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PubMed1996

Insulin release and insulin mRNA levels in rat islets of Langerhans cultured on extracellular matrix.

Primary culture of rat islets of Langerhans lose glucose responsiveness and eventually die when cultured for a long period of time. In this study we evaluated the effect of matrigel, a basement membrane extract, on (i) islet cell survival, (ii) cell responsiveness following a glucose challenge, and (iii) mRNA levels for insulin, glucagon, and somatostatin. Pancreatic islets were isolated by collagenase digestion and plated in culture dishes either coated or not with a matrigel layer. Using the reverse hemolytic plaque assay, we determined the total number of insulin-secreting cells and the amount of insulin secreted by individual beta cells. After 1 h of exposure to 5 mM glucose, beta cells from 6-month-old rat islets cultured for 6 weeks on matrigel showed an equal number of insulin-secreting cells compared to freshly isolated islets cultured for only 3 days in the absence of matrigel (39.5 +/- 2.5 vs. 37.1 +/- 2.6%). Furthermore, the release of insulin by cells cultured on matrigel for 6 weeks increased in a glucose-dependent manner (p < 0.001) and showed an ED50 of 7 mM. However, the amount of insulin released per single beta cell was reduced by 40-60% (p < 0.02) compared to that released from isolated beta cells derived from a 3-day culture of islets. Finally, there was a 35-55% increase (p < 0.05) in the levels of insulin, glucagon, and somatostatin mRNAs in cells cultured for 6 weeks on matrigel. These data suggest a trophic effect of matrigel on the maintenance of normal beta-cell activity and function and may lead the way to the development of a new model for the study of pancreatic islets in long-term culture.

Animals↗
PubMed2005

Basement membrane matrix modifies cytokine interactions between lung cancer cells and fibroblasts.

OBJECTIVE: Proliferation of fibroblasts (desmoplastic reaction) in the lung adenocarcinomas is an important phenomenon that correlates with metastases and poor prognosis. Because basement membranes are often involved in the desmoplastic areas and many cytokines have binding capacity to basement membrane molecules, we hypothesized that basement membrane modify the paracrine effects between cancer cells and fibroblasts via the fibrogenic cytokines and this hypothesis was experimentally investigated. METHODS: The effects of conditioned media derived from ten lung carcinoma cell lines and normal airway epithelial cells on DNA synthesis of fetal lung fibroblasts were determined. We focused on fibroblast growth factor 2 (FGF-2) as the candidate paracrine cytokines and examined their diffusion through an experimental basement membrane matrix model, Matrigel. RESULTS: All the conditioned media promoted DNA synthesis of fetal lung fibroblasts. Detection by ELISA methods and the neutralizing antibodies suggested that FGF-2 was one of the responsible factors for the growth promotion. Diffusion of FGF-2 across the polycarbonate membrane was suppressed by coating with Matrigel. When FGF-2-secreting A549 cells were covered with Matrigel, FGF-2 was stored in Matrigel and its diffusion into the culture media was significantly reduced. Binding of FGF-2 to Matrigel was completely blocked by a basic protein, protamine sulfate. In the presence of protamine sulfate in Matrigel overlaid on A549 cells, diffusion of FGF-2 increased 7-fold as much as that without overlaid Matrigel. CONCLUSION: These results suggest that the basement membrane acts as a barrier to the diffusion and a reservoir of cytokines secreted by cancer cells, and that the subsequent degradation of the basement membrane by cancer cells could release the stored cytokines and promote growth of fibroblasts.

Adenocarcinoma↗
PubMed2000

Matrix-associated transforming growth factor-beta1 primes mouse bone marrow-derived mast cells for increased high-affinity Fc receptor for immunoglobulin E-dependent eicosanoid biosynthesis.

