Aldosterone Antagonism Improves Endothelial-Dependent Vasorelaxation in Heart Failure via Upregulation of Endothelial Nitric Oxide Synthase Production

Journal of Cardiac Failure Vol. 12 No. 3 2006

Basic Science and Experimental Studies

Aldosterone Antagonism Improves Endothelial-Dependent Vasorelaxation in Heart Failure via Upregulation of Endothelial Nitric Oxide Synthase Production HOANG M. THAI, MD, BAO Q. DO, MD, TRUNG D. TRAN, MD, MOHAMED A. GABALLA, PhD, AND STEVEN GOLDMAN, MD Tucson, Arizona

ABSTRACT Background: Altering the renin-angiotensin aldosterone system improve mortality in heart failure (HF) in part through an improvement in nitric oxide (NO)-mediated endothelial function. This study examined if spironolactone affects endothelial nitric oxide synthase (eNOS) and NO-mediated vasorelaxation in HF. Methods and Results: Rats with HF after coronary artery ligation were treated with spironolactone for 4 weeks. Rats with HF had a decrease (P ! .05) in left ventricular (LV) systolic pressure (130 6 7 versus 118 6 6 mm Hg) and LV pressure with respect to time (9122 6 876 versus 4500 6 1971 mm Hg/second) with an increase in LV end-diastolic pressure (4 6 2 versus 23 6 8 mm Hg). Spironolactone did not affect hemodynamics but it improved (P ! .05) endothelial-dependent vasorelaxation at more than 1028 M acetylcholine that was abolished with NG-monomethyl-L-arginine. The eNOS levels were decreased (P ! .05) in the LV and the aorta; spironolactone restored LV and aortic eNOs levels to normal. Conclusion: Spironolactone prevents the decrease in eNOS in the LV and aorta and improves NOdependent vasorelaxation, suggesting that one potential mechanism of spironolactone is an improvement in vasoreactivity mediated though an increase in NO. Key Words: Endothelial-dependent vasorelaxation, nitric oxide, spironolactone.

Successful therapies aimed at reversing neurohormonal activation of the renin-angiotensin-aldosterone system (RAAS), such as angiotensin-converting enzyme (ACE) inhibitors and angiotensin receptor blockers (ARB), normalize endothelial dependent vasorelaxation, reduce symptoms, and improve survival in heart failure (HF).1–4 The Randomized

Aldactone Evaluation Study trial demonstrated a reduction in overall mortality in patients with HF treated with spironolactone, an aldosterone receptor antagonist, in combination therapy with an ACE inhibitor.5 This is not surprising because aldosterone has deleterious effects in patients with HF and has been reported to be persistently elevated after ACE inhibitor treatment.6 Some of these maladaptive effects caused by aldosterone include the initiation of potassium and magnesium excretion, which may result in increased arrhythmias and coronary vasoconstriction, and the promotion of vascular and cardiac fibrosis via fibroblast stimulation.7–13 Aldosterone is also been reported to have adverse effects on vascular endothelial function and has been shown to inhibit nitric oxide (NO) in tissue culture.8 A decrease in NO-mediated endothelial vasorelaxation is believed to be responsible, at least in part, for the increase in vascular tone seen in HF.14–17 Based on these potential adverse effects of aldosterone on endothelial function, this study was designed to determine if spironolactone alters endothelial nitric oxide (eNOS) production and NO-mediated vasorelaxation in HF.

From the Section of Cardiology, Department of Medicine, Southern Arizona VA Health Care System, Sarver Heart Center, University of Arizona, Tucson, Arizona. Manuscript received June 8, 2005; revised manuscript received December 29, 2005; revised manuscript accepted January 4, 2006. Reprint requests: Hoang M. Thai, MD, Assistant Professor of Medicine, Cardiology Section, 1-111C, Southern Arizona VA Health Care System Hospital, 3601 S. 6th Avenue, Tucson, AZ 85723. Supported in part by grants from the Department of Veterans Affairs, the American Heart Association, the WARMER Foundation, the Hansjorg Wyss Foundation, and the Biomedical Research and Education Foundation of Southern Arizona. 1071-9164/$ - see front matter Ó 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.cardfail.2006.01.002

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Spironolactone Increases eNOS

