Artificial Basement Membrane Research Articles

Inhibition of invasion and induction of apoptotic cell death of cancer cell lines by overexpression of TIMP-3.

Dysregulation of matrix degrading metalloproteinase enzymes (MMPs) leads to increased extracellular matrix turnover, a key event in the local invasion and metastasis of many tumours. The tissue inhibitors of metalloproteinases (TIMPs) limit the activity of MMPs, which suggests their use in gene therapy. We have previously shown that overexpression of TIMP-1, -2 or -3 inhibits vascular smooth muscle and melanoma cell invasion, while TIMP-3 uniquely promotes apoptosis. We have therefore sought to determine whether TIMP-3 can inhibit invasion and promote apoptosis in other cancer cell types. Adenoviral-mediated overexpression of TIMP-3 inhibited invasion of HeLa and HT1080 cells through artificial basement membrane to similar levels as that achieved by TIMP-1 and -2. However, TIMP-3 uniquely promoted cell cycle entry and subsequent death by apoptosis. Apoptosis was confirmed by morphological analysis, terminal dUTP nick end labelling (TUNEL) and flow cytometry. The apoptotic phenotype was mimicked by addition of exogenous recombinant TIMP-3 to uninfected cultures demonstrating that the death signal is initiated extracellularly and that a bystander effect exists. These results show that TIMP-3 inhibits invasion in vitro and promotes apoptosis in cancer cell type of differing origin. This clearly identifies the potential of TIMP-3 for gene therapy of multiple cancer types. © 1999 Cancer Research Campaign

Proteinase requirements of epidermal growth factor-induced ovarian cancer cell invasion.

Aberrant expression or activity of the epidermal growth factor (EGF) receptor family of tyrosine kinases has been associated with tumor progression and an invasive phenotype. In this study, we utilized 4 ovarian cancer cell lines, OVCA 432, DOV 13, OVEA6 and OVCA 429, to determine the effects of EGF on the regulation of proteolytic enzymes and their inhibitors, cellular migration and in vitro invasion. Induction of urinary-type plasminogen activator (u-PA) activity and tissue inhibitor of matrix metalloproteinase (TIMP)-1 was observed in all 4 cell lines. OVCA 432 cells showed strong PAI-1 induction; however, the other 3 lines displayed substantial baseline PAI-1 expression that was not induced by EGF. EGF-dependent stimulation of migration and induction of matrix metalloproteinase (MMP)-9 (gelatinase B) was observed in OVEA6 and OVCA 429 cells only. Upon EGF receptor activation, DOV 13, OVEA6 and OVCA 429 cells were induced to invade through an artificial basement membrane (Matrigel); however, no invasion was detected in OVCA 432 cells. Cell lines displaying induction of migration and MMP-9 (OVEA6 and OVCA 429) demonstrated robust EGF-induced invasion (5- to 20-fold), and cell invasion was substantially reduced in the presence of anti-catalytic MMP-9 antibody. Addition of anti-catalytic u-PA antibody inhibited the modest (<2-fold) EGF-induced invasion in a cell line that did not express MMP-9 (DOV 13) and in OVEA6 cells that displayed the highest baseline u-PA activity. Together, our findings indicate that multiple proteinases are important in ovarian cell invasion and implicate EGF induction of MMP-9 and migration as key components of more aggressive ligand-induced invasion.

Adhesion of normal and carcinoma hepatic cells on the membrane containing collagen IV using a micropipette technique

In order to understand the cell rheological mechanism of tumor cell metastasis, the adhesion properties of hepatocellular carcinoma (HCC) and human fetal hepatocytes (HFH) on the artificial basement membrane containing different concentrations of collagen IV were measured by means of a micropipette aspiration technique developed in this paper. Based on 12 measured cases of HCC and HFH cell lines on the artificial basement membrane, the main points are shown as: (1) using the micropipette aspirating system developed, we could measure successfully cell's adhesion force in vitro; (2) under the 18 cases measured, the adhesion force (FHCC) of HCC is higher than that (FHFH) of HFH except for two cases on the artificial basement membrane containing higher concentration (≥5 μg ml −1) of the coating collagen IV and glass only; (3) by comparing the adhesion force of HCC cells and HFH cells and changing the coated collagen IV concentration, we found, in general, that HCC cells have better adhesion on the artificial basement membranae when the concentration of coated collagen IV is 1 and 2 μg ml −1. Therefore, because the HFH cells in tissue have higher binding properties in 5 μg ml −1 of coated collagen IV on the artificial basement membrane, the adhesion force of HFH cells was 1.41 times that of HCC cells.

