Senescence-associated Secretory Phenotype Markers Research Articles

Abstract 2954: Androgen deprivation therapy (ADT) and senescence-associated secretory phenotype (SASP) in vitro: Correlation with SASP in tumor specimens as well as in the serum of patients after ADT

Abstract Introduction: Androgen Deprivation Therapy (ADT) is a highly effective treatment for prostate cancer. However, resistance to ADT develops in most patients, leading to a lethal state of castration-resistant prostate cancer (CRPC). Senescent prostate cancer (SPC) cells can arise after ADT and contribute to therapy resistance. SPCs can restore replicative function and alter the tumor microenvironment through pro-tumorigenic senescence-associated secretory phenotype (SASP). We aimed to characterize changes in SASP in prostate cancer cell lines and serum samples from patients receiving ADT. Methods: SASP factors (Luminex 100/200 System, xMAP Instruments) were analyzed in patients undergoing ADT. To correlate SASP with the sensitivity to ADT, we sought to evaluate the regulation of SASP in vitro using PCa cell lines. The PC3 (AR resistant) and LNCaP (AR sensitive) cell lines were used in the experiments. We performed an analysis of archival tumor tissue (paired) from 18 patients with or without alterations in DNA repair genes (BRCA1, BRCA2, and CDK12). We performed immunohistochemical (IHC) staining of known SASP markers, such as P16, uPAR, gamma-H2AX, and Lamin B1. Results: In a cohort of patients (n=8) with pre and post-ADT matched status, we demonstrate increased levels of SASP factors such as IL-6 (pre-ADT: 1.54±0.24 pg/ml, post-ADT: 5.23±1.3 pg/ml p=0.001), IL-7 (pre-ADT: 3.11±0.6 pg/ml, post-ADT: 13.5±2.7 pg/ml p=0.01), IL-1 alpha (pre-ADT: 1.06±0.3 pg/ml, post-ADT: 15.3±3.5 pg/ml p=0.001), IL-15 (pre-ADT: 15.6±3.1 pg/ml, post-ADT: 37.1±4.8 pg/ml p=0.002), MIF (pre-ADT: 1036±67.6pg/ml, post-ADT: 2362±55.5 pg/ml p=0.003), and IL-15 (pre-ADT: 5±2.1 pg/ml, post-ADT: 37.5±5.1 pg/ml p=0.002). In vitro, the AR-responsive LNCaP cell line, exposed to AR blockade, showed similar findings in upregulating IL-6, IL-7, and IL-15 but not the PC3 cell line. We also demonstrate measurable differences between the expression of u-PAR, gamma-2AX, p16, and loss of Lamin B1 expression in tumor specimens from patients with advanced castration-resistant prostate cancer (n=18). The expression of p16 was significantly upregulated post-treatment irrespective of DDR status (p=0.0417). Other SASP factors showed a trend in upregulation but were insignificant (u-PAR p=0.056, gamma-H2AX p=0.62, Lamin B1 p=0.72) Conclusion: These results provide evidence of systemic increases in SASP-related cytokines following treatment with ADT. The in vitro results suggest that secretion of SASP is promoted by an AR-mediated mechanism. Preliminary results from tumor specimens suggest increased senescence marker p16 expression induced by therapy. Ongoing experiments are further characterizing the senescence markers in prostate tumor specimens and correlated to treatment effect. Citation Format: Maximilian Schwermann, Matthew Hadfield, Shaolei Lu, Praveen Srinivasan, Vida Tajiknia, William MacDonald, Andrew George, Shengliang Zhang, Leiqing Zhang, Kimberly Meza, Andre De Souza, Dragan Golijanin, Elias Hyams, Galina Lagos, Sheldon Holder, Anthony Mega, John Sedivy, Howard Safran, Wafik El-Deiry, Benedito Carneiro. Androgen deprivation therapy (ADT) and senescence-associated secretory phenotype (SASP) in vitro: Correlation with SASP in tumor specimens as well as in the serum of patients after ADT [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2954.

Effect of miR-21 in mesenchymal stem cells-derived extracellular vesicles behavior

BackgroundA challenging new branch of research related to aging-associated diseases is the identification of miRNAs capable of modulating the senescence-associated secretory phenotype (SASP) which characterizes senescent cells and contributes to driving inflammation.MethodsMesenchymal stem cells (MSC) from human umbilical cord stroma were stable modified using lentivirus transduction to inhibit miR-21-5p and shotgun proteomic analysis was performed in the MSC-derived extracellular vesicles (EV) to check the effect of miR-21 inhibition in their protein cargo. Besides, we studied the paracrine effect of those modified extracellular vesicles and also their effect on SASP.ResultsSyndecan-1 (SDC1) was the most decreased protein in MSC-miR21−-derived EV, and it was involved in inflammation and EV production. MSC-miR21−-derived EV were found to produce a statistically significant inhibitory effect on SASP and inflammaging markers expression in receptor cells, and in the opposite way, these receptor cells increased their SASP and inflammaging expression statistically significantly when treated with MSC-miR-21+-derived EV.ConclusionThis work demonstrates the importance of miR-21 in inflammaging and its role in SASP through SDC1.Graphical abstract

