Novel Mechanism and Role of Angiotensin II–Induced Vascular Endothelial Injury in Hypertensive Diastolic Heart Failure
Objective— The mechanism and role of angiotensin II–induced vascular endothelial injury is unclear. We examined the molecular mechanism of angiotensin (AII)-induced vascular endothelial injury and its significance for hypertensive diastolic heart failure.
Methods and Results— We compared the effect of valsartan and amlodipine on Dahl salt-sensitive hypertensive rats (DS rats). Valsartan improved vascular endothelial dysfunction of DS rats more than amlodipine, by inhibiting endothelial apoptosis and eNOS uncoupling more. Moreover, valsartan inhibited vascular apoptosis signal-regulating kinase 1 (ASK1) more than amlodipine. Thus, AT1 receptor contributed to vascular endothelial apoptosis, eNOS uncoupling, and ASK1 activation of DS rats. Using ASK1−/− mice, we examined the causative role of ASK1 in endothelial apoptosis and eNOS uncoupling. AII infusion in wild-type mice markedly caused vascular endothelial apoptosis and eNOS uncoupling accompanied by vascular endothelial dysfunction, whereas these effects of AII were absent in ASK1−/− mice. Therefore, ASK1 participated in AII-induced vascular endothelial apoptosis and eNOS uncoupling. Using tetrahydrobiopterin, we found that eNOS uncoupling was involved in vascular endothelial dysfunction in DS rats with established diastolic heart failure.
Conclusion— AII-induced vascular endothelial apoptosis and eNOS uncoupling were mediated by ASK1 and contributed to vascular injury in diastolic heart failure of salt-sensitive hypertension.
Salt-sensitive hypertensive patients are more prone to cardiovascular diseases than their salt-insensitive counterparts.1,2 Therefore, it is a clinically important issue to determine the mechanism and the therapeutic strategy of cardiovascular diseases in salt-sensitive hypertension. Vascular endothelial function plays a key role in the pathophysiology3,4 and the prognosis5–7 of cardiovascular diseases, including atherosclerosis, ischemic heart disease, and heart failure. However, the detailed molecular mechanism and the pathological significance of vascular endothelial dysfunction in salt-sensitive hypertension are unknown.
Apoptosis signal-regulating kinase 1 (ASK1), a mitogen-activated protein kinase kinase kinase, has been identified as a proapoptotic signaling molecule.8–11 ASK1 is activated in response to a variety of stress stimuli, such as reactive oxygen species (ROS), angiotensin II (AII), or cytokines, etc. Accumulating in vitro evidence indicates that ASK1 participates in not only apoptosis but also various cellular responses, including cell differentiation and growth, or gene expression. Previously, we have shown that ASK1 is responsible for cardiac hypertrophy and fibrosis,12 vascular intimal hyperplasia,13 and ischemia-induced angiogenesis.14 Furthermore, other investigators have also reported that ASK1 is implicated in cardiac myocyte death and remodeling induced by ischemia.15,16 However, the role of ASK1 in vascular endothelial injury is unclear.
In the present study, by using Dahl salt-sensitive hypertensive rats, the useful model of not only salt-sensitive hypertension but also diastolic heart failure,17,18 and ASK1-deficient mice, we have obtained the first evidence that AII-induced vascular endothelial apoptosis and eNOS uncoupling are mediated by the activation of ASK1 and play a key role in exacerbation of vascular injury in salt-sensitive hypertensive rats at the stage of diastolic heart failure.
Materials and Methods
All procedures were in accordance with institutional guidelines for animal research. Dahl salt-sensitive hypertensive rats (DS rats) (Japan SLC Inc, Shizuoka, Japan) were used in the present study.
Male ASK1−/− mice19 and wild-type mice (C57BL/6J) were used in the present study.
Comparative Effect of Valsartan and Amlodipine on Vascular Injury and Survival Rate of DS Rats Fed High-Salt Diet
To elucidate the direct role of angiotensin II (AII) in vascular diseases of salt-sensitive hypertension, we compared the effect of valsartan (Novartis) and amlodipine (Pfizer) on DS rats. Twelve-week-old DS rats, which had fed a high-salt diet from 7 weeks of age, were given vehicle (0.5% carboxymethyl cellulose [CMC]), valsartan (10 mg/kg/d), or amlodipine (1 mg/kg/d), by gastric gavage once a day for 4 weeks (until 16 weeks of age).
