IL-1ß stimulated human umbilical cord mesenchymal stem cells ameliorate rheumatoid arthritis via inducing apoptosis of fibroblast-like synoviocytes.

Rheumatoid arthritis (RA) is characterized by synovial proliferation and lymphocyte accumulation leading to progressive damage of the periarticular bone and the articular cartilage. The hyperplasia of the synovial intima lining mainly consists of fibroblast-like synoviocytes-rheumatoid arthritis (HFLS-RA) which exhibit apoptosis-resistance, hyper-proliferation, and high invasiveness. The therapeutic efficacy of mesenchymal stem cells (MSCs) treatment in RA has been shown to be due to its immuno-regulatory ability. However, the exact factors and mechanisms involved in MSCs treatment in RA remain unclear. In this study, TRAIL receptor-Death receptor 4 (DR4), DR5, and LFA-1 ligand-intercellular adhesion molecule-1 (ICAM-1) were upregulated in IL-1ß-stimulated HFLS-RA. We demonstrated that the total cell number of IL-1ß-stimulated hUCMSCs adhering to IL-1ß-stimulated HFLA-RA increased via LFA-1/ICAM-1 interaction. Direct co-culture of IL-1ß-stimulated hUCMSCs with IL-1ß-stimulated HFLS-RA increased the apoptosis of HFLS-RA. RA symptoms in the CIA mouse model improved after administration of IL-1ß-stimulated hUCMSCs. In conclusion, IL-1ß-stimulated hUCMSCs adhering to HFLS-RA occurred via LFA-1/ICAM-1 interaction, apoptosis of HFLS-RA was induced via TRAIL/DR4, DR5 contact, and RA symptoms and inflammation were significantly improved in a CIA mouse model. The results of this study suggest that IL-1ß-stimulated hUCMSCs have therapeutic potential in RA treatment.

The effects of IL-1ß stimulated human umbilical cord mesenchymal stem cells on polarization and apoptosis of macrophages in rheumatoid arthritis.

Macrophages play an important role in the pathogenesis of rheumatoid arthritis (RA), in which the functions of pro-inflammatory macrophages (M1) and anti-inflammatory macrophages (M2) are different. Our previous studies have demonstrated that interleukin-1ß (IL-1ß) stimulated human umbilical cord mesenchymal stem cells (hUCMSCs) increase the expression of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) and initiate breast cancer cell apoptosis via ligand to death receptor 4 (DR4) and DR5. In this study, we examined the effect of IL-1ß stimulated hUCMSCs (IL-1ß-hUCMSCs) on immunoregulation of M1 and M2 macrophages in vitro and in the RA mouse model. The results showed that IL-1ß-hUCMSCs increased macrophage polarization into M2 macrophages and enhanced apoptosis of M1 macrophages in vitro. Moreover, the intravenous injected IL-1ß-hUCMSCs in RA mice rehabilitated the imbalance of M1/M2 ratio and thus demonstrated the potential to reduce inflammation in RA. This study advances our knowledge of the underlying immunoregulatory mechanisms involved in IL-1ß-hUCMSCs to induce M1 macrophage apoptosis and promote the anti-inflammatory polarization of M2 macrophages and demonstrates the potential of IL-1ß-hUCMSCs to reduce inflammation in RA.

Anti-tumor Effects of IL-1ß Induced TRAIL-Expressing hUCMSCs on Embelin Treated Breast Cancer Cell Lines.

