INTRODUCTION

Hypertension is one of the primary risk factors for cardiovascular and cerebrovascular diseases [1]. The latest data from the survey on China's population shows that the number of people suffering from hypertension is 245 million, among which 21.9%, being then prevalent ones [2]. Long-term ventricular remodeling is one of the most important manifestations of hypertension-mediated organ damage [3]. The prevalence of hypertension and ventricular remodeling are significantly increased in perimenopausal women compared with men of the same age [4]. More importantly, the occurrence of perimenopausal myocardial remodeling and the process of treatment reversal are more complicated than in men [5]. Therefore, an effective reversing of the ventricular remodeling is crucial in patients with perimenopausal hypertension.

Myocardial fibrosis represents strong evidence of poor prognosis in hypertensive patients with ventricular remodeling. No myofibroblasts are present in the normal myocardial tissue [6]. Cardiac fibroblasts at rest are activated into myofibroblasts that express α-smooth muscle actin (α-SMA) and synthesize large amounts of extracellular matrix proteins under pathological conditions [7,8]. The myofibroblast phenotype is intermediate between cardiac fibroblasts and smooth muscle cells and is induced by cytokines, especially the transforming growth factor-β (TGF-β) [9]. Previous studies reported that the promotion of TGF-β1 expression induces cardiac fibroblast activation, myofibroblast differentiation, and excessive production of extracellular matrix [10]. Pro-fibrosis-related factors may also be involved in myocardial remodeling in perimenopausal hypertensive women.

The disorder of sex hormone levels in perimenopausal women contributes to the abnormal activation of the renin–angiotensin–aldosterone system (RAAS) [11,12]. Sacubitril/valsartan is a novel multipathway antihypertensive agent inhibiting the RAAS system and enhancing the natriuretic peptide system, thereby reducing the risk of cardiovascular events. The previous study of our team found that sacubitril/valsartan regulates RAAS in female spontaneous hypertensive rats compared with valsartan, thus exerting its antihypertensive and cardioprotective effects [13]. However, the effect of sacubitril/valsartan in women with perimenopausal hypertension is still uncertain. Therefore, this study evaluated the hypothesis of the superior effects of sacubitril/valsartan compared with valsartan on the reversal of myocardial remodeling in women with perimenopausal hypertension, which would result in a better regulation of biomarkers of myocardial fibrosis.

PATIENTS AND METHODS Study design and participants

A total of 292 women diagnosed with perimenopausal hypertension who were admitted to the Hypertension Department of Lanzhou University Second Hospital (Lanzhou, China) were included in this prospective, randomized, actively controlled open-label study. The inclusion criteria were as follows. age 45–65 years and meeting the definition of perimenopause in the reproductive aging staging criteria (irregular menstrual cycles; more than 60 days of amenorrhea; within 12 months after menopause [14]); diagnostic criteria for hypertension according to the 2018 Chinese guidelines for the management of hypertension (SBP ≥140 mmHg and/or DBP ≥90 mmHg [15]); and no other comorbidities and no antihypertensive treatment.

The main exclusion criteria were the following: secondary hypertension; therapy with hormone replacement or hysterectomy or oophorectomy; history of hypersensitivity to the drugs used in the study or similar drugs; severe disease (tumor), life expectancy less than 1 year; and diabetes mellitus, hyperlipemia, coronary artery disease, hyperkalemia (serum potassium > 5 mmol/l), liver/kidney insufficiency, heart failure, arrhythmia, and thyroid disease.

This study was approved by the ethics committee of Lanzhou University Second Hospital and registered in Clinical trial.gov (Registration No.: NCT04800081). This study was performed according to the Helsinki and drug clinical trial quality management practices.

Randomization

The randomization process involved a validated automatic random number generator to generate a randomization list. The system used the consecutive ascending randomization numbers in the treatment blocks assigned to each study site. Eligible women were treated with sacubitril/valsartan 200 mg once daily (tablet) or valsartan 160 mg once daily (tablet) for 24 weeks in a ratio of 1 : 1 [16].

