Severe steatosis induces portal hypertension by systemic arterial hyporeactivity and hepatic vasoconstrictor hyperreactivity in rats

Published online:


Non-alcoholic fatty liver disease (NAFLD) has become the most prevalent chronic liver disease. The presence of portal hypertension has been demonstrated in NAFLD prior to development of inflammation or fibrosis, and is a result of extrahepatic and intrahepatic factors, principally driven by vascular dysfunction. An increased intrahepatic vascular resistance potentially contributes to progression of NAFLD via intralobular hypoxia. However, the exact mechanisms underlying vascular dysfunction in NAFLD remain unknown. This study investigates systemic hemodynamics and both aortic and intrahepatic vascular reactivity in a rat model of severe steatosis. Wistar rats were fed a methionine-choline-deficient diet, inducing steatosis, or control diet for 4 weeks. In vivo hemodynamic measurements, aortic contractility studies, and in situ liver perfusion experiments were performed. The mean arterial blood pressure was lower and portal blood pressure was higher in steatosis compared to controls. The maximal contraction force in aortic rings from steatotic rats was markedly reduced compared to controls. While blockade of nitric oxide (NO) production did not reveal any differences, cyclooxygenase (COX) blockade reduced aortic reactivity in both controls and steatosis, whereas effects were more pronounced in controls. Effects could be attributed to COX-2 iso-enzyme activity. In in situ liver perfusion experiments, exogenous NO donation or endogenous NO stimulation reduced the transhepatic pressure gradient (THPG), whereas NO synthase blockade increased the THPG only in steatosis, but not in controls. Alpha-1-adrenergic stimulation and endothelin-1 induced a significantly more pronounced increase in THPG in steatosis compared to controls. Our results demonstrate that severe steatosis, without inflammation or fibrosis, induces portal hypertension and signs of a hyperdynamic circulation, accompanied by extrahepatic arterial hyporeactivity and intrahepatic vascular hyperreactivity. The arterial hyporeactivity seems to be NO-independent, but appears to be mediated by specific COX-2-related mechanisms. Besides, the increased intrahepatic vascular resistance in steatosis appears not to be NO-related but rather to vasoconstrictor hyperreactivity.

  • Subscribe to Laboratory Investigation for full access:



Additional access options:

Already a subscriber?  Log in  now or  Register  for online access.


  1. 1.

    Paredes AH, Torres DM, Harrison SA. Nonalcoholic fatty liver disease. Clin Liver Dis. 2012;16:397–419.

  2. 2.

    EASL. EASL–EASD–EASO Clinical Practice Guidelines for the management of non-alcoholic fatty liver disease. J Hepatol. 2016;64:1388–402.

  3. 3.

    Charlton MR, Burns JM, Pedersen RA, et al. Frequency and outcomes of liver transplantation for nonalcoholic steatohepatitis in the United States. Gastroenterology. 2011;141:1249–53.

  4. 4.

    Haas JT, Francque S, Staels B. Pathophysiology and mechanisms of nonalcoholic fatty liver disease. Annu Rev Physiol. 2016;78:181–205.

  5. 5.

    Nascimbeni F, Pais R, Bellentani S, et al. From NAFLD in clinical practice to answers from guidelines. J Hepatol. 2013;59:859–71.

  6. 6.

    Adams LA, Anstee QM, Tilg H, et al. Non-alcoholic fatty liver disease and its relationship with cardiovascular disease and other extrahepatic diseases. Gut. 2017;66:1138–53.

  7. 7.

    Tilg H, Moschen AR. Evolution of inflammation in nonalcoholic fatty liver disease: the multiple parallel hits hypothesis. Hepatology. 2010;52:1836–46.

  8. 8.

    Farrell GC, Teoh NC, McCuskey RS. Hepatic microcirculation in fatty liver disease. Anat Rec (Hoboken). 2008;291:684–92.

  9. 9.

