Treatment Of Proliferative Vitreoretinopathy Research Articles

Bone morphogenetic protein-6 suppresses TGF-β2-induced epithelial-mesenchymal transition in retinal pigment epithelium.

To evaluate the effect of bone morphogenetic protein-6 (BMP-6) on transforming growth factor (TGF)-β2-induced epithelial-mesenchymal transition (EMT) in retinal pigment epithelium (RPE). Adult retinal pigment epithelial cell line (ARPE-19) were randomly divided into control, TGF-β2 (5 µg/L), and BMP-6 small interfering RNA (siRNA) group. The cell morphology was observed by microscopy, and the cell migration ability were detected by Transwell chamber. The EMT-related indexes and BMP-6 protein levels were detected by Western blotting. Furthermore, a BMP-6 overexpression plasmid was constructed and RPE cells were divided into the control group, TGF-β2+empty plasmid group, BMP-6 overexpression group, and TGF-β2+BMP-6 overexpression group. The EMT-related indexes and extracellular regulated protein kinases (ERK) protein levels were detected. Compared with the control group, the migration of RPE cells in the TGF-β2 group was significantly enhanced. TGF-β2 increased the protein expression levels of α-smooth muscle actin (α-SMA), fibronectin and vimentin but significantly decreased the protein levels of E-cadherin and BMP-6 (P<0.05) in RPE. Similarly, the migration of RPE cells in the BMP-6 siRNA group was also significantly enhanced. BMP-6 siRNA increased the protein expression levels of α-SMA, fibronectin and vimentin but significantly decreased the protein expression levels of E-cadherin (P<0.05). Overexpression of BMP-6 inhibited the migration of RPE cells induced by TGF-β2 and prevented TGF-β2 from affecting EMT-related biomarkers (P<0.05). BMP-6 prevents the EMT in RPE cells induced by TGF-β2, which may provide a theoretical basis for the prevention and treatment of proliferative vitreoretinopathy.

Intravitreal Methotrexate for the Prevention and Treatment of Proliferative Vitreoretinopathy in Rhegmatogenous Retinal Detachment: A Systematic Review.

This systematic review evaluates the role of methotrexate (MTX) as an adjunctive intravitreal agent for the prevention and treatment of proliferative vitreoretinopathy (PVR) in rhegmatogenous retinal detachment. Articles investigating the use of MTX in ≥5 patients with PVR or high risk of PVR were identified via searches of OVID MED-LINE, EMBASE, and Cochrane Library. Eight studies and 240 eyes were included. Patients received an average 2.2 MTX injections with doses ranging from 250 to 400 µg. After 10 months of follow-up in the three controlled trials, there was an 80% retinal re-attachment rate and 0.63 logMAR improvement in visual acuity for patients receiving MTX (<i>n</i> = 106), compared to an 83% reattachment rate and 0.30 logMAR improvement for controls (<i>n</i> = 91). Three mild complications related to MTX were reported. Further research is required given the low number of studies and quality of evidence, heterogenous case selection and treatment regimens, high risk of bias, and lack of randomization. <b>[<i>Ophthalmic Surg Lasers Imaging Retina</i> 2022;53:561-568.]</b>.

Systemic and Intraocular Methotrexate for the Prevention and Treatment of Proliferative Vitreoretinopathy in Children With Rhegmatogenous Retinal Detachment and Underlying Inflammatory Disease.

This index study highlights the efficacy and safety of a combined surgical and medical therapy (with intraocular and systemic methotrexate) of pediatric rhegmatogenous retinal detachments (RRD) complicated by proliferative vitreoretinopathy (PVR). Three pediatric patients with RRD complicated by PVR in the setting of underlying atopic dermatitis and/or uveitis who were treated with a combination of vitreoretinal surgery and intraoperative intravitreal methotrexate (MTX) (40mg) infusion, followed by at least one injection of 200 μg of MTX into the silicone oil-filled vitreous cavity, are reported. In addition, the patients received short-term systemic immunosuppression with systemic (oral or subcutaneous 20 mg weekly MTX for 12 weeks) and/or steroids. Retinal reattachment with improvement in visual acuity was achieved in all eyes following a single surgery and remained to the final follow-up examination after oil removal. There were no observed side effects of intraocular or systemic MTX. The safety and efficacy of this combination therapy suggests that it could be a successful strategy for treating pediatric patients with RRD with or without PVR in the presence or absence of underlying inflammatory diseases.

