Jeffery G. Grigsby

The Role of Microglia in Diabetic Retinopathy

There is growing evidence that chronic inflammation plays a role in both the development and progression of diabetic retinopathy. There is also evidence that molecules produced as a result of hyperglycemia can activate microglia. However the exact contribution of microglia, the resident immune cells of the central nervous system, to retinal tissue damage during diabetes remains unclear. Current data suggest that dysregulated microglial responses are linked to their deleterious effects in several neurological diseases associated with chronic inflammation. As inflammatory cytokines and hyperglycemia disseminate through the diabetic retina, microglia can change to an activated state, increase in number, translocate through the retina, and themselves become the producers of inflammatory and apoptotic molecules or alternatively exert anti-inflammatory effects. In addition, microglial genetic variations may account for some of the individual differences commonly seen in patients susceptibility to diabetic retinopathy.

Estrogen-Induced Retinal Endothelial Cell Proliferation: Possible Involvement of Pigment Epithelium-Derived Factor and Phosphoinositide 3-Kinase/Mitogen-Activated Protein Kinase Pathways

PURPOSE Diabetic retinopathy is a leading cause of blindness due to a progressive damage of the retina by neovascularization and other related ocular complications. However, the molecular mechanism underlying the development of diabetic retinopathy is not well understood. An increase in estrogen levels during puberty is associated with an accelerated development of diabetic retinopathy. Previously, we have introduced 17β-estradiol (E2) to rhesus retinal capillary endothelial cells (RhRECs) in culture and observed a dose- and time-dependent increase in the number of viable cells. The purpose of this present study was to investigate the molecular signaling pathway associated with this estrogen-induced proliferation of RhRECs. METHODS Estrogen receptor (ER) ER(α) and ER(β) mRNA expression, and protein synthesis were measured at 0, 3, 6, and 12 h using nested polymerase chain reaction and Western blots. Phosphoinositide 3-kinase (PI3K) and mitogen-activated protein kinase (MAPK) pathway inhibitors were introduced into culture media to study their effects on E2-induced cell proliferation and pigment epithelium-derived factor (PEDF) synthesis. The levels of PEDF in the conditioned media were measured by enzyme-linked immunosorbent assay. RESULTS Exogenous E2 induced a significant increase in the expression of ER(β) along with an increase in the number of viable RhRECs. Cotreatment of E2 with PI3K and MAPK inhibitors significantly reduced the E2-induced effect on cell proliferation and PEDF production in a dose-dependent manner. CONCLUSION Results from the present study suggest that an E2-induced increase in the proliferation of RhRECs may be mediated by the action of ER(β.) Both PI3K and MAPK signaling pathways are involved in this E2-induced cell proliferation, which may follow changes in PEDF levels controlled by these pathways. Further studies will provide additional details on the interaction between these pathways to control changes in PEDF levels and cell proliferation.

A Possible Role of Acrolein in Diabetic Retinopathy: Involvement of a VEGF/TGFβ Signaling Pathway of the Retinal Pigment Epithelium in Hyperglycemia

Purpose: Acrolein has been implicated in retinal pigment epithelium (RPE) cell death, and has been associated with diabetic retinopathy. Our purpose was to investigate the potential effect of high glucose in influencing acrolein-mediated RPE cytokine production and cell death. We investigated the influence of the acrolein effect on ARPE-19 cells in high glucose conditions and quantified the release of transforming growth factor β (TGFβ1 and 2) and vascular endothelial growth factor (VEGF). We assessed the ability of N-benzylhydroxylamine(NBHA) as well as TGFβ pathway inhibitors SIS3 and SB431542 to prevent this effect of acrolein on ARPE-19 cells. Materials and methods: Confluent ARPE-19 cells were treated with acrolein and/or NBHA in both 5.5 and 18.8 mM glucose conditions. Cells were also pretreated with SIS3, a specific inhibitor of the SMAD3 pathway, and SB431542, a specific inhibitor of TGFβ signaling pathway, before treating them with acrolein. Viable cells were counted and ELISAs were performed to measure the cytokines TGFβ1 and 2, and VEGF released into the conditioned media. Results: In ARPE-19 cells exposed to acrolein and hyperglycemia there was reduced cell viability and an increase in the cell media of VEGF, TGFβ1, and TGFβ2, which was reversed by NBHA. Acrolein/hyperglycemia-induced cell viability reduction and cytokine overproduction was also reduced by TGFβ pathway blockade. Conclusions: We conclude that the effect of acrolein on the reduction of viability and VEGF increase by ARPE-19 cells in hyperglycemic media is conducted through the TGFβ signaling pathway. Our results suggest that benefits of sequestering acrolein by NBHA and the blockage of the TGFβ pathway by SB431542 and SIS3 offer suggestions as to potential useful pharmacological drug candidates for the prevention of diabetes-induced complications in the eye.

