Extracellular vesicles (EVs), including exosomes and microvesicles, function in cell-to-cell communication through delivery of proteins, lipids and microRNAs to target cells via endocytosis and membrane fusion. These vesicles are enriched in ceramide, a sphingolipid associated with the promotion of cell senescence and apoptosis. We investigated the ceramide profile of serum exosomes from young (24⁻40 yrs.) and older (75⁻90 yrs.) women and young (6⁻10 yrs.) and older (25⁻30 yrs.) rhesus macaques to define the role of circulating ceramides in the aging process. EVs were isolated using size-exclusion chromatography. Proteomic analysis was used to validate known exosome markers from Exocarta and nanoparticle tracking analysis used to characterize particle size and concentration. Specific ceramide species were identified with lipidomic analysis. Results show a significant increase in the average amount of C24:1 ceramide in EVs from older women (15.4 pmol/sample) compared to those from younger women (3.8 pmol/sample). Results were similar in non-human primate serum samples with increased amounts of C24:1 ceramide (9.3 pmol/sample) in older monkeys compared to the younger monkeys (1.8 pmol/sample). In vitro studies showed that primary bone-derived mesenchymal stem cells (BMSCs) readily endocytose serum EVs, and serum EVs loaded with C24:1 ceramide can induce BMSC senescence. Elevated ceramide levels have been associated with poor cardiovascular health and memory impairment in older adults. Our data suggest that circulating EVs carrying C24:1 ceramide may contribute directly to cell non-autonomous aging.

Ascorbic acid (Vitamin C) has a critical role in bone formation and osteoblast differentiation, but very little is known about the molecular mechanisms of ascorbic acid entry into bone marrow stromal cells (BMSCs). To address this gap in knowledge, we investigated the identity of the transport system that is responsible for the uptake of ascorbic acid into bone marrow stromal cells (BMSCs). First, we examined the expression of the two known isoforms of the sodium-coupled ascorbic acid transporter, namely SVCT1 and SVCT2, in BMSCs (Lin-ve Sca1+ve) and bone at the mRNA level. Only SVCT2 mRNA was detected in BMSCs and bone. Uptake of ascorbic acid in BMSCs was Na(+)-dependent and saturable. In order to define the role of SVCT2 in BMSC differentiation into osteoblasts, BMSCs were stimulated with osteogenic media for different time intervals, and the activity of SVCT2 was monitored by ascorbic acid uptake. SVCT2 expression was up-regulated during the osteogenic differentiation of BMSCs; the expression was maximal at the earliest phase of differentiation. Subsequently, osteogenesis was inhibited in BMSCs upon knock-down of SVCT2 by lentivirus shRNA. We also found that the expression of the SVCT2 could be negatively or positively modulated by the presence of oxidant (Sin-1) or antioxidant (Ascorbic acid) compounds, respectively, in BMSCs. Furthermore, we found that this transporter is also regulated with age in mouse bone. These data show that SVCT2 plays a vital role in the osteogenic differentiation of BMSCs and that its expression is altered under conditions associated with redox reaction. Our findings could be relevant to bone tissue engineering and bone related diseases such as osteoporosis in which oxidative stress and aging plays important role.

Genome-wide association studies (GWAS) have recently identified several new genetic variants associated with obesity. The majority of the variants are within introns or between genes, suggesting they affect gene expression, although it is not clear which of the nearby genes they affect. Understanding the regulation of these genes will be key to determining the role of these variants in the development of obesity and will provide support for a role of these genes in the development of obesity.

Lysyl oxidase like-2 (LOXL2) is a copper-dependent amine oxidase. Our previous studies showed that LOXL2 is elevated during mouse fracture healing. The goal of this study was to evaluate the potential of LOXL2 to act as an anabolic agent in cartilage affected by osteoarthritis (OA).

Leptin receptors are abundant in human skeletal muscle, but the role of leptin in muscle growth, development and aging is not well understood. Here we utilized a novel mouse model lacking all functional leptin receptor isoforms (POUND mouse, Lepr(db/lb)) to determine the role of leptin in skeletal muscle.

