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Retinal Cell Biology  |   June 2017
Overexpression of Angiotensin-Converting Enzyme 2 Ameliorates Amyloid β-Induced Inflammatory Response in Human Primary Retinal Pigment Epithelium
Author Affiliations & Notes
  • Xinyu Fu
    The First Affiliated Hospital of Chongqing Medical University, Chongqing Key Laboratory of Ophthalmology, Chongqing Eye Institute, Chongqing, China
  • Ru Lin
    The First Affiliated Hospital of Chongqing Medical University, Chongqing Key Laboratory of Ophthalmology, Chongqing Eye Institute, Chongqing, China
  • Yiguo Qiu
    The First Affiliated Hospital of Chongqing Medical University, Chongqing Key Laboratory of Ophthalmology, Chongqing Eye Institute, Chongqing, China
  • Peng Yu
    The First Affiliated Hospital of Chongqing Medical University, Chongqing Key Laboratory of Ophthalmology, Chongqing Eye Institute, Chongqing, China
  • Bo Lei
    The First Affiliated Hospital of Chongqing Medical University, Chongqing Key Laboratory of Ophthalmology, Chongqing Eye Institute, Chongqing, China
    People's Hospital of Zhengzhou University and Henan Provincial People's Hospital, Henan Eye Institute, Henan Eye Hospital, Zhengzhou, China
  • Correspondence: Bo Lei, People's Hospital of Zhengzhou University and Henan Provincial People's Hospital, Henan Eye Institute, Henan Eye Hospital, Zhengzhou, China; bolei99@126.com
Investigative Ophthalmology & Visual Science June 2017, Vol.58, 3018-3028. doi:10.1167/iovs.17-21546
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      Xinyu Fu, Ru Lin, Yiguo Qiu, Peng Yu, Bo Lei; Overexpression of Angiotensin-Converting Enzyme 2 Ameliorates Amyloid β-Induced Inflammatory Response in Human Primary Retinal Pigment Epithelium. Invest. Ophthalmol. Vis. Sci. 2017;58(7):3018-3028. doi: 10.1167/iovs.17-21546.

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

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Abstract

Purpose: Amyloid-β (Aβ) is a major constituent of drusen, which is a hallmark of early AMD. The purpose of this study was to investigate whether enhancement of ACE2, an important component of the protective angiotensin-converting enzyme 2 (ACE2)/Ang-(1-7)/Mas axis of the renin angiotensin system (RAS), ameliorates Aβ-induced inflammatory response in human RPE cells.

Methods: Annexin-V FITC/propidium iodide assay for detecting apoptosis rate was used to determine the optimum concentration and incubation time of Aβ1-42. ACE2 plasmid was transfected into primary cultured human RPE (hRPE) and ARPE-19 cells for 6 hours followed by stimulation with Aβ1-42 at the concentration of 1 μM for 48 hours. Gene expression was detected by real-time PCR and protein levels were determined by Western blotting or ELISA. A779, an antagonist of Ang-(1-7), was used to further confirm the involvement of ACE2/Ang-(1-7)/Mas axis.

Results: Flow cytometry showed that the optimal concentration of Aβ1-42 was 1 μM and the optimal incubation time was 48 hours. Aβ1-42 upregulated the expression of IL-1β and monocyte chemoattractant protein-1. ACE2 plasmid significantly upregulated the expression of ACE2 and Ang-(1-7) in hRPE and ARPE-19 cells. Activation of ACE2 reduced the overproduction of inflammatory cytokines at both mRNA and protein levels in hRPE and ARPE-19 cells stimulated with Aβ1-42. Furthermore, an antagonist of Ang-(1-7), A779 reversed the anti-inflammatory effect of ACE2.

Conclusions: Overexpression of ACE2 ameliorates Aβ-induced inflammatory response by activating the ACE2/Ang-(1-7)/Mas axis in human RPE cells. Our data suggest that ACE2/Ang-(1-7)/Mas axis may be a promising target for developing novel therapies for inflammation response in AMD.

