Basic Pathogenic Mechanisms and Epigenetic Players Promoted by Extracellular Vesicles in Vascular Damage

Both progression from the early pathogenic events to clinically manifest cardiovascular diseases (CVD) and cancer impact the integrity of the vascular system. Pathological vascular modifications are affected by interplay between endothelial cells and their microenvironment. Soluble factors, extracellular matrix molecules and extracellular vesicles (EVs) are emerging determinants of this network that trigger specific signals in target cells. EVs have gained attention as package of molecules with epigenetic reversible activity causing functional vascular changes, but their mechanisms are not well understood. Valuable insights have been provided by recent clinical studies, including the investigation of EVs as potential biomarkers of these diseases. In this paper, we review the role and the mechanism of exosomal epigenetic molecules during the vascular remodeling in coronary heart disease as well as in cancer-associated neoangiogenesis.


Introduction
The vasculature controls homeostasis and the functional integration of tissues and organ systems [1,2]. Hemodynamic and bioactive stimuli stimulate the vasculature, which responds with multiple structural and phenotypic changes that are well described during the onset and progression both of cardiovascular diseases (CVDs) and cancer [1][2][3]. Starting from fatty streaks, cell components of the vascular wall change their function, contributing to the progression of atheromatous plaques, chronic inflammation and clinical manifestations of coronary heart disease (CHD) [4][5][6][7][8][9]. Endothelial adaptative remodeling also occurs during cancer growth, representing a key event during the transition from local to systemic and metastasis dissemination [10]. Different metabolic and epigenetic signaling between endothelial cells and the microenvironment are crucial to the initiation and progression of pathological vascular modifications [1][2][3]11]. Therefore, investigation of the pathophysiology of the vascular remodeling mechanism both in CVDs and cancer is of great value for finding new disease targets and biomarkers. A central role in vasculature modifications is the crosstalk between several cell types necessary to transfer information from one site to another. An emerging body of evidence implies that extracellular vesicles (EVs), which circulate in biological fluids, are vehicles of a variety of molecules to recipient endothelial cells during CVD and cancer growth [12][13][14][15][16][17][18]. However, the role of EVs and their epigenetic influences in the pathogenesis of vascular damage are poorly explored. Here, we discuss the epigenetic effects exerted by EVs on the vasculature both in coronary heart disease (CHD) and cancer.

EVs and Peripheral Endothelial Dysfunction
Epigenetic changes contribute to the development of CVDs and begin with progressive alterations of the endothelial tissue [3,7]. Endothelial dysfunction is characterized by impaired vascular function, loss of antithrombotic function, excessive proliferation of vascular smooth muscle cells (VSMCs), platelet activation and macrophage migration [9,39]. In endothelial dysfunctional cells, intercellular and vascular cell adhesion molecule-1 (ICAM-1 and VCAM-1) are overexpressed and promote the leukocytes' adhesion [40]. Several aberrant epigenetic modifiers, mainly miRNAs and lncRNAs, are transported by EVs on dysfunctional endothelial cells (ECs) that modify gene expression and contribute to the acceleration of vascular disease to advanced atherosclerosis [41,42] (Figure 1).
Ding Yu Lee and colleagues demonstrated that the downregulation of miR-10a in both the circulation (serum) and aortic endothelium is highly associated with atherogenesis. miR-10a reduced leukocyte infiltration through GATA6/VCAM-1 signaling pathway, thereby decreasing plaque formation. Thus, this study showed that miR-10a downregulation is associated with atherosclerosis and its induction could prove to be a novel therapeutic target for this disease [43]. Evidence also demonstrated that several miRNAs regulate the leukocytes' infiltration into the endothelium vessel. One example is miR-155-EVs from VSMCs that target ECs down-regulate integrin proteins and tight junctions. These EVs generate leaky membranes, favor leukocytes and other immune cells migration into subendothelium where macrophages can maturate in the foam cells [3,7,40,44]. Moreover, data showed that miRNA-155-and miRNA-223-EVs may also target mononuclear cells, favoring LDL accumulation and their transformation in damaged endothelium [45,46]. Hall and colleagues outlined the importance of an EV-derived circRNA, named Circ-Lpr6, for vascular pathogenesis in both animal and human models. This novel circRNA acts as a sponge for miR-145, thereby regulating its downstream targets. Molecular studies demonstrated that Circ-Lpr6 impairs miR-145-mediated regulation of VSMCs differentiation, migration and proliferation. Furthermore, its overexpression resulted in a decreased intimal area in mouse aortas. Therefore, Circ-Lrp6 could be required to counterbalance miR-145 into Harmful EVs can promote atherogenesis by altering vascular integrity, increasing inflammatory response and promoting thrombus formation. On the other hand, beneficial EVs, containing specific ncRNAs, can play athero-protective roles by promoting the repair of damaged endothelium and repressing the activation of inflammatory cells. The EVs interact with target cells via direct fusion, endocytosis or the binding of surface proteins. After this invagination, cytosolic proteins or RNA content-EVs transfer to the extracellular space. In the target cells, ncRNAs (such as lncRNAs, miRNAs, circRNAs) can (A) control nuclear gene expression via epigenetic changes and/or (B) affect protein function.

