1. Introduction

During the past decade, the interest in the role of exosomes in both physiological and pathological conditions significantly increased. There is growing recognition of their function in CVDs pathogenesis through multifarious intercellular communication mechanisms that, nonetheless, have not yet been fully unraveled ^[1][11]. CVDs are the largest contributor to mortality worldwide, accounting for an estimated 17.9 million deaths and 31% of all global deaths in 2016. According to the World Health Organization (WHO), this number is expected to increase up to 23 million by 2030 [1]. Despite significant advances in medical management over the past decades, the five-year survival rate for CVDs is no longer improving ^[2][58]. Moreover, given their complex nature, including multiple risk factors, such as obesity, hypertension, diabetes, among others, the intricate molecular mechanisms underlying CVDs are far from being fully clarified. The identification of novel biomarkers remains vital to improve diagnosis and prognostic staging, as well as to guide therapy of CVDs with the goal of further reducing morbidity and mortality rates ^[3][59].

The human heart is mostly formed by cardiomyocytes; though, non-myocyte cell types, such as cardiac fibroblasts and endothelial cells, also have relevant roles in cardiac homeostasis ^[4][60]. The presence of diverse cell types within human cardiac tissue results in a complex intercellular network of numerous coordinated signaling pathways. These include cell–cell contacts, cell-extracellular–matrix interactions, and paracrine, autocrine, and endocrine effects of extracellular biologic and/or chemical molecules ^[5][6]. Exosomes contribute to these intercellular communications, although little is known about its regulation within the healthy and diseased hearts ^[6][9]. In recent years, accumulating evidence has implicated exosomes both in normal physiology (cardiac development, reticulocyte maturation, and myocardial angiogenesis) and in pathophysiological processes, including atherosclerosis, ischemia/reperfusion (IR) injury, and cardiac remodeling. For instance, stress conditions, such as hypoxia and inflammation, can modulate exosomal biological content and target cells, contributing to the improvement or impairment of heart function ^[7][8][9][43,61,62]. The biological contents and quantity of released exosomes change in pathological states, reflecting their cellular origin and the pathological disturbance. Since exosomes can be readily isolated from body fluids, allowing the characterization of both exosomes and their mediators, these might serve as non-invasive biomarkers for diagnosis and prognosis of CVDs ^[10][11][63,64]. To date, the potential role of exosomes has not been appraised in cardiovascular clinical research; therefore, further clinical research studies should be conducted to analyze the diagnostic and prognostic value as well as the functional role of exosomes in CVDs ^[12][3].

2. Exosomes in CVDs

Although there is expanding interest in the role of exosomes in CVDs, their use as biomarkers in clinical research is still limited by diverse difficulties in exosome isolation and characterization ^[6][9]. Most of the conducted studies regarding exosomes and its role in CVDs, risk factors, and complications were performed by collecting plasma and isolating the exosomes by precipitation or ultracentrifugation. Mainly, these studies used the Western blotting method to analyze the protein expression and EM techniques to characterize the morphology of the isolated exosomes. In this report, we attempt to link exosomes and exosome-derived mediators to CVDs in an association network approach that gathers literature findings coupled with bioinformatics analysis of the results. The integration of these exosomal markers by the network approach allowed a wide-ranging analysis of the potential biomarker of these mediators in the scope of CVDs.

