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The lens, suspended in the middle of the eye by tendon-like ciliary zonule fibers and facing three different compartments of the eye, is enclosed in what has been described as the thickest basement membrane in the body. While the protein components of the capsule have been a subject of study for many years, the dynamics of capsule formation, and the region-specific relationship of its basement membrane components to one another as well as to other matrix molecules remains to be explored. Through high resolution confocal and super-resolution imaging of the lens capsule and 3D surface renderings of acquired z-stacks, our studies revealed that each of its basement membrane proteins, laminin, collagen IV, nidogen and perlecan, has unique structure, organization, and distribution specific both to the region of the lens that the capsule is located in and the position of the capsule within the eye. We provide evidence of basal membrane gradients across the depth of the capsule as well as the synthesis of distinct basement membrane lamella within the capsule. These distinctions are most prominent in the equatorial capsule zone where collagen IV and nidogen span the capsule depth, while laminin and perlecan are located in two separate lamellae located at the innermost and outermost capsule domains. We discovered that an extracapsular matrix compartment rich in the connective tissue-like matrix molecules fibronectin, tenascin-C, and fibrillin is integrated with the superficial surface of the lens capsule. Each matrix protein in this extracapsular zone also exhibits region-specific distribution with fibrils of fibrillin, the matrix protein that forms the backbone of the ciliary zonules, inserting within the laminin/perlecan lamella at the surface of the equatorial lens capsule., Competing Interests: Declaration of Competing Interest The authors have no competing interests to declare., (Copyright © 2021 The Authors. Published by Elsevier B.V. All rights reserved.)

Microfluidic devices are becoming mainstream tools to recapitulate in vitro the behavior of cells and tissues. In this study, we use microfluidic devices filled with hydrogels of mixed collagen-Matrigel composition to study the migration of lung cancer cells under different cancer invasion microenvironments. We present the design of the microfluidic device, characterize the hydrogels morphologically and mechanically and use quantitative image analysis to measure the migration of H1299 lung adenocarcinoma cancer cells in different experimental conditions. Our results show the plasticity of lung cancer cell migration, which turns from mesenchymal in collagen only matrices, to lobopodial in collagen-Matrigel matrices that approximate the interface between a disrupted basement membrane and the underlying connective tissue. Our quantification of migration speed confirms a biphasic role of Matrigel. At low concentration, Matrigel facilitates migration, most probably by providing a supportive and growth factor retaining environment. At high concentration, Matrigel slows down migration, possibly due excessive attachment. Finally, we show that antibody-based integrin blockade promotes a change in migration phenotype from mesenchymal or lobopodial to amoeboid and analyze the effect of this change in migration dynamics, in regards to the structure of the matrix. In summary, we describe and characterize a robust microfluidic platform and a set of software tools that can be used to study lung cancer cell migration under different microenvironments and experimental conditions. This platform could be used in future studies, thus benefitting from the advantages introduced by microfluidic devices: precise control of the environment, excellent optical properties, parallelization for high throughput studies and efficient use of therapeutic drugs., Competing Interests: The authors have declared that no competing interests exist.

Differentiation of enteric neural stem cells into several appropriate neural phenotypes is crucial while considering transplantation as a cellular therapy to treat enteric neuropathies. We describe the formation of tissue engineered innervated sheets, where intestinal smooth muscle and enteric neuronal progenitor cells are brought into close association in extracellular matrix (ECM) based microenvironments. Uniaxial alignment of constituent smooth muscle cells was achieved by substrate microtopography. The smooth muscle component of the tissue engineered sheets maintained a contractile phenotype irrespective of the ECM composition, and generated equivalent contractions in response to potassium chloride stimulation, similar to native intestinal tissue. We provided enteric neuronal progenitor cells with permissive ECM-based compositional and viscoelastic cues to generate excitatory and inhibitory neuronal subtypes. In the presence of the smooth muscle cells, the enteric neuronal progenitor cells differentiated to functionally innervate the smooth muscle. The differentiation of specific neuronal subtypes was influenced by the ECM microenvironment, namely combinations of collagen I, collagen IV, laminin and/or heparan sulfate. The physiology of differentiated neurons within tissue engineered sheets was evaluated. Sheets with composite collagen and laminin had the most similar patterns of Acetylcholine-induced contraction to native intestinal tissue, corresponding to an increased protein expression of choline acetyltransferase. An enriched nitrergic neuronal population, evidenced by an increased expression of neuronal nitric oxide synthase, was obtained in tissue engineered sheets that included collagen IV. These sheets had a significantly increased magnitude of electrical field stimulated relaxation, sensitive maximally to nitric oxide synthase inhibition. Tissue engineered sheets containing laminin and/or heparan sulfate had a balanced expression of contractile and relaxant motor neurons. Our studies demonstrated that neuronal subtype was modulated by varying ECM composition. This observation could be utilized to derive enriched populations of specific enteric neurons in vitro prior to transplantation., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Collagens are the most abundant components of the extracellular matrix and many types of soft tissues. Elastin is another major component of certain soft tissues, such as arterial walls and ligaments. Many other molecules, though lower in quantity, function as essential components of the extracellular matrix in soft tissues. Some of these are reviewed in this chapter. Besides their basic structure, biochemistry and physiology, their roles in disorders of soft tissues are discussed only briefly as most chapters in this volume deal with relevant individual compounds. Fibronectin with its muldomain structure plays a role of "master organizer" in matrix assembly as it forms a bridge between cell surface receptors, e.g., integrins, and compounds such collagen, proteoglycans and other focal adhesion molecules. It also plays an essential role in the assembly of fibrillin-1 into a structured network. Laminins contribute to the structure of the extracellular matrix (ECM) and modulate cellular functions such as adhesion, differentiation, migration, stability of phenotype, and resistance towards apoptosis. Though the primary role of fibrinogen is in clot formation, after conversion to fibrin by thrombin, it also binds to a variety of compounds, particularly to various growth factors, and as such fibrinogen is a player in cardiovascular and extracellular matrix physiology. Elastin, an insoluble polymer of the monomeric soluble precursor tropoelastin, is the main component of elastic fibers in matrix tissue where it provides elastic recoil and resilience to a variety of connective tissues, e.g., aorta and ligaments. Elastic fibers regulate activity of TGFβs through their association with fibrillin microfibrils. Elastin also plays a role in cell adhesion, cell migration, and has the ability to participate in cell signaling. Mutations in the elastin gene lead to cutis laxa. Fibrillins represent the predominant core of the microfibrils in elastic as well as non-elastic extracellular matrixes, and interact closely with tropoelastin and integrins. Not only do microfibrils provide structural integrity of specific organ systems, but they also provide a scaffold for elastogenesis in elastic tissues. Fibrillin is important for the assembly of elastin into elastic fibers. Mutations in the fibrillin-1 gene are closely associated with Marfan syndrome. Fibulins are tightly connected with basement membranes, elastic fibers and other components of extracellular matrix and participate in formation of elastic fibers. Tenascins are ECM polymorphic glycoproteins found in many connective tissues in the body. Their expression is regulated by mechanical stress both during development and in adulthood. Tenascins mediate both inflammatory and fibrotic processes to enable effective tissue repair and play roles in pathogenesis of Ehlers-Danlos, heart disease, and regeneration and recovery of musculo-tendinous tissue. One of the roles of thrombospondin 1 is activation of TGFβ. Increased expression of thrombospondin and TGFβ activity was observed in fibrotic skin disorders such as keloids and scleroderma. Cartilage oligomeric matrix protein (COMP) or thrombospondin-5 is primarily present in the cartilage. High levels of COMP are present in fibrotic scars and systemic sclerosis of the skin, and in tendon, especially with physical activity, loading and post-injury. It plays a role in vascular wall remodeling and has been found in atherosclerotic plaques as well.

