Visco-Elastic Parameters at Three-Dimensional MR Elastography for Diagnosing Non-Alcoholic Steatohepatitis and Substantial Fibrosis in Mice.

BACKGROUND: Nonalcoholic fatty liver disease (NAFLD) is increasing worldwide and is a growing cause of liver cirrhosis and cancer. The performance of the magnetic resonance elastography (MRE) visco-elastic parameters in diagnosing progressive forms of NAFLD, including nonalcoholic steatohepatitis (NASH) and substantial fibrosis (F ≥ 2), needs to be clarified. PURPOSE: To assess the value of three-dimensional MRE visco-elastic parameters as markers of NASH and substantial fibrosis in mice with NAFLD. STUDY TYPE: Prospective. ANIMAL MODEL: Two mouse models of NAFLD were induced by feeding with high fat diet or high fat, choline-deficient, amino acid-defined diet. FIELD STRENGTH/SEQUENCE: 7T/multi-slice multi-echo spin-echo MRE at 400 Hz with motion encoding in the three spatial directions. ASSESSMENT: Hepatic storage and loss moduli were calculated. Histological analysis was based on the NASH Clinical Research Network criteria. STATISTICAL TESTS: Mann-Whitney, Kruskal-Wallis tests, Spearman rank correlations and multiple regressions were used. Diagnostic performance was assessed with areas under the receiver operating characteristic curves (AUCs). P value <0.05 was considered significant. RESULTS: Among the 59 mice with NAFLD, 21 had NASH and 20 had substantial fibrosis (including 8 mice without and 12 mice with NASH). The storage and loss moduli had similar moderate accuracy for diagnosing NASH with AUCs of 0.67 and 0.66, respectively. For diagnosing substantial fibrosis, the AUC of the storage modulus was 0.73 and the AUC of the loss modulus was 0.81, indicating good diagnostic performance. Using Spearman correlations, histological fibrosis, inflammation and steatosis, but not ballooning, were significantly correlated with the visco-elastic parameters. Using multiple regression, fibrosis was the only histological feature independently associated with the visco-elastic parameters. CONCLUSION: MRE in mice with NAFLD suggests that the storage and loss moduli have good diagnostic performance for detecting progressive NAFLD defined as substantial fibrosis rather than NASH. EVIDENCE LEVEL: 1 TECHNICAL EFFICACY STAGE: 2.

A new NRF2 activator for the treatment of human metabolic dysfunction-associated fatty liver disease.