EVs in Inflammation and Calcification
Cellular damage plays a critical role in the inflammatory response, delivering a high amount of EVs containing pro-and anti-inflammatory signals to target cells [3,7,43]. Although ncRNAs are not classically considered as direct epigenetic modificators, they might have a role in such events. Protective mechanisms are activated by miRNA-126, miRNA-210 and miRNA-214 promoting angiogenesis and vasculogenesis [48][49][50]. In Figure 1. Extracellular vesicles and vascular damage. Schematic representation of atherosclerotic EVs. They are produced by the different cell types involved in the mechanism of plaque formation. Harmful EVs can promote atherogenesis by altering vascular integrity, increasing inflammatory response and promoting thrombus formation. On the other hand, beneficial EVs, containing specific ncRNAs, can play athero-protective roles by promoting the repair of damaged endothelium and repressing the activation of inflammatory cells. The EVs interact with target cells via direct fusion, endocytosis or the binding of surface proteins. After this invagination, cytosolic proteins or RNA content-EVs transfer to the extracellular space. In the target cells, ncRNAs (such as lncRNAs, miRNAs, circRNAs) can (A) control nuclear gene expression via epigenetic changes and/or (B) affect protein function.

EVs in Inflammation and Calcification
Cellular damage plays a critical role in the inflammatory response, delivering a high amount of EVs containing pro-and anti-inflammatory signals to target cells [3,7,43]. Although ncRNAs are not classically considered as direct epigenetic modificators, they might have a role in such events. Protective mechanisms are activated by miRNA-126, miRNA-210 and miRNA-214 promoting angiogenesis and vasculogenesis [48][49][50]. In particular, miRNA126 increased the expression of chemokine 12 ligand (CCL12), reduced macrophage entry into the vascular wall and protected the vascular endothelium [51]. A supportive role for this action mechanism derived from the observation that the silencing of miRNA-126 caused leaky vessels, hemorrhage of blood from arterial vessels and partial embryonic lethality. Silencing also induced a loss of vascular integrity and defects in ECs proliferation and angiogenesis [52]. Although miRNA-214 targeting damaged ECs, it also promoted angiogenesis and thus favored the restoration of vessel integrity [50]. The contribution of EVs to endothelial inflammation has been reported to be due to DC-derived EVs that activated the TNF-α/NF-κβ pathway, contributing to the progression of atherosclerosis in animal model [53]. In response to inflammatory stimuli, ECs transferred EV-miR-92a-3p to VSMC, resulting in cell proliferation, while EV miR-146a inhibited their migration, further exacerbating the inflammatory response [54][55][56]. The RNCR3 ncRNA released from ECs in aortic atherosclerotic lesions showed a protective role, regulating miR-185-5p in VSMC target cells. Specifically, miR-185-5p favored the interaction between RNCR3 and Klf2, promoting ECs vasoprotection [57]. In vivo knockdown of RNCR3 worsens atherosclerosis, hypercholesterolemia and inflammation [57]. Thus, EV-transported ncRNAs have a key role in the onset of CVDs. EVs are also involved in the progression of vascular calcification [58]. Analysis of murine and human vessels showed that macrophage-derived EVs promoted intimal microcalcification [59]. Furthermore, the enrichment of these EVs with the calcium-binding protein S100A9 facilitated the calcification progression together with VSMC-derived EVs [59]. In response to this stimulus, miRNA150-EVs promoted vascular maintenance by activating the endothelial vascular endothelial growth factor (VEGF)-A/VEGFR/PI3K/Akt pathway [60]. Although little data are available on EVs regulating apoptosis during endothelial damage, it was demonstrated that HUVECs treated with macrophage-derived EVs activate NF-κB and TLR4 signaling pathways, causing ECs apoptosis [53].

