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PubMed2005

Role of human cripto-1 in tumor angiogenesis.

BACKGROUND: Human cripto-1 (CR-1) promotes cell transformation and increases migration and invasion of various mouse and human epithelial cell lines. We investigated whether CR-1 also stimulates angiogenesis. METHODS: We used human umbilical vein endothelial cells (HUVECs) to measure in vitro migration with fibronectin-coated Boyden chambers, invasion with Matrigel-coated Boyden chambers, proliferation with a tetrazolium salt, and differentiation with an in vitro Matrigel assay. We investigated new blood vessel formation in vivo by use of Matrigel-filled silicone cylinders implanted under the skin of nude mice and by use of a breast cancer xenograft model with CR-1-transfected or control Neo-transfected MCF-7 human breast cancer cells. We also used a blocking anti-CR-1 monoclonal antibody to investigate the role of CR-1 in angiogenesis in vivo and in vitro. All statistical tests were two-sided. RESULTS: CR-1 stimulated HUVEC proliferation, migration, and invasion and induced HUVEC differentiation into vascular-like structures on Matrigel. In vivo, recombinant CR-1 protein induced microvessel formation in Matrigel-filled silicone cylinders, and microvessel formation was statistically significantly inhibited with a blocking anti-CR-1 monoclonal antibody (CR-1 and antibody = 127% of microvessel formation compared with that in untreated control cylinders and CR-1 alone = 259%; difference = 132%, 95% confidence interval [CI] = 123% to 140%; P<.001). Tumors formed by CR-1-transfected MCF-7 cells in the cleared mammary fat pad of nude mice had higher microvessel density than tumors formed by control Neo-transfected MCF-7 cells (CR-1-transfected cells = 4.66 vessels per field and Neo-transfected cells = 2.33 vessels per field; difference = 2.33 vessels per field, 95% CI = 1.2 to 2.8; P = .004). CONCLUSION: CR-1 appears to have an important role in the multistep process of angiogenesis.

Animals↗
PubMed1993

Extracellular matrix composition and resilience: two parameters that influence the in vitro migration and morphology of rat inner cell mass-derived cells.

Parietal endodermal (PE) migration along rat trophectodermal (TE) cells coincides with the deposition of Reichert's membrane between these two cell layers. In this study, we compared the influences of fibronectin and laminin, two components of Reichert's membrane, on the migration and replication of PE-like cells from cultured rat inner cell masses (ICMs). We also explored the role of substrate nondeformability by comparing cell translocation on gels versus coatings of Matrigel (a tumor cell-derived basement membrane preparation) or of collagen. ICMs, isolated by immunosurgery from Day 5 blastocysts, were cultured on coatings of collagen IV, laminin, fibronectin, collagen I, or Matrigel, or on gels of the latter two substrates. Minimal laminin or fibronectin coating concentrations of 2.5 micrograms/ml were required for ICM attachment and cell migration. Migration was similar during the first 48 h of culture on fibronectin and on laminin; however, by 72 h, the extent of cell translocation on fibronectin was greater (1.5- to 2-fold) than that measured on laminin. Fibronectin-cultured ICM-derived cell clusters also contained 1.5- to 2-fold more cells than those on laminin. Migration did not occur on undiluted gels of Matrigel but was supported by diluted (1:10 and 1:20) Matrigel coatings. Similarly, cell migration on coatings of collagen IV reached almost 3-fold that measured on collagen I gels. Most of the cells migrating on fibronectin or collagen (I or IV) were flattened and elongated. In contrast, a high proportion of the cells migrating on laminin or Matrigel coatings were tall and rounded, with thin cytoplasmic extensions. Fibronectin- and collagen IV-cultured cells stained strongly for both collagen IV and laminin, but contained no fibronectin. In contrast, laminin-cultured cells contained fibronectin but were less immunoreactive for laminin and collagen IV. These findings indicate that substrate composition and resilience influence the in vitro migration and morphology of ICM-derived PE-like cells. A role for the TE cells in anchoring Reichert's membrane during development of the PE cell layer within the blastocyst is postulated. Furthermore, the sensitivity of cell morphology and differentiation to individual basement membrane components provides a potential key mechanism whereby an emerging basement membrane can regulate cell migration and differentiation, two fundamental processes that occur throughout embryonic development.

