Yi-Hao Chen

Cyclin-dependent Kinase Inhibitor Roscovitine Induces Cell Cycle Arrest and Apoptosis in Rabbit Retinal Pigment Epithelial Cells

AIMSnCyclin-dependent kinases (CDKs) play essential roles in the intracellular control of the cell cycle. It has been postulated that roscovitine, a potent CDK2, CDK5, and CDC2 inhibitor, might inhibit cellular proliferation by arresting the cell cycle. This in vitro study investigated the antiproliferative and apoptotic effects of roscovitine in cultured rabbit retinal pigment epithelial (RPE) cells.nnnMETHODSnExperiments using rabbit RPE from young pigmented rabbits were carried out using roscovitine dissolved in dimethylsulfoxide at concentrations ranging from 1 to 100 micromol. Cell proliferation was measured by an MTT assay. The cell cycle response of RPE cells to roscovitine was analyzed by flow cytometry of propidium iodide-stained nuclei. Proteins related to DNA damage in the RPE cells were then assayed by Western blot.nnnRESULTSnRoscovitine inhibited proliferation of RPE cells in a dose-dependent manner. Cell cycle analysis after treatment demonstrated an accumulation of cells arrested in the S- and G2/M phases. Flow cytometry showed that 40 microM of roscovitine increased the cell population in the sub-G1 peak, which is considered a marker of cell death by apoptosis. Western blot analysis revealed Bcl-2 decreased and Bax increased after treatment of RPE cells with roscovitine.nnnCONCLUSIONSnThis study of the response of RPE cells to roscovitine demonstrated a bidirectional relationship between cell cycle control and apoptosis.

Adjunctive Intravitreal Bevacizumab-Combined Trabeculectomy Versus Trabeculectomy Alone in the Treatment of Neovascular Glaucoma

PURPOSEnTo evaluate the effect of adjunctive intravitreal bevacizumab (ivBe) with trabeculectomy versus trabeculectomy alone in the management of patients with neovascular glaucoma (NVG).nnnMETHODSnRetrospective, consecutive, interventional case series. NVG patients were divided into groups by treatment: with adjunctive ivBe and trabeculectomy (ivBe group, n = 14 eyes) and with trabeculectomy only (control group, n = 28 eyes). The main outcome measure was visual acuity. Regression of iris neovascularization (NVI), change(s) in intraocular pressure (IOP), NVI recurrence, additional glaucoma surgeries required, eyes of leading to total blindness, intraoperative and postoperative complications, and number of topical medications required after trabeculectomy were regarded as second outcome measures. In the ivBe group, intravitreal injections of 2.5 mg bevacizumab were delivered using a sharp 27-gauge needle through the inferotemporal quadrant.nnnRESULTSnOf 42 eyes of 42 patients identified, change in IOP, additional glaucoma surgeries required, and number of IOP-lowering topical medications required after trabeculectomy did not differ significantly between groups (P > 0.05 for all). However, the ivBe group had significantly higher frequency and rapidity of iris neovascular regression, improved visual acuity in the logarithm of minimum angle of resolution (logMAR), leading to total blindness in fewer eyes and intraoperative and postoperative complications in others than in the control group (P = 0.015, 0.002, 0.007, 0.023, and 0.008, respectively). The follow-up duration (mean +/- SD) from trabeculectomy surgery was 179 +/- 97 days (range, 93-315 days) and 196 +/- 108 days (range, 92-370 days) in the ivBe and control group (P = 0.324).nnnCONCLUSIONSnIntravitreal bevacizumab might be a useful adjunctive therapy in addition to trabeculectomy in the management of NVG. Large controlled randomized studies for treatment of bevacizumab on NVG are warranted.

The Effects of Ozurdex® (Dexamethasone Intravitreal Implant) on Experimental Proliferative Vitreoretinopathy

Purpose: To investigate a new sustained-release formulation of dexamethasone (Ozurdex®) for inhibiting proliferative vitreoretinopathy (PVR) and its effect on the expression of retinal glial reaction and inflammation in experimental PVR eyes. Methods: We used 30 pigmented rabbits for this study. One week after gas compression, the eyes were injected with 5 × 104 retinal pigment epithelial cells into the vitreous cavity to induce PVR. Concurrently, one eye also received an intravitreal injection of Ozurdex; the other eye was used as a control. PVR was graded by indirect ophthalmoscopy on days 1, 3, 7, 14, 21, and 28. The expression of the retinal glial reaction and inflammation in experimental PVR eyes were evaluated by Western blot analysis. Results: PVR severity increased gradually and peaked after 14 days, and no differences in PVR severity between the study and control groups were observed at any time point. The expression of glial fibrillary acid protein (GFAP) increased on days 7 and 14 in both the PVR control and study groups. While the use of Ozurdex in the study group showed less GFAP expression, this difference was not significant. The expression of tumor necrosis factor (TNF)-α and interleukin (IL)-6 significantly increased on days 7 and 14 in PVR control eyes. There was a significant difference in TNF-α between PVR control eyes and Ozurdex-treated eyes on days 7 (p < 0.001) and 14 (p = 0.019). Ozurdex in the study group showed lower IL-6 expression; however, this difference was not significant on days 7 (p = 0.063) and 14 (p = 0.052). Conclusions: The intravitreal injection of Ozurdex suppressed the expression of inflammatory markers; however, it did not mitigate the severity of experimental PVR in this animal model.