EVs and CHD
Inflammation and calcification have interconnected roles in the pathophysiology of CHD and acute coronary syndrome (ACS) [3,7,11]. For the first time, the transcriptomic analysis of EVs from ACS patients revealed the presence of mRNA coding for epigenetic enzymes, such as DNA methyltransferases (DNMTs) [61]. Notably, ACS-derived EVs absorbed on PBMCs from healthy subjects modulated the expression of several genes, presumably via epigenetic mechanism in agreement with previously reported [62][63][64][65][66]. In particular, EVs induced methylome changes promoting a significant activation of Synaptosin (SYP), Cell Adhesion Molecule L1 Like (CHL1) and SH2 Domain Containing Adaptor Protein B (SHB) genes [61]. These findings underlined the epigenetic contribution in the role of EVs in ACS.
In the clinical practice, CHD diagnosis is made by coronary CT scan [7,67] and coronary angiography [7,68]. However, it is still unclear whether EVs could be a tool to improve CHD risk estimation future investigations should be conducted to combine EVs and clinical parameters. Shi and colleagues demonstrated that human blood samples from healthy subjects have 86% more EVs compared to CHD patients [69,70]. Therefore, both EV count and content are of potential interest as disease-specific biomarkers [71][72][73][74][75].
Nowadays, it is known that, in the cardiovascular system, EVs also play important epigenetic-indirect roles in the acute post-myocardial infarction (MI) damage and repair mechanisms inducing myocardial remodeling and cardiac regeneration [76]. Recently, echocardiographic experiments had shown that exosomes derived from the mesenchymal stem cell of adipose tissue (ADSC) could improve left ventricular ejection fraction, whereas their administration could significantly alleviate MI-induced cardiac fibrosis [77]. Additionally, ADSC-exosomes treatment has been shown to reduce cardiomyocyte apoptosis, increasing angiogenesis. Molecular experiments demonstrated that ADSCs-derived exosomes can promote microvascular ECs proliferation and migration and inhibit cardiomyocytes apoptosis through miRNA-205. This assumption was confirmed via the transfection of ADSC-derived exosomes into MI-induced mice and observing a decrease in cardiac fibrosis, an increase in angiogenesis, and an improvement of cardiac function [77]. Cardiac fibroblast-derived EVs modulate cardiac remodeling, favoring intercellular communication between cardiomyocytes (CM) and cardiac fibroblasts [78,79]. Consistently, exosomal miRNA-21 CM-derived, reduced the expression of sarcoplasmic protein sorbin 2 containing SH3 domain (SORBS2) and PDZ and LIM domain 5 (PDLIM5), producing an increase in cell size and stimulating cardiomyocyte hypertrophy [80,81]. Moreover, miRNA-21 promoted the cardiac fibrosis inducing the endothelial-to-mesenchymal cell transition [82] and altered expression of superoxide dismutase (SOD) in HF patients [83,84]. Increasing research has attracted attention to the roles of EVs-ncRNAs as potential diagnostic biomarkers. Other than miRNAs, several studies have indicated that lncRNAs significantly regulate fibrosis, thereby having a direct effect on ECM gene expression, the TGF-β signaling pathway and the proliferation of fibroblasts or transition to myofibroblast [23,[85][86][87]. These effects are also proposed to be mediated by paracrine communication of EVs between donor and recipient cells, especially in cardiomyocytes and fibroblasts [23]. Exosomes-containing lncRNA ZFAS1 could induce cardiac fibrosis via the Wnt4/β-catenin signal pathway by sponging miR-4711-5p in cardiac fibroblasts [85]. LncRNA MIAT is up-regulated in serum-derived EVs from atrial fibrillation (AF) patients. MIAT aggravated the atrial remodeling and promoted AF by binding with miR-485-5p [86]. Neat1 is obviously up-regulated by P53 and HIF2A in cardiomyocytes in response to hypoxia and is enriched in cardiomyocyte-derived exosomes. Neat1 is essential for cell survival and fibroblast functions. This finding was confirmed via genetic knockout of Neat1 that impaired cardiac function during MI [87]. Although currently studies aim at the analysis of EVs from human samples in AF, HF and MI repair, large studies need to be performed to determine their utility in the clinical routine [88]. In addition to all the findings on basic mechanisms involving EVs, various clinical studies have been initiated to date. Some of these are still at the recruitment stage, and the few completed studies have not yet published their first results (NCT03660683, NCT04142138, NCT03034265, NCT03984006, NCT02822131, NCT00331331, NCT03837470 and NCT04266639). All studies listed in Table 1 will investigate the general cargo of EVs from different biological fluids and tissues, but none have provided help to investigating specific molecules. Trials are evaluating the role of EVs as carriers of anti-inflammatory signal or drug delivery systems. However, these are still few, and their results are not fully available (Table 1).

