Association between non-alcoholic fatty liver disease and subclinical atherosclerosis in Western and Asian cohorts: an updated meta-analysis

Abstract

Background

Non-alcoholic fatty liver disease (NAFLD) is a well-established risk factor for cardiovascular disease, with ethnic and regional differences noted. With the recent surge of research within this field, we re-examine the evidence associating NAFLD with subclinical atherosclerosis, and investigate potential regional differences.

Methods

This is a systematic review and meta-analysis. PubMed and EMBASE were systematically searched for publications from January 1967 to July 2020 using standardised criteria. Original, observational studies investigating the association between NAFLD and either carotid intima-media thickness (CIMT) and/or coronary artery calcification (CAC) were included. Key outcomes included differences in mean CIMT, the presence of increased CIMT, the presence of CAC and the development/progression of CAC. Pooled ORs and pooled standard differences in means were calculated using random-effects models. Between-study heterogeneity was quantified using the Q statistic and I². Subgroup analyses stratified by region of study (Asian vs Western) were also conducted.

Results

64 studies involving a total of 172 385 participants (67 404 with NAFLD) were included. 44 studies assessed the effect of NAFLD on CIMT, with the presence of NAFLD associated with increased CIMT (OR 2.00, 95% CI 1.56 to 2.56). 22 studies assessed the effects of NAFLD on CAC score, with the presence of NAFLD associated with the presence of any coronary calcification (OR 1.21, 95% CI 1.12 to 1.32), and the development/progression of CAC (OR 1.26, 95% CI 1.04 to 1.52). When stratified by region, these associations remained consistent across both Asian and Western populations (p>0.05). The majority (n=39) of studies were classified as ‘high quality’, with the remaining 25 of ‘moderate quality’.

Conclusions

There is a significant positive association between various measures of subclinical atherosclerosis and NAFLD, seen across both Western and Asian populations. These results re-emphasise the importance of early risk evaluation and prophylactic intervention measures to preclude progression to clinical cardiovascular disease in patients with NAFLD.

Key questions.

What is already known about this subject?

• Non-alcoholic fatty liver disease (NAFLD) is a significant, independent risk factor for cardiovascular disease (CVD), with recent evidence positing this association to extend to the preclinical stages of CVD. Previous meta-analyses have quantified positive associations between NAFLD and subclinical atherosclerotic markers before, though the majority of included studies were published before 2016. The last 5 years, however, has experienced a large surge of research in this field, especially within large Asian populations that have not been included in previous meta-analyses. Ethnic and regional differences in the associations between NAFLD and subclinical atherosclerosis have been suggested within individual studies, but have yet to be synthesised across the available literature.

What does this study add?

• This meta-analysis serves as a timely update of the existing literature, incorporating the results of over 21 new studies comprising over 100 000 participants (~50 000 with NAFLD) from both Western and Asian regions. The results reinforce the significant positive association between NAFLD and subclinical atherosclerosis (as defined by increased carotid intima-media thickness and coronary artery calcification scores), and further confirm these associations to be consistent across both Western and Asian populations. Lastly, this is the first meta-analysis to demonstrate that the associations between NAFLD and subclinical atherosclerosis are not just crosssectional but also longitudinal.

Key questions.

How might this impact on clinical practice?

• This study highlights that NAFLD serves as an important atherogenic risk factor in both Western and Asian populations, and reemphasises the role of early risk evaluation and prophylactic intervention measures to preclude progression to clinical CVD in NAFLD. By confirming a longitudinal association between NAFLD and subclinical atherosclerotic markers, these results also provide potential insight into the causal relationship between NAFLD and subclinical atherosclerosis.

Introduction

Non-alcoholic fatty liver disease (NAFLD) encompasses a wide spectrum of pathological hepatic conditions ranging from simple steatosis to non-alcoholic steatohepatitis, and may ultimately progress to advanced fibrosis, cirrhosis, and end-stage liver disease.^1–3 Over the last 20 years, NAFLD has become the leading cause of chronic liver disease, with an estimated 1 billion people affected worldwide.⁴ NAFLD has increasingly been recognised as the hepatic manifestation of the metabolic syndrome (MetS), and is one facet of a multisystem disease, with close relations to abdominal obesity, type 2 diabetes mellitus (T2DM), insulin resistance and hyperlipidaemia.^5–7

Cardiovascular disease (CVD) is the leading cause of mortality in patients with NAFLD, with a large body of evidence demonstrating NAFLD to be a significant, independent risk factor for CVD.2 3 5 8 It is now widely hypothesised that NAFLD is not merely a marker of CVD, but may be actively involved in CVD pathogenesis.^{3 9–11} This association extends to preclinical CVD, with recent work identifying NAFLD as a risk factor for early subclinical atherosclerosis, and as a strong independent predictor of incident CVD.^{3 8 12} This holds highly important implications for the screening and early evaluation of CVD in patients with NAFLD. Carotid intima-media thickness (CIMT) and coronary artery calcification (CAC) are the two most established and widely studied surrogate measures of subclinical atherosclerosis, and a growing body of literature has investigated this relationship between NAFLD and CAC/CIMT over the last decade.^{3 12 13}

NAFLD is no longer considered a disease only prevalent in affluent Western countries, with rapidly growing rates of NAFLD reported within Asia in particular.^{4 14} Ethnic and regional differences in NAFLD prevalence, severity and outcomes have been identified between Western, Hispanic and Asian populations,^{15 16} and have been attributed to factors including lifestyle, environment, insulin resistance, body composition (adipose distribution and muscle bulk) and genetics.14 15 17 18 Asian populations are especially susceptible with cardiometabolic complications such as NAFLD seen to develop within a much shorter period, within younger patient populations and in those with lower body mass index.^{18 19} These disparities may possibly extend to differential associations between NAFLD and subclinical atherosclerosis.^20–22

Previous meta-analyses have quantified the associations between NAFLD and subclinical atherosclerotic markers before, with the majority of included studies published before 2016.^{13 23–25} However, the last 5 years has experienced a large surge in research within this area, especially within large Asian populations that have not been reported in previous meta-analyses.^26–35 We aim to evaluate the relationship between NAFLD and subclinical atherosclerosis including these updated studies, and to further investigate potential regional differences in these associations.

