- Email: [email protected]
Expression of NLRP3 in subcutaneous adipose tissue is associated with coronary atherosclerosis
Accepted Manuscript Expression of NLRP3 in subcutaneous adipose tissue is associated with coronary atherosclerosis Sachiko Bando, Daiju Fukuda, MD, PhD, Takeshi Soeki, Sachiko Nishimoto, Etsuko Uematsu, Tomomi Matsuura, Takayuki Ise, Takeshi Tobiume, Koji Yamaguchi, Shusuke Yagi, Takashi Iwase, Hirotsugu Yamada, Tetsuzo Wakatsuki, Michio Shimabukuro, Masataka Sata PII:
S0021-9150(15)30063-0
DOI:
10.1016/j.atherosclerosis.2015.07.043
Reference:
ATH 14214
To appear in:
Atherosclerosis
Received Date: 8 January 2015 Revised Date:
23 July 2015
Accepted Date: 27 July 2015
Please cite this article as: Bando S, Fukuda D, Soeki T, Nishimoto S, Uematsu E, Matsuura T, Ise T, Tobiume T, Yamaguchi K, Yagi S, Iwase T, Yamada H, Wakatsuki T, Shimabukuro M, Sata M, Expression of NLRP3 in subcutaneous adipose tissue is associated with coronary atherosclerosis, Atherosclerosis (2015), doi: 10.1016/j.atherosclerosis.2015.07.043. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Bando S, et al. Ms. No. ATH-D-15-00026/R2
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Expression of NLRP3 in subcutaneous adipose tissue is associated with coronary atherosclerosis
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Sachiko Bando1, Daiju Fukuda1, Takeshi Soeki1, Sachiko Nishimoto2, Etsuko Uematsu1, Tomomi Matsuura1, Takayuki Ise1, Takeshi Tobiume1, Koji Yamaguchi1, Shusuke Yagi1, Takashi Iwase1, Hirotsugu Yamada1, Tetsuzo Wakatsuki1, Michio Shimabukuro3 and Masataka Sata1
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1. Department of Cardiovascular Medicine, Institute of Biomedical Sciences, Tokushima University Graduate School, Tokushima, Japan 2. Department of Nutrition and Metabolism, Institute of Biomedical Sciences, Tokushima University Graduate School, Tokushima, Japan 3. Department of Cardio-Diabetes Medicine, Institute of Biomedical Sciences, Tokushima University Graduate School, Tokushima, Japan
Running title: NLRP3 in subcutaneous adipose tissue and coronary atherosclerosis
adipose
tissue,
inflammation,
NLRP3
inflammasome,
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Key words: atherosclerosis, lifestyle-related disease
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Word count: 4910 Number of Tables: 3 Number of Figures: 4 Number of Supplementary Figures: 3 Number of Supplementary Tables: 1
Corresponding author: Daiju Fukuda, MD, PhD Department of Cardiovascular Medicine, Institute of Biomedical Sciences, Tokushima University Graduate School 3-18-15, Kuramoto-cho, Tokushima 770-8503, Japan Phone: +81-88-633-7859 Fax: +81-88-633-7894 E-mail: [email protected]
1
Bando S, et al. Ms. No. ATH-D-15-00026/R2
ACCEPTED MANUSCRIPT Abstract
Objectives: The promotion of adipose tissue inflammation by lifestyle-related diseases such as obesity and diabetes accelerates atherogenesis; however, the underlying mechanisms remain
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incompletely understood. Nucleotide-binding domain, leucine-rich-containing family, pyrin domain-containing-3 (NLRP3) inflammasome contributes to chronic inflammation in adipose
tissue (SAT) and the severity of coronary atherosclerosis.
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tissue. Here, we investigated the link between NLRP3 expression in subcutaneous adipose
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Methods and Results: SAT was obtained from 72 patients who underwent heart device implantation and coronary angiography. Expression of NLRP3 inflammasome-related molecules (NLRP3, IL-1β and IL-18) in SAT were evaluated by quantitative RT-PCR. Laboratory markers related to lifestyle-related diseases were measured. Patients with obesity,
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dyslipidemia (P<0.05, respectively), diabetes or hyperuricemia (P<0.01, respectively) had significantly higher expression of NLRP3. Multivariate analysis demonstrated that body mass index and serum level of uric acid were predictors of NLRP3 expression in SAT. The expression
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of NLRP3 in SAT correlated negatively with serum adiponectin level (r= -0.23, P<0.05). Patients
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with coronary artery disease showed higher NLRP3 expression than patients without significant stenosis (P<0.01). Furthermore, the expression of NLRP3 in SAT correlated positively with the severity of coronary atherosclerosis as determined by Gensini score (r = 0.47, P<0.0001) or SYNTAX score (r = 0.55, P<0.0001). Multiple regression analysis revealed that the expression of NLRP3 in SAT remains as an independent predictors for the severity of coronary atherosclerosis. Conclusions: The expression of NLRP3 in SAT, which is affected by lifestyle-related diseases, 2
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is associated with the severity of coronary atherosclerosis. Our results suggest that NLRP3
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inflammasome in SAT may have a role in atherogenesis.
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Bando S, et al. Ms. No. ATH-D-15-00026/R2
ACCEPTED MANUSCRIPT Introduction
There is increasing evidence that lifestyle-related diseases, such as obesity, dyslipidemia and diabetes, promote local pro-inflammatory responses in adipose tissue, accelerating systemic
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inflammation, which contributes to the development of atherosclerosis.[1, 2, 3] Much effort has been made to elucidate the molecular mechanisms of adipose tissue inflammation. Recent studies have shown that danger signals related to metabolic disorders could initiate
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pro-inflammatory responses in adipose tissue. For example, saturated fatty acids activate
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toll-like receptors (TLRs), a family of pattern recognition molecules, and promote inflammation in adipose tissue.[4, 5] More recently, another pattern recognition receptor, nucleotide-binding domain, leucine-rich-containing family, pyrin domain-containing-3 (NLRP3) inflammasome, has been implicated in recognizing endogenous danger signals, leading to the development of
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adipose tissue inflammation through the activation of caspase-1 and subsequent secretion of interleukin (IL)-1β and IL-18.[6, 7, 8] NLRP3 inflammasome also participates in the development of vascular inflammation and atherogenesis.[9, 10] It is widely accepted that
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NLRP3 inflammasome contributes to the development of chronic inflammation in both adipose
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tissue and the vasculature, although, whether the expression of NLRP3 in adipose tissue is associated with atherosclerosis remains unknown. Therefore, in this study, we hypothesized that lifestyle-related diseases affect the expression of NLRP3 in adipose tissue, which associates with the severity of coronary atherosclerosis. To assess our hypothesis, we examined the expression of NLRP3 inflammasome-related molecules (e.g., NLRP3, IL-1β and IL-18) in subcutaneous adipose tissue (SAT) obtained from patients admitted to our hospital for permanent heart device implantation, and investigated its relationship with the severity of 4
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coronary atherosclerosis. The results of our present study suggest that the expression of NLRP3 in SAT may have links with the development of atherosclerosis.
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Methods Study protocol and patient enrollment
Our patient population comprised 72 consecutive patients who were admitted to Tokushima
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University Hospital for implantation of an implantable heart device, such as permanent
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pacemaker, implantable cardiac defibrillator or cardiac resynchronization therapy device because of sick sinus syndrome (n=27), atrio-ventricular block (n=21), ventricular tachycardia/ventricular fibrillation (n=11) and dilated or ischemic cardiomyopathy (n=13). We excluded patients with malignant tumors, sarcoidosis, severe congestive heart failure and active
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inflammatory disease (e.g.; infection or collagen disease). We also excluded patients who were taking immunosuppressants or over-the-counter herbal drugs and patients who had recovered from cardiac arrest.
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Cardiovascular risk factors were defined as follows: hypertension as blood pressure >
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140/90 mmHg in three consecutive readings at rest; dyslipidemia as LDL cholesterol level > 140 mg/dL and/or HDL cholesterol < 40 mg/dL; diabetes as fasting glucose level > 126 mg/dL and/or random blood glucose level > 200 mg/dL and/or HbA1c > 6.5%; obesity as body mass index (BMI) > 25 kg/m2; and hyperuricemia as uric acid level > 7.0 mg/dL. Subjects receiving anti-hypertensive drugs, cholesterol-lowering therapy, anti-diabetic drugs including insulin, or anti-hyperuricemic drugs were also considered to have hypertension, hyperlipidemia, diabetes or hyperuricemia, respectively. Both current smokers and ex-smokers were considered 5
Bando S, et al. Ms. No. ATH-D-15-00026/R2
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Serum concentrations of LDL cholesterol, HDL cholesterol, uric acid, and C-reactive protein (CRP) and plasma concentration of HbA1c were measured in samples collected after
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the patients had fasted overnight. Serum concentration of adiponectin was measured with a commercially available kit (R&D Systems).
