Endothelin 1 Activation of Endothelin A Receptor/NADPH Oxidase Pathway and Diminished Antioxidants Critically Contribute to Endothelial Progenitor Cell Reduction and Dysfunction in Salt-Sensitive Hypertension
Circulating endothelial progenitor cells (EPCs) are reduced in hypertension, which inversely correlates with its mortality. Deoxycorticosterone acetate (DOCA)-salt hypertension features elevated endothelin (ET) 1 and oxidative stress. We tested the hypothesis that ET-1 induces EPC dysfunction by elevating oxidative stress through the ETA/NADPH oxidase pathway in salt-sensitive hypertension. Both ETA and ETB receptors were expressed in EPCs, but only ETA receptors were significantly increased in EPCs of DOCA-salt rats. EPC number and function were reduced in DOCA-salt rats compared with sham controls, and both were reversed by in vivo blockade of ETA receptors or NADPH oxidase. The enzymatic activities of NAPDH oxidase and its subunits gp91phox, p22phox, and Rac1 were augmented in EPCs of DOCA-salt rats, with concomitantly decreased antioxidant enzymes manganese superoxide dismutase, copper-zinc superoxide dismutase, and glutathione peroxidase 1. Reactive oxygen species level was elevated in EPCs from DOCA-salt rats, accompanied by increased EPC telomerase inactivation, senescence, and apoptosis, which were rescued by ETA or NADPH oxidase blockade. Cell therapy of normal or treated DOCA EPCs, but not untreated DOCA EPCs, significantly increased capillary density and blood perfusion in ischemic hindlimbs of DOCA-salt rats. p53 and Bax/Bcl-2 ratios were increased in EPCs of DOCA-salt rats, which were reversed by ETA antagonist, NADPH oxidase inhibitor, or polyethylene glycol-superoxide dismutase. Finally, in ETB-deficient rats, plasma ET-1 was elevated, and EPC number and telomerase activity were diminished. These results demonstrate, for the first time, that both ET-1 activation of ETA/NADPH oxidase pathway and diminished antioxidants critically contribute to EPC reduction and dysfunction via increased oxidative stress in salt-sensitive hypertension.
Endothelial dysfunction contributes to the pathogenesis and progression of hypertension and is an independent predictor of cardiovascular risk.1 The balance between endothelial injury and recovery is important to reduce cardiovascular incidences.1,2 Endothelial progenitor cells (EPCs) can differentiate into mature endothelial cells and regenerate the injured endothelium.2,3 There has been accumulating evidence for reduced availability and impaired function of EPCs in the presence of cardiovascular risks.3–5 Among them, hypertension is a strong predictor of EPC migratory impairment.5,6 Both clinical and animal studies have indicated that hypertension is inversely correlated with EPC reduction and/or dysfunction.7–9 However, investigations of in vivo EPC biology under hypertensive condition are limited, and the mechanisms underlying EPC dysfunction in hypertension remain poorly understood.
Deoxycorticosterone acetate (DOCA)–salt hypertension exhibits low renin, salt-sensitive but elevated arterial endothelin (ET) 1 and oxidative stress,10 due to vascular NADPH oxidase activation and superoxide formation via the ETA receptors,10 resulting in endothelial dysfunction.10 Elevated arterial ET-1 levels in DOCA-salt rats led to NADPH oxidase activation and superoxide formation via the ETA receptors,10 resulting in endothelial dysfunction. In contrast, ETB receptors may protect against vascular injuries in this setting.11 Circulating EPCs are important backups for endothelium integrity and function. In this study, we tested the hypothesis that ET-1 activation of ETA/NADPH oxidase pathway and diminished antioxidants both critically contribute to EPC dysfunction in DOCA-salt hypertension. Our findings may provide a mechanistic basis for restoring EPC number and function to combat endothelial dysfunction in hypertension.
All of the methods and data analysis are described in the online-only Data Supplement.
Characterization of Bone Marrow–Derived EPCs
Bone marrow–derived EPCs used in the present study were characterized by flow cytometry, Dil-acLDL/lectin double staining and Western blot, which were presented in the online-only Data Supplement (Tables S1 and S2 and Figure S1).
