Extracellular vesicles in atherosclerosis and vascular calcification: the versatile non-coding RNAs from endothelial cells and vascular smooth muscle cells

Atherosclerosis (AS) is characterized by the accumulation of lipids, fibrous elements, and calcification in the innermost layers of arteries. Vascular calcification (VC), the deposition of calcium and phosphate within the arterial wall, is an important characteristic of AS natural history. However, medial arterial calcification (MAC) differs from intimal calcification and cannot simply be explained as the consequence of AS. Endothelial cells (ECs) and vascular smooth muscle cells (VSMCs) are directly involved in AS and VC processes. Understanding the communication between ECs and VSMCs is critical in revealing mechanisms underlying AS and VC. Extracellular vesicles (EVs) are found as intercellular messengers in kinds of physiological processes and pathological progression. Non-coding RNAs (ncRNAs) encapsulated in EVs are involved in AS and VC, including microRNAs (miRNAs), long non-coding RNAs (lncRNAs), and circular RNAs (circRNAs). The effects of ncRNAs have not been comprehensively understood, especially encapsulated in EVs. Some ncRNAs have demonstrated significant roles in AS and VC, but it remains unclear the functions of the majority ncRNAs detected in EVs. In this review, we summarize ncRNAs encapsulated in EC-EVs and VSMC-EVs, and the signaling pathways that are involved in AS and VC.


. Introduction
Atherosclerosis (AS) is associated with systemic risk factors, including hypertension, hyperlipidemia, and diabetes mellitus, and is characterized by the accumulation of lipids, fibrous elements, and calcification in the innermost layers of arteries (1,2). Several cell types are directly involved in the pathological progression of AS, such as endothelial cells (ECs), vascular smooth muscle cells (VSMCs), platelets, and foam cells (3). As a cellular monolayer lining the blood vessel wall, ECs first contact endogenous metaboliterelated signals in the bloodstream, in which ECs function as danger signal sensors (4). been demonstrated, which highlights the importance of VSMC phenotypic modulation in AS (17)(18)(19). The switching of VSMCs to macrophage-like cells may be driven by lipids accumulation which was Krüppel-like factor 4 (KLF4) dependent (20, 21).
Vascular calcification (VC), the deposition of calcium and phosphate within the arterial wall, is an important part of AS natural history and an independent predictor of cardiovascular morbidity and mortality (22)(23)(24)(25). VC occurs in both the intimal and medial layers of the arteries, and intimal arterial calcification (IAC) is mainly involved in the calcification of atherosclerotic lesions (26,27). Calcifying vascular cells are derived from local VSMCs, especially in IAC (28). On the contrary, medial arterial calcification (MAC) is a chronic systemic vascular disorder distinct from AS, in which its hallmark is the dissemination and progressive precipitation of calcium phosphate within the medial layer (29). MAC differs in several ways from the IAC seen in atherosclerotic lesions, including cellular aging linked to mechanical stress cooperated and buffered by ECs (27,30,31). Initially, VC was regarded as a passive degenerative process, and now VC is elucidated to be a multifactorial process through the phenotypic modulation of VSMCs (26, 32-36). It means that ECs have a potential role in MAC. Correspondingly, endothelial dysfunction under aging or injured condition promoted VC (37-39). More importantly, it has been demonstrated that endothelial dysfunction aggravated MAC in vivo (40,41). Moreover, extracellular vesicles (EVs) found a novel messenger in the cellular crosstalk between ECs and VSMCs in VC (42, 43) ( Figure 1). Extracellular vesicles, as the generic term for particles, are derived from cells and are delimited by a lipid bilayer (44). Non-coding RNAs (ncRNAs), messenger RNAs (mRNAs), and lipids encapsulated in EVs can involve in intracellular function regulation and intercellular communication. It has been reported that there were a growing number of studies about microRNAs (miRNAs) encapsulated in EVs related to VC (45-50). Nevertheless, there were few special studies aimed to summarize the miRNAs encapsulated in EVs during intercellular communication. Therefore, considering the prospect of cellular communication through EVs, first, we propose to summarize that the miRNAs have appeared in EVs derived from ECs (EC-EVs) and EVs derived from VSMCs (VSMC-EVs) in the AS and VC processes. Subsequently, we will discuss the potential prospect of long non-coding RNAs (lncRNAs) and circular RNAs (circRNAs) in EC-EVs and VSMC-EVs during AS and VC formation.

