Associations of Inflammatory and Osteogenic Activities in Epicardial Adipose Tissue With Aortic Valve Hemodynamic

Abstract

Background: Inflammation in epicardial adipose tissue (EAT) has been hypothesized to influence heart structure and function, thereby contributing to aortic valve (AV) disease. However, it remains unclear how the biological state of EAT is related to AV hemodynamics.

Methods and Results: We studied 50 patients with AV calcification who underwent elective cardiac surgery (cardiac valve surgery and/or coronary artery bypass graft). Echocardiographic data (AV area index [AVAI] and peak transvalvular AV velocity [PAVV]), were acquired before surgery. During cardiac surgery, 2 EAT samples were obtained for immunohistochemistry and the number of CD68- and CD11c-positive macrophages and osteocalcin-positive cells was counted in 6 random high-power fields (×400 magnification). PAVV, but not AVAI, was positively correlated with the number of CD11c-positive macrophages and osteocalcin-positive cells in EAT in patients with clinical AV stenosis (AS), defined as PAVV ≥2.5 m/s. On multivariate analysis adjusted for left ventricular function, the number of osteocalcin-positive cells in EAT was independently correlated with increased PAVV (β=0.42; P=0.013) and the presence of clinical AS (odds ratio per 1-unit increase 1.14; P=0.011), whereas there was no correlation between increased PAVV or the presence of clinical AS and the number of CD68- and CD11c-positive macrophages in EAT.

Conclusions: The biological activities of EAT, which are characterized mainly by osteogenic activity, are associated with AV hemodynamics and may contribute to AS progression.

Central Figure

Mounting evidence suggests that adipose tissue plays a role in the development of a systemic inflammatory state and contributes to obesity-associated vasculopathy and cardiovascular risk.¹ Importantly, chronic systemic inflammation related to obesity is associated with an increase in the amount of epicardial adipose tissue (EAT), the cardiac visceral fat, which is considered a transducer of the adverse effects of systemic inflammation and metabolic disorders on the heart.² Recent studies have underscored the significant correlation between EAT volume and coronary microvascular dysfunction or arrhythmias, including atrial fibrillation and premature ventricular complexes.^3–6 The EAT is located on the myocardium, and there is no fibrous fascial layer between the EAT and the underlying structures of the heart. Thus, it has been hypothesized that the EAT inflammatory state influences the structure and function of the heart, thus contributing to the pathogenesis of several cardiac diseases, including aortic valve (AV) disease.⁷

Several studies have examined the relationship between EAT and AV disease. The thickness of the EAT, assessed via echocardiography, is reportedly greater in patients with calcific AV stenosis (AS) than in healthy individuals and is correlated with the expression of EAT-derived proinflammatory and proatherogenic cytokines.⁸ Another study demonstrated a strong association between EAT thickness on echocardiography and severe AS, independent of traditional risk factors.⁹ In a recent prospective study that evaluated the impact of EAT volume, assessed via cardiac computed tomography (CT), on outcomes in AS patients who underwent transcatheter AV replacement, EAT volume was independently associated with all-cause mortality at 1, 2, and 3 years after the procedure, thus identifying EAT as an important prognostic factor in this population.¹⁰ These studies have focused mainly on the clinical quantification of the amount of EAT, whereas evidence for the clinical implications of the biological attributes of the EAT in AV disease remains scarce.

Using CT, we previously evaluated whether the histological features of EAT were associated with AV calcification (AVC) considering it as an organic etiology of AS.¹¹ Through immunohistochemical staining of EAT, we quantified the number of cells positive for CD68 (indicative of pan-macrophages), CD11c (indicative of proinflammatory macrophages), and osteocalcin (a marker for bone-forming cells [osteoblasts]). We found that the AVC score according to the standard Agatston method on CT was positively correlated with the inflammatory and osteogenic activities of EAT.¹¹ These results suggest the possibility of a specific contribution of EAT to AV degeneration through biological activities. However, the relationship between the biological state of the EAT and AV hemodynamics, which serve as functional and critical determinants of AS severity in clinical practice, remains poorly understood.

