Phytic acid promotes high glucose-mediated bone marrow mesenchymal stem cells osteogenesis via modulating circEIF4B that sponges miR-186-5p and complexes with IGF2BP3.

Diabetes-related hyperglycemia inhibits bone marrow mesenchymal stem cell (BMSC) function, thereby disrupting osteoblast capacity and bone regeneration. Dietary supplementation with phytic acid (PA), a natural inositol phosphate, has shown promise in preventing osteoporosis and diabetes-related complications. Emerging evidence has suggested that circular (circ)RNAs implicate in the regulation of bone diseases, but their specific regulatory roles in BMSC osteogenesis in hyperglycemic environments remain elucidated. In this study, in virto experiments demonstrated that PA treatment effectively improved the osteogenic capability of high glucose-mediated BMSCs. Differentially expressed circRNAs in PA-induced BMSCs were identified using circRNA microarray analysis. Here, our findings highlight an upregulation of circEIF4B expression in BMSCs stimulated with PA under a high-glucose microenvironment. Further investigations demonstrated that circEIF4B overexpression promoted high glucose-mediated BMSC osteogenesis. In contrast, circEIF4B knockdown exerted the opposite effect. Mechanistically, circEIF4B sequestered microRNA miR-186-5p and triggered osteogenesis enhancement in BMSCs by targeting FOXO1 directly. Furthermore, circEIF4B inhibited the ubiquitin-mediated degradation of IGF2BP3, thereby stabilizing ITGA5 mRNA and promoting BMSC osteogenic differentiation. In vivo experiments, circEIF4B inhibition attenuated the effectiveness of PA treatment in diabetic rats with cranial defects. Collectively, our study identifies PA as a novel positive regulator of BMSC osteogenic differentiation through the circEIF4B/miR-186-5p/FOXO1 and circEIF4B/IGF2BP3/ITGA5 axes, which offers a new strategy for treating high glucose-mediatedBMSCosteogenic dysfunction and delayed bone regeneration in diabetes.

Icariin promotes bone marrow mesenchymal stem cells osteogenic differentiation via the mTOR/autophagy pathway to improve ketogenic diet-associated osteoporosis.

BACKGROUND: Icariin, a traditional Chinese medicine, has demonstrated anti-osteoporotic properties in ovariectomized mice. However, its effectiveness in preventing bone loss induced by ketogenic diet (KD), which mimics osteoporosis in human, remains unexplored. This study aims to investigate icariin's impact on KD-induced bone loss in mice. METHODS: Thirty mice were divided into: sham, KD, and KD + icariin groups. Post a 12-week intervention, evaluation including bone microstructures, serum concentrations of tartrate-resistant acid phosphatase (TRAP) and bone-specific alkaline phosphatase (ALP), and femoral tissue expression levels of osteocalcin (OCN) and TRAP. The expression levels of mammalian target of rapamycin (mTOR), ALP, peroxisome proliferator-activated receptor gamma (PPAR-γ), phosphorylated mTOR (p-mTOR), and the autophagy adaptor protein (p62) were also analyzed. Alizarin granule deposition and cellular ALP levels were measured following the induction of bone marrow mesenchymal stem cells (BMSCs) into osteogenesis. RESULTS: The study found that KD significantly impaired BMSCs' osteogenic differentiation, leading to bone loss. Icariin notably increased bone mass, stimulated osteogenesis, and reduced cancellous bone loss. In the KD + icariin group, measures such as bone tissue density (TMD), bone volume fraction (BV/TV), trabecular number (Tb.N), and trabecular thickness (Tb.Th) were significantly higher than in the KD group. Additionally, bone trabecular separation (Tb.Sp) was markedly lower in the KD + icariin group. Moreover, icariin increased OCN and ALP levels while suppressing PPAR-γ, TRAP, p62, and p-mTOR. In cellular studies, icariin encouraged osteogenic development in BMSCs under KD conditions. CONCLUSIONS: Icariin effectively counteracts bone thinning and improves bone microstructure. Its mechanism likely involves stimulating BMSCs osteogenic differentiation and inhibiting bone resorption, potentially through mTOR downregulation. These findings suggest icariin's potential as an alternative treatment for KD-induced bone loss.

