Search

We and others have shown that application of high-level mechanical loading promotes the formation of transient plasma membrane disruptions (PMD) which initiate mechanotransduction. We hypothesized that increasing osteocyte cell membrane fragility, by disrupting the cytoskeleton-associated protein β2-spectrin (Sptbn1), could alter osteocytic responses and bone adaptation to loading in a PMD-related fashion. In MLO-Y4 cells, treatment with the spectrin-disrupting agent diamide or knockdown of Sptbn1 via siRNA increased the number of PMD formed by fluid shear stress. Primary osteocytes from an osteocyte-targeted DMP1-Cre Sptbn1 conditional knockout (CKO) model mimicked trends seen with diamide and siRNA treatment and suggested the creation of larger PMD, which repaired more slowly, for a given level of stimulus. Post-wounding cell survival was impaired in all three models, and calcium signaling responses from the wounded osteocyte were mildly altered in Sptbn1 CKO cultures. Although Sptbn1 CKO mice did not demonstrate an altered skeletal phenotype as compared to WT littermates under baseline conditions, they showed a blunted increase in cortical thickness when subjected to an osteogenic tibial loading protocol as well as evidence of increased osteocyte death (increased lacunar vacancy) in the loaded limb after 2 weeks of loading. The impaired post-wounding cell viability and impaired bone adaptation seen with Sptbn1 disruption support the existence of an important role for Sptbn1, and PMD formation, in osteocyte mechanotransduction and bone adaptation to mechanical loading., (© 2024. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

We and others have seen that osteocytes sense high-impact osteogenic mechanical loading via transient plasma membrane disruptions (PMDs) which initiate downstream mechanotransduction. However, a PMD must be repaired for the cell to survive this wounding event. Previous work suggested that the protein Prkd1 (also known as PKCμ) may be a critical component of this PMD repair process, but the specific role of Prkd1 in osteocyte mechanobiology had not yet been tested. We treated MLO-Y4 osteocytes with Prkd1 inhibitors (Go6976, kbNB 142-70, staurosporine) and generated an osteocyte-targeted (Dmp1-Cre) Prkd1 conditional knockout (CKO) mouse. PMD repair rate was measured via laser wounding and FM1-43 dye uptake, PMD formation and post-wounding survival were assessed via fluid flow shear stress (50 dyn/cm 2 ), and in vitro osteocyte mechanotransduction was assessed via measurement of calcium signaling. To test the role of osteocyte Prkd1 in vivo, Prkd1 CKO and their wildtype (WT) littermates were subjected to 2 weeks of unilateral axial tibial loading and loading-induced changes in cortical bone mineral density, geometry, and formation were measured. Prkd1 inhibition or genetic deletion slowed osteocyte PMD repair rate and impaired post-wounding cell survival. These effects could largely be rescued by treating osteocytes with the FDA-approved synthetic copolymer Poloxamer 188 (P188), which was previously shown to facilitate membrane resealing and improve efficiency in the repair rate of PMD in skeletal muscle myocytes. In vivo, while both WT and Prkd1 CKO mice demonstrated anabolic responses to tibial loading, the magnitude of loading-induced increases in tibial BMD, cortical thickness, and periosteal mineralizing surface were blunted in Prkd1 CKO as compared to WT mice. Prkd1 CKO mice also tended to show a smaller relative difference in the number of osteocyte PMD in loaded limbs and showed greater lacunar vacancy, suggestive of impaired post-wounding osteocyte survival. While P188 treatment rescued loading-induced increases in BMD in the Prkd1 CKO mice, it surprisingly further suppressed loading-induced increases in cortical bone thickness and cortical bone formation. Taken together, these data suggest that Prkd1 may play a pivotal role in the regulation and repair of the PMD response in osteocytes and support the idea that PMD repair processes can be pharmacologically targeted to modulate downstream responses, but suggest limited utility of PMD repair-promoting P188 in improving bone anabolic responses to loading., Competing Interests: Declaration of competing interest The authors state that they have no conflicts of interest., (Copyright © 2024. Published by Elsevier Inc.)

Purpose: Mitofusin 2 (Mfn2) is one of two mitofusins involved in regulating mitochondrial size, shape and function, including mitophagy, an important cellular mechanism to limit oxidative stress. Reduced expression of Mfn2 has been associated with impaired osteoblast differentiation and function and a reduction in the number of viable osteocytes in bone. We hypothesized that the genetic absence of Mfn2 in these cells would increase their susceptibility to aging-associated metabolic stress, leading to a progressive impairment in skeletal homeostasis over time., Methods: Mfn2 was selectively deleted in vivo at three different stages of osteoblast lineage commitment by crossing mice in which the Mfn2 gene was floxed with transgenic mice expressing Cre under the control of the promoter for Osterix (OSX), collagen1a1, or DMP1 (Dentin Matrix Acidic Phosphoprotein 1)., Results: Mice in which Mfn2 was deleted using DMP1-cre demonstrated a progressive and dramatic decline in bone mineral density (BMD) beginning at 10 weeks of age (n = 5 for each sex and each genotype from age 10 to 20 weeks). By 15 weeks, there was evidence for a functional decline in muscle performance as assessed using a rotarod apparatus (n = 3; 2 males/ 1 female for each genotype), accompanied by a decline in lean body mass. A marked reduction in trabecular bone mass was evident on bone histomorphometry, and biomechanical testing at 25 weeks (k/o: 2 male/1 female, control 2 male/2 female) revealed severely impaired femur strength. Extensive regional myofiber atrophy and degeneration was observed on skeletal muscle histology. Electron microscopy showed progressive disruption of cellular architecture, with disorganized sarcomeres and a bloated mitochondrial reticulum. There was also evidence of neurodegeneration within the ventral horn and roots of the lumbar spinal cord, which was accompanied by myelin loss and myofiber atrophy. Deletion of Mfn2 using OSX-cre or Col1a1-cre did not result in a musculoskeletal phenotype. Where possible, male and female animals were analyzed separately, but small numbers of animals in each group limited statistical power. For other outcomes, where sex was not considered, small sample sizes might still limit the strength of the observation., Conclusion: Despite known functional overlap of Mfn1 and Mfn2 in some tissues, and their co-expression in bone, muscle and spinal cord, deletion of Mfn2 using the 8 kB DMP1 promoter uncovered an important non-redundant role for Mfn2 in maintaining the neuromuscular/bone axis., Competing Interests: Declaration of competing interest None of the authors have any conflicts of interest to disclose., (Copyright © 2024. Published by Elsevier Inc.)

A sharp drop in lenticular glutathione (GSH) plays a pivotal role in age-related cataract (ARC) formation. Despite recognizing GSH's importance in lens defense for decades, its decline with age remains puzzling. Our recent study revealed an age-related truncation affecting the essential GSH biosynthesis enzyme, the γ-glutamylcysteine ligase catalytic subunit (GCLC), at aspartate residue 499. Intriguingly, these truncated GCLC fragments compete with full-length GCLC in forming a heterocomplex with the modifier subunit (GCLM) but exhibit markedly reduced enzymatic activity. Crucially, using an aspartate-to-glutamate mutation knock-in (D499E-KI) mouse model that blocks GCLC truncation, we observed a notable delay in ARC formation compared to WT mice: Nearly 50% of D499E-KI mice remained cataract-free versus ~20% of the WT mice at their age of 20 months. Our findings concerning age-related GCLC truncation might be the key to understanding the profound reduction in lens GSH with age. By halting GCLC truncation, we can rejuvenate lens GSH levels and considerably postpone cataract onset.

Kynurenine (Kyn) is a circulating tryptophan (Trp) catabolite generated by enzymes including IDO1 that are induced by inflammatory cytokines such as interferon-gamma. Kyn levels in circulation increase with age and Kyn is implicated in several age-related disorders including neurodegeneration, osteoporosis, and sarcopenia. Importantly, Kyn increases with progressive disease in HIV patients, and antiretroviral therapy does not normalize IDO1 activity in these subjects. Kyn is now recognized as an endogenous agonist of the aryl hydrocarbon receptor, and AhR activation itself has been found to induce muscle atrophy, increase the activity of bone-resorbing osteoclasts, decrease matrix formation by osteoblasts, and lead to senescence of bone marrow stem cells. Several IDO1 and AhR inhibitors are now in clinical trials as potential cancer therapies. We propose that some of these drugs may be repurposed to improve musculoskeletal health in older adults living with HIV., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Sultana, Elengickal, Bensreti, de Chantemèle, McGee-Lawrence and Hamrick.)

