Epicardial Adipocytes Research Articles

Adipokine and Cytokine Profiles of Epicardial and Subcutaneous Adipose Tissue in Patients with Coronary Heart Disease.

The content of adipokines, pro- and anti-inflammatory cytokines were studied in adipocytes isolated from epicardial and subcutaneous adipose tissue of 24 coronary heart disease patients. The content of leptin and soluble leptin receptor in adipocytes of epicardial adipose tissue was higher by 28.6 and 56.9% and the level of adiponectin was lower by 33% than in adipocytes of the subcutaneous fat. In culture of epicardial adipocytes, the levels of proinflammatory cytokines TNF-α and IL-1 were higher. Subcutaneous adipose tissue adipocytes were characterized by higher levels of anti-inflammatory cytokines IL-10 and FGF-β. In epicardial adipocytes of coronary heart disease patients, the concentrations of leptin, TNF-α, and IL-1 were higher, while the levels of defense regulatory molecules (adiponectin, IL-10, and FGF-β) were lower than in subcutaneous adipocytes.

Role of Epicardial Adipose Tissue in Health and Disease: A Matter of Fat?

Epicardial adipose tissue (EAT) is a small but very biologically active ectopic fat depot that surrounds the heart. Given its rapid metabolism, thermogenic capacity, unique transcriptome, secretory profile, and simply measurability, epicardial fat has drawn increasing attention among researchers attempting to elucidate its putative role in health and cardiovascular diseases. The cellular crosstalk between epicardial adipocytes and cells of the vascular wall or myocytes is high and suggests a local role for this tissue. The balance between protective and proinflammatory/profibrotic cytokines, chemokines, and adipokines released by EAT seem to be a key element in atherogenesis and could represent a future therapeutic target. EAT amount has been found to predict clinical coronary outcomes. EAT can also modulate cardiac structure and function. Its amount has been associated with atrial fibrillation, coronary artery disease, and sleep apnea syndrome. Conversely, a beiging fat profile of EAT has been identified. In this review, we describe the current state of knowledge regarding the anatomy, physiology and pathophysiological role of EAT, and the factors more globally leading to ectopic fat development. We will also highlight the most recent findings on the origin of this ectopic tissue, and its association with cardiac diseases. © 2017 American Physiological Society. Compr Physiol 7:1051-1082, 2017.

Flow cytometric single cell analysis reveals heterogeneity between adipose depots

ABSTRACTUnderstanding adipose tissue heterogeneity is hindered by the paucity of methods to analyze mature adipocytes at the single cell level. Here, we report a system for analyzing live adipocytes from different adipose depots in the adult mouse. Single cell suspensions of buoyant adipocytes were separated from the stromal vascular fraction and analyzed by flow cytometry. Compared to other lipophilic dyes, Nile Red uptake effectively distinguished adipocyte populations. Nile Red fluorescence increased with adipocyte size and granularity and could be combined with MitoTracker® Deep Red or fluorescent antibody labeling to further dissect adipose populations. Epicardial adipocytes exhibited the least mitochondrial membrane depolarization and highest fatty-acid translocase CD36 surface expression. In contrast, brown adipocytes showed low surface CD36 expression. Pregnancy resulted in reduced mitochondrial membrane depolarisation and increased CD36 surface expression in brown and epicardial adipocyte populations respectively. Our protocol revealed unreported heterogeneity between adipose depots and highlights the utility of flow cytometry for screening adipocytes at the single cell level.

Investigating interactions between epicardial adipose tissue and cardiac myocytes: what can we learn from different approaches?

This article is part of a themed section on Molecular Mechanisms Regulating Perivascular Adipose Tissue - Potential Pharmacological Targets? To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v174.20/issuetoc.

