Finny Monickaraj

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.

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.

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 upregulation 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 invitro, 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.—Yang, X., Scott, H. A., Monickaraj, F., Xu, J., Ardekani, S., Nitta, C. F., Cabrera, A., McGuire, P. G., Mohideen, U., Das, A., Ghosh, K. Basement membrane stiffening promotes retinal endothelial activation associated with diabetes. FASEB J. 30, 601‐611 (2016). www.fasebj.org

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.

Novel therapeutic targets in diabetic macular edema: Beyond VEGF

&NA; 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.

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 β-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.

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. FASEB J. 30, 1670–1682 (2016). www.fasebj.org

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.

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 proCD demonstrated a significant decrease in the expression of platelet‐derived growth factor receptor‐β, 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. FASEB J. 32, 2539–2548 (2018). www.fasebj.org