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. 2015 Feb 16;17(4):298–305. doi: 10.1111/jch.12493

Increased Endothelial Progenitor Cells and Nitric Oxide in Young Prehypertensive Women

Yang Zhen 1,†, Songhua Xiao 2,†, Zi Ren 3, Hong‐wei Shen 4, Huanxing Su 5, Yong‐Bo Tang 6, Haitao Zeng 7,✉
PMCID: PMC8031957  PMID: 25688720

Abstract

This study investigated the effect of sex differences on circulating endothelial progenitor cells (EPCs) in prehypertension and its underlying mechanism. The authors found that premenopausal women show increased number and activity of circulating EPCs when compared with men, which was similar to enhanced nitric oxide (NO) level in plasma or culture medium. There was no difference in the number and activity of circulating EPCs and NO level between normotensive and prehypertensive premenopausal women. There was also no difference seen in levels of vascular endothelial growth factor and granulocyte macrophage colony‐stimulating factor. Both number and activity of circulating EPCs were correlated with the level of NO. The present study firstly demonstrated that the number and activity of circulating EPCs were preserved in prehypertensive premenopausal women, which was related to the restoration of NO production. The sex differences in EPCs in prehypertension may be involved in the mechanism underlying vascular protection in premenopausal women.

Prehypertension is an intermediate stage for pathogenesis of hypertension, indicating high risk of cardiovascular disease and its future clinical consequences, such as myocardial infarction, stroke, and heart failure.1, 2, 3, 4, 5, 6 The increased cardiovascular risk may be partly associated with endothelial injury and dysfunction accompanied by prehypertension.1, 2, 3, 4, 5, 6, 7 It has been proved that endothelial damage and dysfunction is involved in the pathogenesis and progression of atherosclerotic vascular disease and subsequent cardiovascular events.2, 8, 9, 10 Restoration of endothelial dysfunction in prehypertension may contribute to maintenance of vascular homeostasis and decreased occurrence of cardiovascular events.

It has been demonstrated that circulating endothelial progenitor cells (EPCs) derived from the bone marrow are engaged in the repair process of vascular endothelium.11 Circulating EPCs can promote neovascularization, repair endothelial injury, and improve endothelial function.12 In the presence of cardiovascular risk factors, the number and activity of circulating EPCs are impaired, which plays an important role in the development of endothelial dysfunction.13, 14, 15, 16 Recent studies demonstrated that the activity of circulating EPCs is reduced in prehypertension, indicating that the attenuated endothelial repair capacity may lead to prehypertension‐related vascular injury.1, 17, 18

The prevalence of cardiovascular diseases is lower in premenopausal women than in men of similar age but increases markedly in women after menopause.19, 20 The increased risk of cardiovascular diseases in postmenopausal women is associated with endothelial dysfunction.21, 22 In prehypertension, decreased activity of circulating EPCs has been observed, which is associated with endothelial dysfunction.17, 18 However, whether there is a sex difference in circulating EPCs in the presence of prehypertension is still unclear. It has been proved that sex differences in the number or activity of EPCs exist in middle‐aged adults,23, 24 suggesting that the alteration in endogenous endothelial repair capacity may be engaged in vascular protection concomitant with premenopausal women. Accordingly, we inferred that the number and activity of circulating EPCs in prehypertensive men may also differ from that in prehypertensive premenopausal women. In addition, nitric oxide (NO), vascular endothelial growth factors (VEGFs), and granulocyte macrophage colony‐stimulating factor (GM‐CSF) play important roles in regulating the number and activity of circulating EPCs.25, 26, 27, 28 We further hypothesized that the regulation of sex difference in circulating EPCs might be associated with NO, VEGF, and GM‐CSF. The present study was then designed to measure the number and activity of circulating EPCs in normotensive or prehypertensive premenopausal women and normotensive or prehypertensive men. Simultaneously, we evaluated the levels of NO, VEGF, and GM‐CSF in plasma or culture medium and investigated the underlying mechanism.

