Biomarkers Of Senescence Research Articles

Form follows function: Nuclear morphology as a quantifiable predictor of cellular senescence.

Enlarged or irregularly shaped nuclei are frequently observed in tissue cells undergoing senescence. However, it remained unclear whether this peculiar morphology is a cause or a consequence of senescence and how informative it is in distinguishing between proliferative and senescent cells. Recent research reveals that nuclear morphology can act as a predictive biomarker of senescence, suggesting an active role for the nucleus in driving senescence phenotypes. By employing deep learning algorithms to analyze nuclear morphology, accurate classification of cells as proliferative or senescent is achievable across various cell types and species both invitro and invivo. This quantitative imaging-based approach can be employed to establish links between senescence burden and clinical data, aiding in the understanding of age-related diseases, as well as assisting in disease prognosis and treatment response.

High-Level Secretion of Pregnancy Zone Protein Is a Novel Biomarker of DNA Damage-Induced Senescence and Promotes Spontaneous Senescence.

Identification of unique and specific biomarkers to better detect and quantify senescent cells remains challenging. By a global proteomic profiling of senescent human skin BJ fibroblasts induced by ionizing radiation (IR), the cellular level of pregnancy zone protein (PZP), a presumable pan-protease inhibitor never been linked to cellular senescence before, was found to be decreased by more than 10-fold, while the level of PZP in the conditioned medium was increased concomitantly. This observation was confirmed in a variety of senescent cells induced by IR or DNA-damaging drugs, indicating that high-level secretion of PZP is a novel senescence-associated secretory phenotype. RT-PCR examination verified that the transcription of the PZP gene is enhanced in various cells at senescence or upregulated following DNA damage treatment in a p53-independent manner. Moreover, pretreatment with late pregnancy serum containing a high level of PZP led to inhibition of doxorubicin-induced senescence in A549 cells, and depletion of PZP in the pregnancy serum could enhance such inhibition. Finally, the addition of immuno-precipitated PZP complexes into tissue culture attenuated the growth of A549 cells and promoted the spontaneous senescence. Therefore, we revealed that high-level secretion of PZP is a novel and unique feature associated with DNA damage-induced senescence, and secreted PZP is a positive regulator of cellular senescence, particularly during the late stage of gestation.

Biomarkers of cellular senescence and risk of death in humans.

A robust and heterogenous secretory phenotype is a core feature of most senescent cells. In addition to mediators of age-related pathology, components of the senescence associated secretory phenotype (SASP) have been studied as biomarkers of senescent cell burden and, in turn, biological age. Therefore, we hypothesized that circulating concentrations of candidate senescence biomarkers, including chemokines, cytokines, matrix remodeling proteins, and growth factors, could predict mortality in older adults. We assessed associations between plasma levels of 28 SASP proteins and risk of mortality over a median follow-up of 6.3 years in 1923 patients 65 years of age or older with zero or one chronic condition at baseline. Overall, the five senescence biomarkers most strongly associated with an increased risk of death were GDF15, RAGE, VEGFA, PARC, and MMP2, after adjusting for age, sex, race, and the presence of one chronic condition. The combination of biomarkers and clinical and demographic covariates exhibited a significantly higher c-statistic for risk of death (0.79, 95% confidence interval (CI): 0.76-0.82) than the covariates alone (0.70, CI: 0.67-0.74) (p < 0.001). Collectively, these findings lend further support to biomarkers of cellular senescence as informative predictors of clinically important health outcomes in older adults, including death.

Targeting ribosome biogenesis reinforces ERK-dependent senescence in pancreatic cancer

ABSTRACTPancreatic adenocarcinomas (PDAC) often possess mutations in K-Ras that stimulate the ERK pathway. Aberrantly high ERK activation triggers oncogene-induced senescence, which halts tumor progression. Here we report that low-grade pancreatic intraepithelial neoplasia displays very high levels of phospho-ERK consistent with a senescence response. However, advanced lesions that have circumvented the senescence barrier exhibit lower phospho-ERK levels. Restoring ERK hyperactivation in PDAC using activated RAF leads to ERK-dependent growth arrest with senescence biomarkers. ERK-dependent senescence in PDAC was characterized by a nucleolar stress response including a selective depletion of nucleolar phosphoproteins and intranucleolar foci containing RNA polymerase I designated as senescence-associated nucleolar foci (SANF). Accordingly, combining ribosome biogenesis inhibitors with ERK hyperactivation reinforced the senescence response in PDAC cells. Notably, comparable mechanisms were observed upon treatment with the platinum-based chemotherapy regimen FOLFIRINOX, currently a first-line treatment option for PDAC. We thus suggest that drugs targeting ribosome biogenesis can improve the senescence anticancer response in pancreatic cancer.

