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Erythropoietin-induced excessive erythrocytosis activates the tissue endothelin system in mice ¹

THOMAS QUASCHNING^*,,2, FRANK RUSCHITZKA^*,2, THOMAS STALLMACH, SIDNEY SHAW, HENNING MORAWIETZ^||, WINFRIED GOETTSCH^||, MATTHIAS HERMANN, TORSTEN SLOWINSKI^,, FRANZ THEURING^¶, BERTHOLD HOCHER^,, THOMAS F. LÜSCHER^*,34 and MAX GASSMANN^**,34

^* Cardiology and Cardiovascular Research and Institute of Physiology, University of Zürich, Switzerland;
Department of Medicine, University Hospital of Würzburg, Germany;
Institute of Pathology, University Hospital Zürich, Switzerland;
Clinical Research, Inselspital, University of Bern, Switzerland;
^|| Institute of Pathophysiology, Martin Luther University Halle-Wittenberg, Germany;
Department of Nephrology, University Hospital Charité, Humboldt University, Berlin, Germany;
Molecular Biology and Biochemistry, Free University of Berlin, Germany;
^¶ Institute of Pharmacology and Toxicology, University Hospital Charité, Humboldt University, Berlin, Germany; and
^** Institute of Veterinary Physiology, University of Zürich, Switzerland

⁴Correspondence: Max Gassmann, Institute of Veterinary Physiology, University of Zürich, Winterthurerstr. 260, CH-8057 Zürich, Switzerland. E-mail: maxg{at}access.unizh.ch; and Thomas F. Luscher, Cardiology, University Hospital Zürich, CH-8091 Zürich, Switzerland. E-mail: cardiotfl{at}gmx.ch

SPECIFIC AIMS

Endothelial balance is achieved by controlled expression of nitric oxide (NO), a potent vasodilator, and of its functional counterpart, endothelin-1 (ET-1), which induces vasoconstriction. Although elevated NO levels have been described in excessive erythrocytosis, little is known about ET-1 expression in such conditions. Thus, we examined the endothelin system in transgenic mice, which, due to constitutive overexpression of erythropoietin (Epo) in an oxygen-independent manner, reached hematocrit levels of 80%. We characterized ET_A/B receptor subtype expression, ET-1 promoter activity, ET-1 tissue levels, and vascular contractions to ET-1 in these mice.

PRINCIPAL FINDINGS

1. Excessive erythrocytosis increases ET-1 protein levels in the vascular wall and in liver, heart, and kidney
To our knowledge, our novel Epo-overexpressing transgenic mouse line is the only model available exhibiting reproducible hematocrit levels of 80% in the absence of cardiovascular complications such as hypertension and thrombosis. We previously showed that markedly elevated bioavailability of NO confers vascular protection in these transgenic mice. Immunohistochemical analysis of the pulmonary arteries showed a strong accumulation of ET-1 in the vascular wall of transgenic mice compared with their wild-type littermates (Fig. 1 ). ET-1 promoter analysis demonstrated elevated ET-1 promoter activity in the perivascular tissue. In line with our immunohistochemical observations, quantification by radioimmunoassay revealed a fivefold increase in vascular ET-1 tissue levels in the thoracic aortas of transgenic mice compared with wild-type animals. Similarly, ET-1 protein content was elevated in the transgenic liver, left and right ventricle, renal cortex, and medulla. This indicates a generalized increase in ET-1 production associated with excessive erythrocytosis.

View larger version (89K): [in this window] [in a new window]   Figure 1. Elevated ET-1 protein levels in the pulmonary artery of mice with excessive erythrocytosis. Compared with wild-type (wt) controls, elevated ET-1 protein levels in the vascular wall were demonstrated by immunohistochemical analysis of the pulmonary artery of polycythemic transgenic (tg) mice. As a negative control, the primary antibody was omitted when analyzing transgenic vessels. x400.

