Expression of the laminin γ2 chain in different histological types of lung carcinoma. A study by immunohistochemistry and in situ hybridization

Sixty-four malignant lung tumours and 12 of their regional lymph node metastases were analysed for expression of the laminin γ2 chain by immunohistochemistry and in situ hybridization. Expression of the laminin γ2 chain was strongest in squamous cell carcinomas, followed by adenocarcinomas and large cell carcinomas. Positive cells, except for large cell carcinomas, were located at the epithelial–stromal interface of tumour clusters. An important exception was small cell lung carcinoma, with only a low level of laminin γ2 chain expression. Apart from tumour type, this may reflect the relatively scanty fibrous stroma in these tumours and supports previous observations that small cell lung carcinoma cells, contrary to other types, lack surface expression of α₆β₄ integrin, the specific laminin-5 binding receptor. In frozen sections, immunohistochemistry showed linear basement membranes around tumour clusters in squamous cell carcinomas and adenocarcinomas. This shows that carcinoma cells are capable of heavy deposition of the laminin γ2 chain around tumour clusters and suggests that a laminin γ2 chain-containing substrate may be of significance for the spread and growth of malignant tumours. Copyright © 1999 John Wiley & Sons, Ltd.     

EXPRESSION OF THE LAMININ γ2 CHAIN IN PANCREATIC ADENOCARCINOMA

Forty-two pancreatic adenocarcinomas were investigated immunohistochemically and by in situ hybridization for the expression of the laminin γ 2 chain. In 41 cases, intracytoplasmic immunoreactivity for the γ2 chain was seen. Positive tumour cells were located especially at the epithelial–stromal interface of the tumour cell islands. In 22 cases, diffuse laminin γ2 chain immunoreactivity could also be seen in stroma and in seven cases, occasional positivity was detected in the neoplastic basement membranes. Signals for laminin γ2 chain mRNA in tumour cells displayed a distribution similar to that observed on immunohistochemistry. There were significantly more cases with less than 20 per cent of laminin γ2 chain-positive tumour cells in tumours extending to peripancreatic tissues and/or tumours with regional or distant metastases ( P =0·029). A corresponding statistical significance could also be noted in the mRNA level ( P =0·025). The results show that pancreatic adenocarcinomas display a high activity of laminin γ 2 chain synthesis. Tumours with a strong laminin γ2 chain synthesis show a lower invasive and metastatic potential than tumours with a weak or moderate laminin γ2 chain expression.     

Appearance and distribution of laminin a chain isoforms and integrin α2, α3, α6, β1, and β4 subunits in the developing human small intestinal mucosa

Background: Laminin, a major component of basement membranes, is well known in its classical heterotrimeric form (B1-A-B2) to regulate diverse biological functions, including cell polarization and differentiation. However, the role of merosin, a laminin-like molecule in which an M chain is substituted for its homologous A chain, remains largely unknown. Methods: In the present study, we analyzed by indirect immunofluorescence the expression and distribution of these four laminin chains as well as the integrins α2β1, α3β1,α6β1, and α6β4, four potential recptors, at the epithelial-mesenchymal interface of the developing human small intestine, with a panel of specific monoclonal antibodies. Results: Beginning at 7 weeks of gestation and throughout mucosal organogenesis, the B1 and B2 chains were uniformly detected at the epithelial basement membrane. The A chain also was detected beginning at 7 weeks, and its distribution at the basement membrane remained uniform throughout villus (9+ weeks) and crypt (16+ weeks) formation. In contrast, M chain expression was not observed until 16 weeks; between 16 and 20 weeks, it was exclusively associated with the base of epithelial cells that comprised the forming crypts. Integrins α6β1 and α6β4, as determined by their subunit immunolocalization, appeared to be expressed by all enterocytes from 7 to 20 weeks. In contrast, the expression of the α2β1 and α3β1 integrins was found time- and site-restricted. The α2 subunit was predominantly detected in the epithelial cells of the intervillous area and its derivative, the crypt, whereas the α3 subunit was strongly expressed by all epithelial cells except those located at the bottom of 19–20-week-old crypts. Conclusions: Taken together, these observations demonstrate that both compositional changes in the basement membrane and differential expression of receptors occur during human intestinal organogenesis, suggesting that epithelial cell-matrix interactions play a role during development. © 1995 Wiley-Liss, Inc.     

