Extracellular matrix in renal cell carcinomas

Extracellular matrix (ECM) may be divided into interstitial matrix and the basement membrane (BM). ECM influences a variety of epithelial cell behaviours, including proliferation, differentiation, and morphogenesis, maybe most widely studied in kidney morphogenesis. In carcinomas, including renal cell carcinomas (RCCs), these properties and interactions of cells with interstitial matrix and BM are disturbed. As a carcinoma with a tendency to spread to distant sites, RCC is an interesting target for the study of epithelial-stromal interactions. Among interstitial collagens, type VI collagen appears to be widely distributed in RCCs. Also EDA-fibronectin (EDA-Fn) as well as tenascin-C (Tn) are important stromal components especially in poorly differentiated carcinomas. BMs of RCC islets and those of tumor blood vessel endothelia may merge in poorly differentiated carcinomas. As a dynamic component of BMs, laminins (Ln) are important in kidney development and RCC progression. Type IV collagen and nidogen, other components of BMs in RCCs, are produced by stromal as well as epithelial cells. ECM proteins may function in RCC progression by binding and regulating the activity of growth factors e.g. transforming growth factor beta 1 and basic fibroblast growth factor. Also the expression of cell surface receptors for ECM is disturbed in RCCs. At least alpha v integrin (Int) and CD44 emerge in renal epithelial cells during malignant transformation. Papillary renal neoplasms differ from RCCs by cell adhesion receptor expression and BM composition as well as by ECM avascularity and capacity to bind growth factors, thus suggesting a distinct property for this renal tumor.     

Neonatal estrogen stimulates proliferation of periductal fibroblasts and alters the extracellular matrix composition in the rat prostate

The purpose of this study was to examine whether changes in extracellular matrix (ECM) molecules are associated with the growth inhibition and differentiation defects of the prostate gland following neonatal exposure to estradiol. Using immunocytochemistry (ICC), laminin and collagen IV were localized to the basement membrane (BM) as well to the basal lamina of the periductal smooth muscle of the control developing prostates. In contrast, fibronectin and collagen III were localized throughout the stromal ECM. Exposure to neonatal estrogen altered the staining profile for specific ECM molecules. In the estrogenized rats, a thick layer of cells negative for laminin and collagen IV was observed adjacent to the BM. Electron microscopy and ICC for alpha-actin, fibronectin, and vimentin identified this multicellular layer of periductal cells as differentiated fibroblasts. Peripheral to these fibroblasts, actin-positive smooth muscle formed a second layer of periductal stromal cells. PCNA labeling showed that estrogen exposure increased the fibroblast proliferation. Because many periductal fibroblasts were positive for estrogen receptor alpha (ER alpha) in estrogenized rats, a direct effect of estradiol on their proliferation is suggested. Gelatinolytic gels revealed that estrogen exposure did not alter the activity of matrix metalloproteinases associated with tissue remodeling during prostate morphogenesis. However, the periductal fibroblast layer in estrogenized prostates was devoid of urokinase- and tissue-plasminogen activator, which may potentially alter the localized proteolysis involved in matrix remodeling. It is proposed that proliferation of a multicellular layer of periductal fibroblasts in estrogenized prostates results in a physical barrier that constrains branching morphogenesis and blocks paracrine communications between smooth muscle and epithelial cells which normally regulate differentiation.     

Corneal epithelial basement membrane after experimental anterior stromal puncture in guinea pigs: immunohistochemical study

We investigated alterations in the immunolocalization of the components of epithelial basement membrane (BM), type IV collagen, and laminin in guinea pigs subjected to anterior stromal puncture (ASP) of the cornea performed with a standardized needle. Localization of BM components beneath the corneal epithelium was indicated by laminin immunoreactivity. The BM was interrupted by needle punctures immediately after ASP. During healing, type IV collagen immunoreactivity was detected transiently in the BM of some of the ASP-treated corneas, but no reactivity was observed in normal epithelial BM. Development of type IV collagen immunoreactivity was probably caused by an alteration of the alpha-chains or by an unmasking of the antigenicity of this collagen type, which may be related to an increase in the adhesiveness of the epithelium following ASP.     

