Abstract
Left ventricular hypertrophy (LVH) is the most common target organ damage in hypertension. Abnormal numbers or functions of CD4+CD25+Foxp3+ regulatory T lymphocytes (Tregs) can cause immune disorders, which participates in LVH. This study aimed to explore the role of Tregs in LVH by investigating circulating Tregs and associated cytokine levels in hypertensive patients with or without LVH. Blood samples were collected from 83 hypertensive patients without LVH (essential hypertension group, EH), 91 hypertensive patients with LVH (left ventricular hypertrophy group, LVH), and 69 normotensive controls without LVH (control group, CG). Tregs and cytokines were measured by flow cytometry and enzyme‐linked immunosorbent assays. We found that circulating Tregs were significantly lower in hypertensive patients than in CG subjects. It was lower in LVH than in EH patients. No correlation between blood pressure regulation and Tregs was found in EH or LVH patients. Furthermore, Tregs in older females were lower than those in older males among LVH patients. Additionally, serum interleukin‐10 (IL‐10) and transforming growth factor beta 1 (TGFβ1) decreased in hypertensive patients, and interleukin‐6 (IL‐6) increased in LVH patients. Tregs were negatively correlated with creatine kinase, low‐density lipoprotein cholesterol, apoprotein B, high‐sensitivity C‐reactive protein, and left ventricular mass index (LVMI) values. In general, our study demonstrates significantly decreased circulating Tregs in hypertensive LVH patients. Decreased circulating Tregs in LVH is independent of blood pressure regulation. IL‐6, IL‐10, and TGF‐β1 are related with LVH in hypertension.
Keywords: IL‐10, IL‐6, TGF‐β1, hypertension, left ventricular hypertrophy, Tregs
1. INTRODUCTION
Left ventricular hypertrophy (LVH), which happens in over 30% hypertensive patients, is an independent risk factor associated with adverse outcomes, including myocardial infarction, heart failure, and sudden death. 1 , 2 LVH persists even after the application of antihypertensive drugs, which indicates that LVH is not only a mechanical adaptation caused by the increase of blood pressure. 3 Inflammation and immunoregulation also play roles in hypertension‐associated LVH. 4
CD4+CD25+Foxp3+ regulatory T lymphocytes (Tregs), potent suppressors of effector T cells, can negatively regulate immune responses. 5 The trend of a decreased proportion of Tregs was observed in peripheral blood and multiple organs in hypertensive mice and rats even before the onset of hypertension. 6 , 7 Furthermore, approaches that increase circulating Tregs in vivo, such as adoptive transfer of Tregs and administration of an IL‐2/anti‐IL‐2 monoclonal antibody complex, were shown to effectively prevent the progression of hypertension in animals. 6 , 7 , 8 , 9 These studies suggest that Tregs may regulate the onset and progression of hypertension. Besides, Treg depletion can promote LVH in hypertensive rats 7 while increasing Tregs could attenuate the development of LVH in hypertensive mice, 10 suggesting that Tregs may negatively modulate the progress of hypertension‐associated LVH.
Tregs exert immunosuppressive effects mainly through the secretion of several anti‐inflammatory cytokines, including transforming growth factor (TGF)‐β1 and interleukin‐10 (IL‐10). 5 Recent studies have confirmed that IL‐10 secreted by Tregs plays vital roles in preventing hypertension. 11 Another cytokine, interleukin‐6 (IL‐6), can regulate immune homeostasis between T helper 17 (Th17) cells and Tregs. In hypertensive patients, increased circulating IL‐6 can block the TGF‐β1‐mediated generation of Tregs and enhance Th17 responses, 12 , 13 , 14 , 15 indicating that IL‐6 can negatively regulate the effects of Tregs. However, most of the reliable data are derived from animal models. Much less is known about the levels of circulating Tregs and related cytokines in human hypertension‐associated LVH.
The primary aim of this study was to compare the levels of Tregs and Tregs‐related cytokines between hypertensive patients with or without LVH and control subjects.
2. MATERIALS AND METHODS
In Supplemental Materials.
3. RESULTS
3.1. Clinical characteristics of study groups
Among the patients who provided blood samples for analysis, body weight, body mass index (BMI), incidence rate of smoking, and blood pressure (including systolic blood pressure [SBP] and diastolic blood pressure [DBP]) were significantly higher in the EH and LVH groups compared with the CG group (all p < .05), indicating possible roles of these comorbidities in the pathogenesis of EH and LVH. SBP was higher in the LVH group than in the EH group (p < .05). No significant differences in other characteristics, including age, body weight, BMI, incidence rate of smoking, and DBP, were found between the EH and LVH groups. The clinical data of all patients are listed in Table 1.
