miRNAs are small noncoding RNAs that regulate mRNA targets in a cell-specific manner. miR-29 is expressed in murine and human skin, where it may regulate functions in skin repair. Cutaneous wound healing model in miR-29a/b1 gene knockout mice was used to identify miR-29 targets in the wound matrix, where angiogenesis and maturation of provisional granulation tissue was enhanced in response to genetic deletion of miR-29. Consistently, antisense-mediated inhibition of miR-29 promoted angiogenesis in vitro by autocrine and paracrine mechanisms. These processes are likely mediated by miR-29 target mRNAs released upon removal of miR-29 to improve cell-matrix adhesion. One of these, laminin (Lam)-c2 (also known as laminin γ2), was strongly up-regulated during skin repair in the wound matrix of knockout mice. Unexpectedly, Lamc2 was deposited in the basal membrane of endothelial cells in blood vessels forming in the granulation tissue of knockout mice. New blood vessels showed punctate interactions between Lamc2 and integrin α6 (Itga6) along the length of the proto-vessels, suggesting that greater levels of Lamc2 may contribute to the adhesion of endothelial cells, thus assisting angiogenesis within the wound. These findings may be of translational relevance, as LAMC2 was deposited at the leading edge in human wounds, where it formed a basal membrane for endothelial cells and assisted neovascularization. These results suggest a link between LAMC2, improved angiogenesis, and re-epithelialization.

The LAMB3 and LAMC2 genes encode the laminin-5 β3 and γ2 chains, respectively, which are parts of laminin-5, one of the major components of the basement membrane zone. Here, we report the frequent up-regulation of LAMB3 and LAMC2 by promoter demethylation in gastric cancer. Gene expression data analysis showed that LAMB3 and LAMC2 were up-regulated in various tumor tissues. Combined analyses of DNA methylation and gene expression of both genes in gastric cancer cell lines and tissues showed that DNA hypomethylation was associated with the up-regulation of both genes. Treatment with a methylation inhibitor induced LAMB3 and LAMC2 expression in gastric cancer cell lines in which both genes were silenced. By chromatin immunoprecipitation assay, we showed the activation histone mark H3K4me3 was associated with the expression of both genes. The expression level of LAMB3 affected multiple malignant phenotypes in gastric cancer cell lines. These results suggest that epigenetic activation of LAMB3 and LAMC2 may play an important role in gastric carcinogenesis.

Biomedical implants often exhibit poor clinical performance due to the formation of a periimplant avascular fibrous capsule. Surface modification of synthetic materials has been evaluated to accelerate the formation of functional microcirculation in association with implants. The current study used a flow-mediated protein deposition system to modify expanded polytetrafluoroethylene (ePTFE) with a laminin-5-rich conditioned growth medium and with medium from which laminin-5 had been selectively removed. An in vitro model of endothelial cell adherence determined that laminin-5 modification resulted in significantly increased adhesion of human microvessel endothelial cells to ePTFE. In vivo studies evaluating the periimplant vascular response to laminin-5-treated samples indicated that absorption of laminin-5-rich conditioned medium supported accelerated neovascularization of ePTFE implants. A flow system designed to treat porous implant materials facilitates laminin-5 modification of commercially available ePTFE, resulting in increased endothelial cell adhesion in vitro and increased vascularization in vivo.

The purpose of this study is to evaluate the interactions of rat parotid acinar cells on biomaterials with different surface properties. The biomaterials used in this study included polyvinyl alcohol (PVA), chitosan, poly (ethylene-co-vinyl alcohol) (EVAL), and polyvinylidene fluoride (PVDF). Cell morphology was observed by photomicroscope. Cell growth and differentiated characteristic function were separately assayed with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) reduction activity and amylase activity. Results indicated that behaviors of acinar cells on materials might differ to a great extent depending on the surface hydrophilicity and morphology of the materials. On the relatively hydrophobic materials, the abilities of acinar cells to adhere and proliferate increased simultaneously. In addition, porous PVDF had higher cell growth compared with dense PVDF. Therefore, the hydrophobic PVDF with a porous structure was the best substrate for culturing acinar cells. According to our findings, a tubular PVDF scaffold with dense outer surface to prevent saliva leakage and with porous inner surface for the cell growth was proposed to serve as an artificial salivary gland for future use in the treatment of patients with salivary hypofunction.

