Vascular cells, such as human umbilical vein endothelial cells (HUVECs; American Type Culture Collection, Manassas, VA, USA) and VSMCs (PromoCell GmbH, Heidelberg, Germany), were cultured in high glucose Dulbecco's modified Eagles medium (DMEM) with 10% fetal bovine serum (both from Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA) as previously described (13). Cells at 4–6 passages were used in subsequent experiments. 3T3-L1 adipocytes were differentiated from 3T3-L1 fibroblasts (American Type Culture Collection) as previously described (14). Briefly, 3T3-L1 fibroblasts were cultured to reach confluency in a 12-well plate and were differentiated into adipocytes in the specific differentiation medium, consisting of DMEM supplemented with 10% fetal bovine serum (FBS; Gibco; Thermo Fisher Scientific, Inc.) and 3-isobutyl-1-methylxanthine (5 µM), dexamethasone (0.25 µM) (both from Sigma-Aldrich; Merck KGaA, Darmstadt, Germany) and insulin (5 µg/ml; Gibco; Thermo Fisher Scientific, Inc.) for 2 days, followed by treatment with DMEM supplemented with 10% FBS and insulin (5 µg/ml).

Male, 8–12 week-old C57BL/6J background mice were used in the present study. To induce hypertension, mice (n=5) were subcutaneously infused with Ang II (Sigma-Aldrich; Merck KGaA) at a 1,500 ng/kg/min dose for 14 days with ALZET mini osmotic pumps (DURECT Corporation, Cupertino, CA, USA), and in the sham group mice were treated with saline (n=5). Blood pressure was measured non-invasively every day by the tail-cuff method (Softron BP-98A; Softron Co., Ltd., Tokyo, Japan). To determine the role of APN receptor in hypertensive vascular injury, Ang II-infused mice received APN receptor agonist AdipoRon (n=5, 30 mg/kg/day; Selleck Chemicals, Houston, TX, USA) via a gavage tube. The experiments were performed in adherence with the National Institutes of Health guidelines on the Use of Laboratory Animals and were approved by the Animal Care and Use Committee of Shanghai University of Traditional Chinese Medicine (Shanghai, China).

Thoracic aortas of Ang II-induced hypertensive mice were obtained as previously described (15), and then were fixed in 4% paraformaldehyde at 4°C for 24 h, embedded in paraffin, and 5 µm sections were prepared for hematoxylin (15 min at room temperature) and eosin (5 min at room temperature) (H&E) and picrosirius red staining (30 min at room temperature) to assess the vascular hypertrophy and fibrosis according to the manufacturers (Wuhan Servicebio Technology Co., Ltd., Wuhan, China). Morphometric analysis was performed using Image-Pro Plus 6.0 software to assess vascular hypertrophy by measuring the medium and lumen area. Fibrotic staining was expressed as a percentage of stained areas (red) to the total areas examined. For immunofluorescence staining, paraffin sections were first deparaffinized and rehydrated in xylene (30 min), 100% ethanol (5 min), 90% ethanol (5 min) and 80% ethanol (5 min). Following boiling in 10 mM sodium citrate buffer for 10 min to unmask antigens, the slides were blocked in buffer containing 5% normal goat serum (Invitrogen; Thermo Fisher Scientific, Inc.) at room temperature for 1 h, incubated with primary antibody at 4°C overnight, followed by incubation with the fluorochrome-conjugated secondary antibody (1:200, for 1 h at room temperature; Invitrogen; Thermo Fisher Scientific, Inc.). Primary antibodies included perilin (PLIN1, 1:100, cat no. ab3526; Abcam, Cambridge, UK), APN (1:200, cat no. AF1119; R&D Systems, Inc., Minneapolis, MN, USA), AdipoR1 (1:150, cat no. ab70362), AdipoR2 (1:150, cat no. ab77612) (both from Abcam), α smooth muscle actin (α-SMA, 1:500, cat no. A5228; Sigma-Aldrich; Merch KGaA) and p-p38 (1:100, cat no. 4511; CST Biological Reagents Co., Ltd., Shanghai, China). Images were captured by a laser scanner confocal microscopy system (LSM 710; Zeiss GmbH, Jena, Germany).