Involvement of integrins with adhesion-promoting, heparin-binding peptides of type IV collagen in cultured human corneal epithelial cells

PURPOSE: The aim of this work was to identify the integrin subunits present on the cell surface of human corneal epithelial cells. The authors determined to show whether type IV collagen, heparin-binding peptides of type IV collagen (Hep-I, Hep-II, and Hep-III), fibronectin, and GRGDSP promote cell adhesion of human corneal epithelial cells. Type IV collagen and heparin-binding peptides of type IV collagen may be important in corneal epithelial cell adhesion in normal and pathologic conditions and reepithelialization. The authors assess the role of cell surface integrins in mediating cell adhesion to these proteins and peptides. METHODS: Fluorescence-activated cell sorter (FACS) analysis was used to determine the integrin subunits expressed at the cell surface of the cultured human corneal epithelial cells. Cell adhesion was assessed with type IV collagen, heparin-binding peptides of type IV collagen, fibronectin, and GRGDSP: Antibodies to the integrin subunits were used to determine the potential role of integrins in cell adhesion to the above proteins and peptides. RESULTS: FACS analysis identified the beta 1, beta 4, alpha 2, alpha 3, alpha 5, alpha 6, and alpha v integrin subunits on human corneal epithelial cells grown as primary cultures. The anti-beta 1 antibody inhibited cell adhesion to heparin-binding peptides of type IV collagen, type IV collagen, fibronectin, and GRGDSP: Antibodies to the alpha 2 integrin subunit inhibited cell adhesion to the heparin-binding peptides of type IV collagen and slightly inhibited cell adhesion to intact type IV. Antibodies to the alpha 3 integrin subunit exhibited a somewhat lesser effect compared to the anti-alpha 2 integrin antibody. CONCLUSIONS: These data show that the alpha 2 beta 1 integrin of human corneal epithelial cells recognize heparin-binding peptide sequences derived from human type IV collagen. It seems likely that these sequences play an important role in integrin-mediated corneal epithelial cell adhesion. In addition, the alpha 3 beta 1 integrin may mediate similar events.     

Effects of extracellular matrix constituents on the attachment of human oral epithelial cells at the titanium surface

This in vitro study attempts to delineate the role of extracellular matrix (ECM) constituents at the epithelial tissue-implant interface. To know which ECM constituents have a beneficial influence on the behavior of epithelial cells, the attachment, proliferation, morphologic pattern, and differentiation or cytoskeletal organization of human oral epithelial cells on ECM-coated (type IV collagen, fibronectin, type I collagen, laminin, and vitronectin) and noncoated titanium surface have been evaluated and compared. In each experiment comparing commercially pure titanium and oxygen plasma-cleaned titanium, the same ECM constituents were used. In this study, type IV collagen could provide an excellent substratum for epithelial cell attachment on titanium surface, but vitronectin-coated titanium revealed lower effectiveness for attachment of epithelial cells than noncoated titanium. These results suggested that type IV collagen could be used as a means for obtaining good epithelial seal, whereas vitronectin could be used to restrain the attachment of epithelium to dental implants.     

Corneal wound healing after laser in situ keratomileusis in rabbits

BACKGROUND: The aim of this study was to characterize the cell biology of wound healing in rabbit corneas subjected to laser in situ keratomileusis (LASIK). METHODS: Rabbit corneas underwent LASIK with various multizone photoablations or only a lamellar keratotomy followed by repositioning of the flap. We looked for indications for an active wound healing process. Immunohistochemistry for the extradomain A cellular fibronectin (EDA-cFn) or tenascin (Tn) and routine histology were examined. RESULTS: Four days after LASIK or lamellar keratotomy followed by repositioning of the flap, epithelial plugs and prominent keratocytes as well as Tn and EDA-cFn immunoreactions-indicative of a wound-healing process-appeared in the wound margins. Epithelial plugs were less conspicous, and prominent, presumably activated, keratocytes were no longer identified at the wound margin at 2.5 and 5 months after wounding. However, EDA-cFn and Tn immunoreactivities could still be observed. Only the stromal cells located in the periphery of the flap and in relatively close contact with the epithelium were surrounded by scar tissue expressing immunoreactivity for EDA-cFn or Tn. The central corneal stroma was devoid of scar tissue. CONCLUSION: Results indicate that the wound healing reaction after LASIK takes place only at the periphery of the microkeratome wound, leaving the central optical zone clear.     

