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Abstract: Geldanamycin (GA), a benzoquinone ansamycin, was originally isolated as a natural product with anti-fungal activity. GA and its analogs, including 17-allylamino-demethoxy geldanamycin (17-AAG), are also known to block the function of a molecular chaperone, heat shock protein 90 (Hsp90). In light of their anti-tumor properties through direct cytotoxicity and anti-angiogenicity, GA has been previously demonstrated to suppress hypoxia-induced VEGF production in retinal pigment epithelium (RPE) cells, implicating its applicability in treating intraocular neovascularization. This study aimed at investigating the effectiveness of Hsp90 inhibitor treatment in suppressing proliferation of cultured human RPE cells and elucidating its underlying mechanism. Cultured RPE cells were treated with GA or 17-AAG and subjected for cell proliferation assay and cell cycle analysis. Expression of apoptotic regulators and survival signaling activity were monitored by Western blotting. The results showed that both GA and 17-AAG significantly inhibited RPE cell proliferation at micromolar levels. Treatment with GA and 17-AAG led to growth arrests in G1 and S phases, increased sub-G1 hypodipoid cell population, induced apoptotic cell death, and upregulated P53 and P21 expression, although the drug-induced Bcl-2 upregulation cannot prevent cell death. Additionally, GA and 17-AAG significantly suppressed constitutive contents of phosphorylated ERK1/2 and total Akt proteins, and completely abrogated wortmannin-sensitized Akt phosphorylation. In conclusion, GA and 17-AAG inhibit RPE cell proliferation and induce cytotoxicity, possibly through downregulating Akt- and ERK1/2-mediated signaling activities. They might potentially constitute a therapeutic agent for ocular disorders with RPE over proliferation, such as proliferative vitreoretinopathy. [Copyright &y& Elsevier]

It has been reported that retinoic acid (RA) may inhibit the growth of RPE and be used in the treatment of proliferative vitreoretinopathy (PVR). However, previous reports in this field have been conflicting. The main reason for these contradictory findings is that different methods for evaluating the effects of RA on RPE from different species have been used. In human specimens, only RPE from the donor eye (stationary) but not RPE from the PVR membrane (already at active proliferation status) have been tested. This study tested the effects of RA on the growth of RPE using a novel in vitro model: RPE from the PVR membranes, which simulates the in vivo situation of PVR patients better than RPE from the donor eyes. This study also used various methods to solve the conflicting results reported previously. We found that both all transretinoic acid (all-RA) and 13-cis-retinoic acid (cis-RA) can promptly (though not completely) inhibit proliferation of RPE (inhibition rate of 89%-90%) over a very wide range of concentrations (10-9-10-5 M) and various lengths of periods (2-12 days) in a dosedependent and time-dependent manner and without evident cytotoxic effects. Previously reported disadvantages discovered from the study of RPE from donor eyes, e.g., the absence of inhibitory effects on the early passages of cultured cells and inhibition occurring only after long-term treatment, do not present in RPE cells from the PVR membrane. The proliferation of RPE recovered from the inhibition by RA rapidly after the discontinuation of treatment, indicating that a continuous supply of the drug over a long period, i.e., until the breakdown of the blood-retinal barrier has been repaired, is essential for the success of drug treatment of PVR. [ABSTRACT FROM AUTHOR]

It has been well documented that RPE cells play an important role in the formation of proliferative vitreoretinopathy (PVR). We studied the effects of four most widely used anti-proliferative drugs, i.e., 5-fluorouracil (5-FU), daunomycin (DM), mitomycin C (MMC) and dexamethasone (DEX) in an in vitro model system (cultured human RPE). The cells treated with indicated concentrations of drugs for 48 h were harvested for DNA content analysis. In addition, a time course study with constant dosage of drugs was performed. The results revealed that, after incubation for 48 h, an increased percentage (31.1%) of S phase cells was noted with exposure to MMC (10 ng/ml). It was confirmed by 5-bromodeoxyuridine (BrdU) incorporation test that this increase does not result from higher transition rate from G1 to S phase. The cells of G2/M phase markedly increased from 13.8% to 29.7% with 10 ng/ml DM. We also demonstrated that 5-FU and MMC treatment led to cell accumulation at S phase and DM treatment resulted in cell accumulation at G2/M phase, These findings were compatible with their pharmacological mechanisms. Development of an in vitro model using cultured human RPE to study the effects of various anti-proliferative drugs on cell cycle can provide a rapid, safe and inexpensive method for selection of drugs used for management of PVR. [ABSTRACT FROM AUTHOR]