The performance of biomedical implant devices is often limited by inappropriate tissue responses associated with synthetic materials used in device construction. Adverse healing responses, in particular the lack of an extensive vascular supply in the peri-implant tissue, are believed to lead to the ultimate failure of many of these medical devices. Accelerated formation of new blood vessels in the peri-implant tissue and within porous polymeric implants is hypothesized to improve the performance of such biomedical implant devices. The current study evaluated the use of cell-mediated, extracellular matrix modification of expanded polytetrafluoroethylene (ePTFE) to increase vessel growth in peri-implant tissue and within the pores of the implants. Discs of ePTFE were modified through cell-mediated matrix deposition using epithelial and endothelial cell lines with variable deposition of collagen types, fibronectin, and laminin types. Cell matrix-modified discs, Matrigel-coated discs, and nonmodified discs were implanted in both the adipose and subcutaneous tissues of the rat. Following a 5-week implant period, samples were removed and evaluated histologically and morphometrically for the presence of blood vessels in the peri-implant tissue and within the pores of the polymer as well as for the presence of activated macrophages and monocytes. A significantly increased presence of activated macrophages and monocytes was associated only with the samples modified with the matrix from a human microvessel endothelial cell line. Increased vessel density was identified in association with those ePTFE samples modified with either the 804-G, HaCaT, or II-4 cell matrices, all of which have extracellular matrices enriched in the protein laminin-5.

Relationships between cell-cell/cell-matrix interactions and enamel organ histomorphogenesis were examined by immunostaining and electron microscopy. During the cap-bell transition in the mouse molar, laminin-5 (LN5) disappeared from the basement membrane (BM) associated with the inner dental epithelium (IDE), and nondividing IDE cells from the enamel knot (EK) underwent a tooth-specific segregation in as many subpopulations as cusps develop. In the incisor, the basement membrane (BM) in contact with EK cells showed strong staining for LN5 and integrin alpha 6 beta 4. LN5 seems to provide stable adhesion, while its proteolytic processing might facilitate cell segregation. In both teeth, immunostaining for antigens associated with desmosomes or adherens junctions was similar for EK cells and neighboring IDE cells. Outside the EK, IDE cell-BM interactions changed locally during the initial molar cusp delimitation and on the labial part of the incisor cervical loop. Conversely, cell-cell junctions stabilized the anterior part of the incisor during completion of morphogenesis. Time and space regulation of cell-matrix and cell-cell interactions might thus play complementary roles in allowing plasticity during tooth morphogenesis and stabilization at later stages of epithelial histogenesis.

The laminin family of extracellular matrix (ECM) proteins plays crucial roles in regulating cellular growth, migration, and differentiation. We report here that laminin-5 is expressed in the tunica media of the rat aorta and pulmonary arteries. Using indirect immunofluorescence microscopy, Western blots, and RT-PCR analysis, we found that primary cultures of rat arterial smooth muscle cells express laminin-5 and deposit it into their insoluble ECM. These cells also attach strongly to laminin-5 via beta1 integrin receptors in 30 min adhesion assays. Laminin-5 expression in these cells is upregulated by growth factors in vitro and platelet-derived growth factor (PDGF-BB) stimulation reduces adhesion to laminin-5. As laminin-5 promotes enhanced migration of other cell types, the production of and adhesion to laminin-5 by vascular smooth muscle cells may play a role in the pathological growth and migration of these cells associated with restenosis following vascular injury.

Wounding of the epidermis signals the transition of keratinocytes from quiescent anchorage on endogenous basement membrane laminin 5 to migration on exposed dermal collagen. In this study, we attempt to characterize activation signals that transform quiescent keratinocytes into migratory leading cells at the wound edge. Previously, we reported that adhesion and spreading on collagen via integrin alpha(2)beta(1) by cultured human foreskin keratinocytes (HFKs) requires RhoGTP, a regulator of actin stress fibers. In contrast, adhesion and spreading on laminin 5 requires integrins alpha(3)beta(1) and alpha(6)beta(4) and is dependent on phosphoinositide 3-hydroxykinase (Nguyen, B. P., Gil, S. G., and Carter, W. G. (2000) J. Biol. Chem. 275, 31896-31907). Here, we report that quiescent HFKs do not adhere to collagen but adhere and spread on laminin 5. By using collagen adhesion as one criterion for conversion to a "leading wound cell," we found that activation of collagen adhesion requires elevation of RhoGTP. Adhesion of quiescent HFKs to laminin 5 via integrin alpha(3)beta(1) and alpha(6)beta(4) is sufficient to increase levels of RhoGTP required for adhesion and spreading on collagen. Consistently, adhesion of quiescent HFKs to laminin 5, but not collagen, also promotes expression of the precursor form of laminin 5, a characteristic of leading keratinocytes in the epidermal outgrowth. We suggest that wounding of quiescent epidermis initiates adhesion and spreading of keratinocytes at the wound edge on endogenous basement membrane laminin 5 via alpha(3)beta(1) and alpha(6)beta(4) in a Rho-independent mechanism. Spreading on endogenous laminin 5 via alpha(3)beta(1) is necessary but not sufficient to elevate expression of precursor laminin 5 and RhoGTP, allowing for subsequent collagen adhesion via alpha(2)beta(1), all characteristics of leading keratinocytes in the epidermal outgrowth.

