Publications by authors named "Mohammad Mairaj Siddiquei"

35 Publications

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

Invest Ophthalmol Vis Sci 2021 07;62(9):32

University Hospitals, UZ Gasthuisberg, Leuven, Belgium.

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.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1167/iovs.62.9.32DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8300056PMC
July 2021

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

Front Immunol 2020 20;11:601639. Epub 2021 Jan 20.

Laboratory of Molecular Immunology, Department of Microbiology, Immunology and Transplantation, Rega Institute for Medical Research, University of Leuven, Leuven, Belgium.

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. 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.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3389/fimmu.2020.601639DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7854927PMC
June 2021

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

Mol Cell Biochem 2021 May 30;476(5):2099-2109. Epub 2021 Jan 30.

Department of Ophthalmology, College of Medicine, King Saud University, Riyadh, Saudi Arabia.

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 (CoCl) in the absence or presence of APO. Molecular analysis demonstrates that NOX4 is upregulated in CoCl-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; R = 0.488) and caspase-3 vs ROS (p = 0.04; R = 0.428); whereas a negative correlation in BDNF vs ROS (p = 0.009; R = 0.596) and SIRT1 vs ROS (p = 0.0003; R = 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.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1007/s11010-021-04071-yDOI Listing
May 2021

The Proinflammatory and Proangiogenic Macrophage Migration Inhibitory Factor Is a Potential Regulator in Proliferative Diabetic Retinopathy.

Front Immunol 2019 4;10:2752. Epub 2019 Dec 4.

Department of Microbiology and Immunology, Rega Institute for Medical Research, KU Leuven, Leuven, Belgium.

The macrophage migration inhibitory factor (MIF)/CD74 signaling pathway is strongly implicated in inflammation and angiogenesis. We investigated the expression of MIF and its receptor CD74 in proliferative diabetic retinopathy (PDR) to reveal a possible role of this pathway in the pathogenesis of PDR. Levels of MIF, soluble (s)CD74, soluble intercellular adhesion molecule-1 (sICAM-1) and vascular endothelial growth factor (VEGF) were significantly increased in the vitreous from patients with PDR compared to nondiabetic control samples. We detected significant positive correlations between the levels of MIF and the levels of sICAM-1 ( = 0.43; = 0.001) and VEGF ( = 0.7; < 0.001). Through immunohistochemical analysis of PDR epiretinal membranes, significant positive correlations were also found between microvessel density (CD31 expression) and the numbers of blood vessels expressing MIF ( = 0.56; = 0.045) and stromal cells expressing MIF ( = 0.79; = 0.001) and CD74 ( = 0.59; = 0.045). Similar to VEGF, MIF was induced in Müller cells cultured under hypoxic conditions and MIF induced phosphorylation of ERK1/2 and VEGF production in Müller cells. Intravitreal administration of MIF in normal rats induced increased retinal vascular permeability and significant upregulation of phospho-ERK1/2, NF-κB, ICAM-1 and vascular cell adhesion molecule-1 expression in the retina. MIF induced migration and proliferation of human retinal microvascular endothelial cells. These results suggest that MIF/CD74 signaling is involved in PDR angiogenesis.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3389/fimmu.2019.02752DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6904364PMC
October 2020

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

Ocul Immunol Inflamm 2020 May 12;28(4):575-588. Epub 2019 Aug 12.

Rega Institute for Medical Research, Department of Microbiology and Immunology, University of Leuven , Leuven, Belgium.

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.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1080/09273948.2019.1616772DOI Listing
May 2020

Galectin-1 studies in proliferative diabetic retinopathy.

Acta Ophthalmol 2020 Feb 18;98(1):e1-e12. Epub 2019 Jul 18.

Rega Institute for Medical Research, Department of Microbiology and Immunology, University of Leuven, KU Leuven, Leuven, Belgium.

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 (CoCl ) 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 CoCl -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.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1111/aos.14191DOI Listing
February 2020

Cross-Talk between Sirtuin 1 and the Proinflammatory Mediator High-Mobility Group Box-1 in the Regulation of Blood-Retinal Barrier Breakdown in Diabetic Retinopathy.