Methodology

This meta-analysis was conducted and reported according to the Meta-analysis Of Observational Studies in Epidemiology statement³⁶ and was registered in the International Prospective Register of Systematic Reviews (registration number: CRD42020204784).

Search strategy

A comprehensive literature search was performed via the MEDLINE and EMBASE databases to identify potentially relevant publications in the English language, with a date range from January 1967 to July 2020. The databases were systematically searched using a combination of the following keywords linked with appropriate Boolean logic: (Fatty Liver OR NAFLD OR Hepatic Steatosis OR Non-alcoholic fatty liver disease) AND ((subclinical atherosclerosis OR Preclinical atherosclerosis) OR (Coronary calcium OR Calcium Score OR Coronary Calcification) OR (“Carotid Intima-media thickness” OR CIMT OR IMT OR “‘intima media thickness”’)). Relevant references identified from the bibliographies of pertinent articles or review papers were also retrieved.

Eligibility (inclusion and exclusion) criteria

The eligibility criteria was based on the PICOS framework as recommended by the Preferred Reporting Items for Systematic Reviews and Meta-Analyses statement.³⁷

1. Participants: studies had to be conducted on adult participants. Studies conducted on ‘special populations’ including adolescent/paediatric populations, and those defined by additional pathologies such as HIV, severe CVD or liver transplants were rejected. Populations with existing metabolic conditions such as MetS and diabetes mellitus were accepted.

2. Exposures (intervention): studies had to have a defined exposure of ‘NAFLD’ or ‘fatty liver’ or ‘hepatic steatosis’, as diagnosed by either ultrasound (US), liver biopsy, CT, magnetic resonance spectroscopy (MRS) or Fatty Liver Index.

3. Outcomes: study outcomes had to report on either the (1) presence (cross-sectional) of CAC (CAC score >0), (2) progression (longitudinal) of CAC score, and/or (3) on CIMT. The presence of calcified coronary artery plaques was accepted as a measure of CAC score>0. Studies had to specify how CAC and CIMT were recorded and defined, and also had to quantitatively assess the association between NAFLD and CAC/CIMT, respectively, either via logistic regression for categorical outcomes or via comparison of means techniques (t-test/analysis of variance (ANOVA)) for continuous outcomes.

4. Comparison: studies had to include a ‘healthy’ control group of participants without NAFLD, preferably from the same population as the exposure group.

5. Study design: we included observational studies (cross-sectional, case–control, retrospective, prospective), which reported quantitative outcomes. Descriptive studies, reviews and studies on animals were excluded. Studies with sample sizes <50 were also excluded.

Using our search strategy, a total of 1007 titles were initially identified. Two authors (MYZW and JJLY) assessed the titles independently according to the predefined inclusion and exclusion criteria. Studies were first screened by title and abstract. The full-text articles deemed potentially relevant were then obtained and systematically included after detailed examination. The following data were extracted: (a) study: year, region, design; (b) patients: mean age, gender, sample size; (c) method of NAFLD evaluation: US, CT, MRS, liver biopsy or composite index; (d) outcomes: outcome type (CIMT or CAC) and method of outcome definition; (e) analysis: statistical techniques used, primary outcomes (mean±SD, ORs with 95% CIs), confounders adjustment.

For studies reporting multiple multivariable-adjusted models, we extracted those reflecting the greatest degree of control for potential confounders. Any discrepancies in data quantification were resolved by discussion among the investigators.

Study quality evaluation

The quality of observational studies was assessed using a modified version of the Newcastle–Ottawa Scale (NOS) for cohort and cross-sectional studies.^38–40 The NOS awards a maximum of 9 stars to assess quality based on three main aspects: (a) the selection and representativeness of the participants (maximum 4 stars), (b) the comparability of groups (maximum 2 stars), and (c) the ascertainment of exposure (for case–control) or outcome (for prospective and cross-sectional) (maximum 3 stars). Following previous reviews, studies assigned 0–4, 5–7, and ≥8 stars were considered as low, medium and high quality, respectively.^41–43

Data synthesis and statistical analysis

Outcomes were broadly grouped according to four main categories:

1. Differences in mean CIMT (continuous).

2. Presence of increased CIMT (categorical).

3. Presence of CAC (categorical).

4. Development/progression of CAC (categorical, longitudinal).

All outcomes were pooled using DerSimonian-Laird random-effects model. The continuous and categorical outcome was reported as pooled standard differences (Std Diff) in means and ORs with 95% CI. We further conducted subgroup analysis to look into regional differences between Asian versus Western populations. We defined ‘Western’ studies to comprise of studies conducted in North America, Europe and Australia, while ‘Asian’ studies comprised of those conducted in South Asian, East Asian and Southeast Asian countries. Lastly, additional subgroup analysis on the Std Diff in mean CIMT within the subset of participants with diabetes was conducted.

The heterogeneity of pooled estimates between studies was quantified using the Q statistic and I². A value of I² of 0%–25% indicates no heterogeneity, 26%–50% low heterogeneity, 51%–75% moderate heterogeneity and 76%–100% high heterogeneity. Funnel plots and Egger’s regression test were used to assess publication bias. P<0.05 was considered as statistical significance.

All statistical analyses were conducted using the Comprehensive Meta-Analysis Software V.3.3.

Results

Search strategy and description of studies

The initial search yielded 1007 potentially relevant titles, where 835 articles were excluded on the basis of title and abstract screen. A total of 172 titles underwent full-length review, of which 108 were further excluded (figure 1). A final total of 64 studies, involving 67 404 patients with NAFLD and 104 981 controls were included in the meta-analysis. Tables 1–3 describe the detailed characteristics of the included studies, grouped by study outcome. These included studies were carried out in Asia (n=32), Western Europe (n=15), the Middle East (n=10) and America (n=7; North America: 6, South America: 1). Sixty studies were cross-sectional and four were prospective cohort studies.

Figure 1.

Study selection PRISMA flow diagram. CAC, coronary artery calcification; CIMT, carotid intima-media thickness; PRISMA, Preferred Reporting Items for Systematic Reviews and Meta-Analyses.