Isoliquiritin treatment of osteoporosis by promoting osteogenic differentiation and autophagy of bone marrow mesenchymal stem cells.

Osteoporosis is a chronic progressive bone disease characterized by the decreased osteogenic ability of osteoblasts coupled with increased osteoclast activity. Natural products showing promising therapeutic potential for postmenopausal osteoporosis remain underexplored. In this study, we aimed to analyze the therapeutic effects of isoliquiritin (ISL) on osteoporosis in mice and its possible mechanism of action. An ovariectomy-induced osteoporosis mouse model and bone marrow mesenchymal stem cells (BMSCs) were used to analyze the effects of ISL on bone regeneration in vivo and in vitro, respectively. Mitogen-activated protein kinase (MAPK) and autophagy inhibitors were used, to investigate whether the MAPK signaling pathway and autophagy affect the osteogenic differentiation of BMSCs. ISL significantly improved bone formation and reduced bone resorption in mouse femurs without inducing any detectable toxicity in critical organs such as the liver, kidney, brain, heart, and spleen. In vitro experiments showed that ISL enhanced the proliferation and osteogenic differentiation of BMSCs and that its osteogenic effect was attenuated by p38/extracellular regulated protein kinase (ERK) and autophagy inhibitors. Further studies showed that the inhibition of phosphorylated p38/ERK blocked ISL autophagy in BMSCs. ISL promoted the osteogenic differentiation of BMSCs through the p38/ERK-autophagy pathway and was therapeutically effective in treating osteoporosis in ovariectomized mice without any observed toxicity to vital organs. These results strongly suggest the promising potential of ISL as a safe and efficacious candidate drug for the treatment of osteoporosis.

Aberrant bone marrow-derived microglia in the hypothalamus may dysregulate appetite in diabetes.

Bone marrow derived cells (BMDCs) migrate into the hypothalamus, where those cells give rise to microglia to regulate food intake. Given the fact that diabetes functionally impairs BMDCs, we hypothesized that diabetic microglia would fail to exhibit physiological function, accounting for hyperphagia in diabetes. To examine the role of BMDCs, total bone marrow cells from GFP transgenic mice were transplanted into wild type mice in which diabetes was induced by streptozotocin. We first confirmed that bone marrow transplantation could be utilized to examine BMDCs in the brain parenchyma as GFP positive cells could engraft the brain parenchyma and give rise to microglia even when the BBB was intact in the recipient mice. While diabetic mice manifested hyperphagia, BMDCs were in smaller number in the hypothalamus with less response to fasting in the brain parenchyma compared to nondiabetic mice. This finding was also confirmed by examining nondiabetic chimera mice in which BMDCs were diabetic. Those mice also exhibited less response of BMDCs in response to fasting. In conclusion, diabetic BMDCs had less response of microglia to fasting, perhaps accounting for diabetic hyperphagia.

Novel Peptides from Sea Cucumber Intestinal Enzyme Hydrolysates Promote Osteogenic Differentiation of Bone Mesenchymal Stem Cells via Phosphorylation of PPARγ at Serine 112.

SCOPE: Osteo-adipogenic differentiation imbalance of bone mesenchymal stem cells (BMSCs) has been linked to a variety of pathophysiological processes such as obesity and osteoporosis. Recent studies report that the phosphorylation of peroxisome proliferator-activated receptor gamma (PPARγ) Ser112 affects the fate decision of BMSCs. Novel peptides from the sea cucumber intestinal peptide (SCIP) have been proved to promote the growth of longitudinal bone. However, it is unclear the effect of SCIP on BMSCs differentiation fate. METHODS AND RESULTS: BMSCs in vitro and glucocorticoid induced mice are employed to investigate the effects of SCIP on osteo-adipogenic differentiation of BMSCs. In vitro results show that SCIP supplement significantly promotes the proliferation and osteogenic differentiation of BMSCs, upregulates the expression of osteogenic marker. In vivo results show that SCIP supplement ameliorates the osteo-adipogenic differentiation imbalance in glucocorticoid-treated mice, decreases bone marrow fat, and elevates bone mineral density. Mechanistically, SCIP supplement promotes and maintains the phosphorylation of PPARγ Ser112 through AMPK/ERK and TAZ signals, thereby inducing the osteogenic differentiation of BMSCs. CONCLUSION: Supplement with SCIP promotes BMSCs to differentiate into osteoblasts. These results suggest that SCIP has potential as a functional food to improve obesity and osteoporosis.

