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Matrigel
Terms modified by Matrigel Selected AbstractsRetinoic acid increases the length and volume density of ducts in the rat embryonic pancreasDEVELOPMENT GROWTH & DIFFERENTIATION, Issue 2 2003Carene Erasmus In this study, the role of all -trans retinoic acid (RA) on the proliferation of rat embryonic pancreas ducts and on the proportion of insulin cells was investigated. All- trans RA (10,6 m) was added to Ham's F12. ITS serum-free medium in which 12.5 day rat dorsal pancreatic buds were cultured on Matrigel. Control explants were cultured on Matrigel in Ham's F12. ITS alone or in Ham's F12. ITS containing ethanol (the diluent for RA). After a 7 day culture period, explants were incubated with bromodeoxyuridine (BrdU) for assessment of cell proliferation. Explants were processed for both morphometry and immunocytochemistry. The length density and volume density of the pancreatic ducts were assessed using an image analysis system. Cells positive for insulin, BrdU and glucagon were localized on adjacent serial sections. RA treatment caused a statistically significant increase in the volume density (P < 0.007) and length density (P < 0.008) of the ducts, as well as a 1.2-fold increase (P < 0.0001) in the proportion of insulin to glucagon cells, compared to both control groups. Few insulin cells were BrdU positive, indicating that cells had a low proliferation rate. The increased proportion of insulin cells may relate to the increased volume density and length density of the ducts in RA-treated explants. It is suggested that RA stimulated the production of additional progenitor cells and not proliferation of existing insulin cells. [source] Growth of ameloblast-lineage cells in a three-dimensional Matrigel environmentEUROPEAN JOURNAL OF ORAL SCIENCES, Issue 2006Wu Li Enamel organ epithelial cells grow in culture as two distinct cell populations , either stellate-shaped or polygonal-shaped cells. The polygonal cells have an ameloblast cell phenotype and are difficult to grow in culture beyond two passages. This study was designed to determine the effects of a Matrigel three-dimensional (3D) environment on polygonal cells, as compared with stellate cells, derived from porcine tooth enamel organ. Enamel organs were dissected free from the unerupted molars of 30-kg pigs and then grown in LCH-8e media, either with or without serum. Cells grown in serum-free media were primarily polygonal shaped, whereas cells grown in media containing serum were stellate shaped. Both types of cells were grown in a 3D Matrigel matrix. In addition, polygonal-shaped cells were mixed with hydroxyapatite powder and transplanted subcutaneously into nude mice. Polygonal-shaped epithelial cells formed cell groups, similar to epithelial pearls, both in vitro and in vivo. The stellate-shaped cells, in contrast, did not form similar structures, but remained suspended in the Matrigel and gradually disappeared from the culture. These results suggest that a Matrigel environment, rich in basement membrane and matrix proteins, selects for polygonal-shaped ameloblast-lineage cells and induces the formation of epithelial pearls. [source] Inhibition of urokinase receptor gene expression and cell invasion by anti-uPAR DNAzymes in osteosarcoma cellsFEBS JOURNAL, Issue 14 2005Charles E. De Bock The urokinase-type plasminogen activator (uPA) receptor (uPAR) has been implicated in signal transduction and biological processes including cancer metastasis, angiogenesis, cell migration, and wound healing. It is a specific cell surface receptor for its ligand uPA, which catalyzes the formation of plasmin from plasminogen, thereby activating the proteolytic cascade that contributes to the breakdown of extracellular matrix, a key step in cancer metastasis. We have synthesized three different DNA enzymes (Dz372, Dz483 and Dz720) targeting uPAR mRNA at three separate purine (A or G),pyrimidine (U or C) junctions. Two of these DNAzymes, Dz483 and Dz720, cleaved uPAR transcript in vitro with high efficacy and specificity at a molar ratio (uPAR to Dz) as low as 1 : 0.2. When analyzed over 2 h with a 200-fold molar excess of DNAzymes to uPAR transcript, Dz720 and Dz483 were able to decrease uPAR transcript in vitro by ,,93% and ,,84%, respectively. They also showed an ability to cleave uPAR mRNA in the human osteosarcoma cell line Saos-2 after transfection. The DNAzyme Dz720 decreased uPAR mRNA within 4 h of transfection, and inhibited uPAR protein concentrations by 55% in Saos-2 cells. The decrease in uPAR mRNA and protein concentrations caused by Dz720 significantly suppressed Saos-2 cell invasion as assessed by an in vitro Matrigel assay. The use of DNAzyme methodology adds a new potential clinical agent for decreasing uPAR mRNA expression and inhibiting cancer invasion and metastasis. [source] Chromosome 18 suppresses tumorigenic properties of human prostate cancer cellsGENES, CHROMOSOMES AND CANCER, Issue 3 2006Audrey Gagnon Although prostate cancer is still the most diagnosed cancer in men, most genes implicated in its progression are yet to be identified. Chromosome abnormalities have been detected in human prostate tumors, many of them associated with prostate cancer progression. Indeed, alterations (including deletions or amplifications) of more than 15 human chromosomes have been reported in prostate cancer. We hypothesized that transferring normal human chromosomes into human prostate cancer cells would interfere with their tumorigenic and/or metastatic properties. We used microcell-mediated chromosome transfer to introduce human chromosomes 10, 12, 17, and 18 into highly tumorigenic (PC-3M-Pro4) and highly metastatic (PC-3M-LN4) PC-3-derived cell lines. We tested the in vitro and in vivo properties of these hybrids. Introducing chromosome 18 into the PC-3M-LN4 prostate cancer cell line greatly reduced its tumorigenic phenotype. We observed retarded growth in soft agar, decreased invasiveness through Matrigel, and delayed tumor growth into nude mice, both subcutaneously and orthotopically. This phenotype is associated with a marker in the 18q21 region. Combined with the loss of human chromosome 18 regions often seen in patients with advanced prostate cancer, our results show that chromosome 18 encodes one or more tumor-suppressor genes whose inactivation contributes to prostate cancer