Profiling microRNA expression during senescence and aging: mining for a diagnostic tool of senescent-cell burden.

In the last decade cellular senescence, a hallmark of aging, has come into focus for pharmacologically targeting aging processes. Senolytics are one of these interventive strategies that have advanced into clinical trials, creating an unmet need for minimally invasive biomarkers of senescent cell load to identify patients at need for senotherapy. We created a landscape of miRNA and mRNA expression in five human cell types induced to senescence in-vitro and provide proof-of-principle evidence that miRNA expression can track senescence burden dynamically in-vivo using transgenic p21 high senescent cell clearance in HFD fed mice. Finally, we profiled miRNA expression in seven different tissues, total plasma, and plasma derived EVs of young and 25 months old mice. In a systematic analysis, we identified 22 candidate senomiRs with potential to serve as circulating biomarkers of senescence not only in rodents, but also in upcoming human clinical senolytic trials.

Evaluation of Exploratory Fluid Biomarker Results from a Phase 1 Senolytic Trial in Mild Alzheimer's Disease.

Senescent cell accumulation contributes to the progression of age-related disorders including Alzheimer's disease (AD). Clinical trials evaluating senolytics, drugs that clear senescent cells, are underway, but lack standardized outcome measures. Our team recently published data from the first open-label trial to evaluate senolytics (dasatinib plus quercetin) in AD. After 12-weeks of intermittent treatment, we reported brain exposure to dasatinib, favorable safety and tolerability, and modest post-treatment changes in cerebrospinal fluid (CSF) inflammatory and AD biomarkers using commercially available assays. Herein, we present more comprehensive exploratory analyses of senolytic associated changes in AD relevant proteins, metabolites, lipids, and transcripts measured across blood, CSF, and urine. These analyses included mass spectrometry for precise quantification of amyloid beta (Aß) and tau in CSF; immunoassays to assess senescence associated secretory factors in plasma, CSF, and urine; mass spectrometry analysis of urinary metabolites and lipids in blood and CSF; and transcriptomic analyses relevant to chronic stress measured in peripheral blood cells. Levels of Aß and tau species remained stable. Targeted cytokine and chemokine analyses revealed treatment-associated increases in inflammatory plasma fractalkine and MMP-7 and CSF IL-6. Urinary metabolites remained unchanged. Modest treatment-associated lipid profile changes suggestive of decreased inflammation were observed both peripherally and centrally. Blood transcriptomic analysis indicated downregulation of inflammatory genes including FOS, FOSB, IL1ß, IL8, JUN, JUNB, PTGS2. These data provide a foundation for developing standardized outcome measures across senolytic studies and indicate distinct biofluid-specific signatures that will require validation in future studies. ClinicalTrials.gov: NCT04063124.

Orally-active, clinically-translatable senolytics restore α-Klotho in mice and humans.

