Apoptosis is involved in the senescence of endothelial cells induced by angiotensin II

Vascular endothelial cells have a finite cell lifespan and eventually enter an irreversible growth arrest, cellular senescence. The functional changes associated with cellular senescence are thought to contribute to human aging and age-related cardiovascular disorders, e.g. atherosclerosis. In this study, induction of Angiotensin II (Ang II) promoted a growth arrest with phenotypic characteristics of cell senescence, such as enlarged cell shapes, increased senescence-associated beta-galactosidase (SA-beta-gal) positive staining cell, and depressed cell proliferation. Apoptotic changes were increased in senescent cells, with a small subset of the senescent cells showing aberrant morphology such as pronounced nuclear fragmentation or multiple micronuclei. The results suggest cell apoptosis is possibly an important factor in the process of pathologic and physiologic senescence of endothelial cells as well as vascular aging.     

Cellular senescence contributes to age‐dependent changes in circulating extracellular vesicle cargo and function

Extracellular vesicles (EVs) have emerged as important regulators of inter‐cellular and inter‐organ communication, in part via the transfer of their cargo to recipient cells. Although circulating EVs have been previously studied as biomarkers of aging, how circulating EVs change with age and the underlying mechanisms that contribute to these changes are poorly understood. Here, we demonstrate that aging has a profound effect on the circulating EV pool, as evidenced by changes in concentration, size, and cargo. Aging also alters particle function; treatment of cells with EV fractions isolated from old plasma reduces macrophage responses to lipopolysaccharide, increases phagocytosis, and reduces endothelial cell responses to vascular endothelial growth factor compared to cells treated with young EV fractions. Depletion studies indicate that CD63⁺ particles mediate these effects. Treatment of macrophages with EV‐like particles revealed that old particles increased the expression of EV miRNAs in recipient cells. Transfection of cells with microRNA mimics recapitulated some of the effects seen with old EV‐like particles. Investigation into the underlying mechanisms using bone marrow transplant studies revealed circulating cell age does not substantially affect the expression of aging‐associated circulating EV miRNAs in old mice. Instead, we show that cellular senescence contributes to changes in particle cargo and function. Notably, senolytic treatment of old mice shifted plasma particle cargo and function toward that of a younger phenotype. Collectively, these results demonstrate that senescent cells contribute to changes in plasma EVs with age and suggest a new mechanism by which senescent cells can affect cellular functions throughout the body.     

Differential expression of thymosin beta-10 by early passage and senescent vascular endothelium is modulated by VPF/VEGF: evidence for senescent endothelial cells in vivo at sites of atherosclerosis.

VPF/VEGF acts selectively on the vascular endothelium to enhance permeability, induce cell migration and division, and delay replicative senescence. To understand the changes in gene expression during endothelial senescence, we investigated genes that were differentially expressed in early vs. late passage (senescent) human dermal endothelial cells (HDMEC) using cDNA array hybridization. Early passage HDMEC cultured with or without VPF/VEGF overexpressed 9 and underexpressed 6 genes in comparison with their senescent counterparts. Thymosin beta-10 expression was modulated by VPF/VEGF and was strikingly down-regulated in senescent EC. The beta-thymosins are actin G-sequestering peptides that regulate actin dynamics and are overexpressed in neoplastic transformation. We have also identified senescent EC in the human aorta at sites overlying atherosclerotic plaques. These EC expressed senescence-associated neutral beta-galactosidase and, in contrast to adventitial microvessel endothelium, exhibited weak staining for thymosin beta-10. ISH performed on human malignant tumors revealed strong thymosin beta-10 expression in tumor blood vessels. This is the first report that Tbeta-10 expression is significantly reduced in senescent EC, that VPF/VEGF modulates thymosin beta-10 expression, and that EC can become senescent in vivo. The reduced expression of thymosin beta-10 may contribute to the senescent phenotype by reducing EC plasticity and thus impairing their response to migratory stimuli.     

