Joshua N. Farr

The Achilles' heel of senescent cells: from transcriptome to senolytic drugs

The healthspan of mice is enhanced by killing senescent cells using a transgenic suicide gene. Achieving the same using small molecules would have a tremendous impact on quality of life and the burden of age‐related chronic diseases. Here, we describe the rationale for identification and validation of a new class of drugs termed senolytics, which selectively kill senescent cells. By transcript analysis, we discovered increased expression of pro‐survival networks in senescent cells, consistent with their established resistance to apoptosis. Using siRNA to silence expression of key nodes of this network, including ephrins (EFNB1 or 3), PI3Kδ, p21, BCL‐xL, or plasminogen‐activated inhibitor‐2, killed senescent cells, but not proliferating or quiescent, differentiated cells. Drugs targeting these same factors selectively killed senescent cells. Dasatinib eliminated senescent human fat cell progenitors, while quercetin was more effective against senescent human endothelial cells and mouse BM‐MSCs. The combination of dasatinib and quercetin was effective in eliminating senescent MEFs. In vivo, this combination reduced senescent cell burden in chronologically aged, radiation‐exposed, and progeroid Ercc1−/Δ mice. In old mice, cardiac function and carotid vascular reactivity were improved 5 days after a single dose. Following irradiation of one limb in mice, a single dose led to improved exercise capacity for at least 7 months following drug treatment. Periodic drug administration extended healthspan in Ercc1−/∆ mice, delaying age‐related symptoms and pathology, osteoporosis, and loss of intervertebral disk proteoglycans. These results demonstrate the feasibility of selectively ablating senescent cells and the efficacy of senolytics for alleviating symptoms of frailty and extending healthspan.

In vivo assessment of bone quality in postmenopausal women with type 2 diabetes

Although patients with type 2 diabetes (T2D) are at significant risk for well‐recognized diabetic complications, including macrovascular disease, retinopathy, nephropathy, and neuropathy, it is also clear that T2D patients are at increased risk for fragility fractures. Furthermore, fragility fractures in patients with T2D occur at higher bone mineral density (BMD) values compared to nondiabetic controls, suggesting abnormalities in bone material strength (BMS) and/or bone microarchitecture (bone “quality”). Thus, we performed in vivo microindentation testing of the tibia to directly measure BMS in 60 postmenopausal women (age range, 50–80 years) including 30 patients diagnosed with T2D for >10 years and 30 age‐matched, nondiabetic controls. Regional BMD was measured by dual‐energy X‐ray absorptiometry (DXA); cortical and trabecular bone microarchitecture was assessed from high‐resolution peripheral quantitative computed tomography (HRpQCT) images of the distal radius and tibia. Compared to controls, T2D patients had significantly lower BMS: unadjusted (−11.7%; p < 0.001); following adjustment for body mass index (BMI) (−10.5%; p < 0.001); and following additional adjustment for age, hypertension, nephropathy, neuropathy, retinopathy, and vascular disease (−9.2%; p = 0.022). By contrast, after adjustment for confounding by BMI, T2D patients had bone microarchitecture and BMD that were not significantly different than controls; however, radial cortical porosity tended to be higher in the T2D patients. In addition, patients with T2D had significantly reduced serum markers of bone turnover (all p < 0.001) compared to controls. Of note, in patients with T2D, the average glycated hemoglobin level over the previous 10 years was negatively correlated with BMS (r = −0.41; p = 0.026). In conclusion, these findings represent the first demonstration of compromised BMS in patients with T2D. Furthermore, our results confirm previous studies demonstrating low bone turnover in patients with T2D and highlight the potential detrimental effects of prolonged hyperglycemia on bone quality. Thus, the skeleton needs to be recognized as another important target tissue subject to diabetic complications.

