Aldosterone Impairs Bone Marrow–Derived Progenitor Cell Formation
Aldosterone has been suggested recently to cause vascular injury by directly acting on the vasculature, in addition to causing injury by raising the blood pressure. Bone marrow–derived endothelial progenitor cells (EPCs) have been shown to exert an important role in the repair of the endothelium. In addition, cell-based therapy using EPCs is emerging as a novel therapeutic strategy for myocardial and peripheral vascular diseases. However, impaired formation and function of EPCs has been observed in patients with risk factors for cardiovascular diseases. We evaluated the possible effects of aldosterone on EPCs by examining the progenitor cell formation from bone marrow mononuclear cells ex vivo. Aldosterone (10 to 1000 nmol/L) reduced the formation of progenitor cells in a concentration-dependent manner. This effect of aldosterone was attenuated by cotreatment with spironolactone. Aldosterone reduced the mRNA levels of vascular endothelial growth factor (VEGF) receptor (VEGFR) 2 without having any effect on the production of VEGF or mRNA levels of VEGF and hepatocyte growth factor in the progenitor cells. However, the expression of stromal-derived growth factor 1 mRNA was paradoxically increased. Consistent with the downregulation of VEGFR-2, VEGF-induced phosphorylation of Akt was abolished in the progenitor cells after aldosterone treatment. N-acetylcysteine, an antioxidant, attenuated the inhibitory effects of aldosterone. These data indicate that aldosterone inhibits the formation of bone marrow–derived progenitor cells, at least partly, by attenuating VEGFR-2 expression and the subsequent Akt signaling. Reduction of aldosterone levels, blockade of mineralocorticoid receptor, and/or cotreatment with antioxidants may, therefore, enhance vascular regeneration by EPCs.
Endothelial progenitor cells (EPCs) are mobilized from the bone marrow in response to vascular injury and participate in new vessel formation and re-endothelialization of the injured vessel.1,2 Although EPCs have not yet been strictly defined, partly because of the lack of the specific marker, mononuclear cells from various hematopoietic organs have been shown to contain EPCs.2 Progenitor cells with various characteristics of endothelial lineage have been obtained by cultivation of mononuclear cells from peripheral blood,3,4 bone marrow,5,6 spleen,7,8 or umbilical cord blood9,10 in endothelial differentiation medium containing endothelial growth factors. Vascular endothelial growth factor (VEGF) has been shown to induce the differentiation of mononuclear cells into EPCs by activating the Akt signaling pathway.11 A pivotal role of Akt has also been demonstrated in the recovery of blood flow after ischemia by EPCs.12 After incorporation into the ischemic tissue, EPCs undergo terminal differentiation into endothelial cells in response to angiogenic factors, including VEGF, which are locally upregulated by ischemia. In addition to differentiating into endothelial cells, EPCs also exert angiogenic effects by producing angiogenic factors at the site of incorporation.2,13
Consistent with the significant role of endogenous EPCs on neovascularization, clinical studies have provided an evidence base for a novel and effective cell-based therapeutic strategy using bone marrow–derived progenitor cells for the treatment of myocardial and peripheral ischemia, both major complications of hypertension.1,2 Mononuclear cells derived from the bone marrow or peripheral blood with or without cultivation have been used for such cell-based therapy.1,2
In patients with risk factors for cardiovascular diseases, impaired formation and function of EPCs has been observed,2,13 which could lead to ineffective repair after vascular injury in such patients. In addition, efficacy of cell-based therapy is considered to be limited by EPC dysfunction.2,13 However, the molecular mechanisms underlying the reduced formation and function of EPCs, which can be a potential therapeutic target, have not yet been well characterized.
In addition to the effects attributable to elevation of the blood pressure, aldosterone has been suggested to contribute to vascular damage by directly acting on the vasculature.14 Indeed, aldosterone can cause swelling of endothelial cells,15 stimulate proliferation of vascular smooth muscle cells,16 and induce proinflammatory molecules in peripheral blood mononuclear cells.17 However, the effects of aldosterone on the differentiation of bone marrow precursor cells into EPCs, an early process important for the recovery of injured endothelium, are unknown. In addition, the question of whether aldosterone impairs EPC formation is potentially important, because patients receiving cell-based therapy often have underlying cardiovascular diseases, in which mineralocorticoid receptor (MR) activation is implicated, such as heart failure.18 In the present study, we examined whether aldosterone inhibits the formation of bone marrow–derived progenitor cells (BMPCs) and thereby attempted to uncover the potential target for endothelial recovery and enhancement in cell-based therapy for vascular regeneration.
