Yo-Chen Chang

Effect of Oral 13-Cis-Retinoic Acid Treatment on Postoperative Clinical Outcome of Eyes With Proliferative Vitreoretinopathy

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.

All-trans retinoic acid remodels extracellular matrix and suppresses laminin-enhanced contractility of cultured human retinal pigment epithelial cells.

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 beta1) was profiled. The results indicated that atRA treatment up-regulated de novo synthesis of collagen type I, but decreased that of laminin beta1 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 beta3 but not alphaV subunit, and effectively inhibited the tyrosine phosphorylation of integrin beta3 at residue 747 in RPE cells grown on laminin-coated dish, suggesting that atRA may suppress the RPE contractility through either inhibiting integrin beta3 expression or abrogating the integrin beta3-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.

Methylglyoxal, a reactive glucose metabolite, enhances autophagy flux and suppresses proliferation of human retinal pigment epithelial ARPE-19 cells

Methylglyoxal (MGO), a glycolytic metabolite, induces oxidative injury and apoptotic cell death that play a pathogenetic role in age-related macular degeneration (AMD). This study examined the impact of MGO on cell proliferation and autophagy flux in retinal pigment epithelium (RPE) ARPE-19 cells and elucidated the underlying mechanism. Short-term MGO exposure suppressed cell proliferation without induction of apoptotic cell death, increased production of reactive oxygen species, and potentiated H2O2-exhibited cytotoxicity in ARPE-19 cells. Conversely, pretreatment with N-acetylcysteine, a ROS scavenger, and aminoguanidine, an MGO blocker, prevented MGO-induced growth retardation. MGO significantly enhanced autophagy flux and increased intracellular accumulation of autophagosomes, which was functionally confirmed by addition of autophagy enhancer or inhibitors. Signaling kinetic observation indicated that MGO remarkably triggered phosphorylation of Akt, ERK1/2, p38 MAPK, and JNK1/2. Blockade of kinase activity demonstrated that the hyperphosphorylation of Akt, ERK1/2, JNK, and p38 MAPK were all involved in the MGO-enhanced autophagy and growth-arresting effect in ARPE-19 cells. Moreover, pretreatment with autophagic flux inhibitors including 3-methyladenine, bafilomycin A, and chloroquine effectively ameliorated MGO- but not H2O2-mediated ARPE-19 cytotoxicity. In conclusion, modulation of autophagy flux activity by using autophagic or kinase inhibitors may be an applicable modality to treat AMD.

Geldanamycin and its analog induce cytotoxicity in cultured human retinal pigment epithelial cells

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.

Pleiotropic role of atorvastatin in regulation of human retinal pigment epithelial cell behaviors in vitro

Statins, inhibitors of 3-hydroxy-3-methylglutaryl-coenzyme A reductase, possess pleiotropic effects that have been extended to modulation of various cellular behaviors. This study aimed at examining whether atorvastatin (AVN) modulates cell growth, adhesion, migration, and contraction of cultured human retinal pigment epithelium (RPE) cells. The in vitro effects of AVN on human RPE cells was analyzed in terms of cell proliferation, cell cycle, cell adhesion, migration, and contraction assays. The modulatory effect of AVN on TGF-β2-triggered signaling was determined by Western blotting detection. AVN at submicromolar dose exhibited no prominent morphological alteration and cytotoxicity, whereas it elicited cytostatic effect at concentrations higher than 1 μM. Cell cycle analysis showed that AVN induced growth arrest in both G1 and G2/M phases. AVN at 1 μM or higher concentrations significantly suppressed RPE cell adhesion. Cell migration and 3D collagen contraction assays showed that AVN significantly suppressed RPE cell migration and contractility, respectively. Mechanistically, AVN treatment transiently up-regulated phosphorylation of Akt, ERK1/2, and p38 MAPK, whereas down-regulated that of JNK1. Intriguingly, AVN pretreatment prominently attenuated the TGF-β(2)-mediated non-Smad signaling, including Akt, ERK1/2, p38 MAPK, and JNK1 phosphorylation. Besides, it directly reduced constitutive level of myosin regulatory light chain peptide MYL9 and mitigated the TGF-β(2)-induced phosphorylation of myosin phosphatase-targeting subunit 1, MYPT1. These in vitro findings strongly suggest that AVN possesses pleiotropic function on RPE cells, including anti-proliferation, anti-adhesion, anti-migration as well as anti-contraction. In conclusion, AVN treatment may be considered a useful therapy for proliferative vitreoretinal diseases.

