ABSTRACT
Glioblastoma recruits various nontransformed cells from distant tissues. Although bone marrow-derived mesenchymal stem cells (MSCs) have been observed migrating to glioblastoma, the underlying mechanism driving MSC migration toward glioblastoma remains unclear. Tumor vascularity is critical in the context of recurrent glioblastoma and is closely linked to the expression of stromal cell-derived factor-1 (SDF-1). We demonstrated that cadherin-6 mediated MSC migration both toward SDF-1 and toward glioblastoma cells. Cadherin-6 knockdown resulted in the downregulation of MSCs capacity to migrate in response to SDF-1. Furthermore, MSCs with cadherin-6 knockdown exhibited impaired migration in response to conditioned media derived from glioblastoma cell lines (U87 and U373) expressing SDF-1, thus simulating the glioblastoma microenvironment. Moreover, MSCs enhanced the vasculogenic capacity of U87 cells without increasing the proliferation, cancer stem cell characteristics, or migration of U87. These results suggest that the current strategy of utilizing MSCs as carriers for antiglioblastoma drugs requires careful examination. Furthermore, cadherin-6 may represent a novel potential target for controlling the recruitment of MSCs toward glioblastoma.
Subject(s)
Cadherins , Cell Movement , Chemokine CXCL12 , Glioblastoma , Mesenchymal Stem Cells , Humans , Glioblastoma/metabolism , Glioblastoma/pathology , Glioblastoma/genetics , Mesenchymal Stem Cells/metabolism , Cadherins/metabolism , Cadherins/genetics , Cell Movement/genetics , Chemokine CXCL12/metabolism , Cell Line, Tumor , Cell Proliferation/genetics , Tumor MicroenvironmentABSTRACT
Mesenchymal stem cells are recruited from the bone marrow into breast tumors, contributing to the creation of a tumor microenvironment that fosters tropism for breast tumors. However, the intrinsic mechanisms underlying the recruitment of bone marrow-derived mesenchymal stem cells (MSCs) into the breast tumor microenvironment are still under investigation. Our discoveries identified zonula occludens-1 (ZO-1) as a specific intrinsic molecule that plays a vital role in mediating the collective migration of MSCs towards breast tumor cells and transforming growth factor beta (TGF-ß), which is a crucial factor secreted by breast tumor cells. Upon migration in response to MDA-MB-231 cells and TGF-ß, MSCs showed increased formation of adherens junction-like structures (AJs) expressing N-cadherin and α-catenin at their cell-cell contacts. ZO-1 was found to be recruited into the AJs at the cell-cell contacts between MSCs. Additionally, ZO-1 collaborated with α-catenin to regulate AJ formation, dependently on the SH3 and GUK domains of the ZO-1 protein. ZO-1 knockdown led to the impaired migration of MSCs in response to the stimuli and subsequent downregulation of AJs formation at the cell-cell contacts during MSCs migration. Overall, our study highlights the novel role of ZO-1 in guiding MSC migration towards breast tumor cells, suggesting its potential as a new strategy for controlling and re-engineering the breast tumor microenvironment.
ABSTRACT
Breast tumor cells recruit bone marrow-derived mesenchymal stem cells (BM-MSCs) and alter their cellular characteristics to establish a tumor microenvironment. BM-MSCs enhance tumor angiogenesis through various mechanisms. We investigated the mechanisms by which BM-MSCs promote angiogenesis in response to breast tumor. Conditioned media from MDA-MB-231 (MDA CM) and MCF7 (MCF7 CM) breast tumor cells were used to mimic breast tumor conditions. An in vitro spheroid sprouting assay using human umbilical vein endothelial cells (HUVECs) was conducted to assess the angiogenesis-stimulating potential of BM-MSCs in response to breast tumors. The ROS inhibitor N-acetylcysteine (NAC) and JAK inhibitor ruxolitinib attenuated increased HIF-1α in BM-MSCs in response to MDA CM and MCF7 CM. HIF-1α knockdown or HIF-1ß only partially downregulated VEGF expression and, therefore, the sprouting capacity of HUVECs in response to conditioned media from BM-MSCs treated with MDA CM or MCF7 CM. Inactivation of the VEGF receptor using sorafenib completely inhibited the HUVECs' sprouting. Our results suggest that increased HIF-1α expression under normoxia in BM-MSCs in response to breast tumor cells is mediated by ROS and JAK/Stat3, and that both HIF-1α-dependent and -independent mechanisms increase VEGF expression in BM-MSCs to promote the angiogenic sprouting capacity of endothelial cells in a VEGF-dependent manner.