An analysis of the association between EAT biology and AV hemodynamics would contribute to the elucidation of the pathogenic significance of EAT in the occurrence and progression of AV disease. In addition, this analysis may lead to the identification of novel therapeutic targets for AV disease. Therefore, the aim of the present study was to evaluate the associations of histological features of EAT with AV hemodynamics assessed via echocardiography.

Methods

This study complied with the Declaration of Helsinki. The study protocol was approved by the Ethical Committee for Clinical Research of Hiroshima University (Approval no. C2020-0314), and written informed consent was obtained from all participants. The study has been registered with the University Hospital Medical Information Network (UMIN) Clinical Trials Registry (UMIN000043455).

Study Population

This was a post hoc analysis of our previous study¹¹ in which we focused on the relationship between EAT and AVC, and we expanded the dataset to include additional subjects. Between March 2021 and September 2024, we enrolled 53 patients who underwent cardiac CT examination and transthoracic echocardiography prior to elective cardiac surgery (cardiac valve surgery and/or coronary artery bypass graft [CABG]). All patients had been referred for cardiac CT to diagnose and characterize their coronary artery disease and/or cardiac valve disease and had AVC identified on CT. Patients with a history of cardiac surgery and those receiving dialysis were excluded because these factors could confound the analysis of EAT, AV, and AS. Patients’ clinical information, including their coronary risk factors and the use of statins and sodium-glucose cotransporter 2 inhibitors, was recorded. Laboratory data (serum lipid concentrations, HbA1c levels, and C-reactive protein concentrations) obtained immediately prior to surgery were also recorded. We obtained samples of EAT that were adjacent to the proximal portions of the left anterior descending and right coronary arteries (left and right EAT, respectively) during cardiac surgery (two EAT samples per participant), as described in our previous reports.^11–15 The size of each sample was generalized (longest diameter of 5 mm). These samples were later subjected to immunohistochemical staining.

CT Protocol and Analysis

As in our previous studies,^11–15 cardiac CT imaging was performed via a 320-slice CT scanner (Aquilion ONE; Canon Medical Systems, Otawara, Japan) within the month preceding cardiac surgery. All images were acquired with retrospective electrocardiographic gating. A non-contrast scan (maximum tube current 270 mA; tube voltage 120 kV) was performed to identify the AVC (slice thickness 3.0 mm) and measure EAT volume (slice thickness 0.5 mm). AVC was defined as a calcified lesion (structure with a CT density ≥130 HU) just inferior to the origin of the right coronary artery and located at the aortic leaflets, including the valvular point of attachment.¹⁶ The volume of EAT depots was measured using dedicated software (Virtual Place; AZE Inc., Tokyo, Japan). We defined EAT as the adipose tissue surrounding the myocardium and limited by the epicardium with a density range between −250 and −30 HU on non-contrast CT images. This EAT density was defined according to an earlier study of EAT.¹⁷ We measured the EAT volume of each participant by calculating the total sum of the EAT areas from 1 cm above the left main coronary artery to the left ventricular (LV) apex on images obtained at 1-cm intervals. EAT volume was normalized to body surface area for each participant to generate the EAT volume index (EATI). Datasets of a contrast scan were acquired via the HeartNAVI^® system (collimation 320×0.5 mm; tube current 350–580 mA; tube voltage 120 kV; Canon Medical Systems) and were used to determine AV morphology (tricuspid or bicuspid valve) and assess the coronary lumen for stenosis in all coronary segments >2 mm in diameter on coronary CT angiography. Lumen stenosis ≥70% in any vessel or ≥50% in the left main coronary artery was considered to be clinically obstructive.

