Profiling of vitreous proteomes from proliferative diabetic retinopathy and nondiabetic patients

Diabetes can lead to serious microvascular complications like proliferative diabetic retinopathy (PDR), which is the leading cause of blindness in adults. The proteomic changes that occur during PDR cannot be measured in the human retina for ethical reasons, but could be reflected by proteomic changes in vitreous humor. Thus, we considered that comparisons between the proteome profiles of the vitreous humors of PDR and nondiabetic controls could lead to the discovery of novel pathogenic proteins and clinical biomarkers. In this study, the authors used several proteomic methods to comprehensively examine vitreous humor proteomes of PDR patients and nondiabetic controls. These methods included immunoaffinity subtraction (IS)/2-DE/MALDI-MS, nano-LC-MALDI-MS/MS, and nano-LC-ESI-MS/MS. The identified proteins were subjected to the Trans-Proteomic Pipeline validation process. Resultantly, 531 proteins were identified, i.e., 415 and 346 proteins were identified in PDR and nondiabetic control vitreous humor samples, respectively, and of these 531 proteins, 240 were identified for the first time in this study. The PDR vitreous proteome was also found to contain many proteins possibly involved in the pathogenesis of PDR. The proteins described provide the most comprehensive proteome listing in the vitreous humor samples of PDR and nondiabetic control patients.     

Chemokines in proliferative diabetic retinopathy and proliferative vitreoretinopathy

PURPOSE: To determine levels of the chemokines CCL1/I-309, CCL2/MCP-1, CCL3/MIP-1alpha, CCL4/MIP-1beta, CCL7/MCP-3, CCL8/MCP-2, CXCL5/ENA-78, CXCL6/GCP-2, CXCL10/IP-10, and CXCL11/I-TAC in the vitreous humor and serum, from patients with proliferative diabetic retinopathy (PDR), proliferative vitreoretinopathy (PVR), and rhegmatogenous retinal detachment with no PVR (RD), and to investigate the expression of MCP-1, CXCL12/SDF-1, and the chemokine receptor CXCR3 in epiretinal membranes. METHODS: Paired vitreous humor and serum samples were obtained from patients undergoing vitrectomy for the treatment of RD (57 specimens), PVR (32 specimens), and PDR (88 specimens). The levels of chemokines were measured by enzyme-linked immunosorbent assays. Eighteen PDR and 5 PVR membranes were studied by immunohistochemical techniques. RESULTS: Of all the chemokines studied, only MCP-1 and IP-10 were detected in vitreous humor samples. MCP-1 levels in vitreous humor samples were significantly higher than in serum samples (p < 0.001). MCP-1 levels were significantly higher in vitreous humor samples from patients with PVR and PDR compared with RD (p = 0.0002). MCP-1 levels in vitreous humor samples from patients with active PDR were significantly higher than in inactive PDR cases (p = 0.0224). IP-10 levels in vitreous humor samples were significantly higher than in serum samples (p = 0.0035). IP-10 levels were significantly higher in vitreous humor samples from patients with PVR and PDR compared with RD (p = 0.0083). The incidence of IP-10 detection in vitreous humor samples was significantly higher in active PDR cases compared with inactive cases (p = 0.0214). There was a significant association between the incidence of IP-10 detection and increased levels of MCP-1 in vitreous humor samples from all patients, and patients with RD and PDR (p < 0.001 for all comparisons). MCP-1, and SDF-1 were localized in myofibroblasts in PVR and PDR membranes and in vascular endothelial cells in PDR membranes. CXCR3 was expressed by vascular endothelial cells in PDR membranes. CONCLUSION: MCP-1, IP-10 and SDF-1 may participate in pathogenesis of PVR and PDR. Myofibroblasts and vascular endothelial cells are the major cell types expressing MCP-1, SDF-1, and CXCR3 in epiretinal membranes.     

