Chemerin stimulates aortic smooth muscle cell proliferation and migration via activation of autophagy in VSMCs of metabolic hypertension rats

Abstract

Vascular remodeling is a characteristic pathogenesis of hypertension and a main cause of abnormal construction and function of organs because of hypertension. Chemerin is a new adipokine that is elevated in states of obesity and metabolic syndrome (MS). However, the molecular mechanisms behind these pathological processes are not fully clarified. An animal model of metabolic hypertension was created to evaluate the role of metabolic chemerin in hypertension. In this study, the expression of chemerin/CMKLR-1 and autophagy in the arteries of metabolic hypertension rats undergoing vascular remodeling was investigated and the effect and mechanisms on the regulation of human aortic smooth muscle cells (HA-SMCs) were explored. The vascular remodeling in vivo was more serious in the metabolic hypertensive rat model, and the expression of chemerin and its receptor CMKLR1 were remarkably higher in the media layer of the thoracic aorta and the mesenteric artery in metabolic hypertension rats. In addition, there was an increased number of autophagosomes in SMCs and an up-regulation of the autophagy-related protein LC3 and beclin-1 levels in metabolic hypertension rats. In vitro, chemerin significantly stimulated HA-SMC proliferation and migration, as determined by MTT assay and scratch assay, respectively. Chemerin significantly increased LC3 and beclin-1 levels, as measured by western blot analysis, while this effect was inhibited by the autophagy inhibitor 3-MA. It is demonstrated that chemerin stimulates SMC proliferation and migration via autophagy, which may lead to vascular structural remodeling in metabolic hypertension.

Keywords: Blood pressure; autophagy; chemerin; metabolic hypertension; vascular remodeling.

Figure 1

Morphological changes in the thoracic aorta and mesenteric artery in rats. Hematoxylin-eosin staining: (A, B) 22 male Wistar rats were randomly fed with a standard diet or a high-sucrose/high-fat diet for 6 wk or 30 wk. The thoracic aorta and mesenteric arteries were collected, and the morphological analysis of the arteries was examined by H&E staining. (C, D) Quantification of the wall thickness (IMT) and the ratio of the media thickness and the internal diameter (MT/ID) was performed. The wall thickness and the ratio of MT/ID were higher in MH rats compared with that of the control rats at 30 wk (*P<0.05; n = 5). All of the values are denoted as the mean ± SEM. Magnification = ×200.

Figure 2

Chemerin/CMKLR-1 expression in the arteries of rats. Immunohistochemical analysis: (A, B) 22 male Wistar rats were randomly fed with a standard diet or a high-sucrose/high-fat diet for 6 wk or 30 wk. Chemerin expression was detected by IHC at 6 wk and 30 wk. (C, D) CMKLR-1 expression was detected in the thoracic aorta and mesenteric artery by IHC at 6 wk or 30 wk. (E) Quantification of chemerin expression by Image pro plus software from Panel (A and B). (F) Quantification of CMKLR-1 expression by Image pro plus software from panels (C and D). Compared with control rats, chemerin or CMKLR-1 was significantly up-regulated in the thoracic aorta and mesenteric artery in MH rats. *P<0.05 vs. the control group. All of the values are denoted as the mean ± SEM. Magnification = ×200.

Figure 3

Expression of autophagy-related proteins in VSMCs of the thoracic aorta and mesenteric artery of rats. Immunohistochemical analysis: (A, B) 22 male Wistar rats were randomly fed with a standard diet or a high-sucrose/high-fat diet for 6 wk or 30 wk, and then the thoracic aorta and mesenteric arteries were collected. LC3 expression was detected in the thoracic aorta and mesenteric artery by IHC at 6 wk or 30 wk. (C, D) Beclin-1 expression was detected in the thoracic aorta and mesenteric artery by IHC at 6 wk or 30 wk. (E) Quantification of LC3 expression from panels (A and B). (F) Quantification of LC3 and Beclin-1 expression from panels (C and D). Compared with control rats (n = 8), LC3 or Beclin-1 was significantly up-regulated in the thoracic aorta and mesenteric artery in MH rats (n = 9). * P<0.05 vs. the control group. All of the values are denoted as the mean ± SEM.