Background & Aims: Oxidative stress triggers metabolic-associated fatty liver disease (MAFLD) and fibrosis. Previous animal studies demonstrated that the transcription factor nuclear factor (erythroid-derived 2)-like 2 (NRF2), the master regulator of antioxidant response, protects against MAFLD and fibrosis. S217879, a next generation NRF2 activator has been recently shown to trigger diet-induced steatohepatitis resolution and to reduce established fibrosis in rodents. Our aim was to evaluate the therapeutic potential of S217879 in human MAFLD and its underlying mechanisms using the relevant experimental 3D model of patient-derived precision cut liver slices (PCLS). Methods: We treated PCLS from 12 patients with varying stages of MAFLD with S217879 or elafibranor (peroxisome proliferator-activated receptor [PPAR]α/δ agonist used as a referent molecule) for 2 days. Safety and efficacy profiles, steatosis, liver injury, inflammation, and fibrosis were assessed as well as mechanisms involved in MAFLD pathophysiology, namely antioxidant response, autophagy, and endoplasmic reticulum-stress. Results: Neither elafibranor nor S217879 had toxic effects at the tested concentrations on human PCLS with MAFLD. PPARα/δ and NRF2 target genes (pyruvate dehydrogenase kinase 4 [PDK4], fibroblast growth factor 21 [FGF21], and NAD(P)H quinone dehydrogenase 1 [NQO1], heme oxygenase 1 [HMOX1], respectively) were strongly upregulated in PCLS in response to elafibranor and S217879, respectively. Compared with untreated PCLS, elafibranor and S217879-treated slices displayed lower triglycerides and reduced inflammation (IL-1ß, IL-6, chemokine (C-C motif) ligand 2 [CCL2]). Additional inflammatory markers (chemokine (C-C motif) ligand 5 [CCL5], stimulator of interferon genes [STING], intercellular adhesion molecule-1 [ICAM-1], vascular cell adhesion molecule-1 [VCAM-1]) were downregulated by S217879. S217879 but not elafibranor lowered DNA damage (phospho-Histone H2A.X [p-H2A.X], RAD51, X-ray repair cross complementing 1 [XRCC1]) and apoptosis (cleaved caspase-3), and inhibited fibrogenesis markers expression (alpha smooth muscle actin [α-SMA], collagen 1 alpha 1 [COL1A1], collagen 1 alpha 2 [COL1A2]). Such effects were mediated through an improvement of lipid metabolism, activated antioxidant response and enhanced autophagy, without effect on endoplasmic reticulum-stress. Conclusions: This study highlights the therapeutic potential of a new NRF2 activator for MAFLD using patient-derived PCLS supporting the evaluation of NRF2 activating strategies in clinical trials. Impact and implications: Oxidative stress is a major driver of metabolic-associated fatty liver disease (MAFLD) development and progression. Nuclear factor (erythroid-derived 2)-like 2, the master regulator of the antioxidative stress response, is an attractive therapeutic target for the treatment of MAFLD. This study demonstrates that S217879, a new potent and selective nuclear factor (erythroid-derived 2)-like 2 activator, displays antisteatotic effects, lowers DNA damage, apoptosis, and inflammation and inhibits fibrogenesis in human PCLS in patients with MAFLD.

Endothelial autophagy is not required for liver regeneration after partial hepatectomy in mice with fatty liver.

BACKGROUND & AIMS: Patients with non-alcoholic fatty liver disease (NAFLD) have impaired liver regeneration. Liver endothelial cells play a key role in liver regeneration. In non-alcoholic steatohepatitis (NASH), liver endothelial cells display a defect in autophagy, contributing to NASH progression. We aimed to determine the role of endothelial autophagy in liver regeneration following liver resection in NAFLD. METHODS: First, we assessed autophagy in primary endothelial cells from wild type mice fed a high fat diet and subjected to partial hepatectomy. Then, we assessed liver regeneration after partial hepatectomy in mice deficient (Atg5lox/lox ;VE-cadherin-Cre+ ) or not (Atg5lox/lox ) in endothelial autophagy and fed a high fat diet. The role of endothelial autophagy in liver regeneration was also assessed in ApoE-/- hypercholesterolemic mice and in mice with NASH induced by methionine- and choline-deficient diet. RESULTS: First, autophagy (LC3II/protein) was strongly increased in liver endothelial cells following hepatectomy. Then, we observed at 40 and 48 h and at 7 days after partial hepatectomy, that Atg5lox/lox ;VE-cadherin-Cre+ mice fed a high fat diet had similar liver weight, plasma AST, ALT and albumin concentration, and liver protein expression of proliferation (PCNA), cell-cycle (Cyclin D1, BrdU incorporation, phospho-Histone H3) and apoptosis markers (cleaved Caspase-3) as Atg5lox/lox mice fed a high fat diet. Same results were obtained in ApoE-/- and methionine- and choline-deficient diet fed mice, 40 h after hepatectomy. CONCLUSION: These results demonstrate that the defect in endothelial autophagy occurring in NASH does not account for the impaired liver regeneration occurring in this setting.

Monoacylglycerol lipase reprograms hepatocytes and macrophages to promote liver regeneration.