A bifunctional hybrid molecule of the amino-terminal fragment of urokinase and domain II of bikunin efficiently inhibits tumor cell invasion and metastasis.

Urinary trypsin inhibitor (UTI) inhibits efficiently tumor cell invasion and the formation of metastasis. The anti-metastatic effect is dependent on the COOH-terminal domain II of UTI [UTI-(78-136)-peptide]. To develop a molecule that binds with high affinity to the urokinase (uPA) receptor (uPAR) on tumor cell surfaces, a bifunctional hybrid molecule [uPA-(1-134)-UTI-(78-136)] consisting of the uPAR-binding NH2-terminal fragment [UTI-(78-136)-peptide] of uPA at the NH2-terminus of UTI-(78-136)-peptide was produced in Escherichia coli by genetic engineering. The purified hybrid protein inhibited trypsin and plasmin 2-3-fold less effectively than UTI-(78-136)-peptide and was found to bind to human tumor cells via uPAR, which was confirmed by cell binding and competition experiments. Using a modified Boyden chamber and an artificial basement membrane, Matrigel, it was found that the hybrid protein is very effective at inhibiting invasion by uPAR-expressing human tumor cells. Sensitivities of tumor cells towards the anti-invasive effect of uPA-(1-134)-UTI-(78-136) correlated with the density of uPAR on human tumor cells. Furthermore, in the spontaneous metastasis model, the hybrid protein inhibited the formation of lung and/or lymphatic metastasis by human ovarian carcinoma and choriocarcinoma cells. The hybrid protein was much more effective than uPA-(1-134)-peptide, UTI-(78-136)-peptide, or UTI. We conclude that this approach extends the possibility of applying recombinant protein for therapeutic use in inhibition of human tumor cell metastasis.

Down-regulation of laminin-binding integrins by 1 alpha,25-dihydroxyvitamin D3 in human melanoma cells in vitro.

In the present investigation the effect of 1 alpha,25(OH)2D3 on the expression of the integrin laminin receptor on the melanoma cell line SK-MEL-28 has been examined. The SK-MEL-28 cells were shown to contain high-affinity receptors for 1 alpha,25(OH)2D3 and cell proliferation was found to be inhibited in a dose-dependent manner in response to the hormone. Using monoclonal antibodies against the alpha 6-sub-unit of the integrin laminin receptor, immunocytochemistry demonstrated that exposure of cells to 1 alpha,25(OH)2D3 for 5 days caused a reduced staining intensity. This observation was further confirmed by dot blot analysis, where a dose-dependent decline of alpha 6 expression was obtained after treatment of the cells with 1 alpha,25(OH)2D3 for 6 days. FACS-analysis was performed in order to quantify this decline, and it was found that the level of alpha 6-subunits on the cell surface was reduced by more than 40%. Additional investigations including Northern blot analyses of poly(A)+RNA extracts also showed a dose-dependent reduction of alpha 6 mRNA. Interestingly, the decrease of alpha 6 expression on the surface of SK-MEL-28 melanoma cells was accompanied by a reduced ability of the cells to adhere to an artificial basement membrane. In conclusion, the present investigation shows that besides having an antiproliferative effect on the SK-MEL-28 melanoma cells, 1 alpha,25(OH)2D3 is also able to inhibit the surface expression of the alpha 6-subunit of the integrin laminin receptor. Moreover, the results strongly indicate that 1 alpha,25(OH)2D3 exerts its regulatory effect on the alpha 6-subunit at the transcriptional level rather than at the protein level.