EVs in Neoangiogenesis
Initiation of angiogenesis is an important early event in premalignant lesions to supply nutrients and oxygen [1,9] (Figure 2).
EVs such as extracellular organelles elicit signaling between tumor and vascular cells carrying functional and epigenetic modifications to adapt the microenvironment to tumor growth conditions ( Figure 2). Tumor hypoxia is the most potent stimulus of EVs biogenesis [89]. EV content may activate the proliferative and migratory status of ECs, favoring neovessel formation via different mechanisms, including epigenetic ones [90] (Figure 2). The canonical VEGF (or Notch signaling pathway) was described as being activated by EVs from different tumor types because ECs promoted sprouting and proliferation [91][92][93][94] ( Figure 2). Other metabolic programs have also been proven to influence the migration of ECs [16]. We reported that EVs containing EDIL3 activate a mitochondrial and vesicular trafficking program through the purinergic receptor 4 in normal quiescent ECs. This stimulus promotes endothelial cell survival, proliferation, motility and organization into capillary-like structures in vitro and in vivo [16].
EVs may also regulate endothelial function via epigenetic mechanisms. In preclinical and clinical settings, a variety of ncRNAs and long noncoding RNA (lncRNA) transcripts packaged in EVs have been shown to be capable of modifying gene expression without altering the DNA sequence [95]. Generally, EVs-miRNAs are transferred into ECs, where they are post-transcriptionally downregulating antiangiogenic genes, promoting growth and vessel sprouting. However, the role of individual exosomal miRNAs is dependent on tumor histology and stage. In squamous cell carcinoma (ESCC), miR-181b-5p predicts a poorer overall survival. It was also shown to be involved in vessel formation by promoting endothelial growth via AKT targeting endothelial transcript for PTEN and PHLPP2 [96]. In multiple myeloma, the number of EVs carrying miR-135b is significantly increased compared to normal individuals. Transfer of this miRNA in ECs downregulates the in-hibitor of hypoxia-inducible factor 1 (HIF-1), resulting in the formation of endothelial tubes through the HIF signaling pathway [97]. MiR-9-EVs from glioma play a pivotal role in both pathogenesis and vascularization. When miR-9 is absorbed by ECs, the increased neoangiogenesis activates multiple pathways, among them COL18A1, Thrombospondin-2 (THBS2), Protein patched homolog 1 (PTCH1) and prolyl hydroxylase 3 (PHD3) via MYC and OCT4 [98]. One of the most common miRNAs associated with poor prognosis of several cancer types is miR-210 [99,100]. Exosomal miR-210 uptake from ECs promotes angiogenesis by targeting specific processes depending on the tumor microenvironment [100]. The chromatin epigenetic regulation of proangiogenic genes has also been attributed to different lncRNA packaged in EVs, although the mechanism is not fully elucidated [101][102][103] (Figure 2). LncRNAs can work as molecular scaffold, assembling several chromatin regulator proteins and reshaping the DNA structure in the nucleus and thus favoring the transcription or silencing of target genes [104]. In other cases, lncRNAs can act as ceRNAs or sponges, sequestering endogenous miRNAs and inhibiting their action on target. For example, gastric cancer exosomal lncRNA PVT1 was reported to have the capacity to activate proliferation of ECs by forming a complex with polycomb repressive complex 2 (PRC2), the major histone methyltransferase on the STAT3 promoter, activating the transcription of the VEGFAs [105]. Several exosomal lncRNAs and proteins have been identified that originate from non-small cells lung cancer (NSCLC), some of which have a role in neoangiogenesis, e.g., lncRNA-p21 and Ubiquitin-fold modifier conjugating enzyme 1 (UFC1) protein [105][106][107]. The mechanism identified for UFC1 revealed the binding to the enhancer of Zeste 2 (EZH2) protein in the polycomb repressor complex 2 (PRC2) and their accumulation at the promoter region of the PTEN gene. This complex catalyzes the