Sampathkumar Rangasamy

Diabetic Macular Edema: Pathophysiology and Novel Therapeutic Targets.

Diabetic macular edema (DME) is the major cause of vision loss in diabetic persons. Alteration of the blood-retinal barrier is the hallmark of this disease, characterized by pericyte loss and endothelial cell-cell junction breakdown. Recent animal and clinical studies strongly indicate that DME is an inflammatory disease. Multiple cytokines and chemokines are involved in the pathogenesis of DME, with multiple cellular involvement affecting the neurovascular unit. With the introduction of anti-vascular endothelial growth factor (VEGF) agents, the treatment of DME has been revolutionized, and the indication for laser therapy has been limited. However, the response to anti-VEGF drugs in DME is not as robust as in proliferative diabetic retinopathy, and many patients with DME do not show complete resolution of fluid despite multiple intravitreal injections. Potential novel therapies targeting molecules other than VEGF and using new drug-delivery systems currently are being developed and evaluated in clinical trials.

Diabetic retinopathy and inflammation: novel therapeutic targets.

Most anti-vascular endothelial growth factor (VEGF) therapies in diabetic macular edema are not as robust as in proliferative diabetic retinopathy. Although the VEGF appears to be a good target in diabetic macular edema, the anti-VEGF therapies appear to be of transient benefit as the edema recurs within a few weeks, and repeated injections are necessary. There is new evidence that indicates ‘retinal inflammation’ as an important player in the pathogenesis of diabetic retinopathy. There are common sets of inflammatory cytokines that are upregulated in both the serum and vitreous and aqueous samples, in subjects with diabetic retinopathy, and these cytokines can have multiple interactions to impact the pathogenesis of the disease. The key inflammatory events involved in the blood retinal barrier (BRB) alteration appear to be: (1) Increased expression of endothelial adhesion molecules such as ICAM1, VCAM1, PECAM-1, and P-selectin, (2) adhesion of leukocytes to the endothelium, (3) release of inflammatory chemokines, cytokines, and vascular permeability factors, (4) alteration of adherens and tight junctional proteins between the endothelial cells, and (5) infiltration of leukocytes into the neuro-retina, resulting in the alteration of the blood retinal barrier (diapedesis). VEGF inhibition itself may not achieve neutralization of other inflammatory molecules involved in the inflammatory cascade of the breakdown of the BRB. It is possible that the novel selective inhibitors of the inflammatory cascade (like angiopoietin-2, TNFα, and chemokines) may be useful therapeutic agents in the treatment of diabetic macular edema (DME), either alone or in combination with the anti-VEGF drugs.

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.

A Potential Role for Angiopoietin 2 in the Regulation of the Blood–Retinal Barrier in Diabetic Retinopathy

PURPOSE Although VEGF has been identified as an important mediator of the blood-retinal barrier alteration in diabetic retinopathy, the hypothesis for this study was that that other molecules, including the angiopoietins (Ang-1 and -2), may play a role. The expression of angiopoietins was analyzed in an animal model of diabetic retinopathy, and the role of Ang-2 in the regulation of diabetes-induced alterations of vascular permeability was characterized. METHODS Diabetes was induced in rats, and human retinal endothelial cells (HRECs) were grown in media with 5.5 or 30.5 mM glucose. Levels of Ang-1 and -2 mRNA and protein were analyzed. Fluorescence-based assays were used to assess the effect of Ang-2 on vascular permeability in vivo and in vitro. The effect of Ang-2 on VE-cadherin function was assessed by measuring the extent of tyrosine phosphorylation. RESULTS Ang-2 mRNA and protein increased in the retinal tissues after 8 weeks of diabetes and in high-glucose-treated cells. Intravitreal injection of Ang-2 in rats produced a significant increase in retinal vascular permeability. Ang-2 increased HREC monolayer permeability that was associated with a decrease in VE-cadherin and a change in monolayer morphology. High glucose and Ang-2 produced a significant increase in VE-cadherin phosphorylation. CONCLUSIONS; Ang-2 is upregulated in the retina in an animal model of diabetes, and hyperglycemia induces the expression of Ang-2 in isolated retinal endothelial cells. Increased Ang-2 alters VE-cadherin function, leading to increased vascular permeability. Thus, Ang-2 may play an important role in increased vasopermeability in diabetic retinopathy.

