**4. Chronic kidney disease, a model of chronic pathology that accelerates endothelial aging**

CKD is known to promote cellular senescence and an accelerated aging. It is caused by the accumulation of toxins in the internal medium, and the consequence is the development of elderly associated pathologies, mainly CVD [48]. CKD-associated CVD show similar characteristics to the natural CVD in elderly, and for this reason, several authors propose that the biggest challenge in the treatment of CVD may be to understand why CKD promote the premature aging of the cardiovascular system [49].

Even though the progress in the last few years in the renal replacement therapy is substantial, the mortality of terminal CKD patients remains excessively high, with an incidence between 10 and 20-fold over the general population [50].

Uremic patients have higher rates of cardiovascular morbidity and mortality than would be predicted by Framingham risk factors [50–52]. However, the presence of those factors is not enough to explain the significant increment of the cardiovascular risk in those patients. CKD patients show additional factors associated with uremia that could explain this increased CVD risk [53]. The presence of microalbumin and uremic toxins in blood, hyperhomocysteinemia, anemia, the abnormal calcium/phosphate metabolism, parathyroid hormone (PTH) level alterations, the treatment with vitamin D derived substances, the volume overload, the electrolytic imbalance, oxidative stress, inflammation, malnutrition, thrombogenic factors and the imbalance of NO/endothelin are risk factors intrinsically associated to CKD [54]. The valuation and modulation of those factors are of high importance in CKD patients, as some are variable and the correct treatment may prevent the progression of the pathology.

In CKD patients, the endothelium is exposed to an additional stress because of the presence of factors related to the uremic state. This state can be modified depending on the conservative treatment or renal transplantation, but it has been demonstrated that it relies on a persistent microinflammatory state directly related to endothelial damage, partaking in atherosclerosis processes [7, 55, 56]. Under this hostile uremic-associated state, the endothelium loses its integrity. Some damage substances and molecules will be released as a reflection of the harmful stimuli [56, 57].

Among several inflammatory factors, the subpopulation of monocytes habitually augmented in elderly, increases in the peripheral blood. The contribution of monocytes in inflammation and the CVD development has been widely studied by several groups, including ours [58]. Peripheral blood monocytes show a significant heterogeneity, reflected by the differential expression of the lipopolysaccharide binding receptor (CD14) at their surface and the lowaffinity receptor Fc, FcγRIII (CD16). In the last years, monocytes have been divided into three populations or subsets based on the intensity of CD14 and CD16 expression (cell surface marker phenotype) being functionally differentiated in: classical monocytes (CD14++/ CD16−), present mainly in healthy patients; intermediate monocytes (CD14++/CD16+) and non-classical monocytes (CD14+/CD16++). A possible causal role in the development of atherosclerosis in general population and CKD patients has been attributed to intermediate monocytes (CD14++/CD16+) [59]. CD14+/CD16++ monocytes are inflammatory senescent cells characterized by their increased capacity to produce proinflammatory cytokines and because of their strong function as dendritic cells [60]. CD14+/CD16++ can be differentiated *in vitro* from CD14++/CD16− monocytes by a cellular senescence process. CD14+/CD16++ show senescent cells characteristics, such as an increased content of the enzyme β-gal or a shortened telomere length in comparison to monocytes CD14++/CD16−, and they accumulate in peripheral blood of elderly or CKD patients as a result of their resistance to apoptosis [7, 61]. Intermediate monocytes (CD14++/CD16+) are a developmental step between the classical monocytes (CD14++/CD16−) and non-classical (CD14+/CD16++) and whose activity is related to CVD [62, 63]. Moreover, non-classical CD14+/CD16++ monocytes appear to be involved in the endothelial damage which is usually by elderly people and CKD or others chronic inflamed patients [62, 63] leading to endothelial cells from the neighborhood achieve senescence status. Also, high frequency of CD14+/CD16++ ("non-classical") monocytes is associated with increased vascular superoxide production and apoptosis in endothelial cells [64, 65]. In normal states, the vascular endothelium does not allow the adhesion of leukocytes and prevents their passage. When hemodynamic conditions are altered monocytes, adopt a peripheral position along the endothelial surface producing adhesion of monocytes to the activated endothelium. The injury of endothelial cells is associated with the senescence of endothelial cell [66].

