Introduction
Pressure overload (PO) associated with essential hypertension induces various changes, including sarcomere rearrangement, cardiomyocyte enlargement, and cardiac fibrosis, leading to cardiac hypertrophy and remodeling.1-3 Continuous PO in essential hypertension may be the driving force for recruitment of inflammatory cells and proinflammatory cytokines, which is indispensable for the development of cardiac hypertrophy and remodeling. Moreover, during chronic inflammatory response over long-term PO, a large number of cardiomyocytes are lost, leading to cardiac diastolic and systolic dysfunction with reduced ejection fraction, which is associated with adverse outcomes.
Recently, studies have suggested that type 17 helper T (TH17) cells are involved in many inflammatory cardiovascular diseases and play a critical role in the pathogenesis of myocardial dysfunction.4,5 In our previous studies, TH17 responses amplified the inflammatory responses in the arterial wall and induced the endothelial dysfunction in atherosclerosis. TH17-related cytokines greatly contribute to the accumulation of fibrillar extracellular matrix in atrial fibrillation, and can act as an independent predictor of a higher risk for atrial fibrillation recurrence after catheter ablation.6,7 However, the role of TH17 cells in hypertension-induced PO has not been well defined. We hypothesized that TH17 responses contributed to myocardial fibrosis, elevated filling pressure, increased diastolic stiffness, and reduced distensibility, which could trigger left ventricular hypertrophy (LVH) and remodeling, and subsequently, left ventricular diastolic dysfunction (LVDD) appeared with further progression of heart disease.
In the present study, we aimed to investigate the relationship between TH17 cell differentiation and cardiac remodeling in adults with hypertension. Moreover, we also identified the effect of TH17 responses on the prognosis of hypertensive patients.
Patients and methods
Study population
A total of 187 consecutive patients who were diagnosed with essential hypertension and never received regular antihypertensive therapy, and 70 healthy adults (matched for age and sex) admitted to a health examination center at the same time were enrolled in the present study. All patients had preserved left ventricular ejection fraction (LVEF; >55%). The exclusion criteria were as follows: coronary artery disease, diabetes mellitus, valvular heart disease, hypertrophic cardiomyopathy, and renal disease.
Based on left ventricular mass index (LVMI),8 hypertensive patients were assigned into 2 groups. Left ventricular hypertrophy (LVH) was defined as LVMI greater than 125 g/m2 in men and LVMI greater than 110 g/m2 in women. Those without LVH constituted the NLVH group. The images of echocardiography of the control, NLVH, and LVH groups are presented in Supplementary material, Figure S1.
Follow-up
Blood specimens were obtained from all participants at baseline. All patients underwent routine echocardiography at baseline and after 24 months of follow-up. Hypertensive patients were regularly followed-up at 3, 6, 9, 12, 18, and 24 months by both clinical visits and telephone calls for the occurrence of the primary endpoint (Figure 1). The primary endpoint included new-onset angina pectoris, new-onset arrhythmia, myocardial infarction, myocardial ischemia, new-onset heart failure, and all-cause mortality. As this was an observational study, the diagnostic procedures and treatment were not assessed.
Figure 1. Flow chart showing the enrollment protocol
Abbreviations: LVDD, left ventricular diastolic dysfunction; LVH, left ventricular hypertrophy; LVMI, left ventricular mass index
Two-dimensional Doppler echocardiography
A routine echocardiographic evaluation was performed using a commercially available ultrasound device (Vivid E9; GE Healthcare, Horten, Norway) equipped with an M5S transducer. Images and parameters were obtained in 3 left ventricular short-axis views (basal, middle, and apical) and 3 left ventricular apical views (4-chamber, 2-chamber, and long-axis views). The following parameters were determined according to the recommendations of the American Society of Echocardiography and the European Association of Cardiovascular Imaging9: left ventricular end-diastolic diameter, left ventricular posterior wall diameter, interventricular septal thickness in end-diastolic period, LVEF, left atrial volume index, peak tricuspid regurgitation velocity (TRmax), tissue Doppler velocities in early diastole (e’), E wave deceleration time; change in E/A with Valsalva maneuver (val ∆E/A), duration of pulmonary vein flow and mitral inflow during atrial contraction (Ar–A duration), and stroke volume. The following LVDF parameters were also assessed: 1) LV relaxation; 2) average E/e’ ratio; 3) mitral E/a ratio; left atrial pressure; 4) LA volume index; and 5) peak tricuspid regurgitation (TR) velocity. Different grades of LVDD were identified according to the 2016 American Society of Echocardiography and the European Association of Cardiovascular Imaging guidelines.9
Flow cytometric analysis
Cell suspensions from blood were stained with anti-CD4 mAbs (eBioscience, San Jose, California, United States) followed by permeabilization and incubation with intracellular anti-interleukin (IL)-17 mAbs (eBioscience). The stained cells were analyzed with a FACS Calibur instrument (Becton Dickinson, Shanghai, China).
