Kristiaan Wouters

Dietary cholesterol, rather than liver steatosis, leads to hepatic inflammation in hyperlipidemic mouse models of nonalcoholic steatohepatitis

Nonalcoholic steatohepatitis (NASH) involves liver lipid accumulation (steatosis) combined with hepatic inflammation. The transition towards hepatic inflammation represents a key step in pathogenesis, because it will set the stage for further liver damage, culminating in hepatic fibrosis, cirrhosis, and liver cancer. The actual risk factors that drive hepatic inflammation during the progression to NASH remain largely unknown. The role of steatosis and dietary cholesterol in the etiology of diet‐induced NASH was investigated using hyperlipidemic mouse models fed a Western diet. Livers of male and female hyperlipidemic (low‐density lipoprotein receptor–deficient [ldlr−/−] and apolipoprotein E2 knock‐in [APOE2ki]) mouse models were compared with livers of normolipidemic wild‐type (WT) C57BL/6J mice after short‐term feeding with a high‐fat diet with cholesterol (HFC) and without cholesterol. Whereas WT mice displayed only steatosis after a short‐term HFC diet, female ldlr−/− and APOE2ki mice showed steatosis with severe inflammation characterized by infiltration of macrophages and increased nuclear factor κB (NF‐κB) signaling. Remarkably, male ldlr−/− and APOE2ki mice developed severe hepatic inflammation in the absence of steatosis after 7 days on an HFC diet compared with WT animals. An HFC diet induced bloated, “foamy” Kupffer cells in male and female ldlr−/− and APOE2ki mice. Hepatic inflammation was found to be linked to increased plasma very low‐density lipoprotein (VLDL) cholesterol levels. Omitting cholesterol from the HFC diet lowered plasma VLDL cholesterol and prevented the development of inflammation and hepatic foam cells. Conclusion: These findings indicate that dietary cholesterol, possibly in the form of modified plasma lipoproteins, is an important risk factor for the progression to hepatic inflammation in diet‐induced NASH. (HEPATOLOGY 2008;48:474–486.)

Roles of PPARs in NAFLD: potential therapeutic targets.

Non-alcoholic fatty liver disease (NAFLD) is a liver pathology with increasing prevalence due to the obesity epidemic. Hence, NAFLD represents a rising threat to public health. Currently, no effective treatments are available to treat NAFLD and its complications such as cirrhosis and liver cancer. Peroxisome proliferator-activated receptors (PPARs) are ligand-activated nuclear receptors which regulate lipid and glucose metabolism as well as inflammation. Here we review recent findings on the pathophysiological role of PPARs in the different stages of NAFLD, from steatosis development to steatohepatitis and fibrosis, as well as the preclinical and clinical evidence for potential therapeutical use of PPAR agonists in the treatment of NAFLD. PPARs play a role in modulating hepatic triglyceride accumulation, a hallmark of the development of NAFLD. Moreover, PPARs may also influence the evolution of reversible steatosis toward irreversible, more advanced lesions. Presently, large controlled trials of long duration are needed to assess the long-term clinical benefits of PPAR agonists in humans. This article is part of a Special Issue entitled Triglyceride Metabolism and Disease.

Macrophage MicroRNA-155 Promotes Cardiac Hypertrophy and Failure

Background— Cardiac hypertrophy and subsequent heart failure triggered by chronic hypertension represent major challenges for cardiovascular research. Beyond neurohormonal and myocyte signaling pathways, growing evidence suggests inflammatory signaling pathways as therapeutically targetable contributors to this process. We recently reported that microRNA-155 is a key mediator of cardiac inflammation and injury in infectious myocarditis. Here, we investigated the impact of microRNA-155 manipulation in hypertensive heart disease. Methods and Results— Genetic loss or pharmacological inhibition of the leukocyte-expressed microRNA-155 in mice markedly reduced cardiac inflammation, hypertrophy, and dysfunction on pressure overload. These alterations were macrophage dependent because in vivo cardiomyocyte-specific microRNA-155 manipulation did not affect cardiac hypertrophy or dysfunction, whereas bone marrow transplantation from wild-type mice into microRNA-155 knockout animals rescued the hypertrophic response of the cardiomyocytes and vice versa. In vitro, media from microRNA-155 knockout macrophages blocked the hypertrophic growth of stimulated cardiomyocytes, confirming that macrophages influence myocyte growth in a microRNA-155-dependent paracrine manner. These effects were at least partly mediated by the direct microRNA-155 target suppressor of cytokine signaling 1 (Socs1) because Socs1 knockdown in microRNA-155 knockout macrophages largely restored their hypertrophy-stimulating potency. Conclusions— Our findings reveal that microRNA-155 expression in macrophages promotes cardiac inflammation, hypertrophy, and failure in response to pressure overload. These data support the causative significance of inflammatory signaling in hypertrophic heart disease and demonstrate the feasibility of therapeutic microRNA targeting of inflammation in heart failure.

