Accumulation of free cholesterol and oxidized low-density lipoprotein is associated with portal inflammation and fibrosis in nonalcoholic fatty liver disease

Nonalcoholic fatty liver disease (NAFLD) encompasses a wide spectrum of progressive liver diseases, namely macrovesicular fat accumulation (simple steatosis), nonalcoholic steatohepatitis (NASH), fibrosis, and cirrhosis. Simple steatosis is considered a benign condition, whereas the other conditions are often considered progressive, and they have been increasingly recognized as important risk factors for hepatocellular carcinoma and as conditions that warrant liver transplantation [1, 2]. Although significant data have been obtained to support the “two-hit hypothesis”, important molecular mechanisms underlying NAFLD progression remain elusive [3].

NAFLD is the hepatic manifestation of metabolic syndrome [3, 4]. Patients with NAFLD have a higher risk of cardiovascular mortality than those without NAFLD [5]. Chronic low-grade inflammation mediated by macrophages is a common factor involved in the pathogenesis of a collection of metabolic pathologies, including insulin resistance, NAFLD, atherosclerosis, and dyslipidemia [6‐8]. Specifically, macrophage activation mediated by lipid metabolites, such as oxidized lipoproteins, free fatty acids, free (unesterified) cholesterol, and ceramides, represents a common pathogenic lipotoxic mechanism [9].

Oxidized low-density lipoprotein (oxLDL) is a key pathogenic determinant of vascular atherosclerosis. Moreover, increased levels of oxLDL are associated with an increased incidence of metabolic syndrome [10], acute coronary events [11], and hepatocellular injury in experimental cholestasis and fibrosis [12]. The short-term administration of oxLDL to high-fat diet-fed mice not only aggravated hepatic steatosis but also resulted in inflammatory cell infiltration and subsequent severe hepatic injury, which are typical histological features of NASH [13]. OxLDL has been established to promote the production of inflammatory cytokines and chemotactic factors by macrophages [14] and to stimulate coronary artery smooth muscle cells to overexpress interleukin (IL)-1 [15], a crucial gatekeeper of inflammation and tissue damage [16].

Bieghs et al. demonstrated that, after entering Kupffer cells, oxLDL is trapped inside lysosomes, resulting in the increased expression of inflammatory genes, such as those for tumor necrosis factor (TNF)-α and toll-like receptor (TLR)-4 [17]. Collective evidence suggests that the unregulated uptake of oxLDL by scavenger receptors in Kupffer cells leads to the accumulation of lysosomal free cholesterol (oxLDL-derived lipid metabolites) and triggers an inflammatory response in experimental NASH [18]. We found the accumulation of free cholesterol in the periportal area and detected the colocalization of enlarged Kupffer cells and electronegative LDL (mildly oxidized LDL found in blood) in the portal venous area of high-fat, high-cholesterol (HFC) diet-fed hamsters [19]. These factors were found to be associated with the development of NASH and fibrosis [19]. Moreover, we demonstrated that electronegative LDL, which was isolated from the blood of HFC diet-fed hamsters, induced TNF-α production in rat Kupffer cells through a lectin-like oxLDL receptor (LOX)-1 (a scavenger receptor)-dependent pathway [19]. These results suggest a causal role of electronegative LDL and LOX-1 in the development of NASH. Moreover, the accumulation of free cholesterol can result in cholesterol crystallization in vessel walls. Cholesterol crystals have been shown to induce NLRP3 inflammasome activation and subsequent IL-1β production [20]. However, oxLDL particles, which accumulate in early atherosclerotic lesions, are a prerequisite for NLRP3 inflammasome-mediated IL-1β secretion [21]. Although these key mechanisms of oxLDL and free cholesterol-induced liver toxicity have been described in animal models, their relevance in human NASH remains unclear [18]. In a lipidomic analysis, Puri et al. reported a significant increase in hepatic free cholesterol in human NASH [22], but detailed hepatic distribution remains unknown. According to our review of relevant literature, no study has directly demonstrated both oxLDL and free cholesterol in human liver histology. Thus, the present study investigated the accumulation of oxLDL and free cholesterol in the hepatic microenvironment of NAFLD.

The present study revealed that free cholesterol and oxLDL accumulation in the portal venous wall, instead of the hepatic artery, caused focal narrowing of the intrahepatic portal vein by inducing portal venous atherosclerosis in NAFLD. Ruptured portal venous plaques were closely associated with the local expression of free cholesterol, oxLDL, LOX-1, IL-1β, and Kupffer cells. Free cholesterol and oxLDL accumulation in periportal and sinusoidal fibrosis, which was associated with regional stellate cell activation and chicken-wire fibrosis. Figure 5 presents a graphic summary of the tentative rationale. Our study demonstrated that free cholesterol and oxLDL play specific roles in the pathogenesis of NAFLD. These initial results bridge the knowledge gap between atherosclerosis and NASH in metabolic syndrome and suggest that free cholesterol and oxLDL are the missing link and deserve translational investigation in humans. Moreover, our results implied that additional studies should emphasize atherosclerosis in the portal vein, rather than in the hepatic artery, when addressing oxLDL-related vascular injury in NAFLD.

