Protein composition and biomechanical properties of in vivo-derived basement membranes

Basement membranes (BMs) evolved together with the first metazoan species approximately 500 million years ago. Main functions of BMs are stabilizing epithelial cell layers and connecting different types of tissues to functional, multicellular organisms. Mutations of BM proteins from worms to humans are either embryonic lethal or result in severe diseases, including muscular dystrophy, blindness, deafness, kidney defects, cardio-vascular abnormalities or retinal and cortical malformations. In vivo-derived BMs are difficult to come by; they are very thin and sticky and, therefore, difficult to handle and probe. In addition, BMs are difficult to solubilize complicating their biochemical analysis. For these reasons, most of our knowledge of BM biology is based on studies of the BM-like extracellular matrix (ECM) of mouse yolk sac tumors or from studies of the lens capsule, an unusually thick BM. Recently, isolation procedures for a variety of BMs have been described, and new techniques have been developed to directly analyze the protein compositions, the biomechanical properties and the biological functions of BMs. New findings show that native BMs consist of approximately 20 proteins. BMs are four times thicker than previously recorded, and proteoglycans are mainly responsible to determine the thickness of BMs by binding large quantities of water to the matrix. The mechanical stiffness of BMs is similar to that of articular cartilage. In mice with mutation of BM proteins, the stiffness of BMs is often reduced. As a consequence, these BMs rupture due to mechanical instability explaining many of the pathological phenotypes. Finally, the morphology and protein composition of human BMs changes with age, thus BMs are dynamic in their structure, composition and biomechanical properties.     

Invadopodia and basement membrane invasion in vivo

Over 20 years ago, protrusive, F-actin-based membrane structures, termed invadopodia, were identified in highly metastatic cancer cell lines. Invadopodia penetrate artificial or explanted extracellular matrices in 2D culture conditions and have been hypothesized to facilitate the migration of cancer cells through basement membrane, a thin, dense, barrier-like matrix surrounding most tissues. Despite intensive study, the identification of invadopodia in vivo has remained elusive and until now their possible roles during invasion or even existence have remained unclear. Studies in remarkably different cellular contexts—mouse tumor models, zebrafish intestinal epithelia, and C. elegans organogenesis—have recently identified invadopodia structures associated with basement membrane invasion. These studies are providing the first in vivo insight into the regulation, function, and role of these fascinating subcellular devices with critical importance to both development and human disease.     

Invadopodia and basement membrane invasion in vivo

Over 20 years ago, protrusive, F-actin-based membrane structures, termed invadopodia, were identified in highly metastatic cancer cell lines. Invadopodia penetrate artificial or explanted extracellular matrices in 2D culture conditions and have been hypothesized to facilitate the migration of cancer cells through basement membrane, a thin, dense, barrier-like matrix surrounding most tissues. Despite intensive study, the identification of invadopodia in vivo has remained elusive and until now their possible roles during invasion or even existence have remained unclear. Studies in remarkably different cellular contexts—mouse tumor models, zebrafish intestinal epithelia, and C. elegans organogenesis—have recently identified invadopodia structures associated with basement membrane invasion. These studies are providing the first in vivo insight into the regulation, function, and role of these fascinating subcellular devices with critical importance to both development and human disease.     

Laminin isoforms in endothelial and perivascular basement membranes

Laminins, one of the major functional components of basement membranes, are found underlying endothelium, and encasing pericytes and smooth muscle cells in the vessel wall. Depending on the type of blood vessel (capillary, venule, postcapillary venule, vein or artery) and their maturation state, both the endothelial and mural cell phenotype vary, with associated changes in laminin isoform expression. Laminins containing the α4 and α5 chains are the major isoforms found in the vessel wall, with the added contribution of laminin α2 in larger vessels. We here summarize current data on the precise localization of these laminin isoforms and their receptors in the different layers of the vessel wall, and their potential contribution to vascular homeostasis.     

