Senolytic drugs: from discovery to translation.

Senolytics are a class of drugs that selectively clear senescent cells (SC). The first senolytic drugs Dasatinib, Quercetin, Fisetin and Navitoclax were discovered using a hypothesis-driven approach. SC accumulate with ageing and at causal sites of multiple chronic disorders, including diseases accounting for the bulk of morbidity, mortality and health expenditures. The most deleterious SC are resistant to apoptosis and have up-regulation of anti-apoptotic pathways which defend SC against their own inflammatory senescence-associated secretory phenotype (SASP), allowing them to survive, despite killing neighbouring cells. Senolytics transiently disable these SCAPs, causing apoptosis of those SC with a tissue-destructive SASP. Because SC take weeks to reaccumulate, senolytics can be administered intermittently - a 'hit-and-run' approach. In preclinical models, senolytics delay, prevent or alleviate frailty, cancers and cardiovascular, neuropsychiatric, liver, kidney, musculoskeletal, lung, eye, haematological, metabolic and skin disorders as well as complications of organ transplantation, radiation and cancer treatment. As anticipated for agents targeting the fundamental ageing mechanisms that are 'root cause' contributors to multiple disorders, potential uses of senolytics are protean, potentially alleviating over 40 conditions in preclinical studies, opening a new route for treating age-related dysfunction and diseases. Early pilot trials of senolytics suggest they decrease senescent cells, reduce inflammation and alleviate frailty in humans. Clinical trials for diabetes, idiopathic pulmonary fibrosis, Alzheimer's disease, COVID-19, osteoarthritis, osteoporosis, eye diseases and bone marrow transplant and childhood cancer survivors are underway or beginning. Until such studies are done, it is too early for senolytics to be used outside of clinical trials.

Inflammatory characteristics of adipose tissue collected by surgical excision vs needle aspiration.

Subcutaneous adipose tissue can be obtained for research during an elective, clinically indicated operation by standard surgical excision approaches and by needle aspiration in pure research settings. Whether measurements of inflammatory markers and cells from tissues collected in these two different ways are comparable is debatable. We sought to determine whether these two techniques yield systematically different results for measurements of inflammation, cellular senescence and adipose tissue composition. Twelve subjects undergoing surgery participated. At the time of surgery abdominal subcutaneous adipose tissue from adjacent sites was removed by excision and needle aspiration. Stromovascular cell composition (flow cytometry), the number of senescent cells (senescence-associated-ß-galactosidase staining) and interleukin (IL)-6, IL-1, TNF-α and MCP1 mRNA (reverse transcription-PCR) were measured in each sample. We found no statistically significant differences between the two sample-collection approaches for any of the parameters measured. We conclude that these two methods of obtaining adipose tissue do not systematically differ in the results of cytokine mRNA content, cellular senescence or stromovascular cell composition.

Circulating monocytes expressing senescence-associated features are enriched in COVID-19 patients with severe disease.

Accurate biomarkers for predicting COVID-19 severity have remained an unmet need due to an incomplete understanding of virus pathogenesis and heterogeneity among patients. Cellular senescence and its pro-inflammatory phenotype are suggested to be a consequence of SARS-CoV-2 infection and potentially drive infection-dependent pathological sequelae. Senescence-associated markers in infected individuals have been identified primarily in the lower respiratory tract, while little is known about their presence in more easily accessible bio-specimens. Here, we measured the abundance of senescence-associated signatures in whole blood, plasma and peripheral blood mononuclear cells (PBMCs) of COVID-19 patients and patients without an infection. Bulk transcriptomic and targeted proteomic assays revealed that the level of senescence-associated markers, including the senescence-associated secretory phenotype (SASP), is predictive of SARS-CoV-2 infection. Single-cell RNA-sequencing data demonstrated that a senescence signature is particularly enriched in monocytes of COVID-19 patients, partially correlating with disease severity. Our findings suggest that monocytes are prematurely induced to senescence by SARS-CoV-2 infection, might contribute to exacerbating a SASP-like inflammatory response and can serve as markers and predictors for COVID-19 and its sequelae.