Age-related macular degeneration (AMD) is a progressive retinal degenerative disease that is the leading cause of irreversible vision loss in the elderly and is becoming increasingly prevalent without effective cure worldwide.13 The early stage of AMD is characterized by drusen deposition and RPE dysfunction.4 Drusen, a hallmark of AMD, is abnormal extracellular deposits that build up between the inner layer of Bruch's membrane (BM)5,6 and the basal lamina of the RPE, which plays an essential role in transportation of nutrients and phagocytosis of waste products.5,7 It has been reported that drusen, by provoking an inflammatory event, is a risk factor of early AMD.8 Nevertheless, the detailed mechanisms of drusen in the development of AMD remain poorly understood. 
Amyloid-β (Aβ) is a major component of drusen and is a pathogenic trigger peptide that induces inflammation and neurotoxicity in the retina.9 There is accumulating evidence that inflammatory events in RPE cells are associated with the pathogenesis of early AMD.10,11 Aβ1-40 and Aβ1-42 are the two most common isoforms of Aβ, which are recognized to be the most relevant forms to neurodegeneration in amyloidosis.12 However, Aβ1-42 is considered the most neurotoxic form, which is more prone to oligomerization.13 Increasing Aβ1-42 secretion was found in senescent ARPE-19 cells.14 Moreover, accumulation of Aβ deposition with age may contribute to the development of AMD.9,15 Thus, Aβ1-42 was chosen as a trigger to stimulate human RPE cells to explore the potential mechanisms of inflammatory pathogenesis of early AMD. 
As a trigger, Aβ stimulates RPE cells and results in accelerating the secretion of interleukin (IL)-1β, which is an important inflammatory cytokine that activates macrophages and induces cytokines such as IFN-γ.5 In addition, inflammation of RPE aggravates macrophage infiltration and promotes choroidal neovascularization.16 On the other hand, monocyte chemoattractant protein-1 (MCP-1) is classically considered as the main mediator for neutrophil and monocyte infiltration in ocular diseases.17 MCP-1 also promotes inflammatory disorder and the formation of choroidal neovascularization, which causes progressive blindness.18 Furthermore, Aβ accumulation itself leads to increased release of inflammatory cytokines, including IL-1β and MCP-1.19,20 
The renin angiotensin system (RAS) is a hormone system that consists of a suite of enzymatic reactions. RAS regulates homeostasis of the cardiovascular and renal systems, and also plays a crucial role in inflammatory responses and autoimmune dysfunction.21,22 Two axes, a classic harmful and a newly discovered protective axis, have been identified. Activation of the angiotensin-converting enzyme (ACE)/angiotensin II (Ang II)/Ang II type 1 (AT1) receptor axis, the classic axis of RAS, mediates deleterious effects, including vasoconstriction, fibrosis, migration, fluid retention, and inflammation.23,24 Recent studies have demonstrated the existence of a protective axis. ACE2, a homologue of ACE, cleaves the conversion of Ang II to angiotensin-(1-7) (Ang-[1-7]).25,26 Ang-(1-7) is an endogenous ligand for the G protein–coupled receptor Mas receptor, and is considered a biologically active constituent of the RAS, which has various beneficial effects, such as vasodilation, antithrombosis, antiarrhythmogenic, and anti-inflammation actions.2730 
Most components of RAS, including ACE2/Ang-(1-7)/Mas axis, have been confirmed in various tissues and organs, including the eye.21,31,32 Our previous study showed that activation of ACE2 alleviated the lipopolysaccharide (LPS)-induced inflammatory response in both human RPE cells33 and in endotoxin-induced uveitis (EIU) mouse/rat models.34,35 Moreover, the most recent study from our laboratory demonstrated that adeno-associated virus (AAV)-mediated ACE2 gene delivery alleviated the ocular inflammation in experimental autoimmune uveitis (EAU) mouse model by activating the local ACE2/Ang-(1-7)/Mas axis.36 However, it is unknown whether overexpression of ACE2 could ameliorate Aβ-induced inflammatory responses, which is associated with the pathogenesis of a devastating common eye condition AMD.10,11,37 Here we investigated the protective effect of ACE2 on the inflammatory responses mediated by Aβ1-42 in both human RPE (hRPE) cells and ARPE-19 cells. 
Materials and Methods
Preparation of Aβ1-42 Peptide
Synthetic Aβ1-42 or Aβ42–1 (Chinapeptides, Shanghai, China) oligomeric peptide was prepared as previously described.7,38 Briefly, 1 mg lyophilized Aβ1-42 or Aβ42–1 (reverse peptide) was dissolved in 400 μL 1,1,1,3,3,3-Hexafluoro-2-propanol (HFIP) and incubated overnight at 4°C. Of the resulting seedless solution, 100 μL was subjected to a gentle stream of nitrogen for 5 to 10 minutes to evaporate the HFIP and incubated the peptide in 100% dimethyl sulfoxide at 2 mM for 15 minutes at room temperature with periodic vortexing at moderate speed. Next, the samples were added to 250 μL sterile PBS (without Ca2+) in a siliconized Eppendorf tube. The final concentration was 1 μg/μL. The samples were stirred at 500 rpm using a Teflon-coated microstir bar for 24 to 72 hours at 37°C. Aliquots of this solution were kept at −80°C until use. 
Cell Culture
ARPE-19 cell lines do not act completely the same as the native hRPE cells. Thus, we not only used ARPE-19 cells, but also the low-passage primary hRPE cells to validate the effects. Primary hRPEs were collected from eye cups obtained from the Chongqing Eye Bank. hRPE cells were isolated and cultured using the previous protocol.39 After the cornea, lens, and vitreous were removed, the neuroretina was gently detached from the choroid/RPE layer, and placed in Hanks' balanced salt solution (HBSS; GE Healthcare Life Sciences, Waltham, MA, USA) and digested with 0.05% trypsin and 0.02% EDTA for 30 minutes at 37°C. After centrifugation at 390g for 5 minutes, the cells were suspended in complete Dulbecco's modified Eagle's medium F-12 nutrient mixture (DMEM/F-12; Invitrogen, Carlsbad, CA, USA) containing 10% fetal bovine serum, 100 U/mL penicillin, and 100 μg/mL streptomycin. The cells were incubated in a humidified incubator at 37°C in 5% CO2 and passaged every 5 to 7 days. The homogeneity of cultured RPE cells was confirmed by positive immunostaining with RPE65, a specific marker of RPE cells. The cells used were from passages 2 to 5. 
The hRPE cell line (ARPE-19) was purchased from the American Type Culture Collection (Manassas, VA, USA). Cells were cultured in DMEM/F12 medium containing 10% FBS, 100 U/mL penicillin, and 100 μg/mL streptomycin. The cells were cultured in humidified 5% CO2 atmosphere at 37°C and passaged every 5 to 7 days. At confluence, the cells were detached with 0.25% trypsin-EDTA (Invitrogen) solution, collected by centrifugation, and diluted to 1:3 or 1:4. The ARPE-19 cells used in the experiments were between passages 15 and 23. 
Immunofluorescence Staining
The isolated hRPE and ARPE-19 cells were seeded onto glass coverslips and incubated at 37°C and 5% CO2 until confluent, fixed with 4% paraformaldehyde for 30 minutes at room temperature, followed by three times wash with PBS, permeabilized with 0.1% Triton X-100 for 4 minutes, then washed with PBS three times. The cells were blocked in PBS containing 10% goat serum for 1 hour and incubated with anti-RPE65 antibody (Abcam, Cambridge, MA, USA) with the concentration of 1:100 overnight at 4°C. Cells were washed three times in PBS then incubated with goat anti-mouse IgG secondary antibody (DyLight 594; Abbkine, Wuhan, China) for 1 hour at 37°C, followed by 5 minutes of nuclear staining with 4′,6-diamidino-2-phenylindole (DAPI; Life Technologies, Grand Island, NY, USA). All fluorescent images were taken with a fluorescence microscope (Model DM6000; Leica, Wetzlar, Germany). 
Annexin-V FITC/Propidium Iodide (PI) Apoptosis Assay
This assay was carried out following the manufacturer's instructions from the Annexin-V FITC/PI apoptosis detection kit (Vazyme Biotech Co., Ltd., Nanjing, China). Briefly, ARPE-19 cells (1 × 105 cells/well) were seeded into 24-well plates and incubated at 37°C and 5% CO2 until confluent. The medium was then discarded, and replaced with serum-free medium to starve the cells for 24 hours. The oligomeric form of Aβ1-42 (0.1, 1.0, and 10.0 μM) was added to the wells for 24, 48, and 72 hours. The cells were harvested by digestion with trypsin, followed by washing three times with ice-cold PBS. The cell pellets were resuspended with 100 μL 1X binding buffer and incubated with 5 μL Annexin-V FITC and 5 μL PI at room temperature for 10 minutes in the dark. Afterward, flow cytometry (FCM; FACSAira, Becton Dickinson, Franklin Lakes, NJ, USA) was used to measure the cells with 400 μL 1X binding buffer per tube. 
Plasmid Transfection and Experiment Protocols
The p-EX3-ACE2 (NM_021804.2) plasmid and empty p-EX3 plasmid were constructed by GenePharma (Shanghai, China). The primary human RPE and ARPE-19 cells were seeded in 12-well plates (1 × 105 cells/well) and cultured in 1 mL DMEM culture medium containing 10% FBS without antibiotics until the cells were 70% to 80% confluent. Before transfection, cells were serum-free starved for 12 hours. Lipofectamine 2000 Transfection Reagent (Invitrogen) was used to perform the transfection. The plasmid complex was premixed according to the manufacturer's instructions and added to the 12-well plates. A 1.6-μg amount of ACE2 or empty vector was added per well. Six hours after transfection, the medium was changed to fresh serum-free medium without antibiotics. The concentration of A779 was chosen as previously described.33 Both hRPE and ARPE-19 cells, whether normal or transfected, were pretreated (or not) with 10 μM Ang-(1-7) antagonist A779 (Santa Cruz Biotechnology, Santa Cruz, CA, USA) for 2 hours and then stimulated with Aβ1-42 (1 μM per well) for 48 hours. 
Real-Time PCR Analysis
Total RNA was extracted using Trizol Reagent (Invitrogen) according to the manufacturer's instructions. RNA concentrations were quantified with a Nano instrument (NanoDrop Technologies, Wilmington, DE, USA). cDNA was synthesized using PrimeScript RT reagent kit (Takara Biotechnology, Dalian, China). Real-time PCR (SYBR Green) was performed according to manufacturer's instructions with a sequence detection system (ABI Prism 7500; Applied Biosystems, Foster City, CA, USA). The PCR amplification was conducted in a volume of 20 μL using all-in-one quantitative PCR mix (Takara Biotechnology). The cycling protocol consisted of one cycle of 10 minutes at 95°C followed by 40 cycles of 95°C for 15 seconds and 60°C for 1 minute. To determine the mRNA expression, all samples were tested in duplicate and the average cycle threshold values were used for relative quantification. The mRNA expression was normalized to the endogenous reference gene β-actin. The sequences of the primers are shown in the Table
Table
 