TABLE 1.
Information of clinical characteristics and blood biochemical indexes in CG, EH, and LVH groups
| Characteristics | CG | EH | LVH |
|---|---|---|---|
| Gender (M/F) | 45/24 | 55/28 | 59/32 |
| Age, years | 58.45 ± 12.86 | 58.30 ± 10.88 | 61.34 ± 11.17 |
| Height, cm | 162.34 ± 7.92 | 163.06 ± 7.19 | 163.86 ± 8.78 |
| Weight, kg | 60.58 ± 11.43 | 67.91 ± 11.18* | 69.91 ± 14.08* |
| BMI, kg/m2 | 22.86 ± 3.12 | 25.47 ± 3.30* | 25.88 ± 3.90* |
| SBP, mm Hg | 122.41 ± 16.19 | 141.30 ± 17.75* | 148.40 ± 23.68* , # |
| DBP, mm Hg | 78.45 ± 9.18 | 85.25 ± 13.01* | 87.01 ± 15.74* |
| Smoking, n (%) | 18 (26.1%) | 40 (48.2%)* | 43 (47.3%)* |
| Drinking, n (%) | 21 (30.4%) | 29 (34.9%) | 45 (49.5%)* , # |
| the course of hypertension, year | 0 | 7.0 (3.00, 7.00)* | 9.00 (4.00, 14.25)* |
| Diabetes mellitus, n (%) | 0 (0%) | 15 (18.1%)* | 34 (37.4%)* |
| Coronary artery disease, n (%) | 0 (0%) | 30 (36.1%)* | 43 (47.3%)* |
| WBC, ×109/L | 6.10 ± 1.37 | 6.53 ± 1.57 | 6.68 ± 1.66* |
| N, % | 61.04 ± 9.33 | 63.84 ± 7.84 | 64.79 ± 9.23* |
| hs‐CRP, mg/dL | 0.90 (0.42, 1.45) | 1.60 (0.53, 4.28) | 2.13 (1.22, 4.18)* |
| NT‐proBNP, pg/mL | 95.05 (13.00, 206.10) | 90.90 (31.79, 198.30) | 264.70 (70.85, 599.30)* , # |
| cTnT, μg/L | 4.24 (3.60, 7.52) | 7.87 (5.71, 11.77) | 12.88 (6.57, 26.59) |
| CK, pg/mL | 78.30 (58.45, 92.15) | 88.55 (64.88, 132.90) | 79.70 (54.65, 106.40) |
| CK‐MB, pg/mL | 12.50 (10.50, 16.45) | 15.10 (11.88, 17.63) | 14.00 (10.90, 17.95) |
| Creatinine, mmol/L | 64.60 ± 14.67 | 72.65 ± 25.86 | 100.18 ± 58.81* |
| TG, mmol/L | 1.29 (0.82, 1.99) | 1.63 (1.22, 3.04)* | 1.72 (1.20, 2.47) |
| TC, mmol/L | 4.41 ± 0.97 | 4.03 ± 0.94* | 3.94 ± 0.98* |
| HDL‐C, mmol/L | 1.12 (0.93, 1.32) | 0.91 (0.78, 1.080)* | 0.95 (0.81, 1.10)* |
| LDL‐C, mmol/L | 2.78 ± 0.83 | 2.51 ± 0.76 | 2.42 ± 0.85* |
| HDL‐C/TC | 0.26 ± 0.10 | 0.24 ± 0.07 | 0.26 ± 0.08 |
| free fatty acid, mmol/L | 0.25 (0.15, 0.35) | 0.33 (0.25, 0.43) | 0.36 (0.25, 0.45)* |
| Lp(a), mg/L | 94.00 (33.50, 189.3) | 198.10 (103.20, 479.40)* | 123.00 (61.00, 302.10)* |
| apoA, g/L | 1.04 (0.99, 1.19) | 0.93 (0.85, 1.13) | 0.99 (0.86, 1.07) |
| apoB, g/L | 0.89 ± 0.27 | 0.82 ± 0.22 | 0.79 ± 0.25* |
| HbA1c, % | 5.40 (5.23, 5.60) | 6.05 (5.70, 6.90)* | 6.50 (6.00, 7.60)* , # |
Abbreviations: apoA, apoprotein A; apoB, apoprotein B; BMI, body mass index; CK, creatine kinase; CK‐MB, isoenzyme of creatine kinase‐MB; cTnT, cardiac troponin T; DBP, diastolic blood pressure; WBC, white blood cell count; HbA1c, glycosylated hemoglobin A1; HDL‐C, high‐density lipoprotein cholesterol; hs‐CRP, high‐sensitivity C‐reactive protein; LDL‐C, low‐density lipoprotein cholesterol; Lp(a), lipoprotein(a); N, neutrophil percentage; NT‐proBNP, N‐terminal brain natriuretic peptide; SBP, systolic blood pressure; TC, total cholesterol; TG, triglyceride.
p < .05 vs. the CG group.
p < .05 vs. the EH group.
