ALPL regulates pro-angiogenic capacity of mesenchymal stem cells through ATP-P2X7 axis controlled exosomes secretion.

BACKGROUND: Early-onset bone dysplasia is a common manifestation of hypophosphatasia (HPP), an autosomal inherited disease caused by ALPL mutation. ALPL ablation induces prototypical premature bone ageing characteristics, resulting in impaired osteogenic differentiation capacity of human bone marrow mesenchymal stem cells (hBMMSCs). As angiogenesis is tightly coupled with osteogenesis, it also plays a necessary role in sustaining bone homeostasis. We have previously observed a decrease in expression of angiogenesis marker gene CD31 in the metaphysis of long bone in Alpl+/- mice. However, the role of ALPL in regulation of angiogenesis in bone has remained largely unknown. METHODS: Exosomes derived from Normal and HPP hBMMSCs were isolated and identified by ultracentrifugation, transmission electron microscopy, and nanoparticle size measurement. The effects of ALPL on the angiogenic capacity of hBMMSCs from HPP patients were assessed by immunofluorescence, tube formation, wound healing and migration assay. exo-ELISA and Western Blot were used to evaluate the exosomes secretion of hBMMSCs from HPP, and the protein expression of VEGF, PDGFBB, Angiostatin and Endostatin in exosomes respectively. RESULTS: We verified that ALPL ablation resulted in impaired pro-angiogenic capacity of hBMMSCs, accounting for reduced migration and tube formation of human umbilical vein endothelial cells, as the quantities and proteins composition of exosomes varied with ALPL expression. Mechanistically, loss of function of ALPL enhanced ATP release. Additional ATP, in turn, led to markedly elevated level of ATP receptor P2X7, which consequently promoted exosomes secretion, resulting in a decreased capacity to promote angiogenesis. Conversely, inhibition of P2X7 increased the angiogenic induction capacity by preventing excessive release of anti-angiogenic exosomes in ALPL deficient-hBMMSCs. CONCLUSION: The ALPL-ATP axis regulates the pro-angiogenic ability of hBMMSCs by controlling exosomes secretion through the P2X7 receptor. Thus, P2X7 may be proved as an effective therapeutic target for accelerating neovascularization in ALPL-deficient bone defects.

TC14012 enhances the anti-fibrosis effects of UC-MSCs on the liver by reducing collagen accumulation and ameliorating inflammation.

BACKGROUND: Mesenchymal stem cells (MSCs) are attracting attention as a promising cell-based therapy for the treatment of liver fibrosis or cirrhosis. However, the strategies and potential mechanisms of MSCs therapy need further investigation. The CXCL12/CXCR4/CXCR7 chemokine axis is well known to regulate cell migration and is involved in the regulation of liver fibrosis. This study aims to treat MSCs with a CXCR7-specific agonist to evaluate its therapeutic effects on hepatic fibrosis and potential mechanisms. METHODS: TC14012, a potent agonist of CXCR7, has been used to pretreat human umbilical cord-derived MSCs (UC-MSCs) and assess its effect on proliferation, apoptosis, migration, immunoregulation, and gene regulatory network. Then, CCl4-induced liver fibrosis mice models were used to evaluate the therapeutic effect and mechanism of TC14012-treated UC-MSCs for treating hepatic fibrosis. RESULTS: TC14012 increased CXCR7 expression in UC-MSCs. Notably, co-culture of liver sinusoidal endothelial cells (LSEC) with TC14012-pretreated UC-MSCs increased CXCR7 expression in LSEC. Additionally, TC14012 promoted cell migration and mediated the immunoregulation of UC-MSCs. Compared to UC-MSCs without TC14012 pretreatment, UC-MSCs treated with TC14012 ameliorated live fibrosis by restoring CXCR7 expression, reducing collagen fibril accumulation, inhibiting hepatic stellate cells activation, and attenuating the inflammatory response. CONCLUSION: This study suggests that TC14012 pretreatment can enhance the therapeutic effects of UC-MSCs on liver fibrosis, mainly by promoting the migration and immunoregulation of MSCs.