progression. © 2005 Wiley-Liss, Inc. [source] Fibroblast activation protein increases apoptosis, cell adhesion, and migration by the LX-2 human stellate cell line,HEPATOLOGY, Issue 4 2005Xin Maggie Wang Injury and repair in chronic liver disease involve cell adhesion, migration, apoptosis, proliferation, and a wound healing response. In liver, fibroblast activation protein (FAP) has both collagenase and dipeptidyl peptidase IV (DPIV) activities and is expressed only by activated hepatic stellate cells (HSC) and myofibroblasts, which produce and degrade extracellular matrix (ECM). FAP was colocalized with collagen fibers, fibronectin, and collagen type I in human liver. FAP function was examined in vitro by expressing green fluorescent protein FAP fusion protein in cell lines cultured on collagen-I, fibronectin, and Matrigel. Glutamates at 203 and 204 as well as serine624 of FAP were essential for peptidase activity. Human embryonic kidney 293T cells overexpressing FAP showed reduced adhesion and migration. FAP overexpression in the human HSC line LX-2 caused increased cell adhesion and migration on ECM proteins as well as invasion across transwells in the absence or presence of transforming growth factor beta-1. FAP overexpression enhanced staurosporine streptomyces,stimulated apoptosis in both cell lines. Interestingly, the enzyme activity of FAP was not required for these functions. Overexpressing FAP increased the expression of matrix metalloproteinase-2 and CD44 and reduced integrin-,1 expression in 293T cells, suggesting potential pathways of FAP-mediated impairment of cell adhesion and migration in this epithelial cell line. In conclusion, these findings further support a pro-fibrogenic role for FAP by indicating that, in addition to its enzymatic functions, FAP has important nonenzymatic functions that in chronic liver injury may facilitate tissue remodeling through FAP-mediated enhancement of HSC cell adhesion, migration, and apoptosis. Supplementary material for this article can be found on the HEPATOLOGY website (http://www.interscience.wiley.com/jpages/0270-9139/suppmat/index.html). (HEPATOLOGY 2005;42:935,945.) [source] Derivation, characterization, and phenotypic variation of hepatic progenitor cell lines isolated from adult ratsHEPATOLOGY, Issue 2 2002Li Yin Liver progenitor cells (LPCs) cloned from adult rat livers following allyl alcohol injury express hematopoietic stem cell and early hepatic lineage markers when cultured on feeder layers; under these conditions, neither mature hepatocyte nor bile duct, Ito, stellate, Kupffer cell, or macrophage markers are detected. These phenotypes have remained stable without aneuploidy or morphological transformation after more than 100 population doublings. When cultured without feeder layers, the early lineage markers disappear, and mature hepatocyte markers are expressed; mature hepatocytic differentiation and cell size are also augmented by polypeptide and steroidal growth factors. In contrast to hepatocytic potential, duct-like structures and biliary epithelial markers are expressed on Matrigel. Because they were derived without carcinogens or mutagens, these bipotential LPC lines provide novel tools for models of cellular plasticity and hepatocarcinogenesis, as well as lines for use in cellular transplantation, gene therapy, and bioreactor construction. [source] Autocrine motility factor enhances hepatoma cell invasion across the basement membrane through activation of ,1 integrinsHEPATOLOGY, Issue 1 2001Takuji Torimura Autocrine motility factor/phosphohexose isomerase (AMF/PHI) is a cytokine that is linked to tumor invasion and metastasis. In hepatocellular carcinoma (HCC) tissues, hepatoma cells produce AMF/PHI and its receptor, Mr 78,000 glycoprotein (gp78), is strongly detected in hepatoma cells invading into the stroma and tumor thrombi in the portal vein. Here, we investigated the mechanism of hepatoma cell invasion through Matrigel induced by AMF/PHI using 3 hepatoma cell lines. Production of AMF/PHI, phosphorylation of MEK1/2, and Rho activity were investigated by immunoblotting. Expression of AMF/PHI and gp78 was observed by confocal fluorescence microscopy. The influence of AMF/PHI on activated integrin ,1 subunit expression was evaluated by flow cytometry. Changes in invasion, adhesion, and motility induced by AMF/PHI were evaluated using chemoinvasion, adhesion, and phagokinetic track motility assays. The effect of AMF/PHI on matrix metalloproteinase (MMP) secretion was evaluated by gelatin zymography. Hepatoma cells produced AMF/PHI and expressed gp78. Although AMF/PHI was ubiquitously detected, gp78 was strongly expressed in migrating cells. AMF/PHI induced up-regulation of activated integrin ,1 subunit expression. AMF/PHI stimulated hepatoma cell invasion through Matrigel, and stimulated the adhesion, motility, and MMP-2 secretion of hepatoma cells. The latter effects were suppressed by the function-blocking antibody for integrin ,1 subunit. AMF/PHI also enhanced Rho activity and the phosphorylation of MEK1 and MEK 2. Our results indicate that AMF/PHI enhances hepatoma cell invasion through Matrigel in an autocrine manner by stimulating the adhesion, motility, and MMP-2 secretion of these cells through activation of ,1 integrins. [source] Effect of Matrigel on adenoid cystic carcinoma cell line differentiationINTERNATIONAL JOURNAL OF EXPERIMENTAL PATHOLOGY, Issue 6 2006Márcia M. Marques Summary Adenoid cystic carcinoma (ACC) is a frequent malignant salivary gland neoplasm presenting different growth patterns described as tubular, cribriform and solid, which represent distinct differentiation stages. Cell lines originated from ACCs grown inside three-dimensional environments have not been capable to reproduce all in vivo ACC growth patterns. As ACC cells in vivo present replicated basement membrane, to mimic this situation in vitro ACC cells (CAC2 cells) were grown on the top of a reconstituted basement membrane (Matrigel). Phenotype differences were assessed by light, fluorescence and transmission electron microscopy. The cultures grown on the top of Matrigel presented three-dimensional arrangement of cells intercepted by cellular cords. At these, cell nests pseudocyst formations were observed. This morphological structure entirely reproduced the cribriform growth pattern of ACC. We suggest that the cribriform differentiation of