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PubMed2000

Differential effects of extracellular matrix proteins on human airway smooth muscle cell proliferation and phenotype.

Mature airway smooth muscle cells are characterized by a low proliferative index and expression of contractile marker proteins such as smooth muscle alpha-actin (sm-alpha-actin), calponin, and smooth muscle myosin heavy chain (sm-MHC). In the present study, defined extracellular matrix (ECM) components were examined on the proliferative and phenotypic status of mitogen-stimulated, cultured human airway smooth muscle cells. The results demonstrate that although cells adhered and spread on plates precoated with (1 to 100 microg/ml) of fibronectin (FN), collagen I (Col I), laminin (LN), or Matrigel, their subsequent proliferative response varied qualitatively. FN and Col I enhanced proliferation in response to either platelet-derived growth factor (PDGF)-BB or alpha-thrombin, compared with cells on plastic. LN, however, reduced mitogen-stimulated proliferation. A similar reduction was found in cells cultured on Matrigel. The effect of ECM substrates on contractile phenotype was determined by examining cellular expression of sm-alpha-actin, sm-MHC, and calponin using immunocytochemical and flow cytometric methods. Approximately 75% of PDGF-BB-stimulated cells, cultured on LN or Matrigel, expressed sm-alpha-actin, calponin, and sm-MHC, but only 8 to 10% stained for the Ki67 nuclear antigen proliferation marker. In contrast, more than 75% of cells cultured on FN or Col I were positive for Ki67 antigen, but only 20% were positive for contractile proteins. Flow cytometric analysis of sm-alpha-actin and DNA content confirmed the immunocytochemical findings and showed that the observed reduction in sm-alpha-actin content after culture on FN or Col I, compared with LN and Matrigel, occurred in the majority of the cell population, supporting bidirectional phenotype modulation. Overall, the data suggest that ECM substrates modulate both proliferation and phenotype of human airway smooth muscle cells in culture.

Actins↗
PubMedDate not supplied

Pancreatic cancer cells invasiveness is mainly affected by interleukin-1beta not by transforming growth factor-beta1.

BACKGROUND: We investigated in vitro whether IL-1beta and TGF-beta1 affect pancreatic cancer cell growth, adhesion to the extracellular matrix and Matrigel invasion. MATERIALS AND METHODS: Adhesion to fibronectin, laminin and type I collagen, and Matrigel invasion after stimulation with saline, IL-1beta and TGF-beta1 were evaluated using three primary and three metastatic pancreatic cancer cell lines. RESULTS: Extracellular matrix adhesion of control cells varied independently of the metastatic characteristics of the studied cell lines, whereas Matrigel invasion of control cells was partly correlated with the in vivo metastatic potential. IL-1beta did not influence extracellular matrix adhesion, whereas it significantly enhanced the invasiveness of three of the six cell lines. TGF-beta1 affected the adhesion of one cell line, and exerted contrasting effects on Matrigel invasion of different cell lines. CONCLUSIONS: IL-1beta enhances the invasive capacity of pancreatic cancer cells, whereas TGF-beta1 has paradoxical effects on pancreatic cancer cells; this makes it difficult to interfere with TGF-beta1 signaling in pancreatic cancer treatment.

Cell Adhesion↗
PubMed1996

Altered growth and attachment of rabbit crypt colonocytes isolated from different developmental stages.

