Angiotensin-(1-7) ameliorates high glucose-induced vascular endothelial injury through suppressing chloride channel 3

ABSTRACT Diabetes Mellitus (DM) is a significant risk factor for cardiovascular disease (CVD), which is leading cause of deaths in DM patients. However, there are limited effective medical therapies for diabetic CVD. Vascular endothelial injury caused by DM is a critical risk factor for diabetic CVD. Previous study has indicated that Angiotensin-(1-7) (Ang-(1-7)) may prevent diabetic CVD, whereas it is not clear that Ang-(1-7) whether attenuates diabetic CVD through suppressing vascular endothelial injury. In this study, we found that Ang-(1-7) alleviated high glucose (HG)-induced endothelial injury in bEnd3 cells. Moreover, Ang-(1-7) ameliorated HG-induced endothelial injury through downregulating chloride channel 3 (CIC-3) via Mas receptor. Furthermore, HG-induced CIC-3 enhanced reactive oxygen species (ROS) and cytokine production and reduced the level of nitric oxide (NO), while Ang-(1-7) preserved the impact of HG-induced CIC-3 on productions of ROS, cytokine and NO through inhibiting CIC-3 via Mas receptor. Summarily, the present study revealed that Ang-(1-7) alleviated HG-induced vascular endothelial injury through the inhibition of CIC-3, suggested that Ang-(1-7) may preserve diabetic CVD through suppressing HG-induced vascular endothelial injury.


Introduction
DM is a group of metabolic diseases characterized by hyperglycemia causing by impaired glucose tolerance, which is a significant risk factor for CVD [1][2][3][4]. Diabetic CVD is the leading cause of deaths in DM patients [5,6]. Although outstanding scientific advances have been made in understanding the factors contributed to CVD in patients with DM, there are limited effective medical therapies for CVD. Numerous studies have revealed that vascular endothelial injury caused by DM is a critical risk factor for diabetic CVD [7][8][9][10]. Damage of vascular endothelial cell by HG-induced inflammation, oxidative stress, cell apoptosis is the major cause of vascular endothelial injury [11][12][13]. Suppression of DMinduced vascular endothelial injury may provide a new therapy for diabetic CVD.
As a member of the ClC voltage-gated chloride (Cl − ) channel superfamily, chloride channel 3 (CIC-3) plays important roles in CVD through regulating cell proliferation, inflammation, volume regulation and apoptosis of vascular smooth muscle cells (VSMCs) [23]. For example, hypertension-associated vascular disease induces aberrant VSMC proliferation via upregulating CIC-3 expression [24]. Moreover, activation of CIC-3 channel in VSMC is required for myocardial hypertrophy and heart failure through promoting cytokine-induced reactive oxygen species (ROS) generation [25,26]. These studies suggest that CIC-3 may serve as an inducer of CVD. However, the functions of CIC-3 in diabetic CVD are unknown. Similar to the role in VSMCs, CIC-3 also regulates cell proliferation, apoptosis, inflammation and volume regulation of vascular endothelial cells [27][28][29]. Thus, CIC-3 may contribute to vascular endothelial injury in diabetic CVD.

CCK-8 assay
1 × 10 4 bEnd3 cells were seeded in a well of the 96well plate and then the CCK-8 assay was employed to assess the cell proliferation. After the indicated treatments, 10 µl CCK-8 solution purchased from Beyotime Biotechnology (Shanghai, China) at a 1/ 10 dilution was added to incubate cells for 1.5 h at 37°C. Absorbance at 450 nm was assayed via a microplate reader (Molecular Devices, Sunnyvale, CA, USA). Subsequently, the optical density (OD) of cells were used to calculate the percentage of cell proliferation [31]. This experiment was carried out three times

Immunofluorescence
First, bEnd3 cells were plated in slides covered on a 24well plate and then fixed by 4% paraformaldehyde (PFA) for 1 h at room temperature (RT). After fixation, cells were treated with 0.2% Triton X-100 for 5 min at RT and washed by phosphate buffered solution (PBS) three times. Next, cells were blocked with 10% goat serum for 1 h at RT and the following incubation of anti-CIC-3 antibody (1:200 dilution) (#45,075 Signalway Antibody, College Park, MD, USA) in freshly prepared PBST (0.1 Tween20) with 1% goat serum at 4°C overnight. Cells were washed by PBST three times and incubated with fluorenes-conjugated secondary antibody (1:2000 dilution) in dark for 1 h at RT. After washing with PBST three times, slides were treated with mounting medium contained DAPI and took pictures by the BX50-FLA imaging system (Olympus, Tokyo, Japan). Next, the MFI (Mean fluorescence intensity) was measured by Image J 1.47i software as an index to the amount of CIC-3 expression. This experiment was carried out three times.