Sequences of the Primers for Real-Time PCR
Table
 
Sequences of the Primers for Real-Time PCR
Enzyme-Linked Immunosorbent Assay
The concentrations of MCP-1, IL-1β (R&D Systems, Minneapolis, MN, USA), and Ang-(1-7) (Cloud-Clone, Houston, TX, USA) were determined by ELISA kits for humans according to the manufacturer's instructions. The absorbance at 450 nm wavelength was measured using a multifunction microplate reader (Molecular Devices, Sunnyvale, CA, USA). 
Western Blot Analysis
RPE cells were washed three times with PBS and scraped off with RIPA lysis buffer (Beyotime, Shanghai, China) containing 50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 1% Triton X-100, 1% sodium deoxycholate, 0.1% SDS, 2 mM EDTA, and 100 M phenylmethylsulfonyl fluoride. Protein concentration was detected by bicinchoninic acid assay kit (Beyotime). All samples were diluted in SDS loading buffer (Beyotime) and boiled for 5 minutes. Equal amounts of total protein (60 μg) were separated by 8% SDS-PAGE gel and transferred to polyvinylidene difluoride membranes (Millipore, Bedford, MA, USA), blocked by 5% nonfat skim milk Tris-buffered saline supplemented with Tween 20. Membranes were incubated with primary antibodies ACE2 (1:500; Abcam, Cambridge, MA, USA) and β-actin (1:100; Abcam) overnight at 4°C, and then washed and incubated with horseradish peroxidase–conjugated secondary antibody (1:3000; Abcam) at 37°C for 1 hour. Bands were visualized by ECL kit (Advansta, Menlo Park, CA, USA), and band densitometry was analyzed using ImageJ software (http://imagej.nih.gov/ij/; provided in the public domain by the National Institutes of Health, Bethesda, MD, USA). β-actin was used as loading control. 
Statistical Analysis
All data were expressed as mean ± SEM. Statistical analysis was performed with the GraphPad Prism 5 software (GraphPad Software, Inc., San Diego, CA, USA). A 1-way ANOVA followed by a Bonferroni correction were applied for multiple comparisons; P < 0.05 was considered to be significantly different. 
Results
Identification of RPE Cell
The RPE-specific antibody, anti-human RPE65, was used to confirm the hRPE and the ARPE-19 cells. In hRPE and ARPE-19 cells, RPE65 was localized in the cytoplasm around the DAPI-positive nucleus (Fig. 1), which indicated that both human primary RPE and ARPE-19 cell lines were RPE-derived cells, and were counted in five nonoverlapping fields for two cell lines under a fluorescence microscope. The percentage of the RPE65-positive cell in DAPI-positive cells was calculated. The purity of hRPE and ARPE-19 cells were 95.25% ± 3.13% and 96.28% ± 1.91%, respectively. 
Figure 1
 