Methods

Characteristics of Patients

Twenty normotensive premenopausal women, 21 prehypertensive premenopausal women, 20 normotensive men, and 20 prehypertensive men were recruited. The patients with prehypertension were diagnosed if they had systolic blood pressure (BP) between 120 mm Hg and 139 mm Hg or diastolic BP between 80 mm Hg and 89 mm Hg according to Eighth Joint National Committee (JNC 8) guidelines.29 The normotensive premenopausal women and normotensive men had no cardiovascular risk factors and systolic BP <120 mm Hg and diastolic BP <80 mm Hg. All patients were free of cardiovascular and metabolic disease as evaluated by a full medical history, physical examination, and laboratory tests before being enrolled in the study protocol. The patients with diabetes, malignant disease, infection, or inflammatory disorders and smokers were excluded since all these conditions may influence the number of EPCs. We also excluded pregnant or breastfeeding women and those with irregular menstrual cycles or previous hysterectomy. The experimental protocol was approved by the ethical committee of our hospital. The baseline characteristics of normotensive and prehypertensive patients are shown in Table 1.

Table 1.

Clinical and Biochemical Characteristics

Characteristics Normotensive Women (n=20) Prehypertensive Women (n=21) Normotensive Men (n=20) Prehypertensive Men (n=20)
Age, y 45.3±4.2 45.9±3.7 46.7±3.5 46.4±4.5
Height, cm 160.4±6.3 160.8±6.4 165.4±6.5 a 165.8±6.2 a
Weight, kg 58.7±5.7 59.5±5.7 64.1±4.4 a 63.8±5.1 a
BMI, kg/cm2 22.8±2.0 23.0±2.1 23.6±1.3 23.3±1.9
Systolic blood pressure, mm Hg 111.7±7.1 130.6±5.1 b 112.0±5.5 131.7±5.3 b
Diastolic blood pressure, mm Hg 69.1±5.1 81.3±4.6 b 70.1±4.0 80.9±5.0 b
Heart rate, beats per min 71.9±8.3 73.0±7.6 74.8±7.4 75.4±7.8
AST, mmol/L 25.2±6.4 27.9±6.0 25.7±6.5 24.4±6.8
ALT, mmol/L 23.2±8.8 25.9±5.8 23.4±5.1 22.0±6.2
BUN, mmol/L 5.5±1.2 5.3±1.1 5.1±0.8 5.0±1.0
Cr, mmol/L 70.2±14.5 68.4±16.3 63.8±16.6 66.9±15.9
LDL, mmol/L 2.90±0.46 2.95±0.48 2.85±0.48 3.05±0.36
TC, mmol/L 4.94±0.53 5.01±0.47 4.95±0.53 5.08±0.46
HDL, mmol/L 1.45±0.27 1.41±0.26 1.39±0.23 1.36±0.16
TG, mmol/L 1.40±0.23 1.43±0.19 1.43±0.17 1.53±0.13
FPG, mmol/L 4.81±0.61 4.50±0.49 4.72±0.71 4.65±0.62
hs‐CRP, mmol/L 1.145±0.659 1.326±0.834 1.359±0.897 1.639±0.877
Estradiol, pmol/L 226.1±30.7 221.0±33.9 107.2±22.6 a 104.7±19.4 a
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Abbreviations: ALT, alanine aminotransferase; AST, aspartate aminotransferase; BMI, body mass index; BUN, serum urea nitrogen; Cr, creatinine; FPG, fasting plasma glucose; HDL, high‐density lipoprotein; hs‐CRP, high‐sensitivity C‐reactive protein; LDL, low‐density lipoprotein; TC, total cholesterol; TG, triglyerides. Data are given as mean±standard deviation. avs premenopausal women. bvs normotension.

Blood samples used for determining EPCs, total cholesterol, high‐density lipoprotein (HDL) cholesterol, low‐density lipoprotein (LDL) cholesterol, triglycerides, plasma glucose, high‐sensitivity C‐reactive protein (hs‐CRP), and estradiol were taken from normotensive and prehypertensive patients in the morning after overnight fasting. Patients refrained from consuming alcohol or caffeine for 12 hours before the study. All female volunteers underwent the examination at the menstrual phase of the menstrual cycle (day 2–day 5 after the first day of menstrual bleeding). None of the participants were receiving drug treatment with antiplatelet, anti‐inflammatory, or hypolipidemic agents to exclude the additional influence on the number and activity of circulating EPCs by those treatments.

