Proliferative Diabetic Retinopathy Vitreous Research Articles

Increased Vitreous Heme Oxygenase Activity is Associated with Proliferative Diabetic Retinopathy

Heme oxygenase is an enzyme having three isoforms and is responsible for breakdown of heme into iron, carbon monoxide and bilirubin. There is no study related to heme oxygenase levels in human vitreous. So in this study our objectives were to measure heme oxygenase (HO) enzyme activity in vitreous and plasma of patients with proliferative diabetic retinopathy (PDR) and compare with the HO levels of patients with macular hole (MH). All protocols were approved by the Institution’s Ethics Board. Blood and vitreous samples were collected from 15 patients with PDR and 11 from macular hole who underwent vitrectomy surgery. HO enzyme activity was measured in vitreous and plasma samples by spectrophotometric method. In the PDR group, vitreous HO was 8.03 ± 1.95 pmol of bilirubin/mg protein which is significantly higher when compared to the MH group, 0.67 ± 0.2 pmol of bilirubin/mg protein. However, there was no difference in their HO activity in plasma, between the groups. Total Heme oxygenase enzyme activity is elevated in vitreous of PDR. Vitreous HO activity positively correlated with already reported adiponectin levels in these patients.

Angiogenic potential of vitreous from Proliferative Diabetic Retinopathy and Eales' Disease patients.

PurposeProliferative Diabetic Retinopathy (PDR) and Eales' Disease (ED) have different aetiologies although they share certain common clinical symptoms including pre-retinal neovascularization. Since there is a need to understand if the shared end-stage angiogenic pathology of PDR and ED is driven by common stimulating factors, we have studied the cytokines contained in vitreous from both patient groups and analyzed the angiogenic potential of these samples in vitro.Material and MethodsVitreous samples from patients with PDR (n = 13) and ED (n = 5) were quantified for various cytokines using a cytokine biochip array and sandwich ELISA. An additional group of patients (n = 5) with macular hole (MH) was also studied for comparison. To determine the angiogenic potential of these vitreous samples, they were analyzed for their ability to induce tubulogenesis in human microvascular endothelial cells. Further, the effect of anti-VEGF (Ranibizumab) and anti-IL-6 antibodies were studied on vitreous-mediated vascular tube formation.ResultsElevated levels of IL-6, IL-8, MCP-1 and VEGF were observed in vitreous of both PDR and ED when compared to MH. PDR and ED vitreous induced greater levels of endothelial cell tube formation compared to controls without vitreous (P<0.05). When VEGF in vitreous was neutralized by clinically-relevant concentrations of Ranibizumab, tube length was reduced significantly in 5 of 6 PDR and 3 of 5 ED samples. Moreover, when treated with IL-6 neutralizing antibody, apparent reduction (71.4%) was observed in PDR vitreous samples.ConclusionsWe have demonstrated that vitreous specimens from PDR and ED patients share common elevations of pro-inflammatory and pro-angiogenic cytokines. This suggests that common cytokine profiles link these two conditions.

Roles of elevated intravitreal IL-1β and IL-10 levels in proliferative diabetic retinopathy.

Purpose:To determine the roles of interleukin (IL)-1β and IL-10 in the vitreous of proliferative diabetic retinopathy (PDR).Materials and Methods:Vitreous samples were obtained from 26 eyes of 26 patients with PDR and from eight eyes of eight cases without PDR. The IL-1β and IL-10 concentration in the vitreous was measured by using an enzyme-linked immunosorbent assay (ELISA).Results:Levels of IL-1β and IL-10 in vitreous were higher in PDR patients compared with control group. And there was significantly negative correlation between IL-1β and IL-10 in control group (r = −0.795; P = 0.032), whereas there was no significant correlation in PDR group (r = 0.176; P = 0.391).Conclusion:Levels of IL-1β and IL-10 were upregulated in vitreous of PDR patients, and these two cytokines play roles in regulating the development and progression of PDR.

Identification of chemokines and growth factors in proliferative diabetic retinopathy vitreous.

Associations were investigated between levels of chemokines and growth factors in the vitreous and proliferative diabetic retinopathy (PDR). Enrolled were 58 patients (58 eyes) requiring pars plana vitrectomy (PPV), with PDR (n = 32, none with traction retinal detachment) or not (non-PDR). In the latter, 16 had macular hole (MH) and 10 had epiretinal membrane (ERM). With a multiplex bead immunoassay, levels of 11 chemokines and growth factors were measured from the undiluted vitreous sample from each patient. In the non-PDR eyes, the levels of the 11 chemokines and growth factors tested were similar between patients with MH and those with ERM. However, the levels of all 11 were significantly higher in the PDR eyes relative to the non-PDR; CCL17, CCL19, and TGFβ3 were markedly upregulated and have not been investigated in PDR previously. The significantly higher levels of CCL4 and CCL11 in PDR contradict the results of previous reports. Based on Spearman's nonparametric test, moderate-to-strong correlations were found between VEGF and other mediators. Our results indicate that these chemokines and growth factors could be candidates for research into targeted therapies applied either singly or in combination with anti-VEGF drugs for the treatment of PDR.