This study was conducted in accordance with the Declaration of Helsinki and was
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approved by the Ethics Committee of Tokushima University Hospital. Written informed consent
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was obtained from all subjects. SAT biopsy
SAT biopsy was performed in each patient at the time of devise implantation. A small piece of SAT was obtained when a subcutaneous pocket was created in the infraclavicular region. SAT
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samples were processed for fractionation immediately after biopsy. For RNA extraction, samples were preserved at -80 oC until assay.
Assessment of atherosclerosis in coronary arteries
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Coronary angiography was performed by a standard technique. To prevent coronary spasm, all
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patients received intracoronary injection of isosorbide dinitrate at an optimal dose before angiography. Two independent observers blinded to the results of the analyses of SAT reviewed the coronary angiograms separately. Stenosis > 75% detected angiographically in a major coronary vessel was defined as significant stenosis. Gensini and SYNTAX scores were calculated based on angiographic findings as previously reported.[11, 12] Fractionation of adipose tissue and macrophage selection Fractionation of SAT was performed as described previously.[13] Subcutaneous fat was minced 6
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in PBS and then incubated on a shaking platform for 30 min at 37 oC with collagenase (250 U/ml). The mixture was then passed through a nylon filter (pore size, 250 µm) to remove undigested material, and the filtrate was centrifuged at 200 g for 5 min at 4 oC. Floating cells
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and the pellet were recovered as the mature adipocyte fraction and the stromal vascular fraction, respectively. The stromal vascular fraction was incubated with phycoerythrin-conjugated anti-CD14 antibody (Biolegend) followed by anti-phycoerythrin magnet beads (Miltenyi Biotech).
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CD14-positive and -negative fractions were collected using the MACS® system (Miltenyi
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Biotech). Quantitative RT-PCR
Total RNA was extracted using illustra RNAspin RNA Isolation Kit (GE Healthcare). cDNA was synthesized from 100 ng of total RNA extracted from tissues and cells using a QuantiTect
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Reverse Transcription kit (Qiagen). Real-time quantitative RT-PCR (qPCR) was performed with a Mx3000P (Agilent Technologies) and Power SYBR Green PCR Master Mix (Applied Biosystems). Sequences of gene specific primers are summarized in Supplementary Table 1.
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The validation experiment proved the linear dependency of the threshold cycle (CT) value for
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primers and consistency of change in threshold cycle in a given sample at different RNA concentration. Data are expressed in arbitrary units normalized by G3PDH. Immunohistochemistry
SAT biopsy samples were fixed in formalin, then embedded in paraffin. Thin sections (5 µm) were stained with anti-CD68 antibody (TransGenic, Inc.), anti-NLRP3 antibody (LifeSpan BioSciences, Inc.), anti-CD11c antibody (abcam) and anti-CD206 antibody (abnova), followed by Alexa Fluor488-conjugated anti-rat Ig, Alexa Fluor594-conjugated anti-goat Ig and Alexa 7
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Fluor647-conjugated anti-rabbit Ig staining (Life technologies). Nuclei were counterstained with Hoechst 33258. The sections were mounted with the Prolong® Gold Antifade Reagent (Life technologies) and observed under a confocal microscope (Nikon A1R confocal microscope
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system, Nikon Instruments Inc.). The percentage of NLRP3 positive cells in CD11c-positive macrophages (M1 macrophages) or CD206-positive macrophages (M2 macrophages) was calculated in three randomly selected different fields and the values were averaged in each
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sample.
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Statistics
Data are expressed as mean ± SD for continuous variables. Comparisons between two groups were performed by unpaired Student’s t-test. Comparisons of multiple groups were performed by one-way ANOVA, followed by Scheffe’s test. The association of variables including
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lifestyle-related diseases and medications to the expression of NLRP3 in SAT was assessed by univariate analysis. Multivariate regression analysis was then performed using variables that emerged from univariate analysis. Multiple regression analysis was also performed for
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parameters: age, male sex, BMI, systolic blood pressure, family history of coronary artery
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disease, smoking habit, LDL and HDL cholesterol, HbA1c, uric acid, adiponectin, CRP and the expression of NLRP3 in SAT, predicting the severity of atherosclerosis in coronary arteries as determined by the Gensini score and SYNTAX score. P values<0.05 were considered statistically significant.
Results Patient characteristics 8
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Patient characteristics at enrollment are shown in Table 1. Mean age of the 72 study participants was 70 ± 11 years; 58% were men. In our patient population, 28 subjects (39%) were obese, 50 (69%) had hypertension, 40 (56%) had dyslipidemia, 24 (33%) had diabetes mellitus and 25
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(35%) had hyperuricemia. Expression of NLRP3 in SAT
SAT biopsy was performed without any complications in all patients. Expression of NLRP3
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inflammasome-related molecules (e.g.; NLRP3, IL-1β and IL-18) was observed in subcutaneous
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fat. The expression of NLRP3 showed strong positive correlations with IL-1β and IL-18 expression (Figure 1A). Fractionation of SAT into macrophage, non-macrophage and adipocyte populations demonstrated that macrophages were the major population expressing NLRP3 (Figure 1B). The primary disease for device implantation did not affect the expression of NLRP3
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(Supplementary Figure 1). The result of fluorescent immunohistochemistry demonstrated that the percentage of NLRP3 positive cell was significantly higher in CD11c-positive macrophages than CD206-positive macrophages (Figure 1C).
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Relationship between expression of NLRP3 in SAT and lifestyle-related diseases
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We investigated whether lifestyle-related diseases have an impact on the expression of NLRP3 inflammasome-related molecules in SAT. Patients with obesity, dyslipidemia, diabetes or hyperuricemia had significantly higher expression of NLRP3, although the prevalence of hypertension or smoking habit did not correlate with the expression of NLRP3 (Figure 2A and Supplementary Figure 2). Expression of IL-1β and IL-18 was higher in patients with lifestyle-related diseases (Supplementary Figure 3). Risk stratification (0-1 low risk, 2-3 intermediate risk, ≥4 high risk) revealed that patients with multiple risk factors had elevated 9
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NLRP3 expression in SAT (Figure 2B). Next, we examined the correlation between NLRP3 expression in SAT and patient characteristics including serum levels of risk factors and medications. Results of univariate analysis demonstrated that NLRP3 expression in SAT
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correlated positively with BMI (r = 0.47, P<0.0001), HbA1c (r = 0.25, P<0.05) and uric acid (r = 0.4, P<0.001), and negatively with HDL cholesterol level (r = -0.28, P<0.05) (Table 2). Also, the use of statins, anti-diabetic drugs and anti-hyperuricemic drugs showed positive correlation with NLRP3
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expression (P<0.001). Results of multiple regression analysis using variables that emerged from
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univariate analysis demonstrated that BMI and uric acid level were independent predictors for NLRP3 expression in SAT in our patient population (Table 2).
Relationship between expression of NLRP3 in SAT and circulating inflammatory markers Then, we examined the correlation between the expression of NLRP3 in SAT and markers of
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systemic inflammation. Adiponectin, which is exclusively secreted by adipocytes, has anti-inflammatory effects. Previous studies demonstrated that circulating adiponectin level has an independent negative correlation with the severity of atherosclerosis [14, 15]. In this study, serum
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level of adiponectin showed a significant negative correlation with NLRP3 expression in SAT (r =
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-0.23, P<0.05) (Figure 3A). We also examined the correlation between the expression of NLRP3 in SAT and circulating CRP level, a well-established marker of inflammation. CRP did not correlate with NLRP3 expression in our patient population (Figure 3B). Relationship between expression of NLRP3 and severity of atherosclerosis Coronary angiography was performed in all patients without any complication. In our patient population, 16 patients (22%) had significant coronary artery stenosis (1-vessel disease (VD); 10 patients, 2-VD; 2patients, 3-VD; 4patients). These patients showed higher expression of NLRP3 10
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in SAT than patients without stenosis (Figure 4A). Furthermore, patients with 3-VD showed higher expression of NLRP3 compared with patients without significant stenosis or patients with 1-VD (Figure 4B). Furthermore, the expression of NLRP3 correlated positively with the severity of
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coronary artery disease as determined by the Gensini score (r = 0.47, P<0.001) (Figure 4C) or the SYNTAX score (r = 0.55, P<0.001) (Figure 4D). Multiple regression analysis revealed that the expression of NLRP3 in SAT remains as an independent predictors for the Gensini and SYNTAX
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score (Table 3).