In separate experiments, 12-week-old DS rats, fed a high-salt diet from 7 weeks of age, were orally given vehicle, valsartan (10 mg/kg/d), or amlodipine (1 mg/kg/d) in the same manner as the above experiment, and survival rate was examined until 24 weeks of age.
Effect of Angiotensin II Infusion on Wild-type and ASK1−/− Mice
To examine the role of ASK1 in vascular endothelial injury by angiotensin II (AII), we compared the effects of chronic AII infusion on wild-type and ASK1−/− mice. AII (600 ng/kg/min) was subcutaneously infused to mice via osmotic minipump (ALZA CO) for 4 weeks.
Role of eNOS Uncoupling in End-Stage Heart Failure of DS Rats
Using 20-week-old DS rats with overt heart failure, we examined the role of eNOS uncoupling in end-stage heart failure of DS rats. Tetrahydrobiopterin (BH4; sapropterin hydrochloride, Daiichi Suntory Pharma Co Ltd, Tokyo; 10 mg/kg/d), apocynin (0.3 mmol/kg/d), or hydralazine (20 mg/kg/d) was orally given to 20-week-old DS rats with overt heart failure, for 4 weeks (until 24 weeks of age). Furthermore, as a control, we also examined the effect of tetrahydroneopterin (H4N; Schircks Laboratories) (10 mg/kg/d), which has similar antioxidant properties to BH4 but is not directly linked to eNOS coupling and activity, in DS rats.
The authors had full access to the data and take responsibility for its integrity. All authors have read and agree to the manuscript as written.
Effects of Valsartan and Amlodipine on Vascular Endothelial Function and Remodeling, and Survival of DS Rats
Valsartan and amlodipine slightly and comparably reduced blood pressure of DS rats, throughout 4 weeks of drug treatment (supplemental Figure IA, available online at http://atvb.ahajournals.org). Vascular endothelium-dependent relaxation to acetylcholine of 16-week-old salt-loaded DS rats was remarkably impaired, compared with control DS rats (P<0.01) (supplemental Figure IB), whereas vascular endothelium-independent relaxation by sodium nitroprusside did not differ between the 2 groups of DS rats (data not shown). Despite comparable blood pressure lowering between valsartan and amlodipine, valsartan more potently improved vascular endothelium-dependent relaxation than amlodipine (P<0.05; supplemental Figure IB). Valsartan significantly suppressed coronary arterial thickening, compared with vehicle (P<0.05), whereas amlodipine did not significantly suppress it (supplemental Figure IC).
As shown in supplemental Figure ID, valsartan treatment significantly prolonged survival rate of DS rats, compared with vehicle (P<0.01), whereas amlodipine did not prolong it.
Effects of Valsartan and Amlodipine on Vascular Endothelial Apoptosis of DS Rats
As shown in Figure 1, vascular endothelial apoptosis and ASK1 phosphorylation were significantly enhanced in salt-loaded DS rats. Valsartan attenuated vascular endothelial apoptosis of salt-loaded DS rats more than amlodipine (P<0.05), which was associated with more inhibition of ASK1 by valsartan than amlodipine (P<0.05).
Effects of Valsartan and Amlodipine on Vascular NADPH Oxidase and Superoxide of DS Rats
As shown in supplemental Figure II, 16-week-old salt-loaded DS rats had more NADPH oxidase activity (P<0.01) and vascular superoxide (P<0.01) detected with the fluorescent probe dihydroethidium, than the age-matched control DS rats. The results on preincubation of vascular sections with polyethylene glycol (PEG)-SOD or Tiron confirmed that the increased vascular dihydroethidium fluorescence in salt-loaded DS rats indeed represented superoxide itself (supplemental Figure III). Treatment with valsartan or amlodipine from 12 to 16 weeks of age significantly reduced all the above mentioned parameters of DS rats. However, vascular superoxide levels were reduced by valsartan more than amlodipine (P<0.05; supplemental Figure IIB).