ABSTRACT
Vasculogenic properties of bone marrow-derived mesenchymal stem cells (MSCs) have been reported, but it is still unclear whether the vasculogenic properties are restricted to some populations of MSCs or whether the entire population of MSCs has these properties. We cultured two different populations of MSCs in different culture media and their vasculogenic properties were evaluated using In vitro spheroid sprouting assay. Neither population of MSCs expressed markers of endothelial progenitor cells (EPCs), but they were different in the profiling of angiogenic factor expression as well as vasculogenic properties. One population of MSCs expressed basic fibroblast growth factor (bFGF) and another expressed hepatocyte growth factor (HGF). MSCs expressing HGF exhibited In vitro angiogenic sprouting capacity in response to bFGF derived from other MSCs as well as to their autocrine HGF. The vasculogenic mesenchymal stem cells (vMSCs) derived from the bone marrow also enhanced In vitro angiogenic sprouting capacity of human umbilical vein endothelial cells (HUVECs) in an HGF-dependent manner. These results suggest that MSCs exhibit different vasculogenic properties, and vMSCs that are different from EPCs may contribute to neovascularization and could be a promising cellular therapy for cardiovascular diseases.
Subject(s)
Bone Marrow , Mesenchymal Stem Cells , Humans , Human Umbilical Vein Endothelial Cells , Neovascularization, Physiologic/physiology , Mesenchymal Stem Cells/metabolism , Cell Differentiation/physiology , Cells, CulturedABSTRACT
The prostate tumor microenvironment plays important roles in the metastasis and hormone-insensitive re-growth of tumor cells. Bone marrow-derived mesenchymal stem cells (BM-MSCs) are recruited into prostate tumors to facilitate tumor microenvironment formation. However, the specific intrinsic molecules mediating BM-MSCs' migration to prostate tumors are unknown. BM-MSCs' migration toward a conditioned medium (CM) of hormone-insensitive (PC3 and DU145) or hormone-sensitive (LNCaP) prostate tumor cells was investigated using a three-dimensional cell migration assay and a transwell migration assay. PC3 and DU145 expressed transforming growth factor-ß (TGF-ß), but LNCaP did not. Regardless of TGF-ß expression, BM-MSCs migrated toward the CM of PC3, DU145, or LNCaP. The CM of PC3 or DU145 expressing TGF-ß increased the phosphorylation of Smad2/3 in BM-MSCs. Inactivation of TGF-ß signaling in BM-MSCs using TGF-ß type 1 receptor (TGFBR1) inhibitors, SB505124, or SB431542 did not allow BM-MSCs to migrate toward the CM. The CM of PC3 or DU145 enhanced N-cadherin expression on BM-MSCs, but the LNCaP CM did not. SB505124, SB431542, and TGFBR1 knockdown prevented an increase in N-cadherin expression. N-cadherin knockdown inhibited the collective migration of BM-MSCs toward the PC3 CM. We identified N-cadherin as a mediator of BM-MSCs' migration toward hormone-insensitive prostate tumor cells expressing TGF-ß and introduced a novel strategy for controlling and re-engineering the prostate tumor microenvironment.
ABSTRACT
In vivo phototoxicity testing is important for predicting drug-induced phototoxicity in humans. Currently, there is no internationally validated in vivo test method for the photosafety evaluation of pharmaceuticals. In this study, we evaluated the phototoxicity of systemically administered drugs using SD rats. We first determined the appropriate ultraviolet A (UVA) dose using 8-methoxypsoralen, a well-known phototoxic drug. Compared to lower and higher UVA doses, we found that a UVA dose of 10 J/cm2 allowed for the detection of phototoxic responses in both a dose- and time-dependent manner. We next performed a phototoxicity study using seven pharmaceutical drugs which included known phototoxic and non-phototoxic drugs using a UVA dose of 10 J/cm2. In order to improve the accuracy of our assessment, we evaluated both gross skin findings as well as histopathological findings. Using gross skin findings alone resulted in an accuracy of 85.7% which could be increased to 100% accuracy when the gross skin findings were combined with histopathological findings. This study suggests that the inclusion of histopathological findings increases the accuracy of the phototoxicity evaluation of systemically administered drugs in SD rats. In conclusion, we found that for studying drug-induced phytotoxicity, a 10 J/cm2 UVA dose serves as the optimal radiation dose, and that the inclusion of histopathological findings increases the accuracy of the phototoxicity evaluation of the drugs.
