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Recent studies suggest that chromosomal cohesin complex proteins are important in regulating hematopoiesis and may contribute to myeloid malignancies. To investigate the effects of perturbing the cohesin subunit protein RAD21 on normal hematopoiesis, we used conditional knockout (cKO) mouse models. While cohesin is vital for hematopoietic stem cell (HSC) function, Rad21 haploinsufficiency (Rad21Δ/+) led to distinct hematopoietic phenotypes. Our findings revealed that Rad21Δ/+ cells exhibited decreased hematopoietic reconstitution in competitive bone marrow transplantation assays. This reduction in peripheral blood chimerism was specifically observed in the lymphoid compartment, while the chimerism in the myeloid compartment remained unaffected. Rad21 haploinsufficiency also resulted in changes in the hematopoietic stem and progenitor cells (HSPC) and myeloid progenitor compartments, with a significant accumulation of granulocyte-macrophage progenitors in the bone marrow. We observed differential gene expression in Rad21Δ/+ LSK (Lin- Sca1-Kit+) cells, including genes required for HSPC function and differentiation, such as Setdb1, Hmga2, Ncor1, and Myb. In addition, we observed a notable decrease in the expression of genes related to the interferon response and a significant reduction in the expression of genes involved in the IL2-STAT5 signaling pathways. Our studies suggest that RAD21 protein and level of its post-translational modifications in the bone marrow cells may play a potential role in hematopoiesis. Overall, Rad21 haploinsufficiency impairs hematopoietic differentiation and increases HSC self-renewal.
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Proteínas Cromosómicas no Histona , Trasplante de Células Madre Hematopoyéticas , Ratones , Animales , Diferenciación Celular , Proteínas Cromosómicas no Histona/genética , Proteínas Cromosómicas no Histona/metabolismo , Células Madre Hematopoyéticas/metabolismo , Hematopoyesis/genética , Ratones Endogámicos C57BL , Co-Represor 1 de Receptor Nuclear/metabolismo , CohesinasRESUMEN
Osteoporosis and age-related bone loss increase bone fracture risk and impair bone healing. The need for identifying new factors to prevent or treat bone loss is critical. Previously, we reported that young MRL/MpJ mice have superior bone microarchitecture and biomechanical properties as compared to wild-type (WT) mice. In this study, MRL/MpJ mice were tested for resistance to age-related and long-term ovariectomy-induced bone loss to uncover potential beneficial factors for bone regeneration and repair. Bone tissues collected from 14-month-old MRL/MpJ and C57BL/6J (WT) mice were analyzed using micro-CT, histology, and immunohistochemistry, and serum protein markers were characterized using ELISAs or multiplex assays. Furthermore, 4-month-old MRL/MpJ and WT mice were subjected to ovariectomy (OV) or sham surgery and bone loss was monitored continuously using micro-CT at 1, 2, 4, and 6 months (M) after surgery with histology and immunohistochemistry performed at 6 M post-surgery. Sera were collected for biomarker detection using ELISA and multiplex assays at 6 M after surgery. Our results indicated that MRL/MpJ mice maintained better bone microarchitecture and higher bone mass than WT mice during aging and long-term ovariectomy. This resistance of bone loss observed in MRL/MpJ mice correlated with the maintenance of higher OSX+ osteoprogenitor cell pools, higher activation of the pSMAD5 signaling pathway, more PCNA+ cells, and a lower number of osteoclasts. Systemically, lower serum RANKL and DKK1 with higher serum IGF1 and OPG in MRL/MpJ mice relative to WT mice may also contribute to the maintenance of higher bone microarchitecture during aging and less severe bone loss after long-term ovariectomy. These findings may be used to develop therapeutic approaches to maintain bone mass and improve bone regeneration and repair due to injury, disease, and aging.
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Enfermedades Óseas Metabólicas , Osteoporosis , Femenino , Ratones , Animales , Ratones Endogámicos C57BL , Ratones Endogámicos , Osteoporosis/etiología , Regeneración Ósea , BiomarcadoresRESUMEN
The dystrophin-/-/utrophin-/-/ double knockout (dKO-Hom) mouse is a murine model of human Duchenne muscular dystrophy. This study investigated the bone and muscle abnormalities of dKO-Hom mouse and mechanisms. We collected bone and skeletal muscle samples from control mice and three muscular dystrophic mouse models at different ages and performed micro-computer tomography and histological analyses of both bone and skeletal muscle tissues. Serum receptor activator of nuclear factor kappa-Β ligand (RANKL) and sclerostin (SOST) levels, osteoclastogenesis and serum proteomics were also analyzed. Our results indicated that dKO-Hom mice developed skeletal muscle histopathologies by 5 days of age, whereas bone abnormalities developed at 4 weeks of age. Furthermore, our results indicated that the numbers of osteoblasts and osteoclasts were decreased in the proximal tibia and spine trabecular bone of dKO-Hom mice compared to wild-type (WT) mice, which correlated with a significant reduction in serum RANKL levels. The number of tibia cortical osteocytes also decreased, whereas serum SOST levels increased significantly in dKO-Hom mice than WT mice. Osteoblastic number was significantly lower, but osteoclast number increased, in the spine L6 of dKO-Hom mice than WT mice at 6 weeks of age, resulting in a decrease in bone formation and an increase in bone resorption. Serum proteomics results revealed abnormal proteome profiles in dKO-Hom mice compared to control mice. In conclusion, our study elucidated the timing of development of bone and muscle abnormalities. The bone abnormalities in dKO-Hom mice are correlated with lower serum RANKL and higher SOST levels that resulted in dysregulation of osteogenesis and osteoclastogenesis and bone loss.
