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.

Clinical radiotherapy treats life‐threatening cancers, but the radiation often affects neighboring normal tissues including bone. Acute effects of ionizing radiation include oxidative stress, DNA damage, and cellular apoptosis. We show in this study that a large proportion of bone marrow cells, osteoblasts, and matrix‐embedded osteocytes recover from these insults only to attain a senescent profile. Bone analyses of senescence‐associated genes, senescence‐associated beta‐galactosidase (SA‐β‐gal) activity, and presence of telomere dysfunction‐induced foci (TIF) at 1, 7, 14, 21, and 42 days post–focal radiation treatment (FRT) in C57BL/6 male mice confirmed the development of senescent cells and the senescence‐associated secretory phenotype (SASP). Accumulation of senescent cells and SASP markers were correlated with a significant reduction in bone architecture at 42 days post‐FRT. To test if senolytic drugs, which clear senescent cells, alleviate FRT‐related bone damage, we administered the senolytic agents, dasatinib (D), quercetin (Q), fisetin (F), and a cocktail of D and Q (D+Q). We found moderate alleviation of radiation‐induced bone damage with D and Q as stand‐alone compounds, but no such improvement was seen with F. However, the senolytic cocktail of D+Q reduced senescent cell burden as assessed by TIF⁺ osteoblasts and osteocytes, markers of senescence (p16 ^Ink4a and p21), and key SASP factors, resulting in significant recovery in the bone architecture of radiated femurs. In summary, this study provides proof of concept that senescent cells play a role in radiotherapy‐associated bone damage, and that reduction in senescent cell burden by senolytic agents is a potential therapeutic option for alleviating radiotherapy‐related bone deterioration. © 2020 American Society for Bone and Mineral Research.

BACKGROUND. Evidence from rodent studies indicates that the sympathetic nervous system (SNS) regulates bone metabolism, principally via β₂-adrenergic receptors (β₂-ARs). Given the conflicting human data, we used multiple approaches to evaluate the role of the SNS in regulating human bone metabolism. METHODS. Bone biopsies were obtained from 19 young and 19 elderly women for assessment of ADRB1, ADRB2, and ADRB3 mRNA expression. We examined the relationship of β-blocker use to bone microarchitecture by high-resolution peripheral quantitative CT in a population sample of 248 subjects. A total of 155 postmenopausal women were randomized to 1 of 5 treatment groups for 20 weeks: placebo; propranolol, 20 mg b.i.d.; propranolol, 40 mg b.i.d.; atenolol, 50 mg/day; or nebivolol, 5 mg/day. We took advantage of the β₁-AR selectivity gradient of these drugs (propranolol [nonselective] << atenolol [relatively β₁-AR selective] < nebivolol [highly β₁-AR selective]) to define the β-AR selectivity for SNS effects on bone. RESULTS. ADRB1 and ADRB2, but not ADRB3, were expressed in human bone; patients treated clinically with β₁-AR–selective blockers had better bone microarchitecture than did nonusers, and relative to placebo, atenolol and nebivolol, but not propranolol, reduced the bone resorption marker serum C-telopeptide of type I collagen (by 19.5% and 20.6%, respectively; P < 0.01) and increased bone mineral density of the ultradistal radius (by 3.6% and 2.9%; P < 0.01 and P < 0.05, respectively). CONCLUSIONS. These 3 independent lines of evidence strongly support a role for adrenergic signaling in the regulation of bone metabolism in humans, principally via β₁-ARs. TRIAL REGISTRATION. ClinicalTrials.gov NCT02467400. FUNDING. This research was supported by the NIH (AG004875 and AR027065) and a Mayo Clinic Clinical and Translational Science Award (CTSA) (UL1 TR002377).

The worldwide prevalence of type 2 diabetes (T2D) is increasing. Despite normal to higher bone density, patients with T2D paradoxically have elevated fracture risk resulting, in part, from poor bone quality. Advanced glycation endproducts (AGEs) and inflammation as a consequence of enhanced receptor for AGE (RAGE) signaling are hypothesized culprits, although the exact mechanisms underlying skeletal dysfunction in T2D are unclear. Lack of inducible models that permit environmental (in obesity) and temporal (after skeletal maturity) control of T2D onset has hampered progress. Here, we show in C57BL/6 mice that a onetime pharmacological intervention (streptozotocin, STZ) initiated in adulthood combined with high-fat diet–induced (HFD-induced) obesity caused hallmark features of human adult-onset T2D, including prolonged hyperglycemia, insulin resistance, and pancreatic β cell dysfunction, but not complete destruction. In addition, HFD/STZ (i.e., T2D) resulted in several changes in bone quality that closely mirror those observed in humans, including compromised bone microarchitecture, reduced biomechanical strength, impaired bone material properties, altered bone turnover, and elevated levels of the AGE CML in bone and blood. Furthermore, T2D led to the premature accumulation of senescent osteocytes with a unique proinflammatory signature. These findings highlight the RAGE pathway and senescent cells as potential targets to treat diabetic skeletal fragility.

Estrogen deficiency is a seminal mechanism in the pathogenesis of osteoporosis. Mounting evidence, however, establishes that cellular senescence, a fundamental mechanism that drives multiple age‐related diseases, also causes osteoporosis. Recently, we systematically identified an accumulation of senescent cells, characterized by increased p16^Ink4a and p21^Cip1 levels and development of a senescence‐associated secretory phenotype (SASP), in mouse bone/marrow and human bone with aging. We then demonstrated that elimination of senescent cells prevented age‐related bone loss using multiple approaches, eg, treating old mice expressing a “suicide” transgene, INK‐ATTAC, with AP20187 to induce apoptosis of p16^Ink4a‐senescent cells or periodically treating old wild‐type mice with “senolytics,” ie, drugs that eliminate senescent cells. Here, we investigate a possible role for estrogen in the regulation of cellular senescence using multiple approaches. First, sex steroid deficiency 2 months after ovariectomy (OVX, n = 15) or orchidectomy (ORCH, n = 15) versus sham surgery (SHAM, n = 15/sex) in young adult (4‐month‐old) wild‐type mice did not alter senescence biomarkers or induce a SASP in bone. Next, in elderly postmenopausal women, 3 weeks of estrogen therapy (n = 10; 74 ± 5 years) compared with no treatment (n = 10; 78 ± 5 years) did not alter senescence biomarkers or the SASP in human bone biopsies. Finally, young adult (4‐month‐old) female INK‐ATTAC mice were randomized (n = 17/group) to SHAM+Vehicle, OVX+Vehicle, or OVX+AP20187 for 2 months. As anticipated, OVX+Vehicle caused significant trabecular/cortical bone loss compared with SHAM+Vehicle. However, treatment with AP20187, which eliminates senescent cells in INK‐ATTAC mice, did not rescue the OVX‐induced bone loss or alter senescence biomarkers. Collectively, our data establish independent roles of estrogen deficiency and cellular senescence in the pathogenesis of osteoporosis, which has important implications for testing novel senolytics for skeletal efficacy, as these drugs will need to be evaluated in preclinical models of aging as opposed to the current FDA model of prevention of OVX‐induced bone loss. © 2019 American Society for Bone and Mineral Research.