Estradiol Enhances Recovery After Myocardial Infarction by Augmenting Incorporation of Bone Marrow–Derived Endothelial Progenitor Cells Into Sites of Ischemia-Induced Neovascularization via Endothelial Nitric Oxide Synthase–Mediated Activation of Matrix Metalloproteinase-9
Background— Recent data have indicated that estradiol can modulate the kinetics of endothelial progenitor cells (EPCs) via endothelial nitric oxide synthase (eNOS)–dependent mechanisms. We hypothesized that estradiol could augment the incorporation of bone marrow (BM)–derived EPCs into sites of ischemia-induced neovascularization, resulting in protection from ischemic injury.
Methods and Results— Myocardial infarction (MI) was induced by ligation of the left coronary artery in ovariectomized mice receiving either 17β-estradiol or placebo. Estradiol induced significant increases in circulating EPCs 2 and 3 weeks after MI in estradiol-treated animals, and capillary density was significantly greater in estradiol-treated animals. Greater numbers of BM-derived EPCs were observed at ischemic sites in estradiol-treated animals than in placebo-treated animals 1 and 4 weeks after MI. In eNOS-null mice, the effect of estradiol on mobilization of EPCs was lost, as was the functional improvement in recovery from acute myocardial ischemia. A decrease was found in matrix metalloproteinase-9 (MMP-9) expression in eNOS-null mice under basal and estradiol-stimulated conditions after MI, the mobilization of EPCs by estradiol was lost in MMP-9–null mice, and the functional benefit conferred by estradiol treatment after MI in wild-type mice was significantly attenuated.
Conclusions— Estradiol preserves the integrity of ischemic tissue by augmenting the mobilization and incorporation of BM-derived EPCs into sites of neovascularization by eNOS-mediated augmentation of MMP-9 expression in the BM. Moreover, these data have broader implications with regard to our understanding of the role of EPCs in post-MI recovery and on the sex discrepancy in cardiac events.
Received August 17, 2004; de novo received April 5, 2005; revision received January 16, 2006; accepted January 20, 2006.
Adult organs exhibit virtually no angiogenesis under normal conditions, except in the female reproductive tract. Several lines of experimental evidence have demonstrated that ovarian sex steroid hormones, such as estrogen and progesterone, modulate angiogenesis via effects on endothelial cells. Estradiol induces endothelial proliferation and migration1 mediated by the classic estrogen receptor, which is expressed by endothelial cells.2–4 Previously, endometrial neovascularization throughout the menstrual cycle has been considered to be the result of angiogenesis, ie, proliferation and migration of fully differentiated endothelial cells from preexisting “parent” vessels.5,6 However, normal monthly physiological endometrial proliferation would require that endothelial cells in the uterus replicate >1000 times during the reproductive life span of the average human female. Accordingly, it is unlikely that differentiated endothelial cells in situ could accomplish this mission without the occurrence of replicative senescence.7
Clinical Perspective p 1614
Recently, endothelial progenitor cells (EPCs) isolated from peripheral blood have been shown to incorporate into foci of neovascularization in the adult; this is consistent with the notion of postnatal vasculogenesis.8,9 These circulating EPCs are derived from bone marrow (BM) and are mobilized endogenously in response to tissue ischemia or exogenously by cytokine stimulation.10 Previous findings from our laboratory11 have suggested that cyclic neovascularization of the endometrium involves estradiol-regulated in situ incorporation and differentiation of BM-derived EPCs and that estradiol could also augment the recruitment of EPCs for vascular repair.12,13 These prior studies have also suggested that estradiol exerts its effects on EPCs via an endothelial nitric oxide synthase (eNOS)–dependent mechanism. The effect of estradiol on vascular repair of the heart, and in particular the potential role of EPCs in this process, has not been previously investigated. Accordingly, in the present study, we investigated the hypothesis that estradiol may augment EPC incorporation into sites of myocardial neovascularization after myocardial infarction (MI).
A detailed Methods section is provided in the online Data Supplement available at http://circ.ahajournals.org/cgi/content/full/CIRCULATIONAHA.105.553925/DC1.
All values are expressed as mean±SEM. Statistical significance was evaluated using unpaired Student t tests for comparisons between estradiol- and placebo-treated mice. When multiple time-point measurements were taken over time, repeated-measures analysis was performed, followed by an unpaired t test. Differences in mortality rates were compared by using Fisher exact tests. A value of P<0.05 was considered statistically significant.
The authors had full access to the data and take responsibility for its integrity. All authors have read and agreed to the article as written.