Inhibition of human malignant glioma cell motility and invasion in vitro by hypericin, a potent protein kinase C inhibitor

The effect of hypericin, an antiviral drug and a potent protein kinase C (PKC) inhibitor, on glioma cell invasion was investigated in vitro. Treatment of the established human glioblastoma cell line, T98G, with 1 μM hypericin for 24 h resulted in a significant inhibition of the cell invasion through an artificial basement membrane, but not cell attachment or proliferation. Furthermore, tamoxifen and staurosporine, both PKC inhibitors, also inhibited T98G cell invasion, suggesting that PKC may be the cellular target for hypericin-inhibited glioma cell migration. Similarly, hypericin decreased cell motility significantly in established lines, T98G and U87-MG, and also in a low-passage human malignant glioma cell line. Thus, hypericin may prove useful for studying mechanisms of glioma invasion, and may represent a new agent in malignant glioma therapy.

Epithelial–Stromal Interactions Modulating Penetration of Matrigel Membranes by HPV 16-Immortalized Keratinocytes

The role of epithelial-stromal interactions in the progression of human papillomavirus-associated squamous intraepithelial lesions to invasive cervical cancer is poorly understood. Using the Matrigel artificial basement membrane assay as a model of keratinocyte invasion, the effects of selected growth factors on penetration of human papillomavirus 16-immortalized keratinocytes through Matrigel were studied. Also studied in this model were the effects of conditioned media from fibroblast lines derived from normal cervical tissues (normal fibroblasts) and adjacent cervical cancer biopsies (tumor-associated fibroblasts) and from primary keratinocytes. Addition of basic fibroblast growth factor, transforming growth factor-alpha, and hepatocyte growth factor/scatter factor or conditioned media from tumor-associated fibroblasts to the Matrigel resulted in near-doubling of penetration of human papillomavirus 16-immortalized keratinocytes, whereas transforming growth factor-beta, platelet derived growth factor-B, or conditioned media from primary keratinocytes decreased penetration 10-fold. Antibodies to basic fibroblast growth factor abrogated the stimulatory effects of conditioned media from tumor-associated fibroblasts on keratinocyte penetration, whereas antibodies to transforming growth factor-beta abrogated the inhibitory effects of conditioned media from normal fibroblasts on keratinocyte penetration. S1 nuclease protection and enzyme-linked immunosorbent assay showed increased expression of transforming growth factor-beta and decreased expression of basic fibroblast growth factor in normal compared with tumor-associated fibroblasts. Messenger RNA in situ hybridization of five cervical cancer biopsies demonstrated basic fibroblast growth factor expression in stromal cells surrounding nests of invading keratinocytes. Epithelial-stromal interactions mediated by growth factors such as transforming growth factor-beta and basic fibroblast growth factor modulate penetration of human papillomavirus 16-immortalized keratinocytes through Matrigel in vitro and these interactions may also be operative in vivo.

Effect of NCAM-transfection on growth and invasion of a human cancer cell line.

A cDNA encoding the human transmembrane 140 kDa isoform of the neural cell adhesion molecule (NCAM) was transfected into the highly invasive MDA-MB-231 human breast cancer cell line. Transfectants with a homogeneous expression of NCAM showed a restricted capacity for penetration of an artificial basement membrane. However, when injected into nude mice, both control and NCAM-expressing cell lines produced equally invasive tumors. Tumors generated from NCAM-transfected cells were heterogeneous, containing NCAM-positive as well as NCAM-negative areas, indicating the existence of host factors capable of modulating NCAM expression in vivo. In nude mice, NCAM-transfected cells developed tumors with longer latency periods and slower growth rates than tumors induced by NCAM-negative control cells, implying that NCAM may be involved not only in adhesive and motile behavior of tumor cells but also in their growth regulation. There was no indication of differences in cell proliferative characteristics between the different NCAM-transfected and the control transfected cells as determined by flow cytometric DNA analysis, suggesting an increased cell loss as the reason for decreased in vivo growth rate of the NCAM-transfected cells. The fact that NCAM expression influences growth regulation attributes a pivotal role to this cell adhesion molecule during ontogenesis and tumor development.

