OBJECTIVE: To explore the expression of CCN5 in endometriotic tissues and its impact on proliferation, migration and invasion of human endometrial stromal cells (HESCs). METHODS: We collected ovarian endometriosis samples from 20 women receiving laparoscopic surgery and eutopic endometrium samples from 15 women undergoing IVF-ET for comparison of CCN5 expression. Cultured HESCs were transfected with a recombinant adenovirus Ad-CCN5 for CCN5 overexpression or with a CCN5-specific siRNA for knocking down CCN5 expression, and the changes of cell proliferation, migration and invasion were evaluated using CCK-8 assay, wound healing assay and Transwell chamber assay. RT-qPCR and Western blotting were used to examine the expression levels of epithelial-mesenchymal transition (EMT) markers including E-cadherin, N-cadherin, Snail-1 and vimentin in HESCs with CCN5 overexpression or knockdown. RESULTS: CCN5 expression was significantly decreased in ovarian endometriosis tissues as compared with eutopic endometrium samples (P < 0.01). CCN5 overexpression obviously inhibited the proliferation, migration and invasion of HESCs, significantly increased the expression of E-cadherin and decreased the expressions of N-cadherin, Snail-1 and vimentin (P < 0.01). CCN5 knockdown significantly enhanced the proliferation, migration and invasion of HESCs and produced opposite effects on the expressions of E-cadherin, N-cadherin, Snail-1 and vimentin (P < 0.01). CONCLUSION CCN5 can regulate the proliferation, migration and invasion of HESCs and thus plays an important role in EMT of HESCs, suggesting the potential of CCN5 as a therapeutic target for endometriosis.
Cell Movement ; Cell Proliferation ; Endometriosis/metabolism* ; Endometrium/metabolism* ; Epithelial Cells ; Epithelial-Mesenchymal Transition ; Female ; Humans ; Stromal Cells

Viperin is an IFN-stimulated gene (ISG)-encoded protein that was identified in human primary macrophages treated with IFN-γ and in human primary fibroblasts infected with cytomegalovirus (CMV). This protein plays multiple roles in various cell types. It inhibits viral replication, mediates signaling pathways, and regulates cellular metabolism. Recent studies have shown that viperin inhibits IFN expression in macrophages, while it enhances TLR7 and TLR9-mediated IFN production in plasmacytoid dendritic cells, suggesting that viperin can play different roles in activation of the same pathway in different cell types. Viperin also controls induction of ISGs in macrophages. However, the effect of viperin on induction of ISGs in cell types other than macrophages is unknown. Here, we show that viperin differentially induces ISGs in 2 distinct cell types, macrophages and fibroblasts isolated from wild type and viperin knockout mice. Unlike in bone marrow-derived macrophages (BMDMs), viperin downregulates the expression levels of ISGs such as bone marrow stromal cell antigen-2, Isg15, Isg54, myxovirus resistance dynamin like GTPase 2, and guanylate binding protein 2 in murine embryonic fibroblasts (MEFs) treated with type I or II IFN. However, viperin upregulates expression of these ISGs in both BMDMs and MEFs stimulated with polyinosinic-polycytidylic acid or CpG DNA and infected with murine CMV. The efficiency of viral entry is inversely proportional to the expression levels of ISGs in both cell types. The data indicate that viperin differentially regulates induction of ISGs in a cell type-dependent manner, which might provide different innate immune responses in distinct cell types against infections.
Animals ; Carrier Proteins ; Cytomegalovirus ; Dendritic Cells ; DNA ; Dynamins ; Fibroblasts ; GTP Phosphohydrolases ; Humans ; Immunity, Innate ; Interferons ; Macrophages ; Mesenchymal Stromal Cells ; Metabolism ; Mice ; Mice, Knockout ; Orthomyxoviridae ; Poly I-C

BackgroundCorneal stromal cells (CSCs) are components of the corneal endothelial microenvironment that can be induced to form a functional tissue-engineered corneal endothelium. Adipose-derived mesenchymal stem cells (ADSCs) have been reported as an important component of regenerative medicine and cell therapy for corneal stromal damage. We have demonstrated that the treatment with ADSCs leads to phenotypic changes in CSCs in vitro. However, the underlying mechanisms of such ADSC-induced changes in CSCs remain unclear.

