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Previous studies demonstrated that corneal epithelial cells isolated without basal lamina respond to extracellular matrix (ECM) in an actin dependent manner; the basal cell surface flattens and the actin cortical mat reorganizes. We hypothesize that the actin reorganization is initiated by intracellular signaling mechanisms that includes tyrosine phoshporylation and activation of the Rho, MAP kinase, and PI3 kinase signal transduction pathways. Our goals were to develop a morphological assay to test this hypothesis by answering the following questions: 1) Do the actin bundle formations in the cortical mat have the same configuration in response to different ECM molecules? 2) What is the minimum time ECM molecules need to be in contact with the tissue for the actin to reorganize? 3) Will blocking tyrosine phosphorylation inhibit reorganization of the actin? 4) Are known signal transduction proteins phosphorylated in response to soluble matrix molecules? The actin cortical mat demonstrated distinct bundle configurations in the presence of different ECM molecules. Soluble fibronectin accumulated at the basal cell surfaces 75-fold over 30 min in a clustered pattern. The cells need contact with ECM for a minimum of 10 min to reform the actin bundles at 2 hr. In contrast, two substances that bind to heptahelical receptors to stimulate the Rho pathway, bombesin and lysophosphatidic acid, reorganized the actin bundles in 15-30 min. Focal adhesion kinase, p190 Rho-GAP, tensin, and paxillin were tyrosine phosphorylated in response to soluble fibronectin, type I collagen, or laminin 1. Erk-1, erk-2, and PI3 kinase were activated after 1 hr stimulation by type I collagen. Herbimycin A blocked actin reorganization induced by ECM molecules. In conclusion, we have developed two morphological assays to examine the response of corneal epithelial cells to ECM molecules. In addition, actin bundle reorganization involved tyrosine phosphorylation, MAP kinase, and PI3 kinase activation.
Anat Rec 1999 03
PMID:ECM-stimulated actin bundle formation in embryonic corneal epithelia is tyrosine phosphorylation dependent. 1009 66

In rat uterine epithelium, platelet-derived growth factor (PDGF) and fibronectin (FN) display changes in temporal expression during implantation. PDGF was expressed in the apical epithelium on Day 3, apically, laterally and basally at the time of implantation on Day 6 but was not expressed on Day 7. FN expression was not seen until Day 6, when it was expressed only in the basement membrane. However, this label was markedly increased in the basement membrane on Day 7. We suggest that fibronectin may be upregulated by PDGF in preparation for invasion of the basement membrane by stromal decidual cells and the subsequent attachment of the trophoblast to the maternal extracellular matrix.
Anat Rec 1999 05 01
PMID:Temporal changes in the expression of platelet-derived growth factor and fibronectin in the uterine epithelium during early pregnancy. 1032 87

Aortic distensability is the key to normal aortic function and relates to the lamellar unit in the media. However, the organization of the extracellular matrix components in these lamellar units, which are largely responsible for the distensability, is insufficiently known, especially in the human. We therefore performed a detailed ultrastructural analysis of these components. Thoracic aortas of 56 individuals (age 45-74 years), none of whom suffered from aortic disease, were studied by immunoelectron microscopy of elastin, collagen types I, III, IV, V, and VI, fibronectin, and fibrillin-1, and by ultrastructural histochemistry of proteoglycans, which were further characterized by enzymatic digestion. The elastic lamellae were closely associated with thick collagen fibers containing types I, III, and V collagen. Between these collagen fibers, numerous complex, circumferentially oriented streaks of elastin protruded from the lamellae. In contrast to what is usually reported in the aortas of experimental animals, the smooth muscle cells preferentially adhered to these ill-defined streaks rather than directly to the solid lamellae. Fibrillin-1- and type VI collagen-containing bundles of microfibrils (oxytalan fibers) were also involved in the smooth muscle cell-elastin contact. The smooth muscle cells were invested by basal lamina-like layers connecting them to each other as well as to the oxytalan fibers. Unexpectedly, these layers were abundantly labeled by anti-fibronectin, whereas type IV collagen, a specific basement membrane component, was mainly found in larger, flocculent deposits. The proteoglycans present were collagen-associated dermatan sulfate proteoglycan, cell-associated heparan sulfate proteoglycan, and interstitial chondroitin sulfate proteoglycan. Our observations demonstrate that the extracellular matrix in the human aorta is extremely complex and therefore differs from most descriptions based on experimental animals. They serve as reference for future studies on aortic diseases, such as aneurysmas and dissections.
Anat Rec 2000 01 01
PMID:Extracellular matrix of the human aortic media: an ultrastructural histochemical and immunohistochemical study of the adult aortic media. 1060 43