Brain extracellular vesicles from Alzheimer’s Disease and Down Syndrome patients mediate spread of senescence‐like phenotype in vitro

AbstractBackgroundAlzheimer’s disease (AD) is the prevalent cause of dementia in older adults. Down Syndrome (DS) is the most common chromosomal disorder with strong predisposition to the development of AD. Cellular senescence contributes in the pathophysiology of both AD and DS but the mechanisms are unclear. Senescence associated secretory phenotype (SASP), which refers to a complex of secreted mediators, including soluble factors and extracellular vesicles (EVs) may contribute to the spread of senescence to bystander cells but its role in neurodegenerative disorders has not been evaluated.MethodParietal cortex tissue from patients with AD, DS, and normal/low‐pathology (NL) cases was obtained from the UC Irvine ADRC. The cryopreserved human tissue was used for small EV isolation by ultracentrifugation and size exclusive chromatography (SEC). Purity of the EV fractions was confirmed by immunoblotting against positive (CD63, CD9, Syntenin‐1) and negative (calnexin and GM130) EV/exosomal markers, transmission electron microscopy, and nanoparticle tracking analysis. Cell‐type specific EV fractions were isolated by immunoprecipitation (IP). Senescence transfer potential of brain‐derived EVs were assessed in neuroblastoma and glioma cells using senescence associate beta‐galactosidase activity assay, and confirmed by other senescence markers.ResultOur preliminary data demonstrated the ability of AD and DS, but not NL samples, to transfer senescence to neuroblastoma SH‐SY5Y and glioma U87‐MG cells. Senescence associate beta‐galactosidase activity was on average around 15% higher in cells treated with AD brain derived EVs (AD‐EVs) compared to NL brain EVs (NL‐EVs). This data correlates with around 20% elevation in p21 expression in AD‐EV treated cells. When senescence transfer potentials were compared between AD‐EVs and DS brain derived EVs (DS‐EVs), DS‐EVs exceeded AD‐EVs in their ability to transfer senescence‐like phenotype with around 40% increase in senescence associate beta‐galactosidase activity in cells treated with DS‐EVs. We are currently evaluating levels of senescence‐associated and SASP markers in AD and DS cells and cell‐type specific brain derived small EVs.ConclusionWe demonstrated an ability of AD and DS brain derived EVs to transfer senescence‐like phenotype in vitro. This phenomenon deserves further investigation to estimate its impact on the progression of AD in Down and non‐Down population along with evaluation its therapeutic potential.

The senescence-associated secretome for prediction of mortality in patients with aortic valve stenosis undergoing TAVR

Abstract Background Cellular senescence is an essential mechanism in the process of aging. The senescence-associated secretory phenotype (SASP) includes mainly pro-inflammatory cytokines, proteases, soluble signalling factors, and growth modulators. Degenerative aortic valve stenosis (AVS) is the prototypical inflammatory age-related cardiovascular disease. Purpose Our aim was to assess whether circulating concentrations of SASP factors are associated with prognosis in elderly patients with AVS undergoing transcatheter aortic valve replacement (TAVR), the current standard therapy for severe cases. Methods A total of 24 SASPs were measured using the Luminex Multiplex ELISA Assay (Biotechene, GmbH) in 120 AVS patients undergoing TAVR between February 2017 and February 2020 with a prospective follow-up of up to 4 years. Receiver operating characteristic (ROC) curves were created for each SASP protein in order to identify predictive markers. SASPs were considered positive if their baseline value was above the cut-off value derived from ROC-curves. The only 3 independent predictive factors were selected to develop an easy-to-calculate score based on the number of positive SASP proteins (0 to 3). Primary endpoint was long-term all-cause mortality. Results Median (IQR) age of the population was 83.2 (78.9-87.0) years, 56.7% were women. According to ROC curves, 9 out of 24 SASPs showed a significant area under the curve (AUC), with values above 0.623 (see Table). Only 3 of the markers reflecting potentially different underlying mechanisms remained independent predictors, after including all SASP factors in a Cox Regression analysis: GDF-15, ICAM-1 and Osteoprotegerin (all, p<0.03). A score based on the number of baseline factors being above the AUC-derived threshold level showed an excellent discrimination ability to predict survival, with long-term all-cause mortality substantially increasing for each additional positive marker (see Kaplan-Meier curves). Conclusion In conclusion, the presence of elevated baseline levels for 0, 1, 2, or 3 of the independent SASP factors reflecting different underlying pathophysiological mechanisms predicts increased long-term all-cause mortality with profoundly improved discrimination power in AVS patients undergoing TAVR. If confirmed in further prospective studies, a combination of these 3 SASP markers could serve as a highly useful risk stratification tool in the elderly population with AVS.