Identification of VEGF-Independent Cytokines in Proliferative Diabetic Retinopathy Vitreous

To identify inflammatory cytokines significantly elevated and independent of VEGF levels in the vitreous of proliferative diabetic retinopathy (PDR) patients that may serve as novel diagnostic factors or therapeutic targets. Thirty-nine cytokines and chemokines were measured from the vitreous of 72 patients undergoing vitrectomy (29 controls and 43 PDR) via a magnetic bead-based immunoassay. Patient information, including sex, age, history of smoking, cancer diagnosis and treatment, and presence of diabetes and hypertension were also collected. Univariate and multivariate logistic regression analyses were performed to assess the association of cytokine concentrations and patient demographics with disease. Nineteen cytokines were significantly elevated in the vitreous of PDR patients compared with controls, including five novel cytokines that have not previously been associated with PDR: sCD40L, GM-CSF, IFNα2, IL-12p40, and MCP-3. Sixteen cytokines were found to be statistically independent of VEGF. Of these, 14 show a statistically significant interaction with VEGF, while two do not. With regards to patient demographics, age and hypertension were statistically significant risk factors with the odds of disease decreasing with increasing age and increasing 3-fold for hypertensive patients. This is the first report of a comprehensive multiplex analysis to identify novel VEGF-independent cytokines associated with PDR. Of the 39 inflammatory cytokines tested, 16 are predictive of disease risk, independent of VEGF levels. These PDR-associated cytokines represent potential targets in the treatment of PDR, both in conjunction with anti-VEGF therapy, as well as for patients that are nonresponders to such therapy.

Differentiating vitreous proteomes in proliferative diabetic retinopathy using high-performance liquid chromatography coupled to tandem mass spectrometry

Proliferative diabetic retinopathy (PDR) is a serious microangiopathic complication of diabetes mellitus and a major cause of blindness in working-age adults. Diabetes-induced alterations in the vitreous protein composition in diabetic patients with PDR may be responsible for the presence of PDR. Therefore, we performed a comprehensive proteomic analysis and compared the protein profiles of vitreous humor from type 2 diabetic patients with PDR (n = 8) and that from normal human eyes donated for corneal transplant (n = 8). Using reversed phase high-performance liquid chromatography (RP-HPLC) coupled to electrospray Ionization tandem mass spectrometry (ESI-MS/MS), we identified 96 significant differentially expressed proteins (abundance ratio > 1.5, p < 0.05), including 37 and 59 proteins up- and downregulated in PDR vitreous compared with the control, respectively. Biological pathway analysis revealed 44 proteins involved in 56 biological pathways; among them, the most remarkable pathways differentially represented between PDR and normal vitreous were the glycolysis/gluconeogenesis, complement and coagulation cascades, gap junction, and phagosome pathways. The differential expressions of angiopoietin-related protein 6, apolipoprotein A-I, estrogen receptor alpha, and tubulin were confirmed by western blot analysis. These data provide insight into the molecular events possibly involved in the pathogenesis of PDR and widen the scope of potential avenues for new therapies for PDR.