Discussion
In this study, we demonstrated that lifestyle-related diseases promote expression of NLRP3 in SAT and that the expression of NLRP3 in SAT is associated with the severity of coronary
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atherosclerosis. Previous studies reported that NLRP3 inflammasome participates in chronic inflammation in the vasculature and adipose tissue;[6, 7, 9] however, no study has investigated the association of NLRP3 in SAT and the severity of atherosclerosis in human subjects. Our
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results suggested a link between NLRP3 inflammasome in SAT and coronary atherosclerosis.
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Previous studies using animal models and human subjects reported that multiple lifestyle-related diseases promote the activation of NLRP3 inflammasome in visceral adipose tissue (VAT). Those studies demonstrated that higher levels of metabolic danger signals such as cholesterol or urate crystals, reactive oxygen species, fatty acids and ceramide, which is often observed in patients with lifestyle-related diseases, activate NLRP3 inflammasome.[7, 8, 9, 16] On the other hand, recent clinical studies suggested that statins and anti-hyperuricemic drugs suppress the activation of NLRP3 inflammasome.[17, 18, 19] In accordance with 11
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previous studies, our results of univariate analyses showed that factors related to the lifestyle-related diseases (e.g.; BMI, HbA1c, uric acid, and HDL cholesterol) had significant correlation with NLRP3 expression. In our patient population, however, the use of statins,
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anti-diabetic drugs and anti-hyperuricemic drugs showed positive correlation with NLRP3 expression. Our multivariate analysis demonstrated that only BMI and uric acid levels were
predictors for the expression of NLRP3 in SAT. Differences in study design including patient
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population and target organs/cells might contribute to this discrepancy, although, our present
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study indicated that lifestyle-related diseases also promote the expression of NLRP3 in SAT at least partially, leading to the enhancement of inflammation. In this study, we showed that macrophages, a key player in adipose tissue inflammation, dominantly expressed NLRP3 in SAT. Furthermore, the result of immunohistochemistry indicated that CD11c-positive
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macrophages, one of the features of “M1” polarized macrophage,[2, 20] highly expressed NLRP3. Previous studies demonstrated that excessive polarization of macrophages toward a pro-inflammatory state contributes to the pathogenesis of chronic inflammatory diseases.[13,
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21] Our result suggested that NLPR3 inflammasome participates in this process.
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Another major finding of this study is the association between NLRP3 expression in SAT and the severity of coronary artery disease (CAD). Previous studies demonstrated that NLRP3 inflammasome participates in the process of atherogenesis. Lifestyle-related diseases directly contribute to atherosclerosis, whereas metabolic danger signals such as mentioned above also activate NLRP3 inflammasome in vascular cells and/or immune cells infiltrated in the vasculature, leading to the development of atherosclerosis. In addition, recent clinical studies reported that the activation of NLRP3 inflammasome in circulating mononuclear cells 12
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also related to the severity of CAD.[17] Furthermore, we also found that NLRP3 expression in SAT negatively correlated with the serum level of adiponectin. Numerous studies have reported anti-atherogenic effects of adiponectin and that inflammation of adipose tissue reduces
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adiponectin secretion.[14, 15, 22] Taken together with these findings, the results of our present study suggest that in addition to the direct effects of lifestyle-related diseases on the vasculature which are dependent on or independent of NLRP3 inflammasome, the activation of NLRP3
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inflammasome in SAT by lifestyle-related diseases promotes IL-1β and IL-18 expression,
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probably by macrophages, which stimulates the development of atherosclerosis. Furthermore, a reduction of adiponectin caused by adipose tissue inflammation induced by NLRP3 inflammasome activation might also contribute to this process. In fact, multivariate analysis showed that the expression of NLRP3 in SAT remained as one of the predictors for the severity
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of coronary atherosclerosis in our study. In this study, there was the big jump in NLRP3 expression in patients with 3-VD. The prevalence of risk factors for atherosclerosis in these patients was higher than total population (i.e.; hypertension, diabetes mellitus, dyslipidemia, and
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smoking; 100%, hyperuricemia; 75%, obesity; 50%). Having multiple risk factors is associated
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with higher inflammatory state.[23] Therefore, the prevalence of multiple risk factors might have synergistic effects on the expression of NLRP3. However, in this study, only 4 patients (6%) had 3-VD, because we enrolled patients who need heart devise implantation, but not patients with CAD. The small number of 3-VD patient might also contribute to this result. Thus, further studies which enroll more patients or studies which enroll patients with CAD are needed to determine the role of NLRP3 in SAT in the development of coronary atherosclerosis. Numerous studies have revealed causal roles of VAT in systemic inflammation.[24] 13
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There is no doubt that inflammation in visceral adipose tissue accelerates atherogenesis.[1, 25] Compared with VAT, SAT has been recognized as an inactive tissue. Moreover, several investigators reported protective roles of SAT against metabolic risk.[26] However,
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accumulating evidence suggests that SAT has impacts on the metabolic state, such as insulin resistance and atherosclerosis.[27, 28] Recent studies reported that the features of inflamed adipose tissue, such as macrophage infiltration and crown-like structures, are also observed in
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SAT and that an inflammatory state of SAT is associated with chronic systemic inflammation.[29,
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30] Several studies have shown that total subcutaneous truncal fat mass can be 4-5 times larger than intraperitoneal fat mass.[31, 32] Also, other studies showed that non-visceral adipose tissue contributes to the majority of circulating free fatty acids,[33, 34] which are thought to cause systemic inflammatory responses. Combined with our present results, these
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findings indicate that the inflammation in SAT may partially contribute to the development of atherosclerosis.
This study has several limitations. First, although the data in our study were analyzed
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blindly, the sample size (72 patients) was relatively small. Second, we did not investigate the
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expression of NLRP3 of VAT. Therefore, neither comparison of NLRP3 expression between VAT and SAT, nor examination of the relative contribution of both adipose tissues to the development of atherosclerosis was performed. Thirdly, we performed angiography to evaluate coronary artery atherosclerosis, which did not provide exact plaque volume extension. Fourthly, we
cannot
exclude
the
possibility
that
medications
or
over-the-counter
herbal
drugs/supplements might affect NLRP3 expression and our results. Finally, we did not directly assess the activation of NLRP3 inflammasome in this study. We observed a strong positive 14
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correlation between NLRP3 expression and NLRP3 inflammasome-related cytokines in SAT. This result does not equate to NLRP3 inflammasome activation exactly; however, this result suggests at least partial promotion of the inflammatory environment in SAT. In addition, several
inflammasome is correlated with adipose tissue inflammation.[7]
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recent studies showed that the expression of NLRP3 as well as the activation of NLRP3
In conclusion, the expression of NLRP3 in SAT is associated with the prevalence of
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lifestyle-related diseases and has a link with the severity of coronary atherosclerosis. The
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results of our study suggested a connection between SAT and atherosclerosis through NLRP3 inflammasome. Clarification of the mechanisms of the inflammatory activation of SAT and the interaction between NLRP3 expression in SAT and coronary atherosclerosis may lead to understanding of the relationship between adiposity and atherogenesis, as well as to the
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development of much needed therapy for atherosclerosis.
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All authors are grateful to Yumiko Saga for her expert technical assistance.
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Funding source This work was partially supported by JSPS Kakenhi Grants (Number 25460369 to D.F. and Number 24659392, 22390159, 25670390, 25293184 to M. Sata) and MEXT KAKENHI Grant
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Number 21117007 (M. Sata).
Conflict of interest
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None declared.
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Cardiovascular Disease and Stroke: 2002 Update: Consensus Panel Guide to Comprehensive Risk Reduction for Adult Patients Without Coronary or Other Atherosclerotic Vascular Diseases. American Heart Association Science Advisory and Coordinating Committee. Circulation
Berg AH, Scherer PE. Adipose tissue, inflammation, and cardiovascular disease. Circ
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2002;106: 388-391.
Res 2005;96:939-949. 26
McLaughlin T, Lamendola C, Liu A, et al. Preferential fat deposition in subcutaneous
versus visceral depots is associated with insulin sensitivity. J Clin Endocrinol Metab 2011;96:E1756-60. 27
Ferreira I, Henry RM, Twisk JW, et al. The metabolic syndrome, cardiopulmonary
fitness, and subcutaneous trunk fat as independent determinants of arterial stiffness: the 19
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Amsterdam Growth and Health Longitudinal Study. Arch Intern Med 2005;165:875-882. 28
Goodpaster BH, Thaete FL, Simoneau JA, et al. Subcutaneous abdominal fat and
thigh muscle composition predict insulin sensitivity independently of visceral fat. Diabetes
29
Strissel KJ, Stancheva Z, Miyoshi H, et al. Adipocyte death, adipose tissue remodeling,
and obesity complications. Diabetes 2007;56:2910-2918.