ETA and ETB Receptor Expression in EPCs
The mRNA levels of ETA and ETB receptors were detected in normal EPCs (1.00±0.28 versus 1.27±0.68; P=0.732; n=4–6), which were further confirmed by immunohistochemistry (Figure S2). Furthermore, the expression of ETA receptors in EPCs of DOCA-salt rats was significantly increased compared with sham controls, whereas there were no significant differences in the expressions of ETB receptors in EPCs between DOCA-salt and sham rats (Figure 1).
Activation of NADPH Oxidase and Increased Reactive Oxygen Species in EPCs of DOCA-Salt Rats
Rac1, gp91phox, and p22phox proteins were significantly increased in EPCs of DOCA-salt rats compared with sham controls (Figure S3A). Consistently, NADPH oxidase activity was significantly increased in EPCs from DOCA-salt rats compared with sham rats. In vivo treatment with ETA antagonist (ABT-627) or NADPH oxidase inhibitors (Apocynin) blunted the activation of NADPH oxidase (Figure 2A). When EPCs of DOCA-salt rats were transfected with the dominant-negative Rac1 (DNRac1), which inhibits the key NADPH oxidase subunit Rac1, their NADPH activities were significantly decreased. The same effect was not observed in β-galactosidase–transfected EPCs (Figure 2A). Meanwhile, the intracellular reactive oxygen species (ROS) in DOCA-derived CD34+/Flk-1+ progenitor cells was significantly elevated compared with that in sham rat cells, which was blunted after 4 weeks of treatment with ABT-627 or Apocynin (Figure 2B).
Antioxidant Enzyme in the EPCs of DOCA-Salt Rats
The expressions of major antioxidant enzymes were measured in EPCs from sham and DOCA-salt rats. Except for catalase, the expressions of manganese superoxide dismutase (MnSOD), copper-zinc superoxide dismutase (CuZnSOD), and glutathione peroxidase 1 (GPx-1) in EPCs from DOCA-salt rats were significantly decreased when compared with those from sham rats. In vivo blockade of ABT-627 or Apocynin for 4 weeks significantly reversed the expressions of the decreased antioxidant (Figure 3).
EPC Dysfunctions in DOCA-Salt Rats
The tube formation capacity of EPCs was significantly impaired in DOCA-salt rats compared with sham controls. In vivo treatment with ABT-627 or Apocynin significantly preserved EPC tube formation capacity (Figure 4). In addition, the adhesion activity of EPCs from DOCA-salt rats was decreased by 50% to 60% compared with sham controls, which was reversed by in vivo blockade of ETA receptors (n=4–8; P<0.05) but not by Apocynin (n=3 in Apocynin group and n=5 in DOCA group; P=0.7234).
Increased EPC Apoptosis and Senescence in DOCA-Salt Rats
Both apoptosis and senescence were significantly increased in EPCs from DOCA-salt rats, which were prevented in rats treated with ABT-627 or Apocynin (Figure 5A and 5B). The expressions of apoptosis-related proteins were detected in EPCs. p53 expression in EPCs from DOCA-salt rats was significantly increased and accompanied by upregulation of proapoptotic Bax and downregulation of antiapoptotic Bcl-2 as compared with sham controls. The ratio of Bax/Bcl-2 was significantly increased by ≈2-fold in the EPCs of DOCA-salt rats. The above effects were reversed by chronic blockades of ETA receptors or NADPH oxidase (Figure 5C and Figure S3C and S3D). The ROS scavenger, polyethylene glycol-superoxide dismutase (PEG-SOD, 100 U/mL, 24 hours) can reverse these effects (Figure S3B). Consistently, the telomerase activity in the EPCs of DOCA-salt rats was decreased by ≈70% compared with sham rats, which was rescued after in vivo treatment with ABT-627 or Apocynin for 4 weeks (Figure 5D).