BACKGROUND: Human umbilical cord mesenchymal stem cells (hUCMSCs) have high therapeutic value in cancer treatment. We have found that pre-activating hUCMSCs with IL-1ß promotes tumor necrosis factor-related apoptosis inducing ligand (TRAIL) expression and facilitates anti-tumor effect. Furthermore, embelin has been found to induce apoptosis of different cancer cell lines by upregulating the expression of TRAIL receptor 1 (DR4) and TRAIL receptor 2 (DR5). This study investigated whether IL-1ß induced TRAIL-expressing hUCMSCs, in combination with low-dose embelin, could further induce apoptosis in breast cancer cell lines. MATERIALS AND METHODS: MTT assay was used to examine the cytotoxicity of embelin in MDA-MB-231 and MCF-7. To detect the interested protein expression in cells, Western blot and cell immunofluorescence were used to double-confirm the observed results. Annexin V/PI apoptosis assay was detected by flow cytometry to analyze the apoptosis rate of embelin treated breast cancer cell lines and the effect of co-culturing with breast cancer cells and hUCMSCs. RESULTS: Using Western blot and immunofluorescence, we found that breast cancer cell lines treated with low-dose embelin (2.5-5 µM) increased the expression of apoptosis-related receptor DR4, DR5 and the cleaved caspase 8, 9 and 3. Moreover, TRAIL expression was enhanced in IL-1ß induced hUCMSCs. Combining these observations, we expected that coculturing IL-1ß induced hUCMSCs with low dose embelin treated MDA-MB-231 and MCF-7 cells might enhance the apoptosis of breast cancer cells. We confirmed via flow cytometry that coculture of IL-1ß induced TRAIL-expressing hUCMSCs and embelin treated MDA-MB-231 and MCF-7 cells enhances the apoptosis rate of these breast cancer cells. CONCLUSION: We found that embelin upregulated the expression of DR4 and DR5 to increase the TRAIL-mediated apoptosis in breast cancer cell lines. Low dose embelin treated breast cancer cell lines in combination with IL-1ß induced TRAIL-expressing hUCMSCs may become a potential anti-tumor therapy.

Embelin downregulated cFLIP in breast cancer cell lines facilitate anti-tumor effect of IL-1ß-stimulated human umbilical cord mesenchymal stem cells.

Breast cancer is the leading cause of cancer-related death for women. In breast cancer treatment, targeted therapy would be more effective and less harmful than radiotherapy or systemic chemotherapy. Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) has been shown to induce apoptosis in cancer cells but not in normal cells. Mesenchymal stem cells have shown great therapeutic potential in cancer therapy owing to their ability of homing to tumor sites and secreting many kinds of anti-tumor proteins including TRAIL. In this study, we found that IL-1ß-stimulated human umbilical cord-derived mesenchymal stem cells (hUCMSCs) enhance the expression of membrane-bound and soluble TRAIL. Cellular FADD-like IL-1ß-converting enzyme inhibitory protein (cFLIP) is an important regulator in TRAIL-mediated apoptosis and relates to TRAIL resistance in cancer cells. Previous studies have shown that embelin, which is extracted from Embelia ribes, can increase the TRAIL sensitivity of cancer cells by reducing cFLIP expression. Here we have demonstrated that cFLIPL is correlated with TRAIL-resistance and that embelin effectively downregulates cFLIPL in breast cancer cells. Moreover, co-culture of IL-1ß-stimulated hUCMSCs with embelin-treated breast cancer cells could effectively induce apoptosis in breast cancer cells. The combined effects of embelin and IL-1ß-stimulated hUCMSCs may provide a new therapeutic strategy for breast cancer therapy.

Interleukin-1ß-induced matrix metalloproteinase-3 via ERK1/2 pathway to promote mesenchymal stem cell migration.

Human umbilical cord Wharton's jelly derived mesenchymal stem cells (hUCMSCs), a source of cell therapy, have received a great deal of attention due to their homing or migrating ability in response to signals emanating from damaged sites. It has been found that IL-1ß possesses the ability to induce the expression of matrix metalloproteinase-3 (MMP-3) in bone marrow MSCs. MMP-3 is involved in cell migration in various types of cells, including glioblastoma, vascular smooth muscle, and adult neural progenitor cells. In this study, we proposed that IL-1ß influences hUCMSCs migration involving MMP-3. The expression level of MMP-3 in IL-1ß-induced hUCMSCs was verified using cDNA microarray analysis, quantitative real-time PCR, ELISA and Western blot. Wound-healing and trans-well assay were used to investigate the cell migration and invasion ability of IL-1ß-treated hUCMSCs. In addition, we pre-treated hUCMSCs with interleukin-1 receptor antagonist, MMP-3 inhibitors (ALX-260-165, UK 356618), or transfected with MMP-3 siRNA to confirm the role of MMP3 in IL-1ß-induced cell migration. Our results showed that IL-1ß induced MMP-3 expression is related to the migration of hUCMSCs. Moreover, extracellular signal-regulated protein kinases 1 and 2 (ERK1/2) inhibitor U0126, p38 inhibitor SB205380, JNK inhibitor SP600125 and Akt inhibitor GSK 690693 decreased IL-1ß-induced MMP-3 mRNA and protein expression. The migration and invasion ability analyses showed that these inhibitors attenuated the IL-1ß-induced migration and invasion ability of hUCMSCs. In conclusion, we have found that IL-1ß induces the expression of MMP-3 through ERK1/2, JNK, p38 MAPK and Akt signaling pathways to enhance the migration of hUCMSCs. These results provide further understanding of the mechanisms in IL-1ß-induced hUCMSCs migration to injury sites.