Identification of an anti-aldolase autoantibody as a diagnostic marker for diabetic retinopathy by immunoproteomic analysis

Circulating autoantibodies specific for retinal proteins are associated with retinal destruction in patients with diabetic retinopathy (DR). In this study, we screened diabetic sera for the presence of anti-retinal autoantibodies with an aim of developing diagnostic markers for DR. Immunoblot analysis of DR patients' sera with human retinal cytosolic proteins revealed a higher incidence of anti-retinal autoantibodies, compared to normal blood donors or diabetic patients without DR. Anti-retinal protein autoantibody profiles of DR patient sera were obtained by 2-DE immunoblot analysis. Specifically, 20 protein spots reactive with DR patient sera were identified by ESI-MS/MS. Of these spots, 14 were specific for DR patients, and 4 reacted with both non-proliferative DR (non-PDR) and PDR sera. The anti-aldolase autoantibody was selected as a DR marker candidate, and specific reactivity of DR patient sera was confirmed by immunoblot analysis with rabbit aldolase. The serum anti-aldolase autoantibody level was measured by ELISA. DR patients showed significantly higher autoantibody levels than normal donors or diabetic patients without retinopathy. However, no significant differences were observed between non-PDR and PDR patients, suggesting that the level of anti-aldolase autoantibody is not determined by the severity of retinopathy in diabetic patients. Our data collectively demonstrate that the anti-aldolase autoantibody serves as a useful marker for DR diagnosis.     

Periostin, discovered by nano-flow liquid chromatography and mass spectrometry, is a novel marker of diabetic retinopathy

Diabetes can lead to serious microvascular complications including proliferative diabetic retinopathy (PDR), the leading cause of blindness in adults. Recent studies using gene array technology have attempted to apply a hypothesis-generating approach to elucidate the pathogenesis of PDR, but these studies rely on mRNA differences, which may or may not be related to significant biological processes. To better understand the basic mechanisms of PDR and to identify potential new biomarkers, we performed shotgun liquid chromatography (LC)/tandem mass spectrometry (MS/MS) analysis on pooled protein extracts from neovascular membranes obtained from PDR specimens and compared the results with those from non-vascular epiretinal membrane (ERM) specimens. We detected 226 distinct proteins in neovascular membranes and 154 in ERM. Among these proteins, 102 were specific to neovascular membranes and 30 were specific to ERM. We identified a candidate marker, periostin, as well as several known PDR markers such as pigment epithelium-derived factor (PEDF). We then performed RT-PCR using these markers. The expression of periostin was significantly up-regulated in proliferative membrane specimens. Periostin induces cell attachment and spreading and plays a role in cell adhesion. Proteomic analysis by LC/MS/MS, which permits accurate quantitative comparison, was useful in identifying new candidates such as periostin potentially involved in the pathogenesis of PDR.     

Elevated plasma CD105 and vitreous VEGF levels in diabetic retinopathy

Diabetic retinopathy is the leading cause of blindness in the industrialized world. Hyperglycaemia induces retinal hypoxia that upregulates a range of vasoactive factors which may lead to macular oedema and/or angiogenesis and hence potentially sight threatening retinopathy. In this study, we have focused on the association of CD105 and vascular endothelial growth factor (VEGF) with the development and progression of diabetic retinopathy by means of quantifying their expression in the plasma and vitreous of diabetic patients. CD105 levels were quantified in the plasma of 38 type I diabetic patients at various stages of retinopathy and 15 non-diabetic controls. In an additional cohort of 11 patients with advanced proliferative retinopathy and 23 control subjects, CD105 and VEGF were measured in the vitreous. The values were expressed as median (range) and statistical analysis was carried out using the non-parametric Mann-Whitney U test. Plasma CD105 levels were significantly increased in diabetic patients [1.8 (1.1-2.4) ng/ml] compared with non-diabetic controls [0.7 (0.3-1.8) ng/ml] (p<0.01). Plasma CD105 levels were elevated in diabetic patients with all stages of retinopathy, the highest level was observed in background retinopathy [2.3 (2.1-2.5) ng/ml] followed by proliferative retinopathy [2.1 (0.9-2.8) ng/ml] and advanced proliferative retinopathy [1.4 (0.6-1.8) ng/ml]. Vitreous contents of CD105 did not differ between controls and patients with advanced proliferative retinopathy, but vitreous levels of VEGF were elevated by approximately 3-fold in patients with advanced proliferative retinopathy [7.2 (1.90-15.60) ng/ml] compared with the control subjects [1.80 (1.10-2.210)] (p<0.01). These observations indicate that plasma levels of CD105 and vitreous levels of VEGF are associated with diabetic retinopathy, suggesting that CD105 and the angiogenic factor VEGF may play a critical role in the development and progression of diabetic retinopathy. Further studies are required to determine whether circulating CD105 levels could serve as a surrogate marker for early stage retinopathy and for monitoring disease progression.     