In multicellular organisms, proteins of the extracellular matrix (ECM) play structural and functional roles in essentially all organs, so understanding ECM protein organization in health and disease remains an important goal. Here, we used sub-diffraction resolution stochastic optical reconstruction microscopy (STORM) to resolve the in situ molecular organization of proteins within the kidney glomerular basement membrane (GBM), an essential mediator of glomerular ultrafiltration. Using multichannel STORM and STORM-electron microscopy correlation, we constructed a molecular reference frame that revealed a laminar organization of ECM proteins within the GBM. Separate analyses of domains near the N- and C-termini of agrin, laminin, and collagen IV in mouse and human GBM revealed a highly oriented macromolecular organization. Our analysis also revealed disruptions in this GBM architecture in a mouse model of Alport syndrome. These results provide the first nanoscopic glimpse into the organization of a complex ECM. DOI:http://dx.doi.org/10.7554/eLife.01149.001.

Aims: To identify differences in extracellular matrix contents between idiopathic epiretinal membranes (IEM) of cellophane macular reflex (CMRM) or preretinal macular fibrosis (PMFM) type., Methods and Results: Idiopathic epiretinal membranes were analysed by light and quantitative transmission electron microscopy, immunohistochemistry and Western blotting. Substantial differences between CMRM and PMFM were observed regarding the nature of extracellular fibrils. In CMRM the fibrils were thin, with diameters between 6 and 15 nm. Between the fibrils, aggregates of long-spacing collagen were observed. In PMFM the diameters of fibrils measured either 18-26 or 36-56 nm. Using immunogold electron microscopy, 6-15 nm fibrils in CMRM were labelled for collagen type VI, while the fibrils in PMFM remained unstained. Using Western blotting and immunohistochemistry, a strong signal for collagen type VI was observed in all CMRM, while immunoreactivity was weak or absent in PMFM. In contrast, PMFM showed immunoreactivity for collagen types I and II, which was weak or absent in CMRM. Both types of membranes showed immunoreactivity for collagen types III and IV, laminin and fibronectin with similar intensity., Conclusion: The presence of high amounts of collagen type VI in CMRM and the relative absence of collagen types I and II is the major structural difference to PMFM., (© 2011 Blackwell Publishing Limited.)

Objective: Normal endothelial cells respond to shear stress by elongating and aligning in the direction of fluid flow. Hyperglycemia impairs this response and contributes to microvascular complications, which result in deleterious effects to the endothelium. This work aimed to evaluate cheek pouch microvessel morphological characteristics, reactivity, permeability, and expression of cytoskeleton and extracellular matrix components in hamsters after the induction of diabetes with streptozotocin., Methods: Syrian golden hamsters (90-130 g) were injected with streptozotocin (50 mg/kg, i.p.) or vehicle either 6 (the diabetes mellitus 6 group) or 15 (the diabetes mellitus 15 group) days before the experiment. Vascular dimensions and density per area of vessels were determined by morphometric and stereological measurements. Changes in blood flow were measured in response to acetylcholine, and plasma extravasation was measured by the number of leakage sites. Actin, talin, α-smooth muscle actin, vimentin, type IV collagen, and laminin were detected by immunohistochemistry and assessed through a semiquantitative scoring system., Results: There were no major alterations in the lumen, wall diameters, or densities of the examined vessels. Likewise, vascular reactivity and permeability were not altered by diabetes. The arterioles demonstrated increased immunoreactivity to vimentin and laminin in the diabetes mellitus 6 and diabetes mellitus 15 groups., Discussion: Antibodies against laminin and vimentin inhibit branching morphogenesis in vitro. Therefore, laminin and vimentin participating in the structure of the focal adhesion may play a role in angiogenesis., Conclusions: Our results indicated the existence of changes related to cell-matrix interactions, which may contribute to the pathological remodeling that was already underway one week after induction of experimental diabetes.

The need to identify inhibitors of cancer invasion has driven the development of quantitative in vitro invasion assays. The most common assays used are based on the original Boyden assay system. Today commercially available plastic inserts for multi-well plates, which possess a cell-permeable membrane, as typified by Transwell(®) Permeable Supports, permit accurate repeatable invasion assays. When placed in the well of a multi-well tissue culture plate these inserts create a two-chamber system separated by the cell-permeable membrane. To create an invasion assay the pores in the membrane are blocked with a gel composed of extracellular matrix that is meant to mimic the typical matrices that tumour cells encounter during the invasion process in vivo. By placing the cells on one side of the gel and a chemoattractant on the other side of the gel, invasion is determined by counting those cells that have traversed the cell-permeable membrane having invaded towards the higher concentration of chemoattractant. In this chapter, in addition to protocols for performing Transwell invasion assays, there is consideration of the limitations of current assay designs with regard to available matrices and the absence of tumour microenvironment cells.

Laminins are multidomain glycoproteins that play important roles in development and maintenance of the extracellular matrix via their numerous interactions with other proteins. Several receptors for the laminin short arms revealed their importance in network formation and intercellular signaling. However, both the detailed structure of the laminin γ-1 short arm and its organization within the complexes is poorly understood due to the complexity of the molecule and the lack of a high-resolution structure. The presented data provide the first subatomic resolution structure for the laminin γ-1 short arm in solution. This was achieved using an integrated approach that combined a number of complementary biophysical techniques such as small angle X-ray scattering (SAXS), analytical ultracentrifugation, dynamic light scattering and electron microscopy. As a result of this study, we have obtained a significantly improved model for the laminin γ-1 short arm that represents a major step forward in molecular understanding of laminin-mediated complex formations., (Crown Copyright © 2010. Published by Elsevier B.V. All rights reserved.)

Vitamin A is an essential lipid-soluble nutrient that is crucial for morphogenesis and adult tissue maintenance. The retinoid homeostasis in the liver depends on a regular supply of vitamin A from an adequate dietary intake to preserve the normal organ structure and functions. This study focuses on the effect of vitamin A deficiency on the morphology and extracellular proteins expression of the liver in adult Wistar rats. Animals were fed with a normal (control group) or deficient vitamin A diet for 3 months. At the end of the experimental period, histological examination of the livers under light and electron microscopy revealed that vitamin A deficiency produced a loss of hepatocyte cord disposition with an irregular parenchymal organization. Abundant fat droplets were present in the cytoplasm of the hepatocytes. Elongated myofibroblastic-like cells with an irregular cytoplasmic process and without lipid droplets could be seen at the perisinusoidal space, where an elevated intensity of alpha smooth muscle actin (alpha-SMA) was observed. These results suggest that an activation of hepatic stellate cells (HSCs) occurred. Moreover, immunochemical methods revealed that vitamin A deficiency led to an increased expression of hepatic fibronectin, laminin and collagen type IV. We propose that vitamin A deprivation caused liver injury and that HSCs underwent a process of activation in which they produced alpha-SMA and synthesized extracellular components. These changes may be a factor predisposing to liver fibrosis. In consequence, vitamin A deprivation could affect human and animal health.