GSK3ß rephosphorylation rescues ALPL deficiency-induced impairment of odontoblastic differentiation of DPSCs.

BACKGROUND: Premature exfoliation of the deciduous teeth is a common manifestation in childhood patients with hypophosphatasia (HPP), which is an autosomal inherited disease caused by ALPL mutations. Dysplasia of the cementum, dentin, and alveolar bone has been proposed to be the main reasons for the exfoliation of teeth, while the extraordinarily complex intracellular mechanisms remain elusive. Dental pulp stem cells (DPSCs) have been demonstrated to successfully regenerate functional pulp-dentin-like tissue. Dental pulp cells derived from HPP patients impaired mineralization; however, insight into the deeper mechanism is still unclear. METHODS: The effects of ALPL on odontoblastic differentiation of DPSCs from HPP patient were assessed by Alizarin Red staining, immunofluorescent staining, Western blot and RT-PCR, and micro-CT assays. RESULT: Here, we found DPSCs from HPP patient exhibited low ALP activity and impaired odontoblastic differentiation. Meanwhile, we found that loss of function of ALPL reduced phosphorylation of GSK3ß in DPSCs. While GSK3ß rephosphorylation improved odontoblastic differentiation of HPP DPSCs with LiCl treatment. Finally, we demonstrated systemic LiCl injection ameliorated tooth-associated defects in ALPL+/- mice by enhanced phosphorylation of GSK3ß in the teeth. CONCLUSIONS: Our study indicates that ALPL regulates odontoblastic differentiation of DPSCs and provides useful information for understanding how ALPL deficiency led to tooth dysplasia and, ultimately, may inform efforts at improvement tooth defects in HPP patients.

Impaired autophagy triggered by HDAC9 in mesenchymal stem cells accelerates bone mass loss.

BACKGROUND: Bone mass loss in aging is linked with imbalanced lineage differentiation of bone marrow mesenchymal stem cells (BMMSCs). Recent studies have proved that histone deacetylases (HDACs) are regarded as key regulators of bone remodeling. However, HDACs involve in regulating BMMSC bio-behaviors remain elusive. Here, we investigated the ability of HDAC9 on modulation of autophagy and its significance in lineage differentiation of BMMSCs. METHODS: The effects of HDAC9 on lineage differentiation of BMMSCs and autophagic signaling were assessed by various biochemical (western blot and ChIP assay), morphological (TEM and confocal microscopy), and micro-CT assays. RESULTS: Sixteen-month mice manifested obvious bone mass loss and marrow fat increase, accompanied with decreased osteogenic differentiation and increased adipogenic differentiation of BMMSCs. Further, the expression of HDAC9 elevated in bone and BMMSCs. Importantly, HDAC9 inhibitors recovered the lineage differentiation abnormality of 16-month BMMSCs and reduced p53 expression. Mechanistically, we revealed that HDAC9 regulated the autophagy of BMMSCs by controlling H3K9 acetylation in the promoters of the autophagic genes, ATG7, BECN1, and LC3a/b, which subsequently affected their lineage differentiation. Finally, HDAC9 inhibition improved endogenous BMMSC properties and promoted the bone mass recovery of 16-month mice. CONCLUSIONS: Our data demonstrate that HDAC9 is a key regulator in a variety of bone mass by regulating autophagic activity in BMMSCs and thus a potential target of age-related bone loss treatment.

Increase in HDAC9 suppresses myoblast differentiation via epigenetic regulation of autophagy in hypoxia.

