Laminin Isoforms and Laminin-Producing Cells in Rat Anterior Pituitary

Laminin is a key component of the basement membrane and is involved in the structural scaffold and in cell proliferation and differentiation. Research has identified 19 laminin isoforms, which are assemblies of α, β, and γ chains (eg, the α1, β1, and γ1 chains form the laminin 111 isoform). Although laminin is known to be present in the anterior pituitary, the specific laminin isoforms have not been identified. This study used molecular biological and histochemical techniques—namely, RT-PCR, immunohistochemistry, and in situ hybridization—to identify the laminin isoforms and laminin-producing cells in rat anterior pituitary. RT-PCR showed that laminin α1, α3, and α4 genes were expressed in anterior pituitary. Immunohistochemistry revealed laminin α1 in gonadotrophs and laminin α4 in almost all vascular endothelial cells. Laminin α3 was seen in a subset of vascular endothelial cells. We then performed in situ hybridization to localize β and γ chains in these cells and found that laminin β1, β2, and γ1 were expressed in gonadotrophs and that laminin β1 and γ1 were expressed in endothelial cells. In conclusion, we identified gonadotroph-type (laminin 111 and 121) and vascular-type (laminin 411 and 311) laminin isoforms in rat anterior pituitary.     

The Microvascular Localization of Laminin and Fibronectin in Scleroderma Muscle by Immune Electron Microscopy

Immune electron microscopy using a peroxidase-antiperoxidase (PAP) technique was done on muscle biopsies from patients with scleroderma (PSS), rheumatoid arthritis (RA), systemic lupus erythematosus (SLE), and normal controls, in order to localize laminin and fibronectin in the muscle capillary basement membrane (BM). Three patterns of staining were noted for the 2 antigens: in/out, diffuse and negative. Staining patterns in PSS biopsies were markedly different from both the normal and disease controls. For laminin the expected in/out pattern was rare in PSS, with virtually all biopsies being either diffuse or negative. In the case of fibronectin, the staining patterns were of equal frequency in PSS, whereas in the control groups diffuse staining was rare. These findings suggest that in addition to the morphologic abnormalities previously noted in PSS muscle capillary BM, there is also an alteration in the distribution of specific BM components in PSS.     

Basement Membrane Type IV Collagen and Laminin: An Overview of Their Biology and Value as Fibrosis Biomarkers of Liver Disease

Basement membranes provide structural support to epithelium, endothelium, muscles, fat cells, Schwann cells, and axons. Basement membranes are multifunctional: they modulate cellular behavior, regulate organogenesis, promote tissue repair, form a barrier to filtration and tumor metastasis, bind growth factors, and mediate angiogenesis. All basement membranes contain type IV collagen (Col IV), laminin, nidogen, and perlecan. Col IV and laminin self‐assemble into two independent supramolecular networks that are linked to nidogen and perlecan to form a morphological discernable basement membrane/basal lamina. The triple helical region, 7S domain and NCI domain of Col IV, laminin and laminin fragment P1 have been evaluated as noninvasive fibrosis biomarkers of alcoholic liver disease, viral hepatitis, and nonalcoholic fatty liver disease. Elevated serum Col IV and laminin are related to degrees of fibrosis and severity of hepatitis, and may reflect hepatic basement membrane metabolism. But the serum assays have not been linked to disclosing the anatomical sites and lobular distribution of perisinusoidal basement membrane formation in the liver. Hepatic sinusoids normally lack a basement membrane, although Col IV is a normal matrix component of the space of Disse. In liver disease, laminin deposits in the space of Disse and codistributes with Col IV, forming a perisinusoidal basement membrane. Concomitantly, the sinusoidal endothelium loses its fenestrae and is transformed into vascular type endothelium. These changes lead to capillarization of hepatic sinusoids, a significant pathology that impairs hepatic function. Accordingly, codistribution of Col IV and laminin serves as histochemical marker of perisinusoidal basement membrane formation in liver disease. Anat Rec, 300:1371–1390, 2017. © 2017 Wiley Periodicals, Inc.     