Characterisation of the vitreous proteome in proliferative diabetic retinopathy

BackgroundDiabetes can lead to serious microvascular complications such as proliferative diabetic retinopathy (PDR), which results in severe vision loss. The diabetes-induced alterations in the vitreous protein composition in diabetic patients with PDR may be responsible for the presence of PDR. The vitreous humour can be utilised in a variety of studies aimed toward the discovery of new targets for the treatment or prevention of PDR and the identification of novel disease mechanisms. The aim of this study was to compare the protein profile of vitreous humour from diabetic patients with PDR with that of vitreous humour from normal human eyes donated for corneal transplant.ResultsVitreous humour from type 2 diabetic patients with PDR (n = 10) and from normal human eyes donated for corneal transplant (n = 10) were studied. The comparative proteomic analysis was performed using two-dimensional fluorescence difference gel electrophoresis (2-D DIGE). Differentially produced proteins (abundance ratio > 2 or < -2, p < 0.01) were identified by matrix-assisted laser desorption ionisation time-of-flight mass spectrometry (MALDI-TOF MS) and MALDI-TOF tandem mass spectrometry. A total of 1242 protein spots were detected on the 2-D master gel of the samples, and 57 spots that exhibited statistically significant variations were successfully identified. The spots corresponded to peptide fragments of 29 proteins, including 8 proteins that increased and 21 proteins that decreased in PDR. Excluding the serum proteins from minor vitreous haemorrhage, 19 proteins were found to be differentially produced in PDR patients compared with normal subjects; 6 of these proteins have never been reported to be differentially expressed in PDR vitreous: N(G),N(G)-dimethylarginine dimethylaminohydrolase 1 (DDAH 1), tubulin alpha-1B chain, gamma-enolase, cytosolic acyl coenzyme A thioester hydrolase, malate dehydrogenase and phosphatidylethanolamine-binding protein 1 (PEBP 1). The differential production of pigment epithelium-derived factor (PEDF) and clusterin was confirmed by Western blot analysis.ConclusionsThese data provide an in-depth analysis of the human vitreous proteome and reveal protein alterations that are possibly involved in the pathogenesis of PDR. Further investigation of these special proteins may provide potential new targets for the treatment or the prevention of PDR.

Increased IL‐6 levels in the vitreous of proliferative diabetic retinopathy (PDR)

AbstractPurpose To assess the activity of HIF‐1alpha and NF‐kappaB transcription factors and inflammation markers (IL‐6 and IL‐8) in the vitreous of patients suffering from proliferative diabetic retinopathy.Methods Twenty seven patients were enrolled for the study. The severity of retinopathy was classified (0, 1, 2, 3, 4) and the activity of neovascularization was graded (0, 1, 2, 3, 4) intraoperatively. Samples were collected during surgery and the NF‐κB and HIF‐1alpha and transcription factors activity and expression levels of IL‐6 and IL‐8 were measured.Results The majority of samples fell into the retinopathy classes 3 (n=12) or 4 (n=13). Neither the level of IL‐6 increased from 68.9 + 46.8 pg/ml to 102.7 + 94.1 pg/ml and IL‐8 from 165.1 + 136.0 pg/ml to 521.0 + 870.9 pg/ml (mean + SD, non‐significant change: normality test followed with Mann‐Whitney Rank Sum Test). According to the neovascularization activity the samples fell into the grades 1 (n=7), 2 (n=12) or 3 (n=7). In IL‐6 there was a statistically significant increase (p&lt;0.05) from grade 2 to 3: 58.6 + 40.3 pg/ml and 158.4 + 102.5 pg/ml, respectively (Kruskal‐Wallis One Way ANOVA on Ranks followed with Dunn’s Method). The level of IL‐8 was in grade 1: 118.0 + 62,4 pg/ml, in grade 2: 192.3 + 127.1 pg/ml and in grade 3: 884.3 + 1161.0 pg/ml (statistically non‐significant change). There was a statistically significant Linear Regression between IL‐6 and IL‐8 (P &lt;0.001): IL‐6 = 51.88 pg/ml + (0.092 * IL‐8), r=0.772. Increased activity of the HIF‐1alpha and NF‐κB transcription factors were not observed.Conclusion IL‐6 is a candidate to indicate activity of neovascularization process in PDR. It might be a new molecular therapeutic target to regulate innate immunity response in vitreous.

Increased IL-6 levels are not related to NF-κB or HIF-1α transcription factors activity in the vitreous of proliferative diabetic retinopathy

Purpose The purpose was to assess the activity of nuclear factor (NF)-κB and hypoxia inducible factor (HIF)-1α transcription factors and the expression levels of inflammation markers [interleukin (IL)-6 and IL-8] in the vitreous of patients suffering from proliferative diabetic retinopathy (PDR) scheduled for elective vitreous surgery in a single academic-based retina practice in a prospective clinical study. Methods Twenty-seven patients with PDR were enrolled in the study. The severity of retinopathy was classified (0, 1, 2, 3, 4) and the activity of neovascularization was graded (0, 1, 2, 3, 4) by the surgeon intraoperatively. Samples of the vitreous were collected during surgery, and the activity of NF-κB and HIF-1α transcription factors and the expression levels of IL-6 and IL-8 were measured. Results The majority of samples fell into the retinopathy class 3 ( n=12) or 4 ( n=13). The level of IL-6 increased from 68.9±46.8 pg/ml to 102.7±94.1 pg/ml, and IL-8 increased from 165.1±136.0 pg/ml to 521.0±870.9 pg/ml (mean±S.D., nonsignificant change: normality test followed with Mann–Whitney Rank Sum Test). According to the neovascularization activity, the samples fell into grade 1 ( n=7), 2 ( n=12) or 3 ( n=7). In IL-6, there was a statistically significant increase ( P<.05) from grade 2 to 3: 58.6±40.3 pg/ml and 158.4±102.5 pg/ml, respectively (Kruskal–Wallis One-Way Analysis of Variance on Ranks followed with Dunn's Method). The level of IL-8 was as follows: in grade 1: 118.0±62.4 pg/ml, in grade 2: 192.3±127.1 pg/ml and in grade 3: 884.3±1161.0 pg/ml (statistically nonsignificant change). There was a statistically significant linear regression between IL-6 and IL-8 ( P<.001): IL-6=51.88 pg/ml+(0.092⁎IL-8), r=0.772. Increased activity of the NF-κB and HIF-1α transcription factors was not observed. Conclusion Interleukin-6 is a candidate to indicate activity of neovascularization process in PDR. It might be a new molecular therapeutic target to regulate innate immunity response in vitreous.