Hypoblast cells of chick pre-streak stage embryos invaded basement membrane analogues in vitro: Implications for hypoblast layer formation

In early chick blastodermal morphogenesis, the hypoblast layer is organized beneath the epiblast and induces an axial structure. However, the origin of hypoblast cells and the mechanism of hypoblast layer formation are poorly understood. We hypothesized that the hypoblast layer is formed by an invasive process across the basement membrane of the juxtaposing epiblast, and tested the idea in vitro . Primary and secondary hypoblast cells from embryos at various pre-streak stages were dissociated into single cells and cultured on reconstituted basement membrane gel, laminin gel or fibronectin gel in the culture medium with or without serum for 24–48 h. As a result, we found that after 24 h of serum-supplemented culture, up to 35% of the hypoblast cells dissolved the gel and made holes on it. Similarly, up to 36% of the hypoblast cells showed invasiveness after 48 h in the serum-free culture. Furthermore, it was observed that Koller's sickle cells, which are regarded to be the progenitors of secondary hypoblast cells, penetrated those gels on which they were seeded. The posterior epiblast cells covering Koller's sickle were also invasive. These results suggest that the presumptive primary hypoblast cells that are known to mingle with epiblast cells invade through the basement membrane to form the hypoblast layer. Furthermore, the present results imply that invasion through the basement membrane may be involved in the formation of Koller's sickle, the anlage of secondary hypoblast.     

Laminin extraction and disorganization of collagen fibrils in snail muscles by EDTA treatment

Summary— Snail muscles were extracted by a solution of EDTA and electron microscopy showed that the extract contained dispersed, depolymerized collagen fibrils and cross-shaped laminin-like structures. The extracts were purified by ultracentrifugation followed by two different procedures which enriched the content of laminin-like structures. The laminin-related molecules displayed unique properties when analyzed by biochemical, immunological and morphological methods. Electrophoretic patterns of the molecular form purified primarily by ion exchange chromatography, resembled EHS-tumor laminin and displayed a cruciform shape when viewed by electron microscopy. Immunohistology, using antiserum obtained against the agarose gel-purified protein, showed that this laminin was primarily located in the extracellular matrix surrounding muscle fibers. Western blots using anti-EHS laminin antibody showed reaction of a 300 kDa subunit of this snail laminin. The protein obtained by another procedure, initially using gel filtration, followed by ion exchange chromatography, also appeared to be a laminin. It had a collapsed cruciform appearance when viewed by electron microscopy. It contained several different subunits, one of which, ca 300 kDa, reacted with anti-EHS-laminin antibody and with anti-snail laminin antibody. In contrast, EHS laminin did not react with the anti-snail laminin antibody. The composite results suggest that at least two different forms of laminin are extractable from snail muscle and that they share molecular properties and immune determinants with mouse tumor laminin.     

Basement Membrane Proteins: Structure,Assembly, and Cellular Interactions

Abstract

Basement membranes are thin layers of a specialized extracellular matrix that form the supporting structure on which epithelial and endothelial cells grow, and that surround muscle and fat cells and the Schwann cells of peripheral nerves. One common denominator is that they are always in close apposition to cells, and it has been well demonstrated that basement membranes do not only provide a mechanical support and divide tissues into compartments, but also influence cellular behavior. The major molecular constituents of basement membranes are collagen IV, laminin-entactin/nidogen complexes, and proteoglycans. Collagen IV provides a scaffold for the other structural macromolecules by forming a network via interactions between specialized N-and C-terminal domains. Laminin-entactin/nidogen complexes self-associate into less-ordered aggregates. These two molecular assemblies appear to be interconnected, presumably via binding sites on the entactin/nidogen molecule. In addition, proteoglycans are anchored into the membrane by an unknown mechanism, providing clusters of negatively charged groups. Specialization of different basement membranes is achieved through the presence of tissue-specific isoforms of laminin and collagen IV and of particular proteoglycan populations, by differences in assembly between different membranes, and by the presence of accessory proteins in some specialized basement membranes. Many cellular responses to basement membrane proteins are mediated by members of the integrin class of transmembrane receptors. On the intracellular side some of these signals are transmitted to the cytoskeleton, and result in an influence on cellular behavior with respect to adhesion, shape, migration, proliferation, and differentiation. Phosphorylation of integrins plays a role in modulating their activity, and they may therefore be a part of a more complex signaling system.     