Mast cells at different tissue locations are heterogeneous with respect to histochemical staining characteristics, granule protease and proteoglycan content, and eicosanoid biosynthesis. We used Matrigel, an extract from the Engelbreth-Holm-Swarm mouse sarcoma that is enriched in basement-membrane proteins, to investigate the effect of tissue matrix proteins on the differentiation of mouse mast cells, with particular attention to eicosanoid biosynthesis. Culture of mouse bone-marrow cells in interleukin-3 on Matrigel for 3 to 4 wk provided a population of mast cells with more intense metachromasia and increased safranin counterstaining compared with mast cells derived in the absence of Matrigel (bone marrow-derived mast cells [BMMC]). High-affinity Fc receptor for immunoglobulin E-dependent biosynthesis of prostaglandin D(2) and leukotriene (LT) C(4) was 6- and 11-fold higher, respectively, from mast cells derived in the presence of Matrigel compared with conventional BMMC derived in its absence. BMMC derived in the presence of Matrigel also generated substantial quantities of 6-trans-LTB(4) diastereoisomers and LTB(4), which were minimally generated by conventional BMMC. When conventional BMMC derived in the absence of Matrigel were then cultured on Matrigel for 5 d, eicosanoid biosynthesis was upregulated without any change in granule staining characteristics. This upregulation in eicosanoid biosynthesis was inhibited by neutralizing anti- transforming growth factor (TGF)-beta1-specific antibodies, was reproduced by 1 ng/ml TGF-beta1, and was attributed to increased expression of cytosolic phospholipase A(2).

Animals↗
PubMed1999

Characterization of the collagen phenotype of rabbit proximal tubule cells in culture.

Studies were performed to characterize the collagen phenotype of cultured rabbit proximal tubule (RPT) epithelial cells grown on plastic and on the reconstituted basement membrane preparation, Matrigel. When grown on a plastic substratum, RPT cells display a cobblestone appearance characteristic of glomerular epithelial cells. While initially forming an interlocking network of cells after subculture on Matrigel, this pattern of culture morphology rapidly develops into one characterized by isolated, organized groups of cells. Notwithstanding the effects of Matrigel on culture morphology, total cellular proliferation was reduced only 25% when RPT cells were grown on this substrate. Greater than 90% of the collagen synthesized by RPT cells grown on plastic was secreted into the culture medium. Qualitative analysis by SDS-PAGE revealed components exhibiting electrophoretic mobilities corresponding to the chains present in type IV and type I collagens. Quantitative analysis by CM-Trisacryl chromatography established that approximately 2/3 of the total collagen synthesized by RPT cells grown on plastic was type IV and approximately 1/3 type I. Quantitative analysis of the collagens produced by RPT cells grown on Matrigel again indicated the synthesis of only type IV and type I molecules but in a slightly more equal ratio of both collagen types and in the ratio of secreted to cell-associated molecules. However, the total amount of collagen synthesized by RPT cells grown on Matrigel was reduced to approximately 1% of the level synthesized by the cells grown on plastic. On plastic, approximately 3/4 of the type I collagen produced was recovered as the type I homotrimer, but on Matrigel type I homotrimers represented only approximately 55% of the total type I collagen synthesized. On Matrigel, the majority of the type IV collagen was recovered as heterotrimers containing alpha1(IV) and alpha2(IV) chains. In contrast, RTP cells grown on plastic predominantly produced type IV homotrimers containing only the alpha1(IV) chain. These data represent the initial report describing the collagens produced by nonimmortalized cultured proximal tubule cells. The finding that a significant amount of the total collagen synthesized was type IV (basement membrane) collagen, regardless of culture substrate, suggests that the RPT cells have maintained a significant degree of differentiation in culture, and thus establishes RPT cells as an appropriate model for investigating ECM changes in proximal tubule cells that occur in kidney disease. Finally, the observation that culture of RPT cells on a reconstituted basement membrane preparation results in a significant reduction in total collagen production and alterations in the molecular forms of type IV and type I molecules synthesized indicates that integrity of the tubular basement membrane may represent an important component in preventing the development of tubulointerstitial fibrosis.

Animals↗
PubMed1999

Suppression of growth and dissemination in human pre-B leukemia cells by tumor necrosis factor-alpha in scid mice.