. The flexibility and versatility of miRNAs encapsulated in EC-EVs in AS and VC
The emerging role of EVs and intercellular communication has gained momentum in the past few years. In the process of biological message transferring from ECs to other cells, the presence of EVs has been verified in numerous physiological and pathological pathways, including AS and VC.
Additionally, miR-505, called miR-505-3p at present, is also highly expressed in exosomes from ox-LDL-HUVEC, which was finally proved to be a proatherogenic factor by inducing neutrophil extracellular trap (NET) formation in vitro (55). MiR-505-3p markedly inhibited the proliferation, migration, and tube formation capacity of HUVECs by targeting vascular endothelial growth factor A (VEGFA) (72,73). Therefore, circulating miR-505-3p was a prognostic biomarker of endothelial dysfunction and inflammation in hypertension patients (74,75). However, the biological function of miR-505-3p in VSMCs has not been reported, leaving to further investigation.
Furthermore, miR-4306 encapsulated in EVs derived from ox-LDL condition (ox-LDL-EVs) from human coronary artery vascular endothelial cells (HCAECs) upregulated the AKT/NF-κB signaling pathway enhancing the lipid formation of human monocyte-derived macrophages (HMDMs), while miR-4306 encapsulated in ox-LDL-EVs from HMDMs inhibited capillary tubule formation of HCAECs in vitro (57). Wang et al. (91) found that miR-4306 plays a key role in urban particulate matter-induced endothelial damage. As a result, miR-4306 manifested promotion in AS.
The previously reported MiR-92a-3p, named miR-92 or miR-92a before, performed a different function with its recipient cells during AS (51). MiR-92a-3p encapsulated in EVs from HUVEC treated with TNF-α or subjected to shear flows could be transported to macrophages to suppress KLF4, leading to the atheroprone phenotypes of macrophage in AS (58). The progression of AS was associated with a maladaptive form of angiogenesis which contributed to plaque disruption and intraplaque hemorrhage (92). Independent atherosclerotic stimuli (ox-LDL and IL-6) increased miR-92a-3p expression in donor HCAEC-EVs, promoting an angiogenic phenotype in the recipient ECs through the downregulation of thrombospondin 1 (THBS1) (59). Meaningfully, miR-92a-3p encapsulated in HCAEC-EVs increased in a balloon injury rat model and inhibited the atheroprotective expression of KLF2 (60). Uremic toxins accumulation upregulated the expression level of miR-92a-3p in ECs, contributing to endothelial dysfunction (93). MiR-92a-3p overexpression inhibited VSMCs proliferation and migration, while miR-92a-3p inhibition promoted the proliferation and migration of VSMCs via the KLF4 pathway (94,95). MiR-92a-3p encapsulation in EC-EVs demonstrated a double effect on AS when recipient cells assimilate it: it promoted ECs phenotype switching while it inhibited VSMCs proliferation and migration.
In brief, numerous studies show plenty of potential EC-EV-miRNAs that are involved in the progress of AS and illustrate the multiple signaling pathways. Particularly, miR-126-5p was upregulated in EC-EVs in atherogenic condition, while previous studies showed its atheroprotective effect, addressing the . /fmed. . complicated role of miR-126-5p in the AS process. Further studies are essential to explore the distribution of miR-126-5p between EVs and cells, and how biological function diversities are feasible. In addition, miR-155-5p promoted AS and KLF2 downregulated the expression of miR-155-5p showing its atheroprotective role. Correspondingly, the suppression of KLF2 by miR-92a-3p and the suppression of KLF4 by miR-92a-3p lead to AS. Additionally, the upregulation of miR-143-3p and miR-145-5p encapsulated in EC-EVs was transduced by KLF2 and showed an atheroprotective effect. KLF2, a shear-responsive transcription factor, was critical because it played a major role in exerting atheroprotective effects in ECs, which promised a novel target for AS prevention and treatment (96,97). Similarly, KLF4, as a therapeutic target of atherosclerosis, regulates ECs inflammation, VSMC phenotypic transformation, and foam cell formation during the process of AS (98). Several miRNAs encapsulated in EC-EVs that regulate the process of AS are summarized in Table 1 and  Supplementary Table 1.