Prognosis of Taiwanese patients with isolated optic neuritis after intravenous methylprednisolone pulse therapy.

BACKGROUND/PURPOSE To evaluate the clinical manifestations, prognosis, recurrence rate and development of multiple sclerosis between papillitis group and retrobulbar group in Taiwanese patients with isolated acute optic neuritis (AON) after treatment with intravenous methylprednisolone pulse therapy. METHODS We retrospectively reviewed patients with AON who had received intravenous methylprednisolone pulse therapy. These patients were classified into retrobulbar or papillitis groups. Demographic characteristics, responsiveness to pulse therapy, recurrence rate and incidence of multiple sclerosis were compared between these two groups. RESULTS Of the 43 patients enrolled in this study, 19 patients (44%) were in the retrobulbar group and 24 patients (56%) were in the papillitis group. Seven cases (16%) showed periventricular plaque on magnetic resonance imaging (MRI). Among these seven patients, five developed definite or probable multiple sclerosis. The incidence of multiple sclerosis in patients with positive brain MRI findings was significantly higher than in patients with negative MRI findings (p = 0.002). There was no statistical difference in final visual acuity between the two group (p = 0.353). Sixteen patients suffered from recurrence of AON (21% in the papillitis group and 58% in the retrobulbar group, p = 0.029). Two patients (8%) in the papillitis group and six patients (32%) in the retrobulbar group developed multiple sclerosis (p = 0.061) with a mean interval of 21.6 +/- 11.2 months. CONCLUSION AON in Taiwan has a relatively lower percentage of development of multiple sclerosis than in Western countries. The presence of periventricular plaque on MRI is significantly associated with the later development of multiple sclerosis. The retrobulbar group had a stronger association with recurrence and development of multiple sclerosis.

Correlation Between the Dynamic Postoperative Visual Outcome and the Restoration of Foveal Microstructures After Macular Hole Surgery

PURPOSE To analyze the long-term dynamic healing process of outer retinal changes for 1 year in patients who underwent a standard vitrectomy procedure for idiopathic macular hole (MH) repair. DESIGN Retrospective, consecutive, observational case series. METHODS Data were collected on 60 eyes of 56 patients (30 women, 26 men) that underwent successful pars plana vitrectomy (PPV) and internal limiting membrane (ILM) peeling for idiopathic MH from January 2011 to December 2012. The age distribution ranged from 56 to 85 years (mean: 64 years). Forty eyes underwent combined phacoemulsification, PPV, ILM peeling, and intraocular lens implantation; 20 preoperative pseudophakic eyes underwent PPV and ILM peeling only. The main outcome measures included logMAR best-corrected visual acuity (BCVA) and macular microstructures determined by spectral-domain optical coherence tomography performed pre- and postoperatively during follow-up visits at 1, 3, 6, 9, and 12 months. RESULTS One month after surgery, 24 eyes (40%) showed normal external limiting membrane (ELM), 36 eyes (60%) showed normal ELM at 3 months, and 54 eyes (90%) showed normal ELM 12 months after surgery. Six eyes (10%) revealed a continuous ellipsoid zone (EZ) at 1 month, 18 eyes (30%) at 3 months, and 48 eyes (80%) at 12 months postoperatively. There were no eyes with a disrupted ELM in the presence of an intact EZ line. The eyes with intact ELM and/or intact EZ line showed better BCVA than eyes with defects in ELM or EZ line. On the contrary, glial cell presentation is significantly associated with worse postoperative BCVA. However, the presence of foveal cystoid change is not significantly associated with postoperative BCVA. CONCLUSIONS The ELM and EZ line at the fovea recovered and the presence of glial cells and cystoid space resolved gradually after surgery. The postoperative visual acuity was correlated with resolved glial cells and a restored ELM and EZ line.