Iron deposition at mineralization fronts and osteoid formation following chronic cadmium exposure in ovariectomized rats

Integrins are heterodimeric glycoproteins that have been found to undergo dynamic temporal and spatial changes in distribution in the endometrium during the menstrual cycle in women. Likewise the extracellular matrix (ECM) ligands for these receptors are likely to play a role in the establishment of a receptive endometrium. To develop primate models to study the role of these molecules in the cascade of molecular events leading to implantation, integrin expression and associated changes in ECM were investigated during the menstrual cycle and in early pregnancy in the baboon. Antibodies specific for the integrins (alpha(1-6) and alpha(v); beta1, beta3, and beta4) and ECM (laminin, collagen IV, fibronectin) were utilized. In addition, cytokeratin and alpha-smooth muscle actin were used as epithelial, stromal, and smooth muscle cell markers, respectively. Endometrium was obtained in duplicate or triplicate during the menstrual cycle and early pregnancy. Changes observed during the natural menstrual cycle were confirmed using ovariectomized, steroid-treated animals. Constitutively expressed integrins on the endometrial epithelium included the collagen/laminin receptors: alpha2, alpha3, alpha6, and beta4. The pattern of expression correlated well with the distribution of ECM in this tissue. Collagen IV was confined to the basement membrane of glandular epithelium and blood vessels. Laminin immunostaining was found in the basement membrane, mostly in the stroma of the basal region, in the glandular endometrium and vasculature. Fibronectin was present throughout the stroma but not in the basement membrane. The collagen receptor alpha1 beta1 and fibronectin receptor alpha4 beta1 appeared in the glandular epithelium in the luteal phase. As in the human, alpha1 and alpha4 disappeared from the glandular epithelium with the establishment of pregnancy. In contrast, the alpha4 beta3 vitronectin receptor appeared in the glandular epithelium only in pregnancy or following long-term steroid treatment with estrogen and progesterone but not during the time of uterine receptivity associated with the initial period of embryo attachment. Osteopontin, an ECM ligand for alpha(v) beta3, was coexpressed with this integrin in invading cytotrophoblasts, glandular epithelium, and decidualizing stromal cells. Decidualization in the baboon was associated with changes in integrin expression similar to those found in humans: there was an increase in alpha1, alpha3, alpha6, beta1, and alpha(v) beta3 in the decidualized stromal cells. Laminin and collagen IV expression also increased at the implantation site and throughout the endometrium. In contrast, fibronectin expression was most evident at the implantation site and corresponded to alpha5 expression on the invading cytotrophoblasts. In summary, marked similarities were found in the expression of ECM and the integrin receptors between the baboon and the human endometrium throughout the menstrual cycle and in pregnancy. Cycle-specific integrins, alpha1, and alpha4, were present on epithelial cells during the secretory phase. Delayed expression of alpha(v) beta3 in baboon endometrial glands correlated closely with the time of enhanced glandular secretory activity in this primate. The baboon appears to be an excellent model for the investigation of the role of integrins and ECM leading to successful implantation.     

A quantitative assay for angiogenesis of cultured choroidal tissues in streptozotocin-diabetic Wistar and spontaneously diabetic GK rats

Tenascin-C is an extracellular matrix component which is transiently expressed in association with epithelial cell detachment, proliferation, and migration. This molecule has been identified in respiratory tissue, but little is known about the cellular source of tenascin-C or the factors that regulate its production. Since air pollutants are known to disrupt epithelial integrity, we investigated the regulation of tenascin-C in response to 0.3 ppm ozone in differentiated primate nasal epithelial cells in culture at an air-medium interface. The expression of tenascin-C was upregulated in response to ozone, as determined by Northern blot analysis, Western blotting, and immunofluorescent staining. In contrast, there was no change in the mRNA levels for versican, biglycan, perlecan, or collagen type I. Reduced cellular attachment to the substrate was evident in ozone-treated cultures in association with tenascin-C deposition at the interfaces between cells and basal surfaces. The presence of tenascin-C on denuded areas of the matrix suggests that tenascin-C may have been instrumental in the loss of patches of cells. The modulation of tenascin-C synthesis and distribution may play a significant role in the response of respiratory epithelial cells to ozone exposure.     