Senolytic Therapy to Modulate the Progression of Alzheimer's Disease (SToMP-AD): A Pilot Clinical Trial.

Preclinical studies indicate an age-associated accumulation of senescent cells across multiple organ systems. Emerging evidence suggests that tau protein accumulation, which closely correlates with cognitive decline in Alzheimer's disease and other tauopathies, drives cellular senescence in the brain. Pharmacologically clearing senescent cells in mouse models of tauopathy reduced brain pathogenesis. Compared to vehicle treated mice, intermittent senolytic administration reduced tau accumulation and neuroinflammation, preserved neuronal and synaptic density, restored aberrant cerebral blood flow, and reduced ventricular enlargement. Intermittent dosing of the senolytics, dasatinib plus quercetin, has shown an acceptable safety profile in clinical studies for other senescence-associated conditions. With these data, we proposed and herein describe the objectives and methods for a clinical vanguard study. This initial open-label clinical trial pilots an intermittent senolytic combination therapy of dasatinib plus quercetin in five older adults with early-stage Alzheimer's disease. The primary objective is to evaluate the central nervous system penetration of dasatinib and quercetin through analysis of cerebrospinal fluid collected at baseline and after 12 weeks of treatment. Further, through a series of secondary outcome measures to assess target engagement of the senolytic compounds and Alzheimer's disease-relevant cognitive, functional, and physical outcomes, we will collect preliminary data on safety, feasibility, and efficacy. The results of this study will be used to inform the development of a randomized, double-blind, placebo-controlled multicenter phase II trial to further explore of the safety, feasibility, and efficacy of senolytics for modulating the progression of Alzheimer's disease. Clinicaltrials.gov registration number and date: NCT04063124 (08/21/2019).

Proceedings of the Canadian Frailty Network Workshop: Identifying Biomarkers of Frailty to Support Frailty Risk Assessment, Diagnosis and Prognosis. Toronto, January 15, 2018.

The Canadian Frailty Network (CFN), a pan-Canadian not-for-profit organization funded by the Government of Canada through the Networks of Centres of Excellence Program, is dedicated to improving the care of older Canadians living with frailty. The CFN has partnered with the Canadian Longitudinal Study on Aging (CLSA) to measure potential frailty biomarkers in biological samples (whole blood, plasma, urine) collected in over 30,000 CLSA participants. CFN hosted a workshop in Toronto on January 15 2018, bringing together experts in the field of biomarkers, aging and frailty. The overall objectives of the workshop were to start building a consensus on potential frailty biomarker domains and identify specific frailty biomarkers to be measured in the CLSA biological samples. The workshop was structured with presentations in the morning to frame the discussions for the afternoon session, which was organized as a free-flowing discussion to benefit from the expertise of the participants. Participants and speakers were from Canada, Italy, Spain, United Kingdom and the United States. Herein we provide pertinent background information, a summary of all the presentations with key figures and tables, and the distillation of the discussions. In addition, moving forward, the principles CFN will use to approach frailty biomarker research and development are outlined. Findings from the workshop are helping CFN and CLSA plan and conduct the analysis of biomarkers in the CLSA samples and which will inform a follow-up data access competition.

Muscle-specific differences in expression and phosphorylation of the Janus kinase 2/Signal Transducer and Activator of Transcription 3 following long-term mechanical ventilation and immobilization in rats.

AIM: Muscle wasting is one of the factors most strongly predicting mortality and morbidity in critically ill intensive care unit (ICU). This muscle wasting affects both limb and respiratory muscles, but the understanding of underlying mechanisms and muscle-specific differences remains incomplete. This study aimed at investigating the temporal expression and phosphorylation of the Janus kinase/signal transducer and activator of transcription (JAK/STAT) pathway in muscle wasting associated with the ICU condition to characterize the JAK/STAT proteins and the related changes leading or responding to their activation during exposure to the ICU condition. METHODS: A novel experimental ICU model allowing long-term exposure to the ICU condition, immobilization and mechanical ventilation, was used in this study. Rats were pharmacologically paralysed by post-synaptic neuromuscular blockade and mechanically ventilated for durations varying between 6 hours and 14 days to study muscle-specific differences in the temporal activation of the JAK/STAT pathway in plantaris, intercostal and diaphragm muscles. RESULTS: The JAK2/STAT3 pathway was significantly activated irrespective of muscle, but muscle-specific differences were observed in the temporal activation pattern between plantaris, intercostal and diaphragm muscles. CONCLUSION: The JAK2/STAT3 pathway was differentially activated in plantaris, intercostal and diaphragm muscles in response to the ICU condition. Thus, JAK2/STAT3 inhibitors may provide an attractive pharmacological intervention strategy in immobilized ICU patients, but further experimental studies are required in the study of muscle-specific effects on muscle mass and function in response to both short- and long-term exposure to the ICU condition prior to the translation into clinical research and practice.

