**5. Microvesicles and endothelium**

The endothelial MVs (EMVs) are extracellular vesicles produced by endothelial cells whose essential role is to act as a signaling system between the elements involved in the function and homeostasis of the vessel [71].

In general, the extracellular vesicles can be found in many body fluids, including plasma and urine. They have a variable size, between 0.05 and 5 μm [71], and are involved in physiological and pathophysiological processes, participating as mediators in intercellular communication. They can act directly on the target cells by binding to ligands, cell surface receptors and/or membrane-associated enzymes, delivering or releasing their contents directly into the cytoplasm. Extracellular vesicles are elevated in patients with neurodegenerative, metabolic, pulmonary, autoimmune and vascular diseases, chronic inflammation and cancer [72]. The use of extracellular vesicles as markers for the prediction, diagnosis and prognosis of the disease is increasingly interesting, as well as their potential as new therapeutic targets [73]. There are several types of extracellular vesicles: exosomes, the MVs or microparticles and the apoptotic bodies, which are produced by different mechanisms [65]. The MVs are a heterogeneous population of up 2 μm diameter, which are formed from the cell membrane in a regulated active process, dependent on enzyme activity and calcium.

Recently, it has been demonstrated that MVs may play an essential role in cellular senescence processes [74] since they have been proposed as elements of an endothelial response that can participate in the damaging and repair processes of the endothelium [10–12]. MVs generated from different cell types can induce endothelial dysfunction because they are responsible for increasing oxidative stress, reducing the bioavailability of NO and producing cardiovascular inflammation. The knowledge about their formation and release represent an attractive therapeutic goal to limit MVs levels, but the mechanisms underlying the release are not fully elucidated. On the other hand, a direct or indirect inhibition of the effect of MVs is a more effective proposal [75]. The effect of certain drugs that are used to decrease cardiovascular risk have been shown to affect the MVs plasma levels, suggesting that the beneficial effects of these drugs could, at least in part, be mediated through a reduction of the concentration of MVs [76]. Moreover, different authors have highlighted the importance of diet on MVs release, being perhaps one of the mechanisms involved in the role of diet in the development of CVD [14, 77]. The process of identification and separation of extracellular vesicles is complicated due to their extensive variability. In fact, currently, the absolute separation of exosomes, apoptotic bodies and MVs is not possible because their size ranges may overlap. The most common method for the separation and isolation of extracellular vesicles is the serial centrifugation. In the majority of the studies, a first centrifugation is performed at 200–1500 × g to remove cells and cell debris. Extracellular vesicles more than 100 nm are pelleted at 10,000–20,000 × g and small vesicles of 100 nm at 100,000–200,000 × g [78]. Following these protocols, we can obtain EMVs from supernatants of mature endothelial cells cultures, cellular debris and exosomesfree. The EMVs might also be obtained from plasma by similar processes, but would be found mixed with other MVs derived from other circulating cells.

In addition to the activity of the immune cells in endothelial damage, some other factors could be involved, as some specific molecules are known to be increased in the peripheral blood of CKD. In different models, it has been shown that endothelial cells activated pathologically with uremic serum or uremic toxins enter into a premature senescent state. Also, they reduce their proliferative capability and show shortened telomeres, augmenting the expression of β-gal [69]. Another possible factor in the development of the CKD-associated CVD is the incorrect repair of the damaged endothelium by EPCs. This failure occurs mainly due to two factors: a decreased number of EPCs or their imbalanced function. In our studies, we demonstrated that in CKD patients there is a decrease in the number of EPCs and that this number is considerably lower in severe patients with, for example, vascular calcifications [10, 70]. Also, it has been demonstrated that EPCs lose their angiogenic capability, generally needed in the process of regeneration of harmed vascular structures (vasculogenesis). In this regard, the association between some diseases such as CKD-associated CVD and both number and function of EPCs, accelerate the

processes of EPCs senescence and therefore damage in endothelial cells harboring.