Methods Overall Study Design Adult male Sprague-Dawley rats (8–10 weeks old) were subjected to myocardial infarction by coronary artery ligation and randomized to placebo or treatment with spironolactone, given orally for 4 weeks in the drinking water at a dose of 7 mgkgday. All rats were fed with standard rat chow, given water ad libitum, and housed in a single room of the animal facility with a 12-hour light/dark cycle and independent ventilation, temperature, and humidity control. The study was terminated after 10 rats in each of 3 groups (sham rats without treatment, HF rats without treatment, and HF rats treated with spironolactone) were randomized and successfully studied. Physiologic experiments performed in the study animals included measurements of hemodynamic variables and quantification of the vasorelaxation response of the thoracic aortic cross sectional rings to acetylcholine (ACh). Left ventricular and aortic tissue was analyzed for eNOS levels. The experiments were performed in an American Association for Accreditation of Laboratory Animal Care accredited facility with approval from the animal use committees of the Southern Arizona Veteran’s Health Care System and the University of Arizona. Experimental Myocardial Infarction Myocardial infarction (MI) was created using techniques standard in our laboratory.18,19 Briefly, 3-month-old Sprague-Dawley rats weighing 250–300 g were anesthetized with inactin and a left thoracotomy was performed. The heart was expressed from the thorax and a ligature was placed around the proximal left coronary artery. The heart was returned to the chest cavity and the thorax was closed. Rats received acetaminophen (67 mg/mL) in drinking water as postoperative analgesia. One day after MI, rats were anesthetized with halothane and a 9-lead electrocardiogram performed. Rats with evidence of large MI were selected for study and randomized. Briefly, the presence of Q waves (O1 mV) in the limb leads (I or aVL) and the sum of the R waves in the precordial leads (!10 mV) were used as criteria for large MI. Hemodynamically, rats with large MI and HF had a LV end-diastolic pressure (EDP) O16 mm Hg. Our laboratory has shown that rats selected in this fashion have large MI averaging 40% of the left ventricle.27,28 Rats that had thoracotomy but did not have coronary artery ligation were designed as sham-operated controls. In our laboratory, this procedure has a 40% mortality rate. In this study, there was no mortality in rats after randomization and no obvious changes in clinical presentations of the rats in the treatment groups. LV Hemodynamics Four weeks after randomization, rats were anesthetized with thiobutabarbital (100 mg/kg intraperitoneally). A 1- mm micromanometer-tipped catheter (Millar Instruments, Houston, TX) was inserted into the right carotid artery. The catheter was advanced into the aorta and then into the left ventricle under constant pressure monitoring. The zero pressure baselines were obtained by placing the pressure sensor in 37 C saline before measurements. After a period of stabilization, LV and aortic blood pressure and velocity were recorded and digitized at a rate of 1000 Hz using a PC equipped with analog-digital converter and customized software. From these data, the LV pressure with respect to time (dP/ dt), and LVEDP was measured according to previously described



