A potential novel therapeutic target in diabetic retinopathy: a chemokine receptor (CCR2/CCR5) inhibitor reduces retinal vascular leakage in an animal model.

PURPOSE: We have previously shown that the chemokine CCL2 plays an important role in monocyte trafficking into the retina and alteration of the BRB in an animal model of diabetic retinopathy. In this study, we examined the effect of pharmacologically targeting the chemokine pathway to reduce the increased retinal vascular permeability in this model. METHODS: C57BL/6 J mice were made diabetic using streptozotocin. After 4 months of diabetes, mice (n = 10) were treated by intraperitoneal injections of TAK-779 (dual CCR2/CCR5 inhibitor) (30 mg/kg) daily for 2 weeks. Retinal vascular permeability and protein expression were done using western blot. The SDF-1 levels were measured by ELISA. Immune cell infiltration in the retinas was measured using flow cytometry. RESULTS: The dual inhibitor significantly decreased retinal vascular permeability in diabetic animals. There was a significant reduction in macrophage/microglia infiltration in the retinas of treated animals. Levels of SDF-1 and ICAM-1 were significantly reduced and the tight junction protein ZO-1 level was increased, and phospho-VE-Cad was significantly reduced with drug treatment. CONCLUSIONS: A chemokine receptor inhibitor (CCR2/CCR5) can reduce retinal vascular permeability in diabetic animals. Targeting the chemokine pathway pharmacologically may be used as a novel therapeutic strategy in management of diabetic macular edema.

Transcriptomics analysis of pericytes from retinas of diabetic animals reveals novel genes and molecular pathways relevant to blood-retinal barrier alterations in diabetic retinopathy.

Selective pericyte loss, the histological hallmark of early diabetic retinopathy (DR), enhances the breakdown of the blood-retinal barrier (BRB) in diabetes. However, the role of pericytes on BRB alteration in diabetes and the signaling pathways involved in their effects are currently unknown. To understand the role of diabetes-induced molecular alteration of pericytes, we performed transcriptomic analysis of sorted retinal pericytes from mice model of diabetes. Retinal tissue from non-diabetic and diabetic (duration 3 months) mouse eyes (n = 10 in each group) were used to isolate pericytes through fluorescent activated cell sorting (FACS) using pericyte specific fluorescent antibodies, PDGFRb-APC. For RNA sequencing and qPCR analysis, a cDNA library was generated using template switching oligo and the resulting libraries were sequenced using paired-end Illumina sequencing. Molecular functional pathways were analyzed using differentially expressed genes (DEGs). Differential expression analysis revealed 217 genes significantly upregulated and 495 genes downregulated, in pericytes isolated from diabetic animals. These analyses revealed a core set of differentially expressed genes that could potentially contribute to the pericyte dysfunction in diabetes and highlighted the pattern of functional connectivity between key candidate genes and blood retinal barrier alteration mechanisms. The top up-regulated gene list included: Ext2, B3gat3, Gpc6, Pip5k1c and Pten and down-regulated genes included: Notch3, Xbp1, Gpc4, Atp1a2 and AKT3. Out of these genes, we further validated one of the down regulated genes, Notch 3 and its role in BRB alteration in diabetic retinopathy. We confirmed the downregulation of Notch3 expression in human retinal pericytes exposed to Advanced Glycation End-products (AGEs) treatment mimicking the chronic hyperglycemia effect. Exploration of pericyte-conditioned media demonstrated that loss of NOTCH3 in pericyte led to increased permeability of endothelial cell monolayers. Collectively, we identify a role for NOTCH3 in pericyte dysfunction in diabetes. Further validation of other DEGs to identify cell specific molecular change through whole transcriptomic approach in diabetic retina will provide novel insight into the pathogenesis of DR and novel therapeutic targets.

Genotypes and Phenotypes: A Search for Influential Genes in Diabetic Retinopathy.

