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Retinal Cell Biology  |   May 2015
Decorin Prevents Retinal Pigment Epithelial Barrier Breakdown Under Diabetic Conditions by Suppressing p38 MAPK Activation
Author Affiliations & Notes
  • Shuai Wang
    Department of Ophthalmology Shanghai Jiao Tong University Affiliated Sixth People's Hospital, Shanghai, China
  • Shanshan Du
    Department of Ophthalmology Shanghai Jiao Tong University Affiliated Sixth People's Hospital, Shanghai, China
  • Qiang Wu
    Department of Ophthalmology Shanghai Jiao Tong University Affiliated Sixth People's Hospital, Shanghai, China
    Shanghai Clinical Center for Diabetes Mellitus, Shanghai, China
  • Jianyan Hu
    Department of Ophthalmology Shanghai Jiao Tong University Affiliated Sixth People's Hospital, Shanghai, China
  • Tingting Li
    Department of Ophthalmology Shanghai Jiao Tong University Affiliated Sixth People's Hospital, Shanghai, China
  • Correspondence: Qiang Wu, Department of Ophthalmology, Shanghai Jiao Tong University Affiliated Sixth People's Hospital, 600 Yishan Road, Shanghai, China 200233; qiang.wu@shsmu.edu.cn
  • Footnotes
     SW and SD contributed equally to the work presented here and should therefore be regarded as equivalent authors.
Investigative Ophthalmology & Visual Science May 2015, Vol.56, 2971-2979. doi:10.1167/iovs.14-15874
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      Shuai Wang, Shanshan Du, Qiang Wu, Jianyan Hu, Tingting Li; Decorin Prevents Retinal Pigment Epithelial Barrier Breakdown Under Diabetic Conditions by Suppressing p38 MAPK Activation. Invest. Ophthalmol. Vis. Sci. 2015;56(5):2971-2979. doi: 10.1167/iovs.14-15874.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

Purpose.: The purpose of this study was to determine the effect of decorin on the barrier function of human retinal pigment epithelial (RPE) cells under high-glucose (HG) plus hypoxia conditions.

Methods.: Human RPE (ARPE-19) cells were cultured for 18 days in normal glucose (5.5 mM) or HG (25 mM) medium. In addition, to mimic the hypoxic impact which occurs in diabetic retinopathy, cells were treated with 100 μM CoCl2 during the last 2 days of the experiment. Decorin, 100 nM, was applied 1 hour before CoCl2 was added. Retinal pigment epithelial barrier function was evaluated by measuring transepithelial electrical resistance (TER) and apical-basolateral permeability of fluorescein isothiocyanate (FITC)-dextran. The content and distribution of tight junction proteins (claudin-1, occludin, and zonula occludens-1 [ZO-1]) were examined by Western blotting and immunofluorescence. p38 mitogen-activated protein kinase (MAPK) phosphorylation was evaluated by Western blotting, and small interfering RNA transfection to p38 MAPK was also performed in ARPE-19 monolayers.

Results.: High-glucose plus hypoxia significantly increased FITC-dextran permeability, paralleled by decreased TER. Decorin reversed both of these effects. High-glucose plus hypoxia-induced reduction and disorganization of occludin and ZO-1 were also reversed by decorin. Decorin prevented the activation of p38 MAPK induced by hypoxia. Silence of p38 MAPK by RNA interference also inhibited the breakdown of ARPE-19 cell monolayer induced by HG plus hypoxia.

Conclusions.: Retinal pigment epithelial barrier disruption induced by HG plus hypoxia was prevented by decorin through suppression of p38 MAPK activation, which could present a new therapeutic strategy for inhibition of diabetic macular edema.

