Non-senescent Cells Research Articles

A novel strategy based on targeted cellular metabolomics for quantitatively evaluating anti-aging effect and screening effective extracts of Erzhi Wan

The complexity of ingredients in traditional Chinese medicine (TCM) makes it challenging to clarify its efficacy in an acceptable and scientific approach. The present study was aimed to use quantification results from targeted cellular metabolomics to evaluate anti-aging efficacy of a famous Chinese medicine formula, Erzhi Wan (EZW), and screen possible effective extracts, depending on the developed strategy integrating multivariate receiver operating characteristic (ROC) curve and analytic hierarchy process (AHP). In this study, senescent NRK cells induced by D-galactose were treated with drug-containing serum of EZW and four kinds of extracts (petroleum ether, ethyl acetate, butanol and water). Intermediates of two major metabolic pathways for energy synthesis, tricarboxylic acid (TCA) cycle and glycolysis, were accurately quantified by GC–MS/MS to identify discriminate metabolites for clarifying therapeutic mechanism of EZW based on multivariate statistical analysis. Senescent and non-senescent cells were successfully distinguished using these metabolites by ROC curve analysis. Next, these metabolites were used as evaluation indexes to quantitatively reflect different effect of EZW and its extracts, according to the role of them in distinguishing groups and in conjunction with AHP. In vitro detection of senescence-associated β-galactosidase (SA-β-gal) activity was used to verify the reliability of evaluation results. The reversal after treatment of drug-containing serum of EZW and extracts was observed, and the petroleum ether extract might be the potential active extract responsible for the major anti-aging effect of EZW, which was in agreement with in vitro experiments. Altogether, metabolomics was a powerful approach for evaluation efficacy and elucidation action mechanisms of TCM. The integrated evaluation strategy in this paper with properties of high practicality, feasibility and effectivity was expected to provide a new insight into comprehensive and quantitative efficacy evaluation.

Senolytics reduce coronavirus-related mortality in old mice.

The COVID-19 pandemic has revealed the pronounced vulnerability of the elderly and chronically ill to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-induced morbidity and mortality. Cellular senescence contributes to inflammation, multiple chronic diseases, and age-related dysfunction, but effects on responses to viral infection are unclear. Here, we demonstrate that senescent cells (SnCs) become hyper-inflammatory in response to pathogen-associated molecular patterns (PAMPs), including SARS-CoV-2 spike protein-1, increasing expression of viral entry proteins and reducing antiviral gene expression in non-SnCs through a paracrine mechanism. Old mice acutely infected with pathogens that included a SARS-CoV-2-related mouse β-coronavirus experienced increased senescence and inflammation, with nearly 100% mortality. Targeting SnCs by using senolytic drugs before or after pathogen exposure significantly reduced mortality, cellular senescence, and inflammatory markers and increased antiviral antibodies. Thus, reducing the SnC burden in diseased or aged individuals should enhance resilience and reduce mortality after viral infection, including that of SARS-CoV-2.

The Senolytic Drug JQ1 Removes Senescent Cells via Ferroptosis.

Ferroptosis is an iron-dependent, non-apoptotic programmed cell death. Cellular senescence contributes to aging and various age-related diseases through the expression of a senescence-associated secretory phenotype (SASP). Senescent cells are often resistant to ferroptosis via increased ferritin and impaired ferritinophagy. In this study, we investigated whether treatment with JQ1 could remove senescent cells by inducing ferroptosis. Senescence of human dermal fibroblasts was induced in vitro by treating the cells with bleomycin. The senolytic effects of JQ1 were evaluated using a SA-β gal assay, annexin V analysis, cell counting kit-8 assay, and qRT-PCR. Ferroptosis following JQ1 treatment was evaluated with qRT-PCR and BODIPY staining. At a certain range of JQ1 concentrations, JQ1 treatment reduced the viability of bleomycin-treated cells (senescent cells) but did not reduce that of untreated cells (non-senescent cells), indicating that JQ1 treatment can selectively eliminate senescent cells. JQ1 treatment also decreased SASP expression only in senescent cells. Subsequently, JQ1 treatment reduced the expression of ferroptosis-resistance genes in senescent cells. JQ1 treatment induced lipid peroxidation in senescent cells but not in non-senescent cells. The data indicate that JQ1 can eliminate senescent cells via ferroptosis. This study suggests ferroptosis as a new mechanism of senolytic therapy.