Transthoracic Echocardiography

Transthoracic echocardiography was performed within the month preceding cardiac surgery. An experienced sonographer who was blinded to the patient’s clinical status performed comprehensive echocardiographic assessments according to the American Society of Echocardiography recommendations.¹⁸ In all patients, LV ejection fraction (LVEF) was measured via the biplane Simpson method and peak transvalvular AV velocity (PAVV), an indicator of AV hemodynamics, was measured via the standard Doppler method in apical 3-chamber view. The stroke volume ejected through the LV outflow was measured and normalized to body surface area to obtain the stroke volume index (SVI). Early (E) and late (A) transmitral inflow velocity was measured via pulsed-wave Doppler imaging in the apical 3-chamber view. The early diastolic mitral annular velocity (e′) was measured via tissue Doppler imaging with the sample volume positioned in the septal and lateral mitral annulus in the apical 4-chamber view, and the septal and lateral E/e′ values were averaged to assess LV diastolic dysfunction.¹⁹ For patients who were diagnosed with clinical AS according to the Japanese Circulation Society guidelines for the management of valvular heart disease (PAVV ≥2.5 m/s),²⁰ the AV area was calculated via a continuity equation and normalized to body surface area to obtain the AV area index (AVAI). Furthermore, severe AS was defined as PAVV ≥4.0 m/s according to the Japanese Circulation Society guidelines.²⁰

Immunohistochemical Staining and Image Analysis

As in our previous studies,^11–15 adipose tissue samples (left and right EAT) from each participant) underwent immunohistochemical staining. Samples were fixed in 10% buffered formalin and embedded in paraffin, after which 4-μm sections were cut. Antigen retrieval was performed in citrate buffer (pH 6.0) by heating in a 500-W microwave oven for 15 min. After blocking endogenous peroxidase activity with 3% H₂O₂–methanol for 10 min, the sections were incubated with primary antibodies against CD68 (1 : 50; clone KP-1; Dako, Glostrup, Denmark), CD11c (1 : 100; clone ITGAX/1242; Abcam, Cambridge, UK), or osteocalcin (1 : 100; polyclonal; Abcam, Cambridge, UK) for 60 min at room temperature, and then with horseradish peroxidase-linked anti-mouse or anti-rabbit IgG (Dako EnVision+) for 60 min at 20–25℃. Staining was completed by incubation with the substrate-chromogen solution. Sections were counterstained with 0.1% hematoxylin. Appropriate positive and negative control samples were included as described previously.^11–15

Histological images were analyzed at a magnification of ×400. We counted the number of CD68- and CD11c-positive cells to quantify the levels of pan- and pro-inflammatory macrophage infiltration, respectively, into adipose tissue depots. In addition, we counted the number of osteocalcin-positive cells to quantify the level of calcification in these depots. Cells were counted in 3 random high-power fields (with each field corresponding to a circle with a radius of 250 μm) in each of the left and right EAT samples, and the total number of cells was recorded for each participant.

Statistical Analysis

Continuous variables are expressed as the mean±SD. Student’s t-test or the Mann–Whitney U test was used to compare continuous variables between groups based on the normality of data distribution. Potential correlations between clinical and histological data for EAT and echocardiography-based AV parameters were assessed via Pearson’s correlation coefficient. Linear regression was used to evaluate the relationships of PAVV with clinical and histological data for EAT and echocardiography-based parameters. Logistic regression analyses were used to examine relationships of the histological EAT data with the presence of clinical AS. P<0.05 was considered to indicate statistical significance. Analyses were performed using JMP Pro 17 statistical software (SAS Institute, Cary, NC, USA).

Results

Participants’ Baseline Characteristics

We obtained EAT samples safely and in sufficient quantity for subsequent immunohistochemical staining from 50 of the 53 participants. Table 1 presents baseline clinical characteristics of the 50 participants studied. The mean PAVV was 3.3±1.5 m/s (range 1.0–6.6 m/s). Thirty (60%) participants were diagnosed with clinical AS on the basis of AV velocity.¹⁶ Four (8%) participants had bicuspid AV and the others had tricuspid AV. Thirty-six participants underwent cardiac valve surgery alone (AV repair and/or mitral valve repair and/or tricuspid valve repair), 8 underwent CABG alone, and 6 underwent both cardiac valve surgery and CABG.