Kynurenine (Kyn) is a tryptophan metabolite that increases with age and promotes musculoskeletal dysfunction. We previously found a sexually dimorphic pattern in how Kyn affects bone, with harmful effects more prevalent in females than males. This raises the possibility that male sex steroids might exert a protective effect that blunts the effects of Kyn in males. To test this, orchiectomy (ORX) or sham surgeries were performed on 6-month-old C57BL/6 mice, after which mice received Kyn (10 mg/kg) or vehicle via intraperitoneal injection, once daily, 5×/week, for four weeks. Bone histomorphometry, DXA, microCT, and serum marker analyses were performed after sacrifice. In vitro studies were performed to specifically test the effect of testosterone on activation of aryl hydrocarbon receptor (AhR)-mediated signaling by Kyn in mesenchymal-lineage cells. Kyn treatment reduced cortical bone mass in ORX- but not sham-operated mice. Trabecular bone was unaffected. Kyn's effects on cortical bone in ORX mice were attributed primarily to enhanced endosteal bone resorption activity. Bone marrow adipose tissue was increased in Kyn-treated ORX animals but was unchanged by Kyn in sham-operated mice. ORX surgery increased mRNA expression of the aryl hydrocarbon receptor (AhR) and its target gene Cyp1a1 in the bone, suggesting a priming and/or amplification of AhR signaling pathways. Mechanistic in vitro studies revealed that testosterone blunted Kyn-stimulated AhR transcriptional activity and Cyp1a1 expression in mesenchymal-linage cells. These data suggest a protective role for male sex steroids in blunting the harmful effects of Kyn in cortical bone. Therefore, testosterone may play an important role in regulating Kyn/AhR signaling in musculoskeletal tissues, suggesting crosstalk between male sex steroids and Kyn signaling may influence age-associated musculoskeletal frailty., Competing Interests: Declaration of competing interest The authors state that they have no conflicts of interest., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Ischemia-reperfusion (I/R) injury is a complication impacting multiple organs and tissues in clinical conditions ranging from peripheral arterial disease to musculoskeletal trauma and myocardial infarction. Stem cell-derived extracellular vesicles (EVs) may represent one therapeutic resource for preventing the tissue damage associated with I/R injury. Here we tested the hypothesis that lyophilized extracellular vesicles derived from adipose stem cells could serve as an "off-the-shelf" treatment modality for I/R injury in a mouse hindlimb ischemia model. Ischemia was induced for 90 min using a rubber band tourniquet and extracellular vesicles (0, 50, or 100 µg) administered via tail vein injection immediately prior to reperfusion. Perfusion was measured prior to, during, and after ischemia using laser Doppler imaging. Serum and tissue were collected 24 h after reperfusion. Mass spectrometry (MS)-based proteomics was used to characterize the EV cargo and proteins from the ischemic and non-ischemic hindlimb. Inflammatory cytokines were measured in muscle and serum using a multiplex array. Results indicate that EVs significantly increase reperfusion and significantly increase expression of the anti-inflammatory factor annexin a1 in skeletal muscle; however, the increased reperfusion was also associated with a marked decrease in muscle structural proteins such as dystrophin, plectin, and obscurin. Circulating inflammatory cytokines TNF-alpha and IL-6 were increased with EV treatment, and serum TNF-alpha showed a significant, positive correlation with reperfusion level. These findings suggest that, while EVs may enhance reperfusion, the increased reperfusion can negatively impact muscle tissue and possibly remote organs. Alternative approaches, such as targeting mitochondrial permeability, may be more effective at mitigating I/R injury.

Extracellular vesicles (EVs), including exosomes and microvesicles, are released by numerous cell types. EVs are now acknowledged as playing a critical role in cell-cell communication in healthy aging as well as in age-related diseases. Recently it was shown that senescence, a key hallmark of aging, increases the secretion of EVs. Moreover, EVs can transport proteins and microRNAs (miRNAs) that are key components of the senescence-associated secretory phenotype (SASP). Here we review evidence that SASP-related miRNAs are involved in musculoskeletal degeneration with aging. Specifically, senescence-related miRNAs are elevated in EVs released by skeletal muscle myocytes and fibro-adipogenic progenitor cells with aging and disuse atrophy, respectively. Many of these same senescence-related miRNAs are detected in EVs from the synovial fluid of patients with osteoarthritis, and these miRNAs can contribute to cartilage degeneration. Finally, senescence-associated miRNAs are secreted from bone marrow-derived stem (stromal) cells impacting neighboring hematopoietic stem cells and circulating in the blood. The senescence-associated miRNA mir-34a, which is known to target Wnt and Notch pathways as well as the cell survival factors Sirt1 and Bcl2, is detected in EVs from human and animal subjects with muscle atrophy, bone loss, and osteoarthritis. These findings suggest that suppressing the secretion of EV-derived, senescence-related miRNAs, such as miR-34a, or increasing levels of competing endogenous long noncoding RNAs, such as MALAT1 that inhibit miR-34a, may help to improve musculoskeletal function with aging. © 2022 The Authors. JBMR Plus published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research., (© 2022 The Authors. JBMR Plus published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research.)

The aryl hydrocarbon receptor (AhR) has been implicated in regulating skeletal progenitor cells and the activity of bone-forming osteoblasts and bone-resorbing osteoclasts, thereby impacting bone mass and the risk of skeletal fractures. The AhR also plays an important role in the immune system within the skeletal niche and in the differentiation of mesenchymal stem cells into other cell lineages including chondrocytes and adipocytes. This transcription factor responds to environmental pollutants which can act as AhR ligands, initiating or interfering with various signaling cascades to mediate downstream effects, and also responds to endogenous ligands including tryptophan metabolites. This review comprehensively describes the reported roles of the AhR in skeletal cell biology, focusing on mesenchymal stem cells, osteoblasts, and osteoclasts, and discusses how AhR exhibits sexually dimorphic effects in bone. The molecular mechanisms mediating AhR's downstream effects are highlighted to emphasize the potential importance of targeting this signaling cascade in skeletal disorders.

In this study a manganese-enhanced magnetic resonance imaging (MEMRI) method was developed for mice for measuring axonal transport (AXT) rates in real time in olfactory receptor neurons, which project from the olfactory epithelium to the olfactory neuronal layer of the olfactory bulb. Using this MEMRI method, two major experiments were conducted: 1) an evaluation of the effects of age on AXT rates and 2) an evaluation of the brain-penetrant, microtubule-stabilizing agent, Epothilone D for effect on AXT rates in aged mice. In these studies, we improved upon previous MEMRI approaches to develop a method where real-time measurements (32 time points) of AXT rates in mice can be determined over a single (approximately 100 min) scanning session. In the age comparisons, AXT rates were significantly higher in young (mean age ∼4.0 months old) versus aged (mean age ∼24.5 months old) mice. Moreover, in aged mice, eight weeks of treatment with Epothilone D, (0.3 and 1.0 mg/kg) was associated with statistically significant increases in AXT rates compared to vehicle-treated subjects. These experiments conducted in a living mammalian model (i.e., wild type, C57BL/6 mice), using a new modified MEMRI method, thus provide further evidence that the process of aging leads to decreases in AXT rates in the brain and they further support the argument that microtubule-based therapeutic strategies designed to improve AXT rates have potential for age-related neurological disorders., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Fibro-adipogenic progenitor cells (FAPs) are a population of stem cells in skeletal muscle that play multiple roles in muscle repair and regeneration through their complex secretome; however, it is not well understood how the FAP secretome is altered with muscle disuse atrophy. Previous work suggests that the inflammatory cytokine IL-1β is increased in FAPs with disuse and denervation. Inflammasome activation and IL-1β secretion are also known to stimulate the release of extracellular vesicles (EVs). Here, we examined the microRNA (miRNA) cargo of FAP-derived, platelet-derived growth factor receptor A (PDGFRα + ) EVs from hindlimb muscles of wild-type and IL-1β KO mice after 14 days of single-hindlimb immobilization. Hindlimb muscles were isolated from mice following the immobilization period, and PDGFRα + extracellular vesicles were isolated using size-exclusion chromatography and immunoprecipitation. Microarrays were performed to detect changes in miRNAs with unloading and IL-1β deficiency. Results indicate that the PDGFRα + , FAP-derived EVs show a significant increase in miRNAs, such as miR-let-7c, miR-let-7b, miR-181a, and miR-124. These miRNAs have previously been demonstrated to play important roles in cellular senescence and muscle atrophy. Furthermore, the expression of these same miRNAs was not significantly altered in FAP-derived EVs isolated from the immobilized IL-1β KO. These data suggest that disuse-related activation of IL-1β can mediate the miRNA cargo of FAP-derived EVs, contributing directly to the release of senescence- and atrophy-related miRNAs. Therapies targeting FAPs in settings associated with muscle disuse atrophy may therefore have the potential to preserve muscle function and enhance muscle recovery.

