Deposition Of Basement Membrane Components Research Articles

Pentastatin, a matrikine of the Collagen IVa5, is a novel endogenous mediator of pulmonary endothelial dysfunction.

Deposition of basement membrane components, such as collagen IVα5, is associated with altered endothelial cell function in pulmonary hypertension. Collagen IVα5 harbors a functionally active fragment within its C-terminal non-collageneous (NC1) domain, called pentastatin, whose role in pulmonary endothelial cell behavior remains unknown. Here, we demonstrate that pentastatin serves as a mediator of pulmonary endothelial cell dysfunction, contributing to pulmonary hypertension. In vitro, treatment with pentastatin induced transcription of immediate early genes and pro-inflammatory cytokines and led to a functional loss of endothelial barrier integrity in pulmonary arterial endothelial cells. Mechanistically, pentastatin leads to β1-integrin subunit clustering and Rho/ROCK activation. Blockage of the β1-integrin subunit or the Rho/ROCK pathway partially attenuated the pentastatin-induced endothelial barrier disruption. While pentastatin reduced the viability of endothelial cells, smooth muscle cell proliferation was induced. These effects on the pulmonary vascular cells were recapitulated ex vivo in the isolated-perfused lung model, where treatment with pentastatin induced swelling of the endothelium accompanied by occasional endothelial cell apoptosis. This was reflected by increased vascular permeability and elevated pulmonary arterial pressure induced by pentastatin. This study identifies pentastatin as a mediator of endothelial cell dysfunction, which thus might contribute to the pathogenesis of pulmonary vascular disorders such as pulmonary hypertension.

Characterization of GB73, a new gene involved in the polarized deposition of basement membrane components

Characterization of GB73, a new gene involved in the polarized deposition of basement membrane components

Intestinal epithelial wound healing assay in an epithelial–mesenchymal co-culture system

Rapid and efficient healing of epithelial damage is critical to the functional integrity of the small intestine. Epithelial repair is a complex process that has largely been studied in cultured epithelium but to a much lesser extent in mucosa. We describe a novel method for the study of wound healing using a co-culture system that combined an intestinal epithelial Caco-2/15 cell monolayer cultured on top of human intestinal myofibroblasts, which together formed a basement membrane-like structure that contained many of the major components found at the epithelial-mesenchymal interface in the human intestine. To investigate the mechanism of restitution, small lesions were generated in epithelial cell monolayers on plastic or in co-cultures without disturbing the underlying mesenchymal layer. Monitoring of wound healing showed that repair was more efficient in Caco-2/15-myofibroblast co-cultures than in Caco-2/15 monolayers and involved the deposition of basement membrane components. Functional experiments showed that the addition of type I collagen or human fibronectin to the culture medium significantly accelerated wound closure on epithelial cell co-cultures. This system may provide a new tool to investigate the mechanisms that regulate wound healing in the intestinal epithelium.

Diffusible factors released by fibroblasts support epidermal morphogenesis and deposition of basement membrane components.

Epithelial-mesenchymal interactions play an important role in controlling epidermal morphogenesis and homeostasis but little is known about the mechanisms of these interactions. To examine whether diffusible factors produced by fibroblasts and/or keratinocytes support epidermal morphogenesis and basement membrane formation, organotypic keratinocyte monocultures were established in media collected either from organotypic fibroblast or keratinocyte-monocultures or from keratinocyte-fibroblast cocultures, and the expression of keratin 10, 16, and 17 and basement membrane components (types IV and VII collagen, laminin 5, nidogen, BP 180, LAD-1) were examined. We found that diffusible factors released by keratinocytes were not sufficient to support the establishment of normalized epidermal phenotype and deposition of basement membrane components in contrast to fibroblast- or keratinocyte/fibroblast-derived factors. Keratinocytes appear to affect the spectrum of secreted soluble factors, as keratinocyte/fibroblast-derived factors were more effective to accomplish continuous linear deposition of laminin 5 and of nidogen. The finding that released amounts of keratinocyte growth factor and granulocyte macrophage colony stimulating factor were not sufficient to fully support epidermal morphogenesis and deposition of basement membrane components is suggestive for the involvement of other released diffusible factors. Generation of organotypic keratinocyte monocultures in the presence of fibroblast- or keratinocyte/fibroblast-derived soluble factors resulted in enhanced expression of keratins K16 and K17 and the absence of type IV collagen. This observation indicates that next to paracrine acting factors, epidermal homeostasis is controlled by mutual keratinocyte-fibroblast interaction.

Basement membrane proteins and apolipoprotein E in growth hormone secreting adenomas and their correlation to amyloid: an immunoelectron microscopic study.