Identication and validation of cell senescence biomarkers in idiopathic pulmonary hypertension via integrated transcriptome analyses and machine learning

BackgroundIdiopathic pulmonary hypertension (IPAH) is a rare and severe disease that affects the pulmonary vasculature. As the diagnosis of IPAH requires invasive right heart catheterization surgery, early detection of this condition is notoriously challenging. Therefore, it is of utmost importance to investigate biomarkers present in peripheral blood that could aid physicians in the early identification and management of IPAH. MethodWe speculate that cellular senescence may be involved in the occurrence and development of IPAH through various pathways. In this study, we utilized integrated transcriptome analyses and machine learning-based approach to develop a diagnostic model for IPAH cell senescence. To select genetic features, we employed two machine learning algorithms: the Least Absolute Shrinkage and Selection Operator (LASSO) and Random Forest (RF). Additionally, we validated our findings through both external data sets and qRT-PCR experiments. ResultsThe resulting diagnostic nomogram was able to identify five important biomarkers that can aid in the diagnosis of IPAH, including TNFRSF1B, CCL16, GCLM, IL15, and SOD1. These genes are primarily associated with the immune system, as well as with cell senescence and apoptosis. ConclusionOur study demonstrates the utility of machine learning algorithms in making accurate diagnoses of IPAH, providing clinicians with a more directed approach to the diagnosis and treatment of this disease.

β-Galactosidase activity monitoring and bioimaging by a novel ICT mechanism-based NIR fluorescent probe with large Stokes shift

β-galactosidase (β-gal) is known as a valuable biomarker of ageing and cell senescence, which is closely down-regulated with tumorigenesis and certain diseases. Thus, real-time and in-situ monitoring and bioimaging of β-gal activity is crucial to explore its important biological role in the diseases resulted from ageing and cell senescence. Here, an “off-on” near-infrared fluorescent probe, CPD-gal, was prepared for the monitoring of β-gal activity, which exhibited eye-catching selectivity and anti-interference ability, superior sensitivity (LOD = 6.0 × 10−4 U/mL) and large Stokes shift (133 nm). The fluorescence of CPD-gal was triggered by β-gal-catalyzed cleavage of galactosyl and activating inherent intramolecular charge transfer (ICT) process. Furthermore, the proposed fluorescent probe displayed excellent capacity of monitoring and imaging endogenous β-gal activity in cells and tumor-bearing mice, demonstrating its outstanding prospect in distinguishing senescent cells from normal cells.

Biomarkers of postharvest resilience: unveiling the role of abscisic acid in table grapes during cold storage.

Table grapes are considered non-climacteric fruit, not showing a rapid increase in respiration rate and ethylene production during ripening. Previous research has suggested that abscisic acid (ABA) may have a more crucial role in grape postharvest behaviour. This study aimed to identify biomarkers of postharvest resilience and flavour life of imported table grapes. An experiment was designed to determine i) the role of ABA and catabolites on grape berry senescence; ii) the spatial distribution of these hormones within the grape berry, and iii) the effect of 1-MCP and storage temperature on its postharvest quality. Hence, the use of an ethylene inhibitor, 1-methylcyclopropane (1-MCP), during table grape storage was investigated. Table grapes (Vitis vinifera L.) cv. 'Krissy' were subjected to i) control (untreated); and ii) 1-MCP (1 µL L-1; 12 hours; 15°C) and stored under two scenarios: i) 15 days at 0.5°C, followed by five days at 5.5°C to simulate shelf-life; and ii) 20 days at 5.5°C to simulate a higher storage temperature followed by shelf-life. Physiological (i.e. mould incidence, skin colour, firmness, respiration rate) and biochemical analysis (i.e. individual sugars, organic acids, abscisic acid and catabolites) were performed. Grapes subjected to 5.5°C showed significantly higher mould incidence at the end of the shelf-life compared to 0.5°C storage temperature (12.6% vs. 3.1%). Also, and for the first time, the spatial distribution of ABA during the senescence of table grapes was profiled; the distal section had three times more ABA and metabolites than the proximal. We demonstrated that senescence processes were initiated after a significant increase in respiration rate (from 1 to 2.8 mL CO2 kg-1 h-1), and that ABA could be considered a biomarker for table grapes senescence, since an ABA peak preceded the increase in respiration rate, mould incidence, organic acids, and sucrose hydrolysis during postharvest storage; and coincided with a decrease in berry firmness. These findings are of significant importance for the industry as understanding how ABA regulates both senescence processes and quality changes during postharvest cold storage of tables grapes can improve the consistency and reduce waste and consumer complaints.