Understanding hyperlipidemia and atherosclerosis: lessons from genetically modified apoe and ldlr mice.

Abstract Hyperlipidemia is the most important risk factor for atherosclerosis, which is the major cause of cardiovascular disease. The etiology of hyperlipidemia and atherosclerosis is complex and governed by multiple interacting genes. However, mutations in two genes have been shown to be directly involved, i.e., the low-density lipoprotein receptor (LDLR) and apolipoprotein E (ApoE). Genetically modified mouse models have been instrumental in elucidating the underlying molecular mechanisms in lipid metabolism. In this review, we focus on the use of two of the most widely used mouse models, ApoE- and LDLR-deficient mice. After almost a decade of applications, it is clear that each model has unique strengths and drawbacks when carrying out studies of the role of additional genes and environmental factors such as nutrition and lipid-lowering drugs. Importantly, we elaborate on mice expressing mutant forms of APOE, including the APOE3Leiden ( APOE3L) and the APOE2 knock-in ( APOE2k) mouse models. These models have outstanding potential, as they are highly responsive to dietary factors and pharmacological interventions.

Role of Scavenger Receptor A and CD36 in Diet-Induced Nonalcoholic Steatohepatitis in Hyperlipidemic Mice

BACKGROUND & AIMS Nonalcoholic steatohepatitis (NASH) is a disorder that consists of steatosis and hepatic inflammation. It is not known why only some people with steatosis develop NASH. Recently, we identified dietary cholesterol as a factor that directly leads to hepatic inflammation and hepatic foam cell formation. We propose a mechanism by which Kupffer cells (KCs) take up modified cholesterol-rich lipoproteins via scavenger receptors (SRs). KCs thereby accumulate cholesterol, become activated, and may then trigger an inflammatory reaction. Scavenging of modified lipoproteins mainly depends on CD36 and macrophage scavenger receptor 1. METHODS To evaluate the involvement of SR-mediated uptake of modified lipoproteins by KCs in the development of diet-induced NASH, female low-density lipoprotein receptor-deficient (Ldlr(-/-)) mice were lethally irradiated and transplanted with bone marrow from Msr1(+/+)/Cd36(+/+)or Msr1(-/-)/Cd36(-/-) mice and fed a Western diet. RESULTS Macrophage and neutrophil infiltration revealed that hepatic inflammation was substantially reduced by approximately 30% in Msr1(-/-)/Cd36(-/-)-transplanted mice compared with control mice. Consistent with this, the expression levels of well-known inflammatory mediators were reduced. Apoptotis and fibrosis were less pronounced in Msr1(-/-)/Cd36(-/-)-transplanted mice, in addition to the protective phenotype of natural antibodies against oxidized low-density lipoprotein in the plasma. Surprisingly, the effect on hepatic inflammation was independent of foam cell formation. CONCLUSIONS Targeted inactivation of SR pathways reduces the hepatic inflammation and tissue destruction associated with NASH, independent of hepatic foam cell formation.