Interleukin-1ß Enhances Umbilical Cord Mesenchymal Stem Cell Adhesion Ability on Human Umbilical Vein Endothelial Cells via LFA-1/ICAM-1 Interaction.

The migration of administered mesenchymal stem cells (MSCs) to sites of injury via the bloodstream has been demonstrated. However, the underlying mechanisms of umbilical cord MSC adhesion to endothelial cells during transendothelial migration are still unclear. In this study, our data showed that IL-1ß induced LFA-1 expression on MSCs and ICAM-1 expression on HUVECs. We then pretreated MSCs with protein synthesis inhibitor cycloheximide. The results showed that IL-1ß induced LFA-1 expression on the surface of MSCs via the protein synthesis pathway. Through the p38 MAPK signaling pathway inhibitor SB 203580, we found that IL-1ß induces the expression of LFA-1 through p38 MAPK signaling and enhances ICAM-1 expression in HUVECs. In addition, IL-1ß-induced MSC adhesion to HUVECs was found to be inhibited by IL-1RA and the LFA-1 inhibitor lovastatin. These results indicate that IL-1ß promotes the cell adhesion of MSCs to HUVECs through LFA-1/ICAM-1 interaction. We address the evidence that the cell adhesion mechanism of IL-1ß promotes MSC adhesion to HUVECs. The implications of these findings could enhance the therapeutic potential of MSCs.

Interleukin-1ß induces CXCR3-mediated chemotaxis to promote umbilical cord mesenchymal stem cell transendothelial migration.

BACKGROUND: Mesenchymal stem cells (MSCs) are known to home to injured and inflamed regions via the bloodstream to assist in tissue regeneration in response to signals of cellular damage. However, the factors and mechanisms that affect their transendothelial migration are still unclear. In this study, the mechanisms involved in interleukin-1ß (IL-1ß) enhancing the transendothelial migration of MSCs were investigated. METHODS: Immunofluorescence staining and Western blotting were used to observe IL-1ß-induced CXC chemokine receptor 3 (CXCR3) expression on MSCs. Quantitative real-time PCR and ELISA were used to demonstrate IL-1ß upregulated both chemokine (C-X-C motif) ligand 9 (CXCL9) mRNA and CXCL9 ligand secretion in human umbilical vein endothelial cells (HUVECs). Monolayer co-cultivation, agarose drop chemotaxis, and transwell assay were conducted to investigate the chemotaxis invasion and transendothelial migration ability of IL-1ß-induced MSCs in response to CXCL9. RESULTS: In this study, our immunofluorescence staining showed that IL-1ß induces CXCR3 expression on MSCs. This result was confirmed by Western blotting. Following pretreatment with protein synthesis inhibitor cycloheximide, we found that IL-1ß induced CXCR3 on the surface of MSCs via protein synthesis pathway. Quantitative real-time PCR and ELISA validated that IL-1ß upregulated both CXCL9 mRNA and CXCL9 ligand secretion in HUVECs. In response to CXCL9, chemotaxis invasion and transendothelial migration ability were increased in IL-1ß-stimulated MSCs. In addition, we pretreated MSCs with CXCR3 antagonist AMG-487 and p38 MAPK inhibitor SB203580 to confirm CXCR3-CXCL9 interaction and the role of CXCR3 in IL-1ß-induced chemotaxis invasion and transendothelial migration. CONCLUSION: We found that IL-1ß induces the expression of CXCR3 through p38 MAPK signaling and that IL-1ß also enhances CXCL9 ligand secretion in HUVECs. These results indicated that IL-1ß promotes the transendothelial migration of MSCs through CXCR3-CXCL9 axis. The implication of the finding could enhance the efficacy of MSCs homing to target sites.