Compared with the CG group, the EH group did not show any significant differences in the levels of white blood cell count (WBC), neutrophil percentage, high‐sensitivity C‐reactive protein (hs‐CRP), or N‐terminal brain natriuretic peptide (NT‐proBNP). However, the levels of these characteristics were all significantly higher in the LVH group compared with the CG group (all p < .05), indicating upregulated inflammation in the LVH patients. The level of NT‐proBNP was higher in the LVH group than in the EH group (p < .05), but no significant differences in the WBC, neutrophil percentage, or hs‐CRP level were found (Table 1).
In addition, no significant differences were observed among the three groups for myocardial enzymological indexes, including cardiac troponin T (cTnT), creatine kinase (CK), and an isoenzyme of creatine kinase‐MB (CK‐MB) (Table 1).
Compared with the CG group, the EH group exhibited increased level of triglyceride, and the LVH group showed increased level of free fatty acids (all p < .05). Total cholesterol level was lower while lipoprotein (a) (Lp(a)) level was higher in the EH and LVH groups than in the CG group, and low‐density lipoprotein cholesterol (LDL‐C) and apoprotein B (apoB) levels were lower in the LVH group than in the CG group (all p < .05). High‐density lipoprotein cholesterol (HDL‐C) level was lower in the EH and LVH groups than in the CG group (all p < .05), but there were no differences in apoprotein A (apoA) level or the HDL‐C/total cholesterol ratio among the three groups (Table 1).
Compared with the CG group, the EH and LVH groups showed increased levels of glycosylated hemoglobin A1 (HbA1c) (all p < .05). The level of HbA1c was much higher in LVH patients than in EH patients (p < .05) (Table 1).
3.2. Analysis of patient echocardiograms
The left atrium diameter was higher in the EH group compared with the CG group (p < .05). Compared with the CG and EH groups, the LVH group showed higher left atrium and right atrium diameters (all p < .05).
The interventricular septal diameter (IVSd), posterior wall thickness (PWT), and left ventricular mass index (LVMI) values were significantly higher in the EH group compared with the CG group (all p < .05). Additionally, compared with the CG and EH groups, the LVH group showed higher left ventricular end‐diastolic diameter (LVEDd), IVSd, PWT and LVMI values (all p < .05).
In addition, the ejection fractions of the three groups were in the normal range (50%−70%), but the ejection fractions in the LVH and EH groups were both decreased compared with that in the CG group, and the ejection fraction was lower in the LVH group than in the EH group (all p < .05). These data are listed in Table 2.
TABLE 2.
Characteristics of echocardiogram in CG, EH, and LVH groups
| Characteristics | CG | EH | LVH |
|---|---|---|---|
| IVSd, mm | 9.00 (8.00, 10.00) | 10.00 (10.00, 11.00)* | 12.00 (11.00, 12.00)* , # |
| PWT, mm | 9.00 (8.00, 9.00) | 9.00 (9.00, 10.00)* | 11.00 (10.00, 11.00)* , # |
| LVEDd, mm | 45.43 ± 4.37 | 46.18 ± 3.09 | 50.54 ± 4.18* , # |
| LVMI, g/m2 | 93.40 (78.32, 110.00) | 107.00 (97.30, 113.80)* | 143.80 (133.50, 162.30)* , # |
| LA, mm | 31.50 (28.00, 33.25) | 34.00 (32.00, 37.25)* | 38.00 (35.25, 40.00)* , # |
| RA, mm | 31.00 (28.00, 33.00) | 31.00 (29.00, 33.00) | 33.00 (30.00, 34.00)* , # |
| RV, mm | 30.00 (27.00, 32.00) | 30.00 (29.00, 32.00) | 31.50 (29.00, 33.00) |
| EF, % | 63.93 ± 4.76 | 62.30 ± 3.69* | 57.80 ± 7.98* , # |
Abbreviations: EF, ejection fraction; IVSd, interventricular septum diameter; LA, left atrium; LVEDd, left ventricular end diastolic diameter; LVMI, left ventricular mass index; PWT, posterior wall thickness; RA, right atrium; RV, right ventricular.
p < .05 vs. the CG group.
p < .05 vs. the EH group.