Modified internal limiting membrane peeling technique (maculorrhexis) for myopic foveoschisis surgery.

Myopic foveoschisis occurs in 9–34% of highly myopic eyes with posterior staphyloma. The pathogenesis is still not fully understood. But the relative inflexibility of the inner retina and a tangential traction induced inward traction force in the posterior staphyloma are possible mechanisms. Conventional internal limiting membrane (ILM) peeling generally yields good results. However, a postoperative full‐thickness macular hole happens in 13–28% of cases. Therefore, this study describes a modified ILM peeling technique named ‘ILM maculorrhexis’ to minimize the occurrence of postoperative macular hole in patients with foveoschisis.

Pharmacological implications from the adhesion-induced signaling profiles in cultured human retinal pigment epithelial cells.

Extracellular matrix (ECM) plays an active and complex role in regulating cellular behaviors, including proliferation and adhesion. This study aimed at delineating the adhesion-induced signaling profiles in cultured human retinal pigment epithelium (RPE) cells and investigating the antiadhesion effect of antiproliferative drugs in this context. RPE R-50 cells grown on various ECM molecules, such as type I and IV collagens, fibronectin, and laminin, were used for adhesion assay and for examining the phosphorylation profiles of signaling mediators including Akt, extracellular signal-regulated kinase (ERK) 1/2, and integrin-linked kinase (ILK) using Western blotting. The cells receiving antiproliferative drug treatment at subtoxic doses were used to evaluate their antiadhesive and suppressive effects on kinase activities. ECM coating enhanced adhesion and spreading of RPE cells significantly. The cellular attachment onto ECM-coated surfaces differentially induced Akt, ERK1/2, and ILK phosphorylation, and concomitantly increased p53 phosphorylation and cyclin D1 expression, but decreased Bcl-2/Bax ratios. Treatment with antiproliferative agents, including 5-fluorouracil, mitomycin C, and daunomycin, at subtoxic doses suppressed the ability of RPE cells to adhere to ECM substratum significantly. This suppression was in part mediated through reduction of integrin β1 and β3 expressions and interfering Akt-ILK signaling activity. Mechanistically, blockade of PI3K/Akt signaling resulted in the suppressed adhesion of RPE cells to ECM. These findings support the hypothesis that, in addition to their antimitogenic effect, antiproliferative agents also exhibit suppressive effect on the adhesiveness of cultured RPE cells. Moreover, inhibitors of the PI3K/Akt signaling mediator can potentially be used as therapeutic agents for proliferative vitreoretinopathy.

The effect of combined 5-fluorouracil and dexamethasone on cultured human retinal pigment epithelial cells.

This study was designed to investigate the inhibitory effect of 5-fluorouracil (5-FU) and dexamethasone (DEX) on the proliferation of human retinal pigment epithelial (RPE) cells in vitro. The human RPE cells (R-50 cell line) were cultured and exposed to various concentrations of combined 5-FU (0, 250, 500, 1000, 2000 ng/ml) and DEX (0, 1, 10, 100, 200 micrograms/ml). The cells were incubated for 96 hr and the medium was changed every 48 hr to replenish the drug action. Cell viability was assessed using cell counting and trypan blue exclusion method. Tetrazolium salt, which can be metabolized by mitochondrial dehydrogenase to form a formazan dye, was used to assay cell proliferation. Treatment with 5-FU alone inhibited cell proliferation in a dose-dependent manner. The concentration of 5-FU that inhibited growth by 50% (IC50) was found to be 704.12 ng/ml. There was a bimodal effect of DEX on RPE cells--stimulation at low concentrations (1, 10 micrograms/ml) and inhibition at high concentrations (100, 200 micrograms/ml). When the two drugs were combined, there was additive inhibition in the concentration of 200 micrograms/ml of DEX. These results indicate that a combination of 5-FU and DEX is no more effective in the inhibition of human RPE cells, except in combination with high concentrations of DEX (> or = 200 micrograms/ml).