ACC in culture is dependent of proteins and growth factors associated in a bi-dimensional structure. [source] A subpopulation of peritoneal macrophages form capillary-like lumens and branching patterns in vitroJOURNAL OF CELLULAR AND MOLECULAR MEDICINE, Issue 3 2006Mirela Anghelina Abstract Objective: We have previously shown that monocytes/macrophages (MC/Mph) influence neovascularization by extracellular matrix degradation, and by direct incorporation into growing microvessels. To date, neither the phenotype of these cells, nor the stages of their capillary-like conversion were sufficiently characterized. Methods: We isolated mouse peritoneal Mph from transgenic mice expressing fluorescent proteins either ubiquitously, or specifically in the myelocytic lineage. These Mph were embedded in Matrigel which contained fluorescent protease substrates, exposed to an MCP-1 chemotactic gradient, and then examined by confocal microscopy after various intervals. Results: Within 3 hrs after gel embedding, we detected TIMP-1 and MMP-12 dependent proteolysis of the matrix surrounding Mph, mostly in the direction of high concentrations of MCP-1. After 2 days, Mph developed intracellular vacuoles containing degradation product. At 5 days these vacuoles were enlarged and/or fused to generate trans-cellular lumens in approximately 10% of cells or more (depending on animal's genetic background). At this stage, Mph became tubular, and occasionally organized in three-dimensional structures resembling branched microvessels. Conclusion: Isolated mouse peritoneal Mph penetrate Matrigel and form tunnels via a metalloprotease-driven proteolysis and phagocytosis. Following a morphological adjustment driven by occurrence, enlargement and/or fusion process of intracellular vacuoles, similar to that described in bona fide endothelium, a subpopulation of these cells end up by lining a capillary-like lumen in vitro. Thus we show that adult Mph, not only the more primitive ,endothelial progenitors', have functional properties until now considered defining of the endothelial phenotype. [source] Purification and growth of endothelial progenitor cells from murine bone marrow mononuclear cellsJOURNAL OF CELLULAR BIOCHEMISTRY, Issue 1 2008Qi Ru Wang Abstract This study reports the culture and purification of murine bone marrow endothelial progenitor cells (EPCs) using endothelial cell-conditioned medium (EC-CM). Endothelial-like cells appeared at day 5 in culture of bone marrow mononuclear cells in the presence of EC-CM in the culture system, and these cells incorporated acetylated low-density lipoproteins (Ac-LDL) and reacted with endothelial-specific Ulex Europaeus Lectin. Continued incubation of these cells at low density with EC-CM for longer than 10 days resulted in the formation of endothelial cell colonies which gave rise to colonies of endothelial progeny and can be passed for many generations in the EC-CM culture system. Cells derived from these colonies expressed endothelial cell markers such as vWF and CD31, incorporated Dil-Ac-LDL, stained positive for Ulex Europaeus Lectin, formed capillary-like structures on Matrigel, and demonstrated a high proliferative capacity in culture. These bone marrow-derived adherent cells were identified as EPCs. The purification and the formation of EPC colonies by using EC-CM were associated with the cytokines secreted in the EC-CM. VEGF, bFGF, and GM-CSF in the EC-CM stimulated the proliferation and growth of EPCs, whereas AcSDKP (tetrapeptide NAc-Ser-Asp-Lys-Pro) in EC-CM suppressed the growth of mesenchymal stem cells (MSC) and fibroblasts. This approach is efficient for isolation/purification and outgrowth of bone marrow EPCs in vitro, a very important cell source in angiogenic therapies and regenerative medicine. J. Cell. Biochem. 103: 21,29, 2008. © 2007 Wiley-Liss, Inc. [source] Dominant-negative CREB inhibits heparanase functionality and melanoma cell invasionJOURNAL OF CELLULAR BIOCHEMISTRY, Issue 2 2004Rebecca Aucoin Abstract Heparanase (HPSE-1) is an endo-,- d -glucuronidase involved in the degradation of cell-surface/extracellular matrix heparan sulfate (HS) in normal and neoplastic tissues. HPSE-1 represents the first example of purification and cloning of a mammalian HS-degradative enzyme. Elevated HPSE-1 levels are known to be associated with metastatic cancers, directly implicating HPSE-1 in metastatic events. The purpose of this study was to determine the role of cAMP response element-binding protein (CREB) in modulating HPSE-1-mediated effects on human melanoma cell invasion. Highly invasive, brain-metastatic melanoma cells (70W) were transfected with the dominant-negative CREB (KCREB) and subsequently analyzed for changes in their HPSE-1 content, functionality, and cell invasive properties. KCREB-transfected cells showed a decrease in HPSE-1 mRNA expression and activity. This correlated with a significantly decreased invasion of these cells through MatrigelÔ-coated filters. Furthermore, adenoviral vectors containing the full-length human HPSE-1 cDNA in sense orientation (Ad-S/hep) were constructed to investigate CREB effects on HPSE-1. Restoration of HPSE-1 expression and functionality following Ad-S/hep infection of KCREB-transfected 70W cells recovered melanoma cell invasiveness. These results demonstrate that KCREB inhibits HPSE-1 and suggest that one of the roles CREB plays in the acquisition of melanoma cells metastatic phenotype is affecting HPSE-1 activity. © 2004 Wiley-Liss, Inc. [source] Modulation of VE-cadherin and PECAM-1 mediated cell,cell adhesions by mitogen-activated protein kinasesJOURNAL OF CELLULAR BIOCHEMISTRY, Issue 1 2003Jianqiang Wu Abstract Endothelial cell transition from a differentiated, quiescent phenotype to a migratory, proliferative phenotype is essential during angiogenesis. This transition is dependent on alterations in the balanced production of stimulatory and inhibitory factors, which normally keep angiogenesis in check. Activation of MAPK/ERKs is essential for endothelial cell migration and proliferation. However, its role in regulation of endothelial cell adhesive mechanisms requires further delineation. Here, we show that sustained activation of MAPK/ERKs results in disruption of cadherin-mediated cell,cell adhesion, down-regulation of PECAM-1 expression, and enhanced cell migration in microvascular endothelial cells. Expression of a constitutively active MEK-1 in mouse brain endothelial (bEND) cells resulted in down-regulation of VE-cadherin and catenins expression concomitant with down-regulation of PECAM-1 expression. In contrast, inhibition of MEK-1 restored parental morphology, cadherin/catenins expression and localization. These data are further supported by our observation that sustained activation of MAPK/ERKs in phorbol myristate acetate incubated HUVEC lead to disruption of cadherin-mediate cell,cell interactions and enhanced capillary formation on Matrigel. Thus, sustained activation of MAPK/ERKs plays an important role in disruption of cell,cell adhesion and migration of endothelial cells. J. Cell. Biochem. 