Therapeutic Consequences of Targeting the IGF-1/PI3K/AKT/FOXO3 Axis in Sarcopenia: A Narrative Review.

The high prevalence of sarcopenia in an aging population has an underestimated impact on quality of life by increasing the risk of falls and subsequent hospitalization. Unfortunately, the application of the major established key therapeutic-physical activity-is challenging in the immobile and injured sarcopenic patient. Consequently, novel therapeutic directions are needed. The transcription factor Forkhead-Box-Protein O3 (FOXO3) may be an option, as it and its targets have been observed to be more highly expressed in sarcopenic muscle. In such catabolic situations, Foxo3 induces the expression of two muscle specific ubiquitin ligases (Atrogin-1 and Murf-1) via the PI3K/AKT pathway. In this review, we particularly evaluate the potential of Foxo3-targeted gene therapy. Foxo3 knockdown has been shown to lead to increased muscle cross sectional area, through both the AKT-dependent and -independent pathways and the reduced impact on the two major downstream targets Atrogin-1 and Murf-1. Moreover, a Foxo3 reduction suppresses apoptosis, activates satellite cells, and initiates their differentiation into muscle cells. While this indicates a critical role in muscle regeneration, this mechanism might exhaust the stem cell pool, limiting its clinical applicability. As systemic Foxo3 knockdown has also been associated with risks of inflammation and cancer progression, a muscle-specific approach would be necessary. In this review, we summarize the current knowledge on Foxo3 and conceptualize a specific and targeted therapy that may circumvent the drawbacks of systemic Foxo3 knockdown. This approach presumably would limit the side effects and enable an activity-independent positive impact on skeletal muscle.

Foxo3 Knockdown Mediates Decline of Myod1 and Myog Reducing Myoblast Conversion to Myotubes.

Sarcopenia has a high prevalence among the aging population. Sarcopenia is of tremendous socioeconomic importance because it can lead to falls and hospitalization, subsequently increasing healthcare costs while limiting quality of life. In sarcopenic muscle fibers, the E3 ubiquitin ligase F-Box Protein 32 (Fbxo32) is expressed at substantially higher levels, driving ubiquitin-proteasomal muscle protein degradation. As one of the key regulators of muscular equilibrium, the transcription factor Forkhead Box O3 (FOXO3) can increase the expression of Fbxo32, making it a possible target for the regulation of this detrimental pathway. To test this hypothesis, murine C2C12 myoblasts were transduced with AAVs carrying a plasmid for four specific siRNAs against Foxo3. Successfully transduced myoblasts were selected via FACS cell sorting to establish single clone cell lines. Sorted myoblasts were further differentiated into myotubes and stained for myosin heavy chain (MHC) by immunofluorescence. The resulting area was calculated. Myotube contractions were induced by electrical stimulation and quantified. We found an increased Foxo3 expression in satellite cells in human skeletal muscle and an age-related increase in Foxo3 expression in older mice in silico. We established an in vitro AAV-mediated FOXO3 knockdown on protein level. Surprisingly, the myotubes with FOXO3 knockdown displayed a smaller myotube size and a lower number of nuclei per myotube compared to the control myotubes (AAV-transduced with a functionless control plasmid). During differentiation, a lower level of FOXO3 reduced the expression Fbxo32 within the first three days. Moreover, the expression of Myod1 and Myog via ATM and Tp53 was reduced. Functionally, the Foxo3 knockdown myotubes showed a higher contraction duration and time to peak. Early Foxo3 knockdown seems to terminate the initiation of differentiation due to lack of Myod1 expression, and mediates the inhibition of Myog. Subsequently, the myotube size is reduced and the excitability to electrical stimulation is altered.

Effects of ligandrol as a selective androgen receptor modulator in a rat model for osteoporosis.