Varying capacities for replication of rat adipocyte precursor clones and adipose tissue growth.

Rat adipocyte precursor populations contain clones varying in capacity for replication. In this study we explored factors controlling the frequency of clones of varying replicative capacities (clonal composition). We also explored the relationship between this frequency and fat depot growth. In perirenal and epididymal depots clonal composition was identical bilaterally; perirenal depots contained more extensively replicating clones. Although there were large interanimal differences in clonal composition, variation between animals was always in the same direction for both depots. Clonal composition was unaffected by undernutrition while with animal growth the frequency of the most extensively replicating clones was reduced. Differentiation of precursors occurred in all clones, while differentiation did not occur in skin fibroblasts cloned under identical conditions. Clonal composition and mature fat cell number were related in that fat cell numbers were identical bilaterally in both depots and increased more extensively with growth in perirenal than epididymal tissue. We conclude (a) that clonal composition of adipocyte precursor populations is regulated genetically and by age, (b) that this composition determines, at least in part, the capacity for adipose depot growth.

Molecular pathology endpoints useful for aging studies.

The first clinical trial aimed at targeting fundamental processes of aging will soon be launched (TAME: Targeting Aging with Metformin). In its wake is a robust pipeline of therapeutic interventions that have been demonstrated to extend lifespan or healthspan of preclinical models, including rapalogs, antioxidants, anti-inflammatory agents, and senolytics. This ensures that if the TAME trial is successful, numerous additional clinical trials are apt to follow. But a significant impediment to these trials remains the question of what endpoints should be measured? The design of the TAME trial very cleverly skirts around this based on the fact that there are decades of data on metformin in humans, providing unequaled clarity of what endpoints are most likely to yield a positive outcome. But for a new chemical entity, knowing what endpoints to measure remains a formidable challenge. For economy's sake, and to achieve results in a reasonable time frame, surrogate markers of lifespan and healthy aging are desperately needed. This review provides a comprehensive analysis of molecular endpoints that are currently being used as indices of age-related phenomena (e.g., morbidity, frailty, mortality) and proposes an approach for validating and prioritizing these endpoints.

Effect of Low-Dose Rapamycin on Senescence Markers and Physical Functioning in Older Adults with Coronary Artery Disease: Results of a Pilot Study.

Rapamycin, an mTOR inhibitor affects senescence through suppression of senescence-associated secretory phenotype (SASP). We studied the safety and feasibility of low-dose rapamycin and its effect on SASP and frailty in elderly undergoing cardiac rehabilitation (CR). 13 patients; 6 (0.5mg), 6 (1.0mg), and 1 patient received 2mg oral rapamycin (serum rapamycin <6ng/ml) daily for 12 weeks. Median age was 73.9±7.5 years and 12 were men. Serum interleukin-6 decreased (2.6 vs 4.4 pg/ml) and MMP-3 (26 vs 23.5 ng/ml) increased. Adipose tissue expression of mRNAs (arbitrary units) for MCP-1 (3585 vs 2020, p=0.06), PPAR-γ (1257 vs 1166), PAI-1 (823 vs 338, p=0.08) increased, whereas interleukin-8 (163 vs 312), TNF-α (75 vs 94) and p16 (129 vs 169) decreased. Cellular senescence-associated beta galactosidase activity (2.2% vs 3.6%, p=0.18) tended to decrease. We observed some correlation between some senescence markers and physical performance but no improvement in frailty with rapamycin was noted. (NCT01649960).