Expression of beta 1 integrins changes during transformation of avian lens epithelium to mesenchyme in collagen gels

Remarkably, a number of definitive epithelia, such as that of the anterior lens, give rise when suspended within 3D gels of type I collagen, to elongate, bipolar shaped cells that exhibit the ultrastructure, polarity, and migratory ability of mesenchymal cells. They begin producing type I collagen and stop producing crystallins, type IV collagen, and laminin. Here, we investigated changes in beta 1 integrins and their extracellular matrix (ECM) ligands during this transdifferentiation. The former free surface of the lens epithelium that is now in contact with collagen begins within a day to stain intensely for beta 1 and it is this surface rather than the surface facing the basement membrane that gives rise to mesenchymal cells. Immunoprecipitation experiments reveal a large increase in the beta 1 integrin subunit on mesenchymal cells as compared to the epithelium of origin. The alpha 5 integrin subunit, which is barely detectable in the lens, increases in the mesenchymal cells and alpha 3 continues to be expressed at about the same level as in the epithelium. alpha 6, the epithelial integrin subunit, and laminin, its ECM ligand, are not detected immunohistochemically or biochemically in the mesenchyme. Rather, the mesenchymal cells secrete abundant fibronectin, the major ECM ligand for alpha 5 beta 1. RGD peptides do not inhibit the transformation but antibodies to beta 1 do perturb the emigration of mesenchymal cells from the lens apical surface. We conclude that the beta 1 integrins newly expressed on the apical epithelial surface interact with the surrounding 3D collagen gel to help bring about this unusual epithelial-mesenchymal transition.     

(S)-(-)-[11C]nicotine binding assessed by PET: a dual tracer model evaluated in the rhesus monkey brain

PURPOSE: Determine the tissue distribution patterns for tissue inhibitors of metalloproteinases (TIMP-1, TIMP-2, TIMP-3), gelatinase A and gelatinase B in normal and pathologic corneas. METHODS: Corneas were examined by immunohistochemistry, using antibodies to TIMP-1, TIMP-2, TIMP-3, gelatinase A or gelatinase B. RESULTS: In normal corneas, TIMP-1 antibody stained the epithelium and endothelium. TIMP-2 and TIMP-3 stained the epithelium, keratocytes and endothelium. Gelatinase A staining was weak and restricted to the epithelial cells. Radial keratotomy scars showed increased staining for TIMP-1 and TIMP-2 around the epithelial cell plug and along the incision. Bullous keratopathy corneas showed TIMP staining patterns similar to normal corneas and increased gelatinase A staining in regions of subepithelial fibrosis. Stromal scars of keratoconus corneas also had increased staining with TIMP-1 and TIMP-2 antibodies. In many keratoconus corneas, the TIMP-3 staining pattern was similar to normal corneas. However, in some keratoconus corneas, when Bowman's layer was missing, the stroma beneath was completely devoid of TIMP-3 antibody staining. No gelatinase B was seen in either the normal or diseased corneas. CONCLUSION: These data suggest that TIMP-1 and TIMP-2 are important for scar formation and corneal remodeling, since they were found in increased amounts at radial keratotomy incision sites and keratoconus scars. The significance of the focal stromal defects in TIMP-3 staining, associated with absence of Bowman's layer on keratoconus corneas, needs to be elucidated. At the stages of disease examined in this study, gelatinase B may not play a significant role in these pathological processes, since it was not seen in any of the corneas examined.     

T lymphocyte migration: the influence of interactions via adhesion molecules, the T cell receptor, and cytokines