Epicardial adipocyte hypertrophy: Association with M1-polarization and toll-like receptor pathways in coronary artery disease patients

Background and aimsIn coronary artery disease (CAD) epicardial adipose tissue (EAT) shows an elevated inflammatory infiltrate. Toll-like receptors (TLRs) are important mediators of adipose tissue inflammation and they are able to recognize endogenous products released by damaged cells. Because adipocyte death may be driven by hypertrophy, our aim was to investigate in CAD and non-CAD patients the association between EAT adipocyte size, macrophage infiltration/polarization and TLR-2 and TLR-4 expression. Methods and resultsEAT biopsies were collected from CAD and non-CAD patients. The adipocyte size was determined by morphometric analysis. Microarray technology was used for gene expression analysis; macrophage phenotype and TLRs expression were analyzed by immunofluorescence and immunohistochemical techniques. Inflammatory mediator levels were determined by immunoassays.EAT adipocytes were larger in CAD than non-CAD patients and do not express perilipin A, a marker of lipid droplet integrity. In CAD, EAT is more infiltrated by CD68-positive cells which are polarized toward an M1 state (CD11c positive) and presents an increased pro-inflammatory profile. Both TLR-2 and TLR-4 expression is higher in EAT from CAD and observed on all the CD68-positive cells. ConclusionsOur findings suggested that EAT hypertrophy in CAD promotes adipocyte degeneration and drives local inflammation through increased infiltration of macrophages which are mainly polarized towards an M1 state and express both TLR-2 and TLR-4.

Co-cultivation of human aortic smooth muscle cells with epicardial adipocytes affects their proliferation rate

Co-cultivation of human aortic smooth muscle cells with epicardial adipocytes affects their proliferation rate

Co-cultivation of human aortic smooth muscle cells with epicardial adipocytes affects their proliferation rate.

The abnormal proliferation of vascular smooth muscle cells (VSMC) is thought to play a role in the pathogenesis of atherosclerosis. Adipocytes produce several bioactive paracrine substances that can affect the growth and migration of VSMCs. Our study focuses on the direct effect of the bioactive substances in conditioned media (CM) that was obtained by incubation with primary adipocyte-derived cell lines, including cell lines derived from both preadipocytes and from more mature cells, on the proliferation rate of human aortic smooth muscle cells (HAoSMCs). We used a Luminex assay to measure the adipokine content of the CM and showed that there was a higher concentration of monocyte chemoattractant protein-1 in renal preadipocyte-CM compared with the HAoSMC control (p<0.5). The addition of both renal preadipocyte- and epicardial adipocyte- CM resulted in the elevated production of vascular endothelial growth factor compared with the control HASoSMC CM (p<0.001). The adiponectin content in renal adipocyte-CM was increased compared to all the remaining adipocyte-CM (p<0.01). Moreover, the results showed a higher proliferation rate of HAoSMCs after co-culture with epicardial adipocyte-CM compared to the HAoSMC control (p<0.05). These results suggest that bioactive substances produced by adipocytes have a stimulatory effect on the proliferation of VSMCs.

Differential Association of S100A9, an Inflammatory Marker, and p53, a Cell Cycle Marker, Expression with Epicardial Adipocyte Size in Patients with Cardiovascular Disease

S100A9 (calgranulin B) has inflammatory and oxidative stress properties and was found to be associated with atherosclerosis and obesity. One of the proteins that can regulate S100A9 transcription is p53, which is involved in cell cycle, apoptosis and adipogenesis. Thus, it triggers adipocyte enlargement and finally obesity. Because epicardial adipose tissue (EAT) volume and thickness is related to coronary artery disease (CAD), we studied the gene expression of this pathway in patients with cardiovascular disease and its association with obesity. Adipocytes and stromal cells from EAT and subcutaneous adipose tissue (SAT) from 48 patients who underwent coronary artery bypass graft and/or valve replacement were obtained after collagenase digestion and differential centrifugation. The expression levels of the involved genes on adipogenesis and cell cycle like fatty acid-binding protein (FABP) 4, retinol-binding protein (RBP)4, p53 and S100A9 were determined by real-time polymerase chain reaction (PCR). Adipocyte diameter was measured by optical microscopy. We found that epicardial adipocytes expressed significantly lower levels of adipogenic genes (FABP4 and RBP4) and cell cycle-related genes (S100A9 and p53) than subcutaneous adipocytes. However, in obese patients, upregulation of adipogenic and cell cycle-related genes in subcutaneous and epicardial adipocytes, respectively, was observed. The enlargement of adipocyte size was related to FABP4, S100A9 and p53 expression levels in stromal cells. But only the p53 association was maintained in epicardial stromal cells from obese patients (p=0.003). The expression of p53, but not S100A9, in epicardial stromal cells is related to adipocyte enlargement in obese patients with cardiovascular disease. These findings suggest new mechanisms for understanding the relationship between epicardial fat thickness, obesity and cardiovascular disease.