Bone marrow-derived mesenchymal stem/stromal cells (BMSCs) hold great potential for cell-based therapy, yet the therapeutic efficacy remains uncertain. Transplanted BMSCs often fail to engraft within the bone marrow (BM), in part due to the poor survival of donor cells in response to inflammatory reactions, hypoxia, oxidative stress, or nutrient starvation. Two basic cell processes, apoptosis and autophagy, could potentially be responsible for the impaired survival of transplanted BMSCs. However, the functional relationship between apoptosis and autophagy in BMSC homeostasis is complex and not well understood. The stromal cell-derived factor-1 (SDF-1)/CXC chemokine receptor 4 (CXCR4) signaling axis appears to be critical in maintaining proliferation and survival of BM stem cell populations through improving cell proliferation and survival in response to stress; however, the exact mechanisms remain unclear. We recently described novel genetically engineered Tet-Off-SDF-1β BMSCs, which over-express SDF-1β under tight doxycycline-control, thus providing an ideal model system to investigate the isolated effects of SDF-1β. In this study we tested the hypothesis that SDF-1β can mediate cell survival of BMSCs in vitro through increasing autophagy. We found that SDF-1β had no effect on BMSC proliferation; however, SDF-1β significantly protected genetically engineered BMSCs from H2O2-induced cell death through increasing autophagy and decreasing caspase-3-dependent apoptosis. Taken together, we provide novel evidence that the SDF-1/CXCR4 axis, specifically activated by the SDF-1β isoform, plays a critical role in regulating BMSC survival under oxidative stress through increasing autophagy.

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.

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.

Studies have shown that factors in the blood of young organisms can rejuvenate the old ones. Studies using heterochronic parabiosis models further reinforced the hypothesis that juvenile factors can rejuvenate aged systems. We sought to determine the effect of juvenile plasma-derived factors on the outcome following hemorrhagic shock injury in aged mice. We discovered that pre-pubertal (young) mice subjected to hemorrhagic shock survived for a prolonged period, in the absence of fluid resuscitation, compared to mature or aged mice. To further understand the mechanism of maturational dependence of injury resolution, extracellular vesicles isolated from the plasma of young mice were administered to aged mice subjected to hemorrhagic shock. The extracellular vesicle treatment prolonged life in the aged mice. The treatment resulted in reduced oxidative stress in the liver and in the circulation, along with an enhanced expression of the nuclear factor erythroid factor 2-related factor 2 (Nrf2) and its target genes, and a reduction in the expression of the transcription factor BTB and CNC homology 1 (Bach1). We propose that plasma factors in the juvenile mice have a reparative effect in the aged mice in injury resolution by modulating the Nrf2/Bach1 axis in the antioxidant response pathway.

Aging results in a general decline in function in most systems. This is particularly true with respect to the skeleton and renal systems, impacting mineral homeostasis. Calcium and phosphate regulation requires tight coordination among the intestine, bone, parathyroid gland, and kidney. The role of the intestine is to absorb calcium and phosphate from the diet. The bone stores or releases calcium and phosphate depending on the body's needs. In response to low plasma ionized calcium concentration, the parathyroid gland produces parathyroid hormone, which modulates bone turnover. The kidney reabsorbs or excretes the minerals and serves as the final regulator of plasma concentration. Many hormones are involved in this process in addition to parathyroid hormone, including fibroblast growth factor 23 produced by the bone and calcitriol synthesized by the kidney. Sclerostin, calcitonin, osteoprotegerin, and receptor activator of nuclear factor-κB ligand also contribute to tissue-specific regulation. Changes in the function of organs due to aging or disease can perturb this balance. During aging, the intestine cannot absorb calcium efficiently due to decreased expression of key proteins. In the bone, the balance between bone formation and bone resorption tends toward the latter in older individuals. The kidney may not filter blood as efficiently in the later decades of life, and the expression of certain proteins necessary for mineral homeostasis declines with age. These changes often lead to dysregulation of organismal mineral homeostasis. This review will focus on how mineral homeostasis is impacted by aging with a particular emphasis on the kidney's role in this process. © 2021 The Authors. JBMR Plus published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research.

Aging is a major risk factor for multiple chronic disorders in the elderly population, including Alzheimer's disease (AD) and Osteoporosis. AD is a progressive neurodegenerative disease characterized by memory loss. In addition to dementia, several studies have shown that AD patients experience an increased rate of musculoskeletal co-morbidities, such as osteoporosis. Since tissue-specific macrophages contribute to both diseases, this study analyzed the microglia transcriptome of AD mice to determine a common gene signature involved in osteoclast biology. After comparing differentially regulated genes from GEO data sets (GSE93824 and GSE212277), there were 35 common upregulated genes and 89 common downregulated genes. Of these common genes, seven genes are known to play an important role in bone homeostasis. CSF1, SPP1, FAM20C, and Cst7 were upregulated and are associated with osteoclastogenesis and inflammation. Among the downregulated genes, LILRA6, MMP9, and COL18A1 are involved in bone formation and osteoclast regulation. We further validated some of these genes (CSF1, Cst7, and SPP1) in the cortex and the bone of AD mice models. The dysregulation of these microglial genes in AD might provide insights into the co-occurrence of AD and osteoporosis and offer potential therapeutic targets to combat disease progression.

