Major Constituent Of Basement Membrane Research Articles

Measuring peroxidasin activity in live cells using bromide addition for signal amplification

Peroxidasin (PXDN) is involved in the crosslinking of collagen IV, a major constituent of basement membranes. Disruption of basement membrane integrity as observed in genetic alterations of collagen IV or PXDN can result in developmental defects and diverse pathologies. Hence, the study of PXDN activity in (patho)physiological contexts is highly relevant. So far, measurements of PXDN activity have been reported from purified proteins, cell lysates and de-cellularized extracellular matrix. Here, for the first time we report the measurement of PXDN activity in live cells using the Amplex Red assay with a signal amplifying modification. We observe that bromide addition enhances the obtained signal, most likely due to formation of HOBr. Abrogation of signal amplification by the HOBr scavenger carnosine supports this hypothesis. Both, pharmacological inhibition as well as complementary genetic approaches confirm that the obtained signal is indeed related to PXDN activity. We validate the modified assay by investigating the effect of Brefeldin A, to inhibit the secretory pathway and thus the access of PXDN to the extracellular Amplex Red dye. Our method opens up new possibilities to investigate the activity of PXDN in (patho)physiological contexts.

Acute T-Cell-Driven Inflammation Requires the Endoglycosidase Heparanase-1 from Multiple Cell Types.

It has been accepted for decades that T lymphocytes and metastasising tumour cells traverse basement membranes (BM) by deploying a battery of degradative enzymes, particularly proteases. However, since many redundant proteases can solubilise BM it has been difficult to prove that proteases aid cell migration, particularly in vivo. Recent studies also suggest that other mechanisms allow BM passage of cells. To resolve this issue we exploited heparanase-1 (HPSE-1), the only endoglycosidase in mammals that digests heparan sulfate (HS), a major constituent of BM. Initially we examined the effect of HPSE-1 deficiency on a well-characterised adoptive transfer model of T-cell-mediated inflammation. We found that total elimination of HPSE-1 from this system resulted in a drastic reduction in tissue injury and loss of target HS. Subsequent studies showed that the source of HPSE-1 in the transferred T cells was predominantly activated CD4+ T cells. Based on bone marrow chimeras, two cellular sources of HPSE-1 were identified in T cell recipients, one being haematopoiesis dependent and the other radiation resistant. Collectively our findings unequivocally demonstrate that an acute T-cell-initiated inflammatory response is HPSE-1 dependent and is reliant on HPSE-1 from at least three different cell types.

Dissection of Nidogen function in Drosophila reveals tissue-specific mechanisms of basement membrane assembly.

Basement membranes (BMs) are thin sheet-like specialized extracellular matrices found at the basal surface of epithelia and endothelial tissues. They have been conserved across evolution and are required for proper tissue growth, organization, differentiation and maintenance. The major constituents of BMs are two independent networks of Laminin and Type IV Collagen in addition to the proteoglycan Perlecan and the glycoprotein Nidogen/entactin (Ndg). The ability of Ndg to bind in vitro Collagen IV and Laminin, both with key functions during embryogenesis, anticipated an essential role for Ndg in morphogenesis linking the Laminin and Collagen IV networks. This was supported by results from cultured embryonic tissue experiments. However, the fact that elimination of Ndg in C. elegans and mice did not affect survival strongly questioned this proposed linking role. Here, we have isolated mutations in the only Ndg gene present in Drosophila. We find that while, similar to C.elegans and mice, Ndg is not essential for overall organogenesis or viability, it is required for appropriate fertility. We also find, alike in mice, tissue-specific requirements of Ndg for proper assembly and maintenance of certain BMs, namely those of the adipose tissue and flight muscles. In addition, we have performed a thorough functional analysis of the different Ndg domains in vivo. Our results support an essential requirement of the G3 domain for Ndg function and unravel a new key role for the Rod domain in regulating Ndg incorporation into BMs. Furthermore, uncoupling of the Laminin and Collagen IV networks is clearly observed in the larval adipose tissue in the absence of Ndg, indeed supporting a linking role. In light of our findings, we propose that BM assembly and/or maintenance is tissue-specific, which could explain the diverse requirements of a ubiquitous conserved BM component like Nidogen.

