RESUMEN
Adipocyte differentiation of bone marrow mesenchymal stem/stromal cells (BMSCs) instead of osteoblast formation contributes to age- and menopause-related marrow adiposity and osteoporosis. Vascular calcification often occurs with osteoporosis, a contradictory association called "calcification paradox". Here we show that extracellular vesicles derived from aged bone matrix (AB-EVs) during bone resorption favor BMSC adipogenesis rather than osteogenesis and augment calcification of vascular smooth muscle cells. Intravenous or intramedullary injection of AB-EVs promotes bone-fat imbalance and exacerbates Vitamin D3 (VD3)-induced vascular calcification in young or old mice. Alendronate (ALE), a bone resorption inhibitor, down-regulates AB-EVs release and attenuates aging- and ovariectomy-induced bone-fat imbalance. In the VD3-treated aged mice, ALE suppresses the ovariectomy-induced aggravation of vascular calcification. MiR-483-5p and miR-2861 are enriched in AB-EVs and essential for the AB-EVs-induced bone-fat imbalance and exacerbation of vascular calcification. Our study uncovers the role of AB-EVs as a messenger for calcification paradox by transferring miR-483-5p and miR-2861.
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Vesículas Extracelulares , Células Madre Mesenquimatosas , MicroARNs , Animales , Matriz Ósea , Diferenciación Celular , Femenino , Ratones , MicroARNs/genética , OsteogénesisRESUMEN
[This corrects the article DOI: 10.7150/thno.47408.].
RESUMEN
A differentiation switch of bone marrow mesenchymal stem/stromal cells (BMSCs) from osteoblasts to adipocytes contributes to age- and menopause-associated bone loss and marrow adiposity. Here it is found that osteocytes, the most abundant bone cells, promote adipogenesis and inhibit osteogenesis of BMSCs by secreting neuropeptide Y (NPY), whose expression increases with aging and osteoporosis. Deletion of NPY in osteocytes generates a high bone mass phenotype, and attenuates aging- and ovariectomy (OVX)-induced bone-fat imbalance in mice. Osteocyte NPY production is under the control of autonomic nervous system (ANS) and osteocyte NPY deletion blocks the ANS-induced regulation of BMSC fate and bone-fat balance. γ-Oryzanol, a clinically used ANS regulator, significantly increases bone formation and reverses aging- and OVX-induced osteocyte NPY overproduction and marrow adiposity in control mice, but not in mice lacking osteocyte NPY. The study suggests a new mode of neuronal control of bone metabolism through the ANS-induced regulation of osteocyte NPY.
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Adipocitos/metabolismo , Huesos/metabolismo , Neuropéptido Y/metabolismo , Osteoblastos/metabolismo , Osteoporosis/metabolismo , Adipogénesis/fisiología , Animales , Huesos/fisiopatología , Modelos Animales de Enfermedad , Femenino , Masculino , Ratones , Ratones Endogámicos C57BL , Osteocitos/metabolismo , Osteogénesis/fisiología , Osteoporosis/fisiopatologíaRESUMEN
Serious infection caused by multi-drug-resistant bacteria is a major threat to human health. Bacteria can invade the host tissue and produce various toxins to damage or kill host cells, which may induce life-threatening sepsis. Here, we aimed to explore whether fructose-coated Ångstrom-scale silver particles (F-AgÅPs), which were prepared by our self-developed evaporation-condensation system and optimized coating approach, could kill bacteria and sequester bacterial toxins to attenuate fatal bacterial infections. Methods: A series of in vitro assays were conducted to test the anti-bacterial efficacy of F-AgÅPs, and to investigate whether F-AgÅPs could protect against multi-drug resistant Staphylococcus aureus (S. aureus)- and Escherichia coli (E. coli)-induced cell death, and suppress their toxins (S. aureus hemolysin and E. coli lipopolysaccharide)-induced cell injury or inflammation. The mouse models of cecal ligation and puncture (CLP)- or E. coli bloodstream infection-induced lethal sepsis were established to assess whether the intravenous administration of F-AgÅPs could decrease bacterial burden, inhibit inflammation, and improve the survival rates of mice. The levels of silver in urine and feces of mice were examined to evaluate the excretion of F-AgÅPs. Results: F-AgÅPs efficiently killed various bacteria that can cause lethal infections and also competed with host cells to bind with S. aureus α-hemolysin, thus blocking its cytotoxic activity. F-AgÅPs inhibited E. coli lipopolysaccharide-induced endothelial injury and macrophage inflammation, but not by directly binding to lipopolysaccharide. F-AgÅPs potently reduced bacterial burden, reversed dysregulated inflammation, and enhanced survival in mice with CLP- or E. coli bloodstream infection-induced sepsis, either alone or combined with antibiotic therapy. After three times injections within 48 h, 79.18% of F-AgÅPs were excreted via feces at the end of the 14-day observation period. Conclusion: This study suggests the prospect of F-AgÅPs as a promising intravenous agent for treating severe bacterial infections.