MethodsADSCs and CSCs were isolated from New Zealand white rabbits and cultured in vitro. An Exosome Isolation Kit, Western blotting, and nanoparticle tracking analysis (NTA) were used to isolate and confirm the exosomes from ADSC culture medium. Meanwhile, the optimal exosome concentration and treatment time were selected. Cell Counting Kit-8 and annexin V-fluorescein isothiocyanate/propidium iodide assays were used to assess the effect of ADSC- derived exosomes on the proliferation and apoptosis of CSCs. To evaluate the effects of ADSC- derived exosomes on CSC invasion activity, Western blotting was used to detect the expression of matrix metalloproteinases (MMPs) and collagens.

Results:ADSCs and CSCs were successfully isolated from New Zealand rabbits. The optimal concentration and treatment time of exosomes for the following study were 100 μg/ml and 96 h, respectively. NTA revealed that the ADSC-derived exosomes appeared as nanoparticles (40-200 nm), and Western blotting confirmed positive expression of CD9, CD81, flotillin-1, and HSP70 versus ADSC cytoplasmic proteins (all P < 0.01). ADSC-derived exosomes (50 μg/ml and 100 μg/ml) significantly promoted proliferation and inhibited apoptosis (mainly early apoptosis) of CSCs versus non-exosome-treated CSCs (all P < 0.05). Interestingly, MMPs were downregulated and extracellular matrix (ECM)-related proteins including collagens and fibronectin were upregulated in the exosome-treated CSCs versus non-exosome-treated CSCs (MMP1: t = 80.103, P < 0.01; MMP2: t = 114.778, P < 0.01; MMP3: t = 56.208, P < 0.01; and MMP9: t = 60.617, P < 0.01; collagen I: t = -82.742, P < 0.01; collagen II: t = -72.818, P < 0.01; collagen III: t = -104.452, P < 0.01; collagen IV: t = -133.426, P < 0.01, and collagen V: t = -294.019, P < 0.01; and fibronectin: t = -92.491, P < 0.01, respectively).

Conclusion:The findings indicate that ADSCs might play an important role in CSC viability regulation and ECM remodeling, partially through the secretion of exosomes.

Adipose Tissue ; cytology ; Animals ; Cell Proliferation ; physiology ; Cell Survival ; physiology ; Cells, Cultured ; Exosomes ; metabolism ; Extracellular Matrix ; metabolism ; Fibroblasts ; cytology ; metabolism ; Matrix Metalloproteinases ; metabolism ; Mesenchymal Stromal Cells ; cytology ; metabolism ; Rabbits

BACKGROUND: Use of antidepressant medications has been linked to detrimental impacts on bone mineral density and osteoporosis; however, the cellular basis behind these observations remains poorly understood. The effect does not appear to be homogeneous across the whole class of drugs and may be linked to affinity for the serotonin transporter system. In this study, we hypothesized that antidepressants have a class- and dose-dependent effect on mesenchymal stem cell (MSC) differentiation, which may affect bone metabolism. METHODS: Human MSCs (hMSCs) were committed to differentiate when either adipogenic or osteogenic media was added, supplemented with five increasing concentrations of amitriptyline (0.001–10 µM), venlafaxine (0.01–25 µM), or fluoxetine (0.001–10 µM). Alizarin red staining (mineralization), alkaline phosphatase (osteoblastogenesis), and oil red O (adipogenesis) assays were performed at timed intervals. In addition, cell viability was assessed using a MTT. RESULTS: We found that fluoxetine had a significant inhibitory effect on mineralization. Furthermore, adipogenic differentiation of hMSC was affected by the addition of amitriptyline, venlafaxine, and fluoxetine to the media. Finally, none of the tested medications significantly affected cell survival. CONCLUSIONS: This study showed a divergent effect of three antidepressants on hMSC differentiation, which appears to be independent of class and dose. As fluoxetine and amitriptyline, but not venlafaxine, affected both osteoblastogenesis and adipogenesis, this inhibitory effect could be associated to the high affinity of fluoxetine to the serotonin transporter system.
Adipogenesis ; Alkaline Phosphatase ; Amitriptyline ; Antidepressive Agents ; Bone Density ; Cell Survival ; Fluoxetine ; Humans ; Mesenchymal Stromal Cells ; Metabolism ; Miners ; Osteoblasts ; Osteoporosis ; Serotonin Plasma Membrane Transport Proteins ; Venlafaxine Hydrochloride