3.3. Circulating Tregs and blood pressure control in hypertensive patients
The levels of circulating CD4+ T cells and Tregs were analyzed by flow cytometry, and the results showed that circulating CD4+ T cells were similar among the CG (41.66 ± 8.54%), EH (42.34 ± 8.03%), and LVH (41.14 ± 9.11%) groups (Figure 1A,B). Compared with the levels in the CG group (5.17 ± 2.16%), the Treg levels in the EH (4.29 ± 1.86%) and LVH group (3.68 ± 1.51%) were significantly decreased (Figure 1A,C), and the Treg level was lower in LVH patients than in EH patients (all p < .05) (Table 3). We further graded blood pressure in all hypertensive patients, it shows no significant difference of Tregs in Grade1,2,3 hypertension (Figure S2).
FIGURE 1.
Circulating CD4+ T cells and Tregs in CG, EH and LVH groups. Peripheral blood samples from CG (n = 69), EH (n = 83), and LVH (n = 91) patients were collected and isolated for cell suspensions. Circulating CD4+ T cells and Tregs were analyzed by flow cytometry. (A) Lymphocytes and CD4+ T cells were gated by flow cytometry and intracellular cytokine staining of Tregs were represented for each group. (B,C) The frequencies of CD4+ T cells and Tregs in CG, EH, and LVH groups. *p < .05 vs. the CG group; ***p < .0001 vs. the CG group; #p < .05 vs. the EH group.
TABLE 3.
Tregs and serum cytokine levels in CG, EH, and LVH groups
| Characteristics | CG | EH | LVH |
|---|---|---|---|
| Treg, % | 5.17 ± 2.16 | 4.29 ± 1.86* | 3.68 ± 1.51*** , # |
| IL‐10, pg/mL | 9.14 ± 2.22 | 6.12 ± 0.60* | 6.1 ± 1.45* |
| TGF‐β1, ng/mL | 42.92 ± 8.00 | 37.73 ± 10.40* | 37.09 ± 8.61* |
| IL‐6, pg/mL | 4.20 (2.95, 6.57) | 4.11 (2.16, 9.27) | 8.02 (3.97, 14.63)* , # |
Abbreviations: IL‐10, interleukin‐10; IL‐6, interleukin‐6; TGF‐β1, transforming growth factor beta 1.
p < .05 vs. the CG group.
p < .0001 vs. the CG group.
p < .05 vs. the EH group.
Blood pressure control is closely related to the occurrence of hypertensive myocardial hypertrophy. To explore whether the decreased level of Tregs in hypertensive patients is affected by blood pressure regulation, the EH and LVH groups were further divided into well‐controlled and poorly controlled groups according to each patient's blood pressure control situation using the average of up to three measurements. 16 The well‐controlled group was defined by a mean SBP < 140 mmHg and mean DBP < 90 mmHg, while the poorly controlled group was defined by a mean SBP ≥ 140 mmHg and/or mean DBP ≥ 90 mmHg. The results showed no difference in Treg levels between the well‐controlled and poorly controlled subgroups in either EH patients or LVH patients (Figure 2A). Moreover, no correlation was found between Tregs and the duration year of hypertension in LVH patients (Figure 2B). These results reveal that there is no significant correlation between blood pressure control and circulating Treg levels in hypertensive patients in our study.
FIGURE 2.
Blood pressure control‐ and sex‐difference of circulating Tregs in EH and LVH patients (A) Circulating Tregs in EH and LVH patients with well‐controlled or poor‐controlled blood pressure. (B) The correlation between Tregs and duration of hypertension in LVH patients. (C) Circulating Tregs in hypertensive patients between males (>49 years old) and postmenopausal females aged over 49 years old in EH and LVH patients. ns, means no significance between two groups. *p < .05 vs. EH male; #p < .05 vs. LVH male; △p < .05 vs. EH female.
3.4. Sex difference of circulating Tregs in hypertensive patients
In the EH and LVH groups, we further compared the proportion of circulating Tregs between male and female patients, but no sex difference in circulating Tregs was found (Figure 2C). Several studies have revealed that estrogen could protect the murine heart from pressure overload‐induced LVH. 17 , 18 , 19 Considering the possible role of estrogen in attenuating LVH, we compared the Treg levels in hypertensive patients between males and females aged over 49 years old (the median age at onset of menopause in Asia was 49 years old). 20
The results showed that circulating Tregs were lower in older females than in males (>49 years old) in both the LVH and EH groups (all p < .05) (Figure 2C and Table S1), indicating the possible interaction between Tregs and estrogen to protect female from hypertension and LVH. In addition, in older females, circulating Treg levels were still lower in the LVH group than in the EH group (p < .05), indicating that without the protective effect of estrogen, decreased circulating Treg levels play vital roles in the onset of LVH in female hypertensive patients.