Physical activity levels in patients with early knee osteoarthritis measured by accelerometry

OBJECTIVE Physical activity (PA) is recommended for osteoarthritis (OA) management to reduce pain and improve function. The purpose of this study was to objectively assess the level and pattern of PA in male and female knee OA patients to determine adherence to Centers for Disease Control and Prevention/American College of Sports Medicine and Exercise and Physical Activity Conference recommendations for PA. METHODS Early OA patients (n = 255, 76% women, mean +/- SD age 54.6 +/- 7.1 years, mean +/- SD body mass index 27.8 +/- 4.3 kg/m(2)) with Kellgren/Lawrence-defined grade II (no higher) radiographic OA in at least 1 knee wore an accelerometer for 6-7 contiguous days. Light (LPA), moderate (MPA), and vigorous (VPA) PA intensities were defined as accelerometer recordings of 100-2,224, 2,225-5,950, and >5,950 counts per minute, respectively. RESULTS Patients wore accelerometers for a mean +/- SD of 6.8 +/- 0.3 days and 13.8 +/- 2.2 hours/day, and spent much more time (P < 0.001) in MPA (23.6 +/- 17.2 minutes/day) than VPA (0.95 +/- 3.5 minutes/day). Men spent significantly (P < 0.05) more time in all PA intensities than women. Only 30% of patients achieved recommended PA levels. The proportion of men (47%) achieving the recommendation was significantly (P = 0.04) higher than women (24%). CONCLUSION Knee OA patients accumulate little VPA and most (70%) do not achieve recommended levels for MPA or greater. New strategies to increase levels of PA in this population are needed.

Progressive Resistance Training Improves Overall Physical Activity Levels in Patients With Early Osteoarthritis of the Knee: A Randomized Controlled Trial

Background Prescription of resistance training (RT) exercises is an essential aspect of management for knee osteoarthritis (OA). However, whether patients with knee OA who are randomly assigned to receive RT simply substitute RT for other modes of physical activity remains unclear. Objective The aim of this study was to determine the effect of a structured RT intervention on overall levels of moderate- and vigorous-intensity physical activity (MVPA) in patients with early-onset knee OA. The study compared patients with early-onset OA who participated in an RT program, those who participated in a self-management (SM) program, and those who participated in both RT and SM. Because participants randomly assigned to receive the RT intervention may simply switch activity modes, resulting in little net effect, we assessed total MVPA in addition to tracking changes in strength (force-generating capacity). Design and Intervention This study was a randomized controlled trial comparing the effectiveness of SM alone, RT alone, and combined RT+SM on MVPA in patients with early OA of the knee. Setting The study was conducted on a university campus, with patient recruitment from the local community. Participants The participants in this study were 171 patients (74% women, 26% men) with knee OA. They had a mean age of 55.1 (SD=7.1) years, a mean body mass index of 27.6 (SD=4.2) kg/m2, and radiographic status of grade II OA (and no higher) in at least one knee, as defined by the Kellgren and Lawrence classification. They wore an accelerometer while awake (X̄=14.2 [SD=2.2] hours) for 5 to 7 contiguous days (X̄=6.8 [SD=0.5] days) at baseline and at 3 and 9 months of intervention. Results The participants engaged in MVPA a mean of 26.2 (SD=19.3) minutes per day at baseline. Both groups significantly increased their MVPA from baseline to 3 months (RT group by 18% [effect size (d)=0.26]; SM group by 22% [effect size (d)=0.25]), but only the RT group sustained those changes at 9 months (RT group maintained a 10% increase [effect size (d)=0.15]; SM group maintained a 2% increase [effect size (d)=0.03]). A significant group × time interaction for MVPA indicated that the RT group maintained higher MVPA levels than the SM group. Limitations. Lack of direct measures of energy expenditure and physical function was a limitation of the study. Conclusions Patients with early-onset OA of the knee can engage in an RT program without sacrificing their overall MVPA levels. These results support the value of RT for management of knee OA.

Skeletal muscle fat content is inversely associated with bone strength in young girls

Childhood obesity is an established risk factor for metabolic disease. The influence of obesity on bone development, however, remains controversial and may depend on the pattern of regional fat deposition. Therefore, we examined the associations of regional fat compartments of the calf and thigh with weight‐bearing bone parameters in girls. Data from 444 girls aged 9 to 12 years from the Jump‐In: Building Better Bones study were analyzed. Peripheral quantitative computed tomography (pQCT) was used to assess bone parameters at metaphyseal and diaphyseal sites of the femur and tibia along with subcutaneous adipose tissue (SAT, mm2) and muscle density (mg/cm3), an index of skeletal muscle fat content. As expected, SAT was positively correlated with total‐body fat mass (r = 0.87–0.89, p < .001), and muscle density was inversely correlated with total‐body fat mass (r = −0.24 to −0.28, p < .001). Multiple linear regression analyses with SAT, muscle density, muscle cross‐sectional area, bone length, maturity, and ethnicity as independent variables showed significant associations between muscle density and indices of bone strength at metaphyseal (β = 0.13–0.19, p < .001) and diaphyseal (β = 0.06–0.09, p < .01) regions of the femur and tibia. Associations between SAT and indices of bone strength were nonsignificant at all skeletal sites (β = 0.03–0.05, p > .05), except the distal tibia (β = 0.09, p = .03). In conclusion, skeletal muscle fat content of the calf and thigh is inversely associated with weight‐bearing bone strength in young girls.

