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(Hypertension. 1999;34:e1.)
© 1999 American Heart Association, Inc.

Letters to the Editor - Web

Endothelins and Cardiac Fibrosis

Gian Paolo Rossi; Maurizio Cesari; Achille C. Pessina

Department of Medicina Clinica e Sperimentale, University of Padova Medical School, University Hospital, Padova, Italy

	Introduction

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To the Editor:

In the March issue of Hypertension, Dr Hocher and colleagues reported on the role of the endothelin (ET) system in the cardiac remodeling of renovascular hypertension.¹ According to their findings, both fibrosis of the left ventricle (LV) and hypertrophy of intracardiac arteries were markedly prevented in the 2-kidney, 1-clip (2K1C) rat model, although they found changes neither in the cardiac tissue content of immunoreactive (ir) ET-1 and bigET-1 nor in the cardiac expression of the endothelin ET_A and ET_B receptors, both in the early (10 days) and in the later (4 and 12 weeks) stages after clipping one renal artery. Interestingly, since these effects occurred in spite of the lack of any blood pressure (BP) lowering effect, the authors proposed that the ET system contributes to myocardial fibrosis and arterial hypertrophy via BP-independent mechanisms. Furthermore, on the basis of the differential results obtained with the ET_A antagonist BQ-123 and with an ET_B antagonist IRL-1038, both administered daily intraperitoneally, they concluded that the ET_A receptor is involved in wall hypertrophy of intracardiac arteries while the ET_B plays a role in fibrosis. Although these findings are interesting and obviously important for our understanding of the mechanisms of these common adverse effects of hypertension on the heart, they deserve some comments.

In a recent paper, which Dr Hocher and colleagues quoted twice (References 9 and 33), Dr Mulder and colleagues reported that the mixed ET_A/ET_B receptor antagonist bosentan, administered orally at the same dosage of Dr Hocher and colleagues, dramatically reduced mortality and myocardial fibrosis in a rat model of congestive heart failure.² They suggested an involvement of the ET_A receptor in such detrimental effect of ET-1, in keeping with the results of Nguyen and colleagues, with LU135252, an ET_A selective antagonist.³ Of interest, these investigators found a scar thinning, LV dilatation, an increase in LV end diastolic pressure, and no effect on mortality in the rat treated with the ET_A antagonist after myocardial infarction.

In addition, although other investigators besides Dr Hocher and colleagues have been using IRL-1038 as an ET_B-selective compound over the past few years, it must be pointed out that the selectivity of this compound has been questioned. In 1994, the same investigators who first synthesized IRL-1038,⁴ publicly retreated it on the basis of inconsistent activity because of a high batch-to-batch variability.⁵ This should be widely known by investigators in this field. Thus, based on this consideration, we believe that the contention of Dr Hocher et al must be supported either by data obtained with other ET_B antagonists whose selectivity has been unequivocally proven or by showing that IRL-1038 in fact acted as an ET_B-selective antagonist in their hands. Even if one assumes that IRL-1038 worked as an ET_B specific antagonist, the fact that no decrease of both arterial hypertrophy and fibrosis was seen with bosentan is intriguing, as the authors admitted, given the aforementioned findings of Mulder and colleagues.²

In a recent study on a monogenic form of renin-dependent hypertension in the heterozygote TGREN2 rat, we found no effect of bosentan on BP and cardiovascular damage, as assessed from LV weight and normalized media thickness of the 100 to 200 µm diameter mesenteric arterioles.⁶ Accordingly, our data would not support the contention of Dr Hocher and colleagues that the ET system plays a major role in causing target organ damage in renin-dependent hypertension, although they are consistent with their and other findings⁷ that the ET system does not participate in raising BP in hypertension due to endogenously overproduced angiotensin II. This is at variance with data obtained with the exogenously administered peptide, where ET antagonists were shown to effectively prevent the development of hypertension and target organ damage. The possible explanations of these discrepancies have been recently addressed in detail.⁸