Exosomes, key mediators of cell-cell communication, derived from type 2 diabetes mellitus (T2DM) exhibit detrimental effects. Exercise improves endothelial function in part via the secretion of exosomes into circulation. Extracellular superoxide dismutase (SOD3) is a major secretory copper (Cu) antioxidant enzyme that catalyzes the dismutation of O 2 •- to H 2 O 2 whose activity requires the Cu transporter ATP7A. However, the role of SOD3 in exercise-induced angiogenic effects of circulating plasma exosomes on endothelial cells (ECs) in T2DM remains unknown. Here, we show that both SOD3 and ATP7A proteins were present in plasma exosomes in mice, which was significantly increased after two weeks of volunteer wheel exercise. A single bout of exercise in humans also showed a significant increase in SOD3 and ATP7A protein expression in plasma exosomes. Plasma exosomes from T2DM mice significantly reduced angiogenic responses in human ECs or mouse skin wound healing models, which was associated with a decrease in ATP7A, but not SOD3 expression in exosomes. Exercise training in T2DM mice restored the angiogenic effects of T2DM exosomes in ECs by increasing ATP7A in exosomes, which was not observed in exercised T2DM/SOD3 -/- mice. Furthermore, exosomes overexpressing SOD3 significantly enhanced angiogenesis in ECs by increasing local H 2 O 2  levels in a heparin-binding domain-dependent manner as well as restored defective wound healing and angiogenesis in T2DM or SOD3 -/- mice. In conclusion, exercise improves the angiogenic potential of circulating exosomes in T2DM in a SOD3-dependent manner. Exosomal SOD3 may provide an exercise mimetic therapy that supports neovascularization and wound repair in cardiometabolic disease., (© 2022 Federation of American Societies for Experimental Biology.)

Hallmarks of aging-associated osteoporosis include bone loss, bone marrow adipose tissue (BMAT) expansion, and impaired osteoblast function. Endogenous glucocorticoid levels increase with age, and elevated glucocorticoid signaling, associated with chronic stress and dysregulated metabolism, can have a deleterious effect on bone mass. Canonical glucocorticoid signaling through the glucocorticoid receptor (GR) was recently investigated as a mediator of osteoporosis during the stress of chronic caloric restriction. To address the role of the GR in an aging-associated osteoporotic phenotype, the current study utilized female GR conditional knockout (GR-CKO; GR fl/fl :Osx-Cre+) mice and control littermates on the C57BL/6 background aged to 21 months and studied in comparison to young (3- and 6-month-old) mice. GR deficiency in Osx-expressing cells led to low bone mass and BMAT accumulation that persisted with aging. Surprisingly, however, GR-CKO mice also exhibited alterations in muscle mass (reduced % lean mass and soleus fiber size), accompanied by reduced voluntary physical activity, and also exhibited higher whole-body metabolic rate and elevated blood pressure. Moreover, increased lipid storage was observed in GR-CKO osteoblastic cultures in a glucocorticoid-dependent fashion despite genetic deletion of the GR, and could be reversed via pharmacological inhibition of the mineralocorticoid receptor (MR). These findings provide evidence of a role for the GR (and possibly the MR) in facilitating healthy bone maintenance with aging in females. The effects of GR-deficient bone on whole-body physiology also demonstrate the importance of bone as an endocrine organ and suggest evidence for compensatory mechanisms that facilitate glucocorticoid signaling in the absence of osteoblastic GR function; these represent new avenues of research that may improve understanding of glucocorticoid signaling in bone toward the development of novel osteogenic agents. © 2021 American Society for Bone and Mineral Research (ASBMR)., (© 2021 American Society for Bone and Mineral Research (ASBMR).)

Long non-coding RNAs (lncRNAs) are thought to function as "sponges" for microRNAs, but a role for such competing endogenous RNAs (ceRNAs) in muscle aging is not well understood. We therefore examined in skeletal muscles of young (4-6 months) and aged (22-24) male and female mice the expression of lncRNA MALAT1, which is predicted in silico to bind the senescence-associated microRNA miR-34a-5p. Results indicate a significant decrease in lncRNA MALAT1 expression in mouse skeletal muscle with age that coincides with an age-related increase in miR-34a-5p expression. In vitro studies using mouse C2C12 myoblasts demonstrate that MALAT1 silencing using siRNA increases miR-34a expression, consistent with a role for MALAT1 as an inhibitor of miR-34a-5p activity. Levels of reactive oxygen species (ROS) are known to increase in muscle with age, and so we treated C2C12 cells with hydrogen peroxide (10 and 100 μM) to examine changes in MALAT1 expression. MALAT1 expression decreased significantly with H 2 O 2 treatment, but this effect was attenuated with p53 siRNA. Finally, miR-34a-5p is implicated in tissue fibrosis, and so we assessed the expression of TGF-β1 after MALAT1 silencing. MALAT1 siRNA significantly increased the expression of TGF-β1 in C2C12 cells. These findings suggest that age-related fibrosis and muscle atrophy mediated by ROS may result at least in part from an increase in miR-34a bioavailability resulting from a decline in miR-34a "sponging" due to ceRNA MALAT1 depletion. Crosstalk between MALAT1 and miR-34a may therefore represent a therapeutic target for improving muscle function with aging., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Ruan, Mendhe, Parker, Kent, Isales, Hill, McGee-Lawrence, Fulzele and Hamrick.)

Age-associated osteoporosis is widely accepted as involving the disruption of osteogenic stem cell populations and their functioning. Maintenance of the local bone marrow (BM) microenvironment is critical for regulating proliferation and differentiation of the multipotent BM mesenchymal stromal/stem cell (BMSC) population with age. The potential role of microRNAs (miRNAs) in modulating BMSCs and the BM microenvironment has recently gained attention. However, miRNAs expressed in rapidly isolated BMSCs that are naïve to the non-physiologic standard tissue culture conditions and reflect a more accurate in vivo profile have not yet been reported. Here we directly isolated CD271 positive (+) BMSCs within hours from human surgical BM aspirates without culturing and performed microarray analysis to identify the age-associated changes in BMSC miRNA expression. One hundred and two miRNAs showed differential expression with aging. Target prediction and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses revealed that the up-regulated miRNAs targeting genes in bone development pathways were considerably enriched. Among the differentially up-regulated miRNAs the novel passenger strand miR-29b-1-5p was abundantly expressed as a mature functional miRNA with aging. This suggests a critical arm-switching mechanism regulates the expression of the miR-29b-1-5p/3p pair shifting the normally degraded arm, miR-29b-1-5p, to be the dominantly expressed miRNA of the pair in aging. The normal guide strand miR-29b-1-3p is known to act as a pro-osteogenic miRNA. On the other hand, overexpression of the passenger strand miR-29b-1-5p in culture-expanded CD271+ BMSCs significantly down-regulated the expression of stromal cell-derived factor 1 (CXCL12)/ C-X-C chemokine receptor type 4 (SDF-1(CXCL12)/CXCR4) axis and other osteogenic genes including bone morphogenetic protein-2 (BMP-2) and runt-related transcription factor 2 (RUNX2). In contrast, blocking of miR-29b-1-5p function using an antagomir inhibitor up-regulated expression of BMP-2 and RUNX2 genes. Functional assays confirmed that miR-29b-1-5p negatively regulates BMSC osteogenesis in vitro. These novel findings provide evidence of a pathogenic anti-osteogenic role for miR-29b-1-5p and other miRNAs in age-related defects in osteogenesis and bone regeneration., (Copyright © 2021. Published by Elsevier Inc.)

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) causes coronavirus disease 2019 (COVID-19), affecting multiple organ systems, including the respiratory tract and lungs. Several studies have reported that the tryptophan-kynurenine pathway is altered in COVID-19 patients. The tryptophan-kynurenine pathway plays a vital role in regulating inflammation, metabolism, immune responses, and musculoskeletal system biology. In this minireview, we surmise the effects of the kynurenine pathway in COVID-19 patients and how this pathway might impact muscle and bone biology., Competing Interests: The authors also declare that there is no other conflict of interest regarding the publication of this manuscript. The abovementioned funding did not lead to any conflict of interest regarding the publication of this manuscript., (Copyright © 2021 Sagar Vyavahare et al.)