Laminin α1 orchestrates VEGFA functions in the ecosystem of colorectal carcinoma.

Tumor stroma remodeling is a key feature of malignant tumors and can promote cancer progression. Laminins are major constituents of basement membranes that physically separate the epithelium from the underlying stroma. By employing mouse models expressing high and low levels of the laminin α1 chain (LMα1), we highlighted its implication in a tumor-stroma crosstalk, thus leading to increased colon tumor incidence, angiogenesis and tumor growth. The underlying mechanism involves attraction of carcinoma-associated fibroblasts by LMα1, VEGFA expression triggered by the complex integrin α2β1-CXCR4 and binding of VEGFA to LM-111, which in turn promotes angiogenesis, tumor cell survival and proliferation. A gene signature comprising LAMA1, ITGB1, ITGA2, CXCR4 and VEGFA has negative predictive value in colon cancer. Together, we have identified VEGFA, CXCR4 and α2β1 integrin downstream of LMα1 in colon cancer as of bad prognostic value for patient survival. This information opens novel opportunities for diagnosis and treatment of colon cancer.

Basement membrane collagen IV: Isolation of functional domains.

Collagen IV is a major constituent of basement membranes, specialized form of extracellular matrix that provides a mechanical support for tissues, serves as a polyvalent ligand for cell adhesion receptors and as a scaffold for other proteins, and plays a key role in tissue genesis, differentiation, homeostasis, and remodeling. Collagen IV underlies the pathogenesis of several human disorders including Goodpasture's disease, Alport's syndrome, diabetic nephropathy, angiopathy, and porencephaly. While the isolation of the collagen IV molecules from tissues is an ultimate prerequisite for structural and functional studies, it has been always hampered by the protein insolubility due to extensive intermolecular crosslinking and noncovalent associations with other components of basement membranes. In this chapter, we present methods for the isolation of collagen IV fragments from basement membranes or from extracellular matrix deposited by cultured cells, and the recombinant expression alternative. These methods are useful to address the fundamental questions on the role of collagen IV in tissue genesis under the normal and pathological conditions.

Proprotein Convertase Processing Enhances Peroxidasin Activity to Reinforce Collagen IV

The basement membrane (BM) is a form of extracellular matrix that underlies cell layers in nearly all animal tissues. Type IV collagen, a major constituent of BMs, is critical for tissue development and architecture. The enzyme peroxidasin (Pxdn), an extracellular matrix-associated protein, catalyzes the formation of structurally reinforcing sulfilimine cross-links within the collagen IV network, an event essential to basement membrane integrity. Although the catalytic function of Pxdn is known, the regulation of its activity remains unclear. In this work we show through N-terminal sequencing, pharmacologic studies, and mutational analysis that proprotein convertases (PCs) proteolytically process human Pxdn at Arg-1336, a location relatively close to its C terminus. PC processing enhances the enzymatic activity of Pxdn and facilitates the formation of sulfilimine cross-links in collagen IV. Thus, PC processing of Pxdn is a key regulatory step that contributes to its function and, therefore, supports BM integrity and homeostasis.

Autoantibodies against basement membrane collagen type IV are associated with myocardial infarction