Criteria for termination, shedding, and withdrawal of research interventions (1) Women with severe adverse reactions during the treatment who were unable to follow the study. (2) Women who were treated with other antihypertensive medications during the study. (3) Women who were unable to comply with the clinical trial protocol and for whom the efficacy could not be reliably determined. (4) Women who dropped out of the study or were lost to follow-up during treatment. Ambulatory blood pressure monitoring

Central BP and brachial BP (MOBI-O-GRAPH PWA, Germany) was monitored in all the included women at 24 h before randomization and at 24 weeks of follow-up. The ambulatory BP monitor automatically inflates the cuff and saves the data after automatically measuring BP in the left upper limb. It was measured automatically one time every 30 min during the day and one time every 60 min at night. Perimenopausal women continued to follow their usual routine. Effective measurements accounting for more than 80% of the total measurements were considered as valid.

Echocardiography

Philips IE33 echocardiograph was used to determine the heart structure and function according to the guidelines from the American Society of Echocardiography and the European Association of Cardiovascular Imaging [17,18]. The following parameters were calculated as follows: left ventricular mass (LVM): LVM(g) = 0.8 × 1.04 × [(left ventricular end-diastolic diameter (LVEDd) + interventricular septal thickness + left ventricular posterior wall thickness (LVPWT))3 − LVEDd3] + 0.6. LVM index (LVMI) = LVM/body surface area. Relative wall thickness (RWT) = 2 × LVPWT/LVEDd. LV geometric patterns of perimenopausal hypertensive women were divided into the following four groups according to Ganau classification: normal geometry; concentric remodeling; concentric hypertrophy; and eccentric hypertrophy [19].

Measurements of sex hormone axis

Serum levels of estrogen, progesterone, testosterone, prolactin, follicle-stimulating hormone, and luteinizing hormone (Siemens, San Francisco, California, USA) were determined by chemiluminescence. An electrochemical luminescence analysis system (Roche, CobasE-601; Roche, Basel, Switzerland) was used for detection. The test was always performed by the same inspector. All tested items met the quality control requirements before proceeding to the analysis of the specimens. The internal coefficient of variation was 3.8–5.4% and between batch 4.3–4.6%.

Serum biomarkers of myocardial fibrosis regulation

Fasting blood samples were collected at 8 : 00–10 : 00. They were centrifuged for 15 min at 4000 rpm, the supernatant was collected and frozen at −80 °C for a unified determination. Serum concentrations of TGF-β, α-SMA, and connective tissue growth factor (CT-GF) (JiangLai, Shanghai, China) were measured at baseline and at 24 weeks by ELISA. The coefficient of variation within detection boards was less than 9%, and the coefficient of variation between detection boards was less than 11%.

Statistical analysis

Statistical analysis was performed using SPSS20.0 statistical software (IBM, Armonk, New York, USA). The sample size was calculated based on the observed standard deviation of 20.6 g and considering the difference of 8.1 g as the change in LVM from the baseline after drug administration for both groups. The total target sample size for randomization was 288 women with perimenopausal hypertension assuming a drop-out rate of 10% for each treatment group. This sample size was calculated to ensure the detection of the 90% superiority assessment ability of a statistically significant comparison between sacubitril/valsartan and valsartan at a two-sided significance level of 0.05.

Continuous variables with a normal distribution and homogeneity of variance were expressed as mean ± standard deviation. The independent sample t-test was used for intergroup comparison. The paired-sample t-test was used for the comparison between two time points before and after the same group. Median and interquartile range (P25, P75) were used to represent continuous variables without a conform to normal distribution and homogeneity of variance. The Mann–Whitney U test was used for comparison between groups. Categorical variables were expressed in frequency (percentage) and were compared using the chi-square or Fisher's exact tests. Baseline serum myocardial fibrosis markers and LVMI to compare the difference between the two groups at 24 weeks were calibrated by the analysis of covariance. The confounding factors to determine the effects of sacubitril/ valsartan and valsartan on serum myocardial fibrosis markers and LVMI in perimenopausal hypertension were corrected using the multivariate linear regression model. A value of P less than 0.05 was considered statistically significant.

