Khosla, Sundeep

Abstract Skeletal muscle and bone form highly-integrated systems that undergo significant age-related changes, but the relationships between muscle mass and trabecular versus cortical bone or trabecular microarchitecture have not been systematically investigated. Thus, we examined the association between appendicular skeletal muscle mass (ASM) relative to height squared (relative ASM) and bone parameters at several sites assessed by conventional as well as high-resolution peripheral QCT in a cohort of 272 women and 317 men aged 20 to 97 years. In women, relative ASM was associated with cortical thickness (CtTh) at the femoral neck, lumbar spine, radius, and tibia (age-and physical activity adjusted r = 0.19–0.32; all p < 0.01). Relative ASM was also associated with trabecular volumetric bone mineral density (vBMD) at the femoral neck and spine (all p < 0.05), and trabecular bone volume to tissue volume (BV/TV), number (TbN), thickness (TbTh), and separation (TbSp) at the radius (all p ≤ 0.05). In all men, relative ASM was associated with CtTh at all sites (age- and physical activity–adjusted r = 0.17–0.28; all p < 0.01). Associations between relative ASM and trabecular vBMD at the spine in men were lost after adjusting for age; however, relative ASM was associated with trabecular vBMD at the femoral neck and TbN and TbSp at the radius (all p < 0.01). We also investigated circulating factors associated with bone health that may be indicative of relative ASM and found that serum insulin-like growth factor (IGF) binding protein-2 (IGFBP-2) levels were the most robust negative predictors of relative ASM in both sexes. Collectively, these data add to the growing body of evidence supporting the highly-integrated nature of skeletal muscle and bone, and provide new insights into potential biomarkers that reflect the health of the musculoskeletal system. © 2012 American Society for Bone and Mineral Research.

Abstract Advanced bone imaging with quantitative computed tomography (QCT) has had limited success in significantly improving fracture prediction beyond standard areal bone mineral density (aBMD) measurements. Thus, we examined whether a machine learning paradigm, gradient boosting machine (GBM) modeling, which can incorporate diverse measurements of bone density and geometry from central QCT imaging and of bone microstructure from high-resolution peripheral QCT imaging, can improve fracture prediction. We studied two cohorts of postmenopausal women: 105 with and 99 without distal forearm fractures (Distal Forearm Cohort) and 40 with at least one grade 2 or 3 vertebral deformity and 78 with no vertebral fracture (Vertebral Cohort). Within each cohort, individual bone density, structure, or strength variables had areas under receiver operating characteristic curves (AUCs) ranging from 0.50 to 0.84 (median 0.61) for discriminating women with and without fracture. Using all possible variables in the GBM model, the AUCs were close to 1.0. Fracture predictions in the Vertebral Cohort using the GBM models built with the Distal Forearm Cohort had AUCs of 0.82–0.95, whereas predictions in the Distal Forearm Cohort using models built with the Vertebral Cohort had AUCs of 0.80–0.83. Attempts at capturing a comparable parametric model using the top variables from the Distal Forearm Cohort resulted in resulted in an AUC of 0.81. Relatively high AUCs for differing fracture types suggest that an underlying fracture propensity is being captured by this modeling approach. More complex modeling, such as with GBM, creates stronger fracture predictions and may allow deeper insights into information provided by advanced bone imaging techniques. © 2012 American Society for Bone and Mineral Research.

The trabecular and cortical compartments of bone each contributes to bone strength. Until recently, assessment of trabecular and cortical microstructure has required a bone biopsy. Now, trabecular and cortical microstructure of peripheral bone sites can be determined noninvasively using high-resolution peripheral quantitative computed tomography (HR-pQCT). Studies that have used HR-pQCT to evaluate cohorts of both men and women have provided novel insights into the changes in bone microarchitecture that occur with age between the sexes, which may help to explain the lower fracture incidence in older men relative to women. This review will highlight observations from these studies on both the sex- and age-related differences in trabecular and cortical microstructure that may underlie the differences in bone strength, and thereby fracture risk, between men and women.