The endothelial MVs (EMVs) are extracellular vesicles produced by endothelial cells whose essential role is to act as a signaling system between the elements involved in the function and

In general, the extracellular vesicles can be found in many body fluids, including plasma and urine. They have a variable size, between 0.05 and 5 μm [71], and are involved in physiological and pathophysiological processes, participating as mediators in intercellular communication. They can act directly on the target cells by binding to ligands, cell surface receptors and/or membrane-associated enzymes, delivering or releasing their contents directly into the cytoplasm. Extracellular vesicles are elevated in patients with neurodegenerative, metabolic, pulmonary, autoimmune and vascular diseases, chronic inflammation and cancer [72]. The use of extracellular vesicles as markers for the prediction, diagnosis and prognosis of the disease is increasingly interesting, as well as their potential as new therapeutic targets [73]. There are several types of extracellular vesicles: exosomes, the MVs or microparticles and the apoptotic bodies, which are produced by different mechanisms [65]. The MVs are a heterogeneous population of up 2 μm diameter, which are formed from the cell membrane in a regulated active process, dependent on

Recently, it has been demonstrated that MVs may play an essential role in cellular senescence processes [74] since they have been proposed as elements of an endothelial response that can participate in the damaging and repair processes of the endothelium [10–12]. MVs generated from different cell types can induce endothelial dysfunction because they are responsible for increasing oxidative stress, reducing the bioavailability of NO and producing cardiovascular inflammation. The knowledge about their formation and release represent an attractive therapeutic goal to limit MVs levels, but the mechanisms underlying the release are not fully elucidated. On the other hand, a direct or indirect inhibition of the effect of MVs is a more effective proposal [75]. The effect of certain drugs that are used to decrease cardiovascular risk have been shown to affect the MVs plasma levels, suggesting that the beneficial effects of these

**5. Microvesicles and endothelium**

56 Endothelial Dysfunction - Old Concepts and New Challenges

homeostasis of the vessel [71].

enzyme activity and calcium.

The most common methods to study single MVs are flow cytometry (FC), tunable resistive pulse sensing (TRPS), dynamic light scattering (DLS) and nanoparticle tracking analysis (NTA) [79]. To date, FC is the method most used to establish the cellular origin and the phenotype of the MVs and is based on the detection of light scatter and fluorescence intensity of the labeled MVs [80–82]. To characterize their cellular origin, different antigens expressed on the membrane of the MVs are identified. For this purpose, monoclonal antibodies (mAb) labeled with different fluorochromes that define the phenotype are used. To identify EMVs, specific fluorescent antibodies against endothelial cell can be used to characterize the phenotype. Some markers used to describe EMVs are CD144, CD105 and CD146. Moreover, the phospholipids are a class of lipids that are a major component of all biological membranes and in MVs are externalized. For this reason, these phospholipids present in the MVs membrane have also been used for EMVs detection and characterization [83]. The combination of several mAb simultaneously can facilitate the identification of the origin and the state of activation or apoptosis of the cell from which the MVs originate [84]. The EMVs determination protocol includes some preliminary steps designed to identify sizes, with beads that allow adjustments to the equipment, before the introduction of the samples. However, this method has limitations in identifying the smallest MVs that are below the detection limit of conventional FC equipment (diameter size lower 300 nm) [79]. Recent studies have shown that FC equipment with high sensitivity can amplify the forward scatter parameter capacity, which is used to identify the size of the MVs [85]. On the other hand, it is very helpful to provide information regarding functional activity of the extracellular vesicles [86–89].

In this regard, novel instruments including NTA or DLS have shown their advantages in the analysis of extracellular vesicles. NTA measures the distribution of the absolute size of the vesicles that range from 50 nm to 1 μm [90]. The vesicles in suspension are illuminated by a laser that produces light scattering or fluorescence. A microscope determines the position of individual vesicles, which are continuously moving due to Brownian motion [91]. When a fluorescent marker is used, NTA can also be used to determine the size of a subgroup of vesicles [92]. The principal advantage of this method is the detection of particles below 100 nm in diameter. In contrast, the limitation of this technique, the low resolution, therefore, NTA is incapable of distinguishing MVs from particles in suspension (debris) with the same size [79]. DLS, also known as photon correlation spectroscopy, measures the size distribution of vesicles between 1 nm and 6 μm. However, the absolute concentration of the vesicles cannot be determined by DLS because the average amplitude of the signal depends on the diameter, concentration and the refractive index of the vesicles [93–95].