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methods. Phasic aortic pressure was measured, and the electronic mean was determined after withdrawal of the LV catheter into the aortic root. Vasorelaxation Response of Thoracic Aortic Segments The vasorelaxation response of thoracic aortic segments was examined using standard techniques used in our laboratory. Briefly, a 3.0- to 3.5-mm section of the ascending thoracic aorta was mounted on a ring apparatus attached to a force transducer. The artery segment was attached to stainless steel wire stirrups with one wire fixed in place and the other attached to the transducer. The tissue was suspended in 37 C bath of Krebs-Henseleit solution suffused with 95% oxygen and 5% carbon dioxide. Rings were stretched to a resting tension of 1 gram and allowed to equilibrate for 45 minutes. Rings were precontracted with 60 mM of KCl for 30 minutes and then returned to Krebs-Henseleit solution and allowed to equilibrate again for 45 minutes. Rings were constricted with phenylephrine (3 mM) until a steady-state constriction was obtained. Dose-response studies were performed with increasing concentrations of ACh (1029 to 1024 M) and the resulting vasorelaxation recorded. Determination of eNOS Protein Levels eNOS protein levels are measured using standard immunoblot techniques as described previously. Briefly, LV tissue and aortic tissue were ground up with homogenization buffer (100 mM imidazole buffer and dH2O) at a concentration 1 mL buffer/0.1 gram tissue using a handheld tissue homogenizer (Polytron, Glen Mills, NJ). The homogenized tissue was centrifuged at 10,000g at 4 C for 5 minutes and the supernatant was removed for analysis. In addition, 4 ascending thoracic aortic segments from each treatment group were ground using liquid nitrogen using a mortar and pestle. The ground tissue was mixed with 100 mL of homogenization buffer and centrifuged at 5000g at 4 C for 5 minutes. The remaining supernatant was concentrated using a Minicon B15 concentrator (Amicon). Protein concentration was determined using standard protein analysis with the Lowry method and linear regression (Sigma). The supernatant from the left ventricle and the concentrated supernatant from the thoracic aortic segments were fractionated using 7% sodium dodecyl sulfate polyacrylamide gel electrophoresis. Proteins were then transferred to polyvinylidene difluoride membranes. Membranes were blocked overnight at 20 C in 5% nonfat dry milk in 1X phosphate buffer solution with 0.1% Tween (PBS-T). The membranes were then washed with PBS-T and incubated with a mouse anti-eNOS immunoglobulin G antibody (1:500) (Transduction Laboratories) for 1 hour. The eNOS was then detected by washing the membrane in horseradish peroxidase–labeled rabbit anti-mouse immunoglobulin G secondary antibody (1:40,000) for 1 hour and exposing it to x-ray film for 30 minutes. The levels of eNOS were measured using a photophosphorimaging detection unit, which expressed the degree of exposure in intensity units. Statistical Analysis Data are expressed as mean 6 standard error (SE). In both the physiologic and biochemical measurements, the Student’s t-test was used to compare sham versus HF and spironolactone treated versus untreated HF rats with a significant P value defined as P ! .05.

242 Journal of Cardiac Failure Vol. 12 No. 3 April 2006 These eNOS levels in spironolactone-treated HF animals were similar to noninfarcted sham animals. Figure 2 shows representative immunoblots of aortic eNOS.

Results In Vivo Hemodynamics, Heart Weights

Compared with sham animals, HF animals had a 5-fold increase in LVEDP, a 9% reduction in systolic blood pressure, a 19% reduction in mean arterial blood pressure, and a 51% reduction in LV dP/dt. There were no changes in hemodynamics or heart weights in HF rats treated with spironolactone compared with untreated HF rats (Table 1). The right ventricular weights increased in the HF rats. This is consistent with our previous work showing the increase in right ventricular weight, presumably because of passive pulmonary hypertension in the HF rats.27 Endothelial-Dependent Vasorelaxation

The degree of vasorelaxation in aortic rings of HF animals compared with sham animals in response to ACh was significantly reduced (P ! .05). Treatment of HF animals with spironolactone improved (P ! .05) endothelialdependent vasorelaxation at ACh concentrations greater than log28 10 . This effect of spironolactone was abolished by the addition of NG-monomethyl-L-arginine (LNMMA), an inhibitor of nitric oxide. To examine the maximal vasodilatory effects in our treated groups we compared the vasorelaxation response at the highest ACh concentration of 1024 with that of an exogenous source of nitric oxide, sodium nitroprusside (1025 M). There was no difference in MI rats treated with spironolactone compared with sham treated with spironolactone (50.3 6 7.0% versus 36.4 6 3.3%, P 5 .2). Interestingly, in MI rats, spironolactone decreased (P ! .05) the maximal vasoconstrictor response to phenylephrine (0.1 mM) compared with sham (94.1 6 2.6% versus 78.3 6 3.5%). eNOS Protein Levels

Rats with HF had reduced levels of eNOS protein in their LV myocardium (13.9 6 2.8 versus 36.2 6 9.5 intensity units/mg of protein, P ! .05, Fig. 1) and thoracic aortas (25.8 6 3.6 versus 57.3 6 16.6 intensity units/mg, P 5 .05, Table 2). Treatment with spironolactone was associated with increased eNOS protein levels compared to untreated HF animals in both LV myocardium (35.6 6 5 versus 13.9 6 2.8 intensity units/mg, P ! .05) and thoracic aortas (39.6 6 4.6 versus 25.8 6 3.6 intensity units/mg, P 5 .02).