ABSTRACT
Rationale: Bone marrow-derived mesenchymal stem cells (BM-MSCs) recruited into breast tumors regulate the behavior of tumor cells via various mechanisms and affect clinical outcomes. Although signaling molecules, such as transforming growth factor ß (TGF-ß), are known to transmit signals between BM-MSCs and breast tumor cells for recruiting BM-MSCs, it is unclear which specific intrinsic molecules involved in cell motility mediate the migration of BM-MSCs into breast tumor. It is also unclear as to how specific intrinsic molecules contribute to the migration. Methods: Conditioned medium (CM) from breast tumor cells (MCF-7 and MDA-MB-231) that simulates breast tumor secreting TGF-ß was used to examine the migration of BM-MSCs into breast tumors. A three-dimensional migration assay was performed to investigate the collective migration of BM-MSCs, maintaining cell-cell adhesion, toward breast tumor cells. Results: N-cadherin formed adherens junction-like structures on the intercellular borders of BM-MSCs, and TGF-ß increased the expression of N-cadherin on these borders. Knockdown of Smad4 impaired the TGF-ß-mediated increase in N-cadherin expression in BM-MSCs, but inhibitors of non-canonical TGF-ß pathways, such as extracellular signal-regulated kinases, Akt, and p38, did not affect it. siRNA-mediated knockdown of N-cadherin and Smad4 impaired the migration of BM-MSCs in response to TGF-ß. Conditioned medium from breast tumor cells also enhanced the expression of N-cadherin in BM-MSCs, but inactivation of TGF-ß type 1 receptor (TGFBR1) with SB505124 and TGFBR1 knockdown abolished the increase in N-cadherin expression. BM-MSCs collectively migrated toward CM from MDA-MB-231 in vitro while maintaining cell-cell adhesion through N-cadherin. Knockdown of N-cadherin abolished the migration of BM-MSCs toward the CM from breast tumor cells. Conclusion: In the present study, we identified N-cadherin, an intrinsic transmembrane molecule in adherens junction-like structures, on BM-MSCs as a mediator for the migration of these cells toward breast tumor. The expression of N-cadherin increases on the intercellular borders of BM-MSCs through the TGF-ß canonical signaling and they collectively migrate in response to breast tumor cells expressing TGF-ß via N-cadherin-dependent cell-cell adhesion. We, herein, introduce a novel promising strategy for controlling and re-engineering the breast tumor microenvironment.
