Circulating ErbB3/HER3 Levels are Associated with Increased Risk of Hypertension With Overweight: A Cross-Sectional Study

Background: Hypertension and overweight are both independent risk factors for cardiovascular disease (cid:0) and being overweight can more likely to develop high blood pressure. Recent research has shown that ErbB3/HER3 played a considerable role in the development of cardiovascular disease. However, ErbB3 levels effects in the hypertensive overweight patients are unknown. This study aimed to assess the association between ErbB3 levels and hypertension with overweight in Chinese population. Methods: 128 Chinese adults aged 33-79 years, both sexes, underwent evaluation of height and weight, blood pressure, biochemical indicators and ErbB3 levels. Plasma ErbB3 levels was assessed by the Enzyme-linked immunosorbent assay (ELISA), and body mass index (BMI) was calculated as body weight divided by height squared. Participants were allocated in three groups according to blood pressure and BMI: healthy control group (CNT; n = 31; normotensive and nonoverweight), hypertension group (HT; n = 33; hypertension and nonoverweight) and hypertension with overweight group (HTO; n = 64; hypertension and overweight). A 2-tailed P<0.05 was dened to be statistically signicant. Results: The difference in mean of ErbB3 levels in three groups was not signicant, but had a linear decrease in males, in CNT (1.13±0.36), HT (1.03±0.36) and HTO (0.84±0.26) ng/ml, with P was 0.007. In drinking population, the ErbB3 was signicantly reduced in the HTO group as compared with the CNT and HT groups (0.76±0.23 versus 1.18±0.37 and 1.20±0.30, respectively). ErbB3 levels was negatively correlated with DBP in males(r=-0.293, P=0.012), in smoking populations (r=-0.47, P=0.004) and drinking populations (r=-0.387, P=0.008). Besides, BMI in males and in drinking populations and UA in males presented negatively correlations with ErbB3 (p (cid:0) 0.05). The multivariate conditional logistic regression showed that plasma ErbB3 levels was associated with reduced risk for HTO in males (OR 0.054; 95% CI: 0.007-0.412) and drink group(OR 0.002; 95% CI: 0.000-0.101). The apparent association between lower ErbB3 levels and overweight hypertensive patients suggested that ErbB3 may contribute to the pathogenesis to hypertension with overweight, with BMI, gender, and drinking all potentially modulating the process.

The subjects were recruited via Health Management Center & Physical Examination Center at Yijishan Hospital of Wannan Medical College from July 2019 to August 2019. A total of 128 adults aged 33-79 years were investigated. According to "Seventh report of the joint national committee on prevention, detection, evaluation, and treatment of high blood pressure", Hypertension is generally de ned as a systolic blood pressure of ≥140 mm Hg or a diastolic blood pressure of ≥90 mm Hg or use of antihypertensive medications. For adults, WHO de nes overweight as follows: overweight is a BMI greater than or equal to 25. In this study, healthy control group (CNT) was de ned as both normotensive and BMI < 25; hypertension group (HT) was de ned as both hypertensive and BMI < 25, and hypertension with overweight group (HTO) was de ned as both hypertensive and BMI ≥25.
Finally, 128 adults, including 31 controls, 33 hypertensives, and 64 hypertension with overweight subjects were selected to assess the association between plasma ErbB3 levels and hypertension with overweight.
The study was approved by the Ethics Committee of the First A liated Yijishan Hospital of Wannan Medical College (Wuhu,China). Written informed consent was obtained from all participants.

Data collection
At physical examinations, all subjects were measured for height and weight, and blood pressure. Body mass index (BMI) was calculated as body weight (kg) divided by height squared (m 2 ). A well-trained research staff measured blood pressure once using electronic sphygmomanometer with the participant in the sitting position after at least 5 minutes of rest. Smoking was classi ed as smokers (including current and ex-smokers) or nonsmokers. Drinking alcohol was classi ed as drinkers (including current and ex-drinkers) or non-drinkers.

Biochemical analyses
Blood samples were obtained from the subjects fasted overnight for at least 10h. Total cholesterol (TC), triglyceride (TG), high-density lipoprotein cholesterol (HDL-C), low -density lipoprotein cholesterol (LDL-C) levels, glucose (GLU), and uric acid (UA) were all measured using standard methods by the physical examination institution.
Enzyme-linked immunosorbent assay (ELISA) A 3-ml venous blood samples was collected and centrifugated at 3000r/min for 10 min. The supernatant was collected using a micropipette and stored in the refrigerator at -80°C for use. The plasma samples were then thawed at room temperature for ELISA which performed using a commercial kit (Catalogue No.CSB-EL007765HU).The method is operated according to the manufacturers' instructions, and was repeated twice to calculate the average of two parallel samples.

Statistical analysis
Statistical analysis was carried out with the SPSS software (version 18.0, SPSS Inc., Chicago, IL). The normality assumption of the data was determined using the Kolmogorov Smirnov test. Quantitative data were expressed as mean ± standard deviation (mean ± SD), and categorical data are expressed as frequency (n and %). The differences for variables among the groups were determined by one-way analysis of variance (ANOVA) or Chi-square test. Pearson's correlation was used for parametric correlations. Multiple unconditional logistic regression analysis with effect ratios [odds ratio (OR) and 95% con dence intervals (CI)] was applied to estimate the risk of hypertension, hypertension with overweight after adjusting for potential confounding factors. A 2-tailed P<0.05 was de ned to be statistically signi cant.