Renal function and blood flow decline during aging in association with a decrease in the number of intrarenal vessels, but if loss of estrogen contributes to this microvascular, rarefaction remains unclear. We tested the hypothesis that the decreased renal microvascular density with age is aggravated by loss of estrogen. Six-month-old female C57/BL6 mice underwent ovariectomy (Ovx) or sham operation and then were allowed to age to 18–22 mo. Another comparable group was replenished with estrogen after Ovx (Ovx+E), while a 6-mo-old group served as young controls. Kidneys were then dissected for evaluation of microvascular density (by micro-computed tomography) and angiogenic and fibrogenic factors. Cortical density of small microvessels (20–200 μm) was decreased in all aged groups compared with young controls (30.3 ± 5.8 vessels/mm2, P < 0.05), but tended to be lower in sham compared with Ovx and Ovx+E (9.9 ± 1.7 vs. 17.2 ± 4.2 and 18 ± 3.0 vessels/mm2, P = 0.08 and P = 0.02, respectively). Cortical density of larger microvessels (200–500 μm) decreased only in aged sham ( P = 0.04 vs. young control), and proangiogenic signaling was attenuated. On the other hand, renal fibrogenic mechanisms were aggravated in aged Ovx compared with aged sham, but blunted in Ovx+E, in association with downregulated transforming growth factor-β signaling and decreased oxidative stress in the kidney. Therefore, aging induced in female mice renal cortical microvascular loss, which was likely not mediated by loss of endogenous estrogen. However, estrogen may play a role in protecting the kidney by decreasing oxidative stress and attenuating mechanisms linked to renal interstitial fibrosis.

Abstract A positive association has been reported between greater bone density and higher breast cancer risk, suggesting that these women could be at reduced risk of fracture. To estimate fracture risk among unselected community women with breast cancer and to systematically assess associations with various risk factors including breast cancer treatments, we conducted a population-based historical cohort study of 608 Olmsted County, MN, USA, women with invasive breast cancer first diagnosed in 1990 to 1999 (mean age 61.6 ± 14.8 years), who were followed for 5776 person-years. Altogether, 568 fractures were observed in 270 women (98 per 1000 person-years). Overall fracture risk was elevated 1.8-fold, but the absolute increase in risk was only 9%, and 56% of the women did not experience a fracture during follow-up. Excluding pathologic fractures (15%) and those found incidentally (24%), to allow for ascertainment bias, the standardized incidence ratio was 1.2 (95% confidence interval [CI] 0.99 to 1.3) for total fracture risk and 0.9 (95% CI 0.7 to 1.2) for osteoporotic fracture risk alone. Various breast cancer treatments were associated with an increased risk of fracture, but those associations were strongest for pathologic fractures, which were relatively more common among the women who were premenopausal when their breast cancer was diagnosed. Moreover, underlying clinical characteristics prompting different treatments may have been partially responsible for the associated fracture outcomes (indication bias). These data thus demonstrate that breast cancer patients in general are not at greatly increased risk of fracture but neither are they protected from fractures despite any determinants that breast cancer and high bone density may have in common. © 2012 American Society for Bone and Mineral Research.

Abstract A complex network of transcription factors contributes to the establishment and maintenance of the osteoblastic phenotype. Although relatively few transcription factors, such as Runx2 and osterix, are essential to the process of osteoblastic differentiation, others serve the purpose of fine-tuning in response to various environmental and hormonal cues. The nuclear receptor (NR) superfamily of transcription factors are involved in numerous aspects of bone biology. In this study, we characterized the expression pattern of the entire NR superfamily in differentiating primary murine calvarial cells in order to identify novel NR regulatory patterns. Dynamic patterns of NR expression were observed throughout the differentiation process. Interestingly, retinoic acid receptor-related orphan receptor β (Rorβ) expression was markedly suppressed at later stages of differentiation. To gain further insight into the function of NRs in bone biology, the NR superfamily was also profiled in mouse bone marrow precursor cells isolated from either young (6-month) or aging, osteoporotic (18–22-month) mice. Of interest, Rorβ was potently overexpressed in the aged cohort. Collectively, these data provided evidence that Rorβ expression is inversely correlated with osteogenic potential, suggesting Rorβ may be an important and unexplored regulator of osteogenesis. To validate this hypothesis, a cell model stably expressing Rorβ in mouse osteoblastic MC3T3-E1 cells was produced (MC3T3-Rorβ). These cells displayed markedly suppressed bone nodule formation as well as reduced osteocalcin and osterix gene expression. Because these genes are Runx2 targets, we reasoned that Rorβ may interfere with Runx2 activity. Consistent with this, transient transfection analysis demonstrated that Rorβ inhibited Runx2-dependent activation of a Runx2-reporter construct. In summary, our data provide a comprehensive profile of NR expression during osteoblast differentiation and identify Rorβ as a novel regulator of osteogenesis and potentially of age-related bone loss through antagonism of Runx2 activity. © 2012 American Society for Bone and Mineral Research.

