ALK2使内皮细胞转化为类干细胞的机制

内皮细胞向间充质细胞转分化（endothelial-to-mesenchymal transition,EndMT）过程是血管内皮细胞转化为类干细胞的基础,持续激活状态的ALK2在EndMT过程起到关键的促进作用,EndMT发生后内皮细胞能获得类干细胞的表现型.TGF-β2和BMP4作为特异性配体激活ALK2,下调某些内皮细胞标志物并上调某些间质细胞标志物,促进EndMT过程.此外,编码ALK2的基因序列在206号氨基酸上突变形成R206H ALK2后作用类似持续激活状态ALK2,同样也能达到促进内皮细胞向间充质细胞转分化的目的.     

Potent inhibition of heterotopic ossification by nuclear retinoic acid receptor-γ agonists

Heterotopic ossification consists of ectopic bone formation within soft tissues after surgery or trauma. It can have debilitating consequences, but there is no definitive cure. Here we show that heterotopic ossification was essentially prevented in mice receiving a nuclear retinoic acid receptor-γ (RAR-γ) agonist. Side effects were minimal, and there was no significant rebound effect. To uncover the mechanisms of these responses, we treated mouse mesenchymal stem cells with an RAR-γ agonist and transplanted them into nude mice. Whereas control cells formed ectopic bone masses, cells that had been pretreated with the RAR-γ agonist did not, suggesting that they had lost their skeletogenic potential. The cells became unresponsive to rBMP-2 treatment in vitro and showed decreases in phosphorylation of Smad1, Smad5 and Smad8 and in overall levels of Smad proteins. In addition, an RAR-γ agonist blocked heterotopic ossification in transgenic mice expressing activin receptor-like kinase-2 (ALK2) Q207D, a constitutively active form of the receptor that is related to ALK2 R206H found in individuals with fibrodysplasia ossificans progressiva. The data indicate that RAR-γ agonists are potent inhibitors of heterotopic ossification in mouse models and, thus, may also be effective against injury-induced and congenital heterotopic ossification in humans.     

Fibrodysplasia Ossificans Progressiva-related Activated Activin-like Kinase Signaling Enhances Osteoclast Formation during Heterotopic Ossification in Muscle Tissues

Fibrodysplasia ossificans progressiva is characterized by extensive ossification within muscle tissues, and its molecular pathogenesis is responsible for the constitutively activating mutation (R206H) of the bone morphogenetic protein type 1 receptor, activin-like kinase 2 (ALK2). In this study, we investigated the effects of implanting ALK2 (R206H)-transfected myoblastic C2C12 cells into nude mice on osteoclast formation during heterotopic ossification in muscle and subcutaneous tissues. The implantation of ALK2 (R206H)-transfected C2C12 cells with BMP-2 in nude mice induced robust heterotopic ossification with an increase in the formation of osteoclasts in muscle tissues but not in subcutaneous tissues. The implantation of ALK2 (R206H)-transfected C2C12 cells in muscle induced heterotopic ossification more effectively than that of empty vector-transfected cells. A co-culture of ALK2 (R206H)-transfected C2C12 cells as well as the conditioned medium from ALK2 (R206H)-transfected C2C12 cells enhanced osteoclast formation in Raw264.7 cells more effectively than those with empty vector-transfected cells. The transfection of ALK2 (R206H) into C2C12 cells elevated the expression of transforming growth factor (TGF)-β, whereas the inhibition of TGF-β signaling suppressed the enhanced formation of osteoclasts in the co-culture with ALK2 (R206H)-transfected C2C12 cells and their conditioned medium. In conclusion, this study demonstrated that the causal mutation transfection of fibrodysplasia ossificans progressiva in myoblasts enhanced the formation of osteoclasts from its precursor through TGF-β in muscle tissues.     