In situ-RT-PCR and immunohistochemistry for the localisation of the mRNA of the alpha 3 chain of laminin and laminin-5 during human organogenesis

Laminin-5 is known to be an integral part of the hemidesmosome and therefore responsible for the integrity of the connection of the epithelium to the basement membrane. This is also an important mechanism during embryonic development, as documented by studies in mice. In an attempt to elucidate its implication for human development we localised the mRNA of the alpha3 chain of laminin with the help of in situ RT-PCR, and the laminin-5 protein immunohistochemically. We systematically investigated kidney, lung, skin and intestinal tissue of consecutive developmental stages during human embryogenesis. From gw 6.5 onwards, the mRNA of the alpha3 chain of laminin was found exclusively in the cytoplasm of epithelial cells of the developing kidney, lung, skin and intestine. Interestingly, in the skin and intestine from gw 8 onwards, the superficial cell layers also stained positive for the mRNA, while the protein was still only found in the dermal-epidermal and enteric basement membrane zones. In all developing organs investigated, the mRNA of the alpha3 chain of laminin is strictly of epithelial origin and the corresponding protein localised in the underlying basement membrane zones. Due to this discrepancy, we postulate a broader role for laminin-5 during human embryogenesis, for example, for epithelial cell development, beyond its involvement in hemidesmosome formation and cell adhesion.     

Ultrastructural Characterization of Mesenchymal and Epithelial Cells Co-Cultured from Human Dental Root Apical Explants

Previous studies have shown the role of cell-cell and cell-matrix interactions in the differentiation of the specific secretory cells of the tooth. In order to elucidate the mechanisms implicated in root dentin formation, we developed a co-culture system of human pulpal mesenchymal and epithelial root sheath cells. Root tips of premolars were cultured in Eagle's basal Medium supplemented with fetal calf serum, ascorbic acid, antibiotics and, for some of them, with sodium β-glycerophosphate. After 60 days of culture, cells were prepared for light and electron microscopy. Three main cell types were observed: (1) polygonal mesenchymal cells showing a functional polarity and producing a dense network of tactoid collagenous fibers. The latter had a specific circular organization that delimited small lacunae around the cells and mineralized in the presence of β-glycerophosphate; (2) spindle-shaped mesenchymal cells mainly localized inside epithelial-mesenchymal knots and synthesizing an abundant collagenous matrix; and (3) epithelial cells lying on the plastic culture dish, on the dense collagenous matrix, or on spindle-shaped cells. Epithelial cells deposited a structured basement membrane when they were lying on the plastic culture dish or on spindle-shaped cells. On the contrary, no basement membrane was found when epithelial cells were overlying the dense collagenous network. Immunoelectron microscopic analysis of type IV collagen and laminin indicated that these two specific basement membrane components were produced by all cell types. These results show that the co-culture system should be valuable for (1) studying the in vitro formation of human dental root hard tissues, (2) characterizing cell-cell and cell-matrix interactions implicated in dental basement membrane production, and (3) isolating populations of cells implicated in dental root formation.     

Synthesis of laminin and type IV collagen by trophoblastic cells and fibroblastic stromal cells in the early human placenta

In situ hybridization was used on both routinely processed paraffin embedded tissue sections and cryosections to study the synthesis of the basement membrane (BM) proteins laminin and type IV collagen in early human placentas from 8 to 11 weeks of gestation. Complementary DNA and RNA probes coding for the pro alpha 1 (IV) chain of human type IV collagen and the B1 chain of human laminin were used to detect respective mRNA. The clusters of cytotrophoblastic cells (i.e., cytotrophoblastic cell columns) contained cells that expressed both laminin and type IV collagen mRNA. Nearly all the stromal cells of developing villi also expressed mRNA for these proteins, type IV collagen mRNA levels being more pronounced than those of laminin. Conversely, the cytotrophoblastic cells of the villous trophoblastic epithelium synthesized mRNA for laminin more actively than that for type IV collagen. Decidual cells, endometrial stromal cells, and cells in the wall of spiral arteries all expressed mRNA for both laminin and type IV collagen. The results of in situ hybridization were found to correlate well with immunohistochemical stainings with specific antibodies to laminin and type IV collagen. The trophoblastic cells of the columns and the stromal fibroblasts are shown to contribute significantly to BM protein synthesis in the developing human placenta. The abundance of BM proteins may indicate their significance in guiding tissue organization in the placenta.     