BACKGROUND: α-Klotho is a geroprotective protein that can attenuate or alleviate deleterious changes with ageing and disease. Declines in α-Klotho play a role in the pathophysiology of multiple diseases and age-related phenotypes. Pre-clinical evidence suggests that boosting α-Klotho holds therapeutic potential. However, readily clinically-translatable, practical strategies for increasing α-Klotho are not at hand. Here, we report that orally-active, clinically-translatable senolytics can increase α-Klotho in mice and humans. METHODS: We examined α-Klotho expression in three different human primary cell types co-cultured with conditioned medium (CM) from senescent or non-senescent cells with or without neutralizing antibodies. We assessed α-Klotho expression in aged, obese, and senescent cell-transplanted mice treated with vehicle or senolytics. We assayed urinary α-Klotho in patients with idiopathic pulmonary fibrosis (IPF) who were treated with the senolytic drug combination, Dasatinib plus Quercetin (D+Q). FINDINGS: We found exposure to the senescent cell secretome reduces α-Klotho in multiple nonsenescent human cell types. This was partially prevented by neutralizing antibodies against the senescence-associated secretory phenotype (SASP) factors, activin A and Interleukin 1α (IL-1α). Consistent with senescent cells' being a cause of decreased α-Klotho, transplanting senescent cells into younger mice reduced brain and urine α-Klotho. Selectively removing senescent cells genetically or pharmacologically increased α-Klotho in urine, kidney, and brain of mice with increased senescent cell burden, including naturally-aged, diet-induced obese (DIO), or senescent cell-transplanted mice. D+Q increased α-Klotho in urine of patients with IPF, a disease linked to cellular senescence. INTERPRETATION: Senescent cells cause reduced α-Klotho, partially due to their production of activin A and IL-1α. Targeting senescent cells boosts α-Klotho in mice and humans. Thus, clearing senescent cells restores α-Klotho, potentially opening a novel, translationally-feasible avenue for developing orally-active small molecule, α-Klotho-enhancing clinical interventions. Furthermore, urinary α-Klotho may prove to be a useful test for following treatments in senolytic clinical trials. FUNDING: This work was supported by National Institute of Health grants AG013925 (J.L.K.), AG062413 (J.L.K., S.K.), AG044271 (N.M.), AG013319 (N.M.), and the Translational Geroscience Network (AG061456: J.L.K., T.T., N.M., S.B.K., S.K.), Robert and Arlene Kogod (J.L.K.), the Connor Group (J.L.K.), Robert J. and Theresa W. Ryan (J.L.K.), and the Noaber Foundation (J.L.K.). The previous IPF clinical trial was supported by the Claude D. Pepper Older Americans Independence Centers at WFSM (AG021332: J.N.J., S.B.K.), UTHSCA (AG044271: A.M.N.), and the Translational Geroscience Network.

A new gene set identifies senescent cells and predicts senescence-associated pathways across tissues.

Although cellular senescence drives multiple age-related co-morbidities through the senescence-associated secretory phenotype, in vivo senescent cell identification remains challenging. Here, we generate a gene set (SenMayo) and validate its enrichment in bone biopsies from two aged human cohorts. We further demonstrate reductions in SenMayo in bone following genetic clearance of senescent cells in mice and in adipose tissue from humans following pharmacological senescent cell clearance. We next use SenMayo to identify senescent hematopoietic or mesenchymal cells at the single cell level from human and murine bone marrow/bone scRNA-seq data. Thus, SenMayo identifies senescent cells across tissues and species with high fidelity. Using this senescence panel, we are able to characterize senescent cells at the single cell level and identify key intercellular signaling pathways. SenMayo also represents a potentially clinically applicable panel for monitoring senescent cell burden with aging and other conditions as well as in studies of senolytic drugs.

SARS-CoV-2 causes senescence in human cells and exacerbates the senescence-associated secretory phenotype through TLR-3.

Senescent cells, which arise due to damage-associated signals, are apoptosis-resistant and can express a pro-inflammatory, tissue-destructive senescence-associated secretory phenotype (SASP). We recently reported that a component of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) surface protein, S1, can amplify the SASP of senescent cultured human cells and that a related mouse ß-coronavirus, mouse hepatitis virus (MHV), increases SASP factors and senescent cell burden in infected mice. Here, we show that SARS-CoV-2 induces senescence in human non-senescent cells and exacerbates the SASP in human senescent cells through Toll-like receptor-3 (TLR-3). TLR-3, which senses viral RNA, was increased in human senescent compared to non-senescent cells. Notably, genetically or pharmacologically inhibiting TLR-3 prevented senescence induction and SASP amplification by SARS-CoV-2 or Spike pseudotyped virus. While an artificial TLR-3 agonist alone was not sufficient to induce senescence, it amplified the SASP in senescent human cells. Consistent with these findings, lung p16INK4a+ senescent cell burden was higher in patients who died from acute SARS-CoV-2 infection than other causes. Our results suggest that induction of cellular senescence and SASP amplification through TLR-3 contribute to SARS-CoV-2 morbidity, indicating that clinical trials of senolytics and/or SASP/TLR-3 inhibitors for alleviating acute and long-term SARS-CoV-2 sequelae are warranted.