RESULTS Patients and safety assessment

A total of 301 perimenopausal hypertension women were screened in this trial; six did not receive the assigned intervention, one was lost to follow-up, and two stopped the intervention spontaneously because of adverse events. The remaining 292 women with perimenopausal hypertension were evaluated at baseline and after 24 weeks of therapy (Fig. 1). The average age of the population was 55.10 ± 7.08 years. No statistical difference in BMI and sex hormone levels was found between the two groups (Table 1).

FIGURE 1:

Flow chart.

TABLE 1 - Baseline characteristics of the study population Variable Sacubitril/valsartan (N = 147) Valsartan (N = 145) P value Age (years) 55.42 ± 7.70 54.67 ± 6.18 0.153 BMI (kg/m2) 23.99 ± 2.96 24.21 ± 4.36 0.281 Menopause (%) 26 (17.69) 21 (14.48) 0.456 Kupperman score 8.75 ± 5.06 9.59 ± 4.02 0.128 FPG (mmol/l) 5.83 ± 1.81 6.18 ± 2.64 0.127 BUN 5.85 ± 1.80 5.97 ± 1.68 0.563 CR 61.32 ± 12.79 62.05 ± 13.05 0.637 UA 298.86 ± 60.98 288.45 ± 69.18 0.175 PRL (ng/ml) 8.05 ± 5.68 7.78 ± 4.27 0.657 FSH (mIU/ml) 59.77 ± 31.75 58.61 ± 27.18 0.744 LH (mIU/ml) 30.82 ± 13.38 30.24 ± 10.55 0.751 E2 (pg/ml) 22.66 ± 12.03 22.53 ± 14.93 0.930 PRGE, ng/ml) 0.34 ± 0.29 0.26 ± 0.14 0.351 T (ng/dl) 21.53 ± 7.14 21.79 ± 6.65 0.757 24-h mean SBP (mmHg) 142.83 ± 12.73 140.07 ± 11.96 0.062 24-h mean DBP (mmHg) 83.81 ± 8.91 82.45 ± 8.42 0.191 Daytime mean SBP (mmHg) 149.01 ± 14.02 147.73 ± 13.11 0.424 Daytime mean DBP (mmHg) 84.28 ± 8.59 84.80 ± 7.12 0.612 Night-time mean SBP (mmHg) 128.20 ± 13.62 125.93 ± 14.01 0.145 Night-time mean DBP (mmHg) 81.81 ± 10.39 81.33 ± 9.79 0.690 central SBP (mmHg) 130.60 ± 13.27 129.27 ± 13.17 0.398 central DBP (mmHg) 80.79 ± 9.24 81.17 ± 9.72 0.733 LVEF (%) 69.78 ± 5.83 68.62 ± 8.71 0.176 LVEDD (mm) 44.57 ± 4.35 44.77 ± 5.20 0.726 LAD (mm) 32.94 ± 5.53 33.44 ± 5.25 0.450 LVMI (g/m2) 69.25 ± 19.49 70.86 ± 25.11 0.541 RWT 0.38 ± 0.06 0.38 ± 0.07 0.600 E/A 0.97 ± 0.26 0.92 ± 0.23 0.071 E/e′ 11.15 ± 2.95 11.09 ± 3.32 0.847

BUN, blood urea nitrogen; CR, creatinine; eGFR, estimated glomerular filtration rate; E, estradiol; EVAAs, early vascular aging ambulatory score; FSH, follicle-stimulating hormone; FPG, fasting plasma glucose; E/A, peak velocity during early filling (E), late filling from atrial contraction (A); LAD, diameter of left atrium; LH, luteinizing hormone; LVMI, left ventricular mass index; LVEDD, left ventricular end diastolic diameter; LVEF, left ventricular ejection fraction; RWT, relative wall thickness; PRL, prolactin; PRGE, progesterone; T, testosterone.