EPC Therapy Restored Impaired Angiogenesis in DOCA-Salt Hypertensive Rats
Serial blood flow measurements by laser Doppler showed that limb perfusion recovery was severely delayed and impaired in DOCA-salt rats compared with sham rats (Figure 6A). The ratio of perfusion in ischemia relative to that in nonischemic hindlimb was 0.18±0.04 for DOCA rats versus 0.29±0.02 for sham rats at day 7 and 0.27±0.06% versus 0.62±0.05% at day 14, respectively (Figure 6B). To determine the effects of EPCs on blood flow reperfusions after the DOCA-salt regiment, 1×107 EPCs were injected intramuscularly into anterior tibial muscles 24 hours after femoral artery excision. The sham EPC implanted group significantly improved blood flow perfusion in DOCA-salt ischemia limbs from day 7 until day 14 (0.51±0.03 versus 0.27±0.06). Implantation with EPCs from ABT-627-treated DOCA rats or EPCs treated with DNRac1 significantly accelerated delayed reperfusion in ischemic hind limbs from day 7 to day 14. In contrast, implantation of DOCA EPCs improved the ischemic limb reperfusion at day 7, but they failed to improve the reperfusion in the DOCA-salt ischemia limb at day 14 (0.33±0.04 versus 0.27±0.06; P=0.849; Figure 6B and 6C).
The capillary density of ischemia hindlimb was significantly deceased in DOCA-salt rats compared with sham rats at day 14. EPCs from sham rats significantly increased the capillary number in ischemia hindlimb compared with the untreated DOCA rats (Figure 6D and 6E). Meanwhile, EPCs from ABT-627-treated DOCA rat or EPCs treated with DNRac1 restored capillary density in DOCA rats as well. To investigate whether implanted EPCs incorporate into existing vessels to participate in angiogenesis in hindlimb ischemia, 5-bromodeoxyuridine (BrdU)–labeled EPCs were injected intramuscularly into the anterior tibial muscle. Some BrdU–positive cells (red fluorescence) integrated into the CD31-positive vessels (green fluorescence), whereas the remaining 5-bromodeoxyuridine–positive cells were found in perivascular spaces or the matrix between muscle fibers surrounding the vessels (Figure 4S).
Effects of ETA Receptor Antagonist, NADPH Oxidase Inhibitor, or Diuretic on Blood Pressure and Circulating EPCs in DOCA-Salt Rats
Average systolic blood pressure in DOCA-salt rats began to increase on day 5 after the DOCA regimen compared with sham controls (Figure S5A) accompanied by a significantly elevated level of circulating CD34+/Flk-1+ progenitor cells (Figure S5B). On day 28, average systolic blood pressure was further increased in DOCA-salt rats compared with sham controls (Figure S5A). However, the level of circulating CD34+/Flk-1+ progenitor cells in DOCA-salt rats was reduced by 50% (Figure S5B). In vivo blockade of ETA receptors, inhibition of NADPH oxidase, or diuretic (all in drinking water) for 4 weeks significantly lowered blood pressure and reserved circulating EPCs in DOCA-salt rats (Figure S5C and S5D).
EPC Number and Telomerase Activity in ETB Receptor–Deficient Rats
The circulating CD34+/Flk-1+ progenitor cell level was significantly decreased in ETB receptor-deficient (ETB−/−) rats compared with ETB+/+ rats (Figure S6A). The telomerase activity in EPCs was also significantly reduced in ETB−/− rats (Figure S6B), paralleled with elevated systolic blood pressure (129±0.9 versus 151±1.2 mmHg; n=6; P<0.05) and plasma ET-1 levels (3.61±0.14 versus 5.17±0.26 pg/mL; n=6; P<0.05).