IL-1ß-Induced Matrix Metalloprotease-1 Promotes Mesenchymal Stem Cell Migration via PAR1 and G-Protein-Coupled Signaling Pathway.

Mesenchymal stem cells (MSCs) are known for homing to sites of injury in response to signals of cellular damage. However, the mechanisms of how cytokines recruit stem cells to target tissue are still unclear. In this study, we found that the proinflammation cytokine interleukin-1ß (IL-1ß) promotes mesenchymal stem cell migration. The cDNA microarray data show that IL-1ß induces matrix metalloproteinase-1 (MMP-1) expression. We then used quantitative real-time PCR and MMP-1 ELISA to verify the results. MMP-1 siRNA transfected MSCs, and MSC pretreatment with IL-1ß inhibitor interleukin-1 receptor antagonist (IL-1RA), MMP tissue inhibitor of metalloproteinase 1 (TIMP1), tissue inhibitor of metalloproteinase 2 (TIMP2), MMP-1 inhibitor GM6001, and protease-activated receptor 1 (PAR1) inhibitor SCH79797 confirms that PAR1 protein signaling pathway leads to IL-1ß-induced cell migration. In conclusion, IL-1ß promotes the secretion of MMP-1, which then activates the PAR1 and G-protein-coupled signal pathways to promote mesenchymal stem cell migration.

Stiffness-controlled three-dimensional collagen scaffolds for differentiation of human Wharton's jelly mesenchymal stem cells into cardiac progenitor cells.

Stem cell-based regenerative therapy has emerged as a promising treatment for myocardial infarction. The aim of this study is to develop stiffness-controlled collagen scaffolds to allow proliferation and differentiation of mesenchymal stem cell (MSCs) into cardiac progenitor cells. In this study transforming growth factor ß2 (TGF-ß2), was used to induce stem cell differentiation into cardiac lineage cells. Collagen scaffolds were cross-linked with cross-linkers, 1-Ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC), and N-Hydroxysuccinimide (NHS). The results showed that collagen scaffolds cross-linked with 25/50 and 50/50 of EDC mM/NHS mM cross-linkers exhibited little difference in shape and size, the scaffold cross-linked with 50/50 of cross-linkers demonstrated better interconnectivity and higher Young's modulus (31.8 kPa) than the other (15.4 kPa). SEM observation showed that MSCs could grow inside the scaffolds and interact with collagen scaffolds. Furthermore, greater viability and cardiac lineage differentiation were achieved in MSCs cultured on stiffer scaffolds. The results suggest that three-dimensional type I collagen scaffolds with suitable cross-linking to adjust for stiffness can affect MSC fate and direct the differentiation of MSCs into cardiac progenitor cells with/without TGF-ß2. These stiffness-controlled collagen scaffolds hold great potential as carriers for delivering MSCs differentiated cardiac progenitor cells into infracted hearts. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 104A: 2234-2242, 2016.

Comparisons of Differentiation Potential in Human Mesenchymal Stem Cells from Wharton's Jelly, Bone Marrow, and Pancreatic Tissues.

Background. Type 1 diabetes mellitus results from autoimmune destruction of ß-cells. Insulin-producing cells (IPCs) differentiated from mesenchymal stem cells (MSCs) in human tissues decrease blood glucose levels and improve survival in diabetic rats. We compared the differential ability and the curative effect of IPCs from three types of human tissue to determine the ideal source of cell therapy for diabetes. Methods. We induced MSCs from Wharton's jelly (WJ), bone marrow (BM), and surgically resected pancreatic tissue to differentiate into IPCs. The in vitro differential function of these IPCs was compared by insulin-to-DNA ratios and C-peptide levels after glucose challenge. In vivo curative effects of IPCs transplanted into diabetic rats were monitored by weekly blood glucose measurement. Results. WJ-MSCs showed better proliferation and differentiation potential than pancreatic MSCs and BM-MSCs. In vivo, WJ-IPCs significantly reduced blood glucose levels at first week after transplantation and maintained significant decrease till week 8. BM-IPCs reduced blood glucose levels at first week but gradually increased since week 3. In resected pancreas-IPCs group, blood glucose levels were significantly reduced till two weeks after transplantation and gradually increased since week 4. Conclusion. WJ-MSCs are the most promising stem cell source for ß-cell regeneration in diabetes treatment.