Homocysteine Levels in the Vitreous of Proliferative Diabetic Retinopathy and Rhegmatogenous Retinal Detachment: Its Modulating Role on Lysyl Oxidase

Homocysteine (Hcys), a well-known inducer of vascular endothelial cell damage has been associated with extracellular matrix changes. Lysyl oxidase (LOX) is a copper-dependent amine oxidase that initiates the covalent cross-linking of collagen and elastin in the extracellular matrix (ECM). LOX contributes to the structural integrity of the ECM, and low LOX activity could promote ECM disorganization. Hydroxyproline levels are used to predict collagen turnover status, and most of the endogenous hydroxyproline present in biological fluids is derived from the degradation of various forms of collagen. As Hcys is known to regulate ECM turnover and also inhibit LOX activity, the purpose of this study was to estimate the vitreous levels of Hcys in eyes with proliferative diabetic retinopathy (PDR) and rhegmatogenous retinal detachment (RRD) and to correlate the effect of Hcys, if any on LOX activity. Undiluted human vitreous specimens obtained during vitreoretinal surgeries for PDR (n = 18) and RRD (n = 17) were used. Vitreous specimens from donor eyeballs were used as control (n = 19). Hcys was estimated by HPLC using a fluorescent detector. Hydroxyproline was estimated spectrophotometrically. The total vitreous Hcys level was found to be increased significantly in PDR (P = 0.011) and in RRD (P = 0.001) compared with that in control samples. Hydroxyproline was significantly increased in PDR (P = 0.049) and RRD (P = 0.007) compared with the level in control samples. There was a significant negative correlation between the Hcys level and the specific activity of LOX in PDR (P = 0.040) and in RRD (P = 0.029) This report shows that increased vitreous Hcys in PDR and RRD is associated with a significant decrease in LOX-specific activity along with an increase in collagen turnover.

Lysyl Oxidase Activity in the Ocular Tissues and the Role of LOX in Proliferative Diabetic Retinopathy and Rhegmatogenous Retinal Detachment

Lysyl oxidase (LOX) cross-links the side chain of collagen and elastin and thereby contributes to extracellular matrix (ECM) integrity. ECM remodeling is seen in various ocular diseases. Until now, there have been no reports on the LOX enzyme's activity in ocular tissues. The purpose of this study was to estimate LOX activity and expression in human donor ocular tissues and to measure the specific activity of LOX in the vitreous of proliferative diabetic retinopathy (PDR) and rhegmatogenous retinal detachment (RRD). Human donor eyeballs obtained from an eye bank were used to study tissue distribution of LOX. Human vitreous specimens were obtained during vitreoretinal surgery from PDR (n = 16) and RRD (n = 10). LOX activity was estimated by N-acetyl-3,7-dihydroxyphenoxazine assay, immunohistochemistry, and real-time polymerase chain reaction (RT-PCR). Matrix metalloprotease (MMP)-2 and -9 were quantified in the vitreous by sandwich enzyme-linked immunosorbent assay (ELISA). The specific activity of LOX in ocular tissues was on the order of vitreous, iris ciliary body, lens, choroid RPE, and retina, which were comparable by mRNA expression and immunolocalization. The vitreous level of LOX activity decreased significantly in PDR and RRD, with an increase in total MMP-2 and -9 levels compared with normal donor vitreous. LOX activity showed a statistically significant decrease in the vitreous of PDR and RRD relative to control specimens. This effect can contribute to the inadequate collagen cross-linking that causes the ECM changes that occur in these diseases.