Inhibition of phosphatidylinostol 3-kinase uncouples H2O2-induced senescent phenotype and cell cycle arrest in normal human diploid fibroblasts

Exposure of WI38 human diploid fibroblasts (HDFs) to hydrogen peroxide (H2O2) induced premature senescence. The senescent HDFs were permanently arrested and exhibited a senescent phenotype including enlarged and flattened cell morphology and increased senescence-associated beta-galactosidase (SA-beta-gal) activity. The induction of HDF senescence was associated with an activation of p53, increased expression of p21Cip1/WAF1, and hypophosphorylation of retinoblastoma protein (Rb), while no changes in the expression of p16Ink4a, p27Kip1, and p14Arf were observed. Exposure of WI38 cells to H2O2 also selectively activated phosphatidylinostol 3-kinase (PI3 kinase) and mitogen-activated protein kinase (MAPK) kinase (MEK), while no changes in p38 MAPK and Jun kinase (JNK) activities were observed. Selective inhibition of PI3 kinase activity with LY294002 abrogated H2O2-induced cell enlargement and flattened morphology and significantly attenuated the increase in SA-beta-gal activity, but did not affect H2O2-induced cell cycle arrest. In contrast, selective inhibition of MEK and p38 MAPK with PD98059 and SB203580, respectively, produced no significant effect on H2O2-induced senescent phenotype and cell cycle arrest. These findings demonstrate that expression of the senescent phenotype can be uncoupled from cell cycle arrest in prematurely senescent cells induced by H2O2 and does not contribute to the maintenance of permanent cell cycle arrest.     

ERM proteins and Cdk5 in cellular senescence

Cellular senescence is a tumor-suppressive process instigated by proliferation in the absence of telomere replication, by cellular stresses such as oncogene activation, or by activation of the retinoblastoma tumor suppressor protein, pRb. This process is characterized by an irreversible cell cycle exit, a unique morphology, and expression of senescence-associated-beta-galactosidase (SA-beta-gal). Despite the potential biological importance of cellular senescence, little is known of the mechanisms leading to the senescent phenotype. We have recently discovered that expression of active pRb induces expression and altered localization of the ERM family member ezrin, an actin-binding protein involved in membrane-cytoskeletal signaling. pRb expression results in the stimulation of cdk5-mediated phosphorylation of ezrin with subsequent membrane association and induction of cell shape changes, linking pRb activity to cytoskeletal regulation in senescent cells. Cdk5 activity increases in senescing cells and is required for expression of SA-beta-gal and for actin polymerization accompanying acquisition of the senescent morphology. These results begin to illuminate the mechanisms underlying induction of senescence and the senescent shape change and describe new pathways that may contribute to the ability of senescent cells to influence tumor growth.     

Senescent cells, tumor suppression, and organismal aging: good citizens, bad neighbors

Cells from organisms with renewable tissues can permanently withdraw from the cell cycle in response to diverse stress, including dysfunctional telomeres, DNA damage, strong mitogenic signals, and disrupted chromatin. This response, termed cellular senescence, is controlled by the p53 and RB tumor suppressor proteins and constitutes a potent anticancer mechanism. Nonetheless, senescent cells acquire phenotypic changes that may contribute to aging and certain age-related diseases, including late-life cancer. Thus, the senescence response may be antagonistically pleiotropic, promoting early-life survival by curtailing the development of cancer but eventually limiting longevity as dysfunctional senescent cells accumulate.     

Increased organization of cytoskeleton accompanying the aging of human fibroblasts in vitro

Fibroblastic cells of human origin have a limited lifespan in culture. One of the senescence-associated phenotypic changes is an increase in the abundance of cytoplasmic filaments. Human skin fibroblasts (strain 0011), derived from an 8-week-old male fetus, were passaged according to a predetermined schedule and examined at successive population doubling levels. In young rapidly growing cultures, fluorescence microscopy with NBD-Phallacidin shows a normal organization of the actin-containing fibers, microtubules and intermediate filaments, as has been described previously. At stages close to the end of the in vitro lifespan of the cell strain, large flat fibroblasts are the predominant cell type in culture. These large senescent fibroblasts contain numerous prominent actin fibers traversing the entire long axis of the cytoplasm. The fibers are often located adjacent to each other and appear to form a sheet on the ventral side of the cytoplasm. Staining of senescent cells with anti-tubulin antibody reveals an increase in the abundance of microtubules per cell and the distribution pattern is altered through the increase in the number of organization centers. Intermediate filaments are also more abundant and display tightly packed fibrillar sheets or bundles. Electron microscopic studies have confirmed the increased organization of microfilaments into bundles in senescent cells. These results suggest that during in vitro senescence, the increase in cell size is correlated with increased organization of the cytoskeleton. The presence of a rigid cytoskeletal structure may contribute in part to the inability of the senescent cell to replicate.     