Retinoic acid has been associated with a variety of cardiac defects. A percentage of these defects are related to changes in the endocardial cushions. Studies in mice and older chick embryos have shown a decrease in mesenchymal cell formation attributable to retinoic acid and have suggested that retinoic acid was affecting the extracellular matrix. In this study we have tested the effect of retinoic acid on cardiac mesenchyme formation in vitro and then tested retinoic acid treated myocyte cultures for changes in the expression of hLAMP-1, fibronectin and transferrin members of the particulate matrix that is required for mesenchyme formation. Initial experiments tested the effect of retinoic acid on mesenchymal cell formation first in atrioventricular canal and outflow tract explant cultures and then in AV endothelial monolayer cultures using myocyte conditioned media or the particulate matrix fraction from retinoic acid treated myocyte cultures. In all cases, mesenchymal cell formation was suppressed while no suppression was observed when MyoCM was included with retinoic acid. Protein analysis showed that retinoic acid had a stimulatory effect on protein synthesis. ELISA assays revealed that retinoic acid treated myocyte cultures contained significantly more hLAMP-1 and fibronectin than either normal or DMSO controls. However, transferrin was not affected by retinoic acid treatment in these experiments. Our results suggest that retinoic acid affects the expression of the particulate matrix and that these changes may be responsible for the observed decrease in mesenchymal cell formation.
Anat Rec 2000 02 01
PMID:Retinoic acid inhibition of cardiac mesenchyme formation in vitro correlates with changes in the secretion of particulate matrix from the myocardium. 1064 66

In this study, the presence of cells in the intimal region of normal adult bovine pulmonary artery (BPA) was examined by analysis of longitudinal sections at the level of light and transmission electron microscopy. In addition, the morphological and immunohistochemical phenotype of these cells as well as the presence of particular extracellular matrix (ECM) components in this region were also determined. Since ECM production and cell proliferation have been demonstrated to be regulated by locally released growth factors such as transforming growth factor beta (TGFbeta), the presence of TGFbeta-1 in this region was also investigated. Our findings reveal the presence of immature or "nonmuscle" cells into the subendothelial space of normal adult BPA. These cells were characterized by the presence of abundant cytoplasmic organelles and scanty microfilaments. Such cells were negative to antibodies against smooth muscle alpha actin (SM alpha-actin), 1E12, and vWf, but not to vimentin. Similar cells have recently been detected in normal adult BPA and canine carotid arteries, but in the medial region. Because of their location in these elastic arteries, the nonmuscle cells are involved not only in the remodeling of the medial region, but also in the neointima or intimal thickening formation by migration from the media to the subendothelial space, where they proliferate and secrete ECM components. However, a limited number of morphological studies and the current investigation describe the presence of scattered nonmuscle cells within the intima of some normal elastic arteries. This would suggest an important role for these resident cells within the intima in normal and pathological processes as well. In addition, our results show the presence, in this region, of TGFbeta-1 and of ECM components that include collagen, elastin, fibronectin, and laminin which are present in normal conditions and during the intima formation in vivo.
Anat Rec 2000 03 01
PMID:Characterization of nonmuscle cells present in the intima of normal adult bovine pulmonary artery. 1070 46