Role of LINC01592 in TGF-β1-induced epithelial-mesenchymal transition of retinal pigment epithelial cells.

Regulation of long-chain non-coding RNA01592 (LINC01592) in the process of transforming retinal pigment epithelial (RPE) cells into mesenchymal cells following induction by transforming growth factor beat 1 (TGF-β1) was investigated by interfering with LINC01592 expression in human RPE (hRPE) cells. LINC01592 expression in hRPE cells was significantly increased following treatment with 10 ng/mL TGF-β1 for 48 h. Expression of E-cadherin and Snail were decreased in hRPE cells following induction with TGF-β1 compared with the control group (P < 0.05). Following induction by TGF-β1, expression of E-cadherin, alpha-smooth muscle actin (α-SMA), and Snail were significantly lower in the LINC01592-knockdown group compared with the negative control group (P < 0.05). LINC01592 overexpression significantly enhanced the viability, proliferation, and migration of hRPE cells induced by TGF-β1 (P < 0.05). Following induction by TGF-β1, E-cadherin expression was significantly decreased and α-SMA and Snail expression were significantly increased in the LINC01592-overexpression group compared with the negative control group (P < 0.05). RPE cells induced by TGF-β1 exhibited epithelial-mesenchymal transition (EMT). Inhibiting LINC01592 expression could significantly reduce TGF-β1-induced EMT of hRPE cells. The regulatory effect of LINC01592 on EMT in hRPE cells induced by TGF-β1 provides a novel treatment for proliferative vitreoretinopathy.

Topical delivery of a small molecule RUNX1 transcription factor inhibitor for the treatment of proliferative vitreoretinopathy

Proliferative vitreoretinopathy (PVR) is the leading cause of retinal detachment surgery failure. Despite significant advances in vitreoretinal surgery, it still remains without an effective prophylactic or therapeutic medical treatment. After ocular injury or retinal detachment, misplaced retinal cells undergo epithelial to mesenchymal transition (EMT) to form contractile membranes within the eye. We identified Runt-related transcription factor 1 (RUNX1) as a gene highly expressed in surgically-removed human PVR specimens. RUNX1 upregulation was a hallmark of EMT in primary cultures derived from human PVR membranes (C-PVR). The inhibition of RUNX1 reduced proliferation of human C-PVR cells in vitro, and curbed growth of freshly isolated human PVR membranes in an explant assay. We formulated Ro5-3335, a lipophilic small molecule RUNX1 inhibitor, into a nanoemulsion that when administered topically curbed the progression of disease in a novel rabbit model of mild PVR developed using C-PVR cells. Mass spectrometry analysis detected 2.67 ng/mL of Ro5-3335 within the vitreous cavity after treatment. This work shows a critical role for RUNX1 in PVR and supports the feasibility of targeting RUNX1 within the eye for the treatment of an EMT-mediated condition using a topical ophthalmic agent.

Melfalan in the treatment of proliferative vitreoretinopathy (experimental study)

To study of the effectiveness of the drug Melphalan as an antiproliferative agent during experimental proliferative vitreoretinopathy (PVR). The experimental study used data from 24 eyes of 12 Chinchilla rabbits weighing 2.5-3.0 kg, which had PVR modeled in both eyes by intravitreal injection of a culture of heterogeneous activated fibroblast cells consisting of 200,000 cells in 0.1 ml. Treatment of experimental PVR was performed 1 day after the modeling process. In the first group of animals (6 eyes), 0.02 mg of Melphalan was administered intravitreally. In the second group of animals (6 eyes), 0.005 mg of Melphalan concentrated in 0.1 ml was administered intravitreally. Left eyes in both groups remained without treatment. Animals were observed for 1 month using biomicroscopy and ophthalmoscopy. 30 days after the animals were removed from the experiment, the eyes were enucleated, fixed in 10% buffered formalin and subjected to standard histological examination. The study of paraffin sections of the eyes was performed using the microscopic system «Leica» (Leica Microsystems, Germany) with built-in digital camera at the magnification of 200-600. In groups 1 and 2 of the study in the eyes of rabbits that received treatment, PVR was absent, unlike the eyes without treatment, where PVR remained. In group 1, where the dose of Melphalan was 0.02 mg in 0.1 ml, there were changes in the RPE (retinal pigment epithelium), which was regarded as a retinotoxic effect. Glial degeneration and thinning of the retina with disappearance of the photoreceptor layer (the outer nuclear and plexiform layers) resulted from the disturbance of retinal metabolism caused by RPE destruction. In group 2, structure of the retina remained more intact: isolated foci were noted with a decrease in the volume of the outer nuclear layer, shortening of rods and cones with preservation of the inner layers of the retina. A single intravitreal injection of 0.005 mg Melphalan had a positive therapeutic antiproliferative effect on the PVR model with minimal retinotoxic changes.