Estradiol Preserves Left Ventricular Function After Acute MI
MI was induced in ovariectomized mice receiving either estradiol or placebo via a subcutaneously implanted pellet.13 Left ventricular (LV) function and dimensions (LV diastolic dimension [LVDd], LV systolic dimension [LVDs], fractional shortening, and heart rate) were similar in estradiol- and placebo-treated mice before and for the first 2 weeks after MI (Table). Beginning 3 weeks after MI, however, echocardiography revealed less ventricular dilation in the estradiol-treated versus placebo-treated mice (LVDd, 3.7±0.06 versus 4.0±0.05 mm, respectively, at 3 weeks, and 3.8±0.04 versus 4.2±0.06 mm, respectively, at 4 weeks [P<0.01]; LVDs, 2.5±0.04 versus 3.0±0.08 mm, respectively, at 3 weeks and 2.6±0.05 versus 3.2±0.06 mm, respectively, at 4 weeks [P<0.01]). LV function was also significantly better in the estradiol group than in the placebo group (fractional shortening, 31±0.5 versus 24±0.9 mm, respectively, at 3 weeks and 32±0.7 versus 23±1.2 mm, respectively, at 4 weeks [P<0.01]). Finally, heart rate, an excellent prognostic indicator after MI, was lower (better) in the estradiol group than in the placebo group (492±8 versus 562±10 bpm, respectively [P<0.01]).
Hemodynamic measurements were performed 1 and 4 weeks after MI (Table). There was no significant difference in LV systolic or end-diastolic pressure between the groups at either time point after MI. However, both LV +dP/dt and LV −dP/dt, sensitive indicators of LV function, were significantly better preserved in the estradiol vs the placebo group 4 weeks after MI (LV +dP/dt, 2984±155 versus 2252±180 mm Hg/s, respectively [P=0.01]; LV −dP/dt, 2122±107 versus 1650±83 mm Hg/s, respectively [P<0.01]).
Serum levels of 17β-estradiol in the placebo group 1 and 4 weeks after MI were <2 pg/mL, whereas levels in the estradiol group after 1 and 4 weeks after MI were 580±55 and 559±62 pg/mL, respectively; these values are consistent with the upper range of levels in premenopausal females.13
Acute mortality within 1 week after MI in the estradiol groups (mortality rate, 19%; total number of operated mice=48, including 36 wild-type [WT] and 12 Tie-2/LacZ [LZ]/bone marrow transplant [BMT] mice) was similar to that in the placebo group (mortality rate, 15%; total number of operated mice=40, including 31 WT and 9 Tie-2/LZ/BMT mice).
Histological Assessment of WT Animals
Capillary density 4 weeks after MI was significantly greater in the estradiol group than in the placebo group (2432±110/mm2 versus 1134±39/mm2, respectively [P<0.01]; Figure 1A and 1B). The finding of preserved/improved capillary density has been associated with improved physiological outcome in multiple animal models and has been correlated with a decreased incidence of heart failure.14
Elastic trichrome–stained tissue in the placebo group also indicated marked dilatation of the LV cavity, which is consistent with the echocardiographic measurements. In comparison, the estradiol group showed significantly less LV remodeling (Figure 1C). Moreover, the area of LV fibrosis, indicating the zone of permanent myocardial injury, was significantly less in mice receiving estradiol than in the placebo group (36.0±2.6% versus 47.5±2.4%, respectively [P<0.05]; Figure 1D). These findings of preserved chamber dimensions (less dilation) and decreased LV fibrosis represent positive long-term prognostic findings.15,16
Effect of Estradiol on Circulating EPC Kinetics After Acute MI
As shown in Figure 1A, a greater number of BM-derived EPCs, identified by double staining for 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine perchlorate (DiI)–acetylated LDL and Bandeiraea simplicifolia-1 lectin, were observed 2 and 3 weeks after MI in the estradiol group than in the placebo group. It is notable that the stimulus of MI alone resulted in a significant increase in circulating EPCs in both groups in the first week after MI, indicating that mobilization of these cells is a natural response to myocardial injury. Indeed, this has been documented in humans as well,17 with enhanced mobilization of EPCs correlated with better long-term outcome.18 Within 1 week after MI, however, the number of EPCs decreased in a time-dependent manner in the placebo group (Figure 2B), whereas the estradiol group maintained a significantly higher number of circulating EPCs for 2 and 3 weeks after MI (2 weeks, 430±56/mm2 versus 212±19/mm2 for estradiol versus placebo groups, respectively; 3 weeks, 265±25/mm2 versus 161±17/mm2 for estradiol versus placebo groups, respectively [P<0.01]). These findings were corroborated by fluorescence-activated cell sorter (FACS) analysis of peripheral blood samples collected 1, 2, and 4 weeks after MI, indicating that the number of Sca-1+/Flk-1+ cells was consistently greater in the estradiol-treated mice than in placebo-treated animals (Figure 2C).
