Research Article

Plasma Soluble Lectin-Like Oxidized LDL Receptor 1, A Novel Marker for Atherosclerosis, is Clinically Associated with NAFLD Histological Severity

Ishiba H1, Sumida Y1*, Tanaka S2, Mori K2, Kanemasa K2, Imai S3, Taketani H1, Seko Y1, Hara T1, Okajima A1, Yamaguchi K1, Moriguchi M1, Mitsuyoshi H1, Yasui K1, Minami M1 and Itoh Y1
1Department of Gastroenterology and Hepatology, Kyoto Prefectural University of Medicine, Kyoto, Japan
2Center for Digestive and Liver Diseases, Nara City Hospital, Nara
3Department of Pathology, Nara City Hospital, Nara, Japan


*Corresponding author: Yoshio Sumida, Department of Gastroenterology and Hepatology, Kyoto Prefectural University of Medicine, 465 Kajii-cho, KawaramachiHirokoji, Kamigyo-ku, Kyoto, 602-8566, Japan


Published: 03 Jun, 2016
Cite this article as: Ishiba H, Sumida Y, Tanaka S, Mori K, Kanemasa K, Imai S, et al. Plasma Soluble Lectin-Like Oxidized LDL Receptor 1, A Novel Marker for Atherosclerosis, is Clinically Associated with NAFLD Histological Severity. Remed Open Access. 2016; 1: 1004.

Abstract

Aims: Patients with Non-Alcoholic Fatty Liver Disease (NAFLD), especially steatohepatitis(NASH), are at high risk for Cardio Vascular Disease (CVD). Lectin-like oxidized LDL receptor-1 (LOX-1) is associated with CVD, diabetes, and metabolic syndrome. We aimed to determine whether the level of soluble LOX-1 (sLOX-1), a novel marker for atherosclerosis, is related to the histological severity of NAFLD.
Methods: Plasma sLOX-1 levels were determined in 93 Japanese patients with biopsy-confirmed NAFLD. We evaluated the associations of plasma sLOX-1 with physical and clinical laboratory data and liver histology using the NAFLD Activity Score (NAS) and fibrosis.
Results: In 93 NAFLD patients (76 at fibrosis stage 0–2, 17 at stage 3–4), the plasma levels of sLOX-1 were positively correlated with hyaluronic acid, type IV collagen 7s, and histological fibrosis stage, but not with NAS. The area under the receiver operating characteristic curve of sLOX-1 when separating patients with severe fibrosis was 0.665 with an optimal cutoff at 140 ng/L. The prevalence of patients with >140 ng/L sLOX-1 was significantly higher in those with stage 3–4 (82.4%) than those with stage 0–2 (47.4%, p = 0.03). The association between >140 ng/L sLOX-1 and NASH severe fibrosis persisted after adjusting for age, gender, body mass index, and insulin resistance.
Conclusions: Plasma sLOX-1 level was independently associated with severe fibrosis in NAFLD. This association of sLOX-1 with severe fibrosis implies a link between atherosclerosis and hepatic fibrosis in NAFLD. LOX-1 may be one of targets for drug therapy in NAFLD patients.

Keywords: Lectin-Like Oxidized LDL receptor 1; Liver fibrosis; NASH; Cardiovascular disease

Abbreviations

sLOX-1: soluble Lectin-Like Oxidized LDL Receptor 1; NAFLD: Nonalcoholic Fatty Liver Disease; NASH: Non-Alcoholic Steato-Hepatitis; NAFL: Nonalcoholic Fatty Liver; CVD: Cardio Vascular Disease

