Elsevier

Journal of Hepatology

Volume 64, Issue 4, April 2016, Pages 834-842
Journal of Hepatology

Research Article
Enoxaparin reduces hepatic vascular resistance and portal pressure in cirrhotic rats

https://doi.org/10.1016/j.jhep.2015.12.003Get rights and content

Background & Aims

Increased hepatic vascular resistance due to fibrosis and elevated hepatic vascular tone is the primary factor in the development of portal hypertension. Heparin may decrease fibrosis by inhibiting intrahepatic microthrombosis and thrombin-mediated hepatic stellate cell activation. In addition, heparin enhances eNOS activity, which may reduce hepatic vascular tone. Our study aimed at evaluating the effects of acute, short-, long-term and preventive enoxaparin administration on hepatic and systemic hemodynamics, liver fibrosis and nitric oxide availability in cirrhotic rats.

Methods

Enoxaparin (1.8 mg/kg subcutaneously), or its vehicle, was administered to CCl4-cirrhotic rats 24 h and 1 h before the study (acute), daily for 1 week (short-term) or daily for 3 weeks (long-term) and to thioacetamide-cirrhotic rats daily for 3 weeks with/without thioacetamide (preventive/long-term, respectively). Mean arterial pressure, portal pressure, portal blood flow, hepatic vascular resistance and molecular/cellular mechanisms were evaluated.

Results

No significant changes in hemodynamic parameters were observed in acute administration. However, one-week, three-week and preventive treatments significantly decreased portal pressure mainly due to a decrease in hepatic vascular resistance without significant changes in mean arterial pressure. These findings were associated with significant reductions in liver fibrosis, hepatic stellate cell activation, and desmin expression. Moreover, a reduction in fibrin deposition was observed in enoxaparin-treated rats, suggesting reduced intrahepatic microthrombosis.

Conclusion

Enoxaparin reduces portal pressure in cirrhotic rats by improving the structural component of increased liver resistance. These findings describe the potentially beneficial effects of enoxaparin beyond the treatment/prevention of portal vein thrombosis in cirrhosis, which deserve further investigation.

Introduction

Increased resistance to portal blood flow, derived from architectural alterations of the liver parenchyma and a dynamic increment in the hepatic vascular tone, is the primary factor in the development of portal hypertension [1], [2]. Architectural distortion of the cirrhotic liver is partly due to excessive synthesis of extracellular matrix components performed by dys-regulated fibrogenic cells such as hepatic stellate cells (HSC) and portal myofibroblasts [3]. Several evidences suggest that liver fibrogenesis is positively influenced by inflammation and thrombosis [4]. Indeed, it has been demonstrated that different pro-coagulation factors, such as the serine protease thrombin, dysregulate HSC phenotype through the stimulation of their protease activated receptors (PARs) [5], [6], [7], [8], [9]. Moreover, PARs expression is highly upregulated in human livers undergoing acute and chronic injury [10], and their inhibition results in a significant amelioration in HSC phenotype and hepatic fibrosis progression in experimental models of mild liver damage [11], [12]. Generation of thrombin, from its precursor prothrombin, is efficiently performed by the activated coagulation molecule factor Xa. It is nowadays recognized that either thrombin or factor Xa are agonists of PARs and PAR signaling represents one of the main pathways of HSC activation and collagen deposition [7], [8], [13], [14], [15].

In addition, thrombin-derived clot formation may promote flow disturbances and occlusion of small-sized intrahepatic veins and sinusoids, representing another triggering factor of liver tissue remodelling [16]. This mechanism, named parenchymal extinction, includes progressive vascular obliteration due to thrombosis or inflammation leading to both apoptosis and atrophy of the liver [17].