BackgroundCollagen type IV is the major constituent of basement membranes underlying endothelial cells and is important for endothelial cell attachment and function. Autoantibodies against native collagen type IV have been found in various autoimmune diseases. Oxidation of LDL in the vascular wall results in the formation of reactive aldehydes, which could modify surrounding matrix proteins. Like oxidized LDL, these modified matrix proteins are likely to induce immune responses. We examined whether autoantibodies against native or aldehyde-modified collagen type IV are associated with myocardial infarction. MethodsIgM and IgG against native and aldehyde-modified collagen type IV were measured by ELISA in serum from 387 survivors of a first myocardial infarction and 387 age- and sex-matched controls. ResultsPost-infarction patients had significantly increased levels of IgM against native collagen type IV, and IgG against native collagen type IV was present at detectable level in 17% of patients as opposed to 7% of controls (p<0.001). Controlling for major cardiovascular risk factors demonstrated that the presence of IgG against native collagen type IV was associated with myocardial infarction (OR 2.9 (1.6–5.4), p=0.001). Similarly, subjects in the highest quartile of IgM against native collagen type IV had increased risk of having suffered myocardial infarction (OR 3.11 (1.8–5.4), p<0.001) after adjusting for cardiovascular risk factors. In contrast, IgG against aldehyde-modified collagen type IV was decreased in myocardial infarction patients, but this association was not independent of established cardiovascular risk factors. ConclusionAutoantibodies against collagen type IV are associated with myocardial infarction independently of traditional cardiovascular risk factors.

Proteome array identification of bioactive soluble proteins/peptides in Matrigel: relevance to stem cell responses.

Matrigel and similar commercial products are extracts of the Engelbreth-Holm-Swarm sarcoma that provide a basement-membrane-like attachment substrate or gel that is used to grow cells on or in, respectively. To ascertain further what proteins may be present in Matrigel, besides its major basement-membrane constituents, an analysis of the expressed liquid of gelled Matrigel was performed using proteome array technology. Among the growth factors/cytokines assayed, high positive detection was found for IGFBP1, IGFBP3, LIF, platelet factor 4, PlGF-2, and VEGF; moderate reactivity was found for cyr61, IGFBP2, IGFBP6, IL-1ra, and NOV; and low, but detectable, responses occurred for aFGF, IL-13, IL-23, M-CSF, and VEGF-B. Among the chemokines assayed, high positive detection was found for MIG and serpin E1; moderate reactivity was found for IP-10, MCP-1, and MCP-5, and low, but detectable, responses occurred for CXCL16, I-TAC, and MIP-1α. Among the other biologically active proteins assayed, high positive detection was found for adiponectin, C5a, endocan, lipocalin-2, sICAM-1, MMP-3, and TIMP-1; moderate reactivity was found for C-reactive protein, coagulation factor III, endoglin, endostatin/collagen XVIII, endothelin-1, ICAM-1, MMP-9, osteopontin, pentraxin-3, and RANTES; and low, but detectable, responses occurred for fetuin A, MMP-8, pentraxin-2, RBP4, resistin, and TIMP-4. The study found several growth factors, chemokines, and biologically active proteins not previously identified in Matrigel, and this may have significance to the interpretations of observed cellular responses when cells are grown on or in Matrigel.

Ex Vivo Pathogenicity of Anti–Laminin γ1 Autoantibodies

Autoimmunity against laminins has been described in several autoimmune diseases (including mucous membrane pemphigoid, anti-laminin γ1 pemphigoid, and connective tissue diseases), in pregnancy loss, and in infections such as Chagas disease. Except for anti-laminin-332 mucous membrane pemphigoid, adequate evidence has been lacking for the tissue injury potential of laminin-specific antibodies and the pathogenic epitopes. We evaluated the pathogenic potential of antibodies targeting laminin γ1, a major constituent of basement membranes and the main antigen in anti-laminin γ1 pemphigoid. Rabbit antibodies were generated against fragments of the N-terminus and C-terminus of murine laminin γ1, and their ability to disrupt ligand interactions and/or to activate complement and granulocytes was assessed using previously established exvivo assays. Our findings document a pathogenic potential of antibodies targeting the laminin γ1 N-terminus. These antibodies interfere with the binding of nidogen to laminin and can activate granulocytes and the complement cascade. We detected antibodies with different degrees of reactivity with laminin γ1 N-terminus in patients with anti-laminin γ1 pemphigoid, cutaneous lupus erythematosus, and scleroderma. Our results provide mechanistic insights into the tissue damage associated with laminin autoimmunity and could facilitate development of appropriate diagnostic tools and therapeutic strategies.