Sexual dimorphic metabolic and cognitive responses of C57BL/6 mice to Fisetin or Dasatinib and quercetin cocktail oral treatment

Senolytic treatment in aged mice clears senescent cell burden leading to functional improvements. However, less is known regarding the effects of these compounds when administered prior to significant senescent cell accumulation. From 4–13 months of age, C57BL/6 male and female mice received monthly oral dosing of either 100 mg/kg Fisetin or a 5 mg/kg Dasatinib (D) plus 50 mg/kg Quercetin (Q) cocktail. During treatment, several aspects of healthy aging were assayed including glucose metabolism using an insulin and glucose tolerance test, cognitive performance using Morris water maze and novel object recognition, and energy metabolism using indirect calorimetry. Afterwards, mice were euthanized for plasma, tissue specific markers of senescence-associated secretory phenotype (SASP), and white adipose tissue accumulation (WAT). Sexually dimorphic treatment effects were observed. Fisetin treated male mice had reduced SASP, enhanced glucose and energy metabolism, improved cognitive performance, and increased mRNA expression of adiponectin receptor 1 and glucose transporter 4. D + Q treatment had minimal effects in male C57BL/6 mice, but was detrimental to females causing increased SASP expression along with accumulation of WAT depots. Reduced energy metabolism and cognitive performance were also noted. Fisetin treatment had no effect in female C57BL/6 mice potentially due to a slower rate of biological aging. In summary, the senolytic treatment in young adulthood, has beneficial, negligible, or detrimental effects in C57BL/6 mice dependent upon sex and treatment. These observations should serve as a note of caution in this rapidly evolving and expanding field of investigation.Graphical Male and female C57BL/6 mice were treated with once monthly oral doses of either Dasatinib (D) + Quercetin (Q) or Fisetin from 4–13 months of age. Males treated with Fisetin had reduced SASP markers (blue spheres) as well as improved metabolism (red flame) and cognition. Females treated with D + Q had increased adiposity and SASP markers (red spheres) along with decreased metabolism (blue flame) and cognitive performance. No effects were observed in females treated with Fisetin or males treated with D + Q.

In vitro and in vivo effects of zoledronic acid on senescence and senescence-associated secretory phenotype markers.

In addition to reducing fracture risk, zoledronic acid has been found in some studies to decrease mortality in humans and extend lifespan and healthspan in animals. Because senescent cells accumulate with aging and contribute to multiple co-morbidities, the non-skeletal actions of zoledronic acid could be due to senolytic (killing of senescent cells) or senomorphic (inhibition of the secretion of the senescence-associated secretory phenotype [SASP]) actions. To test this, we first performed in vitro senescence assays using human lung fibroblasts and DNA repair-deficient mouse embryonic fibroblasts, which demonstrated that zoledronic acid killed senescent cells with minimal effects on non-senescent cells. Next, in aged mice treated with zoledronic acid or vehicle for 8 weeks, zoledronic acid significantly reduced circulating SASP factors, including CCL7, IL-1β, TNFRSF1A, and TGFβ1 and improved grip strength. Analysis of publicly available RNAseq data from CD115+ (CSF1R/c-fms+) pre-osteoclastic cells isolated from mice treated with zoledronic acid demonstrated a significant downregulation of senescence/SASP genes (SenMayo). To establish that these cells are potential senolytic/senomorphic targets of zoledronic acid, we used single cell proteomic analysis (cytometry by time of flight [CyTOF]) and demonstrated that zoledronic acid significantly reduced the number of pre-osteoclastic (CD115+/CD3e-/Ly6G-/CD45R-) cells and decreased protein levels of p16, p21, and SASP markers in these cells without affecting other immune cell populations. Collectively, our findings demonstrate that zoledronic acid has senolytic effects in vitro and modulates senescence/SASP biomarkers in vivo. These data point to the need for additional studies testing zoledronic acid and/or other bisphosphonate derivatives for senotherapeutic efficacy.

