BIGH3 protein and macrophages in retinal endothelial cell apoptosis.

Diabetes is a pandemic disease with a higher occurrence in minority populations. The molecular mechanism to initiate diabetes-associated retinal angiogenesis remains largely unknown. We propose an inflammatory pathway of diabetic retinopathy in which macrophages in the diabetic eye provide TGFß to retinal endothelial cells (REC) in the retinal microvasculature. In response to TGFß, REC synthesize and secrete a pro-apoptotic BIGH3 (TGFß-Induced Gene Human Clone 3) protein, which acts in an autocrine loop to induce REC apoptosis. Rhesus monkey retinal endothelial cells (RhREC) were treated with dMCM (cell media of macrophages treated with high glucose and LDL) and assayed for apoptosis (TUNEL), BIGH3 mRNA (qPCR), and protein (Western blots) expressions. Cells were also treated with ΤGFß1 and 2 for BIGH3 mRNA and protein expression. Inhibition assays were carried out using antibodies for TGFß1 and for BIGH3 to block apoptosis and mRNA expression. BIGH3 in cultured RhREC cells were identified by immunohistochemistry (IHC). Distribution of BIGH3 and macrophages in the diabetic mouse retina was examined with IHC. RhRECs treated with dMCM or TGFß showed a significant increase in apoptosis and BIGH3 protein expression. Recombinant BIGH3 added to RhREC culture medium led to a dose-dependent increase in apoptosis. Antibodies (Ab) directed against BIGH3 and TGFß, as well as TGFß receptor blocker resulted in a significant reduction in apoptosis induced by either dMCM, TGFß or BIGH3. IHC showed that cultured RhREC constitutively expressed BIGH3. Macrophage and BIGH3 protein were co-localized to the inner retina of the diabetic mouse eye. Our results support a novel inflammatory pathway for diabetic retinopathy. This pathway is initiated by TGFß released from macrophages, which promotes synthesis and release of BIGH3 protein by REC and REC apoptosis.

Chemical Modification of Ginsenoside on Cell Viability and Cytokine Secretion.

BACKGROUND: Rb1 is a ginsenoside steroid glycoside found exclusively in the plant Panax ginseng. In an earlier report, we showed that Rb1 increased cell proliferation and reduced VEGF (vascular endothelial growth factor) secretion by human retinal pigment epithelial (ARPE19) cells. OBJECTIVE: In the present study, we hypothesized that chemical modification of Rb1 changes the level of VEGF secretion by ARPE19 cells. METHOD: Three derivatives of Rb1 were chemically synthesized by hydrogenation (Rb1-H2), acetylation (Rb1-Acyl), and epoxidation (Rb1-Epoxy). Structural modifications were confirmed by 1H Nuclear Magnetic Resonance (NMR) spectra and Mass Spectrometry (MS). To test the biological activity, chemically modified compounds were added to cell culture media and incubated for 72 hours at a concentration of 250 nM at 37°C. Conditioned media were collected and cells were harvested/ counted after treatment. Viable cell numbers were determined by the trypan blue dye exclusion method and VEGF levels by Enzyme-Linked Immunosorbent Assays (ELISA). RESULTS: Consistent with the prior report, results of the present study show Rb1 increased cell proliferation and decreased VEGF secretion. Similar to Rb1's effect on cell proliferation, treatment with Rb1-H2, Rb1-Acyl and Rb1-Epoxy resulted in an increase in cell numbers. In contrast to Rb1- induced decrease in VEGF secretion, treatment with Rb1-H2, Rb-Acyl and Rb1-Epoxy resulted in increased VEGF levels. CONCLUSION: Chemical modifications of the ginsenoside Rb1 significantly affect the biological activity of VEGF secretion by ARPE19 cells. Additional SAR (Structure Activity Relationship) experiments will be conducted to study the detailed mechanisms by which how specific modifications of Rb1 functional groups alter biological activities.

Oxidative Stress Induces Senescence in Cultured RPE Cells.

The aim of this research is to determine whether oxidative stress induces cellular senescence in human retinal pigment epithelial cells. Cultured ARPE19 cells were subjected to different concentrations of hydrogen peroxide to induce oxidative stress. Cells were seeded into 24-well plates with hydrogen peroxide added to cell medium and incubated at 37°C + 5% CO2 for a 90-minute period [at 0, 300, 400 and 800 micromolar (MCM) hydrogen peroxide]. The number of viable ARPE19 cells were recorded using the Trypan Blue Dye Exclusion Method and cell senescence was measured by positive staining for senescence-associated beta-galactosidase (SA-beta-Gal) protein. Without hydrogen peroxide treatment, the number of viable ARPE19 cells increased significantly from 50,000 cells/well to 197,000 within 72 hours. Treatment with hydrogen peroxide reduced this level of cell proliferation significantly (to 52,167 cells at 400 MCM; to 49,263 cells at 800 MCM). Meanwhile, cells with a high level of positive senescence-indicator SA-Beta-Gal-positive staining was induced by hydrogen peroxide treatment (from a baseline level of 12% to 80% at 400 MCM and at 800 MCM). Our data suggests that oxidative stress from hydrogen peroxide treatment inhibited ARPE19 cell proliferation and induced cellular senescence.