Tumor necrosis factor (TNF) has been shown to inhibit the growth of ALL cells. Since the systemic administration of TNF for malignancy results in poor response and severe toxicity, future efforts should concentrate on local treatment. Here we examined the suppressive effect of TNF alpha on leukemic cells engrafted in scid mice. NALM6 cells derived from pre-B ALL were injected in scid mice subcutaneously with or without Matrigel. In mice with Matrigel, subcutaneous tumors rapidly increased with time, whereas none of the mice without Matrigel showed any obvious signs of disease or apparent tumors. High levels of leukemic infiltration were observed in peripheral organs in mice with Matrigel by flow cytometry and PCR for human beta-actin mRNA expression, while mice without Matrigel showed low or undetectable infiltration in these organs. Human TNF alpha was also coinjected subcutaneously with NALM6 cells and Matrigel into scid mice. Mice with 10 ng of TNF alpha showed small subcutaneous tumors at 8 weeks, which slowly increased. They were found to have a small number of leukemic cells in peripheral organs by flow cytometry. By PCR, all organs with the exception of lung and brain showed low or undetectable expression of beta-actin. However, a large dose of TNF alpha (100 ng) had no suppressive effect on tumor growth and leukemic infiltration in mouse organs. Similar results were obtained in colony formation of leukemic cells in vitro. To examine the mechanism of the suppressive effect of TNF alpha, the expression of TNF receptors in tumor cells was analyzed by flow cytometry. Parental NALM6 expressed both TNF alpha receptors I (TNFR60) and II (TNFR80), but these expressions were suppressed in tumor cells from mice with Matrigel. Only TNFR80 expression was induced in tumor cells of mice with 10 ng of TNF alpha. The induction of Fas expression was also detected, whereas neither DNA fragmentation nor apoptotic change in histology was observed in tumor cells of mice with TNF alpha. These results suggest that the suppressive effect of TNF alpha on the growth of leukemic cells in scid mice is mediated through the activation of TNFR80 without apoptotic signal.

Animals↗
PubMed2002

Promotion of regeneration of corticospinal tract axons in rats with recombinant vascular endothelial growth factor alone and combined with adenovirus coding for this factor.

OBJECT: After spinal cord transection in adult rats, the axons of the corticospinal tract (CST) degenerate retrogradely and do not regenerate. This phenomenon is thought to be related to either secondary ischemia or deficiency of growth factors. To overcome the deficiency of both blood flow and growth factors, the authors added exogenous vascular endothelial growth factor (VEGF165) to the transected spinal cord either as recombinant protein alone or combined with an adenovirus coding for VEGF165. Because most growth factors are rapidly inactivated in the extracellular environment, the authors used an adenovirus coding for VEGF165 to maintain its activity for several days. METHODS: In adult rats, the dorsal two thirds of the spinal cord were transected at the T-8 level. In experimental rats, either human recombinant VEGF165 or a combination of this factor and a replication-defective adenovirus coding for VEGF165 (Ad.CMV.VEGF165) was applied at the lesion site. Both recombinant VEGF165 alone and combined with Ad.CMV.VEGF165 were mixed with Matrigel, which is a reconstituted membrane basement protein extract. Control rats received Matrigel alone or Matrigel plus an adenoviral vector containing the LACZ gene (Ad.CMV.LACZ). Thirty days after spinal cord injury, the number of newly formed blood vessels was assessed in the injured area. In addition, the sensorimotor cortex was injected with anterogradely transported horseradish peroxidase (HRP) to label the CST axons in the spinal cord and to evaluate the extent of retrograde axonal degeneration and regeneration. Gene transfer was assessed using semiquantitative reverse transcription-polymerase chain reaction analysis, enzyme-linked immunosorbent assay for human VEGF and beta-galactosidase expression in injured rats treated with Matrigel plus Ad.CMV.LACZ, Matrigel plus Ad.CMV.VEGF165, and untreated injured rats. A strong gene transfer in the spinal cord tissue of adenovirus-treated rats was found from Day 3 to Day 10 postinjury, confirming infection. In the injured spinal cord area, a significant increase of blood vessels (300% over control, p < 0.005) occurred both in rats treated with recombinant VEGF165 alone and in those treated with the combination of recombinant VEGF165 and Ad.CMV.VEGF165. Also, in both of these groups of animals the retrograde degeneration of CST axons was significantly reduced compared with rats treated with Matrigel alone or Matrigel plus Ad.CMV.LACZ. Furthermore, in rats treated with recombinant VEGF165 alone or combined with Ad.CMV.VEGF165, a few HRP-labeled CST axons, which were not detectable in control rats, were seen distal to the spinal cord injury, indicating some regeneration across the injured area. CONCLUSIONS: These results indicate that locally applied VEGF exerts angiogenic as well as neurotrophic effects in the injured spinal cord of rats.

Adenoviridae↗
PubMed1994

Enhanced proteolytic activity is responsible for the aberrant morphogenetic development of SV40-immortalized normal human salivary gland cells grown on basement membrane components.