CAD is the most common type of heart disease and it results mainly from the activation of inflammatory, oxidative stress, and endothelial dysfunction pathways and enriched-cholesterol plaques in the arteries. The exosomes released from stem cells, endothelial cells, cardiomyocytes, and platelets, among others, include potential valuable biological information for the development and progression of CAD ^[13][101]. Currently, miRNAs are the most studied elements contained in exosomes for their potential as biomarkers. Some miRNAs are reported to be tissue specific, such as miR-208a and miR-499-5p, which are highly present in the heart tissue, and miR-1a and miR-133a, which are expressed in heart and skeletal muscles ^[13][70]. These miRNAs can also be found in the circulation and are thought to play a cardioprotective role, namely by decreasing hypertrophy and fibrosis after AMI ^[14][101]. In this work, we analyzed six circulating exosome-derived miRNAs found to be upregulated in patients with CAD: miR-133a, miR-208a, miR-1, miR-499-5p, miR-92b-5p, and miR-30a. The performed search in Vesiclepedia database uncovers if the evaluated miRNAs have already been identified in exosomes. Since only miR-133a was included in Vesiclepedia database, we can conclude that the other miRNAs analyzed were not yet identified in exosomes or manually curated in this database. Moreover, these non-included miRNAs of exosomal origin can be proposed for further research. Taking into consideration the previously mentioned challenges in exosome isolation and characterization, the main limitation associated to the Vesiclepedia database is some uncertainty associated to the exact origin of the identified mediators ^[15][102]. Nevertheless, the use of this database allowed us to get a better comprehension of the existing literature regarding exosome and exosome-derived mediators.

Circulating exosomal miR-133a increased levels can be considered among the biomarkers for early diagnosis of AMI. miR-133a levels in blood are a result of their release from cardiomyocytes after cardiac injury and cell death. Moreover, the capture of miR-133a by adjacent surviving cells in infarcted cardiac areas contributes to the inhibition of hypertrophy ^[16][17][103,104]. According to Cheng et al. ^[13][70], exosome-derived miR-133a, together with miR-208a, miR-1, and miR-499, are found to be increased in the plasma of AMI patients. Moreover, an available clinical study reported increased circulating miR-133a and miR-1 levels in patients with acute coronary syndrome ^[16][103] despite lacking confirmation of their exosomal origin. Increased plasma levels of miR-208a and miR-499 are associated to cardiac damage in acute HF, acute viral myocarditis, and AMI ^[18][105]. miR-208a is encoded by the α-myosin heavy-chain gene and, therefore, has a particular role in cardiac contractility. High levels of miR-208a are associated to arrythmias, fibrosis, and hypertrophy growth ^[19][20][106,107].

miR-30a is responsible for the regulation of cardiomyocyte autophagy after hypoxia. According to Yang et al. ^[21][75], serum levels of miR30a with exosomal origin were found to be increased in AMI patients. The exosomes enriched with miR-30a in a hypoxic condition were thought to be transferred between cardiomyocytes in order to maintain an autophagic response. Nevertheless, this was still not validated in in vivo cardiac ischemia models ^[4][21][60,75]. Moreover, exosome-derived miR-92b-5p is also considered a putative biomarker since it was determined to be increased in patients of acute HF due to dilated cardiomyopathy ^[22][79] and in patients with HF ^[23][80]. This miRNA is believed to contribute to atrial fibrillation, which often coexists with HF ^[24][108].

Many CADs progress to a state of chronic HF, determined by the complex molecular mechanisms of cardiac remodeling and vascular dysfunction. One of the major challenges in HF management is to identify a reliable approach to evaluate the prognosis of the disease ^[25][109]. Despite the use of functional parameters and resulting risk stratification scores, prognosis evaluation among HF patients would beneficiate by an extended use of exosomes as biomarkers ^[2][58]. We were able to identify four circulating exosome-derived miRNAs upregulated in HF: miR-192, miR-194, miR-146a, and miR-92b-5p. From these, only miR-92b-5p was not included in Vesiclepedia database. Evidence highlights the future clinical applications of miR-192, miR-194, and miR-34a as predictive indicators of HF ^[26][110]. As reported by Matsumoto et al. ^[27][81], circulating increased levels of exosomal miR-192 and miR-194 are highly related to the development of HF after AMI. Additionally, despite not being significantly enriched, exosome fraction of HF patients shows a tendency of an increased level of miR-34a. Exosome-derived miR-92b-5p was found to be increased in patients with acute HF caused by dilated cardiomyopathy ^[11][79] and in patients with HF with reduced ejection fraction hospitalized for acute HF ^[23][80].