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 ß-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.

Pneumocystis cell wall beta-glucan stimulates calcium-dependent signaling of IL-8 secretion by human airway epithelial cells.

BACKGROUND: Respiratory failure secondary to alveolar inflammation during Pneumocystis pneumonia is a major cause of death in immunocompromised patients. Neutrophil infiltration in the lung of patients with Pneumocystis infection predicts severity of the infection and death. Several previous studies indicate that airway epithelial cells release the neutrophil chemoattractant proteins, MIP-2 (rodents) and IL-8 (humans), in response to Pneumocystis and purified Pneumocystis cell wall beta-glucans (PCBG) through the NF-kappaB-dependent pathway. However, little is known about the molecular mechanisms that are involved in the activation of airway epithelium cells by PCBG resulting in the secretion of IL-8. METHOD: To address this, we have studied the activation of different calcium-dependent mitogen-activated protein kinases (MAPKs) in 1HAEo- cells, a human airway epithelial cell line. RESULTS: Our data provide evidence that PCBG induces phosphorylation of the MAPKs, ERK, and p38, the activation of NF-kappaB and the subsequently secretion of IL-8 in a calcium-dependent manner. Further, we evaluated the role of glycosphingolipids as possible receptors for beta-glucans in human airway epithelial cells. Preincubation of the cells with D-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol (PDMP) a potent inhibitor of the glycosphingolipids synthesis, prior to PCBG stimulation, significantly decreased IL-8 production. CONCLUSION: These data indicate that PCBG activates calcium dependent MAPK signaling resulting in the release of IL-8 in a process that requires glycosphingolipid for optimal signaling.

Distribution of major basic protein on human airway following in vitro eosinophil incubation.

Major basic protein (MBP) released from activated eosinophils may influence airway hyperresponsiveness (AHR) by either direct effects on airway myocytes or by an indirect effect. In this study, human bronchi, freshly isolated human eosinophils, or MBP purified from human eosinophil granules were incubated for studying eosinophil infiltration and MBP localization. Eosinophils immediately adhered to intact human airway as well as to cultured human airway myocytes and epithelium. Following incubation 18-24 h, eosinophils migrated into the airway media, including the smooth muscle layer, but had no specific recruitment to airway neurons. Eosinophils released significant amounts of MBP within the airway media, including areas comprising the smooth muscle layer. Most deposits of MBP were focally discrete and restricted by immunologic detection to a maximum volume of approximately 300 microm(3) about the eosinophil. Native MBP applied exogenously was immediately deposited on the surface of the airway, but required at least 1 h to become detected within the media of the airway wall. Tissue MBP infiltration and deposition increased in a time- and concentration-dependent manner. Taken together, these findings suggest that eosinophil-derived cationic proteins may alter airway hyperresponsiveness (AHR) in vivo by an effect that is not limited to the bronchial epithelium.

Senolytics decrease senescent cells in humans: Preliminary report from a clinical trial of Dasatinib plus Quercetin in individuals with diabetic kidney disease.