Direct Effects of ETA Receptor Antagonist and NADPH Oxidase Inhibitor on EPCs
ET-1–induced EPC reduction and NADPH oxidase activation were reversed by pretreatment with the ABT-627 or Apocynin. Transfection of EPCs with DNRac1, which inhibits the key NADPH oxidase subunit Rac1, blunted ET-1–induced EPC reduction and NADPH oxidase activity (Figure S7A through S7C). Consistently, ROS level and apoptosis in normal EPCs were significantly increased after ET-1 treatment in vitro, which was abolished by pretreatment of ABT-627 or Apocynin (Figure S7D and S7E). Telomerase activity was markedly reduced in ET-1–treated normal EPCs, which were rescued by ABT-627 or Apocynin pretreatment (Figure S7F).
The present study demonstrates for the first time that in DOCA-salt hypertension: (1) both ETA and ETB receptors are expressed in rat EPCs, and the expression of ETA receptors is significantly increased; (2) in vivo EPC angiogenesis is significantly impaired; (3) ETA-mediated NADPH oxidase activation leads to decreased EPC number and function; and (4) blockade of the ETA/NADPH oxidase pathway rescues EPC number and function.
Seven-day cultured EPCs used in the present study are heterogeneous populations containing progenitor cells with the potency differentiating into endothelial cells, as we have shown recently.4 A number of clinical and animal studies have shown that the number and function of EPCs are decreased in hypertension. We showed that EPCs from DOCA-salt rats failed to promote capillary formation and blood flow recovery, whereas EPCs from DOCA-salt rats with ETA receptors or NADPH oxidase blockade significantly restored peripheral perfusion. Of note, while the capillary density in DOCA-EPC–implanted hindlimbs was enhanced, blood reperfusion was not proportionally improved after DOCA-EPC implantation on day 14, suggesting that the newly formed capillaries on DOCA-EPC implantation are not functional, leading to impaired angiogenesis and reduced reperfusion. We demonstrated previously that EPCs from DOCA-salt mice secrete thrombospondin 1 (a well-documented antiangiogenic factor) inhibiting Matrigel tube formations, which can be reversed by antioxidant treatments.12 Thus, it is possible that altered EPC paracrine functions impair their ability in angiogenesis in DOCA-salt hypertension.
An important finding in the present study is the presence of ET receptors on rat early cultured EPCs. ET-1, as a major humoral contributor to the development of DOCA-salt hypertension, exerts its biological effects through binding to 2 G protein–coupled membrane receptors in a mature vascular system, namely, ETA and ETB subtypes.13 Decreased circulating EPC number and telomerase activity were found in ETB receptor deficiency (ETB−/−) rats that possess a phenotype of increased plasma ET-1 levels (because of the lack of ET-1 clearance), increased ROS level, and hypertension.14 These data indicate that elevated ET-1 in ETB−/− rats may inactivate EPC telomerase activity and reduce EPC number through the unmasked effect on ETA receptors. Our study provides first evidence that both ETA and ETB receptors are expressed in rat EPCs. More importantly, the upregulated expression of ETA receptors, but not ETB receptors, suggests that ETA receptors may be the therapeutic target for EPC dysfunction in salt-sensitive hypertension. It has been reported that protein kinase C induces ETA receptors at a transcriptional level in rat fibroblasts15 or in cardiac cells from DOCA-salt hypertensive rats.16 However, whether these possibilities exist in EPCs remain unknown.
NADPH oxidase is a complex enzyme with multiple membrane and cytosolic subunits, and pharmacological interventions are rather limited and often difficult for specific inhibition of the enzyme subunits.17 To this end, we transfected EPCs with the DNRac1 by adenoviral vectors to abrogate endogenous Rac1 expression, a key GTPase component of the NADPH oxidase complex.1,10 Our results show that excessive intracellular ROS in EPCs were dominantly generated by activated NADPH oxidase in DOCA-salt rats. Furthermore, blockades of ETA receptors with ABT-627 significantly inhibited NADPH oxidase activity in EPCs from DOCA-salt rats. This evidence from the present study suggests that ETA/NADPH oxidase is a potential pathway involved in EPC dysfunction in DOCA-salt hypertension.
