Corneal neovascularization inhibition and wound healing impregnability of conbercept on rabbit cornea after penetrating keratoplasty.

PURPOSE: The inhibitory effect of conbercept on corneal neovascularization (CNV) after penetrating keratoplasty (PKP) and its effect on postoperative wound healing and corneal strength recovery was investigated. METHODS: New Zealand white rabbits were randomly divided into 3 groups, two experimental arms A and B and one control arm C. Topical conbercept and subconjunctival injection were carried out respectively after PKP. Slit lamp microscope was used to observe the growth of CNV. The expression of vascular endothelial growth factor (VEGF), placental growth factor (PlGF) and vimentin (Vim) were determined via real-time quantitative polymerase chain reaction (RT-qPCR). The placenta growth factor and vimentin, determination of corneal biomechanical machine strength changed. To measure the maximal strength of the corneal, uniaxial tensile test was carried out on the electroforce 3220-AT biomechanics machine. RESULTS: Two weeks after PKP, CNV appeared, inflammatory cell infiltration and new blood vessel formation were observed in the corneal stroma and superficial stroma layer. Compared with the control arm, the expression levels of VEGF and PlGF in the experimental arms were significantly decreased after using conbercept (P < 0.05), and the expression levels reached the maximum at the 4th week and then decreased gradually. The expression level of Vimentin and corneal intensity increased gradually over time. CONCLUSION: Conbercept effectively inhibited the formation of CNV after PKP in rabbits, and did not affect postoperative wound healing, nor did it affect postoperative corneal strength recovery.

Topographical Relationship Between Acute Macular Neuroretinopathy and Choroidal Watershed Zone or Patchy Choroidal Filling.

PURPOSE: To study the topographical relationship between acute macular neuroretinopathy (AMN) lesions and the choroidal watershed zone (CWZ) or patchy choroidal filling (PCF) using multimodal imaging. METHODS: Lesions in patients diagnosed with AMN were clinically examined using multimodal imaging, including fundus photography, near-infrared reflectance imaging, spectral-domain optical coherence tomography (OCT), fluorescein angiography, indocyanine green angiography, OCT angiography, and microperimetry. The topographical relationship between AMN and the CWZ or PCF was evaluated. RESULTS: Seven eyes of six patients were included in the study. The mean age of the patients was 35.8 ± 11.7 years. The AMN lesions were collocated with the CWZ in five eyes and the PCF in one eye. Among these eyes, three had complete patterns, and three had partial patterns. Only one eye showed no topographical relationship between AMN and the CWZ or PCF. CONCLUSION: The colocation of AMN and CWZ/PCF suggests that the AMN lesions were within an area with a dual-watershed zone: the watershed zone between the retinal deep capillary plexus and choriocapillaris, and the choroidal watershed zone or patchy choroidal filling. This retinal area was highly vulnerable to hypoperfusion. Our results suggest a novel pathophysiological mechanism for AMN.

Plumbagin Inhibits Proliferation, Migration, and Invasion of Retinal Pigment Epithelial Cells Induced by FGF-2.

Proliferative vitreoretinopathy (PVR) is a serious ophthalmic disease and characterized by the formation of proliferative membranes by retinal pigment epithelial (RPE) cells. In PVR, the contraction and traction of the fibrocellular membranes cause retinal detachment, which can cause reduction surgery for retinal detachment to fail. Fibroblast growth factor-2 (FGF-2) causes RPE cells to form extracellular matrix (ECM), promotes chemotaxis, mitosis, and positively promotes the disease process of PVR. Plumbagin (PLB) is a plant small molecule naphthoquinone compound. It has the functions in anti-tumor, anti-inflammatory, inhibit proliferation. We tried to investigate the possible effects of PLB on the biological behavior of ARPE-19 cells induced by FGF-2 and its underlying mechanisms. Our study confirmed that proliferation, migration, and invasion of ARPE-19 cells induced by FGF-2 (10 ng/ml) were significantly inhibited by PLB. PLB also significantly inhibits the expression of MMP-2/-9, collagen I Alpha 1 (Col1A1), collagen IV Alpha 1 (Col4A1), collagen VI Alpha 1 (Col6A1), and the phosphorylation of FGF receptor (FGFR)-1, FGFR-2, ERK, p38, JNK of FGF-2-induced ARPE-19 cells. In summary, PLB inhibits FGF-2-stimulated proliferation, migration, and invasion of ARPE-19 cells, which may take place through inhibiting the expression of MMP-2/-9, Col1A1, Col4A1, Col6A1, and the mitogen-activated protein kinase (MAPK) pathway. PLB may have a preventive effect on proliferation, migration, and invasion of FGF-2-induced ARPE-19 cells.