Curr Eye Res 2019 10 14;44(10):1133-1143. Epub 2019 Jun 14.

Department of Ophthalmology, College of Medicine, King Saud University , Riyadh , Saudi Arabia.

: High-mobility group box-1 (HMGB1) mediates inflammation and breakdown of blood-retinal barrier (BRB) in diabetic retina. Sirtuin-1 (SIRT1) has protective effects against inflammation and oxidative stress. The aim of this study was to investigate the interaction between HMGB1 and SIRT1 in regulating BRB breakdown in diabetic retina. : BRB breakdown was assessed in vivo with fluorescein isothiocyanate-conjugated dextran. Vitreous samples from 47 proliferative diabetic retinopathy (PDR) and 19 nondiabetic patients, and epiretinal membranes from 13 patients with PDR were studied by enzyme-linked immunosorbent assay and immunohistochemistry. Retinas from 4-week diabetic rats and from normal rats intravitreally injected with HMGB1 were studied by spectrophotometric assay, Western blot analysis, and RT-PCR. We also studied the effect of the HMGB1 inhibitor glycyrrhizin and the SIRT1 activator resveratrol on diabetes-induced biochemical changes in the retina. : HMGB1 levels in vitreous samples from PDR patients were significantly higher than in nondiabetic controls, whereas SIRT1 levels were significantly lower in vitreous samples from patients with inactive PDR than those in patients with active PDR and nondiabetic controls. In epiretinal membranes, SIRT1 was expressed in vascular endothelial cells and stromal cells. Diabetes and intravitreal injection of HMGB1 in normal rats downregulated SIRT1expression, whereas glycyrrhizin and resveratrol normalized diabetes-induced downregulation of SIRT1. Resveratrol significantly attenuated diabetes-induced downregulation of occludin and upregulation of HMGB1 and receptor for advanced glycation end products in the retina and breakdown of BRB. : Our findings suggest that a functional link between SIRT1 and HMGB1 is involved in regulating of BRB breakdown in diabetic retina.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1080/02713683.2019.1625406DOI Listing
October 2019

The Poly(ADP-Ribose)Polymerase-1 Inhibitor 1,5-Isoquinolinediol Attenuate Diabetes-Induced NADPH Oxidase-Derived Oxidative Stress in Retina.

J Ocul Pharmacol Ther 2018 09 18;34(7):512-520. Epub 2018 Jun 18.

1 Department of Ophthalmology, College of Medicine, King Saud University , Riyadh, Saudi Arabia .

Purpose: To examine the effects of poly(ADP-ribose)polymerase-1 (PARP-1) inhibitor 1,5-isoquinolinediol (IQ) on nicotinamide adenine dinucleotide phosphate (NADPH) oxidase-derived oxidative stress in diabetic retina.

Methods: Streptozotocin-induced diabetic rats were treated with IQ. The NADPH oxidase enzyme activity was determined by luminometer. Expression of gp, P and nitrated proteins was examined by western blot. Interaction between gp and P was determined by coimmunoprecipitation. Enzyme-linked immunosorbent assay was utilized to measure the level of retinal total antioxidant capacity. We also studied the effect of the IQ on hydrogen peroxide (HO)-induced cleavage of PARP-1 and caspase-3 in human retinal Müller glial cells.

Results: Treatment of retinal Müller cells with HO-induced PARP-1 and caspase-3 cleavage that was attenuated by IQ cotreatment. Diabetes upregulated PARP-1, NADPH oxidase enzyme activity, gp, P, nitrated protein expression and interaction between gp and P, and downregulated total antioxidant capacity in the retinas compared with nondiabetic rats. Administration of IQ did not affect the metabolic status of the diabetic rats, but it significantly attenuated diabetes-induced upregulation of NADPH oxidase enzyme activity and expressions of gp, P, and nitrated proteins and interaction between gp and P. In addition, IQ ameliorated diabetes-induced downregulation of total antioxidant capacity in the retina.