Bone Strength and Structural Deficits in Children and Adolescents With a Distal Forearm Fracture Resulting From Mild Trauma

Although distal forearm fractures (DFFs) are common during childhood and adolescence, it is unclear whether they reflect underlying skeletal deficits or are simply a consequence of the usual physical activities, and associated trauma, during growth. Therefore, we examined whether a recent DFF, resulting from mild or moderate trauma, is related to deficits in bone strength and cortical and trabecular bone macro‐ and microstructure compared with nonfracture controls. High‐resolution peripheral quantitative computed tomography was used to assess micro‐finite element‐derived bone strength (ie, failure load) and to measure cortical and trabecular bone parameters at the distal radius and tibia in 115 boys and girls with a recent (<1 year) DFF and 108 nonfracture controls aged 8 to 15 years. Trauma levels (mild versus moderate) were assigned based on a validated classification scheme. Compared with sex‐matched controls, boys and girls with a mild‐trauma DFF (eg, fall from standing height) showed significant deficits at the distal radius in failure load (–13% and –11%, respectively; p < 0.05) and had higher (“worse”) fall load‐to‐strength ratios (both +10%; p < 0.05 for boys and p = 0.06 for girls). In addition, boys and girls with a mild‐trauma DFF had significant reductions in cortical area (–26% and –23%, respectively; p < 0.01) and cortical thickness (–14% and –13%, respectively; p < 0.01) compared with controls. The skeletal deficits in the mild‐trauma DFF patients were generalized, as similar changes were present at the distal tibia. By contrast, both boys and girls with a moderate‐trauma DFF (eg, fall from a bicycle) had virtually identical values for all of the measured bone parameters compared with controls. In conclusion, DFFs during growth have two distinct etiologies: those owing to underlying skeletal deficits leading to fractures with mild trauma versus those owing to more significant trauma in the setting of normal bone strength.

Identification of Senescent Cells in the Bone Microenvironment.

Cellular senescence is a fundamental mechanism by which cells remain metabolically active yet cease dividing and undergo distinct phenotypic alterations, including upregulation of p16Ink4a, profound secretome changes, telomere shortening, and decondensation of pericentromeric satellite DNA. Because senescent cells accumulate in multiple tissues with aging, these cells and the dysfunctional factors they secrete, termed the senescence‐associated secretory phenotype (SASP), are increasingly recognized as promising therapeutic targets to prevent age‐related degenerative pathologies, including osteoporosis. However, the cell type(s) within the bone microenvironment that undergoes senescence with aging in vivo has remained poorly understood, largely because previous studies have focused on senescence in cultured cells. Thus in young (age 6 months) and old (age 24 months) mice, we measured senescence and SASP markers in vivo in highly enriched cell populations, all rapidly isolated from bone/marrow without in vitro culture. In both females and males, p16Ink4a expression by real‐time quantitative polymerase chain reaction (rt‐qPCR) was significantly higher with aging in B cells, T cells, myeloid cells, osteoblast progenitors, osteoblasts, and osteocytes. Further, in vivo quantification of senescence‐associated distension of satellites (SADS), ie, large‐scale unraveling of pericentromeric satellite DNA, revealed significantly more senescent osteocytes in old compared with young bone cortices (11% versus 2%, p < 0.001). In addition, primary osteocytes from old mice had sixfold more (p < 0.001) telomere dysfunction‐induced foci (TIFs) than osteocytes from young mice. Corresponding with the age‐associated accumulation of senescent osteocytes was significantly higher expression of multiple SASP markers in osteocytes from old versus young mice, several of which also showed dramatic age‐associated upregulation in myeloid cells. These data show that with aging, a subset of cells of various lineages within the bone microenvironment become senescent, although senescent myeloid cells and senescent osteocytes predominantly develop the SASP. Given the critical roles of osteocytes in orchestrating bone remodeling, our findings suggest that senescent osteocytes and their SASP may contribute to age‐related bone loss.