Discovery of Salidroside as a Novel Non-Coding RNA Modulator to Delay Cellular Senescence and Promote BK-Dependent Apoptosis in Cerebrovascular Smooth Muscle Cells of Simulated Microgravity Rats.

Cardiovascular aging has been reported to accelerate in spaceflights, which is a great potential risk to astronauts' health and performance. However, current exercise routines are not sufficient to reverse the adverse effects of microgravity exposure. Recently, salidroside (SAL), a valuable medicinal herb, has been demonstrated to display an important role for prevention and treatment in cardiovascular and other diseases. In the present work, Sprague-Dawley rats with four-week tail-suspension hindlimb-unloading were used to simulate microgravity effects on the cardiovascular system. We found that intragastrical administration of SAL not only significantly decreased the expressions of senescence biomarkers, such as P65 and P16, but also obviously increased the expressions of BK-dependent apoptotic genes, including the large-conductance calcium-activated K+ channel (BK), Bax, Bcl-2, and cleaved caspase-3, in vascular smooth muscle cells (VSMCs) in vivo and in vitro. In addition, relative non-coding RNAs were screened, and a luciferase assay identified that SAL increased apoptosis by activating LncRNA-FLORPAR, inhibiting miR-193, and then triggering the activity of the BK-α subunit. Our work indicated that SAL is a novel non-coding RNA modulator for regulating the LncRNA-FLORPAR sponging miR-193 pathway, which significantly promoted BK-dependent apoptosis and delayed cerebrovascular aging-like remodeling during simulated microgravity exposure. Our findings may provide a new approach to prevent cardiovascular aging in future spaceflights.

Impact of autophagy inhibition on intervertebral disc cells and extracellular matrix.

Intervertebral disc degeneration (IDD) is a leading contributor to low back pain (LBP). Autophagy, strongly activated by hypoxia and nutrient starvation, is a vital intracellular quality control process that removes damaged proteins and organelles to recycle them for cellular biosynthesis and energy production. While well-established as a major driver of many age-related diseases, autophagy dysregulation or deficiency has yet been confirmed to cause IDD. In vitro, rat nucleus pulposus (NP) cells treated with bafilomycin A1 to inhibit autophagy were assessed for glycosaminoglycan (GAG) content, proteoglycan synthesis, and cell viability. In vivo, a transgenic strain (Col2a1-Cre; Atg7 fl/fl) mice were successfully generated to inhibit autophagy primarily in NP tissues. Col2a1-Cre; Atg7 fl/fl mouse intervertebral discs (IVDs) were evaluated for biomarkers for apoptosis and cellular senescence, aggrecan content, and histological changes up to 12 months of age. Here, we demonstrated inhibition of autophagy by bafilomycin produced IDD features in the rat NP cells, including increased apoptosis and cellular senescence (p21 CIP1) and decreased expression of disc matrix genes Col2a1 and Acan. H&E histologic staining showed significant but modest degenerative changes in NP tissue of Col2a1-Cre; Atg7 fl/fl mice compared to controls at 6 and 12 months of age. Intriguingly, 12-month-old Col2a1-Cre; Atg7 fl/fl mice did not display increased loss of NP proteoglycan. Moreover, markers of apoptosis (cleaved caspase-3, TUNEL), and cellular senescence (p53, p16 INK4a , IL-1β, TNF-α) were not affected in 12-month-old Col2a1-Cre; Atg7 fl/fl mice compared to controls. However, p21 CIP1and Mmp13 gene expression were upregulated in NP tissue of 12-month-old Col2a1-Cre; Atg7 fl/fl mice compared to controls, suggesting p21 CIP1-mediated cellular senescence resulted from NP-targeted Atg7 knockout might contribute to the observed histological changes. The absence of overt IDD features from disrupting Atg7-mediated macroautophagy in NP tissue implicates other compensatory mechanisms, highlighting additional research needed to elucidate the complex biology of autophagy in regulating age-dependent IDD.

