Superoxide dismutase gene transfer reduces portal pressure in CCl4 cirrhotic rats with portal hypertension.

BACKGROUND: Increased intrahepatic vascular tone in cirrhosis has been attributed to a decrease of hepatic nitric oxide (NO) secondary to disturbances in the post-translational regulation of the enzyme eNOS. NO scavenging by superoxide (O(2)(-)) further contributes to a reduction of NO bioavailability in cirrhotic livers. AIM: To investigate whether removing increased O(2)(-) levels could be a new therapeutic strategy to increase intrahepatic NO, improve endothelial dysfunction and reduce portal pressure in cirrhotic rats with portal hypertension. METHODS: Adenoviral vectors expressing extracellular superoxide dismutase (SOD) (AdECSOD) or beta-galactosidase (Adbetagal) were injected intravenously in control and CCl(4)-induced cirrhotic rats. After 3 days, liver O(2)(-) levels were determined by dihydroethidium staining, NO bioavailability by hepatic cGMP levels, nitrotyrosinated proteins by immunohistochemistry and western blot, and endothelial function by responses to acetylcholine in perfused rat livers. Mean arterial pressure (MAP) and portal pressure were evaluated in vivo. RESULTS: Transfection of cirrhotic livers with AdECSOD produced a significant reduction in O(2)(-) levels, a significant increase in hepatic cGMP, and a decrease in liver nitrotyrosinated proteins which were associated with a significant improvement in the endothelium-dependent vasodilatation to acetylcholine. In addition, in cirrhotic livers AdECSOD transfection produced a significant reduction in portal pressure (17.3 (SD 2) mm Hg vs 15 (SD 1.6) mm Hg; p<0.05) without significant changes in MAP. In control rats, AdECSOD transfection prevents the increase in portal perfusion pressure promoted by an ROS-generating system. CONCLUSIONS: In cirrhotic rats, reduction of O(2)(-) by AdECSOD increases NO bioavailability, improves intrahepatic endothelial function and reduces portal pressure. These findings suggest that scavenging of O(2)(-) might be a new therapeutic strategy in the management of portal hypertension.

The protein SET regulates the inhibitory effect of p21(Cip1) on cyclin E-cyclin-dependent kinase 2 activity.

The cyclin-dependent kinase (CDK) inhibitor p21(Cip1) has a dual role in the regulation of the cell cycle; it is an activator of cyclin D1-CDK4 complexes and an inhibitor of cyclins E/A-CDK2 activity. By affinity chromatography with p21(Cip1)-Sepharose 4B columns, we purified a 39-kDa protein, which was identified by microsequence analysis as the oncoprotein SET. Complexes containing SET and p21(Cip1) were detected in vivo by immunoprecipitation of Namalwa cell extracts using specific anti-p21(Cip1) antibodies. We found that SET bound directly to p21(Cip1) in vitro by the carboxyl-terminal region of p21(Cip1). SET had no direct effect on cyclin E/A-CDK2 activity, although it reversed the inhibition of cyclin E-CDK2, but not of cyclin A-CDK2, induced by p21(Cip1). This result is specific for p21(Cip1), since SET neither bound to p27(Kip1) nor reversed its inhibitory effect on cyclin E-CDK2 or cyclin A-CDK2. Thus, SET appears to be a modulator of p21(Cip1) inhibitory function. These results suggest that SET can regulate G(1)/S transition by modulating the activity of cyclin E-CDK2.

Calmodulin binds to p21(Cip1) and is involved in the regulation of its nuclear localization.

p21(Cip1), first described as an inhibitor of cyclin-dependent kinases, has recently been shown to have a function in the formation of cyclin D-Cdk4 complexes and in their nuclear translocation. The dual behavior of p21(Cip1) may be due to its association with other proteins. Different evidence presented here indicate an in vitro and in vivo interaction of p21(Cip1) with calmodulin: 1) purified p21(Cip1) is able to bind to calmodulin-Sepharose in a Ca(2+)-dependent manner, and this binding is inhibited by the calmodulin-binding domain of calmodulin-dependent kinase II; 2) both molecules coimmunoprecipitate when extracted from cellular lysates; and 3) colocalization of calmodulin and p21(Cip1) can be detected in vivo by electron microscopy immunogold analysis. The carboxyl-terminal domain of p21(Cip1) is responsible for the calmodulin interaction, since p21(145-164) peptide is also able to bind calmodulin and to compete with full-length p21(Cip1) for the calmodulin binding. Because treatment of cells with anti-calmodulin drugs decreases the nuclear accumulation of p21(Cip1), we hypothesize that calmodulin interaction with p21(Cip1) is important for p21(Cip1), and in consequence for cyclin D-Cdk4, translocation into the cell nucleus.