INTRODUCTION: The selective androgen receptor modulator ligandrol (LGD-4033 or VK5211) has been shown to improve muscle tissue. In the present study, the effect of ligandrol on bone tissue was investigated in ovariectomized rat model. MATERIALS AND METHODS: Three-month-old Sprague Dawley rats were either ovariectomized (OVX, n = 60) or left intact (NON-OVX, n = 15). After 9 weeks, OVX rats were divided into four groups: untreated OVX (n = 15) group and three OVX groups (each of 15 rats) treated with ligandrol orally at doses of 0.03, 0.3, or 3 mg/kg body weight. After five weeks, lumbar vertebral bodies (L), tibiae, and femora were examined using micro-computed tomographical, biomechanical, ashing, and gene expression analyses. RESULTS: In the 3-mg ligandrol group, bone structural properties were improved (trabecular number: 38 ± 8 vs. 35 ± 7 (femur), 26 ± 7 vs. 22 ± 6 (L), 12 ± 5 vs. 6 ± 3 (tibia) and serum phosphorus levels (1.81 ± 0.17 vs.1.41 ± 0.17 mmol/l), uterus (0.43 ± 0.04 vs. 0.11 ± 0.02 g), and heart (1.13 ± 0.11 vs. 1.01 ± 0.08 g) weights were increased compared to the OVX group. Biomechanical parameters were not changed. Low and medium doses did not affect bone tissue and had fewer side effects. Body weight and food intake were not affected by ligandrol; OVX led to an increase in these parameters and worsened all bone parameters. CONCLUSION: Ligandrol at high dose showed a subtle anabolic effect on structural properties without any improvement in biomechanical properties of osteoporotic bones. Considering side effects of ligandrol at this dose, its further investigation for the therapy of postmenopausal osteoporosis should be reevaluated.

Chondral/Desmal Osteogenesis in 3D Spheroids Sensitized by Psychostimulants.

Attention deficit hyperactivity disorder (ADHD) affects 6.4 million children in the United States of America. Children and adolescents, the main consumers of ADHD medication, are in the bone growth phase, which extends over a period of up to two decades. Thus, impaired proliferation and maturation of chondrocytes and osteoblasts can result in impaired bone formation. The aim of this study is to investigate, for the first time, the effects of the ADHD-medication modafinil, atomoxetine and guanfacine on bone growth and repair in vitro. Using two-dimensional and three-dimensional cell models, we investigated the chondrogenic/osteogenic differentiation, proliferation and viability of human mesenchymal progenitor cells. Real-time cell proliferation was measured by xCELLigence. Live/dead staining and size measurement of hMSC- and MG63 monolayer and spheroids were performed after administration of therapeutic plasma concentrations of modafinil, atomoxetine and guanfacine. Chondrogenic differentiation was quantified by RTqPCR. The chondrogenic and osteogenic differentiation was evaluated by histological cryo-sections. Modafinil, atomoxetine and guanfacine reduced chondrogenic and osteogenic differentiation terms of transcript expression and at the histological level. Cell viability of the MG63- and hMSC monolayer was not impeded by ADHD-medication. Our in vitro results indicate that modafinil, atomoxetine and guanfacine may impair chondrogenic and osteogenic differentiation in a 3D model reflecting the in vivo physiologic condition.

Psychostimulants Modafinil, Atomoxetine and Guanfacine Impair Bone Cell Differentiation and MSC Migration.