Alpinetin alleviates osteoporosis by promoting osteogenic differentiation in BMSCs by triggering autophagy via PKA/mTOR/ULK1 signaling.

Osteoporosis, a systemic bone disease that is characterized by a reduction in bone mass and destruction of bone microstructure, is becoming a serious problem worldwide. Bone marrow mesenchymal stem cells (BMSCs) can differentiate into bone-forming osteoblasts, and play an important role in maintaining homeostasis of bone metabolism, thus being a potential therapeutic target for osteoporosis. Although the phytochemical alpinetin (APT) has been reported to possess a variety of pharmacological activities, it is still unclear whether APT can influence the osteogenic differentiation of on BMSCs and if it can improve osteoporosis. In this study, we found that APT treatment was able to enhance osteogenic differentiation levels of human BMSCs in vitro and mouse ones in vivo as revealed by multiple osteogenic markers including increased alkaline phosphatase activity and osteocalcin expression. Mechanistically, the protein kinase A (PKA)/mTOR/ULK1 signaling was involved in the action of APT to enhance the osteogenic differentiation of BMSCs. In addition, oral administration of APT significantly mitigated the bone loss in a dexamethasone-induced mouse model of osteoporosis through strengthening PKA signaling and autophagy. Altogether, these data demonstrate that APT promotes osteogenic differentiation in BMSCs by augmenting the PKA/mTOR/ULK1 autophagy signaling, highlighting its potential therapeutic application for treating osteoporotic diseases.

SAG-dihydrochloride enhanced the expression of germ cell markers in the human bone marrow- mesenchymal stem cells (BM-MSCs) through the activation of GLI-independent hedgehog signaling pathway.

Different studies indicated that the enhancing the expression of germ cell markers improved the efficiency of stem cells in the generation of germ line cells. The aim of the present study was to investigate the effect of SAG-dihydrochloride on the expression of germ cell markers in the human bone marrow-mesenchymal stem cells (BM-MSCs). For this purpose, the human BM-MSCs were cultured in the medium containing different concentrations of SAG-dihydrochloride (10, 20 and 30 µM). After RNA extraction and cDNA synthesis, the expression level of PTCH1, GLI1, PLZF, DDX4 and STRA8 genes were determined by using SYBR Green Real time PCR. The analysis of the results obtained from PTCH1 and GLI1 expression indicated that SAG-dihydrochloride had the ability to enhance the expression of germ cell markers in a Gli-independent manner. Furthermore, the significant increased expression of STRA8 was observed in the BM-MSCs treated by 10 µM SAG-dihydrochloride for 4 and 6 days (p < 0.05). There was also the up-regulation of DDX4 in the BM-MSCs following treatment with 20 µM SAG-dihydrochloride for 4 and 6 days. The obtained results suggested that treatment with SAG-dihydrochloride increased the expression of germ cell markers in the human BM-MSCs through the activation of non-canonical sonic hedgehog signaling pathway.

Meteorin links the bone marrow hypoxic state to hematopoietic stem/progenitor cell mobilization.

Hematopoietic stem/progenitor cells (HSPCs) are supported and regulated by niche cells in the bone marrow with an important characterization of physiological hypoxia. However, how hypoxia regulates HSPCs is still unclear. Here, we find that meteorin (Metrn) from hypoxic macrophages restrains HSPC mobilization. Hypoxia-induced factor 1α and Yin Yang 1 induce the high expression of Metrn in macrophages, and macrophage-specific Metrn knockout increases HSPC mobilization through modulating HSPC proliferation and migration. Mechanistically, Metrn interacts with its receptor 5-hydroxytryptamine receptor 2b (Htr2b) to regulate the reactive oxygen species levels in HSPCs through targeting phospholipase C signaling. The reactive oxygen species levels are reduced in HSPCs of macrophage-specific Metrn knockout mice with activated phospholipase C signaling. Targeting the Metrn/Htr2b axis could therefore be a potential strategy to improve HSPC mobilization for stem cell-based therapy.