Diabetic macular edema (DME) is one of the primary causes of visual impairment in patients with diabetes mellitus.1 Breakdown of the blood-retina barrier (BRB) caused by the disruption of tight junctions appears to be the main factor responsible for DME.2,3 The human retina consists of the inner BRB, which is composed mainly of endothelial cells and directly controls the flux into the inner retina, and the outer BRB, which is formed by retinal pigment epithelial (RPE) cells and controls the flow of solutes and fluid from the choroidal vasculature into the outer retina.4 Both of the BRBs are involved in the process of DME. Although extensive work has been performed to identify the factors involved in the disruption of the inner BRB during DME,58 the mechanisms implicated in the regulation of the outer BRB have been poorly studied. The outer BRB separates the neural retina from the choroidal vasculature and fulfills multiple roles that are essential for the maintenance of normal physiological processes in the retina, including (1) transporting nutrients, water, and ions; (2) absorbing light energy; (3) phagocytosing shed photoreceptor outer-segment membranes; (4) secreting essential growth factors such as VEGF and pigmented epithelium-derived factor; and (5) maintaining the immune privilege of the eye.9,10 Diabetes-induced outer BRB dysfunction has been detected in both humans and animals.1114 Breakdown of the outer BRB is increasingly recognized as playing an important role in the development of diabetic retinopathy (DR).4,15,16 
Decorin is a small leucine-rich proteoglycan, which has been found to negatively regulate a variety of cellular functions by binding to extracellular matrix components or cell surface receptors.17,18 In our previous work, we demonstrated that decorin could interfere with angiogenesis by downregulating hypoxia-induced expression of Met, Rac1, hypoxia-inducible factor-1 alpha (HIF-1a), and VEGF in RPE cells.19 However, whether decorin could protect the RPE barrier function during DME remains unclear. The decorin protein core is involved in a number of cellular processes by its interaction with many different molecules such as epidermal growth factor receptor (EGFR),20 transforming growth factor-beta (TGF-β),21 the toll-like receptors (TLRs),22 and vascular endothelial growth factor receptor 2 (VEGFR2).23 In addition, these molecules have all been found to be associated with the p38 mitogen-activated protein kinase (MAPK) pathway,2426 which is one of the three major MAPK signaling pathways triggered by multiple stimuli.27 Because it is difficult to judge which molecules are correlated mainly with outer BRB breakdown, we chose their common activation molecule, p38 MAPK, as the target protein in our research. Experimental studies have recently indicated that p38 MAPK was significantly activated in RPE of diabetic patients.28 Suppression of p38 MAPK could improve the barrier function in mammary epithelial cells29 and inhibit the development of early DR.30 Moreover, Lala et al.18 found that decorin could inhibit VEGF-induced trophoblast migration and endovascular differentiation by interfering with p38 MAPK activation. 
Based on findings of previous studies, the present study investigated the effect of decorin on outer BRB function on cultured human RPE (ARPE-19) cell line. Given that high-glucose (HG) concentration and hypoxia are the two major components in the diabetic milieu, we used hypoxia together with HG concentrations that would mimic the diabetic milieu.28 Fluorescein isothiocyanate (FITC)–dextran permeability, transepithelial electrical resistance (TER), and the expression of tight junction proteins (claudin-1, occludin, and zonula occludens-1 [ZO-1]) were determined. In addition, the role of p38 MAPK in mediating the disruption induced by HG plus hypoxia and the effect of decorin on p38 MAPK activation were also evaluated. 
Materials and Methods
Human RPE Cell Cultures
A human RPE cell line (ARPE-19) was obtained from American Type Culture Collection (Manassas, VA, USA). This cell line forms stable monolayers and has become a good alternative model of outer BRB in vitro.3133 As previously described,32,34 cells from passage 20 were cultured under normal glucose (NG; 5.5 mM d-glucose) or HG (25 mM d-glucose) conditions for 18 days at 37°C under 5% CO2 in Dulbecco's modified Eagle's medium/nutrient mixture F12 (DMEM/F12 medium; Hyclone, Logan, UT, USA) supplemented with 10% fetal bovine serum (FBS; Hyclone) and 100 U/mL penicillin/streptomycin mixture (Sigma-Aldrich Corp., St. Louis, MO, USA). 
At day 16 of the experiments, cells were subjected to serum starvation (1% FBS) and treated with hypoxia (100 μM CoCl2; Sigma-Aldrich Corp.) or recombinant human decorin (R&D Systems, Minneapolis, MN, USA), or their combination for the last 48 hours. According to our previous results, decorin, 100 nM, was applied 1 hour before CoCl2 was added.19 To rule out a potential bias by an osmotic effect, the experiment was performed using mannitol (5.5 mM d-glucose plus 19.5 mM mannitol) as an osmotic control agent. 
Small Interfering RNA Transfection
Small interfering RNA (siRNA) targeted to p38 MAPK was purchased from Cell Signaling Technology (product no. 6243; Danvers, MA, USA). A non-targeted control siRNA was also used in the experiments. Transfection was performed with Lipofectamine RNAiMAX reagent and Opti-MEM I reduced serum medium (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's instruction at day 12. Cell monolayers were treated with 100 nM siRNA for 72 hours, and then the medium was replaced, and the cells were treated with hypoxia and decorin as described above. 
Cell Viability Assay
Cell viability was measured using Cell Counting Kit-8 (CCK-8; Dojindo, Japan) according to the manufacturer's instructions at day 18. Cell Counting Kit-8 assay was performed in a 96-well culture plate with three replicates for each condition at an absorbance of 450 nm. The number of living cells in each well was expressed as the value relative to the control. 
Permeability Assay
Paracellular permeability of ARPE-19 cell monolayers was determined at day 18 by measuring the apical-to-basal movement of FITC-dextran (40 kDa; Sigma-Aldrich Corp.).33,35 Briefly, FITC-dextran was added to the upper chamber at a concentration of 100 μg/ml. Ninety minutes later, the medium from the lower chamber was collected, and the absorbance was measured at 485 nm of excitation and 528 nm of emission, using a multifunctional microplate reader (BioTek Synergy 4, Winooski, VT, USA). Concentrations were calculated by extrapolation in a standard curve. Each experimental condition was assayed in triplicate and repeated in at least three independent experiments. 
Measurement of Transepithelial Electrical Resistance (TER)
The progress of RPE barrier formation and polarization was monitored by measuring TER by using a Millicell electrical resistance system (ERS2; Millipore, Billerica, MA, USA) as described previously.35,36 Transepithelial electrical resistance measures the resistance of the paracellular pathway; the higher the TER, the lower the permeability.37 All TER measurements were begun after a 20-minute equilibration period at room temperature. The net TER was calculated by subtracting the value of a filter without cells from that of a filter with cells. The final unit area resistance (Ω × cm2) was obtained by multiplying the TER by the effective membrane area. Measurements were performed every 3 days and at 24 hours after treatments. Each condition was assayed in triplicate and repeated in three independent experiments. 
Western Blot Analysis
After treatments, cells were washed with ice-cold phosphate-buffered saline (PBS) and lysed in radio immunoprecipitation assay buffer (Shenneng Bocai Biotechnology Co., Ltd., Shanghai, China) containing 1 mM phenylmethanesulfonyl fluoride and 1× phosphatase inhibitor cocktail (Roche, Mannheim, Germany). Protein was extracted, and equal amounts of protein (30 μg per lane) were separated by 8% or 10% SDS-PAGE and transferred onto a polyvinylidene fluoride membrane (Millipore). Blots were probed with primary antibodies overnight at 4°C. After membranes were washed, horseradish peroxidase-conjugated secondary antibody (Proteintech Group, Chicago, IL, USA) was applied, and proteins were visualized by using an enhanced chemiluminescence detection system (Bio-Rad, Hercules, CA, USA). The following primary antibodies were used: anti-occludin and anti-ZO-1 antibodies (Santa Cruz Biotechnology, Dallas, TX, USA), anti-claudin-1 and anti-β-actin antibodies (Abcam, Cambridge, MA, USA), anti-phospho-p38 MAPK (Thr180/Tyr182), and anti-p38 MAPK antibodies (Cell Signaling Technology). Band intensity was analyzed with Quantity One version 4.6.2 software (Bio-Rad) and compared with the internal standard β-actin. 
Immunofluorescence Assay
Human RPE-19 monolayers maintained in glass coverslips were immunostained for tight junction proteins. Cells were washed in PBS, fixed in methanol for 10 min, and blocked with 2% bovine serum albumin (Sigma-Aldrich Corp.) in PBS for 1 hour at room temperature. Mouse anti-ZO-1 and rabbit anti-occludin (Invitrogen) were incubated overnight at 4°C. Afterward, the samples were incubated with Alexa 488 secondary antibodies (1:200 dilution; Invitrogen) for 1 hour at room temperature and mounted with fluorescent mounting medium (Beyotime, Shanghai, China). Images were acquired by using a fluorescence microscope (Olympus, Shinjuku, Japan). 
Statistical Analysis
All experiments were repeated three times. Statistical analysis was performed using SPSS 16.0 software. Data were evaluated statistically, using one-way ANOVA for comparisons among more than two groups. Student-Newman-Keuls test was used to determine the significance of the differences between two different groups. Data are means ± standard error of the mean (SEM). Statistical significance was defined as a P value of <0.05. 
Results
Cell Viability Assay
Under these experimental conditions, we did not observe any significant differences in cell viability as measured by CCK-8 assay (Fig. 1). In addition, we monitored the morphological appearance of ARPE-19 cells, using phase contrast microscopy. Findings suggest that the results obtained are not biased by changes in cell proliferation or cell damage. 
Figure 1
 