Le KA, Mahurkar S, Alderete TL, et al. Subcutaneous adipose tissue macrophage
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1997;46:1579-1585.
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infiltration is associated with hepatic and visceral fat deposition, hyperinsulinemia, and stimulation of NF-kappaB stress pathway. Diabetes 2011;60:2802-2809. 31
Abate N, Garg A. Heterogeneity in adipose tissue metabolism: causes, implications
and management of regional adiposity. Prog Lipid Res 1995;34:53-70. Abate N, Garg A, Peshock RM, et al. Relationship of generalized and regional
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adiposity to insulin sensitivity in men with NIDDM. Diabetes 1996;45:1684-1693. 33
Garg A. Regional adiposity and insulin resistance. J Clin Endocrinol Metab
Jensen MD, Johnson CM. Contribution of leg and splanchnic free fatty acid (FFA)
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2004;89:4206-4210.
kinetics to postabsorptive FFA flux in men and women. Metabolism 1996;45:662-666.
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Figure 1. Expression of NLRP3 and NLRP3 inflammasome-related molecules in SAT. A. Expression of NLRP3 had strong positive correlations with NLRP3 inflammasome-related
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molecules, IL-1β and IL-18, in SAT. B. To investigate cell type-specific expression of NLRP3 inflammasome-related molecules, fractionation of SAT into macrophage, non-macrophage and adipocyte populations was performed. Macrophages were the major population expressing
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NLRP3 inflammasome-related molecules in SAT (n = 7). C. The result of fluorescent
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immunohistochemistry demonstrated that the percentage of NLRP3 positive cells (white, indicated by arrows in panels (a) and (b)) was significantly higher in CD11c-positive macrophages (cyan, indicated by arrow heads in the panel (a)) than CD206-positive macrophages (cyan, indicated by arrow heads in the panel (b)) (n = 7). *; P<0.05, **; P<0.01 and
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***; P<0.001.
Figure 2. Correlation between lifestyle-related diseases and expression of NLRP3 in SAT. A. Expression of NLRP3 in individuals with or without lifestyle-related diseases. B. Relationship
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and **; P<0.01.
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between the number of lifestyle-related diseases and expression of NLRP3 in SAT. *; P<0.05
Figure 3. Relationship between expression of NLRP3 in SAT and circulating inflammatory markers.
A. Serum level of adiponectin negatively correlated with expression of NLRP3. B. Serum level of CRP did not correlate with expression of NLRP3. Figure 4. Relationship between expression of NLRP3 in SAT and atherosclerosis. A. Expression of NLRP3 in patients with CAD was higher than that in patients without CAD. B. 21
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Expression of NLRP3 in patients with 3-VD was higher than that in patients without CAD or with 1-VD. C and D. Severity of coronary artery disease as determined by Gensini score (C) or SXYNTAX score (D) had a significant positive correlation with NLRP3 expression. **; P<0.01
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and ***; P<0.001.
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ACCEPTED MANUSCRIPT Table 1. Patients' Characteristics (N = 72) Age (yrs)
70 ± 11 42 (58) / 30 (42)
Obesity (BMI≥25)
28 (39)
Hypertension
50 (69)
Dyslipidemia
40 (56)
Diabetes mellitus
24 (33)
Hyperuricemia
25 (35)
Smoking
44 (61)
BMI, kg/m2
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23.5 ± 3.2
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Male/Female
Systolic blood pressure, mmHg
119.9 ± 15.2
LDL choletesrol, mg/dL
100.7 ± 32.8
HDL cholesterol, mg/dL
53.3 ± 15.0 5.6 ± 0.8
Uric acid, mg/dL
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HbA1c, %
6.2 ± 1.6
The use of Statins
28 (39)
The use of RAS inhibitors
37 (51)
The use of anti-diabetes drugs
18 (25)
The use of anti-hyperuricemic drugs
21 (29)
Cardiomyopathy Sick sinus syndrome VT or VF
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AVB
13 (18)
13 (18) 27 (38) 11 (15)
Data presented are mean ±SD or number (percentage).
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BMI; body mass index, RAS; renin-angiotensin system, AVB; atrio-ventricular block
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VT; ventricular tachycardia, VF; ventricular fibrillation.
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Table 2. Univariate and Multivariate analyses predicting NLRP3 expression in SAT Univariate analysis
Multivariate analysis R2; 0.40 Standard regression coefficient
P-value
0.26
0.02
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P-value; <0.0001 P-value
0.08
0.53
0.01
0.95
BMI, kg/m
0.47
<0.0001
Systolic blood pressure, mmHg
0.03
0.82
LDL cholesterol, mg/dL
-0.09
0.48
HDL cholesterol, mg/dL
-0.28
0.02
-0.1
0.37
HbA1c, %
0.25
0.03
-0.02
0.87
0.22
0.048
Male sex 2
Uric acid, mg/dL Smoking The use of statins The use of anti-diabetic drugs The use of RAS inhibitors The use of anti-hyperuricemic drugs
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Age
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r
0.4
0.0005
0.09
0.45
0.35
0.002
0.14
0.22
0.41
0.0003
0.17
0.18
0.05
0.7
0.33
0.005
0.15
0.18
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BMI; body mass index, HDL; high density lipoprotein, RAS; renin-angiotensin system.
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Logarithmic value of NLRP3/G3PDH was used in these analyses.
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ACCEPTED MANUSCRIPT Table 3. Multivariate analysis predicting the severity of atherosclerosis Gensini score
SYNTAX score
R2
0.42
0.46
P-value
0.001
0.0002 Standard
Standard P-value
coefficient
P-value
coefficient
-0.12
0.34
-0.03
0.82
0.12
0.35
-0.003
0.98
BMI, kg/m
0.02
0.86
0.01
0.94
Systolic blood pressure, mmHg
-0.23
0.05
-0.16
0.16
LDL cholesterol, mg/dL
-0.24
0.04
-0.24
0.03
HDL cholesterol, mg/dL
-0.08
0.53
-0.16
0.18
HbA1c, %
0.24
0.04
0.15
0.19
Uric acid, mg/dL
-0.01
0.96
-0.02
0.87
Smoking
0.11
0.41
0.13
0.31
0.2
0.08
0.1
0.33
0.15
0.24
0.07
0.57
C-reactive protein, mg/dL
-0.08
0.49
-0.13
0.23
NLRP3 in SAT
0.33
0.02
0.43
0.002
Male sex
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Age
regression
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regression
Family history of CAD Adiponectin, µg/mL
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BMI; body mass index, CAD; coronary artery disease.
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Logarithmic value of NLRP3/G3PDH was used in this analysis.
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Figure 1.
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A
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Figure 3.
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D
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C
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A
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Figure 4.
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Highlights NLRP3 expression in SAT was higher in patients with lifestyle-related diseases.
NLRP3 expression in SAT correlated negatively with serum adiponectin level.
CAD patients showed higher NLRP3 expression in SAT than non-CAD patients.
NLRP3 expression in SAT and HbA1c were predictors of coronary atherosclerosis.
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Supplementary Material for Expression of NLRP3 in subcutaneous adipose tissue
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is associated with coronary atherosclerosis
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Sachiko Bando1, Daiju Fukuda2, Takeshi Soeki1, Sachiko Nishimoto3, Etsuko Uematsu1, Tomomi Matsuura1, Takayuki Ise1, Takeshi Tobiume1, Koji Yamaguchi1, Shusuke Yagi1, Takashi Iwase1, Hirotsugu Yamada1, Tetsuzo Wakatsuki1, Michio Shimabukuro2 and Masataka Sata1
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Number of Supplementary Figures: 3 Number of Supplementary Tables: 1
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1. Department of Cardiovascular Medicine, Institute of Biomedical Sciences, Tokushima University Graduate School, Tokushima, Japan 2. Department of Nutrition and Metabolism, Institute of Biomedical Sciences, Tokushima University Graduate School, Tokushima, Japan 3. Department of Cardio-Diabetes Medicine, Institute of Biomedical Sciences, Tokushima University Graduate School, Tokushima, Japan
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Corresponding author: Daiju Fukuda, MD, PhD Department of Cardiovascular Medicine, Institute of Biomedical Sciences, Tokushima University Graduate School 3-18-15, Kuramoto-cho, Tokushima 770-8503, Japan Phone: +81-88-633-7859 Fax: +81-88-633-7894 E-mail: [email protected]
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Supplementary Figure 1. Comparison of the expression of NLRP3 among the primary diseases. The primary disease for the permanent heart device implantation did not affect the expression of NLRP3. AV; Atrio-ventricular, SSS; sick-sinus syndrome, VT/ VF; ventricular tachycardia/ventricular fibrillation.