Crocetin inhibits PDGF-BB-induced proliferation and migration of retinal pigment epithelial cells.

In proliferative vitreoretinopathy (PVR), the proliferation and migration of retinal pigment epithelial (RPE) cells are important to pathogenesis. Platelet-derived growth factor (PDGF) is an important factor in the underlying mechanism. Several studies have shown that PDGF induced the proliferation and migration effects on RPE cells in PVR. Crocetin-anantioxidant carotenoid that is abundant in saffron-has been shown to suppress the migration and proliferation of many cell types, but studies of the effects on RPE cell migration and proliferation are incomplete. Therefore, we investigated the inhibitory effect of crocetin on the proliferation and migration of ARPE-19 cells induced by PDGF-BB, an isoform of PDGF. The proliferation of cells was assessed by Cell Counting Kit-8 (CCK-8) and 5-ethynyl-2'-deoxyuridine (EdU) assays. The apoptosis of cells was assessed by flow cytometric analysis. The migration of RPE cells was detected by a Transwell migration assay and an in vitro scratch assay. The levels of main regulatory proteins for apoptosis and the PDGF-BB-induced signaling pathway were determined by western blot analysis. The proliferation and migration of ARPE-19 cells treated with crocetin (100-400 µM) and PDGF-BB (20 ng/ml) were significantly inhibited in a concentration- and time-dependent manner. Crocetin exhibited potent inducing effects on the apoptosis of PDGF-BB-induced ARPE-19 cells via the modulation of Bcl-2 family regulators in a concentration-dependent manner. The inhibitory effects of crocetin on PDGF-BB-induced platelet-derived growth factor receptor ß (PDGFRß) and the underlying pathways of PI3K/Akt and ERK, p38, JNK activation were identified. The results showed that crocetin is an effective inhibitor of PDGF-BB-induced proliferation and migration of ARPE-19 cell through the downregulation of regulatory signaling pathways.

Safety, pharmacokinetics, and prevention effect of intraocular crocetin in proliferative vitreoretinopathy.

The study was designed to determine the safety and pharmacokinetics of intraocular crocetin and examine whether crocetin inhibits the development of proliferative vitreoretinopathy (PVR) in a rabbit model. In the toxicity study, the right eyes of rabbits were injected with 0.2 µmol or 0.4 µmol crocetin. The left eyes were injected with 0.1 ml phosphate buffered saline (PBS) containing the same concentration of DMSO. Fundus photography, optical coherence tomography (OCT), and electroretinogram (ERG) were obtained at baseline and 14 days. Afterward, the eyes were enucleated for histopathological analysis and terminal deoxynucleotidyl transferasemediated dUTP nick end labeling (TUNEL) assay. In the pharmacokinetic study, the eyes received an intravitreous injection of 0.4 µmol crocetin to detect vitreous drug levels with HPLC at specific time points. In the efficacy study, PVR was induced with an intravitreal injection of ARPE-19 cells in rabbits. Then ten eyes were injected with 0.4 µmol crocetin, and the other 10 eyes received 0.1 ml PBS. Fundus photography, OCT and ERG were performed at days 3 and 7 and weekly for a total of 4 weeks after injection. Afterward, the eyes were enucleated and subjected to histological analysis and TUNEL staining. The results demonstrated no signs of retinal toxicity. Intravitreal injection of 0.4 µmol crocetin had a half-life of 4.231 h. Treatment with crocetin significantly inhibited the progression of PVR in parallel with a reduced expression of α-SMA, collagen fibers and Ki67. These results indicate that crocetin is an effective and safe inhibitor of PVR in rabbit models.

Effects of Curcumin on Epidermal Growth Factor in Proliferative Vitreoretinopathy.