Cell viability of ARPE-19 cells. At day 16 of the experiments, cells were exposed to hypoxia with or without decorin pretreatment in NG and HG medium. At day 18, as measured by CCK-8 assay, no significant differences in cell viability were found among the different conditions tested. Untreated cells grown in NG medium served as the control. Bars are means ± SEM.
Figure 1
 
Cell viability of ARPE-19 cells. At day 16 of the experiments, cells were exposed to hypoxia with or without decorin pretreatment in NG and HG medium. At day 18, as measured by CCK-8 assay, no significant differences in cell viability were found among the different conditions tested. Untreated cells grown in NG medium served as the control. Bars are means ± SEM.
Effect of Decorin on High-Glucose and Hypoxia-Induced Hyperpermeability in ARPE-19 Cells
The effects of different conditions examined on the permeability of ARPE-19 monolayers are shown in Figure 2. High-glucose alone increased permeability mildly compared with that of NG, whereas hypoxia alone significantly increased permeability. Moreover, both of the conditions (HG plus hypoxia) remarkably increased permeability in what appeared to be a synergistic effect, and this could not be attributed to hyperosmotic effects. When cells grown in HG plus hypoxia medium were treated with 100 nM decorin, a significant reduction in permeability was observed (254.9 ± 24.9 ng/mL/cm2 vs. 408.0 ± 25.4 ng/mL/cm2, respectively, P < 0.05). 
Figure 2
 
Effect of decorin on ARPE-19 cell monolayer permeability. Fluorescein isothiocyanate (FITC)–dextran permeability was examined at 90 minutes. Data indicate that decorin, 100 nM, has a protective effect on HG-plus hypoxia-induced barrier hyperpermeability. Bars are means ± SEM. #P < 0.05 compared with NG. *P < 0.05. **P < 0.05 compared with HG plus hypoxia.
Figure 2
 
Effect of decorin on ARPE-19 cell monolayer permeability. Fluorescein isothiocyanate (FITC)–dextran permeability was examined at 90 minutes. Data indicate that decorin, 100 nM, has a protective effect on HG-plus hypoxia-induced barrier hyperpermeability. Bars are means ± SEM. #P < 0.05 compared with NG. *P < 0.05. **P < 0.05 compared with HG plus hypoxia.
Effect of Decorin on High-Glucose and Hypoxia-Induced Reduction of TER in ARPE-19 Cells
Transepithelial electrical resistance of ARPE-19 monolayers was stable at approximately day 10 of the experiments. The effect of different treatments on TER at 24 hours is shown in Figure 3. High-glucose alone did not affect TER compared with NG, whereas hypoxia alone and HG plus hypoxia significantly decreased TER. Cells treated with 100 nM decorin significantly increased TER compared with cells exposed to HG plus hypoxia alone (146.7 ± 9.3 vs. 105.7 ± 7.5, respectively; P = 0.02). 
Figure 3
 
