ECs form the main components of the intima and act as a barrier between blood and tissues. A growing number of studies suggest that ECs-derived EVs are important in the pathogenesis of atherosclerosis (2). In addition, EVs are implicated in altering lipid metabolism and homeostasis, both of which are important for atherosclerosis.

MiRNAs in ECs-derived EVs play essential roles in atherosclerosis. MiRNA-126 packaged in EVs from ECs transferred to recipient cells can promote ECs proliferation, migration, and reendothelialization through modulating the target protein sprouty-related, EVH1 domain-containing protein 1 (SPRED1). A study analysis of 176 patients with coronary artery disease with and without diabetes mellitus found that the expression of miRNA-126 was decreased in EVs from diabetic patients (24). ECs-derived EVs can promoted anti-inflammation by reducing the expression of ICAM-1 through the transfer of miR-222 into recipient cells. EVs from high glucose-treated ECs exhibit a downregulated level of miR-222 and reduced anti-inflammation (30). It has been reported that EVs released from ECs can attenuate atherosclerosis by inhibiting inflammation. When transferred into monocytes, miRNA-10a in EVs from ECs exert anti-inflammatory effects by down-regulating the production of pro-inflammatory genes, including IRAK4, MAP3K7, and BTRC, and inhibiting the nuclear factor-kappa B (NF-κB) signaling pathway (22). MiR-126 and miR-210 of EVs were upregulated in ECs overexpressing hypoxia-inducible factor (HIF)-1. The transfer of miR-126 and miR-210 inhibited macrophages infiltration and reduced plaque area (3). EVs derived from human umbilical vein ECs (HUVECs) under stimulus, such as shear stress, exhibit a high level of miRNA-143/145 (27). Hergenreider et al. found that ECs-derived EVs overexpressed shear-responsive transcription factor Krüppel-like factor 2 (KLF2) induces miRNA-143/145 accumulation in ECs. Subsequently, EVs from ECs transfer miRNA-143/145 to VSMCs, thereby mediating the communication between ECs and VSMCs. Moreover, they found that overexpression of KLF2 in ECs attenuates the size of atherosclerotic plaque in ApoE(-/-) mice (27). In addition, miRNA-126 was enriched in EVs secreted by ECs. It promotes G protein-coupled receptor (GPCR) signaling by suppressing the function of the regulator of G protein signaling 16, thereby enhancing the production of CXC chemokine ligand 12 (CXCL12) (25). EVs produced from ECs containing miRNA-126 repress the amounts of macrophages and apoptotic cells in plaques, and increase the amount of VSMCs, resulting in less inflammation and small lesions. Ohta et al. reported that elevated IL-6 levels suppress the expression of miRNA-126-3p in EVs derived from ECs and promote monocyte cell line THP-1 cells adhesion, eventually enhancing intercellular adhesion molecule-1 (ICAM-1) levels, leading to exacerbation and deterioration of atherosclerotic plaques. In contrast, elevated the expression level of miRNA-126-3p has the opposite effects (26). The level of miRNA-19b was upregulated in EVs from hypoxia-induced ECs. The transfer of EVs MiRNA-19b can inhibit migration and angiogenesis of ECs through decreasing the expression of transforming growth factor-β2 (TGF-β2) (23). Lombardo et al. found that IL-3 increases the secretion of EVs and transmission of miRNA-126-3P from ECs to wounds by promoting the activity of signal transduction and activator of transcription 5 (STAT5), thereby stimulating angiogenesis (72). Additionally, ECs-derived EVs modulate ECs function, vascular angiogenesis through the delivery of miRNA-214 between ECs (73). van Balkom et al. reported that miRNA-214 enriched in EVs inhibits ataxia telangiectasia mutation and promote angiogenesis. Suppressing miRNA-214 levels shows opposite effects (73). On the contrary, miRNAs encapsulated in EVs from ECs can damage the function of blood vessels and accelerate atherosclerosis progression. Atherosclerosis associated inflammation enhances inflammatory factor accumulation (74), including tumor necrosis factor-α (TNF-α), which promotes the expression of miRNA-155 in ECs-derived EVs. MiRNA-155 of EVs downregulates endothelial Nitric oxide synthase (eNOS) expression and weakens endothelium vasorelaxation. Low production of eNOS contribute to endothelial dysfunction and impair vascular homeostasis (75). Furthermore, simvastatin has been shown to reduce the expression of miRNA-155 in EVs and improve eNOS generation (28, 76). In addition, miRNA-505 in EVs from ox-LDL treated HUVECs have been shown to enhance atherosclerotic development in mice models through impairing blood vessels function. Mechanistically, EVs released from ox-LDL-induced HUVECs co-cultured with neutrophils transfer miRNA-505 to neutrophils, activate the NF-κB pathway and inhibit SIRT3 in neutrophils, resulting in reactive oxygen species (ROS) accumulation and increased formation of neutrophil extracellular traps (NETs) (31).