Lessons from senescence: Chromatin maintenance in non-proliferating cells

Cellular senescence is an irreversible proliferation arrest, thought to contribute to tumor suppression, proper wound healing and, perhaps, tissue and organismal aging. Two classical tumor suppressors, p53 and pRB, control cell cycle arrest associated with senescence. Profound molecular changes occur in cells undergoing senescence. At the level of chromatin, for example, senescence associated heterochromatic foci (SAHF) form in some cell types. Chromatin is inherently dynamic and likely needs to be actively maintained to achieve a stable cell phenotype. In proliferating cells chromatin is maintained in conjunction with DNA replication, but how non-proliferating cells maintain chromatin structure is poorly understood. Some histone variants, such as H3.3 and macroH2A increase as cells undergo senescence, suggesting histone variants and their associated chaperones could be important in chromatin structure maintenance in senescent cells. Here, we discuss options available for senescent cells to maintain chromatin structure and the relative contribution of histone variants and chaperones in this process. This article is part of a Special Issue entitled: Histone chaperones and chromatin assembly.     

ERM Proteins and Cdk5 in Cellular Senescence

Cellular senescence is a tumor-suppressive process instigated by proliferation in the absence of telomere replication, by cellular stresses such as oncogene activation, or by activation of the retinoblastoma tumor suppressor protein, pRb. This process is characterized by an irreversible cell cycle exit, a unique morphology, and expression of senescence-associated-b-galactosidase (SA-b-gal). Despite the potential biological importance of cellular senescence, little is known of the mechanisms leading to the senescent phenotype. We have recently discovered that expression of active pRb induces expression and altered localization of the ERM family member ezrin, an actin-binding protein involved in membrane-cytoskeletal signaling. pRb expression results in the stimulation of cdk5-mediated phosphorylation of ezrin with subsequent membrane association and induction of cell shape changes, linking pRb activity to cytoskeletal regulation in senescent cells. Cdk5 activity increases in senescing cells and is required for expression of SA-b-gal and for actin polymerization accompanying acquisition of the senescent morphology. These results begin to illuminate the mechanisms underlying induction of senescence and the senescent shape change and describe new pathways that may contribute to the ability of senescent cells to influence tumor growth.     

How might replicative senescence contribute to human ageing?

Cell senescence is the limited ability of primary human cells to divide when cultured in vitro. This eventual cessation of division is accompanied by a specific set of changes in cell physiology, morphology, and gene expression. Such changes in phenotype have the potential to contribute to human ageing and age-related diseases. Until now, senescence has largely been studied as an in vitro phenomenon, but recent data have for the first time directly demonstrated the presence of senescent cells in aged human tissues. Although a direct causal link between the ageing of whole organisms and the senescence of cells in culture remains elusive, a large body of data is consistent with cell senescence contributing to a variety of pathological changes seen in the aged. This review considers the in vitro phenotype of cellular senescence and speculates on the various possible routes whereby the presence of senescent cells in old bodies may affect different tissue systems.     

Identification of a novel senolytic agent,navitoclax, targeting the Bcl‐2 family of anti‐apoptotic factors

Clearing senescent cells extends healthspan in mice. Using a hypothesis‐driven bioinformatics‐based approach, we recently identified pro‐survival pathways in human senescent cells that contribute to their resistance to apoptosis. This led to identification of dasatinib (D) and quercetin (Q) as senolytics, agents that target some of these pathways and induce apoptosis preferentially in senescent cells. Among other pro‐survival regulators identified was Bcl‐xl. Here, we tested whether the Bcl‐2 family inhibitors, navitoclax (N) and TW‐37 (T), are senolytic. Like D and Q, N is senolytic in some, but not all types of senescent cells: N reduced viability of senescent human umbilical vein epithelial cells (HUVECs), IMR90 human lung fibroblasts, and murine embryonic fibroblasts (MEFs), but not human primary preadipocytes, consistent with our previous finding that Bcl‐xl siRNA is senolytic in HUVECs, but not preadipocytes. In contrast, T had little senolytic activity. N targets Bcl‐2, Bcl‐xl, and Bcl‐w, while T targets Bcl‐2, Bcl‐xl, and Mcl‐1. The combination of Bcl‐2, Bcl‐xl, and Bcl‐w siRNAs was senolytic in HUVECs and IMR90 cells, while combination of Bcl‐2, Bcl‐xl, and Mcl‐1 siRNAs was not. Susceptibility to N correlated with patterns of Bcl‐2 family member proteins in different types of human senescent cells, as has been found in predicting response of cancers to N. Thus, N is senolytic and acts in a potentially predictable cell type‐restricted manner. The hypothesis‐driven, bioinformatics‐based approach we used to discover that dasatinib (D) and quercetin (Q) are senolytic can be extended to increase the repertoire of senolytic drugs, including additional cell type‐specific senolytic agents.     