ECV304 cells reported as originating from human umbilical vein endothelial cells by spontaneous transformation have been used as a model cell line for endothelia over the last decade. Recently, deoxyribonucleic acid fingerprinting revealed an identical genotype for ECV304 and T24 cells (urinary bladder carcinoma cell line). In order to resolve the apparent discrepancy between the identical genotype and the fact that ECV304 cells phenotypically show important endothelial characteristics, a comparative study was performed. Immortalized porcine brain microvascular endothelial cells/C1-2, and Madin Darby canine kidney cells were included as typical endothelial and epithelial cells, respectively. Various methods, such as confocal laser scanning microscopy. Western blot, and protein activity tests, were used to study the cell lines. ECV304 and T24 cells differ in criteria, such as growth behavior, cytoarchitecture, tight junction arrangement. transmembrane electrical resistance, and activity of gamma-glutamyltransferase. Several endothelial markers (von Willebrand factor, uptake of low-density lipoprotein, vimentin) could clearly be identified in ECV304, but not in T24 cells. Desmoglein and cytokeratin, both known as epithelial markers, were found in ECV304 as well as in T24 tells. However, differences were found for the two cell lines with respect to the type of cytokeratin: in ECV304 cells mainly cytokeratin 18 (45 kDa) is found, whereas in T24 cells cytokeratin 8 (52 kDa) is predominant. As we could demonstrate, the ECV304 cell line exposes many endothelial features which, in view of the scarcity of suitable endothelial cell lines, still make it an attractive in vitro model for endothelia.

Laminin-5 (LN-5), an important basement membrane (BM) protein consisting of laminin alpha3, beta3 and gamma 2 chains, has been suggested to be involved in tumor cell invasion and tissue repair. In this study, the distribution of the LN-5 subunits in atypical adenomatous hyperplasia (AAH) and different types of adenocarcinomas of the lung was examined by immunohistochemical analysis. In AAH and non-sclerosing, well-differentiated adenocarcinomas, the LN gamma 2 chain was frequently detected along with the continuous BMs. These BMs were also positive for both LN alpha3 and beta3 chains, suggesting that LN-5 had been deposited. In contrast, the cytoplasmic staining for the LN gamma 2 chain was frequently observed in tumor cells of sclerosing, well-differentiated adenocarcinomas, as well as of moderately and poorly differentiated adenocarcinomas, without any evidence of co-expression of the LN alpha3 and beta3 chains. This staining pattern of the LN gamma 2 chain was prominent in carcinoma cells invading into interstitial stroma and was associated with the formation of a central scar in the tumor tissues. These results suggest that the LN gamma 2 chain monomer could be an important indicator of progression of lung adenocarcinoma.

During tissue morphogenesis and tumor invasion, epithelial cells must undergo intercellular rearrangement in which cells are repositioned with respect to one another and the surrounding mesenchymal extracellular matrix. Using three-dimensional aggregates of squamous epithelial cells, we show that such intercellular rearrangements can be triggered by activation of beta1 integrins after their ligation with extracellular matrices. On nonadherent substrates, multicellular aggregates (MCAs) formed rapidly via E-cadherin junctional complexes and over time became compacted spheroids exhibiting a more epithelial phenotype. After MCAs were replated on culture substrates, the spheroids collapsed to yield tightly arranged cell monolayers. Cell-cell contact induced rapid elevation in E-cadherin levels, which was due to an increase in the metabolic stability of junctional receptors. During MCA remodeling of cell-cell adhesions, and monolayer formation, their E-cadherin levels fell rapidly. Similar behavior was obtained regardless of which ECM ligand-collagen type I, fibronectin, or laminin 1-MCAs were seeded on. In contrast, when seeded onto a matrix elaborated by squamous epithelial cells, cells in the MCA attached, spread, lost cell-cell junctions, and dispersed. Analysis identified laminin 5 as the active ECM ligand in this matrix, and MCA dispersion required functional beta1 integrin and specifically alpha3beta1. Furthermore, substrate-immobilized anti-integrin antibody effectively reproduced the epithelial-mesenchymal-like transition induced by the laminin 5 matrix. During the early stages of aggregate rearrangement and collapse, cells on laminin 5 substrates, but not those on collagen I substrates, exhibited intense cortical arrays of F-actin, microspikes, and fascin accumulation at their peripheral surfaces. These results suggest that engagement of specific integrin-ligand pairs regulates cadherin junctional adhesions during events common to epithelial morphogenesis and tumor invasion.