Effects of Valsartan and Amlodipine on Vascular eNOS of DS Rats
As shown in Figure 2, the ratio of dimer to monomer of eNOS, eNOS activity, and plasma NOx levels in 16-week-old salt-loaded DS rats were reduced to 27% (P<0.01), 47% (P<0.01), and 37% (P<0.01), respectively, compared with control DS rats. As shown in supplemental Table I, vascular BH4 levels in salt-loaded DS rats were lower than those in control DS rats (P<0.01), whereas vascular oxidized biopterin levels in salt-loaded DS rats were higher than those in control DS rats (P<0.05). As shown in supplemental Figure IV, using lucigenin chemiluminescence, we found that pretreatment of vascular segments with L-NAME significantly decreased superoxide production in salt-loaded DS rats, but not in control salt-unloaded DS rats, indicating that vascular superoxide in salt-loaded DS rats was at least partially derived from eNOS uncoupling. Valsartan prevented the decrease in the ratio of dimer to monomer of eNOS (P<0.01) and the decrease in eNOS activity (P<0.05) in DS rats more than amlodipine (Figure 2). Valsartan significantly prevented the reduction of plasma NOx in DS rats (P<0.01), whereas amlodipine did not.
Effects of Angiotensin II Infusion on Blood Pressure, Vascular ASK1, and Vascular Endothelial Function of ASK1−/− Mice
As shown in supplemental Figure VA, blood pressure elevation in ASK1−/− mice by AII infusion was comparable to that in wild-type mice, throughout 4 weeks of the infusion. As shown in supplemental Figure VB, AII infusion increased the phosphorylation of vascular ASK1×1.7-fold (P<0.01). On the other hand, as expected, ASK1 band was not detected in ASK1−/− mice with or without AII infusion. As shown in Figure 3, there was no significant difference in the dose-response curve of vascular endothelium-dependent relaxation to acetylcholine between wild-type and ASK1−/− mice without AII infusion. However, AII infusion in wild-type mice significantly impaired vascular endothelium-dependent relaxation to acetylcholine (P<0.01). On the other hand, AII infusion in ASK1−/− mice did not at all impair vascular endothelium-dependent relaxation to acetylcholine. Vascular endothelium-independent relaxation by sodium nitroprusside did not differ between wild-type and ASK1−/− mice, regardless of AII infusion (data not shown).
Effects of Angiotensin II Infusion on Vascular NADPH Oxidase Activity, p22phox, and Superoxide of ASK1−/− Mice
As shown in supplemental Figure VIA and VIB, AII infusion significantly increased vascular NADPH oxidase activity and p22phox expression in either wild-type or ASK1−/− mice, to a comparable degree. On the other hand, the increase in vascular superoxide by AII infusion was smaller in ASK1−/− mice than in wild-type mice (P<0.01; supplemental Figure VIC).
Effect of Angiotensin II Infusion on Vascular Endothelial Apoptosis and eNOS of ASK1−/− Mice
As shown in Figure 4A, AII infusion markedly increased vascular endothelial apoptosis in wild-type mice (P<0.01), but not in ASK1−/− mice. AII infusion in wild-type mice significantly reduced the ratio of dimer to monomer of eNOS (P<0.01), whereas AII infusion in ASK1−/− mice did not alter it (Figure 4B). Vascular eNOS activity was reduced by AII infusion in wild-type mice (P<0.05), whereas did not change by AII infusion in ASK1−/− mice (Figure 4C). Vascular phospho-eNOS and total eNOS levels were not altered by AII infusion in wild-type or ASK1−/− mice (supplemental Figure VII).
Effect of Tetrahydrobiopterin, Apocynin, and Hydralazine on Diastolic Heart Failure of 20-Week-Old DS Rats
In the present study, by echocardiography, we confirmed that 20-week-old salt-loaded DS rats used displayed diastolic heart failure (data not shown), being consistent with previous reports by us17,20 and others.18,21 BH4, apocynin, and hydralazine reduced blood pressure of DS rats to a comparable degree, throughout 4 weeks of the treatment (supplemental Figure VIII). BH4 significantly improved diastolic dysfunction (supplemental Figure IX) and prolonged survival rate of DS rats (P<0.01; Figure 5), whereas apocynin or hydralazine did not improve them, despite their comparable hypotensive effects to BH4. Treatment of DS rats with the same dose of H4N as BH4 did not lower blood pressure of DS rats, and did not improve diastolic dysfunction or survival rate of DS rats (data not shown).
Furthermore, after 4 weeks of each drug treatment, we examined vascular endothelial function and coronary arterial thickening of surviving 24-week-old DS rats and compared with those of 20-week-old DS rats without drug treatment. As shown in supplemental Figure XA and XB, 20-week-old salt-loaded DS rats exhibited remarkable impairment of vascular endothelial function (P<0.01) and prominent coronary arterial thickening (P<0.01), compared with control DS rats. BH4 significantly reversed vascular endothelial dysfunction of DS rats (P<0.05), whereas apocynin did not reverse it and hydralazine did not prevent further exacerbation of endothelial dysfunction. Furthermore, BH4, but not apocynin or hydralazine, prevented further progression of coronary arterial thickening of DS rats (supplemental Figure XB).