. . miRNAs encapsulated in EC-EVs regulate the process of VC
We have demonstrated for the first time that high phosphate (HP)-induced EVs from HUVECs directly promoted and accelerated VC through astrocyte elevated gene-1 (AEG-1) (99). Furthermore, miRNAs encapsulated in EC-EVs demonstrated significant messengers in the biogenesis of VC. Lin et al. (100) found that miR-670-3p encapsulated in EC-EVs stimulated by HP could target insulin-like growth factor-1 (IGF-1) resulting in potentiating the VC process in vitro and in vivo. Freise et al. applied EC-EVs with a miR-221 (miR-221-3p) inhibitor which provoked a distinct reduction of VSMC calcification, and miR-143 or miR-145 mimics also provoked a distinct reduction of VSMC calcification (101). MiR-29b (miR-29b-3p) and miR-126-5p in EC-EVs also negatively regulated VC (102,103). Additionally, we detected the profile of miRNAs in HP-EVs from HUVECs by RNA-seq, finding that miR-3182 increased while 13 known miRNAs decreased in HP-EVs from HUVECs (104), indicating the potential function of miRNAs encapsulated in EC-EVs during VC.
MiR-29b-3p was named miR-29b previously according to miRBase. Wang et al. confirmed miR-29b-3p encapsulated in endothelial exosomes mediated anti-calcification and was associated with C-X-C motif chemokine receptor 6 (CXCR6) (102). Similarly, miR-29b-3p downregulated VSMC calcification by targeting matrix metalloproteinase 2 (MMP2) and Wnt family member 7B (Wnt7b)/β-catenin signal (105)(106)(107). The expression of miR-29b in the coronary artery calcification group was significantly lower than that in the control group, indicating that decreased miR-29b-3b level may be a coronary artery calcification risk factor (108). However, it is a paradox that Fang et al. found that miR-29b was increased in calcific aortic tissue and the inhibition of miR-29b mitigated aortic calcification in tissues of calcific aortic valve diseased rats via directly targeting TGF-β3 (109). Additionally, inorganic phosphorus (Pi) stimulation increased miR-29b-3p expression and overexpression of miR-29b-3p facilitated Piinduced VSMC calcification (110). Intriguingly, the level of miR-29b shows no statistical significance in VSMCs during the exposure of 3 mM Pi, compared with normal VSMCs (111). Taken together, miR-29b-3p encapsulated in EC-EVs inhibited VSMC calcification, while the role of miR-29b-3p in VSMC calcification is still ambiguous. Considering specific distribution, further studies explaining the relationship between miR-29b-3p and VC are required in the future.
Freise et al. (101) revealed that three miRNAs (miR-221, miR-222, and miR-126) were significantly enhanced in uremic toxins-induced-EVs from human ECs. Subsequently, VSMC calcification was inhibited by a miR-221 inhibitor and a stronger reduction with a miR-222 inhibitor. Both miR-221 and miR-222 contribute to the atherogenic calcification of VSMCs (112). Correspondingly, increased calcium deposition was observed in the combined treatment of miR-221 and miR-222 that mimics synergistically but not in individual treatments, with significant changes in ectonucleotide phosphodiesterase 1 (Enpp1) and Pit-1 expression (113).
By contrast, Freise et al. (101) confirmed that the levels of miR-143 and miR-145 were significantly decreased during VSMC calcification. MiR-143 and miR-145 can both inhibit AS process as mentioned above (61). Similarly, an individual transfection with miR-143 or miR-145 mimics provoked a distinct reduction of VSMC calcification and a stronger reduction in a concomitant transfection (101). Massy et al. (114) suggested that decreasing levels of miR-143, miR-145, and miR-126, and increasing levels of miR-223 were potential biomarkers of vascular calcification associated with chronic kidney disease (CKD). Elevated Pi significantly downregulated miR-143 and miR-145 and upregulated miR-223 during VSMCs calcification in vitro and in ApoE (−/−) mice (111,115).