BMPCs were cultivated according to a previously described method for the isolation of EPCs from bone marrow.6,19 In brief, bone marrow mononuclear cells were isolated from the femurs and tibias of 6-week-old male SD rats (CLEA Japan, Inc, Tokyo, Japan) by density gradient centrifugation using Ficoll-Paque Plus (GE Healthcare Bio-Sciences). The mononuclear cells were cultured in the endothelial differentiation medium consisting of DMEM supplemented with 10% FBS, 50 μg/mL heparin, 10 ng/mL recombinant human VEGF (R&D Systems), 5 ng/mL fibroblast growth factor 2 (R&D Systems), 100 μg/mL streptomycin, and 500 μg/mL penicillin on fibronectin-coated dishes. Adherent cells after 4 days of culture were used as BMPCs in the present study. We showed previously that BMPCs obtained using this method differentiated into endothelial cells after injection and exerted angiogenic effects on the glomerular capillaries in nephritic rats.20 To evaluate the effects of aldosterone on BMPC formation, bone marrow mononuclear cells were incubated for 4 days in the presence or absence of the hormone (10 to 1000 nmol/L). Thereafter, BMPCs were trypsinized and counted using a hemocytometer.
Bone marrow mononuclear cells obtained from 2 or 3 rats were used for each experiment. Experiments with every treatment performed at least in duplicate with identical protocol were repeated at least twice. For the experiments to determine the effects of spironolactone (0.5 or 1 μmol/L) or N-acetylcysteine (NAC, 0.1 or 0.5 mmol/L), cells were pretreated with the drugs for 1 hour before the addition of aldosterone (100 nmol/L).
For determination of their endothelial lineage characteristics,1,11 BMPCs were evaluated for the uptake of acetylated low-density lipoprotein (LDL) and the binding of Bandeiraea simplicifolia lectin (BS-1). After incubation with 10 μg/mL 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine-labeled acetylated LDL (Biogenesis Ltd) for 1 hour, the cells were fixed in 2% paraformaldehyde, stained with 100 μg/mL fluorescein isothiocyanate–labeled BS-1 (Sigma), and examined by fluorescence microscopy.
Analysis of the mRNA Levels
Total RNA was extracted from BMPCs with an RNA extraction kit, RNeasy Mini (QIAGEN KK). For semiquantitative RT-PCR analysis, the cDNA product was synthesized and amplified by PCR using a commercial kit (QIAGEN OneStep RT-PCR kit). In addition to semiquantitative RT-PCR, expression levels of VEGF receptor 2 (VEGFR-2) mRNA were also determined by real-time RT-PCR. Methods of semiquantitative and real-time RT-PCR analyses are detailed in the online data supplement (available online at http://hyper.ahajournals.org).
Western Blot Analysis
Western blot analysis was conducted according to a method described previously.21 Detailed methods are available in the online data supplement.
After 4-days of incubation of bone marrow mononuclear cells in the differentiation medium described above with or without aldosterone (100 nmol/L), BMPCs were further incubated with or without aldosterone (100 nmol/L) for 24 hours. Culture supernatants were collected and assayed for rat VEGF with a murine VEGF ELISA kit (R&D Systems) using rat VEGF (R&D Systems) as a standard. Cross-reactivity to human VEGF was negligible under our assay conditions. VEGF production was expressed as the amount of VEGF produced per day divided by the cell number counted at the end of the incubation period.
Reactive Oxygen Species Measurement
Reactive oxygen species (ROS) levels in BMPCs were determined by using dichlorodihydrofluorescein (Sigma) according to a method described previously.22 Methods are detailed in the online data supplement.
All of the data were expressed as mean±SEM. Multiple parametric comparisons were performed by ANOVA, followed by Fisher’s protected least-significant difference test. Comparisons between 2 groups were performed by a t test. P values <0.05 were considered to be statistically significant.
Aldosterone Inhibits BMPC Formation via MR
BMPCs incorporated acetylated LDL and stained positive for BS-1 lectin (Figure 1A). These characteristics have been shown to be shared by EPCs from various sources.1,10,11,19,23 BMPCs obtained from cultures exposed to aldosterone exhibited preservation of these characteristics, because >90% incorporated acetylated LDL and stained positive for BS-1 lectin (Figure 1B). Aldosterone (10 to 1000 nmol/L) inhibited the formation of BMPCs in a concentration-dependent manner (P<0.05; Figure 1C).
The inhibitory effect of aldosterone on BMPC formation was significantly attenuated by concomitant treatment with the MR antagonist spironolactone (P<0.05; Figure 2A). MR mRNA was detected in freshly isolated bone marrow mononuclear cells and BMPCs (Figure 2B). Aldosterone did not modify MR mRNA levels in BMPCs (Figure 2C).