Progesterone inhibits the estrogen-induced expression of c-fos messenger ribonucleic acid in the uterus.

Estradiol produces a large increase in the uterine level of c-fos mRNA, which is maximum in 3 h. The administration of progesterone antagonizes this estrogen-induced increase in protooncogene transcript levels in both the rat and mouse. The inhibitory effect of progesterone is observed within 1 h after hormone treatment and persists for 9-18 h. In the rat, this effect can be observed at a dose of 0.25 mg progesterone and is maximum at a dose of 2.5 mg. A similar inhibition of fos mRNA levels after estrogen administration is produced by the glucocorticoid dexamethasone, but not by androgens or mineralocorticoids. Progesterone does not block the induction of c-jun or c-myc mRNA by estradiol. Uterine levels of c-fos mRNA observed after treatment with the phorbol ester phorbol 12-myristate 13-acetate are not decreased by a 3-h pretreatment with progesterone. Under the conditions of our experiments, progesterone does not decrease occupied levels of nuclear estrogen receptors in the uterus after estradiol administration. These findings are consistent with a mechanism in which progesterone inhibits transcriptional activation by the estrogen receptor at the level of the c-fos gene.

Effect of hypophysectomy on rat preadipocyte replication and differentiation.

The effects of hypophysectomy, which results in decreased fat pad weight, fat cell number, and new fat cell formation, on preadipocyte replication were examined. Perirenal and epididymal preadipocytes were cultured from hypophysectomized, sham-operated and unoperated 3-month-old rats. In cloned preadipocytes, hypophysectomy resulted in a 19% decrease in cloning efficiency and a 60% reduction in cell number after 3 weeks in culture. Perirenal cells underwent more extensive replication than epididymal cells. The mechanism of reduced replication following hypophysectomy differed from that of donor site: hypophysectomy resulted in an increased percentage of cells which underwent 2 or fewer population doublings at the expense of cells capable of more than 2 doublings. However, donor site had little effect specifically on these slowly replicating preadipocytes; rather, perirenal preadipocytes underwent more extensive replication than epididymal cells because of the higher percentage of preadipocytes capable of 13 or more doublings in perirenal fat pads. Hypophysectomy did not result in decreased differentiation of preadipocytes. These observations are in accord with the hypothesis that hypophysectomy reduces fat cell number in maturing rats partly through an effect on preadipocyte replicative capacity. Additionally, it seems that more than one form of preadipocyte exists, the various forms having differing susceptibilities to factors such as pituitary function and anatomic site.

Selective blockade of estrogen-induced uterine responses by the antiestrogen nafoxidine.

The response of the immature rat uterus to the antiestrogen, nafoxidine (Upjohn U-11, 100A), is maximum at a dose of 5 micrograms. This dose of nafoxidine sustains the uterine response for at least 72 h. After treatment with 5 micrograms nafoxidine for 24 h, uterine cytosol contains approximately one half the total number of estrogen receptors originally present in uteri of untreated animals, and uterine nuclei also contain approximately one half the receptors originally present in unstimulated tissue. The total amount of uterine estrogen receptor, however, is not altered 24 h after treatment with nafoxidine relative to saline-treated controls, while estradiol treatment doubles the total amount of receptor relative to controls. Pretreatment with 5 micrograms nafoxidine for 24 h does not block the uterine response occurring 4 h after the subsequent administration of estradiol, but does block the uterine response occurring 24 h after the subsequent administration of estradiol. These results suggest that uterine nuclei contain different acceptor sites for regulating short term and long term uterine responses to estrogen.

Hormonal control of uterine growth: alterations in luminal epithelial deoxyribonucleic acid synthesis after intraluminal application of estrogen.

Administration of a single injection of estradiol (E2) causes a maximum increase in DNA synthesis in the uterine luminal epithelium approximately 24 h later. We previously reported that animals receiving a second injection of E2 15-18 h after the first show an apparent decrease in DNA synthesis measured in this cell type at 24 h. This apparent decrease in DNA synthesis is due to a shift in the time course of DNA synthesis rather than an absolute decrease in this parameter. In this report we demonstrate that this inhibitory effect of a second injection of E2 is also observed after intraluminal instillation of the hormone. This effect is dose dependent and appears to result from a shift in the time course of luminal epithelial DNA synthesis. The intraluminal instillation of E2 produces the same pattern of nuclear receptor localization as sc administration of the hormone. These results suggest that this inhibitory effect we previously described results primarily from a direct action of E2 on the luminal epithelium.