Enhanced Contribution of BM-Derived EPCs to Myocardial Neovascularization
Next, Tie-2/LZ/BMT mice were euthanized 1 and 4 weeks after MI. As shown in Figure 3, macroscopic examination of 5-bromo-4-chloro-3-indolyl β-d-galactopyranoside (X-gal)–stained hearts 1 week after MI revealed evidence of abundant recruitment of BM-derived progenitor cells, indicated by the blue areas in the cross sections of the myocardium. Four weeks after MI, macroscopic examination the LVs suggested that a significantly greater number of X-gal–positive cells were present in the estradiol-treated mice than in the placebo-treated mice (Figure 3). Microscopic examination revealed that at 1 and 4 weeks after MI, the number of X-gal–positive cells was significantly greater in the estradiol group than in the placebo group (1 week, 298±24/mm2 versus 220±9/mm2, respectively [P<0.05]; 4 weeks, 82±8/mm2 versus 24±7/mm2, respectively [P<0.01]; Figure 4A and 4B). Furthermore, fluorescence immunohistochemistry performed on frozen sections 1 week after MI documented an increase in cells that were double positive for β-galactosidase (indicating BM origin and Tie-2 expression) and the endothelial cell–specific marker isolectin B4 in the estradiol group (Figure 4C). These data indicate that use of estradiol results in an increase in the kinetics of BM-derived EPCs, not only in the circulation but in the myocardium as well, resulting in enhanced incorporation of BM-derived cells into the neovasculature after MI.
eNOS Is Required for Estradiol-Induced Neovascularization
Prior studies have suggested that eNOS is required for estradiol-mediated mobilization of EPCs after arterial injury.13,19 Figure 5 shows the results of echocardiography before and 1 and 4 weeks after MI in eNOS−/− and WT mice receiving 17β-estradiol pellets. Despite estradiol administration, there was more LV dilation in eNOS−/− mice than in identically treated WT mice, indicating that the beneficial effect of estradiol on LV remodeling after MI was lost in the absence of eNOS (LVDd, 3.9±0.1 versus 4.3±0.1 mm, respectively [P<0.05]; LVDs, 2.9±0.2 versus 3.5±0.2 mm, respectively [P<0.05]). LV function, assessed by fractional shortening, was also significantly worse despite estradiol treatment in eNOS−/− mice as compared with WT mice. Histological assessment also revealed that eNOS−/− mice showed more LV fibrosis than did WT mice (50.0±2.7% versus 40.1±2.6%, respectively [P<0.05]; Figure 5B). Moreover, capillary density 4 weeks after MI was significantly lower in estradiol-treated eNOS−/− mice than in identically treated WT mice (1560±83/mm2 versus 2335±84/mm2, respectively [P<0.01]; Figure 5C).
EPC counts, documented by FACS analysis and culture assay significantly increased 1 week after MI only in estradiol-treated WT mice as compared with WT mice (culture assay, 588±55/mm2 versus 284±24 cells/mm2, respectively [P<0.01]; FACS analysis, 13.2±1.1% versus 7.3±0.7%, respectively [P<0.01]; Figure 5D). Thus, estradiol failed to increase EPC numbers after MI and failed to improve functional and anatomic preservation after MI in the absence of intact eNOS signaling.
