Human memory-like NK cells migrating to tuberculous pleural fluid via IP-10/CXCR3 and SDF-1/CXCR4 axis produce IFN-γ in response to Bacille Calmette Guerin

We have previously shown that human memory-like NK cells were persistent in tuberculous pleurisy but it was unclear how NK cells migrated into the pleural fluids. At present, we found that NK cells from TB pleural fluid cells (PFCs) expressed significantly higher levels of CXCR3 and CXCR4 than NK cells from PBMCs. Migration assay demonstrated that IP-10 and SDF-1 induced more migration of NK cells from PFCs than PBMCs. CD45RO⁺ or CD45RO⁻ NK cells from PFCs were co-cultured with autologous monocytes and stimulated with BCG. The results showed CD45RO⁺ but not CD45RO⁻ NK cells produced significantly higher levels of IFN-γ, which was IL-12-dependent since anti-IL-12Rβ1 mAbs could significantly inhibit the IFN-γ by NK cells. Collectively, our data demonstrated that human Mycobacterium tuberculosis-specific NK cells were migrated into the local site of TB infection mainly via IP-10/CXCR3 and SDF-1/CXCR4 axis, memory-like NK cells might display an important role against M. tuberculosis infection.     

Chondrogenic pre-induction of human mesenchymal stem cells on β-TCP: Enhanced bone quality by endochondral heterotopic bone formation

New techniques to heal bone defects include the combination of bone substitute materials with mesenchymal stem cells (MSC). To find solutions not hampered by low material resorbability or high donor variability of human MSC, the potency of such composites is usually evaluated by heterotopic bone formation assays in immunocompromised animals. The aim of this study was to investigate whether resorbable phase-pure β-tricalcium-phosphate (β-TCP) could support heterotopic bone formation by MSC comparable to partially resorbable hydroxyapatite/tricalcium-phosphate (HA/TCP). Furthermore, in light of disappointing results with osteogenic in vitro priming of MSC, we tested whether chondrogenic pre-induction of constructs may allow for enhanced bone formation by triggering the endochondral pathway. β-TCP granules of three different sizes and HA/TCP were seeded with MSC and transplanted subcutaneously into immunocompromised mice either immediately or after a chondrogenic pre-induction for 6 weeks. After 8 weeks, explants were analysed by histology. β-TCP seeded with unprimed MSC revealed intramembranous bone formation without haematopoietic marrow with 3.8-fold more bone formed with granules smaller than 0.7 mm than with 0.7–1.4 mm particles (p ? 0.018). Chondrogenic pre-induction of β-TCP/MSC composites resulted in collagen type II and proteoglycan-rich cartilage-like tissue which, after transplantation, underwent endochondral ossification, yielding ectopic bone produced by human cells while haematopoietic marrow was derived from the mouse. Transdifferentiation of MSC-derived chondrocytes to osteoblasts or direct osteogenesis of cartilage-resident MSC is postulated to explain the human origin of new bone. In conclusion, β-TCP was significantly more osteo-permissive (p = 0.004) than HA/TCP for human MSC, and chondrogenic priming of β-TCP/MSC represented a superior approach capable of supporting full bone formation, including marrow organization.     

Bone marrow-derived mesenchymal stem cells differentiate to hepatic myofibroblasts by transforming growth factor-β1 via sphingosine kinase/sphingosine 1-phosphate (S1P)/S1P receptor axis

