Distribution of the alpha1 to alpha6 chains of type IV collagen in bovine follicles

During follicular development the proliferative and differentiated state of the epithelioid granulosa cells changes, and the movement of fluid across the follicular basal lamina enables the formation of an antrum. Type IV collagen is an important component of many basal laminae. Each molecule is composed of three alpha chains; however, six different type IV collagen chains have been identified. It is not known which of these chains are present in the follicular basal lamina and whether the type IV collagen composition of the basal lamina changes during follicular development. Therefore, we immunolocalized each of the six chains in bovine ovaries using antibodies directed to the nonconserved non-collagenous (NC) domains. Additionally, dissected follicles were digested with collagenase to release the NC domains, and the NC1 domains were then detected by standard Western immunoblot methods. The follicular basal lamina of almost all primordial and preantral follicles was positive for all type IV collagen alpha chains. Colocalization of type IV collagen and factor VIII-related antigen allowed for discrimination between the follicular and endothelial basal laminae. Type IV collagen alpha1, alpha2, alpha3, alpha4, and alpha5 chains were present within the follicular basal lamina of only a proportion of antral follicles (17 of 22, 20 of 21, 15 of 18, 14 of 28, and 12 of 23, respectively), and staining was less intense than in the preantral follicles. Staining for the alpha1 and alpha2 chains was diffusely distributed throughout the theca in regions not associated with recognized basal laminae. The specificity of this immunostaining for alpha1 and alpha2 chains of type IV collagen was confirmed by Western immunoblots. As well as being detected in the basal lamina of approximately half of the antral follicles examined, type IV collagen alpha4 also colocalized with 3beta-hydroxysteroid dehydrogenase-immunopositive cells in the theca interna. Type IV collagen alpha6 was detected in the basal lamina of only one of the 16 antral follicles examined. Thus, the follicular basal lamina changes in composition during follicular development, with immunostaining levels being reduced for all type IV collagen chains and immunoreactivity for type IV collagen alpha6 being lost as follicle size increases. Additionally, immunoreactivity for alpha1 and alpha2 appears in the extracellular matrix of the theca as it develops.     

Differential expression of alpha 1(IV), alpha 2(IV), alpha 5(IV) and alpha 6(IV) collagen chains in the basement membrane of basal cell carcinoma

Type IV collagen, the major component of basement membrane, consists primarily of alpha 1(IV) and alpha 2(IV) chains. Recently, other types of collagen IV chains, i.e. alpha 3(IV), alpha 4(IV), alpha 5(IV) and alpha 6(IV) chains, have been identified by protein chemistry and molecular cloning. We have examined the diversity of the assembly of alpha (IV) chains of the basement membrane surrounding tumour nests of basal cell carcinomas, in tissues from 11 patients, by immunohistochemical analysis using specific monoclonal antibodies to six alpha (IV) chain. The immunostaining profile of each chain differed with respect to the histological subtypes of basal cell carcinoma. In the morphea-like subtype, which was more invasive, alpha 1(IV) and alpha 2(IV) chains were discontinuously stained, and alpha 5(IV) and alpha 6(IV) chains were entirely absent. However, in the superficial subtype, which was non-aggressive, alpha 1(IV), alpha 2(IV), alpha 5(IV) and alpha 6(IV) chains were well stained compared with the other subtypes of basal cell carcinoma. In addition, in the solid subtype, which showed slow growth and ulceration, alpha 1(IV) and alpha 2(IV) chains were continuously stained, and alpha 5(IV) and alpha 6(IV) chains were discontinuous or absent. The assembly of alpha 5(IV) and alpha 6(IV) chains into the basement membrane was inhibited in the solid and morphea subtypes of BCC. This differential expression of type IV collagen chains seems to be associated with the invasive potential of basal cell carcinoma.     