Matrix Metalloproteinase-9 Is Essential for EPC Mobilization Induced by Estradiol After MI
In BM cells of untreated WT mice, mild matrix metalloproteinase-9 (MMP-9) immunoreactivity was noted, whereas it was undetectable in the BM of untreated eNOS−/− mice. In estradiol-treated mice, MMP-9 was strongly induced 1 day after MI in the WT mice, whereas eNOS-null mice revealed a more modest upregulation of MMP-9 expression (Figure 6A). At least a portion of estradiol-induced MMP-9 expression in the BM is from vascular endothelial growth factor (VEGF) receptor 2–expressing cells (Figure 6B) and results in release of soluble kit ligand (Figure 6C), consistent with the documented role of this pathway in vasculogenesis.20,21
Next, we assessed MMP-9 activity with the use of gelatin zymography. MMP-9 activity was quantified as a band of gelatinolytic activity, which was based on its specific molecular weight (92 kDa), and was confirmed by comparison with commercially available pure MMP-9 (Figure 7A). As shown in Figure 6, estradiol administration resulted in robust induction of MMP-9 activity in WT (FVB/NJ) mice (for WT mice, 40.0±3.0 pg for estradiol versus 21.0±2.0 pg for placebo). Examination of eNOS-null mice and comparison with WT mice revealed that basal MMP-9 activity was significantly reduced in the eNOS-null mice as compared with WT mice and that estradiol-induced MMP-9 activity was also significantly attenuated in the eNOS-null mice as compared with WT control mice (for eNOS−/− mice, 24.0±6.7 pg for estradiol versus 3.0±0.6 pg for placebo). In direct comparison, there was a significant difference in the induced MMP-9 activity between WT mice (C57BL/6J) and eNOS−/− mice receiving 17β-estradiol pellets (76.3±11.7 versus 24.0±6.7 pg, respectively [P<0.05]). These findings suggest that estradiol-induced EPC mobilization after MI might occur by increasing the activity of MMP-9 and that this is, at least in part, dependent on intact eNOS function. This hypothesis was strongly supported by FACS analysis of circulating mononuclear cells from MMP-9−/− mice before and 1 week after MI. Quantification of Sca-1+/Flk-1+ cells revealed that estradiol had no effect on circulating EPC counts after MI in MMP-9–null mice; ie, the MI-induced increase in circulating EPCs was not augmented by estradiol in the absence of MMP-9 (Figure 7B). Most notably, MMP-9–null mice failed to achieve the full benefit of estradiol treatment after MI, showing worse LV dilation and more severely reduced contractile function than was found with WT animals.
The present study provides evidence that estradiol mobilizes circulating EPCs from BM, resulting in incorporation into sites of neovascularization in the adult heart after MI. Moreover, this enhanced neovascularization is associated with a reduction in the extent of LV scarring and with enhanced preservation of LV function in the chronic phase after MI. Most notably, this salutary effect of estradiol appears to be dependent on the ability of estradiol to modulate the kinetics of BM-derived progenitor cells. This latter phenomenon is supported by the fact that eNOS– and MMP-9–null mice fail to mobilize EPCs in response to estradiol administration and also shows significantly worse functional outcome, despite estradiol treatment.
There are now abundant data documenting a role for EPCs in vascular biology. Animal data indicate that BM-derived cells are a part of normal blood vessel homeostasis and can be recruited by a variety of stimuli, including ischemia, tissue injury, and cytokines.8–10,22,23 In humans, the participation of EPCs in vessel formation has been documented in elegant studies in sex-mismatched transplant recipients in which donor cells have been identified in the vasculature of recipient organs.24–26 Most recently, the potential for these cells to induce neovascularization has been exploited in therapeutic trials.27,28
In the context of these discoveries, in humans, observational data linking the quantity and phenotype of circulating progenitor cells to various vascular processes have begun to accumulate. Vasa et al28a first noted a statistical relationship between circulating progenitor cell numbers and phenotype and the presence of various traditional risk factors for coronary artery disease. Others have since corroborated and extended these findings,29 implying that the “health” of the underlying pool of progenitor cells might itself be a major determinant of the overall risk for vascular disease.18 Conversely, EPCs have also been identified as participants in tumor vascularization30,31 and, as such, as possible targets for antitumor therapies.32–34
Juxtaposed against these findings is the current paradox revolving around the role of sex hormones, and in particular estrogen, in cardiovascular health and disease. Animal data and extensive observational human studies have implied a protective role of estradiol against cardiovascular disease.35 Randomized clinical trials, however, have not yielded the anticipated results.36 The reasons for the failure of estrogen replacement to reduce cardiovascular mortality remain to be clarified37; however, our data, along with the body of evidence implicating EPCs as major factors in vascular processes, illuminate another possibility.
The present findings suggest that in the presence of estradiol, the reparative process normally invoked by myocardial injury is markedly enhanced. The enhanced repair process is documented anatomically and functionally and, most notably, appears to be significantly derived via the effect of estradiol on EPC mobilization and recruitment. This latter conclusion is supported by the fact that the therapeutic effect of estradiol has been shown to be reduced in both eNOS-null and MMP-9–null mice, which both showed reduced or absent estradiol-induced EPC mobilization.
VEGF and granulocyte-macrophage colony–stimulating factor can mobilize EPCs from BM into the peripheral circulation, and they have also been shown to enhance recovery after ischemic injury.38,39 Although VEGF-A was initially considered to promote neovascularization through mitogenic and promigratory effects on fully differentiated endothelial cells, it is now clear that VEGF-A also acts to mobilize BM-derived EPCs, resulting in contribution to postnatal neovascularization via vasculogenesis.