    Chu MJJ, Dare AJ, Phillips ARJ, et al. Donor hepatic steatosis and outcome after liver transplantation: a systematic review. J Gastrointest Surg. 2015;19:1713–24.

  10. 10.

    Tashiro H, Kuroda S. Ischemia–reperfusion injury in patients with fatty liver and the clinical impact of steatotic liver on hepatic surgery. Surg Today. 2014;44:1611–25.

  11. 11.

    Hakamada K, Sasaki M, Takahashi K, et al. Sinusoidal flow block after warm ischemia in rats with diet-induced fatty liver. J Surg Res. 1997;70:12–20.

  12. 12.

    Seifalian AM, Chidambaram V, Rolles K, et al. In vivo demonstration of impaired microcirculation in steatotic human liver grafts. Liver Transpl Surg. 1998;4:71–77.

  13. 13.

    Francque S, Verrijken A, Mertens I, et al. Noncirrhotic human nonalcoholic fatty liver disease induces portal hypertension in relation to the histological degree of steatosis. Eur J Gastroenterol Hepatol. 2010;22:1449–57.

  14. 14.

    Francque S, Verrijken A, Mertens I, et al. Visceral adiposity and insulin resistance are independent predictors of the presence of non-cirrhotic NAFLD-related portal hypertension. Int J Obes. 2011;35:270–8.

  15. 15.

    Francque S, Wamutu S, Chatterjee S, et al. Non-alcoholic steatohepatitis induces non-fibrosis-related portal hypertension associated with splanchnic vasodilation and signs of a hyperdynamic circulation in vitro and in vivo in a rat model. Liver Int. 2009;30:365–75.

  16. 16.

    Iwakiri Y. Endothelial dysfunction in the regulation of cirrhosis and portal hypertension. Liver Int. 2012;32:199–213.

  17. 17.

    Mihmanli I, Kantarci F, Yilmaz MH, et al. Effect of diffuse fatty infiltration of the liver on hepatic artery resistance index. J Clin Ultrasound. 2005;33:95–99.

  18. 18.

    Francque S, Laleman W, Verbeke L, et al. Increased intrahepatic resistance in severe steatosis: endothelial dysfunction, vasoconstrictor overproduction and altered microvascular architecture. Lab Invest. 2012;92:1428–39.

  19. 19.

    Oda M, Yokomori H, Han J. Regulatory mechanisms of hepatic microcirculation. Clin Hemorheol Microcirc. 2003;29:167–82.

  20. 20.

    Laleman W, Landeghem L, Wilmer A, et al. Portal hypertension: from pathophysiology to clinical practice. Liver Int. 2005;25:1079–90.

  21. 21.

    McCuskey RS, Ito Y, Robertson GR, et al. Hepatic microvascular dysfunction during evolution of dietary steatohepatitis in mice. Hepatology. 2004;40:386–93.

  22. 22.

    Pasarín M, La Mura V, Gracia-Sancho J, et al. Sinusoidal endothelial dysfunction precedes inflammation and fibrosis in a model of NAFLD. PLoS ONE. 2012;7:e32785.

  23. 23.

    Gonzalez-Paredes FJ, Hernández Mesa G, Morales Arraez D, et al. Contribution of cyclooxygenase end products and oxidative stress to intrahepatic endothelial dysfunction in early non-alcoholic fatty liver disease. PLoS ONE. 2016;11:e0156650.

  24. 24.

    Miyao M, Kotani H, Ishida T, et al. Pivotal role of liver sinusoidal endothelial cells in NAFLD/NASH progression. Lab Invest. 2015;95:1130–44.

  25. 25.

    Leclercq IA. Pathogenesis of steatohepatitis: insights from the study of animal models. Acta Gastroenterol Belg. 2007;70:25–31.

  26. 26.