The rabbit colon was used to establish an in vitro model for examining development-related cellular changes in colonocyte function. Colonic epithelia from newborn, weanling, and adult animals were separated from the muscle and subjected to enzymatic digestion. A mixture of 0.05% Pronase, 0.015% collagenase IV, and 0.023% DTT was determined to be optimal for the isolation of newborn and weanling colonocytes. This solution yielded significantly more cells and of greater viability than a 0.1% Pronase, 0.03% collagenase IV, 0.07% DTT mixture that is optimal for adult colonocytes. The epithelial origin of the colonocytes was confirmed by immunofluorescent staining of cytokeratins. The isolation procedure resulted in a crypt-enriched population and the cell yield/g of mucosa increased with age as did the crypt depth. Colonocyte viability of adults but not of newborns and weanlings, declined from 24 to 72 h. When grown on plastic, the newborn and weanling colonocytes show a approximately 2-fold increase in number, DNA and protein content over 48 h. In contrast, for all three parameters the adult colonocytes revealed only a approximately 10% increase. The colonocytes also showed an age-related decline in attachment to extracellular matrices. Colonocytes showed maximal attachment to Matrigel and collagen IV; newborn and weanling colonocytes show > 80% attachment, whereas adult colonocytes showed only a 45% attachment. The efficacy of attachment to Matrigel compared with that on plastic also differed with age, representing 9.3-, 5.5-, and 4.4-fold increase in adult, weanling, and newborn colonocytes, respectively. Newborn and weanling colonocytes grown on Matrigel for 48 h, showed a significant, 15% increase in cell number, DNA, and protein content compared with those grown on plastic. There was no difference in these parameters when adult colonocytes grown on Matrigel were compared with those grown on plastic. In summary, we have established an in vitro model for studying colonic epithelial cells at different stages of development.

Age Factors↗
PubMed1990

Organotypic arrangement of mouse embryonic lung cells on a basement membrane extract: involvement of laminin.

The behavior of embryonic murine lung cells on a basement membrane extract (Matrigel) was investigated. Single cell suspensions generated by trypsinization of lungs removed from day 12 embryos were plated on Matrigel and cultured for up to one week. The basement membrane extract was used as a gel, and as a wet or dried film. In all of these instances, organotypic arrangement of the embryonic lung cells was observed. This process consisted of cell aggregation, sorting, polarization and formation of a tridimensional organization resembling embryonic lung. The maximal degree of organotypic development was obtained by using a thick gel; minimal reorganization was observed using a dried film. A rabbit polyclonal serum to laminin inhibited organotypic pattern formation while normal rabbit serum did not. Culture of lung cells on laminin gels promoted epithelial cyst formation but poor mesenchymal organization. By studying the behavior of epithelial and/or mesenchymal enriched cell populations on Matrigel, it was concluded that organotypic pattern formation on Matrigel required the presence of both cell populations. Cultivation of dissociated lung cells on a gel consisting of a mixture of collagens type I and III (Vitrogen-100) produced only cell aggregation. Cultivation of lung cells on a thin film of Vitrogen-100 or on uncoated tissue culture plastic produced monolayers of mesenchymal cells alone. Cultivation of lung cells in suspension also failed to induce organotypic arrangement even at maximal cell densities. The present study strongly supports a role for the basement membrane in the organotypic rearrangement of embryonic lung cells and subsequent in vitro cyst formation and budding of the reestablished epithelium. This, in turn, reinforces the concept of the basement membrane as a major regulator of organogenesis.

Animals↗
PubMed1995

Regulation of regional differences in the differentiation of cerebral cortical neurons by EGF family-matrix interactions.

Both lineage-based and epigenetic regulation have been postulated as mechanisms to control the formation of discrete areas in the cerebral cortex, but specific genes or signaling pathways that may be involved have yet to be defined. In this paper, we examine whether progenitors, isolated from the cerebral wall prior to neurogenesis, can respond to exogenous cues by adopting a region-specific phenotype. The expression of the limbic system-associated membrane protein (LAMP), a neuron-specific marker of limbic cortical areas, was monitored in cultured neurons arising from precursors harvested from presumptive perirhinal (limbic) and sensorimotor (nonlimbic) zones at embryonic day 12 in the rat. Neuronal phenotype in all cultures was identified by expression of microtubule-associated protein-2 (MAP2). On a substrate of poly-lysine, over 80% of the precursors from the limbic area that differentiate into neurons express a LAMP+ phenotype. Approximately 20% of the neurons generated from precursors of the sensorimotor region become LAMP+. However, modification of the microenvironment had a significant effect on the differentiation of the sensorimotor precursors. When the nonlimbic precursors are grown on Matrigel, there is a 2-fold increase in the number of MAP2+/LAMP+ double-labeled neurons. Dissection of the Matrigel components reveals that in combination with growth factor-deficient Matrigel or collagen type IV, epidermal growth factor and transforming growth factor-alpha increase LAMP expression in the sensorimotor population. Delaying the addition of growth factor until after most cell division had ceased failed to increase the number of LAMP+ neurons. Another growth factor in Matrigel, platelet-derived growth factor, does not produce the same effect. Our results indicate that local signals can regulate the differentiation of cortical progenitors, providing a potential mechanism for establishing an early commitment to specific regional phenotypes in the developing cerebral wall that relate to future functional domains in the cortex.