BACKGROUND: A reconstituted basement membrane extract, Matrigel, is a potent inducer of cell growth and differentiation in vitro in a number of cell types. In this study, we examined the effect of Matrigel on the morphogenesis of cultured normal human salivary gland cells. EXPERIMENTAL DESIGN: SV40-immortalized normal human salivary gland-cell clones with duct- (NS-SV-DC) or myoepithelial phenotype (NS-SV-MC), which had been established in our laboratory (Azuma M, et al., Lab Invest, 1993;69:24-42), were cultured on Matrigel in serum-free culture conditions, and then morphogenetic behavior of cell clones was examined. RESULTS: When cell clones were seeded on Matrigel, they formed round or zonal clusters on day 1; however, they failed to develop into a salivary gland morphogenesis. Semithin sections of cell clones cultured on Matrigel exhibited multicellular aggregates on day 1, whereas on days 2 and 3, these cells lost both cell-Matrigel and cell-cell interactions and eventually entered crisis. In an attempt to understand the mechanism involved in this phenomenon, we investigated proteolytic enzymes and their inhibitors secreted by cell clones. Although cell clones produced almost identical levels of gelatinases, they released increased amounts of plasminogen activators as compared with a neoplastic human salivary gland cell line, which had already been demonstrated to differentiate into acinar cells when cultured on Matrigel. Obvious difference of expression level of tissue inhibitor of metalloproteinases-1 was not observed in these cells, however, NS-SV-DC produced a relatively small amount of endothelial-type plasminogen activator inhibitor as compared with NS-SV-MC. Neutralization of excess plasminogen activators by exogenously added serine protease inhibitors corrected the aberrant in vitro morphogenesis of NS-SV-DC, but not of NS-SV-MC, and allowed NS-SV-DC to form glandular-like structures.

Animals↗
PubMed1996

[Studies on the mechanism of embryo implantation].

Implantation is a complex process accomplished by synchronization and interactions between embryo and endometrium by local exchange of signals including a number of cytokines and growth factors and direct cell-cell and cell-matrix contact. However, the research in early events of human implantation is still in its infancy. This presentation comprises the results of our attempts to investigate the mechanisms of human implantation process in its early stage by cell-biological method, including establishment of experimental implantation model in vitro. 1. Human trophoblast of early stage of gestation showed active cell locomotion, active endocytosis, and invasion of endometrial cell monolayer in mixed cultures. Trophoblast invasion was later arrested by transformed endometrial cells similar to decidual cells in vivo. These results appeared to indicate the interactions between trophoblast and endometrial cells in implantation. 2. Coculture system of rabbit preimplantation blastocyst and endometrial epithelium reformed from isolated endometrial epithelial cells on basement membrane matrix (Matrigel) simulated the in vivo rabbit implantation processes. This coculture system may provide a useful experimental implantation model. 3. A human trophoblast cell line was established from chorionic tissues of normal early pregnancy. These cells were cytotrophoblast-like morphology and endocrine functions. They formed the villous structures similar to those in vivo in culture on Matrigel and invasion of Matrigel was observed. These indicated the extracellular matrix may affect the morphology and function of invading trophoblast in implantation site. 4. Human endometrial epithelial single cells were cultured on Matrigel. Reconstruction of gland followed by epithelium formation quite similar to in vivo structures by migration and proliferation of isolated cells was demonstrated. Height of gland was promoted by estrogen and initiation of epithelization was upregulated by platelet-derived growth factors. This system revealed the extracellular matrix regulated morphogenesis of endometrial epithelium in vivo and is an essential substrate in experimental implantation model of endometrial epithelium. 5. Parallel cultures of endometrial epithelial cells on Matrigel were carried out with the IVF. ET patients to evaluate the endometrial morphology at time of ET. Endometrial cultures were initiated in previous cycles on Matrigel and the sera of patients were added to her own cultures from 1st day of IVF treatment cycle. Evaluation of reformed epithelium revealed the apparently unsuitable morphology for implantation in group of patients who eventually failed in pregnancy. This system may provide a useful measures in evaluation of endometrial receptivity and modality of treatment for endometrial aberrations. 6. Cyclic changes of extracellular matrix components in endometrium were investigated. Collagen I, III, IV, V were immunohistochemically estimated. Relative levels of all types of collagen except for collagen V declined at early secretory phase. In rodents, not only collagen but also laminin and fibronectin levels declined at early secretory phase. These changes may facilitate trophoblast invasion of endometrium. Collagen V distributed in myometrial surface was found to consist of subunit (alpha 1)2 alpha 2 and trophoblast growth was inhibited on substrate of alpha 1 subunit. Collagen V in myometrial surface may have a role in blocking trophoblast invasion. 7. HGF (hepatocyte growth factor) mRNA was demonstrated to be expressed during menstruation and secretory phase in endometrium distinctly and its receptor in endometrial epithelial cells and decidual cells. Positive correlation between plasma HGF levels and ultrasonographic thickness of endometrium was observed at late secretory phase. Recombinant HGF promoted proliferation of endometrial epithelial cells and decidual cells and upregulated initiation of endometrial epithelization of Matrigel.