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Desarrollo Óseo/genética , Distrofina/genética , Distrofia Muscular de Duchenne/genética , Utrofina/genética , Animales , Huesos/anomalías , Huesos/metabolismo , Modelos Animales de Enfermedad , Humanos , Ratones , Ratones Noqueados , Músculo Esquelético/anomalías , Músculo Esquelético/crecimiento & desarrollo , Distrofia Muscular de Duchenne/patología , FN-kappa B/genética , Osteoclastos/metabolismo , Osteoclastos/patologíaRESUMEN
Cohesin recently emerged as a new regulator of hematopoiesis and leukemia. In addition to cohesin, whether proteins that regulate cohesin's function have any direct role in hematopoiesis and hematologic diseases have not been fully examined. Separase, encoded by the ESPL1 gene, is an important regulator of cohesin's function. Canonically, protease activity of Separase resolves sister chromatid cohesion by cleaving cohesin subunit-Rad21 at the onset of anaphase. Using a Separase haploinsufficient mouse model, we have uncovered a novel role of Separase in hematopoiesis. We report that partial disruption of Separase distinctly alters the functional characteristics of hematopoietic stem/progenitor cells (HSPCs). Although analyses of peripheral blood and bone marrow of Espl1+/Hyp mice broadly displayed unperturbed hematopoietic parameters during normal hematopoiesis, further probing of the composition of early hematopoietic cells in Espl1+/Hyp bone marrow revealed a mild reduction in the frequencies of the Lin- Sca1+ Kit- (LSK) or LSK CD48+ CD150- multipotent hematopoietic progenitors population without a significant change in either long-term or short-term hematopoietic stem cells (HSCs) subsets at steady state. Surprisingly, however, we found that Separase haploinsufficiency promotes regeneration activity of HSCs in serial in vivo repopulation assays. In vitro colony formation assays also revealed an enhanced serial replating capacity of hematopoietic progenitors isolated from Espl1+/Hyp mice. Microarray analysis of differentially expressed genes showed that Separase haploinsufficiency in HSCs (SP-KSL) leads to enrichment of gene signatures that are upregulated in HSCs compared to committed progenitors and mature cells. Taken together, our findings demonstrate a key role of Separase in promoting hematopoietic regeneration of HSCs.
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Given the capacity of self-renewal and multilineage differentiation, stem cells are promising sources for use in regenerative medicines as well as in the clinical treatment of certain hematological malignancies and degenerative diseases. Complex networks of cellular signaling pathways largely determine stem cell fate and function. Small molecules that modulate these pathways can provide important biological and pharmacological insights. However, it is still challenging to identify the specific protein targets of these compounds, to explore the changes in stem cell phenotypes induced by compound treatment and to ascertain compound mechanisms of action. To facilitate stem cell related small molecule study and provide a better understanding of the associated signaling pathways, we have constructed a comprehensive domain-specific chemogenomics resource, called StemCellCKB ( http://www.cbligand.org/StemCellCKB/ ). This new cloud-computing platform describes the chemical molecules, genes, proteins, and signaling pathways implicated in stem cell regulation. StemCellCKB is also implemented with web applications designed specifically to aid in the identification of stem cell relevant protein targets, including TargetHunter, a machine-learning algorithm for predicting small molecule targets based on molecular fingerprints, and HTDocking, a high-throughput docking module for target prediction and systems-pharmacology analyses. We have systematically tested StemCellCKB to verify data integrity. Target-prediction accuracy has also been validated against the reported known target/compound associations. This proof-of-concept example demonstrates that StemCellCKB can (1) accurately predict the macromolecular targets of existing stem cell modulators and (2) identify novel small molecules capable of probing stem cell signaling mechanisms, for use in systems-pharmacology studies. StemCellCKB facilitates the exploration and exchange of stem cell chemogenomics data among members of the broader research community.