Discussion Our data showed a moderate decrease in both systolic blood pressure and LV dP/dt in HF animals treated with spironolactone, but these hemodynamic changes were not accompanied by reduction in the elevated LVEDP. Despite this, heart failure animals treated with spironolactone demonstrated a significant restoration of endothelial-dependent vasorelaxation at low to moderate concentrations of ACh. The NO inhibitor L-NMMA blunted this improvement in endothelial-dependent vasorelaxation. The significant decrease of eNOS in the aorta suggests that the impaired endothelial-dependent vasorelaxation is due to a lack of NO. This is confirmed by the restoration of endothelial-dependent vasorelaxation by spironolactone because it increases eNOS in the left ventricle and aorta. The importance of this process is demonstrated by the attenuation of improved endothelialdependent vasorelaxation in MI animals by L-NMMA. The comparison of ACh-mediated vasorelaxation to the response seen with nitroprusside demonstrates that, although spironolactone is a potent mediator of endothelial-dependent vasorelaxation, it is not the only mechanism that influences the arterial vascular smooth muscle reactivity in HF. This concept is supported by our phenylephrine data showing that, although vasoconstriction is accentuated in MI animals without treatment, reflecting an activated adrenergic milieu in heart failure, the vasoconstrictor response to phenylephrine is attenuated in MI animals treated with spironolactone. Last, from our data, it appears that spironolactone has minimal effects as an afterload reducing agent. There is neurohormonal activation of the RAAS in patients with heart failure. Therapies aimed at reversing neurohormonal activation, such as ACE inhibitor and ARB, improve mortality and symptoms. Unfortunately, ACE inhibitor and ARB only suppress plasma aldosterone levels transiently. The use of spironolactone in patients with HF was an attempt to suppress aldosterone more effectively. This was accompanied by a reduction in overall HF mortality in patients with refractory HF being treated with ACE inhibitor.5,21 These improvements in survival outcome

Table 1. Left Ventricular, Aortic Pressures, and Heart Weights in Sham, Heart Failure Rats, and Heart Failure Rats Treated With Spironolactone LV SP (mm Hg) MAP (mm Hg) LVEDP (mm Hg) LV dP/dt (mm Hg/s) LV weight (grams) RV weight (grams) Sham HF HF 1 spironolactone

130 6 7 118 6 6* 106 6 13*

119 6 9 96 6 5* 94 6 13*

462 23 6 8* 19 6 4*

9122 6 876 4500 6 1971* 4768 6 690*

0.74 6 0.01 0.74 6 0.01 0.67 6 0.09

0.19 6 0.04 0.34 6 0.11* 0.35 6 0.08

Values are mean 6 SE; n 5 10 in each group, except for n 5 8 in the LV and RV weights with spironolactone. HF, heart failure; LV, left ventricular; LVEDP, left ventricular end-diastolic pressure; LV SP, left ventricular systolic blood pressure; MAP, mean arterial pressure. *P ! .05 vs sham.

Spironolactone Increases eNOS

Percent Vasorelaxation (%)

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Heart Failure + Spironolactone Heart Failure Sham

120

*

100

*

*

eNOS + Control HF + Spiro

*

80

40 20 0

-9

-8

-7

-6

-5

-4

HF

Sham

-3

Log10 M (ACh)

Fig. 1. Acetylcholine (ACh)-mediated vasorelaxation response of heart failure untreated (heart failure), heart failure treated with spironolactone alone (heart failure 1 spironolactone), and sham. Data presented as mean 6 SE; n 5 10 in each group. *P ! .05 vs heart failure and heart failure 1 spironolactone. sP ! .05 vs heart failure.

were also accompanied by other clinical benefits of aldosterone inhibition such as: an increase in natriuresis,22 a decrease in circulating levels of atrial natriuretic peptide, B type natriuretic peptide, and pro-collagen type III aminoterminal peptide,23,24 and an improvement in LV ejection fractions while decreasing heart rate variability9,10,25 and ventricular arrhythmias.11 The studies demonstrating spironolactone benefits in HF raise the possibility that aldosterone antagonism may also have a significant role in reversing the endothelial dysfunction seen in HF.5,20,21 This is intriguing because there is clinical evidence that the NO-mediated vasorelaxation is severely curtailed in HF.14–17 Animal studies using an ischemic animal model of HF corroborated the NO-mediated endothelial dysfunction in both large-conduit vessels and smaller resistance vessels.26 Although the mechanisms responsible for the NO-dependent endothelial dysfunction in HF are still under investigation, decreased production Table 2. eNOS Protein Levels in the LV and Thoracic Aorta in Sham, Heart Failure Rats, and Heart Failure Rats Treated With Spironolactone