HYS-32-Induced Microtubule Catastrophes in Rat Astrocytes Involves the PI3K-GSK3beta Signaling Pathway.

HYS-32 is a novel derivative of combretastatin-A4 (CA-4) previously shown to induce microtubule coiling in rat primary astrocytes. In this study, we further investigated the signaling mechanism and EB1, a microtubule-associated end binding protein, involved in HYS-32-induced microtubule catastrophes. Confocal microscopy with double immunofluorescence staining revealed that EB1 accumulates at the growing microtubule plus ends, where they exhibit a bright comet-like staining pattern in control astrocytes. HYS-32 induced microtubule catastrophes in both a dose- and time-dependent manner and dramatically increased the distances between microtubule tips and the cell border. Treatment of HYS-32 (5 µM) eliminated EB1 localization at the microtubule plus ends and resulted in an extensive redistribution of EB1 to the microtubule lattice without affecting the ß-tubulin or EB1 protein expression. Time-lapse experiments with immunoprecipitation further displayed that the association between EB-1 and ß-tubulin was significantly decreased following a short-term treatment (2 h), but gradually increased in a prolonged treatment (6-24 h) with HYS-32. Further, HYS-32 treatment induced GSK3ß phosphorylation at Y216 and S9, where the ratio of GSK3ß-pY216 to GSK3ß-pS9 was first elevated followed by a decrease over time. Co-treatment of astrocytes with HYS-32 and GSK3ß inhibitor SB415286 attenuated the HYS-32-induced microtubule catastrophes and partially prevented EB1 dissociation from the plus end of microtubules. Furthermore, co-treatment with PI3K inhibitor LY294002 inhibited HYS-32-induced GSK3ß-pS9 and partially restored EB1 distribution from the microtubule lattice to plus ends. Together these findings suggest that HYS-32 induces microtubule catastrophes by preventing EB1 from targeting to microtubule plus ends through the GSK3ß signaling pathway.

Undifferentiated Wharton's Jelly Mesenchymal Stem Cell Transplantation Induces Insulin-Producing Cell Differentiation and Suppression of T-Cell-Mediated Autoimmunity in Nonobese Diabetic Mice.

Type 1 diabetes mellitus is caused by T-cell-mediated autoimmune destruction of pancreatic ß-cells. Systemic administration of mesenchymal stem cells (MSCs) brings about their incorporation into a variety of tissues with immunosuppressive effects, resulting in regeneration of pancreatic islets. We previously showed that human MSCs isolated from Wharton's jelly (WJ-MSCs) represent a potential cell source to treat diabetes. However, the underlying mechanisms are unclear. The purpose of this study was to discern whether undifferentiated WJ-MSCs can differentiate into pancreatic insulin-producing cells (IPCs) and modify immunological responses in nonobese diabetic (NOD) mice. Undifferentiated WJ-MSCs underwent lentiviral transduction to express green fluorescent protein (GFP) and then were injected into the retro-orbital venous sinus of NOD mice. Seven days after transplantation, fluorescent islet-like cell clusters in the pancreas were apparent. WJ-MSC-GFP-treated NOD mice had significantly lower blood glucose and higher survival rates than saline-treated mice. Systemic and local levels of autoaggressive T-cells, including T helper 1 cells and IL-17-producing T-cells, were reduced, and regulatory T-cell levels were increased. Furthermore, anti-inflammatory cytokine levels were increased, and dendritic cells were decreased. At 23 days, higher human C-peptide and serum insulin levels and improved glucose tolerance were found. Additionally, WJ-MSCs-GFP differentiated into IPCs as shown by colocalization of human C-peptide and GFP in the pancreas. Significantly more intact islets and less severe insulitis were observed. In conclusion, undifferentiated WJ-MSCs can differentiate into IPCs in vivo with immunomodulatory effects and repair the destroyed islets in NOD mice.

Nickel ions from a corroded cardiovascular stent induce monocytic cell apoptosis: Proposed impact on vascular remodeling and mechanism.