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Biología Computacional/métodos , Descubrimiento de Drogas/métodos , Células Madre , Nube Computacional , Bases de Datos Factuales , Humanos , Bases del Conocimiento , Modelos Moleculares , Mapas de Interacción de Proteínas/efectos de los fármacos , Transducción de Señal/efectos de los fármacos , Células Madre/química , Células Madre/citología , Células Madre/efectos de los fármacos , Células Madre/metabolismoRESUMEN
Cannabinoid receptor-2 (CB2) is expressed dominantly in the immune system, especially on plasma cells. Cannabinergic ligands with CB2 selectivity emerge as a class of promising agents to treat CB2-expressing malignancies without psychotropic concerns. In this study, we found that CB2 but not CB1 was highly expressed in human multiple myeloma (MM) and primary CD138+ cells. A novel inverse agonist of CB2, phenylacetylamide but not CB1 inverse agonist SR141716, inhibited the proliferation of human MM cells (IC50 : 0.62 â¼ 2.5 µM) mediated by apoptosis induction, but exhibited minor cytotoxic effects on human normal mononuclear cells. CB2 gene silencing or pharmacological antagonism markedly attenuated phenylacetylamide's anti-MM effects. Phenylacetylamide triggered the expression of C/EBP homologous protein at the early treatment stage, followed by death receptor-5 upregulation, caspase activation, and ß-actin/tubulin degradation. Cell cycle related protein cdc25C and mitotic regulator Aurora A kinase were inactivated by phenylacetylamide treatment, leading to an increase in the ratio inactive/active cdc2 kinase. As a result, phosphorylation of CDK substrates was decreased, and the MM cell mitotic division was largely blocked by treatment. Importantly, phenylacetylamide could overcome the chemoresistance of MM cells against dexamethasone or melphalan. Thus, targeting CB2 may represent an attractive approach to treat cancers of immune origin.
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Agonistas de Receptores de Cannabinoides/farmacología , Proliferación Celular/efectos de los fármacos , Citoesqueleto/efectos de los fármacos , Mitosis/efectos de los fármacos , Mieloma Múltiple/tratamiento farmacológico , Mieloma Múltiple/genética , Receptor Cannabinoide CB2/genética , Actinas/genética , Apoptosis/efectos de los fármacos , Apoptosis/genética , Proteína Quinasa CDC2/genética , Caspasas/genética , Proteínas de Ciclo Celular/genética , Línea Celular Transformada , Proliferación Celular/genética , Citoesqueleto/genética , Silenciador del Gen/efectos de los fármacos , Humanos , Mitosis/genética , Receptor Cannabinoide CB2/agonistas , Receptores del Ligando Inductor de Apoptosis Relacionado con TNF/genética , Sindecano-1/genética , Tubulina (Proteína)/genética , Regulación hacia Arriba/efectos de los fármacos , Regulación hacia Arriba/genéticaRESUMEN
Ultimate success of hematopoietic stem cell transplantation (HSCT) depends not only on donor HSCs themselves but also on the host environment. Total body irradiation is a component in various host conditioning regimens for HSCT. It is known that ionizing radiation exerts "bystander effects" on nontargeted cells and that HSCs transplanted into irradiated recipients undergo proliferative exhaustion. However, whether irradiated recipients pose a proliferation-independent bystander effect on transplanted HSCs is unclear. In this study, we found that irradiated mouse recipients significantly impaired the long-term repopulating ability of transplanted mouse HSCs shortly (â¼ 17 hours) after exposure to irradiated hosts and before the cells began to divide. There was an increase of acute cell death associated with accelerated proliferation of the bystander hematopoietic cells. This effect was marked by dramatic down-regulation of c-Kit, apparently because of elevated reactive oxygen species. Administration of an antioxidant chemical, N-acetylcysteine, or ectopically overexpressing a reactive oxygen species scavenging enzyme, catalase, improved the function of transplanted HSCs in irradiated hosts. Together, this study provides evidence for an acute negative, yet proliferation-independent, bystander effect of irradiated recipients on transplanted HSCs, thereby having implications for HSCT in both experimental and clinical scenarios in which total body irradiation is involved.
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Efecto Espectador/efectos de la radiación , Trasplante de Células Madre Hematopoyéticas , Células Madre Hematopoyéticas/efectos de la radiación , Traumatismos Experimentales por Radiación/etiología , Enfermedad Aguda , Animales , Efecto Espectador/inmunología , Células Cultivadas , Células HEK293 , Células Madre Hematopoyéticas/fisiología , Humanos , Ratones , Ratones Congénicos , Ratones Endogámicos C57BL , Modelos Biológicos , Traumatismos Experimentales por Radiación/inmunología , Acondicionamiento Pretrasplante/efectos adversos , Irradiación Corporal Total/efectos adversosRESUMEN
High levels of reactive oxygen species (ROS) can exhaust hematopoietic stem cells (HSCs). Thus, maintaining a low state of redox in HSCs by modulating ROS-detoxifying enzymes may augment the regeneration potential of HSCs. Our results show that basal expression of manganese superoxide dismutase (MnSOD) and catalase were at low levels in long-term and short-term repopulating HSCs, and administration of a MnSOD plasmid and lipofectin complex (MnSOD-PL) conferred radiation protection on irradiated recipient mice. To assess the intrinsic role of elevated MnSOD or catalase in HSCs and hematopoietic progenitor cells, the MnSOD or catalase gene was overexpressed in mouse hematopoietic cells via retroviral transduction. The impact of MnSOD and catalase on hematopoietic progenitor cells was mild, as measured by colony-forming units (CFUs). However, overexpressed catalase had a significant beneficial effect on long-term engraftment of transplanted HSCs, and this effect was further enhanced after an insult of low-dose γ-irradiation in the transplant mice. In contrast, overexpressed MnSOD exhibited an insignificant effect on long-term engraftment of transplanted HSCs, but had a significant beneficial effect after an insult of sublethal irradiation. Taken together, these results demonstrate that HSC function can be enhanced by ectopic expression of ROS-detoxifying enzymes, especially after radiation exposure in vivo.