The last method TRPS consists in the movement of the MVs through tunable nanopores which are capable of registering MVs between 80 and 1000 nm [96]. Particles passing the pore generate a change in the electric resistance, thus providing information on diameter, surface charge and concentration of single particles. The major disadvantage of TRPS is that it cannot distinguish between MVs and similarly sized particles [79]. Independently of the method used to study of the MVs, it has been recommended to confirm the presence of MVs by measuring them at least with two different techniques.

processes and/or apoptosis, the number of EMVs increases significantly. Physiological blood

authors have found that mature endothelial cells in culture, exposed to activation by cyto-

MVs concentration in blood from healthy subjects is clinically irrelevant. However, in patients with cardiovascular risk factors and after cardiovascular events, EMVs concentrations are increased significantly [10, 102]. In fact, in patients with CVD, an association between the number of circulating EMVs and the Framingham risk score has been shown [72]. In particular, high levels of EMVs in diseases associated with vascular injury seem to reflect an inflammatory and prothrombotic process. EMVs may participate in the development and amplification of CVD through both cardiac and vascular cells. On the other hand, numerous studies have emphasized the effect of cardioprotective drugs on reducing concentrations of extracellular vesicles [73] which reinforces the evidence about the possible correlation of

EMVs, and in general all extracellular vesicles, carry a specific load that is capable of delivering to other cells, even in remote locations. Extracellular vesicles share characteristics with their parental cells such as cell surface receptors, integral membrane proteins, cytosolic molecules, organelles, mRNAs, miRNAs or small amounts of DNA and proteins, including transcription factors, cytokines and growth factors [103]. Cell receptors and transmembrane proteins can help in the identification of EMVs, and also are indicative of the ability of vesicles to interact directly with receptors on the surface of target cells, resulting in an intracellular signal transmission. In addition to its effect on specific receptors, it has been shown that EMVs may be fused to the target cell and transfer its contents directly inside as a vehicle for transfer of genetic information [11, 67, 104, 105]. Extracellular vesicles are considered as the main source of miRNAs, released into the bloodstream during cell activation or apoptosis [106]. In fact, most miRNAs are associated with extracellular vesicles and only small amounts of them can be found free in plasma. It is thought that extracellular vesicles are necessary to protect circulating miRNAs from degradation by RNases, transferring safely functional miRNAs from the parental cells receptor cells. miRNAs act as regulatory molecules in endothelial cells, vascular smooth muscle cells, platelets and inflammatory cells that contribute to modulate the initiation and progression of atherosclerosis. It is known that the release of miRNAs does not occur randomly but they are produced and released by controlled mechanisms [107, 108]. It has been described that there are several miRNAs involved in the regulation of vascular function and repair. It is expected that in the future, a better understanding of these molecules provides new options both diagnostic and

The MVs from different sources such as endothelial cells, monocytes and lymphocytes can promote oxidative stress in the endothelium through processes that may involve several enzymatic systems [109]. The MVs can regulate the production of reactive oxygen species (ROS), although there are some discrepancies regarding ROS generation systems affected. These contradictory results may be due to the fact that MVs populations studied are from different sources or produced by different stimuli [105, 110]. From the biological point of

and 104

Endothelial Cell Senescence in the Pathogenesis of Endothelial Dysfunction

EMVs/mL and path-

59

EMVs/mL [99]. Several

http://dx.doi.org/10.5772/intechopen.73024

levels of EMVs present in healthy individuals are between 103

kines, released more EMVs [100, 101].

EMVs and vascular injury.

therapeutic in the vascular pathology.

ological concentrations (present in individuals with CVD) are 105

In addition, enzyme-linked immunosorbent assay (ELISA), Western blot or quantitative realtime PCR (qPCR) are useful tools for the detection of proteins or RNA in preparations of purified MVs. Electron microscopy can provide information concerning the vesicular morphology, size and the presence of markers. Moreover, proteomic analysis and profiles of RNA/microRNA (miRNA) may help to determine the composition of the MVs.