Animals↗
PubMed1993

Alveolar type II cell-fibroblast interactions, synthesis and secretion of surfactant and type I collagen.

During alveolar development and alveolar repair close contacts are established between fibroblasts and lung epithelial cells through gaps in the basement membrane. Using co-culture systems we have investigated whether these close contacts influence synthesis and secretion of the principal surfactant apoprotein (SP-A) by cultured rat lung alveolar type II cells and the synthesis and secretion of type I collagen by fibroblasts. The alveolar type II cells remained cuboidal and grew in colonies on fibroblast feeder layers and on Matrigel-coated cell culture inserts but were progressively more flattened on fixed fibroblast monolayers and plastic. Alveolar type II cells cultured on plastic released almost all their SP-A into the medium by 4 days. Alveolar type II cells cultured on viable fibroblasts or Matrigel-coated inserts above fibroblasts accumulated SP-A in the medium at a constant rate for the first 4 days, and probably recycle SP-A by endocytosis. The amount of mRNA for SP-A was very low after 4 days of culture of alveolar type II cells on plastic, Matrigel-coated inserts or fixed fibroblast monolayers: relatively, the amount of mRNA for SP-A was increased 4-fold after culture of alveolar type II cells on viable fibroblasts. Co-culture of alveolar type II cells with confluent human dermal fibroblasts stimulated by 2- to 3-fold the secretion of collagen type I into the culture medium, even after the fibroblasts' growth had been arrested with mitomycin C. Collagen secretion, by fibroblasts, also was stimulated 2-fold by conditioned medium from alveolar type II cells cultured on Matrigel. The amount of mRNA for type I collagen increased only modestly when fibroblasts were cultured in this conditioned medium. This stimulation of type I collagen secretion diminished as the conditioned medium was diluted out, but at high dilutions further stimulation occurred, indicating that a factor that inhibited collagen secretion also was being diluted out. The conditioned medium contained low levels of IGF-1 and the stimulation of type I collagen secretion was abolished when the conditioned medium was pre-incubated with antibodies to insulin-like growth factor 1 (IGF-1). There are important reciprocal interactions between alveolar type II cells and fibroblasts in co-culture. Direct contacts between alveolar type II cells and fibroblasts appear to have a trophic effect on cultured alveolar type II cells, increasing the levels of mRNA for SP-A. Rat lung alveolar type II cells appear to release a factor (possibly IGF-1) that stimulates type I collagen secretion by fibroblasts.

Animals↗
PubMed2001

Formation of insulin-secreting, Sertoli-enriched tissue constructs by microgravity coculture of isolated pig islets and rat Sertoli cells.

Pancreatic islets, isolated from neonatal pigs, and Sertoli cells, isolated from prepubertal rats, were cocultured in simulated microgravity utilizing the NASA-developed highly accelerating, rotating vessel (HARV) biochamber. Following 5 d of incubation, three-dimensional Sertoli-islet cell aggregates (SICA) retained the ability to secrete insulin when exposed to elevated glucose. SICA contained FasL-positive Sertoli cells and insulin-positive beta-cells randomly organized within the spherical construct. The addition of 1% Matrigel induced the reorganization of aggregates (SICAs formed in the presence of Matrigel [SICAmgs]) showing the peripherialization and epithelialization of Sertoli cells and the centralization of islets in association with lumen-like spaces. The Sertoli cells, but not Matrigel, aided in preserving the structural integrity of HARV-incubated islets. Neither Matrigel nor Sertoli cells appeared to interfere with the ability of SICA or SICA mg to secrete insulin and express FasL.

Animals↗
PubMed2006

Common culture conditions for maintenance and cardiomyocyte differentiation of the human embryonic stem cell lines, BG01 and HUES-7.

