Antisenescence as a novel therapeutic strategy for vascular aging

Vascular cells have a finite lifespan when cultured in vitro and eventually enter an irreversible growth arrest state called "cellular senescence." It has been reported that many of the changes in senescent vascular cell behavior are consistent with the changes seen in age-related vascular diseases. Recently, senescent vascular cells have been demonstrated in human atherosclerotic lesions but not non-atherosclerotic lesions. Moreover, these cells express increased levels of proinflammatory molecules and decreased levels of endothelial nitric oxide synthase, suggesting that cellular senescence in vivo contributes to the pathogenesis of human atherosclerosis. One widely discussed hypothesis of senescence is the telomere hypothesis. An increasing body of evidence has established the critical role of the telomere in vascular cell senescence. More recent evidence suggests that telomere-independent mechanisms are implicated in vascular cell senescence. Activation of Ras, an important signaling molecule involved in atherogenic stimuli, induces vascular cell senescence and thereby promotes vascular inflammation in vitro and in vivo. Constitutive activation of Akt also induces vascular cell senescence. This novel role of Akt in regulating the cellular lifespan may contribute to various human diseases including atherosclerosis and diabetes mellitus. Although a causal link between vascular aging and vascular cell senescence remains elusive, a large body of data is consistent with cellular senescence contributing to age-associated vascular disorders. This review considers the clinical relevance of vascular cell senescence in vivo and discusses the potential of antisenescence therapy for human atherosclerosis.     

Endothelial cell senescence in human atherosclerosis: role of telomeres in endothelial dysfunction

BACKGROUND: The functional changes associated with cellular senescence may be involved in human aging and age-related vascular disorders. We have shown the important role of telomeres and telomerase in vascular cell senescence in vitro. Progressive telomere shortening in vivo has been observed in the regions susceptible to atherosclerosis, implicating its contributions to atherogenesis. However, whether senescent vascular cells are present in the vascularture and contribute to the pathogenesis of atherosclerosis remains unclear. METHODS AND RESULTS: Senescence-associated beta-galactosidase (beta-gal) activity was examined in the coronary arteries and the internal mammary arteries retrieved from autopsied individuals who had ischemic heart diseases. Strong beta-gal staining was observed in atherosclerotic lesions of the coronary arteries but not in the internal mammary arteries. An immunohistochemical analysis using anti-factor VIII antibody demonstrated that beta-gal stained cells are vascular endothelial cells. To determine whether endothelial cell senescence causes endothelial dysfunction, we induced senescence in human aortic endothelial cells (HAECs) by inhibiting telomere function and examined the expression of intercellular adhesion molecule (ICAM)-1 and endothelial nitric oxide synthase (NOS) activity. Senescent HAECs exhibited increased ICAM-1 expression and decreased eNOS activity, both of which are alterations implicated in atherogenesis. In contrast, introduction of telomerase catalytic component significantly extended the life span and inhibited the functional alterations associated with senescence in HAECs. CONCLUSIONS: Vascular endothelial cells with senescence-associated phenotypes are present in human atherosclerotic lesions, and endothelial cell senescence induced by telomere shortening may contribute to atherogenesis.     

Vascular cell senescence: contribution to atherosclerosis

Cardiologists and most physicians believe that aging is an independent risk factor for human atherosclerosis, whereas atherosclerosis is thought to be a characteristic feature of aging in humans by many gerontologists. Because atherosclerosis is among the age-associated changes that almost always escape the influence of natural selection in humans, it might be reasonable to regard atherosclerosis as a feature of aging. Accordingly, when we investigate the pathogenesis of human atherosclerosis, it may be more important to answer the question of how we age than what specifically promotes atherosclerosis. Recently, genetic analyses using various animal models have identified molecules that are crucial for aging. These include components of the DNA-repair system, the tumor suppressor pathway, the telomere maintenance system, the insulin/Akt pathway, and other metabolic pathways. Interestingly, most of the molecules that influence the phenotypic changes of aging also regulate cellular senescence, suggesting a causative link between cellular senescence and aging. For example, DNA-repair defects can cause phenotypic changes that resemble premature aging, and senescent cells that show DNA damage accumulate in the elderly. Excessive calorie intake can cause diabetes and hyperinsulinemia, whereas dysregulation of the insulin pathway has been shown to induce cellular senescence in vitro. Calorie restriction or a reduction of insulin signals extends the lifespan of various species and decreases biomarkers of cellular senescence in vivo. There is emerging evidence that cellular senescence contributes to the pathogenesis of human atherosclerosis. Senescent vascular cells accumulate in human atheroma tissues and exhibit various features of dysfunction. In this review, we examine the hypothesis that cellular senescence might contribute to atherosclerosis, which is a characteristic of aging in humans.     