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Catalasa/metabolismo , Células Madre Hematopoyéticas/citología , Especies Reactivas de Oxígeno/metabolismo , Superóxido Dismutasa/metabolismo , Animales , Western Blotting , Catalasa/genética , Femenino , Citometría de Flujo , Regulación de la Expresión Génica , Vectores Genéticos , Trasplante de Células Madre Hematopoyéticas/métodos , Células Madre Hematopoyéticas/efectos de los fármacos , Células Madre Hematopoyéticas/efectos de la radiación , Humanos , Ratones , Ratones Endogámicos C57BL , Oxidación-Reducción , Retroviridae/genética , Células Madre , Superóxido Dismutasa/genética , Transducción GenéticaRESUMEN
Stem cell therapy represents one of the most promising approaches for tissue repair and regeneration. However, the full potential of stem cell therapy remains to be realized. One major challenge is the insufficient homing and retention of stem cells at the desired sites after in vivo delivery. Here, we provide a proof-of-principle demonstration of magnetic targeting and retention of human muscle-derived stem cells (hMDSCs) in vitro through magnetic force-mediated internalization of magnetic iron oxide nanoparticles (MIONs) and the use of a micropatterned magnet. We found that the magnetic force-mediated cellular uptake of MIONs occurs through an endocytic pathway, and the MIONs were exclusively localized in the lysosomes. The intracellular MIONs had no detrimental effect on the proliferation of hMDSCs or their multilineage differentiation, and no MIONs were translocated to other cells in a coculture system. Using hMDSCs and three other cell types including human umbilical vein endothelial cells (HUVECs), human dermal fibroblasts (HDFs), and HeLa cells, we further discovered that the magnetic force-mediated MION uptake increased with MION size and decreased with cell membrane tension. We found that the cellular uptake rate was initially increased with MION concentration in solution and approached saturation. These findings provide important insight and guidance for magnetic targeting of stem cells in therapeutic applications.
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It is widely believed that hematopoiesis after birth is established by hematopoietic stem cells (HSCs) in the bone marrow and that HSC-independent hematopoiesis is limited only to primitive erythro-myeloid cells and tissue-resident innate immune cells arising in the embryo. Here, surprisingly, we find that significant percentages of lymphocytes are not derived from HSCs, even in 1-year-old mice. Instead, multiple waves of hematopoiesis occur from embryonic day 7.5 (E7.5) to E11.5 endothelial cells, which simultaneously produce HSCs and lymphoid progenitors that constitute many layers of adaptive T and B lymphocytes in adult mice. Additionally, HSC lineage tracing reveals that the contribution of fetal liver HSCs to peritoneal B-1a cells is minimal and that the majority of B-1a cells are HSC independent. Our discovery of extensive HSC-independent lymphocytes in adult mice attests to the complex blood developmental dynamics spanning the embryo-to-adult transition and challenges the paradigm of HSCs exclusively underpinning the postnatal immune system.
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Células Endoteliales , Células Madre Hematopoyéticas , Animales , Ratones , Linaje de la Célula , Médula Ósea , HematopoyesisRESUMEN
Recent advances in developmental immunology have revealed a hematopoietic stem cell (HSC)-independent origin for various innate immune lineages, including mast cells (MCs). It is now established that adult bone marrow (BM) long-term HSCs do not regenerate MCs but, instead, the physiological production of MCs starts before the emergence of HSCs in the aorta-gonad-mesonephros (AGM) region and is mostly completed before birth. However, while the AGM region represents a major site of MC generation during ontogeny, whether the first emerging HSCs in the AGM or fetal liver (FL) possess the potential to regenerate MCs is unknown. Here, we combined three fate-mapping mouse models with detailed HSC transplantation assays to determine the potential of AGM and FL HSCs to produce MCs. We show that HSCs from E11.5 AGM and E12.5 FL efficiently repopulated MCs in recipients. In stark contrast, HSCs from ≥E14.5 FL failed to reconstitute MCs. An Endothelial (EC) fate-mapping study confirmed the EC origin of the majority of MCs. Additionally, our HSC-labeling showed that HSCs do not produce MCs in a physiological setting. Hence, although most MCs are generated and maintained via an HSC-independent pathway, the earliest HSCs to emerge in the AGM and seed the early FL can produce MCs, but only during a minimal time window. Our results challenge the stem cell theory in hematology and EC-derived mast cells may contribute to the pathogenesis of postnatal mast cell disorders.
