Effect of PDGF-BB on Human Retinal Pericytes

Background – The majority of ocular conditions seen in patients with diabetic retinopathy are a result of damage to the retinal vasculature, which leads to microaneurysms, hemorrhage, and eventually neovascularization. The first step in the pathogenesis of these conditions is derived from the loss of Human Retinal Pericytes (HRPs), or cells which are essential to the preservation of the integrity of the retinal vasculature, and PDGF-BB is the primary promotor of growth and recruitment for HRPs. Aim - Patients with NPDR produce lower levels of PDGF-BB than in normal conditions, which could contribute to the loss of pericytes early in diabetic retinopathy. The purpose of this study is to evaluate the effect of PDGF-BB on HRPs in vitro to determine whether or not the longevity of the HRPs can be preserved with the intent of contributing to development of therapeutic interventions for patients with diabetic retinopathy. This is the first PDGF-BB study on its effect on the viability and longevity of HRPs. Methods – Three groups of HRPs were treated with three different concentrations of PDGF-BB which correlate to concentrations found in patients with PDR, NPDR, and normal conditions. These three concentrations were also compared with a negative control group which was not treated. After 24 and 48 hours the viable cells were counted by triptan blue measurements. The results were reported by evaluating viable cells at 24 and 48 hours, growth of each group between these time periods, and significant growth differences between groups at each time period. Each group was grown in triplicates and an average from these triplicates was used during analysis. Results – Of the results found, three were clinically significant. Pertaining to viability, the NPDR group was the only one with a significant result at 24 hours. Growth difference analysis was significant for the PDR group at the 24-hour period. Growth difference between all groups was significant at the 24 hours mark but not at 48. Conclusion – Of the results obtained, significant results were only seen at the 24 hours period which is thought to be a result of the well space not being large enough to accommodate growth beyond the 24-hour time frame. The finding of a statistically significant difference at 24 hours but not 48 hours further suggests this as the complication as well. That being said, significant growth difference was seen with the PDR group, which was treated with high levels of PDGF-BB. This finding is consistent with the hypothesis that PDGF-BB levels encourage the growth of HRP cells. Discussion - Previous clinical trials on PDGF-BB inhibitors showed that they did not help patients with diabetic retinopathy due to the inherent deficiency of HRPs found in these patients. We anticipate results from this study will support an opposing approach by supplementing PDGF-BB, rather than blocking leading to innovative clinical outcomes

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

Gemigliptin, a dipeptidyl peptidase-4 inhibitor, inhibits retinal pericyte injury in db/db mice and retinal neovascularization in mice with ischemic retinopathy

AbstractRetinal pericyte loss and neovascularization are characteristic features of diabetic retinopathy. Gemigliptin, a dipeptidyl peptidase-4 (DPP-4) inhibitor, has shown robust blood-glucose lowering effects in type 2 diabetic patients, but its effects on diabetic retinopathy have not yet been reported. We evaluated the efficacy of gemigliptin on retinal vascular leakage in db/db mice, which is an animal model for type 2 diabetes, and neovascularization in oxygen-induced retinopathy (OIR) mice, which is an animal model for ischemic proliferative retinopathy. Gemigliptin (100mg/kg/day) was orally administered to the db/db mice for 12weeks. C57BL/6 mice on postnatal day 7 (P7) were exposed to 75% hyperoxia for 5days, followed by exposure to room air from P12 to P17 to induce OIR. Gemigliptin (50mg/kg/day) was intraperitoneally injected daily from P12 to P17. Retinal neovascularization was analyzed in flat-mounted retinas on P17. We determined the efficacy and possible mechanism of gemigliptin on high glucose-induced apoptosis of primary human retinal pericytes. The oral administration of gemigliptin for 4months significantly ameliorated retinal pericyte apoptosis and vascular leakage in the db/db mice. Gemigliptin also ameliorated retinal neovascularization in the OIR mice. Gemigliptin attenuated the overexpression of plasminogen activator inhibitor-1 (PAI-1) in the retinas of diabetic and OIR mice. Gemigliptin and PAI-1 siRNA significantly inhibited pericyte apoptosis by inhibiting the overexpression of PAI-1, which is induced by high glucose. Our results suggest that gemigliptin has potent anti-angiogenic and anti-apoptotic activities via suppressing DPP-4 and PAI-1, and the results support the direct retinoprotective action of gemigliptin