Sandra Bermeo

Pharmacological inhibition of PPARγ increases osteoblastogenesis and bone mass in male C57BL/6 mice

Infiltration of bone marrow with fat is a prevalent feature in people with age‐related bone loss and osteoporosis, which correlates inversely with bone formation and positively with high expression levels of peroxisomal proliferator‐activated receptor gamma (PPARγ). Inhibition of PPARγ thus represents a potential therapeutic approach for age‐related bone loss. In this study, we examined the effect of PPARγ inhibition on bone in skeletally mature C57BL/6 male mice. Nine‐month‐old mice were treated with a PPARγ antagonist, bisphenol‐A‐diglycidyl ether (BADGE), alone or in combination with active vitamin D (1,25[OH]2D3) for 6 weeks. Micro‐computed tomography and bone histomorphometry indicated that mice treated with either BADGE or BADGE + 1,25(OH)2D3 had significantly increased bone volume and improved bone quality compared with vehicle‐treated mice. This phenotype occurred in the absence of alterations in osteoclast number. Furthermore, the BADGE + 1,25(OH)2D3‐treated mice exhibited higher levels of unmineralized osteoid. All of the treated groups showed a significant increase in circulating levels of bone formation markers without changes in bone resorption markers, while blood glucose, parathyroid hormone, and Ca+ remained normal. Furthermore, treatment with BADGE induced higher levels of expression of vitamin D receptor within the bone marrow. Overall, treated mice showed higher levels of osteoblastogenesis and bone formation concomitant with decreased marrow adiposity and ex vivo adipogenesis. Taken together, these observations demonstrate that pharmacological inhibition of PPARγ may represent an effective anabolic therapy for osteoporosis in the near future.

Interferon Gamma Inhibits Adipogenesis In Vitro and Prevents Marrow Fat Infiltration in Oophorectomized Mice

Interferon gamma (IFNγ) has been reported to induce osteoblastogenesis from mesenchymal stem cells (MSCs) both in vitro and in vivo. With ageing, adipocytes outnumber osteoblasts within the bone microenvironment leading to a decrease in bone formation. Since both osteoblasts and adipocytes are of mesenchymal origin, we hypothesized that IFNγ treatment might negatively affect adipogenesis while stimulating osteoblastogenesis in human MSC. To test this hypothesis, human MSCs were induced to differentiate into adipocytes in the presence or absence of osteogenic doses of IFNγ (1, 10, and 100 ng/ml). IFNγ‐treated MSC showed a decrease in adipocyte differentiation and lipid deposition when compared with vehicle‐treated controls. Additionally, adipogenic markers were significantly decreased by IFNγ treatment at the same doses that have been reported to have a strong osteogenic effect in vitro. Furthermore, DNA binding of peroxisome proliferator‐activated receptor gamma was significantly lower in IFNγ‐treated differentiating MSC. Subsequently, ovariectomized C57BL6 mice were treated with osteogenic doses of IFNγ three times a week for 6 weeks. In distal femur, treated mice showed significantly higher hematopoiesis concomitant with lower levels of fat volume/total volume, adipocyte number, and expression of adipogenic markers when compared with the vehicle‐treated mice. Together, these findings demonstrate that, at osteogenic doses, IFNγ also acts as an inhibitor of adipogenesis in vitro and prevents marrow fat infiltration while favors hematopoiesis in ovariectomized mice. STEM CELLS 2012;30:1042–1048

Fat and Bone Interactions

Fat and bone have a complicated relationship. Although obesity has been associated with low fracture risk, there is increasing evidence that some of the factors that are released by peripheral fat into the circulation may also have a deleterious effect on bone mass, thus, predisposing to fractures. More importantly, the local interaction between fat and bone within the bone marrow seems to play a significant role in the pathogenesis of age-related bone loss and osteoporosis. This “local interaction” occurs inside the bone marrow and is associated with the autocrine and paracrine release of fatty acids and adipokines, which affect the cells in their vicinity including the osteoblasts, reducing their function and survival. In this review, we explore the particularities of the fat and bone cell interactions within the bone marrow, their significance in the pathogenesis of osteoporosis, and the potential therapeutic applications that regulating marrow fat may have in the near future as a novel pharmacologic treatment for osteoporosis.