Effects of telomerase and viral oncogene expression on the in vitro growth of human chondrocytes

Senescent chondrocytes accumulate with aging in articular cartilage, a process that interferes with cartilage homeostasis and increases the risk of cartilage degeneration. We showed previously that chondrocyte telomere length declines with donor age, which suggests that the aging process is telomere dependent. From these results we hypothesized that telomerase should delay the onset of senescence in cultured chondrocytes. Population doubling limits (PDL) were determined for chondrocytes expressing telomerase. We found that telomerase alone did not extend PDL beyond controls that senesced after 25 population doublings. The human papillomavirus 16 oncogenes E6 and E7 were transduced into the same cell population to investigate this telomere-independent form of senescence further. Chondrocytes expressing E6 and E7 grew longer than the telomerase cDNA (hTERT) cells but still senesced at 55 population doublings. In contrast, chondrocytes expressing telomerase with E6 and E7 grew vigorously past 100 population doublings. We conclude that although telomerase is necessary for the indefinite extension of chondrocyte life span, telomere-independent senescence limits PDL in vitro and may play a role in the age-related accumulation of senescent chondrocytes in vivo.     

Cellular senescence and tissue aging in vivo

A long-standing controversy concerns the relevance of cellular senescence, defined and observed as a cell culture phenomenon, to tissue aging in vivo. Here the evidence on this topic is reviewed. The main conclusions are as follows. First, telomere shortening, the principal known mediator of cellular senescence, occurs in many human tissues in aging. Second, it is not clear whether this results in cellular senescence or in some other cell fate (e.g., crisis). Third, rodents probably are not appropriate experimental models for these questions, because of important differences in telomere biology between rodent cells and cells from long-lived mammals (e.g., human or bovine cells). Fourth, better and more comprehensive observations on aging human tissues are needed to answer the question of the occurrence of senescent cells in tissues, and new experimental approaches are needed to elucidate the consequences of telomere shortening in tissues in aging.     

Replicative senescence of human fibroblasts: the role of Ras-dependent signaling and oxidative stress

Replicative senescence of human fibroblasts is a widely used cellular model for human aging. While it is clear that telomere erosion contributes to the development of replicative senescence, it is assumed that additional factors contribute to the senescent phenotype. The free radical theory of aging suggests that oxidative damage is a major cause of aging; furthermore, the expression of activated oncogenes, such as oncogenic Ras, can induce premature senescence in primary cells. The functional relation between the various inducers of senescence is not known. The present study was guided by the hypothesis that constitutive activation of normal, unmutated Ras may contribute to senescence-induced growth arrest in senescent human fibroblasts. When various branches of Ras-dependent signaling were investigated, constitutive activation of the Ras/Raf/MEK/ERK pathway was not observed. To evaluate the role of oxidative stress for the senescent phenotype, we also investigated stress-related protein kinases. While we found no evidence for alterations in the activity of p38, we could detect an increased activity of Jun kinase in senescent fibroblasts. We also found higher levels of reactive oxygen species (ROS) in senescent fibroblasts compared to their younger counterparts. The accumulation of ROS in senescent cells may be related to the constitutive activation of Jun kinase.     

Vascular smooth muscle cells undergo telomere-based senescence in human atherosclerosis: effects of telomerase and oxidative stress

Although human atherosclerosis is associated with aging, direct evidence of cellular senescence and the mechanism of senescence in vascular smooth muscle cells (VSMCs) in atherosclerotic plaques is lacking. We examined normal vessels and plaques by histochemistry, Southern blotting, and fluorescence in situ hybridization for telomere signals. VSMCs in fibrous caps expressed markers of senescence (senescence-associated beta-galactosidase [SAbetaG] and the cyclin-dependent kinase inhibitors [cdkis] p16 and p21) not seen in normal vessels. In matched samples from the same individual, plaques demonstrated markedly shorter telomeres than normal vessels. Fibrous cap VSMCs exhibited markedly shorter telomeres compared with normal medial VSMCs. Telomere shortening was closely associated with increasing severity of atherosclerosis. In vitro, plaque VSMCs demonstrated morphological features of senescence, increased SAbetaG expression, reduced proliferation, and premature senescence. VSMC senescence was mediated by changes in cyclins D/E, p16, p21, and pRB, and plaque VSMCs could reenter the cell cycle by hyperphosphorylating pRB. Both plaque and normal VSMCs expressed low levels of telomerase. However, telomerase expression alone rescued plaque VSMC senescence despite short telomeres, normalizing the cdki/pRB changes. In vivo, plaque VSMCs exhibited oxidative DNA damage, suggesting that telomere damage may be induced by oxidant stress. Furthermore, oxidants induced premature senescence in vitro, with accelerated telomere shortening and reduced telomerase activity. We conclude that human atherosclerosis is characterized by senescence of VSMCs, accelerated by oxidative stress-induced DNA damage, inhibition of telomerase and marked telomere shortening. Prevention of cellular senescence may be a novel therapeutic target in atherosclerosis.     

