Wen-Chuan Wu

Dyslipidemia and diabetic retinopathy.

Diabetic retinopathy (DR) is one of the major microvascular complications of diabetes. In developed countries, it is the most common cause of preventable blindness in diabetic adults. Dyslipidemia, a major systemic disorder, is one of the most important risk factors for cardiovascular disease. Patients with diabetes have an increased risk of suffering from dyslipidemia concurrently. The aim of this article is to review the association between diabetic retinopathy (DR) and traditional/nontraditional lipid markers, possible mechanisms involving lipid metabolism and diabetic retinopathy, and the effect of lipid-lowering therapies on diabetic retinopathy. For traditional lipid markers, evidence is available that total cholesterol and low-density lipoprotein cholesterol are associated with the presence of hard exudates in patients with DR. The study of nontraditional lipid markers is advancing only in recently years. The severity of DR is inversely associated with apolipoprotein A1 (ApoA1), whereas ApoB and the ApoB-to-ApoA1 ratio are positively associated with DR. The role of lipid-lowering medication is to work as adjunctive therapy for better control of diabetes-related complications including DR.

A Comparative Study of Effects of Antiproliferative Drugs on Human Retinal Pigment Epithelial Cells in Vitro

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 ID50 of 5.35 +/- 2.38 x 10(-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 ID50 of 3.73 +/- 0.71 X 10(-9) M. RPE cultured with daunomycin showed an abrupt decrease of cell number from 10(-9) M to 10(-8) M, the ID50 value was 1.07 +/- 0.23 x 10(-8) M. Dexamethasone showed a biphasic effect; it stimulated cell growth at 10(-7) to 10-6 M and inhibited cell growth at 10(-4) M or higher, with an ID50 of 6.05 +/- 1.61 x 10(-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.

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.

INTRAVITREAL TRIAMCINOLONE ACETONIDE FOR PATIENTS WITH MACULAR EDEMA DUE TO BRANCH RETINAL VEIN OCCLUSION

We designed a case series study to evaluate the outcome of intravitreal triamcinolone acetonide for the treatment of macular edema due to branch retinal vein occlusion (BRVO). The prospective comparative nonrandomized clinical interventional study included 27 patients (27 eyes) with macular edema due to BRVO. The study group consisted of 16 patients who had accepted an intravitreal injection (IVI) of 4 mg triamcinolone acetonide. The control group included 11 patients without IVI of triamcinolone acetonide. The mean follow‐up was 103.00 36.24 days in the study group and 94.55 36.31 days in the control group. In the study group, visual acuity measurements improved significantly (p 0.001) from 0.77 0.43 logarithm of minimal angle of resolution (logMAR) preoperatively to a best postoperative visual acuity of 0.44 0.43 logMAR. Fourteen eyes (87.5%) gained improvement in visual acuity, with 10 eyes (62.5%) showing an increase in visual acuity of at least two Snellen lines. All 16 patients showed significant macular edema resolution in optical coherence tomography examination (p 0.001) and perivascular leakage decrease in fluorescein angiography post‐IVI. In the control group, baseline best‐corrected visual acuity and best‐corrected visual acuity during the follow‐up did not vary significantly (p 0.294). In conclusion, IVI of triamcinolone acetonide can lead to an increase in visual acuity and a resolution of macular edema in patients with BRVO.

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.

A DC-81-indole conjugate agent suppresses melanoma A375 cell migration partially via interrupting VEGF production and stromal cell-derived factor-1α-mediated signaling.

