Mehta, G;
(2016)
The Molecular Characterisation of Dimethylarginine Dimethylaminohydrolase-1 and Determination of its Role in Portal Hypertension.
Doctoral thesis , UCL (University College London).
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Abstract
Portal hypertension is a grave step in the progression of cirrhosis, associated with complications and mortality. The pathobiology of portal hypertension involves fibrosis as well as increased intrahepatic vascular tone. Nitric oxide (NO), synthesized by endothelial nitric oxide synthase (eNOS), is a key regulator of intrahepatic vascular tone. Asymmetric dimethylarginine (ADMA) is a competitive inhibitor of eNOS, which has been implicated in the pathobiology of portal hypertension – ADMA levels are elevated in cirrhosis and correlate with portal pressure. The major pathway of elimination of ADMA is the enzyme dimethylarginine dimethylaminohydrolase-1 (DDAH-1). The main aims of this thesis are: (i) delineate the expression and regulation of hepatic DDAH-1 in cirrhosis and portal hypertension, and (ii) determine a causal relationship between hepatic DDAH-1 and portal hypertension in cirrhosis. In initial experiments, hepatic DDAH-1 protein expression was significantly decreased in patients with cirrhosis and bile duct-ligated (BDL) rats. Immunohistochemistry demonstrated that DDAH-1 expression was restricted to the hepatocyte. In vitro work with endothelial cells demonstrated that exogenous ADMA is an inhibitor of NO generation at pathophysiological levels. Subsequent experiments demonstrated that DDAH-1 expression was decreased by exposure to hydrogen peroxide (H2O2), and this mechanism was related to the DDAH-1 3’UTR. Further studies demonstrated the presence of predicted microRNA (miR) binding sites in the DDAH-1 3’UTR, and miR-128 was found to be elevated in BDL rat liver compared with sham controls, as well as being a regulator of DDAH-1 protein expression through gain-of-function and loss-of-function experiments. Finally, a novel truncated transcript of DDAH-1 was demonstrated in human placenta. This transcript was found to be protein-encoding, with bioinformatic evidence of a proximal promoter. Thus, a switch in transcript may play a role in placental vascular disorders such as pre-eclampsia. In summary, hepatic DDAH-1 is reduced in cirrhosis, and is causally related to the development of portal hypertension. DDAH-1 undergoes post-transcriptional regulation through microRNA regulation and alternative transcription.
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