Rescue of glutaric aciduria type I in mice by liver-directed therapies.

Glutaric aciduria type I (GA-1) is an inborn error of metabolism with a severe neurological phenotype caused by the deficiency of glutaryl-coenzyme A dehydrogenase (GCDH), the last enzyme of lysine catabolism. Current literature suggests that toxic catabolites in the brain are produced locally and do not cross the blood-brain barrier. In a series of experiments using knockout mice of the lysine catabolic pathway and liver cell transplantation, we uncovered that toxic GA-1 catabolites in the brain originated from the liver. Moreover, the characteristic brain and lethal phenotype of the GA-1 mouse model was rescued by two different liver-directed gene therapy approaches: Using an adeno-associated virus, we replaced the defective Gcdh gene or we prevented flux through the lysine degradation pathway by CRISPR deletion of the aminoadipate-semialdehyde synthase (Aass) gene. Our findings question the current pathophysiological understanding of GA-1 and reveal a targeted therapy for this devastating disorder.

Dysregulation of the ESRP2-NF2-YAP/TAZ axis promotes hepatobiliary carcinogenesis in non-alcoholic fatty liver disease.

BACKGROUND & AIMS: Non-alcoholic fatty liver disease (NAFLD), the hepatic correlate of the metabolic syndrome, is a major risk factor for hepatobiliary cancer (HBC). Although chronic inflammation is thought to be the root cause of all these diseases, the mechanism whereby it promotes HBC in NAFLD remains poorly understood. Herein, we aim to evaluate the hypothesis that inflammation-related dysregulation of the ESRP2-NF2-YAP/TAZ axis promotes HB carcinogenesis. METHODS: We use murine NAFLD models, liver biopsies from patients with NAFLD, human liver cancer registry data, and studies in liver cancer cell lines. RESULTS: Our results confirm the hypothesis that inflammation-related dysregulation of the ESRP2-NF2-YAP/TAZ axis promotes HB carcinogenesis, supporting a model whereby chronic inflammation suppresses hepatocyte expression of ESRP2, an RNA splicing factor that directly targets and activates NF2, a tumor suppressor that is necessary to constrain YAP/TAZ activation. The resultant loss of NF2 function permits sustained YAP/TAZ activity that drives hepatocyte proliferation and de-differentiation. CONCLUSION: Herein, we report on a novel mechanism by which chronic inflammation leads to sustained activation of YAP/TAZ activity; this imposes a selection pressure that favors liver cells with mutations enabling survival during chronic oncogenic stress. LAY SUMMARY: Non-alcoholic fatty liver disease (NAFLD) increases the risk of hepatobiliary carcinogenesis. However, the underlying mechanism remains unknown. Our study demonstrates that chronic inflammation suppresses hepatocyte expression of ESRP2, an adult RNA splicing factor that activates NF2. Thus, inactive (fetal) NF2 loses the ability to activate Hippo kinases, leading to the increased activity of downstream YAP/TAZ and promoting hepatobiliary carcinogenesis in chronically injured livers.

Pathophysiology and a Rational Basis of Therapy.