Aging Human Endothelial Cells Affect Neuronal Viability and Function

It is estimated that more than 50% of adults over 80 will develop dementia, including Alzheimer's disease (AD), and the primary risk factor for dementia is aging. Aging provides the substrate for neurodegenerative disease through increased inflammation and cellular dysfunction. Despite the fact that aging is a key risk factor in neurodegenerative disease, preclinical studies frequently model age-related diseases using young rodents. In this study we used replicative senescence, a standard model of cell senescence that permits the study of cells as they progress to senescence to examine the differential effects of senescent vs. non-senescent primary human endothelial cells on a human neuronal cell line. We hypothesize that senescent (Sen) endothelial cells (ECs), which accumulate with aging, create a pro-inflammatory environment that adversely affects viability and function of neurons. First, we validated the replicative senescence process in human-derived ECs by quantifying β-galactosidase activity, a marker for cellular senescence, and the protein expression of Lamin B1, which decreases with senescence. After determining senescence phenotypes by passage number, ECs at varying stages of cellular senescence (EP: early passage, ES: early senescence, LS: late senescence) were co-cultured with LUHMES (Lund Human Mesencephalic cell line - dopamine-like neurons) to assess neuronal cell viability by measuring release of lactate dehydrogenase (LDH) into the culture medium and neurite outgrowth. Nitric oxide and extracellular vesicles released into the culture medium were also evaluated as indicators of an inflammatory environment. Co-culturing ECs with LUHMES decreased LUHMES cell viability as evidenced by an increase in LDH in co-cultures of LUHMES LS EC and by a decreased number of nuclei in LUHMES co-cultured with any of the EC types (EP, ES, LS). Co-culture with any of the EC types increased the area labeled by MAP2 and phospho-Neurofilament in LUHMES at 24h, indicating increased neurite outgrowth. At 48h, increased neurite outgrowth was observed in LUHMES+EP ECs and LUHMES+ES ECs co-cultures but not in the LUHMES + LP ECs. Nitric oxide was not increased by co-culture of LUHMES with any of the EC subtypes. The amount of extracellular vesicles as quantified by measuring the activity of acetylcholinesterase (AChE) was increased in co-cultures compared to LUHMES controls, but did not significantly differ from EC controls. In summary, ES ECs promote neurite outgrowth whereas LS ECs promote neurodegeneration. These findings suggest the importance of EC aging on neuronal cell morphology and viability, both parameters affected in age-related dementia, including Alzheimer's disease.

The implication of ROCK 2 as a potential senotherapeutic target via the suppression of the harmful effects of the SASP: Do senescent cancer cells really engulf the other cells?

Chemotherapy-induced senescent cancer cells secrete several factors in their microenvironment called SASP. Accumulated evidence states that SASP is responsible for some of the harmful effects of chemotherapy such as drug resistance and the induction of cancer cell proliferation, migration, and invasion. Therefore, to develop senolytic and/or senomorphic drugs, targeting the senescent cells gains importance as a new strategy for preventing the damage that senescent cancer cells cause. In the current work, we evaluated whether Rho/Rho kinase pathway has the potential to be used as a target pathway for the development of senolytic and/or senomorphic drugs in doxorubicin-induced senescent cancer cell lines. We have determined that inhibition of Rho/Rho kinase pathway with CT04 and Y27632 reduced the secretory activity of senescent cancer cells and changed the composition of SASP. Our results indicate that ROCK 2 isoform was responsible for these observed effects on the SASP. In addition, non-senescent cancer cell proliferation and migration accelerated by senescent cells were set back to the pre-induction levels after ROCK inhibition. Moreover, contrary to the previous observations, another important finding of the current work is that senescent HeLa and A549 cells did not engulf the non-senescent HeLa, A549 cells, and non-cancer HUVEC. These results indicate that ROCK inhibitors, in particular ROCK 2 specific inhibitors, have the potential to be developed as novel senomorphic drugs. In addition, we found that all senescent cancer cells do not share the same engulfment ability, and this process should not be generalized.

The Interaction of the Senescent and Adjacent Breast Cancer Cells Promotes the Metastasis of Heterogeneous Breast Cancer Cells through Notch Signaling.

