Selective endothelin-A receptor blockade attenuates endotoxin-induced pulmonary hypertension and pulmonary vascular dysfunction

Abstract

Endothelin-1 is a potent mediator of sepsis-induced pulmonary hypertension (PH). The pulmonary vascular effects of selective blockade of endothelin receptor subtype A (ETAR) during endotoxemia remain unknown. We hypothesized that selective ETAR antagonism attenuates endotoxin-induced PH and improves pulmonary artery (PA) vasoreactivity. Adult male Sprague-Dawley rats (250-450 g) received lipopolysaccharide (LPS; Salmonella typhimurium; 20 mg/kg intraperitoneally) or vehicle 6 hours before hemodynamic assessment and tissue harvest. The selective ETAR antagonist sitaxsentan (10 or 20 mg/kg) or vehicle was injected intravenously 3 hours after receipt of LPS. Right ventricular systolic pressure, mean arterial pressure (MAP), cardiac output (CO), oxygenation (P/F ratio), and serum bicarbonate were measured. Bronchoalveolar lavage (BAL) cell differential and lung wet-to-dry ratios were obtained. Endothelium-dependent and endothelium-independent vasorelaxations were determined in isolated PA rings. PA interleukin (IL)-1β, IL-6, tumor necrosis factor α (TNF-α), and inducible nitric oxide synthase (iNOS) messenger RNA (mRNA) were measured. LPS caused PH, decreased MAP, CO, and serum bicarbonate, and increased PA IL-1β, IL-6, TNF-α, and iNOS mRNA. Sitaxsentan attenuated sepsis-induced PH and increased MAP. The P/F ratio, CO, serum bicarbonate, and BAL neutrophilia were not affected by sitaxsentan. In isolated PA rings, while not affecting phenylephrine-induced vasocontraction or endothelium-dependent relaxation, sitaxsentan dose-dependently attenuated LPS-induced alterations in endothelium-independent relaxation. PA cytokine mRNA levels were not significantly attenuated by ETAR blockade. We conclude that ETAR blockade attenuates endotoxin-induced alterations in systemic and PA pressures without negatively affecting oxygenation. This protective effect appears to be mediated not by attenuation of sepsis-induced cardiac dysfunction, acidosis, or alveolar inflammation but rather by improved endothelium-independent vasorelaxation.

Keywords: cytokines; endothelium; sepsis; sitaxsentan; vasoreactivity.

Figure 1

Sitaxsentan treatment attenuates endotoxin-induced alterations in pulmonary and systemic hemodynamics. Adult male Sprague-Dawley rats were injected with lipopolysaccharide (LPS; 20 mg/kg intraperitoneally) or LPS vehicle (phosphate-buffered saline [PBS], 1 mL intraperitoneally). Three hours later, animals received either sitaxsentan (10 mg/kg via tail vein) or sitaxsentan vehicle (PBS, 1 mL via tail vein). Right ventricular systolic pressure (RVSP; A), mean arterial pressure (MAP; B), and cardiac output (CO; D) were measured 3 hours after administration of sitaxsentan or its vehicle. Note the LPS-induced decreases in MAP and increases in RVSP (A) and RVSP/MAP (C). Sitaxsentan administration was associated with a significant increase in MAP and a decrease in RVSP and RVSP/MAP. Values are means + SEM. N = 4–5/group. A single asterisk indicates P < 0.05 and 3 asterisks P < 0.001 versus LPS vehicle groups; a single number sign (#) indicates P < 0.05 and 3 number signs P < 0.001 versus sitaxsentan vehicle. P = 0.09 for LPS+sitax versus LPS veh. veh+veh: LPS vehicle + sitxsentan vehicle; veh+sitax: LPS vehicle + sitxsentan; LPS+veh: LPS + sitaxsentan vehicle; LPS+sitax: LPS + sitxsentan.

Figure 2

Sitaxsentan treatment during endotoxemia is not associated with worsening oxygenation. Rats were treated with lipopolysaccharide (LPS), LPS vehicle, sitaxsentan, or sitaxsentan vehicle, as outlined in Figure 1. Partial pressure of arterial oxygen (PaO₂) was measured via arterial blood gas analysis. The fraction of inspired oxygen (FiO₂) was set at 100%. Oxygenation is expressed as the PaO₂/FiO₂ (P/F) ratio. Note that sitaxsentan treatment during endotoxemia did not negatively affect oxygenation, suggesting the lack of additional ventilation-perfusion mismatch. Values are means + SEM. N = 4–5/group. veh+veh: LPS vehicle + sitxsentan vehicle; veh+sitax: LPS vehicle + sitxsentan; LPS+veh: LPS + sitaxsentan vehicle; LPS+sitax: LPS + sitxsentan.