The laminins form a large family of modular proteins found in basement membranes, but also elsewhere. They function as structural components and are essential for morphogenesis, but in addition interact with cell surface receptors such as integrins and alpha-dystroglycan. By virtue of their receptor interactions, they initiate intracellular signalling events that regulate cellular organization and differentiation. The many interactions of laminins are mediated by binding sites, often contributed by single domains, which may differ between different forms of laminin. In the present article, we describe how the diversity of laminins and the genetic regulation of the expression of different laminin forms lead to the formation of extracellular matrices with variable laminin composition and thereby different biological properties.

Laminin 5 (alpha3beta3gamma2) distribution in the human thymus was investigated by immunofluorescence on frozen sections with anti-alpha3, -beta3, and -gamma2 mAbs. In addition to a linear staining of subcapsular basal laminae, the three mAbs give a disperse staining in the parenchyma restricted to the medullary area on a subset of stellate epithelial cells and vessel structures. We also found that laminin 5 may influence mature human thymocyte expansion; while bulk laminin and laminin 2, when cross-linked, are comitogenic with a TCR signal, cross-linked laminin 5 has no effect. By contrast, soluble laminin 5 inhibits thymocyte proliferation induced by a TCR signal. This is accompanied by a particular pattern of inhibition of early tyrosine kinases, including Zap 70 and p59(fyn) inhibition, but not overall inhibition of p56(lck). Using a mAb specific for alpha6beta4 integrins, we observed that while alpha3beta1 are known to be uniformly present on all thymocytes, alpha6beta4 expression parallels thymocyte maturation; thus a correspondence exists between laminin 5 in the thymic medulla and alpha6beta4 on mature thymocytes. Moreover, the soluble Ab against alpha6beta4 inhibits thymocyte proliferation and reproduces the same pattern of tyrosine kinase phosphorylation suggesting that alpha6beta4 is involved in laminin 5-induced modulation of T cell activation.

Hemidesmosomes are cell-substratum adhesion sites that connect the extracellular matrix to the keratin cytoskeleton. Our knowledge of the function of these structures has greatly increased as a result of studies on patients with aberrant expression of hemidesmosome components and studies using targeted inactivation of mouse genes encoding these components. Insight into the formation of hemidesmosomes, as well as into protein-protein interactions that occur in these junctional complexes, has recently been gained by in vitro cell transfections, blot overlay and yeast two-hybrid assays. In addition, recent results indicate that the alpha6 beta4 integrin is involved in the transduction of signals that are induced by the extracellular matrix and which modulate processes as diverse as cell proliferation, differentiation, apoptosis, migration and tissue morphogenesis. Thus it seems that hemidesmosomes do not merely maintain dermo-epidermal adhesion and tissue integrity, but that they are also implicated in intracellular signaling. Here we discuss recently published data on the biology and function of hemidesmosomes.

In the mature gut, laminin-5 is expressed at the basal aspect of the differentiating epithelial cells. In vitro, we show that three more or less differentiated human colonic cancer HT29 cell lines produce and deposit laminin-5; they predominantly synthesize and secrete the 440 kDa form of laminin-5 that comprises the unprocessed 155 kDa gamma2 chain, as determined by immunoprecipitation analysis. In contrast, the highly differentiated colon carcinoma Caco-2 cells produce almost no laminin-5. Using anti-integrin antibodies, we show that adhesion of the two colonic cancer cell lines to laminin-5 is mediated by multiple integrin receptors including those for alpha3beta1, alpha6beta1 and alpha6beta4 integrins like in other cell types. In addition, the implication of integrin alpha2beta1 in this adhesion process is demonstrated for the first time. This has been shown by cell adhesion inhibition experiments, solid phase assays and confocal analysis. Together with previous in situ observations, these data provide a baseline knowledge for the understanding of the regulation of laminin-5 in normal and pathological intestine.