Abstract Previous studies using dual-energy X-ray absorptiometry (DXA) have demonstrated that age is a major predictor of bone fragility and fracture risk independent of areal bone mineral density (aBMD). Although this aBMD-independent effect of age has been attributed to poor bone “quality,” the structural basis for this remains unclear. Because high-resolution peripheral quantitative computed tomography (HRpQCT) can assess bone microarchitecture, we matched younger and older subjects for aBMD at the ultradistal radius and assessed for possible differences in trabecular or cortical microstructure by HRpQCT. From an age-stratified, random sample of community adults, 44 women aged &lt;50 years (mean age 41.0 years) were matched to 44 women aged ≥50 years (mean age 62.7 years) by ultradistal radius aBMD (mean ± SEM, younger and older aBMD 0.475 ± 0.011 and 0.472 ± 0.011 g/cm2, respectively), and 57 men aged &lt;50 years (mean age 41.3 years) were matched to 57 men aged ≥50 years (mean age 68.1 years; younger and older aBMD both 0.571 ± 0.008 g/cm2). In these matched subjects, there were no sex-specific differences in trabecular microstructural parameters. However, significant differences were noted in cortical microstructure (all p &lt; 0.05): Older women and men had increased cortical porosity (by 91% and 56%, respectively), total cortical pore volume (by 77% and 61%, respectively), and mean cortical pore diameter (by 9% and 8%, respectively) compared with younger subjects. These findings indicate that younger and older women and men matched for DXA aBMD have similar trabecular microarchitecture but clearly different cortical microstructure, at least at an appendicular site represented by the radius. Further studies are needed to define the extent to which this deterioration in cortical microstructure contributes to the aBMD-independent effect of age on bone fragility and fracture risk at the distal radius and other sites of osteoporotic fractures. © 2012 American Society for Bone and Mineral Research

In the bone marrow cavity, adipocyte numbers increase, whereas osteoblast progenitor numbers decrease with aging. Because adipocytes and osteoblasts share a common progenitor, it is possible that this shift is due to an increase in adipocyte-lineage cells at the expense of osteoblast-lineage commitment. Estrogens inhibit adipocyte differentiation, and in both men and women, circulating estrogens correlate with bone loss with aging. In bone cells, estrogens stimulate expression of TGF-β and suppress mesenchymal cell adipogenesis. Using a tripotential mesenchymal cell line, we have examined whether estradiol suppression of adipocyte differentiation is due to stimulation of TGF-β and the mechanism by which TGF-β suppresses adipogenesis. We observed that estradiol-mediated suppression of adipogenic gene expression required at least 48 h treatment. TGF-β expression increased within 24 h of estradiol treatment, and TGF-β inhibition reversed estradiol influences on adipogenesis and adipocyte gene expression. Connective tissue growth factor (CTGF) mediates TGF-β suppression of adipogenesis in mouse 3T3-L1 cells. CTGF expression was induced within 24 h of TGF-β treatment, whereas estradiol-mediated induction required 48 h treatment. Moreover, estradiol-mediated induction of CTGF was abrogated by TGF-β inhibition. These data support that estradiol effects on adipogenesis involves TGF-β induction, which then induces CTGF to suppress adipogenesis.

Abstract Recent population-based studies demonstrate an increased fracture risk with monoclonal gammopathy of undetermined significance (MGUS). The etiology of this increased risk remains unclear, however, because areal bone mineral density (aBMD) measurements by dual-energy x-ray absorptiometry cannot assess bone microstructural properties critical to determining bone quality and strength. To better define the skeletal effects of MGUS, we performed aBMD and high-resolution peripheral quantitative computed tomography volumetric bone mineral density (vBMD) measurements in 50 MGUS patients (20 females, 30 males; mean ± SEM age, 70.5 ± 1.4 years) and 100 matched control subjects. Relative to controls, MGUS patients had decreased aBMD at the femoral neck (P = .05) and total femur (P < .05) but no differences at other sites. In contrast, high-resolution peripheral quantitative computed tomography showed markedly diminished cortical thickness (P < .05) and increased endocortical area (P < .01). Average vBMD (P < .01), cortical vBMD (P < .001), and trabecular thickness (P < .01) were all significantly decreased in MGUS patients, suggestive of impaired bone formation. Serum levels of the Wnt pathway inhibitor Dickkopf-related protein 1 (P < .001) and osteoclast-activating factor MIP-1α (P < .05) also were significantly elevated in MGUS patients. Our data provide the first evidence of altered bone microstructure in MGUS and suggest that cytokines elevated in osteolytic myeloma also may be associated with bone loss in MGUS.