Background & Aims: Liver regeneration is a repair process in which metabolic reprogramming of parenchymal and inflammatory cells plays a major role. Monoacylglycerol lipase (MAGL) is an ubiquitous enzyme at the crossroad between lipid metabolism and inflammation. It converts monoacylglycerols into free fatty acids and metabolises 2-arachidonoylglycerol into arachidonic acid, being thus the major source of pro-inflammatory prostaglandins in the liver. In this study, we investigated the role of MAGL in liver regeneration. Methods: Hepatocyte proliferation was studied in vitro in hepatoma cell lines and ex vivo in precision-cut human liver slices. Liver regeneration was investigated in mice treated with a pharmacological MAGL inhibitor, MJN110, as well as in animals globally invalidated for MAGL (MAGL-/-) and specifically invalidated in hepatocytes (MAGLHep-/-) or myeloid cells (MAGLMye-/-). Two models of liver regeneration were used: acute toxic carbon tetrachloride injection and two-thirds partial hepatectomy. MAGLMye-/- liver macrophages profiling was analysed by RNA sequencing. A rescue experiment was performed by in vivo administration of interferon receptor antibody in MAGLMye-/- mice. Results: Precision-cut human liver slices from patients with chronic liver disease and human hepatocyte cell lines exposed to MJN110 showed reduced hepatocyte proliferation. Mice with global invalidation or mice treated with MJN110 showed blunted liver regeneration. Moreover, mice with specific deletion of MAGL in either hepatocytes or myeloid cells displayed delayed liver regeneration. Mechanistically, MAGLHep-/- mice showed reduced liver eicosanoid production, in particular prostaglandin E2 that negatively impacts on hepatocyte proliferation. MAGL inhibition in macrophages resulted in the induction of the type I interferon pathway. Importantly, neutralising the type I interferon pathway restored liver regeneration of MAGLMye-/- mice. Conclusions: Our data demonstrate that MAGL promotes liver regeneration by hepatocyte and macrophage reprogramming. Impact and Implications: By using human liver samples and mouse models of global or specific cell type invalidation, we show that the monoacylglycerol pathway plays an essential role in liver regeneration. We unveil the mechanisms by which MAGL expressed in both hepatocytes and macrophages impacts the liver regeneration process, via eicosanoid production by hepatocytes and the modulation of the macrophage interferon pathway profile that restrains hepatocyte proliferation.

Hepatocyte-derived biomarkers predict liver-related events at 2 years in Child-Pugh class A alcohol-related cirrhosis.

BACKGROUND & AIMS: In patients with compensated alcohol-related cirrhosis, reliable prognostic biomarkers are lacking. Keratin-18 and hepatocyte-derived large extracellular vesicle (lEV) concentrations reflect disease activity, but their ability to predict liver-related events is unknown. METHODS: We measured plasma keratin-18 and hepatocyte lEV concentrations in 500 patients with Child-Pugh class A alcohol-related cirrhosis. The ability of these hepatocyte-derived biomarkers, alone or combined with model for end-stage liver disease (MELD) and FibroTest scores, to predict liver-related events at 2 years was analyzed, taking into account the alcohol consumption at inclusion and during follow-up. RESULTS: Keratin-18 and hepatocyte lEV concentrations increased with alcohol consumption. In patients without active alcohol consumption at enrollment (n = 419), keratin-18 concentration predicted liver-related events at 2 years, independently of FibroTest and MELD. Patients with both keratin-18 concentrations >285 U/L and FibroTest >0.74 had a 24% cumulative incidence of liver-related events at 2 years, vs. 5% to 14% in other groups of patients. Similar results were obtained when combining keratin-18 concentrations >285 U/L with MELD >10. In patients with active alcohol consumption at enrollment (n = 81), hepatocyte lEVs predicted liver-related events at 2 years, independently of FibroTest and MELD. Patients with both hepatocyte lEV concentrations >50 U/L and FibroTest >0.74 had a 62% cumulative incidence of liver-related events at 2 years, vs. 8% to 13% in other groups of patients. Combining hepatocyte lEV concentrations >50 U/L with MELD >10 had a lower discriminative ability. Similar results were obtained when using decompensation of cirrhosis, defined according to Baveno VII criteria, as an endpoint. CONCLUSION: In patients with Child-Pugh class A alcohol-related cirrhosis, combining hepatocyte-derived biomarkers with FibroTest or MELD scores identifies patients at high risk of liver-related events, and could be used for risk stratification and patient selection in clinical trials. IMPACT AND IMPLICATIONS: In patients with compensated alcohol-related cirrhosis, reliable predictors of outcome are lacking. In patients with Child-Pugh class A alcohol-related cirrhosis, combining hepatocyte-derived biomarkers (keratin-18 and hepatocyte-large extracellular vesicles) with FibroTest or MELD scores identifies those at high risk of liver-related events at 2 years. The identified patients at high risk of liver-related events are the target-of-choice population for intensive surveillance (e.g., referral to tertiary care centers; intensive control of risk factors) and inclusion in clinical trials.