In the absence of pathology, the EMVs are involved in the maintenance of vascular homeostasis, participating in the metabolism of the vascular environment [97]. The EMVs can act on the vascular wall, at the endothelial level, and on smooth muscle cells [98], regulating both vasomotor reactivity and angiogenesis. In fact, the formation of EMVs and their elimination seems to reflect a balance between activation and cell damage, cell survival/apoptosis and angiogenesis. Endothelial responses may be immediate; releasing various factors or can be delayed, modulating the expression of genes involved in regulating the structure and function of the vascular system (**Figure 2**). In *in vitro* models, endothelial cell cultures produce EMVs in a meager percentage without additional stimulus. However, in response to activation

**Figure 2.** Mechanisms of endothelial microvesicles (MVs) action upon target cells. SMCs, smooth muscle cells; ECs, endothelial cells.

processes and/or apoptosis, the number of EMVs increases significantly. Physiological blood levels of EMVs present in healthy individuals are between 103 and 104 EMVs/mL and pathological concentrations (present in individuals with CVD) are 105 EMVs/mL [99]. Several authors have found that mature endothelial cells in culture, exposed to activation by cytokines, released more EMVs [100, 101].

The last method TRPS consists in the movement of the MVs through tunable nanopores which are capable of registering MVs between 80 and 1000 nm [96]. Particles passing the pore generate a change in the electric resistance, thus providing information on diameter, surface charge and concentration of single particles. The major disadvantage of TRPS is that it cannot distinguish between MVs and similarly sized particles [79]. Independently of the method used to study of the MVs, it has been recommended to confirm the presence of MVs by measuring

In addition, enzyme-linked immunosorbent assay (ELISA), Western blot or quantitative realtime PCR (qPCR) are useful tools for the detection of proteins or RNA in preparations of purified MVs. Electron microscopy can provide information concerning the vesicular morphology, size and the presence of markers. Moreover, proteomic analysis and profiles of

In the absence of pathology, the EMVs are involved in the maintenance of vascular homeostasis, participating in the metabolism of the vascular environment [97]. The EMVs can act on the vascular wall, at the endothelial level, and on smooth muscle cells [98], regulating both vasomotor reactivity and angiogenesis. In fact, the formation of EMVs and their elimination seems to reflect a balance between activation and cell damage, cell survival/apoptosis and angiogenesis. Endothelial responses may be immediate; releasing various factors or can be delayed, modulating the expression of genes involved in regulating the structure and function of the vascular system (**Figure 2**). In *in vitro* models, endothelial cell cultures produce EMVs in a meager percentage without additional stimulus. However, in response to activation

**Figure 2.** Mechanisms of endothelial microvesicles (MVs) action upon target cells. SMCs, smooth muscle cells; ECs,

RNA/microRNA (miRNA) may help to determine the composition of the MVs.

them at least with two different techniques.

58 Endothelial Dysfunction - Old Concepts and New Challenges

endothelial cells.

MVs concentration in blood from healthy subjects is clinically irrelevant. However, in patients with cardiovascular risk factors and after cardiovascular events, EMVs concentrations are increased significantly [10, 102]. In fact, in patients with CVD, an association between the number of circulating EMVs and the Framingham risk score has been shown [72]. In particular, high levels of EMVs in diseases associated with vascular injury seem to reflect an inflammatory and prothrombotic process. EMVs may participate in the development and amplification of CVD through both cardiac and vascular cells. On the other hand, numerous studies have emphasized the effect of cardioprotective drugs on reducing concentrations of extracellular vesicles [73] which reinforces the evidence about the possible correlation of EMVs and vascular injury.

EMVs, and in general all extracellular vesicles, carry a specific load that is capable of delivering to other cells, even in remote locations. Extracellular vesicles share characteristics with their parental cells such as cell surface receptors, integral membrane proteins, cytosolic molecules, organelles, mRNAs, miRNAs or small amounts of DNA and proteins, including transcription factors, cytokines and growth factors [103]. Cell receptors and transmembrane proteins can help in the identification of EMVs, and also are indicative of the ability of vesicles to interact directly with receptors on the surface of target cells, resulting in an intracellular signal transmission. In addition to its effect on specific receptors, it has been shown that EMVs may be fused to the target cell and transfer its contents directly inside as a vehicle for transfer of genetic information [11, 67, 104, 105]. Extracellular vesicles are considered as the main source of miRNAs, released into the bloodstream during cell activation or apoptosis [106]. In fact, most miRNAs are associated with extracellular vesicles and only small amounts of them can be found free in plasma. It is thought that extracellular vesicles are necessary to protect circulating miRNAs from degradation by RNases, transferring safely functional miRNAs from the parental cells receptor cells. miRNAs act as regulatory molecules in endothelial cells, vascular smooth muscle cells, platelets and inflammatory cells that contribute to modulate the initiation and progression of atherosclerosis. It is known that the release of miRNAs does not occur randomly but they are produced and released by controlled mechanisms [107, 108]. It has been described that there are several miRNAs involved in the regulation of vascular function and repair. It is expected that in the future, a better understanding of these molecules provides new options both diagnostic and therapeutic in the vascular pathology.