Identification of connexin43 in diabetic retinopathy and its downregulation by O-GlcNAcylation to inhibit the activation of glial cells

BackgroundDespite advances in the treatments of diabetic complications, proliferative diabetic retinopathy (PDR) still remains a major cause leading to visual loss, mainly because of the lack of pathological mechanisms and complicated protein expressions in vivo. Current study aimed to investigate the patterns of connexin43 (Cx43) changes and the possible interactions with O-GlcNAcylation in DR.MethodsClinical samples of vitreous and fibrovascular membranes were acquired from PDR patients during pars plana vitrectomy. Brown Norway rats were used to build diabetic animal models; to investigate the effects of O-GlcNAcylation on Cx43 expressions, total retinal O-GlcNAcylation was changed by intravitreal injections. Levels of protein expressions were examined by immunofluorescence staining and western blot.ResultsOur results revealed increased Cx43 expressions in a vessel-shape pattern followed by the distribution of glial fibrillary acidic protein (GFAP) in diabetic fibrovascular membranes. Similarly, Cx43 and GFAP expressions were elevated in PDR vitreous and diabetic animal retinas. Retinal O-GlcNAcylation was effectively regulated by intravitreal injections, and the increase of Cx43 and GFAP was significantly suppressed by O-GlcNAcylation inhibition under hyperglycemia conditions.ConclusionsWe systemically proved the changes of Cx43 with different retinal cells, and reported the effective methods to regulate retinal O-GlcNAcylation by intravitreal injections, and clearly illustrated the downregulated effects of O-GlcNAcylation inhibition on Cx43 and GFAP expressions.General significance:Targeting connexin43 in glial cells reveals a novel mechanism to understand the formation of diabetic fibrovascular membranes and offers a potential therapeutic strategy to interfere the development of PDR.     

A comparison of the levels of hepatocyte growth factor in serum in patients with type 1 diabetes mellitus with different stages of diabetic retinopathy

INTRODUCTION: The aim of this study was to evaluate the blood concentration of hepatocyte growth factor (HGF) in patients at various stages of retinopathy. We hypothesised that the high level of HGF found in diabetic patients may be an important marker of retinopathy progression and that HGF level may be an index of the risk of proliferative retinopathy. MATERIAL AND METHODS: The participants in the study were 76 patients with type 1 diabetes mellitus. Of these, 35 patients were without retinopathy and formed Group 1. Of the remaining 41 patients with retinopathy, 20 patients had non-proliferative diabetic retinopathy (NPDR) and formed Group 2, while 21 patients had proliferative diabetic retinopathy (PDR) and formed Group 3. We evaluated the concentration of HGF In the peripheral blood by an enzyme-linked immunosorbent assay. RESULTS: Mean serum concentrations of HGF in the control group were significantly lower than in the type 1 diabetic patients. We found a significant increase in HGF serum concentrations in diabetic patients with PDR compared with the control group. Mean serum HGF concentrations were significantly higher in diabetic subjects with PDR than in diabetic patients without retinopathy. CONCLUSION: HGF concentration is increased in patients with type 1 diabetes mellitus with proliferative retinopathy, and concentrations increase with the progression of retinopathy, suggesting that HGF plays a role in the pathogenesis of proliferative diabetic retinopathy.     