Alterations of corneal extracellular matrix after multiple refractive procedures: a clinical and immunohistochemical study

PURPOSE: To describe the clinical course and alterations of the corneal extracellular matrix (ECM) and basement membrane (BM) in a cornea after hexagonal keratotomy, transverse keratotomies, and keratomileusis. METHODS: Frozen sections of this cornea and of 12 normal corneas were studied by immunofluorescence with specific antibodies. The patient history was analyzed to allow a clinical correlation. RESULTS: In the treated cornea, keratotomy scars and subepithelial fibrosis with neovascularization were seen. Around and beneath the epithelial plugs and along the keratotomy scars, deposits of types III, VI, VIII, and XIV collagen; fibrillin-1; fibronectin; and tenascin-C were found, together with short streaks of types IV (alpha 1-alpha 2) and VII collagen, laminin-1 and -5, entactin, and perlecan. alpha 3-alpha 4 Type IV collagen chains were abnormally absent from the BM around the epithelial plugs. At the edges of the keratomileusis flap, subepithelial fibrosis areas were found, with abnormal deposits of eight different collagen types, perlecan, fibronectin, fibrillin-1, and tenascin-C. The major part of the flap interface did not show ECM abnormalities. ECM alterations outside the scarred areas included the appearance of tenascin-C in the stroma and of alpha 1-alpha 2 type IV collagen in the epithelial BM, and the disappearance of fibronectin from Descemet's membrane. CONCLUSION: Five years after surgery, the treated cornea still presented BM abnormalities at sites of keratotomy scars and epithelial plugs. Several ECM components were abnormally expressed outside the scarred areas, consistent with an ongoing fibrosis in the treated cornea.     

Distribution and function of laminins in the neuromuscular system of developing, adult, and mutant mice

Laminins, heterotrimers of alpha, beta, and gamma chains, are prominent constituents of basal laminae (BLs) throughout the body. Previous studies have shown that laminins affect both myogenesis and synaptogenesis in skeletal muscle. Here we have studied the distribution of the 10 known laminin chains in muscle and peripheral nerve, and assayed the ability of several heterotrimers to affect the outgrowth of motor axons. We show that cultured muscle cells express four different alpha chains (alpha1, alpha2, alpha4, and alpha5), and that developing muscles incorporate all four into BLs. The portion of the muscle's BL that occupies the synaptic cleft contains at least three alpha chains and two beta chains, but each is regulated differently. Initially, the alpha2, alpha4, alpha5, and beta1 chains are present both extrasynaptically and synaptically, whereas beta2 is restricted to synaptic BL from its first appearance. As development proceeds, alpha2 remains broadly distributed, whereas alpha4 and alpha5 are lost from extrasynaptic BL and beta1 from synaptic BL. In adults, alpha4 is restricted to primary synaptic clefts whereas alpha5 is present in both primary and secondary clefts. Thus, adult extrasynaptic BL is rich in laminin 2 (alpha2beta1gamma1), and synaptic BL contains laminins 4 (alpha2beta2gamma1), 9 (alpha4beta2gamma1), and 11 (alpha5beta2gamma1). Likewise, in cultured muscle cells, alpha2 and beta1 are broadly distributed but alpha5 and beta2 are concentrated at acetylcholine receptor-rich "hot spots," even in the absence of nerves. The endoneurial and perineurial BLs of peripheral nerve also contain distinct laminin chains: alpha2, beta1, gamma1, and alpha4, alpha5, beta2, gamma1, respectively. Mutation of the laminin alpha2 or beta2 genes in mice not only leads to loss of the respective chains in both nerve and muscle, but also to coordinate loss and compensatory upregulation of other chains. Notably, loss of beta2 from synaptic BL in beta2(-/-) "knockout" mice is accompanied by loss of alpha5, and decreased levels of alpha2 in dystrophic alpha2(dy/dy) mice are accompanied by compensatory retention of alpha4. Finally, we show that motor axons respond in distinct ways to different laminin heterotrimers: they grow freely between laminin 1 (alpha1beta1gamma1) and laminin 2, fail to cross from laminin 4 to laminin 1, and stop upon contacting laminin 11. The ability of laminin 11 to serve as a stop signal for growing axons explains, in part, axonal behaviors observed at developing and regenerating synapses in vivo.     