脱细胞猪角膜表面基底膜成分的检测

背景：前期实验显示脱细胞猪角膜具有良好的组织相容性,可以支持角膜细胞和皮肤上皮细胞的生长。 目的：检测脱细胞猪角膜是否保存了利于角膜上皮细胞生长的重要组织结构—基底膜。 方法：利用荧光抗体对脱细胞猪角膜表面的基底膜成分(层粘蛋白和Ⅳ型胶原)进行免疫组织化学检测,荧光显微镜下观察脱细胞猪角膜表面是否保存了基底膜成分。 结果与结论：免疫荧光染色显示脱细胞猪角膜前基质表面层粘蛋白和Ⅳ型胶原呈阳性表达,与新鲜猪角膜表面基底膜的荧光表达相同,表明脱细胞猪角膜保存了利于角膜上皮细胞生长的基底膜。     

Molecular variants of fibronectin and laminin: structure, physiological occurrence and histopathological aspects

This review deals with biological and pathological aspects of various isoforms of the matrix molecules fibronectin and laminin. They are generated by different molecular mechanisms: ED-A⁺ and ED-B⁺ fibronectin by alternative splicing of pre mRNA, de novo-glycosylated fibronectin by alternative post-translational O-linked glycosylation of the IIICS region, and the laminin isoforms by exchange of single chains of the heterotrimeric molecule. In contrast to the common fibronectin, the distribution of ED-B⁺ and de novo-glycosylated fibronectin is restricted to embryonic tissues; they subsequently reappear in granulation tissue, in fibrosing processes and in tumour stroma. The expression of these so-called oncofetal fibronectins is stimulated by growth factors (TGF). The association of the ED-B⁺ fibronectin with proliferative activity and newly formed vessels identifies this fibronectin variant as a marker of cellular activity in the process of fibrosis and as a suitable agent for the evaluation of tumour angioneogenesis. Initial results suggest a correlation between the amount of ED-B⁺ and de novo-glycosylated fibronectin in tumour stroma and the behaviour of carcinomas with regard to their invasiveness and propensity for metastatic dissemination. The current nomenclature of the laminin molecule family is presented. The laminin chain constitution of basement membranes switches from embryonic or proliferatively active to adult terminally differentiated tissues [disappearance of the laminin 2 (s) chain] and depends on the tissue type. The discrepancy between the loss of basement membranes (multiple basement membrane defects) in carcinomas and the recently reported increased laminin chain synthesis in these tumours may be explained by abundant laminin chain deposition outside the basement membrane in the carcinoma invasion front, possibly associated with enhanced adhesion of budding tumour cells.     

Tumor cells of malignant fibrous histiocytomas express mRNA for laminin.

In situ hybridization was used on routinely processed paraffin-embedded tissue sections to study the synthesis of the basement membrane (BM) proteins laminin and type IV collagen in 14 cases of malignant fibrous histiocytoma (MFH). Complementary RNA probes coding for the pro-alpha 1 (IV) chain of human type IV collagen and the B1 chain of human laminin were used to detect the respective mRNAs. The results were correlated with the immunohistochemical reactivity of tumor cells to specific antibodies against the P1 fragment of laminin and the 7S domain of type IV collagen. Signals for the presence of laminin mRNA in atypical neoplastic tumor cells could be detected in 11 MFHs. None of the tumors could be shown to contain signals for type IV collagen mRNA in their cells, although such signals were detected in the endothelial cells of tumor capillaries. In the corresponding immunohistochemical stainings, nine MFHs showed intracytoplasmic staining of tumor cells for laminin and one tumor showed weak staining for type IV collagen in the neoplastic cells. The results show that the laminin immunoreactivity found in MFHs is due to synthesis in the tumor cells and not to endogenous uptake of this protein. Synthesis of laminin in the majority of MFHs is in accordance with the notion that these tumors originate from primitive mesenchymal cells in soft tissues.     

Changes in mesenchymal cell-shape, matrix collagen and tenascin accompany bud formation in the early chick lung

Summary In the chick, lung branches arise as buds from the center of the pre-existing mesobronchial tube. Budding is known to be controlled by the mesenchyme. We have investigated mesenchymal properties in budding vs non-budding regions of the early chick lung, including sources of mesenchyme, cell shapes and densities, morphology and composition of the basement membrane, and distribution of the ECM components collagen, fibronectin and tenascin. We found that at points of outgrowth — the buds and the distal tip of the mesobronchus — mesenchymal cells adjacent to the lung epithelium are flattened, and the basement membrane is markedly thinned. In these basement membranes collagen is largely absent and tenascin redistributed into amorphous clumps. Of these characteristics only the cell-shape change, which results in the flattened mesenchymal cells at the bud tips, is correlated with initiation of the bud. We suggest that the cell-shape change leads to localized loss of collagen, which promotes emergence of buds, and that tenascin, which is found in the mesenchyme only in the budding region, promotes outgrowth and elongation of the bud.     