Keeping zombies alive: The ER-mitochondria Ca2+ transfer in cellular senescence

Cellular senescence generates a permanent cell cycle arrest, characterized by apoptosis resistance and a pro-inflammatory senescence-associated secretory phenotype (SASP). Physiologically, senescent cells promote tissue remodeling during development and after injury. However, when accumulated over a certain threshold as happens during aging or after cellular stress, senescent cells contribute to the functional decline of tissues, participating in the generation of several diseases. Cellular senescence is accompanied by increased mitochondrial metabolism. How mitochondrial function is regulated and what role it plays in senescent cell homeostasis is poorly understood. Mitochondria are functionally and physically coupled to the endoplasmic reticulum (ER), the major calcium (Ca²⁺) storage organelle in mammalian cells, through special domains known as mitochondria-ER contacts (MERCs). In this domain, the release of Ca²⁺ from the ER is mainly regulated by inositol 1,4,5-trisphosphate receptors (IP3Rs), a family of three Ca²⁺ release channels activated by a ligand (IP3). IP3R-mediated Ca²⁺ release is transferred to mitochondria through the mitochondrial Ca²⁺ uniporter (MCU), where it modulates the activity of several enzymes and transporters impacting its bioenergetic and biosynthetic function. Here, we review the possible connection between ER to mitochondria Ca²⁺ transfer and senescence.Understanding the pathways that contribute to senescence is essential to reveal new therapeutic targets that allow either delaying senescent cell accumulation or reduce senescent cell burden to alleviate multiple diseases.     

Microarray analysis of replicative senescence.

BACKGROUND: Limited replicative capacity is a defining characteristic of most normal human cells and culminates in senescence, an arrested state in which cells remain viable but display an altered pattern of gene and protein expression. To survey widely the alterations in gene expression, we have developed a DNA microarray analysis system that contains genes previously reported to be involved in aging, as well as those involved in many of the major biochemical signaling pathways. RESULTS: Senescence-associated gene expression was assessed in three cell types: dermal fibroblasts, retinal pigment epithelial cells, and vascular endothelial cells. Fibroblasts demonstrated a strong inflammatory-type response, but shared limited overlap in senescent gene expression patterns with the other two cell types. The characteristics of the senescence response were highly cell-type specific. A comparison of early- and late-passage cells stimulated with serum showed specific deficits in the early and mid G1 response of senescent cells. Several genes that are constitutively overexpressed in senescent fibroblasts are regulated during the cell cycle in early-passage cells, suggesting that senescent cells are locked in an activated state that mimics the early remodeling phase of wound repair. CONCLUSIONS: Replicative senescence triggers mRNA expression patterns that vary widely and cell lineage strongly influences these patterns. In fibroblasts, the senescent state mimics inflammatory wound repair processes and, as such, senescent cells may contribute to chronic wound pathologies.     

Regulation of ornithine decarboxylase and other cell cycle-dependent genes during senescence of IMR-90 human diploid fibroblasts

Aging of IMR-90 human diploid fibroblasts in vitro is accompanied by significant changes of polyamine metabolism, most notably, a 5-fold decrease of serum-induced activity of ornithine decarboxylase, the key enzyme in the biosynthesis of polyamines (Chen, K. Y., Chang, Z. F., and Liu, A. Y.-C. (1986) J. Cell. Physiol. 129, 142-146). In this paper, we employed Northern blot hybridization and affinity radiolabeling techniques to investigate the molecular basis of this age-associated change of ornithine decarboxylase activity. Since the induction of ornithine decarboxylase by serum is a mid-G1 event, we also examined expressions of other cell cycle-dependent genes that are induced before and after the mid-G1 phase to determine if their expressions may also be age-dependent. Our results demonstrated a 3-fold decrease of the amount of active ornithine decarboxylase molecules that can be labeled by alpha-difluoromethyl[3H]ornithine in senescent IMR-90 cells (population doubling level (PDL) = 52) as compared to young cells (PDL = 22). However, the levels and kinetics of induction of ornithine decarboxylase mRNA in both young and senescent IMR-90 cells were found to be identical throughout a 24-h time period after serum stimulation. The time course and the magnitude of the expression of c-myc, an early G1 gene, were quite similar in young and senescent IMR-90 cells and appeared to be PDL-independent. In contrast, the expression of thymidine kinase, a late G1/S gene, was significantly reduced in senescent IMR-90 cells. Levels of thymidine kinase mRNA and thymidine kinase activity in senescent IMR-90 cells were 6- and 8-fold less than those in young cells, respectively. Based on these data, we proposed that impairment of cell cycling in senescent IMR-90 cells may occur at the late G1/S phase and that decreases of ornithine decarboxylase activity and putrescine accumulation during cell senescence may contribute to this impairment.     