ALK1 signalling analysis identifies angiogenesis related genes and reveals disparity between TGF-β and constitutively active receptor induced gene expression

Background

TGF-β1 is an important angiogenic factor involved in the different aspects of angiogenesis and vessel maintenance. TGF-β signalling is mediated by the TβRII/ALK5 receptor complex activating the Smad2/Smad3 pathway. In endothelial cells TGF-β utilizes a second type I receptor, ALK1, activating the Smad1/Smad5 pathway. Consequently, a perturbance of ALK1, ALK5 or TβRII activity leads to vascular defects. Mutations in ALK1 cause the vascular disorder hereditary hemorrhagic telangiectasia (HHT).

Methods

The identification of ALK1 and not ALK5 regulated genes in endothelial cells, might help to better understand the development of HHT. Therefore, the human microvascular endothelial cell line HMEC-1 was infected with a recombinant constitutively active ALK1 adenovirus, and gene expression was studied by using gene arrays and quantitative real-time PCR analysis.

Results

After 24 hours, 34 genes were identified to be up-regulated by ALK1 signalling. Analysing ALK1 regulated gene expression after 4 hours revealed 13 genes to be up- and 2 to be down-regulated. Several of these genes, including IL-8, ET-1, ID1, HPTPη and TEAD4 are reported to be involved in angiogenesis. Evaluation of ALK1 regulated gene expression in different human endothelial cell types was not in complete agreement. Further on, disparity between constitutively active ALK1 and TGF-β1 induced gene expression in HMEC-1 cells and primary HUVECs was observed.

Conclusion

Gene array analysis identified 49 genes to be regulated by ALK1 signalling and at least 14 genes are reported to be involved in angiogenesis. There was substantial agreement between the gene array and quantitative real-time PCR data. The angiogenesis related genes might be potential HHT modifier genes. In addition, the results suggest endothelial cell type specific ALK1 and TGF-β signalling.     

Stage-specific secretion of HMGB1 in cartilage regulates endochondral ossification

High mobility group box 1 protein (HMGB1) is a chromatin protein that has a dual function as a nuclear factor and as an extracellular factor. Extracellular HMGB1 released by damaged cells acts as a chemoattractant, as well as a proinflammatory cytokine, suggesting that HMGB1 is tightly connected to the process of tissue organization. However, the role of HMGB1 in bone and cartilage that undergo remodeling during embryogenesis, tissue repair, and disease is largely unknown. We show here that the stage-specific secretion of HMGB1 in cartilage regulates endochondral ossification. We analyzed the skeletal development of Hmgb1(-/-) mice during embryogenesis and found that endochondral ossification is significantly impaired due to the delay of cartilage invasion by osteoclasts, osteoblasts, and blood vessels. Immunohistochemical analysis revealed that HMGB1 protein accumulated in the cytosol of hypertrophic chondrocytes at growth plates, and its extracellular release from the chondrocytes was verified by organ culture. Furthermore, we demonstrated that the chondrocyte-secreted HMGB1 functions as a chemoattractant for osteoclasts and osteoblasts, as well as for endothelial cells, further supporting the conclusion that Hmgb1(-/-) mice are defective in cell invasion. Collectively, these findings suggest that HMGB1 released from differentiating chondrocytes acts, at least in part, as a regulator of endochondral ossification during osteogenesis.     

The interplay of osteogenesis and hematopoiesis: expression of a constitutively active PTH/PTHrP receptor in osteogenic cells perturbs the establishment of hematopoiesis in bone and of skeletal stem cells in the bone marrow

The ontogeny of bone marrow and its stromal compartment, which is generated from skeletal stem/progenitor cells, was investigated in vivo and ex vivo in mice expressing constitutively active parathyroid hormone/parathyroid hormone-related peptide receptor (PTH/PTHrP; caPPR) under the control of the 2.3-kb bone-specific mouse Col1A1 promoter/enhancer. The transgene promoted increased bone formation within prospective marrow space, but delayed the transition from bone to bone marrow during growth, the formation of marrow cavities, and the appearance of stromal cell types such as marrow adipocytes and cells supporting hematopoiesis. This phenotype resolved spontaneously over time, leading to the establishment of marrow containing a greatly reduced number of clonogenic stromal cells. Proliferative osteoprogenitors, but not multipotent skeletal stem cells (mesenchymal stem cells), capable of generating a complete heterotopic bone organ upon in vivo transplantation were assayable in the bone marrow of caPPR mice. Thus, PTH/PTHrP signaling is a major regulator of the ontogeny of the bone marrow and its stromal tissue, and of the skeletal stem cell compartment.     

