Extracellular vesicles enriched in connexin 43 promote a senescent phenotype in bone and synovial cells contributing to osteoarthritis progression

The accumulation of senescent cells is a key characteristic of aging, leading to the progression of age-related diseases such as osteoarthritis (OA). Previous data from our laboratory has demonstrated that high levels of the transmembrane protein connexin 43 (Cx43) are associated with a senescent phenotype in chondrocytes from osteoarthritic cartilage. OA has been reclassified as a musculoskeletal disease characterized by the breakdown of the articular cartilage affecting the whole joint, subchondral bone, synovium, ligaments, tendons and muscles. However, the mechanisms that contribute to the spread of pathogenic factors throughout the joint tissues are still unknown. Here, we show for the first time that small extracellular vesicles (sEVs) released by human OA-derived chondrocytes contain high levels of Cx43 and induce a senescent phenotype in targeted chondrocytes, synovial and bone cells contributing to the formation of an inflammatory and degenerative joint environment by the secretion of senescence-associated secretory associated phenotype (SASP) molecules, including IL-1ß and IL-6 and MMPs. The enrichment of Cx43 changes the protein profile and activity of the secreted sEVs. Our results indicate a dual role for sEVs containing Cx43 inducing senescence and activating cellular plasticity in target cells mediated by NF-kß and the extracellular signal-regulated kinase 1/2 (ERK1/2), inducing epithelial-to-mesenchymal transition (EMT) signalling programme and contributing to the loss of the fully differentiated phenotype. Our results demonstrated that Cx43-sEVs released by OA-derived chondrocytes spread senescence, inflammation and reprogramming factors involved in wound healing failure to neighbouring tissues, contributing to the progression of the disease among cartilage, synovium, and bone and probably from one joint to another. These results highlight the importance for future studies to consider sEVs positive for Cx43 as a new biomarker of disease progression and new target to treat OA.Subject terms: Osteoarthritis, Predictive markers     

Insulin-like growth factor binding proteins 4 and 7 released by senescent cells promote premature senescence in mesenchymal stem cells

Cellular senescence is the permanent arrest of cell cycle, physiologically related to aging and aging-associated diseases. Senescence is also recognized as a mechanism for limiting the regenerative potential of stem cells and to protect cells from cancer development. The senescence program is realized through autocrine/paracrine pathways based on the activation of a peculiar senescence-associated secretory phenotype (SASP). We show here that conditioned media (CM) of senescent mesenchymal stem cells (MSCs) contain a set of secreted factors that are able to induce a full senescence response in young cells. To delineate a hallmark of stem cells SASP, we have characterized the factors secreted by senescent MSC identifying insulin-like growth factor binding proteins 4 and 7 (IGFBP4 and IGFBP7) as key components needed for triggering senescence in young MSC. The pro-senescent effects of IGFBP4 and IGFBP7 are reversed by single or simultaneous immunodepletion of either proteins from senescent-CM. The blocking of IGFBP4/7 also reduces apoptosis and promotes cell growth, suggesting that they may have a pleiotropic effect on MSC biology. Furthermore, the simultaneous addition of rIGFBP4/7 increased senescence and induced apoptosis in young MSC. Collectively, these results suggest the occurrence of novel-secreted factors regulating MSC cellular senescence of potential importance for regenerative medicine and cancer therapy.     

Senescence and apoptosis: dueling or complementary cell fates?

In response to a variety of stresses, mammalian cells undergo a persistent proliferative arrest known as cellular senescence. Many senescence‐inducing stressors are potentially oncogenic, strengthening the notion that senescence evolved alongside apoptosis to suppress tumorigenesis. In contrast to apoptosis, senescent cells are stably viable and have the potential to influence neighboring cells through secreted soluble factors, which are collectively known as the senescence‐associated secretory phenotype (SASP). However, the SASP has been associated with structural and functional tissue and organ deterioration and may even have tumor‐promoting effects, raising the interesting evolutionary question of why apoptosis failed to outcompete senescence as a superior cell fate option. Here, we discuss the advantages that the senescence program may have over apoptosis as a tumor protective mechanism, as well as non‐neoplastic functions that may have contributed to its evolution. We also review emerging evidence for the idea that senescent cells are present transiently early in life and are largely beneficial for development, regeneration and homeostasis, and only in advanced age do senescent cells accumulate to an organism's detriment.     