PURPOSE: Angiopoietin-1 (Ang1) is a critical factor for vascular stabilization and endothelial survival via inhibition of endothelial permeability and leukocyte- endothelium interactions. Hence, we hypothesized that treatment with umbilical cord mesenchymal stem cells (UCMSCs) carrying the Ang1 gene (UCMSCs-Ang1) might be a potential approach for acute lung injury (ALI) induced by lipopolysaccharide (LPS). MATERIALS AND METHODS: UCMSCs with or without transfection with the human Ang1 gene were delivered intravenously into rats one hour after intra-abdominal instillation of LPS to induce ALI. After the rats were sacrificed at 6 hours, 24 hours, 48 hours, 8 days, and 15 days post-injection of LPS, the serum, the lung tissues, and bronchoalveolar lavage fluid (BALF) were harvested for analysis, respectively. RESULTS: Administration of fluorescence microscope confirmed the increased presence of UCMSCs in the injured lungs. The evaluation of UCMSCs and UCMSCs-Ang1 actions revealed that Ang1 overexpression further decreased the levels of the pro-inflammatory cytokines TNF-α, TGF-β1, and IL-6 and increased the expression of the anti-inflammatory cytokine IL-10 in the injured lungs. This synergy caused a substantial decrease in lung airspace inflammation and vascular leakage, characterized by significant reductions in wet/dry ratio, differential neutrophil counts, myeloperoxidase activity, and BALF. The rats treated by UCMSCs-Ang1 showed improved survival and lower ALI scores. CONCLUSION: UCMSCs-Ang1 could improve both systemic inflammation and alveolar permeability in ALI. UC-derived MSCs-based Ang1 gene therapy may be developed as a potential novel strategy for the treatment of ALI.
Acute Lung Injury/chemically induced/*therapy ; Angiopoietin-1/*genetics ; Animals ; Bronchoalveolar Lavage Fluid ; Cytokines/metabolism ; Endotoxins ; Genetic Therapy ; Interleukin-10/metabolism ; Interleukin-6/metabolism ; Leukocyte Count ; Lipopolysaccharides ; Lung/metabolism ; Male ; *Mesenchymal Stem Cell Transplantation ; Mesenchymal Stromal Cells/metabolism ; Neutrophils/metabolism ; Rats ; Transforming Growth Factor beta1/metabolism ; Tumor Necrosis Factor-alpha/metabolism ; Umbilical Cord/*cytology

BACKGROUND AND OBJECTIVES: Morinda citrifolia (Noni), an important traditional medicinal plant still used in patients with bone fractures or dislocation to promote connective tissue repair and to reduce inflammation. However, the effects of Noni on bone metabolism and whether it influences the osteogenic differentiation is yet to be clarified. In this study, we investigated the effect of Morinda citrifolia (Noni) juice on the proliferation rate of rat bone marrow derived mesenchymal stem cells (BMSC) and the osteoblastic differentiation as shown by alkaline phosphatase (ALP), Runt-related transcription factor 2 (Runx2) and osteocalcin (OCN) mRNA expression in vitro. METHODS AND RESULTS: Treatment with 200 μg/ml Noni juice enhanced the proliferation rate of the BMSC and also upregulated the osteogenic differentiation marker genes ALP and OCN, and Runx2 measured by RTPCR. Consistent with these results collagen scaffolds implanted in vivo, which were loaded with BMSC pre-exposed to Noni, showed increased bone density measured by computed tomography and histological analysis revealed neo-angiogenesis for bone formation. CONCLUSIONS: These results suggest that Noni stimulates osteoblastogenesis and can be used as adjuvant natural medicine for bone diseases such as osteoporosis.
Alkaline Phosphatase ; Animals ; Bone Density ; Bone Diseases ; Bone Marrow* ; Collagen ; Connective Tissue ; Dislocations ; Fractures, Bone ; Humans ; In Vitro Techniques ; Inflammation ; Mesenchymal Stromal Cells* ; Metabolism ; Morinda* ; Osteoblasts* ; Osteocalcin ; Osteogenesis ; Osteoporosis ; Plants, Medicinal ; Rats* ; RNA, Messenger ; Transcription Factors