Maintaining muscle mass is clinically important as muscle helps to regulate metabolic systems of the body as well as support activities of daily living that require mobility, strength, and power. Losing muscle mass decreases an individual's independence and quality of life, and at the same time increases the risk of disease burden. Fibro-adipogenic progenitor (FAP) cells are a group of muscle progenitor cells that play an important role in muscle regeneration and maintenance of skeletal muscle fiber size. These important functions of FAPs are mediated by a complex secretome that interacts in a paracrine manner to stimulate muscle satellite cells to divide and differentiate. Dysregulation of FAP differentiation leads to fibrosis, fatty infiltration, muscle atrophy, and impaired muscle regeneration. Functional deficits in skeletal muscle resulting from atrophy, fibrosis, or fatty infiltration will reduce biomechanical stresses on the skeleton, and both FAP-derived adipocytes and FAPs themselves are likely to secrete factors that can induce bone loss. These findings suggest that FAPs represent a cell population to be targeted therapeutically to improve both muscle and bone health in settings of aging, injury, and disease., Competing Interests: Conflict of interest statement Nothing declared., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

The gut microflora is a vital component of the gastrointestinal (GI) system that regulates local and systemic immunity, inflammatory response, the digestive system, and overall health. Older people commonly suffer from inadequate nutrition or poor diets, which could potentially alter the gut microbiota. The essential amino acid (AA) tryptophan (TRP) is a vital diet component that plays a critical role in physiological stress responses, neuropsychiatric health, oxidative systems, inflammatory responses, and GI health. The present study investigates the relationship between varied TRP diets, the gut microbiome, and inflammatory responses in an aged mouse model. We fed aged mice either a TRP-deficient (0.1%), TRP-recommended (0.2%), or high-TRP (1.25%) diet for eight weeks and observed changes in the gut bacterial environment and the inflammatory responses via cytokine analysis (IL-1a, IL-6, IL-17A, and IL-27). The mice on the TRP-deficient diets showed changes in their bacterial abundance of Coriobacteriia class, Acetatifactor genus, Lachnospiraceae family, Enterococcus faecalis species, Clostridium sp genus, and Oscillibacter genus. Further, these mice showed significant increases in IL-6, IL-17A, and IL-1a and decreased IL-27 levels. These data suggest a direct association between dietary TRP content, the gut microbiota microenvironment, and inflammatory responses in aged mice models.

Advanced age is one of the important contributing factors for musculoskeletal deterioration. Although the exact mechanism behind this degeneration is unknown, it has been previously established that nutritional signaling plays a vital role in musculoskeletal pathophysiology. Our group established the vital role of the essential amino acid, tryptophan, in aging musculoskeletal health. With advanced age, inflammatory factors activate indoleamine 2,3-dioxygenase (IDO1) and accumulate excessive intermediate tryptophan metabolites such as Kynurenine (KYN). With age, Kynurenine accumulates and suppresses osteogenic differentiation, impairs autophagy, promotes early senescence, and alters cellular bioenergetics of bone marrow stem cells. Recent studies have shown that Kynurenine negatively impacts bone marrow stromal cells (BMSCs) and, consequently, promotes bone loss. Overall, understanding the mechanism behind BMSCs losing their ability for osteogenic differentiation can provide insight into the prevention of osteoporosis and the development of targeted therapies. Therefore, in this article, we review Kynurenine and how it plays a vital role in BMSC dysfunction and bone loss with age., (Copyright © 2021. Published by Elsevier B.V.)

Acute ischemia-reperfusion injury in skeletal muscle is a significant clinical concern in the trauma setting. The mitochondrial permeability transition inhibitor NIM-811 has previously been shown to reduce ischemic injury in the liver and kidney. The effects of this treatment on skeletal muscle are, however, not well understood. We first used an in vitro model of muscle cell ischemia in which primary human skeletal myoblasts were exposed to hypoxic conditions (1% O 2 and 5% CO 2 ) for 6 h. Cells were treated with NIM-811 (0-20 µM). MTS assay was used to quantify cell survival and LDH assay to quantify cytotoxicity 2 h after treatment. Results indicate that NIM-811 treatment of ischemic myotubes significantly increased cell survival and decreased LDH in a dose-dependent manner. We then examined NIM-811 effects in vivo using orthodontic rubber bands (ORBs) for 90 min of single hindlimb ischemia. Mice received vehicle or NIM-811 (10 mg/kg BW) 10 min before reperfusion and 3 h later. Ischemia and reperfusion were monitored using laser speckle imaging. In vivo data demonstrate that mice treated with NIM-811 showed increased gait speed and improved Tarlov scores compared to vehicle-treated mice. The ischemic limbs of female mice treated with NIM-811 showed significantly lower levels of MCP-1, IL-23, IL-6, and IL-1α compared to limbs of vehicle-treated mice. Similarly, male mice treated with NIM-811 showed significantly lower levels of MCP-1 and IL-1a. These findings are clinically relevant as MCP-1, IL-23, IL-6, and IL-1α are all pro-inflammatory factors that are thought to contribute directly to tissue damage after ischemic injury. Results from the in vitro and in vivo experiments suggest that NIM-811 and possibly other mitochondrial permeability transition inhibitors may be effective for improving skeletal muscle salvage and survival after ischemia-reperfusion injury.

The aging process is associated with significant alterations in mitochondrial function. These changes in mitochondrial function are thought to involve increased production of reactive oxygen species (ROS), which over time contribute to cell death, senescence, tissue degeneration, and impaired tissue repair. The mitochondrial permeability transition pore (mPTP) is likely to play a critical role in these processes, as increased ROS activates mPTP opening, which further increases ROS production. Injury and inflammation are also thought to increase mPTP opening, and chronic, low-grade inflammation is a hallmark of aging. Nicotinamide adenine dinucleotide (NAD+) can suppress the frequency and duration of mPTP opening; however, NAD+ levels are known to decline with age, further stimulating mPTP opening and increasing ROS release. Research on neurodegenerative diseases, particularly on Parkinson's disease (PD) and Alzheimer's disease (AD), has uncovered significant findings regarding mPTP openings and aging. Parkinson's disease is associated with a reduction in mitochondrial complex I activity and increased oxidative damage of DNA, both of which are linked to mPTP opening and subsequent ROS release. Similarly, AD is associated with increased mPTP openings, as evidenced by amyloid-beta (A β ) interaction with the pore regulator cyclophilin D (CypD). Targeted therapies that can reduce the frequency and duration of mPTP opening may therefore have the potential to prevent age-related declines in cell and tissue function in various systems including the central nervous system., Competing Interests: The authors declare that they have no conflicts of interest., (Copyright © 2021 Andrew C. Kent et al.)

MicroRNAs (miRNAs) have recently come under scrutiny for their role in various age-related diseases. Similarly, cellular senescence has been linked to disease and aging. MicroRNAs and senescence likely play an intertwined role in driving these pathologic states. In this review, we present the connection between these two drivers of age-related disease concerning mesenchymal stem cells (MSCs). First, we summarize key miRNAs that are differentially expressed in MSCs and other musculoskeletal lineage cells during senescence and aging. Additionally, we also reviewed miRNAs that are regulated via traditional senescence-associated secretory phenotype (SASP) cytokines in MSC. Lastly, we summarize miRNAs that have been found to target components of the cell cycle arrest pathways inherently activated in senescence. This review attempts to highlight potential miRNA targets for regenerative medicine applications in age-related musculoskeletal disease., (Published by Elsevier Inc.)

Traumatic musculoskeletal injuries are common in both the civilian and combat care settings. Significant barriers exist to repairing these injuries including fracture nonunion, muscle fibrosis, re-innervation, and compartment syndrome, as well as infection and inflammation. Recently, extracellular vesicles (EVs), including exosomes and microvesicles, have attracted attention in the field of musculoskeletal regeneration. These vesicles are released by different cell types and play a vital role in cell communication by delivering functional cargoes such as proteins and RNAs. Many of these cargo molecules can be utilized for repair purposes in skeletal disorders such as osteoporosis, osteogenesis imperfecta, sarcopenia, and fracture healing. There are, however, some challenges to overcome in order to advance the successful application of these vesicles in the therapeutic setting. These include large-scale production and isolation of exosomes, long-term storage, in vivo stability, and strategies for tissue-specific targeting and delivery. This paper reviews the general characteristics of exosomes along with their physiological roles and contribution to the pathogenesis of musculoskeletal diseases. We also highlight new findings on the use of synthetic exosomes to overcome the limitations of native exosomes in treating musculoskeletal injuries and disorders.