Vascular media is mainly composed of VSMCs, which are important in maintaining physiological functions and structural integrity of blood vessels. VSMCs proliferation is vital in plaque repair and might be a beneficial process in early atherosclerosis, while VSMCs death and senescence induce plaque instability and promote atherosclerosis (77).

VSMCs-derived EVs exert significant effects in regulation of inflammation, cell migration, vascular calcification and cell coagulation among others. Additionally, EVs secreted by VSMCs can be elevated through stimulation by platelet derived growth factor (PDGF) and TNF-α, leading to VSMCs calcification. VSMCs-derived EVs that are rich in the miRNA-143/145 cluster play important roles in modulating VSMCs differentiation, quiescent vs. proliferative phenotype and vascular homeostasis (78). Climent et al. reported that transforming growth factor-β (TGF-β) or vessel stress-treated VSMCs-derived EVs mediate ECs angiogenesis and vascular stability through the transfer of miR-143/145 (79). Besides, molecules such as TNF-α and PDGF-BB enhance production of miRNA-143 in EVs, resulting in cell proliferation, cell migration, and VSMCs calcification (3, 80, 81). MiRNA-143-3p rather than miRNA-143-5p is enriched in EVs from pulmonary artery smooth muscle cells (PASMCs), and regulated PASMCs migration. Deng et al. revealed that miR-143-3p packaged in EVs derived from PASMC paracrine to ECs and act as a proangiogenic regulator (80). In addition, EVs from VSMCs transfer miRNA-150 to ECs, enhancing the expression of vascular endothelial growth factor-A (VEGF-A) and promoting the VEGF-A/VEGFR/PI3K/Akt pathway, leading to ECs migration (35). Besides, miRNA-150 knockdown in VSMCs attenuate ECs migration (35). Studies have shown that miRNA-150 overexpression induced by either spliced X-box binding protein 1 (XBP1) or PDGF-BB is crucial in regulating ECs and VSMCs function as well as in maintaining vessel homeostasis (35, 82). Dysregulation of autophagy by ECs has an effect on the progression of atherosclerosis. MiRNAs in EVs bind autophagy-related genes to inhibit ECs autophagy. Human aortic SMCs co-cultured with HUVECs were shown to suppress the autophagic activity of HUVECs through upregulating the levels of miRNA-221/222 in EVs from human aortic SMCs. Phosphatase and tensin homolog deleted on chromosome 10 (PTEN) is an anti-inflammatory and antifibrotic modulator, mediating VSMCs differentiation, migration and proliferation (83). The elevated expression levels of miRNA-221/222 in EVs downregulated the expression of PTEN in HUVECs and activated the Akt pathway, resulting in LC3II, Beclin-1, ATG5 downregulation and SQSTM1/p62 upregulation (37). Furthermore, atherosclerosis is associated with downregulated miRNA-133a levels (84). Gao et al. reported that miRNA-133a levels were decreased in ApoE(-/-) atherosclerosis mice models and were associated with inhibited insulin-like growth factor-1 receptor (IGF-1R) levels as well as suppressed VSMC growth (33). In addition, overexpression of miRNA-133a targets proapoptotic genes, including Bim (Bcl2l11) and Bmf (Bcl-2 modifying factor), inhibiting cell apoptosis and promoting proliferation of cardiac progenitor cells (34). On the contrary, overexpression of KLF5 in VSMCs induces miRNA-155 production and secretion (36). KLF5 plays a role in promoting VSMCs proliferation by upregulating the generation of growth factors such as PDGF-A (85). VSMC-derived EVs transfer miRNA-155 to ECs, impairing EC barriers by weakening tight junctions and coherence, leading to increased endothelial permeability and enhanced atherosclerotic development (36). Moreover, miRNA-92a and miRNA-195 in EVs released from VSMCs shuttled into macrophages to regulating inflammation and promoting atherosclerosis through elevating the expression levels of IL-6 and CXCL1 (32).