Heparins, through their interaction with antithrombin, which induces a conformational change in antithrombin molecule, greatly facilitates the interactions between antithrombin and serine protease targets such as thrombin, and also factor Xa, IXa, XIa and XIIa [18]. Potential beneficial effects of the low molecular weight heparin (LMWH)-induced inhibition of the coagulation cascade have been evaluated in liver fibrosis [19], [20]. Previous translational studies, performed in experimental models of mild liver damage induced by CCl4 and common bile duct ligation, have proposed that LMWH may improve hepatic regeneration, by the inhibition of HSC proliferation and/or by inducing a marked reduction in the hepatic cytonecrosis index respectively [19], [21]. Whether the anti-fibrotic effects of the drug is directly due to its anticoagulative effect preventing thrombosis or to the inactivation of cell-mediated fibrogenic mechanisms remains unknown. In addition, it has not yet been evaluated whether these beneficial effects are also observed in more severe liver damage (ie. established cirrhosis). Indeed, a possible beneficial role of LMWH in the treatment of advanced liver disease was recently suggested by a randomised controlled trial investigating the effect of continuous use of the LMWH enoxaparin to prevent portal vein thrombosis (PVT) in patients with cirrhosis. In this study, enoxaparin reduced the incidence of PVT and, although the underlying mechanisms remain largely unknown, also reduced the incidence of liver decompensation and improved survival [22].

In addition, heparins may also have beneficial effects on the vasculature via endothelium-dependent mechanisms. Indeed, LMWH has been shown to upregulate nitric oxide (NO) levels by activation of its synthesis [23], [24], [25].

The present study aimed to characterize the effects, and underlying mechanisms, of acute, short, long-term and preventive administration of the LMWH enoxaparin on the hepatic and systemic hemodynamics and on liver fibrosis in two experimental models of cirrhosis (CCl4 and thioacetamide).

Section snippets

Induction of cirrhosis by CCl4 and LMWH administration

Cirrhosis was induced in male Wistar rats (50–75 g) with CCl4 inhalation three times a week. Phenobarbital (0.3 g/L) was added to drinking water as previously described [26]. When animals developed ascites, after approximately 12–15 weeks of CCl4 inhalation, phenobarbital and CCl4 administration was discontinued. Rats were then randomized to receive either enoxaparin (1.8 mg/kg body weight, subcutaneously; Clexane, Sanofi-Aventis), or its vehicle (saline 0.9%), according to three different

Acute enoxaparin treatment in CCl4-cirrhotic rats

Acute administration of enoxaparin did not affect any systemic or hepatic hemodynamic parameters evaluated (Table 1A). Moreover, acute enoxaparin did not modify hepatic cGMP levels or hepatic oxidative stress content (data not shown).

Short-term enoxaparin administration in CCl4-cirrhotic rats

One-week enoxaparin-treated rats had significantly lower portal pressure (PP) than vehicle-treated rats (Table 1B) without significant changes in portal blood flow (PBF). However, probably due to high variability, we were unable to identify significant changes in

Discussion

Previous studies examining the natural history of liver disease in cohorts of HCV infected patients suggested a role of the coagulation cascade in hepatic fibrogenesis [36], [37]. This hypothesis has gained much more attention after the demonstration that patients with cirrhosis exhibit more frequently a prothrombotic state with increased generation of thrombin than an hypocoagulative state [38], [39]. Indeed, either by activating HSC with the ensuing increase in collagen secretion, and/or by

Conflict of interest

The authors who have taken part in this study declared that they do not have anything to disclose regarding funding or conflict of interest with respect to this manuscript.

Financial support

This work was funded by the Ministerio de Economía y Competitividad, SAF (2013-44723-R) and Fondo Investigaciones Sanitarias (PI14/00029 and PI13/00341), and the European Union (Fondos FEDER, “una manera de hacer Europa”). FC and DMT have a Sheila Sherlock Fellowship from the European Association for the Study of the Liver. JG-S has a Ramón y Cajal contract from the Ministerio de Economía y Competitividad. CIBEREHD is funded by the Instituto de Salud Carlos III.

Authors’ contributions

FC and MV designed the study, performed experiments, analyzed data and wrote the manuscript; HG, DMT, EL and OG-I performed experiments and analyzed data; MA analyzed data and wrote the manuscript; JB conceived ideas, critically reviewed the manuscript and obtained funding; JG-S designed the study, conceived ideas, wrote the manuscript and obtained funding; JCG-P designed the study, conceived ideas, wrote the manuscript, obtained funding and directed the study.

Acknowledgments

Authors are indebted to Montse Monclús and Sergi Vila for their excellent technical assistance.

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