The effectiveness of BQ-123 in preventing hypertrophy of intracardiac arteries despite the lack of any BP lowering effect and any detectable evidence of cardiac activation of the ET system, albeit consistent with a more recent report with LU 127043, a different ET_A selective antagonist,⁷ is even more intriguing. It obviously raises the issue of the accuracy of the measurements of irET-1 and bigET-1 in this study. If the ET system were activated by 2K1C hypertension only in the arterial wall, measurements of tissue extracts are unlikely to detect this upregulation due to a dilution factor. Furthermore, the authors used an enzyme-linked–immunoadsorbent assay (ELISA) method that according to the manufacturer would not require a prior extraction-purification step, which is at variance with practically all the other commercially available assays. From Figure 1¹ it is apparent that a rather large spread of the values was found. Although the authors provided detailed information on the crossreactivity of their antibodies, they failed to provide data on the intra-assay and inter-assay variation coefficients. This piece of information is crucial to enable the reader to critically assess the findings of this study.

In regard to the possible effect of ET on cardiac fibrosis via ET_B receptors, we would like to propose an alternative mechanism, which does not postulate changes in endothelins and ET receptors in the heart. Compelling experimental evidence indicates a role of aldosterone in cardiac fibrosis.^{9 10} We showed that ET-1 is a potent secretagogue of aldosterone¹¹ and that in the rat this effect occurs via ET_B receptors since rat Zona Glomerulosa cells do not express the ET_A receptor.¹² Furthermore, available data suggest a synergistic effect of angiotensin II and ET-1 as secretagogues of aldosterone.¹¹ Thus, it does not seem unreasonable to hypothesize that the activation of the renin-angiotensin system and possibly of the ET system via ET_B receptors in the adrenal cortex markedly enhance aldosterone secretion and therefore the fibrogenic effect of the hormone on cardiac fibroblasts. The beneficial effect of the ET_B antagonist in this paper and of the mixed ET_A/ET_B antagonist bosentan in previous studies, despite no change in the cardiac ET system, could thus be explained on this ground. Unfortunately, no measurements of aldosterone were reported in Dr Hocher’s study and therefore this hypothesis remains to be tested.

	References

Top Introduction References Introduction  References

 

1. Hocher B, George I, Rebstock J, Bauch A, Schwarz A, Neumayer HH, Bauer C. Endothelin system-dependent cardiac remodeling in renovascular hypertension. Hypertension. 1999;33:816–822.[Abstract/Free Full Text]
2. Mulder P, Richard V, Derumeaux G, Hogie M, Henry JP, Lallemand F, Compagnon P, Mace B, Comoy E, Letac B, Thuillez C. Role of endogenous endothelin in chronic heart failure: effect of long-term treatment with an endothelin antagonist on survival, hemodynamics, and cardiac remodeling. Circulation. 1997;96:1976–1982.[Abstract/Free Full Text]
3. Nguyen QT, Cernacek P, Calderoni A, Stewart DJ, Picard P, Sirois P, White M, Rouleau JL. Endothelin A receptor blockade causes adverse left ventricular remodeling but improves pulmonary artery pressure after infarction in the rat. Circulation. 1998;98:2323–2330.[Abstract/Free Full Text]
4. Urade Y, Fujitani Y, Oda K, Watakabe T, Umemura I, Takai M, Okada T, Sakata K, Karaki H. An endothelin B receptor-selective antagonist: IRL 1038, [Cys11-Cys15]-endothelin-1(11–21). FEBS Lett.. 1992;311:12–16.[Medline] [Order article via Infotrieve]
5. Urade Y, Fujitani Y, Oda K, Watakabe T, Umemura I, Takai M, Okada T, Sakata K, Karaki H. An endothelin B receptor-selective antagonist: IRL 1038, [Cys11-Cys15]-endothelin-1(11–21) [retraction of Urade Y, Fujitani Y, Oda K, Watakabe T, Umemura I, Takai M, Okada T, Sakata K, Karaki H. In: FEBS Lett. 1992; 311:12–16]. FEBS Lett.. 1994;342:103–103.[Medline] [Order article via Infotrieve]
6. Rossi GP, Sacchetto A, Rizzoni D, Bova S, Porteri E, Mazzocchi G, Belloni AS, Bachcelioglu M, Nussdorfer GG, Pessina AC. Blockade of AT-1 and not of endothelin receptor prevents hypertension and cardiovascular disease in TGR(mREN2)27 transgenic rats via adrenocortical steroids-independent mechanisms. Arterioscler Thromb Vasc Biol. In press.
7. Ehmke H, Faulhaber J, Munter K, Kirchengast M, Wiesner RJ. Chronic ETA receptor blockade attenuates cardiac hypertrophy independently of blood pressure effects in renovascular hypertensive rats. Hypertension. 1999;33:954–960.[Abstract/Free Full Text]
8. Rossi GP, Sacchetto A, Cesari M, Pessina AC. Interactions between endothelin-1 and the renin-angiotensin-aldosterone system. Cardiovasc Res.. 1999;43:300–307.[Abstract/Free Full Text]
9. Brilla CG. The cardiac structure-function relationship and the renin- angiotensin-aldosterone system in hypertension and heart failure. Curr Opin Cardiol. 1994;9 (suppl 1):S2–S10.
10. Brilla CG, Maisch B, Zhou G, Weber KT. Hormonal regulation of cardiac fibroblast function.. Eur Heart J. 1995;16 (suppl C):45–50.
11. Nussdorfer GG, Rossi GP, Malendowicz LK, Mazzocchi G. Autocrine-paracrine endothelin system in the physiology and pathology of steroid-secreting tissues. Pharmacol Rev. 1999; 51: 1–35.
12. Belloni A, Rossi GP, Andreis PG, Neri G, Albertin G, Pessina AC, Nussdorfer GG. Endothelin adrenocortical secretagogue effect is mediated by the B receptor in rats. Hypertension. 1996;27:1153–1159.[Abstract/Free Full Text]