Tying the collagen-sulfilimine knot

Type IV collagen, a major constituent of basement membranes, contains an unusual intermolecular sulfilimine crosslink whose route of biosynthesis has remained undefined. An oxidative triad consisting of peroxidasin, H(2)O(2) and halide is now shown to drive sulfilimine generation in vivo.

Abnormal Wnt and PI3Kinase Signaling in the Malformed Intestine of lama5 Deficient Mice

Laminins are major constituents of basement membranes and are essential for tissue homeostasis. Laminin-511 is highly expressed in the intestine and its absence causes severe malformation of the intestine and embryonic lethality. To understand the mechanistic role of laminin-511 in tissue homeostasis, we used RNA profiling of embryonic intestinal tissue of lama5 knockout mice and identified a lama5 specific gene expression signature. By combining cell culture experiments with mediated knockdown approaches, we provide a mechanistic link between laminin α5 gene deficiency and the physiological phenotype. We show that laminin α5 plays a crucial role in both epithelial and mesenchymal cell behavior by inhibiting Wnt and activating PI3K signaling. We conclude that conflicting signals are elicited in the absence of lama5, which alter cell adhesion, migration as well as epithelial and muscle differentiation. Conversely, adhesion to laminin-511 may serve as a potent regulator of known interconnected PI3K/Akt and Wnt signaling pathways. Thus deregulated adhesion to laminin-511 may be instrumental in diseases such as human pathologies of the gut where laminin-511 is abnormally expressed as it is shown here.

A novel cell‐adhesive scaffold material for delivering keratinocytes reduces granulation tissue in dermal wounds

Novel peptide-conjugated chitosan membranes were fabricated and used to deliver keratinocytes to dermal wounds in mice. Three active peptides of 12 or 13 amino acids each, RLVSYNGIIFFLK (A5G27), ASKAIQVFLLAG (A5G33), and AGTFALRGDNPQG (A99) were selected from a cell-adhesive peptide library of laminin, a major constituent of basement membrane. The peptides were synthesized and coupled to chitosan membranes, and the resulting peptide-chitosan membranes were tested for keratinocyte attachment. Two of the peptides that bind to cell surface heparin-like receptors (A5G27 and A5G33) were found to promote strong keratinocyte attachment, whereas the one that binds to integrin (A99) was inactive. Subsequently, A5G27- and A5G33-chitosan membranes were tested as vehicles for keratinocyte delivery in a wound model. We found that keratinocytes were delivered into the full-thickness wound with either membrane. Using the A5G33-chitosan membrane, we further evaluated the activity of the delivered keratinocytes in wound healing. Immunohistochemistry for granulation tissue markers, including tenascin and alpha-smooth muscle actin, showed that keratinocyte delivery by the present peptide-chitosan membranes in the wound bed provided a favorable condition for keratinocyte migration along the wound surface and reduced granulation tissue formation.

Essential and overlapping roles for laminin α chains in notochord and blood vessel formation

Laminins are major constituents of basement membranes and have wide ranging functions during development and in the adult. They are a family of heterotrimeric molecules created through association of an α, β and γ chain. We previously reported that two zebrafish loci, grumpy ( gup) and sleepy ( sly), encode laminin β1 and γ1, which are important both for notochord differentiation and for proper intersegmental blood vessel (ISV) formation. In this study we show that bashful ( bal) encodes laminin α1 ( lama1). Although the strongest allele, bal m190, is fully penetrant, when compared to gup or sly mutant embryos , bal mutants are not as severely affected, as only anterior notochord fails to differentiate and ISVs are unaffected. This suggests that other α chains, and hence other isoforms, act redundantly to laminin 1 in posterior notochord and ISV development. We identified cDNA sequences for lama2, lama4 and lama5 and disrupted the expression of each alone or in mutant embryos also lacking laminin α1. When expression of laminin α4 and laminin α1 are simultaneously disrupted, notochord differentiation and ISVs are as severely affected as sly or gup mutants. Moreover, live imaging of transgenic embryos expressing enhanced green fluorescent protein in forming ISVs reveals that the vascular defects in these embryos are due to an inability of ISV sprouts to migrate correctly along the intersegmental, normally laminin-rich regions.