Table 1.

Baseline Characteristics of Participants

	All patients (n=50)	Clinical AS (n=30)	No clinical AS (n=20)	P value
Age (years)	73±8	74±7	72±8	0.2
Male sex	36 (72)	19 (63)	17 (85)	0.09
Body mass index (kg/m2)	24±4	23±3	24±4	0.55
Hypertension	36 (72)	20 (67)	16 (80)	0.3
Hyperlipidemia	31 (62)	19 (63)	12 (60)	0.81
Diabetes	21 (42)	11 (37)	10 (50)	0.35
Current smoking	12 (24)	7 (23)	5 (25)	0.89
Atrial fibrillation	6 (12)	3 (10)	3 (15)	0.59
Obstructive coronary stenosis	17 (34)	8 (27)	9 (45)	0.18
Use of statin	27 (54)	17 (57)	10 (50)	0.64
Use of SGLT2 inhibitors	16 (32)	25 (83)	9 (45)	0.0044
Laboratory results
HDL-C (mg/dL)	65±18	64±15	66±22	0.65
LDL-C (mg/dL)	104±30	106±32	100±30	0.52
Triglyceride (mg/dL)	126±71	128±70	123±74	0.82
HbA1c (%)	6.2±1.0	6.2±1.0	6.3±1.1	0.96
C-reactive protein (mg/L)	1.5±2.0	1.5±2.1	1.5±1.8	0.98
PAVV (m/s)	3.3±1.5	4.4±0.9	1.7±0.4	<0.0001
Bicuspid aortic valve	4 (8)	4 (13)	0 (0)	0.088
AVAI (cm2/m2)	N/A	0.53±0.16	N/A	N/A
LV parameters on echocardiography
LVEF (%)	59±11	62±8	54±13	0.0058
SVI (mL/m2)	54±20	53±18	56±22	0.59
Mean E/e′	14±7	17±8	11± 4	0.0034
Cardiac surgery
Cardiac valve surgery	36 (72)	24 (80)	12 (60)	0.12
CABG	8 (16)	0 (0)	8 (40)	0.0002
Cardiac valve surgery and CABG	6 (12)	6 (20)	0 (0)	0.033

Unless indicated otherwise, data are given as the mean±SD or n (%). AS, aortic stenosis; AVAI, aortic valve area index; CABG, coronary artery bypass graft surgery; HDL-C, high-density lipoprotein cholesterol; LDL-C, low-density lipoprotein cholesterol; LV, left ventricular; LVEF, left ventricular ejection fraction; N/A, not applicable; PAVV, peak transvalvular aortic valve velocity; SGLT2, sodium-glucose cotransporter 2; SVI, stroke volume index.

Among the 50 participants studied, mean EAT volume and EATI obtained via CT were 107±48 mL and 66±28 mL/m², respectively. The mean number of CD68-positive macrophages, CD11c-positive macrophages, and osteocalcin-positive cells in EAT samples was 35±18, 19±10, and 27±20, respectively

Determinants of AV Hemodynamics Among EAT Findings

For all participants, there was no correlation between EATI and PAVV (r=−0.017, P=0.91). The correlation between the number of CD68-positive macrophages in the EAT and PAVV did not reach significance (r=0.27, P=0.061), whereas PAVV was positively correlated with both the number of CD11c-positive macrophages (r=0.44, P=0.0013) and the number of osteocalcin-positive cells (r=0.61, P<0.0001) in the EAT (Figure 1).

Figure 1.