Animals↗
PubMed2000

Establishment of a human lacrimal gland epithelial culture system with in vivo mimicry and its substrate modulation.

PURPOSE AND METHODS: Viable single human lacrimal gland (LG) epithelial cells were obtained by serial incubation in chelating and enzymatic solutions. These cells were evaluated for outgrowth in growth factor-enriched medium using the following culture substrates: Matrigel type I collagen gel with or without fibroblasts, and plastic. Each epithelial outgrowth was characterized morphologically and immunohistochemically, and their growth and viability were examined by bromodeoxyuridine (BrdU) labeling and a quantitative cell viability assay. Synthesized proteins were evaluated by enzyme-linked immunosorbent assay (ELISA), sodium dodecyl sulfate polyacrylamide gradient gels, and 14C-amino acid incorporation. RESULTS: LG epithelial cells plated on Matrigel formed clusters with central cavities that contained lactoferrin, mimicking acinar complexes in vivo. Cells plated on Matrigel containing fibroblasts formed tubuloacinar structures and resembled the human LG in vivo. Cells plated on collagen gel or collagen gel containing fibroblasts formed islands or a monolayer, and lactoferrin was detected in incomplete cavities of epithelia on the latter substrate. Epithelial cells plated on plastic formed a monolayer, and cellular expression of lactoferrin was weak and sporadic. Cellular release of lactoferrin measured by ELISA supported the results of immunohistochemistry. Significant differences in proliferative rate were noted between substrates; cells grown on plastic had the highest proliferative rate, whereas cells grown on Matrigel had the lowest rate. CONCLUSION: Culture substrates modulate LG epithelial cell morphology, proliferative rate, and production of the tear protein lactoferrin. Matrigel promotes acinar differentiation to a greater extent than collagen gel and plastic. Incorporation of fibroblasts in substrates promotes ductal differentiation.

Adolescent↗
PubMed2002

Angiotensin II angiogenic effect in vivo involves vascular endothelial growth factor- and inflammation-related pathways.

Although accumulating lines of evidence indicate the proangiogenic role of angiotensin II (Ang II), little is known about the molecular mechanisms associated with such an effect. This study aimed to identify molecular events involved in Ang II-induced angiogenesis in the Matrigel model in mice. C57Bl/6 female mice received a subcutaneous injection of either Matrigel or Matrigel with Ang II (10(-7) M) alone, with Ang II and an AT1 receptor antagonist (candesartan, 10(-6) M), or with Ang II and AT2 receptor antagonist (PD123319, 10(-6) M). After 14 days, angiogenesis was assessed in the Matrigel-plug by histological evaluation and cellular counting. Ang II increased by 1.9-fold the number of cells within the Matrigel (p < 0.01 versus control). Immunohistological analysis revealed the presence of macrophages, endothelial and smooth muscle cells, and the development of vascular-like structure. Such an angiogenic effect was associated with an increase in vascular endothelial growth factor (VEGF) (1.5-fold, p < 0.01), endothelial nitric oxide (eNOS) (1.7-fold, p < 0.01), and cyclooxygenase-2 (1.4-fold, p < 0.05) protein levels measured by Western blotting. Conversely, Ang II treatment did not affect MMP-9 and MMP-2 activity, assessed by zymography. Blockade of AT1 receptor completely prevented the Ang II-induced angiogenesis and protein regulations, whereas that of AT2 was ineffective. Administration of VEGF neutralizing antibody (2.5 microg ip twice a week) and cyclooxygenase-2 selective inhibitor (nimesulide, 30 mg/L) also hampered Ang II proangiogenic effect. In addition, Ang II-induced cell ingrowth was impaired by treatment with nitric oxide synthase inhibitor (L-NAME, 10 mg/kg/day) and in eNOS-deficient mice. Therefore, in an in vivo model, Ang II induced angiogenesis through AT1 receptor, which involved activation of VEGF/eNOS-related pathway and of the inflammatory process.