Clinical Implications of Serous Retinal Detachment in Branch Retinal Vein Occlusion and Response After Primary Intravitreal Bevacizumab Injection

PURPOSEnTo evaluate the impact of macular serous retinal detachment (SRD) and its relationship to treatment outcome after primary intravitreal bevacizumab (IVB) injection in patients with branch retinal vein occlusion (BRVO) and macular edema (ME).nnnMETHODSnSeventy-three patients with ME secondary to BRVO who received primary IVB (2.5 mg/0.1 mL) were included in this study. The specific ME patterns were investigated using optical coherence tomography (OCT) examination. Visual acuity (VA), central macular thickness (CMT), and macular volume at baseline; at 1, 3, and 6 months; and at final visit after primary IVB were retrospectively analyzed and compared between patients with and without SRD.nnnRESULTSnSRD was found in 25 patients (34.2%). The baseline CMT was significantly thicker in patients with SRD than in those without it (648.4±200.5 μm vs. 440.3±119.6 μm, P<0.001). Six months after primary IVB injection, a greater reduction in CMT change from baseline was observed in the SRD group (412.5±227.2 μm) than in the group without SRD (118.5±175.2 μm) (P<0.001). The improvement of logarithm of the minimum angle of resolution VA was also greater in the SRD group than in the group without SRD (-0.64±0.52 and -0.28±0.62 respectively, P=0.015). Logistic regression analysis showed that the presence of SRD was an independent factor for visual improvement in BRVO (P=0.027).nnnCONCLUSIONnPatients with SRD had greater functional and morphological improvements at 6 months after primary IVB therapy. The results of this study suggest that the presence of SRD observed on OCT may be an indicator of favorable clinical response after IVB injections and that in BRVO patients with SRD, bevacizumab may be a good alternative for treatment.

The upregulation of zinc finger protein 670 and prostaglandin D2 synthase in proliferative vitreoretinopathy

PurposeProteins in the vitreous play an important role on the induction of proliferative vitreoretinopathy (PVR) after retinal detachment. The aim of this study was to investigate the variation of protein patterns in the vitreous of PVR eyes and examine whether differentially expressed protein levels were expressed in experimental PVR retina.MethodsVitreous samples from PVR and macular hole patients were selected for proteomic analysis. The vitreous protein samples were separated by two-dimensional electrophoresis (2-DE). The differentially expressed protein spots in the two groups were excised and subjected to in-gel digestion and identification by electrospray ionization mass spectrometry (ESI-MS) analysis. Two differentially expressed proteins, zinc finger protein 670 (ZFP 670) and prostaglandin D2 synthase (PGD2S), were further validated by immunohistochemical staining and western blotting analysis in the retina of the experimental rabbit PVR model.ResultsIn proteome analysis of human vitreous samples, five proteins had increased expression in PVR, including zinc finger protein 670 (ZFP 670), prostaglandin D2 synthase (PGD2S), IgG (Immunoglobulin G) light chain, transthyretin precursor, and haptoglobin precursor. ZFP 670 and PGD2S levels were expressed significantly higher in the experimental PVR retinas than in the control group.ConclusionsLevels of ZFP 670 and PGD2S were elevated in the vitreous fluid of patients with PVR. In addition, there were higher expressions of ZFP 670 and PGD2S in the experimental PVR retina. This result will expand our knowledge of pathophysiologic characteristics of PVR, and might be helpful for further developing possible treatment on this disorder.

Evaluation of the Effect of Everolimus on Retinal Pigment Epithelial Cells and Experimental Proliferative Vitreoretinopathy

ABSTRACT Purpose: Failure of retinal detachment surgery is most commonly due to the development of proliferative vitreoretinopathy (PVR). Everolimus is an inhibitor of mammalian target of rapamycin (mTOR), and is available as oral tablets. In this study, we investigated the effect of everolimus on retinal pigment epithelial cells and modification of the severity of experimental PVR. Methods: In our in vitro studies, primary culture of retinal pigment epithelium (RPE) cells was obtained from pigmented Rex rabbits. Cell proliferation was assayed with the tetrazolium dye cytotoxicity test, and cell migration assay was performed in 24-well transwell units with 8-μm filters. In the in vivo study, pigmented Rex rabbits weighing between 2 and 2.5 kg were used. Each rabbit eye underwent gas compression; one week later, 5 × 104 RPE cells were injected into the vitreous cavity to induce PVR, and each eye was graded with indirect ophthalmoscopy on days 1, 3, 7, 14, 21, and 28. The rabbits were administered everolimus (0.5 mg/day orally) from the day of PVR induction. Total proteins extracted from RPE cells and dissected retinal samples were processed for Western blotting analysis of mTOR and ribosomal protein S6 (RPS6). Results: The in vitro studies showed that everolimus significantly inhibited the proliferation of RPE cells at 0.1 μg/ml; additionally, at 10 μg/ml, it suppressed the migration of RPE cells and significantly suppressed the expression of mTOR and RPS6 in RPE cells. The in vivo study did not show any benefit of oral everolimus (0.5 mg/day) in suppressing experimental PVR. Thus, everolimus significantly suppressed the expression of mTOR and RPS6 in PVR. Conclusions: Everolimus suppressed the proliferation and migration of RPE cells in vitro. Oral everolimus (0.5 mg/day) suppressed the expression of mTOR and RPS6 in the retina, but showed no effect in suppressing experimental PVR.