Table 1.

Characteristics of included studies which conducted a comparison of carotid-intima media thickness (CIMT) means between those with NAFLD and those without

Name, year	Study region	Study population	Study size	n (%) NAFLD	Age (NAFLD+ vs NAFLD−)	% male (NAFLD+ vs NAFLD−)	NAFLD assessment	Outcome assessment	Confounder adjustment	NOS (max=9)
Oni et al22 2019	North America	Population based	4123	729 (17.7)	61 vs 63	47.0 vs 44.0	CT, LS ratio <1	Ultrasound, mean IMT (L&R)	–	7
Mohammadzadeh et al44 2019	Iran	Hospital based	300	150 (50.0)	49.9 vs 52.5	65.3 vs 57.3	Ultrasound	Ultrasound, mean IMT (L&R)	–	6
Yi et al45 2018	Asia	Outpatient clinic	1981	1888 (95.3)	45.9 vs 44.8	63.4 vs 40.1	Ultrasound	Ultrasound, mean of max IMT (L&R)	–	6
Kim et al30 2018	Asia	Population (health screen)	819	330 (40.3)	53.4 vs 53.1	64.2 vs 41.5	Ultrasound	Ultrasound, mean IMT (L&R)	–	6
Vanjiappan et al46 2018	Asia	Hospital based, patients with T2DM	124	73 (58.9)	Overall=53.8	Overall=54.0	Ultrasound	Ultrasound, mean of max IMT (L&R)	–	6
Gummesson et al47 2018	Europe	Population based	1015	106 (10.4)	58.3 vs 57.5	71.7 vs 52.5	CT, liver HU <40	Ultrasound, mean IMT	–	7
Cetindağlı et al48 2017	Turkey	Outpatient clinic	120	93 (77.5)	34.5 vs 33.8	100 vs 100	Ultrasound and biopsy	Ultrasound, mean IMT (6 measurements)	Age/sex-matched controls	7
Guo et al33 2017	Asia	Hospital based, patients with T2DM	8571	4340 (50.6)	57.4 vs 61.9	54.6 vs 55.9	Ultrasound	Ultrasound, mean IMT (6 measurements)	Age	7
Hong et al35 2016	Asia	Population (health screen)	955	342 (35.8)	53 vs 51 (median)	48.8 vs 42.1	Ultrasound	Ultrasound, mean IMT (99 computer points)	–	7
Zhang et al49 2016	Asia	Outpatient clinic, patients with T1DM	722	123 (17.0)	47.4 vs 46.0	52.8 vs 51.1	Ultrasound	Ultrasound, mean IMT (6 measurements)	Age, sex, BMI, WC, SBP, DBP, total cholesterol, TAG, LDL, HDL, MetS, ALT, AST, GGT, hsCRP, medications	8
Ozturk et al50 2015	Turkey	Outpatient clinic, MetS(−)	82	41 (50.0)	32.8 vs 31.8	100 vs 100	Biopsy	Ultrasound, mean IMT (L&R)	–	6
Asakawa et al55 2014	Asia	Population (health screen)	76	24 (31.6)	61.5 vs 61.0 (median)	91.7 vs 75.0	Ultrasound	Ultrasound, max IMT	–	6
Ayaz et al54 2014	Turkey	Outpatient clinic	90	60 (66.7)	44.5 vs 39.5 (median)	36.7 vs 26.7	Ultrasound	Ultrasound, mean IMT (8 measurements)	–	6
Kim et al53 2014	Asia	Population (health screen), MetS(−)	1285	180 (14.0)	55.7 vs 55.7	58.0 vs 36.0	CT, liver minus spleen <5	Ultrasound, mean IMT (4 measurements)	–	7
Kim et al52 2014	Asia	Hospital based, patients with T2DM	1211	747 (61.7)	56.7 vs 55.6	51.0 vs 41.8	Ultrasound	Ultrasound, mean IMT (6 measurements)	–	6
Nahandi et al51 2014	Iran	Hospital based, patients without diabetes	102	50 (49.0)	43.3 vs 43.1	32.0 vs 40.4	Ultrasound	Ultrasound, mean of max IMT (L&R)	HLP, sex, Smk, HT, obesity, walking, liver enzymes	8
Dogru et al58 2013	Europe	Outpatient clinic	189	115 (60.8)	31 vs 28 (median)	100 vs 100	Liver biopsy	Ultrasound, mean IMT (6 measurements)	–	6
Kucukazman et al57 2013	Europe	Outpatient clinic	161	117 (72.7)	45.8 vs 45.4	44 vs 32	Ultrasound	Ultrasound, mean IMT (6 measurements)	–	6
Mishra et al56 2013	Asia	Population based	645	101 (15.7)	31.6 vs 27.1	100 vs 100	Ultrasound	Ultrasound, mean of max IMT (L&R)	–	7
Huang et al62 2012	Asia	Population based	8632	2590 (30.0)	58.5 vs 58.5	31.4 vs 30.9	Ultrasound	Ultrasound, max IMT (L&R)	–	7
Kang et al61 2012	Asia	Outpatient (health screen), MetS(−)	413	157 (38.0)	52.0 vs 52.5	51.0 vs 41.8	Ultrasound	Ultrasound, mean IMT (L&R)	–	7
Thakur et al59 2012	Asia	Hospital based	80	40 (50.0)	42.1 vs 41.9	67.5 vs 67.5	Ultrasound	Ultrasound, mean IMT (6 measurements)	–	7
Colak et al63 2012	Turkey	Outpatient clinic	87	57 (65.5)	44.2 vs 42.7	45.6 vs 46.7	Liver biopsy	Ultrasound, mean IMT (6 measurements)	–	6
Agarwal et al66 2011	Asia	Hospital based, patients with T2DM	124	71 (57.3)	57 vs 61	52.5 vs 58.5	Ultrasound	Ultrasound, mean IMT	–	6
Mohammadi et al65 2011	Iran	Hospital based	335	250 (74.6)	46.6 vs 44.9	55.6 vs 54.1	Ultrasound	Ultrasound, mean IMT (6 measurements)	HT, DM, HLP, hyperglycaemia	8
Poanta et al64 2011	Europe	Outpatient clinic, patients with T2DM	56	38 (67.9)	59.4 vs 61.5	50.0 vs 83.3	Ultrasound	Ultrasound	–	5
Kilciler et al69 2010	Europe	Outpatient clinic	114	60 (52.6)	31.7 vs 30.3	100 vs 100	Biopsy	Ultrasound, mean IMT (L&R)	Age-matched controls	6
Salvi et al68 2010	Europe	Population based	220	92 (41.8)	50.7 vs 49.3	54.3 vs 36.7	Ultrasound	Ultrasound, mean IMT (6 measurements)	–	7
Vlachopoulos et al67 2010	Europe	Outpatient clinic	51	28 (54.9)	55.4 vs 51.5	52.3 vs 64.3	Biopsy	Ultrasound, mean IMT (L&R)	Age/sex-matched controls	6
Gastaldelli et al73 2009	Europe	Population based	842	234 (27.8)	42 vs 45	69.7 vs 24.0	Fatty liver index >60	Ultrasound, mean IMT (10 measurements)	–	6
Karakurt et al72 2009	Turkey	Not mentioned	66	40 (60.6)	53 vs 53	30.0 vs 42.3	Ultrasound	Ultrasound, mean IMT (6 measurements)	–	5
Petit et al71 2009	Europe	Hospital based, patients with T2DM	101	61 (60.4)	60.3 vs 60.1	44.2 vs 50.0	MR spectroscopy, liver fat content >5.5%	Ultrasound, mean IMT (6 measurements)	–	6
Ramilli et al70 2009	Europe	Outpatient clinic	154	90 (58.4)	59.3 vs 60.1	51.1 vs 45.3	Ultrasound	Ultrasound, mean of max IMT (L&R)	Age, sex, BMI, Smk, HT, dyslipidaemia, DM	8
Fracanzani et al74 2008	Europe	Hospital based	375	125 (33.3)	50.5 vs 52	87.2 vs 87.2	Ultrasound+biopsy	Ultrasound, mean IMT (6 measurements)	–	7
Aygun et al75 2008	Turkey	Hospital based	80	40 (50.0)	43.2 vs 38.8	47.5 vs 50.0	Biopsy	Ultrasound	Age/sex-matched controls	7
Targher et al77 2006 1	Europe	Outpatient clinic, patients with T2DM	200	100 (50.0)	55 vs 56	64.0 vs 67.0	Ultrasound	Ultrasound, mean IMT (6 measurements)	Age/sex-matched controls	7
Targher et al76 2006 2	Europe	Outpatient clinic	245	85 (24.7)	45 vs 45	58.8 vs 59.4	Biopsy	Ultrasound, mean IMT (6 measurements)	Age, sex, BMI, Smk, LDL, HOMA-IR, MetS	8
Brea et al79 2005	Europe	Hospital based	80	30 (50.0)	53.2 vs 51.6	50.0 vs 50.0	Ultrasound	Ultrasound, mean IMT	–	7
Targher et al78 2005	Europe	Outpatient clinic	90	50 (55.5)	46 vs 46	60.0 vs 65	Biopsy	Ultrasound, mean IMT (6 measurements)	Age, Sex, HOMA-IR, MetS	8

