Gender Differences in Endothelial Progenitor Cells and Cardiovascular Risk Profile
The Role of Female Estrogens
Objective— Endothelial progenitor cells (EPCs) participate in vascular homeostasis and angiogenesis. The aim of the present study was to explore EPC number and function in relation to cardiovascular risk, gender, and reproductive state.
Methods and Results— As measured by flow-cytometry in 210 healthy subjects, CD34+KDR+ EPCs were higher in fertile women than in men, but were not different between postmenopausal women and age-matched men. These gender gradients mirrored differences in cardiovascular profile, carotid intima-media thickness, and brachial artery flow-mediated dilation. Moreover, EPCs and soluble c-kit ligand varied in phase with menstrual cycle in ovulatory women, suggesting cyclic bone marrow mobilization. Experimentally, hysterectomy in rats was followed by an increase in circulating EPCs. EPCs cultured from female healthy donors were more clonogenic and adherent than male EPCs. Treatment with 17β-estradiol stimulated EPC proliferation and adhesion, via estrogen receptors. Finally, we show that the proangiogenic potential of female EPCs was higher than that of male EPCs in vivo.
Conclusions— EPCs are mobilized cyclically in fertile women, likely to provide a pool of cells for endometrial homeostasis. The resulting higher EPC levels in women than in men reflect the cardiovascular profile and could represent one mechanism of protection in the fertile female population.
Endothelial progenitor cells (EPCs) are bone marrow–derived cells actively involved in cardiovascular homeostasis.1 In basal conditions, peripheral blood EPCs provide a circulating pool of cells that repair the ongoing endothelial damage. In the setting of ischemia, EPCs are mobilized from bone marrow to peripheral blood, home to the ischemic sites and stimulate compensatory angiogenesis.2 Reduced levels of circulating EPCs have been demonstrated in the presence of classical risk factors for cardiovascular disease3; furthermore, EPC depletion has been shown to predict the development of adverse events.4,5 Therefore, EPCs are an integrated component of the cardiovascular system and are considered a novel biomarker of cardiovascular health.6
Women in the reproductive age are exposed to a lower cardiovascular risk than age-matched men.7 This is generally attributed to the differences in sex hormones and, specifically, to the protective cardiovascular properties of female estrogens.8,9 Besides the effects on plasma lipids and on the vessel wall, other mechanisms may link estradiol to a favorable cardiovascular profile. Preliminary experience has proposed that the hormonal status may modulate EPCs,10,11 but definite data and mechanistic insights in humans are still lacking. Aims of this study were to assess the relationships between EPCs, gender, reproductive state, and cardiovascular risk. Therefore, we explored: (1) gender gradients in EPC number and function in relation to surrogate indexes of cardiovascular risk; (2) EPC variations during menstrual cycle, and (3) effects of 17β-estradiol on EPC function using in vitro and in vivo models.
Materials and Methods
For complete methods, please see the supplemental materials, available online at http://atvb.ahajournals.org.
The study was approved by the local ethic committee, and informed consent was obtained from all subjects. In a population of 210 healthy subjects (104 males and 106 females) we determined cardiovascular parameters, sex hormones, high-sensitive C reactive protein (hCRP), carotid intima-media thickness (c-IMT), and flow mediated dilation (FMD) of the brachial artery. We enrolled also 5 ovulatory and 5 anovulatory young women and determined their levels of circulating EPCs, plasma soluble c-kit ligand (skitL), and sex hormones during follicular, ovulatory, and luteal phases. Additionally, we collected blood samples from 12 male and 9 female prepuberal children, from 10 male and 10 female subjects aged ≥65, and from umbilical cord of 21 male and 21 female newborns.
Quantification of Circulating Progenitor Cells
Human progenitor cells were analyzed for the expression of surface antigens with direct flow cytometry as previously described12 using fluorescein isothiocyanate (FITC)-conjugated anti-CD34, PE-conjugated anti-KDR, and activated protein C (APC)-conjugated anti-CD133 mAbs (supplemental Figure I). Human EPCs were defined as CD34+KDR+ cells, according to recent population-based studies.4,5 Rat progenitor cells were quantified using PE-conjugated anti-mouse sca-1, FITC-conjugated anti–c-kit, and FITC-conjugated anti-rat CD31 mAbs.