Monocyte-endothelial cell adhesion induces numerous monocyte progression to macrophage- or DC- like phenotypes in early atheromata. Risk factors such as hyperlipidemia and hypertension can trigger vascular inflammation and further stimulate the expression of adhesion molecules such as ICAM-1, VCAM-1, and selectin E in ECs. These molecules promote monocyte-ECs adhesion and monocytes infiltration into the subcutaneous space of ECs (86). In early atheromata, the majority of monocyte progression to macrophage- or DC- like phenotypes (87). Many macrophages and dendritic-like cells have cytoplasmic membrane-bound lipid droplets (foam cells). Foam cells secrete numerous pro-inflammatory cytokines and promote monocyte proliferation and accumulation. Accumulating data suggests that macrophages regulate atherosclerosis development by destabilizing atherosclerotic plaques. Ox-LDL induces monocytes/macrophages inflammatory response emerge as a key event in atherosclerosis.

EVs released from macrophages stimulate pro-inflammatory responses by transporting miRNAs and lncRNAs into recipient cells (88). For example, M1-like-type macrophages secrete numerous pro-inflammatory EVs, with anti-angiogenic properties (45). Besides, M2-like-type macrophage-derived EVs have both pro-inflammatory and anti-inflammatory roles (89). Monocyte/macrophage-derived EVs can modulate cell proliferation and migration, inflammation, apoptosis and angiogenesis. Zhang et al. reported that miRNA-150 is transferred from THP-1 cells to ECs through EVs. Mechanistically, THP-1 cells-derived EVs were shown to have an abundance of miRNA-150, which are transferred to human microvascular ECs-1 (HMECs-1), resulting in HMECs-1 migration by downregulating c-Myb (43). Notably, c-Myb, a nuclear transcription factor, was found to be highly expressed in atherosclerotic mice models and acted as a critical regulator of the atherosclerosis process (90). In addition, miRNA-21-3p and miRNA-142a-5p of EVs are secreted by macrophages. Macrophages exposed to nicotine trigger the transfer of miRNA-21-3p to VSMCs via EVs, and induce VSMCs proliferation and migration by directly inhibiting PTEN (38). EVs containing miRNA-106a-3p can be transferred from ox-LDL-treated THP-1 macrophages to VSMCs, thereby promoting VSMCs proliferation and inhibiting cell apoptosis by combining with CASP9 to modulate the Caspase signaling pathway. Contrastingly, treatment with EVs secretion inhibitor, GW4869, suppresses EVs secretion from ox-LDL-induced THP-1 cells, decreasing VSMCs viability and increasing cell apoptosis (40). Wang et al. reported that the transfer of miRNA-222 in EVs from M1 macrophages to VSMCs elevated VSMCs proliferation and migration through the mediation of Cyclin Dependent Kinase Inhibitor 1B (CDKN1B) and CDKN1C in vitro and enhanced neointima formation after carotid artery injury in vivo, which was reversed by 2'OMe-miR-222, a miRNA-222 inhibitor (46). Human THP-1 macrophages exposed to ox-LDL were shown to exhibit elevated expression levels of miRNA-155 in a dose-dependent manner. MiRNA-155 from THP-1 cells is an atheroprotective regulator that inhibits inflammatory responses during the pathogenesis of atherosclerosis (44). Silencing miRNA-155 in THP-1 cells significantly promotes lipid intake, upregulates the secretion of scavenger receptors, leading to the accumulation of inflammatory factors such as IL-6, IL-8, and TNF-α. Furthermore, attenuation of miRNA-155 activation significantly elevates myeloid differentiation primary response gene 88 (MyD88) levels and suppresses the NF-κB pathway. M1 macrophage-derived EVs carrying miRNA-155 shuttle into ECs, playing an anti-angiogenesis role by inhibiting the activity of Sirtuin 1/protein kinase AMP-activated catalytic subunit alpha 2 (AMPKα2), endothelial nitric oxide synthase and Rac family small GTPase 1 (RAC1)/p21-activated kinase 2 (PAK2) signaling pathways (45). MiRNA-210 is a critical anoxic-related miRNA in EVs from cardiac progenitor cell. It promotes angiogenesis and suppresses myocardial cells apoptosis (91). Moreover, HIF-1α in inflammatory and lesional macrophages increases miRNA-210 expression and decreases miRNA-383 production, reduces ATP levels and elevates necroptosis and atherosclerosis through targeting 2,4-dienoyl-CoA reductase and mitochondrial ROS accumulation (92). Moreover, miR-210-3p suppresses the NF-κB pathway by inhibiting IGF2/IGF2R, resulting in amelioration of lipid congestion and inflammation (93). Furthermore, differentiation of macrophages induces EVs secretion and stimulates miRNA-233 delivery to recipient cells. Elevated miRNA-233 levels promote macrophages differentiation and apoptosis (94). It has been reported that miR-223, which is enriched in macrophage-derived EVs, acts as an inhibitor of Pknox1, thereby repressing macrophages inflammatory responses and downregulating adipose tissue inflammation (47). IL-4 induced naive bone marrow macrophages-derived EVs exhibit high expression levels of miRNA-99a/146b/378a, as well as downregulate NF-κB and TNF-α signaling pathways. Therefore, macrophages are polarized toward the M2-like-type, which ameliorates atherosclerosis lesion areas (39). On the contrary, miRNA-146a in EVs derived from macrophages can accelerate the development of atherosclerosis. EVs produced by ox-LDL exposed macrophages are rich in miRNAs, such as miRNA-146a, miR-185, miR-128, miR-503, and miR-365. It has been reported that EVs contained miRNAs transferred from donor macrophages to naive recipient macrophages are involved in the pathogenesis of atherosclerosis. MiRNAs encapsulated in EVs, especially miRNA-146a, from atherogenic macrophages, inhibit recipient macrophages migration and promote macrophages accumulation in vessel walls by suppressing the expression of insulin-like growth factor 2 mRNA-binding protein 1 (IGF2BP1) and human antigen R or ELAV-like RNA-binding protein 1 (HuR), thereby accelerating atherosclerosis progression (41). It has been accepted that IGF2BP1 is associated with cell proliferation and growth while HuR is involved in cell proliferation, differentiation, apoptosis and senescence (95, 96). Zhang et al. found that miRNA-146a was enriched in EVs produced from THP-1 macrophages treated with ox-LDL. MiRNA-146a promoted atherogenesis by targeting superoxide dismutase 2 (SOD2) and increasing the production of ROS and NETs (42).