Lamin A/C Acts as an Essential Factor in Mesenchymal Stem Cell Differentiation Through the Regulation of the Dynamics of the Wnt/β-Catenin Pathway.

Changes in the expression of lamin A/C, a fibrilar protein of the nuclear envelope, are associated with the cellular features of age‐related bone loss. Reduced expression of lamin A/C inhibits osteoblastogenesis while facilitating adipogenic differentiation of mesenchymal stem cells (MSC) in vitro and in vivo. In this study we investigated the regulatory role that lamin A/C plays on the essential elements of the Wnt/β‐catenin pathway, which are pivotal in MSC differentiation. Initially, we assessed the effect of lamin A/C gene (LMNA) overexpression on MSC differentiation while compared it to lamin A/C depleted MSC. Osteogenesis and gene expression of osteogenic factors were higher in LMNA‐transfected MSC as compared to control. Conversely, adipogenesis and expression of adipogenic factors were significantly lower in LMNA transfected cells. Nuclear β‐catenin was significantly higher (∼two fold) in MSC expressing higher levels of LMNA as compared to control with nuclear β‐catenin levels being significantly lower (∼ ‐42%) in siRNA‐treated MSC. Luciferase activity for β‐catenin‐mediated transcriptional activation was significantly higher in cells overexpressing LMNA. These data indicate that MSC overexpressing LMNA have higher osteogenic and lower adipogenic differentiation potential. In conclusion, our studies demonstrate that lamin A/C plays a significant role in the differentiation of both osteoblasts and adipocytes by regulating some of the elements of Wnt/β‐catenin signaling during early MSC differentiation. J. Cell. Biochem. 116: 2344–2353, 2015.

Association Between Circulating Osteogenic Progenitor Cells and Disability and Frailty in Older Persons: The Nepean Osteoporosis and Frailty Study

Circulating osteogenic progenitor (COP) cells are considered as surrogates of the mesenchymal repository in the body. In this study, we hypothesized that COP cells decrease with age and that lower levels of COP cells are associated with greater frailty and disability in older persons. Using well-established clinical criteria, we quantified physical performance and disability and stratified frailty in a random sample of community-dwelling individuals enrolled in the Nepean Osteoporosis and Frailty (NOF) Study (mean age 82.8; N = 77; 70% female; 27 nonfrail, 23 prefrail, and 27 frail). Percentage of COP cells was quantified by flow cytometry. Logistic regression models estimated the relationship between the percentage of COP cells and prevalent disability, poor physical performance, and frailty. We found that aging is associated with a significant decrease in COP cells (p < .001). Lower percentages of COP cells were associated with disability and poor physical performance (p < .001). Older adults with COP cells in the lower quartile were more likely to be frail (odds ratio 2.65, 95% confidence interval 2.72-3.15, p < .001). In conclusion, COP cells in the circulation decrease with age. Lower percentages of COP cells in late life are associated with prevalent frailty and disability. Further longitudinal studies are needed to understand COP cells as a risk stratifier, biomarker, or therapeutic target and to predict disability in frail older persons.

Characterizing the molecular phenotype of an Atp7a(T985I) conditional knock in mouse model for X-linked distal hereditary motor neuropathy (dHMNX).