90: 121,137, 2003. © 2003 Wiley-Liss, Inc. [source] Further characterization of human fetal osteoblastic hFOB 1.19 and hFOB/ER, cells: Bone formation in vivo and karyotype analysis using multicolor fluorescent in situ hybridizationJOURNAL OF CELLULAR BIOCHEMISTRY, Issue 1 2002M. Subramaniam Abstract We have previously generated an immortalized human fetal osteoblastic cell line (hFOB) using stably transfected temperature sensitive SV40 T-antigen (Harris et al. [1995a] J. Bone. Miner. Res. 10:178,1860). To characterize these cells for phenotypic/genotypic attributes desired for a good cell model system, we performed karyotype analysis by multicolor fluorescent in situ hybridization (M-FISH), their ability to form bone in vivo without developing cell transformation, and finally their ability to form extracellular matrix formation in vitro. The karyotype analysis of hFOB cells revealed structural or numeric anomalies involving 1,2 chromosomes. In contrast, the human osteosarcoma MG63 cells displayed multiple, and often complex, numeric, and structural abnormalities. Subcutaneous injection of hFOB cells in the presence of Matrigel into nude mice resulted in bone formation after 2,3 weeks. Electron microscopic analysis of the extracellular matrix deposited by hFOB cells in culture revealed a parallel array of lightly banded fibrils typical of the fibrillar collagens such as type I and III. These results demonstrate that the hFOB cell line has minimal chromosome abnormalities, exhibit the matrix synthetic properties of differentiated osteoblasts, and are immortalized but non-transformed cell line. These hFOB cells thus appear to be an excellent model system for the study of osteoblast biology in vitro. J. Cell. Biochem. 87: 9,15, 2002. © 2002 Wiley-Liss, Inc. [source] Antiangiogenetic Effects of 4 Varieties of Grapes,In VitroJOURNAL OF FOOD SCIENCE, Issue 6 2010Ming Liu Abstract:, The purpose of this study was to investigate the inhibitory effects of grapes on the human umbilical vein endothelial (HUVE) cells' capillary tube formation and matrix metalloproteinase-2 (MMP-2) expression secreted into the medium. Four different grape varieties (Concord, Niagara, Chardonnay, and Pinot Noir) were extracted using 80% acetone and the extracts were stored at ,80 °C. The total amount of phenolics and flavonoids for each of the 4 grape varieties were determined by spectrophotometry. Grape extracts were co-cultured with HUVE cells on Matrigel and inhibitory effects on tube formation were observed under a microscope. The inhibitory effects of grape extracts on MMP-2 expression were examined by zymogram. All 4 grape varieties inhibited the tube formation of HUVE cells in a dose-dependent manner on Matrigel. Except for Chardonnay, the other 3 grape varieties completely inhibited secretion of MMP-2 at 20 mg/mL. There was a significant positive relationship between the total phenolics and flavonoids and antiangiogenetic activities. The grapes tested have the potential to inhibit angiogenesis mainly by their phenolics and flavonoids contents, which partly contribute to their cancer chemopreventive efficacy. [source] Insulin-like growth factor binding protein-3 induces angiogenesis through IGF-I- and SphK1-dependent mechanismsJOURNAL OF THROMBOSIS AND HAEMOSTASIS, Issue 4 2007R. GRANATA Summary. Angiogenesis is critical for development and repair, and is a prominent feature of many pathological conditions. Based on evidence that insulin-like growth factor binding protein (IGFBP)-3 enhances cell motility and activates sphingosine kinase (SphK) in human endothelial cells, we have investigated whether IGFBP-3 plays a role in promoting angiogenesis. IGFBP-3 potently induced network formation by human endothelial cells on Matrigel. Moreover, it up-regulated proangiogenic genes, such as vascular endothelial growth factor (VEGF) and matrix metalloproteinases (MMP)-2 and -9. IGFBP-3 even induced membrane-type 1 MMP (MT1-MMP), which regulates MMP-2 activation. Decreasing SphK1 expression by small interfering RNA (siRNA), blocked IGFBP-3-induced network formation and inhibited VEGF, MT1-MMP but not IGF-I up-regulation. IGF-I activated SphK, leading to sphingosine-1-phosphate (S1P) formation. The IGF-I effect on SphK activity was blocked by specific inhibitors of IGF-IR, PI3K/Akt and ERK1/2 phosphorylation. The disruption of IGF-I signaling prevented the IGFBP-3 effect on tube formation, SphK activity and VEGF release. Blocking ERK1/2 signaling caused the loss of SphK activation and VEGF and IGF-I up-regulation. Finally, IGFBP-3 dose-dependently stimulated neovessel formation into subcutaneous implants of Matrigel in vivo. Thus, IGFBP-3 positively regulates angiogenesis through involvement of IGF-IR signaling and subsequent SphK/S1P activation. [source] Aspirin and salicylate inhibit colon cancer medium- and VEGF-induced endothelial tube formation: correlation with suppression of cyclooxygenase-2 expressionJOURNAL OF THROMBOSIS AND HAEMOSTASIS, Issue 10 2003M. I. Shtivelband Summary., To determine whether aspirin and salicylate suppress colon cancer cell-mediated angiogenesis, we evaluated the effects of aspirin and sodium salicylate on endothelial tube formation on Matrigel. Aspirin and sodium salicylate concentration-dependently inhibited human endothelial cell (EC) tube formation induced by conditioned medium collected from DLD-1, HT-29 or HCT-116 colon cancer cells. Aspirin and sodium salicylate at pharmacological concentrations were equally effective in blocking tube formation. Neutralizing antivascular endothelial growth factor (VEGF) antibodies blocked colon cancer medium-induced tube formation. VEGF receptor 2 but not receptor 1 antibodies