Correlations between epicardial adipose tissue (EAT) findings and aortic valve (AV) hemodynamics. Correlation coefficients (r) and P values are from Pearson’s correlation tests. (A) EAT volume index (EATI) was not correlated with the peak aortic valve velocity (PAVV). (B) There was a tendency for a positive correlation between the number of CD68-positive macrophages in EAT and PAVV, but the correlation did not reach statistical significance. (C,D) The numbers of CD11c-positive macrophages (C) and osteocalcin-positive cells (D) in EAT were positively correlated with PAVV.

Table 2 presents the results of linear regression analysis to identify clinical factors and EAT histological findings associated with PAVV. In univariate analyses, LVEF, mean E/e′, the presence of bicuspid AV, and the number of CD11c-positive macrophages and osteocalcin-positive cells in EAT were positive determinants of PAVV, whereas male sex and the presence of obstructive coronary stenosis were negative determinants of PAVV. In multivariate analysis, which was adjusted for sex, the presence of obstructive coronary stenosis, LVEF, mean E/e′, the presence of bicuspid AV, and EAT histological findings, only the number of osteocalcin-positive cells in EAT was independently correlated with increased PAVV (β=0.42; P=0.013). Figure 2 shows representative images from a patient with increased PAVV and the proliferation of osteocalcin-positive cells in EAT.

Table 2.

Linear Regression Analysis of Clinical Factors and Epicardial Adipose Tissue Histological Findings Associated With Peak Transvalvular Aortic Valve Velocity

	Univariate β	P value	Multivariate β	P value
Age (years)	0.13	0.37
Male sex	−0.29	0.043	−0.22	0.081
Hypertension	−0.13	0.38
Hyperlipidemia	0.010	0.94
Diabetes	−0.22	0.13
Current smoking	−0.077	0.60
Atrial fibrillation	0.15	0.28
Obstructive coronary stenosis	−0.28	0.048	−0.12	0.29
EATI	−0.017	0.91
LVEF	0.31	0.030	0.12	0.33
SVI	0.030	0.83
Mean E/e′	0.35	0.014	0.092	0.48
Presence of bicuspid AV	0.40	0.0036	0.18	0.15
No. CD68-positive macrophages in EAT	0.27	0.061	−0.024	0.85
No. CD11c-positive macrophages in EAT	0.44	0.0013	0.14	0.30
No. osteocalcin-positive cells in EAT	0.61	<0.0001	0.42	0.013

AV, aortic valve; EAT, epicardial adipose tissue; EATI, EAT volume index; LVEF, left ventricular ejection fraction; PAVV, peak transvalvular aortic valve velocity; SVI, stroke volume index.

Figure 2.

Representative images from an 81-year-old male patient. (Top panels) Echocardiography revealed severe aortic valve calcification (AVC), a decreased aortic valve area index (AVAI; 0.19 cm²/m²), and increased aortic valve (AV) velocity (peak aortic valve velocity [PAVV]=6.6 m/s). (Bottom panels) Immunohistochemical staining of epicardial adipose tissue (EAT) revealed a high proliferation of osteocalcin-positive cells and less infiltration of pan- (CD68-positive) macrophages and proinflammatory (CD11c-positive) macrophages.

Impact of EAT Findings on the Presence of AS

Table 1 presents a comparative analysis of the baseline clinical characteristics of participants with and without clinical AS. Individuals diagnosed with clinical AS were more frequently administered sodium-glucose cotransporter 2 inhibitor treatment and had higher LVEF and mean E/e′ values.