Effects of Aldosterone on the Expression of Angiogenic Factors in BMPCs
EPCs obtained from peripheral blood have been shown to secrete angiogenic factors, including VEGF and hepatocyte growth factor (HGF),2 which could enhance growth and differentiation of EPCs in an autocrine manner. Because BMPCs produce a significant amount of VEGF,20 aldosterone may inhibit the formation of BMPCs by reducing VEGF production. Production (Figure 3A) and mRNA levels (Figure 3B) of VEGF were, however, not significantly altered by aldosterone (P>0.05). To analyze the effects of aldosterone on the expression of other endothelial growth factors, we next examined the mRNA levels of HGF and stromal-derived factor (SDF) 1. SDF-1, a growth factor known to be involved in tube formation of vascular endothelial cells in an autocrine manner,24 has been shown recently to enhance EPC differentiation.25 As shown in Figure 3C, whereas there was no significant change in the HGF mRNA level (P>0.05), the SDF-1 mRNA level was paradoxically increased by exposure to aldosterone (P<0.05).
Aldosterone Downregulates VEGFR-2 and Inhibits Akt Phosphorylation in BMPCs
We next examined the effect of aldosterone on the expression of VEGFR-2 mRNA in BMPCs. In contrast to the expression of endothelial growth factors, there was a marked reduction of VEGFR-2 mRNA levels (P<0.05), as determined by semiquantitative RT-PCR, in cells exposed to aldosterone (Figure 3C). Real-time RT-PCR analysis revealed that aldosterone significantly reduced the VEGFR-2 mRNA expression levels at a concentration range similar to that at which its inhibitory effect on BMPC formation was observed (P<0.05; Figure 4A). We next investigated the effect of MR blockade on the reduction in VEGFR-2 mRNA by aldosterone. Real-time RT-PCR analysis revealed that spironolactone (1 μmol/L) significantly attenuated the inhibitory effect of aldosterone (100 nmol/L) on the VEGFR-2 mRNA levels in BMPCs (control: 1.00±0.00, n=6; aldosterone: 0.39±0.09, n=6; P<0.05 versus control values; and aldosterone + spironolactone: 0.58±0.06, n=6; P<0.05 versus control values and values with aldosterone). These results suggest a significant role of MR in the reduction in VEGFR-2 mRNA levels by aldosterone.
To examine the functional consequence of inhibition of VEGFR-2 expression by aldosterone, we next investigated the activation of Akt, a downstream signaling pathway, which has been shown to mediate the formation and proangiogenic function of EPCs.11,12 Consistent with the levels of VEGFR-2 expression, although exposure to VEGF significantly increased phosphorylation of Akt in BMPCs (P<0.05; Figure 4B), cells that had been treated with aldosterone failed to show such response to VEGF stimulation under these conditions (P>0.05; Figure 4C). The basal levels of phosphorylated Akt as determined by the phosphorylated Akt (P-Akt)/Akt ratio were not significantly different between BMPCs with and without aldosterone (control: 1.0±0.0 versus cells with aldosterone: 1.1±0.2; n=6; P>0.05).
NAC Restores BMPC Formation and Expression of VEGFR-2 mRNA
Because ROS have been suggested to be involved in signaling pathways of aldosterone,14 we determined the effects of aldosterone on ROS levels in BMPCs. ROS levels in BMPCs obtained in the presence of aldosterone (100 nmol/L) were significantly increased as compared with those in BMPCs that had not been exposed to aldosterone (control: 100.0±13.3%, n=5; aldosterone: 183.1±22.7% of control, n=5; P<0.05 versus control values). We next examined whether NAC, an antioxidant, could modify the inhibitory effects of aldosterone on BMPC formation and VEGFR-2 mRNA expression. As shown in Figure 5A, NAC attenuated the inhibitory effect of aldosterone on BMPC formation in a concentration-dependent manner (P<0.05). NAC (0.5 mmol/L) alone did not significantly modify BMPC formation in the absence of aldosterone (data not shown). In addition, NAC also attenuated the inhibitory effect of aldosterone on VEGFR-2 mRNA expression (P<0.05; Figure 5B).
Recent studies have suggested that aldosterone may cause vascular injury through direct effects on the vascular cells, including endothelial and smooth muscle cells.14 The present study demonstrated that the bone marrow-derived precursor cells can also serve as a target for aldosterone. It seems that aldosterone may inhibit the early process of differentiation into EPCs from precursor cells in bone marrow by reducing the VEGFR-2 expression.
