Endothelin receptor antagonism prevents hypoxia-induced mortality and morbidity in a mouse model of sickle-cell disease

Abstract

Patients with sickle-cell disease (SCD) suffer from tissue damage and life-threatening complications caused by vasoocclusive crisis (VOC). Endothelin receptors (ETRs) are mediators of one of the most potent vasoconstrictor pathways in mammals, but the relationship between vasoconstriction and VOC is not well understood. We report here that pharmacological inhibition of ETRs prevented hypoxia-induced acute VOC and organ damage in a mouse model of SCD. An in vivo ultrasonographic study of renal hemodynamics showed a substantial increase in endothelin-mediated vascular resistance during hypoxia/reoxygenation-induced VOC. This increase was reversed by administration of the dual ETR antagonist (ETRA) bosentan, which had pleiotropic beneficial effects in vivo. It prevented renal and pulmonary microvascular congestion, systemic inflammation, dense rbc formation, and infiltration of activated neutrophils into tissues with subsequent nitrative stress. Bosentan also prevented death of sickle-cell mice exposed to a severe hypoxic challenge. These findings in mice suggest that ETRA could be a potential new therapy for SCD, as it may prevent acute VOC and limit organ damage in sickle-cell patients.

Figures

Figure 1. PreproET-1 mRNA levels are higher in sickle-cell mice than in WT mice at steady state.

(A) Real-time RT-PCR analysis of preproET-1 from kidneys of C57BL/6J (WT) and SAD mice at steady state (normoxia). n = 5 to 7 animals per condition. *P < 0.05 versus WT. (B–E) In situ hybridization for preproET-1 mRNA in kidneys from WT and SAD mice in steady state. PreproET-1 mRNA in small vessels is restricted to endothelial cells in the kidney cortex of (B) WT and (C) SAD mice. PreproET-1 mRNA is substantially more abundant in (C) afferent arterioles and (E) glomerular capillaries in SAD mice than in (B and D) WT mice. Original magnification, ×400 (B and C); ×600 (D and E).

Figure 2. ETRA reverses and prevents the fall in renal blood flow velocity (RBF) in SAD mice.

(A–C) Reversal of low renal perfusion in sickle mice by infusion of bosentan. Echo-Doppler measurements of (A) CO and (B) RBF velocity in the renal artery at steady state (white bars) and after H/R in SAD and WT mice before (black bars) and 10 minutes after infusion of bosentan (gray bars). H/R-induced vasoocclusive (VOC) events were associated with blunted CO and significant decreases in mean RBF velocity in SAD mice only. Acute infusion of bosentan restored 50% of the initial loss in RBF velocity within 10 minutes, whereas saline had no effect (not shown). n = 13. *P < 0.05 versus SAD at steady state; **P < 0.01 versus SAD at steady state; #P < 0.05 versus SAD before bosentan infusion. (C) Representative RBF waveforms in WT and SAD mice at steady state and after H/R followed by acute infusion of saline or bosentan. End-diastolic and time-averaged mean velocities were lower in saline-treated SAD mice after H/R than in normoxic WT and SAD mice. Acute ETRA restored end-diastolic and time-averaged mean velocities. (D and E) Prevention of H/R-induced renal hypoperfusion in SAD mice treated for 2 weeks with bosentan. (D) CO and (E) mean RBF velocity were unchanged in bosentan-treated SAD mice, whereas mean RBF velocity decreased by 50% in vehicle-treated SAD mice. All data are means ± SEM from n = 2 experiments. n > 10. ***P < 0.001 versus SAD at steady state; ##P < 0.01 versus vehicle-treated SAD in H/R.

Figure 3. Profound vascular congestion as a result of H/R-induced vasoocclusive events in kidneys and lungs from sickle SAD mice.

(A) Vehicle-treated sickle SAD mice had substantial vascular congestion in glomeruli, arterioles, and capillaries in the renal cortex (top) and the renal medulla (bottom). This was less frequently observed in glomeruli, arterioles, and capillaries in bosentan-treated sickle SAD mice. (B) Representative H&E staining showing alleviation of pulmonary vascular congestion and inflammatory infiltration in bosentan-treated hypoxic sickle SAD mice. Original magnification, ×100 (A); ×250 (B).

Figure 4. Bosentan prevents in vitro ET-1 activation of the Gardos channel.

(A) In vitro rubidium (Rb+) influx (mmol/l cell × min) in WT and SAD mouse rbcs in the presence of ET-1 with or without pretreatment with bosentan. *P < 0.05 versus control Rb+ influx; #P < 0.05 versus ET-1–treated rbcs. (B) In vivo effects of 2-week administration of bosentan on density and (C) K+ content in WT and SAD mouse rbcs. *P < 0.05 versus WT mice; #P < 0.05 versus SAD mice at steady state; †P < 0.05 versus vehicle-treated SAD mice; n = 6.

Figure 5. The ETRA bosentan prevents systemic and local inflammation in SAD mice.

(A) Effect of orally administered bosentan on total leukocyte and (B) neutrophil alveolar infiltration in H/R-challenged WT and SAD mice. *P < 0.05 versus steady state; †P < 0.05 versus vehicle-treated mice.

Figure 6. Bosentan prevents increased renal and lung MPO activity, peroxynitrite-induced protein tyrosine nitration in the kidneys, and severe hypoxia–induced death.

MPO activity in (A) kidneys and (B) lungs of WT and SAD mice at steady state and after 18-hour exposure to H/R. *P < 0.05 vs. SAD at steady state; #P < 0.05 vs. SAD in H/R. (C–H) Anti-nitrotyrosine staining of kidney sections is strong in SAD mice in the (E) renal cortex and, to an even greater extent, in the (G) renal medulla under H/R conditions. (F and H) Bosentan administration before H/R prevented protein tyrosine nitration in these 2 renal compartments. Original magnification, ×100. (I) Endothelin receptor antagonism with bosentan protects SAD mice from death due to severe VOC after exposure to a 6% O2 atmosphere for 10 hours. Survival curves for vehicle-treated SAD mice are significantly different (P < 0.001) from those for bosentan-treated SAD mice and WT controls. n = 8–12 mice per group.