Angiotensin II↗
PubMed1993

Modulation of morphological differentiation of human endothelial cells in culture by the synthetic peptide YIGSR and cytochalasin B.

Matrigel is a solubilized tissue basement membrane gel extracted from the mouse Engelbreth-Holm-Swarm sarcoma. Cultures of human endothelial cells could be induced to undergo extremely rapid morphological differentiation into capillary-like structures by Matrigel. The present study demonstrates the following: (a) Matrigel-induced tube formation by endothelial cells is laminin-dependent and, particularly, the YIGSR peptide site in the B1 chain of laminin may be necessary for endothelial cell differentiation resulting in capillary formation; (b) endothelial cells may be preprogrammed for rapid differentiation into blood vessels and do not require DNA, RNA or protein synthesis in order to undergo tube formation on Matrigel; and (c) microfilament assembly and function is important for Matrigel-induced tube formation.

Cell Differentiation↗
PubMed1993

Human neoplastic submandibular intercalated duct cells express an acinar phenotype when cultured on a basement membrane matrix.

Culture of the human neoplastic submandibular gland intercalated duct cell line, HSG, on the basement membrane extract Matrigel induces dramatic morphologic changes and cytodifferentiation. Transmission electron microscopy demonstrated an acinar cell phenotype with polarized cells containing a well-developed Golgi apparatus, multiple microvilli-like projections from the apical surfaces into a lumenal-like area, and numerous granule-like organelles. Amylase, an acinar cell marker, was detected by both immunocytochemical and Northern blot analyses. A 50% reduction in [3H]thymidine incorporation by cells cultured on Matrigel, as compared to cells cultured on tissue culture plates, confirmed the differentiated phenotype of the cells. Multiple components of Matrigel appear to contribute to the morphologic differentiation of the HSG cells since antibodies to both laminin and collagen IV, as well as the laminin-derived bioactive peptide containing SIKVAV, have potent inhibitory effects on HSG cell organization on Matrigel. Collectively, these data indicate that culture of HSG cells on Matrigel is a useful model to study salivary gland acinar development.

Amylases↗
PubMed1989

Matrix-derived soluble components influence type II pneumocytes in primary culture.

Type II pulmonary epithelial cells cultured on a plastic surface fail to retain differentiated form and function. During the first 3 days in primary culture, the cells flatten and lose characteristic lamellar inclusions; they increase in size and exhibit accelerated rates of protein synthesis and thymidine incorporation. These transitions are inhibited markedly if the cells are plated on matrigel (MG), a laminin-rich surface derived from the Englebreth-Holm-Swarm (EHS) sarcoma. Soluble components released from matrigel (MGS) mimic some of the effects of the solid gel. As on a plastic surface, the cells flatten when exposed to MGS during culture. In contrast, MGS inhibits thymidine incorporation and protein synthesis; it is most effective when added early in the culture interval. Direct contact of the cells with the matrigel surface itself is always more effective than maximal MGS activity. The effects of MGS are not reproduced by purified laminin or by transforming growth factor-beta, both of which are present in matrigel. These results indicate that the effects of the solid matrigel surface on cell morphology are caused in part by direct cell-matrix contact but that additional effects, such as decreased DNA synthesis, can be mediated by activity of solubilized gel components. They further provide a model wherein changes in type II cell morphology and function, which typically occur in parallel during primary culture, can be separated experimentally.

Animals↗
PubMed2004

Antiangiogenic properties of 17-(dimethylaminoethylamino)-17-demethoxygeldanamycin: an orally bioavailable heat shock protein 90 modulator.