There was no difference in EATI between participants with and without clinical AS (68±31 vs. 62±23 mL/m², respectively; P=0.50), whereas the numbers of CD68-positive macrophages, CD11c-positive macrophages, and osteocalcin-positive cells in EAT were all significantly greater in participants with than without clinical AS (41±20 vs. 26±10 [P=0.0062], 23±11 vs. 13±6 [P=0.0007], and 36±21 vs. 15±10 [P=0.0001], respectively). Table 3 presents the results of logistic regression analysis to identify EAT histological findings related to the presence of clinical AS. On multivariate analysis adjusted for age, sex, EATI, LVEF, mean E/e′, and SVI, the number of osteocalcin-positive cells in EAT was independently related to the presence of clinical AS (odds ratio per 1-unit increase 1.14; 95% confidence interval 1.03–1.25; P=0.011). However, there was no significant relationship between the presence of clinical AS and the number of CD68-positive or CD11c-positive macrophages in EAT.

Table 3.

Logistic Regression Analysis of Epicardial Adipose Tissue Histological Findings Related to the Presence of Clinical Aortic Valve Stenosis

	Model 1		Model 2		Model 3		Model 4
	OR (95% CI)	P value	OR (95% CI)	P value	OR (95% CI)	P value	OR (95% CI)	P value
CD68+ macrophages (per 1-unit increase)	1.08 (1.02–1.14)	0.010	1.07 (1.01–1.15)	0.015	1.08 (1.01–1.14)	0.014	1.06 (0.98–1.15)	0.15
CD11+ macrophages (per 1-unit increase)	1.15 (1.04–1.27)	0.0054	1.16 (1.04–1.29)	0.0056	1.17 (1.05–1.31)	0.0047	1.14 (0.99–1.32)	0.059
Osteocalcin+ cells (per 1-unit increase)	1.14 (1.05–1.23)	0.0015	1.16 (1.06–1.26)	0.0017	1.17 (1.06–1.29)	0.0022	1.14 (1.03–1.25)	0.011

Model 1 was unadjusted. Model 2 was adjusted for age and sex. Model 3 was adjusted for age, sex, and epicardial adipose tissue volume index (EATI). Model 4 was adjusted for age, sex, EATI, left ventricular ejection fraction, mean E/e′ ratio, and stroke volume index. CI, confidence interval; OR, odds ratio.

Relationship Between EAT Findings and AS Severity

In participants with clinical AS (n=30), there was no correlation between EATI and PAVV (r=−0.27, P=0.15) or AVAI (r=0.23, P=0.21). The number of osteocalcin-positive cells in EAT was positively correlated with PAVV (r=0.40, P=0.027), whereas there was no correlation between PAVV and the number of CD68-positive macrophages (r=−0.16, P=0.39) or CD11c-positive macrophages (r=0.059, P=0.76) in EAT (Figure 3A–C). In this disease population, the number of cells in EAT was not correlated with AVAI (CD68-positive macrophages: r=0.24, P=0.20; CD11c-positive macrophages: r=−0.0046, P=0.98; osteocalcin-positive cells: r=−0.25, P=0.18; Figure 3D–F). In participants with severe AS (n=22), the number of osteocalcin-positive cells in EAT remained positively correlated with PAVV (r=0.63, P=0.018), but not with AVAI (Figure 4). Figure 5 shows representative images from a patient with severe AS based on AV velocity and preserved AVAI.

Figure 3.

Correlations between the number of (A,D) CD68-positive macrophages, (B,E) CD11c-positive macrophages, and (C,F) osteocalcin-positive cells in epicardial adipose tissue (EAT) and echocardiographic aortic valve of parameters peak aortic valve velocity (PAVV; A–C) and aortic valve area index (AVAI; D–F) in patients with clinical aortic valve stenosis (AS). Correlation coefficients (r) and P values are from Pearson’s correlation test. Only the number of osteocalcin-positive cells in EAT was positively correlated with PAVV (C). No other correlations between cell numbers and PAVV or AVAI were observed.

Figure 4.

Correlations between the number of osteocalcin-positive cells in epicardial adipose tissue (EAT) and echocardiographic aortic valve parameters of peak aortic valve velocity (PAVV; A) and aortic valve area index (AVAI; B) in patients with severe aortic valve stenosis. Correlation coefficients (r) and P are from Pearson’s correlation tests. The number of osteocalcin-positive cells in EAT was positively correlated with PAVV (A), but not with AVAI (B).