Although gene-environment interactions are known to play an important role in the inheritance of complex traits, it is still unknown how a genotype and the environmental factors result in an observable phenotype. Understanding this complex interaction in the pathogenesis of diabetic retinopathy (DR) remains a big challenge as DR appears to be a disease with heterogenous phenotypes with multifactorial influence. In this review, we examine the natural history and risk factors related to DR, emphasizing distinct clinical phenotypes and their natural course in retinopathy. Although there is strong evidence that duration of diabetes and metabolic factors play a key role in the pathogenesis of DR, accumulating new clinical studies reveal that this disease can develop independently of duration of diabetes and metabolic dysfunction. More recently, studies have emphasized the role of genetic factors in DR. However, linkage analyses, candidate gene studies, and genome-wide association studies (GWAS) have not produced any statistically significant results. Our recently initiated genomics study, the Diabetic Retinopathy Genomics (DRGen) Study, aims to examine the contribution of rare and common variants in the development DR, and how they can contribute to clinical phenotype, rate of progression, and response to available therapies. Our preliminary findings reveal a novel set of genetic variants associated with proangiogenic and inflammatory pathways that may contribute to DR pathogenesis. Further investigation of these variants is necessary and may lead to development of novel biomarkers and new therapeutic targets in DR.

Cathepsin D plays a role in endothelial-pericyte interactions during alteration of the blood-retinal barrier in diabetic retinopathy.

Inflammation plays an important role in the pathogenesis of diabetic retinopathy. We have previously demonstrated the effect of cathepsin D (CD) on the mechanical disruption of retinal endothelial cell junctions and increased vasopermeability, as well as increased levels of CD in retinas of diabetic mice. Here, we have also examined the effect of CD on endothelial-pericyte interactions, as well as the effect of dipeptidyl peptidase-4 (DPP4) inhibitor on CD in endothelial-pericyte interactions in vitro and in vivo. Cocultured cells that were treated with pro-CD demonstrated a significant decrease in the expression of platelet-derived growth factor receptor-ß, a tyrosine kinase receptor that is required for pericyte cell survival; N-cadherin, the key adherens junction protein between endothelium and pericytes; and increases in the vessel destabilizing agent, angiopoietin-2. The effect was reversed in cells that were treated with DPP4 inhibitor along with pro-CD. With pro-CD treatment, there was a significant increase in the phosphorylation of the downstream signaling protein, PKC-α, and Ca2+/calmodulin-dependent protein kinase II in endothelial cells and pericytes, which disrupts adherens junction structure and function, and this was significantly reduced with DPP4 inhibitor treatment. Increased CD levels, vasopermeability, and alteration in junctional-related proteins were observed in the retinas of diabetic rats, which were significantly changed with DPP4 inhibitor treatment. Thus, DPP4 inhibitors may be used as potential adjuvant therapeutic agents to treat increased vascular leakage observed in patients with diabetic macular edema.-Monickaraj, F., McGuire, P., Das, A. Cathepsin D plays a role in endothelial-pericyte interactions during alteration of the blood-retinal barrier in diabetic retinopathy.

Cathepsin D: an Mϕ-derived factor mediating increased endothelial cell permeability with implications for alteration of the blood-retinal barrier in diabetic retinopathy.

Inflammation plays an important role in the pathogenesis of diabetic retinopathy (DR). We have previously reported increased monocyte (Mono) trafficking into the retinas of diabetic animals. In this study, we have examined the effect of activated Monos on retinal endothelial cells (ECs). The U937 Mϕ-conditioned medium (CM) significantly decreased the transendothelial resistance of EC monolayers as measured by electric cell-substrate impedance sensing (P= 0.007). The CM was fractioned, and the effective fraction (30-100 kDa) was analyzed by liquid chromatography-mass spectrometry, and cathepsin D (CD) was identified as a major secreted product. Immunoprecipitated CD resulted in decreased resistance in ECs (P= 0.006). The specificity of CD in mediating alterations of the EC barrier was confirmed using small interfering RNA. The decreased resistance correlated with a significantly increased gap between ECs. CD altered the Ras homolog gene family, member A/Rho-associated kinase pathway with increased stress actin filament formation in the EC layer. Increased CD levels were found in the retinas of diabetic mice (3-fold) and serum samples of patients with diabetic macular edema (1.6-fold) measured by Western blot and ELISA. These findings suggest an important role for Mϕ-derived CD in altering the blood-retinal barrier and reveal a potential therapeutic target in the treatment of DR.-Monickaraj, F., McGuire, P. G., Nitta, C. F., Ghosh, K., Das, A. Cathepsin D: an Mϕ-derived factor mediating increased endothelial cell permeability with implications for alteration of the blood-retinal barrier in diabetic retinopathy.