The effect of infliximab and octreotide on cytokine levels experimental proliferative vitreoretinopathy

Purpose: To investigate the efficiency of intravitreal octreotide, which has previously been shown to have benefits in the treatment of proliferative vitreoretinopathy (PVR), and intravitreal infliximab as a novel option in an experimental dispase-induced PVR model.Methods: A total of 28 pigmented guinea pigs were divided into four groups, and each group consisted of seven subjects. Group 1 (control) was treated with a 0.2 mL saline solution intravitreally from 1.5 mm behind the limbus. Group 2 (sham) was treated with 0.07 IU/0.1 mL dispase 0.1 mL saline solution using the same method. Group 3(infliximab) received 0.07 IU/0.1 mL dispase and 1 mg/0.1 mL infliximab, and group 4(octreotide) was treated with 0.07 IU/0.1 mL dispase and 1 mg/0.1 mL octreotide. An intravitreal injection of infliximab and octreotide was administered to groups 3 and 4 two times during the experiment. The subjects were held for a 10-week period to await for the formation of PVR. At the end of ten weeks, the eyes were enucleated, and tumour necrosis factor-alpha (TNF-α), interleukin 1(IL-1), interleukin 6 (IL-6), transforming growth factor (TGF-β), and platelet-derived growth factor (PDGF) and levels in homogenised retina tissue were measured using the enzyme linked-immuno-sorbent assay (ELISA) method.Results: Retinal TNF-α, IL-1, IL-6, and PDGF levels had significantly decreased in treatment groups compared to the sham group (p < 0.05). The decrease in the level of TGF-β was not statistically significant between the treatment and the sham groups (p > 0.05).Conclusions: Intravitreal infliximab can inhibit the development of PVR and reduce levels of cytokine, which plays an essential role in the pathogenesis of PVR. The results of our study suggest that it may be possible to identify the ideal adjuvant pharmacological drugs that are effective in preventing PVR.

Plumbagin induces RPE cell cycle arrest and apoptosis via p38 MARK and PI3K/AKT/mTOR signaling pathways in PVR

BackgroundThis study aimed to explore the effects of plumbagin (PLB) on ARPE-19 cells and underlying mechanism.MethodsCultured ARPE-19 cells were treated with various concentrations (0, 5, 15, and 25 μM) of PLB for 24 h or with 15 μM PLB for 12, 24 and 48 h. Then cell viability was evaluated by MTT assay and DAPI staining, while apoptosis and cell cycle progression of ARPE cells were assessed by flow cytometric analysis. Furthermore, the level of main regulatory proteins was examinated by Western boltting and the expression of relative mRNA was tested by Real-Time PCR.ResultsPLB exhibited potent inducing effects on cell cycle arrest at G2/M phase and apoptosis of ARPE cells via the modulation of Bcl-2 family regulators in a concentration- and time-dependent manner. PLB induced inhibition of phosphatidylinositol 3-kinase (PI3K) and p38 mitogen-activated protein kinase (p38 MAPK) signaling pathways contributing to the anti-proliferative activities in ARPE cells.ConclusionsThis is the first report to show that PLB could inhibit the proliferation of RPE cells through down-regulation of modulatory signaling pathways. The results open new avenues for the use of PLB in prevention and treatment of proliferative vitreoretinopathy.