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Supplementary Figure 2. Correlation between the the expression of NLRP3 and degree of risk factors. We divided our patients into quartiles according to the level of each risk factor and examined the expression of NLRP3 in each quartile. There were significant differences between 4th and 1st quartile groups of BMI, HDL-cholesterol. As for HbA1c, the expression of NLRP3 tended to be higher in 4th quartile group than 1st, 2ndand 3rd quartile group (P=0.08, P=0.06 and P=0.09, respectively). BMI; body mass index, LDL; low-density lipoprotein and HDL; high-density lipoprotein. *; P<0.05.
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Supplementary Figure 3. Relationship between lifestyle-related diseases and expression of IL-1β β and IL-18. Expression of NLRP3 inflammasome-related molecules, IL-1β and IL-18, were higher in patients with lifestyle-related diseases. *; P<0.05 **; P<0.01 and ***; P<0.001. 4
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Supplementary Table. Sequence of gene specific primers Gene
Sequence
Reverse 5'- GGGAATGGCTGGTGCTCAATAC -3' IL-1β
Forward 5'- CCAGGGACAGGATATGGAGCA -3'
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NLRP3 Forward 5'- AAGCACCTGTTGTGCAATCTGAAG -3'
Reverse 5'- TTCAACACGCAGGACAGGTACAG -3' IL-18
Forward 5'- GCCTGGACAGTCAGCAAGGA -3'
Reverse 5'- TCTACTGGTTCAGCAGCCATCTTTA -3'
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G3PDH Forward 5'- ACCACAGTCCATGCCATCAC -3'
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Reverse 5'- TCCACCACCCTGTTGCTGTA -3'
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S0021-9150(15)30063-0
DOI:
10.1016/j.atherosclerosis.2015.07.043
Reference:
ATH 14214
To appear in:
Atherosclerosis
Received Date: 8 January 2015 Revised Date:
23 July 2015
Accepted Date: 27 July 2015
Please cite this article as: Bando S, Fukuda D, Soeki T, Nishimoto S, Uematsu E, Matsuura T, Ise T, Tobiume T, Yamaguchi K, Yagi S, Iwase T, Yamada H, Wakatsuki T, Shimabukuro M, Sata M, Expression of NLRP3 in subcutaneous adipose tissue is associated with coronary atherosclerosis, Atherosclerosis (2015), doi: 10.1016/j.atherosclerosis.2015.07.043. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Bando S, et al. Ms. No. ATH-D-15-00026/R2
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Expression of NLRP3 in subcutaneous adipose tissue is associated with coronary atherosclerosis
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Sachiko Bando1, Daiju Fukuda1, Takeshi Soeki1, Sachiko Nishimoto2, Etsuko Uematsu1, Tomomi Matsuura1, Takayuki Ise1, Takeshi Tobiume1, Koji Yamaguchi1, Shusuke Yagi1, Takashi Iwase1, Hirotsugu Yamada1, Tetsuzo Wakatsuki1, Michio Shimabukuro3 and Masataka Sata1
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1. Department of Cardiovascular Medicine, Institute of Biomedical Sciences, Tokushima University Graduate School, Tokushima, Japan 2. Department of Nutrition and Metabolism, Institute of Biomedical Sciences, Tokushima University Graduate School, Tokushima, Japan 3. Department of Cardio-Diabetes Medicine, Institute of Biomedical Sciences, Tokushima University Graduate School, Tokushima, Japan
Running title: NLRP3 in subcutaneous adipose tissue and coronary atherosclerosis
adipose
tissue,
inflammation,
NLRP3
inflammasome,
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Key words: atherosclerosis, lifestyle-related disease
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Word count: 4910 Number of Tables: 3 Number of Figures: 4 Number of Supplementary Figures: 3 Number of Supplementary Tables: 1
Corresponding author: Daiju Fukuda, MD, PhD Department of Cardiovascular Medicine, Institute of Biomedical Sciences, Tokushima University Graduate School 3-18-15, Kuramoto-cho, Tokushima 770-8503, Japan Phone: +81-88-633-7859 Fax: +81-88-633-7894 E-mail: [email protected]
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Bando S, et al. Ms. No. ATH-D-15-00026/R2
ACCEPTED MANUSCRIPT Abstract
Objectives: The promotion of adipose tissue inflammation by lifestyle-related diseases such as obesity and diabetes accelerates atherogenesis; however, the underlying mechanisms remain
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incompletely understood. Nucleotide-binding domain, leucine-rich-containing family, pyrin domain-containing-3 (NLRP3) inflammasome contributes to chronic inflammation in adipose
tissue (SAT) and the severity of coronary atherosclerosis.
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tissue. Here, we investigated the link between NLRP3 expression in subcutaneous adipose
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Methods and Results: SAT was obtained from 72 patients who underwent heart device implantation and coronary angiography. Expression of NLRP3 inflammasome-related molecules (NLRP3, IL-1β and IL-18) in SAT were evaluated by quantitative RT-PCR. Laboratory markers related to lifestyle-related diseases were measured. Patients with obesity,
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dyslipidemia (P<0.05, respectively), diabetes or hyperuricemia (P<0.01, respectively) had significantly higher expression of NLRP3. Multivariate analysis demonstrated that body mass index and serum level of uric acid were predictors of NLRP3 expression in SAT. The expression
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of NLRP3 in SAT correlated negatively with serum adiponectin level (r= -0.23, P<0.05). Patients
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with coronary artery disease showed higher NLRP3 expression than patients without significant stenosis (P<0.01). Furthermore, the expression of NLRP3 in SAT correlated positively with the severity of coronary atherosclerosis as determined by Gensini score (r = 0.47, P<0.0001) or SYNTAX score (r = 0.55, P<0.0001). Multiple regression analysis revealed that the expression of NLRP3 in SAT remains as an independent predictors for the severity of coronary atherosclerosis. Conclusions: The expression of NLRP3 in SAT, which is affected by lifestyle-related diseases, 2
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is associated with the severity of coronary atherosclerosis. Our results suggest that NLRP3
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inflammasome in SAT may have a role in atherogenesis.
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There is increasing evidence that lifestyle-related diseases, such as obesity, dyslipidemia and diabetes, promote local pro-inflammatory responses in adipose tissue, accelerating systemic
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inflammation, which contributes to the development of atherosclerosis.[1, 2, 3] Much effort has been made to elucidate the molecular mechanisms of adipose tissue inflammation. Recent studies have shown that danger signals related to metabolic disorders could initiate
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pro-inflammatory responses in adipose tissue. For example, saturated fatty acids activate
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toll-like receptors (TLRs), a family of pattern recognition molecules, and promote inflammation in adipose tissue.[4, 5] More recently, another pattern recognition receptor, nucleotide-binding domain, leucine-rich-containing family, pyrin domain-containing-3 (NLRP3) inflammasome, has been implicated in recognizing endogenous danger signals, leading to the development of
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adipose tissue inflammation through the activation of caspase-1 and subsequent secretion of interleukin (IL)-1β and IL-18.[6, 7, 8] NLRP3 inflammasome also participates in the development of vascular inflammation and atherogenesis.[9, 10] It is widely accepted that
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NLRP3 inflammasome contributes to the development of chronic inflammation in both adipose
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tissue and the vasculature, although, whether the expression of NLRP3 in adipose tissue is associated with atherosclerosis remains unknown. Therefore, in this study, we hypothesized that lifestyle-related diseases affect the expression of NLRP3 in adipose tissue, which associates with the severity of coronary atherosclerosis. To assess our hypothesis, we examined the expression of NLRP3 inflammasome-related molecules (e.g., NLRP3, IL-1β and IL-18) in subcutaneous adipose tissue (SAT) obtained from patients admitted to our hospital for permanent heart device implantation, and investigated its relationship with the severity of 4
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coronary atherosclerosis. The results of our present study suggest that the expression of NLRP3 in SAT may have links with the development of atherosclerosis.
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Methods Study protocol and patient enrollment
Our patient population comprised 72 consecutive patients who were admitted to Tokushima
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University Hospital for implantation of an implantable heart device, such as permanent
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pacemaker, implantable cardiac defibrillator or cardiac resynchronization therapy device because of sick sinus syndrome (n=27), atrio-ventricular block (n=21), ventricular tachycardia/ventricular fibrillation (n=11) and dilated or ischemic cardiomyopathy (n=13). We excluded patients with malignant tumors, sarcoidosis, severe congestive heart failure and active
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inflammatory disease (e.g.; infection or collagen disease). We also excluded patients who were taking immunosuppressants or over-the-counter herbal drugs and patients who had recovered from cardiac arrest.