Enzyme-linked immunosorbent assay (ELISA)
A blood sample (5 ml) was centrifuged at 2000 g for 15 min at 4 °C within 2 hours of collection. Subsequently, the obtained plasma was aliquoted into 500-μl straws and immediately stored at –80 °C prior to further analysis. Serum levels of IL-17, IL-6, IL-23, matrix metallopeptidase 9, procollagen type I , and procollagen type III were determined using a commercial biotin / avidin-based ELISA kits (eBiosciences).
Intra- and interobserver variability
A single observer analyzed all imaging data twice in random order separated by an interval of 1 month to test intraobserver variability. A second observer analyzed the data without knowledge of the results of the first observer to test interobserver variability. Inter- and intraobserver variability for LVDF parameters were described in Supplementary material, Table S1. The variability of all LVDF parameters was less than 7%.
Statistical analysis
Descriptive data were expressed as means (SD) or as medians with interquartile ranges in case of skewed distribution. Categorical variables were expressed as numbers (percentages). Continuous variables were analyzed using analysis of variance (ANOVA) or the Mann–Whitney test as appropriate. When a significant difference was found in ANOVA, multiple comparisons were made using the Bonferroni procedure with type I error adjustment. The Pearson correlation test was adopted to investigate bivariate correlations. Moreover, the analysis of ROC curves was also conducted. The optimal prognostic cutoff (nonparametric method) of percentage of TH17 was derived from the AUC assessed at baseline. We used the Youden index for best sensitivity and specificity values. The optimal cutoff was calculated as sensitivity + specificity – 1. The independent effect of TH17 on LVH occurrence, after controlling for baseline clinical characteristics and echocardiographic parameters (adopted factors: sex, body surface area, body mass index, heart rate, hyperlipidemia, smoking, systolic blood pressure [SBP], left ventricular end-diastolic volume, LVEF, TRmax, average E/E’, E wave deceleration time, E, A, E/A, val ∆E/A, Ar–A duration, TH17 cells) was assessed via logistic regression analysis. The χ2 was used to compare frequencies of the normal LVDF between-groups comparisons. Survival curves for the primary endpoint as a function over time were obtained using the Kaplan–Meier method and compared using the log-rank statistic. Cox regression analysis was used to assess the correlations between TH17 cells and the considered primary endpoint, baseline clinical and echocardiographic values were used as adjustment variables. All assays were conducted using the SPSS software, version 11.5 (Chicago, Illinois, United States). A P value of less than 0.05 was considered statistically significant.
Ethics
Informed consent was obtained from every participant, and the study was approved by the Ethics Committee of Affiliated Hospital of Jiangsu University.
Results
Patient characteristics and echocardiographic variables
The baseline clinical and echocardiographic characteristics of all participants are presented in Table 1. Hypertensive patients were assigned into 2 groups according to LVMI: LVH group (n = 96) and NLVH group (n = 91). The control group was compared with the NLVH group and LVH group, respectively. SBP, left atrial diameter, the interventricular septal thickness at the end of diastole, thickness of the posterior left ventricular wall at the end of diastole, Ar–A duration, A, and E/A of the NLVH and LVH groups were different from the control group. The pattern in SBP, left atrial diameter, Ar–A duration of the control, NLVH, and LVH groups could be ranked as follows: LVH group, NLVH group, control group. The E/A was the maximal, and the A value was the minimum in the control group. Left atrial volume index, TRmax, average E/e’, val ∆E/A showed higher levels in the LVH group when compared with the NLVH group and the control group (Supplementary material, Table S2).