Development of the peripheral nervous system requires radial axonal sorting by Schwann cells (SCs). To accomplish sorting, SCs must both proliferate and undergo morphogenetic changes such as process extension. Signaling studies reveal pathways that control either proliferation or morphogenesis, and laminin is essential for SC proliferation. However, it is not clear whether laminin is also required for SC morphogenesis. By using a novel time-lapse live-cell-imaging technique, we demonstrated that laminins are required for SCs to form a bipolar shape as well as for process extension. These morphological deficits are accompanied by alterations in signaling pathways. Phosphorylation of Schwannomin at serine 518 and activation of Rho GTPase Cdc42 and Rac1 were all significantly decreased in SCs lacking laminins. Inhibiting Rac1 and/or Cdc42 activities in cultured SCs attenuated laminin-induced myelination, whereas forced activation of Rac1 and/or Cdc42 in vivo improved sorting and hypomyelinating phenotypes in SCs lacking laminins. These findings indicate that laminins play a pivotal role in regulating SC cytoskeletal signaling. Coupled with previous results demonstrating that laminin is critical for SC proliferation, this work identifies laminin signaling as a central regulator coordinating the processes of proliferation and morphogenesis in radial axonal sorting.

The basement membrane protein, laminin I, has been used broadly as a planar two-dimensional film or in a three-dimensional form as a reconstituted basement membrane gel such as Matrigel to support cellular attachment, growth, and differentiation in vitro. In basement membranes in vivo, laminin exhibits a fibrillar morphology, highlighting the electrospinning process as an ideal method to recreate such fibrous substrates in vitro. Electrospinning was employed to fabricate meshes of murine laminin I nanofibers (LNFs) with fiber size, geometry, and porosity of authentic basement membranes. Purified laminin I was solubilized and electrospun in parametric studies of fiber diameters as a function of polymer solution concentration, collecting distance, and flow rate. Resulting fiber diameters ranged from 90 to 300 nm with mesh morphologies containing beads. Unlike previously described nanofibers (NFs) synthesized from proteins such as collagen, meshes of LNFs retain their structural features when wetted and do not require fixation by chemical crosslinking, which often destroys cell attachment and other biological activity. The LNF meshes maintained their geometry for at least 2 days in culture without chemical crosslinking. PC12 cells extended neurites without nerve growth factor stimulation on LNF substrates. Additionally, LNFs significantly enhance both the rate and quantity of attachment of human adipose stem cells (ASCs) compared to laminin films. ASCs were viable and maintained attachment to LNF meshes in serum-free media for at least 3 days in culture and extended neurite-like processes after 24 h in serum-free media conditions without media additives to induce differentiation. LNF meshes are a novel substrate for cell studies in vitro, whose properties may be an excellent scaffold material for delivering cells in tissue engineering applications in vivo.

Hydra, as an early diploblastic metazoan, has a well-defined extracellular matrix (ECM) called mesoglea. It is organized in a tri-laminar pattern with one centrally located interstitial matrix that contains type I collagen and two sub-epithelial zones that resemble a basal lamina containing laminin and possibly type IV collagen. This study used monoclonal antibodies to the three hydra mesoglea components (type I, type IV collagens and laminin) and immunofluorescent staining to visualize hydra mesoglea structure and the relationship between these mesoglea components. In addition, hydra mesoglea was isolated free of cells and studied with immunofluorescence and scanning electron microscopy (SEM). Our results show that type IV collagen co-localizes with laminin in the basal lamina whereas type I collagen forms a grid pattern of fibers in the interstitial matrix. The isolated mesoglea can maintain its structural stability without epithelial cell attachment. Hydra mesoglea is porous with multiple trans-mesoglea pores ranging from 0.5 to 1 microm in diameter and about six pores per 100 microm(2) in density. We think these trans-mesoglea pores provide a structural base for epithelial cells on both sides to form multiple trans-mesoglea cell-cell contacts. Based on these findings, we propose a new model of hydra mesoglea structure.

Cardiac tissue engineering is focused on obtaining functional cardiomyocyte constructs to provide an alternative to cellular cardiomyoplasty. Mechanical stimuli have been shown to stimulate protein expression and the differentiation of mammalian cells from contractile tissues. Our aim was to obtain a flexible scaffold which could be used to apply mechanical forces during tissue regeneration. Poly(1,8-octanediol-co-citric acid) (POC) is an elastomer that can be processed into scaffolds for tissue engineering. We investigated the effect of modifying the porosity on the mechanical properties of the POC scaffolds. In addition, the effects of the storage method and strain rate on material integrity were assessed. The maximum elongation of POC porous films varied from 60% to 160% of their original length. A decrease in the porosity caused a rise in this elastic modulus. The attachment of HL-1 cardiomyocytes to POC was assessed on films coated with fibronectin, collagen and laminin. These extracellular matrix proteins promoted cell adhesion in a protein-type- and concentration-dependent manner. Therefore, POC scaffolds can be optimised to meet the mechanical and biological parameters needed for cardiac culture. This porous material has the potential to be used for cardiac tissue engineering as well as for other soft tissue applications.

The conformation of single laminin molecules adsorbed on synthetic substrates is directly observed making use of the phase magnitude in tapping mode atomic force microscopy (AFM). With AFM, it is not possible to differentiate the proteins on the substrate if use is made of the height signal, since the roughness of the material becomes of the same order of magnitude as the adsorbed protein, typically 10 nm height. This work shows how AFM can be exploited to reveal protein conformation on polymer materials. Different laminin morphologies are observed on a series of different copolymers based on ethyl acrylate and hydroxyethyl acrylate as a function of the surface density of -OH groups: from globular to completely extended morphologies of the protein molecules are obtained, and the onset of laminin network formation on some substrates can be clearly identified. The results stress the importance of the underlying synthetic substrate's surface chemistry for the biofunctional conformation of adsorbed proteins.