3.5. IL‐10, TGF‐β1 and IL‐6 concentrations in hypertensive patients
To investigate the expression of functional cytokines related to Tregs, serum IL‐10, IL‐6, and TGF‐β1 levels were measured by ELISA. Decreased IL‐10 and TGF‐β1 concentrations were observed in the EH and LVH groups compared with the CG group (all p < .05) (Figure 3A,B and Table 3); no significant differences were observed between the EH and LVH groups for these cytokine concentrations. The serum IL‐6 level was increased in the LVH group compared with the EH and CG groups (all p < .05) (Figure 3C and Table 3). No significant difference of IL‐6 level was found between the EH and CG groups.
FIGURE 3.
Serum cytokine levels in CG, EH, and LVH groups. Peripheral blood samples from CG (n = 69), EH (n = 83), and LVH (n = 91) patients were collected using a coagulant. Serum IL‐10, IL‐6, and TGF‐β1 levels were measured by ELISA and the correlations between Tregs and cytokines were analyzed. (A–C) Serum IL‐10, TGF‐β1, and IL‐6 levels in the CG, EH, and LVH patients. (D–F) The correlations of Tregs and IL‐10, TGF‐β1, IL‐6 in CG, EH, and LVH patients. IL‐10, interleukin‐10; TGF‐β1, transforming growth factor beta 1; IL‐6, interleukin‐6. *p < .05 vs. the CG group; #p < .05 vs. the EH group.
We then assessed whether the level of circulating Tregs was associated with serum concentrations of these functional cytokines in CG, EH, and LVH patients, and correlation analysis showed that circulating Tregs were negatively correlated with the serum concentration of IL‐10 and positively correlated with the serum concentration of TGF‐β1 in LVH patients (all p < .05). No correlation was found between the level of Tregs and serum IL‐6 concentration among the three groups (Figure 3D–F).
3.6. Associations between circulating Tregs and clinical characteristics
We assessed whether the level of circulating Tregs were associated with the clinical characteristics of all 243 individuals. Correlation analysis showed that circulating Tregs were negatively correlated with LVMI, hs‐CRP, and CK levels (all p < .05) but not with age, smoking index, BMI, SBP, DBP, WBC, HbA1c, NT‐proBNP, cTnT, CK, CK‐MB, total cholesterol, triglyceride, HDL‐C, LDL‐C, Lp(a), apoA, or apoB levels (Table 4). In addition, the level of circulating Tregs were negatively correlated with CK, LDL‐C, and apoB levels in EH patients (all p < .05) (Table 5). In the LVH group, the level of circulating Tregs were negatively correlated with CK level (p < .05) (Table 5).
TABLE 4.
Correlations between Tregs and clinical characteristics were assessed by Pearson and Spearman's correlation analysis in total 243 patients
| Variables | Correlation coefficient | p‐value |
|---|---|---|
| Age | −0.0313 | .646 |
| BMI | 0.092 | .178 |
| Smoking | 0.0688 | .312 |
| LVMI | −0.1632 | .025* |
| SBP | 0.0083 | .903 |
| DBP | −0.0150 | .825 |
| WBC | 0.1262 | .064 |
| hs‐CRP | −0.1520 | .049* |
| cTnT | −0.0634 | .393 |
| CK | −0.1909 | .011* |
| CK‐MB | −0.1040 | .139 |
| NT‐proBNP | 0.0600 | .402 |
| Lp(a) | −0.1278 | .085 |
| HbA1c | 0.0090 | .922 |
| TG | −0.0378 | .588 |
| TC | 0.0309 | .658 |
| HDL‐C | −0.107 | .123 |
| LDL‐C | 0.0598 | .392 |
| apoA | −0.0260 | .727 |
| apoB | 0.0716 | .334 |
Notes: The Pearson's correlation coefficients were evaluated to detect the variables associated with Tregs, including age, BMI, SBP, DBP, WBC, TC, LDL‐C, apoB. The Spearman's rank correlation coefficients were evaluated to detect the variables associated with Tregs, including smoking, LVMI, hs‐CRP, cTnT, CK, CK‐MB, NT‐proBNP, Lp(a), HbA1c, TG, HDL‐C, apoA. The Tregs was regarded as a continuous normally distributed variable in this analysis.
Abbreviations: apoA, apoprotein A; apoB, apoprotein B; BMI, body mass index; CK, creatine kinase; CK‐MB, isoenzyme of creatine kinase‐MB; cTnT, cardiac troponin T; DBP, diastolic blood pressure; WBC, white blood cell count; HbA1c, glycosylated hemoglobin A1; HDL‐C, high‐density lipoprotein cholesterol; hs‐CRP, high‐sensitivity C‐reactive protein; LDL‐C, low‐density lipoprotein cholesterol; Lp(a), lipoprotein(a); LVMI, left ventricular mass index; NT‐proBNP, N‐terminal brain natriuretic peptide; SBP, systolic blood pressure; TC, total cholesterol; TG, triglyceride.
p < .05 means significant correlation.
