Fucoidan Reduces Secretion and Expression of Vascular Endothelial Growth Factor in the Retinal Pigment Epithelium and Reduces Angiogenesis In Vitro

Dithmer, Michaela; Fuchs, Sabine; Shi, Yang; Schmidt, Harald; Richert, Elisabeth; Roider, Johann; Klettner, Alexa

doi:10.1371/journal.pone.0089150

Cited by 65 publications

(76 citation statements)

References 44 publications

(60 reference statements)

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“…To investigate whether fucoidan can inhibit EMT in RPE cells, we first testified the cell viability of primary RPE cells treated with fucoidan at the recommended concentrations [15]. At 48 h after exposure to fucoidan, no significant influence on cell viability was observed within the range of concentrations tested (Fig.…”

Section: Resultsmentioning

confidence: 99%

Protective Effects of Fucoidan on Epithelial-Mesenchymal Transition of Retinal Pigment Epithelial Cells and Progression of Proliferative Vitreoretinopathy

Zhang¹,

Zhao²,

Yang³

et al. 2018

Cell Physiol Biochem

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Background/Aims: Proliferative vitreoretinopathy (PVR) is a severe blinding complication of rhegmatogenous retinal detachment. Epithelial-mesenchymal transition (EMT) of retinal pigment epithelial (RPE) cells is thought to play a pivotal role in the pathogenesis of PVR. Fucoidan, a marine extract, reportedly has many benefits effects in a variety of tissues and organs such as anti-inflammation, anti-oxidative stress, and anti-carcinogenesis. In this study, we investigated the potential role of fucoidan on EMT in RPE cells and its effect on the development of PVR. Methods: MTS, Transwell, and collagen gel contraction assays were employed to measure the viability, migration, and contraction of RPE cells, respectively. mRNA and protein expression were evaluated via real-time quantitative PCR and western blot analysis, respectively. In vivo, a pigmented rabbit model of PVR was established to examine the anti-PVR effect of fucoidan. Results: Fucoidan reversed the transforming growth factor (TGF)-β1-induced EMT of RPE cells, including the increased expression of α-smooth muscle actin (α-SMA) and fibronectin and down-regulation of E-cadherin in human primary RPE cells. Moreover, the upregulation of phosphorylated Smad2/3 induced by TGF-β1 was suppressed by fucoidan. Fucoidan also inhibited the migration and contraction of RPE cells induced by TGF-β1. In vivo, fucoidan inhibited the progression of experimental PVR in rabbit eyes. Histological findings showed that fucoidan suppressed the formation of α-SMA-positive epiretinal membranes. Conclusion: Our findings regarding the protective effects of fucoidan on the EMT of RPE cells and experimental PVR suggest the potential clinical application of fucoidan as an anti-PVR agent.

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Section: Resultsmentioning

confidence: 99%

Protective Effects of Fucoidan on Epithelial-Mesenchymal Transition of Retinal Pigment Epithelial Cells and Progression of Proliferative Vitreoretinopathy

Zhang¹,

Zhao²,

Yang³

et al. 2018

Cell Physiol Biochem

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“…As such, their roles in the mechanisms leading to PVR have become the current areas of focus. Previous studies have emphasized the important role of epidermal growth factor (EGF) in the migration and proliferation of RPE cells, which promotes the occurrence of PVR [6].…”

Section: Introductionmentioning

confidence: 99%

Effects of Curcumin on Epidermal Growth Factor in Proliferative Vitreoretinopathy

Ren

Zhao

et al. 2018

Cell Physiol Biochem

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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.

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“…A number of recent studies have used RPE isolated from porcine eyes as in vitro models to address questions of trans-epithelial transport, outer segment phagocytosis, regulation of VEGF secretion and inflammation (Ablonczy and Crosson, 2007; Chew et al, 1993; Dintelmann et al, 1999; Dithmer et al, 2014; Hamann et al, 2003; Hammer et al, 2006; Joffre et al, 2007; Klettner et al, 2013; Miura et al, 2010; Muller et al, 2014; Shirasawa et al, 2013; Terasaki et al, 2013). Many of these studies used non-polarized porcine RPE cultures.…”

Section: Introductionmentioning

confidence: 99%

A detailed three-step protocol for live imaging of intracellular traffic in polarized primary porcine RPE monolayers

Toops

Tan

Lakkaraju

2014

Experimental Eye Research

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The retinal pigment epithelium (RPE) performs numerous functions that are indispensable for photoreceptor health and vision. This monolayer of cells is also a major site of insult in inherited and age-related macular degenerations. In vitro models of primary RPE such as human fetal and adult RPE cultures have been invaluable for dissecting disease pathways at the cellular and molecular level. However, numerous studies show that it takes over four weeks for human RPE cell monolayers to become fully polarized after plating on semipermeable membrane supports. Poor persistence of transgene expression over this time period critically limits the applicability of human RPE cultures for live imaging studies required to follow dynamic processes like intracellular trafficking and organelle transport that occur over timescales of milliseconds. Here, we provide a detailed three-step protocol for live imaging of polarized primary RPE using high-speed spinning disk confocal microscopy. Step 1: establish porcine RPE monolayers that undergo differentiation within one week after plating on semipermeable membrane supports; step 2: transfect or transduce RPE using either of two different protocols that result in prolonged transgene expression; and step 3: perform multicolor high-speed live imaging of organelle transport in polarized RPE monolayers. Porcine RPE cells and photoreceptor outer segments were isolated from freshly harvested eyes and plated on collagen-coated Transwell® filters to generate polarized monolayers. After seven days, RPE monolayers were highly pigmented, had TER values ≥ 200 Ω.cm2 and cleared outer segments within 5 hours after phagocytosis. These cells expressed RPE65, localized ZO-1 to the tight junction, Na+,K+-ATPase to the apical membrane and acetylated tubulin to the primary cilium. There was an inverse relationship between initial plating density and the time to differentiation. We used nucleofection to express fluorescently tagged genes in RPE cells prior to plating on filters or baculovirus fusion constructs to transfect polarized monolayers. Both these methods resulted in transfection efficiencies over 40% and transgene expression lasted up to 8 days after plating. These filters were imaged by high-speed spinning disk microscopy to follow tubulovesicular trafficking of lysosomes and actin dynamics in the RPE. Four-dimensional image analysis performed using commercially available software was used to analyze live imaging data. In conclusion, this 3-step protocol describes a powerful method to investigate organelle trafficking and function in real time in the RPE that can be used for answering fundamental questions of RPE cell biology and pathobiology.

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Fucoidan Reduces Secretion and Expression of Vascular Endothelial Growth Factor in the Retinal Pigment Epithelium and Reduces Angiogenesis In Vitro

Cited by 65 publications

References 44 publications

Protective Effects of Fucoidan on Epithelial-Mesenchymal Transition of Retinal Pigment Epithelial Cells and Progression of Proliferative Vitreoretinopathy

Protective Effects of Fucoidan on Epithelial-Mesenchymal Transition of Retinal Pigment Epithelial Cells and Progression of Proliferative Vitreoretinopathy

Effects of Curcumin on Epidermal Growth Factor in Proliferative Vitreoretinopathy

A detailed three-step protocol for live imaging of intracellular traffic in polarized primary porcine RPE monolayers

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