Response

Berthold Hocher, MD; Ines George

Department of Nephrology, Universitätsklinikum Charité der, Humboldt Universität zu Berlin, Berlin, Germany

	Introduction

Top Introduction References Introduction  References

 
We thank Dr Rossi and colleagues for their interest and comments to our recent report.¹ The major goal of this study was to analyze whether the cardiac endothelin system contributes to cardiac remodeling in rats with 2-kidney, 1-clip (2K1C) renovascular hypertension. Our study demonstrated that cardiac tissue concentrations of big ET-1 and ET-1 as well as the expression of the ETA and ETB receptors were similar in the early, middle, and late stage of 2K1C renovascular hypertension as compared to sham-operated controls. Fibrosis of the left ventricle and hypertrophy of intracardiac arteries, on the other hand, were markedly altered after long-term treatment with endothelin receptor antagonists in a blood-pressure independent manner. These two effects are mediated via different subtypes of endothelin receptors. ETA receptor blockade completely normalized the hypertrophy of intracardiac arteries (P<0.01 compared to 2K1C without treatment), whereas the ETB antagonist reduces cardiac fibrosis of the left ventricle (P<0.001 compared to 2K1C without treatment) to baseline values.

The unexpected finding of no activation of the overall cardiac ET system was not due to technical reasons as supposed by Dr Rossi and colleagues, since the size of the groups were large enough and the method to analyze big ET-1 and ET-1 tissue as well as the expression of the ETA and ETB receptor subtypes are well-established methods. Dr Rossi asked for data on interassay and intra-assay variation coefficients of the ELISA’s used in our study: Intra-assay variance was 3.4±0.3% for the ET-1 ELISA and 4.1±0.3% for the big ET-1 ELISA. The corresponding interassay variances were 8.7±1.9% and 9.1±1.7%, respectively. In addition, our data concerning cardiac ET-1 tissue concentrations are in complete agreement with recent reports analyzing cardiac ET-1 gene expression in rats with renovascular hypertension. They demonstrated using in situ hybridization techniques that the myocardial ET-1 gene expression is not enhanced in 2K1C hypertensive rats, whereas the ET-1 gene is upregulated within the coronary arteries in these hypertensive rats.^{2 3} We suggest that the cells that probably mediate effects of endothelin receptor antagonists on cardiac remodeling in renovascular hypertension (cardiac fibroblasts and cells of intracardiac arteries) represent only a small fraction of all cardiac cells. Therefore, an altered activation of these cells in rats with renovascular hypertension might be undetectable when analyzing the overall left ventricular cardiac endothelin system.