Having previously demonstrated the association of basement membrane components (BMC), as well as apolipoprotein E and amyloid in growth hormone (GH)-producing pituitary adenomas, the aim of this study was to further analyze this relationship at the ultrastructural level. Ultrathin sections from four amyloidotic sparsely granulated monohormonal GH-producing adenomas previously investigated light microscopically were selected. Immunoelectron microscopy was performed, using a single labeling postembedding on-grid Protein-A gold method with antisera directed against laminin, fibronectin and apolipoprotein E. In all four adenomas, anti-laminin, anti-fibronectin, and anti-apolipoprotein E reacted with amyloid fibrils. No BMC were demonstrated between amyloid deposits, making it likely that synthesis and deposition of BMC may be secondary to the deposition of amyloid. The intimate spatial relationship between BMC as well as apolipoprotein E and amyloid fibrils may indicate morphological evidence of a particular arrangement of amyloid components in GH-secreting adenoma amyloid and a pathophysiological link.

Chromogranin A Alters Ductal Morphogenesis and Increases Deposition of Basement Membrane Components by Mammary Epithelial Cells in Vitro

The extracellular function of chromogranin A (CgA), a glycoprotein widely distributed in secretory vesicles of neurons and neuroendocrine cells, has not been clearly established. To examine whether CgA might modulate the biological properties of epithelial cells, we used an in vitro model of ductal morphogenesis in which mammary epithelial (TAC-2) cells are grown in three-dimensional collagen gels. Whereas under control conditions TAC-2 cells formed thin, branched cords with pointed ends, in the presence of CgA they formed thicker cords with bulbous extremities, reminiscent of growing mammary ducts in vivo. Immunofluorescence analysis demonstrated that CgA increases the deposition of three major basement membrane components, i.e., collagen type IV, laminin, and perlecan, around the surface of the duct-like structures. Similar effects were observed with CgA partially digested with endoproteinase Lys-C, suggesting that one or more fragments of CgA are endowed with the same activity. These findings reveal a hitherto unsuspected activity for CgA, i.e., the ability to alter ductal morphogenesis and to promote basement membrane deposition in mammary epithelial cells.

Pretelangiectasis and telangiectasis of the bovine liver: a morphological, immunohistochemical and ultrastructural study

Forty-five livers from conventionally slaughtered Holstein-Friesian steers with telangiectasis were studied by histochemical methods, immunolabelling for fibronectin, laminin and type IV collagen, and transmission electron microscopy. None of the previously described changes in telangiectasis (necrosis, hepatitis, thromboembolism, dilatation of the space of Disse by glycogen extruded from hepatocytes and reduced density of the perisinusoidal reticulin framework) were evident. Pretelangiectasis (sinusoidal dilatation) and telangiectasis (blood-filled cavities) were characterized by sinusoidal barrier alterations, leading to sinusoidal capillarization; and there was progressive formation of a true basement membrane and perisinusoidal fibrosis. Comparison of bovine liver telangiectasis and human peliosis hepatis suggests that they have a similar pathogenesis. It is suggested that a primary alteration of the sinusoidal barrier is responsible for an increased deposition of basement membrane components (fibronectin, laminin, type IV collagen) in the perisinusoidal region, and fibrosis. These are likely to render the exchange of oxygen and substrates between blood and hepatocytes more difficult and to produce haemodynamic imbalances, leading to hepatocyte atrophy and eventually to sinusoidal disruption.

Establishment of oral mucosa phenotype in vitro in correlation to epithelial anchorage.

Cell-matrix interactions and the ordered deposition of basement membrane (BM) components are of major importance for the maintenance of tissue homeostasis in complex epithelia. This aspect was studied in vitro in a coculture system designed as an oral mucosa model. As crucial epithelial features the kinetics of proliferation, expression of site-specific keratins as well as integrin patterns in correlation to synthesis of BM components were assessed by immunohistochemistry and in situ hybridization. Comparison with non-cornified gingiva as tissue of origin revealed different stages of epithelial development, eventually leading to complete reconstruction within a time frame of 1-3 weeks. First, the initial activated stage up to 1 week was characterized by (a) high keratinocyte proliferation, (b) extended expression of the basal cell-specific keratin K5 and (c) a patchy pattern of the differentiation-specific keratins K4 and K13. Second, after 2 weeks the improvement of histoarchitecture correlated to (a) predominant K5 expression in the basal and (b) extension of K4 and K13 within the suprabasal cell compartment, (c) high expression of integrins alpha3 beta1 and alpha6 beta4 including their ligand laminin-5 and (d) accumulating deposition of basement membrane components. Third, virtually complete tissue normalization at 3 weeks was indicated by (a) restriction of K5 to the basal cell area, (b) regular suprabasal localization of K4 and K13, (c) polarization of integrins to basal and parabasal cells and (d) linear codistribution of collagen IV, "classical" laminin (-1 or -10) and laminin-5 underneath the basal cells. Thus, these organotypic cocultures represent relevant equivalents for non-keratinized oral mucosa with typical gingival differentiation features and in addition suitable models for preclinical trials such as prospective dental material testing.