2. Vascular response to ET-1 and big ET-1 is reduced in excessive erythrocytosis but is restored by preincubation with the NOS inhibitor L-NAME
Cardiovascular disease accounts for the majority of deaths in western countries. Thus, the evaluation of endothelial function in health and disease is a major contribution to understanding and ultimately preventing cardiovascular morbidity and mortality. Contractile responses of isolated aortic rings to ET-1 and its precursor big ET-1 were determined in quiescent preparations with or without preincubation with the NO synthase (NOS) inhibitor N-nitro-L-arginine methyl ester (L-NAME). Concentration-dependent contractions of aortic rings to ET-1 were reduced in polyglobulic transgenic mice compared with wild-type siblings. However, preincubation with the NOS inhibitor L-NAME augmented the response to ET-1 in transgenic animals, but not in wild-type controls. In analogy, contractions to big ET-1 were most markedly reduced in mice with excessive erythrocytosis but, in agreement with the observed ET-1 contractions, differences were abolished by preincubation with L-NAME and by de-endothelialization of the vessels. These results imply the counter-regulatory properties of NO and ET-1: the detectable change in vessel diameter represents the result of the effect of the agonist, such as the vasoconstrictor ET-1, and its functional antagonist, the vasodilator NO. Reduced reactivity to ET-1 is not due to receptor down-regulation, since semiquantitative PCR of ET_A/B receptor subtypes revealed an increase in vascular ET receptor expression, being more pronounced in ET_B than ET_A receptors. This suggests that ET_B receptors contribute to clearance of ET-1 from the circulation, most suitable in states of elevated ET-1 levels.

3. Pretreatment with the ET_A receptor antagonist darusentan prolongs survival of transgenic mice with excessive erythrocytosis on exposure to L-NAME
To evaluate the effect of the ET system in vivo, transgenic mice with excessive erythrocytosis and wild-type control siblings were given orally the ET_A antagonist darusentan for 3 wk, then exposed to the NOS inhibitor L-NAME or vehicle alone, both compounds being administered in the drinking water under monitoring of systolic blood Transgenic mice on placebo treatment died within 2 days after L-NAME-mediated NO synthesis inhibition began (mean survival time 34±5 h), whereas polyglobulic mice pretreated with the ET_A receptor antagonist darusentan for 3 wk exhibited a doubled survival time during NO depletion (Fig. 2 ). In contrast, wild-type animals pretreated with darusentan and exposed to L-NAME did not exhibit increased mortality.

View larger version (14K): [in this window] [in a new window]   Figure 2. ETA receptor antagonism prolongs survival of transgenic mice with excessive erythrocytosis. Kaplan-Meier curves of wild-type (wt) and polycythemic (tg) mice on long-term treatment with darusentan or placebo, respectively, followed by an acute exposure to L-NAME (40 mg·kg-1·day-1). The NO synthase inhibitor L-NAME (40 mg·kg-1·day-1) was added to drinking water after 3 wk of pretreatment with darusentan or placebo. Data are given as mean ± SE (n=5).

Since treatment with darusentan prolonged survival after acute NO blockade, we proposed that ET-1 contributes to the adverse cardiovascular effects induced by erythrocytosis and thus may have a role as a new target in the treatment of cardiovascular disease associated with severe elevation of the hematocrit.

CONCLUSIONS

Our transgenic mice overexpressing Epo develop excessive erythrocytosis exhibiting hematocrit values of 80% and above without hypoxic stimulus. Adult transgenic animals do not develop hypertension or thromboembolic complications. We recently reported that the expected rise in blood pressure and cardiovascular complications were prevented by a pronounced increase in NO bioavailability in these animals. In the present study, we demonstrate for the first time that excessive erythrocytosis results in marked activation of the tissue endothelin system as indicated by increased ET-1 promoter activity, elevated ET-1 tissue levels, and ET_A/B receptor subtype expression. ET_A receptor antagonism prolonged survival in transgenic mice when NO production was reduced by L-NAME application.

In humans, hematocrit levels tend to be only moderately elevated. Nevertheless, hematocrit levels between 60% and 91% have recently been reported to occur in high altitude dwellers exposed to cobalt. Despite being associated with erythrocytosis, hypertension was not observed in those highlanders with elevated hematocrit. It is tempting to speculate that our novel mouse model with hematocrit levels of 80% and more in the absence of hypertension reflects adaptational mechanisms occurring in humans suffering from severe erythrocytosis.