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Toxinas Bacterianas/antagonistas & inhibidores , Sepsis/tratamiento farmacológico , Plata/farmacología , Animales , Antibacterianos/farmacología , Bacterias/efectos de los fármacos , Modelos Animales de Enfermedad , Escherichia coli/efectos de los fármacos , Fructosa/farmacología , Proteínas Hemolisinas/antagonistas & inhibidores , Inflamación/tratamiento farmacológico , Lipopolisacáridos/antagonistas & inhibidores , Staphylococcus aureus Resistente a Meticilina/efectos de los fármacos , Ratones , Nanopartículas/uso terapéutico , Sepsis/microbiología , Infecciones Estafilocócicas/tratamiento farmacológico , Staphylococcus aureus/efectos de los fármacosRESUMEN
Osteoporosis is one of the most common metabolic bone diseases affecting millions of people. We previously found that harmine prevents bone loss in ovariectomized mice via increasing preosteoclast platelet-derived growth factor-BB (PDGF-BB) production and type H vessel formation. However, the molecular mechanisms by which harmine promotes preosteoclast PDGF-BB generation are still unclear. In this study, we revealed that inhibitor of DNA binding-2 (Id2) and activator protein-1 (AP-1) were important factors implicated in harmine-enhanced preosteoclast PDGF-BB production. Exposure of RANKL-induced Primary bone marrow macrophages (BMMs), isolated from tibiae and femora of mice, to harmine increased the protein levels of Id2 and AP-1. Knockdown of Id2 by Id2-siRNA reduced the number of preosteoclasts as well as secretion of PDGF-BB in RANKL-stimulated BMMs administrated with harmine. Inhibition of c-Fos or c-Jun (components of AP-1) both reversed the stimulatory effect of harmine on preosteoclast PDGF-BB production. Dual-luciferase reporter assay analyses determined that PDGF-BB was the direct target of AP-1 which was up-regulated by harmine treatment. In conclusion, our data demonstrated a novel mechanism involving in the production of PDGF-BB increased by harmine, which may provide potential therapeutic targets for bone loss diseases.
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Becaplermina/metabolismo , Médula Ósea/efectos de los fármacos , Harmina/farmacología , Proteína 2 Inhibidora de la Diferenciación/metabolismo , Macrófagos/efectos de los fármacos , Osteoclastos/metabolismo , Factor de Transcripción AP-1/metabolismo , Animales , Médula Ósea/metabolismo , Células Cultivadas , Alucinógenos/farmacología , Proteína 2 Inhibidora de la Diferenciación/genética , Macrófagos/citología , Macrófagos/metabolismo , Ratones , Osteoclastos/citología , Factor de Transcripción AP-1/genéticaRESUMEN
Recently, the gut microbiota (GM) has been shown to be a regulator of bone homeostasis and the mechanisms by which GM modulates bone mass are still being investigated. Here, it is found that colonization with GM from children (CGM) but not from the elderly (EGM) prevents decreases in bone mass and bone strength in conventionally raised, ovariectomy (OVX)-induced osteoporotic mice. 16S rRNA gene sequencing reveals that CGM reverses the OVX-induced reduction of Akkermansia muciniphila (Akk). Direct replenishment of Akk is sufficient to correct the OVX-induced imbalanced bone metabolism and protect against osteoporosis. Mechanistic studies show that the secretion of extracellular vesicles (EVs) is required for the CGM- and Akk-induced bone protective effects and these nanovesicles can enter and accumulate into bone tissues to attenuate the OVX-induced osteoporotic phenotypes by augmenting osteogenic activity and inhibiting osteoclast formation. The study identifies that gut bacterium Akk mediates the CGM-induced anti-osteoporotic effects and presents a novel mechanism underlying the exchange of signals between GM and host bone.
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Densidad Ósea/fisiología , Huesos/metabolismo , Vesículas Extracelulares/metabolismo , Microbioma Gastrointestinal/fisiología , Osteoporosis/metabolismo , Osteoporosis/fisiopatología , Factores de Edad , Anciano , Animales , Preescolar , Modelos Animales de Enfermedad , Femenino , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Persona de Mediana EdadRESUMEN
PURPOSE: Prostate cancer (PCa) is one of the most common malignancies in males. Despite the success of immunotherapy in many malignant cancers, strategies are still needed to improve therapeutic efficacy in PCa. This study aimed to investigate the effects of Akkermansia muciniphila-derived extracellular vesicles (Akk-EVs) on PCa and elucidate the underlying immune-related mechanism. METHODS: Akk-EVs were isolated by ultracentrifugation and intravenously injected to treat syngeneic PCa-bearing immune-competent mice. Immunophenotypic changes in immune cells, such as cytotoxic T lymphocytes and macrophages, were measured via flow cytometry analysis. Histological examination was used to detect morphological changes in major organs after Akk-EVs treatments. In vitro, flow cytometry was performed to confirm the effects of Akk-EVs on the activation of CD8+ T cells. Quantitative PCR and immunofluorescence staining were carried out to test the impact of Akk-EVs on macrophage polarization. Cell counting kit-8 (CCK-8) analysis, colony formation assays, and scratch wound healing assays were conducted to assess the effects of Akk-EVs-treated macrophages on the proliferation and invasion of PCa cells. CCK-8 assays also confirmed the impact of Akk-EVs on the viability of normal cells. RESULTS: Intravenous injection of Akk-EVs in immune-competent mice reduced the tumor burden of PCa without inducing obvious toxicity in normal tissues. This treatment elevated the proportion of granzyme B-positive (GZMB+) and interferon γ-positive (IFN-γ+) lymphocytes in CD8+ T cells and caused macrophage recruitment, with increased tumor-killing M1 macrophages and decreased immunosuppressive M2 macrophages. In vitro, Akk-EVs increased the number of GZMB+CD8+ and IFN-γ+CD8+ T cells and M1-like macrophages. In addition, conditioned medium from Akk-EVs-treated macrophages suppressed the proliferation and invasion of prostate cells. Furthermore, the effective dose of Akk-EVs was well-tolerated in normal cells. CONCLUSION: Our study revealed the promising prospects of Akk-EVs as an efficient and biocompatible immunotherapeutic agent for PCa treatment.