TGF-beta1 on osteoimmunology and the bone component cells

TGF-β1 is an immunoregulatory cytokine that regulates immune cell proliferation, survival, differentiation, and migration. Compelling evidence has demonstrated a strong association between the immune and skeletal systems (so called Osteoimmunology), such as the critical role of TGF-β1 in the development and maintenance of the skeletal tissue. This review provides an overview of the mechanisms in which TGF-β1 interacts with bone component cells, such as osteoblasts, osteoclasts, chondrocytes, mesenchymal stem cells, and hematopoietic stem cells, in concert with other cytokines and hormones.     

Role of CTGF/HCS24/ecogenin in skeletal growth control

Connective tissue growth factor/hypertrophic chondrocyte-specific gene product 24 (CTGF/Hcs24) is a multifunctional growth factor for chondrocytes, osteoblasts, and vascular endothelial cells. CTGF/Hcs24 promotes the proliferation and maturation of growth cartilage cells and articular cartilage cells in culture and hypertrophy of growth cartilage cells in culture. The factor also stimulates the proliferation and differentiation of cultured osteoblastic cells. Moreover, CTGF/Hcs24 promotes the adhesion, proliferation, and migration of vascular endothelial cells, as well as induces tube formation by the cells and strong angiogenesis in vivo. Because angiogenesis is critical for the replacement of cartilage with bone at the final stage of endochondral ossification and because gene expression of CTGF/Hcs24 predominates in hypertrophic chondrocytes in the physiological state, a major physiological role for this factor should be the promotion of the entire process of endochondral ossification, with the factor acting on the above three types of cells as a paracrine factor. Thus, CTGF/Hcs24 should be called "ecogenin: endochondral ossification genetic factor." In addition to hypertrophic chondrocytes, osteoblasts activated by various stimuli including wounding also express a significantly high level of CTGF/Hcs24. These findings in conjunction with in vitro findings about osteoblasts mentioned above suggest the involvement of CTGF/Hcs24 in intramembranous ossification and bone modeling/remodeling. Because angiogenesis is also critical for intramembranous ossification and bone remodeling, CTGF/Hcs24 expressed in endothelial cells activated by various stimuli including wounding may also play important roles in direct bone formation. In conclusion, although the most important physiological role of CTGF/Hcs24 is ecogenin action, the factors also play important roles in skeletal growth and modeling/remodeling via its direct action on osteoblasts under both physiological and pathological conditions.     

Viscoelastic properties of human mesenchymally-derived stem cells and primary osteoblasts, chondrocytes, and adipocytes

The mechanical properties of single cells play important roles in regulating cell-matrix interactions, potentially influencing the process of mechanotransduction. Recent studies also suggest that cellular mechanical properties may provide novel biological markers, or "biomarkers," of cell phenotype, reflecting specific changes that occur with disease, differentiation, or cellular transformation. Of particular interest in recent years has been the identification of such biomarkers that can be used to determine specific phenotypic characteristics of stem cells that separate them from primary, differentiated cells. The goal of this study was to determine the elastic and viscoelastic properties of three primary cell types of mesenchymal lineage (chondrocytes, osteoblasts, and adipocytes) and to test the hypothesis that primary differentiated cells exhibit distinct mechanical properties compared to adult stem cells (adipose-derived or bone marrow-derived mesenchymal stem cells). In an adherent, spread configuration, chondrocytes, osteoblasts, and adipocytes all exhibited significantly different mechanical properties, with osteoblasts being stiffer than chondrocytes and both being stiffer than adipocytes. Adipose-derived and mesenchymal stem cells exhibited similar properties to each other, but were mechanically distinct from primary cells, particularly when comparing a ratio of elastic to relaxed moduli. These findings will help more accurately model the cellular mechanical environment in mesenchymal tissues, which could assist in describing injury thresholds and disease progression or even determining the influence of mechanical loading for tissue engineering efforts. Furthermore, the identification of mechanical properties distinct to stem cells could result in more successful sorting procedures to enrich multipotent progenitor cell populations.     