Conclusion: PARP-1 inhibition by IQ protects diabetic retina from NADPH oxidase-derived oxidative stress. Thus, inhibition of PARP-1 could have potential therapeutic value in preventing the development of diabetic retinopathy.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1089/jop.2017.0117DOI Listing
September 2018

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

Mol Vis 2018 18;24:394-406. Epub 2018 May 18.

Rega Institute for Medical Research, Department of Microbiology and Immunology, University of Leuven, KU Leuven, Leuven, Belgium.

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.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5957543PMC
November 2018

Rho-Associated Protein Kinase-1 Mediates the Regulation of Inflammatory Markers in Diabetic Retina and in Retinal Müller Cells.

Ann Clin Lab Sci 2018 Mar;48(2):137-145

Department of Ophthalmology, College of Medicine, King Saud University, Riyadh, Saudi Arabia.

Goal: To investigate the effects of blocking Rho kinase pathway on the expression of inflammatory signaling pathways in the retina of diabetic mice and in human retinal Müller glial cells stimulated with high-glucose to replicate hyperglycemia.

Procedures: Retinas from diabetic mice and human retinal Müller glial cells (MIO-M1) were studied. Western blot analysis, immunofluorescence, and enzyme-linked immunosorbent assay were utilized to study the effect of the Rho kinase inhibitor fasudil on the expression of Rho-associated protein kinase-1 (ROCK-1), extracellular signal-regulated kinases1&2(ERK ½), phosphorylated nuclear factor kappa-light-chain-enhancer of activated B cells (p-NF-κB), inducible nitric oxide synthase (iNOS), vascular endothelial growth factor (VEGF), and monocyte chemoattractant protein-1 (MCP-1/CCL2).

Results: Treatment of human retinal Müller cells with high-glucose induced significant upregulation of ROCK-1, VEGF, and MCP-1/CCL2. Fasudil co-treatment normalized the high-glucose-induced upregulation of these mediators. Similarly, fasudil attenuated high-glucose-induced enhanced immunoreactivity for ROCK-1 and VEGF. Diabetes induced upregulation of ROCK-1, p-ERK ½, p-NF-κB and iNOS expression in retinas of mice. Constant fasudil intake from the onset of diabetes did not affect the metabolic status of diabetic mice but it attenuated diabetes-induced upregulation of these inflammatory signaling pathways.

Conclusions: Our finding suggests that Rho-associated protein kinase-1 activation mediates regulation of inflammatory signaling pathways in diabetic retina.
View Article and Find Full Text PDF

Download full-text PDF

Source
March 2018

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

Mol Vis 2017 5;23:853-871. Epub 2017 Dec 5.

Diabetes and Metabolism Research Unit and CIBERDEM (ISCIII).Vall d'Hebron Research Institute. Barcelona, Spain.

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.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5723152PMC
May 2018

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

Acta Ophthalmol 2018 Jun 2;96(4):e460-e467. Epub 2017 Nov 2.

Department of Microbiology and Immunology, Rega Institute for Medical Research, University of Leuven, KU Leuven, Leuven, Belgium.

Purpose: 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).

Methods: Vitreous samples from 40 PDR and 20 non-diabetic patients, epiretinal membranes from 14 patients with PDR, retinas of rats and HRMEC were studied by enzyme-linked immunosorbent assay, immunohistochemistry and Western blot analysis.

Results: We showed a significant increase in expression of VEGF and ORP150 in vitreous samples from PDR patients compared with controls (p < 0.0001 for both comparisons). Total vitreous antioxidant capacity (TAC) levels were significantly lower in patients with PDR than those in controls (p < 0.0001). Vascular endothelial growth factor (VEGF) and ORP150 levels in PDR with active neovascularization were significantly higher than that in inactive PDR (p = 0.016; p = 0.011, respectively). A significant positive correlation was observed between levels of ORP150 and levels of VEGF (r = 0.42; p = 0.001). In epiretinal membranes, ORP150 was expressed in vascular endothelial cells and stromal cells. We also demonstrated colocalization of the nuclear cell proliferation marker Ki67 and ORP150 in endothelial cells of pathologic new blood vessels. 150-kDa oxygen-regulated protein (ORP150) levels were significantly increased in rat retinas after induction of diabetes. Vascular endothelial growth factor (VEGF) and the pro-inflammatory cytokines interleukin-6 (IL-6) and tumour necrosis factor-α (TNF-α) induced upregulation of ORP150 in HRMEC.