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Linfocitos T CD8-positivos/efectos de los fármacos , Vesículas Extracelulares/inmunología , Macrófagos/efectos de los fármacos , Neoplasias de la Próstata/tratamiento farmacológico , Akkermansia/química , Animales , Antineoplásicos Inmunológicos/química , Linfocitos T CD8-positivos/inmunología , Línea Celular Tumoral , Inmunofenotipificación , Inmunoterapia/métodos , Interferón gamma/metabolismo , Macrófagos/inmunología , Masculino , Ratones Endogámicos C57BL , Neoplasias Experimentales/tratamiento farmacológico , Neoplasias Experimentales/inmunología , Neoplasias Experimentales/patología , Neoplasias de la Próstata/inmunología , Neoplasias de la Próstata/patologíaRESUMEN
Alzheimer's disease (AD) is currently ranked as the third leading cause of death for eldly people, just behind heart disease and cancer. Autophagy is declined with aging. Our study determined the biphasic changes of miR-331-3p and miR-9-5p associated with AD progression in APPswe/PS1dE9 mouse model and demonstrated inhibiting miR-331-3p and miR-9-5p treatment prevented AD progression by promoting the autophagic clearance of amyloid beta (Aß). Methods: The biphasic changes of microRNAs were obtained from RNA-seq data and verified by qRT-PCR in early-stage (6 months) and late-stage (12 months) APPswe/PS1dE9 mice (hereinafter referred to as AD mice). The AD progression was determined by analyzing Aß levels, neuron numbers (MAP2+) and activated microglia (CD68+IBA1+) in brain tissues using immunohistological and immunofluorescent staining. MRNA and protein levels of autophagic-associated genes (Becn1, Sqstm1, LC3b) were tested to determine the autophagic activity. Morris water maze and object location test were employed to evaluate the memory and learning after antagomirs treatments in AD mice and the Aß in the brain tissues were determined. Results: MiR-331-3p and miR-9-5p are down-regulated in early-stage of AD mice, whereas up-regulated in late-stage of AD mice. We demonstrated that miR-331-3p and miR-9-5p target autophagy receptors Sequestosome 1 (Sqstm1) and Optineurin (Optn), respectively. Overexpression of miR-331-3p and miR-9-5p in SH-SY5Y cell line impaired autophagic activity and promoted amyloid plaques formation. Moreover, AD mice had enhanced Aß clearance, improved cognition and mobility when treated with miR-331-3p and miR-9-5p antagomirs at late-stage. Conclusion: Our study suggests that using miR-331-3p and miR-9-5p, along with autophagic activity and amyloid plaques may distinguish early versus late stage of AD for more accurate and timely diagnosis. Additionally, we further provide a possible new therapeutic strategy for AD patients by inhibiting miR-331-3p and miR-9-5p and enhancing autophagy.
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Enfermedad de Alzheimer/prevención & control , Autofagia , Modelos Animales de Enfermedad , Regulación de la Expresión Génica , MicroARNs/antagonistas & inhibidores , Enfermedad de Alzheimer/genética , Enfermedad de Alzheimer/patología , Animales , Humanos , Masculino , Ratones , Ratones Transgénicos , MicroARNs/genética , Neuronas/metabolismo , Neuronas/patologíaRESUMEN
Senile osteoporosis (OP) is often concomitant with decreased autophagic activity. OPTN (optineurin), a macroautophagy/autophagy (hereinafter referred to as autophagy) receptor, is found to play a pivotal role in selective autophagy, coupling autophagy with bone metabolism. However, its role in osteogenesis is still mysterious. Herein, we identified Optn as a critical molecule of cell fate decision for bone marrow mesenchymal stem cells (MSCs), whose expression decreased in aged mice. Aged mice revealed osteoporotic bone loss, elevated senescence of MSCs, decreased osteogenesis, and enhanced adipogenesis, as well as optn-/ - mice. Importantly, restoring Optn by transplanting wild-type MSCs to optn-/ - mice or infecting optn-/ - mice with Optn-containing lentivirus rescued bone loss. The introduction of a loss-of-function mutant of OptnK193R failed to reestablish a bone-fat balance. We further identified FABP3 (fatty acid binding protein 3, muscle and heart) as a novel selective autophagy substrate of OPTN. FABP3 promoted adipogenesis and inhibited osteogenesis of MSCs. Knockdown of FABP3 alleviated bone loss in optn-/ - mice and aged mice. Our study revealed that reduced OPTN expression during aging might lead to OP due to a lack of FABP3 degradation via selective autophagy. FABP3 accumulation impaired osteogenesis of MSCs, leading to the occurrence of OP. Thus, reactivating OPTN or inhibiting FABP3 would open a new avenue to treat senile OP.Abbreviations: ADIPOQ: adiponectin, C1Q and collagen domain containing; ALPL: alkaline phosphatase, liver/bone/kidney; BGLAP/OC/osteocalcin: bone gamma carboxyglutamate protein; BFR/BS: bone formation rate/bone surface; CALCOCO2/NDP52: calcium binding and coiled-coil domain 2; CDKN1A/p21: cyclin-dependent kinase inhibitor 1A; CDKN2A/p16: cyclin dependent kinase inhibitor 2A; CDKN2B/p15: cyclin dependent kinase inhibitor 2B; CEBPA: CCAAT/enhancer binding protein (C/EBP), alpha; COL1A1: collagen, type I, alpha 1; Ct. BV/TV: cortical bone volume fraction; Ct. Th: cortical thickness; Es. Pm: endocortical perimeter; FABP4/Ap2: fatty acid binding protein 4, adipocyte; H2AX: H2A.X variant histone; HE: hematoxylin and eosin; MAP1LC3B: microtubule-associated protein 1 light chain 3 beta; MAR: mineral apposition rate; MSCs: bone marrow mesenchymal stem cells; NBR1: NBR1, autophagy cargo receptor; OP: osteoporosis; OPTN: optineurin; PDB: Paget disease of bone; PPARG: peroxisome proliferator activated receptor gamma; Ps. Pm: periosteal perimeter; qRT-PCR: quantitative real-time PCR; γH2AX: Phosphorylation of the Serine residue of H2AX; ROS: reactive oxygen species; RUNX2: runt related transcription factor 2; SA-GLB1: senescence-associated (SA)-GLB1 (galactosidase, beta 1); SP7/Osx/Osterix: Sp7 transcription factor 7; SQSTM1/p62: sequestosome 1; TAX1BP1: Tax1 (human T cell leukemia virus type I) binding protein 1; Tb. BV/TV: trabecular bone volume fraction; Tb. N: trabecular number; Tb. Sp: trabecular separation; Tb. Th: trabecular thickness; µCT: micro computed tomography.