One of the most vital synapomorphic properties of metazoans is the presence of an extracellular matrix (ECM), i.e., a complex of proteoglycans, adhesive glycoproteins, and collagens. The genetically controlled ECM mediates between the respective receptors morphogenesis and cell differentiation and is central to gastrulation, i.e., the process which generates the embryonic germ layers, upon which all metazoan body structures are based. However, the primitive metazoans include a phylum, viz. the Dicyemida, which lacks any kind of typical metazoan ECM structures including a basement membrane, and hence does not develop through gastrulation. Since the ECM components fibronectin, laminin, and type IV collagen, all of which are essential constituents of each basement membrane, have been proved to be evolutionary ancient molecules from the lowest metazoans up to vertebrates, antibodies against the respective vertebrate ECM components were employed by electron microscopy to look for these molecules also in the dicyemid mesozoan Kantharella antarctica. As a result, all three protein families showed an immunolabel which was localized intracellularly and intimately associated with the cell membranes as well as with the submembranously arranged delicate filamentous network. The immunolabel was most intense in the fibronectin-like protein, followed by the type IV collagen-like protein and weakest in the laminin-like protein. From an evolutionary point of view, this kind of distribution of ECM components, primarily found in intracellular regions, seems to reflect a very primitive situation of the structures of the respective ECM molecules having not yet reached their definitive position outside the cell, thus generating the complete biological function of typical ECM. Moreover, these results confirm the long-standing presumption that the dicyemid mesozoan body structure might be the missing link between the Protozoa and Metazoa.
Anat Rec 2000 05 01
PMID:Extracellular matrix (ECM) components in a very primitive multicellular animal, the dicyemid mesozoan Kantharella antarctica. 1076 Jul 43

Retinoic acid has been associated with a number of cardiac defects, some of which seem to be related to changes in the endocardial cushions. Studies in mice and older chick embryos have suggested that these defects may be associated with a decrease in mesenchymal cell formation within the cushion. In a previous report we showed that retinoic acid lowered the number of mesenchymal cells in a culture bioassay of mesenchyme formation and that this response was due to retinoic acid modifying the production of particulate matrix from the myocardium. In this study, we have extended these observations to the embryo by implanting a retinoic acid coated bead into the embryo and examined the effect on cardiac mesenchyme formation and in the production of the particulate matrix. In all cases the addition of retinoic acid resulted in a decrease in the number of mesenchymal cells invading the endocardial cushions. In addition retinoic acid increased the production of hLAMP-1 and fibronectin but not transferrin, confirming our earlier report. Finally, we measured the volume of the cushion and calculated the cell density of both the inferior and superior cushions. The results suggest that the superior cushion is more sensitive to retinoic acid treatment than the inferior cushion. Collectively, these results support our earlier work that suggests that the mechanism of retinoic acid cardiac abnormalities involves a disruption in the production of particulate matrix from the myocardium and a subsequent decrease in cardiac mesenchyme cells that results in a malformed cardiac cushions.
Anat Rec 2001 05 01
PMID:Retinoic acid administration is associated with changes in the extracellular matrix and cardiac mesenchyme within the endocardial cushion. 1133 71

Anastomotic intimal hyperplasia and surface thrombogenicity are the main reasons for the high failure rate of prosthetic small-diameter vascular grafts. While anastomotic intimal hyperplasia is a multifactorial event, ongoing surface thrombogenicity is primarily caused by the lack of an endothelium, even after years of clinical implantation. After decades of poorly performing synthetic artery-grafts, tissue engineering has emerged as a promising approach to generate biologically functional bio-synthetic hybrid grafts mimicking native arteries regarding the presence of an endothelial lining on the blood surface. "In vitro endothelialization" represented the first generation of such tissue-engineered vascular grafts, utilising cell culture techniques for the creation of a confluent autologous endothelium on ePTFE grafts. The clinical long-term results with this method in almost 200 patients are highly encouraging, showing patencies equal to vein grafts. Since "in vitro endothelialization" requires cell culture facilities, it will always be confined to large centres. Therefore, research of the 1990s turned to the development of spontaneously endothelializing implants, to make tissue-engineered grafts amenable to the entire vascular-surgical community. Apart from scaffold designs allowing transmural ingrowth, biological signalling through a facilitating ingrowth matrix holds a key to spontaneous endothelialization. In biological signalling, the increasingly deeper understanding of bio-active molecules and the discovery of domains and peptide sequences during the 1980s created the expectation in the 1990s that peptide signalling may be all that is needed. This present review highlights the possible problems associated with such a reductionist approach. Using the fibronectin molecule, we demonstrated that domains may be more suitable modules in tissue engineering than peptide sequences.
Anat Rec 2001 08 01
PMID:Engineering of vascular ingrowth matrices: are protein domains an alternative to peptides? 1150 Aug 15