Comparison of basement membrane matrix degradation by purified proteases and by metastatic tumor cells

We have examined the nature of biochemical degradation of an isolated basement membrane matrix (bovine lens capsule) using different methodologies. The first strategy was quantitation of the release of surface-bound 125I and a second the documentation by SDS-PAGE of the appearance of putative cleavage products and the loss of high-molecular-weight components from the matrix. Basement membrane matrix bands resolved on SDS-PAGE were identified by their protease sensitivities as well as by Western immunoblots using monoclonal antibodies developed for this study. Radioiodinated components were found predominantly at positions on the gel equivalent to 160-200 kd and 400 kd proteins. Since these labeled moieties were sensitive to bacterial collagenase digestion and stained with anticollagen type IV antibodies, they were determined to represent various configurations of collagen type IV. Several other lower-molecular-weight bands also stained with the anticollagen IV antibodies. Monoclonal antibodies reactive with laminin exhibited a complex staining pattern on the gels, which included the expected 200 and 400 kd components. We confirmed that lens capsule basement membrane contained only a single heparan sulfate glycosaminoglycan species, and tumor cell-induced glycosaminoglycan degradation within the basement membrane matrix was detected using cellulose acetate electrophoresis. Distinctive putative cleavage products were resolved on SDS-PAGE gels from matrices subjected to digestion by a variety of purified proteases as well as by metastatic tumor cells or their conditioned media. Tumor cells of different histiotypes produced different characteristic cleavage patterns, suggestive of the existence of several pathways of matrix degradation. Overall, primary tumor cells exhibited a greater degradative activity towards the basement membrane matrix than did long-term tissue culture-passaged cells. The same tumor cell line could exhibit considerably different patterns of both protein and glycosaminoglycan degradation depending on recent culture history. The relevance of these biochemical studies to the pathogenesis of malignant neoplasms is shown by: 1) the evaluation of degradative activities of B16 tumor cell populations exhibiting enhanced lung-colonizing phenotypes, and 2) the ability of a known antimetastatic moiety with antiprotease activity (Haementeria leech species salivary gland extract) to protect matrix components from degradation by tumor cell-conditioned medium.     

Interaction between Aspergillus fumigatus and basement membrane laminin: Binding and substrate degradation

Summary— Aspergillus fumigatus, the causative agent of human aspergillosis, binds to and degrades basement membrane laminin. Using immunoelectron microscopy, laminin binding appeared to be associated with the cell wall expansions of resting conidia, and progressively extended to the outer electron dense layer of the conidial wall during the germination process. Labeling of thin sections revealed numerous binding sites in the cytoplasm, whereas the inner cell wall and the plasma membrane were not labeled. Attachment of A fumigatus conidia on microtiter plates coated with laminin and its fragments P1 and E8 was also investigated. Conidia cells showed good adhesion to wells coated with laminin. As indicated by inhibition experiments, the interaction was specific and fragment P1 represented the major binding site on the laminin molecule. In addition, since A fumigatus produced an extracellular serine protease, we determined the susceptibility of laminin to this enzyme. We demonstrated that a crude protease extract was capable to degrade laminin in solution as well as in tissue sections. The laminin cleavage products were detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. All the three chains were extensively degraded within 1 h. Treatment of the crude protease extract with the enzyme inhibitors, phenylmethylsulfonyl-fluoride and chymostatin, blocked the degradation of laminin, indicating a chymotrypsin-like serine protease activity. Immunofluorescence microscopy of cryostat sections of mouse and rat kidneys treated with the protease extract showed widespread loss of laminin epitopes from basement membranes. Enzyme treatment also removed immunoreactivity from lungs as observed after immunoperoxidase performed on paraffin sections. Binding and proteolytic degradation of laminin may together facilitate initial interaction of A fumigatus with the host tissues.     