Both hRPE and ARPE-19 cells displayed similar morphology and RPE65 staining. RPE65, the RPE-specific protein, was used to confirm whether the human primary cells separated from the donor eyes, and the RPE cell line were both derived from RPE. Merged images showed that the RPE65 was localized in the cytoplasm of the cells with DAPI-positive nucleus in both hRPE and ARPE-19 cells. The RPE-positive cells were counted in five nonoverlapping fields under a fluorescence microscope. The ratio of RPE65-positive cells was calculated as the percentage of RPE65-positive cells in the DAPI-positive cells. The purity of hRPE and ARPE-19 cells were 95.25% ± 3.13% and 96.28% ± 1.91%, respectively. Scale bar: 25 μm; magnification ×400.
Figure 1
 
Both hRPE and ARPE-19 cells displayed similar morphology and RPE65 staining. RPE65, the RPE-specific protein, was used to confirm whether the human primary cells separated from the donor eyes, and the RPE cell line were both derived from RPE. Merged images showed that the RPE65 was localized in the cytoplasm of the cells with DAPI-positive nucleus in both hRPE and ARPE-19 cells. The RPE-positive cells were counted in five nonoverlapping fields under a fluorescence microscope. The ratio of RPE65-positive cells was calculated as the percentage of RPE65-positive cells in the DAPI-positive cells. The purity of hRPE and ARPE-19 cells were 95.25% ± 3.13% and 96.28% ± 1.91%, respectively. Scale bar: 25 μm; magnification ×400.
RPE Apoptosis After Oligomeric Aβ1-42 Stimulation
The effect of Aβ1-42 on the apoptosis of ARPE-19 cells was tested with different concentrations of Aβ1-42. Confluent ARPE-19 cells were incubated with Aβ1-42 at concentrations of 0.1, 1.0, and 10 μM for 24, 48, and 72 hours (Fig. 2). Treatment with 0.1 and 1 μM Aβ1-42 for 24, 48, and 72 hours did not affect the apoptosis of ARPE-19 cells. However, treatment with 10 μM Aβ1-42 for 24, 48, and 72 hours significantly increased the apoptosis rate of ARPE-19 cells compared with the cells cultured without Aβ1-42. Thus, to limit the times of moving the cells from the incubator and to provide the best balance between inflammatory response and cell toxicity, the concentration of 1 μM and incubation of 48 hours were used for subsequent experiments (***P < 0.001, **P < 0.01, n = 4). 
Figure 2
 
Apoptosis of ARPE-19 cells incubated with Aβ1-42 as measured with Annexin-V FITC/PI assay. Confluent ARPE-19 cells were subjected to stimulation with various concentrations (0.1, 1.0, and 10 μM) of Aβ1-42 for 24, 48, and 72 hours. The apoptosis rate was influenced at 10 μM Aβ1-42 for 24, 48, and 72 hours compared with cells cultured without Aβ1-42, respectively. The quadrant Q2 represents late apoptotic cells (Annexin-FITC and PI positive), which was used to analyze cellular apoptosis rate after Aβ1-42 stimulation. The quadrant Q3 represents viable cells (Annexin-FITC and PI negative). The quadrant Q4 represents early apoptotic cells (Annexin-FITC positive but PI negative). All data are expressed as mean ± SEM (***P < 0.001, **P < 0.01, n = 4).
Figure 2
 
Apoptosis of ARPE-19 cells incubated with Aβ1-42 as measured with Annexin-V FITC/PI assay. Confluent ARPE-19 cells were subjected to stimulation with various concentrations (0.1, 1.0, and 10 μM) of Aβ1-42 for 24, 48, and 72 hours. The apoptosis rate was influenced at 10 μM Aβ1-42 for 24, 48, and 72 hours compared with cells cultured without Aβ1-42, respectively. The quadrant Q2 represents late apoptotic cells (Annexin-FITC and PI positive), which was used to analyze cellular apoptosis rate after Aβ1-42 stimulation. The quadrant Q3 represents viable cells (Annexin-FITC and PI negative). The quadrant Q4 represents early apoptotic cells (Annexin-FITC positive but PI negative). All data are expressed as mean ± SEM (***P < 0.001, **P < 0.01, n = 4).
Aβ1-42 But Not Aβ42–1 Induced the Overproduction of Inflammatory Cytokines
To confirm the proinflammatory effect of Aβ1-42 on human RPE cells, ARPE-19 cells were stimulated with 1 μM Aβ1-42 or 1 μM Aβ42-1 for 48 hours. The mRNA expression of the inflammatory cytokines was determined by real-time PCR. The levels of IL-1β (Fig. 3A) and MCP-1 (Fig. 3B) were significantly increased in Aβ1-42 stimulation, whereas the Aβ42-1 had no effect on expressions of inflammatory cytokines compared with the untreated group (**P < 0.01, *P < 0.05, n = 4). 
Figure 3
 
Aβ1-42 enhanced the expressions of inflammatory cytokines in RPE cells. ARPE-19 cells were incubated with Aβ1-42 or Aβ42-1 for 48 hours. The expressions of IL-1β (A) and MCP-1 (B) were increased in the Aβ1-42 group when compared with the untreated group and the Aβ42-1 group. All data are expressed as mean ± SEM (**P < 0.01, *P < 0.05, n = 4).
Figure 3
 