Attention deficit hyperactivity disorder (ADHD) is one of the most common worldwide mental disorders in children, young and adults. If left untreated, the disorder can continue into adulthood. The abuse of ADHD-related drugs to improve mental performance for studying, working and everyday life is also rising. The potentially high number of subjects with controlled or uncontrolled use of such substances increases the impact of possible side effects. It has been shown before that the early ADHD drug methylphenidate influences bone metabolism negatively. This study focused on the influence of three more recent cognitive enhancers, modafinil, atomoxetine and guanfacine, on the differentiation of mesenchymal stem cells to osteoblasts and on their cell functions, including migration. Human mesenchymal stem cells (hMSCs) were incubated with a therapeutic plasma dosage of modafinil, atomoxetine and guanfacine. Gene expression analyses revealed a high beta-2 adrenoreceptor expression in hMSC, suggesting it as a possible pathway to stimulate action. In bone formation assays, all three cognitive enhancers caused a significant decrease in the mineralized matrix and an early slight reduction of cell viability without triggering apoptosis or necrosis. While there was no effect of the three substances on early differentiation, they showed differing effects on the expression of osterix (OSX), receptor activator of NF-κB ligand (RANKL) and osteoprotegerin (OPG) in the later stages of osteoblast development, suggesting alternative modes of action. All three substances significantly inhibited hMSC migration. This effect could be rescued by a selective beta-blocker (Imperial Chemical Industries ICI-118,551) in modafinil and atomoxetine, suggesting mediation via beta-2 receptor stimulation. In conclusion, modafinil, atomoxetine and guanfacine negatively influence hMSC differentiation to bone-forming osteoblasts and cell migration through different intracellular pathways.

Investigating the Microchannel Architectures Inside the Subchondral Bone in Relation to Estimated Hip Reaction Forces on the Human Femoral Head.

The interplay between articular cartilage (AC) and subchondral bone (SB) plays a pivotal role in cartilage homeostasis and functionality. As direct connective pathways between the two are poorly understood, we examined the location-dependent characteristics of the 3D microchannel network within the SB that connects the basal cartilage layer to the bone marrow (i.e. cartilage-bone marrow microchannel connectors; CMMC). 43 measuring points were defined on five human cadaveric femoral heads with no signs of osteoarthritis (OA) (age ≤ 60), and cartilage-bone cylinders with diameters of 2.00 mm were extracted for high-resolution scanning (n = 215). The micro-CT data were categorized into three groups (load-bearing region: LBR, n = 60; non-load-bearing region: NLBR, n = 60; and the peripheral rim: PR, n = 95) based on a gait analysis estimation of the joint reaction force (young, healthy cohort with no signs of OA). At the AC-SB interface, the number of CMMC in the LBR was 1.8 times and 2.2 times higher compared to the NLBR, and the PR, respectively. On the other hand, the median Feret size of the CMMC were smallest in the LBR (55.2 µm) and increased in the NLBR (73.5 µm; p = 0.043) and the PR (89.1 µm; p = 0.043). AC thickness was positively associated with SB thickness (Pearson's r = 0.48; p < 1e-13), CMMC number. (r = 0.46; p < 1e-11), and circularity index (r = 0.61; p < 1e-38). In conclusion, our data suggest that regional differences in the microchannel architecture of SB might reflect regional differences in loading.

Current State of Bone Adhesives-Necessities and Hurdles.

The vision of gluing two bone fragments with biodegradable and biocompatible adhesives remains highly fascinating and attractive to orthopedic surgeons. Possibly shorter operation times, better stabilization, lower infection rates, and unnecessary removal make this approach very appealing. After 30 years of research in this field, the first adhesive systems are now appearing in scientific reports that may fulfill the comprehensive requirements of bioadhesives for bone. For a successful introduction into clinical application, special requirements of the musculoskeletal system, challenges in the production of a bone adhesive, as well as regulatory hurdles still need to be overcome. In this article, we will give an overview of existing synthetic polymers, biomimetic, and bio-based adhesive approaches, review the regulatory hurdles they face, and discuss perspectives of how bone adhesives could be efficiently introduced into clinical application, including legal regulations.

Radiation-induced sensitivity of tissue-resident mesenchymal stem cells in the head and neck region.