Specnuezhenide suppresses diabetes-induced bone loss by inhibiting RANKL-induced osteoclastogenesis.

Diabetes osteoporosis is a chronic complication of diabetes mellitus (DM) and is associated with osteoclast formation and enhanced bone resorption. Specnuezhenide (SPN) is an active compound with anti-inflammatory and immunomodulatory properties. However, the roles of SPN in diabetic osteoporosis remain unknown. In this study, primary bone marrow macrophages (BMMs) were pretreated with SPN and were stimulated with receptor activator of nuclear factor kappa B ligand (RANKL; 50 ng/mL) to induce osteoclastogenesis. The number of osteoclasts was detected by tartrate-resistant acid phosphatase (TRAP) staining. The protein levels of cellular oncogene fos/nuclear factor of activated T cells c1 (c-Fos/NFATc1), nuclear factor kappa-B (NF-κB), and mitogen-activated protein kinases (MAPKs) were evaluated by western blot analysis. NF-κB luciferase assays were used to examine the role of SPN in NF-κB activation. The DM model group received a high-glucose, high-fat diet and was then intraperitoneally injected with streptozotocin (STZ). Micro-CT scanning, serum biochemical analysis, histological analysis were used to assess bone loss. We found that SPN suppressed RANKL-induced osteoclast formation and that SPN inhibited the expression of osteoclast-related genes and c-Fos/ NFATc1. SPN inhibited RANKL-induced activation of NF-κB and MAPKs. In vivo experiments revealed that SPN suppressed diabetes-induced bone loss and the number of osteoclasts. Furthermore, SPN decreased the levels of bone turnover markers and increased the levels of runt-related transcription factor 2 (RUNX2), osteoprotegerin (OPG), calcium (Ca) and phosphorus (P). SPN also regulated diabetes-related markers. This study suggests that SPN suppresses diabetes-induced bone loss by inhibiting RANKL-induced osteoclastogenesis, and provides an experimental basis for the treatment of diabetic osteoporosis.

Metabolic Switch Under Glucose Deprivation Leading to Discovery of NR2F1 as a Stimulus of Osteoblast Differentiation.

Poor survival of grafted cells is the major impediment of successful cell-based therapies for bone regeneration. Implanted cells undergo rapid death in an ischemic environment largely because of hypoxia and metabolic stress from glucose deficiency. Understanding the intracellular metabolic processes and finding genes that can improve cell survival in these inhospitable conditions are necessary to enhance the success of cell therapies. Thus, the purpose of this study was to investigate changes of metabolic profile in glucose-deprived human bone marrow stromal/stem cells (hBMSCs) through metabolomics analysis and discover genes that could promote cell survival and osteogenic differentiation in a glucose-deprived microenvironment. Metabolomics analysis was performed to determine metabolic changes in a glucose stress metabolic model. In the absence of glucose, expression levels of all metabolites involved in glycolysis were significantly decreased than those in a glucose-supplemented state. In glucose-deprived osteogenic differentiation, reliance on tricarboxylic acid cycle (TCA)-predicted oxidative phosphorylation instead of glycolysis as the main mechanism for energy production in osteogenic induction. By comparing differentially expressed genes between glucose-deprived and glucose-supplemented hBMSCs, NR2F1 (Nuclear Receptor Subfamily 2 Group F Member 1) gene was discovered to be associated with enhanced survival and osteogenic differentiation in cells under metabolic stress. Small, interfering RNA (siRNA) for NR2F1 reduced cell viability and osteogenic differentiation of hBMSCs under glucose-supplemented conditions whereas NR2F1 overexpression enhanced osteogenic differentiation and cell survival of hBMSCs in glucose-deprived osteogenic conditions via the protein kinase B (AKT)/extracellular signal-regulated kinase (ERK) pathway. NR2F1-transfected hBMSCs significantly enhanced new bone formation in a critical size long-bone defect of rats compared with control vector-transfected hBMSCs. In conclusion, the results of this study provide an understanding of the metabolic profile of implanted cells in an ischemic microenvironment and demonstrate that NR2F1 treatment may overcome this deprivation by enhancing AKT and ERK regulation. These findings can be utilized in regenerative medicine for bone regeneration. © 2022 American Society for Bone and Mineral Research (ASBMR).