Sham HF HF 1 spironolactone

HF

Fig. 2. Changes in endothelial nitric oxide synthase (eNOS) protein levels. A typical Western blot for eNOS in aortic tissue with concentrations of eNOS (intensity units/50 mg tissue) in the left ventricle of rats in heart failure (HF), with and without spironolactone treatment. First lane, eNOS-positive control; second lane, HF treated with spironolactone; third and fourth lanes, untreated HF; and fifth lane, untreated sham. Note the decrease in eNOS protein levels in untreated HF compared with sham. Spironolactone treatment restored eNOS level similar to sham control.

*

60

-10



LV eNOS (Intensity Units/mg)

Aorta eNOS (Intensity Units/mg)

36.2 6 9.5 13.9 6 2.8* 35.6 6 5.1**

57.3 6 16.6 25.8 6 3.6* 39.6 6 4.6**

Concentration of eNOS in the LV and thoracic aorta. Values are mean 6 SE; n 5 10 in each group. eNOS, endothelial nitric oxide synthase; HF, heart failure; LV, left ventricular. *P ! .05 HF vs sham. **P 5 .02 HF vs. HF 1 spironolactone.

of NO is thought to be important. The evidence for abnormal NO production in HF comes from studies demonstrating decreased aortic eNOS protein levels in animal models with HF,14,15,27,28 as well as from data showing decreased activity of the L-arginine-nitric oxide pathway in patients with HF.29 In these studies, NO-mediated physiologic effects are diminished. An observation that the decrease in aortic eNOS may have clinical relevance comes from studies where the serum of patients in HF has been shown to downregulate eNOS in umbilical vein endothelial cell culture.30 The possibility that spironolactone may restore the NOmediated endothelial dysfunction in HF was first raised in hypertensive patients, where spironolactone increased blood flow and decreased vascular resistance.31 This was supported by investigators demonstrating that aldosterone antagonism in licorice-induced hypertensive rats restored endothelial vasorelaxation and blunted the decrease in vascular eNOS protein levels.32 This effect on vasorelaxation appears to be central to spironolactone ability to restore arterial elasticity, independent of its diuretic effect.33,34 More intriguing, there is evidence that the impaired NO-mediated endothelial dependent vasorelaxation seen in HF patients may be related to aldosterone level. Studies have demonstrated a significant decrease in vascular compliance that is inversely proportional to plasma aldosterone levels.33,34 In addition, the aldosterone level in HF has been shown to correlate well to a reduction in NO and eNOS.16,35 Finally, in chronic HF patients treated with aldosterone receptor blockers, forearm blood flow increased in response to ACh; this increase was reversed with L-NMMA, a competitive NO inhibitor,36 suggesting that aldosterone inhibition may reverse the NO-mediated endothelial-dependent vasorelaxation seen in HF. We designed our experiments to address the control of vasodilation and eNOS regulation via the direct measurements of ACh-mediated vasorelaxation of large conduit vessels as well as the quantification of both myocardial and arterial eNOS levels in HF rats treated with spironolactone. In our study, we focused on the mechanisms controlling endothelial function in HF. The importance of the association between endothelial dysfunction and mortality risk in HF has been recently

244 Journal of Cardiac Failure Vol. 12 No. 3 April 2006 emphasized by reports showing that endothelial dysfunction can be used to predict mortality risk in patients with HF.37,38 In patients treated on high dose ACE-I, there are elevated aldosterone levels despite inhibition of vascular angiotensin converting enzyme.39 Even with this ‘‘aldosterone escape,’’ patients treated with ACE-I show an improved outcome suggesting that while aldosterone level predicts mortality that it is not the only clinical predictor in patients with heart failure. In part because of this we did not measure aldosterone levels in this study. Furthermore, while activation of cardiac renin-angiotensin system including cardiac aldosterone production has been reported in the rat coronary artery ligation model of heart failure,40–42 this does not result in increases in circulating aldosterone levels.43 We have to reconcile this with data showing that because spironolactone is a non-specific muscarinic inhibitor, its’ use increases aldosterone levels in a rat volume overload model.44 In addition, aldosterone levels are not routinely measured in patients with heart failure since they are influenced by several factors including posture, activity, sodium intake and medication.45 Despite this aldosterone levels have prognostic value clinically since the benefit of an ACE inhibitor is much more robust in heart failure patients with an elevated aldosterone level compared to those with aldosterone levels below the median.46 Potential Limitations