Immunohistochemical Localization of Laminin, Collagen Type IV and Heparan Sulfate Proteoglycan in Human Colorectal Adenocarcinoma: Correlation with Local Invasive Pattern and Lymph Node Metastasis.

The extent of immunohistochemical localization of such basement membrane components (BMs) as laminin (LN), collagen type IV (C-IV) and heparan sulfate proteoglycan (HSPG) were investigated in 78 samples of human colorectal carcinoma, in correlation with histology of the invasive pattern at the parenchymal-stromal border of the invasive margin (PSB) and lymph node metastasis. The immunostaining ratio of BMs at the PSB was significantly lower in the type IV invasive pattern (PSB-IV) than in the other types, and was also significantly related to the incidence of lymphatic invasion and lymph node metastasis. A close relationship was also demonstrated between the PSB-IV and the prognosis of the patient or recurrence of the carcinoma, although there was no statistical significance. Furthermore, the immunostaining ratios of the IN and C-IV in the central areas (CAs) of the carcinoma tissue were also significantly lower in samples with lymph node metastasis than those without metastatic focus. These results thus indicate that the lower degree of immunostaining ratio of BMs at the PSB in human colorectal carcinomas is closely related to both the lymph node metastasis and the patient prognosis, while in addition the varied localization of BMs at the PSB affects diverse histopathological features in the invasive margin of carcinoma tissue. Moreover, the decreased immunostaining deposition of BMs in the CAs of carcinoma tissue also plays an important role in the pathway of lymph node metastasis. Therefore, further attention should be paid to the cellular biology of carcinoma cells localized in the CA, as well as those localized at the PSB, in order to disclose the metastatic cascade of colorectal carcinoma.

Histomorphological and biochemical differentiation capacity in organotypic co-cultures of primary gingival cells.

To establish a three-dimensional in vitro test system mimicking the physiological situation of the oral cavity, organotypic co-cultures consisting of primary gingival cells on a collagen matrix with fibroblasts were generated. The histomorphological development after 7 and 14 d revealed close similarity with the non-keratinized gingiva epithelium. Furthermore, as epithelial specific markers synthesis and localization of keratins as well as the deposition of basement membrane components were assessed on frozen sections by immunofluorescence and keratin expression by in situ hybridization. Primary keratinocytes in conventional culture strained positive for keratin K14 and the mucosal differentiation-specific keratins K4 and K13, while primary fibroblasts, isolated from the same tissue source, and also some keratinocytes, were positive for vimentin. In organotypic co-cultures the keratinocytes formed a multilayered epithelium within 14 d containing basal cells and flattened cells in the uppermost layers. Comparable to native non-keratinized gingiva keratin 14 gene expression was clearly detectable in the basal cell compartment but showed extending immunolocalization. In addition, particularly at the early stage (7 d), basally located keratinocytes were also vimentin positive. According to morphological differentiation K4 and K13 were detectable in suprabasal position a the RNA and protein level. The major basement membrane constituents collagen type IV and laminin increased with time revealing first an interrupted and later a fully extended staining underneath the basal cells. Maintenance of basal cell function was further demonstrated by cell proliferation (BrdU incorporation) which was initially high (7 d) but declined towards the later stages (14-21 d). The results demonstrate i) that this co-culture system leads to a stratified surface epithelium with morphological and biochemical characteristics of the non-keratinized gingiva epithelium and ii) that a state of physiological tissue balance was reached, thus rendering a suitable model for tissue compatibility studies.

Characterization of a new in vitro model for studies of reepithelialization in human partial thickness wounds.