PURPOSE: The purpose of this study was to investigate the antiangiogenic properties of 17-(dimethylaminoethylamino)-17-demethoxygeldanamycin (17-DMAG; NSC707545), a water-soluble benzoquinone ansamycin. EXPERIMENTAL DESIGN: The activity of 17-DMAG, in vivo, was evaluated for inhibition of fibroblast growth factor (FGF)-2-induced angiogenesis in s.c. implanted Matrigel in mice. In vitro, the activity of 17-DMAG on endothelial cells (human umbilical vein endothelial cells; HUVEC) was tested in FGF-2; and vascular endothelial growth factor (VEGF)-induced proliferation and apoptosis, motility, and extracellular matrix invasion; and on the alignment of capillary like structures in Matrigel. The protein level of heat shock protein (Hsp)90 and client proteins was examined by Western blot in FGF-2 and VEGF-stimulated HUVEC. RESULTS: Daily oral administration of 17-DMAG affected the angiogenic response in Matrigel in a dose-dependent manner. The hemoglobin content in the Matrigel implants was significantly inhibited, and the histological analysis confirmed a decrease of CD31(+) endothelial cells and of structures organized in cord and erythrocyte-containing vessels. In vitro, the compound inhibited dose-dependently the migration and the extracellular matrix-invasiveness of HUVEC and their capacity to form capillary like structures in Matrigel. 17-DMAG treatment also inhibited FGF-2 and VEGF-induced HUVEC proliferation and resulted in apoptosis. Accordingly, the expression of Hsp90 direct client proteins (pAkt and c-Raf-1) or their downstream substrates including pERK was also affected. 17-DMAG consistently increased the expression of Hsp70. Throughout the study similar results were obtained with 17-allylamino-17-demethoxygeldanamycin (17-AAG; NSC330507), the analog compound currently undergoing clinical trials. CONCLUSIONS: We show that the Hsp90 targeting agents 17-DMAG and 17-AAG inhibit angiogenesis. The strong effects on endothelial cell functions, in vitro, indicate that the antiangiogenic activity of 17-DMAG/17-AAG could also be due to a direct effect on endothelial cells. The oral bioavailability of 17-DMAG might be of advantage in investigating the potential of this compound in clinical trials with antiangiogenic as well as antiproliferative endpoints.

Administration, Oral↗
PubMed1995

VEGF165 expressed by a replication-deficient recombinant adenovirus vector induces angiogenesis in vivo.

To evaluate the concept that localized delivery of angiogenic factors via virus-mediated gene transfer may be useful in the treatment of ischemic disorders, the replication-deficient adenovirus (Ad) vector AdCMV.VEGF165 (where CMV is cytomegalovirus and VEGF is vascular endothelial growth factor) containing the cDNA for human VEGF165, a secreted endothelial cell-specific angiogenic growth factor, was constructed. Human umbilical vein endothelial cells (HUVECs) and rat aorta smooth muscle cells (RASMCs) infected with AdCMV.VEGF165 (5 and 20 plaque-forming units [pfu] per cell) demonstrated VEGF mRNA expression and protein secretion into the supernatant. Furthermore, the conditioned medium from these cells enhanced vascular permeability in vivo. In contrast, neither VEGF mRNA nor secreted protein was found in uninfected HUVECs or RASMCs or in cells infected with the control vector AdCMV.beta gal (where beta gal is beta-galactosidase). Assessment of starved HUVECs at 14 days demonstrated sixfold more cells for AdCMV.VEGF165-infected HUVECs (20 pfu per cell) than for either infected or uninfected control cells. RASMC proliferation was unaffected by infection with AdCMV.VEGF165. When plated in 2% serum on dishes precoated with reconstituted basement membrane (Matrigel), HUVECs infected with AdCMV.VEGF165 (20 pfu per cell) differentiated into capillary-like structures. Under similar conditions, both uninfected HUVECs and HUVECs infected with AdCMV.beta gal did not differentiate. To evaluate the ability of AdCMV.VEGF165 to function in vivo, either AdCMV. VEGF165 or AdCMV.beta gal (2 x 10(10) pfu) was resuspended in 0.5 mL Matrigel and injected subcutaneously into mice. Immunohistochemical staining demonstrated VEGF in the tissues surrounding the Matrigel plugs containing AdCMV.VEGF165 up to 3 weeks after injection, whereas no VEGF was found in the control plugs with AdCMV.beta gal. Two weeks after injection, there was histological evidence of neovascularization in the tissues surrounding the Matrigel containing AdCMV.VEGF165, whereas no significant angiogenesis was observed in response to AdCMV.beta gal. Furthermore, the Matrigel plugs with AdCMV.VEGF165 demonstrated hemoglobin content fourfold higher than the plugs with AdCMV.beta gal. Together, these in vitro and in vivo studies are consistent with the concept that Ad vectors may provide a useful strategy for efficient local delivery of VEGF165 in the treatment of ischemic diseases.