Zuogui Wan slowed senescence of bone marrow mesenchymal stem cells by suppressing Wnt/ß-catenin signaling.

ETHNOPHARMACOLOGICAL RELEVANCE: In traditional Chinese medicine (TCM), Zuogui Wan (ZGW) is a classical prescription for senile disorders and delay aging. Modern studies show that ZGW promotes central nerve cell regeneration, prevents and cures osteoporosis, enhances the body's antioxidant capacity, regulates the body's immune function, and promotes mesenchymal stem cells (MSCs) proliferation. AIM OF THE STUDY: It has been shown that MSCs aging is closely associated with organism's aging and age-related disorders. The study aimed to define the effects of ZGW on the aging bone marrow mesenchymal stem cells (BMSCs) and to identify the mechanisms of ZGW delaying BMSCs senescence. MATERIALS AND METHODS: Network pharmacology analysis combined with GEO data mining, molecular docking and experimental validation were used to evaluate the mechanisms by which ZGW delays MSCs senescence (MSCS). LC-MS was used for quality control analysis of ZGW. RESULTS: PPI network analysis revealed that EGF, TNF, JUN, MMPs, IL-6, MAPK8, and MYC are components of the core PPI network. GO and KEGG analyses revealed that oxidative stress, regulation of response to DNA damage stimuli, and Wnt signaling were significantly enriched. GEO database validation also indicated that Wnt signaling closely correlated with MSCs aging. Molecular docking analysis of the top-13 active components in the "ZGW-Targets-MSCS" network indicated that most components have strong affinity for key proteins in Wnt signaling, suggesting that modulation of Wnt signaling is an important mechanism of ZGW activity against MSCS. Further experimental validation found that ZGW indeed regulates Wnt signaling and suppresses the expression of age-related factors to enhance cell proliferation, ameliorate DNA damage, and reduce senescence-related secretory phenotype (SASP) secretion, thereby maintaining multidirectional differentiation of rat BMSCs. Similar results were obtained using the Wnt inhibitor, XAV-939. CONCLUSIONS: Together, our data show that ZGW slows BMSCs aging by suppressing Wnt signaling.

Juglans regia L. extract promotes osteogenesis of human bone marrow mesenchymal stem cells through BMP2/Smad/Runx2 and Wnt/ß-catenin pathways.

BACKGROUND: The present study investigates the effects of Juglans regia L. (walnut, JRL) leaves extract on osteogenesis of human bone marrow mesenchymal stem cells (hBMSCs). METHODS: hBMSCs were incubated with different concentrations of JRL extract (10, 20, 40, or 80 µM). Cell proliferation was evaluated by Cell Counting Kit-8 assay (CCK-8) assay. ALP activity and Alizarin Red staining were used to assess the osteogenesis of BMSCs. Western blot was performed to measure the levels of proteins. RESULTS: Our results showed all concentrations of JRL extract had no significant effect on cell proliferation. JRL extract concentration-dependently promoted osteoblastic differentiation and cell autophagy of hBMSCs, characterized by the increased expression of pro-osteogenic markers alkaline phosphatase (ALP), osteocalcin (BGLAP), osterin, and osteoprotegerin (OPG) and autophagy marker proteins (LC3II, Beclin-1, and p62). Furthermore, JRL extract stimulated the activation BMP2/Smad/Runx2 and Wnt/ß-catenin signaling pathways in hBMSCs, which play key roles in osteogenesis differentiation. Meanwhile, BMP inhibitor (Noggin) and Wnt antagonist Dickkopf-1 (DKK1) both reversed the increases of BGLAP, osterin, and OPG expression induced by JRL extract. CONCLUSIONS: Our findings indicate that JRL extract regulated osteogenic differentiation and cell autophagy of hBMSCs through the BMP2/Smad/Runx2 and Wnt/ß-catenin pathways.