Reepithelialization of artificial partial thickness wounds made in biopsies of human skin was determined after 3, 5, or 7 d of incubation, submerged or elevated to the air-liquid interface. The biopsies were reepithelialized within 5-7 d, with a more complete epidermal healing in wounds exposed to air. Both types of wounds showed similar time-course in deposition of basement membrane components, as detected by immunofluorescence labeling. Laminin and collagen type VII were deposited underneath the migrating tips, whereas collagen type IV was detected after reepithelialization. Markers of terminal differentiation showed a pattern close to normal in the air-liquid incubated wounds after reepithelialization. Involucrin was detected in the suprabasal regions of the migrating epidermis and thereafter in the upper half of neo-epidermis in the air-liquid incubated wound. Filaggrin could not be detected in the submerged wounds at any time during healing, whereas wounds exposed to air showed a well-differentiated epidermis by Day 7. Tritiated thymidine-incorporation indicated proliferation of epidermal and dermal cells during reepithelialization and a maintained viability, as shown by cultivation of endothelial- and fibroblast-like cells obtained from the dermis 7 d after wounding. Reepithelialization in this human in vitro model is supported by a matrix close to normal with the possibility of extracellular influences and cell-cell interactions and, in addition, the technique is simple and reproducible. Therefore, we suggest this model for studies of regeneration in culture and as a complement to in vivo studies on epidermal healing.

A novel function for the nm23-H1 gene: overexpression in human breast carcinoma cells leads to the formation of basement membrane and growth arrest.

We have developed a culture system using reconstituted basement membrane components in which normal human mammary epithelial cells exhibit several aspects of the development and differentiation process, including formation of acinar-like structures, production and basal deposition of basement membrane components, and production and apical secretion of sialomucins. Cell lines and cultures from human breast carcinomas failed to recapitulate this process. The data indicate the importance of cellular interactions with the basement membrane in the regulation of normal breast differentiation and, potentially, its loss in neoplasia. Our purpose was to use this assay to investigate the role of the putative metastasis suppressor gene nm23-H1 in mammary development and differentiation. The metastatic human breast carcinoma cell line MDA-MB-435, clones transfected with a control pCMVBamneo vector, and clones transfected with pCMVBamneo vector containing nm23-H1 complementary DNA (the latter of which exhibited a substantial reduction in spontaneous metastatic potential in vivo) were cultured within a reconstituted basement membrane. Clones were examined for formation of acinus-like spheres, deposition of basement membrane components, production of sialomucin, polarization, and growth arrest. In contrast to the parental cell line and control transfectants, MDA-MB-435 breast carcinoma cells overexpressing Nm23-H1 protein regained several aspects of the normal phenotype within reconstituted basement membrane. Nm23-H1 protein-positive cells formed organized acinus-like spheres, deposited the basement membrane components type IV collagen and, to some extent, laminin to the outside of the spheres, expressed sialomucin, and growth arrested. Growth arrest of Nm23-H1 protein-positive cells was preceded by and correlated with formation of a basement membrane, suggesting a causal relationship. The data indicate a previously unidentified cause-and-effect relationship between nm23-H1 gene expression and morphological-biosynthetic-growth aspects of breast differentiation in this model system. While the basement membrane microenvironment is capable of directing the differentiation of normal human breast cells, neoplastic transformation abrogates this relationship, suggesting that intrinsic cellular events are also critical to this process. The data identify nm23-H1 gene expression as one of these events, suggesting an important role in the modulation of cellular responsiveness to the microenvironment. The data also identify previously unknown growth inhibitory effects of nm23-H1 gene overexpression.

Experimental Modulation of the Differentiated Phenotype of Keratinocytes from Epidermis and Hair Follicle Outer Root Sheath and Matrix Cells

Follicles of human anagen hair were separated into morphologically distinct compartments (by sequential trypsinization and microdissection) for the biochemical and immunological analysis of keratins as differentiation markers to diagnose the type of epithelial differentiation. While outer root sheath contained throughout the "soft" (cyto)keratins K5, 6, 14, 16, and 17, and hair cortex contained exclusively a set of acidic and basic "hard" alpha-keratins (consistent up to the hair tip), in inner root sheath and hair cuticle peptides related or derived from suprabasal epidermal keratins K1 and 10 were detected. These keratin profiles served as in vivo correlates for the evaluation of type and degree of differentiation achieved by the respective isolated epithelial cells, comparing different growth or culture conditions. Cultures of ORS cells and hair matrix cells (PHS cells) as well as normal keratinocytes were initiated using postmitotic human dermal fibroblasts as efficient feeder cells. On lifted collagen gels populated with HDF ("surface" cultures), ORS and PHS cells formed stratified epithelial expressing epidermal differentiation markers such as keratins K1 and 10, involucrin, and filaggrin. Compared with NEK "surface" cultures, balance between growth and differentiation was better maintained by both follicular cell types. In contrast, epidermal tissue homeostasis was largely normalized in transplants on nude mice regardless of the epithelial cell type, apparent from orderly tissue structure, regular distribution of keratin K10, filaggrin, and involucrin, and distinct continuous deposition of basement membrane components at the epithelium-collagen interface. Embedded in Matrigel (on top of HDF collagen gels) ORS cells and NEK formed spheroids exhibiting inward-directed epidermoid differentiation, increasing with time. All epidermal maturation products found in "surface" cultures were likewise expressed, and again differentiation greatly outbalanced proliferation in spheroids of NEK but not of ORS cells. PHS cells embedded together with HDF in Matrigel produced similar spheroids as ORS cells. Size of spheroids and degree of epidermoid differentiation were dramatically reduced when HDF were replaced by follicular DP cells, demonstrating the crucial role of the mesenchymal "companion" cells.(ABSTRACT TRUNCATED AT 400 WORDS)