Flow Cytometry Analysis and Cell Culture Assay to Evaluate the Number of Circulating EPCs

Detection of EPCs was performed as previously described.16, 25, 30, 31 The number of circulating EPCs was evaluated by the ratio of CD34+KDR+ cells per 100 peripheral blood mononuclear cells (PBMNCs). The cell culture assay to evaluate the number of circulating EPCs was performed as previously described.16, 25, 31, 32, 33 The number of cultured EPCs were evaluated by DiI‐acLDL/lectin double‐positive cells/×200, and counted manually by two independent observers blinded to the study.

EPC Migration and Proliferation Assay

EPC migration was determined previously repor‐ted.16, 25, 31, 32 EPC proliferation was determined by 3‐(4,5‐dimethylthiazol‐2‐yl) ‐2,5‐diphenyltetrazolium bromide (MTT) assay as our previous studies reported.16, 25, 31, 32

Measurement of Plasma NO, VEGF, and GM‐CSF Levels

Nitrite, the stable metabolite of nitric oxide, was estimated by using the Greiss method as our previous studies reported.25 The results are presented as μmol NOx of NO3−/NO2− per liter of medium. Plasma levels of VEGF and GM‐CSF were measured by high‐sensitivity enzyme‐linked immunosorbent assay (R&D Systems, Wiesbaden, Germany) according to our previous studies.25

Measurement of NO, VEGF, and GM‐CSF Secretion by EPCs

The cultured EPCs were switched to the DMEM/20% fetal bovine serum (no supplemental growth factors) for 48 hours. Then, the conditioned media were assayed for NO, VEGF, and GM‐CSF as previously described.25

Statistical Analysis

The statistical software SPSS version 11.0 (SPSS Inc, Chicago, IL) was used to analyze the data. All data were presented as mean value±standard deviation. Comparisons between the four groups were analyzed by two‐factor analysis of variance (sex and status of normotension or prehypertension). When indicated by a significant F value, a post hoc test using the Newman‐Keuls method identified significant differences among mean values. Univariate correlations were calculated using Pearson's coefficient (r). Statistical significance was assumed if a null hypothesis could be rejected at P<.05.

Results

Baseline Characteristics

The four groups were similar in terms of age and BMI (Table 1. Systolic and diastolic BPs were significantly higher in prehypertensive premenopausal women and prehypertensive men,when compared with normotensive premenopausal women and normotensive men (P<.05 and <.05, respectively). The level of estradiol in normotensive and prehypertensive premenopausal women were higher than that in normotensive and prehypertensive men (P<.05 and P<.05, respectively). The levels of cholesterol, HDL cholesterol, LDL cholesterol, triglycerides, plasma glucose, and hs‐CRP were all similar in the four groups (P>.05).

Number and Activity of Circulating EPCs in the Four Groups

Figure 1 shows the number of circulating EPCs in the four groups. The number of EPCs in prehypertensive premenopausal women was similar to that in normotensive premenopausal women (P>.05). There was also no difference in number of EPCs between normotensive and prehypertensive men (P>.05). However, the number of circulating EPCs in normotensive and prehypertensive men were lower than that in normotensive and prehypertensive premenopausal women (P<.05 and P<.05, respectively). The number of EPCs in normotensive and prehypertensive premenopausal women evaluated by cell culture assay also exhibited no significant difference (P>.05), but both exceeded that in normotensive and prehypertensive men (P<.05).

Figure 1.

Figure 1

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The number of circulating endothelial progenitor cells (EPCs) in the four groups. Evaluated by (A) fluorescence activated cell sorting analysis and (B) phase‐contrast fluorescent microscope, the level of circulating EPCs in prehypertensive premenopausal women was not statistically different from that in normotensive premenopausal women. The number of EPCs in normotensive and prehypertensive men was lower than that in normotensive and prehypertensive premenopausal women. Data are given as mean±standard deviation. #vs premenopausal women.