Conclusion: These results suggest a role for ORP150 in PDR angiogenesis.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1111/aos.13600DOI Listing
June 2018

Unbalanced Vitreous Levels of Osteoprotegerin, RANKL, RANK, and TRAIL in Proliferative Diabetic Retinopathy.

Ocul Immunol Inflamm 2018 15;26(8):1248-1260. Epub 2017 Sep 15.

d Rega Institute for Medical Research, Department of Microbiology and Immunology , University of Leuven, KU Leuven , Leuven , Belgium.

Purpose: We investigated the expression of the proinflammatory and proangiogenic factor osteoprotegerin (OPG) and its ligands, receptor activator of nuclear factor-κB ligand (RANKL), tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), and the receptor RANK in proliferative diabetic retinopathy (PDR).

Materials And Methods: Vitreous samples from PDR and nondiabetic control patients and epiretinal membranes from PDR patients were studied by enzyme-linked immunosorbent assay, immunohistochemistry, and Western blot analysis.

Results: Vascular endothelial growth factor, OPG, and soluble RANK levels in vitreous samples from PDR patients were significantly higher than that in nondiabetic controls. Soluble TRAIL levels were significantly lower in PDR patients than that in nondiabetic control, whereas soluble RANKL levels did not differ significantly. RANKL, RANK, and TRAIL were expressed in vascular endothelial cells, myofibroblasts, and CD45-expressing leukocytes in PDR epiretinal membranes.

Conclusions: Dysregulated expression of OPG/RANKL/RANK pathway and TRAIL might be related to inflammation and angiogenesis in PDR.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1080/09273948.2017.1343855DOI Listing
December 2018

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

Invest Ophthalmol Vis Sci 2017 06;58(7):3189-3201

Diabetes and Metabolism Research Unit and Centro de Investigación Biomédica en Red de Diabetes y Enfermedades Metabólica Asociada (CIBERDEM), Vall d'Hebron Research Institute, Barcelona, Spain.

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.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1167/iovs.16-20993DOI Listing
June 2017

Differential expression and localization of human tissue inhibitors of metalloproteinases in proliferative diabetic retinopathy.

Acta Ophthalmol 2018 Feb 9;96(1):e27-e37. Epub 2017 Apr 9.

Rega Institute for Medical Research, Department of Microbiology and Immunology, University of Leuven, KU Leuven, Leuven, Belgium.

Purpose: Tissue inhibitors of metalloproteinases (TIMPs) block the catalysis by matrix metalloproteinases (MMPs) and have additional biologic activities, including regulation of cell growth and differentiation, apoptosis, angiogenesis and oncogenesis. We investigated the expression levels of all the four human TIMPs and correlated these levels with those of MMP-9 and vascular endothelial growth factor (VEGF) in proliferative diabetic retinopathy (PDR).

Methods: Vitreous samples from 38 PDR and 21 nondiabetic control patients and epiretinal membranes from 14 patients with PDR and 10 patients with proliferative vitreoretinopathy (PVR) were studied by enzyme-linked immunosorbent assay, Western blot analysis and immunohistochemistry.

Results: Tissue inhibitor of metalloproteinases-1, TIMP-4, MMP-9 and VEGF levels were significantly higher in vitreous samples from PDR patients than in nondiabetic controls (p < 0.0001 for all comparisons), whereas TIMP-2 and TIMP-3 levels did not differ significantly. TIMP-1, TIMP-4, MMP-9 and VEGF levels in PDR with active neovascularization were significantly higher than those in inactive PDR (p < 0.0001, 0.001, 0.013, 0.004, respectively). Significant positive correlations existed between levels of TIMP-1 and levels of TIMP-4 (r = 0.37; p = 0.004), MMP-9 (r = 0.65; p < 0.0001) and VEGF (r = 0.59; p < 0.0001), between levels of TIMP-4 and levels of MMP-9 (r = 0.61; p < 0.0001) and VEGF (r = 0.62; p < 0.0001) and between levels of MMP-9 and VEGF (r = 0.62; p < 0.0001). TIMP-1 and TIMP-3 were expressed in vascular endothelial cells in PDR epiretinal membranes and in myofibroblasts and leucocytes in PDR and PVR epiretinal membranes.