inhibited tube formation to a similar extent as anti-VEGF antibodies. These results indicate that VEGF interaction with VEGF receptor 2 is the primary mechanism underlying colon cancer-induced angiogenesis. Aspirin or sodium salicylate inhibited VEGF-induced tube formation in a concentration-dependent manner comparable to that of inhibition of colon cancer medium-induced endothelial tube formation. It has been shown that cyclooxygenase-2 (COX-2) is pivotal in cancer angiogenesis. We found that colon cancer medium-induced COX-2 protein expression in EC and aspirin or sodium salicylate suppressed the cancer-induced COX-2 protein levels at concentrations correlated with those that suppressed endothelial tube formation. Furthermore, aspirin and sodium salicylate inhibited COX-2 expression stimulated by VEGF. These findings indicate that aspirin and other salicylate drugs at pharmacological concentrations inhibit colon cancer-induced angiogenesis which is correlated with COX-2 suppression. [source] Age-dependent vascular endothelial growth factor expression and angiogenic capability of bladder smooth muscle cells: implications for cell-seeded technology in bladder tissue engineeringJOURNAL OF TISSUE ENGINEERING AND REGENERATIVE MEDICINE, Issue 8 2009Joseph Azzarello Abstract Cell seeding technology is commonly used in the field of tissue engineering to enhance the performance of bioscaffolds and promote tissue regeneration. The age of cells used for ex vivo seeding to achieve maximal tissue regeneration has not been defined. Since rapid angiogenesis is the most critical step for tissue graft survival and success, we evaluated passage-dependent vascular endothelial growth factor (VEGF) expression in cultured smooth muscle cells (SMCs) obtained from urinary bladder and endothelial cell response to bladder SMCs. Levels of various VEGF isoforms mRNA expression and total VEGF secretion were determined by a semi-quantitative reverse-transcription polymerase chain reaction (RT-PCR) and an enzyme-linked immunosorbent assay (ELISA) analysis, respectively. In vitro endothelial cell migration in Transwell® and capillary-like tube formation in MatrigelÔ were used to predict the ability of bladder SMCs to promote angiogenesis. VEGF produced by cultured bladder SMCs increased from passages 4 to 7, and decreased from passages 7 to 12 at both mRNA and protein levels. Endothelial cell migration as well as capillary-like tube formation correlated with levels of VEGF expression by bladder SMCs. Pre-incubation of endothelial cells with a VEGF receptor 1/2 inhibitor, SU5416, significantly reduced the number of capillary-like tubes in SMC-endothelial cell MatrigelÔ co-culture, and confirmed the involvement of VEGF in endothelial cell tube formation. Our results demonstrate that cell passage number is related to levels of VEGF production, which may translate to angiogenesis in engineered tissues. Copyright © 2009 John Wiley & Sons, Ltd. [source] Influence of a Self-Assembling Peptide, RADA16, Compared with Collagen I and Matrigel on the Malignant Phenotype of Human Breast-Cancer Cells in 3D Cultures and in vivoMACROMOLECULAR BIOSCIENCE, Issue 5 2009Kun Mi Abstract Cancer-cell phenotype is not only the result of malignant progression, but also dependent on the microenvironment surrounding them, including influences from the extracellular matrix and its structural properties. We have investigated the influence of the nanofiber matrix of the self-assembling peptide, RADA16, in comparison with collagen I and Matrigel on the malignant phenotype of the human breast-cancer cell, MDA-MB-231, in 3D cultures, including the morphology, survival, proliferation rate, migration potential and the effect of these matrices on the malignancy of the cancer cells in vivo. Our data indicate that these tumor cells change their morphology in response to the different 3D matrix in vitro cultures and the RADA16 self-assembling peptide scaffold mimics an extracellular matrix and could effectively reduce the malignant phenotype of the tumor cells in vitro and in vivo. [source] Reduced gap junctional intercellular communication and altered biological effects in mouse osteoblast and rat liver oval cell lines transfected with dominant-negative connexin 43MOLECULAR CARCINOGENESIS, Issue 4 2003Brad L. Upham Abstract Gap junctional intercellular communication (GJIC) maintains normal growth and differentiation of cells in a tissue. The intercellular molecules traversing gap junctions are largely unknown, but the molecular weight (MW) cutoff is normally 1200 Da. No differences in dye transfer were observed in normal or vector controls of WB-F344 rat liver epithelial or mouse osteoblastic MC3T3-E1 cells with either Lucifer Yellow (LY) with a MW of 457 Da (LY-457) or LY with a MW of 649 Da (LY-649). Transfection of a dominant negative-connexin 43 (Cx43) gene decreased GJIC (>50%) when LY-649 was used, however, normal GJIC was observed in both cell lines when LY-457 was used. Therefore, the MW cut off in these clones was considerably less than the wild type. The dominant negative clones of the MC3T3-E1 cells exhibited over 90% less alkaline phosphatase (ALPase) activity and calcium deposition after the induction of differentiation. Similarly, dominant negative Cx43 inhibited gene expression of ALPase and bone sialoprotein but not osteocalcin in MC3T3-E1. WB-F344 cells normally exhibit a biphasic response to 12- O -tetradecanoylphorbol-13-acetate (TPA) where inhibition of GJIC recovers after 2 h, but the dominant negative clones showed no recovery from inhibition of GJIC by TPA. Dominant negative Cx43 also inhibited the formation of network-like structures by WB-F344 cells on Matrigel. These results demonstrate that the dominant negative gene transfected into cell types containing the wild-type connexins result in diminished channel sizes, thus allowing the determination of whether distinct biological endpoints, i.e., differentiation, are dependent upon either small or high MW intercellular signals. © 2003 Wiley-Liss, Inc. [source] Inhibitory effect of magnolol on tumour metastasis in micePHYTOTHERAPY RESEARCH, Issue 8 2003Koji Ikeda Abstract It has previously been reported that magnolol, a phenolic compound isolated from Magnolia obovata, inhibited tumour cell invasion in vitro. The purpose of this study was to investigate the antimetastatic effect of magnolol