Animals↗
PubMed1996

Extracellular matrix upregulates synthesis of glucosylceramide-based glycosphingolipids in primary Schwann cells.

The formation of basement membrane around Schwann cells that are in contact with axons is necessary for Schwann cell differentiation and myelin formation in the peripheral nervous system. However, primary Schwann cells grown on basement membrane in the absence of neuronal influence show increased proliferation rather than differentiation, which implies that the signals generated by Schwann cell-basement membrane interactions are multipotential. We examined the effect of matrigel, an exogenous basement membrane preparation, and other extracellular matrix growth surfaces on primary Schwann cells to determine if the resulting interactions play a role in the control of glycosphingolipid synthesis. Isolated primary Schwann cells grown on a thin layer of matrigel rapidly adhered to the surface and exhibited a greater degree of cell spreading when compared to cells grown on the nonspecific substrate polylysine. Labeling of the cells with [3H]galactose between 24 and 48 hr after plating revealed that the incorporation of [3H]galactose into glucosylceramide-based glycosphingolipids increased from 1.5-3-fold on matrigel in comparison to cells grown on polylysine. The major labeled glycolipids under both conditions were GM3 ganglioside and two neutral glycolipids that comigrated with GbOse4Cer (GalNAc beta 1-3Gal alpha 1-4Gal beta 1-1Cer) and GbOse5Cer (GalNAc alpha 1-3Gal-NAc beta 1-3Gal alpha 1-4Gal beta 1-4Glc beta 1-1Cer) standards. There was little or no increase in the incorporation of [3H]leucine, [3H]galactose, or [3H]glucosamine into proteins or [3H]palmitic acid into phospholipids, free ceramides, or sphingomyelin, suggesting that the matrigel-induced increase in the synthesis of the glycolipids was selective. In the absence of serum, there was little or no difference in the levels of glycolipid labeling between cells grown on the two substrata, demonstrating that serum factors were required for matrigel to have this effect. When cells were grown on surfaces coated with individual extracellular matrix components, those cells grown on laminin and collagen IV showed an increase in glycolipid labeling similar to that produced by matrigel, while labeling increased to a lesser degree for the other components tested. Thus, the signals generated by interactions between Schwann cells and basement membrane, particularly the laminin and collagen IV constituents, contribute to the regulation of glycolipid synthesis which in turn may affect cell morphology and proliferation.

Animals↗
PubMed1990

Proliferation and differentiation of a transfected Schwann cell line is altered by an artificial basement membrane.

Rapidly dividing transfected Schwann cells were grown on Matrigel, a reconstituted basement membrane gel. Matrigel decreased the proliferation of the cells by 75% when compared to sister cultures that were grown on an untreated plastic substrate. Some transfected cells plated onto a Matrigel substrate formed colonies similar to that observed when the cells were plated on a plastic substrate. Additionally, many cells on Matrigel assembled themselves into fascicles projecting away from the colonies. These fascicles were composed of transfected Schwann cells that had assumed a bipolar appearance reminiscent of quiescent secondary Schwann cells in culture. Transfected cells grown on Matrigel contained approximately 10-fold less glial fibrillary acidic protein when compared to sister cultures grown on an untreated plastic substrate. By indirect immunofluorescence laminin immunoreactivity appeared as globules within the cytoplasm of the cells which were cultured on a plastic substrate. However, cells that were grown on the Matrigel substrate appear to organize laminin in a linear array around themselves. These results demonstrate that the presence of an artificial basement membrane alters the morphology, rate of proliferation, and state of differentiation of a transfected Schwann cell line.

Basement Membrane↗
PubMed1990

Long-term maintenance of primary myogenic cultures on a reconstituted basement membrane.