As shown in Figure 2, the migratory activity of EPCs in normotensive and prehypertensive men decreased compared with that in normotensive and prehypertensive premenopausal women (P<.05 and P<.05, respectively). Similarly, the proliferative activity of circulating EPCs in normotensive and prehypertensive men were significantly lower than that in normotensive and prehypertensive premenopausal women (P<.05 and P<.05, respectively). There was no difference in the migratory or proliferative activity of EPCs between normotensive and prehypertensive premenopausal women (P>.05 and P>.05, respectively). However, they were higher in normotensive men than in those in prehypertensive men (P<.05 and P<.05, respectively).

Figure 2.

Figure 2

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The activity of circulating endothelial progenitor cells (EPCs) in the four groups. The migratory (A) and proliferative (B) activities of cultured EPCs decreased in normotensive and prehypertensive men compared with normotensive and prehypertensive premenopausal women. There was no difference in the migratory (A) and proliferative (B) activities between normotensive and prehypertensive premenopausal women. Data are given as mean±standard deviation. ☆vs normotension. #vs premenopausal women.

Plasma NO, VEGF, and GM‐CSF Levels in Four Groups

As shown in Table 2, the plasma NO level in normotensive and prehypertensive premenopausal women were significantly higher than those in normotensive and prehypertensive men (P<.05 and P<.05, respectively). The plasma NO level in normotensive men was also higher than that in prehypertensive men (P<.05). However, the plasma NO level was the same in normotensive premenopausal women as that in prehypertensive premenopausal women (P>.05). No difference was shown among the four groups in terms of either plasma NO level or plasma GM‐CSF level (P>.05).

Table 2.

Levels of NO, VEGF, and GM‐CSF in Plasma and Secretion by EPCs

Normotensive Women (n=20) Prehypertensive Women (n=21) Normotensive Men (n=20) Prehypertensive Men (n=20)
Plasma NOx level, mmol/L 36.9±9.2 34.4±8.1 29.1±7.9 24±6.9 a , b
Plasma VEGF level, pg/mL 41.2±10.2 40.5±9.2 40.3±7.0 39.3±10.1
Plasma GM‐CSF level, pg/mL 0.278±0.049 0.286±0.059 0.301±0.054 0.297±0.049
NO production, nmol/106 cells 49.5±9.6 47.1±10.2 39.7±10.7 33.2±9.1 a , b
VEGF secretion, ng/106 cells 8.95±2.03 9.10±2.00 8.15±1.93 8.69±1.95
GM‐CSF secretion, ng/106 cells 2.03±0.59 1.88±0.45 2.10±0.72 1.98±0.48
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Abbreviations: EPCs, endothelial progenitor cells; GM‐CSF, granulocyte macrophage colony‐stimulating factor; NO, nitric oxide; NOx, nitrate plus nitrite; VEGF, vascular endothelial growth factors. Data are given as mean±standard deviation. avs normotension. bvs premenopausal women.

NO, VEGF, and GM‐CSF Secretion by EPCs in the Four Groups

Table 2 demonstrates NO, VEGF, and GM‐CSF secretion by EPCs in the four groups. Similar to the distribution of plasma NO level, the NO secretion by cultured EPCs in normotensive and prehypertensive men was lower than that in normotensive and prehypertensive premenopausal women (P<.05 and P<.05, respectively). Furthermore, such secretion in normotensive men was higher than that in prehypertensive men (P<.05), and, between normotensive and prehypertensive premenopausal women, it was almost equal (P>.05). Nevertheless, there was no difference in VEGF or GM‐CSF secretion by cultured EPCs in the four groups (P>.05 and P>.05, respectively).

Correlation Between Circulating EPCs and Plasma NO Level

We found a strong univariate correlation between plasma NO level and number or activity of circulating EPCs (Figure 3). The plasma NO level was significantly correlated with either the number of circulating EPCs, which was evaluated by flow cytometry analysis (r=0.33, P<.05) and by cell culture (r=0.36, P<.05), or the migratory activity (r=0.44, P<.05) and proliferative activity (r=0.52, P<.05).

Figure 3.