The antigen I/II family of surface proteins is expressed by most oral streptococci, including Streptococcus mutans, and mediates specific adhesion to, among other things, salivary films and extracellular matrix proteins. In this study we showed that antigen I/II-deficient S. mutans isogenic mutant IB03987 was nearly unable to adhere to laminin films under flow conditions due to a lack of specific interactions (0.8 x 10(6) and 1.1 x 10(6) cells cm(-2) at pH 5.8 and 6.8, respectively) compared with parent strain LT11 (21.8 x 10(6) and 26.1 x 10(6) cells cm(-2)). The adhesion of both the parent and mutant strains was slightly greater at pH 6.8 than at pH 5.8. In addition, atomic force microscopy (AFM) experiments demonstrated that the parent strain experienced less repulsion when it approached a laminin film than the mutant experienced. Upon retraction, combined specific and nonspecific adhesion forces were stronger for the parent strain (up to -5.0 and -4.9 nN at pH 5.8 and 6.8, respectively) than for the mutant (up to -1.5 and -2.1 nN), which was able to interact only through nonspecific interactions. Enthalpy was released upon adsorption of laminin to the surface of the parent strain but not upon adsorption of laminin to the surface of IB03987. A comparison of the adhesion forces in AFM with the adhesion forces reported for specific ligand-receptor complexes resulted in the conclusion that the number of antigen I/II binding sites for laminin on S. mutans LT11 is on the order of 6 x 10(4) sites per organism and that the sites are probably arranged along exterior surface structures, as visualized here by immunoelectron microscopy.

Recent studies, on cells cultured in 3D collagen gels, have shown that, beside from their well known biochemical role, fibronectin (FN) and laminin (LM) affect cell functions via a modification of mechanical and structural properties of matrix due to interaction with collagen molecules. Though biochemical properties of FN and LM have been widely studied, little is known about their role in collagen matrix assembly. The aim of this work was to characterize FN- and LM-based collagen semi-interpenetrating polymer networks (semi-IPNs), in order to understand how these biomacromolecular species can affect collagen network assembly and properties. Morphology, viscoelasticity and diffusivity of collagen gels and FN- and LM-based collagen semi-IPNs were analysed by Confocal Laser Scanning microscopy (CLSM), Environmental Scanning Electron microscopy (ESEM), Transmission Electron microscopy (TEM), Rheometry and Fluorescence Recovery After Photobleaching (FRAP) techniques. It was found that FN and LM were organized in aggregates, interspersed in collagen gel, and in thin fibrils, distributed along collagen fibres. In addition, high FN and LM concentrations affected collagen fibre assembly and structure and induced drastic effects on rheological and transport properties.

Two epithelial cell types cover the alveolar surface of the lung. Type II alveolar epithelial cells produce surfactant and, during development or following wounding, give rise to type I cells that are involved in gas exchange and alveolar fluid homeostasis. In culture, freshly isolated alveolar type II cells assume a more squamous (type I-like) appearance within 4 days after plating. They assemble numerous focal adhesions that associate with the actin cytoskeleton at the cell margins. These alveolar epithelial cells lose expression of type II cell markers including SP-C and after 4 days in culture express the type I cell marker T1alpha. Those cells that express T1alpha also deposit fibers of laminin-311 in their matrix. The latter appears to be related to their development of a type I phenotype because freshly isolated, primary type I cells also assemble laminin-311-rich fibers in vitro. A beta1 integrin antibody antagonist inhibits the assembly of laminin-311 matrix fibers. Moreover, the formation of laminin fibers is dependent on the activity of the small GTPases and is perturbed by ML-7, a myosin light chain kinase inhibitor. In summary, our data indicate that assembly of laminin-311 fibers by lung epithelial cells is integrin and actin cytoskeleton dependent, and that these fibers are characteristic of type I alveolar cells.

Large area nanopatterns of functional proteins are demonstrated. A new approach to analyze atomic force microscopy height histograms is used to quantify protein and antibody binding to nanoscale patches. Arrays of nanopatches, each containing less than 40 laminin molecules, are shown to be highly functional binding close to 1 monoclonal anti-laminin IgG (site by IKVAV sequence) or 3-4 polyclonal anti-laminin IgG's per surface bound laminin. Complementary quartz crystal microbalance measurements indicate higher functionality at nanopatches than on homogeneous surfaces.

Endothelial cells play a crucial role in the pathogenesis of many diseases and are highly sensitive to low gravity conditions. Using a three-dimensional random positioning machine (clinostat) we investigated effects of simulated weightlessness on the human EA.hy926 cell line (4, 12, 24, 48 and 72 h) and addressed the impact of exposure to VEGF (10 ng/ml). Simulated microgravity resulted in an increase in extracellular matrix proteins (ECMP) and altered cytoskeletal components such as microtubules (alpha-tubulin) and intermediate filaments (cytokeratin). Within the initial 4 h, both simulated microgravity and VEGF, alone, enhanced the expression of ECMP (collagen type I, fibronectin, osteopontin, laminin) and flk-1 protein. Synergistic effects between microgravity and VEGF were not seen. After 12 h, microgravity further enhanced all proteins mentioned above. Moreover, clinorotated endothelial cells showed morphological and biochemical signs of apoptosis after 4 h, which were further increased after 72 h. VEGF significantly attenuated apoptosis as demonstrated by DAPI staining, TUNEL flow cytometry and electron microscopy. Caspase-3, Bax, Fas, and 85-kDa apoptosis-related cleavage fragments were clearly reduced by VEGF. After 72 h, most surviving endothelial cells had assembled to three-dimensional tubular structures. Simulated weightlessness induced apoptosis and increased the amount of ECMP. VEGF develops a cell-protective influence on endothelial cells exposed to simulated microgravity.

T cells develop in the thymus in a highly specialized cellular and extracellular microenvironment. The basement membrane molecule, laminin-5 (LN-5), is predominantly found in the medulla of the human thymic lobules. Using high-resolution light microscopy, we show here that LN-5 is localized in a bi-membranous conduit-like structure, together with other typical basement membrane components including collagen type IV, nidogen and perlecan. Other interstitial matrix components, such as fibrillin-1 or -2, tenascin-C or fibrillar collagen types, were also associated with these structures. Three-dimensional (3D) confocal microscopy suggested a tubular structure, whereas immunoelectron and transmission electron microscopy showed that the core of these tubes contained fibrillar collagens enwrapped by the LN-5-containing membrane. These medullary conduits are surrounded by thymic epithelial cells, which in vitro were found to bind LN-5, but also fibrillin and tenascin-C. Dendritic cells were also detected in close vicinity to the conduits. Both of these stromal cell types express major histocompatibility complex (MHC) class II molecules capable of antigen presentation. The conduits are connected to blood vessels but, with an average diameter of 2 mum, they are too small to transport cells. However, evidence is provided that smaller molecules such as a 10 kDa dextran, but not large molecules (>500 kDa), can be transported in the conduits. These results clearly demonstrate that a conduit system, which is also known from secondary lymphatic organs such as lymph nodes and spleen, is present in the medulla of the human thymus, and that it might serve to transport small blood-borne molecules or chemokines to defined locations within the medulla.