Differential localization of laminin chains in bovine follicles

The composition of a basal lamina markedly affects its ability to filter material and affects the fate of adjacent epithelial cells. Therefore, basal laminae differ in composition with tissue development, and between different tissues in the body. Laminins are a component of basal laminae and consist of one alpha, one beta and one gamma chain, of which there are at least five, three and two isoforms, respectively. This is the first study to immunolocalize a range of these individual laminin chains (alpha 1, alpha 2, beta 1, beta 2, gamma 1) in ovarian follicles. Frozen sections of bovine ovaries (n = 6) were immunostained using specific antisera to laminin chains and factor VIII-related antigen (to identify endothelial cells). Secondary antisera were labelled with one of two different fluorochromes (DTAF and Cy3), and dual localization of laminin chains and factor VIII-related antigen was performed. The alpha 1, beta 2 and gamma 1 chains were consistently localized to the follicular basal lamina in all healthy follicles. Staining was less intense in the atretic antral follicles. Conversely, alpha 2 and beta 1 were rarely present in the follicular basal laminae of healthy antral follicles. Two of nine healthy antral follicles observed stained weakly for alpha 2 in their basal lamina, and beta 1 was present at low concentrations in growing preantral follicles. In atretic antral follicles, the follicular basal lamina stained positively for alpha 1, alpha 2, and beta 2 but no beta 1 was detected and the gamma 1 staining was less intense than in healthy follicles. Antisera to Englebreth Holm-Swarm tumour laminin stained basal laminae of all follicles. In the theca of antral follicles, beta 1 and beta 2 chains were both present in the vasculature. Staining for the gamma 1 chain was present in the thecal vasculature and generally throughout the theca of healthy and atretic antral follicles. Therefore, the composition of the follicular basal lamina alters during development and atresia, and potentially plays a role in the changing identity of the granulosa cells and the accumulation of antral follicular fluid.     

Quantitative analysis of Na+ and Ca2+ current contributions on spike initiation in the newt olfactory receptor cell

The developmental localization patterns of collagen type IV alpha1-5 chains, laminin-1, laminin-5, and laminin alpha2 chain were analyzed in the embryonic mouse eye using isoform specific antibodies and immunofluorescence microscopy. Laminin-1 isoform and alpha1-2(IV) were ubiquitously expressed along the ocular surface basement membranes at a very early stage of eye development. Alpha3-5(IV) were first detected at later stages of development, and exhibited a variable distribution pattern along the ocular surface basement membrane. In contrast, expression of the laminin alpha2 chain was restricted to the conjunctival basement membrane, and was first detected during the same developmental period in which keratin K4-positive, differentiated conjunctival epithelial cells were observed. Although laminin-5 was uniformly expressed along the adult ocular surface basement membrane, during embryogenesis it was first incorporated into the conjunctival basement membrane structure. These data suggest that some of the laminin isoforms, including laminin alpha2 and laminin-5, may play a role in the formation of a conjunctival-type basement membrane. The temporal relationship between the localization of these molecules to the conjunctival basement membrane and the appearance of differentiated conjunctival epithelial cells suggests a role for external influence on the differentiation pathways of ocular surface epithelium.     

Roles for laminin in embryogenesis: exencephaly, syndactyly, and placentopathy in mice lacking the laminin alpha5 chain

Laminins are the major noncollagenous glycoproteins of all basal laminae (BLs). They are alpha/beta/gamma heterotrimers assembled from 10 known chains, and they subserve both structural and signaling roles. Previously described mutations in laminin chain genes result in diverse disorders that are manifested postnatally and therefore provide little insight into laminin's roles in embryonic development. Here, we show that the laminin alpha5 chain is required during embryogenesis. The alpha5 chain is present in virtually all BLs of early somite stage embryos and then becomes restricted to specific BLs as development proceeds, including those of the surface ectoderm and placental vasculature. BLs that lose alpha5 retain or acquire other alpha chains. Embryos lacking laminin alpha5 die late in embryogenesis. They exhibit multiple developmental defects, including failure of anterior neural tube closure (exencephaly), failure of digit septation (syndactyly), and dysmorphogenesis of the placental labyrinth. These defects are all attributable to defects in BLs that are alpha5 positive in controls and that appear ultrastructurally abnormal in its absence. Other laminin alpha chains accumulate in these BLs, but this compensation is apparently functionally inadequate. Our results identify new roles for laminins and BLs in diverse developmental processes.     

Oxidant-antioxidant imbalance in the experimental interstitial lung disease induced in sheep by visna-maedi virus

X-linked Alport syndrome (AS) is a heritable disorder which is associated with mutations in the type IV collagen alpha 5 (IV) chain gene (COL4A5) located on chromosome X. Following renal transplantation, an average of 6% of male AS patients develop anti-GBM nephritis. We studied the specificity of the antibodies against type IV collagen in the serum of a patient with COL4A5 partial deletion. The specificity of these alloantibodies was determined against collagenase-digested GBM, as well as against recombinant non-collagenous (NC1) domains of the type IV collagen alpha 1(IV)-alpha 6(IV) chains expressed in escherichia coli. Immunoblotting and ELISA demonstrated that these antibodies bound specifically to the NC1 domain of alpha 5(IV) collagen. There was no binding to the NC1 domain of the other chains, including the Goodpasture antigen. Competitive ELISA confirmed the results obtained by ELISA and immunoblotting. This patient developed alloantibodies directed against antigens present in the grafted kidney, but absent from his Alport kidney. The pathogenesis of post-transplantation glomerulonephritis in the Alport patient studied is thus similar to that of Goodpasture syndrome, with the exception that the pathogenic antibodies are targeted to another alpha chain of type IV collagen.     