Based on Freise et al. (101) and Massy et al. (114) studies, the expression of miR-126 was found to be adverse, which probably was attributed to the different distribution of miR-126 among EC-EVs, VSMCs, and serum of CKD mice. Massy et al. (114) detected that the expression of miRNAs (including miR-126, miR-143, miR-145, and miR-223) in the endothelium or media of the thoracic and abdominal aorta is different. For example, miR-126 expression of VSMCs in the upper abdominal aorta is equal to ECs, lower than ECs in the low thoracic and low abdominal aorta, and higher than ECs in the upper thoracic (114). However, it must be remembered that miR-126 is known as miR-126-3p, and miR-126 * is referred to as miR-126-5p. Due to some reason, the specific sequence of miR-126 was not shown in all studies. As a result, miR-126 refers to miR-126-3p as default. It suggested that miR-126 can be considered a potential cardiovascular risk and VC biomarker in cardiovascular disease (CVD) and cerebral troubles associated with CKD (53, 114,116,117). Additionally, miR-126 expression is increased in CKD and ApoE (−/−) mice aorta (118). Furthermore, miR-126 markedly enhanced the regeneration of vascular smooth muscle (119). In the progress of Pi-induced HASMCs calcification, miR-126 was involved in the Wnt/β-catenin signaling pathway (120). However, miR-126-5p encapsulated in HUVEC-EVs stimulated with advanced glycation end-products led to the reduction of calcium deposition by blocking the smad1/5/9 signaling pathway both in vitro and in a mouse model (103). In short, the biological . /fmed. . effects are different between miR-126-3p and miR-126-5p. The upregulation of miR-126-3p encapsulated in EC-EVs probably enhances VC, while the upregulation of miR-126-5p encapsulated in EC-EVs prohibits VC. However, the versatility of miR-126-5p is still worth exploring because of its atherogenic function. Thus, it is necessary to validate these results in further study.
We have discussed the ambiguous role of miR-29b-3p, anticalcification roles of miR-143-3p, miR-145-5p, and miR-126-5p, and pro-calcification roles of miR-221-3p, miR-222-3p, and miR-126-3p. Overall, the results above show a novel perspective in intercellular communication during VC: miRNAs encapsulated in EC-EVs potentiate an anti-calcification or pro-calcification effect when transmitted to VSMCs. Several miRNAs encapsulated in EC-EVs that regulate the process of VC are summarized in Table 2 and  Supplementary Table 2.

. The flexibility and versatility of miRNAs encapsulated in VSMC-EVs in AS and VC
The phenotype change from the contractible type to the synthetic type of VSMCs plays a crucial role in the biogenesis of AS, VC, and vascular dysfunction. VSMCs are one of the main cells implicated in AS, retaining remarkable plasticity (132). VSMCs can adopt alternative phenotypes resembling foam cells contributing to AS (133). VSMCs are also key players in VC, which transform into calcifying VSMCs and secrete mineralizing EVs that form microcalcifications, subsequently increasing plaque instability and consequential plaque rupture (134). In this review, we summarized miRNAs encapsulated in VSMC-EVs in the process of AS and VC.
. /fmed. .   (143) identified that miR-143-3p was independently associated with time-to-coronary events and that a higher expression in healthy individuals was related to less risk of an incident coronary artery disease (CAD) event at 10 years. By contrast, González et al. (144) suggested that miR-143-3p may be implicated in carotid plaque instability by the modulation of IGF-IIR, contributing to AS.

. . miRNAs encapsulated in VSMC-EVs regulate the process of AS
The characteristics of miR-222-3p in the process of AS were subtle, which were different from the ones in the process of VC, as we summarized in EC-EVs. In 2010, Dentelli et al. demonstrated that miR-222 acted as an anti-angiogenic miRNA that negatively regulated STAT5A, providing a promising perspective to prevent the biogenesis of AS (145). Atherosclerotic plaque rupture exhibited a decrease in miR-221/222 (146). Both miR-221-3p and miR-222-3p decreased significantly in patients with AS, indicating that the downregulation of these two miRNAs may be connected with AS (147). In addition, miR-222 was involved in VSMCs proliferation and upregulated by Ang II in promoting ECs migration and inflammation (148). In 2015, Xue et al. (87) unveiled miR-221/222-induced EC dysfunction by the suppression of peroxisome proliferator-activated receptor-γ coactivator-1α (PGC-1α) in AS. Taraldsen et al. (149) observed that circulating levels of miR-92a-3p, miR-221-3p, and miR-222-3p were associated with baseline coronary necrotic core volume. We speculate that the duality of miR-221-3p and miR-222-3p is due to the different distribution of VC and that miR-221-3p and miR-222-3p probably play key roles in MAC.