We describe a simple technique for maintaining highly contractile long-term chicken myogenic cultures on Matrigel, a gel composed of basement membrane components extracted from the Engelbreth-Holm-Swarm mouse tumor. Cultures grown on Matrigel consist of three-dimensional multilayers of cylindrical, contracting myotubes which endure for at least 60 d without myotube detachment. A Matrigel substrate increases the initial plating efficiency but does not effect cell proliferation. Large-scale differentiation in cultures maintained on Matrigel is delayed by 1 to 2 d, compared to cultures grown on gelatin-coated dishes. Long-term maintenance on Matrigel also results in increased expression of the neonatal and adult fast myosin heavy chain isoforms. Culturing of cells on a Matrigel substrate could thus facilitate the study of later events of in vitro myogenesis.

Animals↗
PubMed1996

CDw49b/CD29 integrin complex mediates the differentiation of human endothelial cells into capillary-like structures in vitro.

We have investigated different beta-1 integrins (CDw49/CD29) on human umbilical vein endothelial cells (HUVEC) with regard to their roles in modifying the morphological structure of these cells on/in matrigel. The inhibition of matrigel-induced capillary formation by antibodies against subunits of beta-1 integrins was examined quantitatively using a digital analyzer. Antibodies to CDw49b and CD29 (common beta chain) caused a marked inhibition of capillary formation (up to 70%) in a dose-dependent manner, whereas antibodies to CDw49d, CDw49e and CDw49f were less inhibitory. We also examined the appearance of HUVEC cultured in matrigel. HUVEC suspended in matrigel for 24 h formed extended cell processes which connected, resulting in the formation of a capillary network. In contrast, fibroblasts cultured in matrigel showed only bipolar extensions without cell-cell contact. After 48 h in culture in matrigel, some HUVEC showed the capillary-unit of a lumen encircled by EC which may mimic the basic putative unit in the formation of capillaries. However, HUVEC pretreated with antibodies to CDw49b and CD29 failed to form significant processes and a hollow lumen. These phenomena may illustrate the importance of endothelial cell-basement membrane matrix interaction (through integrins, especially CDw49b/CD29 complex) occurring during differentiation of endothelial cells in angiogenesis.

Antibodies, Monoclonal↗
PubMed2002

Reversal of activation of human myofibroblast-like cells by culture on a basement membrane-like substrate.

BACKGROUND: Liver injury transforms hepatic stellate cells into myofibroblast (MFB)-like cells. With recovery from injury, MFBs undergo apoptosis, but it is unknown whether they can also revert to quiescence. AIM: To determine whether human (h)MFBs become quiescent if cultured on a basement membrane-like substrate (Matrigel). METHODS: hMFBs obtained from cirrhotic liver were re-cultured on plastic or Matrigel. Expression of genes of collagen metabolism was assayed before and after transforming growth factor beta (TGFbeta) and Oncostatin M (OSM) stimulation. RESULTS: hMFBs had typical MFB-like morphology, with abundant alpha-smooth muscle actin (SMA) but no cytoplasmic lipid droplets. hMFBs re-cultured on Matrigel reverted to alphaSMA-negative, lipid droplet-positive quiescent morphology. alphaSMA, collagen alpha1(1) (COL1A1) and collagen alpha2(1) (COL1A2) messages were upregulated in hMFBs cultured on plastic, but suppressed by Matrigel. The opposite was true for metalloproteinase-1 mRNA. OSM but not TGFbeta reduced alphaSMA mRNA by 30% while TGFbeta but not OSM upregulated COL1A1 mRNA by 48%, in hMFBs on plastic. TGFbeta and OSM stimulated COL1A1 gene expression in Matrigel by 50 and 60%, respectively. CONCLUSIONS: Matrigel culture de-activates hMFBs yet collagen gene expression still responds to fibrogenic cytokines. The responses of hMFB gene expression to TGFbeta and OSM, are regulated differently by the extracellular matrix.

Actins↗
PubMed2004

Angiogenesis model for ultrasound contrast research: exploratory study.

