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Aging and disease    2019, Vol. 10 Issue (5) : 1012-1025     DOI: 10.14336/AD.2018.0919
Orginal Article |
Autoantibodies against AT1 Receptor Contribute to Vascular Aging and Endothelial Cell Senescence
Meili Wang1,2, Xiaochen Yin1,2, Suli Zhang1,2, Chenfeng Mao3,4, Ning Cao1,2, Xiaochun Yang5, Jingwei Bian1,2, Weiwei Hao1,2, Qian Fan5, Huirong Liu1,2,*
1Department of Physiology and Pathophysiology, School of Basic Medical Sciences, Capital Medical University, Beijing, China.
2Beijing Key Laboratory of Metabolic Disorders Related Cardiovascular Disease, Capital Medical University, Beijing, China.
3Department of Physiology and Pathophysiology, School of Basic Medical Sciences, Peking University Health Science Center, Beijing, China.
4Key Laboratory of Molecular Cardiovascular Science, Ministry of Education, Beijing, China.
5Beijing Anzhen Hospital, Capital Medical University, Beijing, China.
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Abstract  

Vascular aging predisposes the elderly to the progression of many aging-related vascular disorders and leads to deterioration of cardiovascular diseases (CVD). However, the underlying mechanisms have not been clearly elucidated. Agonistic autoantibodies against angiotensin II type 1 (AT1) receptor (AT1-AAs) have been demonstrated to be pro-inflammatory and contribute to the progression of atherosclerosis. However, the association between AT1-AAs and vascular aging has not been defined. Peripheral arterial disease (PAD) is an acknowledged vascular aging-related disease. In this study, AT1-AAs were detected in the sera of patients with PAD and the positive rate was 44.44% (n=63) vs. 17.46% in non-PAD volunteers (n=63). In addition, case-control analysis showed that AT1-AAs level was positively correlated with PAD. To reveal the causal relationship between AT1-AAs and vascular aging, an AT1-AAs-positive rat model was established by active immunization. The carotid pulse wave velocity was higher, and the aortic endothelium-dependent vasodilatation was attenuated significantly in the immunized rats. Morphological staining showed thickening of the aortic wall. Histological examination showed that levels of the senescent markers were increased in the aortic tissue, mostly located at the endothelium. In addition, purified AT1-AAs-IgGs from both the immunized rats and PAD patients induced premature senescence in cultured human umbilical vein endothelial cells. These effects were significantly blocked by the AT1 receptor blocker. Taken together, our study demonstrates that AT1-AAs contribute to the progression of vascular aging and induce EC senescence through AT1 receptor. AT1-AA is a novel biomarker of vascular aging and aging-related CVD that acts to accelerate EC senescence.

Keywords AT1 receptor      Autoantibody      Peripheral arterial disease      Vascular aging      EC senescence     
Corresponding Authors: Liu Huirong   
About author:

Jean-Marc Burgunder is currently a visiting professor at Sichuan University (Chengdu), Central South University (Changsha) and Sun Yat Sen University (Guangzhou) in China.