Development of generic differentiation protocols that function in a range of independently-derived human embryonic stem cell (hESC) lines remains challenging due to considerable diversity in culture methods practiced between lines. Maintenance of BG01 and HUES-7 has routinely been on mouse embryonic fibroblast (MEF) feeder layers using manual- and trypsin-passaging, respectively. We adapted both lines to trypsin-passaging on feeders or on Matrigel in feeder-free conditions and assessed proliferation and cardiac differentiation. On feeders, undifferentiated proliferation of BG01 and HUES-7 was supported by all three media tested (BG-SK, HUES-C and HUES-nL), although incidence of karyotypic instability increased in both lines in BG-SK. On Matrigel, KSR-containing conditioned medium (CM) promoted undifferentiated cell proliferation, while differentiation occurred in CM containing Plasmanate or ES-screened Fetal Bovine Serum (FBS) and in unconditioned medium containing 100 ng/ml bFGF. Matrigel cultures were advantageous for transfection but detrimental to embryoid body (EB) formation. However, transfer of hESCs from Matrigel back to feeders and culturing to confluence was found to rescue EB formation. EBs formed efficiently when hESCs on feeders were treated with collagenase, harvested by scraping and then cultured in suspension in CM. Subsequent culture in FBS-containing medium produced spontaneously contracting EBs, for which the mean beat rate was 37.2 +/- 2.3 and 41.1 +/- 3.1 beats/min for BG01-EBs and HUES-7-EBs, respectively. Derived cardiomyocytes expressed cardiac genes and responded to pharmacological stimulation. Therefore the same culture and differentiation conditions functioned in two independently-derived hESC lines. Similar studies in other lines may facilitate development of universal protocols.

Biomarkers↗
PubMed1991

An ultrastructural study of in-vitro interaction of guinea-pig and mouse blastocysts with extracellular matrices.

Guinea-pig (intrusive) and mouse (displacement) blastocysts display different cellular mechanisms of implantation. Blastocysts were placed in CMRL-1066 supplemented with either 10 or 20% fetal calf serum, 0.1M L-glutamine and antibiotics and then transferred to dishes previously coated with either Matrigel or type I collagen. After culture for 48 or 72 h, the dishes were processed for transmission electron microscopy. Blastocysts had attached to both extracellular matrices by 48 h. Matrigel elicited minimal trophoblast cell activity. Trophoblast cell projections were oriented parallel to the Matrigel and displayed little invasive activity, but trophoblast cells displayed active interaction with type I collagen. By 72 h, trophoblast cells exhibited slender, anastomosing projections which extended into the collagen matrix. Bundles of microfilaments running parallel with the long axis of the projections were observed. The morphology of type I collagen was altered in the immediate vicinity of the trophoblast projections. The projections interdigitated and desmosomes developed between processes. Projections appeared to meet, fuse and entrap matrix. These results suggest that trophoblast cells do not significantly interact with Matrigel, but penetrate into type I collagen.

Animals↗
PubMed2006

Receptor for advanced glycation end products is involved in impaired angiogenic response in diabetes.

Angiogenic response is impaired in diabetes. Here, we examined the involvement of receptor for advanced glycation end products (RAGE) in diabetes-related impairment of angiogenesis in vivo. Angiogenesis was determined in reconstituted basement membrane protein (matrigel) plugs containing vascular endothelial growth factor (VEGF) implanted into nondiabetic or insulin-deficient diabetic wild-type or RAGE(-/-) mice. The total, endothelial, and smooth muscle (or pericytes) cells in the matrigel were significantly decreased in diabetes, with the regulation dependent on RAGE. In the matrigel, proangiogenic VEGF expression was decreased, while antiangiogenic thrombospondin-1 was upregulated in diabetic mice, regardless of the presence of RAGE. In wild-type mice, proliferating cell nuclear antigen (PCNA)-positive cells in the matrigel were significantly less in diabetic than in nondiabetic mice, while the numbers of transferase-mediated dUTP nick-end labeling (TUNEL)-positive cells were significantly higher. This alteration in PCNA- and TUNEL-positive cells in diabetes was not observed in RAGE(-/-) mice. Similarly, the percentage of nuclear factor kappaB-activated cells is enhanced in diabetes, with the regulation dependent on the presence of RAGE. Importantly, adenovirus-mediated overexpression of endogenous secretory RAGE, a decoy receptor for RAGE, restores diabetes-associated impairment of angiogenic response in vivo. Thus, RAGE appears to be involved in impairment of angiogenesis in diabetes, and blockade of RAGE might be a potential therapeutic target.