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Cardiovascular risk factors were defined as follows: hypertension as blood pressure >
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140/90 mmHg in three consecutive readings at rest; dyslipidemia as LDL cholesterol level > 140 mg/dL and/or HDL cholesterol < 40 mg/dL; diabetes as fasting glucose level > 126 mg/dL and/or random blood glucose level > 200 mg/dL and/or HbA1c > 6.5%; obesity as body mass index (BMI) > 25 kg/m2; and hyperuricemia as uric acid level > 7.0 mg/dL. Subjects receiving anti-hypertensive drugs, cholesterol-lowering therapy, anti-diabetic drugs including insulin, or anti-hyperuricemic drugs were also considered to have hypertension, hyperlipidemia, diabetes or hyperuricemia, respectively. Both current smokers and ex-smokers were considered 5
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Serum concentrations of LDL cholesterol, HDL cholesterol, uric acid, and C-reactive protein (CRP) and plasma concentration of HbA1c were measured in samples collected after
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the patients had fasted overnight. Serum concentration of adiponectin was measured with a commercially available kit (R&D Systems).
This study was conducted in accordance with the Declaration of Helsinki and was
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approved by the Ethics Committee of Tokushima University Hospital. Written informed consent
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was obtained from all subjects. SAT biopsy
SAT biopsy was performed in each patient at the time of devise implantation. A small piece of SAT was obtained when a subcutaneous pocket was created in the infraclavicular region. SAT
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samples were processed for fractionation immediately after biopsy. For RNA extraction, samples were preserved at -80 oC until assay.
Assessment of atherosclerosis in coronary arteries
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Coronary angiography was performed by a standard technique. To prevent coronary spasm, all
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patients received intracoronary injection of isosorbide dinitrate at an optimal dose before angiography. Two independent observers blinded to the results of the analyses of SAT reviewed the coronary angiograms separately. Stenosis > 75% detected angiographically in a major coronary vessel was defined as significant stenosis. Gensini and SYNTAX scores were calculated based on angiographic findings as previously reported.[11, 12] Fractionation of adipose tissue and macrophage selection Fractionation of SAT was performed as described previously.[13] Subcutaneous fat was minced 6
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in PBS and then incubated on a shaking platform for 30 min at 37 oC with collagenase (250 U/ml). The mixture was then passed through a nylon filter (pore size, 250 µm) to remove undigested material, and the filtrate was centrifuged at 200 g for 5 min at 4 oC. Floating cells
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and the pellet were recovered as the mature adipocyte fraction and the stromal vascular fraction, respectively. The stromal vascular fraction was incubated with phycoerythrin-conjugated anti-CD14 antibody (Biolegend) followed by anti-phycoerythrin magnet beads (Miltenyi Biotech).
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CD14-positive and -negative fractions were collected using the MACS® system (Miltenyi
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Biotech). Quantitative RT-PCR
Total RNA was extracted using illustra RNAspin RNA Isolation Kit (GE Healthcare). cDNA was synthesized from 100 ng of total RNA extracted from tissues and cells using a QuantiTect
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Reverse Transcription kit (Qiagen). Real-time quantitative RT-PCR (qPCR) was performed with a Mx3000P (Agilent Technologies) and Power SYBR Green PCR Master Mix (Applied Biosystems). Sequences of gene specific primers are summarized in Supplementary Table 1.
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The validation experiment proved the linear dependency of the threshold cycle (CT) value for
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primers and consistency of change in threshold cycle in a given sample at different RNA concentration. Data are expressed in arbitrary units normalized by G3PDH. Immunohistochemistry
SAT biopsy samples were fixed in formalin, then embedded in paraffin. Thin sections (5 µm) were stained with anti-CD68 antibody (TransGenic, Inc.), anti-NLRP3 antibody (LifeSpan BioSciences, Inc.), anti-CD11c antibody (abcam) and anti-CD206 antibody (abnova), followed by Alexa Fluor488-conjugated anti-rat Ig, Alexa Fluor594-conjugated anti-goat Ig and Alexa 7
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Fluor647-conjugated anti-rabbit Ig staining (Life technologies). Nuclei were counterstained with Hoechst 33258. The sections were mounted with the Prolong® Gold Antifade Reagent (Life technologies) and observed under a confocal microscope (Nikon A1R confocal microscope
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system, Nikon Instruments Inc.). The percentage of NLRP3 positive cells in CD11c-positive macrophages (M1 macrophages) or CD206-positive macrophages (M2 macrophages) was calculated in three randomly selected different fields and the values were averaged in each
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Statistics
Data are expressed as mean ± SD for continuous variables. Comparisons between two groups were performed by unpaired Student’s t-test. Comparisons of multiple groups were performed by one-way ANOVA, followed by Scheffe’s test. The association of variables including
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lifestyle-related diseases and medications to the expression of NLRP3 in SAT was assessed by univariate analysis. Multivariate regression analysis was then performed using variables that emerged from univariate analysis. Multiple regression analysis was also performed for
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parameters: age, male sex, BMI, systolic blood pressure, family history of coronary artery
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disease, smoking habit, LDL and HDL cholesterol, HbA1c, uric acid, adiponectin, CRP and the expression of NLRP3 in SAT, predicting the severity of atherosclerosis in coronary arteries as determined by the Gensini score and SYNTAX score. P values<0.05 were considered statistically significant.
Results Patient characteristics 8
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Patient characteristics at enrollment are shown in Table 1. Mean age of the 72 study participants was 70 ± 11 years; 58% were men. In our patient population, 28 subjects (39%) were obese, 50 (69%) had hypertension, 40 (56%) had dyslipidemia, 24 (33%) had diabetes mellitus and 25
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(35%) had hyperuricemia. Expression of NLRP3 in SAT
SAT biopsy was performed without any complications in all patients. Expression of NLRP3
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inflammasome-related molecules (e.g.; NLRP3, IL-1β and IL-18) was observed in subcutaneous
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fat. The expression of NLRP3 showed strong positive correlations with IL-1β and IL-18 expression (Figure 1A). Fractionation of SAT into macrophage, non-macrophage and adipocyte populations demonstrated that macrophages were the major population expressing NLRP3 (Figure 1B). The primary disease for device implantation did not affect the expression of NLRP3
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(Supplementary Figure 1). The result of fluorescent immunohistochemistry demonstrated that the percentage of NLRP3 positive cell was significantly higher in CD11c-positive macrophages than CD206-positive macrophages (Figure 1C).
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Relationship between expression of NLRP3 in SAT and lifestyle-related diseases
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We investigated whether lifestyle-related diseases have an impact on the expression of NLRP3 inflammasome-related molecules in SAT. Patients with obesity, dyslipidemia, diabetes or hyperuricemia had significantly higher expression of NLRP3, although the prevalence of hypertension or smoking habit did not correlate with the expression of NLRP3 (Figure 2A and Supplementary Figure 2). Expression of IL-1β and IL-18 was higher in patients with lifestyle-related diseases (Supplementary Figure 3). Risk stratification (0-1 low risk, 2-3 intermediate risk, ≥4 high risk) revealed that patients with multiple risk factors had elevated 9
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NLRP3 expression in SAT (Figure 2B). Next, we examined the correlation between NLRP3 expression in SAT and patient characteristics including serum levels of risk factors and medications. Results of univariate analysis demonstrated that NLRP3 expression in SAT
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correlated positively with BMI (r = 0.47, P<0.0001), HbA1c (r = 0.25, P<0.05) and uric acid (r = 0.4, P<0.001), and negatively with HDL cholesterol level (r = -0.28, P<0.05) (Table 2). Also, the use of statins, anti-diabetic drugs and anti-hyperuricemic drugs showed positive correlation with NLRP3
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expression (P<0.001). Results of multiple regression analysis using variables that emerged from
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univariate analysis demonstrated that BMI and uric acid level were independent predictors for NLRP3 expression in SAT in our patient population (Table 2).
Relationship between expression of NLRP3 in SAT and circulating inflammatory markers Then, we examined the correlation between the expression of NLRP3 in SAT and markers of
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systemic inflammation. Adiponectin, which is exclusively secreted by adipocytes, has anti-inflammatory effects. Previous studies demonstrated that circulating adiponectin level has an independent negative correlation with the severity of atherosclerosis [14, 15]. In this study, serum
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level of adiponectin showed a significant negative correlation with NLRP3 expression in SAT (r =
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-0.23, P<0.05) (Figure 3A). We also examined the correlation between the expression of NLRP3 in SAT and circulating CRP level, a well-established marker of inflammation. CRP did not correlate with NLRP3 expression in our patient population (Figure 3B). Relationship between expression of NLRP3 and severity of atherosclerosis Coronary angiography was performed in all patients without any complication. In our patient population, 16 patients (22%) had significant coronary artery stenosis (1-vessel disease (VD); 10 patients, 2-VD; 2patients, 3-VD; 4patients). These patients showed higher expression of NLRP3 10
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in SAT than patients without stenosis (Figure 4A). Furthermore, patients with 3-VD showed higher expression of NLRP3 compared with patients without significant stenosis or patients with 1-VD (Figure 4B). Furthermore, the expression of NLRP3 correlated positively with the severity of
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coronary artery disease as determined by the Gensini score (r = 0.47, P<0.001) (Figure 4C) or the SYNTAX score (r = 0.55, P<0.001) (Figure 4D). Multiple regression analysis revealed that the expression of NLRP3 in SAT remains as an independent predictors for the Gensini and SYNTAX
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score (Table 3).