Eupatilin prevents H2O2-induced oxidative stress and apoptosis in human retinal pigment epithelial cells

Eupatilin, a pharmacologically active flavone derived from the Artemisia plant species, is known to possess anti-oxidant activity. However, the effects of eupatilin on oxidative stress-induced retinal damage in retinal pigment epithelium (RPE) cells and the potential mechanisms involved have not been explored. Therefore, the aim of this study was to investigate the effects of eupatilin on oxidative stress-induced retinal damage in RPE cells. Our results showed that eupatilin significantly attenuated H2O2-induced cell injury and ROS production in ARPE-19 cells. In addition, eupatilin pretreatment greatly upregulated Bcl-2 expression, downregulated Bax expression, as well as suppressed caspase-3 activity in ARPE-19 cells exposed to H2O2. Furthermore, eupatilin pretreatment markedly enhanced phosphorylation levels of PI3K and Akt in ARPE-19 cells exposed to H2O2. In conclusion, our data showed that eupatilin protected against H2O2-induced oxidative stress and apoptosis through the activation of PI3K/Akt signaling pathway in ARPE-19 cells. Thus, eupatilin may be useful for the prevention or treatment of proliferative vitreoretinopathy (PVR).

Proliferative Vitreoretinopathy: Therapeutic Strategies

Development of proliferative vitreoretinopathy (PVR) is one of the most important complications of vitreoretinal surgery. Reaching the decision to treat and surgical treatment itself are both challenging. Our own data and a review of the literature in PubMed are summarised. Pharmacological approaches to the prevention and treatment of proliferative vitreoretinopathy have been limited to concepts that have been investigated in preclinical and a few clinical studies. Anti-inflammatory and antiproliferative substances may be mentioned in this context. Surgical techniques range from scleral buckling to the gold standard pars plana vitrectomy, preferably with silicone oil endotamponade. Applying an encircling band, retinotomy or retinectomy can be useful in reattaching the tractional shortened retina. Surgery is still the method of choice for the treatment of PVR. Pharmacological strategies to prevent or treat PVR have not been established.

Crosslinked Chitosan/PVA Film, Suturated with 5- Fluorouracil for The Prevention of Proliferative Vitreoretinopathy

5-Fluorouracil (5-FU)-loaded chitosan (Ch) film for chemotherapy were prepared applying a superhydrophobic surfacebased encapsulation technology. The aim of this study was to develop polymeric film with glutaraldehyde (GA) of controlled drug delivery systems for 5 – fluorouracil (FU) as a model drug for the treatment of proliferative vitreoretinopathy. Polymer film of chitosan and polyvinyl alcohol (PVA in 75:25 ratios were prepared and treated with GA. FTIR spectra of 5-FU, Ch/5-FU and Ch/PVA film loaded 5-FU were studied. Physical characteristics such as thickness and swelling coefficient of the film were performed. The thermal of the Ch/PVA film was studied with thermogravimethric analysis. The drug loading efficiency, film size and chemical compositions of the film loaded drug were confirmed by UV–vis spectrophotometer and Fourier transform infrared spectroscopy. In vitro release kinetics of drug from the polymeric films was investigated to determine the drug release properties. In vivo study of PVR was showed the efficacy and no toxicity of this formulation. Further uses of the film loaded 5 - fluorouracil may provide an efficiency deliverable for ophthalmic administration.

The efficacy of proliferative membrane stripping combined with retinal laser photocoagulation for the treatment of proliferative vitreoretinopathy on silicone oil-filled eyes