The MVs from different sources such as endothelial cells, monocytes and lymphocytes can promote oxidative stress in the endothelium through processes that may involve several enzymatic systems [109]. The MVs can regulate the production of reactive oxygen species (ROS), although there are some discrepancies regarding ROS generation systems affected. These contradictory results may be due to the fact that MVs populations studied are from different sources or produced by different stimuli [105, 110]. From the biological point of view, these differences in the production of MVs have a significant for the potential to define MVs populations with different biological activities.

contribute to the release of cytokines and the development of alterations mediated by MVs in

Endothelial Cell Senescence in the Pathogenesis of Endothelial Dysfunction

http://dx.doi.org/10.5772/intechopen.73024

61

Initially, proliferation and migration of adjacent endothelial cells have been identified as a factor of endothelial repair, and subsequent studies have shown that the maintenance of the endothelial structure is associated with EPCs and their ability to differentiate and repair damaged endothelial tissue. Due to the importance of this repair mechanism in the maintenance of vascular homeostasis, it is logical to think about the existence of close communication between damaged endothelial cells and EPCs. Previous studies performed by our group suggest that plasma EMVs, both of healthy subjects and patients with CKD; participate in the activity of the EPCs [10]. Our hypothesis is that EMVs can be an essential and necessary physiological mechanism of signaling to initiate the recruitment of EPCs from bone marrow. In *in vitro* models, we have shown that EMVs may be the key element in the regeneration and maintenance of vascular homeostasis, acting on EPCs [116]. Indeed, in response to different stimuli, the endothelial cells can induce EMV with different membrane characteristics, miRNA and other molecules in your content that reduce the ability of EPC to regenerate and participate in the signaling pathways involved in apoptosis and oxidative stress [117]. These specific

Vascular calcification is an increasingly constant process in developed countries and can contribute significantly to increased cardiovascular risk. The processes and mechanisms involved in the formation of vascular calcifications are poorly understood and are needed to develop new therapeutic strategies to prevent or avoid calcification. Patients CKD have a higher incidence of vascular calcification, and our group has shown that EMVs are increased in patients with an elevated degree of calcification [10]. In *in vitro* studies, EMVs produced in an inflammatory environment or obtained from patients with CKD promoted the calcification of smooth muscle cells, as assessed by some calcification markers (bone morphogenetic protein-2 (BMP-2) and alkaline phosphatase (ALP)) and the phenolsulfonephthalein method [100]. Other authors have also described a role of the MVs in the mineralization of vascular smooth muscle cells [118].

MVs have also been associated with endothelial senescence. As we said before, senescent cells release characteristic molecules and substances composing the SASP. However, some of those substances which are known to be part of this SASP cannot be released as soluble molecules due to their nature, as some transmembrane proteins [119]. It is known that the premature induction of cellular senescence *in vitro* increases the release of extracellular vesicles [120]. Those concepts suggest the contribution of MVs as part of the SASP, which have two important consequences: (1) SASP MVs can be the mechanism by which those insoluble proteins are released and (2) the carrier molecules can activate signaling processes in the target cells. Nevertheless, the specific mechanisms underlying MVs releasing from the senescent cells are still unresolved. It has been described that p53, a tumor-suppressor protein, remarkably upregulated in senescence, modulates the release of extracellular vesicles [121]. Also, p53 takes part in the transcription of some molecules implicated in extracellular vesicles biogenesis, partly explaining how senescence and MVs releasing activation can be related [122–124]. Moreover, the content within those MVs may be necessary in the induction of senescence in the target cells, as it has been shown that some miRNAs can regulate the p53 and pRB pathways [125–127]. Loss of pRB results in deregulated cell proliferation and apoptosis, whereas loss of p53 desensitizes cells to checkpoint signals, including apoptosis [128]. Thus, the presence of those miRNAs in MVs may be associated with

mechanisms may constitute therapeutic objectives in future studies.

the extracellular environment [115].