Hormonal control of uterine growth: temporal relationships between estrogen administration and deoxyribonucleic acid synthesis.

Administration of a single injection of estradiol (E2) causes a maximum increase in DNA synthesis of all major uterine cell types approximately 24 h later. Animals given a second injection of E2 6-12 h after the first show an apparent increase in DNA synthesis in the luminal epithelium. Animals receiving a second injection of E2 15-18 h after the first show an apparent decrease in DNA synthesis at 24 h, which is most prominent in the luminal epithelium. This apparent decrease in DNA synthesis is most apparent if the second injection of E2 is given 18 h after the first, and is due to a shift of 10-12 h in the time course of DNA synthesis rather than to an absolute decrease in this parameter. This shift in the time course of uterine DNA synthesis is a dose-dependent phenomenon and displays a dose-response curve similar to that for the stimulation of DNA synthesis by a single injection of E2. A third injection of E2, 28 h after the initial hormone treatment, again causes a shift of 10-12 h in the time course of DNA synthesis relative to that in animals receiving two injections of hormone. These results suggest that nuclear levels of estrogen receptor, which initially increase after hormone administration, must decrease before the onset of uterine DNA synthesis.

Stimulatory and inhibitory effects of estrogen and antiestrogen on uterine cell division.

Acute administration of 17 beta-estradiol or the antiestrogen nafoxidine to immature rats produces quantitatively similar responses in the uterine stroma and myometrium, although the responses to nafoxidine occur at slightly later times. Both the hormone and drug cause nuclear translocation of equivalent amounts of estrogen receptor in these two cell types, although receptor translocation is slower with nafoxidine, and nuclear receptor levels remain elevated for an extended time. Based upon labeling and mitotic indices, however, the response of the luminal epithelium to a maximum dose of nafoxidine is much lower than that produced by estradiol, although nafoxidine treatment causes massive hypertrophy of luminal epithelial cells and causes nuclear translocation of estrogen receptors in this cell type. If nafoxidine is administered 30 min after administration of estradiol, cell division in the luminal epithelium is inhibited. Furthermore, multiple injections of estradiol itself within a 24-h period decrease cell division in the luminal epithelium relative to controls receiving a single injection of the hormone. These results indicate that estradiol and the antiestrogen can have both inhibitory and stimulatory effects on the luminal epithelium and that cell division in different uterine cell types can be differentially affected.

Decreased cell division of the uterine luminal epithelium of diabetic rats in response to 17 beta-estradiol.

The uterine growth response to estradiol as determined by luminal epithelial cell division was measured in euglycemic and drug induced insulin deficient rats. The results demonstrated that uterine cell division in insulin deficient rats is depressed when compared to euglycemic rats. Replacement of insulin by osmotic minipumps (Alza Corporation) in insulin deficient rats restored the level of luminal cell division to those observed in euglycemic rats.

Regulation of deoxyribonucleic acid polymerase activity in uterine luminal epithelium after multiple doses of estrogen.

Estradiol (E2) exerts both inhibitory and stimulatory effects on DNA synthesis in the rat uterine luminal epithelium (LE). This inhibitory effect is due to a shift in the time course of DNA synthesis, i.e. in animals receiving a single injection of E2, a peak of DNA synthesis occurs 24 h after treatment, but in animals receiving multiple injections of E2, DNA synthesis is suppressed until 10-12 h after hormone treatment ceases. In these previous studies LE DNA synthesis was assessed by measuring tritiated thymidine incorporation. In the present study, we sought to determine if the molecular basis for this decrease in DNA synthesis was due to a suppression of DNA polymerase activity in LE nuclei. Animals receiving a single injection of E2 exhibit a peak of nuclear DNA polymerase activity 20-24 h later. Animals receiving multiple injections of E2 (0, 12, 15, and 18 h) show more than a 50% decrease in DNA polymerase activity at 20-24 h, due to a shift in the maximum increase in enzyme activity to 32-36 h after the initial treatment. The observed differences between these groups are not due to different levels of DNase activity or different degrees of leakage of the nuclear enzyme. The observed enzyme activity is due to DNA polymerase-alpha, since it requires ATP as well as deoxyribonucleoside triphosphates, and is aphidicolin sensitive. These results indicate that the inhibitory effect of E2 on LE DNA synthesis is due at least in part to a suppression of nuclear DNA polymerase-alpha activity.