Changes in Laminin Chain Expression in Pre- and Postnatal Rat Pituitary Gland

Cell–matrix interaction is required for tissue development. Laminin, a major constituent of the basement membrane, is important for structural support and as a ligand in tissue development. Laminin has 19 isoforms, which are determined by combinational assembly of five α, three β, and three γ chains (eg, laminin 121 is α1, β2, and γ1). However, no report has identified the laminin isoforms expressed during pituitary development. We used in situ hybridization to investigate all laminin chains expressed during rat anterior pituitary development. The α5 chain was expressed during early pituitary development (embryonic day 12.5–15.5). Expression of α1 and α4 chains was noted in vasculature cells at embryonic day 19.5, but later diminished. The α1 chain was re-expressed in parenchymal cells of anterior lobe from postnatal day 10 (P10), while the α4 chain was present in vasculature cells from P30. The α2 and α3 chains were transiently expressed in vasculature cells and anterior lobe, respectively, only at P30. Widespread distribution of β and γ chains was also observed during development. These findings suggest that numerous laminin isoforms are involved in anterior pituitary gland development and that alteration of the expression pattern is required for proper development of the gland.     

Laminin and integrin expression in the ventral ectodermal ridge of the mouse embryo: Implications for regulation of BMP signalling

Background: The ventral ectodermal ridge (VER) is an important signalling centre in the mouse tail‐bud following completion of gastrulation. BMP regulation is essential for VER function, but how these signals are transmitted between adjacent tissues is unclear. Results: We investigated the idea that extracellular matrix components might be involved, using immunohistochemistry and in situ hybridisation to detect all known α, β, and γ laminin chains and their mRNAs in the early tail bud. We identified an apparently novel laminin variant, comprising α5, β3 and γ2 chains, as a major component of the VER basement membrane at E9.5. Strikingly, only the mRNAs for these chains were co‐expressed in VER cells, suggesting that lamin532 may be the sole basement membrane laminin at this stage. Since α6 integrin was also expressed in VER cells, this raises the possibility of cell‐matrix interactions regulating BMP signalling at this site of caudal morphogenesis. Conclusions: Laminin532 could interact with α6‐containing integrin to direct differentiation of the specialised VER cells from surface ectoderm. Developmental Dynamics 241:1808–1815, 2012. © 2012 Wiley Periodicals Inc.     

Basement membrane continuity in benign, premalignant and malignant epithelial conditions of the uterine cervix

The pattern of basement membrane deposition in the uterine ectocervix was assessed in benign, premalignant and malignant conditions, using an indirect immunoperoxidase technique to detect laminin, an intrinsic basement membrane component. A semi-quantitative approach was used to assess the frequency of small breaks in the basement membrane. The cervical squamous epithelium in benign epithelial conditions has an almost completely continuous basement membrane, but the development of viral atypia or cervical intraepithelial neoplasia (CIN) is associated with the appearance of small basement membrane breaks. There is a correlation between increasingly severe CIN and increasing numbers of breaks, and there is a concurrent increase in the numbers of subepithelial inflammatory cells. The development of invasive neoplasm is associated with a sudden change to a fragmented pattern of basement membrane deposition, a finding which is of potential diagnostic use.     