BACKGROUND/PURPOSE: Monocytes play important roles in inflammatory responses and vascular remodeling after vascular stenting. This research focused on impacts of nickel (Ni) ions released from a corroded cardiovascular stent on cytotoxicity and monocyte activation. METHODS: A human promonocytic (macrophage-like) cell line (U937) was exposed to graduated concentrations of Ni(2+)in vitro. Cells were observed and harvested at indicated times to determine the effects using histological and biochemical methods. RESULTS: Ni caused U937 cell death in dose- and time-dependent manners. In vitro, high concentrations of Ni(2+) (>240 µM) significantly induced cell apoptosis and increased terminal deoxynucleotidyl transferase (TdT) dUTP nick end labeling (TUNEL)-positive cells according to flow cytometric surveillance and triggered apoptotic cell death. Although no significant changes in Bcl-2 or Bax expressions were detected after 24 hours of Ni(2+) treatment, increasing cleavage of caspase-3 and -8 was present. Results showed that cleavage of caspase-8 was inhibited by the presence of the inhibitor, Z-IETD-FMK, and this suggested the presence of Ni(2+)-induced U937 cell death through a death receptor-mediated pathway. Simultaneously, when treated with a high concentration of Ni(2+) ions, expressions of the vascular remodeling factors, matrix metalloproteinases (MMP)-9 and -2, were activated in dose- and time-dependent manners. Secretion of the proliferative factor, monocyte chemoattractant protein (MCP)-1, significantly increased during the first 6 hours of incubation with 480 µM Ni(2+)-treated medium. CONCLUSION: Our results demonstrated that a high concentration of Ni ions causes apoptotic cell death of circulating monocytes. They may also play different roles in vascular remodeling during the corrosion process following implantation of Ni alloy-containing devices.

IL-1ß-Induced Mesenchymal Stem Cell Migration Involves MLCK Activation via PKC Signaling.

Mesenchymal stem cells (MSCs) migrate via the bloodstream to sites of injury, possibly attracted by inflammatory cytokines. Although many cytokines can induce stem cell migration, the underlying mechanism is not fully understood. We found that tail vein-injected MSCs migrate to the pancreas in nonobese diabetic (NOD) mice. An ELISA assay revealed that hyperglycemic NOD mice have higher pancreatic levels of interleukin-1ß (IL-1ß) than normal NOD mice and that IL-1ß stimulates MSC migration in a Transwell assay and electric cell-substrate impedance sensing system. Microarray analysis showed that myosin light chain kinase (MLCK) is involved in IL-1ß-induced MSC migration, while Western blots showed that IL-1ß stimulates MLCK expression and activation and that MLCK-siRNA transfection reduces MSC migration. Kinase inhibitors, chromatin immunoprecipitation, and a knockdown study revealed that IL-1ß-induced MLCK expression is regulated by the PKCδ/NF-κB signaling pathway, and a kinase inhibitor study revealed that IL-1ß-induced MLCK activation occurs via the PKCα/MEK/ERK signaling pathway. These results show that IL-1ß released from the pancreas of hyperglycemic NOD mice induces MSC migration and that this is dependent on MLCK expression via the PKCδ/NF-κB pathway and on MLCK activation via the PKCα/MEK/ERK signaling cascade. This study increases our understanding of the mechanisms by which MSCs home to injury sites.

Intraportal injection of insulin-producing cells generated from human bone marrow mesenchymal stem cells decreases blood glucose level in diabetic rats.

We studied the process of trans-differentiation of human bone marrow mesenchymal stem cells (hBM-MSCs) into insulin-producing cells. Streptozotocin (STZ)-induced diabetic rat model was used to study the effect of portal vein transplantation of these insulin-producing cells on blood sugar levels. The BM-MSCs were differentiated into insulin-producing cells under defined conditions. Real-time PCR, immunocytochemistry and glucose challenge were used to evaluate in vitro differentiation. Flow cytometry showed that hBM-MSCs were strongly positive for CD44, CD105 and CD73 and negative for hematopoietic markers CD34, CD38 and CD45. Differentiated cells expressed C-peptide as well as ß-cells specific genes and hormones. Glucose stimulation increased C-peptide secretion in these cells. The insulin-producing, differentiated cells were transplanted into the portal vein of STZ-induced diabetic rats using a Port-A catheter. The insulin-producing cells were localized in the liver of the recipient rat and expressed human C-peptide. Blood glucose levels were reduced in diabetic rats transplanted with insulin-producing cells. We concluded that hBM-MSCs could be trans-differentiated into insulin-producing cells in vitro. Portal vein transplantation of insulin-producing cells alleviated hyperglycemia in diabetic rats.