BMI, body mass index; L&R, left and right; MetS, metabolic syndrome; MR, magnetic resonance; NAFLD, non-alcoholic fatty liver disease; NOS, Newcastle–Ottawa Scale; T1DM, type 1 diabetes mellitus; T2DM, type 2 diabetes mellitus.

Table 2.

Characteristics of included studies which investigated the association between NAFLD and Increased CIMT

Name, year	Study region	Study population	Study size	n (%) NAFLD	Age (NAFLD+ vs NAFLD−)	% male (NAFLD+ vs NAFLD−)	NAFLD assessment	Outcome assessment	Outcome definition	Confounder adjustment	NOS (max=9)
Mohammadzadeh et al44 2019	Other: Iran	Hospital based	300	150 (50.0)	49.9 vs 52.5	65.3 vs 57.3	Ultrasound	Ultrasound, mean IMT (L&R)	CIMT >0.8	Age, BMI, HLP, HTN, DM	8
Tan et al80 2019	Asia	Government officials (health screen)	131	84 (64.1)	Overall=47.1	84.0 vs 60.7	Ultrasound (Fibroscan, Controlled attenuation parameter (CAP) ≥263 dB/min)	Ultrasound, mean IMT (6 measurements)	CIMT >0.8	Age, sex, WC, ALT, DM, HT	8
Oni et al22 2019	North America	Population based	4123	729 (17.7)	61 vs 63	47.0 vs 44.0	CT, LS ratio <1	Ultrasound, mean internal carotid IMT (L&R)	CIMT >1.0	Age, sex, ethnicity, SBP, fasting glucose, lipid-lowering meds, HT meds, LDL, Smk, BMI, logCRP	9
Yi et al45 2018	Asia	Outpatient clinic	1981	1888 (95.3)	45.9 vs 44.8	63.4 vs 40.1	Ultrasound	Ultrasound, mean of max IMT (L&R)	–	Sex, SBP, FPG, TG, TC, LDL, ALT, AST, GGT, Cr	6
Zheng et al28 2018	Asia	Population based	4112	1571 (38.2)	56.2 vs 55.6	64.4 vs 35.6	Ultrasound	Ultrasound, max IMT (L&R)	CIMT >0.8	Age, sex, BMI, exercise, Smk, WC, TG, LDL, DM, HT	9
Martínez-Alvarado et al81 2014	Mexican	Population based	429	122 (28.4)	52.1 vs 54.1	0.0 vs 0.0	CT, LS ratio <1	Ultrasound, mean IMT (10 measurements)	>75th sex/age-specific percentile	Age, HT, hypercholesterolaemia, hyperTAG, HDL, WC, HOMA-IR	9
Lankarani et al82 2013	Other: Iran	Population based	580	290 (50.0)	46.4 vs 45.4	44.8 vs 40.0	Ultrasound	Ultrasound, mean IMT (6 measurements)	CIMT >0.8	Age, sex, WC, DM, HT, TAG, HDL	9
Huang et al62 2012	Asia	Population based	8632	2590 (30.0)	58.5 vs 58.5	31.4 vs 30.9	Ultrasound	Ultrasound, max IMT (L&R)	CIMT >0.8	Age, sex, alcohol, Smk, exercise, BMI, LDL, central obesity, FBG, TG, BP, HDL, HOMA-IR	9
Kang et al61 2012	Asia	Outpatient (health screen), MetS(−) participants	413	157 (38.0)	52.0 vs 52.5	51.0 vs 41.8	Ultrasound	Ultrasound, mean IMT (L&R)	CIMT >1.0	Age, BP, BMI, WC, lipid profile, liver enzymes	8
Thakur et al59 2012	Asia	Hospital based	80	40 (50.0)	42.1 vs 41.9	67.5 vs 67.5	Ultrasound	Ultrasound, mean IMT (6 measurements)	CIMT >0.556	Generalised and abdominal obesity, MetS, fasting insulin, dyslipidaemia, SBP, DBP, hsCRP	8
Kim et al83 2009	Asia	Population (health screen)	1021	507 (49.7)	–	62.5 vs 46.5	Ultrasound	Ultrasound, mean of max IMT (L&R)	CIMT >0.8	Age, sex, WC, SBP, fasting glucose, total/HDL cholesterol ratio, Smk, alcohol	9
Fracanzani et al74 2008	Europe	Hospital based	375	125 (33.3)	50.5 vs 52.0	87.2 vs 87.2	Ultrasound+biopsy	Ultrasound, mean IMT (6 measurements)	CIMT >0.64	Sex, Smk, HDL, LDL, TAG, fasting glucose, MetS, DM, BMI, AAT	8
Brea et al79 2005	Europe	Hospital based	80	40 (50.0)	53.2 vs 51.6	50.0 vs 50.0	Ultrasound	Ultrasound, mean IMT	CIMT top quartile	Sex, age, BMI, SBP, DBP, DM, lab serum values	8