Characteristics of the study participants
As shown in Table 1, there was no differences in age, TC, LDL-C, HDL-C, Glu and the distribution of gender, drinking status among the three groups (P > 0.05).The TG signi cantly increased in the HT group in comparison with the CNT group, while was decreased comparison with the HTO group. Compared with the HT group, the UA levels in the HTO group was increased.
Comparison of plasma levels of ErbB3 among the three groups There were no statistical differences in ErbB3 levels between the three groups. By gender subgroup, the plasma concentrations of ErbB3 (1.13±0.36, 1.03±0.36, 0.84±0.26) ng/ml had a linear decrease in CNT group, HT group and HTO group in males, with P was 0.007. In subgroup of drinking population, the ErbB3 was signi cantly decreased in the HTO group as compared with the CNT and HT groups (0.76±0.23 versus 1.18±0.37 and 1.20±0.30, respectively) (Table2). Table 3 summarizes Pearson's correlation coe cients between ErbB3 and hypertension risk factors, strati ed by gender, smoking and drinking. Correlation analysis showed that the ErbB3 levels was negatively correlated with DBP in males(r=-0.293, P=0.012), in smoking populations (r=-0.47, P=0.004) and drinking populations (r=-0.387, P=0.008). Besides, it is noteworthy that BMI both in males and in drinking population and UA in males presented negatively correlations with ErbB3 (all P 0.05).

Multinomial logistic regression
In Table 4, we examined the OR of HT and HTO associated with increasing levels of ErbB3 within subgroups of gender, current smoking and drinking. Plasma ErbB3 levels was negatively associated with hypertension with overweight, with OR (95% CI) was 0.054 0.007-0.412 , and 0.002(0.000-0.101), for males and drink group, respectively. After adjustment for age, gender, the association remains signi cant.

Discussion
Framingham Study have shown that hypertension [18] and overweight [19] are both independent risk factors for cardiovascular disease,and about 30% of hypertensive individuals can be classi ed as being obese [20]. In addition, ErbB3 has been reported to play an important role in the maintenance and development of cardiovascular disorders [11,15]. This study is the rst to demonstrate the importance of ErbB3 levels in hypertension with overweight. We observed the ErbB3 levels had negatively associations with hypertension with overweight in males and drinking populations. These results indicated that the ErbB3 levels might contribute to the development of hypertension and hypertension with overweight.
Animal researches demonstrated that the ErbB3 receptors might be involved in blood pressure regulation through NRG-1/ErbB signaling as an antihypertensive system, but this effect is probably not that strong [21]. We observed a negative association between the ErbB3 and DBP in the males, drinking and smoking groups in this study. Previous reports suggested that males were generally at greater risk for hypertension [22] than were agematched females. Another study reported that environmental exposures to drinking, smoking were associated with adult hypertension in Japanese population [23]. Remarkably,DBP has increased sensitivity to environmental changes, and more strongly predicts cardiovascular disease risk in younger Chinese adulthood [24]. In the present study, BMI both in males and in drinking population and UA in males presented negatively correlations with ErbB3. Males were more overweight, and consumed more alcohol than females [25]. Additionally, Genetic studies have shown ErbB3 gene is responsible for variation in the LDL-C serum concentration, is known to be involved in lipid homeostasis and obesity [26]. Taken together, BMI, gender, and drinking all potentially modulate the association that ErbB3 Levels and hypertension with overweight.
However, the mechanisms that link ErbB3 and hypertension and overweight are currently unclear. Previous studies have explored potential mechanisms linking adiposity and high BP, including sympathetic nervous system activation, activation of the renin-angiotensin system, in ammatory responses and insulin resistance [27]. It may be speculated that ErbB3 participating in networks related to lipid metabolism which is known a major variable in the etiology of overweight and hypertension [28]. Beside,ErbB3 participate in neutrophil survival and ErbB3 inhibitors play positive roles in accelerating in ammation resolution [29], which is known predisposing factor for development and progression of hypertension and overweight. Signaling interactions have been reported between ErbB3 family members and IGF-IR [30], and our group has previously shown that IGF-IR may contribute to the genetic susceptibility to hypertension [31], and the higher IGF-IR mRNA expression observed in obese children [32]. It is reasonable to believe that the ErbB3 levels play an important role in the regulation of hypertension with overweight by effecting on IGF-IR.
There are a few limitations to our study. Firstly, the potential bias in cross-sectional study often distorted results for epidemiological association studies. Secondly, no signi cant differences in the plasma ErbB3 levels were observed among the three groups in the total population, despite the fact that strict standards were used to select representative cases and controls. Relatively small sample size may have led to weak statistical power. Thus, further prospective studies are required to con rm the observations, and increase the sample size and research involving obese population studies are warranted

Conclusions
In summary, our results demonstrated for the rst time that expression of ErbB3 levels was signi cantly down-regulated in hypertension with overweight, and BMI, gender, and drinking all potentially modulating the process. Therefore, it is of great value to prevent the development of hypertension with overweight by guiding the early and correct management of ErbB3 levels.  CNT-healthy control group ;HT-hypertension group ;HTO-hypertension with overweight group; BMI-body mass index; SBP -systolic blood pressure; DBP-diastolic blood pressure ;GLU-glucose; TC-total cholesterol; TG-triglycerides, HDL-C-high-density lipoprotein cholesterol; LDL-Clow-density lipoprotein cholesterol; UA -uric acid. P: All participants had the variables analyzed by one-way ANOVA or Chi-square test among CNT, HT and HTO groups.*: p < 0.05 vs. CNT.# : p < 0.05 vs. HT.  CNT-healthy control group ;HT-hypertension group ;HTO-hypertension with overweight group; BMI-body mass index; SBP -systolic blood pressure; DBP-diastolic blood pressure ;GLU-glucose; TC-total cholesterol; TG-triglycerides, HDL-C-high-density lipoprotein cholesterol; LDL-Clow-density lipoprotein cholesterol; UA -uric acid.