Cellular senescence is associated with trisomies during human lung development

Rationale Human chromosomal anomalies, notably trisomies, induce several alterations in gene expression, leading to various disease phenotypes. Recent studies showed that dermal fibroblasts from patients with trisomy 13 (T13), T18 or T21 present features of increased senescence and oxidative stress. We recently demonstrated that T21 lungs present abnormalities prenatally. Senescence is a state of cell division arrest that plays an important role in organogenesis. However, little is known about cellular senescence in fetal human lung with T13, T18 and T21. We therefore sought to characterize cellular senescence in these lung tissues. Methods Fresh human fetal lung tissues with T13, T18 and T21 were collected along with euploid age and sex matched controls. RT-qPCR and immunofluorescence staining (IF) were performed to assess the expression of senescence markers as well as Senescence-Associated Secretory Phenotype (SASP) elements in the various lung compartments (epithelial and mesenchymal). In addition, fibroblasts were isolated and cultured from each sample to determine the extent of senescence on isolated mesenchyme by IF and RT-qPCR. Staining for oxidative stress detection and Reactive oxygen species were assessed by CellROX and MitoSOX. Results RT-qPCR analysis in T13 whole lung tissues demonstrated no significant differences in the expression of either senescence or SASP markers as compared to control ( P16/ P21/ TP53 and IL6/ IL1 β/ TGF β/ CXCL8 respectively; n=5). Interestingly however, IF staining in T13 tissues showed a significant increase of γ-H2AFX, a marker of senescence, in the mesenchyme (n=4) as well as significantly decreased P21 staining in both the epithelial and mesenchymal compartments. Additionally, isolated fibroblasts from T13 lungs expressed significantly more IL6 as compared to control (n=3), with IF staining demonstrating a significant increase of γ-H2AFX. T18 lungs displayed significantly increased expression of P16 as compared to control (n=6), whereas IF staining revealed an increase of γ-H2AFX in whole tissue (n=4). Fibroblasts isolated from T18 lungs revealed no significant difference in any markers (gene or protein). Lastly, T21 lungs showed a significant increase of CXCL8 expression as compared to control (n=6). IF staining of T21 lungs demonstrated an increase γ-H2AFX in both the epithelium and mesenchyme (n=8). However, isolated T21 lung fibroblasts displayed significantly increased expression of P16, P21, TP53, and CXCL8. Furthermore, IF staining of T21 lung fibroblasts displayed significantly more γ-H2AFX and P21 positive cells as compared to controls (n=5). Interestingly, only cells from T21 lungs presented a significant increase of CellROX and MitoSOX staining (n=5). Conclusion In this study we established that expression of both senescence and SASP markers differ depending on the trisomy. Our results suggest that cellular senescence is more pronounced in lungs from individuals with trisomy 21. These authors acknowledge funding from NIH/NHLBI R01HL141856 (to DAA); NIH/ Office Of The Director, National Institutes Of Health (OD) R01HL155104 (SD); NIH/NHLBI R21HL165411 (SD and DAA); CIRM training grant EDUC4-12837 (RB) and NIH/NICHD 2R24HD000836-52 (IG). This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Premature senescence of the liver in Alagille patients.

Alagille syndrome (ALGS) is an autosomal dominant disease characterized by a multisystem involvement including bile duct paucity and cholestasis, caused by JAG1 or NOTCH2 mutations in most of the cases. Jagged1-Notch2 interactions are known to be crucial for intrahepatic biliary tract development, but the Notch signaling pathway is also involved in the juxtacrine transmission of senescence and in the induction and modulation of the senescence-associated secretory phenotype (SASP). Our aim was to investigate premature senescence and SASP in ALGS livers. Liver tissue from ALGS patients was prospectively obtained at the time of liver transplantation (n = 5) and compared to control livers (n = 5). We evidenced advanced premature senescence in the livers of five JAG1 mutated ALGS pediatric patients through increased senescence-associated beta-galactosidase activity (p<0.05), increased p16 and p21 gene expression (p<0.01), and increased p16 and γH2AX protein expression (p<0.01). Senescence was located in hepatocytes of the whole liver parenchyma as well as in remaining bile ducts. The classical SASP markers TGF-β1, IL-6, and IL-8 were not overexpressed in the livers of our patients. We demonstrate for the first time that ALGS livers display important premature senescence despite Jagged1 mutation, underlying the complexity of senescence and SASP development pathways.

Dynamics of cellular senescence markers after HCV elimination spontaneously or by DAAs in people living with HIV

BackgroundWe identified that acute or chronic Hepatitis C (HCV) infection in people living with HIV (PLWHIV) results in different senescence profiles. However, variations in these profiles after HCV elimination, spontaneously or with direct-acting antivirals (DAAs), remain unclear. MethodsLongitudinal observational study (48 weeks) in 70 PLWHIV: 23 PLWHIV with active HCV-chronic infection (CHC) before and after HCV eradication with DAAs, 12 PLWHIV who spontaneously clarify the HCV (SC), and 35 controls (HIV). Oxidative stress was quantified at DNA, lipid, protein, and nitrate levels, as well as the antioxidant capacity and glutathione enzyme. The replicative senescence was evaluated by relative telomere length measurement by PCR and twenty-six factors related to Senescence-Associated Secretory Phenotype (SASP) were characterized by Luminex. Differences in senescence markers was evaluated by generalized linear models. ResultsDuring follow-up, the SC group achieved a significant improvement in glutathione enzyme and lipid peroxidation. The secretion of SASP markers increased but was still lower than that of the HIV group. Overall, the CHC group reduced the levels of oxidative stress and SASP markers to levels like those of the HIV group. No significant differences in telomere shortening were observed between groups. ConclusionsAs the time since spontaneous resolution of HCV infection increased, patients had an improved senescence profile compared to the HIV group. Elimination of chronic HCV infection by DAAs led to a partial improvement of the senescent profile by restoring oxidative stress levels. However, although some SASP markers reached levels like those of the HIV group, others remained altered.