BMI, body mass index; CIMT, carotid intima-media thickness; Cr, creatinine; DM, diabetes mellitus; L&R, left and right; MetS, metabolic syndrome; NAFLD, non-alcoholic fatty liver disease; NOS, Newcastle–Ottawa Scale.

Table 3.

Characteristics of included studies which investigated the association between NAFLD and CAC presence, development or progression

Name, year	Study region	Study population	Study size	n (%) NAFLD	Age (NAFLD+ vs NAFLD−)	% male (NAFLD+ vs NAFLD−)	NAFLD assessment	Outcome assessment	Outcome definition	Confounder adjustment	NOS (max=9)
CAC presence (CAC >0 and CAC >100)
Jacobs et al85 2016	North America	Population based	250	71 (28.4)	66.8 vs 67.8	43.7 vs 43.0	CT, Liver Spleen ratio ≤1.1	MDCT, Agatston method	CAC >100 & CAC >0	Age, sex, HR, Smk, creatinine, BMI, alcohol, total cholesterol, HDL, TAG, VAT/SAT/WC	9
Chhabra et al92 2013	North America	Population (health screen)	377	43 (11.4)	Overall=57.1	Overall=52.0	CT, spleen minus liver >10	MDCT, Agatston method	CAC >100	Age, sex, Smk, LDL, HT, DM, MetS	9
Kim et al60 2012	Asia	Population (health screen)	4023	1617 (40.2)	57.5 vs 56.4	73.0 vs 52.5	Ultrasound	16 & 64 slice MDCT, Agatston method	CAC >100 & CAC >0	Age, sex, BMI, WC, alcohol, Smk, physical activity, DM, HT, total cholesterol, TAG, HDL, CRP	9
Chen et al94 2010	Asia	Population (health screen)	295	121 (41.0)	Overall=52.6	Overall=65.8	Ultrasound and CT	64 slice MDCT, Agatston method	CAC >100	Age, sex, BMI, Smk, HT, DM, fasting glucose, total cholesterol, TAG, HDL, LDL, ALT, AST, serum uric acid, gallbladder stones	9
Jung et al93 2010	Asia	Population (health screen)	928	219 (34.4)	54.0 vs 51.7	72.8 vs 49.5	Ultrasound	64 slice MDCT, Agatston method	CAC >100	Age, Sex, BMI, WHR, uric acid, SBP, DBP, GGT, TAG, HDL, fasting glucose, Smk, DM, HT, statins	9
Kim et al26 2020	Asia	Population (health screen)	7259	3328 (45.8)	Overall=54	Overall=59.5	Ultrasound	64 slice MDCT, Agatston method	CAC >0	Age, sex, HT, DM, obesity, abdominal obesity, eGFR, CRP, Smk, alcohol, AST, ALT, GGT	9
Oni et al22 2019	North America	Population based	4123	729 (17.7)	61 vs 63	47 vs 44	CT, LS ratio <1	EBCT or MDCT, Agatston method	CAC >0	Age, gender, ethnicity, SBP, fasting glucose, lipid-lowering meds, HT meds, LDL, Smk, BMI, logCRP	9
Chang et al27 2019	Asia	Population (health screen)	86 911	34 382 (39.6)	41.1 vs 40.3	89.1 vs 64.7	Ultrasound	64 slice MDCT, Agatston method	CAC >0	Age, sex, BMI, Smk, physical activity, education, total caloric intake, family history of CVD, DM, HT, LDL, meds, dyslipidaemia, hsCRP, HOMA-IR	9
Gummesson et al47 2018	Europe	Population based	1015	106 (10.4)	58.3 vs 57.5	71.7 vs 52.5	CT, liver HU <40	MDCT, Agatston method	CAC >0	Sex, age, education, BMI, alcohol, Smk, sedentary time, waist, VAT, physical activity, DM, HT, LDL, HDL, TG, CRP, insulin, hsCRP	9
Cho et al31 2018	Asia	Population (health screen)	798	272 (34.1)	53.4 vs 54.1	91.2 vs 72.2	Ultrasound	64 slice MDCT, Agatston method	CAC >0	Age, sex, BMI, Smk, alcohol, exercise, LDL-cholesterol, hsCRP	9
Lee et al29 2018	Asia	Population (health screen)	5121	1979 (38.6)	54.0 vs 53.7	77.6 vs 62.1	Ultrasound	64 slice MDCT, Agatston method	CAC >10	Age, sex, obesity, DM, HT, HLP, Smk, family history of CAD, hsCRP	9
Wu et al32 2017	Asia	Population based	2345	1272 (54.2)	Overall=55.7	Overall=44.1	Ultrasound	64 slice MDCT, Agatston method	CAC >0	Age, sex, Smk, HT, DM, HC, LDL, physical activity, education, income	9
Kim et al84 2016	Asia	Population (health screen)	1473	677 (46.0)	–	68.4 vs 47.1	Ultrasound	64 slice MDCT, Agatston method	CAC >0	Age, sex, Smk, alcohol, exercise, BMI, WC, SBP, total cholesterol, TAG, HDL, LDL, blood urea nitrogen, creatinine, glucose, hsCRP	9
Kang et al88 2015	Asia	Population (health screen)	772	346 (44.8)	50.0 vs 48.6	83.5 vs 55.4	Ultrasound	Presence of calcified coronary plaques	Presence of calcified plaques	Age, Smk, HT, DM, LDL, HDL, MetS	8
Mellinger et al86 2015	North America	Population (health screen)	3014	512 (17.0)	Overall=51.1	Overall=49.5	CT, liver phantom ratio <0.33	MDCT, Agatston method	CAC >0	Age, sex, alcohol, Smk, menopause, HRT, BMI	9