BACKGROUND: Tissue-resident mesenchymal stem cells (MSCs) possess the ability to migrate to areas of inflammation and promote the regeneration of damaged tissue. However, it remains unclear how radiation influences this capacity of MSC in the head and neck region. METHODS: Two types of MSCs of the head and neck region (mucosa [mMSC] and parotid gland [pMSC]) were isolated, cultured and exposed to single radiation dosages of 2 Gy/day up to 10 days. Effects on morphology, colony forming ability, apoptosis, chemokine receptor expression, cytokine secretion, and cell migration were analyzed. RESULTS: Although MSC preserved MSC-specific regenerative abilities and immunomodulatory properties following irradiation in our in vitro model, we found a deleterious impact on colony forming ability, especially in pMSC. CONCLUSIONS: MSC exhibited robustness and activation upon radiation for the support of tissue regeneration, but lost their potential to replicate, thus possibly leading to depletion of the local MSC-pool after irradiation.

Effects of RANKL Knockdown by Virus-like Particle-Mediated RNAi in a Rat Model of Osteoporosis.

Rebalancing of the RANKL/OPG system seems to be an effective treatment strategy in postmenopausal osteoporosis. Here, we evaluate the knockdown of RANKL by in-vivo-delivered siRNA in a rat model of osteoporosis. Virus-like-particles (VLPs) derived from polyoma JC virus were used for delivering RANKL siRNA in ovariectomized (OVX) rats. 48 rats were ovariectomized and treated with either 17ß-estradiol (E2), VLPs containing RANKL siRNA (siRANKL), or VLPs containing non-cognate siRNA (siCtrl). All OVX groups were subdivided into the prophylaxis group (PG) and the therapy group (TG). The PG received treatment directly after being OVX for 10 weeks. The TG received treatment 5 weeks after being OVX for 5 weeks. Rats were sacrificed 10 weeks after being OVX. Bone and blood samples were analyzed. E2 and siRANKL showed a significant knockdown of RANKL mRNA. A protein knockdown was observed with E2 and siRANKL in the TG but not in the PG. No distinct improvements in biomechanical and morphological properties of the bones were observed after siRANKL treatment. In the PG, E2 protected the bone structure. We demonstrated successful mRNA and protein knockdown by VLP-mediated RNAi in vivo. Knockdown of membranous RANKL did not result in significant improvements of bone properties in this model of early-stage postmenopausal osteoporosis.

The Impact of the CD9 Tetraspanin on Lentivirus Infectivity and Exosome Secretion.

Efficient transduction tools are a hallmark for both research and therapy development. Here, we introduce new insights into the generation of lentiviral vectors with improved performance by utilizing producer cells with increased production rates of extracellular vesicles through CD9 overexpression. Most human cells secrete small vesicles from their surface (microvesicles) or intraluminal endosome-derived membranes (exosomes). In particular, enhanced levels of the tetraspanin CD9 result in significantly increased numbers of extracellular vesicles with exosome-like features that were secreted from four different human cell lines. Intriguingly, exosomes and their biogenesis route display similarities to lentivirus and we examined the impact of CD9 expression on release and infectivity of recombinant lentiviral vectors. Although the titers of released viral particles were not increased upon production in high CD9 cells, we observed improved performance in terms of both speed and efficiency of lentiviral gene delivery into numerous human cell lines, including HEK293, HeLa, SH-SY5Y, as well as B and T lymphocytes. Here, we demonstrate that enhanced CD9 enables lentiviral transduction in the absence of any pseudotyping viral glycoprotein or fusogenic molecule. Our findings indicate an important role of CD9 for lentiviral vector and exosome biogenesis and point out a remarkable function of this tetraspanin in membrane fusion, viral infectivity, and exosome-mediated horizontal information transfer.

Dissecting miRNA gene repression on single cell level with an advanced fluorescent reporter system.