Senolytic drugs, dasatinib and quercetin, attenuate adipose tissue inflammation, and ameliorate metabolic function in old age.

Aging results in an elevated burden of senescent cells, senescence-associated secretory phenotype (SASP), and tissue infiltration of immune cells contributing to chronic low-grade inflammation and a host of age-related diseases. Recent evidence suggests that the clearance of senescent cells alleviates chronic inflammation and its associated dysfunction and diseases. However, the effect of this intervention on metabolic function in old age remains poorly understood. Here, we demonstrate that dasatinib and quercetin (D&Q) have senolytic effects, reducing age-related increase in senescence-associated β-galactosidase, expression of p16 and p21 gene and P16 protein in perigonadal white adipose tissue (pgWAT; all p≤ 0.04). This treatment also suppressed age-related increase in the expression of a subset of pro-inflammatory SASP genes (mcp1, tnf-α, il-1α, il-1β, il-6, cxcl2, and cxcl10), crown-like structures, abundance of T cells and macrophages in pgWAT (all p≤ 0.04). In the liver and skeletal muscle, we did not find a robust effect of D&Q on senescence and inflammatory SASP markers. Although we did not observe an age-related difference in glucose tolerance, D&Q treatment improved fasting blood glucose (p= 0.001) and glucose tolerance (p= 0.007) in old mice that was concomitant with lower hepatic gluconeogenesis. Additionally, D&Q improved insulin-stimulated suppression of plasma NEFAs (p= 0.01), reduced fed and fasted plasma triglycerides (both p≤ 0.04), and improved systemic lipid tolerance (p= 0.006). Collectively, results from this study suggest that D&Q attenuates adipose tissue inflammation and improves systemic metabolic function in old age. These findings have implications for the development of therapeutic agents to combat metabolic dysfunction and diseases in old age.

Differential endothelial senescence elicited by AQP1 regulation of epigenetic/metabolic responses

Abstract Introduction Accumulation of senescent endothelial cells with age is an important driver of vascular aging and related cardiovascular disease. However, little is known about the mechanisms and signaling pathways that underpin endothelial cell senescence. Aquaporin-1 (AQP1) has been implicated in sensing hydrogen peroxide (H2O2), which differentially modulates endothelial signal transduction. Nonetheless, the role of AQP1 in the regulation of metabolism and function in young and old vascular cells remains elusive. Purpose We sought to investigate whether 1) AQP1 differentially regulates vascular senescence and angiogenesis through epigenetic/metabolic response, senescence-associated secretory phenotype (SASP), and 2) AQP1 knockdown/inhibition rejuvenates senescent endothelial cells and restores angiogenesis. Methods We assessed the role of AQP1 in endothelial cell senescence and impaired angiogenesis through mitochondrial metabolic disorder, epigenetic alteration, and the SASP in human aortic endothelial cells and aortas from wild-type C57BL/6 mice. For these, we conducted AQP1 knockdown (with siRNA) or blockade of AQP1 (with Bacopaside-II, 10 μM) to inhibit H2O2 transport. Results Here we show the upregulation of AQP1 in the senescent endothelial cells (SEC, p.15) and aorta from old mice (&amp;gt;24 months) (p&amp;lt;0.01 and p&amp;lt;0.0001, respectively), facilitating outside-in H2O2 transport. Interestingly, AQP1 knockdown/inhibition reverses both replicative (increased SA-β-galactosidase positive cells) and stress-induced senescence (increased cell-cycle arrest markers p16, p19, and p21; and SASP markers IL1α, IL1β, and IL6) in SEC, whereas it triggers the senescence processes in the proliferating endothelial cells (PEC, p.5) (for each, p&amp;lt;0.05, n=6). Unlike in PEC or young aorta (3-month-old), AQP1 knockdown/inhibition restores angiogenesis in both SEC and old aortas. The interventions increased mitochondrial OXPHOS (represented by enhanced oxygen consumption rate (OCR)) in SEC, which is accompanied by marked increase in CaMKKβ-regulated AMPKT172 phosphorylation (for each, p&amp;lt;0.05, n=6) and in histone deacetylase-4 (HDAC4) phosphorylation at Ser632 (p&amp;lt;0.05) and further histone 3 (H3) acetylation (p&amp;lt;0.01). The HDAC4 phosphorylation leads to significant increase in Mef2A-mediated eNOS phosphorylation at Ser1177 (p&amp;lt;0.05). By contrast, AQP1 knockdown/inhibition induced a senescent-like state by a decrease in mitochondrial OCR and the phosphorylation of CaMKKβ-AMPK and HDAC4-Mef2A regulation of eNOS activity. Conclusion Our studies identify AQP1 as essential regulator of EC proliferation that differentially regulates angiogenesis via epigenetic/metabolic response and SASP modulation. Moreover, genetic or pharmacological inhibition of AQP1 reverses endothelial senescence and restores angiogenesis, representing a potential therapeutic target for vascular rejuvenation. Funding Acknowledgement Type of funding sources: Public grant(s) – National budget only. Main funding source(s): Swiss National Science Foundation (SNSF)SwissLife Jubiläumsstiftung