Chemotherapy is one of the most common strategies for tumor treatment but often associated with post-therapy tumor recurrence. While chemotherapeutic drugs are known to induce tumor cell senescence, the roles and mechanisms of senescence in tumor recurrence remain unclear. In this study, we used doxorubicin to induce senescence in breast cancer cells, followed by culture of breast cancer cells with conditional media of senescent breast cancer cells (indirect co-culture) or directly with senescent breast cancer cells (direct co-culture). We showed that breast cancer cells underwent the epithelial–mesenchymal transition (EMT) to a greater extent and had stronger migration and invasion ability in the direct co-culture compared with that in the indirect co-culture model. Moreover, in the direct co-culture model, non-senescent breast cancer cells facilitated senescent breast cancer cells to escape and re-enter into the cell cycle. Meanwhile, senescent breast cancer cells regained tumor cell characteristics and underwent EMT after direct co-culture. We found that the Notch signaling was activated in both senescent and non-senescent breast cancer cells in the direct co-culture group. Notably, the EMT process of senescent and adjacent breast cancer cells was blocked upon inhibition of Notch signaling with N-[(3,5-difluorophenyl)acetyl]-l-alanyl-2-phenyl]glycine-1,1-dimethylethyl ester (DAPT) in the direct co-cultures. In addition, DAPT inhibited the lung metastasis of the co-cultured breast cancer cells in vivo. Collectively, data arising from this study suggest that both senescent and adjacent non-senescent breast cancer cells developed EMT through activating Notch signaling under conditions of intratumoral heterogeneity caused by chemotherapy, which infer the possibility that Notch inhibitors used in combination with chemotherapeutic agents may become an effective treatment strategy.

Senescent Tumor Cells Build a Cytokine Shield in Colorectal Cancer.

Cellular senescence can either support or inhibit cancer progression. Here, it is shown that intratumoral infiltration of CD8+ T cells is negatively associated with the proportion of senescent tumor cells in colorectal cancer (CRC). Gene expression analysis reveals increased expression of C‐X‐C motif chemokine ligand 12 (CXCL12) and colony stimulating factor 1 (CSF1) in senescent tumor cells. Senescent tumor cells inhibit CD8+ T cell infiltration by secreting a high concentration of CXCL12, which induces a loss of CXCR4 in T cells that result in impaired directional migration. CSF1 from senescent tumor cells enhance monocyte differentiation into M2 macrophages, which inhibit CD8+ T cell activation. Neutralization of CXCL12/CSF1 increases the effect of anti‐PD1 antibody in allograft tumors. Furthermore, inhibition of CXCL12 from senescent tumor cells enhances T cell infiltration and results in reducing the number and size of tumors in azoxymethane (AOM)/dextran sulfate sodium (DSS)‐induced CRC. These findings suggest senescent tumor cells generate a cytokine barrier protecting nonsenescent tumor cells from immune attack and provide a new target for overcoming the immunotherapy resistance of CRC.

N-Linked glycosylation profiles of therapeutic induced senescent (TIS) triple negative breast cancer cells (TNBC) and their extracellular vesicle (EV) progeny.

Triple negative breast cancer (TNBC) has poor clinical outcomes and limited treatment options. Chemotherapy, while killing some cancer cells, can result in therapeutic-induced-senescent (TIS) cells. Senescent cells release significantly more extracellular vesicles (EVs) than non-senescent cells. Recently, N- and O-linked glycosylation alterations have been associated with senescence. We aimed to profile the N-linked glycans of whole cells, membrane, cytoplasm and EVs harvested from TIS TNBC cells and to compare these to results from non-senescent cells. TIS was induced in the Cal51 TNBC cells using the chemotherapeutic agent paclitaxel (PTX). Ultra-performance liquid chromatography (UPLC) analysis of exoglycosidase digested N-linked glycans was carried out on TIS compared to non-treated control cells. LC-Mass spectrometry (MS) analysis of the N-linked glycans and lectin blotting of samples was carried out to confirm the UPLC results. Significant differences were found in the N-glycan profile of the Cal51 membrane, cytoplasm and EV progeny of TIS compared to non-senescent cells. Protein mass spectrometry showed that the TIS cells contain different glycan modifying enzymes. The lectin, calnexin demonstrated a lower kDa size (∼58 kDa) in TIS compared to control cells (∼90 kDa) while Galectin 3 demonstrated potential proteolytic cleavage with 32 kDa and ∼22 kDa bands evident in TIS compared to non-senescent control cells with a major 32 kDa band only. TIS CAL51 cells also demonstrated a reduced adhesion to collagen I compared to control non-senescent cells. This study has shown that therapeutic-induced-senescent TNBC cells and their EV progeny, display differential N-glycan moieties compared to non-senescent Cal51 cells and their resultant EV progeny. For the future, N-glycan moieties on cancer senescent cells and their EV progeny hold potential for (i) the monitoring of treatment response as a liquid biopsy, and (ii) cancer senescent cell targeting with lectin therapies.

