Plasma auto-antibodies to angiotensin II receptors are correlated with blood pressure and inflammatory factors in hypertension patients

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1 European Heart Journal Supplements (2015) 17 (Supplement B), B65 B70 The Heart of the Matter doi: /eurheartj/suv016 Plasma auto-antibodies to angiotensin II receptors are correlated with blood pressure and inflammatory factors in hypertension patients Wen-Na Zong 1, Xiu-Mei Chen 1, Yu-Qing Yang 1, Jia-Li Cao 1, Hua-Yi-Yang Zou 1, Hui-Wen Sun 1, Mai-Hua Hou 2, Hong-Juan Huang 1, Hong-Jian Zheng 1, Xiao-Yi Qin 1, Hui Zhang 1, Xiang-Qing Kong 1, Jun Huang 1, and Xin-Zheng Lu 1 * 1 Department of Cardiology, the First Affiliated Hospital of Nanjing Medical University, Nanjing , China 2 Department of Dermatology, the First Affiliated Hospital of Nanjing Medical University, Nanjing , China KEYWORDS Auto-antibodies to angiotensin II type 1/2 receptor; Hypertension; Blood pressure; Inflammatory factors Auto-antibodies against to angiotensin II type 1 receptor (AT 1 R-AA) have been discovered in patients with hypertension and they have a close relationship with inflammatory factors. However, auto-antibodies to angiotensin II type 2 receptor (AT 2 R-AA) are seldom investigated in hypertension. Subjects (n ¼ 138) were enrolled and divided into three groups according to their blood pressure levels by using the Seventh Report of the Joint National Committee on Prevention, Detection, Evaluation, and Treatment of High Blood Pressure (JNC-7) criteria: normotensive ( 120/80 mmhg, n ¼ 31), prehypertensive ( /80 89 mmhg, n ¼ 39), and hypertensive ( 140/90 mmhg, n ¼ 68) groups. Plasma AT 1 R-AAs and AT 2 R-AAs were detected by enzyme-linked immunosorbent assay. Plasma tumour necrosis factor-a (TNF-a), endothelin-1 (ET-1), and angiotensin II (Ang II) were measured by radioimmunity assay. (i) Plasma AT 1 R-AAs were positively correlated with systolic blood pressure (SBP), diastolic blood pressure (DBP), mean arterial pressure (MAP), TNF-a, ET-1, and Ang II. (ii) AT 2 R-AAs were negatively correlated with SBP, DBP, MAP, TNF-a, ET-1, Ang II, as well as uric acid and serum creatinine. (iii) TNF-a, ET-1, Ang II (all P, 0.01, when compared with the normotensive group), blood uric acid, and serum creatinine (both P, 0.05, when compared with the normotensive group) increased with BP level. (iv) Multiple linear regression analyses showed that age, AT 1 R-AAs, AT 2 R-AAs, and ET-1 were independent predictors for SBP. AT 1 R-AAs, AT 2 R-AAs, and ET-1 were independent predictors for DBP. AT 1 R-AAs, AT 2 R- AAs, ET-1, and Ang II were independent predictors for MAP. Plasma AT 1 R-AAs and AT 2 R- AAs play an important role in hypertension progression. AT 2 R-AAs are inversely related to renal dysfunction. Introduction The pathophysiology of hypertension is complicated and involves many factors, such as rennin angiotensin system, sympathetic nervous system, inflammatory factors, 1 3 and The first two authors contributed equally to this work. * Corresponding author. Tel: ; Fax: , xzlu@njmu.edu.cn autoimmunity. 4 It has been reported that there are immunoregulatory defects such as elevated plasma concentrations of immunoglobulins (Igs), enhanced secretion of Igs, and increased levels of auto-antibodies against nuclear membranes in hypertensive disease. 5 The auto-antibodies to angiotensin II type 1 receptors (AT 1 R-AAs) were detected in malignant hypertension 5 and pre-eclampsia patients. 6 Recently, new evidence showed that AT 1 R-AAs are also Published on behalf of the European Society of Cardiology. All rights reserved. & The Author For permissions please journals.permissions@oup.com

