Interleukin-10 attenuates vascular responses to endothelin-1 via effects on ERK1/2-dependent pathway

Abstract

Interleukin-10 (IL-10) is an anti-inflammatory cytokine with protective actions on the vasculature. On the other hand, endothelin (ET)-1 has potent vasoconstrictor, mitogenic, and proinflammatory activities, which have been implicated in the pathophysiology of a number of cardiovascular diseases. We hypothesized that, in a condition where ET-1 expression is upregulated, i.e., on infusion of TNF-alpha, IL-10 confers vascular protection from ET-1-induced injury. Aortic rings and first-order mesenteric arteries from male C57BL/6 (WT) and IL-10-knockout (IL-10(-/-)) mice were treated with human recombinant TNF-alpha (220 ng x kg(-1) x day(-1)) or vehicle (saline) for 14 days. TNF-alpha infusion significantly increased blood pressure in IL-10(-/-), but not WT, mice. TNF-alpha augmented vascular ET-1 mRNA expression in arteries from WT and IL-10(-/-) mice. ET type A (ET(A)) receptor expression was increased in arteries from IL-10(-/-) mice, and TNF-alpha infusion did not change vascular ET(A) receptor expression in control or IL-10(-/-) mice. Aorta and mesenteric arteries from TNF-alpha-infused IL-10(-/-) mice displayed increased contractile responses to ET-1, but not the ET type B receptor agonist IRL-1620. The ET(A) receptor antagonist atrasentan completely abolished responses to ET-1 in aorta and mesenteric vessels, whereas the ERK1/2 inhibitor PD-98059 abrogated increased contractions to ET-1 in arteries from TNF-alpha-infused IL-10(-/-) mice. Infusion of TNF-alpha, as well as knockdown of IL-10 (IL-10(-/-)), induced an increase in total and phosphorylated ERK1/2. These data demonstrate that IL-10 counteracts ET(A)-mediated vascular responses to ET-1, as well as activation of the ERK1/2 pathway.

Fig. 1.

Mean arterial pressure (A) and vascular (aorta) preproendothelin-1 mRNA expression (B) in wild-type (WT) mice infused with vehicle (WT + vehicle) and TNF-α-infused WT mice (WT + TNF-α) and IL-10-knockout (IL-10^−/−) mice infused with vehicle (IL-10^−/− + vehicle) and TNF-α-infused IL-10^−/− mice (IL-10^−/− + TNF-α). Values are means ± SE (n = 5 experiments). *P < 0.05 vs. respective control (i.e., vehicle). †P < 0.05, IL-10^−/− vs. WT.

Fig. 2.

Contractile responses to endothelin (ET)-1 are enhanced in arteries from TNF-α-infused IL-10^−/− mice. Cumulative concentration-response curves to ET-1 (0.1 nM–1.0 μM) in 1st-order mesenteric arteries (A) and thoracic aorta (B) were determined in vehicle- and TNF-α-infused WT and vehicle- and TNF-α-infused IL-10^−/− mice. Values are means ± SE (n = 5 experiments). *P < 0.05 vs. other groups.

Fig. 3.

Contractile responses to ET-1 are mediated by ET type A (ET_A) receptor activation. Responses of small mesenteric arteries (A) and aortas (B) to ET-1 were determined in the absence or presence of atrasentan (1 μM), a selective ET_A receptor antagonist. Values are means ± SE (n = 6 experiments). *P < 0.05 vs. ET-1.

Fig. 4.

ET_A receptor expression is augmented in vessels from IL-10^−/− mice. Top: representative immunoblots for ET_A receptor and β-actin expression in murine aorta. Bottom: corresponding bar graphs demonstrating regulatory role of IL-10 in ET_A receptor expression. Densitometric analysis was performed in samples from vehicle- and TNF-α-infused WT and vehicle- and TNF-α-infused IL-10^−/− mice. Values, expressed in arbitrary units, are means ± SE (n = 5 experiments) and were normalized to β-actin protein expression. *P < 0.05 vs. respective control (i.e., WT).

Fig. 5.

ERK1/2 inhibition abrogates augmented contractile responses to ET-1 in arteries from TNF-α-infused IL-10^−/− mice. Cumulative concentration-response curves to ET-1 (0.1 nM–1.0 μM) were determined in the presence of PD-98059 (1 μM), an ERK1/2 inhibitor, in 1st-order mesenteric arteries (A) and aorta (B) from vehicle- and TNF-α-infused WT and vehicle- and TNF-α-infused IL-10^−/− mice. Values are means ± SE (n = 6 experiments).

Fig. 6.

ERK1/2 activity is augmented in vessels from TNF-α-infused IL-10^−/− mice. A and B: representative immunoblots for total ERK1/2, phosphorylated ERK1/2, and β-actin expression in murine aorta (top) and corresponding bar graphs (bottom) demonstrating regulatory role of IL-10 on total and phosphorylated ERK1/2. C: ratio of phosphorylated to total ERK1/2 vascular proteins. Densitometric analysis was performed in samples from vehicle- and TNF-α-infused WT and vehicle- and TNF-α-infused IL-10^−/− mice. Values, expressed in arbitrary units, are means ± SE (n = 5 experiments) and were normalized to β-actin protein expression. *P < 0.05 vs. respective control (i.e., WT). †P < 0.05 vs. respective vehicle-infused group.