Virtual optical biopsy of human adipocytes with third harmonic generation microscopy

Using the sectioning capability of third harmonic generation (THG) microscopy, we assessed the morphologic features of human adipocytes directly without fixation and labeling. At the plane of the largest cross-sectional area, both area-equivalent circular diameters (AECD) and perimeters of adipocytes were measured, and their statistical distributions were examined. We found, in patients with no cardiovascular risk factors, the average AECD of epicardial adipocytes were 70–90 μm with 11–17 μm standard deviations. In contrast, for patients with coronary artery disease, amounts of small-sized (AECD <40 µm) epicardial adipocytes were observed and the corresponding standard deviations of AECD were increased to 20–29 μm. Our results indicate that the THG tomography platform can be used to explore the histopathological features of adipocytes in clinical scenarios based on its superior resolution for virtual optical biopsy.

In vivo Metabolic Imaging of Insulin with Multiphoton Fluorescence of Human Insulin–Au Nanodots

Functional human insulin-Au nanodots (NDs) are synthesized for the in vivo imaging of insulin metabolism. Benefiting from its efficient red to near infrared fluorescence, deep tissue subcellular uptake of insulin-Au NDs can be clearly resolved through a least-invasive harmonic generation and two-photon fluorescence (TPF) microscope. In vivo investigations on mice ear and ex vivo assays on human fat tissues conclude that cells with rich insulin receptors have higher uptake of administrated insulin. Interestingly, the insulin-Au NDs can even permeate into lipid droplets (LDs) of adipocytes. Using this newly discovered metabolic phenomenon of insulin, it is found that enlarged adipocytes in type II diabetes mice have higher adjacent/LD concentration contrast with small-sized ones in wild type mice. For human clinical samples, the epicardial adipocytes of patients with diabetes and coronary artery disease (CAD) also show elevated adjacent/LD concentration contrast. As a result, human insulin-Au nanodots provide a new approach to explore subcellular insulin metabolism in model animals or patients with metabolic or cardiovascular diseases.

Adipocytes modulate the electrophysiology of atrial myocytes: implications in obesity-induced atrial fibrillation

Obesity is an important risk factor for atrial fibrillation (AF). Increased epicardial adipose tissue in obesity can enhance inflammation and plays an important role in the pathophysiology of AF. However, it is not clear whether epicardial adipocytes directly modulate the electrophysiological characteristics of atrial myocytes. Whole-cell patch clamp was used to record the action potentials (APs) and ionic currents in isolated rabbit left atrium (LA) myocytes incubated with and without (control) isolated adipocytes from epicardial, retrosternal, or abdominal adipose tissues, or adipocytes-conditioned supernatant for 2-4h. Compared to control LA myocytes (n=22), LA myocytes incubated with epicardial (n=17), retrosternal (n=18), or abdominal adipocytes (n=22) had longer (80±3, 109±6, 109±6, and 110±7ms, p<0.001) 90% AP durations (APD(90)). LA myocytes incubated with epicardial adipocytes had a more-positive resting membrane potential (RMP) than control LA myocytes (-57±1mV vs. -63.4±1.4mV, p<0.05). However, LA myocytes (n=32) incubated with supernatant had longer APD(90) (93±3ms, p<0.05), but similar RMP values (-62±2mV, p>0.05) in comparison to control myocytes. Epicardial adipocyte-incubated LA myocytes had larger late sodium currents, L-type calcium currents, and transient outward potassium currents, but smaller delayed rectifier potassium and inward rectifier potassium currents than control LA myocytes. Moreover, isoproterenol (10nM) induced a higher incidence (67 vs. 22%, p<0.05) of triggered beats in adipocytes-incubated LA myocytes (n=12) than in control LA myocytes (n=9). In conclusion, adipocytes can directly modulate the electrophysiological properties and ion currents, causing higher arrhythmogenesis in LA myocytes.