Ebselen, Iron Uptake Inhibitor, Alleviates Iron Overload-Induced Senescence-Like Neuronal Cells SH-SY5Y via Suppressing the mTORC1 Signaling Pathway.

Increasing evidence highlights that excessive iron accumulation in the brain plays a vital role in neuronal senescence and is implicated in the pathogenesis of age-related neurodegenerative diseases, including Alzheimer's disease (AD) and Parkinson's disease (PD). Therefore, the chemical compounds that eliminate an iron overload may provide better protection against oxidative stress conditions that cause the accumulation of senescent cells during brain aging. Ebselen has been identified as a strongly useful compound in the research on redox biology mechanisms. We hypothesized that ebselen could alleviate an iron overload-induced oxidative stress and consequently reverses the senescence-like phenotypes in the neuronal cells. In the present study, SH-SY5Y cells were treated with ferric ammonium citrate (FAC) before ebselen, and the evaluation of the cellular iron homeostasis, the indicators of oxidative stress, and the onset of senescence phenotypes and mechanisms were carried out accordingly. Our findings showed that ebselen ameliorated the FAC-mediated iron overload by decreasing the expression of divalent metal transporter 1 (DMT1) and ferritin light chain (FT-L) proteins. In contrast, it increased the expression of ferroportin 1 (FPN1) protein and its correlation led to a decrease in the expression of the cytosolic labile iron pool (LIP). Furthermore, ebselen significantly reduced reactive oxygen species (ROS) and rescued the mitochondrial membrane potential (ΔΨm). Notably, ebselen restored the biomarkers of cellular senescence by reducing the number of senescence-associated β-galactosidase (SA-β-gal) positive cells and senescence-associated secretory phenotypes (SASP). This also suppressed the expression of p53 protein targeting DNA damage response (DDR)/p21 cyclin-dependent kinase (CDK) inhibitor through a mTORC1 signaling pathway. Potentially, ebselen could be a therapeutic agent for treating brain aging and AD by mitigating iron accumulation and restoring senescence in SH-SY5Y cells.

Cellular Senescence in Craniofacial Tissue Regeneration: Inducers, Biomarkers, and Interventions.

Craniofacial defects and dental tissue loss have significant negative impacts on the structure and function of jaws and face, often resulting in psychological issues in patients, emphasizing the urgent need for effective craniofacial tissue reconstruction. Unfortunately, natural regeneration of these tissues is limited. Dental-derived mesenchymal stem cells (MSCs) have emerged as a promising resource for tissue engineering-based therapeutic approaches. However, the clinical outcomes of MSC-based transplantation have not met expectations due to various complex reasons, and cellular senescence is recognized as one of the potential mechanisms contributing to the suboptimal results. The quality of MSC decreases during large-scale in vitro expansion, and it is also influenced by the age and the health status of donors. To address these challenges, extensive efforts have been made to developing strategies to combat senescence in tissue engineering, leveraging on current knowledge of underlying mechanisms. This review aims to elucidate the impact of cell senescence in craniofacial and dental regeneration and provides an overview of state-of-the-art antisenescence strategies. We first discuss the potential factors that trigger cell senescence in craniofacial tissue engineering. Then we describe senescence biomarkers, monitoring methods for senescent MSCs, and their underlying molecular mechanisms. The primary focus of this review is on current strategies to inhibit and alleviate cell senescence in tissue engineering. We summarize the strategies concerning the prevention of cell senescence, senolysis, modulation of the senescent associated secretory phenotype, and reversal of senescent MSCs, offering promising opportunities to overcome the challenges associated with cell senescence in craniofacial tissue engineering.

LncRNA NEAT1 suppresses cellular senescence in hepatocellular carcinoma via KIF11-dependent repression of CDKN2A.