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Envejecimiento , Autofagia , Proteínas de Ciclo Celular , Proteína 3 de Unión a Ácidos Grasos , Proteínas de Transporte de Membrana , Células Madre Mesenquimatosas , Adipogénesis , Animales , Proteínas de Ciclo Celular/metabolismo , Diferenciación Celular , Proteína 3 de Unión a Ácidos Grasos/metabolismo , Proteínas de Transporte de Membrana/metabolismo , Células Madre Mesenquimatosas/metabolismo , Ratones , Osteogénesis , Osteoporosis , Microtomografía por Rayos XRESUMEN
Poor wound healing after diabetes or extensive burn remains a challenging problem. Recently, we presented a physical approach to fabricate ultrasmall silver particles from Ångstrom scale to nanoscale and determined the antitumor efficacy of Ångstrom-scale silver particles (AgÅPs) in the smallest size range. Here we used the medium-sized AgÅPs (65.9 ± 31.6 Å) to prepare carbomer gel incorporated with these larger AgÅPs (L-AgÅPs-gel) and demonstrated the potent broad-spectrum antibacterial activity of L-AgÅPs-gel without obvious toxicity on wound healing-related cells. Induction of reactive oxygen species contributed to L-AgÅPs-gel-induced bacterial death. Topical application of L-AgÅPs-gel to mouse skin triggered much stronger effects than the commercial silver nanoparticles (AgNPs)-gel to prevent bacterial colonization, reduce inflammation, and accelerate diabetic and burn wound healing. L-AgÅPs were distributed locally in skin without inducing systemic toxicities. This study suggests that L-AgÅPs-gel represents an effective and safe antibacterial and anti-inflammatory material for wound therapy.
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Quemaduras , Nanopartículas del Metal , Resinas Acrílicas , Animales , Antibacterianos/farmacología , Quemaduras/tratamiento farmacológico , Inflamación/tratamiento farmacológico , Ratones , Plata/farmacología , Cicatrización de HeridasRESUMEN
Osteosarcoma is a common malignant bone cancer easily to metastasize. Much safer and more efficient strategies are still needed to suppress osteosarcoma growth and lung metastasis. We recently presented a pure physical method to fabricate Ångstrom-scale silver particles (AgÅPs) and determined the anti-tumor efficacy of fructose-coated AgÅPs (F-AgÅPs) against lung and pancreatic cancer. Our study utilized an optimized method to obtain smaller F-AgÅPs and aimed to assess whether F-AgÅPs can be used as an efficient and safe agent for osteosarcoma therapy. We also investigated whether the induction of apoptosis by altering glucose metabolic phenotype contributes to the F-AgÅPs-induced anti-osteosarcoma effects. Methods: A modified method was developed to prepare smaller F-AgÅPs. The anti-tumor, anti-metastatic and pro-survival efficacy of F-AgÅPs and their toxicities on healthy tissues were compared with that of cisplatin (a first-line chemotherapeutic drug for osteosarcoma therapy) in subcutaneous or orthotopic osteosarcoma-bearing nude mice. The pharmacokinetics, biodistribution and excretion of F-AgÅPs were evaluated by testing the levels of silver in serum, tissues, urine and feces of mice. A series of assays in vitro were conducted to assess whether the induction of apoptosis mediates the killing effects of F-AgÅPs on osteosarcoma cells and whether the alteration of glucose metabolic phenotype contributes to F-AgÅPs-induced apoptosis. Results: The newly obtained F-AgÅPs (9.38 ± 4.11 nm) had good stability in different biological media or aqueous solutions and were more effective than cisplatin in inhibiting tumor growth, improving survival, attenuating osteolysis and preventing lung metastasis in osteosarcoma-bearing nude mice after intravenous injection, but were well tolerated in normal tissues. One week after injection, about 68% of F-AgÅPs were excreted through feces. F-AgÅPs induced reactive oxygen species (ROS)-dependent apoptosis of osteosarcoma cells but not normal cells, owing to their ability to selectively shift glucose metabolism of osteosarcoma cells from glycolysis to mitochondrial oxidation by inhibiting pyruvate dehydrogenase kinase (PDK). Conclusion: Our study suggests the promising prospect of F-AgÅPs as a powerful selective anticancer agent for osteosarcoma therapy.