Despite major advances on miRNA profiling and target predictions, functional readouts for endogenous miRNAs are limited and frequently lead to contradicting conclusions. Numerous approaches including functional high-throughput and miRISC complex evaluations suggest that the functional miRNAome differs from the predictions based on quantitative sRNA profiling. To resolve the apparent contradiction of expression versus function, we generated and applied a fluorescence reporter gene assay enabling single cell analysis. This approach integrates and adapts a mathematical model for miRNA-driven gene repression. This model predicts three distinct miRNA-groups with unique repression activities (low, mid and high) governed not just by expression levels but also by miRNA/target-binding capability. Here, we demonstrate the feasibility of the system by applying controlled concentrations of synthetic siRNAs and in parallel, altering target-binding capability on corresponding reporter-constructs. Furthermore, we compared miRNA-profiles with the modeled predictions of 29 individual candidates. We demonstrate that expression levels only partially reflect the miRNA function, fitting to the model-projected groups of different activities. Furthermore, we demonstrate that subcellular localization of miRNAs impacts functionality. Our results imply that miRNA profiling alone cannot define their repression activity. The gene regulatory function is a dynamic and complex process beyond a minimalistic conception of "highly expressed equals high repression".

In Vivo siRNA Delivery Using JC Virus-like Particles Decreases the Expression of RANKL in Rats.

Bone remodeling requires a precise balance between formation and resorption. This complex process involves numerous factors that orchestrate a multitude of biochemical events. Among these factors are hormones, growth factors, vitamins, cytokines, and, most notably, osteoprotegerin (OPG) and the receptor activator for nuclear factor-kappaB ligand (RANKL). Inflammatory cytokines play a major role in shifting the RANKL/OPG balance toward excessive RANKL, resulting in osteoclastogenesis, which in turn initiates bone resorption, which is frequently associated with osteoporosis. Rebalancing RANKL/OPG levels may be achieved through either upregulation of OPG or through transient silencing of RANKL by means of RNA interference. Here, we describe the utilization of a viral capsid-based delivery system for in vivo and in vitro RNAi using synthetic small interfering RNA (siRNA) molecules in rat osteoblasts. Polyoma JC virus-derived virus-like particles are capable of delivering siRNAs to target RANKL in osteoblast cells both in vitro and in a rat in vivo system. Expression levels were monitored using quantitative real-time polymerase reaction and enzyme-linked immunosorbent assay after single and repeated injections over a 14-day period. Our data indicate that this is an efficient and safe route for in vivo delivery of gene modulatory tools to study important molecular factors in a rat osteoporosis model.

Mitogenomic phylogeny of the common long-tailed macaque (Macaca fascicularis fascicularis).

BACKGROUND: Long-tailed macaques (Macaca fascicularis) are an important model species in biomedical research and reliable knowledge about their evolutionary history is essential for biomedical inferences. Ten subspecies have been recognized, of which most are restricted to small islands of Southeast Asia. In contrast, the common long-tailed macaque (M. f. fascicularis) is distributed over large parts of the Southeast Asian mainland and the Sundaland region. To shed more light on the phylogeny of M. f. fascicularis, we sequenced complete mitochondrial (mtDNA) genomes of 40 individuals from all over the taxon's range, either by classical PCR-amplification and Sanger sequencing or by DNA-capture and high-throughput sequencing. RESULTS: Both laboratory approaches yielded complete mtDNA genomes from M. f. fascicularis with high accuracy and/or coverage. According to our phylogenetic reconstructions, M. f. fascicularis initially diverged into two clades 1.70 million years ago (Ma), with one including haplotypes from mainland Southeast Asia, the Malay Peninsula and North Sumatra (Clade A) and the other, haplotypes from the islands of Bangka, Java, Borneo, Timor, and the Philippines (Clade B). The three geographical populations of Clade A appear as paraphyletic groups, while local populations of Clade B form monophyletic clades with the exception of a Philippine individual which is nested within the Borneo clade. Further, in Clade B the branching pattern among main clades/lineages remains largely unresolved, most likely due to their relatively rapid diversification 0.93-0.84 Ma. CONCLUSIONS: Both laboratory methods have proven to be powerful to generate complete mtDNA genome data with similarly high accuracy, with the DNA-capture and high-throughput sequencing approach as the most promising and only practical option to obtain such data from highly degraded DNA, in time and with relatively low costs. The application of complete mtDNA genomes yields new insights into the evolutionary history of M. f. fascicularis by providing a more robust phylogeny and more reliable divergence age estimations than earlier studies.