Physiology: Immunohistochemical characterization of human endometrial microvascular basement membrane components during the normal menstrual cycle

The expression of three basement membrane components [collagenIV (CIV), laminin and heparan sulphate proteoglycan (HSPG)]and platelet endothelial cell adhesion molecule (PECAM) wereexamined by immunohistochemistry in cryostat sections of normalhuman endometrium. Alkaline phosphatase (ALP) was detected usingenzyme histochemistry. Endometrial biopsies from the menstrual(n = 4), mid—late proliferative (n = 5), early—midsecretory (n = 5) and late secretory (n = 5) stages were collectedfrom women with a normal menstrual cycle. At all four stagesof the menstrual cycle, CIV, laminin and HSPG were expressedon basement membranes of both vessels and glands whilst PECAMexpression was localized specifically to endothelial cells.A similar number of vessels/mm² stained for CIV and laminin,as well as for PECAM at each stage of the menstrual cycle, demonstratingthat all vessels in endometrium stain for these two basementmembrane components. By contrast, the number of vessels/ mm²that stained positively for HSPG and ALP was significantly lower,averaging 55% of the total that stained positively for PECAM,CIV and laminin. During the menstrual stage, HSPG staining intensityremained strong in glandular basement membranes but decreaseddramatically in vascular basement membranes. ALP activity wasvariable in both the vessels and glands throughout the fourstages of the menstrual cycle studied. This study demonstratesheterogeneity in basement membrane components within the endometrialmicrovasculature. It is postulated that the disappearance ofHSPG from vascular basement membranes may play a role in theprocess of vascular remodelling during the menstrual stage ofthe cycle.     

Immunocytochemical detection of basement membrane antigens in the histopathological evaluation of laryngeal dysplasia and neoplasia

Immunocytochemical detection of intrinsic components of the basement membrane (type IV collagen and laminin) was performed in biopsies of carcinoma in situ, dysplasia and hyperplasia of the laryngeal mucosa. We found a distinct and continuous basement membrane, containing both laminin and type IV collagen, at the border between epithelial cells and mesenchymal stroma in normal as well as in hyperplastic and dysplastic mucosa. In contrast, irregular discontinuities were found in some cases of carcinoma in situ and in invasive carcinoma. In addition, immunoreactivity for intracytoplasmic basement membrane components was noticed occasionally in neoplastic epithelial cells. In areas of inflammation, infiltration of leucocytes into the epithelium was occasionally accompanied by sharply defined small interruptions of the basement membrane. Our results indicate that immunohistochemical detection of basement membrane components can be of value for the demonstration of microinvasive growth in laryngeal cancer.     

Organisation of basement membrane components in the human adult and fetal pituitary gland and in pituitary adenomas

 Cell–matrix interactions undoubtedly have a role in the development and maintenance of the complex nonrandom structure of the human pituitary gland. We have extended previous studies by documenting the patterns of immunoreactivity for type IV collagen, laminin and fibronectin in the fetal gland, comparing these with the adult patterns. In both we have examined the differences between the anterior lobe and intermediate zone in an attempt to elucidate the apparent differences in functional response between corticotrophs in the two areas. We have also examined expression of these proteins in a series of pituitary adenomas. Finally, we have immunolocalised β₄ integrin, a component of the α₆β₄ laminin receptor, in the adult gland and in adenomas. In the anterior lobe of the adult gland, type IV collagen and laminin were present in both epithelial and vascular basement membrane. Fibronectin was related to the basement membrane but showed a less continuous distribution. β₄ Integrin was expressed on the basal aspects of pituitary cells, in association with laminin, suggesting that this did identify the α₆β₄ laminin receptor. In addition, immunoreactivity was present on the lateral margins of some pituitary cells, which might indicate a role in cell–cell adhesion. None of the proteins showed specific association with any particular cell type, suggesting that these specific interactions do not regulate differentiation. This pattern of expression had developed in the fetal gland by the second trimester, with expression relating to vessels preceding that in epithelial basement membrane. Type IV collagen, laminin and fibronectin were also expressed in epithelial and vascular basement membrane in the intermediate zone of the adult gland, and around Rathke’s cleft in the fetal gland. However, the organisation differed, with larger groups of cells enclosed within a single basement membrane. Possible vascular connections demonstrated between the posterior lobe and the intermediate zone would permit access of posterior lobe hormones to this zone. Our data confirmed disruption of expression in pituitary adenomas, type IV collagen, laminin and β₄ integrin having a mainly perivascular distribution, with more variable immunoreactivity for fibronectin. Received: 17 February 1997 / Accepted: 17 May 1997     