Effect of decorin on TER of ARPE-19 cell monolayer. At 24 hours after treatments, decorin, 100 nM, showed a protective effect on HG-plus hypoxia-induced TER reduction. Bars are means ± SEM. #P < 0.05 compared with NG. *P < 0.05 compared with HG plus hypoxia. TER, transepithelial electrical resistance.
Figure 3
 
Effect of decorin on TER of ARPE-19 cell monolayer. At 24 hours after treatments, decorin, 100 nM, showed a protective effect on HG-plus hypoxia-induced TER reduction. Bars are means ± SEM. #P < 0.05 compared with NG. *P < 0.05 compared with HG plus hypoxia. TER, transepithelial electrical resistance.
Effect of Decorin on High-Glucose and Hypoxia-Induced Downregulation and Disorganization of Tight Junction Proteins in ARPE-19 Cells
Results of Western blotting, examined under different experimental conditions, are shown in Figure 4. There were no significant differences between the contents of claudin-1, occludin, and ZO-1 in cells grown under NG and those grown under HG conditions. However, the expression levels of claudin-1, occludin, and ZO-1 were all significantly reduced in cells exposed to HG plus hypoxia compared to those in NG-cultured cells. Addition of 100 nM decorin significantly increased occludin and ZO-1 protein levels in cells exposed to HG plus hypoxia, reaching values comparable to those of NG-cultured cells, whereas claudin-1 expression was not affected by decorin treatment. 
Figure 4
 
Effect of decorin on the protein expression of claudin-1, occludin, and ZO-1 in ARPE-19 cell monolayers. There were no significant differences in content of claudin-1, occludin, and ZO-1 between cells grown in NG and those grown under HG conditions. However, levels of the claudin-1, occludin, and ZO-1 proteins were all significantly reduced in cells exposed to HG plus hypoxia compared with NG-cultured cells. Treatment with 100 nM decorin reversed the HG-plus hypoxia-induced occludin and ZO-1 protein reduction, whereas claudin-1 expression was not affected by decorin treatment (*P < 0.05). Bars are means ± SEM. β-Actin was used as an internal control.
Figure 4
 
Effect of decorin on the protein expression of claudin-1, occludin, and ZO-1 in ARPE-19 cell monolayers. There were no significant differences in content of claudin-1, occludin, and ZO-1 between cells grown in NG and those grown under HG conditions. However, levels of the claudin-1, occludin, and ZO-1 proteins were all significantly reduced in cells exposed to HG plus hypoxia compared with NG-cultured cells. Treatment with 100 nM decorin reversed the HG-plus hypoxia-induced occludin and ZO-1 protein reduction, whereas claudin-1 expression was not affected by decorin treatment (*P < 0.05). Bars are means ± SEM. β-Actin was used as an internal control.
Immunostaining of occludin and ZO-1 showed a well-structured monolayer in HG-cultured cells (Figs. 5A, 5E) and tight junction disruption in cells cultured under HG plus hypoxia condition (Figs. 5B, 5F). Pretreatment with 100 nM decorin prevented the disorganization of occludin and ZO-1 induced by HG plus hypoxia (Figs. 5C, 5G). 
Figure 5
 
Immunofluorescence of ARPE-19 monolayers shows the beneficial effect of decorin and p38 MAPK siRNA in preventing the HG-plus hypoxia-induced disorganization of occludin and ZO-1. Occludin (AD) and ZO-1 (EH) staining appears in green. (A, E) Immunostaining for occludin and ZO-1 in cells cultured in HG medium showed a well-structured monolayer. (B, F) Disorganization of occludin and ZO-1 induced by HG plus hypoxia. (C, G) Protective effect of decorin 100 nM under HG plus hypoxia conditions. (D, H) Transfection with siRNA targeting p38 MAPK under HG plus hypoxia conditions showed reduced disruption of occludin and ZO-1. Scale bar: 20 μm.
Figure 5
 
Immunofluorescence of ARPE-19 monolayers shows the beneficial effect of decorin and p38 MAPK siRNA in preventing the HG-plus hypoxia-induced disorganization of occludin and ZO-1. Occludin (AD) and ZO-1 (EH) staining appears in green. (A, E) Immunostaining for occludin and ZO-1 in cells cultured in HG medium showed a well-structured monolayer. (B, F) Disorganization of occludin and ZO-1 induced by HG plus hypoxia. (C, G) Protective effect of decorin 100 nM under HG plus hypoxia conditions. (D, H) Transfection with siRNA targeting p38 MAPK under HG plus hypoxia conditions showed reduced disruption of occludin and ZO-1. Scale bar: 20 μm.
Effect of Decorin on High-Glucose and Hypoxia-Induced p38 MAPK Activation in ARPE-19 Cells
p38 MAPK activation was examined by Western blot analysis to study whether this cellular sensor participates in decorin-induced effects on RPE cell barrier function. First, p38 MAPK activation was evaluated in HG-cultured cells at 0, 0.5, 1, 6, 12, and 24 hours after hypoxia exposure. As shown in Figure 6, p38 MAPK was significantly activated at 6 hours after hypoxia as assessed by phosphorylation of the regulatory Thr180/Tyr182 residues. Untreated controls at 0.5- to 24-hour time points were not different from those at 0 minute (data not shown). We next tested p38 MAPK activation in decorin-treated cells at 6 hours after hypoxia. Densitometry analysis showed that HG increased p38 MAPK activation mildly compared with that in NG-cultured cells, whereas HG plus hypoxia significantly increased p38 MAPK activation. Treatment with 100 nM decorin prior to hypoxia exposure strongly reduced the HG plus hypoxia-induced phosphorylation of p38 MAPK, almost to the levels of NG-cultured cells (Fig. 7). 
Figure 6
 