Subject(s)
Antigens, CD/metabolism , Breast Neoplasms/metabolism , Cadherins/metabolism , Mesenchymal Stem Cells/metabolism , Receptor, Transforming Growth Factor-beta Type I/antagonists & inhibitors , Transforming Growth Factor beta/pharmacology , Tumor Microenvironment/drug effects , Antigens, CD/genetics , Benzodioxoles/pharmacology , Breast Neoplasms/genetics , Breast Neoplasms/pathology , Cadherins/genetics , Cell Adhesion , Cell Line, Tumor , Cell Movement/drug effects , Culture Media, Conditioned/pharmacology , Female , Humans , Imidazoles/pharmacology , Immunohistochemistry , Proto-Oncogene Proteins c-akt/metabolism , Pyridines/pharmacology , RNA, Small Interfering , Real-Time Polymerase Chain Reaction , Receptor, Transforming Growth Factor-beta Type I/genetics , Receptor, Transforming Growth Factor-beta Type I/metabolism , Signal Transduction/drug effects , Signal Transduction/genetics , Smad4 Protein/genetics , Smad4 Protein/metabolism , Tumor Microenvironment/genetics , p38 Mitogen-Activated Protein Kinases/metabolismABSTRACT
Cancer cachexia is a multifactorial systemic inflammation disease caused by complex interactions between the tumor and host tissues via soluble factors. However, whether cancer cachexia affects the bone marrow, in particular the hematopoietic stem cells (HSCs) and mesenchymal stem cells (MSCs), remains unclear. Here, we investigated the bone marrow and bone in a cancer cachexia animal model generated by transplanting Lewis lung carcinoma cells. The number of bone marrow mononuclear cells (BM-MNCs) started to significantly decrease in the cancer cachectic animal model prior to the discernable loss of muscle and fat. This decrease in BM-MNCs was associated with myeloid skewing in the circulation and the expansion of hematopoietic progenitors in the bone marrow. Bone loss occurred in the cancer cachexia animal model and accompanied the decrease in the bone marrow MSCs that play important roles in both supporting HSCs and maintaining bone homeostasis. Glucocorticoid signaling mediated the decrease in bone marrow MSCs in the cancer cachectic environment. The cancer cachexia environment also skewed the differentiation of the bone marrow MSCs toward adipogenic fate via JAK/STAT as well as glucocorticoid signaling. Our results suggest that the bone loss induced in cancer cachexia is associated with the depletion and the impaired differentiation capacity of the bone marrow MSCs.
ABSTRACT
Excessive vascular permeability occurs in inflammatory disease processes. Vascular endothelial cadherin (VE-cadherin) is an adhesion protein that controls vascular permeability. We identified monoclonal antibodies (mAbs) to human VE-cadherin that activate cell adhesion and inhibit the increased permeability of endothelial cell monolayers induced by thrombin receptor activator peptide-6 (TRAP-6). Two mAbs, 8A12c and 3A5a, reduce permeability, whereas an inhibitory mAb, 2E11d, enhances permeability. Activating mAbs also reduce permeability induced by tumor necrosis factor-α (TNF-α) and vascular endothelial cell growth factor (VEGF). The activating mAbs also stabilize the organization of the adherens junctions that are disrupted by TRAP-6, VEGF, or TNF-α. The activating mAbs act directly on the adhesive function of VE-cadherin because they did not block the accumulation of actin filaments stimulated by TRAP-6 and enhance physical cell-cell adhesion of VE-cadherin-expressing tissue culture cells. Therefore, VE-cadherin function can be regulated at the cell surface to control endothelial permeability.NEW & NOTEWORTHY Excessive vascular permeability is a serious complication of many inflammatory disease conditions. We have developed monoclonal antibodies that inhibit increases in endothelial monolayer permeability induced by several signaling factors by activating VE-cadherin mediated adhesion and stabilizing cell junctions. These antibodies and/or the mechanisms they reveal may lead to important therapeutics to treat vascular leakiness and inflammation.
Subject(s)
Adherens Junctions/drug effects , Antibodies, Monoclonal/pharmacology , Cadherins/agonists , Capillary Permeability/drug effects , Cell Adhesion/drug effects , Human Umbilical Vein Endothelial Cells/drug effects , Adherens Junctions/metabolism , Antigens, CD/metabolism , Cadherins/antagonists & inhibitors , Cadherins/metabolism , Cell Line, Tumor , Colonic Neoplasms/drug therapy , Colonic Neoplasms/metabolism , Colonic Neoplasms/pathology , Human Umbilical Vein Endothelial Cells/metabolism , Humans , Nocodazole/pharmacology , Oligopeptides/pharmacology , Receptors, Thrombin/agonists , Receptors, Thrombin/metabolism , Signal Transduction , Tubulin Modulators/pharmacology , Tumor Necrosis Factor-alpha/pharmacology , Vascular Endothelial Growth Factor A/pharmacologyABSTRACT
Some drugs cause phototoxicity in humans when exposed to light, thus there is a need for an in vivo phototoxicity test to evaluate them. However, an in vivo phototoxicity test method to evaluate this has not been established. This study aimed to establish an in vivo phototoxicity test method for transdermally administered drugs. For this, we evaluated the phototoxicity using Sprague-Dawley (SD) rats for transdermal administered drugs and we studied the appropriate UVA dose using 8-methoxypsalen, which is a well-known phototoxic drug. We found that a UVA dose of 15 J/cm2 was dose and time dependent response compared to other UVA doses. We performed the Minimum Erythema Dose (MED) test because UVB can cause skin irritation by itself and selected 0.01 J/cm2 as an appropriate dose of UVB. Using the selected UVA and UVB doses, we performed a phototoxicity study of 6 pharmaceutical drugs, which included phototoxic and non-phototoxic drugs. As a result of the phototoxicity test, 100% accuracy was obtained when compared with previous studies. In addition, we performed histopathology to confirm the new findings. We found that histopathology can be used as an additional indicator of phototoxicity test for transdermally administered drugs.