The lncRNA MEG3/miR-16-5p/VGLL4 regulatory axis is involved in etoposide-induced senescence of tumor cells.

The senescence of tumor cells is an important tumor suppressor mechanism. The present study aimed to investigate the role of long non-coding RNA (lncRNA) MEG3 (maternally expressed gene 3) in the senescence process of tumor cells and its potential molecular mechanism by competitively binding with microRNA miR-16-5p to regulate the expression of VGLL4 (encoding vestigial like family member 4). We used etoposide to construct senescence models of tumor cells. The degree of cellular senescence was detected by senescence-associated β-galactosidase, cell cycle and senescence-associated secretory phenotype. The expression of lncRNA MEG3, miR-16-5p and VGLL4 in senescent or non-senescent cells was evaluated using a quantitative real-time reverse transcriptase-PCR (qRT-PCR) or western blotting. Dual luciferase reporter assays were used to detect the binding of miR-16-5p to lncRNA MEG3 and VGLL4. The mRNA and protein expression levels of senescence-related markers (p53, p21 and p16) were detected using qRT-PCR or western blotting. Compared to the control group, the expression of lncRNA MEG3 and VGLL4 was significantly up-regulated in senescent cells. Knockdown of lncRNA MEG3 and VGLL4 reduced the degree of senescence and the expression of p21 and p16. lncRNA MEG3 interfered with the expression of miR-16-5p in senescent A549 and MCF-7 cells. The expression of VGLL4 was regulated by miR-16-5p in senescent A549 and MCF-7 cells. lncRNA MEG3 participated in the senescent progress of tumor cells induced by etoposide via the miR-16-5p/VGLL4 axis. The present study has confirmed the regulatory role of the lncRNA MEG3/miR-16-5p/VGLL4 axis in the low-dose etoposide-induced tumor cell senescence model, which has potential clinical application with respect to treating malignant tumors.

CBIO-11. NOVEL THERAPY TO TARGET PR-RECURRENT GLIOMA

Abstract BACKGROUND Glioblastoma is a fatal infiltrative primary brain tumor, and standard care includes maximal safe surgical resection followed by radiation and Temozolomide (TMZ). Therapy-resistant residual cells persist in a latent state a long time before inevitable recurrence. Conventional radiation and Temozolomide (TMZ) treatment cause oxidative stress and DNA damage resulting senescent-like state of cell-cycle arrest. However, increasing evidence demonstrates escaping senescence leads to tumor recurrence. Thus, the ablation of senescent tumor cells after chemoradiation may be an avenue to limit tumor recurrence. METHODS 100uM TMZ for 7days or 10-20Gy radiation (cesium gamma radiator) was used for senescence induction in human glioblastoma in vitro and confirmed by SA-Beta gal staining and PCR. Replication arrest assessed by automated quantification of cellular confluence (Thermo Scientific Series 8000 WJ Incubator). We evaluated the IC50 for several senolytics targeting multiple SCAPs, including Dasatinib, Quercetin, AMG-232, Fisetin, Onalespib, Navitoclax, and A1331852, and in senescent vs. proliferating cells. RESULTS Among the senolytic tested, the Bcl-XL inhibitors A1331852 and Navitoclax both shown senolytic effect by selectively killing radiated, senescent tumor cells at lower concentrations as compared to 0Gy treated non-senescent cells. Across 12 GBM cell lines, IC50 for senescent cells was 6–500 times lower than non-senescent GBM(p< 0.005). Such differential sensitivity to Bcl-XL inhibition after radiation has also observed by BCL-XL knockdown in radiated glioma. CONCLUSION These findings suggest the potential to harness radiation-induced biology to ablate surviving quiescent cells and demonstrate Bcl-XL dependency as a potential vulnerability of surviving tumor cells after exposure to chemoradiation.