BACKGROUND/AIMS: Proliferative vitreoretinopathy (PVR) is a common refractory eye disease that causes blindness and occurs after retinal detachment or retinal reattachment. Epidermal growth factor (EGF) has been shown to play an important role in the migration and proliferation of retinal pigment epithelium (RPE) cells, which promote PVR. Curcumin inhibits RPE cell proliferation, but it is not known whether it participates in the formation of PVR. Curcumin regulates the biological functions of EGF, which plays important roles in the development of PVR. This study aimed to evaluate the effect of curcumin on the regulation of EGF in PVR. METHODS: Rabbit RPE cells were cultured, and EGF expression was detected by immunocytochemistry. MTT assay was conducted to determine cell proliferation induced by different concentrations of EGF. Immunocytochemical staining was used to detect EGF expression after treatment with curcumin at varying concentrations. Real-time PCR (RT-PCR) and western blot analysis were used to detect the concentrations of EGF mRNA and protein after treatment with curcumin. After RPE cells and curcumin were injected into experimental rabbit eyes, the cornea, aqueous humor, lens, and vitreous opacity were observed and recorded simultaneously by indirect ophthalmoscopy, fundus color photography, and B-ultrasonography. The vitreous body was extracted, and the EGF content in the vitreous humor was measured by enzyme-linked immunosorbent assay (ELISA). RESULTS: At each time point (24, 48, and 72 h), cell proliferation gradually increased with increasing EGF concentrations (0, 3, 6, and 9 ng/mL) in a dose-dependent manner. Cell proliferation between EGF concentrations of 9 and 12 ng/mL were no different, which suggested that 9 ng/mL EGF was the best concentration to use to stimulate RPE cell proliferation in vitro. Under all EGF concentrations (0, 3, 6, 9, and 12 ng/mL), RPE cell proliferation increased with time (from 24 to 72 h), suggesting a time-effect relationship. Curcumin downregulated EGF expression in RPE cells, which also indicated time-effect and dose-effect relationships. The best curcumin concentration for the inhibition of EGF expression was 15 µg/mL. RT-PCR and western blot analyses indicated that the EGF mRNA and expression of the protein in RPE cells treated with curcumin significantly decreased with time. Ocular examinations revealed that the vitreous opacity was lower and the proliferative membrane was thinner in the curcumin group compared with the control group. The PVR grade and the incidence of retinal detachment were significantly lower in the experimental group than in the control group. ELISA results showed that the EGF content in vitreous humor was higher in the control group than in the curcumin group. The curcumin and control groups were significantly different at each time point. CONCLUSION: Curcumin inhibited RPE cell proliferation by downregulating EGF and thus effectively inhibited the initiation and development of PVR.

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

BACKGROUND: This study aimed to explore the effects of plumbagin (PLB) on ARPE-19 cells and underlying mechanism. METHODS: Cultured 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. RESULTS: PLB 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. CONCLUSIONS: This 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.

Inhibitory Effects of Plumbagin on Retinal Pigment Epithelial Cell Epithelial-Mesenchymal Transition In Vitro and In Vivo.

BACKGROUND This study aimed to explore the effects of plumbagin (PLB) on epithelial-to-mesenchymal transition in retinal pigment epithelial (RPE) cells and in proliferative vitreoretinopathy (PVR) rabbit models. MATERIAL AND METHODS Rabbit RPE cells were exposed to various concentrations (0, 5, 15, and 25 µM) of PLB. Motility, migration, and invasion of PLB-treated cells were determined in vitro using Transwell chamber assays and scratch wound assays. The contractile ability was evaluated by cell contraction assay. Expression of matrix metalloproteinases (MMPs) and epithelial-mesenchymal transition (EMT) markers were assessed by western blotting. Furthermore, PLB was injected in rabbit eyes along with RPE cells after gas compression of the vitreous. The presence of PVR was determined by indirect ophthalmoscopy on days 1, 7, 14, and 21 after injection. Also, optical coherence tomography (OCT), ultrasound images, electroretinograms (ERG), and histopathology were used to assess efficacy and toxicity. RESULTS PLB significantly inhibited the migration and invasion of RPE cells. The agent also markedly reduced cell contractive ability. Furthermore, PLB treatment resulted in the decreased expression of MMP-1, MMP2, α-SMA, and the protection of ZO-1. In addition, the PLB-treated eyes showed lower PVR grades than the untreated eyes in rabbit models. PLB exhibited a wide safety margin, indicating no evidence of causing retinal toxicity. CONCLUSIONS PLB effectively inhibited the EMT of rabbit RPE cells in vitro and in the experimental PVR models. The results open new avenues for the use of PLB in prevention and treatment of PVR.

YC-1 Inhibits VEGF and Inflammatory Mediators Expression on Experimental Central Retinal Vein Occlusion in Rhesus Monkey.