ABSTRACT
Endogenous bone marrow-derived mesenchymal stem cells are mobilized to peripheral blood and injured tissues in response to changes in the expression of various growth factors and cytokines in the injured tissues, including substance P (SP), transforming growth factor-beta (TGF-ß), and stromal cell-derived factor-1 (SDF-1). SP, TGF-ß, and SDF-1 are all known to induce the migration of bone marrow-derived mesenchymal stem cells (BM-MSCs). However, it is not yet clear how these stimuli influence or interact with each other during BM-MSC mobilization. This study used mouse bone marrow-derived mesenchymal stem cell-like ST2 cells and human BM-MSCs to evaluate whether SP, TGF-ß, and SDF-1 mutually regulate their respective effects on the mobilization of BM-MSCs. SP pretreatment of ST2 and BM-MSCs impaired their response to TGF-ß while the introduction of SP receptor antagonist restored the mobilization of ST2 and BM-MSCs in response to TGF-ß. TGF-ß pretreatment did not affect the migration of ST2 and BM-MSCs in response to SP, but downregulated their migration in response to SDF-1. SP pretreatment modulated the activation of TGF-ß noncanonical pathways in ST2 cells and BM-MSCs, but not canonical pathways. These results suggest that the migration of mesenchymal stem cells is regulated by complex functional interactions between SP, TGF-ß, and SDF-1. Thus, understanding the complex functional interactions of these chemotactic stimuli would contribute to ensuring the development of safe and effective combination treatments for the mobilization of BM-MSCs.
Subject(s)
Bone Marrow Cells/immunology , Chemotaxis/immunology , Mesenchymal Stem Cells/immunology , Signal Transduction/immunology , Animals , Bone Marrow Cells/cytology , Cell Line , Chemokine CXCL12/immunology , Humans , Mesenchymal Stem Cells/cytology , Mice , Transforming Growth Factor beta/immunologyABSTRACT
This study aimed to analyze the milling accuracy of lithium disilicate and zirconia-reinforced silicate crown fabricated using chairside computer-aided design/manufacturing (CAD/CAM) system. Mandibular left first premolar was selected for abutment. A master model was obtained for digital impression using an intraoral scanner, and crowns were designed using a CAD software design program. Amber Mill (AM), IPS e max CAD (IPS), and CELTRA DUO (CEL) were used in the CAD/CAM system, and a total 45 crowns (15 crowns each for AM, IPS, and CEL) was fabricated. Milling accuracy was analyzed with respect to trueness, measured by superimposing CAD design data and scan data through a three-dimensional program to compare the outer and inner surfaces and internal and external parts, thereby acquiring both quantitative and qualitative data. Data were analyzed using the non-parametric test and Kruskal-Wallis H test. In addition, the Mann-Whitney U test was used by applying the level of significance (0.05/3 = 0.016) adjusted by post-analysis Bonferroni correction. All the measured parts of the lithium disilicate and zirconia-reinforced silicate crowns showed statistically significant differences (p < 0.05). The lithium disilicate (AM and IPS) materials showed superior milling accuracy than the zirconia-reinforced lithium silicate (CEL) materials.