Adenoviridae↗
PubMed1990

Proliferative response and macromolecular synthesis by ocular cells cultured on extracellular matrix materials.

To investigate the effects of extracellular matrix components on cellular function, we cultured several types of ocular cells on substrates composed of extracellular matrix materials that were layered on culture dishes either as dried films or as gels. We measured cellular proliferation on these substrates and on a series of gels composed of varying proportions of rat tail tendon type I collagen and Matrigel, a commercially available extract of a basement membrane-producing murine tumor. In addition, we studied the biosynthesis of collagens and of proteoglycans by these cultured cells using [3H]-L-proline and [35S]-sulfate. The proliferative abilities of the various types of ocular cells on the dried film substrates, on uncoated plastic culture vessels, and on pure type I collagen gel, were similar. However, proliferation of ocular cells cultured on gels composed of greater than or equal to 90% Matrigel was markedly reduced. There was little or no inhibition of growth of two types of non-ocular cells: rat C6 astrocytoma cells, and human dermal fibroblasts. Histologic studies showed that the ocular cells tested often formed long strands and capillary-like tubes, and tended to "burrow" beneath the surface of substrates containing high percentages of Matrigel. Fibroblasts infrequently formed tubes, and exhibited the burrowing property also on gels containing primarily type I collagen, while C6 cells showed neither of these behaviors on any of the matrices tested. The elution pattern of newly synthesized [3H]-labeled and [35S]-labeled macromolecules produced by all of the cultured cell types, and detected by Sepharose CL-4B chromatography in the medium and in the cell layer plus matrix fractions did not vary following culture on the different substrates. Approximately twofold more of the newly synthesized collagens and proteoglycans were deposited in the cell layer plus matrix, and proportionately less appeared in the medium, when cells were cultured on type I collagen gels and on Matrigel than on the dried film substrates. These experiments demonstrate the influence of the extracellular matrix on several aspects of cell behavior, and provide further evidence that modification of the composition of the extracellular matrix may be an important determinant of normal or pathological cell function.

Animals↗
PubMed1993

Effect of recombinant tumor necrosis factor-alpha on three-dimensional growth, morphology, and invasiveness of human glioblastoma cells in vitro.

In order to investigate the antiproliferative and anti-invasive effects of tumor necrosis factor (TNF)-alpha on human glioblastoma cells, an in vitro three-dimensional (anchorage-independent) assay was performed using Matrigel, a mixture of extracellular matrix proteins. Four glioblastoma-derived cell lines, including one cloned line, were cultured in Matrigel with or without TNF-alpha. In the Matrigel containing TNF-alpha, three of the four cell lines, including the cloned line, showed significant growth inhibition in a dose-dependent manner. Dramatic three-dimensional morphological differences were observed between TNF-treated and untreated glioblastoma cells cultured in Matrigel. Untreated cells formed large and highly branched colonies throughout the gel. In contrast, the majority of TNF-treated cells demonstrated truncated branching processes and, at a high TNF-alpha dose, an increasing number of cells remained in relatively small spherical aggregates, their cell processes being significantly reduced. Quantitative invasion assay using a micro-Boyden chamber system confirmed that TNF-treated cells lost invasiveness in a dose-dependent manner. These results suggest that TNF-alpha exerts not only antiproliferative but also anti-invasive effects on human glioblastoma cells in vitro. It is believed that this is the first report showing the anti-invasive effect of TNF-alpha on tumor cells.

Brain Neoplasms↗
PubMed2005

Role of extracellular matrix in regulation of staurosporine-induced apoptosis in breast cancer cells.