Changes in central blood pressure and brachial blood pressure

The 24-h mean SBP, the 24-h mean DBP, daytime mean SBP, daytime mean DBP, night-time mean SBP, night-time mean DBP, central SBP and central DBP at baseline were not statistically significant between the two groups (Table 1). The 24-h mean SBP at 24 weeks of treatment was 120.08 ± 10.47 mmHg in the sacubitril/valsartan group versus 121.00 ± 9.76 mmHg in the valsartan group (P = 0.457). After 24 weeks of treatment, there was no difference in central SBP between the sacubitril/valsartan and valsartan groups (117.17 ± 11.63 versus 116.38 ± 11.58, P = 0.568). BP control was defined as 24-h mean BP below 130/80 mmHg. The BP control rate at 24 weeks was 64% in the sacubitril/valsartan group and 63% in the valsartan group.

Changes in echocardiography

No significant difference in the baseline LVMI was found between the two groups (Table 1). LVMI in the sacubitril/valsartan group was lower than that in the valsartan group at week 24 (P = 0.009) (Table 2). After 24 weeks of treatment, LVMI levels decreased from baseline in the sacubitril/valsartan and valsartan groups. LVMI decreased by 7.23 g/m2 from the baseline in the sacubitril/valsartan group and 3.70 g/m2 in the valsartan group at 24 weeks (P = 0.000 versus 0.017) (Table 2). A statistically significant difference in LVMI was found between the two groups at 24 weeks by covariance analysis after the adjustment for the baseline LVMI (P = 0.001) (Supplementary File 1, https://links.lww.com/HJH/C169). The proportion of centric remodeling, concentric hypertrophy, and eccentric hypertrophy decreased from 23 to 14% in women with perimenopausal hypertension at 24 weeks after sacubitril/valsartan treatment (Fig. 2).

TABLE 2 - Serum biomarkers of myocardial fibrosis and left ventricular mass index at baseline and 24 weeks in the two groups Sacubitril/valsartan Valsartan Mean difference P α-SMA (IU/l) Baseline 175.24 ± 47.38 170.70 ± 40.93 4.54 (−5.94 to 15.03) 0.395 Week 24 143.23 ± 44.08 154.61 ± 46.89 −11.38 (−22.00 to −0.75) 0.036 Mean change from baseline 32.00 (26.29 to 37.72) 16.08 (10.67 to 21.49) P 0.000 0.000 TGF-β (ng/ml) Baseline 157.37 ± 52.35 155.97 ± 48.28 1.400 (−10.48 to 13.28) 0.817 Week 24 92.98 ± 27.35 102.58 ± 47.97 −9.60 (−18.41 to −0.80) 0.033 Mean change from baseline 64.39 (57.17 to 71.62) 53.39 (44.15 to 62.63) P 0.000 0.000 CT-GF (pg/ml) Baseline 1115.24 ± 196.45 1156.32 ± 244.56 −41.09 (−92.27 to 10.09) 0.115 Week 24 1062.96 ± 174.74 1128.54 ± 190.26 −65.58 (−108.14 to −23.02) 0.003 Mean change from baseline 52.27 (15.93 to 88.61) 27.78 (-25.43 to 80.99) P 0.005 0.303 LVMI (g/m2) Baseline 69.26 ± 19.50 70.86 ± 25.12 −1.60 (−6.77 to 3.56) 0.541 Week 24 62.03 ± 15.00 67.16 ± 18.30 −5.13 (−8.99 to −1.27) 0.009 Mean change from baseline 7.23 (5.47 to 8.98) 3.70 (0.68 to 6.73) P 0.000 0.017

Independent sample t test was used for the comparison between the two groups at the same time points above, and paired t-test was used for the comparison between the two time points before and after the same group. α-SMA, α-smooth muscle actin; CT-GF, connective tissue growth factor; LVMI, left ventricular mass index; TGF-β, transforming growth factor-β.

FIGURE 2:

Distribution characteristics of left ventricular geometry patterns in different treatment groups.