Aβ1-42 enhanced the expressions of inflammatory cytokines in RPE cells. ARPE-19 cells were incubated with Aβ1-42 or Aβ42-1 for 48 hours. The expressions of IL-1β (A) and MCP-1 (B) were increased in the Aβ1-42 group when compared with the untreated group and the Aβ42-1 group. All data are expressed as mean ± SEM (**P < 0.01, *P < 0.05, n = 4).
Overexpression of ACE2 at Both mRNA and Protein Levels in hRPE and ARPE-19 Cells
To evaluate the expression of ACE2 after transfection with either the target plasmid (ACE2) or the control plasmid in hRPE and ARPE-19 cells, real-time PCR and Western blotting were applied to detect the ACE2 mRNA and protein expression, respectively. The results showed that the ACE2 plasmid transfection significantly upregulated the expression of ACE2 compared with the cells without transfection or transfected with control vector at the mRNA level in hRPE (Fig. 4A) and ARPE-19 cells (Fig. 4B). Moreover, consistent with the result of real-time PCR, protein expression of ACE2 was also increased in ACE2 plasmid transfected group, as shown by Western blotting in hRPE (Fig. 4A) and ARPE-19 cells (Fig. 4B) (***P < 0.001, n = 4). 
Figure 4
 
Plasmid transfection enhanced the expression of ACE2. Expression of ACE2 was detected in the ACE2 plasmid or the control plasmid transfected hRPE (A) and ARPE-19 (B) cells followed by Aβ1-42 stimulation. The mRNA and protein expressions of ACE2 that were analyzed by real-time PCR and Western blotting showed that the ACE2 was remarkably increased in Aβ1-42+ACE2 group when compared with the untreated group, Aβ1-42 group, and Aβ1-42+control group in both hRPE and ARPE-19 cells. All data are expressed as mean ± SEM (***P < 0.001, n = 4).
Figure 4
 
Plasmid transfection enhanced the expression of ACE2. Expression of ACE2 was detected in the ACE2 plasmid or the control plasmid transfected hRPE (A) and ARPE-19 (B) cells followed by Aβ1-42 stimulation. The mRNA and protein expressions of ACE2 that were analyzed by real-time PCR and Western blotting showed that the ACE2 was remarkably increased in Aβ1-42+ACE2 group when compared with the untreated group, Aβ1-42 group, and Aβ1-42+control group in both hRPE and ARPE-19 cells. All data are expressed as mean ± SEM (***P < 0.001, n = 4).
Overexpression of ACE2 Reduced the Activation of Aβ1-42–Induced Inflammatory Cytokines in hRPE and ARPE-19 Cells
To investigate the effect of ACE2 on the overproduction of inflammatory cytokines induced by Aβ1-42, both hRPE and ARPE-19 cells were transfected with the target plasmid for 6 hours before being stimulated with Aβ1-42 for 48 hours. The gene expression was detected by real-time PCR and the protein levels were determined by ELISA. The inflammatory mediators IL-1β and MCP-1were significantly increased in RPE cells incubated with Aβ1-42. However, overexpression of ACE2 remarkably inhibited the production of IL-1β and MCP-1 induced by Aβ1-42 at both mRNA and protein levels in hRPE (Figs. 5A, 5B) and ARPE-19 (Figs. 5C, 5D) cells (***P < 0.001, **P < 0.01, *P < 0.05, n = 4–5). 
Figure 5
 
Overexpression of ACE2 downregulated the production of inflammatory cytokines induced by Aβ1-42. Overexpression of ACE2 downregulated the abnormal production of IL-1β and MCP-1 in hRPE (A, B) and ARPE-19 (C, D) cells. Total RNA was extracted from the untreated group, Aβ1-42 group, Aβ1-42+ACE2 group, and Aβ1-42+control group to determine the expressions of IL-1β and MCP-1 by real-time PCR in hRPE and ARPE-19 cells. Cell supernatants were collected for ELISA assay to detect the IL-1β and MCP-1 at protein level in hRPE as well as ARPE-19 cells. The expressions of IL-1β and MCP-1 were reduced in the Aβ1-42+ACE2 group when compared with the Aβ1-42 group at both mRNA and protein levels. All data are expressed as mean ± SEM (***P < 0.001, **P < 0.01, *P < 0.05, n = 4–5).
Figure 5
 
Overexpression of ACE2 downregulated the production of inflammatory cytokines induced by Aβ1-42. Overexpression of ACE2 downregulated the abnormal production of IL-1β and MCP-1 in hRPE (A, B) and ARPE-19 (C, D) cells. Total RNA was extracted from the untreated group, Aβ1-42 group, Aβ1-42+ACE2 group, and Aβ1-42+control group to determine the expressions of IL-1β and MCP-1 by real-time PCR in hRPE and ARPE-19 cells. Cell supernatants were collected for ELISA assay to detect the IL-1β and MCP-1 at protein level in hRPE as well as ARPE-19 cells. The expressions of IL-1β and MCP-1 were reduced in the Aβ1-42+ACE2 group when compared with the Aβ1-42 group at both mRNA and protein levels. All data are expressed as mean ± SEM (***P < 0.001, **P < 0.01, *P < 0.05, n = 4–5).
Ang-(1-7) Antagonist A779 Reversed the Protective Effect of ACE2
ACE2, a homologue of ACE, is a key player in the protective axis of RAS, ACE2/Ang-(1-7)/Mas. To determine whether ACE2 played the anti-inflammatory effect via activating the ACE2/Ang-(1-7)/Mas axis, A779 was incubated with both hRPE and ARPE-19 cells for 2 hours. Then the cells were transfected with ACE2 plasmid, followed by stimulation with 1 μM Aβ1-42. A779 abrogated the inhibition effects of ACE2 on IL-1β and MCP-1 at both mRNA and protein levels in hRPE (Figs. 6A, 6B) and ARPE-19 (Figs. 6C, 6D) cells (***P < 0.001, **P < 0.01, *P < 0.05, n = 4–5). 
Figure 6
 
