Diabetic Retinopathy and the Role of VEGF

Diabetic retinopathy (DR) is the leading cause of blindness in the working-age population. It works by disrupting the neural and vascular components in the retina and leads to loss of neural interaction/function, vascular permeability, and angiogenesis. It can be classified into two general stages: non-proliferative diabetic retinopathy (NPDR) and proliferative diabetic retinopathy (PDR). DR can also lead to vascular leakage and cause diabetic macular edema (DME), the most common cause of vision loss in DR (Duh, Sun et al. 2017). There are currently a wide range of therapies, but they are limited in their efficacy and side effects. None of these therapies are as effective as early identification of the disease. Therefore, new studies are being done that focus on this aspect of DR prevention. As of now, the most promising therapy is anti-VEGF intravitreal injections. These are shown to prevent irreversible vision loss in the population of diabetics affected with proliferative diabetic retinopathy. Due to the increasing number of individuals diagnosed with diabetes worldwide, it can be predicted that DR will continue to be a leading cause of vision loss and therefore there is a significant need for new developments in the field. This literature review aims to consolidate the current knowledge on diabetic retinopathy and delve into the role of VEGF in the pathogenesis and treatment of the disease

Laser-Induced Forward Transfer (LIFT) based Bioprinting of the Collagen I with Retina Photoreceptor Cells

This study focuses on the 3D bioprinting of retina photoreceptor cells using a laser-induced forward transfer (LIFT) based bioprinting system. Bioprinting has a great potential to mimic and regenerate the human organoid system, and the LIFT technique has emerged as an efficient method for high-resolution micropatterning and microfabrication of biomaterials and cells due to its capability of creating precise, controlled microdroplets. In this study, the parameters for an effective femtosecond laser-based LIFT process for 3D bioprinting of collagen biomaterial were studied. Different concentrations of collagen I solutions were tested and 0.75 mg/ml to 1 mg/ml collagen Ⅰ was identified as the right concentration that can be transferred through the LIFT system. Then, retinal cone cells were mixed with collagen I and Dulbecco\u27s Modified Eagle\u27s Medium (DMEM) and printed drop-by-drop lines. Some important laser parameters such as pulse energy and pulse pick divider were experimented with to form a successful, smooth, high-resolution deposition. The cell viability in the bioink and printed droplet was measured at different time horizons. A general full factorial design of the experiment was used to analyze and observe the relationship between the droplet quality and the LIFT process parameters. Using 15 µJ and 16µJ pulse energy, the cell-laden bioink was printed successfully. This research study will help to print other retinal neuron cells with collagen Ⅰ in the LIFT system and show the way of constructing layer-by-layer different cell lines that will help to fabricate the retina ultimately

Visual cycle proteins: Structure, function, and roles in human retinal disease

Here, we seek to summarize the current understanding of the biochemical and molecular events mediated by visual cycle molecules in the eye. The structures and functions of selected visual cycle proteins and their roles in human retinal diseases are also highlighted. Genetic mutations and malfunctions of these proteins provide etiological evidence that many ocular diseases arise from anomalies of retinoid (vitamin A) metabolism and related visual processes. Genetic retinal disorders such as retinitis pigmentosa, Leber\u27s congenital amaurosis, and Stargardt\u27s disease are linked to structural changes in visual cycle proteins. Moreover, recent reports suggest that visual cycle proteins may also play a role in the development of diabetic retinopathy. Basic science has laid the groundwork for finding a cure for many of these blindness-causing afflictions, but much work remains. Some translational research projects have advanced to the clinical trial stage, while many others are still in progress, and more are at the ideas stage and remain yet to be tested. Some examples of these studies are discussed. Recent and future progress in our understanding of the visual cycle will inform intervention strategies to preserve human vision and prevent blindness

Involvement of TGFβ signaling pathway in oxidative stress and diabetic retinopathy

Diabetic Retinopathy (DR) is a leading cause of blindness in the U.S. However, not much is known of underlying molecular mechanism and how oxidative stress contributes to its development. In the present study, we investigated the involvement of TGFβ signaling pathway on the effect of oxidative stress on VEGF secretion and viability of retinal cells. VEGF is the hallmark that exacerbates DR progression in prolonged diabetes. Some major concerns that have arisen are the underlying effects of antioxidants in elevating VEGF secretion in diabetes. In this study, we evaluated how hypoxia (or low oxygen) impacts viability and VEGF secretion using 661W cone photoreceptor cells. Confluent 661W cells were grown in 5.5 mM normal or 30 mM high glucose, as well as subjected to CoCl2 to induce hypoxia. After treatment for 24 hours, conditioned media were collected for ELISA measurement to determine the amount of protein (VEGF) secretion. Viable cell numbers were also recorded. High glucose did not induce significant changes in viable cell number nor VEGF concentration in cell media. However, hypoxia condition resulted in a three-fold decrease in viable cell numbers and a three-fold increase in VEGF concentration. Furthermore, treatment with two TGFβ inhibitors: SMAD 3, SIS (or Inhibitor 1) and TGFβ receptor 1 kinase inhibitor (or Inhibitor 2) resulted in a reversal of hypoxia-induced changes. These results strongly suggest that TGFβ signaling pathway mediates hypoxia-induced retinal cell viability and VEGF secretion. Further translational research studies will provide evidence to identify appropriate and effective pharmaceutical targets in this molecular pathway to mitigate the development of DR

Insulin in Combination with N-Acetylcysteine Protects Hypoxia-Induced Toxicity in 661W Cells