Just Accepted Date: 27 September 2018   Issue Date: 27 September 2019
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Wang Meili
Yin Xiaochen
Zhang Suli
Mao Chenfeng
Cao Ning
Yang Xiaochun
Bian Jingwei
Hao Weiwei
Fan Qian
Liu Huirong
Cite this article:   
Wang Meili,Yin Xiaochen,Zhang Suli, et al. Autoantibodies against AT1 Receptor Contribute to Vascular Aging and Endothelial Cell Senescence[J]. Aging and disease, 2019, 10(5): 1012-1025.
URL:  
http://www.aginganddisease.org/EN/10.14336/AD.2018.0919     OR     http://www.aginganddisease.org/EN/Y2019/V10/I5/1012
CharacteristicPAD
χ2P
Yes (%)No (%)
Gendermale46 (73.02)46 (73.02)
female17 (26.98)17 (26.98)
Smoking historyEver16 (25.40)10 (15.87)16.391<0.001
Current34 (53.97)18 (28.57)
Never13 (20.63)35 (55.56)
Drinking historyEver9 (14.29)10 (15.87)0.1840.912
Current18 (28.57)16 (25.40)
Never36 (57.14)37 (58.73)
Hypertension historyNo15 (23.81)32 (50.79)11.0970.004
Yes, good drug control25 (39.68)12 (19.05)
Yes, bad drug control23 (36.51)19 (30.16)
DiabetesNo42 (66.67)60 (95.24)16.676<0.001
Yes21 (33.33)3 (4.76)
Table 1  Chi-square analysis on demographic characteristics of participants.
Figure 1.  High levels of AT1-AAs in the patients with PAD. A) The positive rate of AT1-AAs in PAD group and the non-PAD group was 44.44% and 17.46%, respectively. (n=63 per group). B) The P/N values of AT1-AAs in the sera samples of PAD patients and non-PAD participants measured by ELISA. PAD, peripheral arterial disease, **p<0.01, ***p<0.001.
Figure 2.  Successful establishment of the AT1-AAs-positive rat model. A) The level of AT1-AAs in sera was detected by ELISA. It increased gradually after the first immunization and reached a peak six weeks later and maintained a high value compared with the vehicle group. B) Blood pressure curves of rats immunized with AT1R-ECII peptides or the vehicle group. Data were expressed as means ± SEM, n=8-10 rats per group. ***p<0.001 vs. the vehicle group.
VariablesPAD(n=63)Non-PAD(n=63)Z valueP value
Age65.29±13.1264.81±12.94
AT1-AA(P/N)2.362± 1.8281.533± 0.5299-4.073<0.001
BMI23.72± 3.4824.69± 3.784-1.5060.132
Heart rate80.03± 10.7277.46± 13.04-1.3470.178
SBP146.3±19.32142.7±17.35-1.4120.158
DBP78.78±9.86882.84±13.89-1.4890.136
Blood sugar level5.529± 2.3965.539± 1.232-1.6290.103
TC (mmol/L)4.608± 1.1684.171± 1.070-2.1190.034
TG (mmol/L)1.523± 0.83981.477± 1.975-2.4990.012
HDL (mmol/L)1.077±0.26671.083±0.2648-0.2460.805
LDL (mmol/L)2.956±0.96622.582±0.7198-2.3410.019
Table 2  Wilcoxon rank sum test on the biochemical indexes of PAD patients.
Figure 3.  Functional evidence of vascular aging in immunized rats. A) Pulse wave velocity measured in the left common carotid artery from the vehicle and AT1R-ECII-immunized rats. B) Relaxation curves in response to the vasodilator acetylcholine of phenylephrine (1 μM)-pretreated isolated aortic rings from the vehicle and AT1R-ECII-immunized rats. C) Relaxation in response to SNP. Isolated aortic rings were treated with L-NAME (100 μM) for 30 mins followed by phenylephrine (1 μM) treatment. Data were expressed as mean ± SEM. n = 6-8 rats for each group, *p < 0.05, **p < 0.01, ***p<0.001 vs. the vehicle group.
Figure 4.  Changes in arterial morphology after immunization. A) Microscopic images of the HE-stained aortic sections. Scale bar = 400 μm. There was no significant difference in arterial wall thickness at the 8th weeks, but it increased significantly after 12-week immunization. B) Quantitative analysis of wall thickness of thoracic aortas at 8 and 12 weeks after immunization. C) Representative images of immunohistochemical staining showing p53, p21 and p16INK4a in the rat aorta of the indicated groups. Scale bar = 400 (small views) and 40 μm (enlarged views). Data were expressed as mean ± SEM. n = 6-8 rats for each group, *p < 0.05.
Figure 5.  AT1-AAs induced premature senescence of HUVECs through AT1 receptor. A) Representative images and quantitative graphs of p53, p21 and p16INK4a expressions. HUVECs were incubated with AT1-AAs-IgGs of indicated concentrations for 5 days. B) Representative images and quantitative graphs of p53, p21 and p16INK4a expressions. HUVECs were incubated with AT1-AAs-IgGs (1 μM) for indicated times. **p < 0.01, ***p<0.001 vs. the control group. C) Representative western blot and quantitative graphs of p53, p21 and p16INK4a expressions in HUVECs treated with nIgGs, AT1-AA or valsartan plus AT1-AA for 72 hrs. D. Measurement of cell proliferation using the cck-8 analysis. HUVECs treated with AT1-AA manifested a reduction in cell proliferation by comparison to the nIgG treated group. E) Cell cycle analysis of the nIgGs, AT1-AAs-IgGs or valsartan plus AT1-AAs-IgGs-induced HUVECs by flow cytometry. F) Photographs of typical SA-β-gal-stained HUVECs in the nIgGs, AT1-AA and valsartan+AT1-AA groups (senescent cells are stained blue). Scale bar = 400 μm. H. Quantification of percentages of SA-β-gal-positive HUVECs of the indicated groups. Data in the graphs were from 3 independent experiments and were expressed as mean ± SEM. *p < 0.05, **p < 0.01, ***p<0.001, ****p<0.0001 vs. the nIgGs group; # p < 0.05, ## p < 0.01, ### p < 0.001 vs. the AT1-AAs-IgGs group.
Figure 6.  AT1-AAs from the PAD patients induced HUVECs senescence. A) Representative western blot and quantitative graphs of p53, p21 and p16INK4a expressions in HUVECs treated with nIgGs, AT1-AAs-IgGs or valsartan plus AT1-AAs-IgGs for 72 hrs. B) Photographs of typical SA-β-gal-stained HUVECs in the nIgGs, AT1-AAs-IgGs and valsartan+AT1-AAs-IgGs groups. Scale bar = 400 μm. C) Quantification of percentages of SA-β-gal-positive HUVECs of the indicated groups. Data in the graphs were from 3 independent experiments and were expressed as mean ± SEM. *p < 0.05, **p < 0.01, ***p<0.001 vs. the nIgGs group; # p < 0.05, ## p < 0.01 vs. the AT1-AAs group.
VariablesBWald valueP valueOR95%C.I. for OR
Constant-4.5615.5010.0190.010
AT1-AA (P/N)1.26910.2840.0013.5591.6387.730
Table 3  Logistic regression analysis.
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