Kim et al87 2015	Asia	Population (health screen), postmenopausal women	754	129 (17.1)	59.5 vs 57.1	0.0 vs 0.0	Ultrasound	64 slice MDCT, Agatston method	CAC >0	Age, BMI, SBP, DBP, fasting glucose, total cholesterol, LDL, TAG, HDL, CRP, HOMA-IR	8
VanWagner et al89 2014	North America	Population based	2424	232 (9.57)	50.5 vs 49.9	58.2 vs 41.1	CT, liver HU ≤40	ECG-gated CT, Agatston method	CAC >0	Age, race, sex, study centre, income, education, alcohol, Smk, physical activity, BMI	9
Sung et al90 2012	Asia	Population (health screen)	10 153	3784 (37.3)	Overall=49.1	Overall=76.3	Ultrasound	64 slice MDCT, Agatston method	CAC >0	Age, sex, TAG, HDL, LDL, WC, SBP, alcohol, Smk, activity, Hx CHD, Hx HTN, Hx DM, HOMA-IR	9
Santos et al91 2007	South America	Population (health screen)	505	204 (40.4)	48 vs 46	100 vs 100	Ultrasound	EBCT, Agatston method	CAC >0	Age, pulse pressure, BMI, Smk, alcohol, MetS, LDL, TG/HDL ratio, fasting glucose, BP medication, lipid medication, ALT/AST ratio, GGT	9
CAC development/progression
Cho et al31 2018	Asia	Population (health screen), MetS(−) participants	798	272 (34.1)	53.4 vs 54.1	91.2 vs 72.2	Ultrasound	64 slice MDCT, Agatston method	Incident CAC or increase by >2.5 units between baseline & final square root of CAC score	Age, sex, BMI, Smk, alcohol, exercise, LDL-C, hsCRP, follow-up interval, baseline CAC score	9
Kang et al96 2017	Asia	Population (health screen), non-obese participants	447	105 (23.5)	Overall=54.1	Overall=70.9	Ultrasound	64 slice MDCT, Agatston method	Incident CAC or increase by >2.5 units between baseline & final square root of CAC score	Age, sex, WC, alcohol, Smk, exercise, baseline CAC, LDL, hsCRP, follow-up interval	8
Kim et al95 2017	Asia	Population (health screen)	1575	734 (46.6)	40.0 vs 398	94.8 vs 85.0	Ultrasound	64 slice MDCT, Agatston method	Any development (incidence)	Age, sex, ALT, Smk, FBS, LDL, BMI	9
Park et al34 2016	Asia	Population (health screen)	1732	846 (48.8)	57.1 vs 57.4	81.3 vs 67.7	Ultrasound	256 slice MDCT, Agatston method	Development & progression (>10 CAC from baseline)	Age, sex, BMI, HT, DM, hypercholesterolaemia, TAG, HDL, GFR, Smk, WC, incident DM, lipid- lowering agents	9

BMI, body mass index; CAC, coronary artery calcification; CAD, coronary artery disease; CRP, C reactive protein; CVD, cardiovascular disease; DM, diabetes mellitus; MetS, metabolic syndrome; NAFLD, non-alcoholic fatty liver disease; NOS, Newcastle–Ottawa Scale.

Measurement of exposures and outcomes

The presence of NAFLD was largely determined by US (n=46), with other studies using CT (n=8), biopsy (N=8), Fatty Liver Index (n=1) and MRS (n=1). Twenty-two studies investigated the effects of NAFLD on CAC score, with one study using the presence of calcified coronary artery plaques as a proxy for CAC >0. Forty-four studies investigated the effects of NAFLD on CIMT score. CIMT was assessed via B-mode US of bilateral carotid arteries, with majority of studies (n=18) commonly averaging the mean CIMT over six measurements (three on each carotid artery).

Methodological quality

Tables 1–3 and online supplemental table 1 detail the NOS risk of bias evaluation for the various studies. Of the 60 cross-sectional studies, the majority (n=35) were classified as ‘high quality’ (≥8 stars) with the remaining 25 classified as ‘moderate quality’ (5–7 stars). All four prospective studies were classified as ‘high quality’.

Effect of NAFLD on CIMT

Figures 2 and 3 summarise the studies which investigated the effects of NAFLD on CIMT. Forty-four studies, with a total of 41 189 individuals, assessed the effect of NAFLD on CIMT. Thirty-nine studies investigated the mean differences in CIMT between NAFLD and controls,22 30 33 35 44–79 while 13 studies used logistic regression to quantify the associations between NAFLD and an ‘increased CIMT’.22 28 44 45 59 61 62 74 79–83 Increased CIMT was defined as >0.8 mm in six studies, >1.0 mm in two studies and via other stratification methods in the remaining five studies.