Changes in serum biomarkers of myocardial fibrosis regulation

Changes in the serum indicators of fibrosis from the baseline to 24-week treatment are shown in Table 2. No differences in α-SMA, CT-GF, and TGF-β levels were found between the two groups at baseline. The levels of α-SMA, CT-GF, and TGF-β were reduced in the sacubitril/valsartan group compared with those at the baseline (P = 0.000, 0.005, and 0.000). The levels of α-SMA, TGF-β, and CT-GF in the sacubitril/valsartan group were lower than those in the valsartan group at 24 weeks (P = 0.036, 0.033, and 0.003). A statistically significant difference in α-SMA, CT-GF, and TGF-β was found between the two groups at 24 weeks by covariance analysis after adjusting for baseline (P = 0.000, 0.005 and 0.015) (Supplementary File 1, https://links.lww.com/HJH/C169).

Sensitivity analysis of serum myocardial fibrosis markers and left ventricular mass index changes in perimenopausal hypertension

LVMI between the two groups was statistically significant at 24 weeks after correcting for the confounding factors 24-h mean SBP, 24-h mean DBP, central SBP, and central DBP (P = 0.005). LVMI remained statistically significant between the two groups after further correction of the factors of age, BMI, and sex hormone levels (P = 0.002) (Table 3). α-SMA, CT-GF, and TGF-β were still statistically significant between the two groups after establishing models 1 and 2 for correcting for confounding factors, as shown in Table 3 (P = 0.015, 0.001, and 0.042).

TABLE 3 - Sensitivity analysis of serum myocardial fibrosis markers and left ventricular mass index changes in perimenopausal hypertension Model 1 Model 2 Dependent variable B t P B t P α-SMA 11.768 2.154 0.032 12.182 2.223 0.027 TGF-β 8.224 1.756 0.045 8.786 1.934 0.039 CT-GF 68.066 3.113 0.002 71.855 3.273 0.001 LVMI 5.317 2.728 0.007 5.362 2.826 0.005

Model 1: adjusted for 24-h mean SBP, 24-h mean DBP, central SBP and central DBP; model 2: adjusted for 24-h mean SBP, 24-h mean DBP, central SBP, central DBP, age, BMI, sex hormone levels (estradiol, follicle-stimulating hormone, luteinizing hormone, prolactin, progesterone, and testosterone.) and serum indexes (fasting plasma glucose, blood urea nitrogen, and creatinine). α-SMA, α-smooth muscle actin; CT-GF, connective tissue growth factor; LVMI, left ventricular mass index; TGF-β, transforming growth factor-β.

DISCUSSION

Our study mainly found that sacubitril/valsartan and valsartan exerted different effects on ventricular remodeling in perimenopausal hypertensive women. LVMI improved more after sacubitril/valsartan treatment, and this phenomenon was not related to BP control and sex hormone levels. In addition, TGF-β, α-SMA, and CT-GF levels in perimenopausal hypertensive women in the sacubitril/valsartan group were significantly decreased after 24 weeks of treatment. The factors reversing cardiac remodeling and the down-regulation of fibrosis-related factors observed in this study might provide an explanation for the role of sacubitril/valsartan in perimenopausal hypertensive women.

The central arterial pressure is the lateral pressure of the aortic root, which is one of the important factors to evaluate the progression of cardiovascular and cerebrovascular events. The PARAMETER study found that sacubitril/valsartan could reduce central arterial pressure in elderly patients with systolic hypertension and arteriosclerosis [20]. Our results showed that there was no significant change in central arterial pressure after 24 weeks of sacubitril/valsartan treatment as compared with valsartan. The possible reasons for this difference are related to the included patient population and the age differences. LVM is a risk factor that independently predicts cardiovascular mortality and all-cause mortality. Many studies have explored the relationship between central arterial pressure and LV hypertrophy, but the observed results are not completely consistent. Kollias et al.[21] found that central arterial pressure was a strong predictor of LVH through a meta-analysis of 12 studies. In contrast, Odili et al.[22] found that central artery pressure obtained by noninvasive SphygmoCor devices showed no advantage in predicting the occurrence of LV hypertrophy. Our results also found that improved LVMI effects after 24 weeks of sacubitril/valsartan treatment were independent of central arterial pressure.