    Michielsen P, Boeckxstaens G, Sys SU, et al. The role of increased nitric oxide in the vascular hyporeactivity to noradrenalin in long-term portal hypertensive rats. J Hepatol. 1995;23:341–7.

  27. 27.

    Kanno T, Tsuchiya A, Shimizu T, et al. Indomethacin serves as a potential inhibitor of protein phosphatases. Cell Physiol Biochem. 2012;30:1014–22.

  28. 28.

    Bessems M, Hart NA, Tolba R, et al. The isolated perfused rat liver: standardization of a time-honoured model. Lab Anim. 2006;40:236–46.

  29. 29.

    Ferrigno A, Richelmi P, Vairetti M. Troubleshooting and improving the mouse and rat isolated perfused liver preparation. J Pharmacol Toxicol Methods. 2013;67:107–14.

  30. 30.

    Francque SM, Verrijken A, Mertens I, et al. Noninvasive assessment of nonalcoholic fatty liver disease in obese or overweight patients. Clin Gastroenterol Hepatol. 2012;10:1162–8.

  31. 31.

    Hebbard L, George J. Animal models of nonalcoholic fatty liver disease. Nat Rev Gastroenterol Hepatol. 2011;8:35–44.

  32. 32.

    García-Pagán JC. Complications of cirrhosis. I. Portal hypertension in cirrhosis. J Hepatol. 2012;57:458–61.

  33. 33.

    Abraldes JG, Iwakiri Y, Loureiro-Silva M, et al. Mild increases in portal pressure upregulate vascular endothelial growth factor and endothelial nitric oxide synthase in the intestinal microcirculatory bed, leading to a hyperdynamic state. Am J Physiol Gastrointest Liver Physiol. 2006;290:G980–7.

  34. 34.

    Serna E, Mauricio MD, Lluch P, et al. Basal release of nitric oxide in the mesenteric artery in portal hypertension and cirrhosis: role of dimethylarginine dimethylaminohydrolase. J Gastroenterol Hepatol. 2013;28:880–6.

  35. 35.

    Colle IO, De Vriese AS, Van Vlierberghe HR, et al. Vascular hyporesponsiveness in the mesenteric artery of anaesthetized rats with cirrhosis and portal hypertension: an in-vivo study. J Gastroenterol. 2004;16:139–45.

  36. 36.

    Colle I, Geerts AM, Van Steenkiste C, et al. Hemodynamic changes in splanchnic blood vessels in portal hypertension. Anat Rec (Hoboken). 2008;291:699–713.

  37. 37.

    Hennenberg M, Trebicka J, Sauerbruch T, et al. Mechanisms of extrahepatic vasodilation in portal hypertension. Gut. 2008;57:1300–14.

  38. 38.

    Graupera M, March S, Engel P, et al. Sinusoidal endothelial COX-1-derived prostanoids modulate the hepatic vascular tone of cirrhotic rat livers. Am J Physiol Gastrointest Liver Physiol. 2005;288:G763–70.

  39. 39.

    Laleman W, Van Landeghem L, Van der Elst I, et al. Nitroflurbiprofen, a nitric oxide-releasing cyclooxygenase inhibitor, improves cirrhotic portal hypertension in rats. Gastroenterology. 2007;132:709–19.

  40. 40.

    Abraldes JG, Rodríguez-Vilarrupla A, Graupera M, et al. Simvastatin treatment improves liver sinusoidal endothelial dysfunction in CCl4 cirrhotic rats. J Hepatol. 2007;46:1040–6.

  41. 41.

    Abraldes JG, Albillos A, Bañares R, et al. Simvastatin lowers portal pressure in patients with cirrhosis and portal hypertension: a randomized controlled trial. Gastroenterology. 2009;136:1651–8.

  42. 42.

    Zafra C, Abraldes JG, Turnes J, et al. Simvastatin enhances hepatic nitric oxide production and decreases the hepatic vascular tone in patients with cirrhosis. Gastroenterology. 2004;126:749–55.