Figure 2.

Forest plots showing relationship between NAFLD and presence of increased CIMT. CIMT, carotid intima-media thickness; NFLD, non-alcoholic fatty liver disease.

Figure 3.

(A) Forest plots showing pooled standard differences in unadjusted CIMT means between NAFLD(+) and NAFLD(−) groups. (B) Forest plots showing pooled standard differences in adjusted CIMT means between NAFLD(+) and NAFLD(−) groups. CIMT, carotid intima-media thickness; NFLD, non-alcoholic fatty liver disease.

Compared with participants without NAFLD, the presence of NAFLD was significantly associated with an increased CIMT, with a pooled OR of 2.00 (95% CI 1.56 to 2.56, P_{heterogeneity}<0.001, I²=81.8%, figure 2). Likewise, subjects with NAFLD had a higher mean CIMT than subjects without, both across studies which adjusted for confounders (pooled Std Diff in means: 1.17, 95% CI: 0.49 to 1.85, figure 3B), and in studies which compared unadjusted means (pooled Std Diff in means: 0.68, 95% CI: 0.44 to 0.91, figure 3A). For all CIMT outcomes, a sensitivity analysis including only studies of ‘high quality’ was performed, with similar results obtained.

Subgroup analyses

We further stratified the associations between NAFLD and an increased risk of increased CIMT by study region (figure 4A). The pooled ORs for increased CIMT were (OR: 1.63, 95% CI: 1.19 to 2.22, P_{heterogeneity}=0.06, I²=50%, n=7 studies) in Asian populations vs (OR: 2.70, 95% CI: 1.58 to 4.60, P_{heterogeneity}<0.001, I²=93.6%, n=3 studies) in Western populations (P_difference=0.15). Likewise, the pooled Std Diff in mean CIMT were 0.75 (95% CI: 0.31 to 1.17) in Asian populations (n=12 studies) vs 0.67 (95% CI: 0.25 to 1.09) in Western populations (P_difference=0.83) (figure 4B). Lastly, when analysing the subset of studies conducted on participants with T2DM, no Std Diff in CIMT means were found between those with and without NAFLD (Std Diff in means: 0.99, 95% CI:−0.21 to 2.20, n=7 studies) (online supplemental figure 1).

Figure 4.

(A) Forest plots showing relationship between NAFLD and presence of increased CIMT, stratified by region of study. (B) Forest plots showing pooled standard differences in CIMT means between NAFLD(+) and NAFLD(−) groups, stratified by region of study. CIMT, carotid intima-media thickness; NFLD, non-alcoholic fatty liver disease.

Effect of NAFLD on CAC score

Figure 5 summarises the studies investigating the associations between NAFLD and CAC score. Twenty-two studies, with a total of 136 294 individuals, assessed the effect of NAFLD on CAC score. Sixteen studies investigated the cross-sectional associations between NAFLD and the presence of CAC score >0,22 26 27 29 31 32 47 60 84–91 five studies investigated the cross-sectional associations between NAFLD and the presence of CAC score >100,60 80 85 92–94 and four studies investigated the longitudinal influence of NAFLD on CAC score progression/development.31 34 95 96

Figure 5.

(A) Forest plots showing relationship between NAFLD and CAC scores >0 and >100. (B) Forest plots showing relationship between NAFLD and the development/progression of CAC. CAC, coronary artery calcification; NFLD, non-alcoholic fatty liver disease.

Compared with participants without NAFLD, the presence of NAFLD was significantly associated with the presence of both CAC score >0 (pooled OR: 1.21, 95% CI 1.12 to 1.32, P_{heterogeneity}=0.018, I²=47.7%), and CAC score >100 (pooled OR: 1.28, 95% CI 1.01 to 1.63, P_{heterogeneity}=0.015, I²=67.8%), (figure 5A). Likewise, NAFLD was significantly associated with the development/progression of CAC with a pooled OR of 1.26 (95% CI 1.04 to 1.52, P_{heterogeneity}=0.34, I²=10.6%) (figure 5B).

Subgroup analyses

As with the CIMT analysis, we further stratified the associations of NAFLD with CAC score >0 based on ethnicity (figure 6). The pooled associations between NAFLD and CAC score >0 were (OR: 1.21 95% CI 1.10 to 1.33, P_{heterogeneity}=0.15, I²=31.7%, n=10 studies) in Asian populations vs (OR: 1.20 95% CI 1.03 to 1.38, P_{heterogeneity}=0.004, I²=73%, n=5 studies) in Western populations (P_difference=0.98). There were too few studies to conclusively compare ethnic differences for the associations with CAC score >100, or for the progression/development of CAC.

Figure 6.

(A) Forest plots showing relationship between NAFLD and CAC score >0, stratified by region of study. (B) Forest plots showing relationship between NAFLD and CAC score >100, stratified by region of study. CAC, coronary artery calcification; NFLD, non-alcoholic fatty liver disease.

Evaluation of publication bias

When assessing the studies that investigated the relationships between NAFLD and CIMT, the funnel plot showed asymmetry (online supplemental figures 2 and 3), with studies favouring increased Std Diff in means CIMT (Egger’s, p<0.05) and positive ORs for increased CIMT (Egger’s, p=0.002). For studies investigating the relationships between NAFLD and CAC outcomes (online supplemental figures 4 and 5), the funnel plots excluded bias with symmetrical distribution of studies on both sides of the mean, while the Egger’s test was non-significant (p=0.07 for CAC presence, and p=0.15 for CAC progression/development).

Discussion

In this meta-analysis, we evaluated the associations of NAFLD with two established markers of subclinical atherosclerosis, synthesising the results of 64 published studies with a total of 172 385 patients. In line with existing literature, we have demonstrated that subjects with NAFLD have an increased risk of prevalent subclinical atherosclerosis than those without, even after adjustment for common cardiometabolic risk factors. Our subgroup analyses also revealed these associations to be consistent across both Western and Asian populations. This is also the first meta-analysis to demonstrate that subjects with NAFLD are at increased risk of development and progression of subclinical atherosclerosis. This may provide additional insights into screening and surveillance strategies for patients with NAFLD,² potentially identifying higher-risk NAFLD populations, and may also provide further insight into the role of NAFLD in the development of CVD.