2 B66 W.-N. Zong et al. present in the plasma of hypertension patients, 7,8 and to some extent, they have a relationship with plasma inflammatory factors. Therefore, we hypothesized that the autoantibodies to AT 2 R(AT 2 R-AAs) may also present in hypertensive patients. If so, we are trying to know the relationship among these auto-antibodies, hypertension, inflammatory mediators, and the target organ injury. The present study aimed to explore the possible relationship among the auto-antibodies to angiotensin II (Ang II) type 1/2 receptor, blood pressure, inflammatory factors, and renal function by comparing plasma AT 1 R-AA, AT 2 R-AA, tumour necrosis factor-a (TNF-a), endothelin-1 (ET-1), and Ang II in normotensive, pre-hypertensive, and hypertensive subjects. Methods Subjects Total 138 subjects with age between 35 and 70 (87 males and 51 females) were recruited from the Department of Physical Examination, First Affiliated Hospital, Nanjing Medical University, Nanjing, China. Exclusion criteria were: secondary hypertension; taking angiotensin-converting enzyme inhibitors or angiotensin II receptor blockers within 1 month; previous diagnosis of infections, cancers, liver diseases, kidney diseases, or other chronic diseases. Subjects were divided into three groups according to their blood pressure levels: normotensive ( 120/80 mmhg, n ¼ 31), prehypertensive ( /80 89 mmhg, n ¼ 39), and hypertensive ( 140/90 mmhg, n ¼ 68) groups. The clinical characteristics of patients, such as blood glucose, plasma lipids, blood uric acid, and plasma creatinine, were measured by an automatic biochemical detector (DimensionRxL, IL, USA). Heart rate was measured by electrocardiogram recording; drinking status was assessed by alcohol consumption; alcohol consumption was defined as the weekly consumption of beer, wine, and hard liquor converted into grams of alcohol. Current drinking was defined as alcohol consumption ( 8 g/week). All subjects gave informed consent to participate in the study. Thisstudywasapprovedbytheresearchethicscommittee of Nanjing Medical University. Measurement and criterion of blood pressure Systolic blood pressure (SBP) and diastolic blood pressure (DBP) were measured in the seated position of the subjects by two trained staff members using a mercury sphygmomanometer. Three independent BP measurements were obtained and the reported SBP and DBP (mmhg) were the average of the three readings. Using the SBP and DBP measurements, three levels of BP status were established by the investigators according to the JNC-7 criteria: normotensive (SBP 120 mmhg, DBP 80 mmhg), pre-hypertensive (SBP mmhg, DBP mmhg), and hypertensive (SBP 140 mmhg, DBP 90 mmhg). Ifasubject ssbpordbpputhimor her intwodifferent categories, the higher level determined the subject s BP status. Assays for plasma Ang II, AT 1 R-AA, AT 2 R-AA, TNF-a, and ET-1 Fasting blood samples (4 ml) were collected in the early morning after 30 min of rest and immediately transferred into two tubes that contained disodium EDTA. One tube was used for the measurement of plasma TNF-a, ET-1, and Ang II using immunoradiometric assay kits (Biotechnology Institute of Beijing, Beijing, China) according to the manufacturer s instruction. The other tube was stored at 2208C for the measurement of plasma AT 1 R-AAs and AT 2 R-AAs by using enzyme-linked immunosorbent assay kits (USCNLIFE, TX, USA) according to the manufacturer s instruction. Statistical analysis Results are expressed as mean + standard deviation (SD) and were compared by one-way analysis of variance. Comparisons between two groups were assessed by Sheffé. The relationship between cardiovascular risk factors and BP level was analysed, using both simple and multiple linear regressions. All statistical