Oxidized low‐density lipoprotein in non‐alcoholic steatohepatitis (2016)

Non-alcoholic fatty liver disease (NAFLD) is the most common cause of chronic liver disease worldwide.1, 2 Non-alcoholic steatohepatitis (NASH), the more severe form of NAFLD characterized by inflammation, hepatocyte ballooning and fibrosis, is associated with increased mortality from liver-related complications. With better understanding of the pathogenesis of NAFLD, the original two-hit hypothesis of insulin resistance and oxidative stress has evolved into a multiple-parallel hits hypothesis.3 Lipotoxicity is now thought to play a central role in NASH (and the metabolic syndrome), whereas simple steatosis may reflect a protective mechanism in place to buffer toxic lipid species.4 Oxidized low-density lipoprotein (oxLDL) can be defined as a particle derived from circulating low-density lipoprotein (LDL) that has undergone oxidative changes. The concept of oxidative stress and oxLDL is well established in atherogenesis but has only been recognized recently in the pathogenesis of NASH.5 OxLDL is taken up by macrophages by way of scavenger receptors, which unlike the LDL receptor, is not downregulated when the cholesterol content of the macrophage increases. The excessive accumulation of cholesterol leads to foam cell formation and activation of pro-inflammatory pathways in atherogenesis. Kupffer cells are the liver's resident macrophage population. Similar to foam cell formation in atherogenesis, foamy Kupffer cells were found in a hyperlipidemic mouse model of NASH, and the omission of cholesterol from the diet prevented the formation of the foamy Kupffer cells and hepatic inflammation.6 In a separate study, the administration of oxLDL to mice fed with a high-fat diet also induced NASH, further confirming the role of oxLDL in the pathogenesis of NASH.7 Activated Kupffer cells secrete interleukin-1 and tumor necrosis factor-α; these pro-inflammatory cytokines in turn activate the sterol regulatory element-binding protein 2 (SREBP-2) in hepatocytes. Physiological activation of SREBP-2 under low cholesterol conditions promotes cholesterol synthesis and retention. However, SREBP-2 is inappropriately activated despite cholesterol overload in NASH. The excess free cholesterol interrupts with the function of mitochondria and endoplasmic reticulum, leading to hepatocyte injury and death. In addition, activated Kupffer cells also secrete transforming growth factor-β1 which activates hepatic stellate cells, thus promoting hepatic fibrosis.8 OxLDL may also directly activate hepatic stellate cells via the lectin-like oxLDL receptor-1 (Fig. 1a).9 Anti-oxLDL antibodies (oxLDL-ab) have been shown to correlate negatively with hepatic inflammation.10 However, its potential as a marker for NASH is limited by the fact that the levels may vary from person to person and from time to time and may be influenced by exposure to pathogen due to molecular mimicry.5 On the other hand, immunization to boost oxLDL-ab has been shown to be protective against atherosclerosis and recently against NASH in mouse models, and these deserves further study (Fig. 1b).10 In this issue of the Journal, Ampuero and colleagues explore the possibility of using oxLDL-ab as a biomarker for NAFLD/NASH.11 This cross-sectional study included 72 patients with biopsy-proven NAFLD, among whom 38 had body mass index above 30 kg/m2 and 26 had NASH. By multivariate analysis, the oxLDL-ab level was independently associated with severe steatosis, while the oxLDL-ab-to-high-density lipoprotein cholesterol (HDL-C) ratio was associated with NASH and advanced fibrosis. Interestingly, the association between oxLDL-ab-based biomarkers and histological severity was only observed in NAFLD patients with body mass index below 30 kg/m2. The findings by Ampuero and colleagues are exciting and also raise new questions. While non-invasive tests of liver fibrosis are relatively well developed, the diagnostic accuracy of existing NASH biomarkers is moderate at best. The most widely tested serum marker of cytokeratin-18 fragments, which reflect hepatocyte apoptosis, showed initial promising results but only modest accuracy for diagnosing NASH in subsequent validation studies.12 The lack of a robust NASH biomarker hinders drug development and makes routine management of NAFLD suboptimal. In the current study, while the oxLDL-ab/HDL-C ratio appeared markedly increased in non-obese patients with NASH, the findings were based on a relatively small number of patients, and formal c-statistics and external validation have not been performed.11 On another note, the use of NASH biomarkers to monitor patients and assess treatment response is an even bigger question. At present, most biomarker studies are of cross-sectional design. Whether the change in biomarkers over time reflects histological and clinical progression or response is largely unknown. Nonetheless, oxLDL can potentially be modified by lipid lowering agents and anti-oxidants. For instance, vitamin E reduces oxLDL uptake by aortic smooth muscle cells by down regulating CD36 scavenger receptor, the specific oxLDL receptor.13 It would be interesting to evaluate the serial oxLDL or oxLDL-ab levels against histological changes of NASH. Another intriguing question raised by the study of Ampuero and colleagues is why the association between oxLDL-ab and NAFLD severity was only observed in non-obese patients. NAFLD in non-obese patients has been well described in the Asian population and is recently found to be not uncommon in western countries as well.14 It should be noted, however, that a wide range of body mass index has been used to define obesity in different ethnic groups. In particular, Asians tend to develop central obesity and metabolic complications at a lower body mass index. This is the basis for using lower body mass index cutoffs in some populations, although the definition is not universally agreed. In any case, cutoffs are largely arbitrary. In reality, the higher the adiposity, the higher the prevalence of NAFLD and its associated metabolic diseases. Moreover, body mass index is an imperfect measure of adiposity. Because it is calculated from body weight and body height, it cannot distinguish between muscle and fat mass. Waist circumference may be a better reflection of central or visceral obesity and has been adopted by the Third Adult Treatment Panel of the National Cholesterol Education Program and the International Diabetes Federation as one of the criteria to diagnose metabolic syndrome. Overall, even in the non-obese population, higher body mass index and waist circumference is associated with NASH and liver fibrosis, suggesting this is but the spectrum of disease.15 Data interpretation for non-obese NAFLD in a liver biopsy cohort can be difficult. Unlike population studies, patients undergoing liver biopsy are highly selected. Typical indications for liver biopsy include uncertain diagnosis (i.e., to rule out alternative diagnoses), high likelihood of advanced disease (based on metabolic risk factors or prior assessment using non-invasive tests), and participation in drug trial. The indications for liver biopsy in obese and non-obese patients can be different. In the current study, non-obese patients with NASH or advanced fibrosis had even higher oxLDL-ab/HDL-C ratio than their obese counterparts.11 In our opinion, this finding has two possible explanations. First, the non-obese NASH patients in this study might have higher histological severity or more concomitant metabolic complications. Second, the treatment received by obese patients (e.g., lipid lowering drugs or vitamin E) may have affected oxLDL and the lipid profile. Either of these possibilities will have major bearing on the development of oxLDL-ab as a biomarker and should be evaluated in future studies. In conclusion, the study by Ampuero and colleagues provides new data to support the association between oxLDL-ab and NASH and opens the door to a potential new biomarker. Before this marker can be adopted in clinical practice, bigger studies with longitudinal follow-up are required. Vincent Wong has served as an advisory board member for AbbVie, Gilead, Janssen, Otsuka, and Roche; a consultant for Merck and NovoMedica; and a speaker for Abbott, Echosens, Gilead, Novartis, and Roche. None of these represent significant and direct conflict with this editorial.