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Mastocitos , Mesonefro , Animales , Médula Ósea , Gónadas , Células Madre Hematopoyéticas/metabolismo , RatonesRESUMEN
BACKGROUND: Bone morphogenetic protein 4 (BMP4) promotes the osteogenic differentiation and the bone regenerative potential of muscle-derived stem cells (MDSCs). BMP4 also promotes the self-renewal of both embryonic and somatic stem cells; however, BMP4 signaling activity significantly decreases with age. Cyclin-dependent kinase inhibitors P16INK4A (P16) and P18INK4C (P18) induce early G1-phase cell cycle blockade by targeting cyclin-dependent kinase 4/6. It is still unclear if BMP4 affects the bone regenerative potential of old MDSCs through regulation of P16 and P18 expression. METHODS: Young and old MDSCs were isolated from 3 week (young) and 2-year-old (old) mice. In vitro cell proliferation and multipotent differentiation were performed for young and old MDSCs both before and after BMP4/GFP transduction. Cell cycle genes were analyzed using Q-PCR. The bone regenerative potential of young and old MDSCs transduced with BMP4/GFP were compared using Micro-CT and histological analysis. The bone regenerative potential of young and old MDSCs was also compared between single and double transduction (higher BMP4 levels expression). The cell proliferation, mitochondrial function and osteogenic differentiation was also compared in vitro between cells that have been transduced with BMP4GFP (single and double transduction). The correlation of bone regeneration capacity of young and old MDSCs with P16 and P18 expression was further evaluated at 10 days after cell transplantation using histology and western blot analysis. RESULTS: Old murine MDSCs (MDSCs) exhibit reduced proliferation and multi-lineage differentiation potential with or without BMP4 stimulation, when compared to young murine MDSCs. Old MDSCs express significantly higher P16 and lower P18, with more cells in the G0/1 phase and fewer cells in the G2/M phase, compared to young MDSCs. Old MDSCs retrovirally transduced to express BMP4 regenerated less bone in a critical size skull defect in CD-1 nude mice when compared to young retrovirally transduced MDSCs expressing similar BMP4 levels and contribute less to the new regenerated new bone. Importantly, both young and old MDSCs can regenerate more bone when BMP4 expression levels are increased by double-transduction with the retroviral-BMP4/GFP. However, the bone regeneration enhancement with elevated BMP4 was more profound in old MDSCs (400% at 2 weeks) compared to young MDSCs (200%). Accordingly, P18 is upregulated while P16 is downregulated after BMP4 transduction. Double transduction did not further increase cell proliferation nor mitochondrial function but did significantly increase Osx expression in both young and old MDSCs. Old MDSCs had even significant higher Osx levels as compared to young MDSCs following double transduction, while a similar Alp expression was observed between young and old MDSCs after double transduction. In addition, at 10 days after cell transplantation, old MDSCs having undergone double transduction regenerated bone more rapidly as showed by Alcian blue and Von Kossa staining. Western blot assays demonstrated that old MDSCs after retro-BMP4/GFP double transduction have significantly lower P18 expression levels when compared to young BMP4-transduced MDSCs. In addition, P18 expression was slightly increased in old MDSCs after double transduction when compared to single transduction. P16 expression was not detectable for both young and two old BMP4/GFP transduced MDSCs groups. CONCLUSIONS: In summary, BMP4 can offset the adverse effect of aging on the osteogenic differentiation and the bone regenerative potential of old MDSCs via up-regulation of P18 and down-regulation P16 expression.
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Proteína Morfogenética Ósea 4/metabolismo , Regeneración Ósea , Osteogénesis , Animales , Proteína Morfogenética Ósea 4/genética , Regeneración Ósea/genética , Ciclo Celular , Diferenciación Celular , División Celular , Ratones , Ratones Desnudos , Músculos , Mioblastos , Osteogénesis/genéticaRESUMEN
Nonunion following bone fracture and segmental bone defects are challenging clinical conditions. To combat this clinical dilemma, development of new bone tissue engineering therapies using biocompatible materials to deliver bone growth factors is desirable. This aim of this study is to use a heparin/polycation coacervate sustained-release platform to compare 5 bone morphogenetic proteins (BMPs) for promoting bone defect healing in a critical sized calvarial defect model. The in vitro 3D osteogenic pellet cultures assays demonstrated that BMPs 2, 4, 6, 7 and 9 all enhanced mineralization in vitro compared to the control group. BMP2 resulted in higher mineralized volume than BMP4 and BMP6. All BMPs and the control group activated the pSMAD5 signaling pathway and expressed osterix (OSX). The binding of BMP2 with coacervate significantly increased the coacervate average particle size. BMP2, 4, 6, & 7 bound to coacervate significantly increased the Zeta potential of the coacervate while BMP9 binding showed insignificant increase. Furthermore, using a monolayer culture osteogenic assay, it was found that hMDSCs cultured in the coacervate BMP2 osteogenic medium expressed higher levels of RUNX2, OSX, ALP and COX-2 compared to the control and BMPs 4, 6, 7 & 9. Additionally, the coacervate complex can be loaded with up to 2 µg of BMP proteins for sustained release. In vivo, when BMPs were delivered using the coacervate sustained release system, BMP2 was identified to be the most potent BMP promoting bone regeneration and regenerated 10 times of new bone than BMPs 4, 6 & 9. BMP7 also stimulated robust bone regeneration when compared to BMPs 4, 6 & 9. The quality of the newly regenerated bone by all BMPs delivered by coacervate is equivalent to the host bone consisting of bone matrix and bone marrow with normal bone architecture. Although the defect was not completely healed at 6 weeks, coacervate sustain release BMPs, particularly BMP2 and BMP7, could represent a new strategy for treatment of bone defects and non-unions.