on tumour metastasis in vivo with experimental and spontaneous metastasis models and to clarify the mechanism. The antimetastatic effects of magnolol were evaluated by an experimental liver and spleen metastasis model using L5178Y-ML25 lymphoma, or an experimental and spontaneous lung metastasis model using B16-BL6 melanoma. Intraperitoneal (i.p.) administration of 2 or 10 mg/kg of magnolol signi,cantly suppressed liver and spleen metastasis or lung metastasis. As for the spontaneous lung metastasis model using B16-BL6 melanoma, multiple i.p. administrations of 10 mg/kg of magnolol after and before tumour inoculation signi,cantly suppressed lung metastasis and primary tumour growth. In addition, magnolol signi,cantly inhibited B16-BL6 cell invasion of the reconstituted basement membrane (Matrigel, MG) without affecting cell growth. These data from the in vivo experiments suggest that magnolol possesses strong antimetastatic ability and that it may be a lead compound for drug development. The antimetastatic action of magnolol is considered to be due to its ability to inhibit tumour cell invasion. Copyright © 2003 John Wiley & Sons, Ltd. [source] Matrigel: A complex protein mixture required for optimal growth of cell culturePROTEINS: STRUCTURE, FUNCTION AND BIOINFORMATICS, Issue 9 2010Chris S. Hughes Abstract Numerous cell types require a surface for attachment to grow and proliferate. Certain cells, particularly primary and stem cells, necessitate the use of specialized growth matrices along with specific culture media conditions to maintain the cells in an undifferentiated state. A gelatinous protein mixture derived from mouse tumor cells and commercialized as Matrigel is commonly used as a basement membrane matrix for stem cells because it retains the stem cells in an undifferentiated state. However, Matrigel is not a well-defined matrix, and therefore can produce a source of variability in experimental results. In this study, we present an in-depth proteomic analysis of Matrigel using a dynamic iterative exclusion method coupled with fractionation protocols that involve ammonium sulfate precipitation, size exclusion chromatography, and one-dimensional SDS-PAGE. The ability to identify the low mass and abundance components of Matrigel illustrates the utility of this method for the analysis of the extracellular matrix, as well as the complexity of the matrix itself. [source] Interferon-, Inhibits Metalloproteinase Activity and Cytotrophoblast Cell MigrationAMERICAN JOURNAL OF REPRODUCTIVE IMMUNOLOGY, Issue 1 2010Vanina Andrea Fontana Citation Fontana VA, Sanchez M, Cebral E, Calvo JC. Interferon-, inhibits metalloproteinase activity and cytotrophoblast cell migration. Am J Reprod Immunol 2010; 64: 20,26 Problem, Establishment of a successful pregnancy relies on a complex fetal,mother communication that starts with the embryo adhering and invading the endometrium. This requires remodeling of extracellular matrix, performed by metalloproteinases. Cytokines, such as interferon-, (IFN-,), play a role in implantation and could affect the success of pregnancy. Method of study, Using JEG-3 cell line as model, we cultured the cells in the presence or absence of IFN-, and determined the activities of MMP-2 and MMP-9 using zymography and the secretion of leptin using Western blot. Results, Interferon-, inhibits gelatinase activity from MMP-2 and MMP-9 in a dose-dependent manner, reducing the secretion of leptin (not because of a general inhibition on protein synthesis) and impairs cell migration on Matrigel. Conclusion, Our results correlate with previous reports from our laboratory indicating that IFN- , is deleterious for mouse embryo outgrowth, having an effect on metalloproteinases activity as well as leptin secretion. [source] ORIGINAL ARTICLE: Role of Regulatory and Angiogenic Cytokines in Invasion of Trophoblastic CellsAMERICAN JOURNAL OF REPRODUCTIVE IMMUNOLOGY, Issue 3 2010Valeria Dubinsky Citation Dubinsky V, Poehlmann TG, Suman P, Gentile T, Markert UR, Gutierrez G. Role of regulatory and angiogenic cytokines in invasion of trophoblastic cells. Am J Reprod Immunol 2010; 63: 193,199 Problem, Trophoblast invasion is a temporally and locally restricted process, which regulates implantation and oxygen arrival to the embryo through the dialog with spiral artery endothelium. Trophoblast factors with angiogenic potential are activated by hypoxia. Their capacities to induce proliferation, migration, and invasion of trophoblastic cells have been investigated. Method of study, The expression of interleukin (IL)-6, CD126, CD130, vascular endothelial growth factor (VEGF), and hypoxia inducible factor-1, (HIF-1,) has been silenced in JEG-3 choriocarcinoma cells by using siRNA. Silencing efficacy has been assessed by ELISA, PCR or Western blotting. Proliferation has been measured by flow cytometry, migration by a transwell assay, and invasion by a Matrigel assay. Results, Proliferation was significantly reduced by silencing of CD126 or CD130, migration by silencing of IL-6, VEGF, or HIF-1,, and invasion by silencing of IL-6 and HIF-1,. Conclusion, The expression of IL-6, VEGF, and HIF-1, in trophoblastic cells is involved in the control of trophoblast invasion and migration. [source] Fibroblast Growth Factor 2 Promotes Endothelial Differentiation of Adipose Tissue-Derived Stem CellsTHE JOURNAL OF SEXUAL MEDICINE, Issue 4 2009Hongxiu Ning PhD ABSTRACT Introduction., Adipose tissue-derived stem cells (ADSC) could potentially restore endothelial function in vasculogenic erectile dysfunction (ED). The mechanism for ADSC endothelial differentiation remained unidentified. Aim., To test whether ADSC could differentiate into endothelial cells in the penis and to identify the underlying mechanism of ADSC endothelial differentiation. Methods., For in vivo endothelial differentiation, ADSC were labeled with bromodeoxyuridine (BrdU), injected into rat corpora cavernosa, and localized by immunofluorescence and phase-contrast microscopy. For in vitro endothelial differentiation, ADSC were grown in endothelial growth medium 2 (EGM2), stained for endothelial markers CD31, von Willebrand Factor (vWF), and endothelial nitric oxide synthase (eNOS), and assessed for the ability to form tube-like structures in Matrigel and to endocytose acetylated low-density lipoprotein (Ac-LDL). To identify factors that promote ADSC endothelial