Mesothelial cells (MCs) cover the surface of visceral organs and the parietal walls of cavities, and they synthesize lubricating fluids to create a slippery surface that facilitates movement between organs without friction. Recent studies have indicated that MCs play active roles in liver development, fibrosis, and regeneration. During liver development, the mesoderm produces MCs that form a single epithelial layer of the mesothelium. MCs exhibit an intermediate phenotype between epithelial cells and mesenchymal cells. Lineage tracing studies have indicated that during liver development, MCs act as mesenchymal progenitor cells that produce hepatic stellate cells, fibroblasts around blood vessels, and smooth muscle cells. Upon liver injury, MCs migrate inward from the liver surface and produce hepatic stellate cells or myofibroblast depending on the etiology, suggesting that MCs are the source of myofibroblasts in capsular fibrosis. Similar to the activation of hepatic stellate cells, transforming growth factor β induces the conversion of MCs into myofibroblasts. Further elucidation of the biological and molecular changes involved in MC activation and fibrogenesis will contribute to the development of novel approaches for the prevention and therapy of liver fibrosis.
Epithelial Cells/*physiology ; Epithelium/metabolism ; Hepatic Stellate Cells/*physiology ; Humans ; Liver/*cytology/injuries/*physiology ; Liver Cirrhosis/etiology/prevention & control ; Liver Regeneration/*physiology ; Mesenchymal Stromal Cells/physiology ; Myofibroblasts/physiology

BACKGROUNDSeveral studies have revealed that adipose-derived mesenchymal stem cells (ADSCs) can be used as seed cells for the treatment of spinal cord injury (SCI). Chondroitinase ABC (ChABC) decomposes chondroitin sulfate proteoglycans in the glial scar that forms following SCI, allowing stem cells to penetrate through the scar and promote recovery of nerve function. This study aimed to establish ADSCs that stably express ChABC (ChABC-ADSCs) and evaluate the migratory capability of ChABC-ADSCs in vitro.

METHODSADSCs were obtained from Sprague-Dawley rats using secondary collagenase digestion. Their phenotypes were characterized using flow cytometry detection of cell surface antigens and their stem cell properties were confirmed by induction of differentiation. After successful culture, ADSCs were transfected with lentiviral vectors and ChABC-ADSCs were obtained. Proliferation curves of ChABC-ADSCs were determined using the Cell Counting Kit-8 method, ChABC expression was verified using Western blotting, and the migration of ChABC-ADSCs was analyzed using the transwell assay.

RESULTSSecondary collagenase digestion increased the isolation efficiency of primary ADSCs. Following transfection using lentiviral vectors, the proliferation of ChABC-ADSCs was reduced in comparison with control ADSCs at 48 h (P < 0.05). And the level of ChABC expression in the ChABC-ADSC group was significantly higher than that of the ADSC group (P < 0.05). Moreover, ChABC-ADSC migration in matrigel was significantly enhanced in comparison with the control (P < 0.05).

CONCLUSIONSSecondary collagenase digestion can be used to effectively isolate ADSCs. ChABC-ADSCs constructed using lentiviral vector transfection stably express ChABC, and ChABC expression significantly enhances the migratory capacity of ADSCs.

Adipocytes ; cytology ; metabolism ; Adipose Tissue ; cytology ; Animals ; Cell Differentiation ; physiology ; Cell Movement ; physiology ; Cell Proliferation ; physiology ; Cells, Cultured ; Chondrocytes ; cytology ; metabolism ; Chondroitin ABC Lyase ; metabolism ; Flow Cytometry ; Male ; Mesenchymal Stromal Cells ; cytology ; metabolism ; Osteoblasts ; cytology ; metabolism ; Rats ; Rats, Sprague-Dawley

OBJECTIVEPrevious studies have clarified that calcitonin gene-related peptide (CGRP) can promote the biologi- cal activity of osteoblasts. To further reveal the role of CGRP in bone repair, we studied its influence on osteogenic differentia- tion of mouse bone marrow stromal cells (BMSCs) and initially explored the effect of the Hippo signaling pathway with this process.