Sphingosine kinase (SphK) is involved in numerous biological processes, including cell growth, proliferation, and differentiation. However, whether SphK participates in the differentiation of bone marrow-derived mesenchymal stem cells (BMSCs) to myofibroblasts has been unknown. In a carbon tetrachloride-treated mouse model, SphK1 was expressed in BMSCs in damaged liver. Furthermore, mRNA expression of both SphK1 and transforming growth factor β1 (TGF-β1) was significantly increased after liver injury, with a positive correlation between them. The SphK inhibitor SKI significantly blocked BMSC differentiation to myofibroblasts during liver injury (the proportion of BMSC-derived myofibroblasts decreased markedly, compared with no SKI treatment) and attenuated the extent of liver fibrosis. Using primary mouse BMSCs, we demonstrated that TGF-β1 induced BMSC differentiation to myofibroblasts, accompanied by the up-regulation of SphK1 and modulation of sphingosine 1-phosphate (S1P) receptor (S1PR) expression. Notably, pharmacological or siRNA-mediated inhibition of SphK1 abrogated the prodifferentiating effect of TGF-β1. Moreover, using either S1PR subtype-specific antagonists or specific siRNAs, we found that the prodifferentiating effect of TGF-β1 was mediated by S1PR(1) and S1PR(3). These data suggest that SphK1 activation by TGF-β1 leads to differentiation of BMSCs to myofibroblasts mediated by S1PR(1) and S1PR(3) up-regulation, thus providing new information on the mechanisms by which TGF-β1 gives rise to fibrosis and opening new perspectives for pharmacological treatment of liver fibrosis.     

The delayed addition of human mesenchymal stem cells to pre-formed endothelial cell networks results in functional vascularization of a collagen–glycosaminoglycan scaffold in vivo

This paper demonstrates a method to engineer, in vitro, a nascent microvasculature within a collagen–glycosaminoglycan scaffold with a view to overcoming the major issue of graft failure due to avascular necrosis of tissue-engineered constructs. Human umbilical vein endothelial cells (ECs) were cultured alone and in various co-culture combinations with human mesenchymal stem cells (MSCs) to determine their vasculogenic abilities in vitro. Results demonstrated that the delayed addition of MSCs to pre-formed EC networks, whereby MSCs act as pericytes to the nascent vessels, resulted in the best developed vasculature. The results also demonstrate that the crosstalk between ECs and MSCs during microvessel formation occurs in a highly regulated, spatio-temporal fashion, whereby the initial seeding of ECs results in platelet derived growth factor (PDGF) release; the subsequent addition of MSCs 3 days later leads to a cessation in PDGF production, coinciding with increased vascular endothelial cell growth factor expression and enhanced vessel formation. Functional assessment of these pre-engineered constructs in a subcutaneous rat implant model demonstrated anastomosis between the in vitro engineered vessels and the host vasculature, with significantly increased vascularization occurring in the co-culture group. This study has thus provided new information on the process of in vitro vasculogenesis within a three-dimensional porous scaffold for tissue engineering and demonstrates the potential for using these vascularized scaffolds in the repair of critical sized bone defects.     

Mechanism of bone marrow mesenchymal stem cells secreting miR-26a exosomes affecting high glucose-induced skin fibroblasts function by regulating TLR4/NF-κB signaling

Inflammation Research - The aim of this study was to investigate the molecular mechanism of human bone marrow mesenchymal stem cells (hMSCs) secreting miR-26a exosomes on the function of skin...     

Estrogen regulates osteogenic differentiation of human periodontal ligament stem cells via Wnt/beta-catenin signaling pathway北大核心