trimethylation of H3K27, the inhibition of PTEN expression and the activation of angiogenesis via AKT. UFC1 knockdown inhibited NSCLC growth in a mouse xenograft tumor model [107]. Another pro-angiogenic mechanism is downstream Angiopoietin 2/Tyrosin kinase endothelial receptor 2 (Ang2/Tie2). This pathway is activated by lncRNA EPIC1 in NSCLC and promotes channel formation and the proliferation of ECs [108]. Furthermore, the switch of ECs towards the proliferative phenotype was attributed to exosomal MANTIS. This lncRNA influenced the epigenetic regulation of angiogenic sprouting and alignment of ECs, modifying the chromatin complex, SWI/SNF (Switch/Sucrose non-fermentable), to facilitate the access to RNA polymerase II machinery and gene transcription [109].
Different mechanisms have been attributed to metastasis-associated lung adenocarcinoma transcript 1 (MALAT-1), which may promote angiogenesis in endothelial ovarian carcinoma and in lung cancer [110,111]. Studies have demonstrated the influence of MALAT-1 and other lncRNAs in the complex interplay among lncRNA-miRNA-mRNA governing the sequestration and inactivation of miRNAs. For example, lncNKX2-1 from gastric cancer was reported to increase tumor growth and angiogenesis in both in vitro and in vivo models to direct sponge miR-145-5p, which upregulates Serpin Family E member 1 (SERPINE1) and vascular endothelial growth factor receptor (VEGFR) 2 signaling pathways [112]. A similar mechanism was also defined for receptor activity-modifying protein 2 (RAMP2-AS1) from chondrosarcoma, Urothelial cancer associated (UCA1) for pancreatic cancer, Small nucleolar RNA host gene 1 (SNHG1) from breast [112][113][114], RNA CCAT2 in exosomes from gliomas [115,116] and LINC00707 from cervical or bladder cancer [117,118]. Taken together, these data indicated that EVs have a role in tumor neoangiogenesis through epigenetic mechanisms. Emerging research is evaluating exosomes and their content as potential cancer biomarkers in clinical setting (Table 2). A survey on ClinicalTrials.gov (https://clinicaltrials.gov/, accessed on 15 April 2023) shows a total of 88 trials, of which 60 (68%) EVs' cargo from different source are used as biomarkers of disease or response to therapy applications. In the case of exosome therapy, as a drug-delivery system, seven trials (8%) have been registered and one clinical trial (1.72%) is related to exosome as vaccine study. Although no definitive data are available, a manufacturing practice (GMP) as exosome production method and purification methods is present in the current trial. Furthermore, the European Medicines Agency (https://www.ema.europa.eu/en, accessed on 15 April 2023) has released scientific recommendations on classification of advanced therapy medicinal products. This nascent field of biomedicine offers new opportunities for the treatment of diseases and dysfunctions of the human body. In addition, it provides guidelines, recommendations, for researchers. However, the transition of EVs from basic research to clinical application is still premature. EVs such as extracellular organelles elicit signaling between tumor and vascular cells carrying functional and epigenetic modifications to adapt the microenvironment to tumor growth conditions (Figure 2). Tumor hypoxia is the most potent stimulus of EVs biogenesis [89]. EV content may activate the proliferative and migratory status of ECs, favoring neo-vessel formation via different mechanisms, including epigenetic ones [90] (Figure 2). The canonical VEGF (or Notch signaling pathway) was described as being activated by EVs from different tumor types because ECs promoted sprouting and proliferation [91][92][93][94] (Figure 2). Other metabolic programs have also been proven to influence the migration of ECs [16]. We reported that EVs containing EDIL3 activate a mitochondrial and vesicular trafficking program through the purinergic receptor 4 in normal quiescent ECs. This stimulus promotes endothelial cell survival, proliferation, motility and organization into capillary-like structures in vitro and in vivo [16].
