**2. Biological content of extracellular vesicles**

An increasing body of evidence proves that EVs are not only involved in the waste disposal system, but, more importantly, they function as membrane-bound carriers for intercellular communication [5, 6]. This type of intercellular communication was proven to modulate cellular functions both in homeostatic and pathological conditions [6, 7]. High concentrations of EVs were detected in culture supernatants and biological fluids [8, 9]. For example, EVs have been isolated from CSF and proved to contain overrepresentation of brain-specific proteins derived from cerebral white mater and choroid plexus [10]. Several studies demonstrated that the level and composition of circulating EVs are altered in disease states, neurodegenerative diseases included [10–12]. Endothelial cells (ECs) and platelets have been most studied as sources for EVs [13, 14], but circulating cells as monocytes or lymphocytes may also be a source for EVs.

Protein composition of the EV-enclosing membrane, mainly different types of integrins, cell adhesion molecules, and tetraspanins, guides the interaction with the recipient cells, the targeting, or recruitment once EVs are released into the extracellular environment [15]. Specific molecules allow EVs either to interact with surface receptors on recipient cells to activate signaling cascades, or to promote their docking and uptake. One potential mechanism is direct membrane fusion with direct transfer of the cargo molecules into the recipient cytosol [16]. Endocytosis, including clathrin-dependent endocytosis, lipid raft-dependent pathways, phagocytosis, and even micropinocytosis, was more frequently considered [15].

A recent quantitative proteomic analysis allowing comparison of different EV subpopulations [17] proved that several classic exosome markers such as flotillin-1, heat-shock 70-kDa proteins, actin, and MHC I and II are present in all EV fractions obtained by successive centrifugation. Moreover, classic exosomal tetraspanins CD9, CD63, and CD81 were unreached in the exosomal fraction but also detected in different amounts in larger EVs. The study suggests a further classification of EV pelleting at high speed into four subcategories: (a) EVs enriched in all tetraspanins and endosome markers (bona fide exosomes); (b) EVs devoid of CD63 and CD81 but enriched in CD9 (associated with plasma membrane and an early endocytic signature); (c) EVs devoid of CD63/CD9/CD81; and (d) EVs enriched in extracellular matrix (ECM) or serum-derived factors. They also propose five categories of proteins with different relative distributions in different EV populations that relate them to their intracellular source [17]. Thus, exosomes contain ECM proteins, receptors, heparin-binding, phospholipid-binding, integrins, immune response,

**139**

*Part Two: Extracellular Vesicles as a Risk Factor in Neurodegenerative Diseases*

depleted in exosomes but unchanged or enriched in ectosomes [9].

and cell adhesion molecules, while ectosomes are enriched in endoplasmic reticu-

The amounts of different lipid classes in EVs have been determined in several studies [23, 34], and the enrichment of EV membranes for cholesterol, sphingomyelin, glycosphingolipids, and phosphatidylserine compared with their cellular sources was proved. However, differences in lipid composition were reported between vesicle type and cellular source. Generally, exosomes seem to be enriched in glycolipids, phosphatidylserine, and free fatty acids, while ceramides and sphingomyelins were consistently enriched in ectosomes. Still, phosphatidylcholines were

EVs contain not only proteins and lipids, but several classes of RNAs. Most of the recent *in vitro* studies have proved that EVs contain functional RNA molecules that reflect the cellular status and are involved in intercellular crosstalk [18, 19]. Different species of RNA have been reported to be enclosed in EVs derived from various sources—mRNA, rRNA, and tRNA fragments and especially microRNA (miRs) [5]. Several mechanisms for RNA selection, loading into EVs, and their uptake by various target cells have been proposed [5, 20]. Packing into EVs protects the molecules from RNase degradation once released into the extracellular environment. Thus, RNA molecules can be transferred to distant recipient cells, their protein production can be modulated [8, 21], or they may be used as predictive biomarkers for the occurrence of cardiovascular events as demonstrated by the study of EVs containing miR-199a and miR-126 in patients with stable coronary artery disease [22]. In atherosclerotic disease, miR-containing circulating EVs and apoptotic bodies, along with other bioactive molecules, are released by proinflammatory stimulated monocytes and T cells; ECs and activated platelets initiate hyperplasia of vascular smooth muscle cells (VSMCs) which leads to phenotype switching from

contractile to synthetic and activates their proliferation and migration [23].

such as cytokines, chemokines, growth factors, enzymes or transcription factors, functional organelles, and other bioactive molecules such as lipid mediators, derived from arachidonic acid [5]. Also, some EVs seem to retain the capacity to synthesize eicosanoids using their phospholipid content both by enzymatic and