Perlecan maintains the integrity of cartilage and some basement membranes

Perlecan is a heparan sulfate proteoglycan that is expressed in all basement membranes (BMs), in cartilage, and several other mesenchymal tissues during development. Perlecan binds growth factors and interacts with various extracellular matrix proteins and cell adhesion molecules. Homozygous mice with a null mutation in the perlecan gene exhibit normal formation of BMs. However, BMs deteriorate in regions with increased mechanical stress such as the contracting myocardium and the expanding brain vesicles showing that perlecan is crucial for maintaining BM integrity. As a consequence, small clefts are formed in the cardiac muscle leading to blood leakage into the pericardial cavity and an arrest of heart function. The defects in the BM separating the brain from the adjacent mesenchyme caused invasion of brain tissue into the overlaying ectoderm leading to abnormal expansion of neuroepithelium, neuronal ectopias, and exencephaly. Finally, homozygotes developed a severe defect in cartilage, a tissue that lacks BMs. The chondrodysplasia is characterized by a reduction of the fibrillar collagen network, shortened collagen fibers, and elevated expression of cartilage extracellular matrix genes, suggesting that perlecan protects cartilage extracellular matrix from degradation.     

Importance of Balance between Extracellular Matrix Synthesis and Degradation in Basement Membrane Formation

The epidermal basement membrane (BM) plays important roles in adhesion between epidermis and dermis and in controlling epidermal differentiation. In a skin-equivalent (SE), components of the epidermal BM such as laminin 5 and type IV and VII collagens were detected in conditioned media and in basal keratinocytes. Despite production of these BM components, however, BM was rarely observed at the dermal-epidermal junction. One possible explanation for the absence of BM in SEs is that matrix metalloproteinases (MMPs) degrade newly synthesized extracellular matrices. In fact, several MMPs, such as MMPs-1, 2, 3, and 9, were observed to be present in conditioned media and some of them were in active forms. Tissue inhibitor of metalloproteinase (TIMP)-2 was not detected, although TIMP-1 was present. BM degradation activity presumably exceeds BM formation activity in the SE, resulting in the absence of lamina densa at the dermal-epidermal junction. Synthetic MMP inhibitors CGS27023A and MMP inhibitor I, which inhibit MMPs 1, 2, 3, and 9, markedly augmented deposition of laminin 5 and type IV and VII collagens at the dermal-epidermal junction, resulting in formation of continuous epidermal BM. These results suggest that the balance between synthesis and degradation of BM components is important for BM formation.     

De-differentiation of primary human hepatocytes depends on the composition of specialized liver basement membrane

Despite the understanding of the importance of mitogen-activated protein (MAP) kinase activation in the stimulation of growth, little is known about the role of MAP kinase regulation during contact inhibited growth control. To investigate the role of the MAP kinase extracellular signal-regulated kinase (ERK) during the transition to a contact inhibited state, cultures of normal fibroblasts (BJ) were grown to different stages of confluency. The levels of MAP kinase phosphatase (MKP) expression and the amount of active ERK and MAP ERK kinase (MEK) in these cultures were assessed through western blot analysis and were compared to fibrosarcoma cell cultures (HT-1080), which lack contact inhibition. In normal fibroblasts, the amounts of active MEK and ERK decline at contact inhibition, concurrently with a rise in MKP-1, MKP-2, and MKP-3 protein levels. In contrast, fibrosarcoma cells appear to lack density-dependent regulation of the ERK pathway. Additionally, altering the redox environment of fibrosarcoma cells to a less reducing state, as seen during contact inhibition, results in increased MKP-1 expression. Taken together, these results suggest that the altered redox environment upon contact inhibition may contribute to the regulation of ERK inactivation by MKPs.     