Adenoviridae↗
PubMed1997

Migration of eosinophils through basement membrane components in vitro: role of matrix metalloproteinase-9.

In general, inflammatory cells cross basement membranes by producing proteinases. To investigate the role of proteinases in eosinophil basement membrane migration, we studied peripheral blood eosinophils in Matrigel-coated chemotaxis chambers. Electron microscopy showed degradation of the Matrigel layer when eosinophils, added to the upper chamber, transmigrated the membrane in the presence of both platelet-activating factor (PAF) in the lower chamber and interleukin (IL)-5 in both chambers. In the absence of either or both PAF and IL-5, no changes occurred in the Matrigel layer. Matrigel transmigration of eosinophils induced by PAF and IL-5 was inhibited by 1,10-phenanthroline, batimastat, 3,4-dichloroisocoumarin, chymostatin, and a neutralizing antibody for the matrix metalloproteinase (MMP)-9, indicating that serine proteinase(s) and MMP, specifically MMP-9, were involved in the transmigration of eosinophils through Matrigel. In contrast, eosinophil migration through a bare membrane was not affected by batimastat. Using gelatin zymography and immunoblotting, MMP-9 was detected in the migration upper chamber supernatant of the eosinophil transmigration assay and in the conditioned medium of eosinophils. Release of MMP-9 by eosinophils was increased by IL-5, PAF, or both, but the substrate-degrading activity of MMP-9 was increased only in the presence of both IL-5 and PAF, indicating that the releasing and activating mechanisms of MMP-9 are involved in eosinophil basement membrane migration. This study implicates MMP-9 in basement membrane migration of eosinophils and suggests its involvement in inflammatory diseases where tissue eosinophilia plays a role.

Basement Membrane↗
PubMed2000

Assembly of the exogenous extracellular matrix during basement membrane formation by alveolar epithelial cells in vitro.

We found that immortalized alveolar type II epithelial cells (SV40-T2 cells) that were cultured on dense fibrillar collagen supplemented with Matrigel gel formed a thin and continuous lamina densa beneath them. Immunohistochemical analysis of laminin-1, type IV collagen, entactin (nidogen) and perlecan in the culture indicated that all these components were integrated into a sheet structure of basement membrane beneath the cells. Analysis of the temporal and spatial distribution of the basement membrane macromolecules revealed that the initial deposits of laminin-1 and entactin were significantly greater in area in the presence of Matrigel. These globular deposits and the coarse mesh of basement membrane macromolecules developed into a flat membranous basement membrane. In the absence of Matrigel, the SV40-T2 cells failed to form a continuous lamina densa, and the deposits stayed in the coarse mesh. The major biotinylated Matrigel components that were integrated into the basement membrane were laminin-1 and entactin. Furthermore, SV40-T2 cells supplemented with exogenous laminin-1 alone as well as laminin-1 contaminated with entactin formed a continuous lamina densa. These results indicate that the laminin-1 and entactin supplied from the Matrigel were incorporated into a basement membrane beneath the SV40-T2 cells, and contributed to the formation of basement membrane. Therefore, we concluded that the alveolar epithelial cells synthesize laminin-1, entactin, type IV collagen, and perlecan, but that they also needed to assemble exogenous laminin-1 into the basement membrane to complete its formation in vitro.

