Brandi S. Betts

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.

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

Cultured Müller cells from mammals can synthesize and accumulate retinyl esters

Research reports from our laboratory, as well as those fromothers, have shown that cone-dominant species such as chicken,ground squirrel and zebrafish possess a novel cone cycle which isdependent on the production of 11-cis retinal from Muller cells intheretina(Munizetal.,2006;Munizetal.,2007;WangandKefalov,2011). This visual cycle operates differently from the classical(canonical) visual cycle, which is dependent on the synthesis of11- cis retinal by an isomerohydrolase (RPE65 or Isomerase 1)located in the retinal pigment epithelium (RPE) (Fleisch andNeuhauss, 2010; Takahashi et al., 2011). Using eyecup and iso-lated retina preparations, Kolesnikov et al. recently reported thatthe mouse retina promoted M/L-cone dark adaptation eightfoldfaster than the RPE alone (Kolesnikov et al., 2011) suggesting thatthe initial rapid cone pigment regeneration is highly dependent onthe synthesis of 11-cis retinal from Muller cells in the retina(Kolesnikov et al., 2011). However, it is not clear if IRBP (Inter-photoreceptor Retinoid Binding Protein) is essential for normalretinoidprocessingintheretinalconecycle(Kolesnikovetal.,2011;Parker et al., 2011). Furthermore, it has recently been reported thatdifferent variants of isomerases are expressed in mouse conephotoreceptors (Tang et al., 2011) as well as zebrafish Muller cellsand ganglion cells (RPE65a and RPE65c) (Takahashi et al., 2011).Mullercellsfromthechickenretinahavebeen shownto possessretinol isomerase (Das et al., 1992; Mata et al., 2005; Muniz et al.,2009) and 11-cis retinyl ester synthase (Muniz et al., 2006) activi-ties. Although limited biochemical data are now available on thesevisual cycle enzymes, they are collected from the retina or Mullercellsin cone-dominant species. Atpresent, it is notknown if Mullercellsfromrod-dominantretinasalsopossessaconevisualcycleandspecific visual proteins for the cone cycle.Spontaneously immortalized human Muller cells were obtainedfrom Dr. Astrid Limb (University College; London, UK). Cells werecultured in T25 flasks using Dulbecco’smodified essential medium(DMEM) with 4.5 g/L glucose and 10% fetal bovine serum (FBS) at37 C þ 5% CO