A Splicing Variant of the RON Transcript Induces Constitutive Tyrosine Kinase Activity and an Invasive Phenotype

Volume 16, no. 10, p. 5524: The images in Fig. 10 were inadvertently scrambled in an image-processing program and do not reflect the actual data. Quantitative data obtained from the invasion experiments confirm the conclusions drawn in the paper. The histograms in the revised Fig. 10 show the number of cells that migrated through an 8-μm-pore-size Millipore filter coated with an artificial basement membrane made of polymerized collagen IV, laminin, and glycosaminoglycans (Matrigel). Values represent averages of triplicate determinations of the absorbance of 595 nm of cells stained with crystal violet and solubilized. MOCK indicates mock-transfected cells. The cells expressing recombinant full-size Ron required stimulation by macrophagestimulating protein (MSP) to cross the Matrigel barrier. Cells expressing recombinant Δ-Ron or the C914-A Ron displayed the invasive phenotype constitutively. We apologize for any confusion that this may have caused.

Fibronectin-mediated cell migration promotes glioma cell invasion through chemokinetic activity.

In order to investigate the biological role of fibronectin in glioma cell invasion, we studied the relation between migratory responses or adhesiveness of glioma cells to fibronectin and the in vitro invasion in three human malignant glioma cell lines, A172, T98G and U373MG. All these cell lines chemotactically migrated in a dose-dependent manner to fibronectin in concentrations ranging from 0.5 to 10 microg/ml, with A172 cells showing the strongest migration and U373 cells the weakest. Checkerboard analyses demonstrated that A172 and T98G cells showed much stronger chemokinetic responses to fibronectin than U373MG cells. In contrast to the migratory responses, A172 and U373MG cells showed an almost equally high adhesion to fibronectin and T98G cells a low adhesion. The degree of expression of the integrin alpha5 subunit correlated well with the strength of glioma cell adhesion to fibronectin rather than that of migration to the molecule. Furthermore, the cell adhesion to fibronectin was almost completely inhibited by arginine-glycine-aspartic acid (RGD)-containing peptides, but the fibronectin-stimulated cell migration was only partially inhibited. An in vitro invasion assay disclosed that U373MG cells invaded the artificial basement membrane barrier the most and A172 cells the least. However, addition of fibronectin to the glioma cells markedly enhanced the invasive activity of A172 and T98G cells but had little effect on that of U373MG cells. These results indicate that fibronectin-stimulated migration can be one of the factors promoting invasiveness of glioma cells and that the chemokinetic activity of fibronectin may play a crucial role in glioma invasion through conferring motor-driving force on the glioma cells.

Effect of Tumor Necrosis Factor α on Intrahepatic Bile Duct Epithelial Cell of Rat Liver

Tumor necrosis factor α (TNF–α), which is primarily produced by macrophages, is a cytokine with various biological activities. Macrophage infiltration often accompanies experimental cholangitis in rats, and chronic cholangitis in humans. The pathophysiologic significance of TNF–α in cholangitis is not known. We used cultured, polarized intrahepatic bile duct epithelial cells (IBDECs) from rat liver to determine whether TNF–α directly affects the organization of IBDEC monolayers. The addition of recombinant TNF–α (rTNF–α) to culture media at concentrations from 10 to 200 U/mL lacked cytotoxicity to the IBDECs as judged by trypan blue exclusion and lactate dehydrogenase (LDH) release. rTNF–α transiently reduced transepithelial electrical resistance in a dose–dependent manner. During this decrease in resistance, the cellular tight junctions became leaky, allowing horseradish peroxidase (HRP) penetration. rTNF–α, at concentrations up to 200 U/mL, did not detach IBDECs from Matrigel, an artificial basement membrane. Electron microscopy and immunohistochemistry for F–actin showed a well–preserved cell structure and organization of IBDECs. Results suggest that TNF–α is nontoxic to IBDECs, and that it increases the permeability of tight junctions. TNF–α may thus disturb the barrier function of the bile duct.