BACKGROUND: Senescent cells, which can release factors that cause inflammation and dysfunction, the senescence-associated secretory phenotype (SASP), accumulate with ageing and at etiological sites in multiple chronic diseases. Senolytics, including the combination of Dasatinib and Quercetin (D + Q), selectively eliminate senescent cells by transiently disabling pro-survival networks that defend them against their own apoptotic environment. In the first clinical trial of senolytics, D + Q improved physical function in patients with idiopathic pulmonary fibrosis (IPF), a fatal senescence-associated disease, but to date, no peer-reviewed study has directly demonstrated that senolytics decrease senescent cells in humans. METHODS: In an open label Phase 1 pilot study, we administered 3 days of oral D 100 mg and Q 1000 mg to subjects with diabetic kidney disease (N = 9; 68·7 ±â€¯3·1 years old; 2 female; BMI:33·9 ±â€¯2·3 kg/m2; eGFR:27·0 ±â€¯2·1 mL/min/1·73m2). Adipose tissue, skin biopsies, and blood were collected before and 11 days after completing senolytic treatment. Senescent cell and macrophage/Langerhans cell markers and circulating SASP factors were assayed. FINDINGS: D + Q reduced adipose tissue senescent cell burden within 11 days, with decreases in p16INK4A-and p21CIP1-expressing cells, cells with senescence-associated ß-galactosidase activity, and adipocyte progenitors with limited replicative potential. Adipose tissue macrophages, which are attracted, anchored, and activated by senescent cells, and crown-like structures were decreased. Skin epidermal p16INK4A+ and p21CIP1+ cells were reduced, as were circulating SASP factors, including IL-1α, IL-6, and MMPs-9 and -12. INTERPRETATION: "Hit-and-run" treatment with senolytics, which in the case of D + Q have elimination half-lives <11 h, significantly decreases senescent cell burden in humans. FUND: NIH and Foundations. ClinicalTrials.gov Identifier: NCT02848131. Senescence, Frailty, and Mesenchymal Stem Cell Functionality in Chronic Kidney Disease: Effect of Senolytic Agents.

Thrombin generation profiles as predictors of symptomatic venous thromboembolism after trauma: A prospective cohort study.

BACKGROUND: Reliable biomarkers predictive of venous thromboembolism (VTE) after acute trauma are uncertain. The objective of the study was to identify risk factors for symptomatic VTE after trauma, including individual plasma coagulome characteristics as reflected by thrombin generation. METHODS: In a prospective, case-cohort study, trauma patients were enrolled over the 4.5-year period, 2011 to 2015. Blood was collected by venipuncture into 3.2% trisodium citrate at 0, 6, 12, 24, and 72 hours after injury and at hospital discharge. Platelet poor plasma was stored at -80 °C until analysis. Thrombin generation, as determined by the calibrated automated thrombogram (CAT) using 5 pM tissue factor (TF)/4 µM phospholipid (PS), was reported as peak height (nM thrombin) and time to peak height (ttPeak [minutes]). Data are presented as median [IQR] or hazard ratio with 95% CI. RESULTS: Among 453 trauma patients (injury severity score = 13.0 [6.0, 22.0], hospital length of stay = 4.0 [2.0, 10.0] days, age = 49 [28, 64] years, 71% male, 96% with blunt mechanism, mortality 3.2%), 83 developed symptomatic VTE within 92 days after injury (35 [42%] after hospital discharge). In a weighted, multivariate Cox model that included clinical and CAT characteristics available within 24 hours of admission, increased patient age (1.35 [1.19,1.52] per 10 years, p < 0.0001), body mass index ≥30 kg/m (4.45 [2.13,9.31], p < 0.0001), any surgery requiring general anesthesia (2.53 [1.53,4.19], p = 0.0003) and first available ttPeak (1.67 [1.29, 2.15], p < 0.00001) were independent predictors of incident symptomatic VTE within 92 days after trauma (C-statistic = 0.799). CONCLUSION: The individual's plasma coagulome (as reflected by thrombin generation) is an independent predictor of VTE after trauma. Clinical characteristics and ttPeak can be used to stratify acute trauma patients into high and low risk for VTE. LEVEL OF EVIDENCE: Prognostic, level III.

Thrombin generation and procoagulant microparticle profiles after acute trauma: A prospective cohort study.