PURPOSES: To investigate the therapeutic potential of YC-1 for experimental central retinal vein occlusion (CRVO) of rhesus monkey. METHODS: Six adult rhesus monkeys were recruited in this study. Laser-induced CRVO was established in both eyes of all subjects. Intravitreal injection of YC-1 90 µl (200 µM with 0.01% dimethyl sulfoxide (DMSO) as vehicle) was administrated in right eye and 0.01% DMSO 90 µl in left eye respectively at 1 week after CRVO established. All eyes underwent routine examination at 1 day, 1 week, 2 week, and 1 month after intravitreal injection of YC-1 or DMSO. Meanwhile, vitreous fluid was collected at each time points to analyze concentration of VEGF, HIF-1α, IL-6, IL-8, and MCP-1 mediators by CBA or ELASA method. RESULTS: The experimental CRVO was successfully established in six rhesus monkeys. As expected, the thickness of macular edema significantly decreased at 1 week and 2 weeks after YC-1 injection compared with that of DMSO injection. Subsequently, the central macular thickness in all eyes was recovered to the initial levels at 1 month after photocoagulation. Intraocular pressure (IOP) was not significantly different between two groups during all follow up. Meanwhile, the concentration of IL-6, IL-8, VEGF, and HIF-1α in vitreous fluid significantly decreased after YC-1 injection compared with that of DMSO injection, MCP-1 was not significantly different between both groups. CONCLUSIONS: Intravitreal injection of YC-1 significantly alleviated macular edema compared with that of DMSO control group. Meanwhile, both inflammatory factors and angiogenesis-related factors expression were inhibited in vitreous by YC-1 injection.

Crocetin inhibits the proliferation, migration and TGF-ß2-induced epithelial-mesenchymal transition of retinal pigment epithelial cells.

Retinal pigment epithelial (RPE) cells, the major cell type in the fibrotic membrane of proliferative vitreoretinopathy, display enhanced proliferative and migratory capacities and epithelial-mesenchymal transition (EMT). In this study, we investigated the potential impact of crocetin on the proliferation, migration and EMT of cultured ARPE-19 cells. The cells were treated with crocetin alone or in combination with transforming growth factor-ß2 (TGF-ß2). Cell proliferation was examined using the CCK-8 assay. Cell cycle distribution was analyzed by flow cytometry after propidium iodide staining. The expression levels of proliferating cell nuclear antigen (PCNA), p21 and p53 were examined by Western blot analysis. Cell migration was assessed by in vitro scratch and Transwell assays. Real-time PCR, Western blotting and immunofluorescence were used to assess EMT features. Treatment of ARPE-19 cells with crocetin (50-200µM) significantly inhibited their proliferation and migration in a concentration- and time-dependent manner. Crocetin induced G1 arrest, reduced PCNA protein expression and increased the p21 and p53 accumulation in ARPE-19 cells. Crocetin inhibited TGF-ß2-induced EMT in ARPE-19 cells by maintaining the expression of E-cadherin and ZO-1 and by reducing the expression of vimentin and α-SMA through the suppression of phosphorylation of p38. These results indicate that crocetin is an effective inhibitor of the proliferation, migration and TGF-ß2-mediated EMT of ARPE-19 cells.

Insulin use and risk of diabetic macular edema in diabetes mellitus: a systemic review and meta-analysis of observational studies.

BACKGROUND: Diabetes mellitus is a common and serious disorder. A search of the literature reveals no comprehensive quantitative assessment of the association between insulin use and incidence of diabetic macular edema. Therefore, we performed a meta-analysis of observational studies to evaluate the effect of insulin use on the risk of developing macular edema. MATERIAL/METHODS: Comparative studies published until May 2014 were searched through a comprehensive search of the Medline, Embase, and the Cochrane Library electronic databases. A systematic review and quantitative analysis of comparative studies reporting the effect of insulin use on the incidence of macular edema was performed. All analyses were performed using the Review Manager (RevMan) v.5 (Nordic Cochrane Centre, Copenhagen, Denmark). RESULTS: A total of 202 905 individuals were included in the present meta-analysis. In a random-effects meta-analysis, the use of insulin was found to be associated with increased risk of macular edema (RR, 3.416; 95% CI, 2.417-4.829; I2, 86.6%). Analysis that just included high-quality studies showed that insulin use increased the risk of macular edema (RR, 2.728; 95% CI, 1.881-3.955; I2=77.7%). In cohort studies (RR, 4.509; 95% CI, 3.100-6.559; I2, 77.7%) but not in case-control studies (RR, 1.455; 95% CI, 0.520 to 4.066; I2, 95.9%), increased incidence of macular edema was observed. CONCLUSIONS: The results of this meta-analysis of observational studies demonstrate that insulin use is a risk factor for diabetic macular edema. However, available data are still sparse, and in-depth analyses of the assessed associations in the context of additional longitudinal studies are highly desirable to enable more precise estimates and a better understanding of the role of insulin use in incidence of diabetic macular edema.