One of the best-provided properties of MVs is its ability to promote coagulation [98]. In fact, the MVs are elevated in hypercoagulative disorders probably as a result of their active participation [98]. It is not clear how far MVs contribute to the *in vivo* coagulation, but there are several *in vitro* studies that demonstrate their procoagulant role. This capacity has been extensively studied in platelet-derived MVs, but the fact is that the MVs have two specific and common physical characteristics that may be responsible for this procoagulant activity: firstly, the externalization of phosphatidylserine as coagulation promoter and secondly, the expression of tissue factor, which is a critical component of the early stages of coagulation [11]. Indeed, tissue factor is not expressed under physiological conditions in circulating and endothelial cells, but it is expressed in pathological conditions.

Chronic inflammation is a crucial factor in the development of atherosclerosis, and the effects of EMVs in inflammatory processes have been the subject of numerous studies since they may represent both a cause and a consequence of inflammation [12]. The MVs isolated from human atherosclerotic plaques can transfer intercellular adhesion molecule-1 (ICAM-1) to endothelial cells and could increase the ability to recruit inflammatory cells in a manner dependent of phosphatidylserine, which may increase the progression of the atherosclerotic plaque. The most conclusive evidence of a proinflammatory role for EMVs is that the administration of exogenous EMVs to rats is associated with acute lung injury, with increased levels of proinflammatory cytokines (IL-1β and TNF-α) and neutrophil infiltration on histological lesion perivascular space [111].

Different studies have described a role of MVs in the regulation of angiogenesis [112]. Plateletderived MVs were first involved in the angiogenesis process since platelets contain at least 20 factors that regulate angiogenesis. Platelet-derived MVs stimulate proliferation, survival, migration, and formation of capillary-like structures in endothelial cells *in vitro*. Furthermore, injection of platelet-derived MVs increases myocardial post-ischemic capillary density in rats [113]. Subsequent studies have shown that MVs isolated from atherosclerotic plaques are involved in the formation of new blood vessels and in the progression of the plaques to rupture. Endothelial cells in the culture containing MVs that release matrix metalloproteinases (MMP-2 and MMP-9) and promote matrix degradation and the formation of new blood vessels.

In addition to being a potent stimulus for the formation of MVs, apoptosis can also be a consequence of MVs signaling [112]. Monocyte, erythrocytes, platelets and endothelial cells-derived MVs contain caspase-3. It is thought that the content of caspases may be a mechanism directed to control the apoptosis, suggesting that MVs could release caspase-3 into the target cells, participating in the induction of apoptosis. In addition, caspase-3 is implicated in numerous cellular processes, so the release of this protein could have an even more significant impact on the target cell.

The MVs contain proteolytic enzymes, and then some of its effects could be attributed to alterations in the extracellular matrix or proteolytic cleavage of various signaling molecules. For example, the microvasculature-derived EMVs containing MMP-1, MMP-2, MMP-13 and MMP-7, which degrade fibronectin *in vitro* [114]. Moreover, MVs isolated from human atherosclerotic plaques contain an active form of ADAM17 (metallopeptidase domain 17), an enzyme with a role in the control of inflammation and tissue regeneration. This enzyme could contribute to the release of cytokines and the development of alterations mediated by MVs in the extracellular environment [115].

view, these differences in the production of MVs have a significant for the potential to define

One of the best-provided properties of MVs is its ability to promote coagulation [98]. In fact, the MVs are elevated in hypercoagulative disorders probably as a result of their active participation [98]. It is not clear how far MVs contribute to the *in vivo* coagulation, but there are several *in vitro* studies that demonstrate their procoagulant role. This capacity has been extensively studied in platelet-derived MVs, but the fact is that the MVs have two specific and common physical characteristics that may be responsible for this procoagulant activity: firstly, the externalization of phosphatidylserine as coagulation promoter and secondly, the expression of tissue factor, which is a critical component of the early stages of coagulation [11]. Indeed, tissue factor is not expressed under physiological conditions in circulating and