For understanding the immunological functions of the peritoneum, spatial localization of integrins and their ligands was studied by immuno-SEM on the peritoneal surface of mice with cecal perforation-induced peritonitis. The cecal peritoneum 24 hr after perforation was stained with specific antibodies against LFA-1, Mac-1, VLA-4, ICAM-1, VCAM-1, and fibronectin diluted with cold University of Wisconsin (UW) solution in conjunction with immuno-gold labeling. The spatial localization of those cell adhesion molecules was detected by backscatter electron (BSE) imaging with field emission scanning electron microscope (FESEM). Numerous leukocytes with diverse surface ultrastructure were observed on the peritoneal surface by FESEM. Some leukocytes were in contact with mesothelial cells, and others adhered to the exposed underlying connective tissue. The BSE imaging showed the ubiquitous distribution of Mac-1 on all membrane domains of leukocytes, i.e., cell body, ruffles, and microvilli. In contrast, predominant expressions of LFA-1 and VLA-4 were discernible on ruffles/microvilli of some leukocytes. The mesothelial cells remaining in the inflamed area expressed both ICAM-1 and VCAM-1 on their microvilli. The fibronectin was detected on presumable collagen fibers and/or fibrin over the exposed smooth muscle layer as well as on fibrin extending between leukocyte aggregation. The spatial microlocalization of integrins was clarified on the leukocytes emigrated in peritonitis, and their ligands were detected on the inflamed peritoneum.
Anat Rec 2001 10 01
PMID:Spatial distribution of cell adhesion molecules on the peritoneal surface in the cecal perforation-induced peritonitis. 1159 May 97

In preparation of a clinical phase I/II study in renal cell carcinoma (RCC) patients, we developed a clinically applicable protocol that meets good clinical practice (GCP) criteria regarding the gene transduction and expansion of primary human T lymphocytes. We previously designed a transgene that encodes a single chain (sc) FvG250 antibody chimeric receptor (ch-Rec), specific for a RCC tumor-associated antigen (TAA), and that genetically programs human T lymphocytes with RCC immune specificity. Here we describe the conditions for activation, gene transduction, and proliferation for primary human T lymphocytes to yield: (a) optimal functional expression of the transgene; (b) ch-Rec-mediated cytokine production, and (c) cytolysis of G250-TAA(POS) RCC by the T-lymphocyte transductants. Moreover, these parameters were tested at clinical scale, i.e., yielding up to 5-10 x 10(9) T-cell transductants, defined as the treatment dose according to our clinical protocol. The following parameters were, for the first time, tested in an interactive way: (1) media compositions for production of virus by the stable PG13 packaging cell; (2) T-lymphocyte activation conditions and reagents (anti-CD3 mAb; anti-CD3+anti-CD28 mAbs; and PHA); (3) kinetics of T-lymphocyte activation prior to gene transduction; (4) (i) T-lymphocyte density, and (ii) volume of virus-containing supernatant per surface unit during gene transduction; and (5) medium composition for T-lymphocyte maintenance (i) in-between gene transduction cycles, and (ii) during in vitro T-lymphocyte expansion. Critical to gene transduction of human T lymphocytes at clinical scale appeared to be the use of the fibronectin fragment CH-296 (Retronectin) as well as Lifecell) X-fold cell culture bags. In order to comply with GCP requirements, we used: (a) bovine serum-free human T-lymphocyte transduction system, i.e., media supplemented with autologous patients' plasma, and (b) a closed cell culture system for all lymphocyte processing. This clinical protocol routinely yields 30-65% scFvG250 ch-Rec(POS) T lymphocytes in both healthy donors and RCC patients.
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PMID:Protocol for gene transduction and expansion of human T lymphocytes for clinical immunogene therapy of cancer. 1208 62


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