Basement membrane stiffening promotes retinal endothelial activation associated with diabetes.

Endothelial activation is a hallmark of the high-glucose (HG)-induced retinal inflammation associated with diabetic retinopathy (DR). However, precisely how HG induces retinal endothelial activation is not fully understood. We hypothesized that HG-induced up-regulation of lysyl oxidase (LOX), a collagen-cross-linking enzyme, in retinal capillary endothelial cells (ECs) enhances subendothelial basement membrane (BM) stiffness, which, in turn, promotes retinal EC activation. Diabetic C57BL/6 mice exhibiting a 70 and 50% increase in retinal intercellular adhesion molecule (ICAM)-1 expression and leukocyte accumulation, respectively, demonstrated a 2-fold increase in the levels of BM collagen IV and LOX, key determinants of capillary BM stiffness. Using atomic force microscopy, we confirmed that HG significantly enhances LOX-dependent subendothelial matrix stiffness in vitro, which correlated with an ∼2.5-fold increase in endothelial ICAM-1 expression, a 4-fold greater monocyte-EC adhesion, and an ∼2-fold alteration in endothelial NO (decrease) and NF-κB activation (increase). Inhibition of LOX-dependent subendothelial matrix stiffening alone suppressed HG-induced retinal EC activation. Finally, using synthetic matrices of tunable stiffness, we demonstrated that subendothelial matrix stiffening is necessary and sufficient to promote EC activation. These findings implicate BM stiffening as a critical determinant of HG-induced retinal EC activation and provide a rationale for examining BM stiffness and underlying mechanotransduction pathways as therapeutic targets for diabetic retinopathy.

Transcriptomic Profiling Reveals Chemokine CXCL1 as a Mediator for Neutrophil Recruitment Associated With Blood-Retinal Barrier Alteration in Diabetic Retinopathy.

Inflammation plays an important role in the pathogenesis of diabetic retinopathy (DR). To precisely define the inflammatory mediators, we examined the transcriptomic profile of human retinal endothelial cells exposed to advanced glycation end products, which revealed the neutrophil chemoattractant chemokine CXCL1 as one of the top genes upregulated. The effect of neutrophils in the alteration of the blood-retinal barrier (BRB) was further assessed in wild-type C57BL/6J mice intravitreally injected with recombinant CXCL1 as well as in streptozotocin-induced diabetic mice. Both intravitreally CXCL1-injected and diabetic animals showed significantly increased retinal vascular permeability, with significant increase in infiltration of neutrophils and monocytes in retinas and increased expression of chemokines and their receptors, proteases, and adhesion molecules. Treatment with Ly6G antibody for neutrophil depletion in both diabetic mice as well as CXCL1-injected animals showed significantly decreased retinal vascular permeability accompanied by decreased infiltration of neutrophils and monocytes and decreased expression of cytokines and proteases. CXCL1 level was significantly increased in the serum samples of patients with DR compared with samples of those without diabetes. These data reveal a novel mechanism by which the chemokine CXCL1, through neutrophil recruitment, alters the BRB in DR and, thus, serves as a potential novel therapeutic target. ARTICLE HIGHLIGHTS: Intravitreal CXCL1 injection and diabetes result in increased retinal vascular permeability with neutrophil and monocyte recruitment. Ly6G antibody treatment for neutrophil depletion in both animal models showed decreased retinal permeability and decreased cytokine expression. CXCL1 is produced by retinal endothelial cells, pericytes, and astrocytes. CXCL1 level is significantly increased in serum samples of patients with diabetic retinopathy. CXCL1, through neutrophil recruitment, alters the blood-retinal barrier in diabetic retinopathy and, thus, may be used as a therapeutic target.

Accelerated aging as evidenced by increased telomere shortening and mitochondrial DNA depletion in patients with type 2 diabetes.