EVs may also regulate endothelial function via epigenetic mechanisms. In preclinical

EVs and Vascular Alterations in Tumor Progression
The disruption of vascular integrity and the consequent increase in vascular permeability allows tumor cells to penetrate the vasculature and metastasize. EVs secreted by cancer cells during tumor growth play an important role in vascular leakage (Figure 2). Studies have demonstrated that EVs from metastatic cancer cells may modify ECs' permeability by altering cytoskeletal-associated proteins or downregulating the RhoA/ROCK pathway [119]. EVs containing miRNAs (including miR-200c, miR-141 and miR-429), which correlated with a poor prognosis of colon cancer, were demonstrated to downregulate ZEB protein and modify the membrane permeability of ECs [120]. Similarly, EVs carrying miR-25-3p, more frequent in patients with metastasis than in those without, were demonstrated to induce vascular leakage and enhance colon cancer metastasis [121]. The same is true for EVs miR-105 in breast cancer [122]. Furthermore, EVs miR-939 correlated with poor prognosis of gastric cancer and increased the permeability of ECs by downregulating the transcription of VE-cadherin [123]. A similar mechanism was reported for LncX26 in gastric cancer [124]. Additionally, the loosening of endothelial integrity and ultimately inducing colon cancer metastasis were attributed to post-transcriptional down regulation of p120catenin (p120) mediated by transferring of exosomal miR-27b-3p from colon cancer [125] or miR-638 from HCC [126]. In NSCL cancer patients, exosomal miR-375-3p positively correlated with patient TNM stages. In vivo, miR-375-3p-enriched exosomes destroyed the endothelial structure of lung, liver and brain tissues of mice, leading to increased blood vessel permeability. Exosomal miR-375-3p internalized by breaking the tight junction of ECs through negative regulation of claudin-1 expression [127]. Another important pathway of EVs systemic activity is the degradation of extracellular matrix (ECM), favoring tumor growth and preparation of the metastasis niche. In particular, MMP-1 in EVs derived from cancer cells is a representative indicator of ECM degradation [128] (Figure 2). Evidence shows that the pharmacological reduction of EVs decreases the secretion of MMPs, resulting in an inhibition of tumor growth and metastasis [128,129]. This pathway is epigenetically regulated by exosomal lnc-MMP2-2 released by NSLC. The lncRNA internalized into ECs competed with miR-1207-5p to downregulate EPB41L5 and increased vascular permeability, promoting brain metastasis [130]. These data indicate a key role of EVs in damaging the endothelium and promoting intravasation of tumor cells and extravasation at distal sites ( Figure 2). Moreover, considering the epigenetic roles of some ncRNAs and their capacity to control gene expression, the development of epigenetic-related drugs with higher specificity and fewer side effects would be a possible future approach.