Autocrine and paracrine mechanisms modulate the synthesis and secretion of extracellular matrix (ECM); moreover, each component of the ECM is capable of modulating the synthesis and release of other ECM molecules. Therefore, the synthesis of ECM glycoprotein fibronectin and laminin was studied in the human breast cancer cell lines MCF7 and MDA MB 23, plated on different ECM. Our results showed that the cells plated on a fibronectin substrate increased laminin synthesis: this event correlated with an increase in alpha2 and alpha3 integrin subunits. Staurosporine-induced apoptosis was then analyzed in the cell lines plated on different ECM. Staurosporine treatment determined the apoptosis of 35 and 33% respectively of MDA MB 231 and MCF7; these values increased to 60 and 64% in cells plated on laminin, to 48 and 63% in cells plated on fibronectin and to 64 and 69% in cells plated on matrigel. Moreover, staurosporine treatment decreased bcl-2 expression in the cells plated on fibronectin and laminin. Yet, staurosporine treatment determined PARP cleavage and PARP partial disappearance when the cells were plated on matrigel. Finally, a partial loss of function mutant Ras protein that activated only Raf pathway, was expressed in MCF7, in order to identify whether the increase of apoptosis induced by extracellular matrix involved the Raf/MAP kinase pathway. The increase of apoptosis of the cells plated on matrigel suggested that the activation of the Raf pathway is probably involved in the decrease of survival on matrigel. These data demonstrate that the modification of ECM modulates the apoptotic process of breast cancer cells and suggest that it is worthwhile to dissect the role of ECM in the control of apoptotic process.

Apoptosis↗
PubMed2001

Monocyte-fibronectin interactions, via alpha(5)beta(1) integrin, induce expression of CXC chemokine-dependent angiogenic activity.

Monocyte-derived macrophages are important sources of angiogenic factors in cancer and other disease states. Upon extravasation from vasculature, monocytes encounter the extracellular matrix. We hypothesized that interaction with extracellular matrix proteins leads monocytes to adopt an angiogenic phenotype. We performed endothelial cell chemotaxis assays on conditioned medium (CM) from monocytes that had been cultured in vitro on various matrix substrates (collagen I, laminin, Matrigel, fibronectin), in the presence of autologous serum, or on tissue culture plastic alone. Monocytes cultured on Matrigel and on fibronectin were the most potent inducers of angiogenic activity compared with tissue culture plastic or autologous serum-differentiated monocytes. This increased angiogenic activity was associated with increased expression of angiogenic CXC chemokines (IL-8, epithelial neutrophil-activating peptide-78, growth-related oncogene alpha, and growth-related oncogene gamma) but not of vascular endothelial growth factor. Additionally, CM from monocytes cultured on fibronectin-depleted Matrigel (MG(FN-)) induced significantly less angiogenic activity than CM from monocytes cultured on control-depleted Matrigel. ELISA analysis of CM from monocytes cultured on MG(FN-) revealed a significant decrease in GRO-alpha and GRO-gamma compared with CM from monocytes cultured on MG. Incubation of monocytes before adherence on fibronectin with PHSCN (a competitive peptide inhibitor of the PHSRN sequence of fibronectin binding via alpha(5)beta(1) integrin) results in diminished expression of angiogenic activity and CXC chemokines compared with control peptide. These data suggest that fibronectin, via alpha(5)beta(1) integrin, promotes CXC chemokine-dependent angiogenic activity from monocytes.

Cell Line↗
PubMed2001

Induction of an aging mRNA retinal pigment epithelial cell phenotype by matrix-containing advanced glycation end products in vitro.

PURPOSE: To determine an extensive mRNA phenotype of the established RPE cell line ARPE-19 when grown on a matrix modified by advanced glycation end products (AGEs). METHODS: Growth Factor Reduced Matrigel (Collaborative Biomedical Products, Bedford, MA) was nonenzymatically glycated with glycolaldehyde. ARPE-19 cells were seeded on both AGE-Matrigel and Matrigel and grown to confluence, and serum was withdrawn for 3 days. RNA was extracted, and microarray analysis was performed to characterize the genes, which are altered by a matrix modified by AGEs. Gene expression changes were confirmed by RT-PCR/Southern and Northern blot analysis. Apoptosis was measured by annexin V/propidium iodide labeling. RESULTS: Clusters of genes with altered expression were found related to cell differentiation, growth factors that regulate the RPE cell and basement membrane, and apoptosis. RT-PCR/Southern and Northern blot analysis confirmed the expression patterns of selected genes, and flow cytometry showed increased annexin V/propidium iodide-labeled cells when grown on AGE-Matrigel. CONCLUSIONS: Microarray analysis identified clusters of genes that could promote an aging RPE phenotype in vitro induced by a matrix modified with AGEs.