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Neoplasias Óseas/tratamiento farmacológico , Neoplasias Pulmonares/tratamiento farmacológico , Nanopartículas del Metal/administración & dosificación , Osteosarcoma/tratamiento farmacológico , Plata/administración & dosificación , Adolescente , Animales , Apoptosis/efectos de los fármacos , Neoplasias Óseas/patología , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Cisplatino/administración & dosificación , Femenino , Fructosa/química , Humanos , Lactante , Recién Nacido , Inyecciones Intravenosas , Neoplasias Pulmonares/secundario , Masculino , Nanopartículas del Metal/química , Ratones , Mitocondrias/efectos de los fármacos , Mitocondrias/metabolismo , Osteosarcoma/secundario , Oxidación-Reducción/efectos de los fármacos , Cultivo Primario de Células , Piruvato Deshidrogenasa Quinasa Acetil-Transferidora/antagonistas & inhibidores , Piruvato Deshidrogenasa Quinasa Acetil-Transferidora/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Eliminación Renal , Transducción de Señal/efectos de los fármacos , Plata/farmacocinética , Plata/orina , Distribución Tisular , Efecto Warburg en Oncología/efectos de los fármacos , Ensayos Antitumor por Modelo de Xenoinjerto , Adulto JovenRESUMEN
Osteonecrosis of the femoral head (ONFH) frequently occurs after glucocorticoid (GC) treatment. Extracellular vesicles (EVs) are important nano-sized paracrine mediators of intercellular crosstalk. This study aimed to determine whether EVs from human urine-derived stem cells (USC-EVs) could protect against GC-induced ONFH and focused on the impacts of USC-EVs on angiogenesis and apoptosis to explore the mechanism by which USC-EVs attenuated GC-induced ONFH. The results in vivo showed that the intravenous administration of USC-EVs at the early stage of GC exposure could rescue angiogenesis impairment, reduce apoptosis of trabecular bone and marrow cells, prevent trabecular bone destruction and improve bone microarchitecture in the femoral heads of rats. In vitro, USC-EVs reversed the GC-induced suppression of endothelial angiogenesis and activation of apoptosis. Deleted in malignant brain tumors 1 (DMBT1) and tissue inhibitor of metalloproteinases 1 (TIMP1) proteins were enriched in USC-EVs and essential for the USC-EVs-induced pro-angiogenic and anti-apoptotic effects in GC-treated cells, respectively. Knockdown of TIMP1 attenuated the protective effects of USC-EVs against GC-induced ONFH. Our study suggests that USC-EVs are a promising nano-sized agent for the prevention of GC-induced ONFH by delivering pro-angiogenic DMBT1 and anti-apoptotic TIMP1. STATEMENT OF SIGNIFICANCE: This study demonstrates that the intravenous injection of extracellular vesicles from human urine-derived stem cells (USC-EVs) at the early stage of glucocorticoid (GC) exposure efficiently protects the rats from the GC-induced osteonecrosis of the femoral head (ONFH). Moreover, this study identifies that the promotion of angiogenesis and inhibition of apoptosis by transferring pro-angiogenic DMBT1 and anti-apoptotic TIMP1 proteins contribute importantly to the USC-EVs-induced protective effects against GC-induced ONFH. This study suggests the promising prospect of USC-EVs as a new nano-sized agent for protecting against GC-induced ONFH, and the potential of DMBT1 and TIMP1 as the molecular targets for further augmenting the protective function of USC-EVs.
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Vesículas Extracelulares , Osteonecrosis , Animales , Proteínas de Unión al Calcio , Proliferación Celular , Proteínas de Unión al ADN , Cabeza Femoral , Glucocorticoides , Humanos , Ratas , Células Madre , Inhibidor Tisular de Metaloproteinasa-1 , Proteínas Supresoras de TumorRESUMEN
Healing of the chronic diabetic ulceration and large burns remains a clinical challenge. Therapeutic fasting has been shown to improve health. Our study tested whether fasting facilitates diabetic and burn wound healing and explored the underlying mechanism. Methods: The effects of fasting on diabetic and burn wound healing were evaluated by analyzing the rates of wound closure, re-epithelialization, scar formation, collagen deposition, skin cell proliferation and neovascularization using histological analyses and immunostaining. In vitro functional assays were conducted to assess fasting and refeeding on the angiogenic activities of endothelial cells. Transcriptome sequencing was employed to identify the differentially expressed genes in endothelial cells after fasting treatment and the role of the candidate genes in the fasting-induced promotion of angiogenesis was demonstrated. Results: Two times of 24-h fasting in a week after but especially before wound injury efficiently induced faster wound closure, better epidermal and dermal regeneration, less scar formation and higher level of angiogenesis in mice with diabetic or burn wounds. In vitro, fasting alone by serum deprivation did not increase, but rather reduced the abilities of endothelial cell to proliferate, migrate and form vessel-like tubes. However, subsequent refeeding did not merely rescue, but further augmented the angiogenic activities of endothelial cells. Transcriptome sequencing revealed that fasting itself, but not the following refeeding, induced a prominent upregulation of a variety of pro-angiogenic genes, including SMOC1 (SPARC related modular calcium binding 1) and SCG2 (secretogranin II). Immunofluorescent staining confirmed the increase of SMOC1 and SCG2 expression in both diabetic and burn wounds after fasting treatment. When the expression of SMOC1 or SCG2 was down-regulated, the fasting/refeeding-induced pro-angiogenic effects were markedly attenuated. Conclusion: This study suggests that fasting combined with refeeding, but not fasting solely, enhance endothelial angiogenesis through the activation of SMOC1 and SCG2, thus facilitating neovascularization and rapid wound healing.
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Diabetes Mellitus Experimental/dietoterapia , Ayuno , Neovascularización Fisiológica , Osteonectina/metabolismo , Repitelización , Secretogranina II/metabolismo , Animales , Quemaduras/terapia , Línea Celular , Proliferación Celular , Cicatriz/metabolismo , Células Endoteliales , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Piel/metabolismo , Piel/patologíaRESUMEN
Osteoporosis is a debilitating bone disease affecting millions of people. Here, we used human urine-derived stem cells (USCs), which were noninvasively harvested from unlimited and easily available urine, as a "factory" to obtain extracellular vesicles (USC-EVs) and demonstrated that the systemic injection of USC-EVs effectively alleviates bone loss and maintains bone strength in osteoporotic mice by enhancing osteoblastic bone formation and suppressing osteoclastic bone resorption. More importantly, the anti-osteoporotic properties of USC-EVs are not notably disrupted by the age, gender, or health condition (with or without osteoporosis) of the USC donor. Mechanistic studies determined that collagen triple-helix repeat containing 1 (CTHRC1) and osteoprotegerin (OPG) proteins are enriched in USC-EVs and required for USC-EV-induced pro-osteogenic and anti-osteoclastic effects. Our results suggest that autologous USC-EVs represent a promising novel therapeutic agent for osteoporosis by promoting osteogenesis and inhibiting osteoclastogenesis by transferring CTHRC1 and OPG.