DCs, a group of antigen-presenting cells, are implicated in the pathogenesis of atherosclerosis. During the development of atherosclerosis, DCs play roles in anti-inflammation, antigen presentation, lipid metabolism, and efferocytosis. Normal arteries have been shown to have low levels of DCs while atherosclerotic tissues have been shown to have elevated DC levels, implying that DCs play a critical role in atherosclerosis (97).

EVs secreted by DCs stimulated by other antigens seem to be atherogenic. Mature DCs-derived EVs are associated with ECs inflammation and atherosclerosis progression through the NF-κB signaling pathway. MiRNA-16 and miRNA-21 were found to be enriched in EVs from bone marrow-derived DCs treated with low-intensity pulsed ultrasonography (LIPUS). DCs-derived EVs co-cultured with HUVECs can inhibit TNFα-induced inflammation by ameliorating the expression of ICAM-1 and vascular cell adhesion molecule (VCAM)-1, subsequently inhibiting the NF-κB signaling pathway (48). In addition, it has been admitted that miRNA-155 promotes inflammation while miRNA-146a suppresses it. DCs-derived EVs exhibit elevated levels of miRNA-146a and miRNA-155. MiRNA-146a and miRNA-155 packaged in EVs from DCs, uptaken by recipient DCs, are involved in intercellular communication and modulation of inflammatory responses (50). Zhong et al. co-cultured mature DCs-derived EVs with HUVECs and found that EVs from mature DCs promoted the expression of adhesion molecules, such as VCAM-1 and E-Selectin by a quick activation of NF-κB signaling pathway. Further study indicated that mature DCs-derived EVs shuttled miRNA-146a into HUVECs, inhibiting interleukin 1 receptor associated kinase, which repressed the second stimulation of HUVECs via EVs and inhibiting ECs inflammation (49). The expression of miR-203-3p in EVs was downregulated in ox-LDL-induced bone marrow-derived macrophages (BMDMs). Lin et al. found that the transfer of miR-203-3p targeted cathepsin S in bone marrow-derived macrophages to attenuate atherosclerosis development in mice (51).