We agree with Dr Rossi’s statement that the investigators that first described the synthesis of the ETB receptor antagonist IRL 1038 reported a high batch-to-batch variability. We therefore used only IRL 1038 batches with HPLC checked quality. IRL 1038 can be used as an ETB receptor antagonist when investigators perform control experiments as done in our study.^{4 5 6 7} However, the recent availability of better ETB receptor antagonists will facilitate studies like ours in future.

Our study¹ demonstrates that cardiac fibrosis in the non-failing pressure overloaded heart of rats with renovascular hypertension is at least partially an ETB receptor–dependent process. Dr Rossi and colleagues, on the other hand, suggested by referring to an excellent study performed by Mulder et al⁸ (which analyzed the effects of the nonselective endothelin receptor antagonist bosentan in a chronic heart failure model) that myocardial fibrosis might be ETA receptor dependent. We, however, should keep in mind that the pathophysiology of the cardiac endothelin system in the failing heart after coronary artery ligation is completely different from the pathophysiology in the nonfailing pressure overloaded heart of rats with renovascular hypertension. Thus, our study aimed not and of cause could not really answer the question of which of the endothelin receptor subtypes might be involved in altered cardiac matrix synthesis in the failing heart after coronary artery ligation. Studies comparing the effects of selective ETA and ETB receptor antagonists on cardiac matrix synthesis in the failing heart are missing so far.

We do not agree with Dr Rossi’s and colleagues opinion that analysis of blood pressure and left ventricular weight in TGREN2 rats after bosentan treatment are sufficient to exclude a role of the paracrine cardiac endothelin system in cardiac matrix synthesis in this model. Left ventricular weight is not a good parameter of cardiac fibrosis. We would thus suggest that they analyze cardiac matrix synthesis in their yet unpublished study in more detail before drawing such a conclusion.

Dr Rossi’s statement that the blockade of the ETB receptor in rats with 2K1C renovascular hypertension could block/reduce aldosterone synthesis in Zona Glomerulosa cells thus reducing cardiac fibrosis is an interesting alternative hypothesis possibly explaining our data and should be analyzed in further studies.

	References

Top Introduction References Introduction  References

 

1. Hocher B, George I, Rebstock J, Bauch A, Schwarz A, Neumayer HH, Bauer C. Endothelin system dependent cardiac remodeling in renovascular hypertension. Hypertension. 1999;33:816–822.
2. Li JS, Knafo L, Turgeon A, Garcia R, Schiffrin E. Effect of endothelin antagonism on blood pressure and vascular structure in renovascular hypertensive rats. Am J Physiol. 1996; 271:H88–H93.
3. Deng LY, Schiffrin EL. Endothelin-1 gene expression in blood vessels and kidney of spontaneously hypertensive rats (SHR), L-NAME-treated SHR, and renovascular hypertension. J Cardiovasc Pharmacol. 1998;31:S380–S383.
4. Spatz M, Kawai N, Merkel N, Bembry J, McCarron RM. Functional properties of cultured endothelial cells derived from large microvessels of human brain. Am J Physiol. 1997;272(pt 1):C231–C239.
5. Tack I, Castano EM, Pecher C, Praddaude F, Bascands JL, Bompart G, Ader JL, Girolami JP. Endothelin increases NO-dependent cGMP production in isolated glomeruli but not in mesangial cells. Am J Physiol. 1997;272(pt 2):F31–F39.
6. Alexiou K, Dschietzig T, Simsch O, Laule M, Hundertmark J, Baumann G, Stangl K. Arrhythmogenic effects induced by coronary conversion of pulmonary big endothelin to endothelin: aggravation of this phenomenon in heritable hyperlipidemia. J Am Coll Cardiol. 1998;32:1773–1778.[Abstract/Free Full Text]
7. Boros M, Massberg S, Baranyi L, Okada H, Messmer K. Endothelin 1 induces leukocyte adhesion in submucosal venules of the rat small intestine. Gastroenterology. 1998;114:103–114.[Medline] [Order article via Infotrieve]
8. Mulder P, Richard V, Derumeaux G, Hogie M, Henry JP, Lallemand F, Compagnon P, Mace B, Comoy E, Letac B, Thuillez C. Role of endogenous endothelin in chronic heart failure: effect of long-term treatment with an endothelin antagonist on survival, hemodynamics, and cardiac remodeling. Circulation. 1997;96:1976–1982.

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