Identification of a novel senomorphic agent, avenanthramide C, via the suppression of the senescence-associated secretory phenotype

Senescent cells are deeply involved in the induction of tissue damage and aging-related diseases. The identification of factors that eliminate senescent cells or inhibit the senescence-associated secretory phenotype (SASP) in these cells is necessary. Here, we report an avenanthramice C (Avn C) extracted from oat as a new SASP modulator. Treatment with Avn C led to a significant reduction in the levels of markers of senescent cells, with no toxicity observed. The SASP was also inhibited by Avn C treatment, similar to non-senescent cells, and the suppression of cell division by autocrine signals associated with SASP was restored. To investigate the mechanism underlying SASP inhibition by Avn C, we analyzed the effect of Avn C in lipopolysaccharide (LPS)-induced inflammation in non-senescent cells. Avn C inhibited nuclear factor κB (NF-κB) activity and the secretion of inflammatory cytokines before or after LPS treatment. Although the activity of MAP kinases, which are NF-κB upstream signals, was inhibited by Avn C in LPS-induced inflammation, only p38 activity was specifically inhibited in senescent cells. Interestingly, the inhibition of p38 in senescent cells was observed through Avn C-induced 5′-adenosine monophosphate-activated protein kinase (AMPK) activity. Avn C-induced inhibition of the SASP is triggered by senescence-related stress.

Abstract 5831: Clearance of therapy-induced senescent cancer cells by the senolytic ABT-263 in a model of non-small cell lung cancer

Abstract While most chemotherapies will initially lead to tumor reduction in patients, residual tumor cells that enter a state of dormancy can re-emerge as aggressive, recurrent disease after treatment is terminated. One mechanism by which cancer cells may escape chemotherapy-induced death is senescence. Senescence is a specialized form of cell cycle arrest that is primarily characterized as a durable, prolonged loss of proliferative capacity. While senescence was traditionally considered to be irreversible, evidence has accumulated in recent years supporting the premise that senescence is instead a durable form of growth arrest from which a subpopulation of cells can ultimately escape. Moreover, the accumulation of senescent cells post cancer therapy has been linked with a plethora of unfavorable outcomes. A sequential approach in which chemotherapy is followed by a senolytic, an emerging class of drugs that selectively kill senescent cells, may therefore delay or circumvent recurrent disease by eliminating residual senescent cells. To this end, we tested whether etoposide- or radiation-treated non-small cell lung cancer (NSCL) cells become susceptible to the BH-3 mimetic ABT-263– an established senolytic– following senescence induction. Treatment with etoposide or radiation induced a robust senescent state, as evidenced by the development of multiple senescent phenotypes. Senescent tumor cells demonstrated increased sensitivity to ABT-263 compared to non-senescent cells, undergoing rapid apoptosis subsequent to ABT-263 exposure. We further determined that ABT-263's mechanism of action appears to depend upon the disruption of the BCL-XL/Bax interaction. ABT-263's capacity to eliminate senescent cells is conserved in vivo, as tumor-bearing animals treated with etoposide followed by ABT-263 undergo rapid tumor reduction followed by tumor maintenance, while etoposide-only treated animals develop progressive disease subsequent to treatment completion. These data support the use of senolytics as complementary therapy to eliminate residual tumor cells following chemotherapy or radiation, and thereby potentially delay or reduce recurrent disease. Citation Format: Valerie Carpenter, Tareq Saleh, David Gewirtz. Clearance of therapy-induced senescent cancer cells by the senolytic ABT-263 in a model of non-small cell lung cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5831.