Increased expression of extracellular proteins as a hallmark of human endothelial cell in vitro senescence

A convenient way to study processes of aging in distinct human tissues consists of a molecular analysis of cells from the tissue in question, that were explanted and grown in vitro until they reach senescence. Using human umbilical vein endothelial cells (HUVEC), we have established an in vitro senescence model for human endothelial cells. A major hallmark of HUVEC in vitro senescence is the increased frequency of apoptotic cell death, which occurs as a determining feature of HUVEC senescence. Senescent endothelial cells are also found in vivo in atherosclerotic lesions, suggesting that the presence of such cells may contribute to the development of vascular pathology. To elucidate mechanisms underlying endothelial cell senescence and age-associated apoptosis, gene expression analyses were carried out. In these experiments, we observed the up-regulation of genes coding for extracellular proteins in senescent HUVEC. In particular, a significant upregulation of interleukin-8, VEGI, and the IGF-binding proteins 3 and 5 was observed. Upregulation of these genes was confirmed by both RT-PCR and Western blot. In the case of interleukin-8, a roughly 50-fold upregulation of the protein was also found in cellular supernatants. The extracellular proteins encoded by these genes are well known for their ability to modulate the apoptotic response of human cells, and in the case of interleukin-8, clear links to the establishment of atherosclerotic lesions have been defined. The results described here support a new model, where changes in the secretome of human endothelial cells contribute to vascular aging and vascular pathology.     

Epigenetic aspects of cellular senescence

The limited proliferative potential of normal cells in culture has been proposed as a model for cellular aging in vivo. It is clear that cellular aging has a genetic component but epigenetic processes could also be involved. Insight gained during years of intensive study suggests cellular aging is a multi-step process and that cells possess a counting mechanism that determines the number of doublings the cells can complete. In this paper, we review evidence suggesting a role for epigenetic processes in cell senescence and discuss the possible insights that might be provided by experiments designed to induce a premature senescent like state.     

Aging and atherosclerosis: mechanisms, functional consequences, and potential therapeutics for cellular senescence

Atherosclerosis is classed as a disease of aging, such that increasing age is an independent risk factor for the development of atherosclerosis. Atherosclerosis is also associated with premature biological aging, as atherosclerotic plaques show evidence of cellular senescence characterized by reduced cell proliferation, irreversible growth arrest and apoptosis, elevated DNA damage, epigenetic modifications, and telomere shortening and dysfunction. Not only is cellular senescence associated with atherosclerosis, there is growing evidence that cellular senescence promotes atherosclerosis. This review examines the pathology of normal vascular aging, the evidence for cellular senescence in atherosclerosis, the mechanisms underlying cellular senescence including reactive oxygen species, replication exhaustion and DNA damage, the functional consequences of vascular cell senescence, and the possibility that preventing accelerated cellular senescence is a therapeutic target in atherosclerosis.     

Identification of Hsc70 as target for AGE modification in senescent human fibroblasts

Cellular senescence is known as a potent mechanism of tumor suppression, and cellular senescence in vitro also reflects at least some features of aging in vivo. The Free Radical Theory of aging suggests that reactive oxygen species are important causative agents of aging and cellular senescence. Besides damage of nucleic acids and lipids, also oxidative modifications of proteins have been described as potential causative events in the senescence response. However, the identity of protein targets for post-translational modifications in senescent cells has remained unclear. In the present communication, we analyzed the occurrence of oxidative posttranslational modifications in senescent human endothelial cells and dermal fibroblasts. We found a significant increase in the level of protein carbonyls and AGE modification with senescence in both cell types. Using 2D-Gel electrophoresis and Western Blot we found that heat shock cognate protein 70 is a bona fide target for AGE modification in human fibroblasts.     

Age dependent aneuploidy and telomere length of the human vascular endothelium.