OBJECTIVE: The two sides of trauma-induced coagulopathy, the hypocoagulable and the hypercoagulable states, are poorly understood. To identify potential mechanisms for venous thromboembolism and bleeding after acute trauma, we estimated changes in circulating procoagulant microparticles (MPs) and thrombin activity during hospitalization for trauma. METHODS: Whole blood was collected by venipuncture into 3.2% trisodium citrate at 0, 6, 12, 24, and 72 hours after injury and discharge. Platelet-poor plasma was harvested and stored at -80°C until analysis. Thrombin generation was determined using the calibrated automated thrombogram (CAT), reported as lag time (minutes), peak height (nM thrombin), and time to reach peak height (ttPeak, minutes). The concentration of total procoagulant MPs (number/µL) was measured by flow cytometry. Data are presented as median (interquartile range [IQR]). RESULTS: Among 443 trauma patients (1,734 samples; Injury Severity Score [ISS], 13.0 [IQR, 6.0-22.0]; hospital length of stay, 4.0 days [IQR, 2.0-10.0]; age, 48 years [IQR, 28-65]; 70.7% male; 95% with blunt mechanism; mortality, 3.2%), no discernable patterns in thrombin generation or MP concentration were observed over time. The peak height and MPs were significantly different from healthy volunteers and were 337 nM (IQR, 285-395) and 400/µL plasma (IQR, 211-772), respectively. Extreme (defined as highest or lowest 5%) values reflecting a possible "hypercoagulable state" (lag time ≤ 1.98, peak height ≥ 486.2, ttPeak ≤ 3.61, and total procoagulant MP ≥ 2,278) were reached within 12 hours after acute trauma, while extreme values representing a possible "hypocoagulable state" (lag time ≥ 18.6, peak height ≤ 17.8, and ttPeak ≥ 29.45) were not reached until 1 day to 3 days. CONCLUSION: Although there was no predictable pattern of coagulopathy observed in each patient after trauma, those who reached extreme values did so relatively early after injury. These findings should be taken into account when designing risk model tools involving coagulation laboratory parameters. LEVEL OF EVIDENCE: Epidemiologic study, level III.

Mechanisms of intrinsic force in small human airways.

We quantified the magnitude and investigated mechanisms regulating intrinsic force (IF) in human airway smooth muscle (hASM). IF was identified by reducing extracellular calcium (Ca2+) concentration to nominally zero in freshly isolated isometrically mounted 2mm human bronchi. Our results show: (1) the magnitude of IF is ∼50% of the maximal total force elicited by acetylcholine (10(-5) M) and is epithelial independent, (2) IF can also be revealed by ß-adrenergic activation (isoproterenol), non-specific cationic channel blockade (La3+) or L-type voltage gated Ca2+ channel blockade (nifedipine), (3) atropine, indomethacin, AA-861, or pyrilamine did not affect IF, (4) IF was reduced by the intracellular Ca2+ ([Ca2+]i) chelating agent BAPTA-AM, (5) ω-conotoxin had no effect on IF. In studies in cultured hASM cells nominally zero Ca2+ buffer and BAPTA-AM reduced [Ca2+]i but isoproterenol and nifedipine did not. Taken together these results indicate that rapid reduction of [Ca2+]i reveals a permissive relationship between extracellular Ca2+, [Ca2+]i and IF. However IF can be dissipated by mechanisms effecting Ca2+ sensitivity. We speculate that an increase of IF, a fundamental property of ASM, could be related to human airway clinical hyperresponsiveness and must be accounted for in in vitro studies of hASM.

Role of transient receptor potential C3 in TNF-alpha-enhanced calcium influx in human airway myocytes.

Previous studies have suggested that the proinflammatory cytokine, TNF-alpha, contributes to airway hyperresponsivness by altering airway smooth muscle (ASM) Ca(2+) responses to agonist stimulation. The present study examined the effects of TNF-alpha on Ca(2+) influx pathways in cultured human ASM cells (HASMCs). Proteins encoded by the transient receptor potential (TRP) gene family function as channels through which receptor-operated and store-operated Ca(2+) entry (SOCE) occur. In the present study, the presence of TRPC1, TRPC3, TRPC4, TRPC5, and TRPC6 mRNA and protein expression was confirmed in cultured HASMCs using RT-PCR and Western blot analysis. TNF-alpha treatment significantly increased TRPC3 mRNA and protein levels in HASMCs as well as SOCE. TNF-alpha treatment also increased both the peak and plateau intracellular Ca(2+) concentration responses in HASMCs elicited by acetylcholine and bradykinin. The effects of TNF-alpha treatment on SOCE and agonist-induced intracellular Ca(2+) concentration responses were attenuated using small interfering RNA transfection, which knocked down TRPC3 expression. Thus, in inflammatory airway diseases, TNF-alpha treatment may result in increased myocyte activation due to altered Ca(2+) influx pathways. These results suggest that TRPC3 may be an important therapeutic target in inflammatory airway diseases such as asthma and chronic obstructive pulmonary disease.