Identification of the concentration of chloride
N-(Ethoxycarbonylmethyl)-6-methoxyquinolinium bromide (MQAE) probe (Beyotime Biotechnology) was used to identify the concentration of chloride in bEnd3 cells. Briefly, 10mM MQAE was added into Krebs-HEPES buffer (20 mM HEPES, 128 mM NaCl, 2.5 mM KCl, 2.7 mM CaCl2, 1 mM MgCl2, 16 mM glucose, pH 7.4) to make up the work solution. Next, 1 × 10 8 /ml cells were incubated with the work solution for 1 h at RT in dark. After washing with Krebs-HEPES buffer five times, cells were analyzed by flow cytometry (FACSARIA, BD Biosciences, Franklin Lake, NJ, USA). This experiment was repeated three times.

Analysis of mitochondrial potential
JC-1 dye (Invitrogen) was used to analyze the mitochondrial potential of bEnd3 cells. Briefly, bEnd3 cells were incubated with culture medium containing 1 µg/ml JC-1 dye for 15 min at 37 . Next, cells were harvested and washed with PBS for 3 times. Subsequently, the stained cells were taking pictures using the BX50-FLA imaging system and analyzed by a flow cytometer (FACSARIA, BD Biosciences) [34]. This experiment was repeated three times.

Examination of intracellular ROS generation
The oxidative conversion of cell-permeable oxidation of 2′, 7′-dichlorodihydrofluorescein diacetate (DCFH-DA) to fluorescent DCF was utilized to determine intracellular ROS generation in this study. First, bEnd3 cells were incubated with 10 µmol/L DCFH-DA solution in serum-free medium at 37°C for 30 min. Subsequently, cells were washed five times with PBS, and DCF fluorescence was measured by a flow cytometer (FACSARIA, BD Biosciences) [33]. This experiment was carried out three times.

Detection of nitric oxide (NO)
The level of NO in bEnd3 cells were detected by Nitric Oxide (NO) assay Kit (Nitrate reductase method) (Nanjing Jiancheng Bioengineering Institute, Nanjing, Jiangsu, China) according to the manufacturer's instructions. This experiment was carried out three times.

Determination of intracellular glutathione (GSH)
First, 1 × 10 6 bEnd3 cells were seeded in a 10 cm dish (Corning Inc., Corning, NY, USA). Next, cells were deproteinized using 5% 5-sulfosalicylic acid solution. Subsequently, the cellular level of GSH was identified by Glutathione Assay Kit (Sigma-Aldrich, St. Louis, MO, USA) according to the manufacturer's protocol. This experiment was carried out three times

ELISA for detection of interleukin (IL)-1β, IL-6 and IL-8 in the culture supernatant
First, 1 × 10 4 bEnd3 cells were cultured in a well of the 96-well plate. After indicated treatments, the levels of IL-1β, IL-6 and IL-8 in the culture media were detected by ELISA kits according to the manufacturer's instruction (Solarbio, Beijing, China) [33].
These experiments were repeated three times.

Statistical analysis
All data in the present study were presented as the mean ± standard deviation (SD). The unpaired Student's t-test was utilized for the comparation between two groups, while statistics among multiple groups were analyzed by One way ANOVA. P < 0.05 was considered to indicate a statistically significant difference.

Ang-[1-7] decreases the concentration of chloride in bEnd3 cells under HG condition
To further identify the effect of Ang-[1-7] on CIC-3, the concentration of chloride in bEnd3 cells was measured by MQAE. The fluorescence intensity of MQAE was decreased proportionally with the increase of chloride ion in cells. Compared to NG, HG significantly increased the concentration of chloride (Figure 2a-b). However, Ang-[1-7] treatment and CIC-3 silence alleviated the increase of chloride caused by HG (Figure 2a-b). Furthermore, silence of Mas neutralized the effect of Ang-[1-7] on the increase of chloride (Figure 2a-b). In addition, the concentration of chloride in bEnd3 cells was not modified by mannitol and siRNA NC (Figure 2a and b). Above data suggested that Ang- [1][2][3][4][5][6][7] suppressed the function of CIC-3 through Mas receptor under HG condition.

Ang-[1-7] suppresses HG-induced bEnd3 cell apoptosis through inhibiting CIC-3 via Mas
Consistent to the effect of Ang-[1-7] on bEnd3 cell proliferation, CIC-3 silence enhanced the growth of bEnd3 cell cultured with HG ( Figure 3a). In addition, knockdown of Mas offset the impact of Ang-  apoptosis caused by HG through suppressing CIC-3 via Mas receptor.

Disclosure statement
No potential conflict of interest was reported by the author(s).

Funding
This study was supported by Science and Technology Planning Project of Guangdong Province (2017ZC0474).