Cell Culture, Characterization, and Functional Assays
EPC isolation and culture were performed as previously described.13 To confirm their endothelial phenotype, cells were studied for the uptake of acLDL, binding of Ulex-lectin, and expression of CD34, CD31, KDR, vWf, CXCR4, and CD18. Expression of estrogen receptor and eNOS were also assessed. Cell colonies were counted as an indicator of EPC proliferation. In separated experiments, growth medium was supplemented with 1-10-100 nmol/L 17β-estradiol (E2). Cells cultured under 10 nmol/L E2 were cotreated with a nonselective ER inhibitor (ICI 182.780) or a ERα-selective inhibitor (MPP). The adhesive capacity of EPCs was evaluated on a monolayer of human umbilical vein endothelial cells (HUVECs), as previously described.13 To explore EPC function in vivo, we used a rat model of ischemia-reperfusion (IR) injury14: Sections of IR muscles injected with EPCs were analyzed to quantify capillary density, the total number of EPCs and of apoptotic EPCs, as well as the number of EPCs integrated into the host microvasculature.
EPC Levels Are Related to Cardiovascular Risk and Reproductive State
The study sample was representative of a healthy middle-aged general population. After recruitment, women were retrospectively divided into fertile (n=64) and postmenopausal (n=42). The best random selection of age-matched men (n=45) represented the control group for postmenopausal women. Differences in sex hormones between groups were consistent with gender and reproductive state. Men had a worse cardiovascular risk profile than women (Table), and fertile women had significantly lower c-IMT and higher FMD of the brachial artery than men and postmenopausal women. These differences were abolished when postmenopausal women were compared with age-matched men (Figure 1). The levels of circulating CD34+KDR+ EPCs were significantly higher in fertile women than in young men or postmenopausal women, while not significantly different between postmenopausal women and age-matched men. A quite similar trend was seen for the percent KDR expression on CD34+ cells (CD34+KDR+/CD34+ cell counts ×100), which is taken to represent the extent of endothelial differentiation from generic progenitor cells (Figure 1a and 1b).15 No differences were found in the levels of circulating CD133+KDR+ and CD34+CD133+KDR+ cells (Figure 1i). In the whole sample of 210 subjects, E2 concentration was linearly correlated with %KDR (r=0.14; P=0.04), whereas the E2/T ratio was correlated with both CD34+KDR+ cell count (r=0.20; P=0.002) and %KDR (r=0.21; P=0.002). The EPC difference between fertile women and young men was maximal in subjects with 0 to 1 cardiovascular risk factors, whereas it was tapered in the presence ≥2 risk factors (Figure 1j). To establish whether the lower EPC count in men than in women was actually driven by gender or was simply the result of a higher cardiovascular risk, we ran a multiple linear stepwise regression analysis, which showed that gender and age were significantly correlated with EPC levels, independently of other cardiovascular parameters (supplemental Table I).
EPC Gender Gradient Fluctuates in Lifetime
We determined EPC levels in cord blood of newborns, and in peripheral blood of prepuberal children and elderly individuals to establish whether these cells vary with age. We found that female newborns had far more CD34+KDR+ EPCs than males. Interestingly, this gender difference was inverted in prepuberal children, in favor of males. In the elderly, males and females were matched for age and risk factors, and there was no gender difference in EPC levels (Figure 1k). Across all ages, no significant gender gradients were found in the level of circulating CD133+KDR+ and CD34+CD133+KDR+ cells (not shown).
EPC Are Mobilized During the Hormone/Menstrual Cycle
We hypothesized that increased EPC levels in the female fertile population were related to the menstrual/hormonal cycle. For this purpose, we prospectively enrolled 5 ovulatory and 5 anovulatory young women to determine their levels of circulating EPCs and plasma skitL. Variations of E2 and progesterone (P) were consistent with the presence/absence of ovulation in the 2 groups. In cycling women, a 2-fold increase in CD34+KDR+ EPCs in the ovulatory phase was paralleled by a significant increase in skitL. There was no significant variation of EPCs and skitL in anovulatory women across phases. There were no significant differences in the absolute levels of EPCs during follicular phase between ovulatory and anovulatory women (Figure 2).