Leptin signaling in the central nervous system, and particularly the arcuate hypothalamic nucleus, is important for regulating energy and glucose homeostasis. However, the roles of extra-arcuate leptin responsive neurons are less defined. In the current study, we generated mice with widespread inactivation of the long leptin receptor isoform in the central nervous system via Synapsin promoter-driven Cre (Lepr(flox/flox) Syn-cre mice). Within the hypothalamus, leptin signaling was disrupted in the lateral hypothalamic area (LHA) and ventral premammillary nucleus (PMV) but remained intact in the arcuate hypothalamic nucleus and ventromedial hypothalamic nucleus, dorsomedial hypothalamic nucleus, and nucleus of the tractus solitarius. To investigate the role of LHA/PMV neuronal leptin signaling, we examined glucose and energy homeostasis in Lepr(flox/flox) Syn-cre mice and Lepr(flox/flox) littermates under basal and diet-induced obese conditions and tested the role of LHA/PMV neurons in leptin-mediated glucose lowering in streptozotocin-induced diabetes. Lepr(flox/flox) Syn-cre mice did not have altered body weight or blood glucose levels but were hyperinsulinemic and had enhanced glucagon secretion in response to experimental hypoglycemia. Surprisingly, when placed on a high-fat diet, Lepr(flox/flox) Syn-cre mice were protected from weight gain, glucose intolerance, and diet-induced hyperinsulinemia. Peripheral leptin administration lowered blood glucose in streptozotocin-induced diabetic Lepr(flox/flox) Syn-cre mice as effectively as in Lepr(flox/flox) littermate controls. Collectively these findings suggest that leptin signaling in LHA/PMV neurons is not critical for regulating glucose levels but has an indispensable role in the regulation of insulin and glucagon levels and, may promote the development of diet-induced hyperinsulinemia and weight gain.

Age-associated loss of muscle mass, or sarcopenia, contributes directly to frailty and an increased risk of falls and fractures among the elderly. Aged mice and elderly adults both show decreased muscle mass as well as relatively low levels of the fat-derived hormone leptin. Here we demonstrate that loss of muscle mass and myofiber size with aging in mice is associated with significant changes in the expression of specific miRNAs. Aging altered the expression of 57 miRNAs in mouse skeletal muscle, and many of these miRNAs are now reported to be associated specifically with age-related muscle atrophy. These include miR-221, previously identified in studies of myogenesis and muscle development as playing a role in the proliferation and terminal differentiation of myogenic precursors. We also treated aged mice with recombinant leptin, to determine whether leptin therapy could improve muscle mass and alter the miRNA expression profile of aging skeletal muscle. Leptin treatment significantly increased hindlimb muscle mass and extensor digitorum longus fiber size in aged mice. Furthermore, the expression of 37 miRNAs was altered in muscles of leptin-treated mice. In particular, leptin treatment increased the expression of miR-31 and miR-223, miRNAs known to be elevated during muscle regeneration and repair. These findings suggest that aging in skeletal muscle is associated with marked changes in the expression of specific miRNAs, and that nutrient-related hormones such as leptin may be able to reverse muscle atrophy and alter the expression of atrophy-related miRNAs in aging skeletal muscle.

A positive energy balance promotes white adipose tissue (WAT) expansion which is characterized by activation of a repertoire of events including hypoxia, inflammation and extracellular matrix remodelling. The transmembrane glycoprotein CD248 has been implicated in all these processes in different malignant and inflammatory diseases but its potential impact in WAT and metabolic disease has not been explored.

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.

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.

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.