Effect of tumor necrosis factor alpha on intrahepatic bile duct epithelial cell of rat liver

Tumor necrosis factor alpha (TNF-alpha), which is primarily produced by macrophages, is a cytokine with various biological activities. Macrophage infiltration often accompanies experimental cholangitis in rats, and chronic cholangitis in humans. The pathophysiologic significance of TNF-alpha in cholangitis is not known. We used cultured, polarized intrahepatic bile duct epithelial cells (IBDECs) from rat liver to determine whether TNF-alpha directly affects the organization of IBDEC monolayers. The addition of recombinant TNF-alpha (rTNF-alpha) to culture media at concentrations from 10 to 200 U/mL lacked cytotoxicity to the IBDECs as judged by trypan blue exclusion and lactate dehydrogenase (LDH) release. rTNF-alpha transiently reduced transepithelial electrical resistance in a dose-dependent manner. During this decrease in resistance, the cellular tight junctions became leaky, allowing horseradish peroxidase (HRP) penetration. rTNF-alpha, at concentrations up to 200 U/mL, did not detach IBDECs from Matrigel, an artificial basement membrane. Electron microscopy and immunohistochemistry for F-actin showed a well-preserved cell structure and organization of IBDECs. Results suggest that TNF-alpha is nontoxic to IBDECs, and that it increases the permeability of tight junctions. TNF-alpha may thus disturb the barrier function of the bile duct. (Hepatology 1996 Jun;23(6):1602-7)

Recombinant human uteroglobin suppresses cellular invasiveness via a novel class of high-affinity cell surface binding site.

The mechanism(s) that regulates invasion of trophoblasts through the uterine epithelium during embryo implantation and nidation in hemochorial placental mammals is poorly understood. While limited trophoblast invasion is essential for the establishment of normal pregnancy, dysregulation of this process may contribute to the pathogenesis of choriocarcinoma, a highly invasive and lethal form of cancer arising from the trophoblasts. We have previously demonstrated that rabbit uteroglobin (UG), a cytokine-like, antiinflammatory protein, produced by the endometrial epithelium during pregnancy, has a potent antichemotactic effect on neutrophils and monocytes in vitro. Here, we report that recombinant human UG (hUG) dramatically suppresses invasion of human trophoblasts and NIH 3T3 cells through an artificial basement membrane (Matrigel) in vitro but has no effect on that of human choriocarcinoma cells. We identified a previously unreported high-affinity, high molecular weight (approximately 190 kDa), nonglycosylated hUG-binding protein, readily detectable on human trophoblasts and NIH 3T3 cells but totally lacking on choriocarcinoma cells. Taken together, these results raise the possibility that (i) hUG plays a critical role in regulating cellular invasiveness, at least in part, via its previously unrecognized cell surface binding site, and (ii) some of the numerous biological activities of proteins of the UG family, reported so far, may be mediated via this binding site.

Urokinase plasminogen activator expression by primary and HPV 16-transformed keratinocytes

The molecular events underlying progression of human papillomavirus (HPV) 16-associated intraepithelial neoplasia to invasive cancer have not been studied in detail. Penetration of the basement membrane is an early, but poorly understood step in this process and probably involves the action of one or more metallo- and serine proteinases. Urokinase-type plasminogen activator (uPA) is a serine proteinase that has been implicated in the pathogenesis of several epithelial tumors, but its role in HPV-associated tumors is not known. To examine uPA expression by HPV 16-transformed keratinocytes in vitro, primary foreskin keratinocyte cultures were transfected by HPV 16 DNA. The primary parental cells and the HPV 16-transformed keratinocytes were studied using substrate gel zymography, Western blot analysis and an in vitro assay measuring penetration of a Matrigel artificial basement membrane. Both uPA and its inhibitor, plasminogen activator inhibitor-1 (PAI-1), were overexpressed in the HPV 16-transformed cells relative to the parental cell line. The transformed cells, but not the parental cells, were able to degrade and penetrate the Matrigel membrane and penetration was blocked by both PAI-1 and by antibodies to uPA. Our data suggest that HPV 16-induced transformation of keratinocytes is associated with upregulation of uPA expression. In conjunction with other proteinases, uPA plays an important role in the ability of HPV 16-transformed keratinocytes to penetrate artificial basement membranes.