Introduction

Non-Alcoholic Fatty Liver Disease (NAFLD) is the most common chronic liver disease in many developed countries, affecting 20–30% of the general adult population. NAFLD encompasses a wide spectrum of liver diseases, ranging from Non-Alcoholic Fatty Liver (NAFL), which is typically a benign and non-progressive condition, to Non-Alcoholic Steato-Hepatitis (NASH), which may progress to liver cirrhosis and hepatocellular carcinoma [1-3]. In recent years, a possible role of NAFLD in the development of Cardio Vascular Disease (CVD) has been suggested, and this relationship appears to be independent of obesity and metabolic syndrome [4]. However, the mechanism underlying the relationship between NAFLD and CVD remains unclear. Recently, oxidatively modified low-density lipoprotein (Ox-LDL) cholesterol, compared with unmodified LDL cholesterol, was reported to be a more problematic risk factor for arteriosclerotic disease, including CVD and cerebrovascular disease, among others [5,6]. Lectin-Like Oxidized LDL receptor-1 (LOX- 1) is a type II single-transmembrane protein, which was identified as an endothelial cell surface major receptor for Ox-LDL, but not unmodified LDL [7,8]. LOX- 1 initiates many pathological pathways including inflammation, endothelial dysfunction, apoptosis, and fibrosis [9]. LOX-1 is associated with various arteriosclerotic diseases, including CVD, cerebrovascular disease, and peripheral artery disease [10,11]. A novel sandwich enzyme immunoassay for LOX-1 ligand, which uses a recombinant soluble form of LOX-1 and anti-apo B antibody, was recently developed to detect circulating modified LDL via specific binding to LOX-1 [12]. Estimates of the circulating levels of sLOX-1 were found to be associated with the progress of atherosclerosis and acute coronary syndrome, and with increased serum oxidative stress and inflammatory biomarkers [13]. Additionally, Ox-LDL, which is a ligand of LOX-1, was reported to be associated with hepatic fibrosis in a rat experimental model [14]. LOX-1 polymorphism appears to be associated with histological severity of NASH in humans [15]. However, there have been no reports concerning plasma or serum levels of sLOX-1 in NAFLD patients. The aim of this study was to determine the associations between plasma sLOX-1 and physical and clinical parameters, including liver histology in patients with NAFLD.

Table 1

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Table 1
Characteristics of patients with biopsy-confirmed NAFLD.