Retinoic acid suppresses the adhesion and migration of human retinal pigment epithelial cells.

The study was designed to better understand how retinoic acid (RA) influenced the migration and invasion abilities of retinal pigment epithelial cells (RPE) in vitro and how the related genes of the extracellular matrix (ECM) were expressed. The inhibition effects of RA on proliferative vitreoretinopathy (PVR) formation induced by RPE cells were studied in rabbits. Wound healing and Boyden chamber assays were used to show the abilities of migration and invasion of RPE. Microarray, real-time quantitative PCR (qPCR) and Western blotting showed how RA regulated the ECM genes. RA (10(-5) M) significantly (P < 0.05) inhibited PVR membrane and traction retinal detachment formation (80%). Moreover, RA treatment significantly inhibited the migration (80%) and invasion (65%) behaviors of human RPE cells (P < 0.05) by wound healing and Boyden chamber assays, respectively. Microarray and q PCR analysis showed RA treatment did inhibit the motility of human RPE cells by inhibition of metalloproteinases (MMP) 1, 2, 9, fibronectin-1, transforming growth factor beta, thrombospondin-1, tenascin C, most collagen, integrin, laminin molecules and along enhancing E-cadherin and MMP3 genes expression. And Western blotting indicated the coincident results on protein level of MMP1, 2, 3, 9, 14; fibronectin-1; integrinαM, ß2 and E-cadherin. In conclusions, RA is a vital drug to inhibit the abilities of migration and invasion of RPE and to hamper the PVR formation by regulating some genes expression of ECM.

[The effect of curcumin on DNA content, mitochondrial transmembrane potential and calcium of rabbit cultured retinal pigment epithelial cells].

OBJECTIVE: To investigate the Change of DNA content (apoptosis rate), mitochondrial transmembrane potential (DeltaPsim) and calcium (Ca(2+)) of rabbit retinal pigment epithelial (RPE) cells cultured with curcumin. METHODS: It was an experimental study. The RPE cells were dissociated from rabbit eyes and cultured. The RPE cells in the 4(th) passage were divided into 2 groups: curcumin group and control group (10% FBS-EMDM contains 0.05% dimethyl sulfoxide). The curcumin group contained 3 mass concentration: 10 mg/L, 15 mg/L and 20 mg/L. The MTT assay was used to evaluate the inhibition effect of RPE cells cultured with curcumin after 24 h, 48 h, 72 h and 96 h respectively. The IC(50) value in 24 h, 48 h, 72 h and 96 h were gotten by Linear Regression. Flow cytometry was performed to detect the change of DNA content (apoptosis rate), DeltaPsim and Ca(2+) of RPE cells cultured with curcumin (15 mg/L) after 8 h, 24 h, 48 h and 72 h respectively. RESULTS: RPE cells were significantly inhibited by curcumin in a dose dependent and time dependent manner. The IC(50) value of curcumin at 24 h, 48 h, 72 h and 96 h was 29.31 mg/L, 17.50 mg/L, 13.24 mg/L and 10.99 mg/L respectively. Ca(2+) was significantly increased at 8 h, 24 h, 48 h and 72 h after cultured with curcumin (15 mg/L) than that of the control group respectively (t = 7.50, 10.61, 20.74, 21.14, P < 0.01), and DeltaPsim was significantly decreased at 8 h, 24 h, 48 h and 72 h after cultured with curcumin (15 mg/L) than that of the control group respectively (t = 7.50, 11.74, 14.91, 15.29, P < 0.01). There was no change of DNA content in RPE cells at 8h after cultured with curcumin (15 mg/L), but significantly lower than that of the control group at 24 h, 48 h and 72 h respectively (t = 10.00, 14.68, 13.68, P < 0.01). CONCLUSION: The apoptosis of RPE cells induced by curcumin is caused by increase of Ca(2+) and decrease of DeltaPsim that causes decrease of DNA content. The RPE cells are significantly inhibited by curcumin, which may become a potential drug to prevent and treat proliferative vitreoretinopathy.