A motor neuron disease mouse model reveals a non-canonical profile of senescence biomarkers

ABSTRACTTo evaluate senescence mechanisms, including senescence-associated secretory phenotype (SASP), in the motor neuron disease model hSOD1-G93A, we quantified the expression of p16 and p21 and senescence-associated β-galactosidase (SA-β-gal) in nervous tissue. As SASP markers, we measured the mRNA levels of Il1a, Il6, Ifna and Ifnb. Furthermore, we explored whether an alteration of alternative splicing is associated with senescence by measuring the Adipor2 cryptic exon inclusion levels, a specific splicing variant repressed by TAR DNA-binding protein (TDP-43; encoded by Tardbp). Transgenic mice showed an atypical senescence profile with high p16 and p21 mRNA and protein in glia, without the canonical increase in SA-β-gal activity. Consistent with SASP, there was an increase in Il1a and Il6 expression, associated with increased TNF-R and M-CSF protein levels, with females being partially protected. TDP-43 splicing activity was compromised in this model, and the senolytic drug Navitoclax did not alter the disease progression. This lack of effect was reproduced in vitro, in contrast to dasatinib and quercetin, which diminished p16 and p21. Our findings show a non-canonical profile of senescence biomarkers in the model hSOD1-G93A.

Cellular senescence: a key therapeutic target in aging and diseases.

Cellular senescence is a hallmark of aging defined by stable exit from the cell cycle in response to cellular damage and stress. Senescent cells (SnCs) can develop a characteristic pathogenic senescence-associated secretory phenotype (SASP) that drives secondary senescence and disrupts tissue homeostasis, resulting in loss of tissue repair and regeneration. The use of transgenic mouse models in which SnCs can be genetically ablated has established a key role for SnCs in driving aging and age-related disease. Importantly, senotherapeutics have been developed to pharmacologically eliminate SnCs, termed senolytics, or suppress the SASP and other markers of senescence, termed senomorphics. Based on extensive preclinical studies as well as small clinical trials demonstrating the benefits of senotherapeutics, multiple clinical trials are under way. This Review discusses the role of SnCs in aging and age-related diseases, strategies to target SnCs, approaches to discover and develop senotherapeutics, and preclinical and clinical advances of senolytics.

The cGAS-STING Pathway Affects Vertebral Bone but Does Not Promote Intervertebral Disc Cell Senescence or Degeneration.

The DNA-sensing cGAS-STING pathway promotes the senescence-associated secretory phenotype (SASP) and mediates type-I interferon inflammatory responses to foreign viral and bacterial DNA as well as self-DNA. Studies of the intervertebral disc in humans and mice demonstrate associations between aging, increased cell senescence, and disc degeneration. Herein we assessed the role of STING in SASP promotion in STING gain- (N153S) and loss-of-function mouse models. N153S mice evidenced elevated circulating levels of proinflammatory markers including IL-1β, IL-6, and TNF-α, showed elevated monocyte and macrophage abundance in the vertebral marrow, and exhibited a mild trabecular and cortical bone phenotype in caudal vertebrae. Interestingly, despite systemic inflammation, the structural integrity of the disc and knee articular joint remained intact, and cells did not show a loss of their phenotype or elevated SASP. Transcriptomic analysis of N153S tissues demonstrated an upregulated immune response by disc cells, which did not closely resemble inflammatory changes in human tissues. Interestingly, STING-/- mice also showed a mild vertebral bone phenotype, but the absence of STING did not reduce the abundance of SASP markers or improve the age-associated disc phenotype. Overall, the analyses of N153S and STING-/- mice suggest that the cGAS-STING pathway is not a major contributor to SASP induction and consequent disc aging and degeneration but may play a minor role in the maintenance of trabecular bone in the vertebrae. This work contributes to a growing body of work demonstrating that systemic inflammation is not a key driver of disc degeneration.

Sexual Dimorphism in Brain Cellular Senescence During Angiotensin II‐Induced Hypertension