cellular markers of senescence (upregulation of senescence-associated (SA)-β-galactosidase (gal) and p16) [29] are not robust and might overestimate the numbers of senescent cells that are present at low frequencies [37]. Thus, other cellular markers, such as cyclin D1 and lamin

The use of all these elements to define senescent cells has provided convincing evidence that these senescent cells accumulate in tissues of humans, primates and rodents with advanced age, as well as in sites of tissue injury and remodeling. The most prominent feature of the senescent cells is a cell cycle arrest, which permanently withholds replication and the resistance to apoptosis. An important fact to note is that the cells with senescent characteristics are found in damaged tissues of patients with chronic diseases such as osteoarthritis, pulmonary

CKD is known to promote cellular senescence and an accelerated aging. It is caused by the accumulation of toxins in the internal medium, and the consequence is the development of elderly associated pathologies, mainly CVD [48]. CKD-associated CVD show similar characteristics to the natural CVD in elderly, and for this reason, several authors propose that the biggest challenge in the treatment of CVD may be to understand why CKD promote the

Even though the progress in the last few years in the renal replacement therapy is substantial, the mortality of terminal CKD patients remains excessively high, with an incidence between

Uremic patients have higher rates of cardiovascular morbidity and mortality than would be predicted by Framingham risk factors [50–52]. However, the presence of those factors is not enough to explain the significant increment of the cardiovascular risk in those patients. CKD patients show additional factors associated with uremia that could explain this increased CVD risk [53]. The presence of microalbumin and uremic toxins in blood, hyperhomocysteinemia, anemia, the abnormal calcium/phosphate metabolism, parathyroid hormone (PTH) level alterations, the treatment with vitamin D derived substances, the volume overload, the electrolytic imbalance, oxidative stress, inflammation, malnutrition, thrombogenic factors and the imbalance of NO/endothelin are risk factors intrinsically associated to CKD [54]. The valuation and modulation of those factors are of high importance in CKD patients, as some

are variable and the correct treatment may prevent the progression of the pathology.

In CKD patients, the endothelium is exposed to an additional stress because of the presence of factors related to the uremic state. This state can be modified depending on the conservative treatment or renal transplantation, but it has been demonstrated that it relies on a persistent microinflammatory state directly related to endothelial damage, partaking in atherosclerosis processes [7, 55, 56]. Under this hostile uremic-associated state, the endothelium loses its integrity. Some damage substances and molecules will be released as a reflection of the harmful stimuli [56, 57].

B1 [38, 39], are considered more reliable markers of senescence.

54 Endothelial Dysfunction - Old Concepts and New Challenges

fibrosis, atherosclerosis, Alzheimer's disease or CKD [40].

premature aging of the cardiovascular system [49].

10 and 20-fold over the general population [50].

**accelerates endothelial aging**

**4. Chronic kidney disease, a model of chronic pathology that** 

*In vitro* studies performed with CD14+/CD16++ in mature endothelial cells cultures, we found that those monocytes express high levels of vascular adhesion molecules, have a high adhesion capability to endothelial cells, produce chemokines, angiogenic factors and induce the production of vascular damage-associated MVs [7, 56]. MVs may contain molecules such as proteins, nucleic acids and lipids, which could contribute to the CVD development and also the profile of these molecules, are specific of the cell type of origin [67]. Thus, the accumulation of CD14+/CD16++ monocytes in peripheral blood not only can play a crucial role in the induction and can be responsible for prolonging the inflammatory response in elderly and CKD patients but can be directly related to CVD development. In CKD patients, we found that inflammatory monocytes are increased, mostly in those patients subjected to hemodialysis [68]. Proinflammatory or non-classical monocytes have a high binding affinity for endothelial cells conferred by their high expression of adhesion molecules. As a consequence, CD16-positive monocytes might preferentially adhere to the activated endothelium, enabling the propagation of further vascular damage by secretion of proinflammatory mediators [59].

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.

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

Endothelial Cell Senescence in the Pathogenesis of Endothelial Dysfunction

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

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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].

depends on the diameter, concentration and the refractive index of the vesicles [93–95].

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

mixed with other MVs derived from other circulating cells.