In this paper, we report on engineering 3-D pulmonary tissue constructs in vitro. Primary isolates of murine embryonic day 18 fetal pulmonary cells (FPC) were comprised of a mixed population of epithelial, mesenchymal, and endothelial cells as assessed by immunohistochemistry and RT-PCR of 2-D cultures. The alveolar type II (AE2) cell phenotype in 2-D and 3-D cultures was confirmed by detection of SpC gene expression and presence of the gene product prosurfactant protein C. Three-dimensional constructs of FPC were generated utilizing Matrigel hydrogel and synthetic polymer scaffolds of poly-lactic-co-glycolic acid (PLGA) and poly-L-lactic-acid (PLLA) fabricated into porous foams and nanofibrous matrices, respectively. Three-dimensional Matrigel constructs contained alveolar forming units (AFU) comprised of cells displaying AE2 cellular ultrastructure while expressing the SpC gene and gene product. The addition of tissue-specific growth factors induced formation of branching, sacculated epithelial structures reminiscent of the distal lung architecture. Importantly, 3-D culture was necessary for inducing expression of the morphogenesis-associated distal epithelial gene fibroblast growth factor receptor 2 (FGFr2). PLGA foams and PLLA nanofiber scaffolds facilitated ingrowth of FPC, as evidenced by histology. However, these matrices did not support the survival of distal lung epithelial cells, despite the presence of tissue-specific growth factors. Our results may provide the first step on the long road toward engineering distal pulmonary tissue for augmenting and/or replacing dysfunctional native lung in diseases, such as neonatal pulmonary hypoplasia.

Laminins are components of basement membranes that are required for morphogenesis, organizing cell adhesions and cell signaling. Studies have suggested that laminins function as alpha(x) beta(y) gamma(z) heterotrimers in vivo. In C. elegans, there is only one laminin beta gene, suggesting that it is required for all laminin functions. Our analysis is consistent with the role of the laminin beta as a subunit of laminin heterotrimers; the same cells express the laminin alpha, beta, and gamma subunits, the laminin beta subunit localizes to all basement membranes throughout development, and secretion of the beta subunit requires an alpha subunit. RNAi inhibition of the beta subunit gene or of the other subunit genes causes an embryonic lethality phenotype. Furthermore, a distinctive set of phenotypes is caused by both viable laminin alpha and beta partial loss-of-function mutations. These results show developmental roles for the laminin beta subunit, and they provide further genetic evidence for the importance of heterotrimer assembly in vivo.

A cell-extraction protocol yielding an esophagus acellular matrix (EAM) scaffold for use in tissue engineering of an esophagus, including hypotonic lysis, multiple detergent cell extraction steps, and nucleic acid digestion, was developed in a rat model. Histological techniques, burst pressure studies, in vitro esophageal epithelial cell seeding, and in vivo implantation were used to assess cell extraction, extracellular matrix (ECM) preservation, and biocompatibility. Microscopy demonstrated that cell extraction protocols using sodium dodecyl sulfate (SDS) (0.5%, wt/vol) as a detergent resulted in cell-free EAM with retained ECM protein collagen, elastin, laminin, and fibronectin. Burst pressure studies indicated a loss of tensile strength in EAMs, but at intraluminal pressures that were unlikely to affect in vivo application. In vitro cell seeding studies exhibited epithelial cell proliferation with stratification similar to native esophagi after 11 days, and subcutaneously implanted EAMs displayed neovascularization and a minimal inflammatory response after 30 days of implantation. This study presents an esophagus acellular matrix tissue scaffold with preserved ECM proteins, biomechanical properties, and the ability to support esophageal cell proliferation to serve as the foundation for a tissue-engineered esophagus.

In almost all physiological and pathological situations, cells migrate through three-dimensional environments, yet most studies of cell motility have used two-dimensional substrates. It is clear that two-dimensional substrates do not mimic the in vivo environment accurately, and recent work using three-dimensional environments has revealed many different mechanisms of cell migration (Abbott, 2003; Sahai and Marshall, 2003; Wolf et al., 2003). This chapter will describe methods for generating three-dimensional matrices suitable for studying cell motility, methods for imaging the morphology of motile cells in situ, and methods for quantifying cell migration through three-dimensional environments.

Epithelial cells grown in three-dimensional matrix allows one to investigate biological processes in a setting that is closer to in vivo conditions than those obtained by growing cells on plastic culture dishes. Here we outline procedures that allow one to investigate molecular mechanisms that regulate formation and disruption of three-dimensional epithelial structures.

Purpose: A rare, familial early-onset form of Fuchs corneal dystrophy (FCD) is caused by mutation in the COL8A2 gene. This study describes the aberrant pattern of distribution of collagen type VIII and basement membrane components in Descemet's membrane (DM) and endothelium of three individuals with the same L450W mutation that represent different stages of early-onset FCD., Methods: Immunohistochemical studies with bright field, fluorescence, and confocal microscopy characterized the pathology of sectioned corneal buttons with antibodies against COL8A1, COL8A2, COL4, laminin, and fibronectin. A portion of each was processed for electron microscopy., Results: Histologic examination of pathologic changes in case 1 demonstrated relative preservation of the endothelium, whereas in case 2 much of this layer was atrophic and in case 3 there was complete loss of the endothelium. DM also increased in thickness to 25 mum for case 1, to 31 mum for case 2, and to 38 mum for case 3. Case 1 was the only specimen to reveal shallow warts along the posterior surface of DM, whereas the most advanced specimen, case 3, showed evidence of earlier nodularity that had been buried by the accretion of further extracellular matrix material. The posterior aspect of DM in this specimen had the unusual property of lighter staining relative to the anterior region of DM, laid down earlier in life. Immunocytochemistry revealed increased expression and complex, sharply defined patterns of deposition of collagen VIII, collagen IV, laminin, and fibronectin. Ultrastructurally, the posterior nonbanded layer of DM was intermixed with banded collagen, and the posterior region of DM showed a high density of foci of spindle-shaped structures with intense-staining bands, spaced at approximately 120 nm. Finally, ultrastructural studies of the endothelium in case 1 revealed unusual accumulation of swelling mitochondria. The endothelial cells also had large amounts of abnormal prominent rough endoplasmic reticulum. Type VIII collagen alpha 2 immunogold signal was associated with the highly granular ribosomes of the rough endoplasmic reticulum of these patients., Conclusions: Microscopic and electron microscopic examination revealed pathological changes in DM of L450W COL8A2 mutants that were consistent with several-fold increased growth of the extracellular matrix and progressive deposition and synthesis of extracellular material by endothelial cells. As with late-onset FCD, this is accompanied by attenuation and eventual loss of the endothelium itself. Whether the abnormal deposition of collagen, laminin, and fibronectin contributes to the dysfunction and death of the endothelium remains to be determined.

Laminin-5 (Ln-5) is an important molecule associated with epithelial cell adhesion and migration. In the gingiva around the tooth, Ln-5 localizes within basement membranes between the junctional epithelium (JE) and the tooth or connective tissue. Recently, we reported that in the oral mucosa around a dental implant, Ln-5 is expressed within the basement membranes at the implant-peri-implant epithelium (PIE) interface, and at the PIE-connective tissue interface. However, the ultrastructural localization of Ln-5 within or along the PIE has not yet been reported. Therefore, peri-implant oral mucosa was treated with anti-Ln-5 (gamma2 chain) antibody and examined using immuno-electron microscopy. Ln-5 was localized in the cells of the innermost-third layer and basal layer of the PIE. A 100-nm-wide Ln-5-positive internal basal lamina (basement membrane) and hemidesmosomes as adhesion structures were formed at the apical portion of the implant-PIE interface. However, at the upper-middle portion of the interface, these adhesion structures were not observed. Furthermore, at the PIE-connective tissue interface, the Ln-5-positive external basal lamina (basement membrane) and hemidesmosomes were partially deficient. Judging from these findings, we concluded that Ln-5 contributes to the attachment of the PIE to the titanium surface, and that PIE attached to titanium at the apical portion of the dental implant-PIE interface.