differentiation, ADSC were grown in various media, each of which contained a specific combination of supplemental factors and assessed for LDL-uptake. PD173074, a selective inhibitor of fibroblast growth factor 2 (FGF2) receptor, was used to confirm the importance of FGF2 signaling for ADSC endothelial differentiation. Main Outcome Measures., In vivo endothelial differentiation was assessed by immunofluorescence microscopy. In vitro endothelial differentiation was assessed by immunofluorescence, Matrigel tube formation, and Ac-LDL uptake. Results., Injected ADSC were localized to the sinusoid endothelium, some of which stained positive for both BrdU and endothelial antigen rat endothelial cell antigen. ADSC proliferated at a faster rate in EGM2 than in standard DMEM, expressed endothelial markers CD31, vWF, and eNOS, formed tube-like structures in Matrigel, and endocytosed Ac-LDL. These properties were greatly diminished when ADSC were grown in the absence of FGF2 but were unaffected when grown in the absence of vascular endothelial growth factor, insulin-like growth factor, or epidermal growth factor. Furthermore, ADSC displayed similar endothelial properties when grown in FGF2-supplemented basic medium as in EGM2. Finally, blockade of FGF2 signaling with PD173074 abrogated ADSC endothelial differentiation. Conclusions., ADSC could differentiate into endothelial cells in the penis. FGF2 signaling mediates ADSC endothelial differentiation. Ning H, Liu G, Lin G, Yang R, Lue TF, and Lin CS. FGF2 promotes endothelial differentiation of adipose tissue-derived stem cells. J Sex Med **;**:**,**. [source] A Novel Three-Dimensional In Vitro System to Study Trophoblast,Endothelium Cell InteractionsAMERICAN JOURNAL OF REPRODUCTIVE IMMUNOLOGY, Issue 2 2007Paulomi B. Aldo Introduction Pregnancy complications have been linked to improper trophoblast migration and failure of spiral artery transformation. Endothelial cells play an essential role in directing trophoblast migration and transformation, although by an unknown mechanism. We describe a novel in vitro model to evaluate endothelial,trophoblast interaction and signaling in a three-dimensional system. Method of study Immortalized human endometrial endothelial cell line and first trimester trophoblast cells were co-cultured. Endothelial transformation into vessel-like structures occurred in MatrigelTM OpenLab Image Analysis software was used to monitor labeled trophoblast migration and endothelium transformation. Cytokine/chemokine production was determined using Multiplex. Results Trophoblast migrates toward endothelial cells in Matrigel, aligns on top of the endothelium within 4,8 hr and achieves complete replacement of the endothelium by 72,96 hr. Lipopolysaccharide treatment damages the endothelium and disrupts endothelium,trophoblast interaction. Conclusion We report a novel three-dimensional in vitro and in vivo system of trophoblast,endothelium cell interaction. Significant changes in endothelial cells' phenotype are observed upon differentiation in Matrigel. These changes may be necessary for endothelium to direct trophoblast migration and transformation. [source] The influence of extracellular matrix and prolactin on global gene expression profiles of primary bovine mammary epithelial cells in vitroANIMAL GENETICS, Issue 1 2010L. G. Riley Summary An in vitro bovine mammosphere model was characterized for use in lactational biology studies using a functional genomics approach. Primary bovine mammary epithelial cells cultured on a basement membrane, Matrigel, formed three-dimensional alveoli-like structures or mammospheres. Gene expression profiling during mammosphere formation by high-density microarray analysis indicated that mammospheres underwent similar molecular and cellular processes to developing alveoli in the mammary gland. Gene expression profiles indicated that genes involved in milk protein and fat biosynthesis were expressed, however, lactose biosynthesis may have been compromised. Investigation of factors influencing mammosphere formation revealed that extracellular matrix (ECM) was responsible for the initiation of this process and that prolactin (Prl) was necessary for high levels of milk protein expression. CSN3 (encoding ,-casein) was the most highly expressed casein gene, followed by CSN1S1 (encoding ,S1-casein) and CSN2 (encoding ,-casein). Eighteen Prl-responsive genes were identified, including CSN1S1, SOCS2 and CSN2, however, expression of CSN3 was not significantly increased by Prl and CSN1S2 was not expressed at detectable levels in mammospheres. A number of novel Prl responsive genes were identified, including ECM components and genes involved in differentiation and apoptosis. This mammosphere model is a useful model system for functional genomics studies of certain aspects of dairy cattle lactation. [source] The invasive behaviour of prostatic cancer cells is suppressed by inhibitors of tyrosine kinase,APMIS, Issue 1 2006HAAKON SKOGSETH Proteolytic enzymes, and especially urokinase plasminogen activator (uPA), play an important role in tumour invasion and metastasis. Previously we demonstrated that the production of urokinase plasminogen activator (uPA) was decreased by several tyrosine kinase inhibitors (TKI) in two prostatic carcinoma cell lines. The effect of the two TKI genistein and tyrphostin AG-1478 was investigated in the prostate carcinoma cell lines PC-3 and DU-145. A reconstituted basal lamina (Matrigel) was used as a migration barrier. The production of matrix metalloproteinases (MMP) was also measured. Roles of plasminogen and uPA were examined. Cell invasion was increased by plasminogen, but this enhanced cell migration was counteracted by TKI treatment. The increased cell invasion induced by plasminogen was decreased by at least 60% in both cell lines when ,-2 anti-plasmin was added to the assay. Cells in the absence of plasminogen were not affected by TKI. External uPA failed to regenerate the decreased cell invasion caused by TKI. The production of MMP was inhibited by both TKI. Our results indicate a possible role of TKI as inhibitors of cancer cell invasion by inhibiting uPA and MMP production. [source] Modulation of the angiogenic phenotype of normal and systemic sclerosis endothelial cells by gain,loss of function of pentraxin 3 and matrix metalloproteinase 12ARTHRITIS & RHEUMATISM, Issue 8 2010Francesca