Figure 3

Protective sitaxsentan effects during endotoxemia are not mediated by attenuation of alveolar inflammation. Rats were treated with lipopolysaccharide (LPS), LPS vehicle, sitaxsentan, or sitaxsentan vehicle, as outlined in Figure 1. Lung wet-to-dry ratio (A) was affected by neither LPS nor sitaxsentan. Note, however, that LPS administration was associated with a small but significant increase in bronchoalveolar lavage (BAL) neutrophil count (B), suggesting evolving lung injury. Neutrophilic alveolitis was not attenuated by sitaxsentan. Values are means + SEM. N = 4–5/group. Asterisk indicates P < 0.05 versus LPS vehicle. veh+veh: LPS vehicle + sitxsentan vehicle; veh+sitax: LPS vehicle + sitxsentan; LPS+veh: LPS + sitaxsentan vehicle; LPS+sitax: LPS + sitxsentan; PMN: polymorphonuclear cells.

Figure 4

Protective sitaxsentan effects during endotoxemia are not mediated by increasing serum bicarbonate levels. Rats were treated with lipopolysaccharide (LPS), LPS vehicle, sitaxsentan, or sitaxsentan vehicle, as outlined in Figure 1, and serum bicarbonate levels were measured via arterial blood gas analysis. Note the LPS-induced decrease in bicarbonate levels, indicating development of metabolic acidosis. Bicarbonate levels were not affected by sitaxsentan. Values are means + SEM. N = 4–5/group. A single asterisk indicates P < 0.05 and a double asterisk P < 0.01 versus LPS vehicle groups. veh+veh: LPS vehicle + sitxsentan vehicle; veh+sitax: LPS vehicle + sitxsentan; LPS+veh: LPS + sitaxsentan vehicle; LPS+sitax: LPS + sitxsentan.

Figure 5

Sitaxsentan (sitax) administration attenuates endotoxin-induced decreases in endothelium-independent vasorelaxation in isolated pulmonary artery (PA) rings. Rats were treated with lipopolysaccharide (LPS), LPS vehicle (LPS veh), sitaxsentan 10 mg intravenously (sitax 10), sitaxsentan 20 mg/kg intravenously (sitax 20), or sitaxsentan vehicle (sitax veh; equivalent to a 20 mg/kg dose). Sitaxsentan or vehicle was administered 3 hours after administration of LPS or LPS vehicle. Main PAs were harvested 3 hours after administration of sitaxsentan or its vehicle, cut into 2–3-mm segments, and placed in physiologic organ bath for measurement of phenylephrine (PE)-induced vasocontraction, as well as endothelium-dependent and endothelium-independent vasorelaxation. A, PE-induced vasoconstriction was evaluated by adding increasing concentrations (10⁻⁸M to 10⁻⁵M) to the organ bath and is expressed as percent change from baseline tension. Vasorelaxation was then measured in maximally PE-precontracted rings (10⁻⁵M) and is expressed as percent change from PE precontraction. B, Endothelium-dependent vasorelaxation was evaluated by adding increasing concentrations of acetylcholine (ACh; 10⁻⁸M to 10⁻⁵M) to the organ bath. C, Endothelium-independent vasorelaxation was assessed by adding increasing concentrations of sodium nitroprusside (SNP; 10⁻⁸M to 10⁻⁵M). Note that sitaxsentan dose-dependently improved endothelium-independent vasorelaxation in PA rings from LPS-treated animals. Values are means ± SEM. N = 4 animals/group. A double asterisk indicates P < 0.01, 3 asterisks P < 0.001, and 4 asterisks P < 0.0001 for LPS+sitax veh versus LPS veh+sitax veh; a single number sign (#) indicates P < 0.05 and a double number sign P < 0.01 for LPS+sitax 20 versus LPS+veh.

Figure 6

Effect of sitaxsentan treatment on endotoxin-induced increases in pulmonary artery (PA) proinflammatory cytokine transcripts. Rats were treated with lipopolysaccharide (LPS), LPS vehicle, sitaxsentan, or sitaxsentan vehicle, as outlined in Figure 1. Main PAs were harvested 3 hours after administration of sitaxsentan or its vehicle. Interleukin (IL)-1β (A), tumor necrosis factor α (TNF-α; B), IL-6 (C), and inducible nitric oxide synthase (iNOS; D) messenger RNA (mRNA) was measured in PA homogenates via real-time polymerase chain reaction. Gene expression is expressed as fold change versus LPS vehicle + sitaxsentan vehicle–treated (veh+veh) group. Note the LPS-induced increase in transcript of all cytokines and the trend for sitaxsentan to decrease IL-1β and TNF-α mRNA expression. Error bars represent high- and low-end errors. N = 4/group. A single asterisk indicates P < 0.05, a double asterisk P < 0.01, and 3 asterisks P < 0.001 versus the LPS vehicle groups. veh+veh: LPS vehicle + sitxsentan vehicle; veh+sitax: LPS vehicle + sitxsentan; LPS+veh: LPS + sitaxsentan vehicle; LPS+sitax: LPS + sitxsentan.