Deconstructing B cell tolerance to basement membranes

Basement membrane antigens are frequent targets of autoantibody attack in systemic and organ-restricted autoimmunity. These specialized and highly organized matrices are composed of multiple components with restricted tissue distributions and limited epitope exposure. To dissect mechanisms controlling humoral autoimmunity to nephritogenic basement membrane antigens, we developed autoantibody transgenic models. In mice bearing the LamH Ig transgene encoding B cell receptors specific for laminin, autoreactive B cells are readily generated but actively regulated in vivo. In this model, anti–laminin B cells are immunologically censored by mechanisms that include central deletion, κ light-chain editing, and anergy. Tolerance is maintained when the transgene is established in MRL and BXSB genetic backgrounds with inherited autoimmune susceptibility, and despite provocation with potent environmental stimulants. Collectively, these studies indicate that the pathogenic anti-laminin reactivity characteristic of systemic lupus is tightly regulated. A novel anti-collagen transgenic model is used to assess the tolerogenesis of a structurally distinct pathogenic basement membrane epitope and to determine if reactivity to putative cryptic epitopes targeted in organ-restricted disease is regulated. These studies should provide insight into the molecular mechanisms controlling basement membrane autoreactivity and ultimately facilitate the development of novel strategies to inactivate autoreactive cells and treat autoimmune disease.     

Loss of types XV and XIX collagen precedes basement membrane invasion in ductal carcinoma of the female breast

Ductal and lobular carcinomas comprise most malignancies of the female breast and the morbidity and mortality associated with breast cancer. During the progression from in situ to invasive stages, tumour cells penetrate the epithelial and vascular basement membranes (BM) to realize full metastatic potential. While the definition of these structures has primarily resulted from analysis of laminin and type IV collagen, characterization of newly discovered BM/BM zone (BMZ) proteins will further elucidate the interactions between tumour cells and the host stroma. We have studied the expression of two non-fibrillar BMZ collagens, the type XV proteoglycan and collagen XIX, in breast cancer where a linear, well-formed BM becomes fragmented and even lost in the progression of epithelial malignancy. In the normal breast, types XV and XIX were found in all BMZ: epithelial, muscle, neural, endothelial, and fat. In in situ lesions, these two collagens, and particularly type XV, were often absent from the BM/BMZ displaying a continuous or just focally disrupted type IV/laminin staining pattern. In contrast, infiltrating ductal carcinomas showed only rare traces of laminin and collagen IV reactivity adjacent to the glands and tumour nests, and similarly there was little if any evidence of types XV and XIX collagen. All four molecules were, however, detected in the interstitium associated with some of the invasive carcinomas. The data suggest that types XV and XIX collagen are lost early in the development of invasive tumours, prior to penetration and eventual dissolution of the epithelial BM. Disappearance of these proteins from the BM/BMZ may signal remodelling of the extracellular matrix to promote tumour cell infiltration.     

Codistribution of Collagen Type IV and Laminin in Liver Fibrosis of Elderly Cadavers: Immunohistochemical Marker of Perisinusoidal Basement Membrane Formation

Liver sinusoids are lined by a fenestrated endothelium that lacks a basement membrane. Formation of perisinusoidal basement membranes beneath the endothelium is an integral feature of capillarization of sinusoids that is a significant pathology found in advanced fibrosis. Liver fibrosis is prevalent in elderly cadavers; however, basement membrane formation in these liver samples has yet to be studied. Collagen type IV and laminin are major basement membrane proteins and their codistribution around sinusoids provides an immunohistochemical marker of basement membrane formation. Here, we examined the intralobular sites of perisinusoidal basement membrane formation in elderly cadaveric livers having various stages of fibrosis. Collagen IV and laminin codistributed in basement membranes of portal and septal ductular and vascular structures, providing a positive control. In the parenchyma, collagen IV immunostaining of sinusoids was panlobular in all stages of fibrosis, and the stain was continuous along the sinusoids. In contrast, laminin was not detected in livers, showing minimal fibrotic change. It was rarely seen in perisinusoidal/pericellular fibrosis, but frequently in septa formation, bridging fibrosis, and cirrhosis. The laminin stain was patchy, occurring principally in sinusoids of periportal and periseptal areas, less commonly in mid‐lobular and rarely in centrilobular areas. Consecutive sections revealed that laminin codistributed with collagen IV in these sinusoidal locations, thus marking the sites of perisinusoidal basement membrane formation in aged fibrotic livers. This development is presumably related to aging of the liver and exacerbated by liver injury caused by advanced liver fibrosis, possibly resulting in sinusoidal capillarization. Anat Rec, 296:953–964, 2013. © 2013 Wiley Periodicals, Inc.     