Regulation of the uterine response to estrogen by thyroid hormone.

After estradiol (E2) administration, early increases (within 4 h) in uterine wet weight and the synthesis of 2-deoxyglucose-6-phosphate from 2-deoxyglucose are similar in ovariectomized rats and in ovariectomized rats made hypothyroid by feeding a low iodine diet containing prophylthiouracil. Most late uterine responses occurring 24 h after E2 treatment, however, are greatly diminished in the hypothyroid animals. The diminished responses include increases in uterine wet weight, dry weight, protein content, RNA content, and the incorporation of thymidine into uterine DNA. One response, the synthesis of 2-deoxyglucose-6-phosphate from 2-deoxyglucose is not diminished in hypothyroid rats 24 h after E2 treatment. The diminished uterine response is not due to a shift in the dose-response curve for E2, but results from a decrease in the magnitude of the maximum uterine response. Treatment of hypothyroid rats with exogenous T3 restores the diminished uterine response in a dose-dependent and time-dependent manner. A dose of 0.5 microgram T3/100 g BW for 5 days restores the response completely, while 48-72 h of treatment with higher doses of T3 are required to restore the response. The effect of T3 is not mediated by the pituitary, since exogenous T3 restores diminished uterine responses in ovariectomized, hypophysectomized animals. These results suggest that thyroid hormones may have a direct effect on the uterus which regulates the responsiveness of the organ to E2.

Hormonal control of uterine growth: the effect of hypothyroidism on estrogen-stimulated cell division.

The increase in mitotic indices of uterine luminal epithelium, stroma, and myometrium were determined as a function of time after the administration of a single dose of 17 beta-estradiol to euthyroid and hypothyroid rats. Hypothyroidism reduced the increase in the mitotic index 5-fold in the luminal epithelium, 6-fold in the stroma, and 9-fold in the myometrium. In addition to reducing mitotic indices, hypothyroidism also produced a shift of 12 h in the time course of estrogen-stimulated cell division of all uterine cell types relative to euthyroid animals. This shift in the time course of cell division was preceded by a shift in the time course of uterine DNA synthesis measured by tritiated thymidine incorporation. In contrast, hypothyroidism did not alter the magnitude or the time course of synthesis of 2-deoxyglucose-6-phosphate from 2-deoxyglucose after estrogenic stimulation. These results indicate that hypothyroidism decreases the ability of all major uterine cell types to undergo cell division in response to acute administration of estradiol, and also shifts the time course of the uterine growth response to the hormone.

Polyuria and inappropriate secretion of arginine vasopressin in hypothalamic sarcoidosis.

Inappropriate arginine vasopressin release and polyuria with excessive thirst were found in a patient with hypothalamic sarcoidosis subsequently confirmed at autopsy. He became intensely thirsty during a 5% saline infusion at a plasma osmolality of 274 mosmol/liter. The normal thirst threshold under these conditions is 294 +/- 3 mosmol/liter (+/- SD). An increase in radioimmunoassayable arginine vasopressin was detected at an inappropriately low plasma osmolality, and free water clearance was negative despite a plasma osmolality of 265 +/- 5 mosmol/liter with ad libitum fluid intake. The syndrome of inappropriate antidiuresis has not been described previously in combination with hypothalamic sarcoidosis. Demeclocycline therapy was associated with an exacerbation of the patient's polyuria. Propranolol administration, however, was associated with a reduction of urine output and an increase in plasma osmolality to 279 mosmol/liter on one and 299 mosmol/liter on another occasion.