Extracellular carbonic anhydrase mediates hemorrhagic retinal and cerebral vascular permeability through prekallikrein activation

Excessive retinal vascular permeability contributes to the pathogenesis of proliferative diabetic retinopathy and diabetic macular edema, leading causes of vision loss in working-age adults. Using mass spectroscopy-based proteomics, we detected 117 proteins in human vitreous and elevated levels of extracellular carbonic anhydrase-I (CA-I) in vitreous from individuals with diabetic retinopathy, suggesting that retinal hemorrhage and erythrocyte lysis contribute to the diabetic vitreous proteome. Intravitreous injection of CA-I in rats increased retinal vessel leakage and caused intraretinal edema. CA-I-induced alkalinization of vitreous increased kallikrein activity and its generation of factor XIIa, revealing a new pathway for contact system activation. CA-I-induced retinal edema was decreased by complement 1 inhibitor, neutralizing antibody to prekallikrein and bradykinin receptor antagonism. Subdural infusion of CA-I in rats induced cerebral vascular permeability, suggesting that extracellular CA-I could have broad relevance to neurovascular edema. Inhibition of extracellular CA-I and kallikrein-mediated innate inflammation could provide new therapeutic opportunities for the treatment of hemorrhage-induced retinal and cerebral edema.     

Capillary electrophoretic assay for nitrate levels in the vitreous of proliferative diabetic retinopathy

The determination of nitric oxide (NO) in human vitreous samples is complicated by the relatively short half-life of the analyte and the viscous, high salt and protein biological matrix. In this work, we developed a fast (<5min) and useful CE method to determine the stable metabolite, nitrate, from vitreous samples. This proposed method has been successfully applied to determine the nitrate levels from the vitreous humor of patients undergoing vitrectomy for a variety of conditions. A statistically significant increase (P=0.000001) of the mean level of nitrate was observed in vitreous humor of patients with proliferative diabetic retinopathy (41.17+/-4.09microM, n=27) versus controls (15.22+/-0.86microM, n=35). The elevated levels of nitrate in the vitreous of patients known to have diabetic retinopathy suggests that NO is involved with the pathology of this disease.     

Roles of endothelin-1 and selected proinflammatory cytokines in the pathogenesis of proliferative diabetic retinopathy: Analysis of vitreous samples

PurposeTo investigate the roles of endothelin-1(ET-1), TNF-α, IL-6 in the pathogenesis of proliferative diabetic retinopathy (PDR) in type 2 diabetes.MethodsVitreous and blood serum samples were collected during vitrectomy from 19 patients with PDR and 15 patients who underwent vitrectomy for other reasons. The concentrations of ET-1, TNF-α, IL-6, vWF, sE-selectin were determined by ELISA.ResultsIntraocular and serous concentrations of ET-1, TNF-α, IL-6, vWF, sE-selectin were higher in patients with PDR than in the control group. The vitreous ET-1/plasma ET-1 ratios the group of diabetic patients and in the control group were similar. Also TNF-α, IL-6 vitreous/plasma ratio were not statistically different between the analysed groups. Correlation between intraocular ET-1 and TNF-α concentrations in patients with PDR and between the increases in both factors in the vitreous and HbA₁c concentration were shown. In the vitreous the increase in vWF depended on elevated levels of vWF in the serum. E-selectin concentration correlated with diastolic blood pressure.ConclusionThese data provide evidence of the activation of the local synthesis of ET-1, TNF-α, IL-6 in PDR. The relationship between the increase in vitreous ET-1, TNF-α concentrations and HbA₁c concentration is a important confirmation of the necessity to optimise diabetes treatment.     