A rapid-response near-infrared fluorescent probe with a large Stokes shift for senescence-associated β-galactosidase activity detection and imaging of senescent cells

Detection of senescence-associated β-galactosidase (SA-β-gal) activity at an increased lysosomal pH is one of the most reliable approaches for probing senescent cells. Herein, we developed a novel near-infrared fluorescent probe (KSL0401) for sensing SA-β-gal activity and applying in visualization of aging. This probe was designed by connecting a cleavable d-galactose residue to the coumarin-dicyanoisophorone hybrid fluorophore KSL0401-OH via a β-glycosidic bond. The spectral properties of KSL0401 reaction with β-gal were measured in aqueous solution at pH 6.0, and a remarkable enhancement of fluorescence emission at 706 nm was observed, along with a visible colorimetric change. Probe KSL0401 was highly selective and sensitive to SA-β-gal, and displayed large Stokes shift (218 nm) and good biocompatibility. Moreover, it exhibited extremely rapid response to β-gal within 80 s, which surpassed most of the reported SA-β-gal probes. Additionally, KSL0401 also enabled to detect the endogenous SA-β-gal activity in living cells, and distinguish between senescent cells and non-senescent cells and qualify the degree of aging. These findings demonstrate that KSL0401 is a potential detection tool in the imaging research of aging.

The Premature Senescence in Breast Cancer Treatment Strategy.

Cellular senescence is a permanent blockade of cell proliferation. In response to therapy-induced stress, cancer cells undergo apoptosis or premature senescence. In apoptosis-resistant cancer cells or at lower doses of anticancer drugs, therapy-induced stress leads to premature senescence. The role of this senescence in cancer treatment is discussable. First of all, the senescent cells lose the ability to proliferate, migrate, and invade. In addition, the senescent cells secrete a set of proteins (inflammatory cytokines, chemokines, growth factors) known as the senescence-associated secretory phenotype (SASP), which influences non-senescent normal cells and non-senescent cancer cells in the tumor microenvironment and triggers tumor promotion and recurrence. Recently, many studies have examined senescence induction through breast cancer therapy and potentially using this phenomenon to treat this cancer. This review summarizes the recent in vitro, in vivo, and clinical studies investigating senescence in breast cancer treatments. Senescence inductors, senolytics, as well as their action mechanism are discussed herein. Potential SASP-modulating treatment strategies are also described.

Hydrogel Culture Surface Stiffness Modulates Mesenchymal Stromal Cell Secretome and Alters Senescence.

Current cell culture surfaces used for the expansion and production of mesenchymal stromal cells (MSCs) are not optimized for the production of highly secretory and nonsenescent cells. In this study, we used poly (ethylene glycol) hydrogel substrates with tunable mechanical and biochemical properties to screen the effect of culture surfaces on pro-regenerative secretome by multiplex enzyme-linked immunosorbent assay, proliferation by PicoGreen DNA analysis, and senescence by senescence-associated β-galactosidase activity. We demonstrate that MSCs cultured on 30 kPa hydrogels, regardless of biochemical functionalization, broadly enhanced the secretion of immunomodulatory and regenerative factors versus stiffer 100 kPa or tissue culture plastic surfaces, but did not support robust proliferation. In contrast, culture on 100 kPa hydrogel surfaces promoted proliferation at a similar level and did not substantially alter the amount of secreted factors as compared with tissue culture plastic. Culture on integrin-engaging, cadherin-engaging, and hyaluronic acid-containing 30 kPa substrates enhanced MSC-conditioned media (CM) angiogenic activity in a human umbilical vein endothelial cell tube formation assay and human THP-1 monocyte chemoattraction in a transwell assay. However, 30 kPa substrate culture did not impact the myogenic activity of MSC CM in a C2C12 myoblast tube formation assay. Culture on selected 100 kPa surfaces enhanced CM angiogenic activity and monocyte chemotaxis, but not myogenic activity. Serial culture on 100 kPa RGD hydrogel surfaces significantly reduced senescence in MSCs versus tissue culture plastic, while maintaining the capacity of the cells to enhance their secretome in response to 30 kPa surfaces. Thus, hydrogel substrates that exhibit stiffness orders of magnitude lower than standard tissue culture plastic can serve as novel surfaces for the production of MSCs with an improved therapeutic secretory capacity and reduced senescence. Impact statement The success of mesenchymal stromal cell (MSC)-based therapies is dependent on the manufacture of a large number of cells with high therapeutic potency. Among the culture surfaces tested in this study, we demonstrate that substrate stiffness rather than biochemical functionalization predominantly guides changes in MSC proliferation and secretory capacity. We have identified substrate parameters to support MSC proliferation, enhance secretion of paracrine factors, and to reduce replicative senescence. By maximizing secretory capacity and reducing senescence through the choice of hydrogel culture materials, these findings have great potential to improve the large-scale production of therapeutic MSCs.