Decreased tumorigenicity in vivo when transforming growth factor beta treatment causes cancer cell senescence

We have previously reported that transforming growth factor beta (TGF-beta) triggers two independent senescence programs, 1) replicative senescence dependent upon telomere shortening and 2) premature senescence independent of telomere shortening, in the cell line of A549 human lung adenocarcinoma. In this study, we examined the possibility that cancer cell tumor phenotypes could be suppressed by forced senescence. We used A549 cells treated with TGF-beta for a long time (over 50 days), where senescence was induced in a telomere-shortening-dependent or an independent way. Fully senescent A549 cells were elongated, acquired contact inhibition capabilities when reaching confluence, and secreted the senescence-associated cytokine IL-6. Furthermore, senescent A549 cells had no tumorigenicity in nude mice. These results indicate that the forced induction of senescence in cancer cells may be a novel and potentially powerful method for advancing anti-cancer therapy.     

Senescent cells at the crossroads of aging,disease, and tissue homeostasis

Originally identified as an outcome of continuous culture of primary cells, cellular senescence has moved beyond the culture dish and is now a bona fide driver of aging and disease in animal models, and growing links to human disease. This cellular stress response consists of a stable proliferative arrest coupled to multiple phenotypic changes. Perhaps the most important of these is the senescence-associated secretory phenotype, or senescence-associated secretory phenotype —a complex and variable collection of secreted molecules release by senescent cells with a number of potent biological activities. Senescent cells appear in multiple age-associated conditions in humans and mice, and interventions that eliminate these cells can prevent or even reverse multiple diseases in mouse models. Here, we review salient aspects of senescent cells in the context of human disease and homeostasis. Senescent cells increase in abundance during several diseases that associated with premature aging. Conversely, senescent cells have a key role in beneficial processes such as development and wound healing, and thus can help maintain tissue homeostasis. Finally, we speculate on mechanisms by which deleterious aspects of senescent cells might be targeted while retaining homeostatic aspects in order to improve age-related outcomes.     

Chromatin maintenance and dynamics in senescence: a spotlight on SAHF formation and the epigenome of senescent cells

Senescence is a stable proliferation arrest characterized by profound changes in cellular morphology and metabolism as well as by extensive chromatin reorganization in the nucleus. One particular hallmark of chromatin changes during senescence is the formation of punctate DNA foci in DAPI-stained senescent cells that have been called senescence-associated heterochromatin foci (SAHF). While many advances have been made concerning our understanding of the effectors of senescence, how chromatin is reorganized and maintained in senescent cells has remained largely elusive. Because chromatin structure is inherently dynamic, senescent cells face the challenge of developing chromatin maintenance mechanisms in the absence of DNA replication in order to maintain the senescent phenotype. Here, we summarize and review recent findings shedding light on SAHF composition and formation via spatial repositioning of chromatin, with a specific focus on the role of lamin B1 for this process. In addition, we discuss the physiological implication of SAHF formation, the role of histone variants, and histone chaperones during senescence and also elaborate on the more general changes observed in the epigenome of the senescent cells.     

Cellular senescence and chromatin structure

Cellular senescence is characterized by stable cell cycle arrest that is triggered by various forms of stress stimuli. Senescent cells show a series of morphological and physiological alterations including a flat and enlarged morphology, an increase in acidic β-galactosidase activity, chromatin condensation, and changes in gene expression pattern. These features are not observed in proliferating cells or quiescent cells in vitro. Using these senescence markers, cellular senescence has been shown to occur in benign or premalignant lesions but not in malignant lesions and to act as a tumor-suppressing mechanism in vivo. The onset and maintenance of the senescent state are regulated by two tumor suppressor proteins, p53 and Rb, which mediate senescence signals through p38 mitogen-activated protein kinase and cyclin-dependent kinase inhibitors. Alterations of chromatin structure are believed to contribute to the irreversible nature of the senescent state. Senescent cells form characteristic heterochromatin structure called senescence-associated heterochromatic foci (SAHFs), which may repress the expression of proliferation-promoting genes, such as E2F target genes. Recent studies have provided molecular insights into the structure and the mechanism of SAHF formation. In this paper, we review the role of cellular senescence in tumor suppression in vivo and the molecular mechanism of stable growth arrest in senescent cells, focusing on the special form of heterochromatin, SAHFs.     

