**3. Endothelial cellular senescence as pathophysiological mechanism of vascular pathology**

cells undergo distinctive phenotypic alterations, including profound chromatin and secretome changes, telomere shortening, genomic and epigenomic damage, unbalanced mitogenic signals and tumor-suppressor activation [28, 29]. Also, in human replicative senescence, telomere lengths decline with each cell cycle [35]. Most of these cells are resistant to some apoptosis signals, therefore, they become senescent [31]. Senescence and apoptosis are responses to cellular stress, and both are important in the activation of tumor suppressors [36], but senescence avoids the damage in the stressed cells. To date, some senescence markers have been described (**Table 1**) that are involved in cellular senescence, most of which participate in cell cycle control and DNA repair [31]. Further analysis has highlighted that many common

**Characteristics Markers Regulation Techniques References**

H-dT ↓ Incorporation of radioactivity PCNA ↓ Immunostaining/Western blot Ki-67 ↓ Immunostaining/Western blot

X-gal substrate ↑ Light microscopy (production of

BrdU ↓ Fluorescence microscope [31]

blue precipitate)

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↑ Fluorescence microscopy/Western

blot

↑ Fluorescence microscopy

Hthymidine; PCNA, Proliferating cell nuclear antigen; SA-β-gal, Senescence-associated

(production of green fluorogenic

Fluorescence microscopy [31, 47]

↑ Fluorescence microscopy

color)

p16 ↑ Western blot/immunostaining [43–45] p21 [29, 46]

Cyclin D1 [38] Lamin B1 ↓ [39]

> ↑ Presence of certain heterochromatinassociated histone modifications

β-galactosidase; X-gal substrate, 5-bromo-4-chloro-3-indolyl-D-galactoside; C12FDG, 5-dodecanoylaminofluorescein diβ-D-galactopyranoside; SAHFs, senescence-associated heterochromatin foci; DAPI, 4′,6-diamidino-2-phenylindole; SDF, senescence-associated DNA damage foci; γ-H2AX; phosphorylated histone H2AX; 53BP1, p53-binding protein-1.

[41, 42]

[31]

DNA replication (senescent cells decline in DNA replication)

SA-β-gal activity (the SA-β-gal derives from the lysosomal β-galactosidase and reflects the increased lysosomal biogenesis)

Cell cycle arrest proteins (early markers of DNA damage-induced senescence)

SAHFs (reorganization of chromatin into discrete foci)

SDF (different DNA repair proteins)

BrdU, 5-bromodeoxyuridine; 3

**Table 1.** Senescence markers.

3

C12FDG (fluorogenic substrate)

p53

DNA dyes: DAPI

γ-H2AX: marker of DNA double strand breaks and genomic instability

53BP1: protein associated with DNA damage

H-dT, 3

One of the mechanisms that have been postulated as a possible pathophysiological participant is the cellular senescence of the endothelium. Cellular senescence is an irreversible process typical for all cells in which cells leave the cycle division as a consequence of the cellular damage associated with diseases [28] and aging [29]. Cell senescence processes appear to be involved in physiological processes of control such as cancer protection, biological developmental processes, tissue repair in aging situations and age-related disorders. Although their involvement in the aging process was postulated by Shay and Wright (Hayflick limit) [30], the absence of specific markers of senescence has hampered efforts to characterize senescent cells that accumulate *in vivo* in tissues and organs. Nowadays, the process of cell senescence is becoming better known due to the availability of new techniques to determine and quantify the senescent characteristics. In general, the main characteristic of the senescent phenotype is that cells decline in DNA replication until they cease to proliferate associated with the molecular changes of elements related to the cell cycle [31]. In general, senescent cells exhibit an upregulation and secretion of growth factors, proinflammatory cytokines, and also they release extracellular matrix-degrading proteins, the overall contribution constitutes the senescenceassociated secretory phenotype (SASP) [32] and cells lose the ability to divide at the end of replicative lifespan and decrease their ability to migrate [33]. At a phenotypic level, senescent cells acquire the typical flattened and enlarged morphology [34] (**Figure 1**). Aforementioned

**Figure 1.** Mechanisms by which endothelial cells become senescent and their characteristics. GF, growth factors; MMPs, matrix metalloproteinases; SASP, senescence-associated secretory phenotype; EMVs, endothelial microvesicles; ROS, reactive oxidative species.

cells undergo distinctive phenotypic alterations, including profound chromatin and secretome changes, telomere shortening, genomic and epigenomic damage, unbalanced mitogenic signals and tumor-suppressor activation [28, 29]. Also, in human replicative senescence, telomere lengths decline with each cell cycle [35]. Most of these cells are resistant to some apoptosis signals, therefore, they become senescent [31]. Senescence and apoptosis are responses to cellular stress, and both are important in the activation of tumor suppressors [36], but senescence avoids the damage in the stressed cells. To date, some senescence markers have been described (**Table 1**) that are involved in cellular senescence, most of which participate in cell cycle control and DNA repair [31]. Further analysis has highlighted that many common


BrdU, 5-bromodeoxyuridine; 3 H-dT, 3 Hthymidine; PCNA, Proliferating cell nuclear antigen; SA-β-gal, Senescence-associated β-galactosidase; X-gal substrate, 5-bromo-4-chloro-3-indolyl-D-galactoside; C12FDG, 5-dodecanoylaminofluorescein diβ-D-galactopyranoside; SAHFs, senescence-associated heterochromatin foci; DAPI, 4′,6-diamidino-2-phenylindole; SDF, senescence-associated DNA damage foci; γ-H2AX; phosphorylated histone H2AX; 53BP1, p53-binding protein-1.

**Table 1.** Senescence markers.

**3. Endothelial cellular senescence as pathophysiological mechanism** 

One of the mechanisms that have been postulated as a possible pathophysiological participant is the cellular senescence of the endothelium. Cellular senescence is an irreversible process typical for all cells in which cells leave the cycle division as a consequence of the cellular damage associated with diseases [28] and aging [29]. Cell senescence processes appear to be involved in physiological processes of control such as cancer protection, biological developmental processes, tissue repair in aging situations and age-related disorders. Although their involvement in the aging process was postulated by Shay and Wright (Hayflick limit) [30], the absence of specific markers of senescence has hampered efforts to characterize senescent cells that accumulate *in vivo* in tissues and organs. Nowadays, the process of cell senescence is becoming better known due to the availability of new techniques to determine and quantify the senescent characteristics. In general, the main characteristic of the senescent phenotype is that cells decline in DNA replication until they cease to proliferate associated with the molecular changes of elements related to the cell cycle [31]. In general, senescent cells exhibit an upregulation and secretion of growth factors, proinflammatory cytokines, and also they release extracellular matrix-degrading proteins, the overall contribution constitutes the senescenceassociated secretory phenotype (SASP) [32] and cells lose the ability to divide at the end of replicative lifespan and decrease their ability to migrate [33]. At a phenotypic level, senescent cells acquire the typical flattened and enlarged morphology [34] (**Figure 1**). Aforementioned

**Figure 1.** Mechanisms by which endothelial cells become senescent and their characteristics. GF, growth factors; MMPs, matrix metalloproteinases; SASP, senescence-associated secretory phenotype; EMVs, endothelial microvesicles; ROS,

**of vascular pathology**

52 Endothelial Dysfunction - Old Concepts and New Challenges

reactive oxidative species.

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

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

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*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].

endothelial cell [66].

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