Animals↗
PubMed2000

Role of reconstituted basement membrane in human granulosa cell culture.

The effects of Matrigel, a reconstituted basement membrane, on human granulosa cells were investigated. Cells were obtained from follicular aspirate in the course of oocyte retrieval for in vitro fertilization and were cultured on either a surface coated with Matrigel or uncoated plastic. Light and electron microscopy showed that granulosa cells cultured on Matrigel demonstrated three-dimensional aggregated cells with well differentiated morphology: numerous lipid droplets, microvilli, junctional complexes and lumen-like structures were seen. In contrast, cells cultured on plastic were flattened, poorly differentiated and showed apoptotic cells. Immunocytochemistry showed that the proportion of immunopositive cells for 3beta-hydroxysteroid dehydrogenase was increased in cultures on Matrigel. The results of the present study suggest that culture on Matrigel promotes the differentiation of human granulosa cells and provides a useful tool which may improve the efficiency of in vitro fertilization.

3-Hydroxysteroid Dehydrogenases↗
PubMed1998

Sperm-oviduct epithelial cell monolayer co-culture: an in vitro model of sperm-female tract interactions in a marsupial, the tammar wallaby (Macropus eugenii).

Oviduct epithelial cell (OEC) monolayers were prepared from the isthmic and ampullary parts of the oviducts of FSH-primed tammar wallabies. Co-culture experiments found that 50-60% of wallaby spermatozoa attached immediately to OEC monolayers, tracheal cell monolayer controls, and the surface of culture dishes with and without Matrigel coating. Spermatozoa were considered to be attached if they remained on the culture surface after rapidly pipetting the co-culture medium five times. The percentages of attached and unattached spermatozoa were calculated from the number of spermatozoa recovered in the agitated supernatant. After 2 h co-culture the percentage of attached spermatozoa rose to 60-80%. After 6 h co-culture the number of spermatozoa attached to OEC monolayers derived from the oviductal isthmus remained high and only a small percentage were recovered in the agitated supernatant (unattached spermatozoa 3.85 +/- 0.76%, P = 0.67). However, after 6 h co-culture of spermatozoa with OEC monolayers derived from the ampulla and with the controls the percentage of attached spermatozoa declined significantly (unattached spermatozoa: ampullary monolayer 23.08 +/- 4.80%, P < 0.01; tracheal monolayer 23.23 +/- 5.18%, P < 0.01; Matrigel 27.23 +/- 7.76%, P < 0.01; plastic surface 28.19 +/- 5.30%, P < 0.01). After 6 h co-culture with ampullary and isthmic OEC monolayers, the percentage motility of both attached and unattached spermatozoa was maintained at 64.00 +/- 1.90% and 56.66 +/- 3.18% and 62.00 +/- 3.11% and 52.00 +/- 2.43%, respectively, and was then maintained at > or = 35% after 24 h incubation. In the controls, that is, tracheal monolayer and Matrigel, the motility of attached spermatozoa declined rapidly to 48.66 +/- 2.15% and 33.63 +/- 8.66%, respectively, at 6 h, and all spermatozoa were immotile after 24 h incubation. However, the motility of unattached spermatozoa in the controls (tracheal monolayer and Matrigel) was maintained at 57.33 +/- 3.00% and 34.54 +/- 9.27%, respectively, until 6 h and then declined rapidly, and all spermatozoa were immotile after 24 h incubation. Co-culture of wallaby spermatozoa with OEC monolayers also induced acrosomal modifications that were followed by acrosomal loss. At 6 h incubation 38.92 +/- 3.98% of spermatozoa on ampullary OEC monolayers and 36.50 +/- 3.81% spermatozoa on isthmic OEC monolayers had shed their acrosome. Acrosomal loss during co-culture with both isthmic and ampullary OEC monolayers was significantly (P < 0.01) greater than that observed on tracheal epithelial monolayer (24.42 +/- 1.90%, P < 0.01), Matrigel (20.70 +/- 2.71%, P < 0.01) and plastic (15.54 +/- 2.49%, P < 0.01). Co-culturing spermatozoa with OEC monolayers also induced a transformation from streamlined orientation of sperm head and tail to T-shaped (thumbtack) orientation in a small number (10-15%) of motile spermatozoa after 6 h incubation (data not shown). The significance of these results in relation to the role of the oviduct in sperm capacitation is discussed.