Senescence chips for ultrahigh‐throughput isolation and removal of senescent cells

Cellular senescence plays an important role in organismal aging and age‐related diseases. However, it is challenging to isolate low numbers of senescent cells from small volumes of biofluids for downstream analysis. Furthermore, there is no technology that could selectively remove senescent cells in a high‐throughput manner. In this work, we developed a novel microfluidic chip platform, termed senescence chip, for ultrahigh‐throughput isolation and removal of senescent cells. The core component of our senescence chip is a slanted and tunable 3D micropillar array with a variety of shutters in the vertical direction for rapid cell sieving, taking advantage of the characteristic cell size increase during cellular senescence. The 3D configuration achieves high throughput, high recovery rate, and device robustness with minimum clogging. We demonstrated proof‐of‐principle applications in isolation and enumeration of senescent mesenchymal stem cells (MSCs) from undiluted human whole blood, and senescent cells from mouse bone marrow after total body irradiation, with the single‐cell resolution. After scale‐up to a multilayer and multichannel structure, our senescence chip achieved ultrahigh‐throughput removal of senescent cells from human whole blood with an efficiency of over 70% at a flow rate of 300 ml/hr. Sensitivity and specificity of our senescence chips could be augmented with implementation of multiscale size separation, and identification of background white blood cells using their cell surface markers such as CD45. With the advantages of high throughput, robustness, and simplicity, our senescence chips may find wide applications and contribute to diagnosis and therapeutic targeting of cellular senescence.     

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.     

Quantitative identification of senescent cells in aging and disease

Senescent cells are present in premalignant lesions and sites of tissue damage and accumulate in tissues with age. In vivo identification, quantification and characterization of senescent cells are challenging tasks that limit our understanding of the role of senescent cells in diseases and aging. Here, we present a new way to precisely quantify and identify senescent cells in tissues on a single‐cell basis. The method combines a senescence‐associated beta‐galactosidase assay with staining of molecular markers for cellular senescence and of cellular identity. By utilizing technology that combines flow cytometry with high‐content image analysis, we were able to quantify senescent cells in tumors, fibrotic tissues, and tissues of aged mice. Our approach also yielded the finding that senescent cells in tissues of aged mice are larger than nonsenescent cells. Thus, this method provides a basis for quantitative assessment of senescent cells and it offers proof of principle for combination of different markers of senescence. It paves the way for screening of senescent cells for identification of new senescence biomarkers, genes that bypass senescence or senolytic compounds that eliminate senescent cells, thus enabling a deeper understanding of the senescent state in vivo.     

Oxidative Stress-induced Inhibition of Sirt1 by Caveolin-1 Promotes p53-dependent Premature Senescence and Stimulates the Secretion of Interleukin 6 (IL-6)

Oxidative stress can induce premature cellular senescence. Senescent cells secrete various growth factors and cytokines, such as IL-6, that can signal to the tumor microenvironment and promote cancer cell growth. Sirtuin 1 (Sirt1) is a class III histone deacetylase that regulates a variety of physiological processes, including senescence. We found that caveolin-1, a structural protein component of caveolar membranes, is a direct binding partner of Sirt1, as shown by the binding of the scaffolding domain of caveolin-1 (amino acids 82–101) to the caveolin-binding domain of Sirt1 (amino acids 310–317). Our data show that oxidative stress promotes the sequestration of Sirt1 into caveolar membranes and the interaction of Sirt1 with caveolin-1, which lead to inhibition of Sirt1 activity. Reactive oxygen species stimulation promotes acetylation of p53 and premature senescence in wild-type but not caveolin-1 null mouse embryonic fibroblasts (MEFs). Either down-regulation of Sirt1 expression or re-expression of caveolin-1 in caveolin-1 null MEFs restores reactive oxygen species-induced acetylation of p53 and premature senescence. In addition, overexpression of caveolin-1 induces stress induced premature senescence in p53 wild-type but not p53 knockout MEFs. Phosphorylation of caveolin-1 on tyrosine 14 promotes the sequestration of Sirt1 into caveolar membranes and activates p53/senescence signaling. We also identified IL-6 as a caveolin-1-specific cytokine that is secreted by senescent fibroblasts following the caveolin-1-mediated inhibition of Sirt1. The caveolin-1-mediated secretion of IL-6 by senescent fibroblasts stimulates the growth of cancer cells. Therefore, by inhibiting Sirt1, caveolin-1 links free radicals to the activation of the p53/senescence pathway and the protumorigenic properties of IL-6.     