A779 offset the effect of ACE2 on the expressions of inflammatory cytokines. IL-1β and MCP-1 were analyzed by real-time PCR and ELISA in hRPE (A, B) and ARPE-19 (C, D). Overexpression of ACE2 reduced the expressions of IL-1β and MCP-1, whereas the A779 offset the effect of ACE2. All data are expressed as mean ± SEM (**P < 0.01, *P < 0.05, n = 4–5).
Figure 6
 
A779 offset the effect of ACE2 on the expressions of inflammatory cytokines. IL-1β and MCP-1 were analyzed by real-time PCR and ELISA in hRPE (A, B) and ARPE-19 (C, D). Overexpression of ACE2 reduced the expressions of IL-1β and MCP-1, whereas the A779 offset the effect of ACE2. All data are expressed as mean ± SEM (**P < 0.01, *P < 0.05, n = 4–5).
ACE2 Activated Ang-(1-7), Whereas A779 Reversed It
To further confirm the protective role of ACE2/Ang-(1-7)/Mas axis, an antagonist of Ang-(1-7) A779 was applied. The protein expression of Ang-(1-7) was detected by ELISA assay in both hRPE (Fig. 7A) and ARPE-19 (Fig. 7B) cells. Overexpression of ACE2 significantly increased Ang-(1-7) when compared with the untreated group and Aβ1-42 group; however, A779 abrogated the protective effect (***P < 0.001, **P < 0.01, *P < 0.05, n = 4). 
Figure 7
 
A779 reversed the effect of ACE2 on Ang-(1-7) in hRPE and ARPE-19 cells. The protein expression of Ang-(1-7) was determined by ELISA assay. ACE2 activated Ang-(1-7) when compared with the untreated group and Aβ1-42–stimulated group, whereas A779 reversed the effect of ACE2. All data are expressed as mean ± SEM (***P < 0.001, **P < 0.01, *P < 0.05, n = 4).
Figure 7
 
A779 reversed the effect of ACE2 on Ang-(1-7) in hRPE and ARPE-19 cells. The protein expression of Ang-(1-7) was determined by ELISA assay. ACE2 activated Ang-(1-7) when compared with the untreated group and Aβ1-42–stimulated group, whereas A779 reversed the effect of ACE2. All data are expressed as mean ± SEM (***P < 0.001, **P < 0.01, *P < 0.05, n = 4).
Discussion
In the present study, we investigated the effect of overexpression of ACE2 on Aβ1-42–induced inflammatory response in human RPE and ARPE-19 cells. We found that activation of ACE2 ameliorated Aβ1-42–induced overproduction of IL-1β and MCP-1 at both mRNA and protein levels. Furthermore, the Ang-(1-7) antagonist A779 reversed the protective effect, indicating the protective effect of ACE2 was mediated by upregulation of ACE2/Ang-(1-7)/Mas axis. 
Recently, emerging evidence supported that the pathogenic role of inflammation in response to drusen is associated with the development of AMD, which progressively causes blindness in the elderly. Thus, effective interventions of early AMD and understanding of their underlying mechanisms are highly desirable. Aβ deposition in drusen induces the expression of inflammatory cytokines and other factors that cause cellular dysfunction of RPE, and consequently results in acceleration of AMD.40 It has been reported that a single intraocular injection of oligomeric Aβ leads to overproduction of inflammatory cytokines and activation of nuclear factor (NF)-κB signaling in the RPE in rats.38,41 This model may become useful for exploring the potential mechanisms involved in the early phases of AMD. Here, in agreement with Cao et al.,7 we further demonstrated Aβ is an inducer of inflammatory responses in human RPE cells in vitro. 
RAS plays a crucial role in regulating the inflammatory responses systematically4244 and locally, including the eye.45 Ang II, the major component of RAS, mediates releasing of proinflammatory cytokines, chemokines, cell adhesion molecules, growth factors, reactive oxygen species (ROS), and molecular signaling pathways via the AT1R, and causes cell dysfunction.46 Overproduction of Ang II not only causes dysfunction of RPE,47 but plays a key role in enhancing the invasion of choroidal endothelial cells.48 Furthermore, increasing evidence has demonstrated that Ang II, the main component of RAS, increases expressions of inflammatory cytokines, and even activates nicotinamide adenine dinucleotide phosphate oxidase and enhances production of ROS, whose effects are associated with the pathogenesis of ocular diseases, such as AMD, glaucoma, and diabetic retinopathy (DR).4951 
The protective axis of RAS, namely ACE2/Ang-(1-7)/Mas axis, negatively regulating the classical components of RAS, inhibits leukocyte recruitment, cytokine release, and fibrosis formations.29,5254 ACE2 acts as a counter regulator of ACE in cleaving Ang II into Ang-(1-7), and exerts beneficial effects in several animal models of ocular diseases, including DR,55 glaucoma,56 EIU,34,35 and EAU.21,36 On the other hand, ACE2 deletion exaggerated Ang II-induced expressions of inflammatory cytokines.57 Here, we found that overexpression of ACE2 inhibited Aβ-induced inflammatory responses in human RPE cells. The anti-inflammatory effect of ACE2 appeared to result in decreasing the overproduction of inflammatory cytokines. Moreover, we found that the selective antagonist Ang-(1-7) A779 abolished the anti-inflammation of ACE2, suggesting that the beneficial effects of ACE2 on Aβ-induced inflammatory response in human RPE cells was mediated by regulating the Ang-(1-7)/Mas receptor. However, with the limitation of animal models emulating early AMD, whether activation of Ang-(1-7)/Mas receptor by ACE2 will result in prevention of Aβ-induced acute and chronic inflammation in vivo deserves further investigation. 
Activation of the ACE2/Ang-(1-7)/Mas axis is also relevant to the anti-inflammatory effects of the angiotensin receptor blockers (ARBs), such as telmisartan and olmesartan. ARBs increased the expressions of ACE2 and Ang-(1-7)/Mas; reduced the expression of inflammatory cytokines TNF-α, IFN-γ, IL-1β, and IL-6; and increased the expression of anti-inflammatory cytokine IL-10 in an animal model of autoimmune myocarditis.58,59 Therefore, ACE2 may be another feasible and more specific target in the protective RAS axis to delay the development of the common disease AMD. 
Several other types of cells, including macrophages60,61 and microglia,62,63 are abnormally activated in AMD. Our previous study showed that activating ACE2 significantly reduced the CD45+ macrophages in EIU mouse retina.34 Similarly, AAV-mediated intravitreal delivery of ACE2 decreased the retinal CD11b+ microglia cells in an animal model of DR.55 In addition, our most recent study showed that AAV8-mediated activation of the ACE2/Ang-(1-7)/Mas axis confers a protective effect on EAU mice by inhibiting the local Th1/Th17 cell responses, as well as affecting the shift of M1/M2 macrophages.36 The aforementioned evidence indicates that enhancement of ACE2 may be a promising therapeutic target for ocular diseases by regulating various immune cells, including the RPE. 
Intracellular signaling cascades lead to expression of proinflammatory cytokines in the process of inflammation. The activation of NF-κB and mitogen-activated protein kinase (MAPK) pathways has a crucial role in progression to inflammatory responses after exposure to Aβ.6466 It has been suggested that enhancement of ACE2 significantly suppressed LPS-induced apoptosis of pulmonary microvascular endothelial cells and inflammation by inhibiting the p38 MAPK and jun N-terminal kinase pathways.22 Treatment with Ang-(1-7) alleviated inflammation via regulation of the extracellular signal regulated kinase, one of the MAPK family members, and NF-κB pathways in an allergic asthma mouse model.67 In line with our previous studies, activation of the ACE2/Ang-(1-7)/Mas axis downregulated phosphorylation of MAPKs and NF-κB signaling.33,36 These data indicated that ACE2 may ameliorate the inflammatory response by regulating MAPKs and NF-κB pathways. 
In summary, our study is the first to illustrate the protective effects of ACE2/Ang-(1-7)/Mas in Aβ-induced inflammatory responses in human RPE cells. These results suggest that ACE2 may provide a future therapeutic target for the intervention of AMD. 
Acknowledgments
The authors thank Fengjuan Gu for providing the donor eyes used this experiment and we are grateful to the donors whose generosity made this study possible. 
Supported by National Natural Science Foundation of China Grants 81271033 and 81470621, Chongqing Science and Technology Commission (2014pt-sy10002), and National Key Clinical Specialties Construction Program of China. The authors alone are responsible for the content and writing of the paper. 
Disclosure: X. Fu, None; R. Lin, None; Y. Qiu, None; P. Yu, None; B. Lei, None 
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Figure 1
 