METHODSBMSCs were induced to osteogenic differentiate osteoblasts by different concentrations of CGRP for a screening of the optimal concentration. CGRP was added in BMSCs, then the activity of alkaline phosphatase (ALP) and the number of mineralized nodules were examined by specific ALP kits after 48 hours and alizarin red staining fluid after 7 days, respectively. The protein expression of p-Mst1/2 was measured by Western blot. Verteporfin was used to block the downstream Yap signaling. The mRNA expression of collagen type I (Col I) and runt-related transcription factor 2 (Runx2) were detected by reverse transcription-polymerase chain reaction.

RESULTSCompared to the blank group, different concentrations of CGRP (10⁻⁹, 10⁻⁸, 10⁻⁷ mol · L⁻¹), especially 10⁻⁸ mol · L⁻¹, significantly increased the ALP activity of BMSCs (P < 0.05). Alizarin red staining also showed more mineralized nodules in 10⁻⁸ mol · L⁻¹ group. The expression of p-Mst1/2 increased in the CGRP group (P < 0.05). Verteporfin treatment effectively decreased the mRNA expression of Runx2 and Col I (P < 0.05).

CONCLUSIONThe Hippo signaling pathway plays a role in CGRP-induced osteogenic differentiation in mouse BMSCs.

Alkaline Phosphatase ; Animals ; Calcitonin ; genetics ; metabolism ; Calcitonin Gene-Related Peptide ; metabolism ; Cell Differentiation ; Cells, Cultured ; Collagen Type I ; Core Binding Factor Alpha 1 Subunit ; Mesenchymal Stromal Cells ; physiology ; Mice ; Osteoblasts ; Osteogenesis ; physiology ; Signal Transduction

OBJECTIVEWe explored the expressions of the Notch and Wnt signaling pathways and their significance in the repair process of alveolar bone defects by establishing animal models with a composite of autologous bone marrow mesenchymal stem cells (BMSCs) and platelet-rich fibrin (PRF) to repair bone defects in the extraction sockets of rabbits.

METHODSA total of 36 two-month-old male New Zealand white rabbits were randomly divided into four groups, and the left mandibular incisors of all the rabbits were subjected to minimally invasive removalunder general anesthesia. BMSC-PRF compounds, single PRF, and single BMSC were implanted in Groups A, B, and C. No material was implanted in Group D (blank control). The animals were sacrificed at 4, 8 and 12 weeks after surgery, the bone defect was immediately drawn, and the bone specimens underwent surgery after four, eight, and twelve weeks, with three rabbits per time point. The expressions of Notch1 and Wnt3a in the repair process of the bone defect were measured via immunohistochemical and immunofluorescence detection.

RESULTSImmunohistochemistry showed that the expressions of Notch1 and Wnt3a in Groups A, B, and C were higher than that in Group D at the fourth and eighth week after operation (P<0.05). By contrast, the expressions of Notch1 and Wnt3a in Group D were higher than those in Groups A, B, and C at the twelfth week (P<0.05). Immunofluorescence showed that the expressions of both Notch1 and Wnt3a reached their peaks in the new bone cells of the bone defect after four weeks following surgery and gradually disappeared when the bone was repaired completely.

CONCLUSIONNotch1 and Wnt3a signaling molecules are expressed in the process of repairing bone defects using BMSC-PRF composites and can accelerate the healing by regulating the proliferation and differentiation of BMSCs. Moreover, the expressions of Notch and Wnt are similar, and a crosstalk between them may exist it.

Alveolar Bone Grafting ; methods ; Animals ; Blood Platelets ; Bone Marrow Cells ; cytology ; Bone Transplantation ; methods ; Bone and Bones ; abnormalities ; Cell Differentiation ; Fibrin ; administration & dosage ; Male ; Mesenchymal Stem Cell Transplantation ; methods ; Mesenchymal Stromal Cells ; Platelet-Rich Plasma ; Rabbits ; Random Allocation ; Receptor, Notch1 ; metabolism ; Tissue Engineering ; Wnt Signaling Pathway ; Wnt3A Protein ; metabolism ; Wound Healing