Discussion
In this study, we demonstrated that lifestyle-related diseases promote expression of NLRP3 in SAT and that the expression of NLRP3 in SAT is associated with the severity of coronary
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atherosclerosis. Previous studies reported that NLRP3 inflammasome participates in chronic inflammation in the vasculature and adipose tissue;[6, 7, 9] however, no study has investigated the association of NLRP3 in SAT and the severity of atherosclerosis in human subjects. Our
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results suggested a link between NLRP3 inflammasome in SAT and coronary atherosclerosis.
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Previous studies using animal models and human subjects reported that multiple lifestyle-related diseases promote the activation of NLRP3 inflammasome in visceral adipose tissue (VAT). Those studies demonstrated that higher levels of metabolic danger signals such as cholesterol or urate crystals, reactive oxygen species, fatty acids and ceramide, which is often observed in patients with lifestyle-related diseases, activate NLRP3 inflammasome.[7, 8, 9, 16] On the other hand, recent clinical studies suggested that statins and anti-hyperuricemic drugs suppress the activation of NLRP3 inflammasome.[17, 18, 19] In accordance with 11
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previous studies, our results of univariate analyses showed that factors related to the lifestyle-related diseases (e.g.; BMI, HbA1c, uric acid, and HDL cholesterol) had significant correlation with NLRP3 expression. In our patient population, however, the use of statins,
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anti-diabetic drugs and anti-hyperuricemic drugs showed positive correlation with NLRP3 expression. Our multivariate analysis demonstrated that only BMI and uric acid levels were
predictors for the expression of NLRP3 in SAT. Differences in study design including patient
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population and target organs/cells might contribute to this discrepancy, although, our present
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study indicated that lifestyle-related diseases also promote the expression of NLRP3 in SAT at least partially, leading to the enhancement of inflammation. In this study, we showed that macrophages, a key player in adipose tissue inflammation, dominantly expressed NLRP3 in SAT. Furthermore, the result of immunohistochemistry indicated that CD11c-positive
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macrophages, one of the features of “M1” polarized macrophage,[2, 20] highly expressed NLRP3. Previous studies demonstrated that excessive polarization of macrophages toward a pro-inflammatory state contributes to the pathogenesis of chronic inflammatory diseases.[13,
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21] Our result suggested that NLPR3 inflammasome participates in this process.
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Another major finding of this study is the association between NLRP3 expression in SAT and the severity of coronary artery disease (CAD). Previous studies demonstrated that NLRP3 inflammasome participates in the process of atherogenesis. Lifestyle-related diseases directly contribute to atherosclerosis, whereas metabolic danger signals such as mentioned above also activate NLRP3 inflammasome in vascular cells and/or immune cells infiltrated in the vasculature, leading to the development of atherosclerosis. In addition, recent clinical studies reported that the activation of NLRP3 inflammasome in circulating mononuclear cells 12
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also related to the severity of CAD.[17] Furthermore, we also found that NLRP3 expression in SAT negatively correlated with the serum level of adiponectin. Numerous studies have reported anti-atherogenic effects of adiponectin and that inflammation of adipose tissue reduces
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adiponectin secretion.[14, 15, 22] Taken together with these findings, the results of our present study suggest that in addition to the direct effects of lifestyle-related diseases on the vasculature which are dependent on or independent of NLRP3 inflammasome, the activation of NLRP3
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inflammasome in SAT by lifestyle-related diseases promotes IL-1β and IL-18 expression,
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probably by macrophages, which stimulates the development of atherosclerosis. Furthermore, a reduction of adiponectin caused by adipose tissue inflammation induced by NLRP3 inflammasome activation might also contribute to this process. In fact, multivariate analysis showed that the expression of NLRP3 in SAT remained as one of the predictors for the severity
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of coronary atherosclerosis in our study. In this study, there was the big jump in NLRP3 expression in patients with 3-VD. The prevalence of risk factors for atherosclerosis in these patients was higher than total population (i.e.; hypertension, diabetes mellitus, dyslipidemia, and
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smoking; 100%, hyperuricemia; 75%, obesity; 50%). Having multiple risk factors is associated
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with higher inflammatory state.[23] Therefore, the prevalence of multiple risk factors might have synergistic effects on the expression of NLRP3. However, in this study, only 4 patients (6%) had 3-VD, because we enrolled patients who need heart devise implantation, but not patients with CAD. The small number of 3-VD patient might also contribute to this result. Thus, further studies which enroll more patients or studies which enroll patients with CAD are needed to determine the role of NLRP3 in SAT in the development of coronary atherosclerosis. Numerous studies have revealed causal roles of VAT in systemic inflammation.[24] 13
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There is no doubt that inflammation in visceral adipose tissue accelerates atherogenesis.[1, 25] Compared with VAT, SAT has been recognized as an inactive tissue. Moreover, several investigators reported protective roles of SAT against metabolic risk.[26] However,
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accumulating evidence suggests that SAT has impacts on the metabolic state, such as insulin resistance and atherosclerosis.[27, 28] Recent studies reported that the features of inflamed adipose tissue, such as macrophage infiltration and crown-like structures, are also observed in
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SAT and that an inflammatory state of SAT is associated with chronic systemic inflammation.[29,
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30] Several studies have shown that total subcutaneous truncal fat mass can be 4-5 times larger than intraperitoneal fat mass.[31, 32] Also, other studies showed that non-visceral adipose tissue contributes to the majority of circulating free fatty acids,[33, 34] which are thought to cause systemic inflammatory responses. Combined with our present results, these
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findings indicate that the inflammation in SAT may partially contribute to the development of atherosclerosis.
This study has several limitations. First, although the data in our study were analyzed
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blindly, the sample size (72 patients) was relatively small. Second, we did not investigate the
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expression of NLRP3 of VAT. Therefore, neither comparison of NLRP3 expression between VAT and SAT, nor examination of the relative contribution of both adipose tissues to the development of atherosclerosis was performed. Thirdly, we performed angiography to evaluate coronary artery atherosclerosis, which did not provide exact plaque volume extension. Fourthly, we
cannot
exclude
the
possibility
that
medications
or
over-the-counter
herbal
drugs/supplements might affect NLRP3 expression and our results. Finally, we did not directly assess the activation of NLRP3 inflammasome in this study. We observed a strong positive 14
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correlation between NLRP3 expression and NLRP3 inflammasome-related cytokines in SAT. This result does not equate to NLRP3 inflammasome activation exactly; however, this result suggests at least partial promotion of the inflammatory environment in SAT. In addition, several
inflammasome is correlated with adipose tissue inflammation.[7]
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recent studies showed that the expression of NLRP3 as well as the activation of NLRP3
In conclusion, the expression of NLRP3 in SAT is associated with the prevalence of
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lifestyle-related diseases and has a link with the severity of coronary atherosclerosis. The
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results of our study suggested a connection between SAT and atherosclerosis through NLRP3 inflammasome. Clarification of the mechanisms of the inflammatory activation of SAT and the interaction between NLRP3 expression in SAT and coronary atherosclerosis may lead to understanding of the relationship between adiposity and atherogenesis, as well as to the
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development of much needed therapy for atherosclerosis.
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All authors are grateful to Yumiko Saga for her expert technical assistance.
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Funding source This work was partially supported by JSPS Kakenhi Grants (Number 25460369 to D.F. and Number 24659392, 22390159, 25670390, 25293184 to M. Sata) and MEXT KAKENHI Grant
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Number 21117007 (M. Sata).
Conflict of interest
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None declared.
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ACCEPTED MANUSCRIPT Figure legends
Figure 1. Expression of NLRP3 and NLRP3 inflammasome-related molecules in SAT. A. Expression of NLRP3 had strong positive correlations with NLRP3 inflammasome-related
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molecules, IL-1β and IL-18, in SAT. B. To investigate cell type-specific expression of NLRP3 inflammasome-related molecules, fractionation of SAT into macrophage, non-macrophage and adipocyte populations was performed. Macrophages were the major population expressing
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NLRP3 inflammasome-related molecules in SAT (n = 7). C. The result of fluorescent
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immunohistochemistry demonstrated that the percentage of NLRP3 positive cells (white, indicated by arrows in panels (a) and (b)) was significantly higher in CD11c-positive macrophages (cyan, indicated by arrow heads in the panel (a)) than CD206-positive macrophages (cyan, indicated by arrow heads in the panel (b)) (n = 7). *; P<0.05, **; P<0.01 and
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***; P<0.001.