TABLE 5.
Correlations between Tregs and clinical characteristics were assessed by Pearson and Spearman's correlation analysis in EH or LVH group
| Variables | Group | Correlation coefficient | p‐value |
|---|---|---|---|
| CK (pg/mL) | EH | −0.2621 | 0.028* |
| LDL‐C (mmol/L) | EH | −0.2394 | 0.041* |
| ApoB (g/L) | EH | −0.2398 | 0.046* |
| CK (pg/mL) | LVH | −0.2587 | 0.014* |
Abbreviations: apoB, apoprotein B; CK, creatine kinase; LDL‐C, low‐density lipoprotein cholesterol.
p < .05 means significant correlation.
4. DISCUSSION
In the present study, we observed for the first time that circulating Treg levels were decreased in patients with hypertensive myocardial hypertrophy compared with hypertensive patients without LVH. The Treg level was independent of the blood pressure control status. Among hypertensive patients, the circulating Treg level in older females was lower than that in males (>49 years old). We also are the first to observe that serum IL‐6 levels were increased in hypertensive patients with LVH compared with those without LVH. In addition, the serum levels of the anti‐inflammatory cytokines IL‐10 and TGF‐β1 were decreased in hypertensive patients. In addition, circulating Tregs were positively correlated with the levels of TGF‐β1 and negatively correlated with those of CK, hs‐CRP, LVMI, LDL, and apoB.
Tregs comprise 5%−10% of all peripheral CD4+ T cells in adults. Although CD4+ T cells have no obvious effect on Ang II‐induced blood pressure elevation, 21 Treg depletion or dysfunction can exacerbate endothelial dysfunction and promote the occurrence of hypertension. 7 , 22 , 23 Similarly, no significant differences in circulating CD4+ T cells were found among the CG, EH, and LVH groups in our study, while the proportion of circulating Tregs was significantly decreased in patients with hypertension, indicating that the reduced proportion of circulating Tregs might promote the progression of hypertension. Another study revealed that mice treated with Ang II or Ang II+ Tregs upregulated Foxp3+ cell infiltration in the heart, 24 indicating that circulating Tregs might be recruited to the heart during the development of hypertension. It is possible that the renin‐angiotensin system is activated in patients with hypertension and that Ang II activates chemoattractant signaling in the recruitment of Tregs from the circulation to cardiac tissue for accumulation in the early stage of hypertension; therefore, circulating Treg levels are decreased in patients with hypertension.
Hypertension increases the workload of the heart and causes structural or functional changes in the myocardium. These changes include LVH, which can cause an electric remodeling process and further contribute to increased incidences of ventricular arrhythmias and sudden cardiac death. Previous studies have revealed that a decreased proportion of Tregs is crucial for the onset of hypertension and LVH in mice and rats. 7 , 10 , 22 , 24 Hypertensive mice treated with Treg transfer develop less LVH and exhibit reduced susceptibility to inducible ventricular tachycardia, accompanied by diminished cardiac cell infiltration and a reduced proportion of activated splenic CD4+ cells. 24 Therefore, Tregs might inhibit LVH and electrical remodeling by inhibiting the activation of immune cells and their migration from peripheral immune organs into the heart. However, these results were all generated with animal models and do not reveal whether the proportion or function of Tregs is altered between LVH and EH patients. In this study, we found that circulating Treg levels were significantly lower in hypertensive patients with LVH than in those without LVH, suggesting that decreased circulating Treg levels further facilitate cardiac injury in hypertension.
To explore the correlation between Treg levels and blood pressure regulation, we compared the proportion of Tregs in hypertensive patients according to their blood pressure control condition. The results showed no difference in circulating Tregs between the well‐controlled and poorly controlled subgroups. Similarly, mice treated with Ang II+ Tregs were found to be as hypertensive as Ang II‐treated controls, but cardiac hypertrophy and fibrosis were significantly ameliorated. 24 These findings underscore the notion that Treg‐mediated cardiac protection might be independent of modulatory effects on blood pressure.