As shown in Figure 6A, BH4 significantly increased the ratio of dimer to monomer of vascular eNOS (P<0.01), compared with 20-week-old DS rats, whereas apocynin or hydralazine treatment did not alter it. Compared with 20-week-old DS rats, vascular eNOS activity was also increased by BH4 (P<0.05) but was not altered by apocynin or hydralazine (Figure 6B). BH4 significantly reduced vascular superoxide levels of DS rats (P<0.05), but apocynin or hydralazine could not alter it (Figure 6C). However, vascular NADPH oxide activity was significantly decreased by apocynin (P<0.01), but not altered by BH4 (Figure 6D).
The major purpose of our work was to examine the mechanism of AII-induced vascular endothelial injury and its role in salt-sensitive hypertensive rats with diastolic heart failure. The major findings were that AII-induced vascular endothelial dysfunction was attributed to ASK1-mediated endothelial apoptosis and eNOS uncoupling, and was involved in vascular injury of hypertensive diastolic heart failure.
We17,20 and others21,22 have reported that AII contributes to not only cardiac hypertrophy and remodeling but also the progression of diastolic heart failure in DS rats. However, the precise role of AII in vascular endothelial injury in DS rats is still unknown. In the present work, to determine the potential role of AII in vascular endothelial injury, we compared the effect of valsartan and amlodipine on vascular injury of DS rats (Figures 1 and 2⇑ and supplemental Figures I and II). Recent report by Julius et al23 on subanalysis of the Valsartan Antihypertensive Long-Term Use Evaluation (VALUE) trial has indicated that valsartan is superior to amlodipine in terms of the prevention of hypertensive heart failure, although its mechanism remains to be clarified. Therefore, our present study, comparing between valsartan and amlodipine in DS rats, is of clinical relevance. In the present work, valsartan more prevented the death of DS rats attributable to heart failure than amlodipine, being associated with greater improvement of vascular endothelial function and coronary arterial remodeling by valsartan. Notably, valsartan suppressed vascular endothelial apoptosis to a greater extent than amlodipine, indicating that the improvement of vascular endothelial dysfunction by valsartan was at least in part mediated by the suppression of endothelial apoptosis. Moreover, valsartan more ameliorated vascular eNOS uncoupling than amlodipine, which was accompanied by more decrease in vascular superoxide and more restoration of eNOS activity by valsartan than by amlodipine (supplemental Figure II and Figure 1). These results show that AII participated in vascular endothelial dysfunction of DS rats, by causing not only endothelial apoptosis but also eNOS uncoupling.
In our current work, we found that vascular ASK1 is activated in DS rats in accordance with the occurrence of endothelial apoptosis and eNOS uncoupling and that AII specifically contributed to ASK1 activation in DS rats, as shown by the significant inhibition of ASK1 by valsartan but not amlodipine (Figure 1). Previously, we have reported that ASK1 is involved in vascular neointimal formation induced by balloon injury or cuff injury.13 Furthermore, we have also reported that ASK1 is implicated in AII-induced cardiac hypertrophy and fibrosis.12 Thus, ASK1 seems to be an important signaling molecule responsible for cardiovascular diseases. However, the precise role of ASK1 in AII-induced vascular endothelial injury remains to be defined. Therefore, in the present work, by using mice lacking ASK1, we examined the potential role of ASK1 in vascular endothelial dysfunction, apoptosis, and eNOS uncoupling induced by AII (Figures 3 and 4⇑ and supplemental Figures V through VII). Of note are the observations that AII infusion significantly activated vascular ASK1 and significantly impaired vascular endothelial function, whereas AII infusion did not at all impair vascular endothelial function in mice lacking ASK1. These observations provided the first evidence that ASK1 plays a critical role in AII-induced vascular endothelial dysfunction. To determine the reason for the absence of vascular endothelial dysfunction in ASK1-deficient mice subjected to AII infusion, we measured vascular endothelial apoptosis, NADPH oxidase, ROS, eNOS uncoupling, and eNOS activity. AII infusion markedly caused vascular endothelial apoptosis in wild-type mice, whereas it did not cause apoptosis in ASK1-deficient mice. These observations provide the evidence that ASK1 plays a key role in AII-induced vascular endothelial apoptosis. Furthermore, being consistent with the previous report,24 AII infusion significantly induced vascular eNOS uncoupling in wild-type mice, which was accompanied by the significant increase in vascular superoxide and the significant reduction of eNOS activity. On the other hand, vascular eNOS uncoupling and the reduction of eNOS activity did not apparently occur in ASK1-deficient mice infused with AII, and the increase in vascular superoxide by AII infusion was less in ASK1-deficient mice than wild-type mice. All these results, taken together with the findings that AII infusion increased vascular NADPH oxidase activity and p22phox in wild-type and ASK1-deficient mice to a comparable degree and did not affect phospho-eNOS and total eNOS in either strain of mice, provided the evidence that ASK1 is specifically implicated in AII-induced vascular endothelial dysfunction by causing endothelial apoptosis and eNOS uncoupling.
