A total of 80 subjects were enrolled in the present study, including 20 normotensive postmenopausal females, 20 prehypertensive postmenopausal females, 20 normotensive males and 20 prehypertensive males. The prehypertensive subjects exhibited an SBP between 120 and 139 mmHg, and/or a DBP between 80 and 89 mmHg, according to the guidelines established by the eighth report of the JNC (25). All normotensive subjects had an SBP of <120 mmHg and a DBP of <80 mmHg, and presented no cardiovascular risk factors. The subjects recruited into the present study did not suffer from CVD or metabolic disease, as determined by assessment of their entire clinical history and auxiliary examinations. In addition, subject with conditions that may affect the number of EPCs, such as diabetes mellitus, malignant disease, uncontrolled infection and smoking, were excluded. Subjects with previous hysterectomy were also excluded. All subjects were recruited between January 2016 and January 2017, from Xiangya Hospital, Central South University (Changsha, China), and written informed consent was provided for participation. The experimental protocol was approved by The Ethical committee of Xiangya Hospital (ethical license ID of human clinical trial, no. 201503377). The baseline clinical data of the subjects, which were divided into four groups, are listed in Table I.

Serum samples were collected in the morning after overnight fasting from the study population (n=80; 40 females and 40 males), and the levels of EPCs, total cholesterol (TC), high-density lipoprotein cholesterol (HDL), low-density lipoprotein cholesterol (LDL), triglycerides (TG) and estradiol were measured. All subjects avoided ingesting alcohol or caffeine for half a day prior to specimen collection. Subjects receiving drug treatments, including antiplatelet agents, anti-inflammatory drugs and hypolipidemic agents, were excluded from the study to reduce potential external effects on the number and activity of circulating EPCs.

The number of EPCs in the serum was tested according to previous studies (20,24,26–28). Briefly, Ficoll density gradient centrifugation was used to isolate the peripheral blood mononuclear cells, and then cells were suspended in Endothelial Cell Growth Medium 2 (500 ml; Lonza Group, Ltd., Basel, Switzerland) supplemented with 2% fetal bovine serum (FBS; Sigma-Aldrich; Merck KGaA, Darmstadt, Germany). Next, the cell suspension (2.5×10⁶/ml) was transferred into 25-cm² cell culture flasks (Corning, Inc., Corning, NY, USA), coated with fibronectin (Clonetics Corporation, San Diego, CA, USA), and incubated at 37°C in a humidified atmosphere containing 5% CO₂. After 4 days, nonadherent cells were discarded, while adherent cells were maintained for a further 7 days, and these cells were then utilized in subsequent experiments.

After the 7-day culture, endothelial marker proteins were assessed by flow cytometry. Cell suspension (100 µl) was incubated for 40 min at 4°C with the following primary antibodies: Fluorescein isothiocyanate (FITC) anti-human CD45 (1:10; cat. no. FHF045-025; 4A Biotech, Co., Ltd., Beijing, China), phycoerythrin (PE)-Cy7 anti-human CD34 (1:10; cat. no. FHN034-025; 4A Biotech, Co., Ltd.) and PE-conjugated anti-kinase-insert domain receptor (KDR; 1:20; cat. No. FHK309-025; 4A Biotech, Co., Ltd.). Following erythrocyte lysis, the remaining cells were washed with phosphate-buffered saline (PBS) and fixed in 4% paraformaldehyde for 10 min at 37°C. Flow cytometric analysis was conducted with an ACEA NovoCyte™ flow cytometer (ACEA Biosciences, San Diego, CA, USA), and the results were analyzed with NovoExpress software™ (ACEA Biosciences). The number of circulating EPCs was determined according to the ratio of CD45⁻CD34⁺KDR⁺ cells per 100 peripheral blood mononuclear cells.

In order to determine the EPC phenotype, mononuclear cells (2.5×10⁶/ml) were plated on cell culture flasks with endothelial cell growth medium (EGM^™−2; Lonza Group, Ltd., Basel, Switzerland). Following 7 days of culturing, the attached endothelial cell-like cells were incubated with DiI-labeled acetylated LDL (Molecular Probes; Thermo Fisher Scientific, Inc.) for 1 h at 37°C. Subsequent to fixing in 4% paraformaldehyde for 30 min at 37°C, the cells were incubated with FITC-labeled lectin (Sigma-Aldrich; Merck KGaA) for 4 h at 37°C. Following incubation with FITC-labeled lectin, the samples were observed under a phase-contrast fluorescence microscope (magnification, ×200). Cells presenting double-positive fluorescence were identified as differentiating EPCs by two independent researchers blinded to the study groups.

The EPC migration and proliferation assays were conducted as previously described (20,24,27–29). In order to determine the cell proliferation, EPCs were harvested by centrifugation at 438 × g for 5 min at 4°C and resuspended in 500 µl EGM-2. A total of 2×10⁴ EPCs/well were added into the upper chamber of a modified Boyden chamber (24-well Costar Transwell plate; pore size, 8 µm; Corning, Inc.), while 500 µl EGM-2 supplemented with 50 ng/ml VEGF was added to the lower chamber. Following incubation at 37°C for 24 h, the lower side of the filter was washed with PBS and fixed with 2% paraformaldehyde for 10 min at 37°C. Cell nuclei were then stained with DAPI. Cells that had migrated into the lower chamber were manually counted in three random fields under a fluorescence microscope by two independent blinded investigators.