Objective To evaluate the efficacy of proliferative membrane stripping combined with retinal laser photocoagulation for the treatment of proliferative vitreoretinopathy ( PVR ) on silicone oil-filled eyes. Methods The data of 66 eyes of 66 patients with PVR after vitrectomy combined with silicone oil tamponade surgery from January 2013 to June 2015 in our hospital were retrospectively analyzed. These patients received proliferative membrane stripping combined with retinal laser photocoagulation. The visual acuity before and after the surgery, central macular thickness, retinal reattachment, the timing of silicone oil removal and postoperative complications were observed and analyzed. Results The retinas were reattached in all 66 eyes after the surgery and no serious complication occurred. The removal of the silicone oil was within 3 months in 39 eyes ( 59.09% ) and 4 to 6 months in 27 eyes (40.91%). The membrane stripping operation was performed 2 to 4 times in 10 eyes ( 15.15% ). The recurrent retinal detachment occurred after the oil removal in 2 eyes (3.03%) and the silicone oil tamponade surgery was performed again to reattach the retina. The postoperative visual acuity improved significantly compared with preoperative visual acuity and the difference was statistically significant (χ2=14.85, P=0.0019 ). Before the surgery the mean thickness of central macular area was (352.70 ± 55.52) μm. Three months after the surgery the thickness reduced to (324.37±49.74) μm and the difference was statistically significant (t=7.591, P=0.000 ). Conclusion Proliferative membrane stripping combined with retinal laser photocoagulation for PVR on silicone oil-filled eyes is exactly effective. Key words: Proliferative membrane stripping; Laser photocoagulation, retinal; Vitreoretinopathy, proliferative; Silicone oil-filled eyes; Efficacy

MicroRNA-124 Controls Transforming Growth Factor β1-Induced Epithelial-Mesenchymal Transition in the Retinal Pigment Epithelium by Targeting RHOG.

MicroRNA-124 (miR-124) is thought to be involved in the epithelial-mesenchymal transition (EMT) of RPE. We investigated the regulation of TGF-β1-induced EMT by miR-124 in human RPE cells (ARPE-19). Expression of miR-124 was evaluated after TGF-β1 treatment by quantitative RT-PCR. Phenotypic alterations were analyzed by Western blot analysis and immunocytochemical staining. Target validation was performed by a luciferase reporter assay to identify the putative target of miR-124. The expression level of miR-124 was downregulated during the progression of EMT. Overexpression of miR-124 upregulated the levels of zonular occludens 1 and occludin, and downregulated those of fibronectin, α-smooth muscle actin, and vimentin. Furthermore, inhibition of endogenous miR-124 increased and decreased the levels of mesenchymal and epithelial factors, respectively. TargetScan predicted two well-conserved and two vertebrate-only conserved miR-124 target sequences in the 3' untranslated region (UTR) of the Ras homology Growth-related (RHOG) mRNA. Direct targeting of this 3' UTR by miR-124 was demonstrated using a luciferase assay. Silencing of RHOG using a specific siRNA had identical effects on EMT regulation. Overexpression of miR-124 repressed TGF-β1-induced RPE cell-collagen gel lattice contraction by altering cell spreading/cell-to-cell adhesion. This study describes the regulation of EMT in RPE cells by TGF-β1/miR-124/RHOG signaling and suggests that the supplement of miR-124 exogenously would be a valuable therapeutic approach for the prevention or treatment of proliferative vitreoretinopathy.

MicroRNA‐124 regulates transforming growth factor β1‐induced epithelial‐mesenchymal transition in the retinal pigment epithelium by Down‐regulating expression of the RhoG

PurposeMicroRNA‐124 (miR‐124) is thought to be involved in the epithelial‐mesenchymal transition (EMT) of retinal pigment epithelium (RPE). In the present study, we investigated the regulation of miR‐124 on EMT induced by transforming growth factor β1 (TGF‐β1) in human RPE cells (APRE‐19).MethodsThe expression of miR‐124 was evaluated after treatment of TGF‐β1 by quantitative RT‐PCR. Phenotypic alterations were analyzed by western blot analysis and immunocytochemical staining. Target validation was performed to identify the putative target of miR‐124 by luciferase reporter assay.ResultsThe expression level of microRNA‐124 (miR‐124) was down‐regulated during the progression of EMT. Overexpression of miR‐124 upregulated the levels of zonular occludens 1 and RPE65, and down‐regulated fibronectin, α‐smooth muscle actin, and vimentin. Furthermore, inhibition of endogenous miR‐124 increased and decreased the levels of mesenchymal and epithelial factors, respectively. TargetScan predicted two well‐conserved and two vertebrate‐only conserved miR‐124 target sequences in the 3′ untranslated region (UTR) of the RHOG mRNA. Direct targeting of this 3′ UTR by miR‐124 was demonstrated using a luciferase assay. Silencing of RHOG using a specific siRNA had identical effects on EMT regulation. Overexpression of miR‐124 repressed TGF‐β1‐induced RPE cell‐collagen gel lattice contraction by altering cell spreading/cell‐to‐cell adhesion.ConclusionsThis study describes the regulation of EMT in RPE cells by TGF‐β1/miR‐124/RHOG signaling and suggests that the supplement of miR‐124 expression would be a crucial therapeutic target for the prevention or treatment of proliferative vitreoretinopathy.