Figure 2. Correlation between lifestyle-related diseases and expression of NLRP3 in SAT. A. Expression of NLRP3 in individuals with or without lifestyle-related diseases. B. Relationship
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and **; P<0.01.
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between the number of lifestyle-related diseases and expression of NLRP3 in SAT. *; P<0.05
Figure 3. Relationship between expression of NLRP3 in SAT and circulating inflammatory markers.
A. Serum level of adiponectin negatively correlated with expression of NLRP3. B. Serum level of CRP did not correlate with expression of NLRP3. Figure 4. Relationship between expression of NLRP3 in SAT and atherosclerosis. A. Expression of NLRP3 in patients with CAD was higher than that in patients without CAD. B. 21
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Expression of NLRP3 in patients with 3-VD was higher than that in patients without CAD or with 1-VD. C and D. Severity of coronary artery disease as determined by Gensini score (C) or SXYNTAX score (D) had a significant positive correlation with NLRP3 expression. **; P<0.01
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and ***; P<0.001.
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ACCEPTED MANUSCRIPT Table 1. Patients' Characteristics (N = 72) Age (yrs)
70 ± 11 42 (58) / 30 (42)
Obesity (BMI≥25)
28 (39)
Hypertension
50 (69)
Dyslipidemia
40 (56)
Diabetes mellitus
24 (33)
Hyperuricemia
25 (35)
Smoking
44 (61)
BMI, kg/m2
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23.5 ± 3.2
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Male/Female
Systolic blood pressure, mmHg
119.9 ± 15.2
LDL choletesrol, mg/dL
100.7 ± 32.8
HDL cholesterol, mg/dL
53.3 ± 15.0 5.6 ± 0.8
Uric acid, mg/dL
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HbA1c, %
6.2 ± 1.6
The use of Statins
28 (39)
The use of RAS inhibitors
37 (51)
The use of anti-diabetes drugs
18 (25)
The use of anti-hyperuricemic drugs
21 (29)
Cardiomyopathy Sick sinus syndrome VT or VF
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AVB
13 (18)
13 (18) 27 (38) 11 (15)
Data presented are mean ±SD or number (percentage).
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BMI; body mass index, RAS; renin-angiotensin system, AVB; atrio-ventricular block
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VT; ventricular tachycardia, VF; ventricular fibrillation.
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Table 2. Univariate and Multivariate analyses predicting NLRP3 expression in SAT Univariate analysis
Multivariate analysis R2; 0.40 Standard regression coefficient
P-value
0.26
0.02
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P-value; <0.0001 P-value
0.08
0.53
0.01
0.95
BMI, kg/m
0.47
<0.0001
Systolic blood pressure, mmHg
0.03
0.82
LDL cholesterol, mg/dL
-0.09
0.48
HDL cholesterol, mg/dL
-0.28
0.02
-0.1
0.37
HbA1c, %
0.25
0.03
-0.02
0.87
0.22
0.048
Male sex 2
Uric acid, mg/dL Smoking The use of statins The use of anti-diabetic drugs The use of RAS inhibitors The use of anti-hyperuricemic drugs
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Age
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r
0.4
0.0005
0.09
0.45
0.35
0.002
0.14
0.22
0.41
0.0003
0.17
0.18
0.05
0.7
0.33
0.005
0.15
0.18
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BMI; body mass index, HDL; high density lipoprotein, RAS; renin-angiotensin system.
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Logarithmic value of NLRP3/G3PDH was used in these analyses.
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SYNTAX score
R2
0.42
0.46
P-value
0.001
0.0002 Standard
Standard P-value
coefficient
P-value
coefficient
-0.12
0.34
-0.03
0.82
0.12
0.35
-0.003
0.98
BMI, kg/m
0.02
0.86
0.01
0.94
Systolic blood pressure, mmHg
-0.23
0.05
-0.16
0.16
LDL cholesterol, mg/dL
-0.24
0.04
-0.24
0.03
HDL cholesterol, mg/dL
-0.08
0.53
-0.16
0.18
HbA1c, %
0.24
0.04
0.15
0.19
Uric acid, mg/dL
-0.01
0.96
-0.02
0.87
Smoking
0.11
0.41
0.13
0.31
0.2
0.08
0.1
0.33
0.15
0.24
0.07
0.57
C-reactive protein, mg/dL
-0.08
0.49
-0.13
0.23
NLRP3 in SAT
0.33
0.02
0.43
0.002
Male sex
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Age
regression
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regression
Family history of CAD Adiponectin, µg/mL
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BMI; body mass index, CAD; coronary artery disease.
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Logarithmic value of NLRP3/G3PDH was used in this analysis.
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B
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C
Figure 1.
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Figure 2.
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A
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Figure 3.
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D
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C
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A
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Figure 4.
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Highlights NLRP3 expression in SAT was higher in patients with lifestyle-related diseases.
NLRP3 expression in SAT correlated negatively with serum adiponectin level.
CAD patients showed higher NLRP3 expression in SAT than non-CAD patients.
NLRP3 expression in SAT and HbA1c were predictors of coronary atherosclerosis.
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Supplementary Material for Expression of NLRP3 in subcutaneous adipose tissue
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is associated with coronary atherosclerosis
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Sachiko Bando1, Daiju Fukuda2, Takeshi Soeki1, Sachiko Nishimoto3, Etsuko Uematsu1, Tomomi Matsuura1, Takayuki Ise1, Takeshi Tobiume1, Koji Yamaguchi1, Shusuke Yagi1, Takashi Iwase1, Hirotsugu Yamada1, Tetsuzo Wakatsuki1, Michio Shimabukuro2 and Masataka Sata1
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Number of Supplementary Figures: 3 Number of Supplementary Tables: 1
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1. Department of Cardiovascular Medicine, Institute of Biomedical Sciences, Tokushima University Graduate School, Tokushima, Japan 2. Department of Nutrition and Metabolism, Institute of Biomedical Sciences, Tokushima University Graduate School, Tokushima, Japan 3. Department of Cardio-Diabetes Medicine, Institute of Biomedical Sciences, Tokushima University Graduate School, Tokushima, Japan
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Corresponding author: Daiju Fukuda, MD, PhD Department of Cardiovascular Medicine, Institute of Biomedical Sciences, Tokushima University Graduate School 3-18-15, Kuramoto-cho, Tokushima 770-8503, Japan Phone: +81-88-633-7859 Fax: +81-88-633-7894 E-mail: [email protected]
1
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Supplementary Figure 1. Comparison of the expression of NLRP3 among the primary diseases. The primary disease for the permanent heart device implantation did not affect the expression of NLRP3. AV; Atrio-ventricular, SSS; sick-sinus syndrome, VT/ VF; ventricular tachycardia/ventricular fibrillation.
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Supplementary Figure 2. Correlation between the the expression of NLRP3 and degree of risk factors. We divided our patients into quartiles according to the level of each risk factor and examined the expression of NLRP3 in each quartile. There were significant differences between 4th and 1st quartile groups of BMI, HDL-cholesterol. As for HbA1c, the expression of NLRP3 tended to be higher in 4th quartile group than 1st, 2ndand 3rd quartile group (P=0.08, P=0.06 and P=0.09, respectively). BMI; body mass index, LDL; low-density lipoprotein and HDL; high-density lipoprotein. *; P<0.05.
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Supplementary Figure 3. Relationship between lifestyle-related diseases and expression of IL-1β β and IL-18. Expression of NLRP3 inflammasome-related molecules, IL-1β and IL-18, were higher in patients with lifestyle-related diseases. *; P<0.05 **; P<0.01 and ***; P<0.001. 4
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Supplementary Table. Sequence of gene specific primers Gene
Sequence
Reverse 5'- GGGAATGGCTGGTGCTCAATAC -3' IL-1β
Forward 5'- CCAGGGACAGGATATGGAGCA -3'
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NLRP3 Forward 5'- AAGCACCTGTTGTGCAATCTGAAG -3'
Reverse 5'- TTCAACACGCAGGACAGGTACAG -3' IL-18
Forward 5'- GCCTGGACAGTCAGCAAGGA -3'
Reverse 5'- TCTACTGGTTCAGCAGCCATCTTTA -3'
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G3PDH Forward 5'- ACCACAGTCCATGCCATCAC -3'
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Reverse 5'- TCCACCACCCTGTTGCTGTA -3'
5