MAIT cell inhibition promotes liver fibrosis regression via macrophage phenotype reprogramming.

Recent data have shown that liver fibrosis can regress even at later stages of cirrhosis and shifting the immune response from pro-inflammatory towards a resolutive profile is considered as a promising option. The immune regulatory networks that govern the shift of the inflammatory phenotype and thus potential reversal of liver fibrosis are lesser known. Here we show that in precision-cut human liver slices obtained from patients with end-stage fibrosis and in mouse models, inhibiting Mucosal-Associated Invariant T (MAIT) cells using pharmacological or antibody-driven approaches, limits fibrosis progression and even regresses fibrosis, following chronic toxic- or non-alcoholic steatohepatitis (NASH)-induced liver injury. Mechanistic studies, combining RNA sequencing, in vivo functional studies (performed in male mice) and co-culture experiments indicate that disruption of the MAIT cell-monocyte/macrophage interaction results in resolution of fibrosis both by increasing the frequency of restorative Ly6Clo at the expenses of pro-fibrogenic Ly6Chi monocyte-derived macrophages and promoting an autophagic phenotype in both subsets. Thus, our data show that MAIT cell activation and the consequential phenotype shift of liver macrophages are important pathogenic features of liver fibrosis and could be targeted by anti-fibrogenic therapy.

Selective disruption of NRF2-KEAP1 interaction leads to NASH resolution and reduction of liver fibrosis in mice.

Background & Aims: Oxidative stress is recognized as a major driver of non-alcoholic steatohepatitis (NASH) progression. The transcription factor NRF2 and its negative regulator KEAP1 are master regulators of redox, metabolic and protein homeostasis, as well as detoxification, and thus appear to be attractive targets for the treatment of NASH. Methods: Molecular modeling and X-ray crystallography were used to design S217879 - a small molecule that could disrupt the KEAP1-NRF2 interaction. S217879 was highly characterized using various molecular and cellular assays. It was then evaluated in two different NASH-relevant preclinical models, namely the methionine and choline-deficient diet (MCDD) and diet-induced obesity NASH (DIO NASH) models. Results: Molecular and cell-based assays confirmed that S217879 is a highly potent and selective NRF2 activator with marked anti-inflammatory properties, as shown in primary human peripheral blood mononuclear cells. In MCDD mice, S217879 treatment for 2 weeks led to a dose-dependent reduction in NAFLD activity score while significantly increasing liver Nqo1 mRNA levels, a specific NRF2 target engagement biomarker. In DIO NASH mice, S217879 treatment resulted in a significant improvement of established liver injury, with a clear reduction in both NAS and liver fibrosis. αSMA and Col1A1 staining, as well as quantification of liver hydroxyproline levels, confirmed the reduction in liver fibrosis in response to S217879. RNA-sequencing analyses revealed major alterations in the liver transcriptome in response to S217879, with activation of NRF2-dependent gene transcription and marked inhibition of key signaling pathways that drive disease progression. Conclusions: These results highlight the potential of selective disruption of the NRF2-KEAP1 interaction for the treatment of NASH and liver fibrosis. Impact and implications: We report the discovery of S217879 - a potent and selective NRF2 activator with good pharmacokinetic properties. By disrupting the KEAP1-NRF2 interaction, S217879 triggers the upregulation of the antioxidant response and the coordinated regulation of a wide spectrum of genes involved in NASH disease progression, leading ultimately to the reduction of both NASH and liver fibrosis progression in mice.

