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.
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.
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.
Blood sugar level
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.
95%C.I. for OR
Table 3 Logistic regression analysis.
North BJ, Sinclair DA (2012). The intersection between aging and cardiovascular disease. Circ Res, 110:1097-1108.
Sindler AL, Fleenor BS, Calvert JW, Marshall KD, Zigler ML, Lefer DJ, et al. (2011). Nitrite supplementation reverses vascular endothelial dysfunction and large elastic artery stiffness with aging. Aging Cell, 10:429-437.
Harvey A, Montezano AC, Touyz RM (2015). Vascular biology of ageing-Implications in hypertension. J Mol Cell Cardiol, 83:112-121.
Lakatta EG, Levy D (2003). Arterial and cardiac aging: major shareholders in cardiovascular disease enterprises: Part I: aging arteries: a "set up" for vascular disease. Circulation, 107:139-146.
Fitch RM, Vergona R, Sullivan ME, Wang YX (2001). Nitric oxide synthase inhibition increases aortic stiffness measured by pulse wave velocity in rats. Cardiovasc Res, 51:351-358.
Tian XL, Li Y (2014). Endothelial cell senescence and age-related vascular diseases. J Genet Genomics, 41:485-495.
Minamino T, Miyauchi H, Yoshida T, Ishida Y, Yoshida H, Komuro I (2002). Endothelial cell senescence in human atherosclerosis: role of telomere in endothelial dysfunction. Circulation, 105:1541-1544.
Te Riet L, van Esch JH, Roks AJ, van den Meiracker AH, Danser AH (2015). Hypertension: renin-angiotensin-aldosterone system alterations. Circ Res, 116:960-975.
Ng K, Hildreth CM, Avolio AP, Phillips JK (2011). Angiotensin-converting enzyme inhibitor limits pulse-wave velocity and aortic calcification in a rat model of cystic renal disease. Am J Physiol Renal Physiol, 301:F959-966.
Shan H, Zhang S, Li X, Yu K, Zhao X, Chen X, et al. (2014). Valsartan ameliorates ageing-induced aorta degeneration via angiotensin II type 1 receptor-mediated ERK activity. J Cell Mol Med, 18:1071-1080.
Unal H, Karnik SS (2014). Constitutive activity in the angiotensin II type 1 receptor: discovery and applications. Adv Pharmacol, 70:155-174.
Li T, Yu B, Liu Z, Li J, Ma M, Wang Y, et al. (2018). Homocysteine directly interacts and activates the angiotensin II type I receptor to aggravate vascular injury. Nat Commun, 9:11.
Wallukat G, Homuth V, Fischer T, Lindschau C, Horstkamp B, Jupner A, et al. (1999). Patients with preeclampsia develop agonistic autoantibodies against the angiotensin AT1 receptor. J Clin Invest, 103:945-952.
Fu ML, Herlitz H, Schulze W, Wallukat G, Micke P, Eftekhari P, et al. (2000). Autoantibodies against the angiotensin receptor (AT1) in patients with hypertension. J Hypertens, 18:945-953.
Zhu F, Sun YX, Liao YH, Wei YM, Chen M, Wang M, et al. (2008). Agonistic AT(1) receptor autoantibody increases in serum of patients with refractory hypertension and improves Ca(2+) mobilization in cultured rat vascular smooth muscle cells. Cell Mol Immunol, 5:209-217.
Dragun D, Muller DN, Brasen JH, Fritsche L, Nieminen-Kelha M, Dechend R, et al. (2005). Angiotensin II type 1-receptor activating antibodies in renal-allograft rejection. N Engl J Med, 352:558-569.
Li W, Li Z, Chen Y, Li S, Lv Y, Zhou W, et al. (2014). Autoantibodies targeting AT1 receptor from patients with acute coronary syndrome upregulate proinflammatory cytokines expression in endothelial cells involving NF-kappaB pathway. J Immunol Res, 2014:342693.