The requirement for eNOS for EPC release from the BM has been noted previously,19 as has its role in mediating a variety of the effects of estradiol on the vasculature.13,40–47 The present study advances the notion that the cardioprotective effect of estradiol is mediated, at least in part, by its ability to augment the mobilization and recruitment of BM progenitors for vascular repair. This concept may offer important clues about the failure of hormone replacement in clinical trials. Moreover, because the eNOS-null mouse used in our studies has been shown to be deficient in mobilizing EPCs under multiple stimuli,19 the specific mechanism involved in estradiol-mediated EPC release remains to be ascertained.
Our previous findings11 have also revealed that estradiol administration induces the recruitment and incorporation of EPCs into the endometrial neovasculature in ovariectomized mice. The present study reveals that estradiol can augment EPC mobilization not only into physiological (eg, reproductive) neovascularization but also into sites of neovascularization in adult organs under pathologic conditions. In addition to the obvious application of these findings to the understanding of sex differences in recovery from MI, they may also have important implications for understanding the pathophysiology of certain hormone-dependent solid tumors that have also been shown to be angiogenesis dependent.48
In the present study, the beneficial effects of estradiol were not observed in the acute phase after MI, which is consistent with prior reports.49 These findings may provide clues toward understanding the seemingly conflicting experimental, epidemiological, and clinical data on sex differences in outcome after MI.50,51 On one hand, female sex and intact ovarian function are associated with a lower risk of death from MI before menopause, whereas in postmenopausal life, female sex has been consistently associated with a worse prognosis after MI. Could EPC function be a factor to explain this apparent discrepancy? In this context, it is noteworthy that stem cell mobilization is markedly attenuated in older patients52 and that EPC numbers have also been shown to be reduced with age. We speculate that, among other reasons, the failure of postmenopausal estrogen replacement could be linked to the decreased availability of EPCs, thereby obviating some of the potential benefits of estradiol while still exposing patients to the risks of increased thrombosis, among others.53 It is further tempting to speculate about a potential role for EPCs in the protection against MI that has been documented in premenopausal females and the markedly accelerated incidence of cardiac events that occurs after menopause. Estradiol mobilizes EPCs on a cyclic basis, in part (apparently) for uterine vascularization. However, a side effect of the increased circulating supply of these cells might be the incidental repair of sites of vascular injury, thereby deterring the progression of atherosclerosis.54 If it is assumed that the population of BM cells capable of differentiation into EPCs is finite, it is possible that the monthly consumption of these progenitors for uterine vascularization during premenopause results in depletion of these cells over time, so that after menopause the availability of EPCs is relatively deficient in females as compared with males.
In the present study, estradiol attenuated the impairment of LV function in the chronic phase after MI. The data provide clear evidence that this is due, at least in part, to an estradiol-mediated increase in incorporation of BM-derived EPCs into sites of neovascularization. However, these findings do not exclude other potential mechanisms whereby estradiol may inhibit the adverse LV remodeling after MI and subsequent congestive heart failure, such as inhibition of angiotensin-converting enzyme activity and regulation of the endothelin system.55 Further investigation will clarify these mechanisms and their relative contributions to post-MI recovery.
The results of these experiments may have clinical relevance from 2 perspectives. First, and most direct, there are implications in the ongoing debate about hormone replacement therapy in postmenopausal women, inasmuch as our results could suggest a potential benefit for the management of MI in postmenopausal women, preventing LV dysfunction in the chronic phase after MI. Second, and more comprehensive, the present study has implications for our general understanding of recovery from MI, suggesting that enhanced recruitment and incorporation of BM-derived EPCs may play a significant role in restoring myocardial perfusion, salvaging jeopardized tissue, and attenuating late-phase remodeling and loss of function after MI.
This study was supported in part by National Institutes of Health grants HL-53354, HL-77428, HL-63414, HL-57516, HL-80137, and P01 HL-66957. The authors gratefully acknowledge the secretarial assistance of Mickey Neely and Dierdre Couchon in the preparation of this manuscript.
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From a clinical perspective, the findings in the present study may have several implications. Most general, but perhaps most important, these findings provide another piece of evidence indicating that preservation of the myocardial microvasculature in the peri-infarct zone is associated with preservation of overall left ventricular function. These data also indicate that mobilizing endothelial progenitor cells and enhancing the contribution of these bone marrow–derived stem cells into the myocardial microvasculature can achieve improved outcome after myocardial infarction. Finally, the mechanisms involving endothelial nitric oxide synthase signaling and matrix metalloproteinase-9 that are revealed may provide suitable therapeutic targets for human study.
The online-only Data Supplement can be found at http://circ.ahajournals.org/cgi/content/full/CIRCULATIONAHA.105.553925/DC1.