Figure 4

Autophagosome formation and extensive vacuolization in VSMCs of the thoracic aorta in rats. Microscopic evaluation of autophagy and ultrastructure in VSMCs of the thoracic aorta: (A-F) Representative transmission electron micrographs of cells in the thoracic aorta in the control group (A-C) or MH group (D-F). In (F), the light red arrows indicate the autophagic vacuoles or early double membrane structures. Magnification = ×200.

Figure 5

Effect of chemerin on cell proliferation and cell migration in VSMCs. Scratch wound healing assay: (A) VSMCs were plated in 6-well plates, and a straight line was scratched to create a cross in each well. The cells were treated with a vehicle control, chemerin 1 ug/L, 10 ug/L or 100 ug/L as indicated for 24 hours. The gap distance was evaluated using Image J software. (B) Quantification of gap distance closure from panel (A). *P<0.05 vs. the control group. MTT assay: (C) VSMCs plated in 96-well plates were treated with vehicle control (0), chemerin 0.1 ug/L, 1 ug/L, 10 ug/L or 100 ug/L as indicated for 12 hours, 24 hours or 48 hours. The viable cells number was determined at the end of experiments by the MTT assay. The data are expressed as the fold of the corresponding media control of each experiment. **P<0.01, *P<0.05 vs. the control group at the corresponding time. All of the values are denoted as the mean ± SEM.

Figure 6

Effect of chemerin on autophagy in VSMCs. Immunoblot analysis: (A) VSMCs were stimulated with chemerin (100 ug/ul) for 6 h, 12 h and 24 h. The abundance of LC3 and Beclin-1 was detected by western blotting; (B) Quantification of LC3 expression from panel (A), the data was expressed as the fold of the GAPDH. (C) Quantification of Beclin-1 expression from panel (A), the data was expressed as the fold of GAPDH. Immunofluorescence analysis: (D) VSMCs were treated with control or chemerin for 24 h, and LC-3 expression was detected by immunofluorescence. *P<0.05 vs. 0 h; All of the values are denoted as the mean ± SEM. Magnification = ×200.

Figure 7

Effect of the autophagy inhibitor 3-MA on chemerin-induced cell proliferation and migration in VSMCs. Scratch wound healing assay: (A) VSMCs were plated in 6-well plates and were pre-treated with or without 3-MA (5 mM, 30 min), and a straight line was scratched to create a cross in each well. The cells were treated with vehicle control or chemerin 100 ug/L as indicated for 24 hours. The gap distance was quantitatively evaluated using Image J software. (B) Quantification of gap distance closure from panel (A). MTT assay: (C) VSMCs were pre-treated with or without 3-MA (5 mM, 10 min) in 96-well plates and were treated with vehicle control or chemerin 100 ug/L as indicated for 24 hours. The viable cell number was determined at the end of the experiments by the MTT assay. *P<0.05 vs. the control group. #P<0.05 vs. the chemerin group. Magnification = ×200. All of the values are denoted as the mean ± SEM.

Figure 8

Chemerin down-regulated AKT and mTOR expression in HA-VSMC. Immunoblot analysis of VSMCs after chemerin treatment: (A) Representative Western blots of p-Akt and p-mTOR phosphorylation in VSMCs treated with vehicle control or chemerin 100 ug/L as indicated for 0 min, 15 min, 30 min, 1 hour or 2 hours. Each phospho protein was normalized to its non-phosphorylated total protein band intensity. (B) Quantification of AKT changes in band intensity from groups shown in panel (A) *P<0.05 vs. the 0 min time point. (C) Quantification of mTOR changes in band intensity from groups shown in panel (A). *P<0.05 vs. the 0 min time point.