Integrins as receptors for laminins.

Laminins are a family of trimeric glycoproteins present in the extracellular matrix and the major constituents of basement membranes. Integrins are alpha beta transmembrane receptors that play critical roles in both cell-matrix and cell-cell adhesion. Several members of the integrin family, including alpha 1 beta 1, alpha 2 beta 1, alpha 3 beta 1, alpha 6 beta 1, alpha 7 beta 1 and alpha 6 beta 4 heterodimers serve as laminin receptors on a variety of cell types. This review summarizes recent advances in understanding the involvement of individual integrins in cell interactions with laminins and the roles of laminin-binding integrins in adhesion-mediated events in vertebrates, including embryonic development, cell migration and tumor cell invasiveness, cell proliferation and differentiation, as well as basement membrane assembly. We discuss the regulation of integrin function via alternative splicing of cytoplasmic domains of alpha and beta subunits of the integrin receptors for laminins and present examples of functional collaboration between laminin-binding integrins and non-integrin laminin receptors. Advances in our understanding of the laminin-binding integrins continue to demonstrate the essential roles these receptors play in maintaining cell polarity and tissue architecture.

Rupture of the anterior lens capsule in Alport syndrome

J AAPOS 1999;3:381-2.

Laminin and Heparan Sulfate Proteoglycan Mediate Epithelial Cell Polarization in Organotypic Cultures of Embryonic Lung Cells: Evidence Implicating Involvement of the Inner Globular Region of Laminin β 1 Chain and the Heparan Sulfate Groups of Heparan Sulfate Proteoglycan

The extracellular matrix and in particular the basement membrane (BM) play an important role in the induction of organotypic rearrangement of cells in culture. This process involves cell aggregation, sorting into epithelial and mesenchymal components, epithelial cell polarization, and lumen formation. In this study, a combination of laminin (LM) and heparan sulfate proteoglycan (HSPG), two major BM constituents, induced organotypic rearrangement of embryonic mouse lung cells. In the absence of LM/HSPG supplementation, the cells sorted into epithelial and mesenchymal compartments but epithelial cell polarization and lumen formation did not occur. Neither LM nor HSPG alone could trigger this process. Synthetic peptide F-9, representing an amino acid sequence from the inner globular region of the laminin β1 chain (RYVVLPRPVCFEKGMNYTVR) induced organotypic cell rearrangement when substituted for LM. Exogenous LM as well as peptide F-9 were localized at the epithelial–mesenchymal interface of organotypic cultures, where a BM-like structure is formedde novo.Organotypic cell rearrangement was blocked by heparin, heparan sulfate, or antibodies against peptide F-9. Binding assays indicated that peptide F-9 interacts with HSPG but not with LM or type IV collagen. Preincubation of embryonic lung cells with peptide F-9 resulted in a significant increase in cell attachment to HSPG but not to other major BM constituents. These findings suggest that the interaction between LM and BM HSPG is critical for the development of epithelial cell polarization and lumen formation. This interaction occurs at the epithelial–mesenchymal interface and is mediated by a site in the LM molecule represented by peptide F-9 and the heparan sulfate groups of HSPG.

T cell adhesion to extracellular matrix molecules secreted by endothelial cells cultured on a substrate of type IV collagen

T cells emigrating from the bloodstream into lymphoid organs or sites of inflammation in the connective tissue must adhere to, and traverse, the subendothelial basement membrane (BM). The goal of the current investigation was to develop a method to study the adhesion of T cells to endothelial cell (EC)-derived extracellular matrix (ECM) as a model for the interaction of T cells with the subendothelial BM in vivo. To be certain that we were truly measuring T cell adhesion to ECM molecules secreted by the EC, it was necessary to culture the EC on a substrate to which T cells could not attach. Non-tissue culture-treated microtiter plate wells which had been coated with type IV collagen (tIVC), a major constituent of BM in vivo, were found to be suitable for this purpose since EC, but very few T cells, adhered to such wells. After incubating the EC on a substrate of tIVC in non-treated wells for a period of 48 h, the EC were gently removed from their underlying ECM and T cell adhesion to that ECM was examined. Using this system, it was observed that approximately 15–40% of human peripheral blood T cells specifically adhered to ECM molecules produced by the EC. This method should be useful as a model for the interactions of T cells and other leukocytes with the vascular BM in vivo.