EPC proliferation was determined using an MTT assay. Briefly, EPCs were digested with 0.25% trypsin and then cultured in serum-free medium in a 96-well culture plate (2,000 cells/well) for 24 h. Next, the EPCs were supplemented with 10 µl MTT (5 g/l; Sigma-Aldrich; Merck KGaA, Darmstadt, Germany) and incubated for a further 4 h. The supernatant was then aspirated and discarded. Subsequent to mixing the EPC preparation with 200 µl dimethyl sulfoxide by shaking for 10 min, the optical density value at 490 nm was measured (20,24,28,29).

The Griess method was used to determine the inactive metabolite of NO in the plasma, as reported in earlier studies (20,24), and the results are expressed as µmol NO_x of NO₃-/NO₂- per liter of medium. In addition, in order to measure the levels of VEGF and GM-CSF in the plasma, high-sensitivity enzyme-linked immunosorbent assay (ELISA; R&D Systems, Inc., Wiesbaden, Germany) was conducted according to the manufacturer's protocol.

EPCs were cultured in Dulbecco's modified Eagle's medium (Gibco; Thermo Fisher Scientific, Inc.) supplemented with 20% FBS (Sigma-Aldrich; Merck KGaA) for 48 h. Subsequently, in order to measure the levels of NO, VEGF and GM-CSF secretion in the conditioned media of EPCs, the Griess method and ELISA assays were conducted in accordance with the aforementioned protocol.

The assessment of FMD was performed as described in previous studies (30,31). The brachial artery FMD was assessed by a trained investigator with high-resolution ultrasonography using a 5–12-MHz linear transducer on an HDI 5000 system (Philips Medical Systems, Inc., Bothell, WA, USA). After a 15-min rest, the brachial artery was studied at 20–100 mm proximal to the antecubital fossa in supine position. An upper-forearm sphygmomanometer cuff was inflated to raise the pressure to 250 mmHg, and this was maintained for 5 min. The FMD was then calculated as the percentage of increase in the mean diastolic diameter reactive to hyperemia at 55–65 sec after deflation to baseline. After a further 15 min, 400 µg sublingual glyceryl trinitrate was administered, and the diastolic diameter was measured again after 5 min to determine the endothelial independent dilation.

All data were analyzed using the SPSS statistical software package, version 11.0 (SPSS, Inc., Chicago, IL, USA), and are presented as the mean ± standard deviation. Two-way analysis of variance was used to compare between the four groups. When a significant F-value was observed, the Newman-Keuls method was applied as a post hoc test to identify differences among mean values. Univariate correlations were examined by Pearson's correlation coefficient method. A P-value of <0.05 was considered to denote a difference that was statistically significant.

The essential characteristics of the study population are summarized in Table I. No marked differences were observed with regard to the patient age and body mass index among the subjects. The SBP and DBP values were markedly lower in normotensive males and normotensive postmenopausal females as compared with those in prehypertensive subjects of the same sex, respectively. However, the levels of TC, HDL, LDL, TG and plasma glucose were similar among the four groups. Furthermore, FMD in prehypertensive postmenopausal females was significantly lower in comparison with that in normotensive postmenopausal females. Similarly, FMD was lower in prehypertensive males as compared with that in normotensive males. Additionally, FMD in postmenopausal females was increased compared with male subjects, regardless of the level of blood pressure.

To determine whether the circulating EPC was associated with prehypertension, their number and activity was determined. The results shown in Fig. 1 indicated that there was no significant difference in circulating EPCs between normotensive and prehypertensive postmenopausal females, or between normotensive and prehypertensive males. In addition, no notable difference was detected in circulating EPCs between normotensive males and females (postmenopausal), or between prehypertensive males and females (postmenopausal). Furthermore, the number of EPCs determined by the cell culture assay exhibited no significant difference among the four groups.

The migration and proliferation of EPCs were then examined, and the results are shown in Fig. 2. As compared with normotensive postmenopausal females or males, the migration of EPCs was attenuated in the prehypertensive postmenopausal females and males, respectively. Similarly, the proliferation of EPCs in normotensive subjects was significantly higher compared with that of prehypertensive subjects in the same sex group, respectively. However, no marked difference in migration and proliferation of circulating EPCs was observed between normotensive males and females, or between prehypertensive males and females.

To investigate the mechanism leading to differences in the activity of circulating EPCs, the levels of certain factors present in the plasma influencing the function of EPCs were examined. The levels of NO, VEGF and GM-CSF detected in the plasma of the four groups are displayed in Fig. 3. As shown in Fig. 3A, the plasma NO level in postmenopausal females with prehypertension or prehypertensive males was significantly lower compared with that in normotensive postmenopausal females or normotensive males, respectively. However, the plasma level of NO presented no notable difference between the two prehypertensive males or postmenopausal females, and between the two normotensive groups. As exhibited in Fig. 3B, the plasma VEGF level did not significantly differ in any of the four groups. Similar to VEGF, the plasma GM-CSF level shown in Fig. 3C exhibited no evident difference among the four groups.

As displayed in Fig. 4, the secretion of NO, VEGF and GM-CSF by EPCs among the four groups was consistent with their distribution in the plasma. In brief, NO production in prehypertensive postmenopausal females declined in comparison with that in normotensive postmenopausal females. Similar results were observed when comparing prehypertensive with normotensive males. By contrast, VEGF and GM-CSF secretion by EPCs did not differ among the four groups.

The study then sought to explore the correlation of FMD with the NO level or circulating EPCs. As exhibited in Fig. 5A, a strong univariate correlation between FMD and the migratory activity of circulating EPCs was ascertained. Similarly, as shown in Fig. 5B, the FMD enhancement was concordant with EPC proliferation promotion. As presented in Fig. 5C and D, the FMD significantly increased with the increase in plasma NOx level and the NO production secreted by EPCs.