analyses were carried out with the Stata 7.0 (Stata Corp, TX, USA) and Excel software (Microsoft, WA, USA). Statistical significance was set as P, Results Clinical characteristics of subjects Table 1 shows the clinical characteristics, including age, drinking, body mass index (BMI), waist, hips, waist hip ratio, heart rate, BP, blood glucose, cholesterol, triglycerides, urea nitrogen, uric acid, and plasma creatinine, in the normotensive, pre-hypertensive, and hypertensive groups. There were no significant differences in drinking, waist, waist hip ratio, heart rate, cholesterol (total, high-density lipoprotein, and low-density lipoprotein), or triglycerides among the three groups. Interestingly, we found that (i) the age, BMI, and blood uric acid increased significantly in prehypertensive and hypertensive groups when compared with those of normotensive group; (ii) hips, blood glucose, urea nitrogen, and serum creatinine increased significantly in hypertensive groups when compared with those of normotensive group; (iii) BMI, blood glucose, and urea nitrogen increased significantly in hypertensive groups when compared with those of pre-hypertensive group. Plasma levels of AT 1 R-AAs, AT 2 R-AAs, TNF-a, ET-1, and Ang II Enzyme-linked immunosorbent assay and immunoradiometric assays were used to measure the plasma levels of AT 1 R-AAs, AT 2 R-AAs, TNF-a, ET-1, and Ang II in the three groups, respectively (Table 2). Plasma AT 1 R-AAs increased in the pre-hypertensive subjects when compared with that of normotensive group (P, 0.05). The hypertensive patients had elevated plasma AT 1 R-AAs when compared with both the pre-hypertensive (P, 0.05) and normotensive (P, 0.01) groups. However, the levels of AT 2 R-AAs had an opposite pattern in the three groups. They were significantly lower in the pre-hypertensive and the hypertensive groups when compared with that of the normotensive group (both P, 0.01). And they were significantly lower in the hypertensive group when compared with that of the pre-hypertensive group (P, 0.01). And the difference existed after age, sex, BMI, hips, and free plasma glucose adjustments. The levels of TNF-a, ET-1, and Ang II increased significantly with BP level (all P, 0.01). Linear regression analysis Figures 1 and 2 showedthatplasma AT 1 R-AAs were positively correlated with SBP, DBP, mean arterial pressure (MAP), Ang II, TNF-a, and ET-1, whereas AT 2 R-AAs were inversely

3 Role of plasma auto-antibodies in hypertension progression B67 Table 1 Clinical characteristics of patients Normotensive Pre-hypertensive Hypertensive Subjects (male/female) 31 (12/19) 39 (24/25) 68 (51/17) Age ** ** Alcohol (%) 4 (12.9) 13 (33.3) 24 (35.3) BMI (kg/m 2 ) ** **## Waist (cm) Hips (cm) * Waist hip ratio Heart rate (b.p.m.) Systolic BP (mmhg) ** **## Diastolic BP (mmhg) ** **## Free plasma glucose (mmol/l) **# Cholesterol (mmol/l) Low-density lipoprotein (mmol/l) High-density lipoprotein (mmol/l) Triglycerides (mmol/l) Urea nitrogen (mmol/l) *# Serum creatinine (mmol/l) * Uric acid (mmol/l) * ** Subjects are number and alcohol is number (%). Other data were expressed as mean + SD. * P, 0.05, ** P, 0.01 vs. normotensive group; # P, 0.05, ## P, 0.01 vs. pre-hypertensive group. Table 2 Changes of AT 1 R-AA, AT 2 R-AA, TNF-a, ET-1, and Ang II in plasma Normotensive (n ¼ 31) Pre-hypertensive (n ¼ 39) Hypertensive (n ¼ 68) AT 1 R-AA (ng/ml) * **# AT 2 R-AA (ng/ml) ** **## TNF-a (pmol/l) ** **## ET-1 (ng/l) ** **## Ang II (pg/ml) ** **## Data were expressed as mean + SD. * P, 0.05, ** P, 0.01 vs. normotensive group; # P, 0.05, ## P, 0.01 vs. pre-hypertensive groups. AT 1 R-AA, auto-antibodies to angiotensin II type 1 receptor; AT 2 R-AA, auto-antibodies to angiotensin II type 