A panel of human tumor cell lines was screened for selective expression of laminin alpha5 chain, a newly identified laminin subunit comprising laminin-10 (alpha5 beta1 gamma1) and -11 (alpha5 beta2 gamma1). The lung adenocarcinoma cell line A549 was found to express the alpha5 chain at relatively high levels but no detectable amounts of other alpha chains. The laminin variants containing alpha5 chain were purified from the conditioned medium of A549 cells by immunoaffinity chromatography using the anti-laminin monoclonal antibody 4C7 which was shown recently to recognize the laminin alpha5 chain (Tiger, C.-F., Champliaud, M.-F., Pedrosa-Domellof, F., Thornell, L.-E., Ekblom, P., and Gullberg, D. (1997) J. Biol. Chem. 272, 28590-28595). The purified laminin variants consisted of three chains with molecular masses of 350, 220, and 210 kDa. The 350-kDa chain was specifically recognized by another anti-alpha5 chain monoclonal antibody capable of recognizing denatured alpha5 chain on immunoblots, whereas the 210-kDa chain was recognized by an anti-gamma1 chain antibody. The purified alpha5 chain-containing laminin variants (hereafter referred to as laminin-10/11) were highly active in mediating adhesion of A549 cells to the substratum with potency as high as that of laminin-5 and significantly higher than those of laminin-1, laminin-2/4, or fibronectin. Adhesion to substrata coated with laminin-10/11 was specifically inhibited by anti-integrin antibodies directed against the integrin alpha3 or beta1 subunit but not by those against alpha2 or alpha6 subunit, indicating that laminin-10/11 is specifically recognized by integrin alpha3 beta1. Given the wide distribution of laminin-10/11 in the basement membrane of various tissue types and dominant expression of integrin alpha3 beta1 in most epithelial cells, specific interaction of laminin-10/11 with integrin alpha3 beta1 may play an important role in in vivo regulation of proliferation and differentiation of epithelial cells through the basement membrane.

The generation and maintenance of cell polarity in endothelial cells is poorly understood, partly because of a lack of a permanent endothelial in vitro model system. Here we evaluated the spontaneously immortalized human endothelial-derived cell line ECV304 as an in vitro model system for the study of the polarized expression of heterologous membrane proteins. Several stable ECV304 clones were generated by calcium phosphate transfection/G418 selection with cDNAs encoding membrane proteins of known cell surface distribution in the epithelial Madin Darby canine kidney (MDCK) cell line: influenza hemagglutinin and uvomorulin/E-cadherin were used as markers for the apical, respectively lateral cell membrane, the human lymphocyte surface marker CD7 served as an example of a circumferentially distributed membrane protein. Analysis of the transfected ECV304 clones using conventional and confocal immunofluorescence microscopy and immunoelectron microscopy revealed the same membrane distribution of the heterologous proteins in ECV304 cells as in MDCK cells. This polarized expression of heterologous membrane proteins in the endothelial-derived ECV304 cell line indicates efficient protein sorting/membrane trafficking mechanisms. The apical, lateral and basal cell membrane domains could be distinguished in ECV304 cells by confocal immunofluorescence microscopy. The permanent endothelial-derived ECV304 cell line may be a useful in vitro model system for the study of endothelial cell polarity.

Matrilysin is believed to play important roles in tumor progression and metastasis. In the present study, we analyzed matrilysin-producing cells in various human cancer tissues by immunohistochemistry and in situ hybridization. Tumor cells in colorectal carcinomas, pancreatic carcinomas, transitional-cell carcinomas of the kidney and small-cell lung carcinomas were frequently positive for matrilysin. In addition, we found that endothelial cells of arterioles and venules adjacent to matrilysin-positive tumors expressed matrilysin mRNA and protein. The endothelial cells adjacent to matrilysin-negative tumors and those in normal tissues were negative for matrilysin. Furthermore, analyses by casein zymography, Western blotting and RT-PCR showed that matrilysin was weakly expressed by cultured human umbilical vein endothelial cells. Our results suggest that the expression of matrilysin in vascular endothelial cells and in tumor cells may be regulated by common soluble factors, and that endothelial cell-derived matrilysin may contribute to tumor angiogenesis and tumor metastasis.

Proteinases produced by vascular endothelial cells are expected to play important roles in many biological processes. Here we report that human vascular endothelial cells express trypsinogen-2 mRNA and its protein product in culture. The trypsinogen production was stimulated by a tumor promoter and associated with cell growth. In situ hybridization analysis showed that the trypsinogen gene was significantly expressed in vascular endothelial cells around gastric tumors and in patients with disseminated intravascular coagulation (DIC). These results suggest the possible roles of endothelial cell-derived trypsin in tumor angiogenesis and abnormal blood coagulation.