Hepatocellular carcinoma (HCC) is the third leading cause of cancer-related deaths worldwide. Therapeutic options for advanced HCC are limited, which is due to a lack of full understanding of pathogenesis. Cellular senescence is a state of cell cycle arrest, which plays important roles in the pathogenesis of HCC. Mechanisms underlying hepatocellular senescence are not fully understood. LncRNA NEAT1 acts as an oncogene and contributes to the development of HCC. Whether NEAT1 modulates hepatocellular senescence in HCC is unknown. The role of NEAT1 and KIF11 in cellular senescence and tumor growth in HCC was assessed both in vitro and in vivo. RNA pulldown, mass spectrometry, Chromatin immunoprecipitation (ChIP), luciferase reporter assays, RNA FISH and immunofluorescence (IF) staining were used to explore the detailed molecular mechanism of NEAT1 and KIF11 in cellular senescence of HCC. We found that NEAT1 was upregulated in tumor tissues and hepatoma cells, which negatively correlated with a senescence biomarker CDKN2A encoding p16INK4a and p14ARF proteins. NEAT1 was reduced in senescent hepatoma cells induced by doxorubicin (DOXO) or serum starvation. Furthermore, NEAT1 deficiency caused senescence in cultured hepatoma cells, and protected against the progression of HCC in a mouse model. During senescence, NEAT1 translocated into cytosol and interacted with a motor protein KIF11, resulting in KIF11 protein degradation and subsequent increased expression of CDKN2A in cultured hepatoma cells. Furthermore, KIF11 knockdown caused senescence in cultured hepatoma cells. Genetic deletion of Kif11 in hepatocytes inhibited the development of HCC in a mouse model. Conclusively, NEAT1 overexpression reduces senescence and promotes tumor progression in HCC tissues and hepatoma cells, whereas NEAT1 deficiency causes senescence and inhibits tumor progression in HCC. This is associated with KIF11-dependent repression of CDKN2A. These findings lay the foundation to develop potential therapies for HCC by inhibiting NEAT1 and KIF11 or inducing senescence.

Molecularly Targeted Fluorescent Sensors for Visualizing and Tracking Cellular Senescence.

Specific identification and monitoring of senescent cells are essential for the in-depth understanding and regulation of senescence-related life processes and diseases. Fluorescent sensors providing real-time and in situ information with spatiotemporal resolution are unparalleled tools and have contributed greatly to this field. This review focuses on the recent progress in fluorescent sensors for molecularly targeted imaging and real-time tracking of cellular senescence. The molecular design, sensing mechanisms, and biological activities of the sensors are discussed. The sensors are categorized by the types of markers and targeting ligands. Accordingly, their molecular recognition and fluorescent performance towards senescence biomarkers are summarized. Finally, the perspective and challenges in this field are discussed, which are expected to assist future design of next-generation sensors for monitoring cellular senescence.

Senescence ofbone marrow mesenchymal stem cells in Wistar malerats receiving normal chow/high-calorie diets with/without vitamin D.

Bone marrow mesenchymal stem cells (BM-MSCs) have a momentous function in the composition of the bone marrow microenvironment because of their many valuable properties and abilities, such as immunomodulation and hematopoiesis. The features and actions of MSCs are influenced by senescence, which may be affected by various factors such as nutritional/micronutrients status, e.g., vitamin D. This study aimed to examine the effects of a high-calorie diet (HCD) with/without vitamin D on BM-MSCs senescence. In the first phase, 48 middle-aged rats were fed a normal chow diet (NCD, n = 24) and an HCD (n = 24) for 26 weeks. Afterward, the rats in each group were randomly divided into three equal subgroups. Immediately, eight-rat from each diet group were sacrificed to assess the HCD effects on the first phase measurements. In the second phase, the remaining 4 groups of rats were fed either NCD or HCD with (6 IU/g) or without vitamin D (standard intake: 1 IU/g); in other words, in this phase, the animals were fed (a) NCD, (b) NCD plus vitamin D, (c) HCD, and (d) HCD plus vitamin D for 4 months. BM-MSCs were isolated and evaluated for P16INK4a, P38 MAPK, and Bmi-1 gene expression, reactive oxygen species (ROS) levels, SA-β-gal activity, and cell cycle profile at the end of both phases. After 26 weeks (first phase), the ROS level, SA-β-gal-positive cells, and cells in the G1 phase were significantly higher in HCD-fed rats than in NCD-fed ones (P < 0.05). HCD prescription did not significantly affect cells in the S and G2 phases (p > 0.05). Compared with the NCD-fed animals, P16INK4a and P38 MAPK gene expression were up-regulated in the HCD-fed animals; also, Bmi-1 gene expression was down-regulated (P < 0.05).BM-MSCs from vitamin D-treated rats (second phase) exhibited reduced mRNA levels of P16INK4a and P38 MAPK genes and increased Bmi-1 mRNA levels (all P<0.05). Vitamin D prescription also declined the percentage of SA-β-gal-positive cells, ROS levels, and the cells in the G1 phase and increased the cells in the S phase in both NCD and HCD-fed animals (P <0.05). The reduction of the cells in the G2 phase in rats fed with an NCD plus vitamin D was statistically non-significant (P=0.128) and significant in HCD plus vitamin D rats (P=0.002).HCD accelerates BM-MSCs senescence, and vitamin D reduces BM-MSCs senescence biomarkers.