Extremely reduced oxygen (O2) levels are detrimental to myogenic differentiation and multinucleated myotube formation, and chronic exposure to high-altitude hypoxia has been reported to be an important factor in skeletal muscle atrophy. However, how chronic hypoxia causes muscle dysfunction remains unknown. In the present study, we found that severe hypoxia (1% O2) significantly inhibited the function of C2C12 cells (from a myoblast cell line). Importantly, the impairment was continuously manifested even during culture under normoxic conditions for several passages. Mechanistically, we revealed that histone deacetylases 9 (HDAC9), a member of the histone deacetylase family, was significantly increased in C2C12 cells under hypoxic conditions, thereby inhibiting intracellular autophagy levels by directly binding to the promoter regions of Atg7, Beclin1, and LC3. This phenomenon resulted in the sequential dephosphorylation of GSK3ß and inactivation of the canonical Wnt pathway, impairing the function of the C2C12 cells. Taken together, our results suggest that hypoxia-induced myoblast dysfunction is due to aberrant epigenetic regulation of autophagy, and our experimental evidence reveals the possible molecular pathogenesis responsible for some muscle diseases caused by chronic hypoxia and suggests a potential therapeutic option.

Genome­wide analysis and prediction of functional long noncoding RNAs in osteoblast differentiation under simulated microgravity.

Long noncoding RNAs (lncRNAs) have been regarded as important regulators in numerous biological processes during cell development. However, the holistic lncRNA expression pattern and potential functions during osteoblast differentiation under simulated microgravity remain unknown. In the present study, a high throughput microarray assay was performed to detect lncRNA and mRNA expression profiles during MC3TC­E1 pre­osteoblast cell osteo­differentiation under simulated microgravity. The expression of 857 lncRNAs and 2,264 mRNAs was signi-ficantly altered when MC3T3­E1 cells were exposed to simulated microgravity. A relatively consistent distribution pattern on the chromosome and a co­expression network were observed between the differentially­expressed lncRNAs and mRNAs. Genomic context analysis further identified 132 differentially­expressed lncRNAs and nearby coding gene pairs. Subsequently, 3 lncRNAs were screened out for their possible function in osteoblast differentiation, based on their co­expression association and potential cis­acting regulatory pattern with the deregulated mRNAs. The present study aimed to provide a comprehensive understanding of and a foundation for future studies into lncRNA function in mechanical signal­mediated osteoblast differentiation.

Moderate exercise based on artificial gravity preserves orthostatic tolerance and exercise capacity during short-term head-down bed rest.

Numerous countermeasures have been proposed to minimize microgravity-induced physical deconditioning, but their benefits are limited. The present study aimed to investigate whether personalized aerobic exercise based on artificial gravity (AG) mitigates multisystem physical deconditioning. Fourteen men were assigned to the control group (n=6) and the countermeasure group (CM, n=8). Subjects in the CM group were exposed to AG (2 Gz at foot level) for 30 min twice daily, during which time cycling exercise of 80-95 % anaerobic threshold (AT) intensity was undertaken. Orthostatic tolerance (OT), exercise tests, and blood assays were determined before and after 4 days head-down bed rest (HDBR). Cardiac systolic function was measured every day. After HDBR, OT decreased to 50.9 % and 77.5 % of pre-HDBR values in control and CM groups, respectively. Exercise endurance, maximal oxygen consumption, and AT decreased to 96.5 %, 91.5 % and 91.8 % of pre-HDBR values, respectively, in the control group. Nevertheless, there were slight changes in the CM group. HDBR increased heart rate, sympathetic activity, and the pre-ejection period, but decreased plasma volume, parasympathetic activity and left-ventricular ejection time in the control group, whereas these effects were eliminated in the CM group. Aldosterone had no change in the control group but increased significantly in the CM group. Our study shows that 80-95 % AT aerobic exercise based on 2 Gz of AG preserves OT and exercise endurance, and affects body fluid regulation during short-term HDBR. The underlying mechanisms might involve maintained cardiac systolic function, preserved plasma volume, and improved sympathetic responses to orthostatic stress.

Simulated Microgravity Promotes Angiogenesis through RhoA-Dependent Rearrangement of the Actin Cytoskeleton.