The effects of various antiproliferative drugs have been tested in numerous cell types in vitro, but a comparison of effects of these drugs on cultured human retinal pigment epithelium (RPE) has not been reported. We studied the effects of four most widely used antiproliferative drugs (5-FU, daunomycin, mitomycin and dexamethasone) in a new in vitro model system (cultured human RPE). Various concentrations and exposure periods were tested in four human RPE cell lines. 5-FU showed a dose-dependant growth inhibition, with an ID[sub 50] of 5.35 ± 2.38 × 10[sup -7] M after 4 days culture. A decrease of cell number occurred relatively later, the slope of the dose-response curve was less steep than that of others. Mitomycin C showed an immediate and strong growth inhibition and cytotoxic effects, with an ID[sub 50] of 3.73 ± 0.71 × 10[sup -9] M. RPE cultured with daunomycin showed an abrupt decrease of cell number from 10[sup -9] M to 10[sup -8] M, the ID value was 1.07 ± 0.23 × 10[sup -8] M. Dexamethasone showed a biphasic effect; it stimulated cell growth at 10[sup -7] to 10[sup -6] M and inhibited cell growth at 10[sup -4] M or higher, with an ID[sub 50] of 6.05 ± 1.61 × 10[sup -3] M. The advantages and disadvantages of these drugs and the prospective clinical application of these drugs for management of proliferative vitreoretinopathy (PVR) were discussed. Development of an in vitro model using cultured human RPE to study the effects of various antiproliferative drugs can provide a rapid, safe and inexpensive method for selection of drugs used for management of proliferative vitreoretinopathy. [ABSTRACT FROM AUTHOR]

Purpose: To determine whether postoperative oral 13-cis-retinoic acid (RA) treatment could improve the outcome of vitreoretinal surgery with silicone oil for the management of proliferative vitreoretinopathy (PVR). Design: Prospective controlled randomized interventional case series. Methods: This study included 35 eyes of 35 patients with primary rhegmatogenous retinal detachment and PVR. All patients underwent surgical repair by similar procedures. The RA group consisted of 16 patients who received 10 mg oral RA twice daily for eight weeks postoperatively. The control group included 19 patients without taking RA. The outcome measure included the rate of retinal attachment, macular pucker formation, ambulatory vision, and RA–related side effects. Results: At last follow-up (at least one year postoperatively), 15 of 16 eyes (93.8%) in the RA group and 12 of 19 eyes (63.2%) in the control group maintained retinal attachment (P = .047). The rate of macular pucker formation was significantly lower in the RA group (18.8% vs 78.9% in the control group; P = .001). A higher rate of ambulatory vision was achieved in the RA group as compared to the control group (56.3% vs 10.5%; P = .009). Conclusions: Postoperative administration with oral moderate dosage of RA for eight weeks appears to maintain retinal attachment, decrease the macular pucker, and improve vision after surgical repair for eyes with PVR. [Copyright &y& Elsevier]

Abstract: All-trans retinoic acid (atRA) has been reported to inhibit the proliferation of retinal pigment epithelial (RPE) cells and used in treatment of proliferative vitreoretinopathy (PVR) in animal model. This study aimed at examining the effectiveness of atRA in inhibiting the extracellular matrix (ECM) biosynthesis by RPE cells and the RPE cell-mediated collagen gel contraction. Cultured RPE cells were treated with atRA and the expression of four ECM proteins (collagen types I, III, IV and laminin β1) was profiled. The results indicated that atRA treatment up-regulated de novo synthesis of collagen type I, but decreased that of laminin β1 in a dose-dependent manner. Moreover, the effect of atRA on RPE cell contraction was evaluated by measuring the area of collagen gel where RPE cells populated. Treatment with atRA significantly inhibited RPE cell-mediated collagen gel contraction. Addition of exogenous laminin nonapeptide into gels promoted RPE cell contraction, while atRA reversed the laminin-enhanced contractility. atRA treatment significantly suppressed the gene expression of integrin β3 but not αV subunit, and effectively inhibited the tyrosine phosphorylation of integrin β3 at residue 747 in RPE cells grown on laminin-coated dish, suggesting that atRA may suppress the RPE contractility through either inhibiting integrin β3 expression or abrogating the integrin β3-mediated signaling. In conclusion, atRA pharmacologically possesses a tissue-remodeling capacity and inhibits contractility of RPE cells. Therefore, atRA might be potentially a therapeutic agent for certain ocular disorders such as PVR. [Copyright &y& Elsevier]