Western blot of p38 MAPK phosphorylation after hypoxia in HG-cultured cells. p38 MAPK activity was expressed as the phosphorylated-to-total p38 MAPK ratio. Maximum activation occurred at 6 hours after hypoxia. Bars are means ± SEM. #P < 0.05. *P < 0.05 compared with the control (0 hours).
Figure 6
 
Western blot of p38 MAPK phosphorylation after hypoxia in HG-cultured cells. p38 MAPK activity was expressed as the phosphorylated-to-total p38 MAPK ratio. Maximum activation occurred at 6 hours after hypoxia. Bars are means ± SEM. #P < 0.05. *P < 0.05 compared with the control (0 hours).
Figure 7
 
Effect of decorin in preventing hypoxia-induced p38 MAPK phosphorylation. p38 MAPK activity was expressed as the phosphorylated-to-total p38 MAPK ratio. Treatment with 100 nM decorin before hypoxia exposure significantly reduced hypoxia-induced p38 MAPK phosphorylation at 6 hours after hypoxia. Bars are means ± SEM. #P < 0.05 compared with NG. *P < 0.05 compared with HG plus hypoxia.
Figure 7
 
Effect of decorin in preventing hypoxia-induced p38 MAPK phosphorylation. p38 MAPK activity was expressed as the phosphorylated-to-total p38 MAPK ratio. Treatment with 100 nM decorin before hypoxia exposure significantly reduced hypoxia-induced p38 MAPK phosphorylation at 6 hours after hypoxia. Bars are means ± SEM. #P < 0.05 compared with NG. *P < 0.05 compared with HG plus hypoxia.
p38 MAPK Activation Mediates High-Glucose and Hypoxia-Induced RPE Barrier Disruption
To confirm whether p38 MAPK was responsible for the HG plus hypoxia-induced RPE barrier disruption, ARPE-19 cells were transfected with siRNA targeting p38 MAPK. Small interfering RNA to p38 MAPK significantly reduced p38 MAPK protein levels by approximately 58% (Fig. 8). To examine the functional effects of those results, we measured TER and FITC-dextran permeability of ARPE-19 monolayers. High-glucose plus hypoxia-induced increase of permeability and decrease of TER were almost prevented in p38 MAPK siRNA-transfected cells (Figs. 9A, 9B). Finally, the results of Western blot analysis (Fig. 10) and immunofluorescence (Figs. 5D, 5H) showed that the content and distribution of tight junction proteins (occludin and ZO-1) in p38 MAPK siRNA-transfected cells treated with HG plus hypoxia were greatly preserved compared with those of untransfected cells treated with HG plus hypoxia. 
Figure 8
 
Western blot analysis shows the effectiveness of siRNA in reducing protein expression of p38 MAPK. Transfection with p38 MAPK siRNA resulted in a significant decrease in p38 protein expression (*P < 0.05 versus control). Transfection with control siRNA did not affect p38 expression. Bars are means ± SEM. β-Actin was used as an internal control.
Figure 8
 
Western blot analysis shows the effectiveness of siRNA in reducing protein expression of p38 MAPK. Transfection with p38 MAPK siRNA resulted in a significant decrease in p38 protein expression (*P < 0.05 versus control). Transfection with control siRNA did not affect p38 expression. Bars are means ± SEM. β-Actin was used as an internal control.
Figure 9
 
Permeability and TER of ARPE-19 monolayers after p38 MAPK siRNA transfection. (A) High-glucose–plus hypoxia-induced increase of FITC-dextran permeability was prevented by p38 siRNA. (B) High-glucose–plus hypoxia-induced decrease of TER was prevented by p38 siRNA. Bars are means ± SEM. *P < 0.05 compared with the other conditions.
Figure 9
 
Permeability and TER of ARPE-19 monolayers after p38 MAPK siRNA transfection. (A) High-glucose–plus hypoxia-induced increase of FITC-dextran permeability was prevented by p38 siRNA. (B) High-glucose–plus hypoxia-induced decrease of TER was prevented by p38 siRNA. Bars are means ± SEM. *P < 0.05 compared with the other conditions.
Figure 10
 
Protein expression of occludin and ZO-1 in ARPE-19 cell monolayers after p38 MAPK siRNA transfection. High-glucose–plus hypoxia-induced reduction of occludin and ZO-1 protein expression was prevented by p38 siRNA. Bars are means ± SEM. β-Actin was used as an internal control. *P < 0.05 compared with the other conditions.
Figure 10
 