Adipose tissue depot-specific differences in adipocyte apolipoprotein E expression

Important differences in gene expression have been documented in adipocytes derived from specific adipose tissue depots. We have previously documented an important role for adipocyte apolipoprotein E (apoE) in modulating adipocyte and adipose tissue triglyceride and lipoprotein metabolism. We now evaluate the endogenous expression of apoE in adipocytes isolated from unique adipose tissue depots in 4 different species. Adipocyte apoE expression is higher in subcutaneous fat compared with visceral fat in humans, mice, rats, and baboons. In baboons, evaluation of apoE expression in 5 adipose tissue depots (subcutaneous abdominal, subcutaneous gluteal, visceral, pericardial, epicardial) showed that, compared with subcutaneous abdominal adipocytes, the level of apoE expression is similar in subcutaneous gluteal, lower in visceral and pericardial, and higher in epicardial adipocytes. Consistent with previously demonstrated suppression of adipocyte apoE by adipose tissue inflammation, adipose tissue depots with lower apoE expression demonstrated greater infiltration of macrophages and an increased expression of tumor necrosis factor–α messenger RNA. Depot-specific differences in apoE expression were maintained after in vitro differentiation. Adipocytes isolated from depots with lower apoE expression manifested lower rates of triglyceride synthesis in the absence and presence of triglyceride-rich lipoproteins. Adenoviral-mediated increase of apoE expression in omental adipocytes increased triglyceride synthesis in these cells. Our results demonstrate significant heterogeneity in adipocyte apoE expression across adipose tissue depots in several species. Because of its role in modulating adipocyte triglyceride and lipoprotein metabolism, depot-specific differences in endogenous adipocyte apoE could have important implications for modulating the accumulation of lipid in these depots.

Adiponectin gene expression and adipocyte diameter: a comparison between epicardial and subcutaneous adipose tissue in men

Interest has recently focused on epicardial fat, but little is known about epicardial adipocyte size and its relation with insulin resistance and adipokines. Biopsies were collected from subcutaneous, epicardial-, and peritoneal fat from 21 males undergoing elective cardiac surgery either for coronary artery bypass grafting (n=11) or for valve replacement (n=10). We assessed epicardial adipocyte size, comparing it with that from subcutaneous fat and peritoneal fat. The adipocyte size was determined by using collagenase digestion of adipose tissue, separation of adipocytes by centrifugation, methylene blue staining of the nuclei, and measurement of the cell diameter. Patient's weight, height, body mass index, waist, as well as glucose, insulin, homeostatic model assessment index, adiponectin, and leptin serum levels were determined. Adiponectin mRNA levels were determined by real-time polymerase chain reaction on subcutaneous fat and epicardial fat biopsies. Adipocytes in epicardial fat were significantly smaller than those in subcutaneous and peritoneal fat. The adipocyte size in epicardial fat correlated positively with insulin resistance and serum leptin, and correlated negatively with serum and mRNA expression of adiponectin. Adiponectin mRNA expression in epicardial fat was significantly lower than in subcutaneous fat. Adipocyte size in epicardial fat was significantly smaller in valve-replacement patients than in coronary artery bypass graft patients. Adiponectin gene expression was lower in the latter than in the former, although not significantly. Adipocytes in epicardial fat are smaller than those in peritoneal and subcutaneous fat. Adipocyte size, both in epicardial and in subcutaneous fat, is positively related with insulin resistance, shows negative association with local adiponectin gene expression, and is decreased in subjects with coronary artery disease. Adiponectin gene expression is significantly lower in epicardial- than in subcutaneous fat.