ABSTRACT
This study aims to compare the torque values for various lengths of the titanium-based orthodontic anchor screw (OAS), different anchorage methods and varying artificial bone densities after predrilling. Furthermore, the effects of these parameters on bone stability are evaluated. A total of 144 OASs were prepared with a diameter of 1.6 mm and heights of 6, 8 and 10 mm. Artificial bones were selected according to their density, corresponding to Grades 50, 40 and 30. Torque values for the automatic device and manual anchorage methods exhibited a statistically significant difference for the same-sized OAS, according to the bone density of the artificial bones (p < 0.05). However, when insertion torque was at the maximum rotations, there was no significant difference in the torque values for the Grade 30 artificial bone (p > 0.05). When the torque values of both anchorage methods were statistically compared with the mean difference for each group, the results of the manual anchorage method were significantly higher than those of the automatic device anchorage method (p < 0.05). A statistically significant difference was observed in the bone stability resulting from different OAS anchorage methods and artificial bone lengths. These findings suggest that the automatic anchorage method should be used when fixing the OAS.
ABSTRACT
Diabetes induces cellular dysfunction in dermal fibroblasts, such as impairment in migration, which is a major cause of chronic wound. Here, we demonstrated that the migration of human dermal fibroblasts was impaired under a high glucose culture condition. Substance P (SP) rescued the impaired migration of the fibroblasts. The activity of Rac1, Rho-associated kinase (ROCK), and Src was required for SP-mediated rescue of fibroblast migration. SP activated Rac1 and Src, whereas, NSC23766, a Rac1 inhibitor, and PP1 and PP2, Src inhibitors, inhibited SP-mediated enhancement of fibroblast migration. Y-27632, a ROCK inhibitor, inhibited the SP-mediated rescue of fibroblast migration. Senescence-associated ß-galactosidase activity increased in human dermal fibroblasts cultured in a high glucose environment, but SP inhibited the ß-galactosidase activity of the fibroblasts. These results suggest that SP promotes the migration of human dermal fibroblasts in diabetic-condition-mimicking cultures via the activity of Rac1, ROCK, and Src, and inhibits fibroblast senescence in hyperglycemic cultures.
Subject(s)
Cell Movement/drug effects , Cellular Senescence/drug effects , Dermis/pathology , Fibroblasts/pathology , Hyperglycemia/pathology , Substance P/pharmacology , Cells, Cultured , Cyclin-Dependent Kinase Inhibitor p21/metabolism , Fibroblasts/drug effects , Fibroblasts/metabolism , Glucose/toxicity , Humans , rac1 GTP-Binding Protein/metabolism , rho-Associated Kinases/metabolism , src-Family Kinases/metabolismABSTRACT
In order to overcome the limitations of single in vitro eye irritation tests, Integrated Approaches to Testing Assessment strategies have been suggested for evaluating eye irritation. This study developed two tiered approaches combining alternative test methods. They were designed in consideration of the solubility property of test chemicals and to use the RhCE tests at final steps. The tiered approach A is composed of the STE, BCOP, HET-CAM or RhCE tests, whereas the tiered approach B is designed to perform simultaneously two in vitro test methods at the first stage and the RhCE test at the final stage. The predictive capacity of the two tiered approaches was estimated using 47 chemicals. The accuracy, sensitivity, and specificity value of the tiered approach A were 95.7% (45/47), 100% (34/34), and 84.6% (11/13), respectively, whereas those of the tiered approach B were 95.7% (45/47), 97.1% (33/34), and 92.3% (12/13), respectively. The approach A and B were considered to be available methods for distinguishing test chemicals of Category 1 (all 73.3%) and No Category (84.6% and 92.3%), respectively. Especially, the approach B was considered as an efficient method as the Bottom-Up approach, because it predicted correctly test chemicals classified as No Category.