Lipopolysaccharide induces degradation of connexin43 in rat astrocytes via the ubiquitin-proteasome proteolytic pathway.

The astrocytic syncytium plays a critical role in maintaining the homeostasis of the brain through the regulation of gap junction intercellular communication (GJIC). Changes to GJIC in response to inflammatory stimuli in astrocytes may have serious effects on the brain. We have previously shown that lipopolysaccharide (LPS) reduces connexin43 (Cx43) expression and GJIC in cultured rat astrocytes via a toll-like receptor 4-mediated signaling pathway. In the present study, treatment of astrocytes with LPS resulted in a significant increase in levels of the phosphorylated forms of stress-activated protein kinase/c-Jun N-terminal kinase (SAPK/JNK) -1, -2, and -3 for up to 18 h. An increase in nuclear transcription factor NF-κB levels was also observed after 8 h of LPS treatment and was sustained for up to 18 h. The LPS-induced decrease in Cx43 protein levels and inhibition of GJIC were blocked by the SAPK/JNK inhibitor SP600125, but not by the NF-κB inhibitor BAY11-7082. Following blockade of de novo protein synthesis by cycloheximide, LPS accelerated Cx43 degradation. Moreover, the LPS-induced downregulation of Cx43 was blocked following inhibition of 26S proteasome activity using the reversible proteasome inhibitor MG132 or the irreversible proteasome inhibitor lactacystin. Immunoprecipitation analyses revealed an increased association of Cx43 with both ubiquitin and E3 ubiquitin ligase Nedd4 in astrocytes after LPS stimulation for 6 h and this effect was prevented by SP600125. Taken together, these results suggest that LPS stimulation leads to downregulation of Cx43 expression and GJIC in rat astrocytes by activation of SAPK/JNK and the ubiquitin-proteasome proteolytic pathway.

Matrix metalloproteases and tissue inhibitors of metalloproteinases in medial plica and pannus-like tissue contribute to knee osteoarthritis progression.

Osteoarthritis (OA) is characterized by degradation of the cartilage matrix, leading to pathologic changes in the joints. However, the pathogenic effects of synovial tissue inflammation on OA knees are not clear. To investigate whether the inflammation caused by the medial plica is involved in the pathogenesis of osteoarthritis, we examined the expression of matrix metalloproteinases (MMPs), tissue inhibitors of metalloproteinases (TIMPs), interleukin (IL)-1ß, and tumor necrosis factor (TNF)-α in the medial plica and pannus-like tissue in the knees of patients with medial compartment OA who underwent either arthroscopic medial release (stage II; 15 knee joints from 15 patients) or total knee replacement (stage IV; 18 knee joints from 18 patients). MMP-2, MMP-3, MMP-9, IL-1ß, and TNF-α mRNA and protein levels measured, respectively, by quantitative real-time PCR and Quantibody human MMP arrays, were highly expressed in extracts of medial plica and pannus-like tissue from stage IV knee joints. Immunohistochemical staining also demonstrated high expression of MMP-2, MMP-3, and MMP-9 in plica and pannus-like tissue of stage IV OA knees and not in normal cartilage. Some TIMP/MMP ratios decreased significantly in both medial plica and pannus-like tissue as disease progressed from stage II to stage IV. Furthermore, the migration of cells from the pannus-like tissue was enhanced by IL-1ß, while plica cell migration was enhanced by TNF-α. The results suggest that medial plica and pannus-like tissue may be involved in the process of cartilage degradation in medial compartment OA of the knee.

Antofine-induced connexin43 gap junction disassembly in rat astrocytes involves protein kinase Cß.