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Proteína Morfogenética Ósea 2 , Heparina , Proteína Morfogenética Ósea 2/metabolismo , Proteínas Morfogenéticas Óseas , Regeneración Ósea , Preparaciones de Acción Retardada , Osteogénesis , PolielectrolitosRESUMEN
Radiation therapy (RT) to the chest increases the patients' risk of cardiovascular disease (CVD). A complete understanding of the mechanisms by which RT induces CVD could lead to specific preventive, therapeutic approaches. It is becoming evident that both genotoxic chemotherapy agents and radiation induce mitochondrial dysfunction and cellular senescence. Notably, one of the common phenotypes observed in cancer survivors is accelerated senescence, and immunosenescence is closely related to both cancer risk and CVD development. Therefore, suppression of immunosenescence can be an ideal target to prevent cancer treatment-induced CVD. However, the mechanism(s) by which cancer treatments induce immunosenescence are incompletely characterized. We isolated peripheral blood mononuclear cells (PBMCs) before and 3 months after RT from 16 thoracic cancer patients. We characterized human immune cell lineages and markers of senescence, DNA damage response (DDR), efferocytosis, and determinants of clonal hematopoiesis of indeterminant potential (CHIP), using mass cytometry (CyTOF). We found that the frequency of the B cell subtype was decreased after RT. Unsupervised clustering of the CyTOF data identified 138 functional subsets of PBMCs. Compared with baseline, RT increased TBX21 (T-bet) expression in the largest B cell subset of Ki67-/DNMT3a+naïve B cells, and T-bet expression was correlated with phosphorylation of p90RSK expression. CD38 expression was also increased in naïve B cells (CD27-) and CD8+ effector memory CD45RA T cells (TEMRA). In vitro, we found the critical role of p90RSK activation in upregulating (1) CD38+/T-bet+ memory and naïve B, and myeloid cells, (2) senescence-associated ß-gal staining, and (3) mitochondrial reactive oxygen species (ROS) after ionizing radiation (IR). These data suggest the crucial role of p90RSK activation in immunosenescence. The critical role of p90RSK activation in immune cells and T-bet induction in upregulating atherosclerosis formation has been reported. Furthermore, T-bet directly binds to the CD38 promoter region and upregulates CD38 expression. Since both T-bet and CD38 play a significant role in the process of immunosenescence, our data provide a cellular and molecular mechanism that links RT-induced p90RSK activation and the immunosenescence with T-bet and CD38 induction observed in thoracic cancer patients treated by RT and suggests that targeting the p90RSK/T-bet/CD38 pathway could play a role in preventing the radiation-associated CVD and improving cancer prognosis by inhibiting immunosenescence.
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Background. Fibrin sealant has been used as a scaffold to deliver genetically modified human muscle-derived stem cells (hMDSCs) for bone regeneration. Alternatively, autologous blood clots are safe, economic scaffolds. This study compared autologous blood clot (BC) with fibrin sealant (FS) as a scaffold to deliver lenti-BMP2/GFP-transduced hMDSCs for bone regeneration. Methods. In vitro osteogenic differentiation was performed using 3D pellet culture and evaluated using microCT and Von Kossa staining. The lenti-GFP transduced cells were then mixed with human blood for evaluation of osteogenic differentiation. Furthermore, a murine critical- sized calvarial defect model was utilized to compare BC and FS scaffolds for lenti-BMP2/GFP-transduced hMDSCs mediated bone regeneration and evaluated with micro-CT and histology. Results. Lenti-BMP2/GFP transduced hMDSCs formed significantly larger mineralized pellets than non-transduced hMDSCs. hMDSCs within the human blood clot migrated out and differentiated into ALP+ osteoblasts. In vivo, BC resulted in significantly less new bone formation within a critical-sized calvarial bone defect than FS scaffold, despite no difference observed for GFP+ donor cells, osteoclasts, and osteoblasts in the newly formed bone. Conclusions. Human lenti-BMP2/GFP-transduced hMDSCs can efficiently undergo osteogenic differentiation in vitro. Unexpectedly, the newly regenerated bone in BC group was significantly less than the FS group. The autologous blood clot scaffold is less efficacious for delivering stem cells for bone regeneration than fibrin sealant.