Aging↗
PubMed2001

Autotaxin (NPP-2), a metastasis-enhancing motogen, is an angiogenic factor.

Autotaxin [ATX (NPP-2)], originally isolated as a tumor motility-stimulating protein, has recently been shown to augment tumor aggressiveness. Specifically, atx-transfected, ras-transformed NIH3T3 cell lines have been shown to be more invasive, tumorigenic, and metastatic than mock-transfected ras-transformed control cells. In addition, the atx-transfected ras-transformed cell lines appeared to produce tumors that were much more hyperemic than those formed by appropriate control cells. This observation led to the present study, in which we demonstrate that ATX modulates angiogenesis both directly and indirectly. We have used a murine in vivo angiogenesis model in which treated Matrigel plugs are injected s.c. into athymic nude BALB/c mice. Using the same transfected cell lines as before, we found that mixing atx-transfected ras-transformed NIH3T3 cells into the Matrigel resulted in greater new blood vessel formation than control cells. Similarly, mixing purified ATX into the Matrigel resulted in new blood vessel formation within the plug, similar to that produced by vascular endothelial growth factor. Mechanistically, ATX is not a strong chemoattractant for human endothelial cells (HUVECs); however, it strongly stimulates motility in human coronary artery smooth muscle cells. In addition, ATX stimulates HUVECs grown on Matrigel to form tubules, much like vascular endothelial growth factor. Both of these normal cell types are shown to express and secrete ATX. In HUVECs, ATX expression is up-regulated by basic fibroblast growth factor in a time-dependent manner. This up-regulation also extends to secretion of enzymatically active protein, as demonstrated by Western blot analysis and quantification of type-1 phosphodiesterase activity. These results establish the presence of ATX in HUVECs and coronary artery smooth muscle cells and specify ATX as a novel angiogenic factor, suggesting that ATX could contribute to the metastatic cascade through multiple mechanisms, perhaps by supporting an invasive microenvironment for both normal and tumor cells.

3T3 Cells↗
PubMed2000

[The invasive ability of bladder cancer].

OBJECTIVE: To find a direct method for testing the invasive ability of bladder cancer. METHOD: The invasive ability of 49 specimens of primarily cultured bladder cancer, transfected BIU-87 cells with pcDNA3-E1AF and untransfected BIU-87 cells was evaluated by Matrigel invasion assay. RESULTS: Sixteen of the 49 specimens were found to penetrate the matrigel barrier, and the result of the Matrigel invasion was related to the grade of the tumor cells. The number of positive cells of transfected BIU-87 was remarkably larger than that of untransfected BIU-87. CONCLUSIONS: Matrigel invasion assay is a simple, direct and dependable method, and could be used clinically to test the invasive ability of bladder cancer.

Adenovirus E1A Proteins↗
PubMedDate not supplied

Matrix-dependent plasticity of the malignant phenotype of bladder cancer cells.

The purpose of this study was to investigate the effect of cancer- and normal basement membrane-derived extracellular matrix to modulate the phenotype of bladder cancer cell lines. Five lines, varying in malignancy from papilloma to highly undifferentiated and invasive and immortalized human urothelial cells, were grown on two extracellular matrix preparations, Matrigel and SISgel. Matrigel represents matrix remodeled by malignancy while SISgel, obtained from small intestine submucosa (SIS), represents the normal matrix supporting differentiated cell growth. On Matrigel, regardless of the content of growth factors, the invasive lines displayed an invasive phenotype, while the low grade lines grew as papillary structures. In contrast, when the same cells were grown on SISgel, they grew as a layer of cells one to 5 cells thick, failed to invade, and expressed cell-surface E-cadherin. Unlike breast cancer cells, neutralization of beta 1, beta 4 and alpha 6 integrins altered cell-cell and cell-matrix adhesiveness but did not alter the phenotype. When invasive cells were grown on mixtures of SISgel and Matrigel, the phenotype changed gradually, not abruptly, indicating that factors within the gel reversibly alter the phenotypic expression of invasion. In summary, the phenotype of bladder cancer cells growing in tissue-like 3-dimensional culture is highly plastic, and malignant properties such as invasion and papillary growth can be suppressed by the matrix.

Cadherins↗