2 receptor; TNF-a, tumour necrosis factor-a; ET-1, endothelin-1; Ang II, angiotensin II. correlated with all of them as well as uric acid and plasma creatinine. The relationship existed even after age, sex, BMI, and other variable adjustments by covariance analysis. Multiple linear regression analysis For multiple linear regression, SBP was dependant variable, and age, AT 1 R-AAs, AT 2 R-AAs, TNF-a, ET-1, and Ang II were independent variables. The results showed that R 2 ¼ After age (b ¼ 0.243, P, 0.01), AT 1 R-AAs (b ¼ 0.431, P, 0.01), AT 2 R-AAs (b ¼ , P, 0.01), and ET-1 (b ¼ 0.072, P, 0.01) entered the equation, they correlated with SBP level. When the dependant variable was DBP and the independent variables were age, AT 1 R-AAs, AT 2 R-AAs, TNF-a, ET-1, and Ang II, R 2 ¼ After AT 1 R-AAs (b ¼ 0.315, P, 0.01), AT 2 R-AAs (b ¼ , P, 0.01), and ET-1 (b ¼ 0.123, P, 0.05) entered the equation, they correlated with DBP level. When the dependant variable was MAP and the independent variables were age, AT 1 R-AAs, AT 2 R-AAs, TNF-a, ET-1, and Ang II, R 2 ¼ After AT 1 R-AAs (b ¼ 0.353, P, 0.01), AT 2 R-AAs (b ¼ , P, 0.01), ET-1 (b ¼ 0.070, P, 0.01), and Ang II (b ¼ , P, 0.05) entered the equation, they correlated with MAP level. Multiple linear regression analysis showed that age, ET-1, AT 1 R-AAs, and AT 2 R-AAs were independent predictors for SBP and ET-1, AT 1 R-AAs, and AT 2 R-AAs were independent predictors for DBP. Ang II, ET-1, AT 1 R-AAs, and AT 2 R-AAs were independent predictors for MAP. Discussion In this study, we showed that AT 1 R-AAs and AT 2 R-AAs are present in the plasma of hypertensive patients. Plasma levels of AT 1 R-AAs positively correlated with BP levels, TNF-a, ET-1, and Ang II, whereas AT 2 R-AAs inversely correlated with BP levels, TNF-a, ET-1, Ang II, uric acid, and plasma creatinine. Ang II type 1/2 receptors (AT 1 R/AT 2 Rs) are known as G-protein-coupled receptors (GPCRs). AT 1 R-AAs, as the auto-antibodies to GPCRs, can result in an activation

4 B68 W.-N. Zong et al. Figure 1 The regression analysis shows the relationships between (A) AT 1 R-AA and systolic blood pressure, (B) AT 1 R-AA and diastolic blood pressure, (C) AT 1 R-AA and MAP, (D)AT 1 R-AA and TNF-a, and (E)AT 1 R-AA and ET-1, and (F)AT 1 R-AA and Ang II. AT 1 R-AA, auto-antibodies to angiotensin II type 1 receptor; SBP, systolic blood pressure; DBP, diastolic blood pressure; MAP, mean arterial pressure; TNF-a, tumour necrosis factor-a; ET-1, endothelin-1; Ang II, angiotensin II. effect via binding to the second extracellular loop of AT 1 R. 9 Dechend et al. 9 found that AT 1 R-AAs belong to the Ig G3 subclass. Several animal studies showed that AT 1 R-AAs have an agonist effect similar to that of Ang II in vascular smooth muscle cells proliferation, during which NF-kB and JAK-STAT proteins play essential roles. 10 Yang et al. 11 reported that AT 1 R-AAs causes significant vascular constriction in large conduit vessels as well as small resistant vessels through activation of the AT 1 R and induces vasoconstriction in a concentration-dependent fashion in isolated thoracic aortic rings, middle cerebral artery, and coronary artery segments. LaMarca reported that a correlation between AT 1 R-AAs and blood pressure during pregnancy by an ET-1-dependent mechanism, 12 but the mechanisms underlying AT 1 R-AA production are still unclear. Recently, some researchers reported that placental ischaemia and TNF-a are important stimuli of AT 1 R-AA production. 13 They also found that the plasma TNF-a level increased in uterine perfusion pressure rats and that chronic infusion of TNF-a to pregnant rats increases arterial pressure. 