Senescence as an Amyloid Cascade: The Amyloid Senescence Hypothesis.

Due to their postmitotic status, the potential for neurons to undergo senescence has historically received little attention. This lack of attention has extended to some non-postmitotic cells as well. Recently, the study of senescence within the central nervous system (CNS) has begun to emerge as a new etiological framework for neurodegenerative diseases such as Alzheimer’s disease (AD) and Parkinson’s disease (PD). The presence of senescent cells is known to be deleterious to non-senescent neighboring cells via development of a senescence-associated secretory phenotype (SASP) which includes the release of inflammatory, oxidative, mitogenic, and matrix-degrading factors. Senescence and the SASP have recently been hailed as an alternative to the amyloid cascade hypothesis and the selective killing of senescence cells by senolytic drugs as a substitute for amyloid beta (Aß) targeting antibodies. Here we call for caution in rejecting the amyloid cascade hypothesis and to the dismissal of Aß antibody intervention at least in early disease stages, as Aß oligomers (AßO), and cellular senescence may be inextricably linked. We will review literature that portrays AßO as a stressor capable of inducing senescence. We will discuss research on the potential role of secondary senescence, a process by which senescent cells induce senescence in neighboring cells, in disease progression. Once this seed of senescent cells is present, the elimination of senescence-inducing stressors like Aß would likely be ineffective in abrogating the spread of senescence. This has potential implications for when and why AßO clearance may or may not be effective as a therapeutic for AD. The selective killing of senescent cells by the immune system via immune surveillance naturally curtails the SASP and secondary senescence outside the CNS. Immune privilege restricts the access of peripheral immune cells to the brain parenchyma, making the brain a safe harbor for the spread of senescence and the SASP. However, an increasingly leaky blood brain barrier (BBB) compromises immune privilege in aging AD patients, potentially enabling immune infiltration that could have detrimental consequences in later AD stages. Rather than an alternative etiology, senescence itself may constitute an essential component of the cascade in the amyloid cascade hypothesis.

Impaired Myofibroblast Dedifferentiation Contributes to Nonresolving Fibrosis in Aging.

Idiopathic pulmonary fibrosis (IPF) is a fatal age-associated disease with no cure. Although IPF is widely regarded as a disease of aging, the cellular mechanisms that contribute to this age-associated predilection remain elusive. In this study, we sought to evaluate the consequences of senescence on myofibroblast cell fate and fibrotic responses to lung injury in the context of aging. We demonstrated that nonsenescent lung myofibroblasts maintained the capacity for dedifferentiation, whereas senescent/IPF myofibroblasts exhibited an impaired capacity for dedifferentiation. We previously demonstrated that the transcription factor MyoD acts as a critical switch in the differentiation and dedifferentiation of myofibroblasts. Here, we demonstrate that decreased levels of MyoD preceded myofibroblast dedifferentiation and apoptosis susceptibility in nonsenescent cells, whereas MyoD expression remained elevated in senescent/IPF myofibroblasts, which failed to undergo dedifferentiation and demonstrated resistance to apoptosis. Genetic strategies to silence MyoD restored the susceptibility of IPF myofibroblasts to undergo apoptosis and led to a partial reversal of age-associated persistent fibrosis in vivo. The capacity for myofibroblast dedifferentiation and subsequent apoptosis may be critical for normal physiologic responses to tissue injury, whereas restricted dedifferentiation and apoptosis resistance in senescent cells may underlie the progressive nature of age-associated human fibrotic disorders. These studies support the concept that senescence may promote profibrotic effects via impaired myofibroblast dedifferentiation and apoptosis resistance, which contributes to myofibroblast accumulation and ultimately persistent fibrosis in aging.

Galacto-conjugation of Navitoclax as an efficient strategy to increase senolytic specificity and reduce platelet toxicity.