Efforts would be essential to understand the conflicting role of miR-222-3p in AS. As a result, increasing number of evidence is required to address its time-based and spatial location in ECs, VSMCs, and EVs. The protective function of miR-143-3p seemed more specific than miR-222-3p in AS. Nevertheless, further studies are required to elucidate the relationship between the functional and distributional diversity of miR-143-3p and miR-222-3p encapsulated in EV. signaling pathways in AS mouse models, as shown by the evidence. In the future, the distinction and differential expression of miR-155 in different conditions still need to be elucidated, and potential signaling pathways associated with AS also need to be uncovered. Gonzalo et al. (159) discovered that atherogenic lipoproteins significantly reduced miR-15b-5p, −24-3p, −29b-3p, −130a-3p, −143-3p, −146a-3p, −222-3p, and −663a in microvesicles (MVs) released by HCASMC. MiR-143-3p, miR-222, and miR-29b have been discussed above. MiR-24-3p and miR-130a-3p were also reduced in circulating MVs from familial hypercholesterolemia (FH) patients. In addition, the expression of miR-130a-3p was reduced in uremia toxin indoxyl sulfate-induced HCAEC-EVs (160). Downregulation of miR-130a-3p expression leads to endothelial barrier dysfunction in the development of AS (161). Paradoxically, serum miR-130a-3p was elevated in cerebral AS patients and provided a possible method to predict cerebrovascular events (162). The different expressions of miR-130a-3p in AS period indicated the potential ability in AS diagnosis. However, we suppose that the signaling pathways of miR-130a-3p that implicate AS need to be discovered.
We have discussed the protective effect of miR-143-3p and the versatility of miR-222-3p, miR-155-5p, and miR-130a-3p in the process of AS. On the whole, versatile distributions between EVs and intracellular location probably perform profound effects on cellular metabolism, which implicated a variety of signaling pathways (Figure 2). Table 3 and Supplementary Table 3 indicate several miRNA-encapsulated VSMC-EVs that regulate the AS process.

. The emerging role of lncRNAs encapsulated in EVs in AS and VC processes
LncRNAs, another type of non-coding RNA larger than 200 base pairs, have significant enrichment in the nucleus compared with mRNAs, emerging as crucial regulators of tissue physiology and pathology mechanism (176-179). Many lncRNAs modulated gene expression in neuronal disorders and cancer (180). In turn, therapeutic targeting of lncRNAs provided a promising approach to the treatment of multiple diseases (181). Furthermore, lncRNA encapsulated in EVs represented an approach to utilizing lncRNAs in disease diagnostics and therapy (182). Here, we aimed to review studies related to lncRNAs encapsulated in EV in the process of AS and VC.

. . lncRNAs encapsulated in EVs involved in the process of AS
Indeed, different lncRNAs involved in AS showed opposite effects, such as lncRNA RNCR3 has an atheroprotective effect while lncRNA MIAT has a proinflammatory effect on VSMCs (183,184). Moreover, lncRNAs encapsulated in EVs were also demonstrated to be tightly associated with AS (185). In mesenchymal stem cells (MSCs)-EVs, lncRNA FENDRR induced by ox-LDL alleviated HUVECs injury and AS by binding with miR-28 (186); knockdown of LOC100129516 alleviated the progression of AS (187). Encapsulated in ox-LDL-EVs from THP-1 cells, lncRNA GAS5 enhanced the apoptosis of HUVECs and HAECs and lncRNA LIPCAR-enhanced AS (188)(189)(190).
In conclusion, lncRNAs encapsulated in EVs provided a new perspective on the intercellular communication of AS. The potential role of lncRNAs in the prevention and progress of AS still needs to be illuminated. Several lncRNAs encapsulated in EVs involved in the process of AS are summarized in Table 5.