Circulating EPCs Increase After Hysterectomy in Rats
To evaluate the impact of endometrial regeneration on EPC levels, we measured sca-1+c-kit+ and sca-1+CD31+ cells before and after hysteroctomy in rats. Hysterectomy was performed with care to preserve anatomically and functionally the ovaries, and to favor maintenance of the ovarian hormonal cycle. Two weeks later, sca-1+c-kit+ and sca-1+CD31+ cells were increased significantly, suggesting that removal of the target organ with preservation of the hormonal cycle prevented EPCs to home at sites of endometrial regeneration/remodeling, thus increasing their levels in the circulation (supplemental Figure II).
Genuine Phenotype of Isolated Human EPCs
Human EPC isolation was performed according to a validated protocol. During growth in endothelial medium, a subset of PBMCs form colonies of endothelial cells which, after 2 weeks, display function and phenotype of endothelial cells: all survived cells bind lectin, take up LDL and are brightly positive for the surface expression of CD34, vWf, CD31, and KDR. Isolated EPCs were also positive for CXCR4 (SDF-1α receptor) and CD18 (ICAM receptor), which have been previously shown to be relevant to EPC function. Finally, cultured EPCs expressed eNOS (Figure 3a through 3d).
EPCs Express Estrogen Receptors
Immunofluorescence and Western blot analyses showed that EPCs express estrogen receptor α (Figure 3 days). Real-time polymerase chain reaction (PCR) revealed that the mRNAs of both α and β isoforms were present in cultured EPCs. The relative expression of both ER isoforms normalized to the housekeeping gene 18S was more abundant in male than in female EPCs cultured from peripheral blood and cord blood. Although expression of ER α and β isoforms did not differ in female EPCs, male EPCs form peripheral blood expressed much more ERα than ERβ, whereas ERβ was more abundant in male EPCs from cord blood (supplemental Figure III).
Gender Differences in EPC Function and Effects of E2
EPC Colony Formation
In basal conditions, the number of endothelial colonies 15 days after plating was significantly higher in samples derived from women than from men. Culture supplementation with E2 progressively increased colony yield from male samples, an effect that was prevented by ER inhibitors. E2 had little effect on female samples, but ER inhibition substantially decreased female EPC colonies from baseline in the setting of both E2-stimulated and unstimulated conditions (Figure 4a).
Adhesive Properties of EPCs
In basal conditions, female EPCs showed increased adherence to a mature endothelial layer with respect to male EPCs. After exposure to E2, adherence of male EPCs progressively increased, matching adhesion of female EPCs at 1 to 10 nmol/L E2. Adhesive properties of female EPCs increased significantly versus baseline when exposed to 100 nmol/L E2. Nonselective (ICI) and α-selective (MPP) ER inhibitors prevented the effects of E2 on EPC adhesion (Figure 4b).
In Vivo Vasculogenic Capacity of EPCs
To provide further evidence for gender differences in EPC function, we explored the vasculogenic capacity of male and female EPCs in a rat model of ischemia/reperfusion injury (Figure 4c and 4d). Two weeks after implantation into ischemic rat muscles, a similar number of male and female EPCs were found in the cryosections, and injected cells were not apoptotic (supplemental Figure IV), suggesting no difference in cell survival. However, the number of chimeric vessels (containing human CMTMR-labeled EPCs lining the vessel lumen), as well as the total capillary density, were higher with transplanted female than male EPCs (Figure 4 days).
The main findings in this study are the following: (1) gender differences in EPC number and function correlate with surrogate indexes of cardiovascular risk; (2) EPC gender gradient is present at birth and fluctuates during lifetime; (3) worsening of the cardiovascular risk profile after menopause is associated with EPC decline; and (4) EPCs are regulated by E2 in vitro and in vivo.
