Extracellular vesicles (EVs) are a cluster of nano-sized vesicles of different sizes, cargo, and surface markers that are secreted into the extracellular environment through a variety of mechanisms. They carry various components of the cytoplasm and cell membrane that are selectively loaded into these vesicles. They are secreted by all forms of living cells and play essential roles in different physiological functions and pathological processes. They also have been utilized as Diagnostic markers and therapeutic tools in several conditions.

Three types of EVs are biologically distinguishable from one another via the distinct processes through which they are released by the cell. However, the experimental classification of these vesicles is less clear as there is no consensus on what criteria to use for their differentiation. The first type is the microvesicles (MVs), or the ectosomes. These vesicles arise by direct budding through the cell membrane to the outside of the cell.[1] The second type is the exosomes, which are first formed by budding into the endosomes to create what is called the multivesicular bodies (MVBs). These MVBs either fuse with lysosomes, resulting in content digestion, or fuse with the cell membrane, which results in the content released as exosomes.[2] The third type arises from the cells during the process of apoptosis, hence its name apoptotic bodies (APBs). APBs emerge either by separation of the membrane blebs or from apoptopodia that arise during the process of apoptosis. There are two types of apoptopodia: non-beaded and beaded. The distinction in the course and the roles of these different routes is still under investigation.[3]

Source

EVs are secreted by all ranges of organisms, from prokaryotes to unicellular eukaryotes - like protozoa, yeasts, and fungi - to mammals. They can be isolated from all bodily fluids, including blood, plasma, serum, saliva, urine, sweat, cerebrospinal fluid, breast milk, and semen. In humans, these EVs have been reported to be secreted by nearly all types of cells in our physique, including but not limited to immune cells, endothelial cells, red blood cells, erythrocytes, liver cells, and epithelial cells.

Characterization

After their isolation, it is necessary to confirm that the required population of EVs has been obtained. Scientists utilize several techniques to do this confirmation. First is the electron microscope. The regular scanning electron microscope shows EVs as irregular shaped membrane-enclosed vesicles. Alternatively, cryo-electron microscopy can be utilized. Despite being time-consuming and expensive, it gives more accurate information about the shape and size of the isolated vesicles. The second method of characterization is nano-track analysis, which provides a general idea about the concentration and size distribution of the isolated vesicles. The third method is a western blot, which is used to recognize protein markers associated with the membrane of the EVs. Despite some distinction of specific proteins on the surfaces of different EVs, there is still no consensus on which proteins can be used reliably as markers for the different types.[4] Recently, flow cytometry procedures have been developed to characterize different types of EVs based on their surface markers.[5]

The vesicles are surrounded by phospholipid bilayer membrane similar to the one that surrounds the cells.[6] However, lipidemic and proteomic studies showed that the membrane of the vesicles has its natural lipid and protein profile. One of the unique features of this profile, for example, is the high level of the lysoglycerophospholipid, which is absent in the other cellular membranes.[7] They also reach in cholesterol, ceramides, and sphingomyelin. Regarding the protein content of the vesicle membrane, it shows heterogeneity not only between EVs isolated from different cells but even between EVs isolated from the same type of cells under various conditions. However, some proteins are common in different EVs like tetraspanins and integrins families, which are used as markers for characterization of the isolated EVs.[8]

Size

Although the different types of vesicles have distinct size ranges, there is still overlap, making classification based solely on size not possible.[9] Exosomes range in size from 50 to 150 nm, while microvesicles range from 100 to 1000 nm.[10] The range of the APBs is much broader, from 50 to 5000 nm in diameter.[11]

Cargo

EVs may contain any content found in their origin cell’s cytoplasm; this includes proteins, mRNA, miRNA, long noncoding RNAs (lncRNA), circular RNAs (circRNA), metabolites, and lipids.[12][13]. Some research showed an association between DNA and EVs.[14][15] Furthermore, some APBs contain intact cell organelles like mitochondria, endoplasmic reticulum, and ribosomes.[11]