Platelets, inflammatory cells with a well-established role in hemostasis and thrombosis, play central roles in the progression of atherosclerosis. Vascular exposure to stimuli such as hyperlipidemia and hyperglycemia initiates platelet activation as well as interactions with ECs and leukocytes, which has been implicated in atherosclerosis.

Platelet-derived EVs interact with monocytes, ECs and VSMCs, thereby promoting atherosclerosis progression. EVs secreted by thrombin-activated platelets inhibit ECs inflammation, cell proliferation and migration, angiogenesis and atherogenesis through miRNAs. MiRNA-25-3p has been shown to be enriched in EVs derived from thrombin induced platelets in atherosclerosis mice models. Peripheral blood platelet-derived EVs inhibited the expression of Adam 10 and further suppressed coronary vascular ECs inflammation and lipid deposition through reducing the levels of IL-1β, IL-6, TNF-α, α-smooth muscle actin, Collagen I a1, Collagen III a1, total cholesterol, and triglycerides through the delivery of miR-25-3p. In addition, miRNA-25-3p packaged in EVs from platelets alleviates ECs inflammation by suppressing the NF-κB signaling pathway (52). Li et al. found that thrombin-activated platelet-derived EVs from atherosclerotic animal models were rich in miRNA-21, miRNA-223, and miRNA-339. MiRNA-223 transferred to HUVECs to suppressed ICAM-1 production, which can be reversed by a miRNA-223 inhibitor. Thrombin-activated platelets-derived EVs carry miRNA-223 and inhibit ECs inflammation by modulating MAPK and NF-κB signaling pathways (56). Moreover, platelets-derived EVs contain miRNA-223, which regulates ECs apoptosis by decreasing IGF-1R levels (55). The expression levels of miRNA-21, miRNA-223, and miRNA-339 are associated with platelet activation and are enriched in platelet-derived EVs. They inhibited co-cultured VSMCs proliferation by suppressing PDGFRβ production, which is associated with VSMCs invasion and migration (57, 98). Additionally, in acute coronary syndrome patients, miRNA-126 has been shown to be significantly elevated in platelet-derived EVs. Isolated EVs, incubated with HUVECs, promote cell proliferation and migration by increasing the production of angiogenic factors, such as VEGF, basic Fibroblast Growth Factor and TGF-β1 (53). Thrombin-activated platelets-derived EVs contained a high level of miRNA-142-3p and shuttled miR-142-3p into ECs to promoted ECs proliferation through targeting Bcl-2-associated transcription factor-1(BCLAF-1) and its downstream genes (54). MiRNA Let-7a was enriched in platelet-derived EVs. The transfer of miRNA let-7a from platelet to HUVECs promoted angiogenesis through downregulating the generation of thrombospondin-1 (THBS-1), an anti-angiogenic factor (99). MiR-320b secreted by activated platelets was slightly elevated in the circulation of myocardial infarction patients. Activated platelet-derived EVs transfer miRNA-320b to human microvascular endothelial cell-1 cells to mediate inflammatory responses through downregulating the expression of ICAM-1 (58, 84).

MSCs with the characteristics of self-renewal and multipotent differentiation as well as the capability of anti-inflammation may be promising therapeutic strategies for atherosclerosis. For example, bone marrow-derived MSCs (BMSCs) enhance the generation of anti-inflammatory cytokines, such as TGF-β1 and IL-10 and inhibit the expression of pro-inflammatory cytokines, such as TNF-α and IL-6 (100). An increasing number of publications indicate that EVs secreted by MSCs are served as atheroprotective regulators through regulating intercellular communications.