Although lymphocytes have been studied extensively with respect to a number of motile aspects the understanding of directed lymphocyte motility and its regulation has increased relatively slowly. T lymphocyte migration/translocation in vivo and in vitro are critically dependent on the avidity of adhesive lymphocyte receptors for endothelial cell ligands and extracellular matrix (ECM) components and on the capacity of the lymphocytes to undergo a motile response. Lymphocytes are rendered motile by adhesion to endothelial cells and ECM components. Thus, T lymphocytes exhibit chemotactic and haptotactic migration to the ECM components fibronectin, laminin, and collagen type IV. This directed migration is mediated by beta 1-integrins and separate T-lymphocyte lines have a functional specialization using either alpha 4 beta 1 or alpha 5 beta 1 during chemo- and haptotaxis to ECM components, although the same cell line may use both integrins for adhesion. Noteworthy, signals triggering T cell migration to ECM components seem to be delivered preferentially via alpha 4 beta 1 or alpha L beta 2. The T cell antigen receptor cannot by itself trigger T lymphocyte migration to fibronectin, laminin, or collagen type IV but does so in collaboration with signals via alpha 4 beta 1. It follows that the migration-triggering signals can be separated from the integrin interactions with matrix components that mediate the chemo- and haptotactic migration per se. This suggests that T cell recruitment to inflammatory sites is induced by antigen receptor signals and beta 1- and beta 2-integrin signals in synergy. Cytokines with chemokinetic properties may collaborate with lymphocyte counterreceptors on endothelial cells and with ECM components in control of the lymphocyte migratory pathways and specifically attract lymphocyte subsets to different compartments. T lymphocytes are endowed with multiple enzymes, classified as serine proteinases or metalloproteinases, which can degrade extracellular matrix components. These enzymes may play an important role for the capacity of T cells to migrate and infiltrate tissues.     

Multi-component T1 relaxation and magnetisation transfer in peripheral nerve

Corneas of diabetic patients have abnormal healing and epithelial adhesion, which may be due to alterations of the corneal extracellular matrix (ECM) and basement membrane (BM). To identify such alterations, various ECM and BM components and integrin receptors were studied by immunofluorescence on sections of normal and diabetic human corneas. Age-matched corneas from 15 normal subjects, 10 diabetics without diabetic retinopathy (DR), and 12 diabetics with DR were used. In DR corneas, the composition of the central epithelial BM was markedly altered, compared to normal or non-DR diabetic corneas. In most cases the staining for entactin/nidogen and for chains of laminin-1 (alpha1beta1gamma1) and laminin-10 (alpha5beta1gamma1 was very weak, discontinuous, or absent over large areas. Other BM components displayed less frequent changes. The staining for alpha3beta1 (VLA-3) laminin binding integrin was also weak and discontinuous in DR corneal epithelium. Components of stromal ECM remained unchanged even in DR corneas. Therefore, distinct changes were identified in the composition of the epithelial BM in DR corneas. They may be due to increased degradation or decreased synthesis of BM components and related integrins. These alterations may directly contribute to the epithelial adhesion and wound healing abnormalities found in diabetic corneas.     

Changes in extracellular matrix components and steroidogenic enzymes during growth and atresia of antral ovarian follicles in the sheep

To investigate the involvement of extracellular matrix (ECM) in folliculogenesis in the sheep, parallel changes in ECM components and key steroidogenic enzymes were studied by quantitative immunohistochemistry and immunoblotting during follicular growth and atresia. Growth of ovarian follicles from 1 to 5 mm in diameter was characterized by a progressive increase in P450 cholesterol sidechain cleavage levels in both thecal (p < 0.001) and granulosa cells (p < 0.001), an increase in P450 aromatase levels in granulosa cells of follicles larger than 3.5 mm (p < 0.001), and an increase in levels of P450 17 alpha-hydroxylase C17,20 lyase (P450(17 alpha)) in the theca interna. In addition, during follicular growth, a change in localization of cells expressing P450(17 alpha) within the theca interna was observed, positive cells being sparse within the theca interna of small follicles and specifically located close to the basal laminae in large follicles. In parallel, follicular growth was associated with an increase in levels of type I collagen in granulosa cell layers (p < 0.01) and an increase in levels of fibronectin (p < 0.05), particularly the specific ED-A alternatively spliced variant of fibronectin, in the theca externa. Follicular atresia was characterized by a loss of P450 aromatase in granulosa cells (p < 0.001) and a decrease in levels of P450(17 alpha) in the theca interna (p < 0.05). Simultaneously, levels of fibronectin (p < 0.05), particularly the ED-A variant of fibronectin, decreased in the theca externa of atretic follicles. Within the wall of granulosa cells, levels of fibronectin (p < 0.05), laminin, type IV collagen, and heparan sulfate proteoglycans strongly increased during follicular atresia. Overall, these results show that follicular growth and atresia were associated with distinct changes in levels of ECM components, suggesting that ECM components may play a role in the regulation of proliferation, differentiation, and apoptosis of follicular cells.     