Fibroblasts Induce the Assembly of the Macromolecules of the Basement Membrane

The mechanism regulating the deposition of basement membrane components (BMCs) in a polymeric structure at the junction with the connective tissues is not yet understood. Cultures and cocultures of epithelial BMC-producing cells (L2 or PER cells) and fibroblasts were prepared in several experimental conditions and the organization of BMCs was studied by immunofluorescence. The pattern of BMCs in pure cultures of L2 or pulmonary epithelial rat (PER) cells consisted of intra- and extracellular granular deposits. At very high density, the cell contours were also underlined by a disrupted network of BMC deposits. A different fibrillar plexus--containing laminin, collagen type IV, and heparan-sulfate proteoglycan resistant to deoxycholate treatment and distant from the cell membrane--was observed in cocultures of L2 or PER cells with fibroblasts. Fibrils of fibronectin and/or collagen type I were most often dissociated from this plexus of BMCs. Similar results were obtained by adding a conditioned medium of L2 or PER cells to confluent fibroblasts, even when the cells were killed. Pure laminin also bound to the fibroblast layer. A coated film of fibronectin or polymeric collagen type I was unable to bind BMC provided by a conditioned medium. It is suggested that molecule(s) synthesized by fibroblasts and deposited in the pericellular matrix are involved in the assembly of BMCs.

Differential deposition of basement membrane components during formation of the caudal neural tube in the mouse embryo.

The distribution of basement membrane and extracellular matrix components laminin, fibronectin, type IV collagen and heparan sulphate proteoglycan was examined during posterior neuropore closure and secondary neurulation in the mouse embryo. During posterior neuropore closure, these components were densely deposited in basement membranes of neuroepithelium, blood vessels, gut and notochord; although deposition was sparse in the midline of the regressing primitive streak. During secondary neurulation, mesenchymal cells formed an initial aggregate near the dorsal surface, which canalized and merged with the anterior neuroepithelium. With aggregation, fibronectin and heparan sulphate proteoglycan were first detected at the base of a 3- to 4-layer zone of radially organized cells. With formation of a lumen within the aggregate, laminin and type IV collagen were also deposited in the forming basement membrane. During both posterior neuropore closure and secondary neurulation, fibronectin and heparan sulphate proteoglycan were associated with the most caudal portion of the neuroepithelium, the region where newly formed epithelium merges with the consolidated neuroepithelium. In regions of neural crest migration, the deposition of basement membrane components was altered, lacking laminin and type IV collagen, with increased deposition of fibronectin and heparan sulphate proteoglycan.

Partial characterization of collagenous and noncollagenous basement membrane proteins synthesized by the 14.5-day rat embryo parietal yolk sac in vitro.

Parietal yolk sacs isolated from 14.5-day rat embryos and incubated in vitro with either [14C]proline, [3H]mannose or 3H-labeled amino acid mixture synthesized and secreted basement membrane collagenous and noncollagenous glycoprotein components with relative molecular weights of 350,000 (350K), 220,000 (220K), 185,000 (185K), 175,000 (175K) and 150,000 (150K). The 185K and 175K components appeared to be similar to the pro-alpha 1 (IV) and pro-alpha 2(IV) chains, respectively, which have been isolated from other sources. These components were completely susceptible to bacterial collagenase, but were only partially susceptible to alpha-chymotrypsin digestion. The 350K and 220K components appeared to be similar to subunits of laminin (or PYS A and PYS B, respectively) which have been characterized by others, while the 150K component may be similar to entactin (or PYS C). These components were completely resistant to bacterial collagenase and completely susceptible to alpha-chymotrypsin digestion. In addition, the basement membrane of the parietal yolk sac (Reichert's membrane) stained intensely with antibodies directed against either rat laminin or mouse basement membrane procollagen. The results of these experiments suggest that the 14.5-day rat embryo parietal yolk sac is a useful system for studying the structure, biosynthesis and deposition of basement membrane components.