/fmed. .    reason that lncRNA expressions are relatively low in EVs so their transcripts cannot be analyzed in RNA-seq. More importantly, the lncRNA sequence-function relationship needs to be explored in the future (212). LncRNAs for the indepth study often follow a specific physiologic or pathological state (213). The relationship between EVs and lncRNAs has the potential to advance our understanding of AS, VC, and other diseases.
. The emerging role of circRNAs encapsulated in EVs in AS and VC processes CircRNAs, generated from intronic lariats during colinear splicing and characterized by their covalently closed circular structure, showed tissue-specific and development stage-specific expression (214-216). Hansen et al. (217) first revealed that circRNAs were regarded as miRNAs sponges generally in gene regulation. Some endogenous circRNAs were highly abundant and evolutionarily conserved, providing potential implications for research and treatment applications (218). We have discussed miRNA encapsulated in EVs in AS and VC so far and subsequently aimed to summarize circRNA encapsulated in EVs in AS and VC.
. . circRNAs encapsulated in EVs involved in the process of AS CircRNAs have been identified in the process of AS, such as circ_USP36, circ_0007478, circCHFR, and circ_0001879 (219)(220)(221)(222)(223). CircRNAs encapsulated in EVs were also involved in the process of AS (224). CircNPHP4 in monocyte-EVs regulated heterogeneous adhesion in coronary heart atherosclerotic disease by the modulation of miR-1231 (225). Hsa_circ_0001445 acted as a biomarker in CAD patients and was decreased in VSMCs-EVs on AS condition (226). Hsa_circ_0005699 was downregulated in the blood exosomes of patients with coronary heart disease and upregulated in ox-LDL-treated macrophage, providing candidate targets for the diagnosis of AS (227). Encapsulation in serum EVs of patients with unstable/vulnerable plaque AS, upregulated circRNA_0006896 plays a crucial role in carotid plaque destabilization by modulation of miR-1264 (228); circ_0043837 and circ_0001801 were independent predictive factors for artery atherosclerotic stroke (229). Hsa_circ_0086296 was also observed to be higher in AS patient serum EVs and ox-LDL-treated HUVECs, which promoted ECs injury and AS by the modulation of miR-576-3p (230). Although many circRNAs in AS have been revealed, circRNAs encapsulated in EVs during AS between intercellular communication will be focused, discussing issues and trends in studies in the future. Several circRNAs .
/fmed. . encapsulated in EVs involved in the process of AS are summarized in Table 6.
. . circRNAs involved in the process of VC Ryu et al. discovered that circSamd4a reduced VC by modulating miR-125a-3p and miR-483-5p; circSmoc1-2 reduced VC by the modulation of miR-874-3p (231,232). Moreover, CircSamd4a could act as a biomarker for the diagnosis of VC (233). In contrast, circRNA CDR1 promoted VC by sponging miR-7-5p (234). Similarly, hsa_circRNA_0008028modulated miR-182-5p and promoted high glucose-induced VC (235). Notably, there were few articles concentrated on circRNAs participating in VC. Further studies are essential to elucidate the emerging role of circRNAs in the process of VC.

. Discussion
In this review, we have summarized almost all miRNAs encapsulated in EVs secreted by ECs and VSMCs in the process of AS and VC. We concluded on the function of different miRNAs encapsulated in EVs which are effective to inhibit or aggravate AS and VC, depending on the stimuli and distributions. In addition, miRNAs encapsulated in EVs were expected to be indicators in the diagnosis and prognosis of cardiovascular diseases. There is still a long way to go. Different methods (direct electroporation, cell transfection, and chemical transfection) were developed to vehicle therapeutic molecules through EVs (236). Nevertheless, limitations, such as EV disruption and aggregation, still exist. The application of the atheroprotective miRNAs encapsulated in EVs in the treatment is still needed to be further investigated. In addition to elucidating the versatility of miRNAs in different signaling pathways, the complicated and subtle effects in the animal model should be illustrated.
Moreover, lncRNAs and circRNAs are expressed differentially during AS and VC. We have discussed the current understanding of lncRNAs and circRNAs in AS and VC. We have noticed the potential prospect of lncRNAs and circRNAs in the prevention, diagnosis, and treatment of AS/VC.
In conclusion, the versatile non-coding RNAs encapsulated in EVs provide a novel perspective on the biogenesis mechanisms of AS and VC.

Conflict of interest
The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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Supplementary material
The