Besides membrane proteins and RNA cargo, EVs may contain cytosolic proteins,

All neural cells from rodent [25, 26] and human [25, 27], even immortalized human brain microvascular ECs [25, 28], release EVs which contain mRNA and miRs for epigenetic reprogramming of neural cells or post-transcriptional control of specific genes [25]. In vitro studies of brain angiogenesis revealed that EVs deliver proangiogenic protein, mRNAs, and miRs from cultured glioblastoma cells into cerebral ECs [25, 29], especially increased VEGFR-B from immortalized mouse cerebral ECs stimulated with LPS and cytokines into targeted cerebral vascular pericytes [25, 30]. In vitro and in vivo studies showed that neuronal exosomes containing miR-132 could mediate neuronal regulation of brain vascular integrity. Thus, in zebrafish larvae and cultured rodent brain cells, it has been shown that neurons transfer miR-132, a highly conserved and neuron-enriched miR, via secreting exosomes to ECs to maintain brain vascular integrity. Following translocation to ECs through exosome internalization, miR-132 regulates the expression of vascular ECs cadherin (VE-cadherin), an important adherens junction protein, by directly targeting eukaryotic elongation factor 2 kinase [31]. In addition, two proteins found in peripherally circulating plasma EVs, cystatin C and CD14, have been linked to the development of brain atrophy and to cerebral white matter lesions, a small vessel disease within the brain [32]. Because exosomes contain transferrin and insulin receptor [25, 28], which mediate macromolecular passing through the blood-brain

*DOI: http://dx.doi.org/10.5772/intechopen.86604*

nonenzymatic processes [24].

lum proteasome and mitochondrial proteins [9].

#### *Part Two: Extracellular Vesicles as a Risk Factor in Neurodegenerative Diseases DOI: http://dx.doi.org/10.5772/intechopen.86604*

and cell adhesion molecules, while ectosomes are enriched in endoplasmic reticulum proteasome and mitochondrial proteins [9].

The amounts of different lipid classes in EVs have been determined in several studies [23, 34], and the enrichment of EV membranes for cholesterol, sphingomyelin, glycosphingolipids, and phosphatidylserine compared with their cellular sources was proved. However, differences in lipid composition were reported between vesicle type and cellular source. Generally, exosomes seem to be enriched in glycolipids, phosphatidylserine, and free fatty acids, while ceramides and sphingomyelins were consistently enriched in ectosomes. Still, phosphatidylcholines were depleted in exosomes but unchanged or enriched in ectosomes [9].

EVs contain not only proteins and lipids, but several classes of RNAs. Most of the recent *in vitro* studies have proved that EVs contain functional RNA molecules that reflect the cellular status and are involved in intercellular crosstalk [18, 19]. Different species of RNA have been reported to be enclosed in EVs derived from various sources—mRNA, rRNA, and tRNA fragments and especially microRNA (miRs) [5]. Several mechanisms for RNA selection, loading into EVs, and their uptake by various target cells have been proposed [5, 20]. Packing into EVs protects the molecules from RNase degradation once released into the extracellular environment. Thus, RNA molecules can be transferred to distant recipient cells, their protein production can be modulated [8, 21], or they may be used as predictive biomarkers for the occurrence of cardiovascular events as demonstrated by the study of EVs containing miR-199a and miR-126 in patients with stable coronary artery disease [22]. In atherosclerotic disease, miR-containing circulating EVs and apoptotic bodies, along with other bioactive molecules, are released by proinflammatory stimulated monocytes and T cells; ECs and activated platelets initiate hyperplasia of vascular smooth muscle cells (VSMCs) which leads to phenotype switching from contractile to synthetic and activates their proliferation and migration [23].

Besides membrane proteins and RNA cargo, EVs may contain cytosolic proteins, such as cytokines, chemokines, growth factors, enzymes or transcription factors, functional organelles, and other bioactive molecules such as lipid mediators, derived from arachidonic acid [5]. Also, some EVs seem to retain the capacity to synthesize eicosanoids using their phospholipid content both by enzymatic and nonenzymatic processes [24].