Conclusion: The differential expression of TIMPs in PDR suggests that among the 4 TIMPs, TIMP-1 and TIMP-4 may be possible biomarkers of disease activity.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1111/aos.13451DOI Listing
February 2018

Extracellular matrix metalloproteinase inducer (EMMPRIN) is a potential biomarker of angiogenesis in proliferative diabetic retinopathy.

Acta Ophthalmol 2017 Nov 18;95(7):697-704. Epub 2016 Nov 18.

Laboratory of Immunobiology, Rega Institute for Medical Research, Department of Microbiology and Immunology, University of Leuven, KU Leuven, Leuven, Belgium.

Purpose: Extracellular matrix metalloproteinase inducer (EMMPRIN) promotes angiogenesis through matrix metalloproteinases (MMPs) and vascular endothelial growth factor (VEGF) production. We investigated the expression levels of EMMPRIN and correlated these levels with VEGF, MMP-1 and MMP-9 in proliferative diabetic retinopathy (PDR). In addition, we examined the expression of EMMPRIN in the retinas of diabetic rats and the effect of EMMPRIN on the induction of angiogenesis regulatory factors in human retinal microvascular endothelial cells (HRMECs).

Methods: Vitreous samples from 40 PDR and 19 non-diabetic patients, epiretinal membranes from 12 patients with PDR, retinas of rats and HRMECs were studied by enzyme-linked immunosorbent assay (ELISA), immunohistochemistry, Western blot analysis, zymography analysis and RT-PCR.

Results: We showed a significant increase in the expression of EMMPRIN, VEGF, MMP-1 and MMP-9 in vitreous samples from PDR patients compared with non-diabetic controls (p < 0.0001; p = 0.001; p = 0.009; p < 0.0001, respectively). Significant positive correlations were found between the levels of EMMPRIN and the levels of VEGF (r = 0.38; p = 0.003), MMP-1 (r = 0.36; p = 0.005) and MMP-9 (r = 0.46; p = 0.003). In epiretinal membranes, EMMPRIN was expressed in vascular endothelial cells and stromal cells. Significant increase of EMMPRIN mRNA was detected in rat retinas after induction of diabetes. EMMPRIN induced hypoxia-inducible factor-1α, VEGF and MMP-1 expression in HRMEC.

Conclusions: These results suggest that EMMPRIN/MMPs/VEGF pathway is involved in PDR angiogenesis.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1111/aos.13284DOI Listing
November 2017

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.

Ophthalmic Res 2017 25;57(3):150-160. Epub 2016 Aug 25.

Department of Ophthalmology, College of Medicine, King Saud University, Riyadh, Saudi Arabia.

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.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1159/000448115DOI Listing
April 2017

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

Curr Eye Res 2016 12 3;41(12):1590-1600. Epub 2016 Jun 3.

d Department of Microbiology and Immunology , Rega Institute for Medical Research, University of Leuven , KU Leuven , Belgium.

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 (CoCl).

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 CoCl, 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.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3109/02713683.2016.1141964DOI Listing
December 2016

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

Mol Vis 2016 30;22:424-35. Epub 2016 Apr 30.

Rega Institute for Medical Research, Department of Microbiology and Immunology, University of Leuven, KU Leuven, Leuven, Belgium.

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.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4851855PMC
November 2016

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

Invest Ophthalmol Vis Sci 2015 Dec;56(13):8239-47

Rega Institute for Medical Research, Department of Microbiology and Immunology, University of Leuven, KU Leuven, Leuven, Belgium.