Potential atrial arrhythmogenicity of adipocytes: Implications for the genesis of atrial fibrillation

Atrial fibrillation is the most common sustained cardiac arrhythmia. Obesity and metabolic syndrome are independent risk factors for atrial fibrillation, and epicardial adipose tissues are highly associated with the genesis of atrial fibrillation, compared to visceral fat. Adipocytes can produce inflammatory cytokines and adipocytokines, which may enhance inflammation and oxidative stress in patients with obesity or metabolic syndrome. Moreover, it is possible that local interactions between epicardial adipose tissue and the adjacent myocardium can directly produce electrical or structural remodelings in the atrium. Taken together, we hypothesized that epicardial adipocytes contain distinctive arrhythmogenicity through increases in inflammatory cytokines, adipocytokines, and adipocyte-cardiomyocyte interactions to induce atrial fibrillation.

Weight of Pericardial Fat on Coronaropathy

The regional distribution of adipose tissue (AT) is a major determinant of metabolic and cardiovascular diseases. The mass of fat in the visceral area associates independently of obesity with the development and progression of cardiovascular diseases in a series of clinical and epidemiological studies.1 This led to the concept of a pathophysiological link between abdominal obesity and metabolic syndrome. More recently, fat depots localized around the heart, highly variable among individuals, were proposed to contribute to the pathogenesis of coronaropathy independently of other visceral depots (ie, in the omental and mesenteric area).2,3 The study by Greif et al3a in this issue of Arteriosclerosis, Thrombosis, and Vascular Biology highlights the association between pericardial adipose tissue (PAT) and the number of atherosclerotic plaques evaluated concomitantly by Dual source CT scan. This measurement was qualitatively interpretable in 264 consecutive patients with a large range of age, a normal or moderately increased body mass index (BMI), and no a priori coronary disease. An estimated volume of pericardial fat more than 300 cm3 provided an incremental value for the presence of coronary atherosclerosis (odds ratio 4.1) independently of well known risk factors (hyperglycemia or diabetes, hypercholesterolemia, hypertension, and smoking). Ninety-five percent of patients with PAT volume >300 cm3 had one or more atherosclerotic plaques by ROC estimation. In univariate statistical analysis, PAT …

Human epicardial adipocytes have distinct functional and structural properties as compared to subcutaneous and perirenal adipocytes

Growing evidence suggests that adipose tissue is an important endocrine organ, and that adipocytes (Ads) from different fat depots have distinct functional properties. Epicardial (EC) fat has been shown to produce inflammatory mediators, but the functional properties of isolated EC Ads have not been determined. For our studies, we compared isolated human EC Ads with subcutaneous (SQ) and perirenal (PR) Ads from the same donors (n = 3). EC fat was more highly vascularized and had a greater density of extracellular matrix than did SQ fat. Upon differentiation, both SQ and PR Ads exhibited significant accumulation of large lipid droplets, whereas EC Ads exhibited slower lipid accumulation and smaller lipid droplets. Microarray analysis showed that EC Ads expressed higher levels of pro‐inflammatory genes, including MMP1, MMP9, IL6, IL8, MCP1, and COX2. Conversely, EC Ads expressed lower levels of leptin and of the anti‐inflammatory, cardioprotective adipokine adiponectin. EC Ads also released less adiponectin as measured by ELISA. These results suggest that EC Ads exhibit structural and functional differences as compared to SQ and PR Ads. Furthermore, the increased adiponectin expression and release by SQ Ads suggests a potential basis for the observed cardioprotective effect of gluteofemoral fat. Funding for these studies was provided by the NIH (NHLBI) and the University of Iowa Department of Emergency Medicine.