Although shortened telomeres were shown associated with several risk factors of diabetes, there is lack of data on their relationship with mitochondrial dysfunction. Therefore, we compared the relationship between telomere length and mitochondrial DNA (mtDNA) content in patients with type 2 diabetes mellitus (T2DM; n = 145) and in subjects with normal glucose tolerance (NGT; n = 145). Subjects were randomly recruited from the Chennai Urban Rural Epidemiology Study. mtDNA content and telomere length were assessed by Real-Time PCR. Malonodialdehyde, a marker of lipid peroxidation was measured by thiobarbituric acid reactive substances (TBARS) using fluorescence methodology. Adiponectin levels were measured by radioimmunoassay. Oxidative stress as determined by lipid peroxidation (TBARS) was significantly (p < 0.001) higher in patients with T2DM compared to NGT subjects. In contrast, the mean telomere length, adiponectin and mtDNA content were significantly (p < 0.001) lower in patients with T2DM compared to NGT subjects. Telomere length was positively correlated with adiponectin, HDL, mtDNA content and good glycemic/lipid control and negatively correlated with adiposity and insulin resistance. On regression analysis, shortened telomeres showed significant association with T2DM even after adjusting for waist circumference, insulin resistance, triglyceride, HDL, adiponectin, mtDNA & TBARS. mtDNA depletion showed significant association with T2DM after adjusting for waist circumference and adiponectin but lost its significance when further adjusted for telomere length, TBARS and insulin resistance. Our study emphasizes the clustering of accelerated aging features viz., shortened telomeres, decreased mtDNA content, hypoadiponectinemia, low HDL, and increased oxidative stress in Asian Indian type 2 diabetes patients.

Transcriptional regulation of cytokines and oxidative stress by gallic acid in human THP-1 monocytes.

Increased inflammation/prooxidation has been linked not only to Type 2 diabetes but also in prediabetes state. In this study we investigated hyperglycemia-mediated proinflammatory/prooxidant effects in THP-1 monocytes and tested whether gallic acid could attenuate changes in gene expression induced by high-glucose. Cells were treated either with 5.5mM glucose or 25mM glucose in the absence and presence of gallic acid. While oxidative DNA damage was assessed by COMET assay, GSH and GSSG levels were estimated fluorimetrically. Gene expression patterns were determined by RT-PCR. Cells treated with high-glucose showed increased DNA damage and glutathione depletion and this was attenuated in the presence of gallic acid. High-glucose treated cells exhibited increased mRNA expression of TNF-alpha, IL-6, NADPH oxidase and TXNIP and gallic acid attenuated these proinflammatory and prooxidant effects. Cells treated with high-glucose revealed a deficiency in mounting SOCS-3 expression and gallic acid upregulates this feedback regulatory signal. Gallic acid attenuates DNA damage, maintains glutathione turnover, and suppresses hyperglycemia-induced activation of proinflammatory and prooxidant gene expression. Gallic acid beneficially modulate transcription of functionally diverse groups of genes and its regulation of SOCS-3 and TXNIP signals is a newly identified mechanism that has therapeutic implications.

Endoplasmic reticulum stress in diabetes: New insights of clinical relevance.

The endoplasmic reticulum (ER) is a cellular compartment responsible for multiple important cellular functions including the biosynthesis and folding of newly synthesized proteins destined for secretion, such as insulin. A myriad of pathological and physiological factors perturb ER function and cause dysregulation of ER homeostasis, leading to ER stress. Accumulating evidence suggests that ER stress plays a role in the pathogenesis of diabetes, contributing to pancreatic ß-cell loss and insulin resistance. ER stress may also link obesity, inflammation and insulin resistance in type 2 diabetes. In this review, we address the transition from physiology to pathology, namely how and why the physiological UPR evolves to a proapoptotic ER stress response in diabetes and its complications. Special attention was given to elucidate how ER stress could explain some of the 'clinical paradoxes' such as secondary sulfonylurea failure, initial worsening of retinopathy during tight glycemic control, insulin resistance induced by protease inhibitors and other clinically relevant observations.

Do Genomic Factors Play a Role in Diabetic Retinopathy?

Although there is strong clinical evidence that the control of blood glucose, blood pressure, and lipid level can prevent and slow down the progression of diabetic retinopathy (DR) as shown by landmark clinical trials, it has been shown that these factors only account for 10% of the risk for developing this disease. This suggests that other factors, such as genetics, may play a role in the development and progression of DR. Clinical evidence shows that some diabetics, despite the long duration of their diabetes (25 years or more) do not show any sign of DR or show minimal non-proliferative diabetic retinopathy (NPDR). Similarly, not all diabetics develop proliferative diabetic retinopathy (PDR). So far, linkage analysis, candidate gene studies, and genome-wide association studies (GWAS) have not produced any statistically significant results. We recently initiated a genomics study, the Diabetic Retinopathy Genetics (DRGen) Study, to examine the contribution of rare and common variants in the development of different phenotypes of DR, as well as their responsiveness to anti-VEGF treatment in diabetic macular edema (DME). Our preliminary findings reveal a novel set of genetic variants involved in the angiogenesis and inflammatory pathways that contribute to DR progression or protection. Further investigation of variants can help to develop novel biomarkers and lead to new therapeutic targets in DR.