RESUMEN
Poor wound healing affects millions of people worldwide each year and needs better therapeutic strategies. Synechococcus elongatus PCC 7942 is a naturally occurring photoautotrophic cyanobacterium that can be easily obtained and large-scale expanded. Here, we investigated the therapeutic efficacy of this cyanobacterium in a mouse model of acute burn injury and whether the secretion of extracellular vesicles (EVs), important mediators of cell paracrine activity, is a key mechanism of the cyanobacterium-induced regulation of wound healing. Methods: The effects of Synechococcus elongatus PCC 7942 on burn wound healing in mice under light or dark conditions were evaluated by measuring wound closure rates, histological and immunofluorescence analyses. A series of assays in vivo and in vitro were conducted to assess the impact of the cyanobacterium on angiogenesis. GW4869 was used to interfere with the secretion of EVs by the cyanobacterium and the abilities of the GW4869-pretreated and untreated Synechococcus elongatus PCC 7942 to regulate endothelial angiogenesis were compared. The direct effects of the cyanobacterium-derived EVs (S. elongatus-EVs) on angiogenesis, wound healing and expressions of a class of pro-inflammatory factors that have regulatory roles in wound healing were also examined. Results: Synechococcus elongatus PCC 7942 treatment under light and dark conditions both significantly promoted angiogenesis and burn wound repair in mice. In vitro, the cyanobacterium enhanced angiogenic activities of endothelial cells, but the effects were markedly blocked by GW4869 pretreatment. S. elongatus-EVs were capable of augmenting endothelial angiogenesis in vitro, and stimulating new blood vessel formation and burn wound healing in mice. The expression of interleukin 6 (IL-6), which has an essential role in angiogenesis during skin wound repair, was induced in wound tissues and wound healing-related cells by S. elongatus-EVs and Synechococcus elongatus PCC 7942. Conclusion: Synechococcus elongatus PCC 7942 has the potential as a promising strategy for therapeutic angiogenesis and wound healing primarily by the delivery of functional EVs, not by its photosynthetic activity. The promotion of IL-6 expression may be a mechanism of the cyanobacterium and its EVs-induced pro-angiogenic and -wound healing effects.
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Quemaduras/terapia , Células Endoteliales/efectos de los fármacos , Vesículas Extracelulares/fisiología , Piel/efectos de los fármacos , Synechococcus/fisiología , Cicatrización de Heridas/efectos de los fármacos , Compuestos de Anilina/farmacología , Animales , Compuestos de Bencilideno/farmacología , Quemaduras/genética , Quemaduras/metabolismo , Quemaduras/patología , Línea Celular , Línea Celular Transformada , Células Endoteliales/citología , Células Endoteliales/metabolismo , Células Endoteliales/efectos de la radiación , Vesículas Extracelulares/química , Femenino , Regulación de la Expresión Génica , Humanos , Interleucina-1alfa/genética , Interleucina-1alfa/metabolismo , Interleucina-1beta/genética , Interleucina-1beta/metabolismo , Interleucina-6/genética , Interleucina-6/metabolismo , Queratinocitos/citología , Queratinocitos/efectos de los fármacos , Queratinocitos/metabolismo , Queratinocitos/efectos de la radiación , Luz , Ratones , Ratones Endogámicos C57BL , Células 3T3 NIH , Neovascularización Fisiológica/efectos de los fármacos , Neovascularización Fisiológica/efectos de la radiación , Piel/irrigación sanguínea , Piel/lesiones , Piel/efectos de la radiación , Synechococcus/química , Factor de Necrosis Tumoral alfa/genética , Factor de Necrosis Tumoral alfa/metabolismo , Cicatrización de Heridas/fisiología , Cicatrización de Heridas/efectos de la radiaciónRESUMEN
Recently, researchers identified a distinct vessel subtype called type H vessels that couple angiogenesis and osteogenesis. We previously found that type H vessels are reduced in ovariectomy (OVX)-induced osteoporotic mice, and preosteoclasts are able to secrete platelet-derived growth factor-BB (PDGF-BB) to stimulate type H vessel formation and thereby to promote osteogenesis. This study aimed to explore whether harmine, a ß-carboline alkaloid, is capable of preventing bone loss in OVX mice by promoting preosteoclast PDGF-BB-induced type H vessel formation. METHODS: The impact of harmine on osteoclastogenesis of RANKL-stimulated RAW264.7 cells was verified by gene expression analysis and tartrate-resistant acid phosphatase (TRAP) staining. Enzyme-linked immunosorbent assay (ELISA) was conducted to test PDGF-BB production by preosteoclasts. A series of angiogenesis-related assays in vitro were performed to assess the pro-angiogenic effects of the conditioned media from RANKL-stimulated RAW264.7 cells treated with or without harmine. Meanwhile, the role of PDGF-BB in this process was determined. In vivo, OVX mice were intragastrically administrated with harmine emulsion or an equal volume of vehicle. 2 months later, bone samples were collected for µCT, histological, immunohistochemical and immunofluorescent analyses to evaluate bone mass, osteogenic and osteoclastic activities, as well as the numbers of type H vessels. Bone marrow PDGF-BB concentrations were assessed by ELISA. RESULTS: Exposure of RANKL-stimulated RAW264.7 cells to harmine enhanced the formation of preosteoclasts and the production of PDGF-BB. Harmine augmented the ability of RANKL-stimulated RAW264.7 cells to promote angiogenesis of endothelial cells, whereas the effect was blocked by PDGF-BB inhibition. In vivo, the oral administration of harmine emulsion to OVX mice resulted in enhanced trabecular bone mass and osteogenic responses, increased numbers of preosteoclasts, as well as reduced numbers of osteoclasts and fat cells. Moreover, OVX mice treated with harmine exhibited higher levels of bone marrow PDGF-BB and much more type H vessels in bone. CONCLUSION: Harmine may exert bone-sparing effects by suppression of osteoclast formation and promotion of preosteoclast PDGF-BB-induced angiogenesis.