Glycolytic enzyme PKM2 regulates cell senescence but not inflammation in the process of osteoarthritis.

Chondrocyte senescence is an important mechanism underlying osteoarthritis in the senile population and is characterized by reduced expressions of the extracellular matrix proteins. The involvement of glycolysis and the tricarboxylic acid cycle in the development of osteoarthritis is inclusive. The present study aims to investigate the role of the glycolytic enzyme M2 isoform of pyruvate kinase (PKM2) in chondrocytes in senescence and inflammation. Primary chondrocytes are isolated from the knee joints of neonatal mice. Small interfering RNAs (siRNAs) against PKM2 are transfected using lipofectamine. RNA sequencing is conducted in primary chondrocytes with the PKM2 gene deleted. Cell apoptosis, autophagy, reactive oxygen species measurement, and senescent conditions are examined. The glycolytic rate in cells is measured by Seahorse examination. Interleukin 1-β (IL-1β) increases the protein expressions of matrix metallopeptidases (MMP)13 and PKM2 and reduces the protein expression of collagen type II (COL2A1) in primary chondrocytes. Silencing of PKM2 alters the protein expressions of MMP13, PKM2, and COL2A1 in the same pattern in quiescent and stimulated chondrocytes. RNA sequencing analysis reveals that PKM2 silencing reduces senescent biomarker p16 INK4a expression. Compared with low-passage chondrocytes, high-passage chondrocytes exhibit increased expression of p16 INK4a and reduced expression of COL2A1. Silencing of PKM2 reduces SA-β-Gal signals and increases COL2A1 expression in high-passage chondrocytes. Seahorse assay reveals that PKM2 deletion favors the tricarboxylic acid cycle in mitochondria in low- but not in high-passage chondrocytes. In summary, the glycolytic enzyme PMK2 modulates chondrocyte senescence but does not participate in the regulation of inflammation.

The intensities of canonical senescence biomarkers integrate the duration of cell-cycle withdrawal

Senescence, a state of irreversible cell-cycle withdrawal, is difficult to distinguish from quiescence, a state of reversible cell-cycle withdrawal. This difficulty arises because quiescent and senescent cells are defined by overlapping biomarkers, raising the question of whether these states are truly distinct. To address this, we use single-cell time-lapse imaging to distinguish slow-cycling cells that spend long periods in quiescence from cells that never cycle after recovery from senescence-inducing treatments, followed by staining for various senescence biomarkers. We find that the staining intensity of multiple senescence biomarkers is graded rather than binary and reflects the duration of cell-cycle withdrawal, rather than senescence per se. Together, our data show that quiescent and apparent senescent cells are nearly molecularly indistinguishable from each other at a snapshot in time. This suggests that cell-cycle withdrawal itself is graded rather than binary, where the intensities of senescence biomarkers integrate the duration of past cell-cycle withdrawal.

Inhibition of tyrosine kinase Fgr prevents radiation-induced pulmonary fibrosis (RIPF)

Cellular senescence is involved in the development of pulmonary fibrosis as well as in lung tissue repair and regeneration. Therefore, a strategy of removal of senescent cells by senolytic drugs may not produce the desired therapeutic result. Previously we reported that tyrosine kinase Fgr is upregulated in ionizing irradiation-induced senescent cells. Inhibition of Fgr reduces the production of profibrotic proteins by radiation-induced senescent cells in vitro; however, a mechanistic relationship between senescent cells and radiation-induced pulmonary fibrosis (RIPF) has not been established. We now report that senescent cells from the lungs of mice with RIPF, release profibrotic proteins for target cells and secrete chemotactic proteins for marrow cells. The Fgr inhibitor TL02-59, reduces this release of profibrotic chemokines from the lungs of RIPF mice, without reducing numbers of senescent cells. In vitro studies demonstrated that TL02-59 abrogates the upregulation of profibrotic genes in target cells in transwell cultures. Also, protein arrays using lung fibroblasts demonstrated that TL02-59 inhibits the production of chemokines involved in the migration of macrophages to the lung. In thoracic-irradiated mice, TL02-59 prevents RIPF, significantly reduces levels of expression of fibrotic gene products, and significantly reduces the recruitment of CD11b+ macrophages to the lungs. Bronchoalveolar lavage (BAL) cells from RIPF mice show increased Fgr and other senescent cell markers including p16. In human idiopathic pulmonary fibrosis (IPF) and in RIPF, Fgr, and other senescent cell biomarkers are increased. In both mouse and human RIPF, there is an accumulation of Fgr-positive proinflammatory CD11b+ macrophages in the lungs. Thus, elevated levels of Fgr in lung senescent cells upregulate profibrotic gene products, and chemokines that might be responsible for macrophage infiltration into lungs. The detection of Fgr in senescent cells that are obtained from BAL during the development of RIPF may help predict the onset and facilitate the delivery of medical countermeasures.