Background: Proliferative diabetic retinopathy (PDR) is the leading cause of blindness among working-age adults. Photoreceptors are the most numerous and metabolically demanding cells in the retina thus oxygen is essential for retinal function. It has been reported that photoreceptors found in rat retina are specifically vulnerable to hypoxia. Hypoxia-induced metabolic stress leads to photoreceptor atrophy and retinopathy. Furthermore, photoreceptor cell death is known to occur mainly through apoptosis. However, the protection of hypoxia-induced-cytotoxicity in cone photoreceptor cells has not been investigated extensively. The aim of this study was to determine whether co-treatment of insulin and the N-Acetyl-L-Cysteine (NAC) (a free radical scavenger) efficiently protects against hypoxia-induced cytotoxicity in 661W cells. Methods: 661W, an immortalized mouse cone photoreceptor cells, were cultured at 5% CO2 at 37˚C in Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 10% FBS, penicillin (100 units/mL), and streptomycin (100μg/mL). Cobalt (II) Chloride hexahydrate (CoCl2) was used to induce hypoxia. Insulin was suspended in sterile water, and NAC was diluted in the culture medium. For recovery experiment, cells were pretreated with CoCl2 for 24hrs, and then followed by replacing of medium with insulin (100nM) and NAC (3mM) alone, or with a combination of the two reagents for another 24hrs. Cell viability was determined by MTT assay in a 96 well culture plate. Morphological changes of the cells were observed and photographed under phase-contrast microscope and protein expression was measured by Western blot analysis. Statistical analysis was undertaken using independent two-tailed Students’ t-test and determined with SPSS Statistics software. Results: Treatment with CoCl2 significantly inhibited cell proliferation, reduced the number of viability cells, and induced apoptosis, initiated (poly (ADP-ribose) polymerase (PARP) cleavage, and increased caspase 3 activation. In addition, CoCl2 treatment led to oxidative stress, autophagy, and ubiquitination in the 661W cells. All of these effects, including cell proliferations were significantly reversed by the combination treatment of Insulin and NAC. In contrast, treatment with Insulin alone did not result in a similar protective effect and NAC partially protects against hypoxia induced toxicity. Conclusion: Hypoxia induces significant apoptosis, oxidative stress, and protein ubiquitination in 661W cone photoreceptors. A combination treatment of Insulin and NAC completely reversed such hypoxia-induced cytotoxicity. Additional research on a combination therapy employing insulin and NAC may provide a novel and promising therapeutic strategy for hypoxia-mediated cone photoreceptor cell damage

Expression of Integrin and TGFBI in Human Retinal Pericytes

Purpose: The aim for this study is to investigate the expression of integrin α3, β1 and TGF-β induced protein (TGFBI) and the secretion of TGFBI by primary culture of human retinal pericytes (pHRP). Evidence suggests that chronic diabetes associate with HRP apoptosis leading to the development of diabetic retinopathy. Methods: pHRP (Cell Systems) were cultured in complete media (15mM glucose) in a humidified, 5% CO2, 37°C condition. Cells were seeded at passage 6 to 8 into a 24 well-plate with coverslips or P10 dishes. Cells (85% confluence) media were then replaced by DMEM media with euglycemic glucose (5.5mM) or hyperglycemic glucose (30mM) and cells were incubated for 48 or 72 hours. Gene and protein expressions of α3, β1 were detected by Real-Time PCR and flow cytometry. TGFBI gene expression was detected by Real-Time PCR and ELISA was used to measure protein level in cell media. Results: Real-Time PCR showed expression of α3, β1 and TGFBI in pHRP at 48 hrs of incubation in both glucose concentrations. Expression of a3 in pHRP in 30 mM glucose was 1.3 times higher than cells in 5.5mM glucose whereas expressions of b1 and TGFBI were comparable in two glucose concentrations. Flow cytometry results also showed expression of integrin subunits in pHRP at 72 hr of incubation. Expression of a3 in pHRP in 30mM glucose was similar to those in cells in 5.5m M (MFI of 251 vs 221 respectively). However, expression of b1 was higher in cells in the higher glucose concentration (MFI: 422 vs 343). ELISA data showed secretion TGFBI protein by HRP at 48 hr of incubation. Protein concentration in media of cell in 30mM glucose was significantly higher than those in 5.5mM (97 vs 57 pg/ml; p=0.0318). Conclusions: This is the first report on the expression of integrin subunits in HPR in euglycemic and hyperglycemic conditions. Both RT-PCR and flow cytometry results show α3, β1 subunits expressions, the level of which may be affected by glucose concentration in the cell media. Furthermore, our ELISA results confirm the secretion of TGFB1 by HRP and a significantly higher protein secretion in hyperglycemic condition. Overall, our data support the hypothesis of integrin and TGFBI expression in HRP. The increase in TGFBI secretion in hyperglycemia suggest a possible role of diabetes. Further studies will provide insight into the role of integrin and TGFBI interaction on the signaling pathway of HRP apoptosis and diabetic retinopathy

Diabetic Retinopathy: Targeting BIGH3 to Develop Novel Molecular Therapies

Diabetic retinopathy (DR) is a complication of diabetes due to damage of blood vessels in the retina. Aside from being a major cause of blindness in the world, DR also has a significant impact on quality of life. Although there are methods to delay the progression of DR, there are no existing therapeutic regimens for early intervention. Thus, it is critical to develop cost-effective therapies towards preventing DR development

Ginsenoside-Rb1 Inhibition of VEGF Release – Structure and Activity Relations (SAR) Perspective

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