BACKGROUND: Estrogen can promote the osteogenic differentiation of human periodontal ligament stem cells (hPDLSCs), but the molecular mechanism is unclear. OBJECTIVE: To study the regulatory effect of estrogen on the osteogenic differentiation of hPDLSCs via Wnt/β-catenin signaling pathway. METHODS: The hPDLSCs were isolated and purified by digestion method combined with limited dilution clone method. Three experimental groups were set as follows: osteogenic induction only (control group); 1×10-7 mol/L estrogen with osteogenic induction (estrogen group); and 100 µg/L Wnt3a protein with osteogenic induction (Wnt3a group). Alkaline phosphatase activity was detected at 1, 3, 5, 7 days of osteogenic induction. Western blot was used to detect the expression of Wnt/β-catenin signaling pathway related proteins β-catenin, P-GSK-3β, GSK-3β, CyclinD1 and osteoblast-related proteins Runx2 and OCN after 7 days of osteogenic induction. RESULTS AND CONCLUSION: The activity of ALP in all groups increased with time. The expression level of ALP in the estrogen group and Wnt3a group was higher than that in the control group at 1, 3, 5 and 7 days of induction (P < 0.05), while there was no significant difference between the former two groups (P > 0.05). The western blot results showed that the expression levels of β-catenin, P-GSK-3β, CyclinD1, Runx2 and OCN in the estrogen group and Wnt3a group were higher than those in the control group (P < 0.05), while the expression of GSK-3β was lower than that in the control group (P < 0.05). But there were no differences in the expression of Wnt/β-catenin signaling pathway related proteins and mid-late osteogenic markers between estrogen group and Wnt3a group (P > 0.05). To conclude, estrogen can enhance the osteogenic differentiation of hPDLSCs, and the underlying mechanism is likely to activate the Wnt/β-catenin signaling pathway in activated hPDLSCs exposed to estrogen. © 2018, Journal of Clinical Rehabilitative Tissue Engineering Research. All rights reserved.     

SIRT6/NF-κB signaling axis in ginsenoside Rg1-delayed hematopoietic stem/progenitor cell senescence

Objective: To investigate the role of SIRT6/NF-κB signaling axis in ginsenoside Rg1-delayed hematopoietic stem/progenitor cell senescence and to provide theoretical and experimental evidence for delaying HSC/HPC senescence pathway. Methods: After the separation and purification by immunomagnetic sorting, Sca-1⁺HSC/HPC was divided into: normal control group; aging group; positive control group; Rg1 delaying group and Rg1 treatment group. Senescence-associated β-galactosidase (SA-β-Gal) staining, flow cytometry analysis of cell cycle and hematopoietic progenitor cells mixed colony (CFU-Mix) culture were performed to determine the delaying or curing roles of Rg1 in Sca-1⁺HSC/HPC senescence. Quantitative PCR and Western blotting were used to detect the mRNA and protein expression of senescence regulatory molecules, such as SIRT6 and NF-κB. Results: Compared with the aging group, the positive rate of SA-β-gal staining cells and the proportion of cells in G1 phase decreased; the number of CFU-Mix increased; mRNA and protein expression of SIRT6 increased; mRNA and protein expression of NF-κB was down-regulated in Rg1 delaying and treatment groups; the changes of the indicators in Rg1 delaying group were more significant than those in Rg1 treatment group. Conclusion: Rg1 may fight against Sca-1⁺HSC/HPC senescence induced by t-BHP through regulating SIRT6-NF-κB signaling pathway.     

Repair of critical-sized rat calvarial defects using genetically engineered bone marrow-derived mesenchymal stem cells overexpressing hypoxia-inducible factor-1α

The processes of angiogenesis and bone formation are coupled both temporally and spatially during bone repair. Bone marrow-derived mesenchymal stem cells (BMSCs) have been effectively used to heal critical-size bone defects. Enhancing their ability to undergo angiogenic and osteogenic differentiation will enhance their potential use in bone regeneration. Hypoxia-inducible factor-1α (HIF-1α) has recently been identified as a major regulator of angiogenic-osteogenic coupling. In this study, we tested the hypothesis that HIF-1α gene therapy could be used to promote the repair of critical-sized bone defects. Using lentivirus-mediated delivery of wild-type (HIF) or constitutively active HIF-1α (cHIF), we found that in cultured BMSCs in vitro, HIF and cHIF significantly enhanced osteogenic and angiogenic mRNA and protein expression when compared with the LacZ group. We found that HIF-1α-overexpressing BMSCs dramatically improved the repair of critical-sized calvarial defects, including increased bone volume, bone mineral density, blood vessel number, and blood vessel area in vivo. These data confirm the essential role of HIF-1α modified BMSCs in angiogenesis and osteogenesis in vitro and in vivo.     