A study found that administration of valsartan significantly increased the frequencies of Treg cells in Ang II‐treated mice, 25 indicating that some type of antihypertensive drugs may also affect the level of circulating Tregs. To explore whether the Tregs was affected by different types of antihypertensive drugs, we collected the detailed medication (Table S2). Calcium channel blockers (CCB) and angiotensin‐converting enzyme inhibitors/Ang II receptor blockers (ACEI/ARB) were most frequently used in the treatment of hypertension in our study, but Tregs shows no significant difference compared with the untreated group. However, Tregs decreased in the beta‐blocker‐treated group. A recent study also shows that propranolol treatment alleviates asthma while reducing Treg number. 26
We also explored the sex difference in Tregs in hypertensive patients. We found lower Treg levels in females than in males among hypertensive patients over 49 years old, while no sex difference was found between males and females of all ages. It may be related to the postmenopausal status of older females. In previous studies, administration of 17β‐estradiol, the main circulating form of estrogen in premenopausal females, limited hypertension and LVH in ovariectomized female mice, 18 , 19 indicating that estrogen may protect premenopausal females from hypertension and LVH. These results indicate that Tregs may mediate the modulatory effect of estrogen in attenuating EH and LVH.
The protective effect of Tregs on microvascular endothelial function in hypertension may be attributed to the release of Treg‐associated anti‐inflammatory cytokines, such as TGF‐β1 and IL‐10, thereby inhibiting NADPH oxidase activity and improving endothelium‐dependent relaxation of the arteries. 27 , 28 , 29 We found for the first time that serum IL‐10 and TGF‐β1 levels were decreased in hypertensive patients with or without LVH, indicating a role for anti‐inflammatory cytokines in the development of EH and LVH. In addition, circulating Tregs were positively correlated with the levels of TGF‐β1 in our study. There are two potential explanations for this. First, Tregs can secret TGF‐β1. 30 Second, TGF‐β1 can induce the differentiation of naive T cells into Tregs and thus increase the number of Tregs. 31 TGF‐β1‐deficient mice exhibit a significantly decreased proportion of peripheral Tregs. 32
Surprisingly, we found that circulating Tregs were negatively correlated with the serum levels of IL‐10. Similarly, Barhoumi and colleagues. 33 found that adoptive transfer of Tregs normalized the increased level of IL‐10 in Ang II‐induced hypertensive mice. They speculated that both Tregs and IL‐10 could downregulate the levels of proinflammatory cytokines. After Treg adoptive transfer, feedback loops that modulate proinflammatory cytokines by increasing IL‐10 were inhibited, and therefore, the level of IL‐10 was decreased. In fact, IL‐10 can be produced by multiple cell types, such as neutrophils, dendritic cells (DCs), mast cells, monocytes, macrophages, eosinophils, and natural killer cells, in addition to B cells and CD8+ and CD4+ T cells. 34 We could not rule out the secretory function of other cells in terms of IL‐10. In addition, the infiltration of proinflammatory cytokines may act in different ways between the serum and heart tissue. In our study, circulating Tregs were negatively correlated with serum IL‐10, while in another study, cardiac IL‐10 was elevated after adoptive transfer of Tregs, 35 so serum and cardiac IL‐10 may have different correlations with circulating Tregs.
IL‐6 is produced by a variety of cell types, including monocytes, T cells, B cells, and endothelial cells. IL‐6 signaling is critical for cardiomyocyte hypertrophy and Th17/Treg balance regulation. 14 Cardiac IL‐6 expression is increased in pressure‐overloaded hearts. 36 Similarly, serum IL‐6 was increased in LVH patients in our study. Studies targeting IL‐6 further support an essential role for this cytokine as a driver of inflammatory disease mechanisms in animal models of hypertension, preeclampsia, and LVH. In previous studies, IL‐6−/− mice were protected from both AngII‐induced hypertension 37 , 38 and LVH. 39 Pressure overload‐induced LV cardiac remodeling and functional deterioration were shown to be attenuated in the absence of IL‐6, indicating an important role for this cytokine in hypertensive myocardial remodeling. 39 In addition, bazedoxifene, a drug for anti‐inflammatory therapy, can protect the heart from cardiac remodeling by inhibiting IL‐6/gp130 signaling in transverse aortic constriction mice. 40 Thus, IL‐6 may be a potential therapeutic target in LVH.