RATIONALE AND OBJECTIVES: To optimize an angiogenesis model for imaging research that is stable and can be imaged several times over the angiogenic time course. MATERIALS AND METHODS: Mice and rats received two injections of 0.4 mL of extract of basement membrane matrix (Matrigel; Becton Dickinson Labware, Bedford, MA) in the subcutaneous spaces on either side of the spine. One of the two Matrigel plugs in each animal had either 0.1 microg/mL of basic fibroblast growth factor (bFGF) (11 mice), 1.0 microg/mL of bFGF (12 mice, 5 rats), or 1.0 microg/mL of bFGF and 60 U/mL of heparin (11 mice). Three to 12 days after implantation, animals were imaged before and after the administration of up to four injections of 0.1 mL AF0150. Phase inversion imaging was used on a Siemens Elegra (Siemens ultrasound, Issaquah, WA) equipped with a 13 MHz VFX transducer. Three observers subjectively assessed the pattern of enhancement using a four-point scale. The Matrigel plugs were then removed and two observers graded the angiogenic response on a four-point scale. Ten Matrigel plugs, five with 1.0 microg/mL bFGF and five without, were evaluated histologically following immunohistochemical staining with anti-CD31. RESULTS: The angiogenic response was greater in Matrigel plugs with 1.0 than with 0.1 microg/mL of bFGF. Heparin did not increase the angiogenic response. Vessels were predominantly at the periphery of the plugs with variable central penetration. Plugs appeared anechoic and homogeneous on ultrasound. Contrast enhancement within the plug occurred in 44% of mice with an angiogenic response at or after day 6 and the enhancement increased with the angiogenic response. In the others, peripheral enhancement could not be distinguished from the enhancement of surrounding tissues that were also hyperemic. The thicker rat skin interfered with plug assessment. CONCLUSION: A stable angiogenesis model without the complexity of tumors is described. This model offers the opportunity to image the development and/or inhibition of angiogenesis. Neovasculature in Matrigel was detectable using ultrasound contrast. Quantitative studies correlating the degree of enhancement to microvascular density will be determined in subsequent studies.

Animals↗
PubMed2002

Extracellular domain of TGFbeta type III receptor inhibits angiogenesis and tumor growth in human cancer cells.

TGFbeta overexpression in human cancer cells has been shown to promote tumor progression. In the present study, we sought to determine whether sequestration of endogenous TGFbeta by the expression of a soluble TGFbeta type III receptor (sRIII), can reduce malignancy in human carcinoma cells and whether the tumor-suppressive activity of sRIII is associated with the inhibition of angiogenesis. Ectopic expression of sRIII significantly inhibited the growth of tumors formed by human colon carcinoma HCT116 and breast carcinoma MDA-MB-435 cells in nude mice. It also reduced the metastatic potential of the MDA-MB-435 cells. Thus, endogenous TGFbeta appears to be necessary for the progression of these two carcinomas. Furthermore, when the tumor cells were mixed with Matrigel and embedded subcutaneously in nude mice, the blood volume in Matrigel plugs containing sRIII-expressing cells as indicated by hemoglobin levels was significantly lower than that in Matrigel plugs containing the respective control cells. Blood vessel counts in paraffin sections of the Matrigel plugs containing sRIII-expressing cells were also significantly lower than those in paraffin sections of the Matrigel plugs containing control cells. Treatment of human endothelial cells with a recombinant sRIII significantly inhibited their ability to form a capillary web structure on Matrigel. These results for the first time indicate that the sRIII-induced tumor suppression appears to be in part due to the inhibition of angiogenesis.

Angiogenesis Inhibitors↗
PubMed2002

Image analysis of Transwell assays in the assessment of invasion by malignant cell lines.

This study aims to determine if layering of extracellular matrix (ECM) can achieve a physiological basement membrane thickness of 8 microm and to assess the use of paraffin wax-embedded Transwell plates coupled with digital image analysis as a means of determining invasion by malignant cell lines. Layers of Matrigel, a sarcoma-derived ECM was built to a concentration of 7.4 microg/mm2 in the upper chamber of a Transwell plate invasion assay. Two cell lines from extrahepatic bile duct adenocarcinoma were tested in serum-free growth medium. Conditioned medium was added to the lower chamber to act as a chemoattractant. Following attachment, cells were incubated for 48 h and the Matrigel-coated insert cut from its holder and fixed in 10% unbuffered formalin saline. Each insert was bisected and processed to paraffin wax. Serial levels were stained by haematoxylin and eosin. A Kontron image analysis system was used to measure the mean thickness of Matrigel for each cell line and the degree of invasion was assessed by measuring the depth to which cells had degraded the Matrigel. A mean thickness of 8 microm was achieved using 5.0 microg/mm2 for the OCUCh-LM1 cell line and 7.4 microg/mm2 for the SKChA-1 cell line. No significant difference was seen in the ability of either cell line to degrade Matrigel. Immunocytochemistry for laminin and cytokeratin helped to identify ECM components and cells, respectively. In conclusion, digital image analysis of paraffin wax-embedded inserts can be used to determine the invasive capacity of various cell lines; immunocytochemistry may help to identify ECM components and cells; and the assay used to assess different cell lines and their ability to degrade Matrigel.