Metformin attenuates trauma‐induced heterotopic ossification via inhibition of Bone Morphogenetic Protein signalling

AMP‐activated protein kinase (AMPK) is an intracellular sensor of energy homoeostasis that is activated under energy stress and suppressed in energy surplus. AMPK activation leads to inhibition of anabolic processes that consume ATP. Osteogenic differentiation is a process that highly demands ATP during which AMPK is inhibited. The bone morphogenetic proteins (BMPs) signalling pathway plays an essential role in osteogenic differentiation. The present study examines the inhibitory effect of metformin on BMP signalling, osteogenic differentiation and trauma‐induced heterotopic ossification. Our results showed that metformin inhibited Smad1/5 phosphorylation induced by BMP6 in osteoblast MC3T3‐E1 cells, concurrent with up‐regulation of Smad6, and this effect was attenuated by knockdown of Smad6. Furthermore, we found that metformin suppressed ALP activity and mineralization of the cells, an event that was attenuated by the dominant negative mutant of AMPK and mimicked by its constitutively active mutant. Finally, administration of metformin prevented the trauma‐induced heterotopic ossification in mice. In conjuncture, AMPK activity and Smad6 and Smurf1 expression were enhanced by metformin treatment in the muscle of injured area, concurrently with the reduction of ALK2. Collectively, our study suggests that metformin prevents heterotopic ossification via activation of AMPK and subsequent up‐regulation of Smad6. Therefore, metformin could be a potential therapeutic drug for heterotopic ossification induced by traumatic injury.     

Heparanase mRNA expression during fracture repair in mice

Bone fracture healing takes place through endochondral ossification where cartilaginous callus is replaced by bony callus. Vascular endothelial growth factor (VEGF) is a requisite for endochondral ossification, where blood vessel invasion of cartilaginous callus is crucial. Heparanase is an endoglucuronidase that degrades heparan sulfate proteoglycans (HSPG) and releases heparin-binding growth factors including VEGF as an active form. To investigate the role of heparanase in VEGF recruitment during fracture healing, the expression of heparanase mRNA and VEGF, and vessel formation were examined in mouse fractured bone. On days 5 and 7 after the fracture, when mesenchymal cells proliferated and differentiated into chondrocytes, heparanase mRNA was detected in osteo(chondro)clasts and their precursors, but not in the inflammatory phase (day 3). On day 10, both VEGF and HSPG were produced by hypertrophic chondrocytes of the cartilaginous callus and by osteoblasts of the bony callus; numerous osteo(chondro)clasts resorbing the cartilage expressed strong heparanase signals. Adjacent to the cartilage resorption sites, angiogenesis with CD31-positive endothelial cells and osteogenesis with osteonectin-positive osteoblasts were observed. On days 14 and 21, osteoclasts in the woven bone tissue expressed heparanase mRNA. These data suggest that by producing heparanase osteo(chondro)clasts contribute to the recruitment of the active form of VEGF. Thus osteo(chondro)clasts may promote local angiogenesis as well as callus resorption in endochondral ossification during fracture healing.     