Clinical islet transplantation in liver has achieved normoglycemia. However, this site may not be ideal for islet survival. To create a more optimal site for islet transplantation, we have developed a construct with biodegradable scaffolds. Islets were seeded in scaffolds and transplanted into the epididymal fat pad of diabetic BALB/c mice. Controls included islets transplanted underneath the kidney capsule or into the fat pad without scaffolds. All animals with islets in scaffolds or the kidney became normoglycemic and maintained this metabolic state. When islets were transplanted without scaffolds the time to achieve normoglycemia was significantly increased and less than 45% of mice survived. An oral glucose tolerance test was performed on the scaffold and kidney groups with similar blood glucose levels and area under the curve values between the groups. Grafts were removed at more than 100 days posttransplantation and all animals became hyperglycemic. There was no significant difference in insulin content between the grafts and all grafts were well vascularized with insulin-positive beta cells. Therefore, islets in scaffolds function and restore diabetic animals to normoglycemic levels, similar to islets transplanted underneath the kidney capsule, suggesting scaffolds can be used to create a site for islet transplantation.

Embryonic stem (ES) cells could potentially serve as an excellent cell source for various applications in regenerative medicine and tissue engineering. Our laboratory is particularly interested in generating a reproducible endothelial cell source for the development of prevascularized materials for tissue/organ reconstruction. After developing methods to isolate highly purified (>96%) proliferating populations of endothelial cells from mouse embryonic stem cells, we tested their ability to form three-dimensional (3-D) vascular structures in vitro. The ES cell-derived endothelial cells were embedded in 3-D collagen gel constructs with rat tail collagen type I (2 mg/mL) at a concentration of 10(6) cells/mL of gel. The gels were observed daily with a phase-contrast microscope to analyze the time course for endothelial cell assembly. The first vessels were observed between days 3 and 5 after gel construct formation. The number and complexity of structures steadily increased, reaching a maximum before beginning to regress. By 2 weeks, all vessel-like structures had regressed back to single cells. Histology and fluorescent images of the vessel-like structures verified that tube structures were multicellular and could develop patent lumens. We have shown that endothelial cells derived, purified and expanded in vitro from ES cells sustain an important endothelial cell function, the ability to undergo vasculogenesis in collagen gels, indicating that endothelial products derived in vitro from stem cells could be useful in regenerative medicine applications.

Physiological patterns of the extracellular matrix protein, laminin-1, were obtained on glass substrates by physisorption-assisted microcontact printing. Besides the well-retained antigenicity confirmed by indirect immunofluorescence assays, we investigated the supramolecular organization of the proteins by atomic force microscopy. We found the characteristic protein self-assembling in polygonal networks with well-defined sub-100 nm quaternary structures of laminin. The formation of these physiological mesh-like protein matrices was obtained by means of one-step soft lithography without any preliminary functionalization of glass, which can be exploited for many possible applications for cell cultures and biomolecular devices.

In this brief introductory paper the general structure and the molecular composition of the extracellular matrix are outlined. Ultrastructural morphology of the extracellular matrix is introduced and subsequently the molecular structure of each of the main protein families, which together make up the extracellular matrix, is reviewed. Collagens, laminins, tenascins, and proteoglycans are addressed. An important common feature is the domain structure of these in general very large proteins. Several families have domains in common, which favours extensive interactions. Integrins play an important role in these interactions and also in the communication between cells and the matrix. The extracellular matrix appears to be a very dynamic structure, which has a prominent role in normal development as well as in a variety of disease processes. Matrix metalloproteinases are essential actors in this complex interplay between cells and the extracellular matrix., (Copyright 2003 John Wiley & Sons, Ltd.)

Sulfur mustard (SM; 2,2(')-dichloroethyl sulfide) is a percutaneous alkylating agent first used as a chemical weapon at Ypres, Belgium, in World War I. Despite its well-documented history, the primary lesions effecting dermal-epidermal separation and latent onset of incapacitating blisters remain poorly understood. By immunofluorescent imaging of human epidermal keratinocytes (HEK) and epidermal tissues exposed to SM (400 microM for 5 min), we have amassed unequivocal evidence that SM disrupts adhesion complex molecules, which are also disrupted by epidermolysis bullosa-type blistering diseases of the skin. Images of keratin 14 (K14) in control cells showed tentlike filament networks linking the HEK's basolateral anchoring sites to the dorsal surface of its nuclei. Images from 6-h postexposure profiles revealed early disruption (

Proteoglycans are considered to be important molecule in cell-microenvironment interactions. They are overexpressed in neoplastic cells modifying their growth and migration in hosts. In this work we verified that undersulfation of proteoglycans and other sulfated molecules, induced by sodium chlorate treatment, inhibited C6 glioma cells proliferation in a dose-dependent way. This effect was restored by the addition of exogenous heparin. We could not detect significant cell mortality in our culture condition. The treatment also impaired in a dose-dependent manner, C6 cell adhesion to extracellular matrix (ECM) proteins (collagen IV, laminin and fibronectin). In addition, sodium chlorate treatment altered C6 glioma cell morphology, from the fibroblast-like to a more rounded one. This effect was accompanied by increased synthesis of fibronectin and alterations in its extracellular network organization. However, we could not observe modifications on laminin organization and synthesis. The results suggest an important connection between sulfation degree with important tumor functions, such as proliferation and adhesion. We suggest that proteoglycans may modulate the glioma microenvironment network during tumor cell progression and invasion.

The laminin (LN) family of large heterotrimeric extracellular matrix glycoproteins has multiple functions: LNs take part in the regulation of processes such as cell migration, differentiation, and proliferation, in addition to contributing to the structure of basement membranes. LN-10, composed of alpha5, beta1, and gamma1 chains, is widely distributed in most basement membranes of both epithelia and endothelia. We determined the complete human cDNA sequence for the LN alpha5 chain and produced recombinant human LN-10 (rLN-10) in HEK293 cells by triple transfection of full-length cDNAs encoding the human LN alpha5, beta1, and gamma1 chains. The rLN-10 was purified using affinity chromatography and had an apparent molecular mass of approximately 800 kDa in SDS-PAGE and a native domain structure in rotary shadowing electron microscopy. By using function-blocking monoclonal antibodies, integrin alpha(3)beta(1) was found to be a major mediator of adhesion of HT-1080 and human saphenous vein endothelial cells. Human saphenous vein endothelial cells adhered more strongly to rLN-10 than to LN-1 and LN-8 and showed better migration on rLN-10, compared with several other matrices. Considering the cell adhesive and migration-promoting properties of rLN-10 on endothelial cells, this molecule could be useful in improving the biocompatibility and endothelialization of vascular grafts.