Isoforms and levels of transferrin, antithrombin, alpha(1)-antitrypsin and thyroxine-binding globulin in 48 patients with carbohydrate-deficient glycoprotein syndrome type I

To improve our understanding of the role of extracellular matrix (ECM) proteins and integrins during the processes of granuloma formation in sarcoidosis, we examined the distribution of ECM proteins and the expression of integrins in sarcoid lymph nodes by immunohistochemical methods. We also examined the expression of transforming growth factor-beta1 (TGF-beta1), which is one of major regulators for synthesis of ECM proteins. Most ECM proteins were detected in the periphery of the granulomas in a concentric pattern, and fibronectin was diffusely detected from an early to a regressive stage. Compared with normal lymph nodes, most beta1-integrin subfamilies (alpha1, alpha4, alpha5 and alpha6) were more strongly expressed on lymphocytes around the granulomas. Epithelioid cells exhibited strong expression of the alpha5 molecule. Fibroblasts exhibited the expression of the alpha2 and alpha5 molecules surrounding ECM proteins. The alpha5beta1 molecule had a distribution similar to that of fibronectin. TGF-beta1 was detected in epithelioid cells throughout the various evolutional stages and its expression was especially marked in mature granulomas. Interaction of fibronectin and the alpha5beta1 molecule may have an important role in the process of formation of sarcoid granuloma. The expression of TGF-beta1 may be involved in the regression of sarcoid granuloma by initiating fibrosis and atrophy of epithelioid cells.     

The reversed-flow medio-distal fasciocutaneous island thigh flap: anatomic basis and clinical applications

To investigate changes in retinal pigment epithelial (RPE) cells during wound healing, we evaluated the deposition of newly synthesized extracellular matrix (ECM) over time during wound healing in rat RPE cultures. We also estimated the effect of growth factors on the healing rate and ECM synthesis. After preparing rat RPE cell sheet cultures, we made round 1-mm defects in the cultures. Fibronectin, laminin, and collagen IV synthesis were evaluated with immunocytochemistry every 12 hours after wounding. S-phase cell distribution was analyzed every 12 hours by 5-bromodeoxyuridine uptake. We added either platelet-derived growth factor (PDGF), epidermal growth factor (EGF), or transforming growth factor- beta2 (TGF-beta2) to cultures at concentrations of 1, 10, and 100 ng/mL and immunocytochemically analyzed the effects on ECM and estimated the rate of wound closure. Although approximately 50% closure was achieved 24 hours after wounding, fibronectin deposits first appeared at that time. Laminin and collagen IV were first detected at 36 hours and fibronectin staining had extended toward the wound center. S-phase cells were distributed in concentric rings that moved centripetally over time and corresponded to the leading edge of the area stained with anti-ECM antibodies. TGF-beta2 enhanced ECM deposition, but EGF and PDGF did not. TGF-beta2 decreased the healing rate in a dose-dependent manner, whereas PDGF promoted wound closure. EGF enhanced closure at the highest concentration only. In summary, wound healing in RPE may be initiated when cells at the wound edge slide or migrate toward the wound center, which is followed by cell proliferation and then ECM synthesis. ECM components may be produced in a specific sequence during healing. TGF-beta2 may promote RPE cell differentiation, and PDGF may enhance proliferation during wound healing of the RPE.     

Diabetic pigmented spots on the extrapretibial region: immunohistochemical study of type IV collagen in dermal vessel wall

A case of diabetes mellitus associated with multiple pigment spots is reported. The patient had multiple pigmented atrophic patches on the abdominal area and thighs. There were no pigmented spots on the pretibial area. Histologically, dermal vessels showed intimal thickening and deposition of periodic acid-Schiff (PAS)-positive fibrillar material in vessel walls. Clinical and histological features indicated that these pigment spots were diabetic pigmented pretibial patches occurring in the extrapretibial area. Immunohistochemical studies of lesional and non-lesional skin using the antibodies for alpha1-alpha6 chains of type IV collagen revealed that PAS-positive material of vessel walls in both lesional and non-lesional skin was stained with alpha1 and alpha2 chain antibodies but not with alpha3-alpha6 chain antibodies, indicating that alpha1 and alpha2 chains of type IV collagen, which are normal components of the dermal vessel basement membrane, accumulate in the vessel walls in diabetes mellitus.