Patients and Methods

Patients
This study was a cross-sectional analysis of the relationship between plasma sLOX-1 concentration and clinical characteristics, including liver histology, in adult NAFLD patients. In this study, plasma sLOX-1 concentration was measured in 93 patients with biopsy-confirmed NAFLD. Patients received a liver biopsy at the Center for Digestive and Liver Diseases, Nara City Hospital from November 2012 to March 2013. The diagnosis of NAFLD was based on the following criteria: (1) persistent elevation of transaminase activities for more than 6 months, (2) a liver biopsy showing steatosis in at least 5% of hepatocytes [16], and (3) the appropriate exclusion of liver diseases with separate etiologies, including viral hepatitis, autoimmune hepatitis, drug-induced liver disease, primary biliary cirrhosis, biliary obstruction, hemochromatosis, Wilson's disease, and α-1-antitrypsin-deficiency-associated liver disease, or suspected or decompensated liver cirrhosis and hepatocellular carcinoma. We excluded all patients with an average alcohol consumption of more than 30g daily for men and more than 20g daily for women during the prior two years. Written informed consent was obtained from all patients at the time of their liver biopsy, and the protocol for this study protocol was approved by the Institutional Review Board of Nara City Hospital and conformed to the ethical guidelines of the 1975 Declaration of Helsinki.
Covariates
Physical characteristics and clinical laboratory data were examined for each patient. The physical factors included age, gender, and Body Mass Index (BMI), and the clinical characteristics included a confirmed diagnosis of hypertension and Diabetes Mellitus (DM). Blood samples were taken in the morning after a 12-hour overnight fast. Plasma sLOX-1 concentrations were measured by sandwich ELISA using two specific monoclonal antibodies against sLOX-1 with recombinant sLOX-1 as an assay standard [10]. Plasma sLOX- 1 was measured by using commercially available ELISA kit. The measurement was performed at Biomarker Science Co. Limited (Kyoto, Japan). The plasma samples were stored at -80oC until assays were performed. Repeated freezing and thawing of plasma was excluded. The following clinical laboratory data were also collected: a blood cell count and values for Aspartate Aminotransferase (AST), Alanine Aminotransferase (ALT), gamma Glutamyl Transpeptidase (γGT), Cholinesterase (ChE), Albumin (Alb), creatinine, estimated Glomerular Filtration Rate (eGFR), total cholesterol, triglyceride, High-Density Lipoprotein Cholesterol (HDL-C), Low-Density Lipoprotein Cholesterol (LDL-C), Fasting Plasma Glucose (FPG), prothrombin time, hyaluronic acid, type IV collagen 7S, Immune Reactive Insulin (IRI), Homeostasis Model Assessment-Insulin Resistance (HOMA-IR), and HbA1c. These were measured using standard techniques in clinical laboratories. BMI was calculated as (weight in kilograms) / (height in meters). Obesity was defined as a BMI >25.0, according to the criteria of the Japan Society for the Study of Obesity [17]. Patients were assigned a diagnosis of DM if a documented use of oral hypoglycemic medication, a random glucose level in excess of 200 mg/dL, or an FPG >126 mg/dL was present [18]. Dyslipidemia was diagnosed if the cholesterol level was higher than 220 mg/dL and/or the triglyceride level was >160 mg/dL. Hypertension was diagnosed if the patient was on antihypertensive