Pharmacologically active compounds with preferential cytotoxic activity for senescent cells, known as senolytics, can ameliorate or even revert pathological manifestations of senescence in numerous preclinical mouse disease models, including cancer models. However, translation of senolytic therapies to human disease is hampered by their suboptimal specificity for senescent cells and important toxicities that narrow their therapeutic windows. We have previously shown that the high levels of senescence‐associated lysosomal β‐galactosidase (SA‐β‐gal) found within senescent cells can be exploited to specifically release tracers and cytotoxic cargoes from galactose‐encapsulated nanoparticles within these cells. Here, we show that galacto‐conjugation of the BCL‐2 family inhibitor Navitoclax results in a potent senolytic prodrug (Nav‐Gal), that can be preferentially activated by SA‐β‐gal activity in a wide range of cell types. Nav‐Gal selectively induces senescent cell apoptosis and has a higher senolytic index than Navitoclax (through reduced activation in nonsenescent cells). Nav‐Gal enhances the cytotoxicity of standard senescence‐inducing chemotherapy (cisplatin) in human A549 lung cancer cells. Concomitant treatment with cisplatin and Nav‐Gal in vivo results in the eradication of senescent lung cancer cells and significantly reduces tumour growth. Importantly, galacto‐conjugation reduces Navitoclax‐induced platelet apoptosis in human and murine blood samples treated ex vivo, and thrombocytopenia at therapeutically effective concentrations in murine lung cancer models. Taken together, we provide a potentially versatile strategy for generating effective senolytic prodrugs with reduced toxicities.

Alteration of fatty acid oxidation by increased CPT1A on replicative senescence of placenta-derived mesenchymal stem cells

BackgroundHuman placenta-derived mesenchymal stem cells (PD-MSCs) are powerful sources for cell therapy in regenerative medicine. However, a limited lifespan by senescence through mechanisms that are well unknown is the greatest obstacle. In the present study, we first demonstrated the characterization of replicative senescent PD-MSCs and their possible mitochondrial functional alterations.MethodsHuman PD-MSCs were cultured to senescent cells for a long period of time. The cells of before passage number 8 were early cells and after passage number 14 were late cells. Also, immortalized cells of PD-MSCs (overexpressed hTERT gene into PD-MSCs) after passage number 14 were positive control of non-senescent cells. The characterization and mitochondria analysis of PD-MSCs were explored with long-term cultivation.ResultsLong-term cultivation of PD-MSCs exhibited increases of senescent markers such as SA-β-gal and p21 including apoptotic factor, and decreases of proliferation, differentiation potential, and survival factor. Mitochondrial dysfunction was also observed in membrane potential and metabolic flexibility with enlarged mitochondrial mass. Interestingly, we founded that fatty acid oxidation (FAO) is an important metabolism in PD-MSCs, and carnitine palmitoyltransferase1A (CPT1A) overexpressed in senescent PD-MSCs. The inhibition of CPT1A induced a change of energy metabolism and reversed senescence of PD-MSCs.ConclusionsThese findings suggest that alteration of FAO by increased CPT1A plays an important role in mitochondrial dysfunction and senescence of PD-MSCs during long-term cultivation.

Small extracellular vesicles deliver miR-21 and miR-217 as pro-senescence effectors to endothelial cells

ABSTRACT The role of epigenetics in endothelial cell senescence is a cutting-edge topic in ageing research. However, little is known of the relative contribution to pro-senescence signal propagation provided by microRNAs shuttled by extracellular vesicles (EVs) released from senescent cells. Analysis of microRNA and DNA methylation profiles in non-senescent (control) and senescent (SEN) human umbilical vein endothelial cells (HUVECs), and microRNA profiling of their cognate small EVs (sEVs) and large EVs demonstrated that SEN cells released a significantly greater sEV number than control cells. sEVs were enriched in miR-21-5p and miR-217, which target DNMT1 and SIRT1. Treatment of control cells with SEN sEVs induced a miR-21/miR-217-related impairment of DNMT1-SIRT1 expression, the reduction of proliferation markers, the acquisition of a senescent phenotype and a partial demethylation of the locus encoding for miR-21. MicroRNA profiling of sEVs from plasma of healthy subjects aged 40–100 years showed an inverse U-shaped age-related trend for miR-21-5p, consistent with senescence-associated biomarker profiles. Our findings suggest that miR-21-5p/miR-217 carried by SEN sEVs spread pro-senescence signals, affecting DNA methylation and cell replication.