Histone acetyltransferase inhibitor C646 reverses epithelial to mesenchymal transition of human peritoneal mesothelial cells via blocking TGF-β1/Smad3 signaling pathway in vitro

Peritoneal fibrosis resulting from long-term peritoneal dialysis is a major cause of failure of peritoneal ultrafiltration function and main reason of dropout from peritoneal dialysis. Epithelial-mesenchymal transition (EMT) of peritoneal mesochelial cells (HPMCs) is a major contributor of peritoneal fibrosis. Recently, the association between histone acetylation and kinds of fibrosis including liver, lung and kidney fibrosis is well established. Thus, in this study we tried to profile whether histone acetylation is also operates EMT process in HPMCs and what’s the regulatory mechanism. We established an EMT model of HPMCs through high glucose treatment. And hyperacetylation of H3 histone was found using western blot in EMT model. After treated with C646, a histone acetyltransferase (HAT) inhibitor, high glucose-induced EMT in HPMCs was counteracted. To further understand the molecular mechanism of C646 rescues high glucose-induced EMT, CHIP-qPCRwas used to examine the modulation of histone H3 acetylation at promoters of series signaling target genes. We found that the H3 acetylation level at TGF-β1 gene promoter was down-regulation by C646 treatment. Moreover, we also found that TGF-β1/Smad3 signaling was blocked. Hence, our results suggest that histone H3 acetylation activated TGF-β1/Smad3 signaling during EMT of HPMCs, and C646 can rescue the mesenchymal phenotype transition. These findings may provide a novel pathogenic mechanism and therapeutic target for peritoneal fibrosis.     

黄芪甲苷对EPCs中SDF-1α/CXCR4的调控作用

目的:探讨黄芪甲苷(AS-IV)对内皮祖细胞(EPCs)中CXC趋化因子受体4(CXCR4)和基质细胞衍生因子1α(SDF-1α)的调控作用及其作用机制。方法:体外培养大鼠骨髓源性EPCs,观察应用AS-IV及CXCR4的特异性阻断剂AMD3100后EPCs的增殖、黏附、迁移、凋亡和管状结构形成能力的变化,并分析AS-IV对EPCs中SDF-1α及CXCR4的mRNA和蛋白,以及p-CXCR4蛋白水平变化的影响。结果:AS-IV可以显著提升EPCs的增殖、黏附、迁移和管状结构形成能力,减轻EPCs的凋亡,上调EPCs中SDF-1α和CXCR4的mRNA和蛋白及p-CXCR4蛋白的水平(P0.05);AMD3100可以阻断AS-IV对CXCR4的mRNA和蛋白及p-CXCR4蛋白水平的上调作用,但不影响AS-IV对SDF-1α的mRNA和蛋白水平的上调作用。结论:AS-IV可能通过调控EPCs中SDF-1α/CXCR4的表达而增强EPCs的生物学作用。     

Phenotypic Knockout of CXCR4 on Molt-4 with SDF-1α/54 Attached with KDEL

Objective ： To investigate the mechanism of phenotypic knockout of CXCR4 on T-cell leukemia cell line Molt-4 via SDF-1α/54/KDEL intrakine technology, which the mutant SDF-1α/54, human stromal cell-derived Faceor-1 （SDF-1α） was deleted its C- terminal α-helix and attached with a endoplasimc reticulum retention signal 4-peptide- KDEL encoding gene, so that retain the newly synthesized receptor CXCR4 within the Molt-4 cells endoplasmic reticulum. Methods： The recombinant vector pEGFP-C3/SDF- 1α/54/KDEL were transfected into Cos-7 cells by liposome, SDF-1α/54/KDEL fusion protein was confirmed with western blot. The recombinant plasmids were transfected transiently into Molt-4 by electroporation. Results：Western blot confirmed SDF-1α/54/KDEL expression in Cos-7. A dramatic downregulation of CXCR4 expression on Molt-4 was demonstrated by flow cytometric （FCM） analysis. Conelusion：SDF-1α/54/KDEL and SDF- 1αKDEL have no significant deviation for phenotypic knockout of CXCR4. These suggest that the phenotypic knockout effects of SDF-1α/54 against CXCR4 are not influenced by deleting of SDF-1α helix in the C-terminal.     