Subclinical inflammation/oxidation as revealed by altered gene expression profiles in subjects with impaired glucose tolerance and Type 2 diabetes patients.

Although subclinical inflammation and oxidative stress are implicated in the aetiology of diabetes, there are hardly any studies in prediabetes. Therefore, we made an attempt to study the gene expression pattern of certain inflammatory/oxidative genes using lymphocytes from Type 2 diabetic patients, impaired glucose tolerance (IGT), and normal glucose tolerance (NGT) subjects. Compared to NGT group, interleukin-6, tumor necrosis factor-alpha (TNF-alpha), p(22)Phox NADPH oxidase, and thioredoxin interacting protein (TXNIP) mRNA levels were higher and suppressor of cytokine signaling (SOCS-3) mRNA was lower in subjects with IGT and diabetes. The mean (+/-SE) levels of thiobarbituric acid reactive substances and protein carbonyl content were also elevated in glucose intolerant subjects. In multiple linear regression analysis, TXNIP and TNF-alpha showed a significant association with HbA1c even after adjusting for TBARS and PCO (TXNIP: beta = 1.70, P < 0.01; TNF-alpha: beta = 1.86, P < 0.01). Increased subclinical inflammation/oxidation is seen in Asian Indians with not only Type 2 diabetes but also IGT.

Novel therapeutic targets in diabetic macular edema: Beyond VEGF.

The leading cause of major vision loss in diabetic persons is diabetic macular edema (DME). The hallmark feature of diabetic retinopathy is the alteration of the blood-retinal barrier (BRB). Inflammation plays a crucial role in DME with involvement of several chemokines and cytokines including vascular endothelial growth factor (VEGF). VEGF is a potent cytokine and vaso-permeability factor that has been targeted in multiple, large clinical trials. Multiple anti-VEGF drugs are widely used in the treatment of diabetic macular edema (DME) as the first line of treatment, and have been shown to be effective in vision improvement and prevention of vision loss. However, many DME patients do not show complete response to anti-VEGF drugs despite multiple intravitreal injections with these drugs. Also, the effect seems to be transient in those responders, and many patients do not show complete resolution of fluid. This article summarizes the mechanisms other than VEGF, and how these novel factors can be targeted as promising therapies of DME.

Association of increased levels of MCP-1 and cathepsin-D in young onset type 2 diabetes patients (T2DM-Y) with severity of diabetic retinopathy.

AIM: Young onset type 2 diabetes patients (T2DM-Y) have been shown to possess an increased risk of developing microvascular complications particularly diabetic retinopathy. However, the molecular mechanisms are not clearly understood. In this study, we investigated the serum levels of monocyte chemotactic protein 1 (MCP-1) and cathepsin-D in patients with T2DM-Y without and with diabetic retinopathy. METHODS: In this case-control study, participants comprised individuals with normal glucose tolerance (NGT=40), patients with type 2 diabetes mellitus (T2DM=35), non-proliferative diabetic retinopathy (NPDR=35) and proliferative diabetic retinopathy (PDR=35). Clinical characterization of the study subjects was done by standard procedures and MCP-1 and cathepsin-D were measured by ELISA. RESULTS: Compared to control individuals, patients with T2DM-Y, NPDR and PDR exhibited significantly (p<0.001) higher levels of MCP-1. Cathepsin-D levels were also significantly (p<0.001) higher in patients with T2DM-Y without and with diabetic retinopathy. Correlation analysis revealed a positive association (p<0.001) between MCP-1 and cathepsin-D levels. There was also a significant negative correlation of MCP1/cathepsin-D with C-peptide levels. The association of increased levels of MCP-1/cathepsin-D in patients with DR persisted even after adjusting for all the confounding factors. CONCLUSION: As both MCP-1 and cathepsin-D are molecular signatures of cellular senescence, we suggest that these biomarkers might be useful to predict the development of retinopathy in T2DM-Y patients.