Rac1 modulates the vitreous‐induced plasticity of mesenchymal movement in retinal pigment epithelial cells

The vitreous has been shown to induce epithelial-mesenchymal transdifferentiation because it induces fibroblast-like morphology, enhanced migration and invasion in retinal pigment epithelial cells in proliferative vitreoretinopathy. Rac1 is the principal mediator of cell migration. In the current study, the relationship between Rac1 and cell migration, and invasion in vitreous-transformed retinal pigment epithelial cells was investigated using NSC23766, a specific inhibitor of Rac guanosine-5'-triphosphatase activity, and the involvement of a Rac1 guanosine-5'-triphosphatase-dependent pathway was detected. One-way design with multiple levels and repeated measurement design. The vitreous humor was collected from 20 healthy donor eyes and the retinal pigment epithelial cells were obtained from 9 healthy donor eyes. Human low-passage retinal pigment epithelial cells were treated with normal medium or 25% vitreous medium. Rac1 activity was measured using a pull-down assay. The cytotoxicity of NSC23766 was measured using the trypan blue dye exclusion test. Cell migration was measured using a wound healing assay. Cell invasion was determined using a transwell invasion assay. Protein expression of Rac1 and phosphorylation of LIM kinase 1 and cofilin were detected by Western blot analysis. Cell migration, invasion, Rac1 activity and phosphorylation of LIM kinase 1 and cofilin. Rac1guanosine-5'-triphosphatase was activated in vitreous-transformed retinal pigment epithelial cells. A Rac inhibitor suppressed vitreous-induced migration and invasion in retinal pigment epithelial cells. Cofilin phosphorylation was activated by vitreous treatment but blocked by NSC23766. Rac1 mediates vitreous-transformed retinal pigment epithelial cells' plasticity of mesenchymal movement via Rac1 guanosine-5'-triphosphatase-dependent pathways that modulate LIM kinase 1 and cofilin activity. Rac inhibition may be considered a novel treatment for proliferative vitreoretinopathy.

Novel intraocular and systemic absorption drug delivery and efficacy of N‐acetylcarnosine lubricant eye drops or carcinine biologics in pharmaceutical usage and therapeutic vision care

The latest estimates of the World Health Organization indicate that there are 161 million visually impaired individuals worldwide, 37 million of whom are blind, with a yearly increase of 1-2 million. The scientists developed and patented the lubricant eye drops formulated as 1%N-acetylcarnosine prodrug of l-carnosine containing a mucoadhesive cellulose-based compound combined with corneal absorption promoters in an ocular drug delivery system. Carcinine is suitable for the systemic administration (per oral) for ophthalmic therapeutic indications. The HPLC analysis was developed to search the pathways of ocular metabolic activities of 1%N-acetylcarnosine and the bioactivation of this drug molecule promoting transcorneal uptake of l-carnosine in the aqueous humor. A meta-analysis of phase 2 randomized double-blind placebo-controlled clinical trial data was conducted. The intraocular absorbed l-carnosine demonstrated a number of pharmacological mechanisms of prevention and reversal of cataracts. Results of systemic absorption of l-carnosine provide tuberomammillary activation that regulates neuronal functions such as hypothalamic control promoting sensory input in the primary vision perceptual pathway. The parabulbar, subconjunctival, and intravitreal injection of carcinine with most of the vehicle removed is not toxic to intraocular structures, reduces postoperative intraocular inflammation, is a potentially useful tool in the treatment of proliferative vitreoretinopathy as well as considered as the antiapoptotic drug for the protection of photoreceptor cells from oxidative light-induced stress. The discovery of naturally occurring carnosine derivatives introduces N-acetylcarnosine and carcinine as effective medical treatment for sight-threatening eye disorders.

