Murine study of portal hypertension associated endothelin-1 hypo-response

Abstract

Aim: To investigate endothelin-1 hypo-responsive associated with portal hypertension in order to improve patient treatment outcomes.

Methods: Wild type, eNOS(-/-) and iNOS(-/-) mice received partial portal vein ligation surgery to induce portal hypertension or sham surgery. Development of portal hypertension was determined by measuring the splenic pulp pressure, abdominal aortic flow and portal systemic shunting. To measure splenic pulp pressure, a microtip pressure transducer was inserted into the spleen pulp. Abdominal aortic flow was measured by placing an ultrasonic Doppler flow probe around the abdominal aorta between the diaphragm and celiac artery. Portal systemic shunting was calculated by injection of fluorescent microspheres in to the splenic vein and determining the percentage accumulation of spheres in liver and pulmonary beds. Endothelin-1 hypo-response was evaluated by measuring the change in abdominal aortic flow in response to endothelin-1 intravenous administration. In addition, thoracic aorta endothelin-1 contraction was measured in 5 mm isolated thoracic aorta rings ex-vivo using an ADI small vessel myograph.

Results: In wild type and iNOS(-/-) mice splenic pulp pressure increased from 7.5 ± 1.1 mmHg and 7.2 ± 1 mmHg to 25.4 ± 3.1 mmHg and 22 ± 4 mmHg respectively. In eNOS(-/-) mice splenic pulp pressure was increased after 1 d (P = NS), after which it decreased and by 7 d was not significantly elevated when compared to 7 d sham operated controls (6.9 ± 0.6 mmHg and 7.3 ± 0.8 mmHg respectively, P = 0.3). Abdominal aortic flow was increased by 80% and 73% in 7 d portal vein ligated wild type and iNOS when compared to shams, whereas there was no significant difference in 7 d portal vein ligated eNOS(-/-) mice when compared to shams. Endothelin-1 induced a rapid reduction in abdominal aortic blood flow in wild type, eNOS(-/-) and iNOS(-/-) sham mice (50% ± 8%, 73% ± 9% and 47% ± 9% respectively). Following portal vein ligation endothelin-1 reduction in blood flow was significantly diminished in each mouse group. Abdominal aortic flow was reduced by 19% ± 9%, 32% ± 10% and 9% ± 9% in wild type, eNOS(-/-) and iNOS(-/-) mice respectively.

Conclusion: Aberrant endothelin-1 response in murine portal hypertension is NOS isoform independent. Moreover, portal hypertension in the portal vein ligation model is independent of ET-1 function.

Keywords: Endothelin-1; Hyper-dynamic circulation; Liver disease; Nitric oxide synthase isoforms; Portal hypertension.

Figure 1

Illustration of interrelationship between Endothelin-1 induced vasoconstriction and nitric oxide mediated vasodilation. Endothelin-1, PGI2 and NO are closely related in relation to vascular smooth muscle cell tone. (Reprinted with permission Ohkita et al[42] 2002).

Figure 2

Partial portal vein ligation induces chronic hyperemia and persistent portal hypertension in wild type and iNOS^-/- mice but not eNOS^-/- mice. Wild type (A, D, G), iNOS^-/- (B, E, H) and eNOS^-/- (C, F, I) mice were subjected to partial portal vein ligation surgery. 0-7 d thereafter-splenic pulp pressure (A-C), aortic blood flow (D-F) and portal systemic shunting (G-I) were determined. A-C: Splenic pulp pressure was increased acutely in all mouse groups following ligation (0-1 d). After which pressure was increased further in wild type and iNOS^-/- but not in eNOS^-/- mice; D, E: Aortic flow was significantly reduced in wild type, iNOS^-/- and eNOS^-/- mice (0-1 d). In wild type and iNOS^-/- mice this low blood flow converted to hyperemia and increased steadily. In eNOS mice flow returned to pre-surgical baseline and was not increased; G-I: Portal systemic shunting increased steadily in wild type and iNOS^-/- mice (G, H). There was a significant delay in the development of collateral circulation in eNOS^-/- mice (I).

Figure 3

Endothelin-1 hypo-response develops in wild type, iNOS^-/- and eNOS^-/- mice following portal vein ligation. A: Aortic blood flow was monitored in unadulterated wild type (squares), iNOS^-/- (diamonds) and eNOS^-/- (triangle) mice prior to and following IV administration of 5 pmol/kg endothelin-1 (ET1). ET1 induced a rapid vessel contraction and subsequent reduction in flow. Response to ET1 was significantly greater in eNOS^-/- when compared to wild type controls; B: Wild type, iNOS^-/- and eNOS^-/- mice were subjected to sham (open bars) or portal vein ligation surgery (PVL) (shaded bars). After 7 d changes in aortic flow was recorded following IV administration of 5 pmol/kg ET1. In all mouse groups the response to ET1 was markedly reduced following PVL (iNOS > wild type > eNOS^-/-); C: ET1 induced contraction of isolated aortic segments from unadulterated wild type (triangle) iNOS^-/- (square) and eNOS^-/- (triangle) mice were determined using an ADI 610M small animal myograph. Aortic vessel segments contracted to exogenous ET1. At high ET1 dose (10^-4 mol/L) aortic vessel segments from eNOS^-/- mice contracted significantly greater then segments from wild type controls; D: Wild type, iNOS^-/- and eNOS^-/- mice were subjected to sham (open bars) or portal vein ligation surgery (shaded bars). After 7 d the aorta was carefully dissected and ET1 contractility was measured. Ex-vivo aorta ET1 (10^-4 mol/L) contractility was significantly decreased in vessels from 7 d wild type, iNOS^-/- and eNOS^-/- PVL mice when compared to shams (PVL vs sham, P < 0.01).