The low cardiovascular risk in fertile women is attributable to the antiatherosclerotic effects of estrogens, which improve endothelial function.7,8 Fertile women included in our study had a healthier risk profile than men and postmenopausal women. Parallely, FMD and c-IMT indicated a better vascular homeostasis in females than in males, and fertile women had higher levels of circulating CD34+KDR+ EPCs than men. This difference was likely related to gender per se rather than to the effects of concomitant risk factors, as shown by the independent association between gender and EPCs in the multivariate analysis. The impact of risk factors was stronger in women, and the EPC gender gradient was tapered in the presence of at least 2 cardiovascular risk factors. After menopause, there were no gender differences in the risk profile, FMD, c-IMT, and CD34+KDR+ EPCs. This is in compliance with a recent small study showing lower CD34+KDR+ EPCs in postmenopausal versus premenopausal women.10 Given that EPCs are actively involved in endothelial healing and reflect the global cardiovascular health,6 the gender-related difference in EPCs represents a plausible explanation for the difference in endothelial function and c-IMT. After menopause, EPC reduction, attributable to aging, to the change in the reproductive state, and to the worsened risk profile, may cause endothelial dysfunction and predispose to atherosclerosis. Our data contradict a recent small study showing that, in comparison with men, postmenopausal women had higher EPCs,11 which were however isolated with an outdated method (CFU-ECs) which is now known to select a subpopulation of monocytes/macrophages, instead of endothelial cells.16 It is worth remarking that a clear gender difference in EPC levels has not been reported previously because very few studies have been conducted with healthy subjects, so that any gender difference was masked by the underlying cardiovascular risk and advanced age.
Studying ovulatory women, we found that EPCs and soluble c-kit ligand vary in phase with menstrual/hormonal cycle. As bone marrow membrane–bound c-kit ligand is cleaved by SDF-1–stimulated MMP9 activity during progenitor cells mobilization,17 we suggest that EPCs are mobilized to the peripheral circulation of fertile women on a monthly basis. These results confirm and integrate a recent study by Masuda et al, showing cyclic E2-regulated bioactivity of EPCs.18 In this view, the higher steady-state levels of circulating EPCs in fertile women than in men may reflect their cyclic mobilization, possibly related to the vascular regeneration and remodeling taking place in the endometrium. Endometrial homing is likely to be directed by the local production of growth factors and chemokines (such as VEGF and SDF-1) that favors recovery after menstrual discharge, and governs vascular proliferation and remodeling. As an indirect demonstration that EPCs are mobilized for endometrial recruitment, we show in rats that removing the uterus while preserving the ovarian cycle increased sca-1+CD31+ EPCs, which probably accumulated in the circulation because they could not find their target organ. Consistently with this scenario, after human HLA- or sex-mismatched bone marrow transplantation, marrow cells contributed for up to 50% of stromal cells and to 14% of endometrial endothelial cells,19,20 whereas the contribution of bone marrow–derived EPCs to cyclic endometrial vascular turnover has been recently demonstrated in mice.18
Uncertainties still exist on the exact antigenic definition of EPCs.21 For ex vivo human studies, we defined EPCs as CD34+KDR+ cells because this phenotype was previously shown to correlate with cardiovascular risk and subclinical atherosclerosis better than other antigenic combinations,12,22 and proved as an independent predictor of future cardiovascular events.4,5 Additionally, CD34+KDR+ cells are believed to represent adult hemangioblasts and behave like EPCs in vivo.23,24 Consistently, CD34+KDR+ were selectively modulated, whereas other putative EPC phenotypes, such as CD133+KDR+ and CD34+CD133+KDR+ cells, showed no gender gradients throughout ages.