Changes in the level and distribution of Ku proteins during cellular senescence

Aging is associated with accumulation of genomic rearrangements consistent with aberrant repair of DNA breaks. We have shown previously that DNA repair by non-homologous end joining (NHEJ) becomes less efficient and more error-prone in senescent cells. Here, we show that the levels of Ku70 and Ku80 drop approximately twofold in replicatively senescent cells. Intracellular distribution of Ku also changes. In the young cells roughly half of Ku is located in the nucleus and half in the cytoplasm. In senescent cells the nuclear levels of Ku do not change, while the cytoplasmic Ku fraction disappears. Upon treatment with gamma-irradiation, in the young cells cytoplasmic Ku moved into the nuclear and membrane fractions, while no change in the Ku distribution occurred in senescent cells. Upon treatment with UVC Ku moved out of the nucleus in the young cells, while most Ku remained nuclear in senescent cells. This suggests that the nuclear Ku in senescent cells is unable to respond to DNA damage. We hypothesize that overall decline in Ku levels changes in Ku intracellular distribution, and the loss of appropriate response of Ku to DNA damage in senescent cells contribute to the decline of NHEJ and to age-related genomic instability.     

Reduction in flippase activity contributes to surface presentation of phosphatidylserine in human senescent erythrocytes

Mature human erythrocytes circulate in blood for approximately 120 days, and senescent erythrocytes are removed by splenic macrophages. During this process, the cell membranes of senescent erythrocytes express phosphatidylserine, which is recognized as a signal for phagocytosis by macrophages. However, the mechanisms underlying phosphatidylserine exposure in senescent erythrocytes remain unclear. To clarify these mechanisms, we isolated senescent erythrocytes using density gradient centrifugation and applied fluorescence‐labelled lipids to investigate the flippase and scramblase activities. Senescent erythrocytes showed a decrease in flippase activity but not scramblase activity. Intracellular ATP and K⁺, the known influential factors on flippase activity, were altered in senescent erythrocytes. Furthermore, quantification by immunoblotting showed that the main flippase molecule in erythrocytes, ATP11C, was partially lost in the senescent cells. Collectively, these results suggest that multiple factors, including altered intracellular substances and reduced ATP11C levels, contribute to decreased flippase activity in senescent erythrocytes in turn to, present phosphatidylserine on their cell membrane. The present study may enable the identification of novel therapeutic approaches for anaemic states, such as those in inflammatory diseases, rheumatoid arthritis, or renal anaemia, resulting from the abnormally shortened lifespan of erythrocytes.     

Elimination of senescent osteoclast progenitors has no effect on the age‐associated loss of bone mass in mice

Both an increase in osteoclast and a decrease in osteoblast numbers contribute to skeletal aging. Markers of cellular senescence, including expression of the cyclin inhibitor p16, increase with aging in several bone cell populations. The elimination of p16‐expressing cells in old mice, using the INK‐ATTAC transgene, increases bone mass indicating that senescent cells contribute to skeletal aging. However, the identity of the senescent cells and the extent to which ablation of p16‐expressing cells may prevent skeletal aging remain unknown. Using mice expressing the p16‐3MR transgene, we examined whether elimination of p16‐expressing cells between 12 and 24 months of age could preserve bone mass; and whether elimination of these cells from 20 to 26 months of age could restore bone mass. The activation of the p16‐3MR transgene by ganciclovir (GCV) greatly diminished p16 levels in the brain, liver, and osteoclast progenitors from the bone marrow. The age‐related increase in osteoclastogenic potential of myeloid cells was also abrogated by GCV. However, GCV did not alter p16 levels in osteocytes—the most abundant cell type in bone—and had no effect on the skeletal aging of p16‐3MR mice. These findings indicate that the p16‐3MR transgene does not eliminate senescent osteocytes but it does eliminate senescent osteoclast progenitors and senescent cells in other tissues, as described previously. Elimination of senescent osteoclast progenitors, in and of itself, has no effect on the age‐related loss of bone mass. Hence, other senescent cell types, such as osteocytes, must be the seminal culprits.