Manfredi Rizzo, Giuseppe Montalto and Manlio Vinciguerra Pages 741 - 744 ( 4 )
The liver plays a major role in lipid metabolism, importing free fatty acids (FFA) and manufacturing, storing and exporting lipids: derangements in any of these processes can lead to non-alcoholic fatty liver disease (NAFLD) . NAFLD can be seen as the result of an imbalance between lipid availability and lipid disposal resulting in hepatic steatosis . NAFLD is considered by many as the hepatic manifestation of insulin resistance (IR) and is strongly associated with the metabolic syndrome . The rapid increase in obesity and diabetes mellitus (DM) during the last decade is associated with an increase in the prevalence of NAFLD, making it the most common cause of chronic liver disease in the Western countries with up to 30% of the population affected . Histological evaluation is the gold standard for precisely estimating the degree of liver damage caused by simple steatosis or by non-alcoholic steatohepatitis (NASH). NASH is a disturbance at the end of NAFLD spectrum characterized by hepatocellular injury/inflammation/macrophage infiltration with or without fibrosis . The individuals with NAFLD develop NASH in 10% of the cases, 8-26% progress to cirrhosis and these patients are also at risk of developing hepatocellular carcinoma (HCC) . As recently highlighted by the guidelines of the American Association for the Study of Liver Diseases (AASLD) , patients with NAFLD and NASH are at increased risk for cardiovascular disease (CVD) (their most common cause of death) . Therefore, patients with NAFLD and NASH should be stratified for such risk and their CVD factors, including dyslipidemia, should be managed accordingly . At the cellular level, defects in the insulin signaling pathways contribute to the increase of FFA flux in the liver which in turn activates a series of signaling cascades which lead to the phosphorylation of several substrates; in the case of IR there is a decreased insulin receptor kinase activity resulting in lower AKT activity . Insulin is the main driver of the global response to nutrient ingestion, and acts on hepatic fat metabolism through the phosphatidylinositol kinase signaling pathway by accumulating the intracellular lipid small messenger phosphatidylinositol [3,4,5]-trisphosphate. This situation is reproduced faithfully by phosphatase and tensin homologue (PTEN) loss, which similarly accompanies fatty liver development [11-17]. Besides the PTEN/AKT and downstream effectors, de novo fatty acid synthesis is regulated by a plethora of other transcription factors . The mechanisms behind the progression of steatosis to NASH are not completely understood; in particular the factors that lead to increasing hepatocellular damage after triglyceride (TG) accumulation. According to the ‘two-hit’ theory, the ‘first hit’ is an imbalance in hepatic lipid accumulation the likely cause being IR which leads to an increased traffic of FFA in the hepatocytes; then follows the ‘second hit’, represented by inflammatory cytokines/adipokines, mitochondrial dysfunction and oxidative stress, promoting apoptosis through several pathways that lead to inflammatory infiltrates in the liver that may progress to fibrosis . This old model is now revitalized by recent studies analyzing the role of the gut microbiota. The nucleotide-binding domain and leucine-rich-repeat-containing (NLR) family of pattern-recognition molecules NLRP6 and NLRP3 take part to cytosolic protein complexes termed inflammasomes, which in turn have various roles in immune defense . The NLRP6 and NLRP3 inflammasomes and the downstream cytokine interleukin-18 (IL-18) negatively regulate NAFLD/NASH progression, by regulating the activity of the gut microbiota: changed interactions between the gut microbiota and the host, produced by defective NLRP3 and NLRP6 inflammasome sensing may govern NAFLD progression . Inflammasome sensing has also been proposed as a mechanism in macrophage activation in atherosclerotic lesions . In this context, the hypothesis that NASH and atherosclerosis are 2 aspects of the same disease, involving the local presence of activated macrophages, has been put forward . NASH may promote pro-inflammatory and pro-atherogenic factors that are likely to play a major role in CVD pathogenesis [8, 23, 24]. Oxidative stress is a strong atherogenic stimulus, associated with the secretion of inflammation biomarkers and endothelial dysfunction such as interleukin-6 (IL-6) and tumour-necrosis factor-?? (TNF-??). NASH is associated with a systemic release of pro-inflammatory mediators from the liver including C-reactive protein (CRP), fibrinogen and plasminogen activator inhibitor-1 (PAI-1) [8, 23, 