Both hRPE and ARPE-19 cells displayed similar morphology and RPE65 staining. RPE65, the RPE-specific protein, was used to confirm whether the human primary cells separated from the donor eyes, and the RPE cell line were both derived from RPE. Merged images showed that the RPE65 was localized in the cytoplasm of the cells with DAPI-positive nucleus in both hRPE and ARPE-19 cells. The RPE-positive cells were counted in five nonoverlapping fields under a fluorescence microscope. The ratio of RPE65-positive cells was calculated as the percentage of RPE65-positive cells in the DAPI-positive cells. The purity of hRPE and ARPE-19 cells were 95.25% ± 3.13% and 96.28% ± 1.91%, respectively. Scale bar: 25 μm; magnification ×400.
Figure 1
 
Both hRPE and ARPE-19 cells displayed similar morphology and RPE65 staining. RPE65, the RPE-specific protein, was used to confirm whether the human primary cells separated from the donor eyes, and the RPE cell line were both derived from RPE. Merged images showed that the RPE65 was localized in the cytoplasm of the cells with DAPI-positive nucleus in both hRPE and ARPE-19 cells. The RPE-positive cells were counted in five nonoverlapping fields under a fluorescence microscope. The ratio of RPE65-positive cells was calculated as the percentage of RPE65-positive cells in the DAPI-positive cells. The purity of hRPE and ARPE-19 cells were 95.25% ± 3.13% and 96.28% ± 1.91%, respectively. Scale bar: 25 μm; magnification ×400.
Figure 2
 
Apoptosis of ARPE-19 cells incubated with Aβ1-42 as measured with Annexin-V FITC/PI assay. Confluent ARPE-19 cells were subjected to stimulation with various concentrations (0.1, 1.0, and 10 μM) of Aβ1-42 for 24, 48, and 72 hours. The apoptosis rate was influenced at 10 μM Aβ1-42 for 24, 48, and 72 hours compared with cells cultured without Aβ1-42, respectively. The quadrant Q2 represents late apoptotic cells (Annexin-FITC and PI positive), which was used to analyze cellular apoptosis rate after Aβ1-42 stimulation. The quadrant Q3 represents viable cells (Annexin-FITC and PI negative). The quadrant Q4 represents early apoptotic cells (Annexin-FITC positive but PI negative). All data are expressed as mean ± SEM (***P < 0.001, **P < 0.01, n = 4).
Figure 2
 
Apoptosis of ARPE-19 cells incubated with Aβ1-42 as measured with Annexin-V FITC/PI assay. Confluent ARPE-19 cells were subjected to stimulation with various concentrations (0.1, 1.0, and 10 μM) of Aβ1-42 for 24, 48, and 72 hours. The apoptosis rate was influenced at 10 μM Aβ1-42 for 24, 48, and 72 hours compared with cells cultured without Aβ1-42, respectively. The quadrant Q2 represents late apoptotic cells (Annexin-FITC and PI positive), which was used to analyze cellular apoptosis rate after Aβ1-42 stimulation. The quadrant Q3 represents viable cells (Annexin-FITC and PI negative). The quadrant Q4 represents early apoptotic cells (Annexin-FITC positive but PI negative). All data are expressed as mean ± SEM (***P < 0.001, **P < 0.01, n = 4).
Figure 3
 