Ldl receptor knock-out mice are a physiological model particularly vulnerable to study the onset of inflammation in non-alcoholic fatty liver disease

Background & Aims Non-alcoholic steatohepatitis (NASH) involves steatosis combined with inflammation, which can progress into fibrosis and cirrhosis. Exploring the molecular mechanisms of NASH is highly dependent on the availability of animal models. Currently, the most commonly used animal models for NASH imitate particularly late stages of human disease. Thus, there is a need for an animal model that can be used for investigating the factors that potentiate the inflammatory response within NASH. We have previously shown that 7-day high-fat-high-cholesterol (HFC) feeding induces steatosis and inflammation in both APOE2ki and Ldlr−/− mice. However, it is not known whether the early inflammatory response observed in these mice will sustain over time and lead to liver damage. We hypothesized that the inflammatory response in both models is sufficient to induce liver damage over time. Methods APOE2ki and Ldlr−/− mice were fed a chow or HFC diet for 3 months. C57Bl6/J mice were used as control. Results Surprisingly, hepatic inflammation was abolished in APOE2ki mice, while it was sustained in Ldlr−/− mice. In addition, increased apoptosis and hepatic fibrosis was only demonstrated in Ldlr−/− mice. Finally, bone-marrow-derived-macrophages of Ldlr−/− mice showed an increased inflammatory response after oxidized LDL (oxLDL) loading compared to APOE2ki mice. Conclusion Ldlr−/− mice, but not APOE2ki mice, developed sustained hepatic inflammation and liver damage upon long term HFC feeding due to increased sensitivity for oxLDL uptake. Therefore, the Ldlr−/− mice are a promising physiological model particularly vulnerable for investigating the onset of hepatic inflammation in non-alcoholic steatohepatitis.

Higher levels of advanced glycation endproducts in human carotid atherosclerotic plaques are associated with a rupture-prone phenotype

AIMS Rupture-prone atherosclerotic plaques are characterized by inflammation and a large necrotic core. Inflammation is linked to high metabolic activity. Advanced glycation endproducts (AGEs) and their major precursor methylglyoxal are formed during high metabolic activity and can have detrimental effects on cellular function and may induce cell death. Therefore, we investigated whether plaque AGEs are increased in human carotid rupture-prone plaques and are associated with plaque inflammation and necrotic core formation. METHODS AND RESULTS The protein-bound major methylglyoxal-derived AGE 5-hydro-5-methylimidazolone (MG-H1) and N(ε)-(carboxymethyl)lysine (CML) were measured in human carotid endarterectomy specimens (n = 75) with tandem mass spectrometry. MG-H1 and CML levels were associated with rupture-prone plaques, increased protein levels of the inflammatory mediators IL-8 and MCP-1 and with higher MMP-9 activity. Immunohistochemistry showed that AGEs accumulated predominantly in macrophages surrounding the necrotic core and co-localized with cleaved caspase-3. Intra-plaque comparison revealed that glyoxalase-1 (GLO-1), the major methylglyoxal-detoxifying enzyme, mRNA was decreased (-13%, P < 0.05) in ruptured compared with stable plaque segments. In line, in U937 monoctyes, we found reduced (GLO-1) activity (-38%, P < 0.05) and increased MGO (346%, P < 0.05) production after stimulation with the inflammatory mediator TNF. Direct incubation with methylglyoxal increased apoptosis up to two-fold. CONCLUSION This is the first study showing that AGEs are associated with human rupture-prone plaques. Furthermore, this study suggests a cascade linking inflammation, reduced GLO-1, methylglyoxal- and AGE-accumulation, and subsequent apoptosis. Thereby, AGEs may act as mediators of the progression of stable to rupture-prone plaques, opening a window towards novel treatments and biomarkers to treat cardiovascular diseases.

Internalization of Modified Lipids by CD36 and SR-A Leads to Hepatic Inflammation and Lysosomal Cholesterol Storage in Kupffer Cells