Change in ventricular geometry is an independent predictor of the increased risk of cardiovascular disease in hypertensive patients [23,24]. Nowadays, multiple antihypertensive drugs reverse LV hypertrophy and improve adverse configurations [25]. A previous study performed by our research team showed that valsartan treatment in perimenopausal women controls BP in a better manner and improves cardiac function [26]. As a novel mechanism of antihypertensive drugs, sacubitril/valsartan inhibits RAAS and degradation of the natriuretic peptide system, which improves better fibrosis and ventricular remodeling compared to the effect of angiotensin-converting enzyme inhibitors and angiotensin receptor blockers [27]. The results of this study showed that the mean LVMI reduction level in perimenopausal hypertensive women of the sacubitril/valsartan treatment was higher than that in the valsartan group at 24 weeks. In addition, the proportion of women with concentric remodeling and concentric hypertrophy in the sacubitril/valsartan group decreased from 23 to 14% at 24 weeks of follow-up, versus 30 to 27% in the valsartan group, which further demonstrated that sacubitril/valsartan was superior to valsartan in reversing ventricular remodeling in women with perimenopausal hypertension.

This is the first study investigating the role of pro-fibrosis-related factors in myocardial remodeling in perimenopausal hypertensive patients. Cardiac fibroblasts play a key role in the process of myocardial fibrosis, leading to collagen remodeling by the activation and induction of the phenotypic transformation into myofibroblasts [28]. TGF-β is a cytokine secreted by immune cells, cardiac fibroblasts, and cardiomyocytes that induce trans-differentiation of cardiac fibroblasts and matrix collagen deposition [29]. The TGF-β signaling pathway in animal models of myocardial infarction and volume load heart disease promotes the transformation of cardiac fibroblasts into myofibroblasts by controlling the transient receptor potential channel 6 [30,31]. In addition, α-SMA contractility proteins are expressed during the trans-differentiation of cardiac fibroblasts to myofibroblasts during myocardial remodeling, with the synthesis of a large amount of extracellular matrix proteins [32,33]. This study found that LVMI was decreased, and the ventricular structure was improved after 24 weeks of treatment with sacubitril/valsartan in perimenopausal hypertensive women, which was accompanied with a reduction in serum TGF-β, CT-GF, and α-SMA levels. The above results suggested that sacubitril/valsartan might play a role in the inhibition of the pro-fibrosis-related factors in perimenopausal hypertensive women.

In addition, some limitations should also be considered in this study. Firstly, changes in BP and ventricular structure were observed only at two time points, without a dynamic analysis of the effect of sacubitril/valsartan on the left ventricular and left atrium structure over time. Secondly, the patient sample size was small, and the results need a confirmation in multiple centers and more perimenopausal hypertensive women. Finally, deficiencies in short-term randomized controls should also be considered. Despite the above limitations, the results observed in our study have at least the following important clinical implications. The use of sacubitril/valsartan in perimenopausal hypertensive women reversed ventricular remodeling more effectively than valsartan, potentially helping clinicians the plan of more effective and individualized treatment protocols for perimenopausal hypertension women. Moreover, the pro-fibrotic regulation might be the cellular basis of ventricular remodeling in perimenopausal hypertension women, potentially opening up new research area. Finally, the observed reversal of the ventricular remodeling might provide an explanation for the role of sacubitril/valsartan in perimenopausal hypertension women.

In conclusion, these findings suggest that sacubitril/valsartan may have certain advantages over valsartan in reversing ventricular remodeling, probably as a result of its superior effect on reducing pro-fibrosis-related factors. However, more evidence may be needed in the future to confirm the effect of sacubitril/valsartan on myocardial remodeling in perimenopausal hypertensive women.

ACKNOWLEDGEMENTS

We would like to thank our patients as well as the doctors and nurses who helped recruit the participants for this study. We are also grateful to statistics expert Guiyun Wu for providing statistical guidance.

Sources of funding: this study was supported by the National Natural Science Foundation of China (NSFC 82160089,81960086). This study was also supported by the project of the Science and Technology Commission of Gansu Province (17CXIFJ063).

Conflicts of interest

There are no conflicts of interest.

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