Chronic inflammation is a crucial factor in the development of atherosclerosis, and the effects of EMVs in inflammatory processes have been the subject of numerous studies since they may represent both a cause and a consequence of inflammation [12]. The MVs isolated from human atherosclerotic plaques can transfer intercellular adhesion molecule-1 (ICAM-1) to endothelial cells and could increase the ability to recruit inflammatory cells in a manner dependent of phosphatidylserine, which may increase the progression of the atherosclerotic plaque. The most conclusive evidence of a proinflammatory role for EMVs is that the administration of exogenous EMVs to rats is associated with acute lung injury, with increased levels of proinflammatory cytokines (IL-1β and TNF-α) and neutrophil infiltration on histological lesion perivascular space [111]. Different studies have described a role of MVs in the regulation of angiogenesis [112]. Plateletderived MVs were first involved in the angiogenesis process since platelets contain at least 20 factors that regulate angiogenesis. Platelet-derived MVs stimulate proliferation, survival, migration, and formation of capillary-like structures in endothelial cells *in vitro*. Furthermore, injection of platelet-derived MVs increases myocardial post-ischemic capillary density in rats [113]. Subsequent studies have shown that MVs isolated from atherosclerotic plaques are involved in the formation of new blood vessels and in the progression of the plaques to rupture. Endothelial cells in the culture containing MVs that release matrix metalloproteinases (MMP-2 and MMP-9) and promote matrix degradation and the formation of new blood vessels. In addition to being a potent stimulus for the formation of MVs, apoptosis can also be a consequence of MVs signaling [112]. Monocyte, erythrocytes, platelets and endothelial cells-derived MVs contain caspase-3. It is thought that the content of caspases may be a mechanism directed to control the apoptosis, suggesting that MVs could release caspase-3 into the target cells, participating in the induction of apoptosis. In addition, caspase-3 is implicated in numerous cellular processes, so the release of this protein could have an even more significant impact

The MVs contain proteolytic enzymes, and then some of its effects could be attributed to alterations in the extracellular matrix or proteolytic cleavage of various signaling molecules. For example, the microvasculature-derived EMVs containing MMP-1, MMP-2, MMP-13 and MMP-7, which degrade fibronectin *in vitro* [114]. Moreover, MVs isolated from human atherosclerotic plaques contain an active form of ADAM17 (metallopeptidase domain 17), an enzyme with a role in the control of inflammation and tissue regeneration. This enzyme could

MVs populations with different biological activities.

60 Endothelial Dysfunction - Old Concepts and New Challenges

endothelial cells, but it is expressed in pathological conditions.

on the target cell.

Initially, proliferation and migration of adjacent endothelial cells have been identified as a factor of endothelial repair, and subsequent studies have shown that the maintenance of the endothelial structure is associated with EPCs and their ability to differentiate and repair damaged endothelial tissue. Due to the importance of this repair mechanism in the maintenance of vascular homeostasis, it is logical to think about the existence of close communication between damaged endothelial cells and EPCs. Previous studies performed by our group suggest that plasma EMVs, both of healthy subjects and patients with CKD; participate in the activity of the EPCs [10]. Our hypothesis is that EMVs can be an essential and necessary physiological mechanism of signaling to initiate the recruitment of EPCs from bone marrow. In *in vitro* models, we have shown that EMVs may be the key element in the regeneration and maintenance of vascular homeostasis, acting on EPCs [116]. Indeed, in response to different stimuli, the endothelial cells can induce EMV with different membrane characteristics, miRNA and other molecules in your content that reduce the ability of EPC to regenerate and participate in the signaling pathways involved in apoptosis and oxidative stress [117]. These specific mechanisms may constitute therapeutic objectives in future studies.

Vascular calcification is an increasingly constant process in developed countries and can contribute significantly to increased cardiovascular risk. The processes and mechanisms involved in the formation of vascular calcifications are poorly understood and are needed to develop new therapeutic strategies to prevent or avoid calcification. Patients CKD have a higher incidence of vascular calcification, and our group has shown that EMVs are increased in patients with an elevated degree of calcification [10]. In *in vitro* studies, EMVs produced in an inflammatory environment or obtained from patients with CKD promoted the calcification of smooth muscle cells, as assessed by some calcification markers (bone morphogenetic protein-2 (BMP-2) and alkaline phosphatase (ALP)) and the phenolsulfonephthalein method [100]. Other authors have also described a role of the MVs in the mineralization of vascular smooth muscle cells [118].

