1. Introduction

For decades, adipose tissue (AT) was not deeply researched, as it was thought to be merely an energy storage depot. The secretory function of AT was only recognized in the mid-1980s, and an increasing number of scientific works involving adipocytes and AT have been published since then. Currently, AT is accepted as the largest endocrine organ, secreting over 600 proteins ^[1][2][1,2]. To date, numerous adipocyte-derived secreted hormones (leptin, adiponectin, and resistin) and effectors have been demonstrated to modulate a plethora of physiological and pathological metabolic processes, spanning from food intake, gluconeogenesis, glucose uptake, fatty-acid oxidation and insulin resistance, to reproductive axes and immune responses. Cardiovascular diseases (CVDs) are a group of disorders altering heart functionality, which are characterized by a high fatality rate, and are the driving cause of premature death in humans. Among the main risk factors leading to the onset of CVDs are obesity and diabetes ^[3][4][5][3,4,5]. These pathologies are associated with a low-grade inflammatory state that differentially affects the homeostasis and the secretome of specific AT depots. This leads to an alteration in the release of extracellular vesicles (EVs) in terms of both number and cargo, which is strictly dependent on the type and state of the cellular and tissue origin ^[6][7][6,7]. The role of epicardial adipose tissue (EAT) in this framework is of particular interest, as it has a significantly different transcriptome and secretome compared to other fat depots, and is in direct contact with the heart muscle [8].

2. Epicardial Adipose Tissue Cellular Components

The cellular components of EAT include copies of white adipocytes, which are smaller than those of VAT, and are specialized in energy storage ^[9][22], stroma-vascular cells, neurons, and immune cells including lymphocytes (CD3+), macrophages (CD68+), and mast cells ^[10][19]. As an adipose depot, EAT is characterized by a secretory activity that, in physiological conditions, ensures myocardium health ^[11][12][23,24]. The direct interaction of EAT with the myocardium, due to the total absence of fascia between the two tissues and their shared microcirculation, favors the vasocrine and paracrine secretions, with these being of particular importance in explaining the role that EAT plays in the development of cardiovascular pathophysiology. Several studies have demonstrated that a dysfunctional EAT mass expansion, together with its pro-inflammatory state and secretome, is associated with coronary artery diseases (CAD) ^[13][25], metabolic syndrome ^[14][26], chronic heart failure ^[15][16][27,28], and atrial fibrillation (AF) ^[17][18][19][29,30,31].

3. Epicardial Adipose Tissue Functions

EAT performs many different functions. Due to its elasticity and compressibility, it confers onto the coronary artery a mechanical protection against excessive distortion and compression induced by myocardial contractions [20]. Thanks to the similarity to brown adipose tissue (BAT) and to the molecular features of beige adipocytes, EAT can defend the myocardium against hypothermia and unfavorable hemodynamic conditions, contributing to cardiac cryoprotection ^[21][22][23][32,33,34]. Moreover, EAT serves as an energy reservoir for the myocardium, having a high capability for mobilization, deposition, and synthesis of free fatty acids (FFAs) ^[24][35]. It performs a pivotal function in lipid and glucose homeostasis regulation ^[25][18], and pro-inflammatory and anti-inflammatory cytokine production, showing a unique expression profile for genes linked to coagulation, endothelial function, apoptosis, immune response, and a specific secretome ^{[25][26][27][28]}[18,36,37,38].

4. Extracellular Vesicles

EVs are membrane-packed vesicles that can be secreted by many mammalian cell types, and can be found in almost all body fluids, including plasma, saliva, and even breast milk. EVs can be divided into two large subgroups, depending on their dimensions and specific membrane markers. The microvesicles (or ectosomes), ranging from 100 nm to 1000 nm, are positive for CD40 ligand, and Annexin A1 or Annexin V ^[29][30][31][39,40,41]. The smallest EVs, exosomes, ranging from 40 nm to 100 nm, show increased levels of CD63, CD9, and CD81, among others, along with proteins involved in their biogenesis (e.g., Alix, TSG101, FLOT-1) ^[32][42]. The biogenesis of these two classes of vesicles is profoundly different, because larger vesicles are generated by the external budding of the plasma membrane, while exosomes are generated in a multistep process, by inward budding of the endocytic cisternae membrane. Firstly, exosome precursors are accumulated in the cytoplasm into multivesicular bodies (MVBs) that, upon proper stimulation, fuse with the plasma membrane, and release mature exosomes in the extracellular space. The cargo of EVs is enriched in proteins, lipids, and various RNAs (mRNA, miRNA, and circular RNA) that are peculiar for parental cells, and for the developmental and functional state of the generating cells. Once in the extracellular fluids, vesicles are internalized into neighboring or even distant target cells by different mechanisms, and upon content release into the intracellular space, their regulatory effects are exerted. However, it is worth considering that, although many studies are aimed at the identification of the exact content of EVs ^[33][34][43,44], a complete understanding of the intricate biological effects and functionality transferred to recipient cells by the EV cargo has still not been reached. An aberrant production and/or cargo of EVs has been established in the context of many different pathological scenarios, including cancer, diabetes, insulin resistance, and CVDs ^[35][36][37][45,46,47]. Thus, a multitude of different vesicles are present in extracellular space and in body fluids; however, to date, although several separation methods based on an EV’s specific features have been developed, only a mixed EV population can be isolated.