Laminin alpha 5 is required for lobar septation and visceral pleural basement membrane formation in the developing mouse lung

Laminin alpha/beta/gamma heterotrimers are the major noncollagenous components of all basement membranes. To date, five alpha, three beta, and three gamma chains have been identified. Laminin alpha 5 is expressed early in lung development and colocalizes with laminin alpha1. While laminin alpha1 expression in the lung is restricted to the embryonic period, laminin alpha 5 expression persists throughout embryogenesis and adulthood. Targeted mutation of the mouse laminin alpha 5 gene Lama5 causes embryonic lethality at E14-E17 associated with exencephaly, syndactyly, placentopathy, and kidney defects, all attributable to abnormal basement membranes. In this investigation, lung development in Lama5(-/-) mice up to E16.5 was examined. We observed normal lung branching morphogenesis and vasculogenesis, but incomplete lobar septation and absence of the visceral pleura basement membrane. Preservation of branching morphogenesis was associated with ectopic deposition of laminin alpha 4 in the airway basement membrane. Perturbation of pleural basement membrane formation and right lung septation correlated with absence of laminin alpha 5, which was found to be the only laminin alpha chain present in the normal visceral pleura basement membrane. Our finding of normal lung branching morphogenesis with abnormal lobar septation demonstrates that these processes are not obligatorily linked.     

Intracellular mechanisms involved in basement membrane induced blood vessel differentiation in vitro

Summary The extracellular matrix, particularly basement membranes, plays an important role in angiogenesis (blood vessel formation). Previous work has demonstrated that a basement membranelike substrate (Matrigel) induces human umbilical vein endothelial cells to rapidly form vessel-like tubes (Kubota, et al., 1988; Grant et al., 1989b); however, the precise mechanism of tube formation is unclear. Using this in vitro model, we have investigated morphologic changes occurring during tube formation and the cytoskeletal and protein synthesis requirements of this process. Electron microscopy showed that endothelial cells attach to the Matrigel surface, align, and form cylindrical structures that contain a lumen and polarized cytoplasmic organelles. The cytoskeleton is reorganized into bundles of actin filaments oriented along the axis of the tubes and is located at the periphery of the cells. The addition of colchicine or cytochalasin D blocked tube formation, indicating that both microfilaments and microtubules are involved in this process. Cycloheximide blocked tube formation by 100%, indicating that the process also required protein synthesis. In particular, collagen synthesis seems to be required for tube formation because cis-hydroxyproline inhibited tube formation, whereas either the presence of ascorbic acid or the addition of exogenous collagen IV to the Matrigel increased tube formation. Our results indicate that endothelial cell attachment to Matrigel induces the reorganization of the cytoskeleton and elicits the synthesis of specific proteins required for the differentiated phenotype of the cells.     

To form and function: on the role of basement membrane mechanics in tissue development,homeostasis and disease

The basement membrane (BM) is a special type of extracellular matrix that lines the basal side of epithelial and endothelial tissues. Functionally, the BM is important for providing physical and biochemical cues to the overlying cells, sculpting the tissue into its correct size and shape. In this review, we focus on recent studies that have unveiled the complex mechanical properties of the BM. We discuss how these properties can change during development, homeostasis and disease via different molecular mechanisms, and the subsequent impact on tissue form and function in a variety of organisms. We also explore how better characterization of BM mechanics can contribute to disease diagnosis and treatment, as well as development of better in silico and in vitro models that not only impact the fields of tissue engineering and regenerative medicine, but can also reduce the use of animals in research.     

