Vasoconstrictor antagonism improves functional and structural vascular alterations and liver damage in rats with early NAFLD

Abstract

Background & aims: Intrahepatic vascular resistance is increased in early non-alcoholic fatty liver disease (NAFLD), potentially leading to tissue hypoxia and triggering disease progression. Hepatic vascular hyperreactivity to vasoconstrictors has been identified as an underlying mechanism. This study investigates vasoconstrictive agonism and antagonism in 2 models of early NAFLD and in non-alcoholic steatohepatitis (NASH).

Methods: The effects of endothelin-1 (ET-1), angiotensin II (ATII) and thromboxane A₂ (TxA₂) agonism and antagonism were studied by in situ ex vivo liver perfusion and preventive/therapeutic treatment experiments in a methionine-choline-deficient diet model of steatosis. Furthermore, important results were validated in Zucker fatty rats after 4 or 8 weeks of high-fat high-fructose diet feeding. In vivo systemic and portal pressures, ex vivo transhepatic pressure gradients (THPG) and transaminase levels were measured. Liver tissue was harvested for structural and mRNA analysis.

Results: The THPG and consequent portal pressure were significantly increased in both models of steatosis and in NASH. ET-1, ATII and TxA₂ increased the THPG even further. Bosentan (ET-1 receptor antagonist), valsartan (ATII receptor blocker) and celecoxib (COX-2 inhibitor) attenuated or even normalised the increased THPG in steatosis. Simultaneously, bosentan and valsartan treatment improved transaminase levels. Moreover, bosentan was able to mitigate the degree of steatosis and restored the disrupted microvascular structure. Finally, beneficial vascular effects of bosentan endured in NASH.

Conclusions: Antagonism of vasoconstrictive mediators improves intrahepatic vascular function. Both ET-1 and ATII antagonists showed additional benefit and bosentan even mitigated steatosis and structural liver damage. In conclusion, vasoconstrictive antagonism is a potentially promising therapeutic option for the treatment of early NAFLD.

Lay summary: In non-alcoholic fatty liver disease (NAFLD), hepatic blood flow is impaired and the blood pressure in the liver blood vessels is increased as a result of an increased response of the liver vasculature to vasoconstrictors. Using drugs to block the constriction of the intrahepatic vasculature, the resistance of the liver blood vessels decreases and the increased portal pressure is reduced. Moreover, blocking the vasoconstrictive endothelin-1 pathway restored parenchymal architecture and reduced disease severity.

Keywords: ALT, alanine aminotransferase; ARB, angiotensin receptor blocker; AST, aspartate aminotransferase; ATII, angiotensin II; COX, cyclooxygenase; ET, endothelin; HFHFD, high-fat high-fructose diet; IHVR, intrahepatic vascular resistance; Jak2, Janus-kinase-2; MCD, methionine-choline deficient diet; Mx, methoxamine; NAFLD, non-alcoholic fatty liver disease; NASH, non-alcoholic steatohepatitis; NO, nitric oxide; PP, portal pressure; PR, pulse rate; SEM, scanning electron microscopy; TBW, total body weight; TEM, transmission electron microscopy; TXAS, thromboxane synthase; TxA2, thromboxane A2; ZFR, Zucker fatty rats; angiotensin II; endothelin-1; non-alcoholic fatty liver disease; portal hypertension; thromboxane A2; transhepatic pressure gradient.

Graphical abstract

Fig. 1

Histological liver sections (H&E stain, original magnification 10x) of rats fed control or methionine-choline-deficient diet (steatosis) after preventive placebo, bosentan, valsartan or celecoxib treatment.

Fig. 2

Representative scanning electron microscopy images of hepatic vascular corrosion casts of rats fed control or methionine-choline-deficient diet (steatosis) after preventive placebo, bosentan, valsartan or celecoxib treatment (original magnification 300x). In controls, sinusoids are regular with small and even diameters. In steatosis, the number of sinusoids is reduced, the vascular pattern is irregular and the remaining sinusoids appear to have uneven and larger diameters with numerous blebs (dead-ending vessel stumps). After bosentan treatment, the number of sinusoids remains reduced, but the diameter is more comparable to control livers and the pattern appears more regular with less blebs.