Hydroxyapatite-hybridized double-network hydrogel surface enhances differentiation of bone marrow-derived mesenchymal stem cells to osteogenic cells.

Recently, we have developed a hydroxyapatite (HAp)-hybridized double-network (DN) hydrogel (HAp/DN gel), which can robustly bond to the bone tissue in the living body. The purpose of this study is to clarify whether the HAp/DN gel surface can differentiate the bone marrow-derived mesenchymal stem cells (MSCs) to osteogenic cells. We used the MSCs which were harvested from the rabbit bone marrow and cultured on the polystyrene (PS) dish using the autogenous serum-supplemented medium. First, we confirmed the properties of MSCs by evaluating colony forming unit capacity, expression of MSC markers using flow cytometry, and multidifferential capacity. Secondly, polymerase chain reaction analysis demonstrated that the HAp/DN gel surface significantly enhanced mRNA expression of the eight osteogenic markers (TGF-ß1, BMP-2, Runx2, Col-1, ALP, OPN, BSP, and OCN) in the cultured MSCs at 7 days than the PS surfaces (p < 0.0001), while the DN gel and HAp surfaces provided no or only a slight effect on the expression of these markers except for Runx2. Additionally, the alkaline phosphatase activity was significantly higher in the cells cultured on the HAp/DN gel surface than in the other three material surfaces (p < 0.0001). Thirdly, when the HAp/DN gel plug was implanted into the rabbit bone marrow, MSC marker-positive cells were recruited in the tissue generated around the plug at 3 days, and Runx2 and OCN were highly expressed in these cells. In conclusion, this study demonstrated that the HAp/DN gel surface can differentiate the MSCs into osteogenic cells.

Calycosin-7-O-ß-Glucoside Isolated from Astragalus membranaceus Promotes Osteogenesis and Mineralization in Human Mesenchymal Stem Cells.

Stem cells have received attention in various diseases, such as inflammatory, cancer, and bone diseases. Mesenchymal stem cells (MSCs) are multipotent stem cells that are critical for forming and repairing bone tissues. Herein, we isolated calycosin-7-O-ß-glucoside (Caly) from the roots of Astragalus membranaceus, which is one of the most famous medicinal herbs, and investigated the osteogenic activities of Caly in MSCs. Caly did not affect cytotoxicity against MSCs, whereas Caly enhanced cell migration during the osteogenesis of MSCs. Caly increased the expression and enzymatic activities of ALP and the formation of mineralized nodules during the osteogenesis of MSCs. The osteogenesis and bone-forming activities of Caly are mediated by bone morphogenetic protein 2 (BMP2), phospho-Smad1/5/8, Wnt3a, phospho-GSK3ß, and phospho-AKT, inducing the expression of runt-related transcription factor 2 (RUNX2). In addition, Caly-mediated osteogenesis and RUNX2 expression were attenuated by noggin and wortmannin. Moreover, the effects were validated in pre-osteoblasts committed to the osteoblast lineages from MSCs. Overall, our results provide novel evidence that Caly stimulates osteoblast lineage commitment of MSCs by triggering RUNX2 expression, suggesting Caly as a potential anabolic drug to prevent bone diseases.

PAC1 Receptor Mediates Electroacupuncture-Induced Neuro and Immune Protection During Cisplatin Chemotherapy.

Platinum-based chemotherapy is an effective treatment used in multiple tumor treatments, but produces severe side effects including neurotoxicity, anemia, and immunosuppression, which limits its anti-tumor efficacy and increases the risk of infections. Electroacupuncture (EA) is often used to ameliorate these side effects, but its mechanism is unknown. Here, we report that EA on ST36 and SP6 prevents cisplatin-induced neurotoxicity and immunosuppression. EA induces neuroprotection, prevents pain-related neurotoxicity, preserves bone marrow (BM) hematopoiesis, and peripheral levels of leukocytes. EA activates sympathetic BM terminals to release pituitary adenylate cyclase activating polypeptide (PACAP). PACAP-receptor PAC1-antagonists abrogate the effects of EA, whereas PAC1-agonists mimic EA, prevent neurotoxicity, immunosuppression, and preserve BM hematopoiesis during cisplatin chemotherapy. Our results indicate that PAC1-agonists may provide therapeutic advantages during chemotherapy to treat patients with advanced neurotoxicity or neuropathies limiting EA efficacy.