Subject(s)
Cornea/drug effects , Epithelium/drug effects , Irritants/toxicity , Toxicity Tests , Animal Testing Alternatives , Animals , Cattle , Chick Embryo , Chorioallantoic Membrane/drug effects , Corneal Opacity/chemically induced , Humans , Sensitivity and SpecificityABSTRACT
Bone morphogenetic protein (BMP) signaling and Notch signaling play important roles in tumorigenesis in various organs and tissues, including the breast. BMP-4 enhanced epithelial mesenchymal transition (EMT) and stem cell properties in both mammary epithelial cell line and breast carcinoma cell line. BMP-4 increased the expression of EMT biomarkers, such as fibronectin, laminin, N-cadherin, and Slug. BMP-4 also activated Notch signaling in these cells and increased the sphere forming efficiency of the non-transformed mammary epithelial cell line MCF-10A. In addition, BMP-4 upregulated the sphere forming efficiency, colony formation efficiency, and the expression of cancer stem cell markers, such as Nanog and CD44, in the breast carcinoma cell line MDA-MB-231. Inhibition of Notch signaling downregulated EMT and stem cell properties induced by BMP-4. Down-regulation of Smad4 using siRNA impaired the BMP-4-induced activation of Notch signaling, as well as the BMP-4-mediated EMT. These results suggest that EMT and stem cell properties are increased in mammary epithelial cells and breast cancer cells through the activation of Notch signaling in a Smad4-dependent manner in response to BMP-4.
Subject(s)
Bone Morphogenetic Protein 4/genetics , Epithelial-Mesenchymal Transition/genetics , Neoplasms/etiology , Neoplasms/metabolism , Neoplastic Stem Cells/metabolism , Receptors, Notch/metabolism , Signal Transduction , Animals , Bone Morphogenetic Protein 4/metabolism , Cell Line, Tumor , Cell Movement/genetics , Disease Models, Animal , Female , Humans , Mice , Neoplasms/pathology , Neoplastic Stem Cells/pathology , Smad4 Protein/genetics , Smad4 Protein/metabolism , Xenograft Model Antitumor AssaysABSTRACT
The need for in vitro eye irritation test replacing in vivo is steadily increasing. The MCTT HCE™ eye irritation test (EIT) using 3D reconstructed human cornea-like epithelium, was developed to identify ocular irritants from non-irritants those that are not requiring classification and labelling for eye irritation. Here, we report the results of me-too validation study, which was conducted to evaluate the reliability and relevance of the MCTT HCETM EIT, according to performance standards (PS) of OECD TG 492. The optimal cutoff to determine irritation in the prediction model was preliminarily established at 45% with the receiver operation characteristics (ROC) curve for 141 reference substances. To demonstrate the reproducibility of within- and between-laboratory (WLR and BLR), a set of 30 PS reference chemicals were tested in three laboratories three times. The WLR and BLR concordance with the binary decision of whether non-irritant or irritant was estimated to be 90-100% and 90%, respectively, and both met the PS requirements. The predictive capacity of the respective laboratories for the 30 reference chemicals were evaluated based on three different estimation methods, and the results were comparable, with sensitivity ranging from 89.6 to 93.3%, the specificity ranging from 62.2 to 66.7%, and the accuracy ranging from 75.9 to 80.0%. Additional test with the new set of 30 PS substances in the revised OECD GD 216 yielded a performance of sensitivity ranging from 92.6-93.3%, the specificity 62.2-66.7% and the accuracy 77.4-80.0%. 95.0% sensitivity, 67.2% specificity, and 83.0% accuracy were obtained for 141 reference substances in total. Furthermore, separate cutoffs for liquids and solids, 35% and 60%, respectively, produced better predictivity, which was established as a final prediction model. Collectively, our study demonstrated that MCTT HCETM EIT meets the reproducibility and predictivity criteria stated in OECD TG 492 PS.
Subject(s)
Animal Testing Alternatives , Epithelium, Corneal/drug effects , Irritants/toxicity , Toxicity Tests/methods , Humans , Reproducibility of ResultsABSTRACT
Endogenous bone marrow-derived mesenchymal stem cells (BM-MSCs) are mobilized into peripheral blood and injured tissues by various growth factors and cytokines that are expressed in the injured tissues, such as substance P (SP), stromal cell derived factor-1 (SDF-1), and transforming growth factor-beta (TGF-ß). Extracellular bioactive lipid metabolites such as ceramide-1-phosphate and sphingosine-1-phosphate also modulate BM-MSC migration as SP, SDF-1, and TGF-ß. However, the roles of intrinsic lipid kinases of BM-MSCs in the stem cell migration are unclear. Here, we demonstrated that ceramide kinase mediates the chemotactic migration of BM-MSCs in response to SP, SDF-1, or TGF-ß. Furthermore, a specific inhibitor of ceramide kinase inhibited TGF-ß-induced migration of BM-MSCs and N-cadherin that is necessary for BM-MSCs migration in response to TGF-ß. Therefore, these results suggest that the intracellular ceramide kinase is required for the BM-MSCs migration and the roles of the intrinsic ceramide kinase in the migration are associated with N-cadherin regulation.
