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Non-alcoholic fatty liver disease (NAFLD) and its complications are an expanding health problem associated with the metabolic syndrome. Liver sinusoidal endothelial cells (LSECs) are highly specialized endothelial cells localized at the interface between the blood derived from the gut and the adipose tissue on the one side, and other liver cells on the other side. In physiological conditions, LSECs are gatekeepers of liver homeostasis. LSECs display anti-inflammatory and anti-fibrogenic properties by preventing Kupffer cell and hepatic stellate cell activation and regulating intrahepatic vascular resistance and portal pressure. This review focusses on changes occurring in LSECs in NAFLD and on their consequences on NAFLD progression and complications. Capillarization, namely the loss of LSEC fenestrae, and LSEC dysfunction, namely the loss of the ability of LSECs to generate vasodilator agents in response to increased shear stress both occur early in NAFLD. These LSEC changes favour steatosis development and set the stage for NAFLD progression. At the stage of non-alcoholic steatohepatitis, altered LSECs release inflammatory mediators and contribute to the recruitment of inflammatory cells, thus promoting liver injury and inflammation. Altered LSECs also fail to maintain hepatic stellate cell quiescence and release fibrogenic mediators, including Hedgehog signalling molecules, promoting liver fibrosis. Liver angiogenesis is increased in NAFLD and contributes to liver inflammation and fibrosis, but also to hepatocellular carcinoma development. Thus, improving LSEC health appears to be a promising approach to prevent NAFLD progression and complications.

Background Nonalcoholic fatty liver disease (NAFLD) is the leading cause of chronic liver disease, which is associated with features of metabolic syndrome. NAFLD may progress in a subset of patients into nonalcoholic steatohepatitis (NASH) with liver injury resulting ultimately in cirrhosis and potentially hepatocellular carcinoma. Today, there is no approved treatment for NASH due to, at least in part, the lack of preclinical models recapitulating features of human disease. Here, we report the development of a dietary model of NASH in the Gottingen minipig. Methods First, we performed a longitudinal characterization of diet-induced NASH and fibrosis using biochemical, histological, and transcriptional analyses. We then evaluated the pharmacological response to Obeticholic acid (OCA) treatment for 8 weeks at 2.5mg/kg/d, a dose matching its active clinical exposure. Results Serial histological examinations revealed a rapid installation of NASH driven by massive steatosis and inflammation, including evidence of ballooning. Furthermore, we found the progressive development of both perisinusoidal and portal fibrosis reaching fibrotic septa after 6 months of diet. Histological changes were mechanistically supported by well-defined gene signatures identified by RNA Seq analysis. While treatment with OCA was well tolerated throughout the study, it did not improve liver dysfunction nor NASH progression. By contrast, OCA treatment resulted in a significant reduction in diet-induced fibrosis in this model. Conclusions These results, taken together, indicate that the diet-induced NASH in the Gottingen minipig recapitulates most of the features of human NASH and may be a model with improved translational value to prioritize drug candidates toward clinical development

Rationale: Inflammation is a key contributor to atherosclerosis. MicroRNA-146a (miR-146a) has been identified as a critical brake on proinflammatory nuclear factor κ light chain enhancer of activated B cells signaling in several cell types, including endothelial cells and bone marrow (BM)–derived cells. Importantly, miR-146a expression is elevated in human atherosclerotic plaques, and polymorphisms in the miR-146a precursor have been associated with risk of coronary artery disease. Objective: To define the role of endogenous miR-146a during atherogenesis. Methods and Results: Paradoxically, Ldlr −/− (low-density lipoprotein receptor null) mice deficient in miR-146a develop less atherosclerosis, despite having highly elevated levels of circulating proinflammatory cytokines. In contrast, cytokine levels are normalized in Ldlr −/− ;miR-146a −/− mice receiving wild-type BM transplantation, and these mice have enhanced endothelial cell activation and elevated atherosclerotic plaque burden compared with Ldlr −/− mice receiving wild-type BM, demonstrating the atheroprotective role of miR-146a in the endothelium. We find that deficiency of miR-146a in BM-derived cells precipitates defects in hematopoietic stem cell function, contributing to extramedullary hematopoiesis, splenomegaly, BM failure, and decreased levels of circulating proatherogenic cells in mice fed an atherogenic diet. These hematopoietic phenotypes seem to be driven by unrestrained inflammatory signaling that leads to the expansion and eventual exhaustion of hematopoietic cells, and this occurs in the face of lower levels of circulating low-density lipoprotein cholesterol in mice lacking miR-146a in BM-derived cells. Furthermore, we identify sortilin-1( Sort1 ), a known regulator of circulating low-density lipoprotein levels in humans, as a novel target of miR-146a. Conclusions: Our study reveals that miR-146a regulates cholesterol metabolism and tempers chronic inflammatory responses to atherogenic diet by restraining proinflammatory signaling in endothelial cells and BM-derived cells.