Distribution of laminins in the basement membranes of the upper gastrointestinal tract and Barrett's oesophagus

Barrett's oesophagus predisposes to oesophageal adenocarcinoma. In vitro, laminin, a component of the epithelial basement membrane (BM), is important in regulation of cell differentiation. There is limited information on the distribution of laminin chains in the upper gastrointestinal tract (GIT) and none in Barrett's oesophagus. This study aimed to investigate qualitatively the distribution of laminins in the normal upper GIT mucosa and Barrett's oesophagus in order to understand the role of laminins in metaplasia. Immunoperoxidase staining for laminin chains alpha1, alpha2, alpha3, alpha5, beta1, beta2, beta3, gamma1, and gamma2 was performed on frozen endoscopic squamous and Barrett's oesophageal biopsies and surgical resection specimens from squamous oesophagus (in resection specimens for oesophageal cancer), and in oesophageal and gastric biopsies from control subjects. alpha1 laminin was expressed in the BM of submucosal glands and ducts in squamous oesophagus and Brunner's glands in the duodenum, but not in Barrett's oesophagus or elsewhere in the upper GIT. alpha2 laminin chain was expressed in a granular distribution in the BM of squamous epithelium. In columnar epithelium, including Barrett's oesophagus, alpha2 laminin chain was expressed continuously in the BM of glands and deeper pits, but expression was reduced and granular in the surface epithelial BM. beta2 laminin was continuous in squamous epithelial BM, but in Barrett's and cardia, gastric body, and duodenum, it was expressed faintly in the surface but continuously in the BM of glands and deeper pits. The constituents of laminin-5 were continuously expressed in the BM of squamous epithelium, but in the cardia, gastric body, duodenum, and Barrett's, they were expressed only in the BM of surface epithelium, with a sharp decline in the glandular and deeper pit BM. Site-specific distribution of the alpha2 and beta2 laminin chains may therefore have an important role in Barrett's metaplasia. However, the absence of alpha1 laminin in Barrett's mucosa suggests that this is unlikely to play an important role in columnar metaplasia.     

Histological and biological characteristics of microinvasion in mammary carcinomas ≤ 2 cm in diameter

Fifty-two mammary carcinomas, 2 cm or less in diameter, were examined in order to clarify the morphology and biology of microinvasion. The morphological characteristics of microinvasion of carcinomas include: (i) a loss of myoeplthelial cells and a rupture with concomitant loss of collagen IV and lamlnin in the basement membrane of involved mammary glands; and (ii) budding of carcinomas from the rupture into the stroma. When microinvasion was defined as a rupture of < 200 pm In the basement membrane with invasion, the number of microinvasions per 1 mm of basement membrane was larger in the tumors in which the area of invasion was larger. The prevalence of microinvasion showed a significant correlation with lymph node metastasis and the rate of histological deviation, while no correlation of expression of either estrogen receptors or progesterone receptors and c-erbB-2 protein was found. The study clarified that the early invasion of mammary carcinomas could be detected by the immunohistochemical method using anti-smooth muscle actin, laminin and collagen IV antibodies. The study also suggested that microinvasion might be an indicator of lymph node metastasis in mammary carcinomas ≤ 2 cm diameter.     

Pericellular deposition of basement membrane material in myxoid meningioma: Immunohistochemical evidence for unbalanced production of type IV collagen and laminin

Myxold meningioma seen In a 25-year-old man is presented. Hlstologlcally, Leu 7-positive meningotheliomatous tumor cells were embedded in the alclanophilic myxoid matrix. Characteristically, eosinophilic granular deposition was detected around the tumor cells and the boundary of tumor cells was not clearly defined. The pericellular deposits revealed the nature of the basement membrane with positive reactions by periodic acid-Schlff (PAS) sequence and immunostaining for type IV collagen, which is the major structural component of basement membrane. However, laminin, which is a non-collagenous glycoprotein of the basement membrane, was undetectable, and silver was not impregnated. Similar abnormal deposition of PAS-positive basement membrane-like material was observed in the myxoid stroma of a microcystic meningioma among 72 meninglomas additionally examined. The significance of the discrepant localization of immunoreactive type IV collagen and laminin is discussed.     