24]. CRP levels and the degree of carotid atherosclerosis were found to be higher in NASH patients than in control subjects, suggesting a link between hepatic injury, inflammation and development and progression of CVD . An important step in atherosclerosis progression is the transfer of oxidised (ox) low density lipoprotein (LDL) into the arterial wall . OxLDL represents a variety of modifications of both the lipid and protein components of LDL and this oxidation process is thought to occur mainly in the arterial wall, rather than in plasma . LDL are heterogeneous particles with several distinct subclasses that differ in physicochemical composition, metabolic and oxidative properties as well as atherogenicity . Oxidative susceptibility increases and antioxidant concentrations decrease with decreasing LDL size , so that small dense (sd) LDL particles are those with enhanced susceptibility to oxidation and greater atherogenicity. sdLDL represent an emerging cardiovascular risk factor, as highlighted by several international guidelines  and expert panels . It is plausible that the more sdLDL in plasma, the more oxLDL in the arterial wall; oxLDL have pro-inflammatory, pro-thrombotic and pro-apoptotic potential . On this basis, sdLDL/oxLDL levels have been proposed as a novel biomarker of CVD risk beyond LDL-cholesterol concentrations . Recently, studies by Bieghs et al. have shown that sdLDL receptor knock-out mice developed hepatic inflammation and liver damage upon high-fat feeding due to increased oxLDL uptake . Models for atherosclerosis include the sdLDL receptor knock-out (Ldlr-/-) mouse, which display defects in the clearance of apo B- and apo E-containing lipoproteins and the apo E2 knock-in (APOE2ki) mouse, in which the murine apo E gene is substituted with the human apo E2. Apo E2 has a decreased affinity for the LDL receptor, leading to a lipoprotein profile resembling human type III hyperlipoproteinaemia . Both APOE2ki mice and Ldlr-/- mice develop early hepatic inflammation and steatosis when fed a high-fat-high-cholesterol diet, whereas wild type mice only developed fatty liver . The lipoprotein profile of the Ldlr-/- mouse and APOE2ki is comparable with the human, in which cholesterol is mainly present in the LDL fraction . When fed a high-fat-high-cholesterol diet, both APOE2ki and Ldlr-/- mice are useful to study the factors that augment the inflammatory response during NASH. Bieghs et al. recently hypothesized that the inflammatory response in these mice models would be strong enough to induce hepatic fibrosis . Upon feeding APOE2ki and Ldlr-/- mice with a high-fat-high-cholesterol diet for 3 months, a pronounced inflammatory response was observed only in Ldlr-/- mice, as well as increased apoptosis and hepatic fibrosis. These differences were dependent on an increased sensitivity for oxLDL-induced inflammation in Ldlr-/-mice compared with APOE2ki mice . The Ldlr-/- model is thus a promising option to study the onset of NASH in the context of steatosis and to assess therapeutic strategies for hepatic inflammation. Accordingly, it is plausible to think that specific immunization strategies against oxLDL may reduce NASH. Interestingly, the levels of immunoglobulin M (IgM) autoantibodies to oxLDL/sdLDL are inversely correlated with atherosclerotic plaques [37-39]. Specific epitopes present in oxLDL are recognized by circulating IgM . In another recent study, Bieghs et al. used the Ldlr-/- model for immunization with heat-inactivated pneumococci to investigate whether anti-oxLDL antibodies can exert a protective effect on NASH . Immunized Ldlr-/- mice showed reduced NASH compared with non-immunized mice. Although no difference in liver lipids were observed between immunized and nonimmunized Ldlr-/- mice, decreased cholesterol, inflammation and expression of hepatic fibrosis-associated genes were observed upon a high-fat-high-cholesterol diet . These findings in mice demonstrate the potential role of antibodies to oxLDL in the pathogenesis of NASH, in line with recent data on lipids implicated in lipotoxicity to the liver and hepatocytes . Understanding the relationship between NAFLD/NASH, sdLDL/oxLDL and atherogenic processes is of clinical relevance. These recent studies suggest that vaccination strategies against sdLDL/oxLDL could be explored as an option for the prevention and treatment of NASH. Such research would help uncover new therapeutic approaches to prevent the development of CVD associated with NAFLD/NASH. Since NAFLD is often associated with obesity and IR/type 2 diabetes, lifestyle modification (diet and aerobic exercise) and weight reduction remain the cornerstones of treatment because they can ameliorate or even reverse