The major limitations of our study are the high dose of spironolactone we used and the fact that the rats were not treated with other neurohormonal-blocking agents in addition to spironolactone as would be done in patients with HF. Thus our data may not be directly applicable to clinical medicine. In addition, spironolactone lowers blood pressure such that it is possible that the observed changes in endothelial function are load-dependent and not specific to aldosterone receptor blockade. This question can not be addressed in these types of studies because it is difficult to study the effects of decreasing load on endothelial function in the intact animal without pharmacologic agents. Thus the lack of statistical significance for the between-group comparisons in these ventricular loading measures is could be due to beta error. Unfortunately, we did not perform a dose-response study to nitroprusside in the current study; this may have given us an idea as to how differences in loading conditions may affect endothelial and vascular smooth muscle control of vasorelaxation. Our use of a single dose challenge of nitroprusside was intended to demonstrate the viability of the vascular smooth muscle surrounding the aortic rings. Finally we did not test any non-muscarinic receptor agonist in our study so it is possible that spironolactone-induced changes in muscarinic signaling may have contributed to our findings. Summary The focus of our work was to explore potential mechanisms of action of aldosterone inhibition in HF. In summary,

we have shown that one potential mechanism of action of spironolactone appears to be an increase in eNOS in the vessel wall that leads to restoration of the impaired endothelial dependent vasorelaxation seen in HF. Acknowledgments The authors would like to thank Howard Byrne, Maribeth Stansifer, and Nicholle Johnson, BS, for their contributions.

References 1. Swedberg K, Eneroth P, Kjekshus J, Snapinn S. Effects of enalapril and neuroendocrine activation on prognosis in severe congestive heart failure. Am J Cardiol 1990;66:40D–5D. 2. Swedberg K, Eneroth P, Kjekshus J, Wilhelmsen L. Hormones regulating cardiovascular function in patients with severe congestive heart failure and their relation to mortality. Circulation 1990;82: 1730–6. 3. The SOLVD Investigators. Effect of enalapril on survival in patients with reduced left ventricular ejection fraction and congestive heart failure. N Engl J Med 1988;319:293–302. 4. Cohn J, Tognoni G, the Valsartan Heart Failure Trial Investigators. A randomized trial of the angiotensin receptor valsartan in chronic heart failure. N Engl J Med 2001;345:1667–75. 5. Pitt B, Zannad F, Remme W, Cody R, Castaigne A, Perez A, et al. The effect of spironolactone on morbidity and mortality in patients with severe heart failure. N Engl J Med 1999;341:709–17. 6. Struthers AD. Aldosterone escape during ACE inhibitor therapy in chronic heart failure. Eur Heart J 1995;16:N103–6. 7. Davis KL, Nappi JM. The cardiovascular effects of eplerenone, a selective aldosterone receptor antagonist. Clin Ther 2003;25:2647–68. 8. Ikeda U, Kanbe T, Nakayama I, Kawahara Y, Yokoyama M, Shimada K. Aldosterone inhibits nitric oxide synthesis in rat vascular smooth muscle cells induced by interleukin-1B. Eur J Pharmacol 1995;290:69–73. 9. Muller JE. Spironolactone in the management of congestive heart failure. Am J Cardiol 1990;65:51K–3K. 10. MacFadyen RJ, Barr CS, Struthers AD. Aldosterone blockade reduces vascular collagen turnover, improves heart rate variability and reduces early morning rise in heart rate in heart failure patients. Cardiovasc Res 1996;35:30–4. 11. Ramires FJA, Mansur A, Coelho O, Marahnao M, Gruppi CJ, Mady C, et al. Effect of spironolactone on ventricular arrhythmias in congestive heart failure secondary to idiopathic dilated or ischemic cardiomyopathy. Am J Cardiol 2000;85:1207–11. 12. Zannad F, Alla F, Dousset Brigitte, Perez A, Pitt B, on behalf of the RALES investigators. Limitation of excessive extracellular matrix turnover may contribute to survival benefit of spironolactone therapy in patients with congestive heart failure. Circulation 2000;102:2700–6. 13. Lacolley P, Safar ME, Lucet B, Ledudal K, Labat C, Benetos A. Prevention of aortic and cardiac fibrosis by spironolactone in old normotensive rats. J Am Coll Cardiol 2001;37:662–7. 14. Katz SD, Biasucci L, Sabba C, Strom JA, Jondeau G, Galvao M, et al. Impaired endothelium mediated vasodilation in the peripheral vasculature of patients with congestive heart failure. J Am Coll Cardiol 1992; 19:918–25. 15. Kubo SH, Rector TS, Bank AJ, Williams RE, Heifetz SM. Endothelium dependent vasorelaxation is attenuated in patients with heart failure. Circulation 1991;84:1589–96. 16. Ontkean M, Gay R, Greenberg B. Diminished endothelium-derived relaxing factor activity in an experimental model of chronic heart failure. Circ Res 1991;69:1088–96.