Stimulatory effects of hepatocyte growth factor on normal and neoplastic human bronchial epithelial cells

We examined the mitogenic, chemoinvasive, and chemotactic effects of hepatocyte growth factor (HGF) toward normal and neoplastic human epithelial cells derived from the bronchial mucosa. Primary cultures of human bronchial epithelial cells (HBE cells), immortalized bronchial epithelial cells (IB3-1 cells), and cells derived from a squamous cell carcinoma of the lung (128-88T cells) were used as targets. HGF was mitogenic for all three cell types as measured by bromodeoxyuridine (BrdU) labeling and colony-forming efficiency (CFE). With the use of BrdU labeling, 9.8-16.8% of nuclei were labeled in controls vs. 56.9-65.6% labeled nuclei in cells treated with HGF. HGF stimulated colony formation 3.6-6.2-fold over untreated control. Analysis by reverse transcription-polymerase chain reaction demonstrated the presence of the c-met gene, the receptor for HGF, in all three cell types. Cell lysates from all three cell types contained proteins that were recognized by a c-met antibody as determined by Western blotting. The gene for HGF was not expressed in any of the cell types, although it was expressed in control MRC5 fibroblasts. No HGF protein could be detected by Western blotting in the conditioned medium from epithelial cells, although it was readily detectable in medium conditioned by lung fibroblasts. HGF proved to be a powerful chemotactic agent for all three cell types and also stimulated invasion into Matrigel, an artificial basement membrane. The results indicate HGF acts mainly as a paracrine growth factor for cells derived from the human bronchus, and may play a role in the growth and progression of lung tumors.

Glioblastoma cells do not intravasate into blood vessels.

Glioblastoma is very rarely found outside the central nervous system. The ability of rat C6 glioblastoma cells to intravasate into central nervous system and pial blood vessels is tested using a rat homografting model and two in vitro models. In vivo, scanning electron microscopy demonstrates that upon grafting C6 cells into implantation pockets in rat cortex, blood vessels can be spared in large digestion cysts formed in host brain parenchyma. Immunocytochemistry of the grafted rat cortex reveals that the glioblastoma cells are upon the blood vessel basement membrane, surrounded by the extracellular matrix material, fibronectin. The endothelial cells of the blood vessel are inside the laminin and fibronectin, and there were areas of endothelial cell hyperplasia. C6 cells are not observed inside blood vessels. In vitro, C6 cell cultures seeded with blood vessels from fresh rat pia exhibit the same relationship of the C6 glioblastoma cells to the blood vessel as those in the other models. The C6 cells migrate upon the pial blood vessel basement membrane but do not intravasate into the blood vessel. To ascertain whether structure and components of the blood vessel basement membrane are important factors in glioblastoma cell exclusion from blood vessels, C6 cells are seeded upon artificial basement membrane hydrated gel wafers. C6 cells migrate into the artificial basement membrane gel wafer by 1 day after seeding. These data indicate that glioblastoma cells are confined to the central nervous system by an inability to pass through vital basement membrane.

Extracellular matrix proteins inhibit proliferation, upregulate migration and induce morphological changes in human glioma cell lines.

The influence of an artificial basement membrane (BM), Matrigel, and four individual extracellular matrix proteins, fibronectin, laminin, collagen I and vitronectin, on cell proliferation, morphology and migration was assessed in four glioma cell lines. Matrigel and individual BM proteins differentially inhibited cell proliferation of all cell lines studied. In addition, Matrigel was found to induce extensive morphological changes in glioma cells. Polycarbonate filters, of 8-microns porosity in modified Boyden chambers, were used to assess the chemoattraction activity of Matrigel and the individual proteins on glioma cells. All these components were found to stimulate cell migration, albeit to different extents but laminin proved to be the most effective chemoattractant for glioma cells in vitro. These data suggest that basement membrane proteins may inhibit proliferation and stimulate migration in order to facilitate invasion.