Aβ1-42 enhanced the expressions of inflammatory cytokines in RPE cells. ARPE-19 cells were incubated with Aβ1-42 or Aβ42-1 for 48 hours. The expressions of IL-1β (A) and MCP-1 (B) were increased in the Aβ1-42 group when compared with the untreated group and the Aβ42-1 group. All data are expressed as mean ± SEM (**P < 0.01, *P < 0.05, n = 4).
Figure 3
 
Aβ1-42 enhanced the expressions of inflammatory cytokines in RPE cells. ARPE-19 cells were incubated with Aβ1-42 or Aβ42-1 for 48 hours. The expressions of IL-1β (A) and MCP-1 (B) were increased in the Aβ1-42 group when compared with the untreated group and the Aβ42-1 group. All data are expressed as mean ± SEM (**P < 0.01, *P < 0.05, n = 4).
Figure 4
 
Plasmid transfection enhanced the expression of ACE2. Expression of ACE2 was detected in the ACE2 plasmid or the control plasmid transfected hRPE (A) and ARPE-19 (B) cells followed by Aβ1-42 stimulation. The mRNA and protein expressions of ACE2 that were analyzed by real-time PCR and Western blotting showed that the ACE2 was remarkably increased in Aβ1-42+ACE2 group when compared with the untreated group, Aβ1-42 group, and Aβ1-42+control group in both hRPE and ARPE-19 cells. All data are expressed as mean ± SEM (***P < 0.001, n = 4).
Figure 4
 
Plasmid transfection enhanced the expression of ACE2. Expression of ACE2 was detected in the ACE2 plasmid or the control plasmid transfected hRPE (A) and ARPE-19 (B) cells followed by Aβ1-42 stimulation. The mRNA and protein expressions of ACE2 that were analyzed by real-time PCR and Western blotting showed that the ACE2 was remarkably increased in Aβ1-42+ACE2 group when compared with the untreated group, Aβ1-42 group, and Aβ1-42+control group in both hRPE and ARPE-19 cells. All data are expressed as mean ± SEM (***P < 0.001, n = 4).
Figure 5
 
Overexpression of ACE2 downregulated the production of inflammatory cytokines induced by Aβ1-42. Overexpression of ACE2 downregulated the abnormal production of IL-1β and MCP-1 in hRPE (A, B) and ARPE-19 (C, D) cells. Total RNA was extracted from the untreated group, Aβ1-42 group, Aβ1-42+ACE2 group, and Aβ1-42+control group to determine the expressions of IL-1β and MCP-1 by real-time PCR in hRPE and ARPE-19 cells. Cell supernatants were collected for ELISA assay to detect the IL-1β and MCP-1 at protein level in hRPE as well as ARPE-19 cells. The expressions of IL-1β and MCP-1 were reduced in the Aβ1-42+ACE2 group when compared with the Aβ1-42 group at both mRNA and protein levels. All data are expressed as mean ± SEM (***P < 0.001, **P < 0.01, *P < 0.05, n = 4–5).
Figure 5
 
Overexpression of ACE2 downregulated the production of inflammatory cytokines induced by Aβ1-42. Overexpression of ACE2 downregulated the abnormal production of IL-1β and MCP-1 in hRPE (A, B) and ARPE-19 (C, D) cells. Total RNA was extracted from the untreated group, Aβ1-42 group, Aβ1-42+ACE2 group, and Aβ1-42+control group to determine the expressions of IL-1β and MCP-1 by real-time PCR in hRPE and ARPE-19 cells. Cell supernatants were collected for ELISA assay to detect the IL-1β and MCP-1 at protein level in hRPE as well as ARPE-19 cells. The expressions of IL-1β and MCP-1 were reduced in the Aβ1-42+ACE2 group when compared with the Aβ1-42 group at both mRNA and protein levels. All data are expressed as mean ± SEM (***P < 0.001, **P < 0.01, *P < 0.05, n = 4–5).
Figure 6
 
A779 offset the effect of ACE2 on the expressions of inflammatory cytokines. IL-1β and MCP-1 were analyzed by real-time PCR and ELISA in hRPE (A, B) and ARPE-19 (C, D). Overexpression of ACE2 reduced the expressions of IL-1β and MCP-1, whereas the A779 offset the effect of ACE2. All data are expressed as mean ± SEM (**P < 0.01, *P < 0.05, n = 4–5).
Figure 6
 
A779 offset the effect of ACE2 on the expressions of inflammatory cytokines. IL-1β and MCP-1 were analyzed by real-time PCR and ELISA in hRPE (A, B) and ARPE-19 (C, D). Overexpression of ACE2 reduced the expressions of IL-1β and MCP-1, whereas the A779 offset the effect of ACE2. All data are expressed as mean ± SEM (**P < 0.01, *P < 0.05, n = 4–5).
Figure 7
 
A779 reversed the effect of ACE2 on Ang-(1-7) in hRPE and ARPE-19 cells. The protein expression of Ang-(1-7) was determined by ELISA assay. ACE2 activated Ang-(1-7) when compared with the untreated group and Aβ1-42–stimulated group, whereas A779 reversed the effect of ACE2. All data are expressed as mean ± SEM (***P < 0.001, **P < 0.01, *P < 0.05, n = 4).
Figure 7
 
A779 reversed the effect of ACE2 on Ang-(1-7) in hRPE and ARPE-19 cells. The protein expression of Ang-(1-7) was determined by ELISA assay. ACE2 activated Ang-(1-7) when compared with the untreated group and Aβ1-42–stimulated group, whereas A779 reversed the effect of ACE2. All data are expressed as mean ± SEM (***P < 0.001, **P < 0.01, *P < 0.05, n = 4).
Table
 
Sequences of the Primers for Real-Time PCR
Table
 
Sequences of the Primers for Real-Time PCR
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