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Huesos/efectos de los fármacos , Harmina/farmacología , Neovascularización Fisiológica/fisiología , Osteoporosis/fisiopatología , Animales , Becaplermina/metabolismo , Enfermedades Óseas Metabólicas/tratamiento farmacológico , Enfermedades Óseas Metabólicas/metabolismo , Resorción Ósea/tratamiento farmacológico , Resorción Ósea/metabolismo , Huesos/metabolismo , Diferenciación Celular/efectos de los fármacos , Línea Celular , Medios de Cultivo Condicionados/metabolismo , Ratones , Osteoclastos/efectos de los fármacos , Osteoclastos/metabolismo , Osteogénesis/efectos de los fármacos , Osteoporosis/metabolismo , Ovariectomía/métodos , Células RAW 264.7RESUMEN
Chronic non-healing wounds represent one of the most common complications of diabetes and need advanced treatment strategies. Exosomes are key mediators of cell paracrine action and can be directly utilized as therapeutic agents for tissue repair and regeneration. Here, we explored the effects of exosomes from human urine-derived stem cells (USC-Exos) on diabetic wound healing and the underlying mechanism. Methods: USCs were characterized by flow cytometry and multipotent differentiation potential analyses. USC-Exos were isolated from the conditioned media of USCs and identified by transmission electron microscopy and flow cytometry. A series of functional assays in vitro were performed to assess the effects of USC-Exos on the activities of wound healing-related cells. Protein profiles in USC-Exos and USCs were examined to screen the candidate molecules that mediate USC-Exos function. The effects of USC-Exos on wound healing in streptozotocin-induced diabetic mice were tested by measuring wound closure rates, histological and immunofluorescence analyses. Meanwhile, the role of the candidate protein in USC-Exos-induced regulation of angiogenic activities of endothelial cells and diabetic wound healing was assessed. Results: USCs were positive for CD29, CD44, CD73 and CD90, but negative for CD34 and CD45. USCs were able to differentiate into osteoblasts, adipocytes and chondrocytes. USC-Exos exhibited a cup- or sphere-shaped morphology with a mean diameter of 51.57 ± 2.93 nm and positive for CD63 and TSG101. USC-Exos could augment the functional properties of wound healing-related cells including the angiogenic activities of endothelial cells. USC-Exos were enriched in the proteins that are involved in regulation of wound healing-related biological processes. Particularly, a pro-angiogenic protein called deleted in malignant brain tumors 1 (DMBT1) was highly expressed in USC-Exos. Further functional assays showed that DMBT1 protein was required for USC-Exos-induced promotion of angiogenic responses of cultured endothelial cells, as well as angiogenesis and wound healing in diabetic mice. Conclusion: Our findings suggest that USC-Exos may represent a promising strategy for diabetic soft tissue wound healing by promoting angiogenesis via transferring DMBT1 protein.
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Medios de Cultivo Condicionados/farmacología , Diabetes Mellitus Experimental/terapia , Exosomas/química , Neovascularización Fisiológica/efectos de los fármacos , Receptores de Superficie Celular/genética , Cicatrización de Heridas/efectos de los fármacos , Heridas no Penetrantes/terapia , Adipocitos/citología , Adipocitos/efectos de los fármacos , Adipocitos/metabolismo , Animales , Antígenos CD/genética , Antígenos CD/metabolismo , Proteínas de Unión al Calcio , Diferenciación Celular , Movimiento Celular , Proliferación Celular , Condrocitos/citología , Condrocitos/efectos de los fármacos , Condrocitos/metabolismo , Medios de Cultivo Condicionados/química , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Diabetes Mellitus Experimental/inducido químicamente , Diabetes Mellitus Experimental/metabolismo , Diabetes Mellitus Experimental/patología , Complejos de Clasificación Endosomal Requeridos para el Transporte/genética , Complejos de Clasificación Endosomal Requeridos para el Transporte/metabolismo , Células Endoteliales/citología , Células Endoteliales/efectos de los fármacos , Células Endoteliales/metabolismo , Femenino , Expresión Génica , Humanos , Ratones , Ratones Endogámicos C57BL , Osteoblastos/citología , Osteoblastos/efectos de los fármacos , Osteoblastos/metabolismo , Receptores de Superficie Celular/metabolismo , Piel/efectos de los fármacos , Piel/lesiones , Piel/metabolismo , Células Madre/química , Células Madre/citología , Células Madre/metabolismo , Estreptozocina , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Proteínas Supresoras de Tumor , Orina/química , Heridas no Penetrantes/metabolismo , Heridas no Penetrantes/patologíaRESUMEN
Liraglutide, a synthetic analogue of glucagon-like peptide1, is utilized in the treatment of type 2 diabetes and obesity. Liraglutide has been previously demonstrated to prevent osteoblastic differentiation of human vascular smooth muscle cells, resulting in the slowing of arterial calciï¬cation, however, its effect on bone formation remains unclear. The present study investigated the effect of liraglutide on osteoblastic differentiation using Alizarin Red S staining, and examined the molecular mechanisms underlying the regulatory effect by western blot analysis. The present study demonstrated that protein expression levels of phosphorylated adenosine monophosphateactivated protein kinase (pAMPK) were downregulated in MC3T3E1 cells during osteoblastic differentiation in commercial osteogenic differentiation medium, whereas protein expression levels of transforming growth factorß (TGFß) and phosphorylated mammalian target of rapamycin (pmTOR) increased. Liraglutide was subsequently demonstrated to dosedependently attenuate the osteoblastic differentiation of MC3T3E1 cells, to upregulate pAMPK, and downregulate pmTOR and TGFß protein expression levels. Treatment with an AMPKspecific inhibitor, Compound C, eradicated the effect of liraglutide on osteoblastic differentiation, and pmTOR and TGFß downregulation. An mTOR activator, MHY1485, also abolished the inhibitory effect of liraglutide on osteoblastic differentiation, and resulted in pmTOR and TGFß downregulation, but did not attenuate the liraglutideinduced increase in pAMPK protein expression levels. The results of the present study demonstrate that liraglutide attenuates osteoblastic differentiation of MC3T3E1 cells via modulation of AMPK/mTOR signaling. The present study revealed a novel function of liraglutide, which contributes to the understanding of its pharmacological and physiological effects in clinical settings.