Replication stress triggered by nucleotide pool imbalance drives DNA damage and cGAS-STING pathway activation in NAFLD.

Non-alcoholic steatotic liver disease (NAFLD) is the most common cause of chronic liver disease worldwide. NAFLD has a major effect on the intrinsic proliferative properties of hepatocytes. Here, we investigated the mechanisms underlying the activation of DNA damage response during NAFLD. Proliferating mouse NAFLD hepatocytes harbor replication stress (RS) with an alteration of the replication fork's speed and activation of ATR pathway, which is sufficient to cause DNA breaks. Nucleotide pool imbalance occurring during NAFLD is the key driver of RS. Remarkably, DNA lesions drive cGAS/STING pathway activation, a major component of cells' intrinsic immune response. The translational significance of this study was reiterated by showing that lipid overload in proliferating HepaRG was sufficient to induce RS and nucleotide pool imbalance. Moreover, livers from NAFLD patients displayed nucleotide pathway deregulation and cGAS/STING gene alteration. Altogether, our findings shed light on the mechanisms by which damaged NAFLD hepatocytes might promote disease progression.

Characterization and Pharmacological Validation of a Preclinical Model of NASH in Göttingen Minipigs.

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 Göttingen 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 Göttingen 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.

Procollagen C-Proteinase Enhancer-1 (PCPE-1) deficiency in mice reduces liver fibrosis but not NASH progression.

BACKGROUND AND AIMS: Nonalcoholic Steatohepatitis (NASH) is a major cause of end-stage liver diseases such as cirrhosis and hepatocellular carcinoma resulting ultimately in increased liver-related mortality. Fibrosis is the main driver of mortality in NASH. Procollagen C-Proteinase Enhancer-1 (PCPE-1) plays a key role in procollagen maturation and collagen fibril formation. To assess its role in liver fibrosis and NASH progression, knock-out mice were evaluated in a dietary NASH model. METHODS: Global constitutive Pcolce-/- and WT male mice were fed with a Choline Deficient Amino acid defined High Fat Diet (CDA HFD) for 8 weeks. Liver triglycerides, steatosis, inflammation and fibrosis were assessed at histological, biochemical and gene expression levels. In addition, human liver samples from control and NASH patients were used to evaluate the expression of PCPE-1 at both mRNA and protein levels. RESULTS: Pcolce gene deficiency prevented diet-induced liver enlargement but not liver dysfunction. Furthermore, liver triglycerides, steatosis and inflammation were not modified in Pcolce-/- male mice compared to WT under CDA HFD. However, a significant decrease in liver fibrosis was observed in Pcolce-/- mice compared to WT under NASH diet, associated with a decrease in total and insoluble collagen content without any significant modifications in the expression of genes involved in fibrosis and extracellular matrix remodeling. Finally, PCPE-1 protein expression was increased in cirrhotic liver samples from both NASH and Hepatitis C patients. CONCLUSIONS: Pcolce deficiency limits fibrosis but not NASH progression in CDA HFD fed mice.

A defect in endothelial autophagy occurs in patients with non-alcoholic steatohepatitis and promotes inflammation and fibrosis.

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.

Role of liver sinusoidal endothelial cells in non-alcoholic fatty liver disease.

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.

Primary cilia sensitize endothelial cells to BMP and prevent excessive vascular regression.

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.

Endothelial autophagic flux hampers atherosclerotic lesion development.

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.

Autophagy is required for endothelial cell alignment and atheroprotection under physiological blood flow.

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.

Paradoxical Suppression of Atherosclerosis in the Absence of microRNA-146a.

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.