Sex differences in the development of hypertension across the lifespan are well established. Women are at lower risk of hypertension compared to age‐matched men until menopause, when the risk of hypertension sharply rises for women. The peptide hormone angiotensin II (Ang II) is a well‐recognized contributor to hypertension, particularly through its actions in the brain. Importantly, Ang II is too large to cross into the central nervous system (CNS) and influences blood pressure regulation by acting at circumventricular nuclei located outside of the blood‐brain‐barrier, particularly the forebrain subfornical organ. For instance, in males, Ang II drives pro‐hypertensive cellular stressors in the SFO while females are resistant to Ang II‐induced hypertension. However, the underlying CNS (i.e. SFO) mechanisms that contribute to hypertension in a sexually dimorphic manner remain unclear. Intriguingly, Ang II‐induced stressors can culminate in cellular senescence. Cellular senescence is a complex cellular phenotype characterized by marked changes in cell metabolism, macromolecular damage, and a pro‐inflammatory environment known as the senescence associated secretory phenotype (SASP). The role of senescence in the CNS as related to the development of hypertension remains unclear. Based on this, we hypothesized that brain cellular senescence, particularly in the SFO, may be a novel mechanism for the sexually dimorphic nature of Ang II‐induced hypertension. To test this, 8‐week‐old male and female C57Bl/6J mice (n=7‐10/group) were implanted with subcutaneous mini‐osmotic pumps for the infusion of Ang II (600 ng/kg/min). Micropunches of the SFO were collected at baseline and following 14 days of Ang II infusion for quantitative real‐time PCR analysis. In males, infusion of Ang II resulted in a robust increase in key senescent genes including p16 (1.9±0.3 fold baseline, p&lt;0.05) and p21 (2.5±0.5 fold baseline, p&lt;0.05) in the SFO. In parallel with the induction of senescence, inflammatory SASP indicators were also upregulated in the SFO of males following 14 days of Ang II infusion, including interleukin‐6 (IL‐6: 2.4±0.4 fold baseline, p&lt;0.05) and interleukin‐10 (IL‐10: 2.8±0.7 fold baseline, p&lt;0.05). However, when examining females, clear sexual dimorphism in Ang II‐induced SFO cellular senescence was apparent. Specifically, no changes in p16 (1.1±0.4 fold baseline, p=0.9) or p21(1.2±0.2 fold baseline, p=0.2) were noted in the SFO following Ang II administration. Similarly, SASP markers that accompany senescence remained unchanged in the SFO (e.g. IL‐6: 1.3±0.5 fold baseline, p=0.8 and IL‐10: 0.98±0.2 fold baseline, p=0.6). Together, these findings indicate that: 1) Cellular senescence in the SFO is associated with Ang II‐induced hypertension in males; and 2) Females are protected against Ang II‐driven cellular senescence in the SFO. Importantly, cellular senescence increases with advancing age. Thus, our data suggests that cellular senescence may underlie the sexually dimorphic nature of hypertension at younger ages, as well as the increased hypertensive risk in women as they age.

α-1 Adrenergic receptor antagonist doxazosin reverses hepatic stellate cells activation via induction of senescence

BackgroundActivated hepatic stellate cells (aHSCs) are the main effector cells during liver fibrogenesis. α-1 adrenergic antagonist doxazosin (DX) was shown to be anti-fibrotic in an in vivo model of liver fibrosis (LF), but the mechanism remains to be elucidated. Recent studies suggest that reversion of LF can be achieved by inducing cellular senescence characterized by irreversible cell-cycle arrest and acquisition of the senescence-associated secretory phenotype (SASP). AimTo elucidate the mechanism of the anti-fibrotic effect of DX and determine whether it induces senescence. MethodsPrimary culture-activated rat HSCs were used. mRNA and protein expression were measured by qPCR and Western blot, respectively. Cell proliferation was assessed by BrdU incorporation and xCelligence analysis. TGF-β was used for maximal HSC activation. Norepinephrine (NE), PMA and m-3M3FBS were used to activate alpha-1 adrenergic signaling. ResultsExpression of Col1α1 was significantly decreased by DX (10 μmol/L) at mRNA (−30 %) and protein level (−50 %) in TGF-β treated aHSCs. DX significantly reduced aHSCs proliferation and increased expression of senescence and SASP markers. PMA and m-3M3FBS reversed the effect of DX on senescence markers. ConclusionDoxazosin reverses the fibrogenic phenotype of aHSCs and induces the senescence phenotype.

Radiation-Induced Osteocyte Senescence Alters Bone Marrow Mesenchymal Stem Cell Differentiation Potential via Paracrine Signaling.

Cellular senescence and its senescence-associated secretory phenotype (SASP) are widely regarded as promising therapeutic targets for aging-related diseases, such as osteoporosis. However, the expression pattern of cellular senescence and multiple SASP secretion remains unclear, thus leaving a large gap in the knowledge for a desirable intervention targeting cellular senescence. Therefore, there is a critical need to understand the molecular mechanism of SASP secretion in the bone microenvironment that can ameliorate aging-related degenerative pathologies including osteoporosis. In this study, osteocyte-like cells (MLO-Y4) were induced to cellular senescence by 2 Gy γ-rays; then, senescence phenotype changes and adverse effects of SASP on bone marrow mesenchymal stem cell (BMSC) differentiation potential were investigated. The results revealed that 2 Gy irradiation could hinder cell viability, shorten cell dendrites, and induce cellular senescence, as evidenced by the higher expression of senescence markers p16 and p21 and the elevated formation of senescence-associated heterochromatin foci (SAHF), which was accompanied by the enhanced secretion of SASP markers such as IL-1α, IL-6, MMP-3, IGFBP-6, resistin, and adiponectin. When 0.8 μM JAK1 inhibitors were added to block SASP secretion, the higher expression of SASP was blunted, but the inhibition in osteogenic and adipogenic differentiation potential of BMSCs co-cultured with irradiated MLO-Y4 cell conditioned medium (CM- 2 Gy) was alleviated. These results suggest that senescent osteocytes can perturb BMSCs’ differential potential via the paracrine signaling of SASP, which was also demonstrated by in vivo experiments. In conclusion, we identified the SASP factor partially responsible for the degenerative differentiation of BMSCs, which allowed us to hypothesize that senescent osteocytes and their SASPs may contribute to radiation-induced bone loss.