ATP7A is a P-type ATPase essential for cellular copper (Cu) transport and homeostasis. Loss-of-function ATP7A mutations causing systemic Cu deficiency are associated with severe Menkes disease or its milder allelic variant, occipital horn syndrome. We previously identified two rare ATP7A missense mutations (P1386S and T994I) leading to a non-fatal form of motor neuron disorder, X-linked distal hereditary motor neuropathy (dHMNX), without overt signs of systemic Cu deficiency. Recent investigations using a tissue specific Atp7a knock out model have demonstrated that Cu plays an essential role in motor neuron maintenance and function, however the underlying pathogenic mechanisms of ATP7A mutations causing axonal degeneration remain unknown. We have generated an Atp7a conditional knock in mouse model of dHMNX expressing Atp7a(T985I), the orthologue of the human ATP7A(T994I) identified in dHMNX patients. Although a degenerative motor phenotype is not observed, the knock in Atp7a(T985I/Y) mice show altered Cu levels within the peripheral and central nervous systems, an increased diameter of the muscle fibres and altered myogenin and myostatin gene expression. Atp7a(T985I/Y) mice have reduced Atp7a protein levels and recapitulate the defective trafficking and altered post-translational regulatory mechanisms observed in the human ATP7A(T994I) patient fibroblasts. Our model provides a unique opportunity to characterise the molecular phenotype of dHMNX and the time course of cellular events leading to the process of axonal degeneration in this disease.

Age, gender, and percentage of circulating osteoprogenitor (COP) cells: The COP Study

Abstract Circulating osteoprogenitor (COP) cells are blood‐borne cells which express a variety of osteoblastic markers and are able to form bone nodules in vivo. Whereas a high percentage of COP cells (%COP) is associated with vascular calcification, low %COP has been associated with disability and frailty. However, the reference range of %COP in age‐ and gender‐matching populations, and the age‐related changes in %COP remain unknown. A cross‐sectional study was undertaken in 144 healthy volunteers in Western Sydney (20–90 year‐old, 10 male and 10 female subjects per decade). %COP was quantified by flow cytometry. A high inter‐and intra‐rater reliability was found. In average, in this healthy population average of %COP was 0.42. There was no significant difference in %COP among the age groups. Similarly, no significant difference was found in %COP with gender, weight, height or BMI. In addition, we identified a normal reference range of %COP of 0.1–3.8%. In conclusion, in addition to the identification of steady levels of COP cells with age, we also identified a normal reference range of %COP, which could be used in future studies looking at musculoskeletal diseases in older populations. HighlightsPercentage of circulating osteoprogenitor (COP) cells is not affected by age.The normal reference range of %COP in the blood of a normal population is 0.1–3.8%.Age‐related changes in osteogenic differentiation of COP cells remain to be elucidated.

The Role of the Nuclear Envelope Protein MAN1 in Mesenchymal Stem Cell Differentiation

Mutations in MAN1, a protein of the nuclear envelope, cause bone phenotypes characterized by hyperostosis. The mechanism of this pro‐osteogenic phenotype remains unknown. We increased and decreased MAN1 expression in mesenchymal stem cells (MSC) upon which standard osteogenic and adipogenic differentiation were performed. MAN1 knockdown increased osteogenesis and mineralization. In contrast, osteogenesis remained stable upon MAN1 overexpression. Regarding a mechanism, we found that low levels of MAN1 facilitated the nuclear accumulation of regulatory smads and smads‐related complexes, with a concurrently high expression of nuclear β‐Catenin. In addition, we found adipogenesis to be decreased in both conditions, although predominantly affected by MAN1 overexpression. Finally, lamin A, a protein of the nuclear envelope that regulates MSC differentiation, was unaffected by changes in MAN1. In conclusion, our studies demonstrated that lower levels of MAN1 in differentiating MSC are associated with higher osteogenesis and lower adipogenesis. High levels of MAN1 only affected adipogenesis. These effects could have an important role in the understanding of the role of the proteins of the nuclear envelope in bone formation. J. Cell. Biochem. 118: 4425–4435, 2017.