MiR-21 suppresses endothelial progenitor cell proliferation by activating the TGFβ signaling pathway via downregulation of WWP1

Endothelial damage is strongly associated with cardiovascular diseases such as atherosclerosis. Bone marrow-derived endothelial progenitor cells (EPCs) play an important role in the maintenance of endothelial homeostasis and contribute to re-endothelialization of injured vessels as well as revascularization of ischemic tissues. MicroRNAs (miRNAs) have been reported to regulate EPC biological functions. In this study, we found that EPCs of atherosclerosis patients and EPCs exposed to hypoxia have increased expression of miRNA-21 (miR-21) as well as diminished ability to proliferate. MiR-21 knockdown rescued hypoxia-induced growth arrest in EPCs. Next, we used a luciferase reporter assay to demonstrate that miR-21 downregulates the expression of WW domain-containing protein 1 (WWP1), a negative regulator of TGFβ signaling, by directly targeting the 3’-UTR of WWP1. Finally, miR-21 overexpression or WWP1 knockdown in EPCs significantly activates the TGFβ signaling pathway and inhibits cell proliferation. Taken together, our results indicate that miR-21 suppresses EPC proliferation by activating the TGFβ signaling pathway via downregulation of WWP1. These findings may help the development of strategies to enhance the vitality of EPCs for therapeutic applications.     

BDNF promotes human neural stem cell growth via GSK-3β-mediated crosstalk with the wnt/β-catenin signaling pathway

Brain-derived neurotrophic factor (BDNF) plays important roles in neural stem cell (NSC) growth. In this study, we investigated whether BDNF exerts its neurotrophic effects through the Wnt/β-catenin signaling pathway in human embryonic spinal cord NSCs (hESC-NSCs) in vitro. We found an increase in hESC-NSC growth by BDNF overexpression. Furthermore, expression of Wnt1, Frizzled1 and Dsh was upregulated, whereas GSK-3β expression was downregulated. In contrast, hESC-NSC growth was decreased by BDNF RNA interference. BDNF, Wnt1 and β-catenin components were all downregulated, whereas GSK-3β was upregulated. Next, we treated hESC-NSCs with 6-bromoindirubin-3′-oxime (BIO), a small molecule inhibitor of GSK-3β. BIO reduced the effects of BDNF upregulation/downregulation on the cell number, soma size and differentiation, and suppressed the effect of BDNF modulation on the Wnt signaling pathway. Our findings suggest that BDNF promotes hESC-NSC growth in vitro through crosstalk with the Wnt/β-catenin signaling pathway, and that this interaction may be mediated by GSK-3β.     

Beta-naphthoflavone inhibits LPS-induced inflammation in BV-2 cells via AKT/Nrf-2/HO-1-NF-κB signaling axis