Laminins are important for Schwann cell basement membrane assembly and axonal function. In this study, we found that exogenous laminin-1, like neuromuscular laminins-2/4, formed two distinct extracellular matrices on Schwann cell surfaces, each facilitated by laminin polymerization. Assembly of one, a densely-distributed reticular matrix, was accompanied by a redistribution of cell-surface dystroglycan and cytoskeletal utrophin into matrix-receptor-cytoskeletal complexes. The other, a fibrillar matrix, accumulated in separate zones associated with pre-existing beta1-integrin arrays. The laminin-1 fragment E3 (LG-modules 4-5), which binds dystroglycan and heparin, inhibited reticular-matrix formation. By contrast, beta1-integrin blocking antibody (Ha2/5) prevented fibrillar assembly. Ultrastructural analysis revealed that laminin treatment induced the formation of a linear electron-dense extracellular matrix (lamina densa) separated from plasma membrane by a narrow lucent zone (lamina lucida). This structure was considerably reduced with non-polymerizing laminin, fully blocked by E3, and unaffected by Ha2/5. Although it formed in the absence of type IV collagen, it was nonetheless able to incorporate this collagen. Finally, cell competency to bind laminin and form a basement membrane was passage-dependent. We postulate that laminin induces the assembly of a basement membrane on competent cell surfaces probably mediated by anchorage through LG 4-5. Upon binding, laminin interacts with dystroglycan, mobilizes utrophin, and assembles a 'nascent' basement membrane, independent of integrin, that is completed by incorporation of type IV collagen. However, the fibrillar beta1-integrin dependent matrix is unlikely to be precursor to basement membrane.

The ability of carcinomas to invade and metastasize depends in part on their passage through basement membranes. Two of the major components of basement membranes are type IV collagen and laminin and these have been extensively studied by light microscopical immunocytochemical techniques to investigate alterations in their distribution in human breast carcinomas [1-3]. Breaks in the epithelial basement membrane associated with malignant epithelial tumors have been reported [3, 4]. However, these breaks are based on immunocytochemical observations and have not been correlated with basement membrane morphology. By light microscopical techniques there is no evidence of type IV collagen or laminin between myoepithelial cells or between myoepithelial and epithelial cells and they appear to be restricted to the basement membrane surrounding the entire ductule of the breast. By electron microscopy the basement membrane region contains a linear, homogeneous, electron-dense region (lamina densa) beneath a clear zone (lamina lucida) directly beneath the epithelial and myoepithelial cells of the breast ductule. These two regions constitute the basal lamina. Hemidesmosomes are present on the basal aspect of myoepithelial cells where fine filaments connect these regions to the adjacent basal lamina. The extracellular matrix components laminin and type IV collagen have both been localized in the basement membrane of the normal human breast ductule. Breaks in the continuity of these components occur in breast carcinomas and have been implicated in tumor metastasis. Using a postembedding ultrastructural immunogold technique, laminin and type IV collagen were distributed within the basal lamina surrounding the normal human breast ductule. Both components were present diffusely along the basal lamina and were not localized to particular regions, and neither were present between epithelial and myoepithelial cells. Laminin binding of these cells thus probably occurs only at the basal aspect where they are in contact with the basal lamina and is not involved in the cell-cell adhesion between epithelial and myoepithelial cells. This study provides a basis for further ultrastructural investigations of extracellular matrix components in normal and neoplastic breast tissue.

Background: The dento-epithelial junction forms the primary periodontal defense structure against oral microbes. The cells of the junctional epithelium (JE) attach both to a basement membrane (BM) facing the connective tissue and to a hard dental tissue by structurally similar but molecularly distinct mechanisms. Here we describe a new organotypic cell culture model for the dento-epithelial junction comprising not only epithelial and mesenchymally derived components, but also a tooth surface equivalent., Methods: Rat palatal keratinocytes were seeded on fibroblast-collagen gels. A tooth slice was placed on top of the epithelial cells and the multilayer cultures were grown at the air-liquid interface. Formation of the epithelial structures, BM components, and the epithelial attachment to the tooth surface were studied by immunofluorescence and light and electron microscopy. The findings were compared to the structure of the dento-epithelial junction in vivo., Results: A well-differentiated stratified epithelium was formed. Under the tooth slice the epithelium remained thin and non-differentiated. Attachment of the epithelial cells to the tooth surface was mediated by hemidesmosomes (HDs) as in vivo. Laminin-5 (Ln-5) was present in the extracellular matrix (ECM) between the tooth and the epithelium as well as in the BM structure between the epithelium and the fibroblast-collagen matrix. Instead, Ln-10/11 was present only at the mesenchymal tissue side as is known to be the case in vivo., Conclusions: The organotypic model presented expresses the characteristic structural and molecular features of the dento-epithelial junction and may be applied for studying physiological and pathological processes in the epithelial attachment.

Purpose: To determine whether differences in the ultrastructural characteristics or composition of the basement membranes of the trabecular lamellae and Schlemm's canal exist in normal eyes and eyes with primary open-angle glaucoma (POAG). Basement membranes play key roles in the attachment of the overlying trabecular cells and Schlemm's canal cells., Methods: Electron microscopy used in conjunction with immunogold labeling was used to examine the ultrastructure of the basement membranes in the trabecular meshwork and to determine the presence of collagen IV, laminin, and fibronectin in 6 normal eyes and 6 eyes with POAG. To determine which cells in the meshwork synthesized these molecules in situ hybridization was studied in an additional 8 normal eyes., Results: No distinctive ultrastructural changes were found in the basement membranes of glaucomatous eyes, whether early or advanced disease, when compared with normal eyes. Label for all three proteins was present in the basement membranes of the trabecular lamellae, Schlemm's canal, and in scattered patches within the juxtacanalicular tissue. Laminin and fibronectin were most abundant in the periphery of the sheath material surrounding the elastic tendons in the juxtacanalicular tissue. In contrast to previously published light microscopic studies, no increase in fibronectin was found in glaucoma. Regions of the basement membrane of the canal underlying giant vacuoles were similar to regions without giant vacuoles in both appearance and labeling. In situ hybridization revealed that mRNA for all three proteins was present in most trabecular cells throughout the meshwork; no regional differences in cellular labeling within were observed., Conclusion: The ultrastructural characteristics and immunogold labeling of basement membranes were similar in normal and glaucomatous eyes; no additional structures were labeled in POAG eyes that were not also labeled in normal eyes. Label of the patches of amorphous fibrogranular material within the juxtacanalicular tissue suggests it is basement membrane in origin, while the sheath material which is known to accumulate in POAG was not heavily labeled and does not appear to be basement membrane in origin., (Copyright 2001 S. Karger AG, Basel)

Assembly of the extracellular matrix (ECM) has been tightly linked to compact myelin formation in the peripheral nervous system. We recently demonstrated that myelination of dorsal root ganglion (DRG) axons by Schwann cells may occur in the absence of basal lamina. We have now determined whether laminin deposition occurs around myelinating SC, even though basal lamina has not been assembled. DRG/SC co-cultures were prepared from E15 rat embryos and incubated in fully defined medium (B27) with and without ascorbic acid for 21-24 days. Cultures were stained with a rabbit anti-laminin antibody and examined by laser confocal fluorescence microscopy. Myelination occurred in both groups. In the presence of ascorbic acid, there was dense even laminin staining around myelinating SC. In the absence of ascorbic acid, laminin staining was also present but was irregular and less dense. DRG and SC were co-cultured without ascorbic acid in the presence or absence of a function blocking anti-beta(1) integrin receptor antibody. The antibody completely inhibited myelination. Finally, DRG/SC co-cultures were prepared both with and without ascorbic acid and incubated under control conditions or in the presence of continual, gentle motion. Movement in the absence of ECM significantly inhibited myelination. This demonstrates that laminin deposition on the surface of SC but not ECM assembly is required for formation of compact myelin. ECM is required to provide mechanical stability during the process of myelination., (Copyright 2001 Wiley-Liss, Inc.)