The Effect of Antidepressants on Mesenchymal Stem Cell Differentiation

Background Use of antidepressant medications has been linked to detrimental impacts on bone mineral density and osteoporosis; however, the cellular basis behind these observations remains poorly understood. The effect does not appear to be homogeneous across the whole class of drugs and may be linked to affinity for the serotonin transporter system. In this study, we hypothesized that antidepressants have a class- and dose-dependent effect on mesenchymal stem cell (MSC) differentiation, which may affect bone metabolism. Methods Human MSCs (hMSCs) were committed to differentiate when either adipogenic or osteogenic media was added, supplemented with five increasing concentrations of amitriptyline (0.001–10 µM), venlafaxine (0.01–25 µM), or fluoxetine (0.001–10 µM). Alizarin red staining (mineralization), alkaline phosphatase (osteoblastogenesis), and oil red O (adipogenesis) assays were performed at timed intervals. In addition, cell viability was assessed using a MTT. Results We found that fluoxetine had a significant inhibitory effect on mineralization. Furthermore, adipogenic differentiation of hMSC was affected by the addition of amitriptyline, venlafaxine, and fluoxetine to the media. Finally, none of the tested medications significantly affected cell survival. Conclusions This study showed a divergent effect of three antidepressants on hMSC differentiation, which appears to be independent of class and dose. As fluoxetine and amitriptyline, but not venlafaxine, affected both osteoblastogenesis and adipogenesis, this inhibitory effect could be associated to the high affinity of fluoxetine to the serotonin transporter system.

Lamin A expression in circulating osteoprogenitors as a potential biomarker for frailty: The Nepean Osteoporosis and Frailty (NOF) Study

&NA; Lamin A is a protein of the nuclear lamina. Low levels of lamin A expression are associated with osteosarcopenia in mice. In this study, we hypothesized that low lamin A expression is also associated with frailty in humans. We aimed to develop a non‐invasive method to quantify lamin A expression in epithelial and circulating osteoprogenitor (COP) cells, and to determine the relationship between lamin A expression and frailty in older individuals. COP cells and buccal swabs were obtained from 66 subjects (median age 74; 60% female; 26 non‐frail, 23 pre‐frail, and 17 frail) participating at the Nepean Osteoporosis and Frailty (NOF) Study. We quantified physical performance and disability, and stratified frailty in this population. Lamin A expression in epithelial and COP cells was quantified by flow cytometry. Linear regression models estimated the relationship between lamin A expression in buccal and COP cells, and prevalent disability and frailty. Lamin A expression in buccal cells showed no association with either disability or frailty. Low lamin A expression values in COP cells were associated with frailty. Frail individuals showed 60% lower levels of lamin A compared to non‐frail (95% CI −36 to −74%, p < 0.001) and 62% lower levels compared to pre‐frail (95%CI −40 to −76%, p < 0.001). In summary, we have identified lamin A expression in COP cells as a strong indicator of frailty. Further work is needed to understand lamin A expression as a risk stratifier, biomarker, or therapeutic target in frail older persons. HighlightsQuantification of lamin A expression in COP cells is feasibleLow levels of lamin A expression in COP cells are associated with disability and frailtyThis novel method has the potential to become a risk stratifier, biomarker, or therapeutic target in frail older persons. Abbreviations: COP: circulating osteoprogenitor; NOF: Nepean Osteoporosis and Frailty; HGPS: Hutchinson Gilford Progeria Syndrome; MMSE: mini mental state examination; GDS: geriatric depression scale; CCI: Charlson comorbidity index; BMI: body mass index; OARS: Older Americans Resource Scale; ADL: activities of daily living; IADL: instrumental activities of daily living; PASE: Physical Activity Scale for the Elderly; CES‐D: f; CSHA: Canadian Study of Health and Aging; QUS: quantitative ultrasound; SOS: speed of sound; BUA: broadband ultrasound attenuation; SI: stiffness index; TSH: thyroid stimulating hormone; EDTA: ethylenediaminetetraacetic acid; PBMC: peripheral blood mononuclear cells; FMO: fluorescence minus one; PMT: photomultiplier tube; G‐MFI: general median fluorescence intensity; IL‐6: interleukin 6.