Background & Aims Previous studies demonstrated that autophagy is protective in hepatocytes and macrophages, but detrimental in hepatic stellate cells in chronic liver diseases. The role of autophagy in liver sinusoidal endothelial cells (LSECs) in non-alcoholic steatohepatitis (NASH) is unknown. Our aim was to analyze the potential implication of autophagy in LSECs in NASH and liver fibrosis. Methods We analyzed autophagy in LSECs from patients using transmission electron microscopy. We determined the consequences of a deficiency in autophagy: (a) on LSEC phenotype, using primary LSECs and an LSEC line; (b) on early stages of NASH and on advanced stages of liver fibrosis, using transgenic mice deficient in autophagy specifically in endothelial cells and fed a high-fat diet or chronically treated with carbon tetrachloride, respectively. Results Patients with NASH had half as many LSECs containing autophagic vacuoles as patients without liver histological abnormalities, or with simple steatosis. LSECs from mice deficient in endothelial autophagy displayed an upregulation of genes implicated in inflammatory pathways. In the LSEC line, deficiency in autophagy enhanced inflammation (Ccl2, Ccl5, Il6 and VCAM-1 expression), features of endothelial-to-mesenchymal transition (α-Sma, Tgfb1, Col1a2 expression) and apoptosis (cleaved caspase-3). In mice fed a high-fat diet, deficiency in endothelial autophagy induced liver expression of inflammatory markers (Ccl2, Ccl5, Cd68, Vcam-1), liver cell apoptosis (cleaved caspase-3) and perisinusoidal fibrosis. Mice deficient in endothelial autophagy treated with carbon tetrachloride also developed more perisinusoidal fibrosis. Conclusions A defect in autophagy in LSECs occurs in patients with NASH. Deficiency in endothelial autophagy promotes the development of liver inflammation, features of endothelial-to-mesenchymal transition, apoptosis and liver fibrosis in the early stages of NASH, but also favors more advanced stages of liver fibrosis. Lay summary Autophagy is a physiological process controlling endothelial homeostasis in vascular beds outside the liver. This study demonstrates that autophagy is defective in the liver endothelial cells of patients with non-alcoholic steatohepatitis. This defect promotes liver inflammation and fibrosis at early stages of non-alcoholic steatohepatitis, but also at advanced stages of chronic liver disease.

How endothelial cells sense and react to flow during vascular remodeling is poorly understood. Vion et al. show that endothelial cells utilize their primary cilia to stabilize vessel connections during vascular remodeling. Molecularly, they identify enhanced sensitivity to BMP9 in ciliated endothelial cells, selectively under low flow., Blood flow shapes vascular networks by orchestrating endothelial cell behavior and function. How endothelial cells read and interpret flow-derived signals is poorly understood. Here, we show that endothelial cells in the developing mouse retina form and use luminal primary cilia to stabilize vessel connections selectively in parts of the remodeling vascular plexus experiencing low and intermediate shear stress. Inducible genetic deletion of the essential cilia component intraflagellar transport protein 88 (IFT88) in endothelial cells caused premature and random vessel regression without affecting proliferation, cell cycle progression, or apoptosis. IFT88 mutant cells lacking primary cilia displayed reduced polarization against blood flow, selectively at low and intermediate flow levels, and have a stronger migratory behavior. Molecularly, we identify that primary cilia endow endothelial cells with strongly enhanced sensitivity to bone morphogenic protein 9 (BMP9), selectively under low flow. We propose that BMP9 signaling cooperates with the primary cilia at low flow to keep immature vessels open before high shear stress–mediated remodeling., Graphical Abstract