Protein expression of occludin and ZO-1 in ARPE-19 cell monolayers after p38 MAPK siRNA transfection. High-glucose–plus hypoxia-induced reduction of occludin and ZO-1 protein expression was prevented by p38 siRNA. Bars are means ± SEM. β-Actin was used as an internal control. *P < 0.05 compared with the other conditions.
Discussion
Recent studies suggest the prospective role of multikinase inhibitors as potential treatment for retinal neovascularization38 and retinal edema.39,40 Decorin is a novel multikinase inhibitor,41 and it has exhibited a protective effect in RPE cells under hypoxic conditions in our previous work.19 However, whether decorin could protect the RPE barrier function after diabetic injury remains to be clarified. Findings from the present study indicate that decorin is able to prevent the HG plus hypoxia-induced breakdown of RPE cell monolayer, and this beneficial effect is mainly mediated by inhibition of p38 MAPK activation. 
Retinal pigment epithelium is an epithelium lying between the neural retina and the choriocapillaris, where it forms the outer BRB. Compared with the effects of DR on the inner BRB, those on the outer BRB have received less attention. Hypoxia can stimulate increased secretion of cytokines and growth factors such VEGF and hepatocyte growth factor (HGF), resulting in breakdown of the BRB.1 Because both neuroretina as well as RPE are affected by hypoxia in DR,42 we cultured ARPE-19 cells in the presence of HG and then treated them with hypoxia to mimic the major pathological events of diabetic milieu. 
In this study, we demonstrated that exposure to HG plus hypoxia is a good method for inducing breakdown of the RPE cell monolayer. Following treatment with HG plus hypoxia, the permeability of ARPE-19 cells was significantly increased as determined by flux of FITC-dextran, and it was accompanied by a significant reduction of TER values. In addition, our results demonstrated that cell permeability, TER, tight junction protein expression, and p38 MAPK activation were all minimally affected in the presence of HG but significantly altered when hypoxia was added to HG condition. Similarly, in the study by Trudeau et al.,33 HG alone significantly increased fibronectin and collagen type IV expression in ARPE-19 cells but minimally affected cell permeability. However, Villarroel et al.32 showed that HG strengthened rather than weakened the integrity of ARPE-19 cell monolayers. It is worth noting that Busik et al.43 reported that in vivo diabetes-related endothelial injury in the retina may be due primarily to glucose-induced cytokine release by other retinal cells but not a direct effect of HG. Therefore, we considered that HG per se was not an important factor responsible for the disruption of outer BRB in DR but that HG plus hypoxia can significantly impair the outer BRB. These findings agree with the clinical concept that hypoxia accelerates the progression of DR and DME.44 
Treatment with decorin significantly reversed the increased permeability and decreased TER induced by HG plus hypoxia. This effect was mainly associated with the regulation of tight junction protein content or distribution.45,46 Among the 40 proteins related to tight junctions, claudin-1, occludin, and ZO-1 are the proteins most studied.34,47,48 In our experiment, we observed that exposure of cells to HG plus hypoxia significantly reduced the protein content of claudin-1, occludin, and ZO-1. Decorin was able to restore the content of occludin and ZO-1 to almost control levels, whereas the content of claudin-1 was not affected. Additionally, recent studies have shown that, after claudin-1 expression was blocked by siRNA, no changes in permeability and TER in ARPE-19 cell monolayers were detected.32 Therefore, we considered that claudin-1 may not be involved in the mechanisms of hyperpermeability in ARPE-19 cells. Apart from the content of tight junction proteins, the ordered protein distribution is also important for the efficient function of RPE barrier. Although several studies using interleukin-1 beta (IL-1β) to provoke the RPE barrier breakdown showed no change in the contents of occludin and ZO-1, we all observed abnormal distribution of these proteins when hyperpermeability occurred.33,45 Decorin in our study was able to prevent hypoxia-induced disorganization of occludin and ZO-1. However, tight junctions are highly complex structures, and the relative contribution of the various proteins to RPE barrier function remains unclear. Therefore, in addition to preventing the downregulation and disorganization of occludin and ZO-1, decorin might also modulate other tight junction proteins or other pathways associated with RPE cell permeability. 
p38 MAPK is one of the major MAPK pathways activated by a variety of cellular stresses and cytokines, such as proinflammatory cytokines, growth factors, and reactive oxygen species.27 Induction of p38 MAPK activation by hypoxia and the effect of decorin in preventing this activation were detected in our study. We found that HG plus hypoxia-induced hyperpermeability, TER reduction, and tight junction protein (occludin and ZO-1) downregulation and disorganization can be prevented by inhibiting p38 MAPK activation. These findings suggested that decorin could reduce RPE barrier breakdown via inhibition of p38 MAPK activation. In addition, oxidative stress and inflammation are also the important factors for DR and DME. A number of articles have demonstrated that decorin core protein can inhibit inflammation by downregulating receptors such as TLRs, which are also associated with p38 MAPK pathway.22 However, little literature could prove the ability of decorin to regulate oxidative stress presently.49 Therefore, the effects of decorin on inflammation and oxidative stress in RPE cells need further investigation. Another weakness of our study is that we only demonstrated the inhibitory effect of decorin on p38 MAPK activation, but we did not explore the downstream mechanism following the inhibition of p38 MAPK. p38 MAPK has multiple downstream molecules, including the activation of nuclear factor-kappa B (NF-κB), signal transducer and activator of transcription 3 (STAT3), and CCAAT/enhancer-binding protein-homologous protein (CHOP).50 Previous studies have found that NF-κB was significantly upregulated in HG-treated ARPE-19 cells and type 2 diabetic retinas, and the BRB permeability of the diabetic retinas increased.51 STAT3 was also found activated in human RPE cells cultured with HG.52 Both NF-κB and STAT3 activation can upregulate the expression of vascular endothelial growth factor,52,53 which is thought to be one of the pivotal factors responsible for BRB breakdown.54 Moreover, HG and hypoxia could also trigger induction of the pro-apoptotic transcription factor CHOP in ARPE-19 cells and disrupt the tight junction integrity.28 The downstream mechanisms following the inhibition of p38 MAPK are so complicated that we cannot figure out which is the main downstream mechanism correlated with tight junction protein alterations. Further studies are needed to elucidate the specific mechanisms of decorin on the protection of outer BRB in diabetic condition. 
In addition, because existing methods presently cannot clearly distinguish outer BRB-specific leakage from inner BRB-specific leakage under pathologic conditions in live animals, we only investigated the effect of decorin on outer BRB function in vitro. A fluorescent microscopy imaging assay for quantifying the outer BRB-specific leakage in diabetic and ischemic rodents has recently been developed by Xu et al.,15 but the cumulative effect of outer BRB breakdown may be larger than what is reflected in cryosections.15 Therefore, our in vitro results cannot be easily transferred to clinical practice. However, p38 MAPK phosphorylation was found increased in RPE from diabetic patients compared to levels found in nondiabetic controls,28 which is consistent with our results. Moreover, another study has delivered decorin gene in vivo and proved that decorin could fulfill its biofunction through p38 MAPK pathway.26 We are now trying new animal models to investigate the effect of decorin on the outer BRB function. 
In summary, our study demonstrated that decorin can significantly reduce the increment of permeability and the breakdown of outer BRB under diabetic conditions in an in vitro model of DME. This effect was mainly mediated by suppression of p38 MAPK activation induced by hypoxia. These findings suggested that decorin may have beneficial effects on DME development. 
Acknowledgments
The authors thank the Laboratory of Oncology, Shanghai Sixth People's Hospital for excellent technical assistance. 
Supported by National Natural Science Foundation of China Grants 81400414 and 81271031. 
Disclosure: S. Wang, None; S. Du, None; Q. Wu, None; J. Hu, None; T. Li, None 
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Figure 1
 