Characterization of the Vitreous Proteome in Diabetes without Diabetic Retinopathy and Diabetes with Proliferative Diabetic Retinopathy

An understanding of the diabetes-induced alterations in vitreous protein composition in the absence and in the presence of proliferative diabetic retinopathy (PDR) may provide insights into factors and mechanisms responsible for this disease. We have performed a comprehensive proteomic analysis and comparison of vitreous samples from individuals with diabetes but without diabetic retinopathy (noDR) or with PDR and nondiabetic individuals (NDM). Using preparative one-dimensional SDS-PAGE and nano-LC/MS/MS of 17 independent vitreous samples, we identified 252 proteins from human vitreous. Fifty-six proteins were differentially abundant in noDR and PDR vitreous compared with NDM vitreous, including 32 proteins increased and 10 proteins decreased in PDR vitreous compared with NDM vitreous. Comparison of noDR and PDR groups revealed increased levels of angiotensinogen and decreased levels of calsyntenin-1, interphotoreceptor retinoid-binding protein, and neuroserpin in PDR vitreous. Biological pathway analysis revealed that vitreous contains 30 proteins associated with the kallikrein-kinin, coagulation, and complement systems. Five of them (complement C3, complement factor I, prothrombin, alpha-1-antitrypsin, and antithrombin III) were increased in PDR vitreous compared with NDM vitreous. Factor XII was detected in PDR vitreous but not observed in either NDM or noDR vitreous. PDR vitreous also had increased levels of peroxiredoxin-1 and decreased levels of extracellular superoxide dismutase, compared with noDR or NDM vitreous. These data provide an in depth analysis of the human vitreous proteome and reveal protein alterations that are associated with PDR.

Novel role of erythropoietin in proliferative diabetic retinopathy

Diabetic retinopathy is a leading cause of visual disturbance in adults. In proliferative diabetic retinopathy, ischemia-induced pathologic growth of new blood vessels often causes catastrophic loss of vision. Besides VEGF, the existence of another potent ischemia-induced angiogenic factor is postulated. Since ischemia-inducible erythropoietin (Epo) has recently been identified its angiogenic properties, we investigated its potential role during retinal angiogenesis in proliferative diabetic retinopathy (PDR). The vitreous Epo level in patients with PDR was significantly higher than that in nondiabetic patients. Multivariate logistic regression analyses indicated that Epo and VEGF were independently associated with PDR and that Epo was more strongly associated with PDR than VEGF. Blockade of Epo inhibits retinal neovascularization in vivo, and inhibits endothelial cell proliferation response to PDR vitreous in vitro. Our data provide strong evidence that erythropoietin is a potent retinal angiogenic factor independent of VEGF and is capable of stimulating ischemia-induced retinal angiogenesis in proliferative diabetic retinopathy. Inhibition of such molecular mechanisms in the retinal angiogenesis could be a new therapeutical strategy in halting or preventing pathologic angiogenesis in diabetic retinopathy.

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 (<5 min) 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 ± 4.09 μM, n = 27) versus controls (15.22 ± 0.86 μM, 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.

Interleukin (IL)-6, Interleukin (IL)-8 Levels and Cellular Composition of the Vitreous Humor in Proliferative Diabetic Retinopathy, Proliferative Vitreoretinopathy, and Traumatic Proliferative Vitreoretinopathy

Purpose: To investigate the interleukin (IL)-6 levels, IL-8 levels, and cellular composition of the vitreous humor in patients with proliferative diabetic retinopathy (PDR), proliferative vitreoretinopathy (PVR), and traumatic PVR. Methods: Vitreous samples from 14 patients with PDR, 10 patients with PVR, and 10 patients with traumatic PVR were analyzed. Fifteen cadaver eyes were used as controls. Cytokine levels were measured by ELISA. Results: Elevated IL-6 levels were detected in the vitreous of 12 (85.7%) of the PDR patients, eight (80%) of the PVR patients, and all (100%) of the traumatic PVR patients. None of the control IL-6 results were elevated. Vitreous IL-8 levels were elevated in 12 (85.7%) of the PDR patients, six (60%) of the PVR patients, all (100%) of the traumatic PVR patients, and one (6.7%) of the control eyes. Cytological examination of the vitreous specimens revealed a predominance of macrophages (50%) in the PDR samples and a predominance of retinal pigment epithelial (RPE) cells (60%) in the PVR samples. In contrast, neutrophils predominated (88%) in the traumatic PVR samples. Conclusion: The findings suggest that IL-6 and IL-8 may be involved in the pathogenesis of PDR, PVR, and traumatic PVR. High proportions of RPE cells and macrophages are associated with elevated IL-6 and IL-8 levels in the vitreous of PDR and PVR patients; however, the fact that these cells are not predominant in traumatic PVR suggests that different immune response mechanisms may be active in the pathogenesis of these disorders.