Figure 3

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The correlation between circulating endothelial progenitor cells (EPCs) and plasma nitrate plus nitrate (NOx) level. (A, B) The number of circulating EPCs evaluated by flow cytometry analysis or by cell culture correlated positively with plasma NOx level. (C, D) There was a correlation between the proliferatory or migratory activity of circulating EPCs and plasma NOx level.

Correlation Between Circulating EPCs and NO Secretion by EPCs

Figure 4 presents the correlation between the number or activity of circulating EPCs and NO secretion by EPCs. The NO secretion by EPCs positively correlated with the number of circulating EPCs evaluated by flow cytometry analysis (r=0.32, P<.05) and by cell culture (r=0.30, P<.05). There was also a significant correlation between such secretion and the migratory (r=0.38, P<.05) or proliferative activity (r=0.42, P<.05) of circulating EPCs.

Figure 4.

Figure 4

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The correlation between circulating endothelial progenitor cells (EPCs) and nitric oxide (NO) secretion by EPCs. (A, B) The number of circulating EPCs evaluated by flow cytometry analysis or by cell culture positively correlated with NO secretion by EPCs. (C, D) There was a correlation between the proliferatory or migratory activity of circulating EPCs and NO secretion by EPCs.

Discussion

In our study, we found that the number and activity of circulating EPCs in prehypertensive premenopausal women were preserved when compared with prehypertensive men. Similarly, the NO level in plasma or culture medium in prehypertensive premenopausal women was also restored. There was a significant correlation between the number and activity of circulating EPCs and plasma NO level or NO secretion by EPCs. Sex differences in the number and activity of circulating EPCs in prehypertension was firstly demonstrated in the present study, which is at least in part related to the enhanced NO production.

Recent studies have proved that the activity of circulating EPC is reduced in prehypertension, supporting the role of suppressed endothelial repair capacity in the pathogenesis of prehypertension.17, 18 However, until now there have been no data to investigate sex differences in EPCs under the condition of prehypertension. It is well established that premenopausal women have a low risk of cardiovascular disease compared with age‐matched men and postmenopausal women. Further studies confirmed that the number or activity of EPCs in women is higher than that in male middle‐aged adults or healthy volunteers,23, 24, 34, 35 suggesting that the vascular protection in premenopausal women may be the result of enhanced endothelial repair capacity. Hence, we hypothesized that the sex differences in circulating EPCs might also exist in prehypertension. We then observed the quantitative and qualitative changes of circulating EPCs in the four groups. In this study,we chose CD34+KDR+ cells to define the antigenic phenotype of EPCs by fluorescence activated cell sorting analysis. Although CD34+CD133+KDR+ could be used as a restrictive EPC phenotype, previous studies have proved that CD34+KDR+ is also a common and reliable method to detect putative antigenic phenotypes of EPCs.17, 31, 36 Our results revealed that the quantitative and qualitative differences in circulating EPCs were observed in prehypertensive men and prehypertensive premenopausal women. Both number and activity of circulating EPCs in prehypertensive premenopausal women were almost identical to those in normotensive premenopausal women. The present study indicates that the endogenous repair capacity of vascular endothelium is reversed in prehypertensive premenopausal women, which may counter against prehypertension‐related endothelial dysfunction.

In addition, we found that in the male population, prehypertension reduces the activity of circulating EPCs, but does not affect the number of circulating EPCs. Previous studies have reported similar changes of circulating EPCs in mixed‐sex populations with prehypertension,17, 18 but whether this phenomena occurs in the male or female population is still not understood. Our results further reveal that the activity of circulating EPCs decreases only in prehypertensive men but not in prehypertensive premenopausal women, indicating the sex differences in endogenous endothelial repair capacity in prehypertension. This phenomenon suggests that prehypertensive men may have a higher risk for cardiovascular events than prehypertensive premenopausal women.