Met5‐enkephalin‐Arg6‐Phe7 (MEAP) attenuates pro‐inflammatory responses in human epicardial adipocytes

Adipocytes are previously unrecognized sources of proinflammatory mediators that contribute to metabolic syndrome, diabetes, and atherosclerosis. Epicardial fat overlying coronary arteries produces higher levels of inflammatory mediators than subcutaneous fat. Because of their unique properties and location, we hypothesize that epicardial adipocytes (EpAd) may play an important role in transducing inflammatory responses to coronary arteries. Enkephalins are naturally occurring endogenous delta opioids that improve myocardial ischemic tolerance. We found that EpAd express CD14, which plays a crucial role in transducing signaling by endotoxin and other inflammatory mediators, and release significant levels of IL-8 in response to both endotoxin and TNF-alpha. Met5-enkephalin-Arg6-Phe7 (MEAP; 1 μM) inhibited this IL-8 release by 50% or more, as did PCP-enkephalin. Microarray analysis revealed that MEAP blocked LPS-induced expression of a number of immunomodulatory genes. Together, these data suggest that EpAd may contribute to proinflammatory responses in coronary arteries, and that endogenous enkephalins may blunt these effects, thus playing a protective role in coronary artery disease.

Isolation and characterization of human epicardial adipocytes: potential role in vascular inflammation

Adipose tissue is an important endocrine and secretory organ. There is growing evidence that adipocytes from different fat depots have distinct functional properties. Epicardial adipose tissue has been shown to produce inflammatory mediators, but the functional properties of isolated epicardial adipocytes (EA) have not been studied. We characterized human EA isolated from freshly harvested hearts, along with subcutaneous and perirenal adipocytes from the same donors. Differentiation from preadipocytes was associated with greatly increased adipokine and proinflammatory cytokine expression; of note, the proinflammatory mediator CD14 was expressed at 50-fold higher levels by EA compared to preadipocytes. EA have substantial cyclooxygenase and epoxide hydrolase activity. The major COX metabolite is PGE2, which plays an important role in angiogenesis and inflammation. LPS caused increased expression of adiponectin, VEGF, and CD14 in both epicardial adipose tissue explants and cultured EA. Conditioned medium from EA (but not control medium or medium from epicardial preadipocytes or subcutaneous adipocytes) strongly promoted growth and tubule formation by human coronary artery endothelial cells. These findings suggest that EA may play an important role in promoting coronary artery inflammation and growth of vasa vasorum and collateral coronary vessels in ischemia. (Supported by NIH HL70860 [NLW])

Adipose tissue in the mammalian heart and pericardium: structure, foetal development and biochemical properties.

1. The occurrence and relative abundance of adipose tissue around the heart and in the pericardium of wild and domesticated mammals are reviewed and some new data reported. 2. For macaque monkeys and a wide range of other adult mammals, the mean volume of epicardial adipocytes is constant at about half the average of that of other depots, although the relative mass of this depot is unrelated to the abundance of adipose tissue in the rest of the body. 3. In young adult guinea-pigs, the maximum rate of fatty acid synthesis is significantly higher in epicardial adipose tissue than that in the pericardial, perirenal and popliteal depots. 4. The rate of fatty acid release by epicardial adipose tissue is approximately twice that of the pericardial and perirenal depots. 5. The protein contents of guinea-pig epicardial and pericardial adipose tissue are similar, and are significantly higher than those of the perirenal and popliteal adipose tissue and there are no site-specific differences in the abundance of mitochondria. 6. In adult Macaca monkeys, the capacity of the epicardial adipose tissue for glucose utilization is about half that of the intra-abdominal depots. 7. The principal difference between epicardial adipose tissue and that elsewhere in the body is its greater capacity for fatty acid release. 8. It is suggested that cardiac adipose tissue may act as a local energy supply for adjacent myocardium and/or as a buffer against toxic levels of free fatty acids.