Figure 5.

Representative images from an 84-year-old male patient. (Top panels) Echocardiography revealed increased aortic valve velocity (peak aortic valve velocity [PAVV]=5.0 m/s) and a preserved aortic valve area index (AVAI; 0.94 cm²/m²). (Bottom panels) Immunohistochemical staining of epicardial adipose tissue (EAT) revealed a high proliferation of osteocalcin-positive cells and less infiltration of pan- (CD68-positive) macrophages and proinflammatory (CD11c-positive) macrophages.

Discussion

To investigate the biological significance of EAT in the development of AV disease, we sought to explore associations between AV hemodynamics assessed via echocardiography and the volume and histological characteristics of EAT. Our findings are as follows: (1) CT-based EATI was not correlated with AV velocity; (2) inflammatory and osteogenic activities of EAT were positively correlated with AV velocity, but not with AVAI, in patients with clinical AS diagnosed on the basis of AV velocity; (3) after adjustment for LV function, the osteogenic activity of EAT was independently associated with increased AV velocity, but the inflammatory activity of EAT was not; and (4) after adjustment for LV function, higher osteogenic activity of EAT was independently associated with the presence of clinical AS. Our findings suggest that the biological activities of EAT, which are characterized mainly by osteogenic activity, may affect AV hemodynamics. This study provides new insights into the biological significance of EAT in AV dysfunction and indicates that EAT has the potential to be a new therapeutic target for AV diseases.

An increase in visceral adipose tissue is associated with a higher incidence of cardiovascular disease, including AS.²¹ Abdominal visceral adipose tissue exerts its deleterious effects through the production and secretion of various inflammatory cytokines, which is a fundamental characteristic of metabolic syndrome. In the Multi-Ethnic Study of Atherosclerosis (MESA), metabolic syndrome was reported to be associated with a significant increase in the incidence of AVC, suggesting that metabolic syndrome could be a therapeutic target to prevent AV diseases.²² Another study reported an association of metabolic syndrome with more pronounced LV hypertrophy and worse myocardial function in patients with calcific AS.²³ These findings indicate that visceral adipose tissue contributes to the pathogenesis and progression of AV disease through its effect on the inflammatory state. EAT is the cardiac visceral adipose tissue and a metabolically active organ that generates various bioactive molecules. Thus, it may significantly affect cardiac function. In the present study we demonstrated that the biological activities of EAT, based on immunohistochemistry, are associated with AV function. Interestingly, the osteogenic activity of EAT, as well as its inflammation, was correlated with AV hemodynamics, and the correlation between osteogenic activity and AV hemodynamics remained after adjusting for LV parameters. To the best of our knowledge, this is the first report providing evidence of the correlation between the specific biology of EAT and AV hemodynamics.

Whether EAT contributes to the degeneration of the AV has been explored. Parisi et al. reported proinflammatory activation of EAT in patients with isolated AS, together with a strong association between EAT thickness and AS.⁸ We found that inflammatory and osteogenic activities, assessed histologically, in EAT are associated with the severity of AVC, as assessed using CT.¹¹ These findings support the hypothesis that cardiac visceral adipose tissue is involved in causing and promoting AV diseases and degeneration. Furthermore, in a preliminary immunohistochemical study of EAT samples,¹¹ we found high expression of fibroblast markers, in addition to increased proliferation of osteocalcin-positive cells. In the present study, the number of osteocalcin-positive cells in EAT was related to AV velocity, but was not correlated with the normalized morphological AV area (AVAI) in patients with clinical AS. Calcific AV disease is understood to progresses through 3 interconnected pathological stages: inflammation, fibrosis, and calcification.²⁴ The biological activities of EAT, such as the proliferation of osteoblasts and fibroblasts, may influence the progression of these stages and affect the flexibility and mobility of AV leaflets through tissue degeneration, including sclerosis and calcification. We hypothesize that this mechanism is responsible for the relationship between the osteogenic activity of EAT and AV hemodynamics; however, this hypothesis requires further investigation.