Zhang SL, Du YH, Wang J, Yang LH, Yang XL, Zheng RH, et al. (2010). Endothelial dysfunction induced by antibodies against angiotensin AT1 receptor in immunized rats. Acta Pharmacol Sin, 31:1381-1388.
Li W, Chen Y, Li S, Guo X, Zhou W, Zeng Q, et al. (2014). Agonistic antibody to angiotensin II type 1 receptor accelerates atherosclerosis in ApoE-/- mice. Am J Transl Res, 6:678-690.
Abadir PM, Jain A, Powell LJ, Xue QL, Tian J, Hamilton RG, et al. (2017). Discovery and Validation of Agonistic Angiotensin Receptor Autoantibodies as Biomarkers of Adverse Outcomes. Circulation, 135:449-459.
Fowkes FG, Rudan D, Rudan I, Aboyans V, Denenberg JO, McDermott MM, et al. (2013). Comparison of global estimates of prevalence and risk factors for peripheral artery disease in 2000 and 2010: a systematic review and analysis. Lancet, 382:1329-1340.
Scandale G, Dimitrov G, Recchia M, Carzaniga G, Minola M, Perilli E, et al. (2018). Arterial stiffness and subendocardial viability ratio in patients with peripheral arterial disease. J Clin Hypertens (Greenwich), 20:478-484.
Vlachopoulos C, Aznaouridis K, Stefanadis C (2010). Prediction of cardiovascular events and all-cause mortality with arterial stiffness: a systematic review and meta-analysis. J Am Coll Cardiol, 55:1318-1327.
Walther T, Stepan H (2007). Agonist autoantibodies against the angiotensin AT1 receptor in renal and hypertensive disorders. Curr Hypertens Rep, 9:128-132.
Okruhlicova L, Morwinski R, Schulze W, Bartel S, Weismann P, Tribulova N, et al. (2007). Autoantibodies against G-protein-coupled receptors modulate heart mast cells. Cell Mol Immunol, 4:127-133.
Wang B, Liao YH, Zhou Z, Li L, Wei F, Wang M, et al. (2005). Arterial structural changes in rats immunized by AT1-receptor peptide. Heart Vessels, 20:153-158.
Hirsch AT, Haskal ZJ, Hertzer NR, Bakal CW, Creager MA, Halperin JL, et al. (2006). ACC/AHA 2005 Practice Guidelines for the management of patients with peripheral arterial disease (lower extremity, renal, mesenteric, and abdominal aortic): a collaborative report from the American Association for Vascular Surgery/Society for Vascular Surgery, Society for Cardiovascular Angiography and Interventions, Society for Vascular Medicine and Biology, Society of Interventional Radiology, and the ACC/AHA Task Force on Practice Guidelines (Writing Committee to Develop Guidelines for the Management of Patients With Peripheral Arterial Disease): endorsed by the American Association of Cardiovascular and Pulmonary Rehabilitation; National Heart, Lung, and Blood Institute; Society for Vascular Nursing; TransAtlantic Inter-Society Consensus; and Vascular Disease Foundation. Circulation, 113:e463-654.
Wakabayashi I, Sotoda Y, Hirooka S, Orita H (2015). Association between cardiometabolic index and atherosclerotic progression in patients with peripheral arterial disease. Clin Chim Acta, 446:231-236.
Fu ML, Leung PS, Wallukat G, Bergstrom G, Fu H, Schulze W, et al.(1999). Agonist-like activity of antibodies to angiotensin II receptor subtype 1 (AT1) from rats immunized with AT1 receptor peptide. Blood Press, 8:317-324.
Shirwany NA, Zou MH (2010). Arterial stiffness: a brief review. Acta Pharmacol Sin, 31:1267-1276.
Tousoulis D, Kampoli AM, Tentolouris C, Papageorgiou N, Stefanadis C (2012). The role of nitric oxide on endothelial function. Curr Vasc Pharmacol, 10:4-18.