Cell viability of ARPE-19 cells. At day 16 of the experiments, cells were exposed to hypoxia with or without decorin pretreatment in NG and HG medium. At day 18, as measured by CCK-8 assay, no significant differences in cell viability were found among the different conditions tested. Untreated cells grown in NG medium served as the control. Bars are means ± SEM.
Figure 1
 
Cell viability of ARPE-19 cells. At day 16 of the experiments, cells were exposed to hypoxia with or without decorin pretreatment in NG and HG medium. At day 18, as measured by CCK-8 assay, no significant differences in cell viability were found among the different conditions tested. Untreated cells grown in NG medium served as the control. Bars are means ± SEM.
Figure 2
 
Effect of decorin on ARPE-19 cell monolayer permeability. Fluorescein isothiocyanate (FITC)–dextran permeability was examined at 90 minutes. Data indicate that decorin, 100 nM, has a protective effect on HG-plus hypoxia-induced barrier hyperpermeability. Bars are means ± SEM. #P < 0.05 compared with NG. *P < 0.05. **P < 0.05 compared with HG plus hypoxia.
Figure 2
 
Effect of decorin on ARPE-19 cell monolayer permeability. Fluorescein isothiocyanate (FITC)–dextran permeability was examined at 90 minutes. Data indicate that decorin, 100 nM, has a protective effect on HG-plus hypoxia-induced barrier hyperpermeability. Bars are means ± SEM. #P < 0.05 compared with NG. *P < 0.05. **P < 0.05 compared with HG plus hypoxia.
Figure 3
 
Effect of decorin on TER of ARPE-19 cell monolayer. At 24 hours after treatments, decorin, 100 nM, showed a protective effect on HG-plus hypoxia-induced TER reduction. Bars are means ± SEM. #P < 0.05 compared with NG. *P < 0.05 compared with HG plus hypoxia. TER, transepithelial electrical resistance.
Figure 3
 
Effect of decorin on TER of ARPE-19 cell monolayer. At 24 hours after treatments, decorin, 100 nM, showed a protective effect on HG-plus hypoxia-induced TER reduction. Bars are means ± SEM. #P < 0.05 compared with NG. *P < 0.05 compared with HG plus hypoxia. TER, transepithelial electrical resistance.
Figure 4
 
Effect of decorin on the protein expression of claudin-1, occludin, and ZO-1 in ARPE-19 cell monolayers. There were no significant differences in content of claudin-1, occludin, and ZO-1 between cells grown in NG and those grown under HG conditions. However, levels of the claudin-1, occludin, and ZO-1 proteins were all significantly reduced in cells exposed to HG plus hypoxia compared with NG-cultured cells. Treatment with 100 nM decorin reversed the HG-plus hypoxia-induced occludin and ZO-1 protein reduction, whereas claudin-1 expression was not affected by decorin treatment (*P < 0.05). Bars are means ± SEM. β-Actin was used as an internal control.
Figure 4
 
Effect of decorin on the protein expression of claudin-1, occludin, and ZO-1 in ARPE-19 cell monolayers. There were no significant differences in content of claudin-1, occludin, and ZO-1 between cells grown in NG and those grown under HG conditions. However, levels of the claudin-1, occludin, and ZO-1 proteins were all significantly reduced in cells exposed to HG plus hypoxia compared with NG-cultured cells. Treatment with 100 nM decorin reversed the HG-plus hypoxia-induced occludin and ZO-1 protein reduction, whereas claudin-1 expression was not affected by decorin treatment (*P < 0.05). Bars are means ± SEM. β-Actin was used as an internal control.
Figure 5
 