MVs have also been associated with endothelial senescence. As we said before, senescent cells release characteristic molecules and substances composing the SASP. However, some of those substances which are known to be part of this SASP cannot be released as soluble molecules due to their nature, as some transmembrane proteins [119]. It is known that the premature induction of cellular senescence *in vitro* increases the release of extracellular vesicles [120]. Those concepts suggest the contribution of MVs as part of the SASP, which have two important consequences: (1) SASP MVs can be the mechanism by which those insoluble proteins are released and (2) the carrier molecules can activate signaling processes in the target cells. Nevertheless, the specific mechanisms underlying MVs releasing from the senescent cells are still unresolved. It has been described that p53, a tumor-suppressor protein, remarkably upregulated in senescence, modulates the release of extracellular vesicles [121]. Also, p53 takes part in the transcription of some molecules implicated in extracellular vesicles biogenesis, partly explaining how senescence and MVs releasing activation can be related [122–124]. Moreover, the content within those MVs may be necessary in the induction of senescence in the target cells, as it has been shown that some miRNAs can regulate the p53 and pRB pathways [125–127]. Loss of pRB results in deregulated cell proliferation and apoptosis, whereas loss of p53 desensitizes cells to checkpoint signals, including apoptosis [128]. Thus, the presence of those miRNAs in MVs may be associated with hormonal changes driving aging (endocrine senescence induction) playing a critical role in the aging process and adding a new perspective on the mechanisms involved in aging.

under different pathologic conditions, its regulation is modified, as in CVD or CKD. Senescent endothelial cells change their morphological and functional characteristics (**Figure 3**) and cannot correctly regulate the repairing and regenerative activity of EPCs. In the endothelial senescence context, the role of EMVs appears to be important. EMVs are considered as biomarkers of endothelial injury and are associated with an inflammatory and prothrombotic state. However, the perspectives of their study are beyond their role as biomarkers, as they are capable of transmitting biologic information in several physiologic and physiopathologic processes. EMVs are increased in elderly, but also in patients with CVD and CKD. Many questions remain unresolved to understand the role of EMVs in the endothelial function and damage. To comprehend and characterize the mechanisms by which the senescent endothelial cells show an imbalanced functionality is of great interest, opening new perspectives to increase our knowledge and to identify useful bio-

Endothelial Cell Senescence in the Pathogenesis of Endothelial Dysfunction

http://dx.doi.org/10.5772/intechopen.73024

63

This work was supported by Plan Nacional Proyectos de Investigación en Salud of Instituto de Salud Carlos III (ISCIII) Fondos Feder European Grants (PI14/00806 and PI17/01029); Red de Investigación Renal (REDinREN; RD16/0009/0034) Junta de Andalucía Grants, P12-CTS-7352 and Santander Universidad Complutense de Madrid PR41/17-20964. Matilde Alique is a fellow of the program "Ayuda Postdoctoral Programa Propio" from Universidad de Alcalá, Madrid, Spain. Rafael Ramírez-Carracedo is a fellow of the program FPI (Formación de

, Matilde Alique3

and

markers in the timely diagnostics and to design therapeutic objectives in CVD.

Personal Universitario) from Universidad Francisco de Vitoria, Madrid, Spain".

1 Department of Genetic, Physiology and Microbiology, Faculty of Biology, Complutense University/Instituto de Investigación Sanitaria Hospital 12 de Octubre (imas12), Madrid,

2 Cardiovascular Joint Research Unit, University Francisco de Vitoria/University Hospital

3 Biology Systems Department, Physiology, Alcala University, Alcala de Henares, Madrid,

[1] Widlansky ME, Gokce N, Keaney JF, Vita JA. The clinical implications of endothelial dysfunction. Journal of the American College of Cardiology. 2003;**42**(7):1149-1160

\*, Rafael Ramírez-Carracedo2

\*Address all correspondence to: julcar01@ucm.es

Ramon y Cajal Research Unit (IRYCIS), Madrid, Spain

**Acknowledgements**

**Author details**

Julia Carracedo1

Spain

Spain

**References**

Rafael Ramírez-Chamond3