Women are at a significantly higher risk of developing osteoarthritis (OA) compared to males. The pathogenesis of osteoarthritis (OA) in women is poorly understood. Extracellular vesicles (EVs) have been shown to play an essential role in numerous signaling processes during the pathogenesis of age-related diseases via paracrine signaling. Molecular profiling of the synovial fluid-derived EVs cargo in women may help in the discovery of novel biomarkers and therapeutics for the treatment of OA in women. Previously, we reported that synovial fluid-derived EV miRNA cargo differs in a sex-specific manner. This study aims to characterize synovial fluid-derived EV protein cargo in OA patients. Our data showed sex-specific EVs protein content in OA. We found haptoglobin, orosomucoid, and ceruloplasmin significantly up-regulated, whereas apolipoprotein down-regulated in female OA EVs. In males, we discovered β-2-glycoprotein, and complement component 5 proteins significantly up-regulated and Spt-Ada-Gcn5 acetyltransferase (SAGA)-associated factor 29 down-regulated in male OA EVs. Database for Annotation, Visualization, and Integrated Discovery (DAVID) and QuickGO analysis revealed OA-specific protein involvement in several biological, molecular, and cellular pathways, specifically in inflammatory processes. In conclusion, synovial fluid EV protein content is altered in a sex-specific manner with OA, explaining the increased prevalence and severity of OA in women.

Inquiry into relationships between energy metabolism and brain function requires a uniquely interdisciplinary mindset, and implementation of anti-aging lifestyle strategies based on this work also involves consistent mental and physical discipline. Dr. Mark P. Mattson embodies both of these qualities, based on the breadth and depth of his work on neurobiological responses to energetic stress, and on his own diligent practice of regular exercise and caloric restriction. Dr. Mattson created a neurotrophic niche in his own laboratory, allowing trainees to grow their skills, form new connections, and eventually migrate, forming their own labs while remaining part of the extended lab family. In this historical review, we highlight Dr. Mattson's many contributions to understanding neurobiological responses to physical exercise and dietary restriction, with an emphasis on the mechanisms that may underlie neuroprotection in ageing and age-related disease. On the occasion of Dr. Mattson's retirement from the National Institute on Aging, we highlight his foundational work on metabolism and neuroplasticity by reviewing the context for these findings and considering their impact on future research on the neuroscience of aging., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Despite the accumulating evidence from in vitro and animal experiments supporting the role of kynurenine (a tryptophan metabolite) in a number of degenerative age-related changes, the relationship between kynurenine and frailty in older adults is not well understood. We collected blood samples from 73 participants who underwent a comprehensive geriatric assessment, measuring kynurenine levels using liquid chromatography-tandem mass spectrometry. We assessed the phenotypic frailty and the deficit accumulation frailty index using widely validated approaches proposed by Fried et al. and Rockwood et al., respectively. After adjusting for sex, age, and body mass index, the frail participants presented 52.9% and 34.3% higher serum kynurenine levels than those with robustness and prefrailty, respectively ( P = 0.005 and 0.014, respectively). Serum kynurenine levels were positively associated with the frailty index, time to complete 5 chair stands, and patient health questionnaire-2 score and inversely associated with grip strength and gait speed ( P = 0.042 to <0.001). Furthermore, the odds ratio per increase in serum kynurenine level for phenotypic frailty was approximately 2.62 (95% confidence interval = 1.22-5.65, P = 0.014). These data provide clinical evidence that circulating kynurenine might be a potential biomarker for assessing the risk of frailty in humans.

There is increasing evidence of the involvement of the tryptophan metabolite kynurenine (KYN) in disrupting osteogenesis and contributing to aging-related bone loss. Here, we show that KYN has an effect on bone resorption by increasing osteoclastogenesis. We have previously reported that in vivo treatment with KYN significantly increased osteoclast number lining bone surfaces. Here, we report the direct effect of KYN on receptor activator of nuclear factor kappa-B ligand (RANKL)-induced osteoclastogenesis in Raw 264.7 macrophage cells, and we propose a potential mechanism for these KYN-mediated effects. We show that KYN/RANKL treatment results in enhancement of RANKL-induced osteoclast differentiation. KYN drives upregulation and activation of the key osteoclast transcription factors, c-fos and NFATc1 resulting in an increase in the number of multinucleated TRAP+ osteoclasts, and in hydroxyapatite bone resorptive activity. Mechanistically, the KYN receptor, aryl hydrocarbon receptor (AhR), plays an important role in the induction of osteoclastogenesis. We show that blocking AhR signaling using an AhR antagonist, or AhR siRNA, downregulates the KYN/RANKL-mediated increase in c-fos and NFATc1 and inhibits the formation of multinucleated TRAP + osteoclasts. Altogether, this work highlights that the novelty of the KYN and AhR pathways might have a potential role in helping to regulate osteoclast function with age and supports pursuing additional research to determine if they are potential therapeutic targets for the prevention or treatment of osteoporosis.

Background: Apelin, an active endogenous peptide, has been recently receiving great attention as a promising target for antiaging intervention, primarily based on results from genetically altered mice. To validate previous experimental data and investigate the possible role of apelin in humans, in this study, we examined serum apelin level in relation to frailty and its associated parameters in a cohort of ambulatory, community-dwelling older adults., Methods: Blood samples were collected from 80 participants who underwent a comprehensive geriatric assessment, and apelin level was measured using an enzyme immunoassay kit. Phenotypic frailty and deficit-accumulation frailty index (FI) were assessed using widely validated approaches, proposed by Fried and Rockwood groups, respectively., Results: After adjustment for sex, age, and body mass index, serum apelin level was found to be not significantly different according to phenotypic frailty status (P = 0.550) and not associated with FI, grip strength, gait speed, time to complete 5 chair stands, and muscle mass (P = 0.433 to 0.982). To determine whether the association between serum apelin level and frailty has a threshold effect, we divided the participants into quartiles according to serum apelin level. However, there were no differences in terms of frailty-related parameters and the risk for frailty among the quartile groups (P = 0.248 to 0.741)., Conclusions: The serum apelin level was not associated with both phenotypic frailty and functional parameters in older adults, despite its beneficial effects against age-related physiologic decline in animal models. Further large-scale longitudinal studies are necessary to understand the definite role of circulating apelin in frailty risk assessment.

Although aging is considered a normal process, there are cellular and molecular changes that occur with aging that may be detrimental to health. Osteoporosis is one of the most common age-related degenerative diseases, and its progression correlates with aging and decreased capacity for stem cell differentiation and proliferation in both men and women. Tryptophan metabolism through the kynurenine pathway appears to be a key factor in promoting bone-aging phenotypes, promoting bone breakdown and interfering with stem cell function and osteogenesis; however, little data is available on the impact of tryptophan metabolites downstream of kynurenine. Here we review available data on the impact of these tryptophan breakdown products on the body in general and, when available, the existing evidence of their impact on bone. A number of tryptophan metabolites (e.g., 3-hydroxykynurenine (3HKYN), kynurenic acid (KYNA) and anthranilic acid (AA)) have a detrimental effect on bone, decreasing bone mineral density (BMD) and increasing fracture risk. Other metabolites (e.g., 3-hydroxyAA, xanthurenic acid (XA), picolinic acid (PIA), quinolinic acid (QA), and NAD+) promote an increase in bone mineral density and are associated with lower fracture risk. Furthermore, the effects of other tryptophan breakdown products (e.g., serotonin) are complex, with either anabolic or catabolic actions on bone depending on their source. The mechanisms involved in the cellular actions of these tryptophan metabolites on bone are not yet fully known and will require further research as they are potential therapeutic targets. The current review is meant as a brief overview of existing English language literature on tryptophan and its metabolites and their effects on stem cells and musculoskeletal systems. The search terms used for a Medline database search were: kynurenine, mesenchymal stem cells, bone loss, tryptophan metabolism, aging, and oxidative stress., Competing Interests: The authors declare no conflicts of interest.