Consistent with the results of our previous animal study [19], the current histopathological observation in human NAFLD showed an association between LOX-1 and Kupffer cell-associated inflammation, which was closely attributed to oxLDL. In our animal model of NASH, active inflammation was documented in the liver parenchyma [19]. Compared with the previous animal model study, in the current study, human surgical specimens of NAFLD showed low-grade parenchymal inflammation but active inflammation at focal fragile portal venous plaques. The phenotypic difference may be explained by the longer duration and lower dose of exposure of oxLDL and free cholesterol in humans than in animals. Furthermore, the specimens were selectively sampled from patients having a low inflammatory status of the liver who were scheduled to undergo partial hepatectomy. Selection bias cannot be avoided. However, the findings revealed that portal venous atherosclerosis, which is novel, occurred in actual clinical scenarios and warrant corresponding studies. Consistently, a previous study reported that increased periportal chronic inflammation is associated with the multiple clinical and pathological features of progressive NAFLD [36]. Altogether, these findings suggest that portal venous atherosclerosis and inflammation in NAFLD are attributed to free cholesterol and oxLDL accumulation. However, additional studies are warranted to consolidate our understanding of the phenomenon.

We found several LOX-1-containing oxLDL-rich fragments with activated macrophages in the portal venous lumen. These particles may be cleaved by enhanced protease activity locally [37] and separated from fragile portal venous plaques, as seen in coronary atherosclerosis concurrent with cardiovascular accidents. Moreover, a previous study reported that the level of soluble LOX-1 is increased in NAFLD patients with steatohepatitis [38]. It remains unknown whether the separated fragments of portal venous plaques cause distal downstream liver damage, thus warranting further investigation.

In the cirrhotic stage of NAFLD, oxLDL presents a chicken-wire pattern in addition to activated stellate cells and fibrosis. LOX-1 expression was not obvious in this stage. Liver sinusoidal endothelial cells and Kupffer cells constitute the largest scavenger cell system for oxLDL in the body [39]. They can effectively eliminate oxLDL in noncirrhotic livers, which might explain the rarity of oxLDL particles in the noncirrhotic sinusoidal parenchyma. Therefore, the accumulation of oxLDL and free cholesterol in the cirrhotic parenchyma suggests the dysfunction of the hepatic scavenger system. Liver fibrosis involves complex dynamic interactions among stellate cells, sinusoidal endothelial cells, Kupffer cells, hepatocytes, and cholangiocytes [40]. oxLDL can induce the activation of stellate cells by increasing the expression of α(I) collagen; α-SMA; and profibrogenic genes, such as those for connective tissue growth factor and transforming growth factor-β receptors in vitro [41]. Furthermore, the accumulation of free cholesterol in cells can cause the progression of liver fibrosis through stellate cells activation by disrupting the integrity of mitochondrial and endoplasmic reticulum (ER) membranes and by triggering mitochondrial oxidative injury and ER stress [42]. Altogether, patients with NAFLD can develop liver fibrosis through alternative pathways other than those mediated by LOX-1.

The limitation of this study is the potential coexistence of native LDL and oxLDL. However, the coexistence of native LDL and oxLDL would not be substantial because the extensively coaccumulated free cholesterol was not likely to have originated from native LDL [42]. The possible presence of other modified forms of LDL (e.g., aggregated LDL or LDL-containing immune complexes), which can also contribute to atherogenesis [43], was not excluded in this study. However, our previous study revealed that periportal oxLDL contributed to the development of experimental NASH, and that anti-LOX-1 agent blocked TNF-α production by Kupffer cells [19]. CD36 is another scavenger receptor for oxLDL and is associated with NLRP3 inflammasomes in NAFLD [7, 21]. We cannot exclude the possibility that and degrees to which IL-1β was contributed to by CD36. Besides, LOX-1 is a non-specific multiligand receptor sensing multiple danger signals, including oxLDL, C-reactive protein and C1q [26]. However, we qualitatively identified the involvement of LOX-1 and Kupffer cells in portal venous plaque inflammation. This is a novel finding in humans and support our observation in the experimental NASH model [19].

Portal venous atherosclerosis induced by free cholesterol and oxLDL accumulation is a unique feature of NAFLD. A ruptured portal venous plaque closely associates oxLDL, LOX-1, and Kupffer cells with portal venous inflammation. In the cirrhotic stage, free cholesterol and oxLDL are associated with chicken-wire fibrosis through stellate cell activation. The present findings reveal a direct association between cholesterol accumulation, portal venous inflammation and fibrosis in NAFLD.