Carbidopa is a drug that blocks conversion of levodopa to dopamine outside of central nervous system (CNS) and thus inhibits unwanted side effects of levodopa on organs located outside of CNS during management of Parkinson's Disease (PD). PD is associated with increased expression of inflammatory genes in peripheral and central nervous system (CNS), infiltration of immune cells into brain, and increased numbers of activated/memory T cells. Animal models of PD have shown a critical role of T cells in inducing pathology in CNS. However, the effect of carbidopa on T cell responses in vivo is unknown. In this report, we show that carbidopa strongly inhibited T cell activation in vitro and in vivo. Accordingly, carbidopa mitigated myelin oligodendrocyte glycoprotein peptide fragment 35-55 (MOG-35-55) induced experimental autoimmune encephalitis (EAE) and collagen induced arthritis in animal models. The data presented here suggest that in addition to blocking peripheral conversion of levodopa, carbidopa may inhibit T cell responses in PD individuals and implicate a potential therapeutic use of carbidopa in suppression of T cell mediated pathologies.

Indoleamine-2,3-dioxygenase (IDO) degrades the essential amino acid tryptophan resulting in tryptophan depletion and the accumulation of catabolites such as kynurenine. The expression/activity of IDO in various cells, including macrophages and dendritic cells, results in an inhibition of T-cell responses in a number of situations, such as toward allogeneic fetuses and tissue grafts. Psoriasis is an immune-mediated skin disease involving T cells; kynureninase and its generation of catabolites downstream of IDO are reported to play an important role in this disease. We hypothesized that mice lacking the IDO1 gene would exhibit a hyperactive immune response and an exacerbation of skin lesions in the imiquimod-induced mouse model of psoriasis. Littermate wild-type and IDO1-knockout mice were treated with imiquimod for 5 days, and the severity of psoriasiform skin lesions assessed using the psoriasis area and severity index (PASI), ear edema measured using a digital caliper, and thickness of the epidermis determined by histology. Expression of pro-inflammatory mediators and tryptophan-metabolizing enzymes was monitored using quantitative RT-PCR. Imiquimod increased ear edema, PASI scores, and epidermal thickness in both WT and IDO1 knockout mice; however, there were no differences observed between the 2 genotypes. There were also no differences in imiquimod's induction of skin inflammatory mediators, indicating no effect of IDO1 gene loss in this psoriasis model. Although these data suggest a lack of involvement of IDO1 in psoriatic skin inflammation, other possible mechanisms, such as compensatory changes in other pathways and the involvement of the IDO2 isoform, must also be considered.

The long evolutionary battle between humans and pathogens has played an important role in shaping the current network of host-pathogen interactions. Each organ brings new challenges from the perspective of a pathogen to establish a suitable niche for survival while subverting the protective mechanisms of the host. Lungs, the organ for oxygen exchange, have been an easy target for pathogens due to its accessibility. The organ has evolved diverse capabilities to provide the flexibility required for an organism's health and at the same time maintain protective functionality to prevent and resolve assault by pathogens. The pathogenic invasions are strongly challenged by healthy lung architecture which includes the presence and activity of the epithelium, mucous, antimicrobial proteins, surfactants, and immune cells. Competitively, the pathogens in the form of viruses, bacteria, and fungi have evolved an arsenal of strategies that can over-ride the host's protective mechanisms. While bacteria such as Mycobacterium tuberculosis (M. tuberculosis) can survive in dormant form for years before getting active in humans, novel pathogens can wreak havoc as they pose a high risk of morbidity and mortality in a very short duration of time. Recently, a coronavirus strain SARS-CoV-2 has caused a pandemic which provides us an opportunity to look at the host manipulative strategies used by respiratory pathogens. Their ability to hide, modify, evade, and exploit cell's processes are key to their survival. While pathogens like M. tuberculosis have been infecting humans for thousands of years, SARS-CoV-2 has been the cause of the recent pandemic. Molecular understanding of the strategies used by these pathogens could greatly serve in design of predictive, preventive, personalized medicine (PPPM). In this article, we have emphasized on the clinically relevant evasive strategies of the pathogens in the lungs with emphasis on M. tuberculosis and SARS-CoV-2. The molecular basis of these evasive strategies illuminated through advances in genomics, cell, and structural biology can assist in the mapping of vulnerable molecular networks which can be exploited translationally. These evolutionary approaches can further assist in generating screening and therapeutic options for susceptible populations and could be a promising approach for the prediction, prevention of disease, and the development of personalized medicines. Further, tailoring the clinical data of COVID-19 patients with their physiological responses in light of known host-respiratory pathogen interactions can provide opportunities to improve patient profiling and stratification according to identified therapeutic targets.