Senescence is a cellular defense mechanism that helps cells prevent acquired damage, but chronic senescence, as in aging, can contribute to the development of age-related tissue dysfunction and disease. Previous studies clearly show that removal of senescent cells can help prevent tissue dysfunction and extend healthspan during aging. Senescence increases with age in the skeletal system, and selective depletion of senescent cells or inhibition of their senescence-associated secretory phenotype (SASP) has been reported to maintain or improve bone mass in aged mice. This suggests that promoting the selective removal of senescent cells, via the use of senolytic agents, can be beneficial in the treatment of aging-related bone loss and osteoporosis. Navitoclax (also known as ABT-263) is a chemotherapeutic drug reported to effectively clear senescent hematopoietic stem cells, muscle stem cells, and mesenchymal stromal cells in previous studies, but its in vivo effects on bone mass had not yet been reported. Therefore, the purpose of this study was to assess the effects of short-term navitoclax treatment on bone mass and osteoprogenitor function in old mice. Aged (24 month old) male and female mice were treated with navitoclax (50 mg/kg body mass daily) for 2 weeks. Surprisingly, despite decreasing senescent cell burden, navitoclax treatment decreased trabecular bone volume fraction in aged female and male mice (-60.1% females, -45.6% males), and BMSC-derived osteoblasts from the navitoclax treated mice were impaired in their ability to produce a mineralized matrix (-88% females, -83% males). Moreover, in vitro administration of navitoclax decreased BMSC colony formation and calcified matrix production by aged BMSC-derived osteoblasts, similar to effects seen with the primary BMSC from the animals treated in vivo . Navitoclax also significantly increased metrics of cytotoxicity in both male and female osteogenic cultures (+1.0 to +11.3 fold). Taken together, these results suggest a potentially harmful effect of navitoclax on skeletal-lineage cells that should be explored further to definitively assess navitoclax's potential (or risk) as a therapeutic agent for combatting age-related musculoskeletal dysfunction and bone loss., (Copyright © 2020 Sharma, Roberts, Benson, Pierce, Yu, Hamrick and McGee-Lawrence.)

Tryptophan is an essential amino acid catabolized initially to kynurenine (kyn), an immunomodulatory metabolite that we have previously shown to promote bone loss. Kyn levels increase with aging and have also been associated with neurodegenerative disorders. Picolinic acid (PA) is another tryptophan metabolite downstream of kyn. However, in contrast to kyn, PA is reported to be neuroprotective and further, to promote osteogenesis in vitro. Thus, we hypothesized that PA might be osteoprotective in vivo. In an IACUC-approved protocol, we fed PA to aged (23-month-old) C57BL/6 mice for eight weeks. In an effort to determine potential interactions of PA with dietary protein we also fed PA in a low-protein diet (8%). The mice were divided into four groups: Control (18% dietary protein), +PA (700 ppm); Low-protein (8%), +PA (700 ppm). The PA feedings had no impact on mouse weight, body composition or bone density. At sacrifice bone and stem cells were collected for analysis, including μCT and RT-qPCR. Addition of PA to the diet had no impact on trabecular bone parameters. However, marrow adiposity was significantly increased in PA-fed mice, and in bone marrow stromal cells isolated from these mice increases in the expression of the lipid storage genes, Plin1 and Cidec, were observed. Thus, as a downstream metabolite of kyn, PA no longer showed kyn's detrimental effects on bone but instead appears to impact energy balance., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Mechanisms leading to age-related reductions in bone formation and subsequent osteoporosis are still incompletely understood. We recently demonstrated that kynurenine (KYN), a tryptophan metabolite, accumulates in serum of aged mice and induces bone loss. Here, we report on novel mechanisms underlying KYN's detrimental effect on bone aging. We show that KYN is increased with aging in murine bone marrow mesenchymal stem cells (BMSCs). KYN reduces bone formation via modulating levels of CXCL12 and its receptors as well as histone deacetylase 3 (Hdac3). BMSCs responded to KYN by significantly decreasing mRNA expression levels of CXCL12 and its cognate receptors, CXCR4 and ACKR3, as well as downregulating osteogenic gene RUNX2 expression, resulting in a significant inhibition in BMSCs osteogenic differentiation. KYN's effects on these targets occur by increasing regulatory miRNAs that target osteogenesis, specifically miR29b-1-5p. Thus, KYN significantly upregulated the anti-osteogenic miRNA miR29b-1-5p in BMSCs, mimicking the up-regulation of miR-29b-1-5p in human and murine BMSCs with age. Direct inhibition of miR29b-1-5p by antagomirs rescued CXCL12 protein levels downregulated by KYN, while a miR29b-1-5p mimic further decreased CXCL12 levels. KYN also significantly downregulated mRNA levels of Hdac3, a target of miR-29b-1-5p, as well as its cofactor NCoR1. KYN is a ligand for the aryl hydrocarbon receptor (AhR). We hypothesized that AhR mediates KYN's effects in BMSCs. Indeed, AhR inhibitors (CH-223191 and 3',4'-dimethoxyflavone [DMF]) partially rescued secreted CXCL12 protein levels in BMSCs treated with KYN. Importantly, we found that treatment with CXCL12, or transfection with an miR29b-1-5p antagomir, downregulated the AhR mRNA level, while transfection with miR29b-1-5p mimic significantly upregulated its level. Further, CXCL12 treatment downregulated IDO, an enzyme responsible for generating KYN. Our findings reveal novel molecular pathways involved in KYN's age-associated effects in the bone microenvironment that may be useful translational targets for treating osteoporosis., Competing Interests: Drs. William Hill, Sergio Mas Herrero, and Sudharsan Periyasamy-Thandavanis are inventors on U.S. Patent No. 9,267,139, “Compositions and Methods for Treating Musculoskeletal Disorders” issued. Ahmed Elmansi, Galina Kondrikova, Jessica Pierce, Helen Kaiser, Drs. Khaled Hussein, Xue Jiang, Alexandra Aguilar-Pérez, Dmitry Kondrikov, Nada H. Eisa, Ke-Hong Ding, Aisha Walker, Sadanand Fulzele, Wendy B. Bollag, Mohammed Elsalanty, Qing Zhong, Xing-ming Shi, Yun Su, Maribeth Johnson, Monte Hunter, Charles Reitman, Brian Volkman, Mark Hamrick, Carlos Isales, Meghan McGee-Lawrence have no conflicts of interest or financial ties to disclose., (© 2020 The Author(s).)

Bone loss in aging is linked with chronic low-grade inflammation and the accumulation of marrowfat in animals and humans. Peroxisome proliferator-activated receptor gamma (PPARγ), an adipogenic regulator, plays key roles in these biological processes. However, studies of the roles of PPARγ in age-related bone loss and inflammation are lacking. We hypothesized that deletion of PPARγ in bone marrow mesenchymal lineage cells would reduce bone loss with aging, potentially through a reduction in fat-generated inflammatory responses and an increase in osteoblastic activity. In the present study, we show that mice deficient of PPARγ in Dermo1-expressing mesenchymal lineage cells (Dermo1-Cre:PPARγ fl/fl) have reduced fat mass and increased cortical bone thickness but that deficiency of PPARγ had limited effect on protection of trabecular bone with aging as demonstrated by dual-energy X-ray absorptiometry, µCT, and histomorphometric analyses. Conditional knockout of PPARγ reduced serum concentrations of adipokines, including adiponectin, resistin, and leptin, and reduced marrow stromal cell expression levels of inflammation-related genes. Inflammation genes involved in the interferon signaling pathway were reduced the most. These results demonstrate that disruption of the master adipogenic regulator, PPARγ, has a certain protective effect on aging-induced bone loss, suggesting that regulation of adipose function and modulation of interferon signaling are among the key mechanisms by which PPARγ regulates bone homeostasis during aging process., (© The Author(s) 2020. Published by Oxford University Press on behalf of The Gerontological Society of America. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Cellular therapies have tremendous potential for the successful treatment of major extremity wounds in the combat setting, however, the challenges associated with transplanting stem cells in the prolonged field care (PFC) environment are a critical barrier to progress in treating such injuries. These challenges include not only production and storage but also transport and handling issues. Our goal is to develop a new strategy utilizing extracellular vesicles (EVs) secreted by stem cells that can resolve many of these issues and prevent ischemic tissue injury. While EVs can be preserved by freezing or lyophilization, both processes result in decrease in their bioactivity. Here, we describe optimized procedures for EVs production, isolation, and lyophilization from primary human adipose-derived stem cells (hADSCs). We compared two isolation approaches that were ultrafiltration (UF) using a tangential fluid filtration (TFF) system and differential ultracentrifugation (UC). We also optimized EVs lyophilization in conjunction with trehalose and polyvinylpyrrolidone 40 (PVP40) as lyoprotectants. Bioactivity of EVs was assessed based on reversal of hypoxia-induced muscle cell injury. To this end, primary human myoblasts were subjected to hypoxic conditions for 6 h, and then treated with hADSC-derived EVs at a concentration of 50 μg/mL. Subsequently, muscle cell viability and toxicity were evaluated using MTS and LDH assays, respectively. Overall, nanoparticle tracking data indicated that UF/TFF yields threefold more particles than UC. Lyophilization of EVs resulted in a significantly reduced number of particles, which could be attenuated by adding lyoprotections to the freeze-drying solution. Furthermore, EVs isolated by UF/TFF and freeze-dried in the presence of trehalose significantly increased viability ( P < 0.0193). Taken together, our findings suggest that the isolation and preservation methods presented in this study may enhance therapeutic applications of EVs., (Copyright © 2020 El Baradie, Nouh, O’Brien, Liu, Fulzele, Eroglu and Hamrick.)