Developmental regulation of the expression of 72 and 92 kd type IV collagenases in human trophoblasts: A possible mechanism for control of trophoblast invasion

During early pregnancy fetal cytotrophoblast cells invade the uterus and penetrate the basement membrane, a property that is characteristic of malignant cells. However, unlike tumor invasion, trophoblast invasion of the uterus is under strict control. This control limits invasion, so that it primarily remains confined to the endometrial aspect of the myometrium and continues only until midgestation. The invasive properties of the trophoblast cells are made possible by the activity of proteolytic enzymes that belong to the metalloproteinases and serine proteinases. Type IV collagenase (metalloproteinase) is considered crucial in the extracellular matrix remodeling that takes place during the invasion process. In this study we set out to characterize the invasive properties of trophoblast cells at different stages of pregnancy. Human trophoblast cells were isolated from first- and third-trimester placentas by trypsin digestion and Percoll fractionation and were then cultured under serum-free conditions. The invasive ability of trophoblast cells was determined by the in vitro invasion assay, in which the ability of cells to penetrate an artificial basement membrane was examined. Metalloproteinase activity was measured by zymography, and the expression of messenger ribonucleic acid transcripts of 72 and 92 kd type IV collagenases was examined by reverse transcriptase polymerase chain reaction. First-trimester trophoblasts were 3.5 time more invasive in vitro than were third-trimester trophoblast cells (p < 0.005). Although first-trimester trophoblasts secreted both species of type IV collagenase, 72 and 92 kd, in large amounts, third-trimester cells secreted the 92 kd and only minimal amounts of 72 kd type IV collagenase. Moreover, first-trimester trophoblasts secreted significantly more (p < 0.05) 92 kd type IV collagenase than did third-trimester trophoblast. The messenger ribonucleic acid transcript expression of 72 and 92 kd type IV collagenases correlated with the activity of these enzymes secreted by first- and third-trimester trophoblasts. The described high in situ invasive capacity of first-trimester trophoblast might be explained by the increased expression and production of 72 kd type IV collagenase and the higher expression of 92 kd type IV collagenase by first-trimester trophoblast cells.

An integrated approach to the design and engineering of hybrid arterial prostheses.

A newly devised hybrid small-caliber graft was developed. The graft consisted of three components: a microporous polyurethane graft (inside diameter 3 mm; length 5 cm) with compliance close to that of a natural artery; an artificial basement membrane composed of a complex gel of type I collagen and dermatan sulfate, which showed enhanced adhesion and growth of endothelial cells (ECs) and reduced adhesion of platelets in vitro; and an autogenous EC monolayer with high degrees of cell-substrate and cell-cell interactions, which was performed before implantation. Twenty EC-seeded grafts were implanted bilaterally into carotid arteries of dogs without anticoagulant. The implantation period was up to 26 weeks. The overall patency rate for seeded grafts was 75%. The percentage of endothelial coverage of seeded grafts was 98% as implanted, 92% at 2 weeks, and 100% after 12 weeks. The mean intimal thickness of grafts was around 80 microns at 12 weeks. Little additional increase was observed at 26 weeks. It appears that the complete endothelialization as implanted, high cell-to-substrate adhesive strength that resists hydrodynamic shear stress, and biomechanical compatibility of the polyurethane graft functioned cooperatively to provide a vascular graft with high antithrombogenicity and minimal hyperplasia. The integrated approach of combining biomechanical and cellular engineering designs leading to an important functional smaller-caliber graft is discussed.

Association of glucocerebroside homolog biosynthesis with Schwann cell proliferation

The biosynthesis of myelin-associated glycolipids was studied in quiescent secondary cultures of Schwann cells and in rapidly proliferating population of transfected Schwann cells (TSC) by in vitro incorporation of [3H]galactose. The TSC demonstrated a marked increase (> 10-fold) in [3H]galactose incorporation when compared to quiescent Schwann cells. The level (or amount) of [3H]galactose incorporation into lipids is dependent upon the number of TSC in culture. The majority of 3H-labeled lipids were oligohexosylceramides (GL-2, GL-3, and GL-4). Substrates that inhibit TSC proliferation, collagen type I and Matrigel, and artificial basement membrane, decrease the [3H]galactose incorporation by 25% and 80%, respectively. Our results indicate that the synthesis of glucocerebroside and its homologs is associated with Schwann cell proliferation.