Inhibitory Effect of Epigallocatechin Gallate (EGCG), Resveratrol, and Curcumin on Proliferation of Human Retinal Pigment Epithelial Cells In Vitro

Purpose: To investigate potential inhibitory effects of three polyphenolic agents, epigallocatechin gallate (EGCG; from green tea), resveratrol (from red wine), and curcumin (from turmeric), on the proliferation of human retinal pigment epithelial (RPE) cells and to elucidate unwanted effects.Methods: ARPE19 cells and primary human RPE cells were cultured in the presence of various concentrations of EGCG, resveratrol, or curcumin, and compared with controls. The number of viable cells was determined after 24, 48, and 72 hr by flow cytometrical enumeration. Furthermore, cell division was measured by dye dilution assay using carboxyfluorescein succinimidyl ester (CFSE), cell death by Hoechst 33258 staining, and apoptosis by staining for active caspase 3/7 and 8.Results: The three drugs inhibited the increase of RPE cell numbers at all time points, with resveratrol being the most efficient and curcumin being the least efficient. EGCG inhibited cell proliferation with intermediate efficiency, and showed little induction of cell death. Resveratrol almost completely suppressed cell proliferation, and induced RPE cell necrosis and caspase 3/7- and caspase 8-dependent apoptosis. Curcumin inhibited RPE cell increase exclusively by inducing caspase 3/7-dependent but caspase 8-independent cell death and necrosis.Conclusions: All three polyphenols tested reduced the absolute number of cells, but had different effects on cell proliferation, apoptosis, and necrosis. Resveratrol was most potent and EGCG induced the least cell death. These polyphenols may aid treatment of proliferative vitreoretinopathy (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.

ACTA‐EVER lecture 2007 The retinal pigment epithelium: friend or foe?

The retinal pigment epithelium (RPE) plays an important role in the physiology and pathophysiology of the vertebrate retina. The RPE absorbs fluid from the retinal extracellular space, via a proton-lactate-water co-transport mechanism located in the apical membrane of the epithelium. This mechanism can account for the apparent capability of the RPE to absorb water against an osmotic gradient. RPE cells participate in retinal wound healing. We have created a porcine model of experimental choroidal neovascularization (CNV). In this model, the CNV eventually becomes enveloped by seemingly proliferating RPE cells. By means of 5-bromo-2-deoxyuridine (BrdU) labelling, we studied the proliferation of RPE cells in the porcine eye after experimental posterior pole injury. Surprisingly, we found that only the peripheral RPE cells incorporated the BrdU label, indicating that central injury elicits peripheral RPE proliferation. This might suggest the existence of a peripheral pool of RPE stem cells. RPE cell proliferation plays a role in the pathological wound healing known as proliferative vitreoretinopathy. Antiproliferative agents have been tried to treat this condition but with little success so far. We report on a drug delivery system under development where a prodrug of the antimetabolite 5-fluoro-uracil (5-FU) is suspended in the silicone oil used as a surgical device in the treatment of proliferative vitreoretinopathy (PVR). The theoretical advantage of this approach is that it allows for long contact times between therapeutic, and non-toxic, concentrations of 5-FU and the RPE.

Lab research on the prevent and treatment of proliferative Vitreoretinopathy with Chuanxiongzin

目的 通过定量检测增殖性玻璃体视网膜病变玻璃体血管内皮细胞生长因子及肿瘤坏死因子的含量,探讨川芎嗪对PVR的防治作用.方法 采用经睫状体平坦部玻璃体腔注入自体新鲜血制造动物模型.应用浓度为5 mg/ml川芎嗪治疗实验组.应用酶连免疫吸附实验定量检测血管内皮细胞生长因子及肿瘤坏死因子-α含量.结果 实验组中血管内皮细胞生长因子及肿瘤坏死因子-α的含量较模型组降低,具有显著性差异,P＜0.01.结论 川芎嗪能够降低玻璃体中血管内皮细胞生长因子和肿瘤坏死因子-α的含量,具有防治PVR的作用.