International audience; It has been known for some time that atherosclerotic lesions preferentially develop in areas exposed to low SS and are characterized by a proinflammatory, apoptotic, and senescent endothelial phenotype. Conversely, areas exposed to high SS are protected from plaque development, but the mechanisms have remained elusive. Autophagy is a protective mechanism that allows recycling of defective organelles and proteins to maintain cellular homeostasis. We aimed to understand the role of endothelial autophagy in the atheroprotective effect of high SS. Atheroprotective high SS stimulated endothelial autophagic flux in human and murine arteries. On the contrary, endothelial cells exposed to atheroprone low SS were characterized by inefficient autophagy as a result of mammalian target of rapamycin (mTOR) activation, AMPKα inhibition, and blockade of the autophagic flux. In hypercholesterolemic mice, deficiency in endothelial autophagy increased plaque burden only in the atheroresistant areas exposed to high SS; plaque size was unchanged in atheroprone areas, in which endothelial autophagy flux is already blocked. In cultured cells and in transgenic mice, deficiency in endothelial autophagy was characterized by defects in endothelial alignment with flow direction, a hallmark of endothelial cell health. This effect was associated with an increase in endothelial apoptosis and senescence in high-SS regions. Deficiency in endothelial autophagy also increased TNF-α-induced inflammation under high-SS conditions and decreased expression of the antiinflammatory factor KLF-2. Altogether, these results show that adequate endothelial autophagic flux under high SS limits atherosclerotic plaque formation by preventing endothelial apoptosis, senescence, and inflammation.

Objective: Preferential development of atherosclerotic lesions in areas of low shear stress is associated with increased endothelial inflammation, apoptosis and senescence. On the contrary, high shear stress areas are protected from plaque development, but the mechanisms remain elusive. Autophagy is a protective mechanism allowing recycling of defective organelles and proteins to maintain cellular homeostasis. Our aim was to understand the role of autophagy in athero-protective effects of high shear stress. Approach and Results: We used the parallel plate chamber system in vitro to generate different shear stress conditions on endothelial cell deficient or not in autophagy (shRNA Atg5 vs. control) and examined autophagic flux in cells transfected with RFP-GFP-LC3 plasmid. Endothelial autophagy was silenced in Atg5 flox/flox ; VE-cadherin-cre mice. We first demonstrated in human and murine arteries and in cultured endothelial cells that atheroprotective shear stress activates endothelial autophagic flux. On the opposite, endothelial cells exposed to atheroprone low shear stress displayed inefficient autophagy, associated with activation of mTOR, inhibition of AMPKα pathways and blockade of the autophagic flux. Interestingly, plaque burden augmented specifically in areas usually atheroresistant in ApoE -/- hypercholesterolemic mice deficiency in endothelial autophagy. Both in cultured endothelial cells and in transgenic mice, deficiency in endothelial autophagy was associated with a defect in endothelial alignment with flow direction, a hallmark of endothelial cell health. Deficiency in endothelial autophagy increased endothelial senescence and TNFα-induced inflammation under high shear stress conditions. These effects were associated with impaired KLF2 activation. In transgenic mice, endothelial senescence and apoptosis was augmented in high shear stress areas of the descending thoracic aorta when compared to control littermate mice. Conclusions: Altogether, these results show that adequate endothelial autophagic flux under high shear stress limits atherosclerotic plaque formation by preventing inflammation, senescence and apoptosis.

International audience; Blood flowing in arteries generates shear forces at the surface of the vascular endothelium that control its anti-atherogenic properties. However, due to the architecture of the vascular tree, these shear forces are heterogeneous and atherosclerotic plaques develop preferentially in areas where shear is low or disturbed. Here we review our recent study showing that elevated shear forces stimulate endothelial autophagic flux and that inactivating the endothelial macroautophagy/autophagy pathway promotes a proinflammatory, prosenescent and proapoptotic cell phenotype despite the presence of atheroprotective shear forces. Specific deficiency in endothelial autophagy in a murine model of atherosclerosis stimulates the development of atherosclerotic lesions exclusively in areas of the vasculature that are normally resistant to atherosclerosis. Our findings demonstrate that adequate endothelial autophagic flux limits atherosclerotic plaque formation by preventing endothelial apoptosis, senescence and inflammation.

Atherosclerotic plaques form preferentially in arterial areas exposed to low shear stress (LSS) where endothelial cells express senescence-associated phenotypes. We tested the hypothesis that endothelial autophagy is an anti-senescence and anti-atherogenic process regulated by shear stress. Endothelial cells exposure to LSS (2 dyn/cm2, 24h) decreased LC3II/I ratio compared to high shear stress (HSS, 20 dyn/cm2) (1.6±0.2 vs. 2.0±0.3, respectively p