Autocrine and Paracrine Secretion of Vascular Endothelial Growth Factor in the Pre-Hypoxic Diabetic Retina.

Vascular endothelial growth factor (VEGF) is well established as the main agent responsible for vascular leakage and angiogenesis in the diabetic retina. While VEGF can have positive effects on hyperglycemia stressed retinal tissues, it also plays a role in events progressing to the oxygen- stressed, i.e. hypoxic, diabetic retina. Some VEGF makes its way to the retina from systemic sources and some is produced locally within the eye. Hyperglycemia, oxidants, inflammation, and advanced glycation end-products are all stimulants to VEGF production, both in the hypoxic and the pre-hypoxic retina. Endothelial cells, pericytes, Müller cells, microglia, astrocytes, retinal pigment epithelium and neurons have all been known to produce VEGF at some point in retinal development or in disease. Excessive VEGF production in the early diabetic retina can lead to retinal exposure or mechanisms which exacerbate further damage. While Müller cells are likely the most significant producer of VEGF in the pre-hypoxic retina, other VEGF producing cells may also play a role due to their proximity to vessels or neurons. Study of the release of VEGF by retinal cells in hyperglycemia conditions, may help identify targets for early treatment and prevent the serious consequences of diabetic retinopathy.

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 patient's 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 17ß-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(ß) 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(ß) 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(ß.) 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.

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 ß 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.