Characteristic | Control (n = 70) | NLVH (n = 96) | LVH (n = 91) |
|---|---|---|---|
Male sex, n (%) | 43 (61) | 58 (60) | 56 (62) |
BSA, m2, mean (SD) | 1.79 (0.4) | 1.81 (0.1) | 1.76 (0.2) |
BMI, kg/m2, mean (SD) | 22.5 (2.1) | 22.3 (2.7) | 22.1 (1.9) |
Heart rate, bpm, mean (SD) | 66 (13) | 68 (12) | 67 (13) |
Hyperlipidemia, n (%) | 11 (16) | 19 (20) | 15 (16) |
Smoking, n (%) | 16 (23) | 23 (24) | 19 (21) |
SBP, mm Hg, mean (SD) | 121 (13) | 137 (17) | 151 (14) |
DBP, mm Hg, mean (SD) | 76 (9) | 77 (11) | 80 (10) |
LAD, mm, mean (SD) | 34 (3) | 38 (4) | 41 (5) |
IVSD, mm, mean (SD) | 8.9 (1.1) | 9.2 (0.4) | 11.6 (1.4) |
LVPWD, mm, mean (SD) | 8.7 (0.8) | 9.1 (0.6) | 11.9 (1.6) |
LVEDV, ml, mean (SD) | 79 (16) | 81 (11) | 82 (14) |
LVESV, ml, mean (SD) | 31 (7) | 32 (12) | 32 (8) |
LVEF, %, mean (SD) | 66 (3) | 65 (8) | 66 (5) |
LAVI, ml/m2, mean (SD) | 25 (5) | 25 (7) | 34 (8) |
TR max, cm/s, mean (SD) | 238 (27) | 240 (33) | 269 (35) |
Average E/e’, median (IQR) | 10.3 (10–18.9) | 11.2 (11–19.6) | 15.9 (10.3–19.8) |
DT, cm/s, mean (SD) | 203 (32) | 206 (37) | 207 (42) |
E, m/s, mean (SD) | 0.82 (0.13) | 0.84 (0.15) | 0.76 (0.11) |
A, m/s, mean (SD) | 0.41 (0.04) | 0.53 (0.07) | 0.87 (0.08) |
E/A, median (IQR) | 2.13 (1.56–2.91) | 1.76 (1.02–2.32) | 1.31 (0.67–1.93) |
val ∆E/A, median (IQR) | 0.41 (0.3–0.65) | 0.40 (0.32–0.61) | 0.81 (0.46–1.02) |
Ar-A duration, ms, median (IQR) | 9 (6–19) | 18 (11–31) | 30 (18–43) |
Abbreviations: A, peak velocity during late diastole of anterior mitral leaflet; Ar–A duration, duration of pulmonary vein flow and mitral inflow during atrial contraction; BSA, body surface area; BMI, body mass index; DBP, diastolic blood pressure; DT, E wave deceleration time; E, peak velocity during early diastole of anterior mitral leaflet; e’, Tissue Doppler velocities in early diastole; IVSD, interventricular septal thickness in end-diastolic period; IQR, interquartile range; LAD, left atrial diameter; LAEF, left atrial emptying fraction; LAVI, left atrial volume index; LVEDV, left ventricular end-diastolic volume; LVEF, left ventricular ejection fraction; LVESV, left ventricular end-systolic volume; LVPWD, left ventricular posterior wall thickness in end-diastolic period; NLVH, patients without left ventricular hypertrophy; SBP, systolic blood pressure; TR max, peak tricuspid regurgitation velocity; val ∆E/A, change in E/A with Valsalva maneuver; others, see Figure 1 | |||
Higher circulating TH17 cell percentages in adults with hypertension
We measured the percentage of TH17 cells and serum TH17-related cytokines (IL-17, IL-6, and IL-23) in patients at baseline. A higher TH17 expression was observed in hypertensive patients when compared with the control group, and the percentage of TH17 cells was significantly higher in the LVH group compared with the NLVH group (Figure 2A and 2B). Using ROC curves, we observed that TH17 differentiation (AUC, 0.943; 95% CI, 0.914–0.971; P <0.001) showed a significant AUC (Figure 2C), and the cutoff value was 2.3%. On logistic regression analysis, the percentage of TH17 cells was an independent predictor of LVH (odds ratio [OR], 1.47, 95% CI, 2.23–2.28; P = 0.005). Factors associated with LVH in hypertensive patients are depicted in Supplementary material, Table S3. Univariable logistic regression analysis showed that SBP, TRmax, average E/e’, A, E/A, val ∆E/A, Ar–A duration, TH17 cells were significantly associated with LVH. Multivariable logistic regression analysis showed that SBP and TH17 cells were independent predictors of LVH in hypertensive patients, after adjusting for potential confounders.