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.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1167/iovs.15-18025DOI Listing
December 2015

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

J Physiol Biochem 2015 Sep 4;71(3):359-72. Epub 2015 Jun 4.

Department of Ophthalmology, College of Medicine, King Saud University, Riyadh, Saudi Arabia,

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.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1007/s13105-015-0416-xDOI Listing
September 2015

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

Invest Ophthalmol Vis Sci 2015 Feb 24;56(3):1956-64. Epub 2015 Feb 24.

Laboratory of Molecular Immunology, Rega Institute for Medical Research, Department of Microbiology and Immunology, University of Leuven, KU Leuven, Leuven, Belgium.

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.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1167/iovs.14-16144DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4365984PMC
February 2015

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

Ophthalmic Res 2015 7;53(3):122-30. Epub 2015 Feb 7.

Department of Ophthalmology, College of Medicine, King Saud University, Riyadh, Saudi Arabia.

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.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1159/000369300DOI Listing
September 2015

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

Lipids Health Dis 2014 Dec 11;13:187. Epub 2014 Dec 11.

Department of Ophthalmology, College of Medicine, King Saud University, Riyadh, Saudi Arabia.

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.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1186/1476-511X-13-187DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4293108PMC
December 2014

High-Mobility Group Box-1 Modulates the Expression of Inflammatory and Angiogenic Signaling Pathways in Diabetic Retina.

Curr Eye Res 2015 11;40(11):1141-52. Epub 2014 Dec 11.

a Department of Ophthalmology , College of Medicine, King Saud University, King Abdulaziz University Hospital , Riyadh , Saudi Arabia.

Purpose: The expression of high-mobility group box-1 (HMGB1) is upregulated in epiretinal membranes and vitreous fluid from patients with proliferative diabetic retinopathy and in the diabetic retina. HMGB1 mediates inflammation, breakdown of the blood-retinal barrier and apoptosis in the diabetic retina. Here, we investigated inflammatory and angiogenic signaling pathways activated by HMGB1 in diabetic retina.

Methods: Human retinal microvascular endothelial cells (HRMEC) and retinas from 1-month diabetic rats and normal rats intravitreally injected with HMGB1 were studied using RT-PCR, Western blot analysis and co-immunoprecipitation. We also studied the effect of the HMGB1 inhibitor glycyrrhizin on diabetes-induced biochemical changes in the retina.

Results: Diabetes and intravitreal injection of HMGB1 in normal rats induced significant upregulation of the mRNA levels of the chemokine stromal cell-derived factor-1 (SDF-1/CXCL12) receptor CXCR4 and protein levels of hypoxia-inducible factor-1α, early growth response-1, tyrosine kinase 2 and the CXCL12/CXCR4 chemokine axis. Constant glycyrrhizin intake from onset of diabetes did not affect the metabolic status of the diabetic rats, but it restored these increased mediators to control values. Stimulation of HRMEC with HMGB1 and intraviteral injection of HMGB1 significantly increased the expression of vascular endothelial growth factor (VEGF) and VEGF receptor-2. Co-immunoprecipitation studies showed that diabetes increased the interaction between CXCL12 and CXCR4 and between HMGB1 and receptor for advanced glycation end products (RAGE), but not between HMGB1 and the CXCL12/CXCR4 chemokine axis.

Conclusions: Our findings suggest that HMGB1 activates inflammatory and angiogenic signaling pathways in diabetic retina mediated by RAGE.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3109/02713683.2014.982829DOI Listing
June 2016

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

Mol Vis 2014 10;20:1209-24. Epub 2014 Sep 10.

Rega Institute for Medical Research, Department of Microbiology and Immunology, University of Leuven, KU Leuven, Belgium.

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.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4160443PMC
December 2015

Functional links between gelatinase B/matrix metalloproteinase-9 and prominin-1/CD133 in diabetic retinal vasculopathy and neuropathy.

Prog Retin Eye Res 2014 Nov 28;43:76-91. Epub 2014 Jul 28.