Portal hypertension is a common complication of chronic liver disease. Its relevance comes from the fact that it determines most complications leading to death or liver transplantation in patients with cirrhosis of the liver: bleeding from esophageal or gastric varices, ascites and renal dysfunction, sepsis and hepatic encephalopathy. Portal hypertension results from increased resistance to portal blood flow through the cirrhotic liver. This is caused by two mechanisms: (1) distortion of the liver vascular architecture due to the liver disease causing structural abnormalities (nodule formation, remodeling of liver sinusoids, fibrosis, angiogenesis and vascular occlusion), and (2) increased hepatic vascular tone due to sinusoidal endothelial dysfunction, which results in a defective production of endogenous vasodilators, mainly nitric oxide (NO), and increased production of vasoconstrictors (thromboxane A2, cysteinyl leukotrienes, angiotensin II, endothelins and an activated adrenergic system). Hepatic endothelial dysfunction occurs early in the course of chronic liver disease as a consequence of inflammation and oxidative stress, and determines loss of the normal phenotype of liver sinusoidal endothelial cells (LSECs) that become proliferative, prothrombotic, proinflammatory and vasoconstrictor. The cross-talk between LSECs and hepatic stellate cells (HSCs) induces activation of the latter, which in turn proliferate, migrate and increase collagen deposition around the sinusoids, contributing to fibrogenesis, architectural disruption and angiogenesis, which further increase the hepatic vascular resistance and worsen liver failure by interfering with the blood perfusion of the liver parenchyma. An additional factor further worsening portal hypertension is an increased blood flow through the portal system due to splanchnic vasodilatation. This is an adaptive response to decreased effective hepatocyte perfusion, and is maximal once portal pressure has increased sufficiently to promote the development of intrahepatic shunts and portal-systemic collaterals, including varices, through which portal blood flow bypasses the liver. In human portal hypertension collateralization and hyperdynamic circulation start at a portal pressure gradient >10 mm Hg. Rational therapy for portal hypertension aims at correcting these pathophysiological abnormalities: liver injury, fibrogenesis, increased hepatic vascular tone and splanchnic vasodilatation. Continuing liver injury may be counteracted specifically by etiological treatments (the best example being the direct-acting antivirals for hepatitis C viral infection), while architectural disruption and fibrosis can be ameliorated by a variety of antifibrotic drugs and antiangiogenic strategies. Several drugs in this category are currently under investigation in phase II-III randomized controlled trials. Sinusoidal endothelial dysfunction is ameliorated by statins as well as by other drugs increasing NO availability. It is of note that simvastatin has already been proven to be clinically effective in two randomized controlled trials. Splanchnic hyperemia can be counteracted by nonselective ß-blockers (NSBBs), vasopressin analogs and somatostatin analogs, drugs that until recently were the only available treatments for portal hypertension, but that are not very effective in the initial stages of cirrhosis. There is experimental and clinical evidence indicating that a more effective reduction of portal pressure is obtained by combining agents acting on these different pathways. It is likely that the treatment of portal hypertension will evolve to use etiological treatments together with antifibrotic agents and/or drugs improving sinusoidal endothelial function in the initial stages of cirrhosis (preprimary prophylaxis), while NSBBs will be added in advanced stages of the disease.

KLF2 exerts antifibrotic and vasoprotective effects in cirrhotic rat livers: behind the molecular mechanisms of statins.

OBJECTIVE: In the liver, the transcription factor, Kruppel-like factor 2 (KLF2), is induced early during progression of cirrhosis to lessen the development of vascular dysfunction; nevertheless, its endogenous expression results insufficient to attenuate establishment of portal hypertension and aggravation of cirrhosis. Herein, we aimed to explore the effects and the underlying mechanisms of hepatic KLF2 overexpression in in vitro and in vivo models of liver cirrhosis. DESIGN: Activation phenotype was evaluated in human and rat cirrhotic hepatic stellate cells (HSC) treated with the pharmacological inductor of KLF2 simvastatin, with adenovirus codifying for this transcription factor (Ad-KLF2), or vehicle, in presence/absence of inhibitors of KLF2. Possible paracrine interactions between parenchymal and non-parenchymal cells overexpressing KLF2 were studied. Effects of in vivo hepatic KLF2 overexpression on liver fibrosis and systemic and hepatic haemodynamics were assessed in cirrhotic rats. RESULTS: KLF2 upregulation profoundly ameliorated HSC phenotype (reduced α-smooth muscle actin, procollagen I and oxidative stress) partly via the activation of the nuclear factor (NF)-E2-related factor 2 (Nrf2). Coculture experiments showed that improvement in HSC phenotype paracrinally ameliorated liver sinusoidal endothelial cells probably through a vascular endothelial growth factor-mediated mechanism. No paracrine interactions between hepatocytes and HSC were observed. Cirrhotic rats treated with simvastatin or Ad-KLF2 showed hepatic upregulation in the KLF2-Nrf2 pathway, deactivation of HSC and prominent reduction in liver fibrosis. Hepatic KLF2 overexpression was associated with lower portal pressure (-15%) due to both attenuations in the increased portal blood flow and hepatic vascular resistance, together with a significant improvement in hepatic endothelial dysfunction. CONCLUSIONS: Exogenous hepatic KLF2 upregulation improves liver fibrosis, endothelial dysfunction and portal hypertension in cirrhosis.