eNOS uncoupling25 and the increase in NADPH oxidase activity26 have been reported in patients with heart failure. Therefore, the investigation on the relative role of eNOS uncoupling and NADPH oxidase in vascular endothelial injury in heart failure is of great clinical relevance. To further elucidate the potential role of eNOS uncoupling in hypertensive heart failure, we initiated treatment with BH4,27,28 the essential cofactor of eNOS, or apocynin, a specific NADPH oxidase inhibitor, in 20-week-old DS rats with overt heart failure (Figures 5 and 6⇑ and supplemental Figures VIII through X). Of note are the observations that the suppression of vascular eNOS uncoupling by BH4 treatment at advanced stage of heart failure significantly improved cardiac diastolic dysfunction and prolonged survival rate of DS rats. Furthermore, BH4 treatment in DS rats with overt heart failure significantly reversed vascular endothelial dysfunction, and these beneficial effects were associated with the significant reduction of vascular superoxide and the restoration of eNOS activity. On the other hand, the significant NAPDH oxidase inhibition by apocynin treatment or the vasodilation by hydralazine treatment did not significantly improve survival rate of DS rats, despite their similar blood pressure lowering effects to BH4. Differing from BH4 treatment, apocynin significantly inhibited NADPH oxidase activity, but did not improve vascular endothelial function, not prevent the progression of coronary remodeling, not diminish vascular superoxide, and not restore eNOS activity. These results provided the solid evidence that eNOS uncoupling, via the production of superoxide, is involved in the exacerbation of vascular endothelial injury in DS rats, and suggested that eNOS uncoupling may play some role in the pathophysiology of diastolic heart failure in DS rats.
NADPH oxidase is reported to be involved in angiotensin II–induced ROS generation, as reviewed.4,29 However, differing from our present study, previous studies have not examined animals at the stage of advanced vascular remodeling or heart failure. Therefore, the difference in the source of ROS between our present finding and previous findings might be explained by the difference in the stage of progression of vascular remodeling or cardiac dysfunction. Another possible reason is that our present findings might be specific for salt-sensitive hypertension or heart failure. Thus the relative role of eNOS uncoupling and NADPH oxidase in the generation of ROS seems to depend on the stage of vascular remodeling or the type of cardiovascular diseases.
BH4 treatment of DS rats with diastolic heart failure improved cardiac diastolic dysfunction and survival rate, suggesting that vascular endothelial dysfunction caused by eNOS uncoupling may participate in the exacerbation of diastolic heart failure. However, the present study did not allow us to elucidate the potential role of vascular endothelial dysfunction in the pathogenesis of diastolic heart failure, because the main purpose of our present work was to examine the molecular mechanism of vascular endothelial injury in hypertensive diastolic heart failure. Hence, further study is needed to elucidate the accurate role of vascular endothelial injury in the pathophysiology of diastolic heart failure. Moreover, it remains to be determined whether or not our present findings are specific for diastolic heart failure.
In conclusion, in our current work, we obtained the evidence that AII-induced endothelial apoptosis and eNOS uncoupling are mediated by ASK1 activation and play a key role in the exacerbation of vascular injury in diastolic heart failure of salt-sensitive hypertension. Thus, our present work provided novel molecular mechanism underlying AII-induced vascular injury. Furthermore, ASK1 appears to be potentially the useful target for treatment of hypertensive diastolic heart failure.
Sources of Funding
This work was supported by Grants-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science, and Technology.
Original received March 29, 2007; final version accepted September 27, 2007.
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