Figure 2. Type 17 helper T (TH17) cell differentiation in the study groups. A, B – flow cytometric analysis of TH17 cells; C – receiver operating characteristic curve for the prediction of left ventricular hypertrophy using TH17 cell differentiation; D – expression of interleukin (IL) 17, IL-6, and IL-23 in the study groups.
a P <0.05
Abbreviations: AUC, area under the receiver operating characteristic curve; others, see Figure 1 and Table 1
We observed that the levels of IL-17 and IL-6 in the LVH group were higher in hypertensive patients compared with the control group, and the levels of IL-17 and IL-6 in the LVH group were significantly higher compared with the NLVH group (P <0.001). There was no difference in the levels of IL-23 among the 3 groups (Figure 2D).
Relationship between TH17 cells and fibrotic parameters
We analyzed the correlations between TH17 cells and fibrotic parameters in hypertensive patients at baseline. The proportion of TH17 cells was positively associated with the levels of matrix metallopeptidase 9 (r = 0.35; P <0.001), procollagen type I (r = 0.19; P = 0.008), and procollagen type III (r = 0.18; P = 0.01) (Figure 3).
Figure 3. Association of helper T 17 (TH17) cell differentiation with fibrosis parameters in adults. A – correlation between the proportion of TH17 cells and matrix metallopeptidase 9 (MMP-9); B – correlation between the proportion of TH17 cells and procollagen type I; C – correlation between the proportion of TH17 cells and procollagen type III
Abbreviations: see Figure 2
Assessment of the prognosis of hypertensive patients
According to the cutoff value of the percentage of TH17 cells, hypertensive patients were divided into 2 groups: those with the cutoff of less than 2.3% (the <2.3% group; n = 101) and those with the cutoff of 2.3% and greater (the ≥2.3% group; n = 86). Based on the antihypertensive therapy, we reevaluated the LVDF at 24-month follow-up.
Even in asymptomatic patients, the 3-to-5-year mortality rate of patients with grade I LVDD is 5-fold higher than that of patients with normal diastolic function.9 We compared the proportion of patients with normal LVDF in different groups at baseline and after 24-months of follow-up. We observed that the proportion of patients with normal LVDF in the <2.3% group was higher compared with the patients in the ≥2.3% group at baseline (P <0.001). At 24-month follow-up, the proportion of patients with normal LVDF changed from 49% to 64% (P = 0.03) in the <2.3% group, and when compared with the <2.3% group, the proportion gap was further widened (P <0.001). However, the proportion of patients with normal LVDF in the ≥2.3% group had no significant improvement at 24-month follow-up (Figure 4A and 4B).
Figure 4
The prognostic impact of T helper 17 (TH17) cell differentiation in adults with hypertension. A – left ventricular diastolic function at baseline and at last follow-up; B – normal left ventricular diastolic function at baseline and at last follow-up; C – survival analysis according to the proportion of TH17 cells. Kaplan–Meier event-free rates of the adverse cardiac events and all-cause mortality.
Abbreviations: LVDF, left ventricular diastolic function; others, see Figure 1
During the follow-up period of 24 months, 29 patients (15%) met the composite primary endpoint. The higher percentage of TH17 cells (≥2.3%) was associated with a higher probability of the primary endpoint in unadjusted analysis (P <0.001). Importantly, the percentage of TH17 cells was also significantly associated with an increased risk of the primary endpoint after multivariable adjustment for LVMI, LVH, and baseline clinical characteristics (P = 0.003, hazard ratio, 1.52; 95% CI, 1.02–1.87). The higher the percentage of TH17 cells, the poorer the prognosis (Figure 4C).
Discussion
In the present study, we demonstrated that TH17 responses were indispensable for hypertension-induced PO, which contributed to cardiac hypertrophy and remodeling, and a high percentage of TH17 cells could be used as a potential inflammatory marker to reflect myocardial fibrosis in hypertensive patients. Moreover, TH17 responses could be used as an independent predictor of LVDF in the development of hypertensive structural heart disease.
Hypertension-induced chronic inflammation activation and infiltration into LV negatively contributed to the progression of myocardial dysfunction through myocardial fibrosis, LVH, and remodeling. TH17 responses have been identified as a new prognostic biomarker of cardiac abnormalities that can promote stiffness of arteries and fibrosis in the myocardium, playing a critical role in cardiac remodeling.10-12 Consistent with previous studies, our results demonstrated that the percentage of TH17 cells was significantly higher in patients with LVH, and strongly correlated with fibrotic parameters in hypertensive patients.