Catalogue of soluble proteins in the human vitreous humor: comparison between diabetic retinopathy and macular hole

Two-dimensional gel electrophoresis and mass spectrometry were used to make a catalogue of soluble proteins in the human vitreous humor (VH). Fifty-one different proteins were identified on silver-stained two-dimensional (2D) gel patterns with VH proteins obtained from diabetic retinopathy and macular hole. Thirty of these have not been listed in the reported 2D profiles of plasma. Immunoglobulin (Ig), alpha1-antitrypsin, alpha2-HS glycoprotein,and complement C(4) fragment showed stronger spots in VH with diabetic retinopathy patient samples than those with macular hole. Pigment epithelium-derived factor, a potent inhibitor of angiogenesis in the cornea and vitreous, was clearly detected in VH with diabetes. It is impressive that the inhibitor increases in the vitreous with proliferative angiogenesis.     

Quantitative analysis of proteins in the tear fluid of patients with diabetic retinopathy

Diabetic retinopathy is the leading cause of new cases of legal blindness among adults in the developed countries. Approximately 40% of all people with diabetes have diabetic retinopathy and 5% of these have sight-threatening form. As the advanced stage, where there is a high risk for vision loss, can develop without any serious symptoms, sometimes it is hard to detect it. A non invasive method to detect biomarkers characteristic for diabetic retinopathy from the tear fluid was developed. Tear samples from diabetic patients with no retinopathy, non proliferative and proliferative stages of diabetic retinopathy were analyzed and the protein content of each sample was compared to the protein content of tear pool from healthy volunteers. The samples were labeled with iTRAQ fourplex labels and were analyzed with nanoHPLC coupled ESI-MS/MS mass spectrometry. The lipocalin 1, lactotransferrin, lacritin, lysozyme C, lipophilin A and immunoglobulin lambda chain were identified as possible biomarker candidates with significantly higher relative levels in the tear of patients with diabetic retinopathy.     

The angio-fibrotic switch of VEGF and CTGF in proliferative diabetic retinopathy

Background

In proliferative diabetic retinopathy (PDR), vascular endothelial growth factor (VEGF) and connective tissue growth factor (CTGF) cause blindness by neovascularization and subsequent fibrosis, but their relative contribution to both processes is unknown. We hypothesize that the balance between levels of pro-angiogenic VEGF and pro-fibrotic CTGF regulates angiogenesis, the angio-fibrotic switch, and the resulting fibrosis and scarring.

Methods/Principal Findings

VEGF and CTGF were measured by ELISA in 68 vitreous samples of patients with proliferative DR (PDR, N = 32), macular hole (N = 13) or macular pucker (N = 23) and were related to clinical data, including degree of intra-ocular neovascularization and fibrosis. In addition, clinical cases of PDR (n = 4) were studied before and after pan-retinal photocoagulation and intra-vitreal injections with bevacizumab, an antibody against VEGF. Neovascularization and fibrosis in various degrees occurred almost exclusively in PDR patients. In PDR patients, vitreous CTGF levels were significantly associated with degree of fibrosis and with VEGF levels, but not with neovascularization, whereas VEGF levels were associated only with neovascularization. The ratio of CTGF and VEGF was the strongest predictor of degree of fibrosis. As predicted by these findings, patients with PDR demonstrated a temporary increase in intra-ocular fibrosis after anti-VEGF treatment or laser treatment.

Conclusions/Significance

CTGF is primarily a pro-fibrotic factor in the eye, and a shift in the balance between CTGF and VEGF is associated with the switch from angiogenesis to fibrosis in proliferative retinopathy.     

Systemic levels contribute significantly to increased intraocular IGF-I, IGF-II and IGF-BP3 [correction of IFG-BP3] in proliferative diabetic retinopathy.