Objectives: Degeneration of bioprosthetic heart valves has been suggested to be at least partly an immunogenic reaction toward the xenogeneic tissue. An autologous endothelial lining has been proposed to overcome this problem. We examined in vitro endothelialization of such tissue and retention of endothelial cells after exposure to flow resembling the in vivo situation., Methods: Cultured human saphenous vein endothelial cells were used to in vitro endothelialize photo-oxidized bioprosthetic heart valves. The endothelialized valves were mounted in a specially designed flow device, creating a pulsatile flow through the valve. Maintenance of a confluent cell layer and deposition of basement membrane markers were determined with immunohistochemical labeling., Results: Labeling of the main components of the basement membrane, laminin and collagen type IV, was verified within 6 hours after in vitro endothelialization. Under static conditions, 4-mm wide denudations were completely re-endothelialized in 4 days, which was similar to the growth rate on gelatin-coated cell culture plastic, which served as a control material. After exposure of endothelialized valves to pulsatile flows for 24 hours (80 beats/min, 3.4 L/min), there were minimal cell losses from the bioprostheses. The cell layer adapted to the pulsatile flow, as verified by rearrangement of morphology and intracellular stress fibers., Conclusions: This study shows the feasibility of in vitro endothelialization of photo-oxidized bioprosthetic heart valves. The cells are able to withstand a pulsatile flow in vitro, to develop basement membrane-like structures, and to re-endothelialize denuded areas. This technology may be used to enhance the performance of bioprosthetic heart valve prostheses.

Papillary thyroid carcinomas (PTCs) have characteristic nuclear shape changes compared to follicular-type thyroid epithelium. We tested the hypothesis that the altered nuclear shape results from altered distribution or expression of the major structural proteins of the nuclear envelope. Lamin A, lamin B1, lamin C, lamin B receptor (LBR), lamina-associated polypeptide 2 (LAP2), emerin, and nuclear pores were examined. PTC's with typical nuclear features by H&E were compared to non-neoplastic thyroid and follicular neoplasms using confocal microscopy, and semi-quantitative immunoblotting. Lamin A/C, lamin B1, LAP2, emerin, and nuclear pores all extend throughout the grooves and intranuclear inclusions of PTC. Their distribution and fluorescent intensity is not predictably altered relative to nuclear envelope irregularities. By immunoblotting, the abundance (per cell) and electrophoretic mobilities of lamin A, lamin B1, lamin C, emerin, and LAP2 proteins do not distinguish PTC, normal thyroid, or follicular neoplasms. These results do not support previously published predictions that lamin A/C expression is related to a loss of proliferative activity. At least three LAP2 isoforms are identified in normal and neoplastic thyroid. LBR is sparse or undetectable in all the thyroid samples. The results suggest that the irregular nuclear shape of PTC is not determined by these nuclear envelope structural proteins per se. We review the structure of the nuclear envelope, the major factors that determine nuclear shape, and the possible functional consequences of its alteration in PTC.

The major macromolecules of basement membranes-collagen IV, laminin-1, and heparan sulfate proteoglycan (HSPG)-have been analyzed by atomic force microscopy (AFM), both individually and in combination with each other. The positions of laminin binding to collagen IV were mapped and compared with the positions of imperfections in the amino acid sequence of collagen IV; the apparent molecular volumes of the HSPG proteoglycans were measured and used to estimate the corresponding molecular weights. Even the thin, thread-like strands of the polyanion heparan sulfate can be visualized with AFM without staining, coating, or fixation. These strands are single polysaccharide chains and are thus thinner than single-stranded DNA. The heparan sulfate strands in HSPG are necessary for protein filtration in kidney basement membranes. We propose that these thin strands filter proteins by functioning as an entropic brush-i.e., that they filter proteins by their constant thermally driven motion in the basement membrane. These AFM analyses in air are a step toward AFM analyses under fluid of basement membrane macromolecules interacting with each other., (Copyright 2000 Academic Press.)

Laminins are a large family of heterotrimeric extracellular matrix glycoproteins that, in addition to having structural roles, take part in the regulation of processes such as cell migration, differentiation, and proliferation. The laminin alpha(4) chain is widely distributed both in adults and during development in tissues such as cardiac, skeletal and smooth muscle fibers, vascular endothelia, lungs, and in peripheral nerves. It can associate with laminin beta(1)/gamma(1) chains to form laminin-8 and with the beta(2)/gamma(1) chains to form laminin-9. Functional studies on these laminins have been hampered by poor availability of the protein in pure and soluble forms. To facilitate studies on laminin-8, recombinant laminin-8 was produced in a mammalian expression system, purified and shown to form native Y-shaped molecules in rotary shadowing electron microscopy. Integrins mediating cell adhesion to laminin-8 were identified using function-blocking mAbs. The integrin specificities were found to differ somewhat from that of laminin-1. Integrin alpha(6)beta(1) was found to be a major mediator of adhesion of HT-1080 and cultured capillary endothelial cells to laminin-8. Considering the expression patterns of laminin-8 and integrin alpha(6)beta(1) it is likely that the former is a ligand for the latter in vivo as well.

Medullary thyroid carcinoma (MTC) originates from C cells, which secrete calcitonin (CT) and CT gene-related peptide (CGRP), the two splice peptide products of the CALC I gene. Normal and hyperplastic C cells are intrafollicular, in contact with the basement membrane (BM) that is maintained around the differentiated tumors. To investigate the relationships between MTC evolution and BM constituents, we examined the modifications induced by laminin-1 and -2 (merosin), two isoforms colocalized in the follicular BM, on three MTC cell lines: murine rMTC 6-23 and CA-77 cells, and human TT cells. Laminin exerted a mitogenic activity on rMTC 6-23 and on TT cells, causing a concurrent decrease in both CT and CGRP mRNA levels and production of the peptides. Conversely, laminin reduced the proliferation rate and enhanced CGRP synthesis and secretion in CA-77 cells. This antiproliferative response, which coincides with an increase in differentiation markers, is comparable to that reported in normal cells and also in the neoplastic Caco-2 cell line. This suggests that laminin could exert opposite effects depending on the stage of tumor evolution.