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BACKGROUND: Bone marrow stimulation (BMS) via microfracture historically has been a first-line treatment for articular cartilage lesions. However, BMS has become less favorable because of resulting fibrocartilage formation. Previous studies have shown that angiogenesis blockade promotes cartilage repair. Bevacizumab is a Food and Drug Administration-approved medication used clinically to prevent angiogenesis. HYPOTHESIS: The intra-articular injection of bevacizumab would prevent angiogenesis after BMS and lead to improved cartilage repair with more hyaline-like cartilage. STUDY DESIGN: Controlled laboratory study. METHODS: The dose of bevacizumab was first optimized in a rabbit osteochondral defect model with BMS. Then, 48 rabbits (n = 8/group/time point) were divided into 3 groups: osteochondral defect (defect), osteochondral defect + BMS (BMS group), and osteochondral defect + BMS + bevacizumab intra-articular injection (bevacizumab group). Rabbits were sacrificed at either 6 or 12 weeks after surgery. Three-dimensional (3D) micro-computed tomography (microCT), macroscope score, modified O'Driscoll histology scores, collagen type 2, Herovici staining, and hematoxylin and eosin staining were performed. Angiogenesis markers were also evaluated. RESULTS: The intra-articular dose of 12.5 mg/0.5 mL bevacizumab was found to be effective without deleteriously affecting the subchondral bone. Intra-articular injection of bevacizumab resulted in significantly improved cartilage repair for the bevacizumab group compared with the BMS or the defect group based on 3D microCT, the macroscope score (both P < .05), the modified O'Driscoll histology score (P = .0034 and P = .019 vs defect and BMS groups, respectively), collagen type 2, Herovici staining, and hematoxylin and eosin staining at 6 weeks. Cartilage in the bevacizumab group had significantly more hyaline cartilage than did that in other groups. At 12 weeks, the cartilage layer regenerated in all groups; however, the bevacizumab group showed more hyaline-like morphology, as demonstrated by microCT, histology scores (P < .001 and .0225 vs defect and BMS groups, respectively), histology, and immunohistochemistry. The bevacizumab injection did not significantly change mRNA expressions of smooth muscle actin, vascular endothelial growth factor, or hypoxia-inducible factor-1 alpha. CONCLUSION: Intra-articular injection of bevacizumab significantly enhanced the quality and quantity of hyaline-like cartilage after BMS in a rabbit model. Future large-animal and human studies are necessary to evaluate the clinical effect of this therapy, which may lead to improved BMS outcomes and thus the durability of the regenerated cartilage. CLINICAL RELEVANCE: The use of bevacizumab may be an important clinical adjunct to improve BMS-mediated cartilage repair.
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Médula Ósea , Cartílago Articular , Animales , Bevacizumab/farmacología , Inyecciones Intraarticulares , Conejos , Factor A de Crecimiento Endotelial Vascular , Microtomografía por Rayos XRESUMEN
RAD21 (also known as KIAA0078, NXP1, HR21, Mcd1, Scc1, and hereafter called RAD21), an essential gene, encodes a DNA double-strand break (DSB) repair protein that is evolutionarily conserved in all eukaryotes from budding yeast to humans. RAD21 protein is a structural component of the highly conserved cohesin complex consisting of RAD21, SMC1a, SMC3, and SCC3 [STAG1 (SA1) and STAG2 (SA2) in metazoans] proteins, involved in sister chromatid cohesion. This function is essential for proper chromosome segregation, post-replicative DNA repair, and prevention of inappropriate recombination between repetitive regions. In interphase, cohesin also functions in the control of gene expression by binding to numerous sites within the genome. In addition to playing roles in the normal cell cycle and DNA DSB repair, RAD21 is also linked to the apoptotic pathways. Germline heterozygous or homozygous missense mutations in RAD21 have been associated with human genetic disorders, including developmental diseases such as Cornelia de Lange syndrome (CdLS) and chronic intestinal pseudo-obstruction (CIPO) called Mungan syndrome, respectively, and collectively termed as cohesinopathies. Somatic mutations and amplification of the RAD21 have also been widely reported in both human solid and hematopoietic tumors. Considering the role of RAD21 in a broad range of cellular processes that are hot spots in neoplasm, it is not surprising that the deregulation of RAD21 has been increasingly evident in human cancers. Herein, we review the biology of RAD21 and the cellular processes that this important protein regulates and discuss the significance of RAD21 deregulation in cancer and cohesinopathies.
