Sorafenib targets dysregulated Rho kinase expression and portal hypertension in rats with secondary biliary cirrhosis

Abstract

Background and purpose: Extrahepatic vasodilation and increased intrahepatic vascular resistance represent attractive targets for the medical treatment of portal hypertension in liver cirrhosis. In both dysfunctions, dysregulation of the contraction-mediating Rho kinase plays an important role as it contributes to altered vasoconstrictor responsiveness. However, the mechanisms of vascular Rho kinase dysregulation in cirrhosis are insufficiently understood. They possibly involve mitogen-activated protein kinase/extracellular signal-regulated kinase (ERK)-dependent mechanisms in extrahepatic vessels. As the multikinase inhibitor sorafenib inhibits ERK, we tested the effect of sorafenib on haemodynamics and dysregulated vascular Rho kinase in rats with secondary biliary cirrhosis.

Experimental approach: Secondary biliary cirrhosis was induced by bile duct ligation (BDL). Sorafenib was given orally for 1 week (60 mg.kg(-1).d(-1)). Messenger RNA levels were determined by quantitative real time polymerase chain reaction, protein expressions and protein phosphorylation by Western blot analysis. Aortic contractility was studied by myographic measurements, and intrahepatic vasoregulation by using livers perfused in situ. In vivo, haemodynamic parameters were assessed invasively in combination with coloured microspheres.

Key results: In BDL rats, treatment with sorafenib decreased portal pressure, paralleled by decreases in hepatic Rho kinase expression and Rho kinase-mediated intrahepatic vascular resistance. In aortas from BDL rats, sorafenib caused up-regulation of Rho kinase and an improvement of aortic contractility. By contrast, mesenteric Rho kinase remained unaffected by sorafenib.

Conclusions and implications: Intrahepatic dysregulation of vascular Rho kinase expression is controlled by sorafenib-sensitive mechanisms in rats with secondary biliary cirrhosis. Thus, sorafenib reduced portal pressure without affecting systemic blood pressure.

Figure 1

Vascular and intrahepatic targets of sorafenib in BDL rats: intracellular effects of sorafenib in the liver and different vessels. (A) Activity of ERK1 and 2 was monitored as Tyr202-Tyr204 phosphorylation. Shown are representative Western blots and densitometric quantification of all experiments (means ± SEM, n = 9 for each group). (B) The activity of the PDGF-Rβ was assessed as its phosphorylation state at the indicated autophosphorylation sites (Tyr751, Tyr857). Representative Western blots and densitometric quantification of all experiments (means ± SEM, n = 9 for each group) are shown. BDL, bile duct ligation; ERK, extracellular signal-regulated kinase; PDGF-R, platelet-derived growth factor receptor.

Figure 2

Vascular and intrahepatic effects of sorafenib on Rho kinase in BDL rats. (A) Aortic and hepatic Rho kinase mRNA levels. The higher the −ΔCt, the higher the mRNA concentration. Results from all experiments are shown. (B) Western blot analysis for Rho kinase protein expression and activity. The latter was assessed as the phosphorylation state of the Rho kinase substrate moesin at Thr558. Representative Western blots and densitometric quantification of all experiments (means ± SEM, n = 9 for each group for livers and mesenteric arteries, n = 12 for each group for aortas) are shown. BDL, bile duct ligation; Ct, number of cycles required to exceed a threshold; mRNA, messenger RNA.

Figure 3

Effects of sorafenib on aortic contractility and regulators of Rho kinase in BDL rats. (A) Contractility of isolated aortic rings to the α₁-adrenoceptor agonist methoxamine. All vessels were incubated with the NOS inhibitor L-NAME 30 min before and during the entire measurements. Concentration response curves are shown in the right panel and the EC₅₀ values in the left panel. Data are means ± SEM from experiments with eight sham-operated rats, seven BDL rats and eight sorafenib-treated BDL rats. #P < 0.05 for BDL versus BDL + sorafenib. (B) Effect of sorafenib on aortic mRNA and protein expression of β-arrestin 2 in BDL rats. Results from all experiments for mRNA expression (n = 5 for each group), and representative Western blots and densitometric quantification of all experiments for protein expression (n = 8 for each group) are shown. BDL, bile duct ligation; L-NAME, Nω-nitro-L-arginine methyl ester; mRNA, messenger RNA; NOS, nitric oxide synthase.

Figure 4

Haemodynamic effects of sorafenib in sham-operated and BDL rats. Results from experiments with five sham-operated rats and seven BDL rats (means ± SEM) are shown. BDL, bile duct ligation.

Figure 5

Effects of sorafenib on the perfusion pressure of livers perfused in situ. (A) Basal perfusion pressure of livers perfused in situ of sorafenib-treated and-untreated BDL rats. Shown are data from all experiments (means ± SEM, nine sham-operated rats, five sorafenib-treated sham-operated rats, eight BDL rats, seven sorafenib-treated BDL rats). (B) Methoxamine-induced increase in perfusion pressure (methoxamine-induced perfusion pressure – basal perfusion pressure) in sorafenib-treated and-untreated sham-operated and BDL rats. #P < 0.05 versus untreated sham-operated rats; †P < 0.05 versus sorafenib-treated BDL. Data from all experiments (means ± SEM, eight sham-operated rats, seven sorafenib-treated sham-operated rats, six BDL rats, six sorafenib-treated BDL rats) are shown. (C) Y27632-induced decrease in perfusion pressure in sorafenib-treated and-untreated BDL rats. Data from all experiments (means ± SEM, six BDL rats, six sorafenib-treated BDL rats) are shown. BDL, bile duct ligation; Y27632, (R)-(+)-trans-N-(4-pyridyl)-4-(1-aminoethyl)-cyclohexanecarboxamide.

Figure 6

Effects of sorafenib on HSC markers in BDL rats. (A) α-SMA protein expression in livers from BDL and sorafenib-treated BDL rats. Representative Western blot and densitometric quantification of all experiments (means ± SEM, n = 9 for each group) are shown. (B) Hepatic mRNA levels of PDGF-Rβ in BDL and sorafenib-treated BDL rats. Data from all experiments are shown. BDL, bile duct ligation; Ct, number of cycles required to exceed a threshold; HSC, hepatic stellate cell(s); mRNA, messenger RNA; PDGF-R, platelet-derived growth factor receptor.

Figure 7

Assumed mechanisms of post-transcriptional Rho kinase down-regulation and its correction by sorafenib in hypocontractile vessels. Given an β-arrestin 2-dependent activation mode – which is likely to occur in cirrhosis due to up-regulation of β-arrestin 2 expression – ERK may cause post-transcriptional Rho kinase down-regulation in cultured fibroblasts and in vivo in the internal anal sphincter. We assume that aberrant signalling by usually G-protein-coupled vasoconstrictor receptors cause the Rho kinase down-regulation of hypocontractile vessels at least from BDL rats: changing the coupling from G-proteins to the overexpressed β-arrestin 2, these receptors may lead to down-regulation of Rho kinase which can be targeted by ERK inhibition through sorafenib. BDL, bile duct ligation; ERK, extracellular signal-regulated kinase.