Spironolactone Increases eNOS 17. Drexler H, Hayoz D, Munzel T, Just H, Zelis R, Brunner HR. Endothelial function in congestive heart failure. Am Heart J 1993;126: 761–4. 18. Thai HM, Van HT, Gaballa MA, Goldman S, Raya TE. Effects of AT1 receptor blockade after myocardial infarct on myocardial fibrosis, stiffness, and contractility. Am J Physiol 1999;276:H873–80. 19. Thai H, Goldman S, Gaballa MA. AT1 receptor blockade improves vasorelaxation in heart failure by up-regulation of endothelial nitric oxide synthase via activation of the AT2 receptor. J Pharmacol Exp Therapeutics 2003;307:1171–8. 20. Barr CS, Lang CC, Hanson J, Arnott M, Kennedy N, Struthers AD. Effects of adding spironolactone to an angiotensin-converting enzyme inhibitor in chronic congestive heart failure secondary to coronary artery disease. Am J Cardiol 1995;76:1259–65. 21. Pitt B, Remme W, Zannad F, Neaton J, Martinez F, Roniker B, et al. Eplerenone, a selective aldosterone blocker, in patients with left ventricular dysfunction after myocardial infarction. N Engl J Med 2003; 348:1309–21. 22. Bauersachs J, Fraccarollo D, Ertl G, Gretz N, Wehling M, Christ M. Striking increase of natriuresis by low-dose spironolactone in congestive heart failure only in combination with ACE inhibition. Circulation 2000;102:2325–8. 23. Tsutamoto T, Wada A, Maeda K, Mubuchi N, Hayashi M, Tsutsui T, et al. Effects of spironolactone on plasma brain natriuretic peptide and left ventricular remodeling in patients with congestive heart failure. J Am Coll Cardiol 2001;37:1228–33. 24. Kinugawa T, Ogino K, Kato M, Furuse Y, Shimoyama M, Mori M, et al. Effects of spironolactone on exercise capacity and neurohormonal factors in patients with heart failure treated with loop diuretics and ACEI. Gen Pharmacol 1998;31:93–9. 25. Korkmaz ME, Muderrisoglu H, Ulucam M, Ozin B. Effects of spironolactone on heart rate variability and left ventricular systolic function in severe ischemic heart failure. Am J Cardiol 2000;86:649–53. 26. Furchgott RF, Zawadski V. The obligatory role of endothelial cells in the relaxation of arterial smooth muscle by acetylcholine. Nature 1980;288:373–6. 27. Litwin SE, Litwin CM, Raya TE, Warner A, Goldman S. Contractility and stiffness of noninfarcted myocardium following coronary ligation in rats: Effects of chronic angiotensin converting enzyme inhibition. Circulation 1991;83:1028–37. 28. Gaballa MA, Goldman S. Overexpression of endothelium nitric oxide synthase reverses the diminished vasorelaxation in the rat hindlimb in heart failure. J Mol Cell Cardiol 1999;31:1243–52. 29. Katz SD, Khan T, Zeballos GA, Mathew L, Potharlanka P, Knecht M, et al. Decreased activity of the L-arginine-nitric oxide metabolic pathway in patients with congestive heart failure. Circulation 1999;99: 2113–7. 30. Agnoletti L, Curello S, Bacheti T, Malacarne F, Gaia G, Comini L, et al. Serum from patients with severe heart failure downregulates eNOS and its proapoptotic: Role of tumor necrosis factor-alpha. Circulation 1999;100:1983–91. 31. Clement DL. Peripheral action of spironolactone: plethysmographic studies. Am J Cardiol 1990;65:12K–3K.