Our meta-analysis serves as a timely update to build on the previous work of Zhou et al, Kapuria et al and Jaruvongvanich et al,^23–25 incorporating the results of over 21 new studies published from 2016 and 2020, comprising over 100 000 participants (~50 000 of which have NAFLD). The inclusion of these new studies enables us to conduct a more robust analysis of the differences between ethnic populations, with a larger number of studies conducted in both Western and Asian populations. Our overall findings of the associations between NAFLD and an increased risk of subclinical atherosclerosis (as measured by CIMT and/or CAC score) are in agreement with existing literature, further reinforcing the findings of previous studies and meta-analyses.11 12 23–25 97 In addition to these associations with subclinical atherosclerosis, other meta-analyses have also found NAFLD to be significantly associated with increased cardiovascular mortality, coronary artery disease (CAD), incident CVD events, and other subclinical manifestations of CVD including abnormalities in myocardial metabolism, ventricular structure and function.^98–100 Our findings reiterate how the increased risk of CVD in patients with NAFLD can be attributed to an increased underlying subclinical atherosclerotic burden, and suggest that patients with NAFLD should be considered at high risk of atherosclerotic CVD.

Interestingly, we did not observe differential associations between NAFLD and both CAC or CIMT across Asian and Western populations. Our subgroup analyses found similar associations between NAFLD and CAC in both Asian (OR: 1.21 (1.10 to 1.33)) and Western regions (OR: 1.20 (1.03 to 1.38)), with a P_difference=0.98. Likewise, similar associations between NAFLD and increased CIMT were found across both regions. Despite literature suggesting ethnic differences in the pathogenesis, severity and outcomes of NAFLD,^{14 17 101} remarkably few studies have specifically investigated these ethnic differences in the context of associations with subclinical atherosclerosis. The Multi-Ethnic Study of Atherosclerosis found a positive association between NAFLD and both CAC and increased CIMT in white and Hispanic individuals, but not in Chinese individuals.^{21 22} While we did not specifically look at ethnic differences, our results show that NAFLD serves as an important atherogenic risk factor in both Western and Asian populations.

The associations between NAFLD and atherosclerotic CVD were originally considered epiphenomena due to a shared confluence of metabolic risk factors.¹⁰² However, increasingly, evidence has now recognised that NAFLD is an independent risk factor for CVD, with NAFLD thought to play an active role in the systemic release of proatherogenic and proinflammatory mediators, with additional contributions to insulin resistance and abnormal atherogenic lipid profiles, all of which increase the risk of atherogenesis.^{3 5 8} These potential pathways and mechanisms are covered in detail in other reviews.^{2 3 103} Nonetheless, the interplay between NAFLD, MetS, diabetes and CVD remains complex. Evidence on the effect of NAFLD on subclinical atherosclerosis within subjects with T2DM, for example, remains equivocal.33 52 71 77 In our subgroup analysis of studies conducted within populations with T2DM, our forest plots did not show significant Std Diff in mean CIMT between those with NAFLD and those without (p=0.107). Diabetes is a potent risk factor for both CAD and CVD, and may have thus masked subtler associations between NAFLD and subclinical atherosclerosis. Alternatively, this may also highlight the role of insulin resistance in mediating the relationship between NAFLD and atherosclerosis.¹⁰⁴

Only recently have studies begun to investigate the longitudinal associations between NAFLD and CAC progression/development, with this paper being the first meta-analysis to synthesise the results of four studies published from 2016 onwards.31 34 95 96 We demonstrated that patients with NAFLD are at greater risk of development/progression of CAC, even after adjustment for known confounders. While our results do not elucidate the exact pathophysiological mechanisms by which NAFLD may affect CAC development/progression, they do provide insight into the causal relationship between NAFLD and subclinical atherosclerosis. It should be noted that Park et al found differential associations between NAFLD and CAC development and progression, reporting that NAFLD might play a role in the early development of atherosclerosis, but not in the progression to more severe degrees of atherosclerosis.³⁴ Future studies may be warranted to confirm such observations.

Strengths of our study include the large participant numbers, the assessment of various modalities of subclinical atherosclerosis including CIMT and CAC, the large number of studies from both Western and Asian populations enabling robust analysis of regional differences, and our analysis of not just cross-sectional, but longitudinal outcomes (CAC development and progression). Nonetheless, our results should be interpreted with caution, taking into consideration certain limitations. Heterogeneity was consistently present across the different subclinical atherosclerotic outcomes. This can be attributed to differences in study design, population characteristics, the use of different cut-off definitions for both CAC and increased CIMT, the adjustment for different cardiometabolic confounders and the different modalities of NAFLD diagnosis. In addition, even though liver biopsy remains the gold standard for NAFLD evaluation, US was the most common modality used in the NAFLD assessment in the included studies, and is cited to have diminished accuracy when it comes to the diagnosis of milder hepatic steatosis.^{105 106} While we did not find regional differences in the results, we could not perform actual ethnic comparisons as these data were not available. Whether these regional data accurately reflect ethnic data is uncertain and also the influence of cultural and socioeconomic factors cannot be quantified. Nevertheless, this provides one of the first combined regional comparison of such results. Finally, potential publication bias exists with regard to the studies investigating CIMT-related outcomes.

Conclusion

In conclusion, this meta-analysis reports a significant positive association between NAFLD and subclinical atherosclerosis, as defined by increased CIMT and CAC scores. These observed associations are not just cross-sectional, but also longitudinal, and are seen across both Western and Asian populations. These results re-emphasise the importance of early risk evaluation and prophylactic intervention measures to preclude progression to clinical CVD in NAFLD.

Data availability statement

Data are available upon reasonable request.

Ethics statements

Patient consent for publication

Not required.

Ethics approval

Ethical approval is not applicable for this systematic review/meta-analysis.