Kynurenine, a metabolite of tryptophan breakdown, has been shown to increase with age, and plays a vital role in a number of age-related pathophysiological changes, including bone loss. Accumulation of kynurenine in bone marrow stromal cells (BMSCs) has been associated with a decrease in cell proliferation and differentiation, though the exact mechanism by which kynurenine mediates these changes is poorly understood. MiRNAs have been shown to regulate BMSC function, and accumulation of kynurenine may alter the miRNA expression profile of BMSCs. The aim of this study was to identify differentially expressed miRNAs in human BMSCs in response to treatment with kynurenine, and correlate miRNAs function in BMSCs biology through bioinformatics analysis. Human BMSCs were cultured and treated with and without kynurenine, and subsequent miRNA isolation was performed. MiRNA array was performed to identify differentially expressed miRNA. Microarray analysis identified 50 up-regulated, and 36 down-regulated miRNAs in kynurenine-treated BMSC cultures. Differentially expressed miRNA included miR-1281, miR-330-3p, let-7f-5p, and miR-493-5p, which are important for BMSC proliferation and differentiation. KEGG analysis found up-regulated miRNA targeting glutathione metabolism, a pathway critical for removing oxidative species. Our data support that the kynurenine dependent degenerative effect is partially due to changes in the miRNA profile of BMSCs., (Published by Elsevier Inc.)

The tryptophan metabolite kynurenine increases with aging and inflammation, and appears to contribute directly to the development and progression of several age-related conditions. Kynurenine is now known to signal through the aryl hydrocarbon receptor (Ahr) to modulate levels of reactive oxygen species (ROS). The Ahr promoter region contains several sites for NF-kB binding, indicating that inflammation is a key factor modulating Ahr expression. Furthermore, kynurenine activation of Ahr is observed to stimulate expression of the enzyme IDO1, which generates kynurenine by degrading tryptophan, representing a positive feedback loop that may link inflammation with ROS production. On the other hand, the antioxidant system-inducing transcription factor Nrf2 can be stimulated by Ahr, and Nrf2 can itself activate Ahr expression. The balance between pro- and antioxidant functions of Ahr mediated by kynurenine may therefore regulate healthy versus unhealthy aging in different tissues and organ systems. Potential therapeutic approaches to target this pathway include exercise to alter kynurenine production or molecules such as metformin or resveratrol that may suppress Ahr activity., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Aging is a progressive process associated with declining tissue function over time. Kynurenine, an oxidized metabolite of the essential amino acid tryptophan that increases in abundance with age, drives cellular processes of aging and dysfunction in many tissues, and recent work has focused on understanding the pathways involved in the harmful effects of kynurenine on bone. In this study, we sought to investigate the effects of controlled kynurenine administration on osteoblast bioenergetics, in vivo osteoblast abundance, and marrow fat accumulation. Additionally, as an extension of earlier studies with dietary administration of kynurenine, we investigated the effects of kynurenine on Hdac3 and NCoR1 expression and enzymatic deacetylase activity as potential mechanistic contributors to the effects of kynurenine on osteoblasts. Kynurenine administration suppressed cellular metabolism in osteoblasts at least in part through impaired mitochondrial respiration, and suppressed osteoblastic numbers in vivo with no concurrent effects on marrow adiposity. Deleterious effects of kynurenine treatment on osteoblasts were more pronounced in female models as compared to males. However, kynurenine treatment did not inhibit Hdac3's enzymatic deacetylase activity nor its repression of downstream glucocorticoid signaling. As such, future work will be necessary to determine the mechanisms by which increased kynurenine contributes to aging bone bioenergetics. The current study provides novel further support for the idea that kynurenine contributes to impaired osteoblastic function, and suggests that impaired matrix production by kynurenine-affected osteoblasts is attributed in part to impaired osteoblastic bioenergetics. As circulating kynurenine levels in increase with age, and human bone density inversely correlates with the serum kynurenine to tryptophan ratio, these mechanisms may have important relevance in the etiology and pathogenesis of osteoporosis in humans., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Transient plasma membrane disruptions (PMD) occur in osteocytes with in vitro and in vivo loading, initiating mechanotransduction. The goal here was to determine whether osteocyte PMD formation or repair is affected by aging. Osteocytes from old (24 months) mice developed fewer PMD (-76% females, -54% males) from fluid shear than young (3 months) mice, and old mice developed fewer osteocyte PMD (-51%) during treadmill running. This was due at least in part to decreased pericellular matrix production, as studies revealed that pericellular matrix is integral to formation of osteocyte PMD, and aged osteocytes produced less pericellular matrix (-55%). Surprisingly, osteocyte PMD repair rate was faster (+25% females, +26% males) in osteocytes from old mice, and calcium wave propagation to adjacent nonwounded osteocytes was blunted, consistent with impaired mechanotransduction downstream of PMD in osteocytes with fast PMD repair in previous studies. Inducing PMD via fluid flow in young osteocytes in the presence of oxidative stress decreased postwounding cell survival and promoted accelerated PMD repair in surviving cells, suggesting selective loss of slower-repairing osteocytes. Therefore, as oxidative stress increases during aging, slower-repairing osteocytes may be unable to successfully repair PMD, leading to slower-repairing osteocyte death in favor of faster-repairing osteocyte survival. Since PMD are an important initiator of mechanotransduction, age-related decreases in pericellular matrix and loss of slower-repairing osteocytes may impair the ability of bone to properly respond to mechanical loading with bone formation. These data suggest that PMD formation and repair mechanisms represent new targets for improving bone mechanosensitivity with aging., (© 2019 The Authors. Aging Cell published by the Anatomical Society and John Wiley & Sons Ltd.)

Musculoskeletal disorders are the leading cause of disability worldwide; two of the most prevalent of which are osteoporosis and sarcopenia. Each affect millions in the aging population across the world and the associated morbidity and mortality contributes to billions of dollars in annual healthcare cost. Thus, it is important to better understand the underlying pathologic mechanisms of the disease process. Regulatory chemokine, CXCL12, and its receptor, CXCR4, are recognized to be essential in the recruitment, localization, maintenance, development and differentiation of progenitor stem cells of the musculoskeletal system. CXCL12 signaling results in the development and functional ability of osteoblasts, osteoclasts, satellite cells and myoblasts critical to maintaining musculoskeletal homeostasis. Interestingly, one suggested pathologic mechanism of osteoporosis and sarcopenia is a decline in the regenerative capacity of musculoskeletal progenitor stem cells. Thus, because CXCL12 is critical to progenitor function, a disruption in the CXCL12 signaling axis might play a distinct role in these pathological processes. Therefore, in this article, we perform a review of CXCL12, its physiologic and pathologic function in bone and muscle, and potential targets for therapeutic development., (Published by Elsevier Ltd.)

We assessed whether circulating ceramides, which play a role in a number of degenerative changes with aging, significantly differed according to fragility hip fracture (HF) status. We also performed a human study using bone marrow (BM) aspirates, directly reflecting the bone microenvironment, in addition to in vitro experiments. Peripheral blood and BM samples were simultaneously collected from 74 patients 65 years or older at hip surgery for either HF ( n = 28) or for other causes ( n = 46). Ceramides were measured by liquid chromatography-tandem mass spectrometry. Age was correlated positively with circulating C16:0, C18:0, and C24:1 ceramide levels. Patients with fragility HF had 21.3%, 49.5%, 34.3%, and 22.5% higher plasma C16:0, C18:0, C18:1, and C24:1 ceramide levels, respectively, than those without HF. Higher C16:0, C18:0, C18:1, and C24:1 ceramide levels were positively related to bone resorption markers in both blood and BM samples. Furthermore, in vitro studies showed that C18:0 and C24:1 ceramides directly increased osteoclastogenesis, bone resorption, and expression levels of osteoclast differentiation markers. These results suggested that the association of increased ceramides, especially C18:0 and C24:1, with adverse bone phenotypes in elderly people could be explained mainly by the increase in osteoclastogenesis and bone resorption.