Effects of Tamoxifen Versus Raloxifene on Retinal Capillary Endothelial Cell Proliferation

PURPOSE Endothelial cell proliferation in angiogenesis is active in conditions such as cancers and diabetic retinopathy. Tamoxifen (T) and raloxifene (R) have been compared in numerous studies as a prophylaxis for breast cancer, and T is used to treat breast cancer. T, unlike R, has been linked to an increase in uterine cancers, thrombo-embolic events, and cataract. The purpose of our study was to evaluate the efficacies of T and R in reducing estrogen-induced retinal capillary endothelial cell proliferation. METHODS Rhesus monkey retinal capillary endothelial cells (ATCC RF/6A) were used to assay cell proliferation when treated with 0.0, 0.1, 1.0, and 10.0 nM 17 β estradiol (E2) for 24 and 48 h. Viable cells were counted using a Neubauer hemocytometer with a trypan blue exclusion method to determine the number of viable cells. Cell counts were also performed using 1.0 nM E2 with 0.01, 0.1, 1.0, and 10.0 nM concentrations of either T or R. Cell medium, collected at 24 h, was evaluated for vascular endothelial growth factor and pigment epithelium-derived factor. RESULTS Viable cells were significantly greater in cultures treated with 1.0 or 10.0 nM E2, compared to cells treated with 0.0 or 0.1 nM E2 both at 24 and 48 h. Viable cell counts were reduced significantly in cultures treated with 0.1, 1.0, or 10.0 nM T or R in addition to the 1.0 nM E2. Cell counts were not significantly different when comparing equal concentrations of T and R, that is, 1.0 nM E2+1 nM T or R. Vascular endothelial growth factor and pigment epithelium-derived factor protein/10,000 cells was reduced by 1.0 nM E2, but returned to higher levels with the introduction of T and R to growth media. CONCLUSIONS T and R showed similar potency in inhibiting estrogen-induced retinal capillary endothelial cell proliferation. Considering drug safety profiles, our results, when extended to animals and humans, suggest that R is preferable to T in treating angiogenic retinal diseases. Further studies on the signaling mechanism of estrogen-induced endothelial cell proliferation may lead to new treatment strategies in the treatment of ocular angiogenic diseases.

Ginsenoside-Rb1 Induces ARPE-19 Proliferation and Reduces VEGF Release

Rb1, a ginsenoside from ginseng root extract, possesses antiangiogenic effects, but its role on ocular cells has not been studied. We hypothesize that Rb1 inhibits the production of the angiogenic cytokine VEGF from ARPE-19 cells, leading to a significant reduction in the proliferation of ocular vasculatures. Data from our experiments show that Rb1 induced an increase in the number of ARPE cells in culture, while VEGF release (pg/10,000 viable cells) was significantly reduced. Treatment with VEGF and cotreatment with Rb1 and VEGF showed that this Rb1-induced cell proliferation was mediated by VEGF. Because VEGF from RPE plays a major role in promoting angiogenesis in ocular vasculatures. Our finding that Rb1 inhibits the release of VEGF from RPE cells suggests that Rb1 has a significant role in the eye to protect against angiogenic diseases such as age-related macular degeneration.

The Role of Sex Hormones in Diabetic Retinopathy

Jeffery G. Grigsby1, Donald M. Allen2, Richard B. Culbert3,4, Gerardo Escobedo4, Kalpana Parvathaneni1, Brandi S. Betts1 and Andrew T.C. Tsin1 1Department of Biology, University of Texas at San Antonio, San Antonio, Texas 2Department of Biology, University of Texas of the Permian Basin, Odessa, Texas 3Department of Surgery, Texas Tech School of Medicine, Midland, Texas 4Premier Retina Specialists, Midland, Texas 1, 2,3,4United States of America