Background & Aims Non-alcoholic steatohepatitis (NASH) is characterized by steatosis and inflammation, which can further progress into fibrosis and cirrhosis. Recently, we demonstrated that combined deletion of the two main scavenger receptors, CD36 and macrophage scavenger receptor 1 (MSR1), which are important for modified cholesterol-rich lipoprotein uptake, reduced NASH. The individual contributions of these receptors to NASH and the intracellular mechanisms by which they contribute to inflammation have not been established. We hypothesize that CD36 and MSR1 contribute independently to the onset of inflammation in NASH, by affecting intracellular cholesterol distribution inside Kupffer cells (KCs). Methods & Results Ldlr−/− mice were transplanted with wild-type (Wt), Cd36−/− or Msr1−/− bone marrow and fed a Western diet for 3months. Cd36−/−- and Msr1−/−- transplanted (tp) mice showed a similar reduction in hepatic inflammation compared to Wt-tp mice. While the total amount of cholesterol inside KCs was similar in all groups, KCs of Cd36−/−- and Msr1−/−-tp mice showed increased cytoplasmic cholesterol accumulation, while Wt-tp mice showed increased lysosomal cholesterol accumulation. Conclusion CD36 and MSR1 contribute similarly and independently to the progression of inflammation in NASH. One possible explanation for the inflammatory response related to expression of these receptors could be abnormal cholesterol trafficking in KCs. These data provide a new basis for prevention and treatment of NASH.

Intrahepatic cholesterol influences progression, inhibition and reversal of non-alcoholic steatohepatitis in hyperlipidemic mice

Hepatic inflammation is the key factor in non‐alcoholic steatohepatitis (NASH) and promotes progression to liver damage. We recently identified dietary cholesterol as the cause of hepatic inflammation in hyperlipidemic mice. We now show that hepatic transcriptome responses are strongly dependent on cholesterol metabolism during diet‐induced NASH and its inhibition by fenofibrate. Furthermore, we show that, despite doubling hepatic steatosis, pharmacological LXR activation reverses hepatic inflammation, in parallel with reversing hepatic cholesterol levels. Together, the results indicate a prominent role of cholesterol during the development, inhibition and reversal of hepatic inflammation in NASH and reveal potential new therapeutic strategies against NASH.

Peroxisome Proliferator–Activated Receptor-α Gene Level Differently Affects Lipid Metabolism and Inflammation in Apolipoprotein E2 Knock-In Mice

Objective—Peroxisome proliferator–activated receptor-&agr; (PPAR&agr;) is a ligand-activated transcription factor that controls lipid metabolism and inflammation. PPAR&agr; is activated by fibrates, hypolipidemic drugs used in the treatment of dyslipidemia. Previous studies assessing the influence of PPAR&agr; agonists on atherosclerosis in mice yielded conflicting results, and the implication of PPAR&agr; therein has not been assessed. The human apolipoprotein E2 knock-in (apoE2-KI) mouse is a model of mixed dyslipidemia, atherosclerosis, and nonalcoholic steatohepatitis (NASH). The aim of this study was to analyze, using homo- and heterozygous PPAR&agr;-deficient mice, the consequences of quantitative variations of PPAR&agr; gene levels and their response to the synthetic PPAR&agr; agonist fenofibrate on NASH and atherosclerosis in apoE2-KI mice. Methods and Results—Wild-type (+/+), heterozygous (+/−), and homozygous (−/−) PPAR&agr;-deficient mice in the apoE2-KI background were generated and subjected to a Western diet supplemented with fenofibrate or not supplemented. Western diet–fed PPAR&agr;−/− apoE2-KI mice displayed an aggravation of liver steatosis and inflammation compared with PPAR&agr;+/+ and PPAR&agr;+/− apoE2-KI mice, indicating a role of PPAR&agr; in liver protection. Moreover, PPAR&agr; expression was required for the fenofibrate-induced protection against NASH. Interestingly, fenofibrate treatment induced a similar response on hepatic lipid metabolism in PPAR&agr;+/+ and PPAR&agr;+/− apoE2-KI mice, whereas, for a maximal antiinflammatory response, both alleles of the PPAR&agr; gene were required. Surprisingly, atherosclerosis development was not significantly different among PPAR&agr;+/+, PPAR&agr;+/−, and PPAR&agr;−/− apoE2-KI mice. However, PPAR&agr; gene level determined both the antiatherosclerotic and vascular antiinflammatory responses to fenofibrate in a dose-dependent manner. Conclusion—These results demonstrate a necessary but quantitatively different role of PPAR&agr; in the modulation of liver metabolism, inflammation, and atherogenesis.