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Proteínas Quinasas Activadas por AMP/metabolismo , Diferenciación Celular/efectos de los fármacos , Hipoglucemiantes/farmacología , Liraglutida/farmacología , Osteoblastos/efectos de los fármacos , Transducción de Señal/efectos de los fármacos , Serina-Treonina Quinasas TOR/metabolismo , Animales , Línea Celular , Ratones , Osteoblastos/citología , Osteoblastos/metabolismo , Osteogénesis/efectos de los fármacosRESUMEN
PURPOSE: We present a retrospective study of patients with multilevel contiguous tuberculous spondylitis of thoracic region that underwent single-stage posterolateral debridement and fusion and following posterior instrumentation. METHODS: From June 2000 to March 2009, 870 consecutive spinal tubercular patients including 36 patients who were diagnosed and treated as multilevel contiguous thoracic spinal tuberculosis in our institution. Apart from five patients being treated conservatively, the 31 cases received surgery by single-stage posterolateral debridement, fusion, following posterior instrumentation and postural drainage. The patients were evaluated based on the Frankel scoring system, kyphotic Cobb angle, and visual analog scale (VAS) pain score. RESULTS: The mean duration of postoperative follow-up was 79.2 ± 9.9 months (range 62-98 months). Neither mortalities nor any major complications were found. Solid bony fusion was achieved in all patients. No patients with neurological deficit deteriorated postoperatively. According to Frankel scoring system, 7 cases were rated as Grade D, 24 cases as Grade E at last follow-up. The average preoperative Cobb's angle was 32° (range 21°-39°). The average early postoperative Cobb's angle was 23° (range 15°-32°). The mean latest postoperative Cobb's angle was 26° (range 20°-32°), with a small loss of correction at last follow-up. Pre-op VAS was 8.8 ± 0.7 (range 7-10) and final follow-up was 1.8 ± 1.1. There was a significant difference of VAS between preoperation and the final follow-up. CONCLUSIONS: One-stage surgical treatment for multilevel contiguous spinal tuberculosis by posterolateral debridement, fusion, posterior instrumentation can be an effective and feasible treatment method.
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Desbridamiento/métodos , Drenaje Postural/métodos , Fusión Vertebral/métodos , Vértebras Torácicas/cirugía , Tuberculosis de la Columna Vertebral/cirugía , Adulto , Anciano , Desbridamiento/efectos adversos , Femenino , Estudios de Seguimiento , Humanos , Masculino , Persona de Mediana Edad , Dimensión del Dolor , Periodo Posoperatorio , Estudios Retrospectivos , Fusión Vertebral/efectos adversos , Vértebras Torácicas/patología , Resultado del TratamientoRESUMEN
PURPOSE: There are few papers in the literature comparing outcomes between antero-posterior and posterior-only approaches for treating thoracolumbar tuberculosis (T10L2) in children. METHODS: We performed a retrospective review of 47 children who were diagnosed and treated as thoracolumbar tuberculosis (T10L2) in our department from January 2005 to June 2009. Forty-seven cases of thoracolumbar tuberculosis were treated by two different surgical approaches. All the cases were divided into two groups: 25 cases in group A underwent one-stage posterior debridement, transforaminal fusion, and instrumentation, and 22 cases in group B underwent anterior debridement, bone graft, and posterior instrumentation in a single- or two-stage procedure. Two approaches were compared in terms of average operative time, blood loss, hospitalizations, bony fusion, intraoperative and postoperative complications, the Oswestry disability index score, neurological status, and the angle of kyphosis. RESULTS: All 47 patients (24 M/23F), averaged 9.1 ± 2.6 years old (range 5 to 14 years), who were followed up for mean of 49.3 ± 8.6 months (range 36 to 65 months). Spinal tuberculosis (TB) was completely cured, and the grafted bones were fused in 9 months in all cases. It was obviously that the average operative time, blood loss, hospitalization, and complication rate of group A was less than those of group B. Good clinical outcomes were achieved in both groups. CONCLUSIONS: Both the antero-posterior and posterior approaches can effectively heal T10L2 vertebral tuberculosis, but the average surgical time, blood loss, complications, and hospital stay following the posterior approach are prominently less than those following the antero-posterior approach. It might be a better surgical treatment for thoracic spinal tuberculosis in children with poor health status, especially for cases in early phase of bone destruction and/or mild and moderate kyphosis.