Multi-omic analysis of mandibuloacral dysplasia type A patient iPSC-derived MSC senescence reveals miR-311 as a novel biomarker for MSC senescence.

Mandibuloacral dysplasia type A (MADA) is a rare genetic progeroid syndrome associated with lamin A/C (LMNA) mutations. Pathogenic mutations of LMNA result in nuclear structural abnormalities, mesenchymal tissue damage and progeria phenotypes. However, it remains elusive how LMNA mutations cause mesenchymal-derived cell senescence and disease development. Here, we established an in vitro senescence model using induced pluripotent stem cell-derived mesenchymal stem cells (iMSCs) from MADA patients with homozygous LMNA p.R527C mutation. When expanded to passage 13 in vitro, R527C iMSCs exhibited marked senescence and attenuation of stemness potential, accompanied by immunophenotypic changes. Transcriptome and proteome analysis revealed that cell cycle, DNA replication, cell adhesion and inflammation might play important roles in senescence. In-depth evaluation of changes in extracellular vesicle (EV) derived iMSCs during senescence revealed that R527C iMSC-EVs could promote surrounding cell senescence by carrying pro-senescence microRNAs (miRNAs), including a novel miRNA called miR-311, which can serve as a new indicator for detecting chronic and acute mesenchymal stem cell (MSC) senescence and play a role in promoting senescence. Overall, this study advanced our understanding of the impact of LMNA mutations on MSC senescence and provided novel insights into MADA therapy as well as the link between chronic inflammation and aging development.

730-P: Effects of Obstructive Sleep Apnea Treatment on Whole-Body and Adipose Tissue Insulin Sensitivity—Role of Senescence and Mitigation by Adjuvant Metformin Therapy

Introduction: Obstructive sleep apnea (OSA) is a prevalent sleep disorder linked to high type 2 diabetes risk. Positive airway pressure (PAP) therapy is the standard of care for OSA, but it inconsistently improves glucose homeostasis. Senescent cells are common in adipose tissue (AT) of patients with OSA and may impair insulin signaling. However, the effects of PAP therapy on AT senescence are not known. Addition of metformin, an insulin-sensitizer with anti-senescent effects, may be a good adjuvant therapeutic strategy to improve glycemic control in patients with OSA. Methods: We randomly assigned 16 nondiabetic participants with OSA (Mean± SD; age: 50.9 ± 6.7 y, BMI: 36.5 ± 2.9 kg/m2) in a 1:1 ratio to receive either 2g metformin or placebo daily along with PAP therapy for 3 months in a double-blind pilot study. Whole body and AT insulin sensitivity was determined by 2h oral glucose tolerance test (OGTT). AT biopsy was obtained to estimate tissue insulin signaling and senescence. Results: The metformin and placebo groups had comparable age, BMI and OSA severity at baseline. PAP compliance was 38% and 75% in the metformin and placebo groups, respectively. Matsuda Index, glucose area under the curve (2h AUC), and free-fatty acid suppression at baseline and follow-up were not different in metformin or placebo treated groups. Compared to baseline, insulin AUC and insulin to glucose AUC ratio during OGTT was unchanged in the metformin but increased in the placebo group. Metformin, but not placebo, was associated with increased insulin mediated AKT phosphorylation and decreased senescence biomarkers (p21 and MCP1) in AT during the follow-up visit. Conclusion: Metformin, as adjunct to PAP therapy, attenuated insulin rises during OGTT, improved AT insulin signaling, and lowered senescence biomarkers in OSA patients. Larger trials of longer treatment duration are needed to determine if metformin can prevent diabetes in OSA patients. Disclosure S. Rodrigues: None. W.S. Dantas: None. R.A. Beyl: None. R.C. Hebert: None. I. Griffith: None. M. Tanksley: None. E.C. Mader: None. J.P. Kirwan: None. C.L. Axelrod: None. E. Ravussin: Research Support; Eli Lilly and Company, Novartis AG. Advisory Panel; Novartis. P. Singh: None. Funding National Institutes of Health (P30DK072476, U54GM104940); CAPES (88887.470405/2019-00 to S.R.)