We isolated EPCs from healthy young male and female donors to analyze gender differences in EPC function. There is no consensus on the protocols to isolate EPCs, but our culture method underwent an extensive validation, by showing that cultured cells display antigenic and functional characteristics of endothelial cells, including eNOS expression (which is considered a stringent criterion),25 thus corresponding to “true late EPCs,” rather than to the so-called “monocytic EPCs.”26 Moreover, being CD34+KDR+CD31+vWf+ CXCR4+CD18+ and acLDL+Lectin+, cells isolated in vitro represent a subpopulation of the CD34+KDR+ population addressed in the ex vivo study. We found that female blood cells gave rise to a higher number of endothelial colonies than male cells, suggesting a gender difference in EPC generation. Moreover, a gender difference was clearly seen in the capacity of EPCs to adhere to a mature endothelium, a fundamental property required to reconstitute in vivo the dysfunctional intimal layer. Finally, using a rat model of ischemia/reperfusion injury, we show that female EPCs, when transplanted into male rat ischemic muscles, stimulated compensatory angiogenesis more efficiently than male EPCs, and were more frequently integrated into the host microvasculature. We were able to detect only a small proportion of chimeric vessels bearing human EPCs, which could not explain the strong increase in capillary density after ischemia. The intramuscular route of administration is believed to cause early apoptosis of injected cells, and may explain in part this paradox. Nonetheless, our results agree with previous observations that the proangiogenic effect of EPCs occurs in a paracrine fashion and not only through the direct integration into new vessels.27
The quantitative and functional differences in EPCs between young men and women, together with the observation that EPCs are mobilized during the hormonal cycle, strongly indicated that EPCs are influenced by female sex hormones. In fact, available experimental studies support a link between estrogens and EPCs. In ovariectomized animals, estrogen deficiency decreased circulating EPCs, whereas estrogen replacement increased EPC levels and stimulated EPC-mediated reendothelization after carotid injury.28,29 Moreover, E2 enhanced functional and anatomic recovery after experimental myocardial infarction through recruitment of EPCs.30 We confirm that E2 has a potent ability to stimulate human EPCs. Available data indicate that these effects are mediated through inhibition of apoptosis, stimulation of telomerase, and bone marrow mobilization.28,31,32 Herein, we report that KDR expression on CD34+ cells was higher in fertile women than in men, although it was similar in postmenopausal women and in age-matched men. This may indicate that E2 promotes differentiation of circulating CD34+ progenitor cells into CD34+KDR+ EPCs in vivo, but we failed to confirm this in vitro, as E2 did not modify the expression of endothelial markers during EPC culture (supplemental Figure V).
We demonstrate that EPCs express both ER isoforms and that E2 dose-dependently enhances EPC generation and adhesion in vitro. Interestingly, male EPCs were more responsive to the mitogenic effects of E2, whereas female EPCs seemed to be maximally stimulated in basal conditions. To explore possible reasons for this different responsiveness to E2, we quantified gene expression of ER isoforms: male EPCs expressed larger amounts of ERs than female EPCs, with a prevalence of the vasculoprotective α isoform, which may mediate the potent functional stimulation induced by E2, perhaps through upregulation of VEGF.30 Nonselective and α-selective ER inhibition prevented the ability of E2 to promote generation and adhesion of EPCs, confirming that these effects are mediated by classical ER pathways. Interestingly, ER inhibition abolished the higher generation of female EPCs in both basal conditions and E2-stimulation, suggesting a sort of “constitutive” activation of ERs in female cells, as pharmacological inhibition downregulates both ligand-dependent and -independent ER activity.33
As sex hormone concentrations vary not only during the fertile cycle in women, but also with age, we investigated EPC gender differences throughout human ages. We report a marked difference in EPC levels in the cord blood of newborns in favor of females. The predominance of ERβ over ERα in male cord blood EPCs may be responsible for the lower levels of circulating EPCs: in fact, the 2 receptor isoforms act as antagonists and their relative expression drives downstream events that determine the net effect of E2.33 This may represent a sort of prenatal imprinting of the endogenous vascular regenerative potential. In prepuberal children, EPC gender difference was inverted, in favor of males, suggesting that female EPCs are more sensitive to the fall in sex hormone concentrations during the prepuberal phase. Finally, there was no EPC gender gradient in the elderly. As already known from other studies,34 we confirm that age is one major determinant of EPC level in the multivariate analysis, but EPC level tended to be relatively stable over time in males, whereas it widely fluctuated in females. This trend, apparently inconsistent with the observation that E2 influenced male EPCs more than female EPCs in vitro, could be attributed to a differential expression of ER isoforms, and to the different effects of continuous versus cyclic hormonal stimulation in men versus women.
In conclusion, we provide a series of data indicating that EPCs are regulated by sex hormones in humans. Cyclic EPC mobilization may be related to endometrial regeneration; the resulting gender gradients in EPC number and function reflect the cardiovascular protection of the fertile female population. After menopause, on cessation of both ovarian and endometrial function, female EPCs decrease to the levels of coeval males, thus hampering vascular homeostasis and increasing cardiovascular risk.
Sources of Funding
This work was partially supported by a grant from the Heart Repair Consortium – IP 018630.
Original received May 28, 2007; final version accepted January 31, 2008.
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