the disease [42, 43], as indicated also by the AASLD guidelines . The effects of lifestyle intervention were examined by Promrat et al. in a randomized controlled trial: patients who achieved a weight loss >7% of initial weight had significant improvement in steatosis and NASH activity score compared with patients who lost less than 7% of initial . Drugs potentially effective against NAFLD are the insulin sensitizers, metformin and pioglitazone, which all reduce oxidative stress . Studies on overweight or obese patients with ultrasonographic diagnosis of hepatic steatosis, metformin was found to be more effective than dietary treatment alone in ameliorating metabolic derangements and NAFLD . Studies on NAFLD patients as well as on murine models have suggested some beneficial roles of natural antioxidant molecules [47, 48]. Vitamin D deficiency exacerbated NAFLD and inflammation in a Western diet rat model, suggesting that its supplementation might be beneficial . Vitamin E treatment compared with placebo was associated with a significantly higher rate of improvement in NAFLD patients, and it did not cause a weight gain as observed with pioglitazone and rosiglitazone . Despite these interesting findings, the impact of vitamin D supplementation in human NAFLD awaits validation and treatment with high dose vitamin E should be carefully considered due to its association with higher risk for stroke and mortality . The thiazolidinediones, pioglitazone and rosiglitazone, activate peroxisome proliferator-activated receptor-gamma PPAR??, which regulates TG homeostasis, contributing to hepatic steatosis . Unfortunately, the use of thiazolidinediones has been hampered by important side effects [53-55]; rosiglitazone has been taken off the market. The AASLD statement  suggests that pioglitazone can be used to treat steatohepatitis in patients with biopsy-proven NASH, although the long-term safety and efficacy of pioglitazone in these patients is not established. Fibrates are potent peroxisome proliferator activator receptors alpha (PPAR??) agonists and they can significantly increase the hepatic oxidation of free fatty acids . A controlled trial with the gemfibrozil 600 mg daily for 4 weeks showed some lipid improvement in NAFLD , and similar results have been shown with fenofibrate . Overall, fibrates are an attractive option to treat hypertriglyceridemia in patients with NAFLD but, as recently highlighted , their effect on liver histology is unclear. Another option to treat hypertriglyceridemia is supplementation with omega-3 polyunsaturated fatty acid (PUFA). Several studies have shown that PUFA supplementation can improve biochemical and ultrasonographic steatosis [59-61]. Yet, as recently highlighted in a comprehensive review , no clear conclusion can be made, since published human studies are of small sample size and have several methodological flaws. However, the experimental evidence strongly supports the use of PUFA in NAFLD. On the basis of this evidence, the AASLD guidelines  state that it is premature to recommend omega-3fatty acids for the specific treatment of NAFLD or NASH, but they may be considered as the first line agents to treat hypertriglyceridemia in NAFLD. There is reluctance to use statins in patients with suspected or established chronic liver disease (e.g. NAFLD, NASH or elevated aminotransferase activity) . All statins seems to lower cholesterol levels in NAFLD patients  and atorvastatin showed a reduced CVD morbidity: the Greek Atorvastatin and Coronary Heart Disease Evaluation (GREACE) study showed a reduction of CVD events with statins in patients with NAFLD . In addition, a review that examined all available evidence about potential statin hepatotoxicity concluded that patients with NAFLD are not at significantly increased risk of severe hepatic toxicity with standard doses of statins . The AASLD statement  suggests that statins can be used to treat dyslipidemia in patients with NAFLD and NASH, although these drugs should not be used specifically to treat NASH. In conclusion, patients with NAFLD and NASH have increased CVD risk. NAFLD and NASH patients have several alterations in lipid metabolism that are associated with atherogenic dyslipidemia, including increased levels of TGs, high levels of sdLDL particles and decreased levels of HDL-cholesterol . Lipid-lowering drugs are not fully recommended in NASH patients; therefore, the recent studies suggesting that vaccination strategies against sdLDL/oxLDL could be explored as an option for the prevention and treatment of NASH are fascinating.Editorial:, Exploring, Lipid-related, Treatment, Options, for, the, Treatment, of, NASH
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