Fig. 3

Transhepatic pressure gradient with modulation of endothelin-1 pathways. (A) Dose-response curves of the transhepatic pressure gradient to ET_A-receptor blocker BQ-123 with endothelin-1. (B) Dose-response curves of the transhepatic pressure gradient to ET_B-receptor blocker BQ-788 with endothelin-1. (C) Flow-pressure curves of control and steatotic rat hepatic vasculature after preventive bosentan. (D) Flow-pressure curves of control and steatotic rat hepatic vasculature after therapeutic bosentan. All data were analysed using the generalised estimating equation model. Significances between controls with or without compound/treatment are indicated by black signs. Significances between steatosis with or without compound/treatment are indicated by red signs. ∗p <0.05; ∗∗p <0.01; n.s., not significant.

Fig. 4

Serum ALT and AST in rats fed control or methionine-choline-deficient diet (steatosis) after preventive/therapeutic placebo, bosentan, valsartan or celecoxib treatment. Statistic comparison by two-way ANOVA and post hoc Scheffé. ∗p <0.05; ∗∗p <0.01; ∗∗∗p <0.001. ALT, alanine aminotransferase; AST, aspartate aminotransferase.

Fig. 5

Effect of bosentan, celecoxib and valsartan treatment on mRNA expression of genes related to endothelin-1-, angiotensin II- and cyclooxygenase-related pathways. Heat maps were generated using the nSolver Analysis Software (NanoString). Normalised gene counts and all other results were analysed with a two-way ANOVA (with the diet as the first factor [between], the treatment used as the second factor [within]) and Scheffé post hoc testing when appropriate. BOS, bosentan; CD, control diet; CEL, celecoxib; MCDD, methionine-choline-deficient diet; VAL, valsartan.

Fig. 6

Transhepatic pressure gradient with modulation of angiotensin II pathways. (A) Dose-response curves of the transhepatic pressure gradient to angiotensin II or Krebs in livers of control and methionine-choline-deficient diet-fed rats (steatosis). (B) Dose-response curves of the transhepatic pressure gradient to the angiotensin receptor blocker valsartan or Krebs with angiotensin II. (C) Flow-pressure curves of control and steatotic hepatic vasculature after preventive valsartan. (D) Flow-pressure curves after therapeutic valsartan. All data were analysed using the generalised estimating equation model. Significances between controls with or without compound/treatment are indicated by black signs. Significances between steatosis with or without compound/treatment are indicated by purple signs. ∗p <0.05; ∗∗p <0.01; ∗∗∗p <0.001; n.s., not significant.

Fig. 7

Transhepatic pressure gradient with modulation of thromboxane pathways. (A) Dose-response curves of the transhepatic pressure gradient to thromboxane agonist U-46619 or Krebs in control and methionine-choline-deficient diet-fed rats (steatosis). (B) Dose-response curves of the transhepatic pressure gradient to COX-1 antagonist SC-560 or Krebs with methoxamine. (C) Dose-response curves of the transhepatic pressure gradient to COX-2 antagonist SC-236 or Krebs with methoxamine. (D) Flow-pressure curves of control and steatotic hepatic vasculature with preventive celecoxib. (E) Flow-pressure curves with therapeutic celecoxib. All data were analysed using the generalised estimating equation model. Significances between controls with or without compound/treatment are indicated by black signs. Significances between steatosis with or without compound/treatment are indicated by purple signs. ∗p <0.05; ∗∗p <0.01; ∗∗∗p <0.001; n.s., not significant.

Fig. 8

Lean Zucker rats on control diet (controls) compared to 8-week high-fat high-fructose diet-fed Zucker fatty rats (NASH) receiving bosentan or placebo treatment. (A) Serum ALT and AST levels. Statistical comparison by two-way ANOVA and post hoc Scheffé. n.s., not significant. (B) Liver histological liver sections (H&E and Masson’s trichrome stains [TM], original magnification 10x). (C) Flow-pressure curves of the transhepatic pressure gradient, analysed using the generalised estimating equation model. Significances between controls and NASH are indicated by black signs. Significances between NASH with placebo or bosentan are indicated by purple signs. ∗∗∗p <0.001. ALT, alanine aminotransferase; AST, aspartate aminotransferase; NASH, non-alcoholic steatohepatitis.