Effect of Glucose on Retinal Endothelial Cell Viability and VEGF Secretion.

Previous studies have shown that in diabetic patients, there is an increase of retinal capillaries associated with the development of diabetic retinopathy in the eye. The objective of current study is to investigate the effect of glucose on retinal endothelial cell viability and VEGF secretion. 20,000 cells per well were treated without glucose or with 5.5mM (euglycemic), 18.5mM and 30mM (hyperglycemic) glucose for 24 hours. Viable cells were counted using Trypan blue dye exclusion method. ELISA was used to measure VEGF secretion from cells into the cell medium. The number of viable cells incubated with 5.5mM glucose (physiological control) increased by 53.7% after 24 hours. In comparison, cells treated with 18.5mM glucose decreased by 2.8% while cells treated with 30mM glucose decreased by 20% after 24 hours of incubation. Cells without glucose treatment (0mM control) decreased by 33.3%. In contrast to the decrease of viable cell numbers after treatment with high glucose, there is an increase in VEGF secretion (pg/mL) to the cell medium with increase in glucose concentration from 5.5mM to 0, 18.5, and 30mM. The amount of VEGF secreted per cell also increased with increasing glucose concentrations. Our results show that viability of retinal endothelial cells and VEGF release are highly responsive to changes in glucose concentration. Such glucose-induced changes in retinal endothelial cells may negatively impact the integrity of the microvasculature in the diabetic retina leading to angiogenesis and microaneursyms.

Interphotoreceptor retinoid-binding protein (IRBP) promotes retinol uptake and release by rat Müller cells (rMC-1) in vitro: implications for the cone visual cycle.

PURPOSE: Interphotoreceptor retinoid-binding protein's (IRBP) role in facilitating the exchange of retinoids between rod and cone photoreceptors, RPE, and Müller cells in the visual cycle remains a mystery. Interphotoreceptor retinoid-binding protein's ability to bind the pericellular matrix of the cone outer segment and Müller cell villi suggests a function in all-trans and 11-cis retinol targeted trafficking in the cone visual cycle. We hypothesize that IRBP facilitates delivery and uptake of all-trans retinol to and release of 11-cis retinol from rat Müller cells (rMC-1). METHODS: Rat Müller cells were incubated with all-trans retinol and BSA or bovine IRBP (bIRBP). Retinoids in the cell homogenates and conditioned media were analyzed by high performance liquid chromatography (HPLC). RESULTS: Cells incubated with 10 µM retinol and BSA had 2100 pmol of all-trans retinol per milligram homogenate protein compared with 3450 pmol when retinol was delivered by bIRBP; these cells also had 450 pmol all-trans retinyl ester per milligram when retinol was delivered by BSA compared with 270 pmol when retinol was delivered by bIRBP. Conditioned media from cells incubated with retinol delivered by BSA did not contain11-cis retinol. However, cells with retinol delivered by bIRBP released 130 pmol/mL of 11-cis retinol into the cell media. Incubation with 5.0 mM deferoxamine (an iron chelator) reduced IRBP-dependent 11-cis retinol retrieval by 60%. CONCLUSIONS: Promoting Müller cell uptake of all-trans retinol and release of 11-cis retinol is a previously unrecognized function of IRBP that may be critical to cone function and integrity.

Effect of Silver Coating on Barium Titanium Oxide Nanoparticle Toxicity.

Nanoparticles are presently being studied for optical and biomedical applications such as medical imaging and drug delivery. Nanoparticles impact the cellular environment due to many variables such as size, shape, and composition. How these factors affect cell viability is not fully understood. The purpose of this study is to test the toxicity effects of silver coating (Ag@) Barium Titanium Oxide (BaTiO3) nanoparticles on Rhesus Monkey Retinal Endothelial cells (RhREC's) in culture. The addition of silver to the nanoparticles increases their nonlinear optical properties significantly, making the Ag@BaTiO3 nanoparticles good candidates for nonlinear microscopy contrast agents. We hypothesize that by silver coating nanoparticles, there will be an increase in cell viability at higher concentrations when compared to non-silver coated nanoparticles. RhREC's were treated with BaTiO3 and Ag@BaTiO3 at concentrations of 0, 1.0, 10.0, and 100µg/ml for 24 hours at 37°C + 5%CO2. After 24 hour incubation with respective nanoparticles, cell viability was determined using the trypan blue dye-exclusion method. Treatment with 0, 1.0 and 10.0µg/ml of Ag@BaTiO3 had minimal effect on cell viability, with 90% viable cells remaining at the end of the 24 hours treatment period. However, cells treated with 100µg/ml of Ag@BaTiO3 resulted in a decrease to 51% viable cells. Comparatively, cells treated with 0, 1.0 and 10µg/ml of BaTiO3 had no significant effect on cell viability (90% viable cells after treatment) while the 100µg/ml treatment resulted in a decrease to 29% viable cells. These results show that silver coating of BaTiO3 nanoparticles has a protective effect on cellular toxicity at high concentrations.