Pericyte-Derived Sphinogosine 1-Phosphate Induces the Expression of Adhesion Proteins and Modulates the Retinal Endothelial Cell Barrier

Objective—The mechanisms that regulate the physical interaction of pericytes and endothelial cells and the effects of these interactions on interendothelial cell junctions are not well understood. We determined the extent to which vascular pericytes could regulate pericyte-endothelial adhesion and the consequences that this disruption might have on the function of the endothelial barrier. Methods and Results—Human retinal microvascular endothelial cells were cocultured with pericytes, and the effect on the monolayer resistance of endothelial cells and expression of the cell junction molecules N-cadherin and VE-cadherin were measured. The molecules responsible for the effect of pericytes or pericyte-conditioned media on the endothelial resistance and cell junction molecules were further analyzed. Our results indicate that pericytes increase the barrier properties of endothelial cell monolayers. This barrier function is maintained through the secretion of pericyte-derived sphingosine 1-phosphate. Sphingosine 1-phosphate aids in maintenance of microvascular stability by upregulating the expression of N-cadherin and VE-cadherin, and downregulating the expression of angiopoietin 2. Conclusion—Under normal circumstances, the retinal vascular pericytes maintain pericyte-endothelial contacts and vascular barrier function through the secretion of sphingosine 1-phosphate. Alteration of pericyte-derived sphingosine 1-phosphate production may be an important mechanism in the development of diseases characterized by vascular dysfunction and increased permeability.

New treatments for diabetic retinopathy.

Diabetic retinopathy is the major cause of vision loss in middle‐aged adults. Alteration of the blood–retinal barrier (BRB) is the hallmark of diabetic retinopathy and, subsequently, hypoxia may result in retinal neovascularization. Tight control of systemic factors such as blood glucose, blood pressure and blood lipids is essential in the management of this disease. Vascular endothelial growth factor (VEGF) is one of the most important factors responsible for alteration of the BRB. The introduction of anti‐VEGF agents has revolutionized the therapeutic strategies used in people with diabetic retinopathy, and the use of laser therapy has been modified. In the present article, we examine the clinical features and pathophysiology of diabetic retinopathy and review the current status of new treatment recommendations for this disease, and also explore some possible future therapies.

The role of monocyte subsets in myocutaneous revascularization

BACKGROUND The controlled recruitment of monocytes from the circulation to the site of injury and their differentiation into tissue macrophages are critical events in the reconstitution of tissue integrity. Subsets of monocytes/macrophages have been implicated in the pathogenesis of atherosclerosis and tumor vascularity; however, the significance of monocyte heterogeneity in physiologic neovascularization is just emerging. MATERIALS AND METHODS A cranial-based, peninsular-shaped myocutaneous flap was surgically created on the dorsum of wild-type mice (C57BL6) and populations of mice with genetic deletion of subset-specific chemokine ligand-receptor axes important in monocyte trafficking and function (CCL2(-/-) and CX3CR1(-/-)) (n=36 total; 12 mice per group, nine with flap and three unoperated controls). Planimetric analysis of digital photographic images was utilized to determine flap surface viability in wild-type and knockout mice. Real-time myocutaneous flap perfusion and functional revascularization was determined by laser speckle contrast imaging. Image analysis of CD-31 immunostained sections confirmed flap microvascular density and anatomy. Macrophage quantification and localization in flap tissues was determined by F4/80 gene and protein expression. Quantitative reverse transcription-polymerase chain reaction was performed on nonoperative back skin and postoperative flap tissue specimens to determine local gene expression. RESULTS Myocutaneous flaps created on wild type and CX3CR1(-/-) mice were engrafted to the recipient site, resulting in viability. In contrast, distal full thickness cutaneous necrosis and resultant flap dehiscence was evident by d 10 in CCL2(-/-) mice. Over 10 d, laser speckle contrast imaging documented immediate graded flap ischemia in all three groups of mice, functional flap revascularization in wild type and CX3CR1(-/-) mice, and lack of distal flap reperfusion in CCL2(-/-) mice. Immunostaining of serial histologic specimens confirmed marked increases in microvascular density and number of macrophages in wild type mice, intermediate increases in CX3CR1(-/-) mice, and no significant change in vessel count or macrophage quantity in CCL2(-/-) mice over the study interval. Finally, quantitative reverse transcriptase polymerase chain reaction demonstrated that the loss of function of chemokine ligand and receptor genes influenced the transcription of local genes involved in monocyte chemotaxis and wound angiogenesis. CONCLUSIONS In a graded-ischemia wound healing model, monocyte recruitment was severely impaired in CCL2(-/-) mice, resulting in failure of flap revascularization and concomitant cutaneous necrosis. Analysis of CX3CR1-deficient mice revealed adequate monocyte recruitment and revascularization for flap survival; however, the myeloid cell response and magnitude of neovascularization were dampened compared with wild type mice.