Skeletal changes through the lifespan--from growth to senescence.

Age-related fragility fractures are an enormous public health problem. Both acquisition of bone mass during growth and bone loss associated with ageing affect fracture risk late in life. The development of high-resolution peripheral quantitative CT (HRpQCT) has enabled in vivo assessment of changes in the microarchitecture of trabecular and cortical bone throughout life. Studies using HRpQCT have demonstrated that the transient increase in distal forearm fractures during adolescent growth is associated with alterations in cortical bone, which include cortical thinning and increased porosity. Children with distal forearm fractures in the setting of mild, but not moderate, trauma also have increased deficits in cortical bone at the distal radius and in bone mass systemically. Moreover, these children transition into young adulthood with reduced peak bone mass. Elderly men, but not elderly women, with a history of childhood forearm fractures have an increased risk of osteoporotic fractures. With ageing, men lose trabecular bone primarily by thinning of trabeculae, whereas the number of trabeculae is reduced in women, which is much more destabilizing from a biomechanical perspective. However, age-related losses of cortical bone and increases in cortical porosity seem to have a much larger role than previously recognized, and increased cortical porosity might characterize patients at increased risk of fragility fractures.

Applications of a New Handheld Reference Point Indentation Instrument Measuring Bone Material Strength

A novel, hand-held Reference Point Indentation (RPI) instrument, measures how well the bone of living patients and large animals resists indentation. The results presented here are reported in terms of Bone Material Strength, which is a normalized measure of how well the bone resists indentation, and is inversely related to the indentation distance into the bone. We present examples of the instruments use in: (1) laboratory experiments on bone, including experiments through a layer of soft tissue, (2) three human clinical trials, two ongoing in Barcelona and at the Mayo Clinic, and one completed in Portland, OR, and (3) two ongoing horse clinical trials, one at Purdue University and another at Alamo Pintado Stables in California. The instrument is capable of measuring consistent values when testing through soft tissue such as skin and periosteum, and does so handheld, an improvement over previous Reference Point Indentation instruments. Measurements conducted on horses showed reproducible results when testing the horse through tissue or on bare bone. In the human clinical trials, reasonable and consistent values were obtained, suggesting the Osteoprobe® is capable of measuring Bone Material Strength in vivo, but larger studies are needed to determine the efficacy of the instruments use in medical diagnosis.

Relationship of Sympathetic Activity to Bone Microstructure, Turnover, and Plasma Osteopontin Levels in Women

CONTEXT Studies in rodents have demonstrated that sympathetic activity reduces bone formation and bone mass; these effects are mediated by the noncollagenous matrix protein, osteopontin. OBJECTIVE The objective of the study was to relate sympathetic activity (measured using microneurography at the peroneal nerve) to bone microstructure (assessed by high resolution peripheral quantitative computed tomography), bone turnover, and plasma osteopontin levels. DESIGN, SETTING, AND PATIENTS Twenty-three women aged 20-72 yr (10 premenopausal and 13 postmenopausal) were studied in the Clinical Research Unit. RESULTS Sympathetic activity (bursts per 100 heart beats) was 2.4-fold higher in postmenopausal as compared with premenopausal women (P < 0.001). In the two groups combined and after age adjustment, sympathetic activity was inversely correlated with trabecular bone volume fraction (r = -0.55, P < 0.01) and thickness (r = -0.59, P < 0.01) and positively correlated with trabecular separation (r = 0.45, P < 0.05). Sympathetic activity was negatively correlated with serum amino-terminal propeptide of type I collagen in postmenopausal women (r = -0.65, P = 0.015), with a similar trend in premenopausal women (r = -0.58, P = 0.082). Sympathetic activity was also negatively correlated with plasma osteopontin levels (r = -0.43, P = 0.045), driven mainly by the correlation in postmenopausal women (r = -0.76, P = 0.002). CONCLUSION These findings represent the first demonstration in humans of a relationship between sympathetic activity and bone microstructure and circulating levels of amino-terminal propeptide of type I collagen and osteopontin. Given the critical role of osteopontin in mediating the effects of β-adrenergic signaling on bone, the inverse association between sympathetic activity and plasma osteopontin levels may reflect a negative feedback loop to limit the deleterious effects of sympathetic activity on bone metabolism. Based on the higher sympathetic activity observed in postmenopausal women, additional human studies are needed to define the role of increased sympathetic activity in mediating postmenopausal bone loss.