人胎盘层粘连蛋白单克隆抗体的制备与鉴定

目的获得能特异性识别人层粘连蛋白（hLN）的单克隆抗体（McAb）。方法用纯化的人层粘连蛋白（hLN）作抗原免疫Balb/C小鼠,以细胞融合、酶联免疫吸附实验（ELISA）筛选和克隆化技术获得抗hLN的杂交瘤细胞株;用生物学方法鉴定杂交瘤细胞,用免疫学方法鉴定McAb的特异性。结果获得4株稳定分泌抗人LN的杂交瘤细胞株（2A1、3B5、2C4、4D1）,培养上清的ELISA效价分别为：1∶512、1∶1024、1∶512、1∶256;腹水效价分别为：1×10^6、1×10^7、1×10^6、1×10^5;采用ELISA相加法表明2A1、4D1与3B5、2C4识别的hLN上的抗原决定簇和识别的不同,4株单抗均属实IgG。结论成功建立了4株稳定分泌抗人LNMcAb的杂交瘤细胞株,它们分别识别hLN上2个不同的抗原位点,有望作为hLN定量检测的特异性抗体。     

Mucinous biliary papillomatosis: a tumour in need of wider recognition

We report three cases of mucinous biliary papillomatosis occurring high in the extrahepatic bile ducts. Histological assessment of malignancy is difficult and subjective because there is no unequivocal evidence of stromal invasion. Using anti-laminin antibodies to assess the basement membranes of these tumours we have found a large number of discontinuities in the epithelial basement membrane. On this basis we consider that mucinous biliary papillomatosis should be considered a low-grade malignancy. This would correlate with the natural history of these tumours, namely recurrence but with no widespread metastasis, death resulting from liver failure usually with 5-6 years of presentation.     

Changes in vascular basement membrane in the endometrium of Norplant users

Progestogen-only contraception is almost invariably associated with changes in menstrual bleeding patterns. Changes in the endometrial vasculature, and in particular an increase in vascular fragility, may contribute to this bleeding. In this study, endometrial vascular density and endothelial cell basement membrane components were examined using immunohistochemistry before and after insertion of Norplant. Endometrial vascular density was increased from a mean (+/- SEM) of 189.6 +/- 7.0 vessels/mm2 during the control cycle to 253.9 +/- 80.7 vessels/mm2 at 2-13 weeks of Norplant exposure, and to 212.7 +/- 12.9 vessels/mm2 at 14-42 weeks. During the control cycle, a mean of 161.4 +/- 4.5 vessels/mm2 stained for collagen IV (85% of all vessels), while at 2-13 weeks, 144.5 +/- 13.0 vessels/mm2 stained for collagen IV (57% of all vessels) (t ratio = 2.08, P = 0.0057). By 14-42 weeks, 71% of vessels (151.0 +/- 9.8) vessels/mm2 were surrounded by collagen IV. This was not significantly different from control values (t ratio = 2.03). Endometrial vascular laminin was also reduced following Norplant insertion, from a mean of 176.0 +/- 4.2 vessels/mm2 in the control cycle (93% of vessels), to 156.3 +/- 6.7 vessels/mm2 at 2-13 weeks of exposure (57% of vessels) (t ratio = 2.08, P = 0.01). By 14-42 weeks of exposure to Norplant, 162.5 +/- 9 vessels/mm2 (76%) stained for laminin. This was not significantly different from control values (t ratio = 2.04). Endometrial vascular heparan sulphate proteoglycan (HSPG) was reduced from 58.6 +/- 3.0 vessels/mm2 during the control cycle (31% of vessels) to 43.6 +/- 5.6 vessels/mm2 (only 17% of vessels) at 2-13 weeks (t ratio = 2.08, P = 0.025). At 14-42 weeks, only 19% of vessels stained for HSPG (41.3 +/- 5.8 vessels/mm2; t ratio = 2.04, P = 0.009).     