Novel pharmacotherapies in diabetic retinopathy: Current status and what's in the horizon?

The blood-retinal barrier (BRB) alteration is the hallmark feature of diabetic retinopathy. Vascular endothelial growth factor (VEGF) is a potent vasopermeability factor that has been implicated in the pathogenesis of BRB alteration. Inflammation also plays a crucial role in this process with involvement of several chemokines and cytokines. Multiple anti-VEGF drugs are widely used as in the treatment of diabetic macular edema (DME) as well as proliferative diabetic retinopathy. Several clinical trials have proved the beneficial effects of these drugs in improvement of vision and prevention of vision loss. However, the response to anti-VEGF drugs in DME is not complete in a significant number of patients. The effect seems transient in this latter group, and many patients do not show complete resolution of fluid. Potential novel therapies targeting molecules beyond VEGF are being developed and examined in clinical trials.

Chemokine mediated monocyte trafficking into the retina: role of inflammation in alteration of the blood-retinal barrier in diabetic retinopathy.

Inflammation in the diabetic retina is mediated by leukocyte adhesion to the retinal vasculature and alteration of the blood-retinal barrier (BRB). We investigated the role of chemokines in the alteration of the BRB in diabetes. Animals were made diabetic by streptozotocin injection and analyzed for gene expression and monocyte/macrophage infiltration. The expression of CCL2 (chemokine ligand 2) was significantly up-regulated in the retinas of rats with 4 and 8 weeks of diabetes and also in human retinal endothelial cells treated with high glucose and glucose flux. Additionally, diabetes or intraocular injection of recombinant CCL2 resulted in increased expression of the macrophage marker, F4/80. Cell culture impedance sensing studies showed that purified CCL2 was unable to alter the integrity of the human retinal endothelial cell barrier, whereas monocyte conditioned medium resulted in significant reduction in cell resistance, suggesting the relevance of CCL2 in early immune cell recruitment for subsequent barrier alterations. Further, using Cx3cr1-GFP mice, we found that intraocular injection of CCL2 increased retinal GFP+ monocyte/macrophage infiltration. When these mice were made diabetic, increased infiltration of monocytes/macrophages was also present in retinal tissues. Diabetes and CCL2 injection also induced activation of retinal microglia in these animals. Quantification by flow cytometry demonstrated a two-fold increase of CX3CR1+/CD11b+ (monocyte/macrophage and microglia) cells in retinas of wildtype diabetic animals in comparison to control non-diabetic ones. Using CCL2 knockout (Ccl2-/-) mice, we show a significant reduction in retinal vascular leakage and monocyte infiltration following induction of diabetes indicating the importance of this chemokine in alteration of the BRB. Thus, CCL2 may be an important therapeutic target for the treatment of diabetic macular edema.

Accelerated fat cell aging links oxidative stress and insulin resistance in adipocytes.

Telomere shortening is emerging as a biological indicator of accelerated aging and aging-related diseases including type 2 diabetes. While telomere length measurements were largely done in white blood cells, there is lack of studies on telomere length in relation to oxidative stress in target tissues affected in diabetes. Therefore, the aim of this study is to induct oxidative stress in adipocytes and to test whether these adipocytes exhibit shortened telomeres, senescence and functional impairment. 3T3-L1 adipocytes were subjected to oxidative stress and senescence induction by a variety of means for 2 weeks (exogenous application of H2O2, glucose oxidase, asymmetric dimethylarginine (ADMA) and glucose oscillations). Cells were probed for reactive oxygen species generation (ROS), DNA damage, mRNA and protein expression of senescent and pro-inflammatory markers, telomere length and glucose uptake. Compared to untreated cells, both ROS generation and DNA damage were significantly higher in cells subjected to oxidative stress and senescence. Adipocytes subjected to oxidative stress also showed shortened telomeres and increased mRNA and protein expression of p53, p21, TNF alpha and IL-6. Senescent cells were also characterized by decreased levels of adiponectin and impaired glucose uptake. Briefly, adipocytes under oxidative stress exhibited increased ROS generation, DNA damage, shortened telomeres and switched to senescent/pro-inflammatory phenotype with impaired glucose uptake.

Imbalanced levels of angiogenic and angiostatic factors in vitreous, plasma and postmortem retinal tissue of patients with proliferative diabetic retinopathy.