Acrosome Reaction↗
PubMed2003

Extracellular matrix-regulated p53 expression and nuclear localization in cultured Detroit 562 cells derived from pharyngeal carcinoma.

Tumor suppressor p53 functions as a transcriptional factor that regulates the cell cycle and apoptosis. A mutated p53 gene can result in decreased sequence-specific DNA binding and transcriptional activity of the p53 protein. In this study, we examined the regulatory role of the extracellular matrix components in p53 expression and nuclear localization in a Detroit 562 cell line derived from a pharyngeal carcinoma. When cultured on a polystyrene surface, type I collagen gel, or Matrigel containing basement membrane components, Detroit 562 cells showed a distinct response to extracellular matrix components morphologically. As shown by Western blotting, Detroit 562 cells cultured on Matrigel displayed an increased expression of p53 protein as well as an elevated nuclear p53 level, as compared with the cells cultured on the polystyrene surface or type I collagen gel. When cultured on Matrigel, nuclear p53-positive cells were exclusively localized to the outer surface layer of the cell clusters, whereas most of the inner cells showed no p53 expression. In Detroit 562 cell clusters on Matrigel, proliferative activities, as evaluated by proliferationcell nuclear antigen staining and bromo-deoxyuridine incorporation assay, were evenly distributed; virtually no apoptotic cells, as evaluated by the fluorescence TUNEL assay, were detected in the cell clusters, suggesting that the peculiar localization of nuclear p53-positive cells was not directly related to cell proliferation or apoptosis. These results indicate that p53 expression and its localization in Detroit cells were modulated by extracellular matrix signals, particularly by the basement membrane components in Matrigel.

Biocompatible Materials↗
PubMed2000

Investigation of an in vitro model of trophoblast invasion.

OBJECTIVES: To re-investigate methods for visualising cytotrophoblast invasion using the Matrigel invasion model. STUDY DESIGN: Cytotrophoblast cells were isolated from pooled first trimester placentae and cultured on Matrigel-coated transwells in media supplemented with either 5 ng/ml vascular endothelial growth factor (VEGF) or 10 ng/ml epidermal growth factor (EGF). Invasion was visualised using standard and confocal fluorescent microscopy. RESULTS: The purity of the enriched cytotrophoblast cells was 84-100%. Immunofluorescent cytokeratin staining examined with an inverted fluorescent microscope suggested that cytokeratin-positive cells were present on the underside of the membrane, having invaded through the Matrigel on the upper surface. However, confocal microscopy indicated that these cells did not invade through the Matrigel, and no viable cells were identified in the culture media below the membrane. CONCLUSION: These results suggest that cytokeratin-positive staining on Matrigel-coated transwells is not necessarily indicative of cell invasion, and that similar studies should be interpreted with caution depending on the method of quantification.

Biocompatible Materials↗
PubMed2002

Effect of normal endometrial stroma on growth and differentiation in Ishikawa endometrial adenocarcinoma cells.

