VEGF) in proliferative diabetic retinopathy (PDR). In addition, we examined the expression of PAR1 and thrombin in the retinas of diabetic rats and PAR1 in human retinal microvascular endothelial cells (HRMEC) following exposure to high-glucose, the proinflammatory cytokines interleukin-1β (IL-1β), tumor necrosis factor-α (TNF-α), and the hypoxia mimetic agent cobalt chloride (CoCl2). Methods: Vitreous samples from 32 PDR and 23 nondiabetic patients, epiretinal membranes from 10 patients with PDR, retinas of rats, and HRMEC were studied by enzyme-linked immunosorbent assay (ELISA), immunohistochemistry, and Western blot analysis. An assay for in vitro cell migration angiogenesis was performed in HRMEC. Results: In epiretinal membranes, PAR1 was expressed in vascular endothelial cells, CD45-expressing leukocytes, and myofibroblasts. ELISA and Western blot assays revealed significant increases in the expression levels of thrombin, MMP-1, and VEGF in vitreous samples from PDR patients compared to nondiabetic controls. Significant positive correlations were found between the levels of VEGF and the levels of thrombin (r = 0.41; p = 0.006) and MMP-1 (r = 0.66; p < 0.0001). Significant increases of cleaved PAR1 (approximately 50 kDa) and the proteolytically active thrombin (approximately 50 kDa) were detected in rat retinas after induction of diabetes. The proinflammatory cytokines IL-1β and TNF-α, but not high-glucose and CoCl2, induced upregulation of cleaved PAR1 (approximately 30 kDa) in HRMEC. In addition, thrombin and MMP-1 induced VEGF in HRMEC and vorapaxar, a PAR1 inhibitor, inhibited thrombin-induced migration in HRMEC. Conclusions: Interactions among thrombin, MMP-1, PAR1, and VEGF might facilitate angiogenesis in PDR.

Osteoprotegerin Is a New Regulator of Inflammation and Angiogenesis in Proliferative Diabetic Retinopathy

Purpose Osteoprotegerin (OPG) is a novel regulator of endothelial cell function, angiogenesis, and vasculogenesis. We correlated expression levels of OPG with those of the angiogenic and inflammatory factors vascular endothelial growth factor (VEGF) and monocyte chemoattractant protein-1 (MCP-1/CCL2) in proliferative diabetic retinopathy (PDR). We also examined expression of OPG in retinas from diabetic rats and diabetic patients and measured production of OPG by human retinal microvascular endothelial cells (HRMEC) and investigated its angiogenic activity. Methods Vitreous samples from 47 PDR and 28 nondiabetic patients, epiretinal membranes from 14 patients with PDR, human retinas (10 from diabetic patients and 10 from nondiabetic subjects), and rat retinas and HRMEC were studied by using enzyme-linked immunosorbent assay, immunohistochemistry, immunofluorescence, Western blot analysis, and RT-PCR. In vitro and in vivo angiogenesis assays were performed. Results We showed a significant increase in the expression of OPG, VEGF, and MCP-1/CCL2 in a comparison between vitreous samples from PDR patients and those from nondiabetic controls. Significant positive correlations were found between levels of OPG and levels of VEGF and MCP-1/CCL2. In epiretinal membranes, OPG was expressed in vascular endothelial cells and stromal cells. Significant increases of OPG mRNA and protein were detected in the retinas from diabetic patients. The proinflammatory cytokines TNF-α and IL-1β, but not VEGF, MCP-1/CCL2 or thrombin, induced upregulation of OPG in HRMEC. Osteoprotegerin induced ERK1/2 and Akt phosphorylation in HRMEC and stimulated their migration. Osteoprotegerin potentiated the angiogenic effect of VEGF in the in vivo protein gelatin plug assay. Conclusions These results suggest that OPG is involved in PDR angiogenesis.

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.

Myeloid-Related Protein-14/MRP-14/S100A9/Calgranulin B is Associated with Inflammation in Proliferative Diabetic Retinopathy

ABSTRACT Purpose: To investigate the expression of the leukocyte proteins myeloid-related protein (MRP)-8 and MRP-14 in proliferative diabetic retinopathy (PDR) and the effect of MRP-8/MRP-14 (calprotectin) heterodimer on induction of proinflammatory factors in human retinal microvascular endothelial cells (HRMEC). Methods: Epiretinal membranes from 20 patients with PDR and 10 patients with proliferative vitreoretinopathy (PVR), vitreous fluid samples from PDR and non-diabetic subjects and HRMEC were studied by immunohistochemistry and Western blot analysis. Results: MRP-14 expression was localized in endothelial cells, leukocytes and myofibroblasts in all PDR membranes. MRP-8 expression was limited to intravascular leukocytes in 42% of the studied membranes. In PVR membranes, MRP-14 was expressed in leukocytes and myofibroblasts, whereas MRP-8 immunoreactivity was limited to leukocytes. MRP-14 was significantly upregulated in vitreous from PDR patients. MRP-8/MRP-14 (calprotectin) increased expression of intercellular adhesion molecule-1, but attenuated vascular cell adhesion molecule-1 expression in HRMEC. Conclusions: Increased MRP-14 levels are associated with inflammation in PDR.

Association of 150‐kDa oxygen‐regulated protein with vascular endothelial growth factor in proliferative diabetic retinopathy

150‐kDa oxygen‐regulated protein (ORP150), a member of heat‐shock protein family located in endoplasmic reticulum (ER), has a critical role in secretion of vascular endothelial growth factor (VEGF). We investigated expression levels of ORP150 and correlated these levels with VEGF and total vitreous antioxidant capacity (TAC) in proliferative diabetic retinopathy (PDR). We also examined expression of ORP150 in retinas of diabetic rats and in human retinal microvascular endothelial cells (HRMEC).