Basement membrane formation and re-distribution of the β1 integrins in a human intestinal co-culture system

We have developed a co-culture system suited for the study of epithelial-mesenchymal interactions in the human fetal small intestine. As the epithelial component of this model, we used the human intestinal cell line Caco-2 that is unique in its property to differentiate in vitro into a mature fetal enterocyte-like cell type. A sheet of human intestinal mesenchymal cells, which we derived from an 18-week-old fetus, was used as mesenchymal element. Expression and distribution of cell-specific markers (cytokeratin 18 and dipeptidyl peptidase IV), major basement membrane components, and β1 integrins were analyzed. In 14-day co-cultures, Caco-2 cells formed a cytokeratin 18-positive epithelial-like sheet covering the vimentin-positive HIM cell layers. As assessed by brush border dipeptidyl peptidase IV expression, co-cultured Caco-2 cells achieved cytodifferentiation as when cultured on plastic. A complete deposition of all known major human fetal intestinal basement membrane components occurred at the Caco-2/HIM interface. Type IV collagen and tenascin were produced from the mesenchymal compartment, whereas laminin and fibronectin were contributed by both cell types. Interestingly, synthesis and deposition of basement membrane heparan sulfate proteoglycan were exclusively observed in co-cultures, suggesting modulation of epithelial expression of this molecule by HIM cells. Finally, we observed that epithelial integrin-β1 chains redistributed at the basal domain of co-cultured Caco-2 cells. Taken to gether, these observations indicate that the Caco-2/HIM co-culture model is a valuable system to study in vitro human basement membrane formation in the context of intestinal epithelial-mesenchymal interactions. © 1993 Wiley-Liss, Inc.     

Hedgehog signaling and laminin play unique and synergistic roles in muscle development

Hedgehog (Hh) signaling and laminin‐111, a basement membrane protein, are required for early muscle development. Hh signaling specifies different populations of muscle fibers and laminin‐111 is critical for early muscle morphogenesis. However, additional requirements for Hh signaling and laminin during later phases of muscle development are not known. Furthermore, interactions between Hh signaling and laminin in this context are unknown. We used laminin gamma1 mutant zebrafish and cyclopamine to block Hh signal transduction separately and in combination to investigate their functions and interactions. We found that both Hh signaling and laminin are required for normal myosin chain expression. In addition, Hh signaling and laminin act synergistically during fast‐twitch fiber elongation: fast muscle cells do not elongate in embryos deficient for both Hh signaling and laminin. Finally, we present evidence that suggests that Hh signaling is indirectly required via slow fiber specification for recovery of fast fiber elongation in laminin gamma1 mutant embryos. Developmental Dynamics 239:905–913, 2010. © 2010 Wiley‐Liss, Inc.     

Laminin expression in the mouse lung increases with development and stimulates spontaneous organotypic rearrangement of mixed lung cells.

The recent establishment of a role for laminin in mouse lung organogenesis (Schuger et al. 1990a,b, 1991) prompted us to study its expression in the developing lung. Laminin A and B chains were detected in the murine lung from the first hours of development onward. In situ hybridization of mRNA as well as SDS-PAGE studies of lung cells in monoculture indicated that both epithelium and mesenchyme produce complete laminin molecules. Quantitative analysis of the in situ hybridization studies showed a gradual increase in laminin expression during development which was further supported by immunohistochemistry and ELISA. The overall pattern of expression suggested that the effects of laminin in morphogenesis were not restricted to a particular stage of development. Furthermore, the increase in expression during late development supported a role for the molecule in the fetal lung, which was not previously established. We next determined whether the increase in laminin production modulated the behavior of fetal lung cells as compared with their embryonic counterparts. We previously showed that organotypic pattern formation does not occur in cultures of mixed embryonic lung cells unless exogenous laminin is added (Schuger et al., 1990b). Organotypic pattern formation is the result of cell sorting into epithelial and mesenchymal compartments and further rearrangement in a pattern resembling the tissue of origin. In the present study, we demonstrated that organotypic pattern formation occurs spontaneously in cultures of mixed fetal lung cells, which express high laminin levels. Pattern formation was abolished by antibodies to laminin. These studies suggest a correlation between laminin expression and the ability of lung cells in culture to reproduce normal tissue patterns. We conclude that laminin is critical for epithelial-mesenchymal recognition and further morphogenic interaction during both the embryonic and fetal stages of lung development.