Adipocytes, VSMCs and HUVECs were lysed in a radioimmune precipitation assay buffer (0.1% sodium dodecyl sulfate, 1.0% Triton X-100, 2 µg/ml aprotonin and 2 ng/ml leupeptin) supplemented with protease inhibitor cocktail (Roche Diagnostics, Basel, Switzerland) on ice for 10 min and sonicated. Cell lysates were centrifuged (12,000 × g, 15 min at 4°C) and the supernatant was preserved at 20°C for western blot analysis. Protein concentrations were determined using the Bradford method. Aliquots of protein (20 µg) were run on a 10% sodium dodecyl sulfate-polyacrylamide gel followed by blotting onto polyvinylidene fluoride membranes by wet transfer. Membranes were blocked in blocking buffer (5% nonfat milk in TBS buffer with 0.1% Tween-20) and were incubated with primary antibody at 4°C overnight. The primary antibodies included APN (1:2,000, cat no. AF1119, R&D Systems, Inc.), AdipoR1 (1:1,000, cat no. ab70362), AdipoR2 (1:1,000, cat no. ab77612) (both from Abcam), α-SMA (1:5,000, cat no. A5228, Sigma-Aldrich; Merch KGaA), p-p38 (1:1,000, cat no. 4511), t-p38 (1:1,000, cat no. 9212) (both from CST Biological Reagents Co., Ltd.) and β-actin (1:5,000, cat no. ab8226; Abcam). The membranes were washed with TBS with Tween-20 for 3×10 min followed by incubation with the horseradish peroxidase-conjugated secondary antibodies [rabbit anti-mouse IgG (1:5,000, cat no. ab6728); goat anti-rabbit IgG (1:5,000, cat no. ab6721); rabbit anti-goat IgG (1:5,000, cat no. ab6741) (all from Abcam)] for 1 h at room temperature. Protein bands were detected by chemiluminescence (EMD Millipore, Billerica, MA, USA).

VSMCs migration was measured using a Transwell chamber with a polycarbonate membrane (6.5 mm diameter and 8.0 µm pore size; Corning Incorporated, Corning, NY, USA). Briefly, serum-starved VSMCs were trypsinized and resuspended in DMEM with 0.5% bovine serum albumin (Sigma Sigma-Aldrich; Merch KGaA). Cell suspension (100 µl, 1.0×10⁶ cells/ml) was loaded in the upper compartment of the chamber. DMEM with APN (10 µg/ml) were added to lower compartment and incubated at 37°C. Following 2 h incubation, Ang II (100 nM) was added. After 6 h, the cells remaining on the upper surface of the chamber were removed with a cotton swap, migrated cells on the lower surface of the chamber were fixed in 4% paraformaldehyde at room temperature for 1 h and stained with hematoxylin (15 min at room temperature) and eosin (5 min at room temperature) and three random microscopic fields (magnification, ×200) per well were quantified by a fluorescence microscope (DM3000; Leica, Wetzlar, Germany).

Statistical analysis was performed using SPSS 19.0 (SPSS; IBM Corp., Armonk, NY, USA). Data are expressed as the mean ± standard deviation. Comparisons of experimental groups were analyzed by Student's t-test (between two groups) or one-way analysis of variance, followed by the post-hoc Dunnett's test for data with more than two groups (Levene's tests for equal variance and Welch's and Brown-Forsythe's test for unequal variances). P<0.05 was considered to indicate a statistically significant difference.

APN and AdipoR expression were detected in adipocytes, VSMCs and endothelial cells (ECs) by western blot analysis. As demonstrated in Fig. 1, APN expression was highest in the adipocytes, with the expression of APN in the VSMCs and ECs less than in the adipocytes. Additionally, AdipoR1 and AdipoR2 were expressed in the VSMCs, ECs and adipocytes.

To detect APN and AdipoR expression in the blood vessels, immunostaining of cross-sections of thoracic aorta in Sham and Ang II-induced hypertensive mice was performed. Ang II infusion resulted in a dramatic increased blood pressure including systolic and diastolic blood pressure (Fig. 2A). In accordance with the in vitro results, APN was mainly distributed in the perivascular adipocytes (Fig. 2B and C, perilipin-positive cells), while AdipoR1 and AdipoR2 were expressed in both VSMCs (α smooth muscle actin-positive cells) and adipocytes (Fig. 2D and E). Ang II-induced hypertension resulted in decreased APN and AdipoR expression in the vasculature.

To detect the direct effects of Ang II on APN, APN levels in the 3T3-L1 adipocytes pretreated with Ang II were measured. The result demonstrated that APN expression was decreased in a time-dependent manner following Ang II treatment (Fig. 3A). In addition, it was also illustrated that AdipoR1 and AdipoR2 expression was decreased in a time-dependent manner following Ang II treatment in the VSMCs (Fig. 3B).

Vascular active substance-induced VSMCs migration serves a pivotal role in hypertension-associated vascular remodeling (16). Whether APN affects Ang II-induced VSMCs migration was examined by utilizing the Transwell chamber assay. As depicted in Fig. 4A, APN (10 µg/ml) significantly attenuated Ang II-induced VSMCs migration. The p38 signaling pathway is known to be involved in the regulation of VSMC migration (17) In the present study, the phosphorylation of p38 mitogen-activated protein kinase (MAPK) was reduced following APN treatment in Ang II-stimulated VSMCs (Fig. 4B).

AdipoRon is an orally active APN receptor activator, which potentially exerts protective effects against cardiac remodeling (18,19). In order to detect the effects of AdipoRon in hypertension-associated vascular remodeling, the Ang II-infused mice were orally treated with AdipoRon (30 mg/kg/day). As demonstrated in Fig. 5A, AdipoRon treatment resulted in decreased p38 phosphorylation. In addition, AdipoRon attenuated Ang II-induced perivascular collagen deposition and vascular hypertrophy as detected by Sirious Red and H&E stainings (Fig. 5B and C, respectively).