Besides the analysis regarding the circulating levels of exosome-derived miRNAs, we also analyzed the proteins of exosomal origin found to be up- or downregulated in plasma or serum samples of patients with CAD. We analyzed 14 upregulated proteins: angiotensinogen (AGT), complement C4-B (C4B), haptoglobin (HP), FGG, FGB, vimentin (VIM), inter-alpha-trypsin inhibitor heavy chain H4 (ITIH4), complement component C9 (C9), immunoglobulin kappa constant (IGKC), myosin-binding protein C, cardiac-type (MYBPC3), alpha-1 antichymotrypsin (SERPINA3), leucine-rich alpha-2-glycoprotein (LRG1), complement factor H-related protein 1 (CFHR1), and immunoglobulin kappa variable 4-1 (IGKVA-1). From these, only five proteins were identified in the Vesiclepedia database: AGT, C4B, HP, FGG, and FGB. Moreover, FGB, FGG, ITIH4, and SERPINA3 were found to be involved in the platelet degranulation molecular mechanism.

One of the main pathophysiological mechanisms associated to CAD is endothelial dysfunction. It is frequently observed in patients with cardiovascular risk factors and contributes to the development of atherosclerosis and myocardial ischemia. Endothelial dysfunction is characterized by impaired fibrinolysis, prothrombotic, and proinflammatory responses, which leads to the activation of coagulation factors and platelets ^[28][111]. According to Zhang et al. ^[10][78], the exosomal proteins FGB, FGG, ITIH4, and SERPINA3 were found to be upregulated in CAD. These proteins are associated to an alteration of the platelet degranulation and activation ^[29][112]. FGB is cleaved by thrombin to yield monomers that polymerize into insoluble fibrin, usually associated with coagulation disorders. Both exosomal FBG and FGG were found to be significantly increased in patients with malignant pulmonary nodules and evidence reveals its increasing interest of candidate diagnostic biomarkers for bladder and prostate cancer, among others ^[30][113]. ITIH4 is a protein involved in inflammatory responses and host–virus interaction processes. For instance, ITIH4 is elevated in urine samples of patients with type 2 diabetes mellitus and microalbuminuria and it is considered as a putative biomarker for diabetic kidney disease ^[31][114]. SERPINA 3 gene codes for alpha-1-antichymotrypsin, a powerful inhibitor of proteolytic enzymes. An increase in alpha-1-antichymotrypsin occurs during tissue damage and it is considered to be one of the “acute phase reactant” ^[32][115]. Moreover, it contributes to the impairment of the coagulation cascade and fibrinolysis in the development of calcific aortic stenosis and could be useful as a biomarker for this disease with considerable clinical value ^[33][116].

The downregulated circulating proteins CLU, TF, ALB, tetranectin (CLEC3B), kininogen-1 (KNG1), alpha-1B-glycoprotein (A1BG), vitamin K-dependent protein S (PROS1), and alpha-1-antitrypsin (SERPINA1) were found to participate in the platelet degranulation molecular mechanism. Exosomal CLU is a heterodimeric glycoprotein highly expressed after AMI in order to induce vascular growth and cardiac tissue regeneration ^[34][117]. ALB is the most abundant circulatory protein, associated with various physiological functions, such as the maintenance of microvascular integrity, regulating metabolic and vascular functions, and anticoagulant effects, among others ^[35][118]. Indeed, the association between low levels of ALB and increased risk of CVD ^[36] [119] and HF ^[37][120] is reported in several studies, as well as the prognostic significance of this mediator in CAD ^[38][121]. Together with CLU and ALB, DBP was found to be associated to the negative regulation of protein oligomerization. DBP, also associated to the negative regulation of protein oligomerization, is found to be downregulated in plasma samples of patients with coronary artery atherosclerosis and can be used as a potential predictor factor of the severity of the disease ^[39][122].

The downregulated proteins C3, TF, and CLU participate in the positive regulation of receptor-mediated endocytosis, which suggests the association with exosomal biogenesis ^[40][10]. C3 is an important component of the complement system, involved in the immune response. When considering local and systemic inflammation, not only an increase in extracellular vesicles is observed but also an increase in complement activation products. This link between vesicles and the complement signaling pathway leads to innate and adaptive immune responses ^[41][123].