BACKGROUND/AIMS: Microgravity leads to hydrodynamic alterations in the cardiovascular system and is associated with increased angiogenesis, an important aspect of endothelial cell behavior to initiate new vessel growth. Given the critical role of Rho GTPase-dependent cytoskeleton rearrangement in cell migration, small GTPase RhoA might play a potential role in microgravity-induced angiogenesis. METHODS: We examined the organization of actin filaments by FITC-conjugated phalloidin staining, as well as the expression and activity of RhoA by quantitative PCR and Western blot, in human umbilical vein endothelial cells (HUVECs) under normal gravity and simulated microgravity. Effect of simulated microgravity on the wound closure and tube formation in HUVECs, and their dependence on RhoA, were also analyzed by cell migration and tube formation assays. RESULTS: We show that in HUVECs actin filaments are disorganized and RhoA activity is reduced by simulated microgravity. Blocking RhoA activity either by C3 transferase Rho inhibitor or siRNA knockdown mimicked the effect of simulated microgravity on inducing actin filament disassembly, followed by enhanced wound closure and tube formation in HUVECs, which closely resembled effects seen on microgravity-treated cells. In contrast, overexpressing RhoA in microgravity-treated HUVECs restored the actin filaments, and decreased wound closure and tube formation abilities. CONCLUSION: These results suggest that RhoA inactivation is involved in the actin rearrangement-associated angiogenic responses in HUVECs during simulated microgravity.

Mechanical stress regulates osteogenic differentiation and RANKL/OPG ratio in periodontal ligament stem cells by the Wnt/ß-catenin pathway.

BACKGROUND: The balance between osteoblastic and osteoclastic activity is critical in orthodontic tooth movement (OTM). Mesenchymal stem cells (MSCs) play an important role in maintaining bone homeostasis, and periodontal ligament stem cells (PDLSCs) are tissue-specific MSCs in the periodontal ligament. However, whether PDLSCs are required for periodontal tissue remodeling during OTM is not fully understood. METHODS: Here, we used PDGFRα and Nestin to trace PDLSCs during OTM in rats. We treat human PDLSCs with 100kpa static pressure for 1h or 12h in vitro, and examined the phenotypic changes and expression of RANKL and OPG in these cells. RESULTS: In vivo, we found that positive signals of PDGFRα and Nestin in the PDL gradually increased and then decreased on the pressure side to which pressure was applied. In vitro, the osteogenic differentiation of PDLSCs was significantly increased after force treatment for 1h relative to 12h. In contrast, the expression ratio of RANKL/OPG was reduced at 1h and significantly increased at 12h. Furthermore, we found that the Wnt/ß-catenin pathway was dynamically activated in the PDL and in PDLSCs after mechanical stimulation. Importantly, the canonical Wnt pathway inhibitor DKK1 blocked the osteogenesis effect and rescued the ratio of RANKL/OPG in PDLSCs under force treatment for 1h. CONCLUSIONS: Our findings reveal that PDLSCs participate in OTM and that the Wnt/ß-catenin pathway maintains bone homeostasis during tooth movement by regulating the balance between osteoblastic and osteoclastic activity. GENERAL SIGNIFICANCE: We describe a novel potential mechanism related to tooth movement.

The Impact of Simulated Weightlessness on Endothelium-Dependent Angiogenesis and the Role of Caveolae/Caveolin-1.

BACKGROUND/AIMS: The potential role of caveolin-1 in modulating angiogenesis in microgravity environment is unexplored. METHODS: Using simulated microgravity by clinostat, we measured the expressions and interactions of caveolin-1 and eNOS in human umbilical vein endothelial cells. RESULTS: We found that decreased caveolin-1 expression is associated with increased expression and phosphorylation levels of eNOS in endothelial cells stimulated by microgravity, which causes a dissociation of eNOS from caveolin-1 complexes. As a result, microgravity induces cell migration and tube formation in endothelial cell in vitro that depends on the regulations of caveolin-1. CONCLUSION: Our study provides insight for the important endothelial functions in altered gravitational environments.