Increased intraocular levels of angiogenic growth factors such as insulin-like growth factor I (IGF-I) have been demonstrated in proliferative diabetic retinopathy (PDR). It is unclear whether increased leakage of the blood retina barrier or local synthesis primarily determine intraocular levels of IGFs in man, which is of special interest regarding possible therapeutic options with somatostatin analogues in PDR. This is the first study investigating parallelly serum and vitreous levels of IGF-I/II, IGF-BP3 and the liver-derived permeability marker albumin to determine in vivo the amount of circulation-derived intraocular IGFs. A control group without retinal proliferation and patients with PDR were compared. Levels of IGF-I/II, IGF-BP3 and albumin were determined by immunological methods. Vitreous levels of albumin were 2.2-fold elevated in patients with PDR (254.1 +/- 37.2mg/dl; n = 27; p = 0.0027) compared to controls (115.7 +/- 36.2mg/dl; n =10), whereas serum levels were slightly decreased in diabetes patients (5049 +/- 196 mg/dl vs. 4330 +/- 186 mg/dl; p = 0.0283). This was comparable to an increase of IGF-I/11 and IGF-BP3 in vitreous from PDR patients (IGF-I: 2.3 +/- 1.1 ng/ml p = 0.005. IGF-II: 37.9 +/- 4.9 ng/ml; p = 0.0003. IGF-BP3: 97.9 +/- 26.9 ng/ml; p = 0.0001; n = 34) compared to controls (IGF-I: 0.7 +/- 0.1 ng/ml. IGF-II: 21.3 +/- 4.2 ng/ml. IGF-BP3: 31.3 +/- 4.9 ng/ml: n = 19). Serum levels did not differ significantly among the groups regarding IGF-I, II and IGF-BP3. Intraocular albumin and IGF-I levels calculated as percentage of the respective serum levels correlated significantly (r = 0.42; p = 0.012). This study demonstrates that influx of IGF-I, II and IGF-BP3 in PDR quantitatively parallels influx of the liver derived serum protein albumin suggesting that leakage of the blood retina barrier and serum levels of IGF primarily determine intravitreal IGF levels rather than local synthesis. Suppression of systemic IGF levels by new, highly effective somatostatin-analogues therefore provides a promising approach to prevent PDR.     

Vitamin D insufficiency in diabetic retinopathy

ObjectiveTo assess the relationship between vitamin D status and diabetic retinopathy.MethodsA clinic-based, cross-sectional study was conducted at Emory University, Atlanta, Georgia. Overall, 221 patients were classified into 5 groups based on diabetes status and retinopathy findings: no diabetes or ocular disease (n = 47), no diabetes with ocular disease (n = 51), diabetes with no background diabetic retinopathy (n = 41), nonproliferative diabetic retinopathy (n = 40), and proliferative diabetic retinopathy (PDR) (n = 42). Patients with type 1 diabetes and those taking > 1,000 IU of vitamin D daily were excluded from the analyses. Study subjects underwent dilated funduscopic examination and were tested for hemoglobin A_1c, serum creatinine, and 25-hydroxyvitamin D [25(OH)D] levels between December 2009 and March 2010.ResultsAmong the study groups, there was no statistically significant difference in age, race, sex, or multivitamin use. Patients with diabetes had lower 25(OH)D levels than did those without diabetes (22.9 ng/mL versus 30.3 ng/mL, respectively; P < .001). The mean 25(OH)D levels, stratified by group, were as follows: no diabetes or ocular disease = 31.9 ng/mL; no diabetes with ocular disease = 28.8 ng/mL; no background diabetic retinopathy = 24.3 ng/ mL; nonproliferative diabetic retinopathy = 23.6 ng/mL; and PDR = 21.1 ng/mL. Univariate analysis of the 25(OH) D levels demonstrated statistically significant differences on the basis of study groups, race, body mass index, multivitamin use, hemoglobin A_1c, serum creatinine level, and estimated glomerular filtration rate. In a multivariate linear regression model with all potential confounders, only multivitamin use remained significant (P < .001).ConclusionThis study suggests that patients with diabetes, especially those with PDR, have lower 25(OH)D levels than those without diabetes. (Endocr Pract. 2012; 18:185-193)     

Substance P and secretoneurin in vitreous aspirates of patients with various vitreoretinal diseases