Neuroblastoma Formation Requires Unconventional CD4 T Cells and Arginase-1-Dependent Myeloid Cells.

Immune cells regulate tumor growth by mirroring their function as tissue repair organizers in normal tissues. To understand the different facets of immune-tumor collaboration through genetics, spatial transcriptomics, and immunologic manipulation with noninvasive, longitudinal imaging, we generated a penetrant double oncogene-driven autochthonous model of neuroblastoma. Spatial transcriptomic analysis showed that CD4+ and myeloid populations colocalized within the tumor parenchyma, while CD8+ T cells and B cells were peripherally dispersed. Depletion of CD4+ T cells or CCR2+ macrophages, but not B cells, CD8+ T cells, or natural killer (NK) cells, prevented tumor formation. Tumor CD4+ T cells displayed unconventional phenotypes and were clonotypically diverse and antigen independent. Within the myeloid fraction, tumor growth required myeloid cells expressing arginase-1. Overall, these results demonstrate how arginine-metabolizing myeloid cells conspire with pathogenic CD4+ T cells to create permissive conditions for tumor formation, suggesting that these protumorigenic pathways could be disabled by targeting myeloid arginine metabolism. SIGNIFICANCE: A new model of human neuroblastoma provides ways to track tumor formation and expansion in living animals, allowing identification of CD4+ T-cell and macrophage functions required for oncogenesis.

Nicotinamide riboside attenuates age-associated metabolic and functional changes in hematopoietic stem cells.

With age, hematopoietic stem cells (HSC) undergo changes in function, including reduced regenerative potential and loss of quiescence, which is accompanied by a significant expansion of the stem cell pool that can lead to haematological disorders. Elevated metabolic activity has been implicated in driving the HSC ageing phenotype. Here we show that nicotinamide riboside (NR), a form of vitamin B3, restores youthful metabolic capacity by modifying mitochondrial function in multiple ways including reduced expression of nuclear encoded metabolic pathway genes, damping of mitochondrial stress and a decrease in mitochondrial mass and network-size. Metabolic restoration is dependent on continuous NR supplementation and accompanied by a shift of the aged transcriptome towards the young HSC state, more youthful bone marrow cellular composition and an improved regenerative capacity in a transplant setting. Consequently, NR administration could support healthy ageing by re-establishing a more youthful hematopoietic system.

Bone marrow-derived AXL tyrosine kinase promotes mitogenic crosstalk and cardiac allograft vasculopathy.

Cardiac Allograft Vasculopathy (CAV) is a leading contributor to late transplant rejection. Although implicated, the mechanisms by which bone marrow-derived cells promote CAV remain unclear. Emerging evidence implicates the cell surface receptor tyrosine kinase AXL to be elevated in rejecting human allografts. AXL protein is found on multiple cell types, including bone marrow-derived myeloid cells. The causal role of AXL from this compartment and during transplant is largely unknown. This is important because AXL is a key regulator of myeloid inflammation. Utilizing experimental chimeras deficient in the bone marrow-derived Axl gene, we report that Axl antagonizes cardiac allograft survival and promotes CAV. Flow cytometric and histologic analyses of Axl-deficient transplant recipients revealed reductions in both allograft immune cell accumulation and vascular intimal thickness. Co-culture experiments designed to identify cell-intrinsic functions of Axl uncovered complementary cell-proliferative pathways by which Axl promotes CAV-associated inflammation. Specifically, Axl-deficient myeloid cells were less efficient at increasing the replication of both antigen-specific T cells and vascular smooth muscle cells (VSMCs), the latter a key hallmark of CAV. For the latter, we discovered that Axl-was required to amass the VSMC mitogen Platelet-Derived Growth Factor. Taken together, our studies reveal a new role for myeloid Axl in the progression of CAV and mitogenic crosstalk. Inhibition of AXL-protein, in combination with current standards of care, is a candidate strategy to prolong cardiac allograft survival.