A role for vascular endothelial growth factor (VEGF) has been clearly implicated in the pathogenesis of proliferative diabetic retinopathy (PDR). However, other molecules and mechanisms may be operating independently, or in conjunction with VEGF in the pathogenesis of this disease. Therefore, we made an attempt to comparatively investigate the levels of angiogenic and angiostatic factors in vitreous, plasma and postmortem retinal tissue of subjects with Proliferative Diabetic Retinopathy (PDR) compared to control subjects. The vitreous and plasma concentrations of VEGF, EPO (Erythropoietin) and PEDF (Pigment Epithelium Derived Factor) were measured using Enzyme Linked Immunosorbent Assay (ELISA) and the postmortem retinal tissue was subjected to Western blot analysis. The mean vitreous and plasma levels of VEGF and EPO in patients with PDR were significantly (p<0.001) higher than those in subjects without diabetes. Conversely, the vitreous and plasma levels of PEDF were significantly (p<0.001) lower in the PDR patients compared to control subjects. Multivariate logistic-regression analyses indicated that EPO was more strongly associated with PDR than VEGF. The protein expression of the VEGF and EPO in the retinal tissue was significantly higher in PDR and diabetes without complication groups compared to controls. Compared to controls, the protein expression of PEDF was significantly lower in retinal tissues from diabetes patients without complications and in patients with PDR. The fact that the vitreous and plasma levels and the retinal tissue protein expression of EPO were strongly associated with PDR implies a definite role of 'hypererythropoietinemia' in neovascularization processes.

Convergence of adipocyte hypertrophy, telomere shortening and hypoadiponectinemia in obese subjects and in patients with type 2 diabetes.

OBJECTIVE: Although telomere shortening has been linked with type 2 diabetes and most variables of adiposity, a shortcoming of such studies is the measurement of telomere length in leukocytes. Therefore, we tested the association among adipocyte cell size, telomere length (both subcutaneous and visceral adipose tissue) and systemic levels of adiponectin in obese subjects and patients with type 2 diabetes compared to control subjects. METHODS: Human subcutaneous and visceral adipose tissues were obtained from the subjects who have undergone bariatric surgery or other abdominal surgeries. The study groups comprised: i) control subjects, ii) type 2 diabetes patients, iii) obese subjects without diabetes and iv) obese subjects with diabetes. Adipocyte cell size was measured by histological staining. Adiponectin levels were measured by ELISA. Telomere length was determined by Real-time PCR and lipid peroxidation was assessed by fluorimetry. RESULTS: Compared to control subjects, adipocyte size (both subcutaneous and visceral) from obese, diabetic and obese-diabetic subjects was significantly larger [p<0.001]. Individuals with adipose hypertrophy also exhibited shortened telomeres and hypoadiponectinemia. Pearson correlation analysis revealed that both visceral and subcutaneous fat cell size showed a positive correlation with FBS, HbA1c, HOMA-IR, LDL, total cholesterol, triglycerides and negatively correlated with HDL and adiponectin. Regression analysis revealed that the association between shortened telomeres and hypoadiponectinemia was lost when adjusted for adipocyte cell size. CONCLUSION: Adipocyte hypertrophy appears to be strongly associated with shortened telomeres, hypoadiponectinemia and poor glycemic and lipid control. Interestingly, these molecular alterations seen in lean diabetics reflect a state of 'metabolic obesity'.

Novel pharmacotherapies in diabetic retinopathy: Current status and what’s in the horizon.

The blood–retinal barrier (BRB) alteration is the hallmark feature of diabetic retinopathy. Vascular endothelial growth factor (VEGF) is a potent vasopermeability factor that has been implicated in the pathogenesis of BRB alteration. Inflammation also plays a crucial role in this process with involvement of several chemokines and cytokines. Multiple anti‑VEGF drugs are widely used as in the treatment of diabetic macular edema (DME) as well as proliferative diabetic retinopathy. Several clinical trials have proved the beneficial effects of these drugs in improvement of vision and prevention of vision loss. However, the response to anti‑VEGF drugs in DME is not complete in a significant number of patients. The effect seems transient in this latter group, and many patients do not show complete resolution of fluid. Potential novel therapies targeting molecules beyond VEGF are being developed and examined in clinical trials.