An Experimental Study Of Akt Gene Transfer To Cirrhotic Ratic Liver Ameliorates Portal Hypertension

Background and Objective: Increased intrahepatic vascular resistance to portal blood flow is the primary factor in the pathophysiology of portal hypertension and is the main cause of morbidity and mortality of patients with cirrhosis. This increased resistance is the consequence of anatomic abnormalities caused by fibrosis and the formation of regenerating nodules in cirrhotic liver. It is well established that this is not a purely mechanical phenomenon and different vasoactive agents such as nitric oxide(NO) play an important role in the development of the intrahepatic resistance associated with cirrhosis and portal hypertension. In this regard, there are studies showing that NO production and endothelial nitric oxide synthase(eNOS) protein activity are decreased in cirrhotic ratic liver, which is deteriorates intrahepatic vascular resistance. There are experimental evidences showing that serine/threonine kinase Akt is a major activator of the eNOS enzyme, but its potential role in the setting of cirrhosis and portal hypertension is unknown. For this reason the aims of the present study were to determine whether there is an impaired Akt activation in cirrhotic livers and how this phenomenon relates to the decrease in NO production associated with portal hypertension.Methods: Cirrhosis was induced in rats by a complex method of injection carbon tetrachloride(CCl4). Replication-defective recombinant adenovirus (Ad-myr-Akt) was produced by homologous recombination with AdMax in the 293 cells. The roles of protein kinase Akt in cirrhotic ratic livers were investigated through infection with adenoviruses encoding either constitutively active Akt (myr-Akt) or enhanced green fluorescent protein (EGFP). Protein abundance and phosphorylation status of Akt and eNOS were identified by Western blot. The NO levels were measured by enzymatically reducing nitrate to nitrite using nitrate reductase. The maximal flow speed and interior diameter of portal vein, portal pressure, mean arterial pressure , and heart rate were measured in all rats. The hepatic histology was evaluated by H&E staining in all rats. Albumin, alanine transaminase and aspartic transaminase levels were measured by the LX20 automatic analyzer. The EGFP expression was examined by fluorescence microscope.Results:1,Cirrhosis modal animals were successfully induced by a complex method of injection CCl₄ in 64 adult male Wistar rats, and adenovirus carrying target gene myr-Akt with 5.5×10¹¹ vp/ml dose was successfully constructed in our experimental study.2,The serine/threonine kinase Akt phosphorylation is reduced significantly in the liver homogenate of cirrhotic rats in comparison with control animals; Moreover, the eNOS phosphorylation also is diminished significantly in cirrhotic ratic livers as shown in the Akt enzyme. No changes were observed in the amount of Akt or eNOS protein levels between either control or cirrhotic animals.3,Adenoviral delivery of myr-Akt restored eNOS phosphorylation and increased the intrahepatic NO concentration. Moreover, normalization in portal pressure and improvement in portal hemodynamics after 3 days of adenoviral infection in comparison with EGFP-transduced cirrhotic rats. In addition, animals infected with EGFP adenovirus did not experience any change in these parameters, as compared with noninfected(injected with physiological saline) cirrhotic rats.4,Intravenous administration of adenoviral vectors encoding myr-Akt gene preferentially target the liver and effectively express in it. Moreover, this effect can be observed within 30 days after adenoviral infection.5,We failed to detect significant increase in albumin, alanine transaminase and aspartic transaminase levels with either EGFP or myr-Akt adenovirus in cirrhotic rats. Adenoviral vectors(Ad5) result in minimal liver damage in normal rats as shown in transaminase elevated transitorily. After an adenoviral delivery of myr-Akt gene, 30 days survival was improved in the cirrhotic rats induced by prolonged CCl₄.Conclusion: There is an impaired Akt activation in cirrhotic ratic livers, and the intravenous administration of myr-Akt adenovirus to cirrhotic rats resulted in transgene expression, increased eNOS phosphorylation, and enhanced intrahepatic release of NO. Furthermore, restoring eNOS activity by myr-Akt gene delivery normalizes portal pressure, and improves portal hemodynamics in cirrhotic rats after 3 days of adenoviral infection. Collectively, these data suggest that Akt gene transduction may be useful for patients with cirrhosis and portal hypertension in the future.