Reduced oxygen supply is known to stabilize hypoxia-inducible factor-1 (HIF-1), which in turn binds to the HIF-1 binding site present upstream of the ET-1 gene, thereby increasing ET-1 transcription. Considering that erythrocytosis increases the blood’s oxygen transport capacity, thereby reducing the level of HIF-1 in the periphery, we predicted reduced ET-1 expression. However, one should keep in mind that excessive erythrocytosis as occurring in the present mouse model might decrease tissue oxygenation by a certain sludge phenomenon in the capillaries that might hinder erythrocytes from delivering their oxygen to the tissue properly. Thus, localized tissue hypoxia and enhanced local ET-1 production may occur.

In the present study, we found enhanced promoter activity of ET-1 and ET-1 tissue levels, suggesting that excessive erythrocytosis induces long-term activation of the tissue endothelin system in vivo. This is in line with previous findings demonstrating up-regulation of ET-1 production by pulsatile stretching in cell culture experiments. Overexpression of ET-1 in perivascular tissue but not in the entire vascular wall suggests that NO primarily down-regulated ET-1 production on the luminal side of the vessel wall, underscoring the paracrine character of ET-1 in perivascular tissue. Increased promoter activity and tissue levels of ET-1 were paralleled by increased gene expression of both ET receptor subtypes even though one would have expected ET receptor down-regulation in the presence of elevated ET tissue levels.

Levels of ET_B receptor mRNA were more pronounced than that of the ET_A receptor subtype. This may be linked to the fact that ET_B receptors mediate the pulmonary clearance of ET, especially when ET-1 levels are elevated. Despite overexpression of ET_A receptors, contractile responses of aortic rings to ET-1 in vitro were reduced. Vascular reactivity to ET-1, however, was increased after preincubation of the vessels with the NO synthase inhibitor L-NAME. As the response to KCl and norepinephrine was comparable in transgenic and wild-type mice, this shows that in vivo the vascular effects of ET-1 are offset by NO, the endothelial counterpart of ET-1. These counter-regulatory effects of endothelium-derived NO were disclosed by experiments with de-endothelialized aortic rings in which ET-1 led to a moderate but not significant increase in contractile response in transgenic mice compared with wild-type littermates. Correspondingly, survival time of transgenic mice with excessive erythrocytosis after acute NO blockade was increased by > twofold by treatment with the ET_A receptor antagonist darusentan compared with placebo, indicating the effect of the activated ET system. This finding is in line with our previous observation that in transgenic animals acutely exposed to L-NAME, blood pressure is elevated due to peripheral vasoconstriction, followed by acute left ventricular dysfunction and cardiac death. Since darusentan, a selective ET_A receptor antagonist, leaves the beneficial ET_B receptor-mediated release of endothelial NO unaltered but reduces ET_A receptor-mediated vasoconstriction, these data suggest a potential therapeutic use of selective ET_A antagonism in excessive erythrocytosis, particularly in the context of endothelial dysfunction.

In conclusion, we provide here for the first time evidence that the tissue endothelin system is activated in excessive erythrocytosis despite an enhanced bioavailability of NO. Since treatment with darusentan prolonged survival after acute NO blockade, therapeutical modification of the tissue endothelin system may advance as a new target in the treatment of cardiovascular disease associated with erythrocytosis.

View larger version (46K): [in this window] [in a new window]   Figure 3. Endothelial regulation associated with high hematocrit. In states of excessive erythrocytosis, laminar shear stress is enhanced resulting in induction of endothelial NO synthase (eNOS), which in turn augments NO production. In parallel with these adaptive processes, ET-1 production is elevated due to enhanced conversion of the ET-1 precursor big ET-1 by the endothelin-converting enzyme (ECE) to the potent vasoconstrictor ET-1. This compensatory process may be induced by pathways linked to elevated NO production (green arrows) or may be mediated directly by erythrocytosis-induced increase in laminar shear stress. The fragile balance of endothelial regulation may be influenced inversely by the NOS inhibitor L-NAME or by treatment with ETA receptor antagonists. EC, endothelial cell; VSMC, vascular smooth muscle cell; L-Arg, L-arginin; sGC, soluble guanylate cyclase; ETA-R, endothelinA receptor.

FOOTNOTES

¹ To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.02-0296fje; to cite this article, use FASEB J. (December 18, 2002) 10.1096/fj.02-0296fje

² Both authors contributed equally to this work

³ The contribution of both senior authors was equivalent.

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