#4455 DISCOVERY AND VALIDATION OF NOVEL RENAL EPITHELIAL SENESCENCE ASSOCIATED BIOMARKERS

Abstract Background and Aims Cellular senescence is characterised by irreversible cell cycle arrest and marked changes in transcriptional and secretory activity. It can occur as a physiological component of healthy organismal development or in response to a range of cellular insults including DNA damage, oncogenic mutations and metabolic stress. Renal tubular senescence in response to ageing and injury is proposed as a driver of kidney fibrosis. Senescent cell depletion in mice improves outcomes in multiple organs including the kidney. There are currently no non-invasive biomarkers for quantifying renal senescence available. We are using a multi-omics approach and utilising human renal proximal tubular epithelial cells (hRPTECs) in culture, a murine model of renal senescence and human samples, to identify urinary biomarkers of renal tubular senescence and to determine if they can predict decline in kidney function. Method In vitro: We optimised a model of induced senescence in hRPTECs in vitro using 10Gy irradiation and treatment with MDM2-antagonist Nutlin 3A. Transcriptomic studies using bulk RNAseq were performed comparing senescent cells with proliferating controls (each group n = 5). Genes with &amp;gt;x2 fold-change between groups and adjusted p values &amp;lt;0.01 were regarded as differentially expressed. In vivo: Senescence was induced using 7Gy total body irradiation (TBI) in mice whilst the Bcl2/w/xL inhibitor ABT-263, was used to deplete senescent cells. We performed LC-MS proteomic studies on urine from mice comparing healthy controls with mice exposed to TBI with or without subsequent senolytic therapy. In human kidney biopsy samples from 55 patients with CKD, we performed immunofluorescence staining for senescence marker p21CIP1, proliferation marker Ki67 (absent in senescent cells) and tubular markers CD10 and CKPAN and quantified tubular senescence using machine learning algorithms in the software QuPath (see figure). Tubular cells were classified as senescent when p21CIP1 positive and Ki67 negative and expressed as a percentage of all tubular cells. Results Irradiated and Nutlin 3A treated cells had increased mRNA levels of CDKN1A and reduced LMNB1 and MKI67 in keeping with senescence induction. Other transcripts including CXCL8 and IL6 rose in irradiated cells compared to controls but fell in Nutlin 3A treated cells. 1272 genes where differentially expressed in irradiated cells compared to controls; 760 of these genes were differentially expressed in the same direction in Nutlin 3A treated samples. Over representation analysis highlighted pathways relating to the cell cycle, consistent with senescence induction. LC-MS studies on murine urine samples identified 15 proteins that fell in mice exposed to TBI compared to healthy controls but reverted towards baseline with senolytic treatment. By combining my datasets with publicly available data, we identified several candidate biomarkers of senescence. This includes urokinase plasminogen activator surface receptor, a protein linked with senescence and ageing as well as other novel senescence markers (not named due to pending patent applications). In the patients where senescence was quantified, the mean age at the time of biopsy was 54 years (range 19-81 years) and the mean eGFR was 56 ml/min/1.73 m2 [s.d. 34]). Renal tubular senescence correlated with age (rho = 0.64, p&amp;lt;0.001) and inversely with baseline eGFR (rho = -0.51, p&amp;lt;0.001). LC-MS and ELISA studies in matched urine samples are ongoing to determine which molecules most closely correlate with senescence histologically in the human kidney; these will be presented at the meeting. Conclusion Through a combination of transcriptomic studies and LC-MS proteomics, we have identified several candidate urinary biomarkers of senescence; ongoing studies will determine which molecules correlate with renal senescence histologically. Further studies in a cohort of &amp;gt;380 patients with &amp;gt;4 years follow-up will determine if the most promising biomarkers predict renal outcomes and will also be presented at the meeting.