Margheri Objective Studies have shown that in systemic sclerosis (SSc) endothelial cells, overproduction of matrix metalloproteinase 12 (MMP-12) and pentraxin 3 (PTX3) is associated with defective angiogenesis. This study was undertaken to examine whether overexpression of the relevant molecules could inhibit angiogenesis of normal microvascular endothelial cells (MVECs), and whether silencing of these molecules in SSc MVECs could restore the lost angiogenic properties of the cells in vitro and in vivo. Methods Transient transfection of MVECs with human MMP12 and PTX3 was performed by electroporation. Silencing of MMP12 and PTX3 was obtained by treatment with small interfering RNA, and treatment effects were validated by Western blotting with specific antibodies and a fluorimetric assay. In vitro cell migration and capillary morphogenesis were studied on Matrigel substrates. In vivo angiogenesis was studied using a Matrigel sponge assay in mice. Results Transfection of MMP12 and PTX3 in normal MVECs resulted in loss of proliferation, invasion, and capillary morphogenesis in vitro, attributed to truncation of the urokinase-type plasminogen activator receptor by MMP12 and to the anti,fibroblast growth factor 2/anti,vascular endothelial growth factor activity of PTX3. These effects were particularly evident in mixed populations of transfected normal MVECs (50% transfected with MMP12 and 50% with PTX3). Silencing of the same molecules in SSc MVECs increased their invasion in Matrigel. Single-gene silencing did not increase the capillary morphogenesis of SSc MVECs, whereas double-gene,silenced cells showed a burst of capillary tube formation. Culture medium of silenced SSc MVECs stimulated angiogenesis in assays of Matrigel sponge invasion in mice. Conclusion Overexpression of either MMP12 or PTX3 in normal MVECs blunts their angiogenic properties. Loss of function of MMP12 and PTX3 in SSc MVECs restores the ability of the cells to produce capillaries in vitro and induces vascularization in vivo on a Matrigel sponge. [source] Decreased expression of heparin-binding epidermal growth factor,like growth factor as a newly identified pathogenic mechanism of antiphospholipid-mediated defective placentationARTHRITIS & RHEUMATISM, Issue 5 2010N. Di Simone Objective Heparin-binding epidermal growth factor,like growth factor (HB-EGF) plays a role in blastocyst implantation and is down-regulated in preeclampsia and in hypertensive pregnancy disorders associated with defective extravillous trophoblast invasion. Defective placentation and severe preeclampsia are also features of the antiphospholipid syndrome (APS). The purpose of this study was to investigate whether abnormal HB-EGF expression plays a pathogenic role in antiphospholipid antibody (aPL),mediated defective placentation. Methods HB-EGF expression in placental tissue was evaluated by Western blotting and messenger RNA analysis in normal and APS placentae. Polyclonal IgG fractions or monoclonal ,2 -glycoprotein I,dependent aPL and their respective controls were investigated for the following 4 features: their binding to human trophoblast monolayers, as determined by cell enzyme-linked immunosorbent assay (ELISA); their effect on HB-EGF expression by Western blotting in trophoblast cell extracts as well as by ELISA as a protein secreted in the culture supernatants; their inhibitory effect on in vitro trophoblast invasiveness, as evaluated by Matrigel assay; and their inhibitory effect on matrix metalloproteinase (MMP) levels, as measured by gelatin zymography. Experiments were also performed in the presence of serial concentrations of heparin or recombinant HB-EGF. Results Placental APS tissue displayed reduced expression of HB-EGF. Polyclonal and monoclonal aPL bound to trophoblast monolayers and significantly reduced the in vitro synthesis and secretion of HB-EGF. Heparin inhibited aPL binding and restored HB-EGF expression in a dose-dependent manner. Addition of recombinant HB-EGF reduced the in vitro aPL-induced inhibition of Matrigel invasiveness as well as MMP-2 levels. Conclusion These preliminary findings suggest that the reduction of aPL-mediated HB-EGF represents an additional mechanism that is responsible for the defective placentation associated with APS and that heparin protects from aPL-induced damage by inhibiting antibody binding. [source] Abnormal basement membrane type IV collagen ,-chain composition in labial salivary glands in Sjögren's syndromeARTHRITIS & RHEUMATISM, Issue 4 2009P. Poduval Objective Sjögren's syndrome (SS) is characterized by atrophy and malfunction of the acinar cells. The aim of this study was to investigate whether type IV collagen ,-chain composition of acinar cell compartments could be abnormal in diseased glands. Methods Messenger RNA (mRNA) from human submandibular gland (HSG) cells, cultured with or without growth factor,depleted Matrigel, was analyzed using quantitative reverse transcription,polymerase chain reaction (RT-PCR). Labial salivary glands were analyzed using quantitative RT-PCR and immunohistochemistry. Results HSG cells of both the ductal and acinar phenotypes synthesized all ,-chain mRNA, in particular those of the ,1 and ,2 chains. Labial salivary glands (LSGs) contained ,1/2 chains but also contained mRNA of all the other ,-chains, although the mRNA copy numbers for the ,3 and ,4 chains were low, and the corresponding proteins were absent. Type IV collagen ,1/2-chains were observed in all tubuloalveolar basement membranes. In healthy glands, ,5 and ,6 chains were continuous around ducts but discontinuous around acini. In SS glands, these chains were absent or patchy around the ducts and absent around the acini. Conclusion Ductal and acinar epithelial cells are able to locally produce mRNA for all 6 different ,-chains. Type IV collagen ,1/2-chains seem to form the backbone in the tubuloalveolar basement membrane in salivary glands. Type IV collagen ,3 and ,4 chain mRNA were found in cultured salivary epithelial cells and LSG explants but were not translated to the corresponding ,-chains in LSGs. Both ,5 and ,6 mRNA were observed in salivary epithelial cells and glands. In healthy glands, immunolabeling always disclosed corresponding ,-chains around ducts, but their synthesis and/or degradation seemed to be locally regulated around acinar cells. [source] | ||||||||||||||||||||||||||||||||||