TH17 responses trigger early-phase ventricular remodeling as demonstrated by higher expressions of fibrosis-related cytokines, interstitial fibrosis, and microvascular endothelial dysfunction. Then, TH17 responses aggravate late-phase ventricular remodeling as presented by ventricular fibrosis and apoptosis, impair ventricular relaxation, and lead to cardiac dysfunction.12,13 Increasing evidence has linked TH17 responses with myocardial fibrosis in hypertensive structural heart disease,14,15 and here, we showed that activation of TH17 responses could promote the expression of fibrosis-related cytokines in the progression of ventricular remodeling. In this way, a malignant circle was created in hypertension-induced PO, which contributed to cardiac hypertrophy and remodeling. If we could intervene at the early-phase of ventricular remodeling, the cardiac abnormalities might be reversed. Otherwise, systolic and diastolic dysfunctions and adverse cardiovascular events would happen.
TH17 responses induces hypertension in a rat model when the disease aggravates,16 hypertension-induced PO causes several changes in hemodynamics, and the fluid shear stress stimulates a systemic inflammatory state in the circulation. Our study also showed coherent relationships between hypertension-induced PO, TH17 responses, and TH17-related cytokines in the immune systems. Therefore, systemic inflammation occurs before myocardial fibrosis, and the increased percentage of TH17 cells is observed before a higher expression of fibrosis-related cytokines. In the initiation of cardiac disorder, diastolic dysfunction usually appears first, and it contributes to the adverse clinical outcome.17-19 In this study, we found that the differentiation of TH17 cells was strongly correlated with the levels of fibrosis-related cytokines in hypertensive patients, indicating that TH17 responses played a profibrotic role in promoting myocardial fibrosis and LV remodeling, eventually leading to LVDD.
Left ventricular hyperthrophy and remodeling were identified as important contributors to high myocardial diastolic stiffness, especially in LVDD. Based on the above-mentioned data, we hypothesized that TH17 responses induced LV remodeling, and thus regulated LVDF in hypertensive structural heart disease.
According to the cutoff value of the percentage of TH17 cells, the higher percentage of TH17 cells was dramatically associated with poor prognosis, suggesting its effect on the development of myocardial fibrosis, and irreversible ventricular remodeling. Therefore, TH17 responses were a critical factor affecting cardiac hypertrophy and remodeling in hypertensive patients, and it could be used as a more reliable diagnostic indicator for prognosis of hypertension-induced PO.
However, this study is a single-center trail, and the sample size was relatively small. Therefore, it is necessary to carry out further studies with larger sample size and multicenter trails. Second, in the present study, we demonstrated that TH17 responses induced cardiac hypertrophy and remodeling in essential hypertension. However, the underlying molecular mechanism remains largely unexplored. Further investigation on the molecular mechanisms is still ongoing.
Collectively, the differentiation of TH17 cells reflected cardiac hypertrophy and remodeling response to hypertension-induced PO, and contributed to LVDD by chronic inflammation and myocardial fibrosis. Moreover, the differentiation of TH17 cells could be a potential inflammatory marker to predict the prognosis of hypertensive patients.
Wei Yuan, PhD, Department of Cardiology, Affiliated Hospital of Jiangsu University, 438 Jiefang Road, Zhenjiang, Jiangsu Province, China, phone: +86 0511 85026387, email: dryuanwei123@163.com
September 22, 2020.
February 7, 2021.
February 11, 2021.
This study was supported by the project of preventive medicine of Jiangsu Province (no. Y2018110; to LX), the medical research project of Jiangsu provincial health committee (no. H2019075; to LX), the project of Social Development of Zhenjiang (no. SH2019087; to TF], and the Zhenjiang Cardiovascular Clinical Research Center Project (no. SS2018008; to WY).
WY and LX conceived the concept of the study. FZ, TP, and GC contributed to the design of the research and statistical analysis. HZ and XC were involved in data collection. LX and CZ analyzed the data. YL coordinated the project tasks. All authors edited, revised, and approved the final version of the manuscript.
None declared.
Xu L, Chen G, Liang Y, et al. T helper 17 cell responses induce cardiac hypertrophy and remodeling in essential hypertension. Pol Arch Intern Med. 2021; 131: 257-265. doi:10.20452/pamw.15811
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