Senescence,Wound Healing,and Cancer: the PAI-1 Connection

Prolonged propagation of primary diploid fibroblasts in culture activates an ageing process known as replicative senescence, which is considered to provide a barrier against oncogenic transformation. Remarkably, both cell autonomous tumor-suppressive and cell non-autonomous tumor-promoting effects of senescent cells have been reported. Recently, we described that the p53 target gene plasminogen activator inhibitor-1 (PAI-1) is an essential mediator of replicative senescence. PAI-1 antagonizes the protease urokinase-type plasminogen activator (uPA). Both are secreted factors and involved in heterotypic signaling processes such as wound healing, angiogenesis and metastasis. Both uPA and PAI-1 are expressed in senescent cells and their relative abundance controls proliferation downstream of p53. Here, we present data that the effects of PAI- 1 and uPA in the senescence response are not strictly cell autonomous. We discuss these findings in the context of the emerging roles of PAI-1 and uPA in heterotypic cellular signaling in senescence, wound healing and metastasis.     

Increased expression of SIRT2 is a novel marker of cellular senescence and is dependent on wild type p53 status

Sirtuins (SIRT) belonging to the NAD+ dependent histone deacetylase III class of enzymes have emerged as master regulators of metabolism and longevity. However, their role in prevention of organismal aging and cellular senescence still remains controversial. In the present study, we now report upregulation of SIRT2 as a specific feature associated with stress induced premature senescence but not with either quiescence or cell death. Additionally, increase in SIRT2 expression was noted in different types of senescent conditions such as replicative and oncogene induced senescence using multiple cell lines. Induction of SIRT2 expression during senescence was dependent on p53 status as depletion of p53 by shRNA prevented its accumulation. Chromatin immunoprecipitation revealed the presence of p53 binding sites on the SIRT2 promoter suggesting its regulation by p53, which was also corroborated by the SEAP reporter assay. Overexpression or knockdown of SIRT2 had no effect on stress induced premature senescence, thereby indicating that SIRT2 increase is not a cause of senescence; rather it is an effect linked to senescence-associated changes. Overall, our results suggest SIRT2 as a promising marker of cellular senescence at least in cells with wild type p53 status.     

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.     

Molecular signaling and genetic pathways of senescence: Its role in tumorigenesis and aging

In response to progressive telomere shortening in successive cell divisions, normal somatic cells enter senescence, during which they cease to proliferate irreversibly and undergo dramatic changes in gene expression. Senescence can also be activated by various types of stressful stimuli, including aberrant oncogenic signaling, oxidative stress, and DNA damage. Because of the limited proliferative capacity imposed by senescence, as well as the ability of senescent cells to influence neighboring non-senescent cells, senescence has been proposed to play an important role in tumorigenesis and to contribute to aging. Considerable effort has been put into elucidating the molecular mechanisms of senescence, including the signals that trigger senescence, the molecular pathways by which cells enter senescence, and evidence that supports its role in tumorigenesis and aging.     

Aged‐senescent cells contribute to impaired heart regeneration

Aging leads to increased cellular senescence and is associated with decreased potency of tissue‐specific stem/progenitor cells. Here, we have done an extensive analysis of cardiac progenitor cells (CPCs) isolated from human subjects with cardiovascular disease, aged 32–86 years. In aged subjects (>70 years old), over half of CPCs are senescent (p16^INK4A, SA‐β‐gal, DNA damage γH2AX, telomere length, senescence‐associated secretory phenotype [SASP]), unable to replicate, differentiate, regenerate or restore cardiac function following transplantation into the infarcted heart. SASP factors secreted by senescent CPCs renders otherwise healthy CPCs to senescence. Elimination of senescent CPCs using senolytics abrogates the SASP and its debilitative effect in vitro. Global elimination of senescent cells in aged mice (INK‐ATTAC or wild‐type mice treated with D + Q senolytics) in vivo activates resident CPCs and increased the number of small Ki67‐, EdU‐positive cardiomyocytes. Therapeutic approaches that eliminate senescent cells may alleviate cardiac deterioration with aging and restore the regenerative capacity of the heart.     

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.