medication and/or had a resting recumbent blood pressure≥140/90 mmHg on at least two occasions. The HOMA-IR was calculated based on fasting values of plasma glucose and insulin according to the HOMA model formula: HOMA-IR = (IRI [μU/mL] × FPG [mg/dL] / 405 [19]. eGFR was calculated as eGFR = 194 × Creatinine−1.094 × age−0.287. For female patients, this formula was multiplied by 0.739 [20]. FIB4 index, one of the non-invasive fibrosis scores for predicting severe fibrosis, was calculated using the following formula: [age (years) × AST (IU/L)] / [platelet counts (×109/L) × ALT1/2 (IU/L)] [21].
Liver histology
All patients enrolled in this study underwent a percutaneous liver biopsy under ultrasonic guidance. The liver specimens were embedded in paraffin and stained with hematoxylin and eosin, Masson-trichrome, and reticulin silver stain. Two hepatopathologists (S.I and Y.S.) were blinded to the clinical data and reviewed the liver biopsy specimens. If the diagnosis is discordant, final diagnoses were determined through discussion between the two hepatopathologists. An adequate liver biopsy sample was defined as a biopsy specimen with length >1.5 cm and/or having more than 6 portal tracts. NASH was defined as steatosis with lobular inflammation and ballooning degeneration with or without Mallory–Denk bodies or fibrosis. Patients whose liver biopsy specimens showed steatosis or steatosis with nonspecific inflammation were identified as the NAFL cohort [2,3]. The score, named NAS (NAFLD Activity Score) proposed by Kleiner et al. [16] is the unweighted sum of the scores for steatosis (0-3), lobular inflammation (0-3), and ballooning degeneration (0-2). NAS is ranging from 0 to 8. The severity of hepatic fibrosis (stage) was defined as Stage 1: zone 3 perisinusoidal fibrosis; Stage 2: zone 3 perisinusoidal fibrosis with portal fibrosis; Stage 3: zone 3 perisinusoidal fibrosis and portal fibrosis with bridging fibrosis; or Stage 4: cirrhosis [22].
Statistical analysis
Data are expressed as median and ranges for quantitative date or as numbers of patients with percentages in parentheses for qualitative data. Statistical differences between the two groups were analyzed by Mann–Whitney U tests for quantitative data and by Fisher's or chi-square tests for qualitative data (Table 1 and 3). Normality was confirmed using Shapiro–Wilk analysis. The correlation coefficients between sLOX-1 and clinical parameters were calculated using Spearman rank correlation analysis (Table 2, Figure 1 and 2). Multiple logistic regression analysis was used to identify variables independently associated with advanced liver fibrosis of NASH (Table 4). To assess the accuracy of clinical parameters in differentiating advanced fibrosis (stage 3-4) from no or mild fibrosis (stage 0-2), we calculated the sensitivity and the specificity for each value of each test and then constructed ROC curves by plotting the sensitivity against the reverse specificity (1 minus specificity) at each value. The diagnostic performance of the scoring systems was assessed by analysis of the ROC curves. The most commonly used index of accuracy is the Area Under the ROC curve (AUROC), with values close to 1.0 indicating high diagnostic accuracy. The Youden index was used to identify the optimal cut-off points. All analyses were performed using SPSS (v22). Nominal, two-sided P values were used and were considered statistically significant for P values less than 0.05, a priori.