VEGFR-2, expressed on the cells of endothelial lineage, has been shown to mediate the major physiological effects of VEGF, including cell proliferation, survival, migration, and differentiation.26,27 Activation of VEGFR-2 is known to stimulate Akt, a key signaling molecule known to regulate cell survival, activation of endothelial NO, and formation of EPCs.11,12,26 The present study demonstrated that reduction of VEGFR-2 mRNA levels in BMPCs by aldosterone was accompanied by impaired phosphorylation of Akt in response to VEGF. These findings indicate that aldosterone probably inhibits the formation of BMPCs by downregulating VEGFR-2 expression, thereby also inhibiting the downstream signaling pathways, including Akt activation, that are important for maturation of EPCs. The precise mechanism by which aldosterone reduces VEGFR-2 expression still remains to be determined.
Some molecular mechanisms underlying the reduced formation of EPCs in patients with cardiovascular risk factors have been suggested recently. Tumor necrosis factor α, high glucose,28 and C-reactive protein4 have been shown to reduce EPC formation, as determined by the same ex vivo assay system as that used in the present study using peripheral blood mononuclear cells. In addition to these proinflammatory molecules, the present study revealed that aldosterone can also act as an inhibitory factor. Activation of p38 mitogen-activated protein kinase has been reported to be involved in the reduced EPC formation by tumor necrosis factor α and high glucose.28 Because aldosterone is also able to stimulate p38 mitogen-activated protein kinase in vascular smooth muscle cells,29 these inhibitory factors may act via a common pathway. In addition, blockade of angiotensin II receptor type I, which potentially decreases aldosterone levels, has been demonstrated to increase the number of circulating EPCs in patients with type 2 diabetes mellitus.30 Investigation of the possible interaction of aldosterone with these inhibitory factors on EPC formation is underway in our laboratory.
Conflicting data about the role of MR activation in the angiogenic response to ischemia have been reported. Blockade of MR by eplerenone preserved the capillary density after myocardial ischemia,31 whereas MR activation was shown to enhance angiogenesis in an ischemic limb.32 Such discrepancies may be explained by the differential effects of MR activation on each process of vascular repair, in which multiple cell types and proinflammatory molecules are involved. Although the present study indicated that aldosterone inhibited BMPC formation, an early differentiation step in the maturation process of bone marrow–derived precursor cells into EPCs in vitro, the in vivo effect of aldosterone-signaling blockade on EPC formation remains to be determined.
Recent evidence indicates that angiogenic cell therapy may be effective for treatment of myocardial and peripheral vascular diseases refractory to the conventional treatment. However, there are some patients who do not respond to cell therapy for unexplained reasons. Freshly isolated bone marrow mononuclear cells are one of the major preparations used for cell-based therapy.2 The injected cells are considered to adhere and differentiate into endothelial lineage cells with angiogenic properties in response to angiogenic factors, including VEGF, at the site of incorporation in the ischemic organ. The present study suggests that inhibition of differentiation of bone marrow mononuclear cells by aldosterone may be potentially involved in some patients who fail to respond to angiogenic cell therapy. In patients with MR activation, such as those with hyperaldosteronism and heart failure, concomitant MR blockade by spironolactone or eplerenone may enhance the response to cell-based therapy.
The present finding that NAC, an antioxidant that can increase intracellular glutathione levels, attenuated inhibitory effect of aldosterone on BMPC formation suggests a significant role of ROS in the reduction of BMPC formation. The protective effect of NAC may be partly attributable to attenuated reduction of VEGFR-2 expression. Consistent with the notion that antioxidants may exert protective effects on VEGFR-2 expression, a recent study demonstrated that expression levels of VEGFR-2 in EPCs obtained from mice deficient of glutathione peroxidase 1, an enzyme with antioxidant properties, was reduced after ischemic limb injury, as compared with those in EPCs isolated from wild-type mice.33 In addition, the observation that NAC attenuated the inhibitory effect of aldosterone on BMPC formation suggests that coadministration of antioxidants may improve the outcome of angiogenic cell therapy. In accordance with this notion, cotreatment with antioxidants has been demonstrated to enhance angiogenesis after bone marrow cell therapy in limb ischemia.34
The present study showed that aldosterone inhibited the formation of BMPCs by reducing the expression of VEGFR-2 and inhibiting activation of the subsequent signaling pathways, including Akt phosphorylation. These data reveal a novel biological activity of aldosterone and suggest an additional mechanism by which this hormone may participate in the progression of vascular injury. Reduction of aldosterone levels, blockade of MR, and/or cotreatment with antioxidants may enhance vascular regeneration induced by EPCs.
We thank Hidemasa Onoda for expert technical assistance.
Sources of Funding
This work was supported by Mochida Pharmaceutical Co, Ltd, and a Grant-in-Aid for Scientific Research from the Japan Society for the Promotion of Science.
- Received May 22, 2006.
- Revision received June 9, 2006.
- Accepted June 27, 2006.
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