Numerous studies have shown that over-activation of microglia could cause neuroinflammation and release pro-inflammatory mediators, which could result in neurodegenerative diseases, like Parkinson’s disease, Alzheimer’s disease etc. Beta-naphthoflavone (BNF) has anti-oxidant and anti-inflammatory effects in borderline tissues, but BNF has not been reported the effect associated with neuroinflammation. Therefore, the purpose of this experiment is to inquiry the impact and mechanism of BNF on neuroinflammation. The results indicated that BNF significantly inhibited the production of pro-inflammatory mediators (inducible nitric-oxide synthase (iNOS), Cyclooxygenase-2 (COX-2), tumor necrosis factor-α (TNF-α) andinterleukin-6 (IL-6)) in LPS-exposed BV-2 cells. Analysis of western blot results found that BNF accelerated the activation of AKT/Nrf-2/HO-1 signaling pathway and suppressed NF-κB pathway activation. Further study showed that BNF inhibited activation of NF-κB pathway via promoting HO-1, and SnPP IX (a HO-1 inhibitor) could inhibit anti-inflammatory function of BNF. We also found that BNF reduced the apoptosis rate of Human neuroblastoma cells (SHSY5Y) and mouse hippocampal neuron cell line (HT22) by inhibiting release of inflammatory mediators in LPS-exposed BV2 cells. In a word, our results suggested that BNF could inhibit inflammatory response via AKT/Nrf-2/HO-1-NF-κB signaling axis in BV-2 cells and exerts neuroprotective impact via inhibiting the activation of BV2 cells.     

Near-infrared fluorescence imaging of cancer mediated by tumor hypoxia and HIF1α/OATPs signaling axis

Near-infrared fluorescence (NIRF) imaging agents are promising tools for noninvasive cancer imaging. Here, we explored the mechanistic properties of a specific group of NIR heptamethine carbocyanines including MHI-148 dye we identified and synthesized, and demonstrated these dyes to achieve cancer-specific imaging and targeting via a hypoxia-mediated mechanism. We found that cancer cells and tumor xenografts exhibited hypoxia-dependent MHI-148 dye uptake in vitro and in vivo, which was directly mediated by hypoxia-inducible factor 1α (HIF1α). Microarray analysis and dye uptake assay further revealed a group of hypoxia-inducible organic anion-transporting polypeptides (OATPs) responsible for dye uptake, and the correlation between OATPs and HIF1α was manifested in progressive clinical cancer specimens. Finally, we demonstrated increased uptake of MHI-148 dye in situ in perfused clinical tumor samples with activated HIF1α/OATPs signaling. Our results establish these NIRF dyes as potential tumor hypoxia-dependent cancer-targeting agents and provide a mechanistic rationale for continued development of NIRF imaging agents for improved cancer detection, prognosis and therapy.     

Improved endothelialization and reduced thrombosis by coating a synthetic vascular graft with fibronectin and stem cell homing factor SDF-1α

Failure of synthetic small-diameter vascular grafts is determined mainly by the lack of endothelial cells, as these cells inhibit thrombosis and intimal hyperplasia. Coating of graft material with homing factors for circulating stem cells has the potential to improve endogenous endothelialization of these grafts and to reduce graft failure. Synthetic knitted polyester grafts (6mm diameter) were coated with FN and SDF-1α before surgical interposition in the carotid artery of sheep. Similar uncoated vascular grafts were implanted in the contralateral side as internal controls. To study the early attraction of stem cells, grafts were implanted in a first series of nine sheep and explanted after 1 or 3 days. In coated grafts, four times higher fractions of CD34(+) and three to four times higher fractions of CD117(+) cells adhering to the vessel walls were found than in control grafts (P<0.05). When such coated and non-coated grafts were implanted in 12 other sheep and explanted after 3 months, all coated grafts were patent, while one control graft was occluded. EcNOS staining revealed that FN-SDF-1α coating significantly increased coverage with endothelial cells from 27 ± 4% of the graft to 48 ± 4% compared with the controls (P=0.001). This was associated with a significant reduction of intimal hyperplasia (average thickness 1.03 ± 0.09 mm in controls vs. 0.69 ± 0.04 mm in coated grafts; P=0.009) and significantly less adhesion of thrombotic material in the middle part of the graft (P=0.029). FN-SDF-1α coating of synthetic small-caliber vascular grafts stimulated the attraction of stem cells and was associated with improved endothelialization and reduced intimal hyperplasia and thrombosis.