Distribution of collagen type IV in brain tumors: an immunohistochemical study.

One hundred-twenty seven human brain tumors were examined by an immunoperoxidase technique for the expression of collagen Type IV, a major constituent of basement membrane. The parenchymal components were negative for the marker protein in all tumors except for neurilemmomas which were positively stained. In every case, the antibody to collagen Type IV showed distinct staining of the vascular pattern. In gliomas, capillaries increased in number and the vascular staining increased in intensity. Fine branching capillaries and endothelial glomeruloid proliferations characteristic each of oligodendrogliomas and glioblastomas could be distinctly illustrated. In two ependymomas, marked capillary proliferation was noted in periventricular areas. Fibrillar staining was observed between the tumor cells in seven of 34 meningiomas. Pericapillary lamellar deposition of collagen Type IV suggests a vascular origin of psammoma bodies. In some malignant tumors, pial-glial membranes were disrupted and the Virchow-Robin spaces were filled with malignant cells. Collagen Type IV was absent around the stromal cells of hemangioblastomas, suggesting that these stromal cells were unrelated histogenetically with endothelial cells. Collage Type IV may be useful in the differential diagnosis between meningiomas and neurilemmomas.

A role for the laminin receptor in leukocyte chemotaxis.

Previous studies on the mechanism of leukocyte traversal of basement membranes showed that rabbit peritoneal exudate polymorphonuclear cells (PMN) preferentially used laminin, a major constituent of basement membrane, to attach to another component, type IV collagen. PMN also responded chemotactically to nanomolar levels of laminin. We have now determined that PMN possess a receptor for laminin. Scatchard analysis using 125I laminin indicates a single class of saturable high affinity binding sites (kd = 6.15 nM/L) on PMN and 3.6 X 10(4) sites per cell. A chymotryptically derived fragment of laminin, C1, which lacks both the long arm and the globular end regions of the short arm (i.e., matrix binding sites) but retains the laminin receptor binding region, gave similar results. Immunoperoxidase studies using monoclonal antibodies to the laminin receptor (mAbLR) indicated the presence of the receptor on the surface of PMN. These cells responded chemotactically to nanomolar levels of C1 and laminin, a result consonant with binding data. PMN chemotaxis to a formyl peptide was markedly inhibited by mAbLR, suggesting that the laminin receptor may be required for PMN chemotaxis in general. Our results suggest that PMN extravasation across basement membranes is aided both by reversible attachment of the cells to laminin in the matrix and by chemotaxis to a gradient of soluble intact and possibly degraded laminin. These characteristics have much in common with those of highly metastatic tumor cells.

Type IV collagen and laminin-related antigens in human serum in alcoholic liver disease.

The two major constituents of basement membranes are type IV collagen and laminin. Specific radioimmunoassays are described here for two structural domains of these proteins (7-S collagen and the fragment P1, respectively) that allow the related antigens to be quantified in human serum. The serum 7-S collagen antigen was uniform in size, whereas the laminin P1 antigenicity was heterogeneous. These proteins were measured in sera from sixty-three alcoholics, divided on the basis of liver histology into four groups: normal light microscopy, fatty liver, alcoholic cirrhosis with hepatitis and inactive cirrhosis. The group with cirrhosis and hepatitis had clearly elevated values in both assays, differing significantly from the others. A few pathological results were also seen in the other groups. The increases noted in 7-S collagen concentration were larger than those in laminin P1. During follow-up of a patient with cirrhosis and hepatitis the 7-S collagen level in particular seemed to reflect the course of the disease. The elevated basement membrane protein concentrations in serum may be associated with the formation of real basement membranes in the perisinusoidal space, a process known as capillarization of the sinusoids which is found during the development of liver cirrhosis.