Isolation of colorectal cancer stem-like cells

This study is aimed at isolating colorectal cancer stem-like cells in vitro using a neurosphere assay method employed in isolating gliobastoma multiforme tumor cells. This was followed with confirmation of the isolated cells by flow cytometry, pluripotent genes expression and in vivo tumorigenicity assay. Using this culture assay, stem-like and non-stem-like CRC cells were isolated and expanded in vitro from purchased Balb/c mice induced with CT26 colorectal cancer (CRC) cell line. The procedure includes an initial mechanical dissociation and chemical digestion of tumor tissue and subsequently plating the resulting single cell suspension in serum-free medium (SFM) or serum-containing medium (SCM). This selectively permits growth of cancer stem-like cells in SFM and eliminates non-stem-like cancer cells through the process of anoikis or apoptosis. CRC stem cells derived cultures proliferated as non-adherent spheres in vitro in different shapes and sizes. These cells expressed cell surface markers previously reported for tumor stem cells, including CD44, CD133, CD166 and CD26 and formed tumors when implanted in severe combined immunodeficient mice in a concentration dependent manner. Importantly, the stem-like cells had self-renewal properties with significantly higher expression of the pluripotent stem cell genes NANOG, OCT4, and SOX2 compared to the adherent non-stem cells. Collectively, the results of this study indicate that SFM is a defined culture medium that enriches for CRC stem-like cells and represents a suitable in vitro model for the study of CRC stem-like cells. This finding may be useful in developing therapeutic strategies aimed at eradicating the tumorigenic subpopulation within colorectal cancer.     

Critical roles of the TGF-β type I receptor ALK5 in perichondrial formation and function, cartilage integrity, and osteoblast differentiation during growth plate development

TGF-β has been implicated in the proliferation and differentiation of chondrocytes and osteoblasts. However, the in vivo function of TGF-β in skeletal development is unclear. In this study, we investigated the role of TGF-β signaling in growth plate development by creating mice with a conditional knockout of the TGF-β type I receptor ALK5 (ALK5^CKO) in skeletal progenitor cells using Dermo1-Cre mice. ALK5^CKO mice had short and wide long bones, reduced bone collars, and trabecular bones. In ALK5^CKO growth plates, chondrocytes proliferated and differentiated, but ectopic cartilaginous tissues protruded into the perichondrium. In normal growth plates, ALK5 protein was strongly expressed in perichondrial progenitor cells for osteoblasts, and in a thin chondrocyte layer located adjacent to the perichondrium in the peripheral cartilage. ALK5^CKO growth plates had an abnormally thin perichondrial cell layer and reduced proliferation and differentiation of osteoblasts. These defects in the perichondrium likely caused the short bones and ectopic cartilaginous protrusions. Using tamoxifen-inducible Cre-ER™-mediated ALK5-deficient primary calvarial cell cultures, we found that TGF-β signaling promoted osteoprogenitor proliferation, early differentiation, and commitment to the osteoblastic lineage through the selective MAPKs and Smad2/3 pathways. These results demonstrate the important roles of TGF-β signaling in perichondrium formation and differentiation, as well as in growth plate integrity during skeletal development.     

HIF-1α在骨组织细胞代谢及骨疾病中的调控作用

低氧诱导因子-1α(HIF-1α)是调节细胞对低氧应答的关键因子,可在氧含量降低时被激活,能够调节氧代谢、糖酵解等多种生理活动。骨代谢主要包括骨形成和骨吸收作用,均受到氧浓度等多种因素的调控。HIF-1α在细胞代谢、骨组织生理及病理过程的调控中起着重要的作用,能够增加骨组织的低氧耐受能力,调节骨形成和矿化过程。该文主要综述了HIF-1α对成骨细胞、破骨细胞、骨髓间充质干细胞、软骨细胞等骨组织细胞的调控,对骨血管形成过程的影响,以及对肿瘤骨转移、股骨头坏死、异位骨化等病理过程的调节作用,为探讨HIF-1α对骨代谢的调控和相关疾病的治疗提供参考。     

Wnt/beta-catenin signaling in mesenchymal progenitors controls osteoblast and chondrocyte differentiation during vertebrate skeletogenesis