An increased level of CRP or hs‐CRP is closely related to the onset of LVH in patients with hypertension and can be an independent predictor of LVH. 41 , 42 We also found elevated hs‐CRP levels in hypertensive patients with LVH compared with hypertensive patients without LVH and healthy individuals, indicating the hyperinflammatory state in patients with LVH and a possible application for hs‐CRP in predicting the occurrence of LVH. In addition, the plasma NT‐proBNP level was significantly elevated in patients with LVH in our study. Similarly, plasma NT‐proBNP has been found to rise progressively with increasing hypertension severity, particularly when LVH is present. 43 In a previous study on Black patients with hypertension, however, even though NT‐proBNP was effective in differentiating hypertensive subjects with or without LVH from those with hypertensive heart failure, it could not differentiate hypertensive patients with LVH from those without LVH. 44 Therefore, whether NT‐proBNP is an ideal marker for LVH is still controversial. The LVMI is commonly used to identify patients with cardiac hypertrophy. We also found a negative correlation between circulating Tregs and LVMI values in all 243 subjects. In patients with rheumatic heart disease, Tregs were found to have a negative relationship with CK‐MB. 45 In this study, we also found a negative correlation between circulating Tregs and serum CK. However, the detailed mechanism of their negative relationship needs to be further explored.
Moreover, dyslipidemia and abnormal blood glucose levels were previously found in hypertensive patients with or without LVH. 46 , 47 Increased body weight, BMI, HbA1c, and Lp(a) values as well as a decreased HDL‐C level were also found in hypertensive patients in our study. We also found that the proportion of circulating Tregs was negatively related to LDL‐C and apoB in patients with hypertension. LDL, the shell of which contains apoB‐100, can carry cholesterol to form LDL‐C. However, oxidized LDL, not native LDL, can promote cholesterol accumulation in monocytes and macrophages. 48 The Fas/Fas ligand pathway in activation‐induced cell death serves as the major mechanism of peripheral Treg apoptosis, 49 and increased oxidized LDL levels may activate Fas/Fas ligand/Caspase‐3‐mediated Treg apoptosis. 49 , 50 These results may provide clues about the negative correlation between Tregs and LDL‐C in hypertension. Negative correlations between circulating Tregs and HbA1c or BMI were previously found in obese patients, 51 while no significant correlation was found in our study. There were only 5 obese patients (BMI > 30), which might explain why circulating Tregs was not related to BMI in our study. A recent study found that diabetic patients have a higher prevalence of ECG‐LVH. 52 Considering that both the EH and LVH groups have a significant cohort of diabetic patients in our study, we did Spearman correlation analysis between LVMI and HbA1c in all hypertensive patients (both EH and LVH groups). There is no correlation, which means that ventricular hypertrophy in hypertensive patients was not related to the difference of HbA1c in our study (Figure S3). Since there are 37.4% diabetic patients in LVH group, we also compared the Tregs between diabetic and non‐diabetic patients in LVH group, there is no difference (Figure S4). It might because those diabetic patients were controlled on medications or insulin or because the sample size is relatively small in our study.
The present study had several limitations. First, macrophages, neutrophils, and other subsets of CD4+ T cells are important inflammatory cells, but we did not evaluate their proportions. In addition, the 243 patients were all from the Second Xiangya Hospital, so the conclusion should be verified in a large, multicenter study.
In conclusion, altered levels of circulating Tregs, IL‐10, TGF‐β1, and IL‐6 were found in EH and LVH patients. The morbidity and mortality in patients with LVH were significantly increased, but the current treatment approach for LVH still follows standard hypertension guidelines and has unclear benefits. 53 Our study provides possible molecular targets for immunotherapy for hypertension and hypertensive myocardial hypertrophy. In contrast to the administration of traditional antihypertensive drugs, increasing the proportion of functional Tregs and supplementing anti‐inflammatory cytokines or reducing proinflammatory cytokine levels may be new approaches for the treatment of hypertension and LVH.
AUTHOR CONTRIBUTIONS
Ying Tang and Li Shen carried out the molecular genetic studies, participated in the sequence alignment, and drafted the manuscript. Jing‐hui Bao carried out the immunoassays. Ying Tang and Jinghui Bao participated in the sequence alignment and collected clinical characteristics from electronic medical record. Ying Tang and Dan‐yan Xu participated in the design of the study and performed the statistical analysis. Dan‐yan Xu conceived of the study and participated in its design and coordination and helped to draft the manuscript. All authors read and approved the final manuscript.
CONFLICT OF INTEREST STATEMENT
The authors declare no conflicts of interest.
Supporting information
Supporting Information
ACKNOWLEDGMENTS
The authors have nothing to report. This work was supported by the National Natural Science Foundation of China (No. 81871858, 82172550).
Tang Y, Shen L, Bao J‐H, Xu D‐Y. Deficiency of Tregs in hypertension‐associated left ventricular hypertrophy. J Clin Hypertens. 2023;25:562–572. 10.1111/jch.14660
Ying Tang and Li Shen are co‐first authors.
DATA AVAILABILITY STATEMENT
The data used in the current study are available upon reasonable request to the corresponding author.
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Supplementary Materials
Supporting Information
Data Availability Statement
The data used in the current study are available upon reasonable request to the corresponding author.