Increased apoptotic and senescence EPCs were observed in DOCA-salt rats, suggesting that some molecules controlling cell survival and cell cycle may be the downstream targets in the ETA/NADPH oxidase pathway. Telomerase protects EPCs from senescence and improves their survival and regenerative properties against excessive ROS,8,9,18 whereas the activity of telomerase in EPCs from DOCA-salt rats was markedly decreased, which partially contributes to impaired EPC angiogenic functions. In addition, the apoptosis-related molecules (p53 and Bax) were upregulated in DOCA EPCs, which promote EPC apoptosis in this hypertensive setting. Blockades of the ETA receptors or NADPH oxidase significantly blunted telomerase activity and inhibited p53 expression and Bax/Bcl-2 ratio. Moreover, PEG-SOD, a membrane-permeable superoxide scavenger, reversed the above changes. Together, oxidative stress induced by ETA/NADPH oxidase activation leads to EPC apoptosis and senescence via blunting telomerase activity, downregulated p53 expression, and Bax/Bcl-2 ratio in DOCA-salt hypertension.
Although ETA receptor antagonism or NADPH oxidase inhibition could preserve the EPC number and its resistance to oxidative stress in DOCA-salt rats, the question remained regarding their direct antihypertensive effect on EPCs. To address this issue, we treated the DOCA-salt rats with the diuretic trichlormethiazide, a nonselective blood pressure–lowering agent, for 4 weeks and found that trichlormethiazide failed to rescue the circulating EPC number in DOCA-salt rats despite its blood pressure–lowering effect. A recent study also showed that diuretics did not reverse the EPC number in spontaneously hypertensive rats when the blood pressure is reduced.19 In addition, it is important to note that, although the systolic blood pressure was markedly reduced in DOCA-salt rats after in vivo ETA receptor blockade or NADPH oxidase inhibition, it was significantly higher (ie, ≈30 mmHg) compared with sham rats. In contrast, the number of circulating EPCs in the 2 groups of pharmacological intervention maintained the similar level to that of sham rats. Moreover, inhibition of ETA receptors or NADPH oxidase also ameliorated ET-1–induced EPC reduction in vitro, without the compounding effect of the blood pressure. Finally, we found that the circulating EPCs were increased at the beginning of the blood pressure elevation, which suggests that high blood pressure itself may not decrease circulating EPCs in DOCA-salt rats and the elevation in the number of EPCs in the beginning of the DOCA-salt regimen may be attributed to increased compensation of EPC mobilization from the bone marrow. Thus, inhibition of ET-1/ETA-NADPH oxidase–induced oxidative stress, more than blood pressure reduction, may account for the preservation of circulating EPC number in DOCA-salt rats.
The present study demonstrates, for the first time, that ET-1 activation of ETA/NADPH oxidase pathway and diminished antioxidants both critically contribute to EPC reduction and dysfunction via increased oxidative stress in salt-sensitive hypertension. Oxidative stress–induced telomerase inactivation, senescence, and apoptosis may represent important cellular mechanisms underlying ET-1–induced EPC reduction and dysfunction in low-renin, salt-sensitive hypertension. Hypertension with exacerbated systemic oxidative stress impairs EPC function, resulting in loss of its regenerative capacity, with obvious implications for endothelial dysfunction. Our finding on how the ETA/NADPH oxidase pathway and diminished antioxidants affect EPC function may provides a mechanistic basis for EPC genetic modification and therapeutic rejuvenation in hypertension.
Sources of Funding
This work was supported by National Institutes of Health grant R01 GM077352 and the American Heart Association Grant-in-Aid 0855601G (to A.F.C.). D.-D.C. is the receipt of the American Heart Association postdoctoral fellowship 0720114Z and the National Science Foundation of China grant 30900519.
We thank Drs Eric Marrotte and Xiao-Ling Dai for their technical support.
D.-D.C. and Y.-G.D. are joint first authors on this article.
The online-only Data Supplement is available with this article at http://hyper.ahajournals.org/lookup/suppl/doi:10.1161/HYPERTENSIONAHA.111.183368/-/DC1.
- Received September 14, 2011.
- Revision received October 11, 2011.
- Accepted February 26, 2012.
- © 2012 American Heart Association, Inc.
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