NO, VEGF, and GM‐CSF play important roles in regulating the number and activity of circulating EPCs.25, 26, 27, 28 It has been reported that prehypertension is associated with impaired NO‐mediated endothelium‐dependent vasodilation.37 Therefore, we hypothesized that the effect of sex differences on circulating EPCs in preypertension might be associated with NO, VEGF, and GM‐CSF. To verify this hypothesis, we first measured the levels of NO, VEGF, and GM‐CSF in plasma in the four groups. Plasma NO level was restored in prehypertensive premenopausal women compared with normotensive premenopausal women, which is similar to the change of circulating EPCs in response to sex differences. The present observation is similar to prior reports supporting the sex differences in plasma NO level in prehypertension.38 Furthermore, there is a significant correlation between plasma NO level and the number or activity of circulating EPCs. The present study indicates that restored exogenous NO production contributes to reversed number or activity of circulating EPCs in prehypertension. However, no significant difference in plasma VEGF or GM‐CSF level among the four groups was found, indicating that the change of EPCs in response to sex differences may be not be caused by the alteration in plasma VEGF or GM‐CSF level.

Endogenous NO biosynthase is also important in the function of EPCs.39 A previous study reported that NO production in early EPCs is markedly reduced in prehypertension,17 and the endothelial repair capacity of EPCs is associated with NO production by early EPCs.40, 41 Accordingly, we further evaluated the sex differences in NO secretion by EPCs. NO secretion by EPCs was restored in prehypertensive premenopausal women compared with normotensive premenopausal women, which is similar to the change in plasma NO level. There was a significant correlation between NO secretion by EPCs and the number or activity of circulating EPCs. These results indicated that enhanced endogenous NO biosynthase may contribute to reversed number or activity of circulating EPCs in prehypertensive premenopausal women. No marked difference in VEGF and GM‐CSF secretion by cultured EPCs between the four groups was observed, indicating that sex differences in circulating EPCs were independent of their release of VEGF and GM‐CSF.

The mechanisms underlying the sex differences in NO production and circulating EPCs in prehypertension can be postulated as follows. The present study observed a higher plasma estradiol level in women than in men. Estrogen appears to be responsible for the sex differences in endothelial NO release,42, 43, 44, 45, 46 which may influence NO production by increasing NO synthase expression,42, 43, 44 preventing destabilization of endothelial NO synthase mRNA,47 and exhibiting antioxidant effects.48 NO can promote the mobilization, proliferative, and migratory activities of circulating EPCs, which increases the number and activity of circulating EPCs.25, 40 Therefore, it can be inferred that the estrogen‐induced enhanced NO production by either vascular endothelium or EPCs contributes to increased number and activity of circulating EPCs in prehypertension.

The present study may have important implications for the evaluation and treatment of prehypertension‐related vascular injury. First, the present data revealed the sex differences in circulating EPCs in prehypertension, indicating that circulating EPCs can be an important biomarker for detecting prehypertension‐related vascular injury in men. Compared with premenopausal women, it would be more necessary to enhance the number and activity of circulating EPCs in prehypertensive men, which may serve as a novel therapeutic target for improvement of prehypertension‐related endothelial damage. Second, our results showed that increased NO biosynthesis may be responsible for sex differences in circulating EPCs in prehypertension. The strategies to induce NO production in prehypertension, such as exercise,49 may contribute to the upregulation in the number and activity of circulating EPCs and the subsequent strengthening of endogenous endothelial repair capacity.

Conclusions

The present study for the first time demonstrates that the number and activity of circulating EPCs in prehypertensive premenopausal women are preserved, which is associated with restored NO production. The augmented endogenous endothelial repair capacity may be an important mechanism underlying vascular protection in premenopausal women. Understanding the sex differences in circulating EPCs in prehypertension will be of great significance to explore more effective evaluation and therapeutic approaches for prehypertension‐related vascular injury.

Disclosures

This study was financially supported by grants from the National Natural Scientific Foundation of China (31270992 and 30800215), the project of Zhu Jiang Science and Technology new star of Guangzhou City (2013J2200019), the project of Guangdong Province Science and Technology Plan (2013B021800275), and the Fundamental Research Funds for the Central Universities in Sun Yat‐sen University (13ykpy24).

J Clin Hypertens (Greenwich). 2015;17:298–305. DOI: 10.1111/jch.12493. © 2015 Wiley Periodicals, Inc.

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