  43. 43.

    Wang W, Zhao C, Zhou J, et al. Simvastatin ameliorates liver fibrosis via mediating nitric oxide synthase in rats with non-alcoholic steatohepatitis-related liver fibrosis. PLoS ONE. 2013;8:1–11.

  44. 44.

    Chang C-C, Wang S-S, Hsieh H-G, et al. Rosuvastatin improves hepatopulmonary syndrome through inhibition of inflammatory angiogenesis of lung. Clin Sci (Lond). 2015;129:449–60.

  45. 45.

    Fiorucci S, Antonelli E, Brancaleone V, et al. NCX-1000, a nitric oxide releasing derivative of ursodeoxycholic acid, ameliorates portal hypertension and lowers norepinephrine-induced intrahepatic resistance in the isolated and perfused rat liver. J Hepatol. 2003;39:932–9.

  46. 46.

    Biecker E, Trebicka J, Kang A, et al. Treatment of bile duct-ligated rats with the nitric oxide synthase transcription enhancer AVE 9488 ameliorates portal hypertension. Liver Int. 2008;28:331–8.

  47. 47.

    Elliot AJ, Vo LT, Grossman VL, et al. Endothelin-induced vasoconstriction in isolated perfused liver preparations from normal and cirrhotic rats. J Gastroenterol Hepatol. 1997;12:314–8.

  48. 48.

    Shibamoto T, Kamikado C, Koyama S. Increased sinusoidal resistance is responsible for the basal state and endothelin-induced venoconstriction in perfused cirrhotic rat liver. Pflug Arch Eur J Physiol. 2008;456:467–77.

  49. 49.

    Chan C-C, Lee K-CL, Lin H-C, et al. Increased expression of hepatic endothelin-1 during fibrosis progression in non-alcoholic fatty liver disease. J Gastroenterol Hepatol. 2014;1:1–7.

Download references


W.J.K. and S.M.F. received funding from the Fund for Scientific Research (FWO) Flanders (11J9513N, 1802154N).


The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Author information

Author notes

  1. Van der Graaff Denise and Kwanten Wilhelmus J contributed equally to this work.


  1. Department of Gastroenterology and Hepatology, Antwerp University Hospital, Antwerp, Belgium

    • Denise Van der Graaff
    • , Wilhelmus J Kwanten
    • , Wim Verlinden
    • , Peter P Michielsen
    •  & Sven M Francque
  2. Laboratory of Experimental Medicine and Pediatrics (LEMP), Division of Gastroenterology-Hepatology, Faculty of Medicine and Health Sciences, University of Antwerp, Antwerp, Belgium

    • Denise Van der Graaff
    • , Wilhelmus J Kwanten
    • , Filip J Couturier
    • , Jesse S Govaerts
    • , Wim Verlinden
    • , Isabel Brosius
    • , Michiel D’Hondt
    • , Benedicte Y De Winter
    • , Joris G De Man
    • , Peter P Michielsen
    •  & Sven M Francque
  3. Department of Pathology, Antwerp University Hospital, Laboratory of Pathology, Faculty of Medicine and Health Sciences, University of Antwerp, Antwerp, Belgium

    • Ann Driessen


  1. Search for Denise Van der Graaff in:

  2. Search for Wilhelmus J Kwanten in:

  3. Search for Filip J Couturier in:

  4. Search for Jesse S Govaerts in:

  5. Search for Wim Verlinden in:

  6. Search for Isabel Brosius in:

  7. Search for Michiel D’Hondt in:

  8. Search for Ann Driessen in:

  9. Search for Benedicte Y De Winter in:

  10. Search for Joris G De Man in:

  11. Search for Peter P Michielsen in:

  12. Search for Sven M Francque in:

Conflict of interest

The authors declare that they have no conflict of interest.

Corresponding author

Correspondence to Sven M Francque.

Electronic supplementary material