Pyrrolo[2,1-c][1,4]benzodiazepine (PBD) chemicals are antitumor antibiotics inhibiting nucleic acid synthesis. An indole carboxylate-PBD hybrid with six-carbon spacer structure (IN6CPBD) has been previously demonstrated to induce melanoma cell apoptosis and reduce metastasis in mouse lungs. This study aimed at investigating the efficacy of the other hybrid compound with four-carbon spacer (IN4CPBD) and elucidating its anti-metastatic mechanism. Human melanoma A375 cells with IN4CPBD treatment underwent cytotoxicity and apoptosis-associated assays. Transwell migration assay, Western blotting, and ELISA were used for mechanistic study. IN4CPBD exhibited potent melanoma cytotoxicity through interrupting G1/S cell cycle progression, increasing DNA fragmentation and hypodipoidic DNA contents, and reducing mitochondrial membrane potential. Caspase activity elevation suggested that both intrinsic and extrinsic pathways were involved in IN4CPBD-induced melanoma apoptosis. IN4CPBD up-regulated p53 and p21, thereby concomitantly derailing the equilibrium between Bcl-2 and Bax levels. Transwell migration assay demonstrated that stromal cell-derived factor-1α (SDF-1α) stimulated A375 cell motility, while kinase inhibitors treatment confirmed that Rho/ROCK, Akt, ERK1/2, and p38 MAPK pathways were involved in SDF-1α-enhanced melanoma migration. IN4CPBD not only abolished the SDF-1α-enhanced chemotactic motility but also suppressed constitutive MMP-9 and VEGF expression. Mechanistically, IN4CPBD down-regulated Akt, ERK1/2, and p38 MAPK total proteins and MYPT1 phosphorylation. In conclusion, beyond the fact that IN4CPBD induces melanoma cell apoptosis at cytotoxic dose, the interruption in the VEGF expression and the SDF-1α-related signaling at cytostatic dose may partially constitute the rationale for its in vivo anti-metastatic potency.

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.

Comparison of Vitrectomy Alone and Combined Vitrectomy, Phacoemulsification and Intraocular Lens Implantation for Proliferative Diabetic Retinopathy

There have been concerns that there may be an increased incidence of iris neovascularization (NV) following lens removal in patients with proliferative diabetic retinopathy (PDR). In this study, we retrospectively compared vitrectomy alone and vitrectomy combined with phacoemulsification (phacovitrectomy) and intraocular lens implantation regarding both complications and results. Fifty‐three eyes for vitrectomy group and 31 eyes for phacovitrectomy group were included. Postoperative iris and angle NV were found in eight (15.1%) eyes in the first group and no (0%) eyes in the second. The incidence was significantly lower (p < 0.05) in the phacovitrectomy group. The final vision gain of one or more lines was found in 17 (32.1%) and 21 (67.7%) eyes, respectively. There was significantly better vision improvement in the phacovitrectomy group. We consider the combined procedure to be useful as an alternative surgical treatment for patients with PDR and cataract formation.

Relationship between Outcome of Proliferative Vitreoretinopathy and Results of Tissue Culture of Excised Preretinal Membranes

The purpose of this study was to explore the relationship between clinical post-surgery outcome of proliferative vitreoretinopathy (PVR) and the laboratory results of tissue culture, the specimens of which were excised of pre- or subretinal membranes from PVR patients. Surgically excised membranes from 25 PVR patients were microdissected into small pieces and plated into culture dishes with F12 medium supplemented with 30% fetal bovine serum. After primary culture became confluent, cells were passaged in subculture with F12 medium (10% fetal bovine serum). PVR patients were followed-up after surgery for an average of 21 months. The clinical outcome was compared, according to the growth pattern of the cells derived from the explanted membranes. In 25 PVR patients, 16 cases showed cell migration in the membrane, and cells grew rapidly to confluence in the primary culture in 7 cases. All active growing cells were identified as retinal pigment epithelial (RPE) cells by immunocytochemistry. In 7 cases with active cell growth, all had recurrent retinal detachment. In 18 cases without active cell growth, only 4 cases had the same outcomes. Statistical study showed that the difference between these two groups was significant (P < 0.01). These results indicate that the growth capacity of cultured RPE derived from excised membranes of PVR patients strongly influenced the prognosis for surgery.