5. Epicardial Adipose Tissue EVs in Cardiovascular Diseases

The inflammatory conditions that characterize different pathologies such as obesity and diabetes are responsible for the fibrotic and inflammatory state of the heart ^[38][39][77,78]. Recently, the epicardial fatty depot has been considered as an important regulator of cardiovascular health status, and its pathological phenotype has been associated with the onset and exacerbation of CVDs ^[8][19][8,31]. As evidence of this, the worsening of coronary atherosclerosis disease (CAD) has been shown to be significantly associated with a reduced adiponectin mRNA level, and with an increased IL-6 mRNA level in EAT ^[40][79]. EAT thickness has been correlated with insulin resistance and many other risk factors of cardiovascular pathologies ^[41][42][43][80,81,82], but the exact mechanism linking EAT to cardiac dysfunction has yet to be fully elucidated. Lately, the role of the EAT secretome, particularly of EVs released by the depot, in the pathogenesis of CVD, has been greatly explored. From the latest studies published in the literature, the hypothesis has been established of a unique secretome characterizing pathological EAT that is involved in the development and propagation of CVD. Exosomal miRNA-802-5p released by hypertrophic 3T3-L1 cells has been demonstrated to cause insulin resistance in neonatal rat ventricular myocytes through the reduction in the expression of intracellular HSP60, a mitochondrial chaperone already known to be involved in CVDs among obese and diabetic patients ^[44][83]. Insulin resistance has been considered the main factor linking the diabetic conditions to the occurrence of many CVDs and heart failure. The close anatomic proximity between the EAT and the myocardium could be the basis of the paracrine crosstalk between the two tissues, explaining a possible mechanism through which EAT impairs insulin signaling, and consequently induces the structural and functional alteration in cardiomyocytes. Moreover, in CAD patients, the EAT microenvironment plays a pivotal role in modulating the cargo of EAT exosomes that in turn is responsible for an impaired adipogenic differentiation in stem cells lying in the depot ^[45][84]. Indeed, although epicardial adipose stem cells (EASCs) from CAD and non-CAD patients have an identical adipogenic potential, Wankei Y. et al. have recently demonstrated that it decreases significantly after exposure to EAT-derived exosomes of CAD subjects. This evidence indicates that various factors triggering CAD (e.g., insulin resistance and inflammation) are responsible for an alteration in the EAT secretome. In particular, the effects observed were ascribed to the down-regulation of Neuronatin protein targeted by miR-3064-5p, enriched in EAT-exosomes of CAD patients. In addition, a direct role of EVs released by EAT in AF onset and propagation was demonstrated for the first time, in a very comprehensive study by Shaihov-Teper et al. ^[46][85]. AF is a multifactorial atrial arrhythmia, very often interconnected with CVD. Histological examinations of EAT explant from AF and non-AF patients were analyzed, and an excess of extracellular matrix deposition, and inflammatory cell infiltrations, were found in AF patients. The EVs isolated from EAT explant cultured in vitro have revealed that the vesicles from AF (AF-EVs) were enriched in pro-inflammatory cytokines (IL-6, IL-1a, TNF-α, IL-4), with lower levels of IL-10 (anti-inflammatory and pro-fibrotic cytokine), VEGF and soluble VEGF receptor. The proteomic profile of AF-EVs corroborated the pro-inflammatory and pro-fibrotic outline that may be ascribed to the upregulation of miR-146b, and to a reduction in miR-133a and miRNA-29a expression. Furthermore, the pro-arrhythmic feature of AF-EVs in a two-dimensional hiPSC-derived cardiac cell sheet was demonstrated. Remarkably, the AF-EV profile was independent from the method used for isolation; both ultracentrifugation and size exclusion chromatography isolation preserve the EV signature. Other possible EAT-EVs-dependent pathways implicated in AF pathogenesis involve circular RNAs. Circular RNAs are non-coding RNAs characterized by a closed-loop structure that provides a high stability released in the extracellular space within exosomes ^[47][48][86,87]. They regulate gene expression, acting as sponges by buffering specific miRNA, and impeding their target gene’s silencing ^[47][49][50][86,88,89]. Recently, the circular RNAs from the EAT of patients with AF were profiled, and an unique expression profile was shown ^[51][90]. The reconstruction of a circular RNA–miRNA–mRNA interactional network has demonstrated that hsa_circRNA_000932 and hsa_circRNA_0078619 modulate the expression of many genes involved in the CVD frame, through the interaction with various miRNAs such as miR-103a-2-5p and miR-199a-5p, providing a direct link between EAT exosomal circular RNA, and the structural and functional remodeling of the heart in AF development. The contribution of the secretome deriving from EAT and AT in inflammatory processes and in the onset of CVDs are summarized in Table 1.

Tissue	Effects	Mediators	Ref
AT	insulin resistance and macrophage activation	IL-6; TNF-α	[52][72]
	M1 polarization and formation of macrophage foam cells	IL-6; TNF-α	[53][75]
	Adipogenesis	miR-450a-5p	[54][70]
	macrophages M2/M1 phenotypic switching and insulin resistance	TNF-α; MCP-1	[55][56][57][55,91,92]
	secretion of extracellular matrix protein	plasminogen activator; proteases; type VI collagen	[58][59][58,59]
	angiogenesis	VEGF; MMPs; SDF-1; miR-126	[60][61][62][63][60,71,73,74]
	cell cycle and apoptosis regulation	hsa-miR-222-5p; miR-888-5p; hsa-miR-6752-5p; has-miR-6338-3p; miR-130b-3p	[62][64][73,76]
EAT	insulin resistance	miR-802-5p	[44][83]
	arterial damage	adiponectin; IL-6	[40][79]
	adipogenic differentiation	miR-3064-5p	[45][84]
	inflammation, fibrosis, and apoptosis promotion	IL-1α; IL-6; TNF-α; IL-4; VEGF; IL-10; sFLT-1	[46][85]
	Modulation of inflammation and cell proliferation	hsa_circ_0099634; hsa_circ_0000932; hsa_circ_0097669; hsa_circ_0135289; hsa_circ_0098155; hsa_circ_0079672	[51][90]