Subject(s)
Cell Movement/physiology , Mesenchymal Stem Cells/physiology , Phosphotransferases (Alcohol Group Acceptor)/metabolism , Animals , Antigens, CD/genetics , Cadherins/genetics , Cell Line , Ceramides/metabolism , Chemokine CXCL12/metabolism , Chemotaxis/physiology , Gene Expression Regulation , Humans , Mice , RNA, Messenger/genetics , RNA, Messenger/metabolism , Substance P/metabolism , Transforming Growth Factor beta/metabolismABSTRACT
Preservation of pancreatic ß-cells is required for the development of therapies for type 1 and type 2 diabetes (T1D and T2D, respectively). Our previous study demonstrated that substance P (SP) preserves ß-cell populations in mice with streptozotocin-induced T1D. Here, we demonstrated that chronic systemic treatment with SP restored the mass of ß-cells both in nonobese diabetic (NOD) mice with T1D or db/db mice with T2D. SP delayed the onset of T1D in NOD mice via immune modulation. SP inhibited immune infiltration into islets and the salivary glands of NOD mice. In db/db mice, SP treatment rescued glucose intolerance. Moreover, SP inhibited apoptosis, as well as the activation of pancreatic stellate cells in pancreatic islets of db/db mice. SP downregulated the number of α-smooth muscle actin (α-SMA) expressing cells in db/db pancreatic islets. Cleaved-caspase-3 expression was reduced in islets of SP-treated db/db mice compared to that in the control. Therefore, these results suggested that SP may preserve pancreatic ß-cells through immune modulation and protection from the stimulated activation of pancreatic stellate cells and apoptosis in T1D and T2D, respectively.
Subject(s)
Diabetes Mellitus, Experimental/pathology , Diabetes Mellitus, Type 1/pathology , Diabetes Mellitus, Type 2/pathology , Insulin-Secreting Cells/pathology , Substance P/pharmacology , Animals , Apoptosis/drug effects , Diabetes Mellitus, Experimental/complications , Diabetes Mellitus, Experimental/drug therapy , Diabetes Mellitus, Type 1/complications , Diabetes Mellitus, Type 1/drug therapy , Diabetes Mellitus, Type 1/immunology , Diabetes Mellitus, Type 2/complications , Diabetes Mellitus, Type 2/drug therapy , Female , Hyperglycemia/complications , Hyperglycemia/drug therapy , Hyperglycemia/pathology , Immunomodulation/drug effects , Insulin-Secreting Cells/drug effects , Insulin-Secreting Cells/metabolism , Male , Mice, Inbred NOD , Pancreatic Stellate Cells/drug effects , Pancreatic Stellate Cells/metabolism , Pancreatic Stellate Cells/pathology , Substance P/therapeutic useABSTRACT
Atopic dermatitis (AD) is a chronic inflammatory skin disorder characterized by intense pruritus and eczematous lesion. In this study, topically applied substance P (SP) significantly alleviated AD-like clinical symptoms in 2, 4, 6-trinitrochlorobenzene (TNCB)-induced dermatitis in NC/Nga mice. This effect was nullified by pretreatment of the neurokinin-1 receptor (NK-1R) antagonist CP99994. SP treatment significantly reduced the infiltration of mast cells and CD3-positive T cells as well as inflammatory cytokines, such as tumor necrosis factor-α (TNF-α) and thymic stromal lymphopoietin (TSLP), in AD-like skin lesions and decreased the levels of IgE and thymus and activation-regulated chemokine in serum. This SP-induced alleviation of allergic inflammatory responses was also confirmed as reduced activation in the axillary lymph nodes (aLN) and spleen, suggesting the systemic effect of SP on immune responses in TNCB-induced NC/Nga mice. Furthermore, SP-mediated TSLP reduction was confirmed in human keratinocyte culture under pro-inflammatory TNF-α stimulation. Taken together, these results suggest that topically administered SP may have potential as a medication for atopic dermatitis.