Table 2

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Table 2
Correlations between sLOX-1 and clinical parameters in 93 patients with biopsy-confirmed NAFLD.

Figure 1

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Figure 1
Variation in sLOX-1 levels with total NAFLD activity score, steatosis, inflammation, and hepatocyte ballooning for patients with NAFLD. The box includes the values between the 25th and 75th percentiles, and the bold horizontal line represents the median. The error bars extend from the 10th to the 90th percentile.

Figure 2

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Figure 2
Variation in sLOX-1 levels with fibrosis stage for patients with NAFLD. The box includes the values between the 25th and 75th percentiles, and the bold horizontal line represents the median. The error bars extend from the 10th to the 90th percentile.

Table 3

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Table 3
Characteristics for each parameter separated into fibrosis stage 0–2 and stage 3–4.

Table 4

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Table 4
Logistic regression models for the association of advanced liver fibrosis (stage 3–4) with sLOX-1 (≥140 ng/L) and other clinical variables.

Results

Demographic, clinical, and laboratory characteristics in NASH compared to NAFLD
Among the 93 patients with biopsy-confirmed NAFLD, 70 patients (75%) were histologically classified as NASH and 23 patients (25%) were classified as NAFL. The characteristics of patients with NASH and NAFL are shown in (Table 1). Compared with NAFL patients, NASH patients were more likely to be female and older, and had higher levels of AST, hyaluronic acid, type IV collagen 7S, IRI, and HOMA-IR, but lower levels of creatinine and prothrombin time. There was no statistical difference in sLOX-1 levels between NASH and NAFL patients. Nor were there any statistical differences in sLOX-1 levels according to gender (male: 154.5 ng/L vs. female: 149.4 ng/L; p = 0.668) or the existence of DM (with DM: 148.2 ng/L vs. without DM: 151.7 ng/L; p = 0.939). Patients with insulin resistance (HOMA-IR>2.5) had significantly higher levels of sLOX-1 (154.6 ng/L) compared to those without (HOMA-IR<2.5, 139.1 ng/L; p = 0.031).
Spearman's rank correlation between sLOX-1 levels and physical and clinical variables and liver histology
Spearman's rank correlations between sLOX-1 levels and different physical, metabolic, and hepatic variables were calculated. Plasma sLOX-1 levels were negatively correlated with ChE (r = −0.230, p = 0.045), and positively correlated with hyaluronic acid (r = 0.248, p = 0.021), type IV collagen 7S (r = 0.255, p = 0.014) and FIB4 index (r = 0.269, p = 0.012) (Table 2). No correlations were found between the other physical and clinical variables. Evaluated with liver histology, the sLOX-1 level tended to increase in parallel with increased NAS (r = 0.210, p = 0.076), but not with the grade of steatosis, inflammation, or ballooning (Figure 1). The sLOX-1 level was only positively correlated with fibrosis stage (r = 0.225, p = 0.03, (Figure 2)).
Mann–Whitney U test comparing patients with fibrosis stages 0–2 or 3–4
Clinical characteristics were compared between NAFLD patients with liver fibrosis stage 0–2 (n = 76) and stage 3–4 (n = 17) (Table 3). No statistical differences in gender, age, BMI, or the prevalence of DM were found between the two groups. Patients with stage 3–4 exhibited lower levels of platelet count, ChE, cholesterol, and prothrombin time, and higher levels of γGT, hyaluronic acid, type IV collagen 7s, IRI, and HOMA-IR. There were no statistical differences in HDL-C and LDL-C between the two groups. The AUROC for plasma sLOX- 1 levels in separating patients with and without severe fibrosis was 0.665, with an optimal cutoff value of 140 ng/L. A significantly higher percentage of patients exhibited plasma sLOX-1 levels >140 ng/mL in the advanced stage (82.4%) compared to that in mild stage (52.6%, p = 0.03). The sensitivity of the sLOX-1 value of >140 ng/mL for the presence of advanced fibrosis was 94.1% (16/17) and the specificity was 47.4% (36/76). The positive predictive value of the cutoff was 28.6% (16/56), and the negative predictive value was 97.3% (36/37).
Multiple logistic regression analysis of the advanced NASH groups
Several multiple logistic regression models were used to determine the association of sLOX-1 levels with advanced fibrosis in NAFLD characteristics. The variables were selected from life background, for example, age, gender, the prevalence of DM, insulin resistance, and sLOX-1. The liver enzymes and fibrosis markers were excluded because these variables were strongly collated with liver fibrosis. As shown in (Table 4), the unadjusted (model 1) association of sLOX- 1 levels with advanced fibrosis remained highly significant when after adjusting for gender and age (model 2). The sLOX-1 levels also remained highly significantly associated with liver fibrosis after being adjusted for BMI, insulin resistance, and the presence of DM (models 3, 4, and 5).