Immunofluorescence of ARPE-19 monolayers shows the beneficial effect of decorin and p38 MAPK siRNA in preventing the HG-plus hypoxia-induced disorganization of occludin and ZO-1. Occludin (AD) and ZO-1 (EH) staining appears in green. (A, E) Immunostaining for occludin and ZO-1 in cells cultured in HG medium showed a well-structured monolayer. (B, F) Disorganization of occludin and ZO-1 induced by HG plus hypoxia. (C, G) Protective effect of decorin 100 nM under HG plus hypoxia conditions. (D, H) Transfection with siRNA targeting p38 MAPK under HG plus hypoxia conditions showed reduced disruption of occludin and ZO-1. Scale bar: 20 μm.
Figure 5
 
Immunofluorescence of ARPE-19 monolayers shows the beneficial effect of decorin and p38 MAPK siRNA in preventing the HG-plus hypoxia-induced disorganization of occludin and ZO-1. Occludin (AD) and ZO-1 (EH) staining appears in green. (A, E) Immunostaining for occludin and ZO-1 in cells cultured in HG medium showed a well-structured monolayer. (B, F) Disorganization of occludin and ZO-1 induced by HG plus hypoxia. (C, G) Protective effect of decorin 100 nM under HG plus hypoxia conditions. (D, H) Transfection with siRNA targeting p38 MAPK under HG plus hypoxia conditions showed reduced disruption of occludin and ZO-1. Scale bar: 20 μm.
Figure 6
 
Western blot of p38 MAPK phosphorylation after hypoxia in HG-cultured cells. p38 MAPK activity was expressed as the phosphorylated-to-total p38 MAPK ratio. Maximum activation occurred at 6 hours after hypoxia. Bars are means ± SEM. #P < 0.05. *P < 0.05 compared with the control (0 hours).
Figure 6
 
Western blot of p38 MAPK phosphorylation after hypoxia in HG-cultured cells. p38 MAPK activity was expressed as the phosphorylated-to-total p38 MAPK ratio. Maximum activation occurred at 6 hours after hypoxia. Bars are means ± SEM. #P < 0.05. *P < 0.05 compared with the control (0 hours).
Figure 7
 
Effect of decorin in preventing hypoxia-induced p38 MAPK phosphorylation. p38 MAPK activity was expressed as the phosphorylated-to-total p38 MAPK ratio. Treatment with 100 nM decorin before hypoxia exposure significantly reduced hypoxia-induced p38 MAPK phosphorylation at 6 hours after hypoxia. Bars are means ± SEM. #P < 0.05 compared with NG. *P < 0.05 compared with HG plus hypoxia.
Figure 7
 
Effect of decorin in preventing hypoxia-induced p38 MAPK phosphorylation. p38 MAPK activity was expressed as the phosphorylated-to-total p38 MAPK ratio. Treatment with 100 nM decorin before hypoxia exposure significantly reduced hypoxia-induced p38 MAPK phosphorylation at 6 hours after hypoxia. Bars are means ± SEM. #P < 0.05 compared with NG. *P < 0.05 compared with HG plus hypoxia.
Figure 8
 
Western blot analysis shows the effectiveness of siRNA in reducing protein expression of p38 MAPK. Transfection with p38 MAPK siRNA resulted in a significant decrease in p38 protein expression (*P < 0.05 versus control). Transfection with control siRNA did not affect p38 expression. Bars are means ± SEM. β-Actin was used as an internal control.
Figure 8
 
Western blot analysis shows the effectiveness of siRNA in reducing protein expression of p38 MAPK. Transfection with p38 MAPK siRNA resulted in a significant decrease in p38 protein expression (*P < 0.05 versus control). Transfection with control siRNA did not affect p38 expression. Bars are means ± SEM. β-Actin was used as an internal control.
Figure 9
 
Permeability and TER of ARPE-19 monolayers after p38 MAPK siRNA transfection. (A) High-glucose–plus hypoxia-induced increase of FITC-dextran permeability was prevented by p38 siRNA. (B) High-glucose–plus hypoxia-induced decrease of TER was prevented by p38 siRNA. Bars are means ± SEM. *P < 0.05 compared with the other conditions.
Figure 9
 
Permeability and TER of ARPE-19 monolayers after p38 MAPK siRNA transfection. (A) High-glucose–plus hypoxia-induced increase of FITC-dextran permeability was prevented by p38 siRNA. (B) High-glucose–plus hypoxia-induced decrease of TER was prevented by p38 siRNA. Bars are means ± SEM. *P < 0.05 compared with the other conditions.
Figure 10
 
Protein expression of occludin and ZO-1 in ARPE-19 cell monolayers after p38 MAPK siRNA transfection. High-glucose–plus hypoxia-induced reduction of occludin and ZO-1 protein expression was prevented by p38 siRNA. Bars are means ± SEM. β-Actin was used as an internal control. *P < 0.05 compared with the other conditions.
Figure 10
 
Protein expression of occludin and ZO-1 in ARPE-19 cell monolayers after p38 MAPK siRNA transfection. High-glucose–plus hypoxia-induced reduction of occludin and ZO-1 protein expression was prevented by p38 siRNA. Bars are means ± SEM. β-Actin was used as an internal control. *P < 0.05 compared with the other conditions.
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