Sickle cell adhesion to laminin: potential role for the alpha5 chain

Sickle red blood cell (RBC) adhesion to the endothelium and to exposed, underlying subendothelial proteins is believed to contribute to vascular occlusion in sickle cell disease. Laminin, a major component of the subendothelium, supports significant adhesion of sickle, but not normal RBCs. The purpose of this study was to define the adhesive region for sickle RBCs within a human laminin preparation using a flow adhesion assay designed to mimic physiologic flow through postcapillary venules. Because sickle RBCs did not adhere to the common laminin contaminants entactin or collagen type IV, neither of these proteins are likely to contribute to the observed adhesion to laminin. Known adhesive regions of laminin neither supported nor inhibited sickle RBC adhesion to laminin, suggesting a mechanism of adhesion previously uncharacterized in other laminin adhesion studies. Moreover, sickle RBCs did not adhere to mouse EHS laminin or to human laminin-2 (merosin), eliminating the alpha1, alpha2, beta1, and gamma1 chains as mediators of sickle cell adhesion. The monoclonal antibody 4C7, which binds at or near the G-domain of the laminin alpha5 chain, significantly inhibited sickle RBC adhesion. These results suggest that an adhesive region for sickle RBCs is contained within the laminin alpha5 chain.     

Secular trend in body height since the Neolithic period

Rat mesangial cells express two unique isoforms of laminin which can be modulated by culture medium composition. To define further the nature of laminin expressed by cultured rat mesangial cells, synthesis of individual laminin chains, as well as their trimeric association, was examined. Based on data from Northern analysis of mRNA expression, immunoblots, immunofluorescence staining and radioimmunoprecipitation of biosynthetically labeled proteins, mesangial cells express laminin beta1, beta2, and gamma1 chains. Mesangial cells do not express laminin alpha1 or alpha2. MC produce a unique alpha chain, designated alpha'm. These laminin chains assemble into two major isoforms. One contains alpha'mbeta1gamma1, co-precipitates with entactin and is assembled into the fibrillar extracellular matrix. The second isoform contains alpha'mbeta2 and a presumed gamma chain that migrates in gel slightly ahead of gamma1. The beta2-containing isoform is concentrated in punctate sites on the cell surface. In addition, mesangial cells display different phenotypes when plated on laminin-1 (alpha1beta1gamma1), as compared to purified beta2. An LRE-containing peptide of laminin beta2 serves as an attachment site for mesangial cells and is sufficient to induce the phenotype observed with intact beta2. These data suggest that laminin isoform expression plays an important role in mesangial cell phenotype and function.     

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.     

Laminin interactions important for basement membrane assembly are promoted by zinc and implicate laminin zinc finger-like sequences

Laminin is an abundant basement membrane (BM) glycoprotein which regulates specific cellular functions and participates in the assembly and maintenance of the BM superstructure. The assembly of BM is believed to involve the independent polymerization of collagen type IV and laminin, as well as high affinity interactions between laminin, entactin/nidogen, perlecan, and collagen type IV. We report here that Zn2+ can influence laminin binding activity, in vitro. Laminin contains 42 cysteine-rich repeats of which 12 contained nested zinc finger consensus sequences. Recently, the entactin binding site was mapped to one of these zinc finger-containing repeats on the laminin gamma chain (Mayer, U., Nischt, R., Poschl, E., Mann, K., Fukuda, K., Gerl, M., Yamada, Y., and Timpl, R. (1993) EMBO J. 12, 1879-1885). Based on these observations, the effect of a series of essential ions (Ca2+, Cd2+, Cu2+, Mg2+, Mn2+, and Zn2+) on laminin binding activity was evaluated. Zn2+ was found to be the most effective at enhancing laminin-entactin and laminin-collagen type IV binding. Laminin-bound Zn2+ was detected by flame atomic absorption spectroscopy at a maximum of 8 mol/mol of laminin. Furthermore, Ca2+-dependent laminin polymerization was unaffected by Zn2+, an observation consistent with the lack of zinc finger-containing repeats in the terminal globular domains required for polymerization. We conclude that Zn2+-laminin complexes may generate high affinity binding sites which contribute to BM cross-linking important for its assembly and homeostasis. Zinc is likely a cofactor for 2 kinds of cross-linking interactions; one involving direct binding between laminin and collagen type IV and the other a ternary complex of laminin-entactin-collagen type IV.     