Discussion

The results of the present study demonstrated that the plasma sLOX-1 concentration was associated with histological severity of NAFLD by multivariate analysis, independent of gender, age, BMI, and insulin resistance.
To date, LOX-1 has been implicated in the pathogenesis of several different diseases, including CVDs, such as atherosclerosis, hypertension, myocardial infarction, and congestive heart failure [7,23,24]. And more, serum levels of sLOX-1 levels are reported to be elevated in coronary artery disease [13,25]. Although the exact mechanisms underlying the elevation of sLOX-1 in advanced stages of NASH remain unknown, we can suggest several plausible explanations. First, LOX-1 may promote liver disease indirectly through the modulation of pro- and anti-inflammatory adipokines, which play a key role in the pathogenesis of liver injury in NASH. In the present study, however, the plasma sLOX-1 level was not associated with histological inflammation, which tended to increase with NAS. There were no significant differences in sLOX-1 levels between NASH and NAFL. Unfortunately, inflammatory cytokines or adipokines were not evaluated in this study. Another plausible explanation is that sLOX-1 promotes Reactive Oxygen Species (ROS), which contribute to hepatic fibrosis by activating Hepatic Stellate Cells (HSCs). During oxidative stress, ROS acts on LDL, which undergoes oxidative modification forming Ox-LDL. Elevated Ox-LDL concentrations have been reported to correlate with histologic severity in liver tissues samples from NAFLD patients [26]. These Ox-LDL moieties activate the LOX-1 receptor, which further induces oxidative stress by increasing ROS generation via the expression of the NADPH oxidase system [27], creating a positive feedback loop. Functional regulation of LOX-1 activity may modulate not only atherogenesis in the vessel wall, but also fibrosis of organs, such as cardiac or hepatic fibrosis [28]. Extracellular Ox-LDL is transported into HSCs, mediated by LOX- 1, leading to the stimulation of HSC activation. In mouse models of NAFLD, LOX-1 expression is a required step to activate HSCs and to trigger hepatic fibrogenesis [29]. Furthermore, the association of LOX-1 SNP (G allele carriers) with severe liver histology suggests that LOX-1 function may directly modulate the progression of liver diseases in NASH [15]. In that study, the G allele was associated with lipoprotein metabolism (higher triglyceride rich lipoprotein), adipokine imbalance (higher resistin and lower adiponectin), and increased cytokeratin-18 fragments (CK-18), though the plasma or serum levels of sLOX-1 were not determined.
Higher levels of sLOX-1 in the advanced fibrosis of NASH suggest a possible link between liver disease progression and atherosclerosis in NAFLD. Such a relationship between liver disease severity and atherosclerosis is controversial. Previous studies suggest there is a protective effect of (at time mostly viral) cirrhosis on CVD [30], possibly caused by reduced cholesterol, fibrinogen, and platelet count. In contrast, recent studies described a higher prevalence of major risk factors for atherosclerosis and coronary artery disease in NASH- and ALD-related cirrhosis [31].
Although an association between NAFLD and CVD has recently been established, the biological mechanisms by which NAFLD may contribute to the acceleration of atherosclerosis remain poorly understood. The accumulating evidence suggests that NASH patients are at a higher CVD risk compared to NAFL patients because of their more atherogenic lipid profile [32-34]. Based on data from 85 patients with biopsy-confirmed NAFLD, the severity of the histopathological features in NAFLD is strongly associated with early carotid atherosclerosis, independent of classical risk factors, insulin resistance, and metabolic syndrome [35]. NASH patients also had higher circulating levels of c-reactive protein, fibrinogen, and Plasminogen Activator-1 (PAI-1), and lower levels of adiponectin compared to NAFL patients [33]. The present study suggests that the mechanism underlying the association between atherosclerosis and the severity of NAFLD may be explained, at least partly, by the elevated sLOX-1 levels.
Several important limitations of this study should be addressed. First, there may be a patient selection bias because we selected patients who were likely to have NASH when we considered liver biopsy for the NAFLD patients. Second, the proportion of subjects with advanced fibrosis was small (18.3%). We acknowledge that the pathological diagnosis was mainly determined using liver tissues received by percutaneous liver biopsy, a method which is prone to sampling error and inter-observer variability [36]. We did not measure the levels of sLOX-1 in patients with other liver disease or healthy people. According to a recent report, the levels of plasma sLOX-1 levels (medians [interquartile ranges]) were 113 (93, 141) ng/L in healthy men (n=310) and 116 (100-140) ng/L in healthy women (n=205) [37]. Though it was impossible to compare those values with ours by statistical analyses, NAFLD patients seem to have higher levels of sLOX-1 compared to healthy population. The association of sLOX-1 with severe fibrosis suggests a possible link between hepatic fibrosis and atherosclerosis in NAFLD, although the status of atherosclerosis such as carotid intima-media thickness, ankle-brachial index, or pulse wave velocity was not evaluated. In the future, it is necessary to estimate the association between liver fibrosis and the status of atherosclerosis. sLOX-1 as not been established yet to estimate Ox-LDL cholesterol and is not wide used in clinical practice now. However in above mentioned, sLOX-1 has potential to represent the existence of Ox-LDL and ROS and to be an important surrogate marker or target in therapy. Because of these limitations, validation and longitudinal studies are required to reach firm conclusions. However, this study is the first to clarify the relationship between hepatic fibrosis and sLOX-1 in patients with biopsy-confirmed NAFLD.

Conclusion

The concentration of circulating plasma sLOX-1, a novel marker for atherosclerosis, was found to be associated with severe fibrosis in NAFLD patients. The association of sLOX-1 and severe fibrosis implies a link between atherosclerosis and hepatic fibrosis in NAFLD. LOX-1 may be one of targets for drug therapy in NAFLD patients.

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