Jin Z, Wang J, Zhang W, Zhang G, Jiao X, Zhi J (2011). Changes in cardiac structure and function in rats immunized by angiotensin type 1 receptor peptides. Acta Biochim Biophys Sin (Shanghai), 43:970-976.
Yin H, Pickering JG (2016). Cellular Senescence and Vascular Disease: Novel Routes to Better Understanding and Therapy. Can J Cardiol, 32:612-623.
Minamino T, Komuro I (2007). Vascular cell senescence: contribution to atherosclerosis. Circ Res, 100:15-26.
Hayflick L (1965). The Limited In Vitro Lifetime Of Human Diploid Cell Strains. Exp Cell Res, 37:614-636.
Chen J, Patschan S, Goligorsky MS (2008). Stress-induced premature senescence of endothelial cells. J Nephrol, 21:337-344.
Marcelo KL, Goldie LC, Hirschi KK (2013). Regulation of endothelial cell differentiation and specification. Circ Res, 112:1272-1287.
Griendling KK, Sorescu D, Ushio-Fukai M (2000). NAD(P)H oxidase: role in cardiovascular biology and disease. Circ Res, 86:494-501.
Harman D (1981). The aging process. Proc Natl Acad Sci U S A, 78:7124-7128.
Gryglewski RJ, Palmer RM, Moncada S (1986). Superoxide anion is involved in the breakdown of endothelium-derived vascular relaxing factor. Nature, 320:454-456.
Marui N, Offermann MK, Swerlick R, Kunsch C, Rosen CA, Ahmad M, et al. (1993). Vascular cell adhesion molecule-1 (VCAM-1) gene transcription and expression are regulated through an antioxidant-sensitive mechanism in human vascular endothelial cells. J Clin Invest, 92:1866-1874.
Meijles DN, Sahoo S, Al Ghouleh I, Amaral JH, Bienes-Martinez R, Knupp HE, et al. (2017). The matricellular protein TSP1 promotes human and mouse endothelial cell senescence through CD47 and Nox1. Sci Signal, 10.
Gray SP, Jandeleit-Dahm KA (2015). The role of NADPH oxidase in vascular disease--hypertension, atherosclerosis & stroke. Curr Pharm Des, 21:5933-5944.
Dinh QN, Drummond GR, Sobey CG, Chrissobolis S (2014). Roles of inflammation, oxidative stress, and vascular dysfunction in hypertension. Biomed Res Int, 2014:406960.
Csiszar A, Wang M, Lakatta EG, Ungvari Z (2008). Inflammation and endothelial dysfunction during aging: role of NF-kappaB. J Appl Physiol (1985), 105:1333-1341.
Haller H, Ziegler EM, Homuth V, Drab M, Eichhorn J, Nagy Z, et al. (1997). Endothelial adhesion molecules and leukocyte integrins in preeclamptic patients. Hypertension, 29:291-296.
Dechend R, Homuth V, Wallukat G, Kreuzer J, Park JK, Theuer J, et al. (2000). AT(1) receptor agonistic antibodies from preeclamptic patients cause vascular cells to express tissue factor. Circulation, 101:2382-2387.
Min LJ, Mogi M, Iwai M, Horiuchi M (2009). Signaling mechanisms of angiotensin II in regulating vascular senescence. Ageing Res Rev, 8:113-121.
Xia Y, Kellems RE (2013). Angiotensin receptor agonistic autoantibodies and hypertension: preeclampsia and beyond. Circ Res, 113:78-87.
Zhang S, Zheng R, Yang L, Zhang X, Zuo L, Yang X, et al. (2013). Angiotensin type 1 receptor autoantibody from preeclamptic patients induces human fetoplacental vasoconstriction. J Cell Physiol, 228:142-148.
Wenzel K, Rajakumar A, Haase H, Geusens N, Hubner N, Schulz H, et al. (2011). Angiotensin II type 1 receptor antibodies and increased angiotensin II sensitivity in pregnant rats. Hypertension, 58:77-84.