The EVs exert their functions through different mechanisms. They can transfer their cargo or membrane constituents from one cell to another, thus transferring functions between cells. In doing so, they perform several roles based on the type of donor and recipient cells.[16] Also, they carry molecules on their surface, which act as ligands that would stimulate surface receptors in other cells activating intracellular signaling.[17] Alternatively, these surface molecules can directly perform functions in the extracellular milieu. For example, exosomes may carry on their surface matrix remodeling enzymes such as matrix metalloproteinases, heparanases, and hyaluronidases, which play roles in several physiological and pathological conditions.[18] 

Another mechanism of EVs function is what we can call "the decoy" function in which surface proteins on the EVs membrane capture external molecules or pathogens to neutralize their effects. Whatever is the mechanism of action, the final role of EVs, either physiological or pathological, depends on their cell of origin and the conditions through which they have been stimulated. These factors determine the final cargo of the EVs and the lipid and protein content of their membranes, which, by turn, decides their ultimate destination and actions.

EVs play essential roles in several physiological processes, like embryonic growth, pregnancy, blood hemostasis, and body metabolism. With regards to coagulation functions, EVs were first identified as "platelet dust," which were described as factors in the plasma that enhance the coagulation process. These platelet dust were recognized later as the "platelet-derived EVs."  Molecules exposed on the surface of these EVs, such as the tissue factor or phosphatidylserine, work as activators of the coagulation cascade. These types of EVs are available in the blood during injury or endothelial damage, but they are absent in healthy circulating blood. On the other hand, normal circulating EVs carry plasminogen activators, so they induce fibrinolytic activity, preventing thrombus formation. Additionally, exosomes released from platelets under normal conditions inhibit platelet aggregation.[19]

EVs also play essential metabolic functions; this occurs by transferring enzymes and metabolites between cells or by performing extracellular metabolic activities. This reason is why some scientists call them "metabolic nanomachines."[20]  Studies showed that EVs secreted by the liver cells contain hundreds of enzymes that belong to the different metabolic pathways. When these EVs were incubated with rat serum, they changed the metabolic profile of the serum, indicating a functional role of these EVs. However, the specific functional roles of these enzymes outside the cells are yet to be identified.[21]

The role of EVs in the Host-pathogen Relationship

EVs plays several roles in the war between the human body and its invaders. They are utilized by both sides, endeavoring to win the war. This process applies to all types of pathogens, including viruses, bacteria, fungi, and parasites. The pathogens, for instance, could use their EVs to evade the immune system through several mechanisms like complement inhibition and antibody degradation.  This process is in addition to the direct utilization of the EVs in their pathogenic actions like delivery of virulence factors, disruption of natural barriers, cytotoxicity, and modulation of host cells.[22][23] 

Proteomic studies on bacterial EVs suggested that the protein cargo of these EVs have been loaded selectively from the cytoplasm, and not randomly, to perform these functions.[24] Also, bacterial EVs mediates the non-genetic acquisition of antibacterial resistance. One example is the ability of EVs to transfer b-lactamase protein to bacteria that are not genetically expressing it.[25]

On the other hand, EVs play essential roles in the recognition and destruction of the microbes by the immune system. Different components of the innate and the adaptive immune systems communicate EVs among each other to orchestrate their actions aiming at combating the invaders. This process involves EVs from several sources, including both the pathogen and the host immune and non-immune cells. Besides, the immune system utilizes these EVs as direct tools in their war against these attackers. For example, decoy exosomes carry molecules that neutralize bacterial toxins like diphtheria toxins and toxins released by Staphylococcus aureus.[26]

Another example is that MVs released from neutrophils under the effect of specific bacterial stimulation play direct antibacterial actions against the stimulating bacteria.[27][28] Likewise, mesenchymal stem cells (MSCs) and epithelial cells derived exosomes exert a direct antiviral activity through different mechanisms, including neutralization of the virus through the specific virus receptor loaded on their surface. Recent research illustrated such an effect against HIV by EVs derived from human placental trophoblast or CD4+ T-lymphocytes.[29][30]

EVs isolated from different body fluids carry immune response-related and immune-modulatory molecules. These molecules include proteins, lipids, and nucleic acids. EVs released by different types of immune cells vary in their content and surface markers and perform different roles in the immune system.[31] A prominent example is that EVs from microbes, host dendritic cells, or B lymphocytes have been acknowledged as "miniature antigen-presenting cells surrogates" as they transfer antigens directly to the T-lymphocytes leading to their activation.[32]