Vildagliptin improves vascular smooth muscle relaxation and decreases cellular senescence in the aorta of doxorubicin-treated rats

IntroductionDoxorubicin (DOX) is a chemotherapeutic agent used in cancer treatment. Its use is limited by later toxicity to the cardiovascular system (CVS). Cellular senescence has been proposed as one mechanism of DOX toxicity. It has also been suggested that senescence reduction can improve the condition in many pathologies. We hypothesised that vildagliptin treatment can reduce senescence and thus improve the relaxation of vascular smooth muscle (VSM) in the aorta of a rat DOX model. MethodsThe rats received DOX and were treated with vildagliptin for 6 weeks. Thereafter, the rats were sacrificed, and the aorta prepared for measurements of VSM relaxation and RNA isolation to detect the level of senescence markers. To further prove the antisenescence effect of the main vildagliptin effector glucagon-like peptide 1(GLP-1), VSM cells (VSMCs) were incubated with DOX and treated with GLP-1. Subsequently, senescence was detected by senescence-associated beta-galactosidase (SA-β-gal) and by the presence of senescence markers. ResultsDOX in rats caused diminished relaxation of VSM to sodium nitrate and caused an increase in the senescence mRNA markers p16Ink4a and p27Kip1 and the senescence-associated secretory phenotype (SASP) IL-6 and IL-8. Vildagliptin treatment led to improved relaxation and a reduction in senescence and SASP markers. Furthermore, in VSMCs DOX increased SA-β-gal activity, p16Ink4a, p27Kip1, IL-6 and IL-8, and GLP1 treatment led to a decrease of both senescence and SASP markers. ConclusionIn summary we conclude that vildagliptin can reduce senescence and improve relaxation of vascular smooth muscle in the aorta of DOX-treated rats, and GLP-1 can reduce senescence of DOX-treated VSMCs. These data suggest that incretin-based drugs are promising candidates for patients suffering from late doxorubicin cardiovascular toxicity.

B Cells with a Senescent-Associated Secretory Phenotype Accumulate in the Adipose Tissue of Individuals with Obesity

Senescent cells accumulate in the adipose tissue (AT) of individuals with obesity and secrete multiple factors that constitute the senescence-associated secretory phenotype (SASP). This paper aimed at the identification of B cells with a SASP phenotype in the AT, as compared to the peripheral blood, of individuals with obesity. Our results show increased expression of SASP markers in AT versus blood B cells, a phenotype associated with a hyper-metabolic profile necessary to support the increased immune activation of AT-derived B cells as compared to blood-derived B cells. This hyper-metabolic profile is needed for the secretion of the pro-inflammatory mediators (cytokines, chemokines, micro-RNAs) that fuel local and systemic inflammation.

Renovascular Disease Induces Senescence in Renal Scattered Tubular-Like Cells and Impairs Their Reparative Potency.

Scattered tubular-like cells (STCs), dedifferentiated renal tubular epithelial cells, contribute to renal self-healing, but severe injury might blunt their effectiveness. We hypothesized that ischemic renovascular disease (RVD) induces senescence in STC and impairs their reparative potency. CD24+/CD133+ STCs were isolated from swine kidneys after 16 weeks of RVD or healthy controls. To test their reparative capabilities in injured kidneys, control or RVD-STC (5×105) were prelabeled and injected into the aorta of 2 kidneys, 1-clip (2k,1c) mice 2 weeks after surgery. Murine renal function and oxygenation were studied in vivo 2 weeks after injection using micro-magnetic resonance imaging, and fibrosis, tubulointerstitial injury, capillary density, and expression of profibrotic and inflammatory genes ex vivo. STC isolated from swine RVD kidneys showed increased gene expression of senescence and senescence-associated secretory phenotype markers and positive SA-β-gal staining. Delivery of normal pig STCs in 2k,1c mice improved murine renal perfusion, blood flow, and glomerular filtration rate, and downregulated profibrotic and inflammatory gene expression. These renoprotective effects were blunted using STC harvested from RVD kidneys, which also failed to attenuate hypoxia, fibrosis, tubular injury, and capillary loss in injured mouse 2k,1c kidneys. Hence, RVD may induce senescence in endogenous STC and impair their reparative capacity. These observations implicate cellular senescence in the pathophysiology of ischemic kidney disease and support senolytic therapy to permit self-healing of senescent kidneys.