Previous studies have reported associations between clinical quantification of the amount of EAT, assessed via echocardiography and CT, and severe AS and its outcome.^9,10 In the present study, we found no associations between the volumetric parameter of EAT (EATI) and AV hemodynamics or the presence or severity of AS. This inconsistency may be attributed to the small sample size of this study and our use of EAT volume normalized to body surface area (EATI). In addition, associations of EAT volume with the severity of AS are likely to be affected by EAT-related unfavorable LV remodeling. This cardiac visceral adipose tissue may drive LV hypertrophy and fibrosis causing diastolic dysfunction.⁷ We found a significant correlation between EATI and the mean E/e′ (r=0.50, P=0.0002) in the 50 participants enrolled in this study (Supplementary Figure). An increase in the amount of EAT could result in enhanced hypertrophic stimuli and contribute to severe LV remodeling.²⁵ We did find a significant association between the specific biology of EAT and the presence and severity of AS on the basis of AV velocity. This finding indicates that the biological state of EAT, rather than the amount of EAT, is a critical factor in the development of AS.

Study Limitations

We acknowledge that this study has several limitations. First, although this was a post hoc analysis of our previous study, the sample size was relatively small and there may have been bias because of the patient selection method used. The small sample size and potential selection bias represent important limitations. Our non-significant results (e.g., correlations of cell numbers in EAT with AVAI in participants with clinical AS) and multivariate analyses need to be further investigated in more subjects. Second, we assessed PAVV using the standard Doppler method in the apical 3-chamber view. This approach is routinely used at Hiroshima University Hospital to ensure the reproducibility of PAVV measurements. Nevertheless, maximum AV velocity can be obtained using various methods, including the right parasternal approach. Third, we cannot draw conclusions regarding the direct effect of EAT on AV dysfunction and degeneration on the basis of the present results. Histological studies of human AV leaflets or preclinical studies using animal models may be helpful in elucidating the effects of EAT on AV dysfunction and degeneration. A study involving patients with AS revealed that the valvular uptake of¹⁸ F-sodium fluoride, indicative of active calcification, colocalized with regions that exhibited histological staining for osteocalcin.²⁶ This imaging marker may prove beneficial. In addition, a molecular imaging modality that enables serial assessment of EAT characteristics would be desirable, and may contribute to the elucidation of EAT pathogenicity in cardiovascular diseases. Finally, there is no currently available pharmacological therapy to prevent the occurrence and progression of AV degeneration and disease. Although our results highlight the potential of EAT as a new therapeutic target for AV diseases, how to treat its pathogenicity remains unclear. Further studies in preclinical models and clinical patients are needed to establish a causative mechanism between biological modifications of EAT and pathophysiological changes in AV diseases and to propose a novel treatment. The potential impact of surgical intervention on the histological characteristics of EAT may provide new insights in this field.

Conclusions

The biological activities of EAT, which are characterized mainly by osteogenic activity, are associated with AV hemodynamics. The findings of the present study support the hypothesis that the biological attributes of EAT may contribute to the progression of AS; however, the mechanism responsible and the potential of EAT as a therapeutic target for AV diseases require further investigation.

Acknowledgments

We have no specific acknowledgments other than the funding.

Sources of Funding

This study was supported, in part, by the Suzuki Memorial Foundation and a JSPS KAKENHI Grant-in-Aid for Scientific Research (Grant no. 21K08127).

Disclosures

The authors declare that there are no conflicts of interest.

IRB Information

This study was approved by the Ethical Committee for Clinical Research of Hiroshima University (Reference no. C2020-0314).

Supplementary Files

Please find supplementary file(s);

https://doi.org/10.1253/circrep.CR-25-0189

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