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Endothelial- and Immune Cell-Derived Extracellular Vesicles in the Regulation of Cardiovascular Health and Disease

Summary Intercellular signaling by extracellular vesicles (EVs) is a route of cell-cell crosstalk that allows cells to deliver biological messages to specific recipient cells. EVs convey these messages through their distinct cargoes consisting of cytokines, proteins, nucleic acids, and lipids, which they transport from the donor cell to the recipient cell. In cardiovascular disease (CVD), endothelial- and immune cell-derived EVs are emerging as key players in different stages of disease development. EVs can contribute to atherosclerosis development and progression by promoting endothelial dysfunction, intravascular calcification, unstable plaque progression, and thrombus formation after rupture. In contrast, an increasing body of evidence highlights the beneficial effects of certain EVs on vascular function and endothelial regeneration. However, the effects of EVs in CVD are extremely complex and depend on the cellular origin, the functional state of the releasing cells, the biological content, and the diverse recipient cells. This paper summarizes recent progress in our understanding of EV signaling in cardiovascular health and disease and its emerging potential as a therapeutic agent.

C ardiovascular disease (CVD) still represents the leading cause of mortality worldwide.
The underlying disease, atherosclerosis, is initiated and propagated by continuous damage of the vascular endothelium, leading to endothelial activation and apoptosis, the development of endothelial dysfunction, and subsequent atherosclerotic lesion formation (1). Endothelial cell (EC) injury is a key element in the complex pathophysiology of atherogenesis and triggers the release of EC-derived extracellular vesicles (EVs) such as exosomes and microvesicles (MVs) (2). Accordingly, patients with vascular diseases associated with systemic endothelial damage, such as atherosclerosis, show significantly increased levels of circulating EVs (3,4).
However, EVs are not simply inactive debris that reflect cellular activation or injury. EVs can transfer proteins, cytokines, mRNA, or noncoding RNA such as microRNA (miRNA) or long noncoding RNA to target cells and influence their function and phenotype (5,6). Accordingly, the role of EVs has changed from being only a marker of vascular integrity toward being relevant effectors in intercellular vascular signaling (7,8). In CVD, EVs have been shown to contribute to disease development and progression by promoting initial lesion formation, intravascular calcifications, plaque progression, and thrombus formation after rupture. In contrast, numerous studies have demonstrated that certain subtypes of EVs can mediate vascular protection and endothelial regeneration (9). In line with these findings, EVs released by progenitor or mesenchymal stem cells have been shown to improve cardiac function after myocardial infarction in experimental studies, highlighting the therapeutic potential of EVs in cardiovascular pathologies (10). This review summarizes current knowledge of EVs as regulators of cardiovascular health and disease and potential opportunities for therapeutic use.

BIOGENESIS OF EXTRACELLULAR VESICLES AND THEIR INTERACTIONS WITH TARGET CELLS
EVs are membrane vesicles secreted from cells that contain intracellular contents (11). Cells can release a broad range of vesicles with diverse features. This review focuses on 2 major types: MVs and exosomes.
MVs are large (>150 nm) vesicles that are released by budding from the plasma membrane, whereas exosomes are smaller (30 to 100 nm) and originate from the endosome (12). However, there is no strict cutoff value that distinguishes MVs from exosomes by vesicle size, which can differ in diverse studies (12).
Exosomes represent a homogeneous population of vesicles that are formed by inward budding of the multivesicular body (MVB) membrane. Exosome biogenesis is mediated mainly by the endosomal sorting complex required for transport (ESCRT) protein (13) or lipid ceramide and neutral sphingomyelinase, the enzyme that converts sphingomyelin to ceramide (14). Cargo sorting into exosomes involves ESCRT and associated proteins such as tumor susceptibility gene 101 protein (TSG101) and ALG-2interacting protein X (ALIX) and small GTPases such as Rab7a and Rab27b (15)(16)(17). Exosomes are liberated into the extracellular space following fusion of MVBs with the cell membrane, regulated by Rab27A, Rab11, and Rab31 (18,19). MVs represent a relatively heterogeneous population of vesicles formed by outward budding of the cell membrane. This process is regulated by membrane lipid microdomains and regulatory proteins such as ADP-ribosylation factor 6 (ARF6) (20). EVs can be regarded as intercellular messengers for various biological processes. Several routes of interaction between EVs and recipient cells have been described. First, EVs can directly activate target cell surface receptors by bioactive ligands and proteins (21)(22)(23). Second, EVs are able to transfer their biological content by membrane fusion with the recipient cell. The fusion process is regulated by the lipid composition of EV membrane, and several reports indicate that the presence of phosphatidylserine contributes to membrane fusion (24). Third, incorporation of EVs into target cells is mediated by endocytosis, pinocytosis, or phagocytosis (25). Through these interaction routes, EVs transfer their biological contents containing nucleic acids such as mRNA (26), noncoding RNAs (miRNAs [27], long noncoding RNAs [6]), proteins (28), cytokines (29), or bioactive lipids (30) (Figure 1).

EFFECTORS OF CARDIOVASCULAR DISEASES
In cardiovascular biology, EVs have various physiological functions, including activation of platelets and ECs, as well as regulation of inflammation and coagulation (31)(32)(33)(34). Therefore, EVs are emerging as key players in different stages of CVD development (31,32,35). The effects of EVs in CVD are extremely complex and depend on the cellular origin, the functional state of the releasing cells, the intravesicular content, and the recipient cells (36,37    Exosome formation starts with endocytosis, a process in which the cell membrane is pinched inward and captures bioactive molecules, resulting in the formation of the endosome. These molecules are sorted into smaller vesicles that bud from the perimeter membrane into the endosome lumen, forming vesicles; this leads to the multivesicular appearance of late endosomes and so they are also known as MVBs. From MSBs, exosome formation occurs by an ESCRT-and ceramide-dependent pathway. Cargo sorting into exosomes involves ESCRT and TSG101, ALIX, and Rab7a, and Rab27b. Exosomes are released into the extracellular space following the fusion of MVBs with the cell membrane, which is regulated by Rab27A, Rab11, and Rab31. Microvesicles are formed by the outward budding of the cell membrane, a process that is regulated by ARF6. Several routes of interaction between EVs and recipient cells have been described. First, EVs can directly activate target cell surface receptors. Second, EVs are able to transfer their biological content by membrane fusion with the recipient cell. Third, incorporation of EVs into target cells is mediated by endocytosis, pinocytosis, or phagocytosis. Using these interaction routes, EVs transfer their biological content containing nucleic acids such as mRNA, noncoding RNAs (microRNAs, long noncoding RNAs), proteins, cytokines, or bioactive lipids. mRNA ¼ messenger RNA; MVB ¼ multivesicular bodies. Jansen et al. represents an important translational approach to exploring the relationship between EVs and certain diseases. However, comorbidities as well as patients' age and sex should be taken into consideration as possible confounders. Therefore, inclusion and exclusion criteria should be thoughtfully determined, and control groups must be carefully selected.    In summary, EVs derived from endothelium, platelets, or endothelium-regenerating cells play a fundamental role by facilitating regenerative processes after vascular or myocardial injury (10,110,111). an early stage of inflammation could contribute to its resolution (112). This effect seems to be mediated by annexin I expression on the surface of these EVs (113).
EVs are also taken up by monocytes and B cells through diverse mechanisms and affect target cells toward an anti-inflammatory phenotype (114).

Mesenchymal stem cells contribute to inflammatory
Continued on the next page Jansen et al.     (Figure 4).      Endothelial and Immune Cell EVs in CV Health and Disease