Endometrial cancer is characterized by alterations in the stromal cells and the supporting extracellular matrix in addition to the intrinsic alterations of the malignant epithelial cells. We have developed a cell culture model that demonstrates the role of stromal cells in the regulation of proliferation, hormone responsiveness, and differentiation of an endometrial adenocarcinoma cell line (Ishikawa). Conditioned medium (CM) was collected from normal primary human endometrial stromal cells grown on plastic or within the basement membrane extract, Matrigel. The CM produced by stromal cells cultured in contact with Matrigel markedly inhibited Ishikawa cell proliferation compared with CM from stromal cells cultured on plastic. Ishikawa cell proliferation varied with steroid hormone treatment in the presence of CM from stromal cells embedded in Matrigel. When the Ishikawa cells were placed in coculture in contact with stromal cells in Matrigel, production of a differentiated epithelial secretory product, glycodelin, was induced. Gene expression of stromal cell hormone receptors, growth factors, and integrins was analyzed by reverse transcription-PCR in the presence of Matrigel to determine the potential factors involved in stromal regulatory function. These combined studies imply that the phenotype of the Ishikawa cells can be induced to differentiate to more closely resemble normal endometrial epithelium by reintroduction of stromal factors and appropriate extracellular matrix. Additionally, the study shows that basement membrane proteins influence the regulatory function of stromal cells as they mediate epithelial cell growth.

Adenocarcinoma↗
PubMed2003

Endothelial precursor cells as a model of tumor endothelium: characterization and comparison with mature endothelial cells.

Human umbilical vein endothelial cells (HUVEC) and human microvascular endothelial cells (HMVEC) have been the standards for cell-based assays in the field of angiogenesis research and in antiangiogenic drug discovery. These normal mature endothelial cells may not be most representative of human tumor endothelial cells. Human AC133+/CD34+ bone marrow progenitor cells were established in cell culture media containing vascular endothelial growth factor, basic fibroblast growth factor (bFGF), and heparin to drive differentiation toward the endothelial phenotype. The resulting cells designated endothelial precursor cells (EPC) have many of the same functional properties as mature endothelial cells represented by HUVEC and HMVEC. By SAGE analysis, the genes expressed by EPC are more similar to the genes expressed by endothelial cells isolated from fresh surgical specimens of human tumors than are the genes expressed by HUVEC and HMVEC. Analysis of several cell surface markers by flow cytometry showed that EPC, HUVEC, and HMVEC have similar expression of P1H12, vascular endothelial growth factor 2, and endoglin but that EPC have much lower expression of ICAM1, ICAM2, VCAM1, and thrombomodulin than do HUVEC and HMVEC. The EPC generated can form tubes/networks on Matrigel, migrate through porous membranes, and invade through thin layers of Matrigel similarly to HUVEC and HMVEC. However, in a coculture assay using human SKOV3 ovarian cancer cell clusters in collagen as a stimulus for invasion through Matrigel, EPC were able to invade into the malignant cell cluster, whereas HMVEC were not able to invade the malignant cell cluster. In vivo, a Matrigel plug assay where human EPC were suspended in the Matrigel allowed tube/network formation by human EPC to be carried out in a murine host. EPC may be a better model of human tumor endothelial cells than HUVEC and HMVEC and, thus, may provide an improved cell-based model for second generation antineoplastic antiangiogenic drug discovery.

Antigens, CD34↗
PubMed1991

Invasion of reconstituted basement membrane matrix is not correlated to the malignant metastatic cell phenotype.

Interactions between tumor cells and basement membranes represent a critical step in the progression of neoplasia and in the metastatic process. Reconstituted basement membrane matrix, matrigel, has been recently used with the aim of developing an in vitro assay of tumor cell invasiveness. We have extended these studies by comparing the invasiveness of a large series of normal and malignant epithelial and mesenchymal cells of human and animal origin cultured on matrigel. Normal cells (fibroblasts, glomerular mesangial cells, keratinocytes), human fibrosarcoma cells (HT1080), and reticular sarcoma cells (M5076) clearly established invasive capabilities in the matrix. However, all the other tested cell lines, malignant or virally transformed cells invasive in vivo (MCF7, T47D, SA52, SW613, MO4, A431, BeWo), as well as normal nontransformed cells (MOH22) were incapable of penetration. The morphological features of matrigel invasion by normal fibroblasts and HT1080 cells are described at the light and electron microscope levels. The extent of degradation of a radiolabeled matrigel is minimal and similar in several cell lines reported to be noninvasive or invasive in vivo. Our data suggest that matrigel does not provide a universal model to correlate the invasiveness of cells in vivo and in vitro.

Animals↗