Exposure to a continuous low dose of tetrachlorodibenzo-p-dioxin impairs the development of the tooth root in lactational rats and alters the function of apical papilla-derived stem cells.

OBJECTIVES: Ubiquitous environmental pollutants such as 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) cause abnormalities in reproduction and development. TCDD inhibits the development of teeth, and its effects depend on its dose and the developmental stage of the tooth. Our aim here was to investigate the effect of lower doses of TCDD on the development of the tooth root in vivo and in vitro. DESIGN: We observed tooth root development in lactational rats exposed to continuous low doses of TCDD starting on postnatal day 6 using Mico-CT analyses and histopathological examinations. And then the characteristics of stem cells derived from the apical papilla (SCAPs) were evaluated and compared with SCAPs induced by lower doses of TCDD both in vitro and in vivo. RESULTS: The results of experiments showed that rat pups exposed to low dose TCDD at prenatal stage developed, dentine hypoplasia, and hypomineralization. Further, TCDD impaired the functions of SCAPs in vivo by inhibiting cell proliferation and osteogenic and odontogenic differentiation. The impairment of SCAPs after TCDD exposure was accompanied by increased expression of AHR, down-regulation of the expression of Runx2, and alkaline phosphatase, suggesting that the AHR pathway mediated the effects of TCDD. CONCLUSION: These results provide the first insights into the toxicity of TCDD, which adversely affects the development of the tooth root through indirectly altering the function of SCAPs.

Clinorotation enhances autophagy in vascular endothelial cells.

Individuals exposed to extended periods of spaceflight or prolonged 6° head-down-tilt bed rest often suffer from health hazards represented by cardiovascular deconditioning. Many studies have reported that alterations in vascular endothelial cells contribute to cardiovascular dysfunction induced by microgravity. Autophagy, a lysosomal degradation pathway, serves an adaptive role for survival, differentiation, and development in cellular homeostasis, and can be triggered by various environmental stimuli. However, whether autophagy can be induced in endothelial cells by real or simulated microgravity remains to be determined. This study was designed to investigate the effects of simulated microgravity on the activation of autophagy in human umbilical vein endothelial cells (HUVECs). We report here that clinorotation, a simulated model of microgravity, enhances autophagosome formation, increases LC3 and beclin-1 expression, and promotes the conversion of LC3-I to LC3-II in HUVECs. These results demonstrate that simulated microgravity for 48 h activates autophagy of vascular endothelial cells.

Spatiotemporal expression of D10Wsu52e in the developing mouse pancreas.

D10Wsu52e is a recently discovered and highly conserved mouse gene. FAAP, the protein encoded by D10Wsu52e, participates in regulation of integrin-based focal adhesions. To explore the function of FAAP in pancreas development, we assessed the spatiotemporal expression of D10Wsu52e, paxillin and vinculin in the developing mouse pancreas through quantitative RT-PCR, in situ hybridization and histochemistry methods. Our results show that, at e9.5 and e10.5, D10Wsu52e mRNA is mainly expressed in the brain, optic vesicles, otic vesicles, limb buds, somites and gut, and also in the pancreatic buds at e10.5. Subsequently, D10Wsu52e mRNA is expressed mainly in the epithelial progenitors at e12.5, then decreases in the endocrine precursors and ductal cells, whereas still maintains in the exocrine precursors until e15.5. At e17.5, D10Wsu52e mRNA is highly expressed in exocrine acinar cells, but weakly in ductal and endocrine cells. Furthermore, the expression patterns of paxillin and vinculin mRNAs are similar to D10Wsu52e from e12.5 to e15.5, which suggests that D10Wsu52e may be related to the acinar development, adhesion and migration of progenitors and endocrine precursors.