Neoexpression of the epithelial adhesion complex antigens in thyroid tumours is associated with proliferation and squamous differentiation markers

Integrin dimer α₆β₄ is a transmembrane component of an epithelial cell adhesion complex that consists of hemidesmosomes (HDs), basement membrane (BM)-associated laminin-5 (Ln-5), and anchoring filaments/type VII collagen, all of which are absent from normal thyroid follicular epithelium. In the present study, the expression of epithelial cell adhesion complex antigens in thyroid tumours was investigated using immunohistochemistry. In addition to integrin subunits α₆ and β₄, immunoreactivity was found for all chains of Ln-5, α3, β3 and γ2, type VII collagen and hemidesmosomal antigen, HD1, in most thyroid carcinomas associated with tumour anaplasia and papillary growth pattern and located at the border of parenchymal cells and connective tissue or blood vessel walls. In addition, a more restricted expression of bullous pemphigoid antigens 180 and 230 (BP180 and BP230), constituents of HDs, was found in some papillary and anaplastic carcinomas and atypical adenomas. Adhesion complex antigens were located to regions of cells which were immunoreactive for cytokeratin (ck)-5 and proliferating cell nuclear antigen Ki-67. The results suggest that in thyroid carcinomas, the emergence of adhesion complex antigens is associated with squamous differentiation and high proliferative activity. © 1998 John Wiley & Sons, Ltd.     

Integrin α6 expression in human prostate carcinoma cells is associated with a migratory and invasive phenotypein vitro andin vivo

Cell adhesion and migration are important features in tumor invasion, being mediated in part by integrins (extracellular matrix receptors). Integrins are significantly decreased in human prostate cancer. An exception is 6 integrin (laminin receptor) which persists during prostate tumor progression. We have selected high (DU-H) and low (DU-L) expressors of 6 integrin from a human prostate tumor cell line, DU145, to assess experimentally the importance of 6 integrin in tumor invasion. DU-H cells exhibited a four-fold increased expression of 6 integrin on the surface compared to DU-L cells. Both cell types contained similar amounts of 3 and 5 integrin. The DU-H cells contained 6 subunits complexed with both the 1 and 4 subunits whereas DU-L cells contained 6 complexed only with 4. DU-H cells were three times more mobile on laminin as compared to DU-L, but adhered similarly on laminin. Adhesion and migration were inhibited with anti-6 antibody. Each subline was injected intraperitoneally into SCID mice to test its invasive potential. Results showed greater invasion of DU-H compared to DU-L cells, with increased expression of a6 integrin on the tumor at the areas of invasion. These data suggest that 6 integrin expression is advantageous for prostate tumor cell invasion.     

MICROVASCULAR ANGIOPATHY IN ADVANCED PERIODONTAL DISEASE

Previous studies have shown a perivascular hyaline thickening affecting restricted regions of the microcirculation in gingivitis and moderate periodontitis and in the pulpal vessels in chronic pulpitis. In the present study of the lesion of advanced periodontitis, immunostaining for type IV collagen and laminin demonstrated widespread deposition of basement membrane material, with manifest involvement of the venous network. Some vessels were associated with an increased deposition of both basement membrane proteins, while others showed preferential deposition of either laminin or type IV collagen. Immunostaining also revealed an extensive trabecular network of type IV collagen throughout the affected gingival tissue that was not related to recognizable vessels but was co-extensive with less intense staining for laminin. This network was not associated with viable endothelial cells demonstrable by staining with the endothelial marker Ulex agglutinin (UEA-1). The results indicate extensive vascular pathology in advanced periodontitis that could explain the attenuation of the inflammatory reaction and the restricted ability to develop reparative granulation tissue in this disease.