EVs in Metastatic Niche: And Other Types of Vessels
Tumor vasculatures increase the tumor capability to metastasize by acquiring mesenchymal characteristics [131,132]. Both normal and tumor endothelial ECs are capable of acquiring a mesenchymal phenotype, increasing the tumor's ability to metastasize. TECs comparing to ECs exhibiting cytogenetic abnormalities, such as aneuploid chromosomes, are differ metabolically, more resistant to starvation and release more angiocrine molecules and EVs [133,134]. The presence of TEC is associated with conversion of indolent tumor to more aggressive phenotype, and their presence was correlated with reactivation of dormant CD44v6+ tumor stem cells [135,136]. ECs and TECs within the vasculature gain a mesenchymal phenotype (vimentin alfa-smooth muscle actin positive), in some tumor types, shed from the endothelial lining layer of neoplastic vasculature into peripheral blood to the colonizing distal organ [135][136][137][138]. The mechanism is not fully understood. Exosomal Annexin A1 is one of the triggers inducing endothelial-to-mesenchymal transition (EndEMT) and activating RAC1/PAK2 in the gastric cancer model, whereas TGF-β on the surface of EVs triggers the same phenotypic switch in melanoma [139][140][141]. Moreover, data from EVs containing miR-92a-3p, derived from colon cancer cells, showed upregulation of mesenchymal markers, such as snail and vimentin, and downregulated the tight junction marker ZO-1 in HUVECs referred as "partial-EndoMT" [142]. EVs released from tumor cells during hypoxia participate in the EndMT switch of TECs, particularly EVs loaded with lysyl oxidase-like 2 protein (LOXL2) [143]. LOXL2 is a member of the LOX family and regulates extracellular matrix (ECM) remodeling, angiogenesis and premetastatic niche formation. LOXL2 acts as deaminase enzyme into the nucleus contributing to transcription, alternative splicing and miRNA regulation. All these pathways contribute to EndoEMT transition, proliferation and migration of ECs and TECs to distant organs and vasculogenic mimicry mechanism (Figure 2) [143]. Exosomal lncRNA antisense transcript of GATA6 (GATA6-AS) has also been reported to bind LOXL2, which regulates endothelial gene expression underlying EndoEMT transition [144]. The authors demonstrated that GATA6-AS associated with LOXL2 activates H3K4me3 marks and positively regulates the angiogenesis-related genes periostin and cyclooxygenase-1 in ECs. [144]. According to recent studies a multitude of other forms of neovascularization have been observed in cancer settings, including vascular looping and splitting (intussusception), vasculogenic mimicry and remodeling of larger 'feeding' vessels (arterio/venogenesis). An epigenetic mechanism mediated by EVs was also involved in vascular mimicry ( Figure 2). Here, tumor cells undergoing epithelial-mesenchymal transition (EMT) form microvascular channels contributing to tumor vascularization [145]. These vessel types are negative to CD31 antigen and insensitive to anti angiogenic therapy. The mechanism involved the transferring of EVs containing lncRNAs into target cells, where they act as sponge of selective miRNAs or as scaffold for chromatin remodeling [145][146][147][148][149][150][151]. These mechanisms have been described for HULC in glioblastoma MALAT-1 in gastric cancer and in lung cancer Linc0155 [152][153][154]. Vasculogenic cooption (VCO) is another way by which tumor cells incorporate vessels from normal tissue to gain access to blood supply and growth locally ( Figure 2). It is reported that tumor cells can migrate along blood vessels beyond the advancing front of the tumor and may spread to distant metastases [143]. Vessel cooption was initially described in gliomas and lung metastasis [155,156]. Then, VCO was reported in breast cancer metastasis and gastric metastasis from colon [143]. The mechanism of VCO is mediated by VEGF, but it is reasonable to think that EVs rich in VEGF are also involved [155].

Conclusions and Perspectives
Some pathogenic aspects of cancer and CHD are defined by the pathophysiology of vascular damage induced by key events in which EVs might play a role. EV counts, types and contents tend to vary, depending on collection methods. By analyzing literature data, we found that only three ncRNAs, specifically, miR-92a-3p, miR-145 and miR-210, were involved in vascular damage, both in the context of CVDs and in tumor neoangiogenesis, although with different mechanisms, underling the possible common pathway. Epigenetic tissue-specific reversible changes induced by exosomal cargo could play a role in the initiation and progression of disease complications. In terms of biomarkers, exosomal ncRNAs could be superior compared to non-exosomal ncRNAs for their specify in methods preparation, the protection from degradation and stability. Moreover, exosomal ncRNAs exist in considerable amounts in different body fluids that can be non-invasively sampled. Considering the epigenetic reversible roles of some ncRNAs, the establishment of novel approaches for modulating their expression or counteract their action could be a future goal [18]. To this end, in the cardiovascular field, 28 clinical studies were started which include exosome analyses (Table 1). In the field of oncology, 88 trials have begun in order to investigate the potential prognostic role of EVs. The most promising application appears to be biomarkers for gastrointestinal cancers (Table 2). However, larger, randomized, prospectively enrolling multicenter trials are necessary to identify EVs as potential clinical biomarkers to better guide clinical treatments.