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Proteínas de Ciclo Celular/genética , Proteínas Cromosómicas no Histona/genética , Reparación del ADN/genética , Proteínas de Unión al ADN/genética , Neoplasias/genética , Apoptosis/genética , Esófago de Barrett/genética , Roturas del ADN de Doble Cadena , Síndrome de Cornelia de Lange/genética , Hematopoyesis/genética , Humanos , Seudoobstrucción Intestinal/genética , Meiosis/genética , Neoplasias/patología , CohesinasRESUMEN
This study investigated the role of muscle damage in bone defect healing using skull and tibial double-defect and tibial fracture models in dystrophin-/-/Utrophin-/- double-knockout (dKO-Hom) mice. The skull and tibia bone defect and fracture healing was monitored using micro-CT, histology, immuohistochemistry and quantitative PCR. We found the skull defect healing is not impaired while the tibial defect healing was delayed at day 7 in the dKO-Hom group compared to wild-type (WT) group as revealed by micro-CT. Mechanistically, the number of osteoclasts and osteoblasts significantly decreased in the defect area in dKO-Hom group compared to WT group on day 21. DKO-Hom mice showed higher mortality after fracture (6/12) and significantly impaired fracture healing compared to the other groups as revealed by the micro-CT parameters of the calluses. Histology showed higher osteoclast number in the calluses of dKO-Hom mice than other groups. Furthermore, dKO-Hom mice showed down-regulation of 15-Pgdh, Il-4, Bmp7, and Bmp9 at 10 days after tibia fracture and BMP6 and 7 in the muscle. In conclusion, the long bone defect and fracture healing are impaired in dKO-Hom mice which demonstrated significantly muscle sarcopenia and related with disturbance of osteoclastogenesis and osteoblastogenesis. The impaired tibial fracture healing was associated with down-regulation of several genes in the muscle.
RESUMEN
BACKGROUND: Microfracture or bone marrow stimulation (BMS) is often the first choice for clinical treatment of cartilage injuries; however, fibrocartilage, not pure hyaline cartilage, has been reported because of the development of fibrosis in the repair tissue. Transforming growth factor ß1 (TGF-ß1), which can promote fibrosis, can be inhibited by losartan and potentially be used to reduce fibrocartilage. HYPOTHESIS: Blocking TGF-ß1 would improve cartilage healing in a rabbit knee BMS model via decreasing the amount of fibrocartilage and increasing hyaline-like cartilage formation. STUDY DESIGN: Controlled laboratory study. METHODS: An osteochondral defect was made in the patellar groove of 48 New Zealand White rabbits. The rabbits were divided into 3 groups: a defect group (defect only), a BMS group (osteochondral defect + BMS), and a BMS + losartan group (osteochondral defect + BMS + losartan). For the rabbits in the BMS + losartan group, losartan was administrated orally from the day after surgery through the day of euthanasia. Rabbits were sacrificed 6 or 12 weeks postoperatively. Macroscopic appearance, microcomputed tomography, histological assessment, and TGF-ß1 signaling pathway were evaluated at 6 and 12 weeks postoperatively. RESULTS: The macroscopic assessment of the repair revealed that the BMS + losartan group was superior to the other groups tested. Microcomputed tomography showed superior healing of the bony defect in the BMS + losartan group in comparison with the other groups. Histologically, fibrosis in the repair tissue of the BMS + losartan group was significantly reduced when compared with the other groups. Results obtained with the modified O'Driscoll International Cartilage Repair Society grading system yielded significantly superior scores in the BMS + losartan group as compared with both the defect group and the BMS group (F value: 15.8, P < .001, P = .012, respectively). TGF-ß1 signaling and TGF-ß-activated kinase 1 of the BMS + losartan group were significantly suppressed in the synovial tissues. CONCLUSION: By blocking TGF-ß1 with losartan, the repair cartilage tissue after BMS was superior to the other groups and consisted primarily of hyaline cartilage. These results should be easily translated to the clinic because losartan is a Food and Drug Administration-approved drug and it can be combined with the BMS technique for optimal repair of chondral defects. CLINICAL RELEVANCE: Biologically regulated marrow stimulation by blocking TGF-ß1 (oral intake of losartan) provides superior repair via decreasing fibrocartilage formation and resulting in hyaline-like cartilage as compared with outcomes from BMS only.
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Bloqueadores del Receptor Tipo 1 de Angiotensina II , Cartílago Articular , Cartílago Hialino , Losartán , Factor de Crecimiento Transformador beta1 , Administración Oral , Bloqueadores del Receptor Tipo 1 de Angiotensina II/farmacología , Animales , Médula Ósea , Cartílago Articular/efectos de los fármacos , Hialina , Cartílago Hialino/efectos de los fármacos , Losartán/farmacología , Conejos , Factor de Crecimiento Transformador beta1/fisiología , Microtomografía por Rayos XRESUMEN
Mechanisms underlying the generation of induced pluripotent stem cells (iPSC) and keeping iPSC stability remain to be further defined. Accumulated evidences showed that iPSC reprogramming may be controlled by the cell-division-rate-dependent model. Here we reported effects of absence of mouse p27 or p18 on iPSC generation efficiency and genomic stability. Expression levels of cyclin-dependent kinases inhibitors (CDKIs), p21, p27, and p18 decreased during iPSC reprogramming. Like p21 loss, p27 or p18 deficiency significantly promoted efficiency of iPSC generation, whereas ectopic expression of p27, p18, or treatment with CDK2 or CDK4 inhibitors repressed the reprogramming rate, suggesting that CDKIs-regulated iPSC reprogramming is directly related with their functions as CDK inhibitors. However, unlike p21 deletion, absence of p27 or p18 did not increase DNA damage or chromosomal aberrations during iPSC reprogramming and at iPSC stage. Our data not only support that cell cycle regulation is critical for iPSC reprogramming, but also reveal the distinction of CDKIs in somatic cell reprogramming.