Adenocarcinoma↗
PubMed1999

Function of alpha3beta1-tetraspanin protein complexes in tumor cell invasion. Evidence for the role of the complexes in production of matrix metalloproteinase 2 (MMP-2).

Tumor cell migration through the three- dimensional extracellular matrix (ECM) environment is an important part of the metastatic process. We have analyzed a role played by the integrin-tetraspanin protein complexes in invasive migration by culturing MDA-MB-231 cells within Matrigel. Using time-lapse video recording, we demonstrated that the Matrigel-embedded cells remain round and exhibit only limited ability for migration by extending short, highly dynamic pseudopodia. The alpha3beta1-tetraspanin protein complexes were clustered on the thin microvilli-like protrusions extending from both the main cell body and pseudopodia. Ligation of the alpha3beta1-tetraspanin protein complexes with monoclonal antibodies specifically stimulates production of matrix metalloproteinase 2 (MMP-2) and induces formation of long invasive protrusions within Matrigel. Accordingly, treatment with the monoclonal antibodies to various tetraspanin proteins and to the alpha3 integrin subunit increases invasive potential of the MDA-MB-231 cells in the Matrigel-penetration assay. A specific inhibitor of phosphoinositide 3-kinase (PI3K), LY294002, negated the effect of the monoclonal antibodies on the morphology of the Matrigel-embedded cells and on production of MMP-2. Interestingly, broad-spectrum inhibitors of protein tyrosine kinases (genistein) and protein tyrosine phosphatases (orthovanadate), and actin filament stabilizing compound (jasplakinolide), also block protrusive activity of the Matrigel-embedded cells but have no effect on the production of MMP-2. These results indicate that alpha3beta1-tetraspanin protein complexes may control invasive migration of tumor cells by using at least two PI3K-dependent signaling mechanisms: through rearrangement of the actin cytoskeleton and by modulating the MMP-2 production.

Actins↗
PubMed1997

Characterization of the lactoferrin-dependent inhibition of the adhesion of Actinobacillus actinomycetemcomitans, Prevotella intermedia and Prevotella nigrescens to fibroblasts and to a reconstituted basement membrane.

Lactoferrin was previously shown to inhibit the adhesion of A. actinomycetemcomitans, P. intermedia and P. nigrescens to human cells. Lactoferrin was also shown to competitively inhibit the binding of these bacteria to the basement membrane protein laminin. The present study aimed to determine the type of interactions inhibited by lactoferrin. Lactoferrin binds to fibroblast monolayers and Matrigel, a reconstituted basement membrane, through ionic interactions. The adhesion of A. actinomycetemcomitans to these substrata was mainly dependent on the ionic strength of the environment. P. intermedia and P. nigrescens also adhere to fibroblasts mainly by ionic interactions, while their adhesion to Matrigel seems to be mediated by specific mechanisms. Lectin-type interactions were not found to be involved in the binding of these bacteria to the substrata. Treatment of either A. actinomycetemcomitans or fibroblasts with lactoferrin decreased the adhesion in a dose-dependent manner, while lactoferrin treatment of Matrigel alone had no adhesion-counteracting effect. Adhesion of P. intermedia and P. nigrescens to Matrigel was not significantly affected by the ionic strength, but the presence of lactoferrin inhibited the adhesion. Lactoferrin bound to Matrigel, P. intermedia and P. nigrescens was rapidly released, while lactoferrin bound to A. actinomycetemcomitans and fibroblasts was retained. These findings indicate that lactoferrin-dependent inhibition of the adhesion of A. actinomycetemcomitans, P. intermedia and P. nigrescens to fibroblasts and Matrigel can involve binding of lactoferrin to both the bacteria and substrata. The decreased adhesion may be due to blocking of both specific adhesin-ligand as well as non-specific charge-dependent interactions.

Aggregatibacter actinomycetemcomitans↗