By means of highly sensitive radioimmunoassays, the levels of substance P (SP) and secretoneurin (SN) were detected in vitreous aspirates of patients with macular holes which served as controls, in patients with nonproliferative diabetic retinopathy (DR), active proliferative diabetic retinopathy (active PDR), inactive PDR, rhegmatogenous retinal detachment and proliferative vitreoretinopathy (PVR). Furthermore, SN-like immunoreactivities were characterized by reversed phase-HPLC. The concentration of SN was more than 20-fold higher in macular holes when compared with SP and reversed phase HPLC revealed evidence that the vitreous levels of SN represent authentic SN. SN was significantly decreased in patients with nonproliferative DR, active PDR and inactive PDR by more than 70% which seems to result from a reduced expression and/or secretion from the cilary epithelium and a reduced release from the retina both due to diabetes mellitus. By contrast SP was increased in rhegmatogenous retinal detachment most obviously due to an enhanced outflow of the peptide through retinal breaks. Despite their proangiogenic activities, SP and SN are unlikely to be involved in the pathogenesis of neovascularizations in DR because of their unchanged and reduced levels, respectively, but the low levels of both peptides may facilitate the regression of vasoproliferations following laser photocoagulation.     

Relationship between Vitreous Levels of Matrix Metalloproteinases and Vascular Endothelial Growth Factor in Proliferative Diabetic Retinopathy

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.     

Plasma angiogenesis and oxidative stress markers in patients with diabetic retinopathy

Abstract

Background: Neovascularization in the retina and hyperglycaemia-induced oxidative stress are implicated in the pathogenesis of diabetic retinopathy (DR). In this study, we hypothesized that the plasma angiogenic and oxidative stress markers associated with these derangements could aid in the screening of diabetic patients who are at an increased risk of developing retinopathy.

Methods: This study included normal (n?=?148), type2 diabetes without retinopathy (DNR; n?=?148), proliferative DR (PDR; n?=?74) and non-PDR (NPDR; n?=?148) subjects. Plasma concentrations of vascular endothelial growth factor-A (VEGF-A), hypoxia-inducible factor-1α (HIF-1α), matrix metalloproteinase-9 (MMP-9), pigment epithelium-derived factor (PEDF), nitric oxide (NO), soluble receptors for advanced glycation end products (sRAGE), malondialdehyde (MDA) and protein thiols were estimated.

Results: A statistically significant increase was observed in the plasma concentrations of pro-angiogenic factors and markers of oxidative stress in both retinopathy groups. By contrast, the concentrations of anti-angiogenic factors and antioxidants were decreased significantly in these groups. Receiver operating characteristic analysis indicated that the plasma thresholds of HIF-1α and PEDF can be suitable markers in case of NPDR. However, in PDR, HIF-1α, NO, MMP-9 and PEDF showed high sensitivity and specificity.

Conclusions: The factors associated with hypoxia, matrix degradation and angiogenic inhibition play a crucial role in predicting DR.     

ProMMP-9 (92 kDa gelatinase) in vitreous fluid of patients with proliferative diabetic retinopathy.

Matrix metalloproteinases (MMPs) are implicated in tissue destruction during various pathophysiologic conditions. The vitreous body is a gel-like extracellular matrix that undergoes liquefaction during aging and pathological processes. To investigate the pathogenic role of MMPs in proliferative diabetic retinopathy (PDR), we studied 73 eyes from PDR patients and 25 eyes from patients with non-diabetic ocular diseases. Vitreous MMPs were measured by zymography. Retinopathy was assessed by ophthalmoscopy and PDR was classified into 3 stages, 'naked', 'active', and 'quiescent'. Although proMMP-9 was expressed in only 8% (2/25) of non-diabetic patients, it was expressed in more than 80% (38/47) of 'active' PDR patients and still expressed in 60% (9/15) of those with 'quiescent' PDR. Vascular endothelial growth factor (VEGF) in vitreous fluids was undetectable (<0.16 ng/ml) in most of the non-diabetic patients, and was maximally elevated in the 'active' PDR patients (mean=2.20 ng/ml, range; 0.16-7.61), declining in patients with 'quiescent' PDR (1.04 ng/ml, 0.16-3.77). These results suggest that MMP-9 is one of the noteworthy factors in relation to the progress of PDR, as well as angiogenic cytokines such as VEGF.