Molecular Intricacies and the Role of ER Stress in Diabetes

Diabetes mellitus is a metabolic disease caused by both genetic and environmental factors. The pathogenic mechanism(s) of diabetes are complex, and the complicated networks related to this disease involve distinct signaling pathways. Evidence has recently been provided that ER stress might be involved in the pathogenesis of diabetes and its complications. Early steps in the maturation of secretory proteins take place in the ER, for example, the folding of the nascent polypeptide chains and posttranslational modifications important for proper folding and function of the protein. At a stage (due to several metabolic disturbances), when unfolded polypeptide exceeds the folding and/or processing capacity of the ER, cells are susceptible to a phenomenon referred to as “ER stress.” Under these conditions, specific signaling pathways, termed the unfolded protein response (UPR), are activated to return the ER to its normal physiological state. Prolonged activation of the ER stress and the UPR can lead to cell pathology and subsequent tissue dysfunction. There is now ample evidence that the UPR is chronically activated in many disease states including diabetes and its complications. Therefore, a better understanding of the pathways regulating ER stress and UPR is warranted in order to be instrumental for the design of novel therapies for diabetes and its complications. In this focused issue of the journal, we have assembled several invited reviews, from well-recognized experts in their fields, as well as original research articles. These reviews provide state-of-the-art knowledge dealing with several mechanisms not only related to the genesis of diabetes but also to its progression to diabetic complications, all of which potentially originate or converge from chronic ER stress. In addition, several excellent original research articles demonstrate novel pathophysiologic aspects of diabetes with mechanistic studies central to ER stress and give hope and directionality for identifying new drug targets and developing newer therapeutic measures. Of all the professional secretory cells we possess, β-cells are the most sensitive to ER stress because of the large fluctuations in protein synthesis (including insulin) they face daily. M.-K. Kim et al. have reviewed how this “protein quality-control machinery” of the cell is responsible for appropriate insulin biosynthesis and how ER stress plays an important role in the impairment of insulin biosynthesis. J. Zhong et al. have summarized the status on how ER stress plays an essential role in autoimmune-mediated β-cell destruction and also pointed out how ER stress regulates the functionality of immune cells relevant to autoimmune progression during Type 1 diabetes development. In an attempt to improve islet transplantation in humans, the molecular mechanism of apoptosis in β cells of islets in the transplantation setting needs to be clearly understood. In this context, M. Wang et al. have discussed their original research work on human islets subjected to multiple stressors and delineated several apoptotic pathways originating from oxidative stress, autophagy, and ER stress. While ER stress is emerging as a unifying paradigm in diabetes and its complications, several recent studies, emphasized a definite role of ER stress in retinal, podocyte, and neuronal cell apoptosis. G. Jing et al. have summarized the recent progress on ER stress and apoptosis in retinal diseases, focusing on various proapoptotic and antiapoptotic pathways that are activated by the UPR and discussed how these pathways contribute to ER stress-induced apoptosis in retinal cells. Considering the fact that ER stress is initially an adaptive response, studying ER stress-related factors appear to unravel novel drug targets to prevent and treat diabetic retinopathy. In this connection, W. -K. Hu et al. have explained the role of P58IPK and ER-associated degradation (ERAD) of unfolded protein which prevents ER stress and reduce retinal vascular leakage under high-glucose conditions. While thioredoxin interacting protein (TXNIP) has been recently identified as an early response gene highly induced by diabetes and hyperglycemia, its role in the pathogenesis of diabetic retinopathy is not clearly understood. Using appropriate animal model and retinal Muller cell line and several molecular biology techniques, T. S. Devi et al. have described how upregulation of TXNIP evokes a program of cellular defense and survival mechanism(s) that ultimately lead to oxidative stress, ER-stress, inflammation, and apoptosis. Despite a great deal of research, the mechanisms that may link high-glucose