MSCs-derived EVs are implicated in regulating cell proliferation, migration, and apoptosis, and alleviating atherosclerotic plaque formation via miRNAs. Expression levels of miRNA-30b are highly elevated in MSCs-derived EVs. Co-cultures with HUVECs revealed that EVs transfer miRNAs from MSCs into HUVECs and enhance angiogenesis by suppressing delta-like 4 (DLL4) levels, a negative modulator of angiogenesis (59, 101). In addition, human adipose MSCs-derived EVs exhibit high expression of miRNA-125a that inhibit the production of DLL4, leading to ECs angiogenesis and the improvement of endothelial tip cell formation (61). MiRNA-31 was up-regulated in EVs from endothelial differentiation medium-treated adipose-derived stem cells. EVs containing miRNA-31 shuttled from SCs into HUVECs to regulate cell migration and tube formation through suppressing HIF-1 (60). Overexpression of miRNA-126 in MSCs-derived EVs were shown to promote cell proliferation and migration by enhancing the production of EGF, VEGF, bFGF, and PDGF in Hypoxia/Reoxygenation-injured ECs, as well as MSCs. Besides, miRNA-126-3p was found to be enriched in EVs from human umbilical cord MSCs. In rat models, miRNA-126-3p was shown to enhance HUVECs proliferation, migration, and tube formation through the SPRED-1/PIK3R2/AKT/ERK1/2 pathways (102). In contrast, Wang et al. reported that MSCs-derived miRNA-125b transfer to VSMCs through EVs suppresses VSMCs proliferation and migration as well as neointimal hyperplasia by ameliorating the expression of myosin 1E (Myo1e) (62). MSCs-derived EVs carrying miRNA-145 shuttled into HUVECs. The transfer of miRNA-145 inhibits cell migration and ameliorates atherogenesis plaque development in vivo by suppressing junctional adhesion molecule A (JAM-A), which plays an essential role in maintaining endothelial barrier functions (66, 103). Furthermore, MSCs-derived EVs containing miRNA-126 shuttled into ECs to attenuated ECs apoptosis by increasing the activity of PI3K/Akt/eNOS signaling pathway (65). Additionally, miRNA-342-5p in EVs from adipose-derived MSCs inhibits HUVECs apoptosis and protects ECs function against atherosclerosis by inhibiting PPP1R12B expression (69). Moreover, miRNA-125b-1-3p in EVs was highly expressed in human adipose-derived MSCs and reduced B-cell chronic lymphocytic leukemia (CLL)/lymphoma 11B (BCL11B) levels in lymphocyte, leading to lymphocyte apoptosis in atherosclerotic arterial tissues and in amelioration of atherosclerosis (63). EVs secreted by BMSCs from aorta tissues of ApoE(-/-) atherosclerosis mice models were shown to contain significantly elevated miRNA-125b-5p levels, which induced the suppression of mitogen-activated protein 4 kinase 4 (Map4k4), down-regulated MMP-9 expression, inflammation, blood lipid accumulation and plaque size, as well as increased α-SMA expression, thereby alleviating apoptosis in atherosclerosis mice (64). Furthermore, MSC-derived EVs can also attenuate atherosclerosis by modulating inflammation. ECs endocytosis of EVs with highly expressed miRNA-512-3p from MSCs was shown to suppress ox-LDL induced EC injury, Caspase-3 activation and cell apoptosis, decreased the secretion of inflammatory cytokines such as oxidative factor MDA, TNF-α, IL-1β and IL-6, increased GSH-PX and SOD levels and promoted ECs proliferation. Mechanistically, the transfer of miRNA-512-3p in EVs from MSCs inhibits Keleh-like ECH-associated protein 1 (Keap1) to alleviate ox-LDL-induced ECs dysfunction (70). In addition, EVs released from MSCs transfer miRNA-let7 into macrophages, where they promote M2 macrophage polarization, inhibit M1 macrophage polarization and alleviates atherosclerotic plaque in ApoE-/- mice models through the miR-let7/HMGA2/NF-κB signaling pathway (71). Moreover, MSCs-derived EVs suppress macrophage infiltration in the atherogenesis plaque through the miR-let7/IGF2BP1/PTEN pathway (71). Furthermore, EVs containing miRNA-301 transferred from BMSCs can protect against atherosclerosis through inhibiting myocardial autophagy (68). BMSCs-derived EVs carrying miR-223 to suppress the expression of nod-like receptor protein 3 (NLRP3) and inhibit macrophages pyroptosis, thereby inhibiting the rupture of atherosclerotic plaques (67).