RATIONALE: Aneuploidy and telomere length are two major parameters that have been associated with cellular senescence in vitro. In order to explore the role of aneuploidy and telomere length in aging of the human vasculature, we studied these two parameters in direct preparations of endothelial cells of the human abdominal aorta. METHODS: Using fluorescent in situ hybridization on 'touch prep' slides, we evaluated aneuploidy of two autosomes (chromosomes 6 and 16) and sex chromosomes in non cultured endothelial cells of the abdominal aorta as a function of the donor's age. RESULTS: We found that the frequency of aneuploidy of vascular endothelial cells significantly increased with age. This was expressed by age-dependent tetrasomy (r(s)=0.56, P=0.006 for chromosome 6; and r(s)=0.54, P=0.008 for chromosome 16), and age dependent loss of the Y chromosome (r(s)=0.85, P=0.0003). In addition, we found that telomere length was inversely correlated with age (r=-0.38, P=0.008). DATA INTERPRETATION: These findings suggest that indicators of cellular senescence, earlier observed in vitro, are also expressed in the human vascular endothelium in vivo. Aneuploidy and telomere attrition might thus play a role in the aging of the human vasculature.     

Sustained inhibition of oxidative phosphorylation impairs cell proliferation and induces premature senescence in human fibroblasts

The mitochondrial theory of aging predicts that functional alterations in mitochondria contribute to the aging process. Whereas this hypothesis implicates increased production of reactive oxygen species (ROS) as a driving force of the aging process, little is known about molecular mechanisms by which mitochondrial impairment might contribute to aging. Using cellular senescence as a model for human aging, we have recently reported partial uncoupling of the respiratory chain in senescent human fibroblasts. In the present communication, we address a potential cause-effect relationship between mitochondrial impairment and the appearance of a senescence-like phenotype in young cells. We found that treatment by antimycin A delays proliferation and induces premature senescence in a subset of the cells, associated with increased reactive oxygen species (ROS) production. Quenching of ROS by antioxidants did however not restore proliferation capacity nor prevent premature senescence. Premature senescence is also induced upon chronic exposure to oligomycin, irrespective of ROS production, and oligomycin treatment induced the up-regulation of the cdk inhibitors p16, p21 and p27, which are also up-regulated in replicative senescence. Thus, besides the well-established influence of ROS on proliferation and senescence, a reduction in the level of oxidative phosphorylation is causally related to reduced cell proliferation and the induction of premature senescence.     

Further studies on the genetic and biochemical basis of cellular senescence

Cell fusion analysis, exploiting the fact that the phenotype of immortality is recessive in hybrids, has allowed the assignment of 26 different immortal human cell lines to at least four complementation groups for indefinite division. This indicates that there are at least four sets of genes or processes involved in the mechanisms leading to cellular senescence. We have also observed alterations in gene expression accompanying senescence that induce the the expression of a protein inhibitor of DNA synthesis, expression of new cell surface epitopes as identified by monoclonal antibodies specific to senescent cells, and changes in the extracellular matrix. We have yet to determine whether these changes in gene expression are casual or the result of senescence. The assignment of immortal cell lines to specific complementation groups now allow for a focused approach to identify the normal growth regulatory genes that have been modified to yield immortal cells and determine whether certain senescent cell specific patterns of gene expression continue to be expressed in immortal cells within a group. In addition, the isolation of senescent cell-specific antibodies provides for the first time the tools with which to probe the relationship between in vitro and in vivo aging.     

Abrupt telomere shortening in normal human fibroblasts

Aging is one of the most basic properties of living organisms. Abundant evidence supports the idea that cell senescence underlies organismal aging in higher mammals. Therefore, examining the molecular mechanisms that control cell and replicative senescence is of great interest for biology and medicine. Several discoveries strongly support telomere shortening as the main molecular mechanism that limits the growth of normal cells. Although cultures gradually approach their growth limit, appearance of individual senescent cells is sudden and stochastic. A theoretical model of abrupt telomere shortening has been proposed in order to explain this phenomenon, but until now there was no reliable experimental evidence supporting this idea. Here, we have employed novel methodology to provide evidence for the generation of extrachromosomal circular telomeric DNA as a result of abrupt telomere shortening in normal human fibroblasts. This mechanism ensures heterogeneity in growth potential among individual cells, which is crucial for gradual progression of the aging process.     