Rega Institute for Medical Research, Department of Microbiology and Immunology, University of Leuven, KU Leuven, Belgium. Electronic address:

Retinopathy, a common cause of blindness, is a hallmark of diabetes and depends on two pathological mechanisms: vasculopathy and neuropathy. Whereas vasculopathy is well understood and has been associated with changes in gelatinase B/matrix metalloproteinase-9 (MMP-9) and other vasculotropic factors, specific markers for diabetes-induced retinal neuropathy are not yet described. Neuropathy may result from damages to the blood-retinal barrier (BRB) and from loss of neuroprotective factors. We studied diabetes-induced changes in vascular, inflammatory and regenerative markers and demonstrated that MMP-9 was increased, whereas prominin-1/CD133 was decreased in retinal extracts. In vitro, MMP-9 specifically destroyed prominin-1/CD133. Streptozotocin-induced diabetes resulted in BRB breakdown as a sign of vasculopathy and in prominin-1/CD133 destruction in photoreceptors as an in situ parameter of diabetic neuropathy. Both in vivo phenotypes were completely reversed in single MMP-9 gene knockout mice, demonstrating that MMP-9 mediates both diabetes-induced retinal vasculopathy and neuropathy, with prominin-1/CD133 being a critical and specific substrate of MMP-9. This functional link between gelatinase B/MMP-9 and prominin-1/CD133 explains mechanistically both the vasculopathy and neuropathy of diabetic retinopathy and suggests that specific MMP-9 inhibition is an interesting therapeutic avenue to investigate.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.preteyeres.2014.07.002DOI Listing
November 2014

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

Mediators Inflamm 2014 10;2014:746415. Epub 2014 Mar 10.

Department of Ophthalmology, College of Medicine, King Saud University, Riyadh, Saudi Arabia.

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.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1155/2014/746415DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3964896PMC
December 2014

Relationship between vitreous levels of matrix metalloproteinases and vascular endothelial growth factor in proliferative diabetic retinopathy.

PLoS One 2013 31;8(12):e85857. Epub 2013 Dec 31.

Rega Institute for Medical Research, Department of Microbiology and Immunology, University of Leuven, KU Leuven, Belgium.

To investigate which matrix metalloproteinases (MMPs) are more likely to be involved in the angiogenic process in proliferative diabetic retinopathy (PDR), we measured the levels of MMPs in the vitreous fluid from patients with PDR and controls and correlated these levels with the levels of vascular endothelial growth factor (VEGF). Vitreous samples from 32 PDR and 24 nondiabetic patients were studied by mosaic multiplex MMPs enzyme-linked immunosorbent assay (ELISA), single ELISA, Western blot and zymography analysis. Epiretinal membranes from 11 patients with PDR were studied by immunohistochemistry. MMP-8 and MMP-13 were not detected. ELISA, Western blot and gelatin ymography assays revealed significant increases in the expression levels of MMP-1, MMP-7, MMP-9 and VEGF in vitreous samples from PDR patients compared to nondiabetic controls, whereas MMP-2 and MMP-3 were not upregulated in vitreous samples from PDR patients. Significant correlations existed between ELISA and zymography assays for the quantitation of MMP-2 (r=0.407; p=0.039) and MMP-9 (r=0.711; p<0.001). Significant correlations were observed between levels of VEGF and levels of MMP-1 (r=0.845; P<0.001) and MMP-9 (r=0.775; p<0.001), and between levels of MMP-1 and MMP-9 (r=0.857; p<0.001). In epiretinal membranes, cytoplasmic immunoreactivity for MMP-9 was present in vascular endothelial cells and stromal monocytes/macrophages and neutrophils. Our findings suggest that among the MMPs measured, MMP-1 and MMP-9 may contribute to the angiogenic switch in PDR.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0085857PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3877391PMC
August 2014

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

Mediators Inflamm 2013 21;2013:510451. Epub 2013 Nov 21.

Department of Ophthalmology, College of Medicine, King Saud University, P.O. Box 245, Riyadh 11411, Saudi Arabia.

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.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1155/2013/510451DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3857786PMC
October 2014
-->