Chondrocytes and osteoblasts are two primary cell types in the skeletal system that are differentiated from common mesenchymal progenitors. It is believed that osteoblast differentiation is controlled by distinct mechanisms in intramembranous and endochondral ossification. We have found that ectopic canonical Wnt signaling leads to enhanced ossification and suppression of chondrocyte formation. Conversely, genetic inactivation of beta-catenin, an essential component transducing the canonical Wnt signaling, causes ectopic formation of chondrocytes at the expense of osteoblast differentiation during both intramembranous and endochondral ossification. Moreover, inactivation of beta-catenin in mesenchymal progenitor cells in vitro causes chondrocyte differentiation under conditions allowing only osteoblasts to form. Our results demonstrate that beta-catenin is essential in determining whether mesenchymal progenitors will become osteoblasts or chondrocytes regardless of regional locations or ossification mechanisms. Controlling Wnt/beta-catenin signaling is a common molecular mechanism underlying chondrocyte and osteoblast differentiation and specification of intramembranous and endochondral ossification.     

Antisense-Oligonucleotide Mediated Exon Skipping in Activin-Receptor-Like Kinase 2: Inhibiting the Receptor That Is Overactive in Fibrodysplasia Ossificans Progressiva

Fibrodysplasia ossificans progressiva (FOP) is a rare heritable disease characterized by progressive heterotopic ossification of connective tissues, for which there is presently no definite treatment. A recurrent activating mutation (c.617G→A; R206H) of activin receptor-like kinase 2 (ACVR1/ALK2), a BMP type I receptor, has been shown as the main cause of FOP. This mutation constitutively activates the BMP signaling pathway and initiates the formation of heterotopic bone. In this study, we have designed antisense oligonucleotides (AONs) to knockdown mouse ALK2 expression by means of exon skipping. The ALK2 AON could induce exon skipping in cells, which was accompanied by decreased ALK2 mRNA levels and impaired BMP signaling. In addition, the ALK2 AON potentiated muscle differentiation and repressed BMP6-induced osteoblast differentiation. Our results therefore provide a potential therapeutic approach for the treatment of FOP disease by reducing the excessive ALK2 activity in FOP patients.     

The expression of the fibrillar collagen genes during fracture healing: heterogeneity of the matrices and differentiation of the osteoprogenitor cells

The cells that express the genes for the fibrillar collagens, types I, II, III and V, during callus development in rabbit tibial fractures healing under stable and unstable mechanical conditions were localized. The fibroblast-like cells in the initial fibrous matrix express types I, III and V collagen mRNAs. Osteoblasts, and osteocytes in the newly formed membranous bone under the periosteum, express the mRNAs for types I, III and V collagens, but osteocytes in the mature trabeculae express none of these mRNAs. Cartilage formation starts at 7 days in calluses forming under unstable mechanical conditions. The differentiating chondrocytes express both types I and II collagen mRNAs, but later they cease expression of type I collagen mRNA. Both types I and II collagens were located in the cartilaginous areas. The hypertrophic chondrocytes express neither type I, nor type II, collagen mRNA. Osteocalcin protein was located in the bone and in some cartilaginous regions. At 21 days, irrespective of the mechanical conditions, the callus consists of a layer of bone; only a few osteoblasts lining the cavities now express type I collagen mRNA.We suggest that osteoprogenitor cells in the periosteal tissue can differentiate into either osteoblasts or chondrocytes and that some cells may exhibit an intermediate phenotype between osteoblasts and chondrocytes for a short period. The finding that hypertrophic chondrocytes do not express type I collagen mRNA suggests that they do not transdifferentiate into osteoblasts during endochondral ossification in fracture callus.     

Stem cells from adipose tissue

This is a review of the growing scientific interest in the developmental plasticity and therapeutic potential of stromal cells isolated from adipose tissue. Adipose-derived stem/stromal cells (ASCs) are multipotent somatic stem cells that are abundant in fat tissue. It has been shown that ASCs can differentiate into several lineages, including adipose cells, chondrocytes, osteoblasts, neuronal cells, endothelial cells, and cardiomyocytes. At the same time, adipose tissue can be harvested by a minimally invasive procedure, which makes it a promising source of adult stem cells. Therefore, it is believed that ASCs may become an alternative to the currently available adult stem cells (e.g. bone marrow stromal cells) for potential use in regenerative medicine. In this review, we present the basic information about the field of adipose-derived stem cells and their potential use in various applications.