deFective Autophagie in Sinusoidal endothelial Cells In Non Alcoholic sTeatohEpatitis Development – FASCINATED

This project is divided into 3 tasks.
TASK 1: REGULATION OF LIVER SINUSOIDAL ENDOTHELIAL CELL AUTOPHAGY IN NASH

TASK 2: ROLE OF ENDOTHELIAL AUTOPHAGY IN NASH DEVELOPMENT

TASK 3: CONSEQUENCES OF THE DEFECT IN AUTOPHAGY IN LIVER SINUSOIDAL ENDOTHELIAL CELLS ON THE MECHANISMS OF NASH DEVELOPMENT

1. We analyzed by electron microscopy autophagic vacuoles in LSEC in patients with NASH, bland steatosis and in controls with abnormal liver blood tests but no or mild abnormalities at liver histological analysis. We observed less autophagic vacuoles in LSECs from patients with NASH than in controls

2. We observed that TNFa and IL6 reduced autophagy levels in TSECs exposed to shear stress while glucose and insulin do not

3. Development of mouse models of NASH :
We identified mice with an efficient recombination in LSECs :
- constitutive Cdh5-Cre associated with bone marrow transplantation and clodronate injection.
- inductible recombination in Cdh5(PAC)-CreERT2 (but not Cdh5-CreERT2 mice)

4. Identification of the mechanisms responsible for autophagy deficiency in NASH :
We observed that TSECs express the primary cilium

We are breeding mice to study the role of LSEC autophagy in NASH. We will use 2 complementary mouse models of NASH (high fat diet or MCD), of deficiency in autophagy (Atg5flox/flox or Atg7flox/flox) and of endothelial recombination (VE-Cadherin-cre or Cdh5(PAC)-CreERT2)

Poisson J, Lemoinne S, Boulanger CM, Durand F, Moreau R, Valla D, Rautou PE. Liver sinusoidal endothelial cells: physiology and role in liver disease. J Hepatol 2016; in press

In the past decade, an epidemic increase in Non-Alcoholic Fatty Liver Diseases (NAFLD) prevalence has been observed. NAFLD is closely associated with the metabolic syndrome, defined by a constellation of features including central obesity, diabetes mellitus, raised blood pressure, increased triglyceride levels and decreased high-density lipoprotein-cholesterol. The prevalence of NAFLD increases with age. Nowadays, in western countries, NAFLD is among the most common causes of chronic liver disease with a prevalence ranging between 17 and 46%. NAFLD encompasses a spectrum of liver injuries associated with liver fat accumulation in the absence of significant alcohol consumption or viral exposure. Nonalcoholic steatohepatitis (NASH) is a part of the NAFLD spectrum. In addition to a marked presence of excess fat in the liver (steatosis), NASH is characterized by variable liver inflammation with hepatocyte injury (ballooning) with or without scarring (fibrosis). NASH is an important health problem. Indeed, this disease affects 5% of the general population and will potentially be the most common cause of advanced liver disease in coming decades. Moreover, contrary to bland steatosis, NASH is a serious disease since it can progress to cirrhosis, liver failure, and liver cancer and may require transplantation. NASH patients have a higher risk of death compared with the general population and with patients with bland steatosis. Importantly, efficacious therapeutic agents for the treatment of NASH are lacking.

Although, some recent data have shown microvascular abnormalities and liver endothelial dysfunction in animal models of fatty liver, as well as a possible role of angiogenesis in the pathophysiology of NASH, the precise role of liver sinusoidal endothelial cells (LSEC) in NASH remains to be determined.

Autophagy is a general term for processes by which cytoplasmic materials reach lysosomes for degradation. Interestingly, aging is a cause of decreased autophagy. If it has been shown that hepatocyte autophagy plays a key role in the metabolic syndrome, the role of endothelial autophagy in this syndrome, and in particular in NASH, has not been investigated to date.

We hypothesize that a defect in autophagy in LSEC occurs in NASH and contributes to the development of this disease.
We obtained preliminary results supporting this hypothesis. Indeed, we observed that patients with NASH have a lower number of LSEC containing autophagic vacuoles, suggesting a defect in autophagy in their LSEC. Moreover, we generated mice deficient in endothelial autophagy and fed these mice a diet inducing NASH features. We observed that mice deficient in endothelial autophagy have much more liver fibrosis, more liver inflammation and features suggesting capillarization of the sinusoids.

This proposal includes 3 specific aims.
(1) To determine the level of autophagy in LSEC in the liver of patients with NASH as compared to patients with bland steatosis and to controls, and to highlight the intracellular mechanisms regulating autophagy level in LSEC in NASH.
(2) To establish in vivo whether or not endothelial autophagy plays a role in NASH development. Two complementary mouse models of NASH (high fat diet in wild type and in ApoE-/- mice) and of deficiency in endothelial autophagy (Atg5flox/flox ;VE-Cadherin-cre); Atg7flox/flox ;VE-Cadherin-cre) will be used.
(3) To highlight the consequences of the defect in autophagy in LSEC on the mechanisms of NASH development. In particular, we will assess in vitro and in the liver of mice deficient in endothelial autophagy and fed a high fat diet LSEC apoptosis, activation of hepatic stellate cells, angiogenesis, changes in endothelial phenotype (capillarization) and endocytotic capacity and endothelial dysfunction.

The Principal Investigator has extensive expertise in vascular biology, autophagy and in liver diseases. Several preliminary results have been obtained attesting the feasibility of this project.