Spatiotemporal patterns of expression of extracellular matrix molecules in the developing and adult rat olfactory system

Using immunocytochemical methods, we have examined extensively the spatial and temporal patterns of expression of three extracellular matrix molecules-laminin, fibronectin, and type IV collagen-in the embryonic, postnatal (days 2 and 11) and adult rat olfactory system. The study started at embryonic day 14 when olfactory fibres and their associated migrating cells course through the nasal mesenchyme. From embryonic day 14 to the adult, a sheet-like pattern of labelling for laminin, fibronectin and type IV collagen was observed along the basal surface of the olfactory epithelium and around the telencephalon. This type of labelling was continuous around the telencephalic vesicle, whereas it appeared disrupted in the basal lamina of the olfactory epithelium to permit exit of the olfactory axons and their associated migrating cells into the mesenchyme. From embryonic day 14 to day 20, punctate labelling for the three molecules studied was observed along the mesenchymal olfactory pathway, the ventral part of the olfactory bulb, the olfactory nerve layer and the presumptive glomerular layer, respectively. By embryonic day 17, the punctate labelling initially detected in the mesenchymal olfactory pathway was replaced by a sheet-like pattern related to the mature basal lamina surrounding the olfactory axon fascicles. Punctate labelling for laminin and type IV collagen persisted in the olfactory nerve layer and around the glomeruli through adult life whereas that of fibronectin declined and disappeared by postnatal day 2. The spatiotemporal distribution of the punctate pattern for laminin, fibronectin and type IV collagen observed in the embryonic olfactory system suggests a role in delineating the pathway for olfactory axon elongation. The continuous expression of laminin and type IV collagen in the adult olfactory bulb may be related to the regenerative activity and high plasticity of the olfactory system.     

Differential expression of five laminin alpha (1-5) chains in developing and adult mouse kidney

The nature of the laminin alpha chains in the embryonic and adult kidney is still being debated. The present study attempted to clarify this issue by immunofluorescence study using monoclonal antibodies against mouse alpha1, alpha2, and alpha5 chains and in situ hybridization for the alpha2, alpha3B, alpha4, and alpha5 mRNAs. Novel alpha1 chain-specific monoclonal antibodies against E8 fragment revealed a restricted distribution of alpha1 chain in a subset of epithelial basement membranes in the embryo, in agreement with previous mRNA data. The alpha2 mRNA was produced by mesenchyme, although the protein was deposited in epithelial basement membranes. The alpha3B mRNA was found only in a small subset of endothelial cells. The alpha4 mRNA was found transiently in embryonic mesenchyme, with particularly high levels in condensed mesenchyme, close to the tips of the ureteric tree where tubulogenesis is initiated. The alpha5 mRNA was strongly expressed by ureter epithelium but not expressed at early stages of tubulogenesis. Immunofluorescence verified low levels of the alpha5 chain in the early stages of tubulogenesis. However, during the capillary loop stage, the alpha5 chain became strongly expressed in the developing glomerular basement membrane, which matches the in situ hybridization results. During subsequent maturation of the kidney, the alpha5 chain became ubiquitously expressed in basement membranes. Overall, the alpha5 chain exhibited the broadest pattern of expression, followed by the alpha1 chain, particularly in the adult stage. These chains were the only ones produced by epithelial cells. Although some basement membranes contained several alpha chains, we failed to detect any of the five studied chains in some basement membranes. Thus, the identity of the alpha chains of many embryonic kidney blood vessels and several basement membranes in the inner medulla in the developing and adult kidney remain unclear.     

Removal of extracellular materials by HCL-tween treatment

Glutaraldehyde-fixed tissues were treated with HCl and tween 20 to remove such extracellular materials as collagen fibers and basal laminae. In the HCl-treatment the collagen fibers were digested, while in the tween-treatment the basal laminae were removed. By this method the basal interdigitations of cells of the proximal tubule and the pericytes on th capillary wall were clearly demonstrable by scanning electron microscopy (SEM).