concentrations to the molecular and cellular pathways of diabetic atherogenesis are not fully understood. D. R. Beriault and G. H. Werstuck have summarized the current state of our knowledge of pathways and mechanisms that may link diabetes and hyperglycemia to atherogenesis highlighting the recent work from their lab (and others) that supports a role for ER stress in these processes. Although recent studies have shown that perturbations in lipid metabolism cause an ER stress response, very little is known about the mechanism of UPR activation by perturbations in glucose and lipid metabolism. Moreover, it has been demonstrated that 4-phenylbutyrate (4-PBA) and tauroursodeoxycholic acid (TUDCA), which are two different chemical structures having chemical chaperone activity in common, relieve ER stress. Using THP-1 human monocytes as a surrogate cell model and utilizing several molecular biology techniques, R. Lenin et al. have demonstrated that monocytes subjected to glucolipotoxicity exhibited increased UPR responses (as evidenced by increased mRNA expression of several ER stress markers) along with increased oxidative stress and apoptosis. Interestingly, ER stress inducted by glucolipotoxicity was shown resisted by PBA. These observations constitute an important proof of principle that manipulation of the ER system to decrease ER stress by chemical agents may have therapeutic implications for diabetes and its complications. Lastly, the prevalence of nonalcoholic fatty liver disease (NAFLD) has increased in parallel with the epidemics of obesity and type 2 diabetes, which are risk factors for NAFLD. Whereas the association of type 2 diabetes with microvascular complications and macrovascular disease is well established, the association of type 2 diabetes with NAFLD is only recently recognized and so are the inter-related pathogenic mechanisms. Using steatohepatitis animal model and HepB3 cells, M. K. Chae et al. have demonstrated that Pentoxifylline (a known anti-inflammatory agent) attenuates methionine and choline-1-deficient diet-induced steatohepatitis by suppressing ER stress. These papers, hopefully, will provide better understanding of ER stress and UPR pathway involvement in the pathogenesis of diabetes and its complications and bring forward new and innovative ideas with respect to the development of efficient and adjuvant treatment modalities. Considering the involvement of ER stress in multiple tissues and their convergence in multiple pathogenic pathways (oxidative stress, inflammation, apoptosis, autophagy, and proteasomal degradation), targeting the ER stress pathway appears as a promising therapeutic strategy. The significant side effects with existing drugs and the demand for newer molecules with improved safety and a different mode of action justifies this directionality. In fact, it has been reported that chemical ER chaperones can reduce ER stress, suggesting that small molecules can affect ER stress signaling in disease states. There is also much hope in investigating the traditional plant principles of medicinal claims to see whether they act as beneficial ER stress modulators. Given the possible development of novel UPR-targeted therapies for diabetes and its complications, it is essential to know which components of the ER stress response to target and which particular disease stage will be most amenable to therapy. Although there is an enormous progress in studying the ER stress aspects, the list of unresolved queries in the stress mediated pathway of ER dysfunction in diabetes and its complications warrant continued research efforts. Obviously not all aspects of this exciting ER stress field could be addressed in one issue and we extend our apologies to many contributors of this field whose work has not been covered. We thank all the authors who contributed to this special issue of EDR and the reviewers for the highly constructive and helpful comments. Muthuswamy Balasubramanyam Lalit P. Singh Sampathkumar Rangasamy

Proteases in Diabetic Retinopathy

The human retinal structure along with the neuronal component develops from a single layer of undifferentiated neuroepithelial cells during embryonic ontogenesis. During this process, retinal vasculature develops to form an elaborate vascular tree that matches the metabolic need of tissues. Retinal vascular development involves a complex process of vasculogenesis and angiogenesis. Vasculogenesis describes the de novo formation of vessels from vascular endothelial precursor cells (angioblasts), which migrate to or differentiate at the location of future vessels, coalesce into cords, and differentiate into endothelial cells leading to the formation of ultimate vessels.