Thai et al

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32. Quashning T, Ruschitzka F, Shaw S, Luscher TF. Aldosterone receptor antagonism normalizes vascular function in liquorice-induced hypertension. Hypertension 2001;37:801–5. 33. Lagrue G, Ansquer JC, Meyer-Heine A. Peripheral action of spironolactone: improvement in arterial elasticity. Am J Cardiol 1990;65: 9K–11K. 34. Schohn DC, Jahn HA, Pelletier BC. Dose related cardiovascular effects of spironolactone. Am J Cardiol 1993;71:40A–5A. 35. Duprez DA, De Buyzere ML, Rietzschel ER, Taes Y, Clement DL, Morgan D, et al. Increase relationship between aldosterone and large artery compliance in chronically treated heart failure patients. Eur Heart J 1998;19:1371–6. 36. Farquharson CAJ, Struthers AD. Spironolactone increases nitric oxide bioactivity, improves endothelial vasodilator dysfunction, and suppresses vascular AT1/AT2 conversion in patients with chronic heart failure. Circulation 2000;101:594–7. 37. Heitzer T, Baldus S, van Kodolitsch Y, Rudolph V, Meinertz T. Systemic endothelial dysfunction as an early predictor of adverse outcomes in heart failure. Arterioscler Thromb Vasc Biol 2005;25: 1174–9. 38. Katz SD, Hryniewicz K, Hrilijac I, Balidemaj K, Dimayuga C, Hudaihed A, et al. Vascular endothelial dysfunction and mortality risk in patients with chronic heart failure. Circulation 2005;111: 310–4. 39. Jorde UP, Vittorio T, Katz SD, Colombo PC, Latif F, LeJemtel TH. Elevated plasma aldosterone levels despite complete inhibition of the vascular angiotensin-converting enzyme in chronic heart failure. Circulation 2002;106:1055–7. 40. Hirsch AT, Talsness CE, Schunkert H, Paul M, Dzau VJ. Tissuespecific acativation of cardiac angiotensin converting enzyme in experimental heart failure. Circ Res 1991;69:475–82. 41. Hirsch AT, Opsahl JA, Lunzer MM, Katz AS. Active renin and angiotensinogen in cardiac interstitial fluid after myocardial infarction. Am J Physiol 2005;276:H1818–26. 42. Silvestre J-S, Heymes C, Oubenaissa A, Valerie R, Aupetit-Faisant B, Carayon A, et al. Activation of cardiac aldosterone production in rat myocardial infarction. Circulation 1999;99:2694–701. 43. Veldhuisen DJ, van Gilst WH, de Smet BJG, de Graeff PA, Scholtens E, Buikema H, et al. Neurohumoral and hemodynamic effects of ibopamine in a rat model of chronic myocardial infarction and heart failure. Cardiovasc Drugs Therapy 1994;8:245–50. 44. Karram T, Abbasi A, Keidar S, Golomb E, Hochberg I, Winaver J, et al. Effects of spironolactone and eprosartan on cardiac remodeling and angiotensin-converting enzyme isoform in rats with experimental heart failure. Am J Physiol 2005;289:H1351–8. 45. Vasan RS, Evans JC, Benjamin EJ, Levy D, Larson MG, Sundstrom J, et al. Relations of serum aldosterone to cardiac structure: genderrelated differences in the Framingham Heart Study. Hypertension 2004;43:957–62. 46. Swedberg K, Eneroth P, Kjekshus J, Wilhelmsen L, for the CONSENSUS Trial Study Group. Hormones regulating cardiovascular function in patients with severe congestive heart failure and their relation to mortality. Circulation 1990;82:1730–6.