Functional differentiation of mouse uterine epithelial cells grown on collagen gels or reconstituted basement membranes

Summary Epithelial cells were isolated from mouse endometrium and cultured on two types of extracellular matrix, namely, rat-tail collagen (type I) gels and basement membrane extract (BME) derived from the Engelbreth-Holm-Swarm murine sarcoma. Cell attachment in serum-free medium during the initial 24 h after seeding was approximately twofold higher on BME compared with collagen type I. Addition of serum to the medium enhanced cell attachment on both matrices. On both collagen and BME, uterine cells grew as smooth-bordered colonies, and within a week of culture the cells became cuboidal to columnar in shape. Electron microscopy revealed the presence of apical microvilli associated with a glycocalyx, junctional complexes, tonofilaments, short strands of undilated endoplasmic reticulum, Golgi complex, and lipid droplets. However, cells on BME showed a higher degree of differentiation as assessed by occasional formation of small patches of basement membranelike structure subjacent to the flattened basal surface and formation of glandlike structures within the matrix. Proliferation of these cells as measured by radioactive thymidine incorporation into DNA was increased threefold by addition of epidermal growth factor (EGF) and insulin to the medium, but was not changed by 17β-estradiol. The expression of progesterone receptors by uterine epithelial cells grown on both matrices was doubled by addition of EGF and estradiol to the medium. This work was supported in part by a Rockefeller Foundation postdoctoral fellowship (D.G.), and NIh grant 23511.     

Podosomes and invadopodia: tools to breach vascular basement membrane

The vascular basement membrane (BM) is a thin and dense cross-linked extracellular matrix layer that covers and protects blood vessels. Understanding how cells cross the physical barrier of the vascular BM will provide greater insight into the potentially critical role of vascular BM breaching in cancer extravasation, leukocyte trafficking and angiogenic sprouting. In the last year, new evidence has mechanistically linked the breaching of vascular BM with the formation of specific cellular micro-domains known as podosomes and invadopodia. These structures are specialized cell-matrix contacts with an inherent ability to degrade the extracellular matrix. Specifically, the formation of podosomes or invadopodia was shown as an important step in vascular sprouting and tumor cell extravasation, respectively. Here, we review and comment on these recent findings and explore the functions of podosomes and invadopodia within the context of pathological processes such as tumor dissemination and tumor angiogenesis.     

Esophageal muscle physiology and morphogenesis require assembly of a collagen XIX-rich basement membrane zone

Collagen XIX is an extremely rare extracellular matrix component that localizes to basement membrane zones and is transiently expressed by differentiating muscle cells. Characterization of mice harboring null and structural mutations of the collagen XIX (Col19a1) gene has revealed the critical contribution of this matrix protein to muscle physiology and differentiation. The phenotype includes smooth muscle motor dysfunction and hypertensive sphincter resulting from impaired swallowing-induced, nitric oxide-dependent relaxation of the sphincteric muscle. Muscle dysfunction was correlated with a disorganized matrix and a normal complement of enteric neurons and interstitial cells of Cajal. Mice without collagen XIX exhibit an additional defect, namely impaired smooth-to-skeletal muscle cell conversion in the abdominal segment of the esophagus. This developmental abnormality was accounted for by failed activation of myogenic regulatory factors that normally drive esophageal muscle transdifferentiation. Therefore, these findings identify collagen XIX as the first structural determinant of sphincteric muscle function, and as the first extrinsic factor of skeletal myogenesis in the murine esophagus.     

Endothelial basement membrane limits tip cell formation by inducing Dll4/Notch signalling in vivo

How individual components of the vascular basement membrane influence endothelial cell behaviour remains unclear. Here we show that laminin α4 (Lama4) regulates tip cell numbers and vascular density by inducing endothelial Dll4/Notch signalling in vivo. Lama4 deficiency leads to reduced Dll4 expression, excessive filopodia and tip cell formation in the mouse retina, phenocopying the effects of Dll4/Notch inhibition. Lama4-mediated Dll4 expression requires a combination of integrins in vitro and integrin β1 in vivo. We conclude that appropriate laminin/integrin-induced signalling is necessary to induce physiologically functional levels of Dll4 expression and regulate branching frequency during sprouting angiogenesis in vivo.     

MYO10-filopodia support basement membranes at pre-invasive tumor boundaries

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