14 Our findings showed that AT 1 R-AAs positively correlated with TNF-a, ET-1, and Ang II. Taken together, we speculated that AT 1 R-AAs and inflammatory factors can influence each other and regulate blood pressure together. AT 1 R-AAs were detected in malignant hypertension, pregnancy-induced hypertension, and refractory hypertension. In addition, they are related with hypertensioninduced injury of target organs, such as kidney However, the correlation of AT 2 R-AAs, blood pressure, and target organ injury is uncertain. The present study found that plasma levels of AT 2 R-AAs are significantly different among the three groups (P, 0.01), and the difference exists even after age, sex, BMI, hips, and free plasma glucoseadjustment(p, 0.05). Unlike AT 1 R-AAs, we found that AT 2 R-AAs inversely correlated with SBP, DBP, MAP, TNF-a, ET-1, and Ang II. At the same time, we also found that AT 2 R-AAs are an independent risk factor of SBP, DBP, and MAP. As previously described, AT 1 R-AAs exhibited an agonist effect on AT 1 R. 6 Thus, we presumed that AT 2 R-AAs may have a similar agonist effect on AT 2 R to protect vascular endothelium, decrease blood pressure, benefit vascular repair, reduce inflammation, and so on by reducing vessel inflammation and plasma levels of ET-1 and TNF-a. Inflammation and hypertension affect each other. The latter can cause the activation of a pro-inflammatory state by increasing the expression of Ang II, TNF-a, ET-l, cell adhesion molecules, chemokines, growth factors, cardiac shock proteins, and other factors. 18 The present

5 Role of plasma auto-antibodies in hypertension progression B69 Figure 2 The regression analysis shows the relationships between (A) AT 2 R-AA and systolic blood pressure, (B) AT 2 R-AA and diastolic blood pressure, (C) AT 2 R-AA and MAP, (D) AT 2 R-AA and TNF-a, (E) AT 2 R-AA and ET-1, (F) AT 2 R-AA and Ang II, (G) AT 2 R-AA and uric acid, and (H )AT 2 R-AA and creatinine. AT 2 R-AA, auto-antibodies to angiotensin II type 2 receptor; SBP, systolic blood pressure; DBP, diastolic blood pressure; MAP, mean arterial pressure; TNF-a, tumour necrosis factor-a; ET-1, endothelin-1; Ang II, angiotensin II. study showed that Ang II, TNF-a, and ET-l significantly increased in the hypertensive group when compared with those of normotensive group, which is consistent with previous reports. 3,19,20 Moreover, we found that they also significantly increased in the pre-hypertensive group. Blood uric acid and creatinine are important indicators of renal function, and plasma uric acid was closely related to the occurrence and development of hypertension. 21 Our study showed that the levels of blood uric acid and creatinine significantly increased with BP level (P, 0.05) after age, sex, BMI, hips, and free plasma glucose adjustments by covariance analysis, suggesting that renal function may be affected even in the early stage of hypertension. The present study also found that AT 2 R-AAs were inversely correlated with uric acid and plasma creatinine. It has been reported that AT 2 R can improve renal function by protecting the glomerular filtration barrier. 22 We speculate that AT 2 R-AAs have a similar role as that of AT 2 R in protecting renal function. Hips, BMI, and free plasma glucose also increased with BP level, which is consistent with previous studies. 23 But waist, blood cholesterol, and triglycerides had no significant difference among the three groups in our study. As a summary, AT 1 R-AAs, AT 2 R-AAs, Ang II, TNF-a, and ET-l are significantly different in different levels of blood pressure. The changes existed in pre-hypertension status and progressed gradually, which may provide the theoretic basis for early intervention in pre-hypertensive people.

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