All neural cells from rodent [25, 26] and human [25, 27], even immortalized human brain microvascular ECs [25, 28], release EVs which contain mRNA and miRs for epigenetic reprogramming of neural cells or post-transcriptional control of specific genes [25]. In vitro studies of brain angiogenesis revealed that EVs deliver proangiogenic protein, mRNAs, and miRs from cultured glioblastoma cells into cerebral ECs [25, 29], especially increased VEGFR-B from immortalized mouse cerebral ECs stimulated with LPS and cytokines into targeted cerebral vascular pericytes [25, 30]. In vitro and in vivo studies showed that neuronal exosomes containing miR-132 could mediate neuronal regulation of brain vascular integrity. Thus, in zebrafish larvae and cultured rodent brain cells, it has been shown that neurons transfer miR-132, a highly conserved and neuron-enriched miR, via secreting exosomes to ECs to maintain brain vascular integrity. Following translocation to ECs through exosome internalization, miR-132 regulates the expression of vascular ECs cadherin (VE-cadherin), an important adherens junction protein, by directly targeting eukaryotic elongation factor 2 kinase [31]. In addition, two proteins found in peripherally circulating plasma EVs, cystatin C and CD14, have been linked to the development of brain atrophy and to cerebral white matter lesions, a small vessel disease within the brain [32]. Because exosomes contain transferrin and insulin receptor [25, 28], which mediate macromolecular passing through the blood-brain

*Extracellular Vesicles and Their Importance in Human Health*

**2. Biological content of extracellular vesicles**

lymphocytes may also be a source for EVs.

The classification of EVs is based on their size and mechanism of biogenesis and includes: exosomes, less than 100 nm small vesicles released from multivesicular bodies after endocytosed materials have been sorted in the endolysosomal compartment [1, 2]; ectosomes, up to 500 nm larger vesicles budding from the plasma membrane [2, 3]; and multivesicular cargos, consisting of numerous vesicles, about 150 nm, enclosed in a plasma membrane-derived shield [4]. Although many medical fields experienced real progress with newly discovered diagnostic tools or treatments for several diseases, smaller steps are taken in the field of cerebrovascular and neurodegenerative diseases. Age-related changes, cardiac diseases, and atherosclerosis are known to contribute to the pathogenic mechanism of cerebrovascular and neurodegenerative diseases affecting the

An increasing body of evidence proves that EVs are not only involved in the waste disposal system, but, more importantly, they function as membrane-bound carriers for intercellular communication [5, 6]. This type of intercellular communication was proven to modulate cellular functions both in homeostatic and pathological conditions [6, 7]. High concentrations of EVs were detected in culture supernatants and biological fluids [8, 9]. For example, EVs have been isolated from CSF and proved to contain overrepresentation of brain-specific proteins derived from cerebral white mater and choroid plexus [10]. Several studies demonstrated that the level and composition of circulating EVs are altered in disease states, neurodegenerative diseases included [10–12]. Endothelial cells (ECs) and platelets have been most studied as sources for EVs [13, 14], but circulating cells as monocytes or

Protein composition of the EV-enclosing membrane, mainly different types of integrins, cell adhesion molecules, and tetraspanins, guides the interaction with the recipient cells, the targeting, or recruitment once EVs are released into the extracellular environment [15]. Specific molecules allow EVs either to interact with surface receptors on recipient cells to activate signaling cascades, or to promote their docking and uptake. One potential mechanism is direct membrane fusion with direct transfer of the cargo molecules into the recipient cytosol [16]. Endocytosis, including clathrin-dependent endocytosis, lipid raft-dependent pathways, phagocytosis, and even micropinocytosis, was more frequently

A recent quantitative proteomic analysis allowing comparison of different EV subpopulations [17] proved that several classic exosome markers such as flotillin-1, heat-shock 70-kDa proteins, actin, and MHC I and II are present in all EV fractions obtained by successive centrifugation. Moreover, classic exosomal tetraspanins CD9, CD63, and CD81 were unreached in the exosomal fraction but also detected in different amounts in larger EVs. The study suggests a further classification of EV pelleting at high speed into four subcategories: (a) EVs enriched in all tetraspanins and endosome markers (bona fide exosomes); (b) EVs devoid of CD63 and CD81 but enriched in CD9 (associated with plasma membrane and an early endocytic signature); (c) EVs devoid of CD63/CD9/CD81; and (d) EVs enriched in extracellular matrix (ECM) or serum-derived factors. They also propose five categories of proteins with different relative distributions in different EV populations that relate them to their intracellular source [17]. Thus, exosomes contain ECM proteins, receptors, heparin-binding, phospholipid-binding, integrins, immune response,

**138**

considered [15].

elderly.

**Figure 1.**

*Transmission electron microscopy of the isolated extracellular vesicles: (A) negative staining and (B) cryo-electron microscopy.*

barrier (BBB), peripherally infused modified exosomes containing specific RNA were used to knockdown a specific gene in mouse brain [32–34]. Considering the extraordinary intricate cytoarchitecture of the brain, the presence of EVs in the adult brain is hard to be documented. Fetal brain and neurospheres contain cells which seem to release vesicles into the extracellular space (**Figure 1**). EVs are easier to be seen near ependymal cells floating in the ventricles from where they can be isolated [11].