With age, joints become subject to chronic inflammatory processes that lead to degeneration of articular cartilage. Although multifactorial, cytokines have been shown to play a role in the pathogenesis of these chronic disease states. Stromal cell-derived factor 1 (SDF-1) is a chemokine that has been shown to be active in homeostatic mechanisms and developmental processes throughout the body, such as endochondral bone formation. SDF-1 plays a role in the transition from cartilage to bone. Although it has been shown to be a factor in normal development, it has also been shown to involve in the pathogenesis of rheumatoid arthritis (RA) and osteoarthritis (OA). In RA, SDF-1 has been shown to stimulate the recruitment of proinflammatory cells, as well as osteoclasts to the synovium, aiding in the facilitation of synovial degradation. Similarly, in OA, SDF-1 has been shown to regulate key proteins involved in the degradation of the cartilage of the joint. Because of its role in degenerative joint disease, SDF-1 has been investigated as a potential therapeutic target. Animal studies have been employing SDF-1 inhibitors, such as AMD3100 and T140, to study their effects on attenuating degenerative joint disease. These studies have shown promising results in slowing the progression of cartilage degradation and could potentially be used as therapeutic target for humans OA and RA., Competing Interests: Conflict of interest statement: The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article., (© The Author(s), 2019.)

The cellular and molecular mechanisms underlying loss of muscle mass with age (sarcopenia) are not well-understood; however, heterochronic parabiosis experiments show that circulating factors are likely to play a role. Kynurenine (KYN) is a circulating tryptophan metabolite that is known to increase with age and is a ligand of the aryl hydrocarbon receptor (Ahr). Here, we tested the hypothesis that KYN activation of Ahr plays a role in muscle loss with aging. Results indicate that KYN treatment of mouse and human myoblasts increased levels of reactive oxygen species (ROS) 2-fold and KYN treatment in vivo reduced muscle size and strength and increased muscle lipid peroxidation in young mice. PCR array data indicate that muscle fiber size reduction with KYN treatment reduces protein synthesis markers whereas ubiquitin ligase gene expression is not significantly increased. KYN is generated by the enzyme indoleamine 2,3-dioxygenase (IDO), and aged mice treated with the IDO inhibitor 1-methyl-D-tryptophan showed an increase in muscle fiber size and muscle strength. Small-molecule inhibition of Ahr in vitro , and Ahr knockout in vivo , did not prevent KYN-induced increases in ROS, suggesting that KYN can directly increase ROS independent of Ahr activation. Protein analysis identified very long-chain acyl-CoA dehydrogenase as a factor activated by KYN that may increase ROS and lipid peroxidation. Our data suggest that IDO inhibition may represent a novel therapeutic approach for the prevention of sarcopenia and possibly other age-associated conditions associated with KYN accumulation such as bone loss and neurodegeneration., Competing Interests: Carlos M. Isales and Mark W. Hamrick are founders of Gerologix Inc. which seeks to target the KYN pathway for the treatment of age-related diseases. Helen Kaiser and Mark Hamrick hold intellectual property related to KYN inhibition in sarcopenia. These conflicts are managed in accordance with the university policy., (Copyright © 2019 Helen Kaiser et al.)

Recently, muscle has received much attention as an endocrine organ regulating other biological targets, including the pancreas, liver, and adipose tissue. Although there is a possibility that muscle-secreting factors biochemically affect bone metabolism in a paracrine manner, the net effects of myokines on the biology of osteoclasts and osteoblasts, particularly on bone mass in vivo, have not yet been thoroughly investigated. Therefore, we performed in vitro as well as animal experiments using conditioned media (CM) collected from C2C12 myoblast and myotube cultures to better understand the interactions between muscle and bone. Compared with non-CM (i.e., control) and myoblast CM, myotube CM markedly inhibited in vitro bone resorption through the suppression of osteoclast differentiation and resorptive activity of individual osteoclasts. Consistently, the expressions of osteoclast differentiation markers, such as tartrate-resistant acid phosphatase (Trap) and calcitonin receptor (Ctr), decreased with myotube CM. Myotube CM significantly stimulated preosteoblast viability and migration and reduced apoptosis, thereby resulting in an increase in calvaria bone formation. Importantly, systemic treatment with myotube CM for 4 weeks increased bone per tissue volume by 30.7% and 19.6% compared with control and myoblast CM, respectively. These results support the hypothesis that muscle plays beneficial roles in bone health via secretion of anabolic factors, in addition to mechanical stimuli, and importantly indicate that muscle-derived factors can be potential therapeutic targets against metabolic bone diseases., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Stromal cell-derived factor-1 (SDF-1 or CXCL12) is a cytokine secreted by cells including bone marrow stromal cells (BMSCs). SDF-1 plays a vital role in BMSC migration, survival, and differentiation. Our group previously reported the role of SDF-1 in osteogenic differentiation in vitro and bone formation in vivo; however, our understanding of the post-transcriptional regulatory mechanism of SDF-1 remains poor. MicroRNAs are small noncoding RNAs that post-transcriptionally regulate the messenger RNAs (mRNAs) of protein-coding genes. In this study, we aimed to investigate the impact of miR-141-3p on SDF-1 expression in BMSCs and its importance in the aging bone marrow (BM) microenvironment. Our data demonstrated that murine and human BMSCs expressed miR-141-3p that repressed SDF-1 gene expression at the functional level (luciferase reporter assay) by targeting the 3'-untranslated region of mRNA. We also found that transfection of miR-141-3p decreased osteogenic markers in human BMSCs. Our results demonstrate that miR-141-3p expression increases with age, while SDF-1 decreases in both the human and mouse BM niche. Taken together, these results support that miR-141-3p is a novel regulator of SDF-1 in bone cells and plays an important role in the age-dependent pathophysiology of murine and human BM niche., (© The Author(s) 2018. Published by Oxford University Press on behalf of The Gerontological Society of America. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Although both liver and muscle are metabolically active endocrine organs, and non-alcoholic fatty liver disease (NAFLD) and sarcopenia may share common pathogenic determinants, there have been few clinical studies of the relationship between NAFLD and muscle strength, especially in the elderly. We conducted a nationally representative population-based, cross-sectional study using data from the Korea National Health and Nutrition Examination Survey, which involved 1,897 men aged ≥50 years and 2,206 postmenopausal women. NAFLD was defined using the hepatic steatosis index (HSI) and low muscle strength was defined using the Korea-specific cut-off point of hand grip strength (HGS). Men and women with NAFLD had 7.3% and 7.9% lower HGS than controls, respectively. The odds ratios for low muscle strength in the presence of NAFLD were 2.51 in men and 2.34 in women. HSI inversely correlated with HGS in both men and women. Consistently, compared with men and women in the lowest HSI quartile, those in the highest quartile had 7.6% and 12.4% lower HGS, respectively, and were 5.63- and 3.58-times more likely to have low muscle strength, respectively. These results provide the first clinical evidence that NAFLD can be associated with muscular impairment in older adults, as demonstrated by lower muscle strength.

Excess fat within bone marrow is associated with lower bone density. Metabolic stressors such as chronic caloric restriction (CR) can exacerbate marrow adiposity, and increased glucocorticoid signaling and adrenergic signaling are implicated in this phenotype. The current study tested the role of glucocorticoid signaling in CR-induced stress by conditionally deleting the glucocorticoid receptor (GR) in bone marrow osteoprogenitors (Osx1-Cre) of mice subjected to CR and ad libitum diets. Conditional knockout of the GR (GR-CKO) reduced cortical and trabecular bone mass as compared to wildtype (WT) mice under both ad libitum and CR conditions. No interaction was detected between genotype and diet, suggesting that the GR is not required for CR-induced skeletal changes. The lower bone mass in GR-CKO mice, and the further suppression of bone by CR, resulted from suppressed bone formation. Interestingly, treatment with the -adrenergic receptor antagonist propranolol mildly but selectively improved metrics of cortical bone mass in GR-CKO mice during CR, suggesting interaction between adrenergic and glucocorticoid signaling pathways that affects cortical bone. GR-CKO mice dramatically increased marrow fat under both ad libitum and CR-fed conditions, and surprisingly propranolol treatment was unable to rescue CR-induced marrow fat in either WT or GR-CKO mice. Additionally, serum corticosterone levels were selectively elevated in GR-CKO mice with CR, suggesting the possibility of bone-hypothalamus-pituitary-adrenal crosstalk during metabolic stress. This work highlights the complexities of glucocorticoid and β-adrenergic signaling in stress-induced changes in bone mass, and the importance of GR function in suppressing marrow adipogenesis while maintaining healthy bone mass.