Human osteoclastogenesis in Epstein-Barr virus-induced erosive arthritis in humanized NOD/Shi-scid/IL-2Rγnull mice.

Many human viruses, including Epstein-Barr virus (EBV), do not infect mice, which is challenging for biomedical research. We have previously reported that EBV infection induces erosive arthritis, which histologically resembles rheumatoid arthritis, in humanized NOD/Shi-scid/IL-2Rγnull (hu-NOG) mice; however, the underlying mechanisms are not known. Osteoclast-like multinucleated cells were observed during bone erosion in this mouse model, and therefore, we aimed to determine whether the human or mouse immune system activated bone erosion and analyzed the characteristics and origin of the multinucleated cells in hu-NOG mice. Sections of the mice knee joint tissues were immunostained with anti-human antibodies against certain osteoclast markers, including cathepsin K and matrix metalloproteinase-9 (MMP-9). Multinucleated cells observed during bone erosion stained positively for human cathepsin K and MMP-9. These results indicate that human osteoclasts primarily induce erosive arthritis during EBV infections. Human osteoclast development from hematopoietic stem cells transplanted in hu-NOG mice remains unclear. To confirm their differentiation potential into human osteoclasts, we cultured bone marrow cells of EBV-infected hu-NOG mice and analyzed their characteristics. Multinucleated cells cultured from the bone marrow cells stained positive for human cathepsin K and human MMP-9, indicating that bone marrow cells of hu-NOG mice could differentiate from human osteoclast progenitor cells into human osteoclasts. These results indicate that the human immune response to EBV infection may induce human osteoclast activation and cause erosive arthritis in this mouse model. Moreover, this study is the first, to our knowledge, to demonstrate human osteoclastogenesis in humanized mice. We consider that this model is useful for studying associations of EBV infections with rheumatoid arthritis and human bone metabolism.

Mitigation of X-ray induced DNA damages and expression of DNA-repair genes by antioxidative Potentilla fulgens root extract and its ethyl-acetate fraction in mammalian cells.

Potentilla fulgens is a medicinal plant in North-East India whose root is reported to have anti-diabetic, anticarcinogenic and antioxidant properties. The potential of hydro-alcoholic extract of P. fulgens root (PRE) for providing protection to mammalian cells exposed to ionising radiation was investigated in this study. The methanolic extract of PRE shows an enhanced radical scavenging ability in a concentration dependent manner. PRE-pre-treatment to stimulated human blood lymphocytes (HBLs) reduced the frequency of deletion and exchange aberrations induced by X-irradiation. Similar protection of chromosome aberrations was also observed in mouse bone marrow cells (BMCs) where mice were given PRE extract (1 mg extract/day/mice) ad libitum in the drinking water for 45 days before whole-body X-irradiation. Of the various extracts prepared by partitioning of the methanol extract, the ethyl-acetate (EA) fraction was found to possess better antioxidant, radical scavenging and DNA-damage reduction activities. PRE-pre-treatment also reduced the radiation-induced cell-cycle delay effectively in HBL. In HEK-293 cells, PRE reduced radiation-induced G2-block in cell kinetics. Interestingly, PRE-treatment alone increased the concentration of endogenous glutathione (GSH) in mouse BMC and in stimulated HBL along with the elevated expression of γ-glutamyl-cysteine synthetase heavy/catalytic subunit, a key determinant of GSH synthesis. Studies on expression of two DNA-repair genes revealed that there was a marked increase in the expression of GADD45 and H2AX genes after X-irradiation in stimulated HBL, and such expression was reduced significantly if PRE-treatment was given prior to radiation. The present findings show the ability of PRE to reduce radiation-induced DNA damages probably by free radical scavenging whereas modulation of expression of DNA-repair genes' and endogenous GSH-increment emerge as effective strategies. The present study is the first report on the selected medicinal plant species that suggests it to be a potential natural radioprotector when used as root extract or its EA fraction for mitigating radiation toxicity.