Senescence research from historical theory to future clinical application

Historically, the findings from cellular lifespan studies have greatly affected aging research. The discovery of replicative senescence by Hayflick developed into research on telomeres and telomerase, while stress-induced senescence became known as a telomere-independent event. Senescence-inducing signals comprise several tumor suppressors or cell cycle inhibitors, e.g., p53, cyclin-dependent kinase inhibitor p16 Ink4a and others. Stress-induced senescence serves as a physiological barrier to oncogenesis in vivo, while it activates senescence-associated secretary phenotype, inducing chronic inflammation. Thus, beside telomere length, p16, p53 and inflammatory cytokines have been utilized as biomarkers for cellular senescence. Telomere lengths in human leukocytes correlate well with events of aging-related lifestyle diseases, indicating the importance of cellular senescence in organismal aging. As such, the development of senescence research will have significant future clinical applications, e.g., senolysis. Geriatr Gerontol Int 2021; 21: 125–130 .     

Premature aging/senescence in cancer cells facing therapy: good or bad?

Normal and cancer cells facing their demise following exposure to radio-chemotherapy can actively participate in choosing their subsequent fate. These programmed cell fate decisions include true cell death (apoptosis-necroptosis) and therapy-induced cellular senescence (TIS), a permanent “proliferative arrest” commonly portrayed as premature cellular aging. Despite a permanent loss of proliferative potential, senescent cells remain viable and are highly bioactive at the microenvironment level, resulting in a prolonged impact on tissue architecture and functions. Cellular senescence is primarily documented as a tumor suppression mechanism that prevents cellular transformation. In the context of normal tissues, cellular senescence also plays important roles in tissue repair, but contributes to age-associated tissue dysfunction when senescent cells accumulate. Theoretically, in multi-step cancer progression models, cancer cells have already bypassed cellular senescence during their immortalization step (see hallmarks of cancer). It is then perhaps surprising to find that cancer cells often retain the ability to undergo TIS, or premature aging. This occurs because cellular senescence results from multiple signalling pathways, some retained in cancer cells, aiming to prevent cell cycle progression in damaged cells. Since senescent cancer cells persist after therapy and secrete an array of cytokines and growth factors that can modulate the tumor microenvironment, these cells may have beneficial and detrimental effects regarding immune modulation and survival of remaining proliferation-competent cancer cells. Similarly, while normal cells undergoing senescence are believed to remain indefinitely growth arrested, whether this is true for senescent cancer cells remains unclear, raising the possibility that these cells may represent a reservoir for cancer recurrence after treatment. This review discusses our current knowledge on cancer cell senescence and highlight questions that must be addressed to fully understand the beneficial and detrimental impacts of cellular senescence during cancer therapy.     

A Natural Model of Mouse Cardiac Myocyte Senescence

Many cardiac aging studies are performed on mice first and then, due to difficulty in mouse cardiomyocyte culture, applied the rat neonatal cardiomyocytes to further determine the mechanisms in vitro. Now, the technological challenge of mouse cardiomyocyte culture has been overcome and there is an increasing need for the senescence models of mouse cardiomyocytes. In this study, we have demonstrated that the senescence of mouse cardiomyocytes occurred with the extended culture time as shown by the increased β-galactosidase staining, increased p53 expression, decreased telomere activity, shorted telomere length, increased production of ROS, increased cell apoptosis, and impaired mitochondrial ΔΨm. These senescent responses shared similar results in aged mouse heart tissues in vivo. In summary, we have established and characterized a novel senescence model of mouse cardiomyocytes induced by the extended culture time in vitro. The cell model could be useful for the increased cardiac aging studies worldwide.     

Cellular senescence after single and repeated balloon catheter denudations of rabbit carotid arteries

The hypothesis that increased cellular proliferation in the vasculature may lead to replicative senescence has been tested in a model of neointima formation. We have used a biomarker of replicative senescence, senescence-associated beta-galactosidase (SA-beta-gal), to detect senescence in rabbit carotid arteries subjected to single and double balloon denudations. We found an accumulation of senescent cells in the neointima and media of all injured vessels, in contrast to the near absence of such cells in control vessels. The relative area occupied by SA-beta-gal-positive cells was higher in vessels subjected to double denudation than in those subjected to single denudation, both in the neointima (0.99% versus 0.06%, respectively; P:<0.001) and in the media (0.11% versus 0.01%, respectively; P:<0.02). The majority of SA-beta-gal-positive cells were vascular smooth muscle cells, and a minority were endothelial cells. SA-beta-gal-positive cells showed no evidence of apoptosis by use of terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling. Our results indicate that the proliferative response that follows intraluminal injury to the artery leads to the emergence of senescent endothelial and smooth muscle cells. The demonstration that vascular cell senescence can occur in vivo suggests that this process may be involved in cardiovascular pathologies that have a proliferative component.