Opposite alterations of DNA methyltransferase gene expression in endometrioid and serous endometrial cancers.

OBJECTIVE: To examine the DNA methyltransferase (DNMT) mRNA and protein levels in endometrioid and serous cancers and to study the relationship between DNA methyltransferase expression and endometrial cancer development. METHODS: Normal endometrium, Grade I and Grade III endometrioid carcinoma tissues and cell lines, as well as serous cancer tissues, were analyzed for DNMT expression. Real-time PCR and Western blot techniques were employed to measure the mRNA and protein levels of the four DNA methyltransferases, DNMT1, DNMT2, DNMT3A, and DNMT3B. Immunohistochemistry was performed to detect alterations in DNMT nuclear localization and spatial organization patterns. RESULTS: While DNMT2 and DNMT3A expression appear to be normal, two- to fourfold increase in DNMT1 and DNMT3B were found in both Grade I and Grade III endometrioid cancers. In addition, the poorly differentiated cell lines expressed relatively higher DNMT levels than well-differentiated cells. In contrast to endometrioid carcinomas, serous cancers expressed substantially lower levels of DNMT1 and DNMT3B than normal controls, with four- and twofold reduction observed in DNMT1 and DNMT3B mRNA levels, respectively. Western blot analysis confirmed opposite expression patterns of DNMT1 and DNMT3B protein in endometrioid and serous cancers. Immunohistochemistry showed normal nuclear localization of DNMT1 and DNMT3B in Type I and Type II cancer specimens as well as cell cultures. CONCLUSION: Two opposite DNMT expression patterns were identified in endometrioid and serous cancers. The concerted upregulation in maintenance and de novo DNA methyltransferases in endometrioid carcinomas is consistent with a tendency for gene-specific hypermethylation observed in this histologic subtype, and may be implicated in tumor suppressor silencing. In contrast, the downregulation of maintenance and de novo DNA methyltransferases in serous cancers suggests that these tumors may contain hypomethylated genomic DNA, which has been associated with a higher mutation rate and is consistent with the known pathogenesis of serous-specific phenotypes. Taken together, the data suggest that divergent DNA methylation pathways may be implicated in the development of Type I and Type II endometrial cancers.

HLA-DQ8 is a predisposing molecule for detergent enzyme subtilisin BPN'-induced hypersensitivity.

Several million individuals are exposed to agents in the workplace associated with atopy and asthma. Detergent enzymes have been implicated in occupationally induced hypersensitivity. However, the genetic susceptibility and T cell responses to detergent enzymes are undefined. We generated and used HLA-DQ6, -DQ8, -DR2, -DR3, and -DR4 transgenic mice to examine the immune and inflammatory components involved in the response to the detergent enzyme subtilisin BPN'. Based on in vitro and in vivo studies, for the first time, we present evidence that DQ8 is a strong susceptibility marker for BPN'-induced hypersensitivity. Only DQ8 mice showed consistent T cell responses to five immunodominant regions of BPN' comprising peptides #14 to 16, 36-37, 42-43, 62-63, and 80-81. The DQ8 mice also developed allergic eosinophilic inflammatory reactions in the airways following intranasal instillations of this enzyme. The DQ8 mice also responded to BPN' with a significant IgG1 and IgE production. We propose that the HLA Class II tg mice are useful for understanding allergenic responses to enzymes in humans, screening of allergenic and immunogenic properties of detergent enzymes, and for the development of modified enzymes to maintain efficient detergent qualities without allergic properties.