Antofine, a phenanthroindolizidine alkaloid derived from Cryptocaryachinensis and Ficusseptica in the Asclepiadaceae milkweed family, is cytotoxic for various cancer cell lines. In this study, we demonstrated that treatment of rat primary astrocytes with antofine induced dose-dependent inhibition of gap junction intercellular communication (GJIC), as assessed by scrape-loading 6-carboxyfluorescein dye transfer. Levels of Cx43 protein were also decreased in a dose- and time-dependent manner following antofine treatment. Double-labeling immunofluorescence microscopy showed that antofine (10ng/ml) induced endocytosis of surface gap junctions into the cytoplasm, where Cx43 was co-localized with the early endosome marker EEA1. Inhibition of lysosomes or proteasomes by co-treatment with antofine and their respective specific inhibitors, NH4Cl or MG132, partially inhibited the antofine-induced decrease in Cx43 protein levels, but did not inhibit the antofine-induced inhibition of GJIC. After 30min of treatment, antofine induced a rapid increase in the intracellular Ca(2+) concentration and activation of protein kinase C (PKC)α/ßII, which was maintained for at least 6h. Co-treatment of astrocytes with antofine and the intracellular Ca(2+) chelator BAPTA-AM prevented downregulation of Cx43 and inhibition of GJIC. Moreover, co-treatment with antofine and a specific PKCß inhibitor prevented endocytosis of gap junctions, downregulation of Cx43, and inhibition of GJIC. Taken together, these findings indicate that antofine induces Cx43 gap junction disassembly by the PKCß signaling pathway. Inhibition of GJIC by antofine may undermine the neuroprotective effect of astrocytes in CNS.

Enhanced membrane-type 1 matrix metalloproteinase expression by hyaluronan oligosaccharides in breast cancer cells facilitates CD44 cleavage and tumor cell migration.

Hyaluronan (HA), a component of the extracellular matrix, plays an important role in cell-cell adhesion and cell migration. Membrane type 1-matrix metalloproteinase (MT1­MMP) is often expressed in invasive cancer cells. CD44, a transmembrane receptor for HA, is implicated in various adhesion-dependent cellular processes including cell migration, tumor cell metastasis and invasion. Previous studies have shown that CD44 is highly expressed in cancer cells and may be proteolytically cleaved at the ectodomain by MT1-MMP; this process of inducing CD44 cleavage plays a critical role in cancer cell migration. We hypothesized that HA modulates MT1-MMP expression to facilitate breast cancer cell migration. Flow cytometry, real-time PCR, western blotting and immunofluorescence staining were used to quantify HA-induced MT1-MMP expression in breast cancer cells. In order to validate the relevance of cell migration and HA-induced MT1-MMP, we analyzed the cell migration via matrigel-coated transwell. We found that after HA oligosaccharide (6.5 kDA) stimulation, MT1-MMP expression in the membrane of breast cancer cells was increased. In response to HA oligosaccharide stimulation, significant upregulation of MT1-MMP mRNA occurred. Our data also provide evidence that HA oligosaccharide enhances MT1-MMP; the elevated expression of MT1-MMP confers enhanced CD44 cleavage and cell migration. In conclusion, we have identified a new function of HA in the induction of MT1-MMP expression in breast cancer cell lines and CD44 cleavage to increase cell migration during the invasion process. The HA oligosaccharide-induced MT1-MMP expression in breast cancer cells may be a critical step in the formation of metastatic colonies.

Transplantation of insulin-producing cells from umbilical cord mesenchymal stem cells for the treatment of streptozotocin-induced diabetic rats.

BACKGROUND: Although diabetes mellitus (DM) can be treated with islet transplantation, a scarcity of donors limits the utility of this technique. This study investigated whether human mesenchymal stem cells (MSCs) from umbilical cord could be induced efficiently to differentiate into insulin-producing cells. Secondly, we evaluated the effect of portal vein transplantation of these differentiated cells in the treatment of streptozotocin-induced diabetes in rats. METHODS: MSCs from human umbilical cord were induced in three stages to differentiate into insulin-producing cells and evaluated by immunocytochemistry, reverse transcriptase, and real-time PCR, and ELISA. Differentiated cells were transplanted into the liver of diabetic rats using a Port-A catheter via the portal vein. Blood glucose levels were monitored weekly. RESULTS: Human nuclei and C-peptide were detected in the rat liver by immunohistochemistry. Pancreatic ß-cell development-related genes were expressed in the differentiated cells. C-peptide release was increased after glucose challenge in vitro. Furthermore, after transplantation of differentiated cells into the diabetic rats, blood sugar level decreased. Insulin-producing cells containing human C-peptide and human nuclei were located in the liver. CONCLUSION: Thus, a Port-A catheter can be used to transplant differentiated insulin-producing cells from human MSCs into the portal vein to alleviate hyperglycemia among diabetic rats.