In vitro studies on the expansion of endothelial cell monolayers on components of the basement membrane

The purpose of the present study was to observe the expansion of a monolayer of endothelial cells over specific components of the basement membrane. This was performed in vitro in a monolayer expansion assay over 5 days. The control surface was uncoated glass in the form of coverslips. Test substances were coated at a concentration of 10 μg/ml. The highest expansion was obtained with a high molecular weight fragment mixture of collagen type IV (IV-F, consisting of 75, 120 and 140 KD fragments), followed by fibronectin. Collagens type I, III and IV tetramer gave similar results, less than fibronectin or collagen type IV-F, although all of the above basement membrane coatings promoted expansion significantly above that of the control (P<0.01). The poorest expansion was obtained with laminin, which was significantly less than the control. The pentapeptide GRGDS, related to the fibronectin cell binding region, gave expansion significantly below that of the intact fibronectin molecule, as did the intact collagen type IV molecule compared with type IV-F (P<0.025). This indicates that sequences of the fibronectin molecule other than the cell binding sequence may be involved in promoting endothelial cell expansion. In addition, the integrity of the collagen type IV molecule does not appear necessary for this effect. On the contrary, the higher movement on IV-F may represent an inherent repair mechanism in damaged endothelium. Autoradiographic studies show that endothelial cell proliferation at the expanding front is involved in the migration assay.     

Interaction between endothelial cells and basement membrane components. In vitro studies on endothelial cell adhesion to collagen types I, III, IV and high molecular weight fragments of IV

Understanding the mechanisms involved in maintaining the integrity of the vascular endothelium is fundamental to studies on atherosclerosis, thrombosis, inflammation and tumor invasion. One of the essential aspects is the relationship between the endothelial cell (EC) layer and the underlying components of the basement membrane (BM). The importance of the biological role of the individual components of the BM in the promotion of EC adhesion is investigated. In this study suspensions of bovine corneal ECs (BCECs; 5 x 10(4)/ml) were used to investigate the adhesion of EC to collagen type IV and a mixture of fragments of the tetrameric molecule (IV-F, consisting of 75, 120 and 140 kD fragments), as well as collagen types I and III, coated at a 10-micrograms/ml concentration onto glass coverslips in vitro. Adhesion was quantified after 2 h of interaction by direct counting in the light microscope following fixation of the adherent cells. Collagens type IV and IV-F markedly promoted BCEC adhesion both in the presence or absence of 10 or 50% fetal calf serum, indicating that the integrity of the tetrameric molecule is not required for EC adhesion to collagen type IV, but can be replaced by high molecular weight fragments. Collagens type I and III increased EC adhesion in the absence of serum, although not in the presence of serum. Indirect evidence for a possible role of fibronectin in EC adhesion to type-IV collagen is given by the ability of the tetrapeptide (Arg-Gly-Asp-Ser (10 micrograms) to temporarily block (15-30 min) the adhesion-promoting effect of type-IV collagen. The nature of the adhesion sequences on the fragments of type-IV collagen remains to be elucidated.     

Clonal tumorigenic endodermal cell lines producing basement membrane components

Tumors were developed from the mouse teratocarcinoma-derived endodermal cell line PF HR-9 by subcutaneous injections in syngeneic mice of large numbers of cells previously cultured for several years at high cell density. Cell cultures were established from the tumors and the cells were cloned. The cloned sublines were highly malignant in vivo and tumor metastases were occasionally observed. The tumors contained abundant extracellular material, which was distinctly laminated and contained type IV collagen, laminin, and heparan sulfate proteoglycan. The tumorigenic sublines were also shown to have retained markers of the original cells, such as the cytoskeletal proteins Endo A and B. These cell lines should be useful for biosynthetic studies on basement membrane and cytoskeletal components and the tumors for isolation of these macromolecules and their mRNAs.     

Interactions of basement membrane components

The binding of laminin, type IV collagen, and heparan sulfate proteoglycan to each other was assessed. Laminin binds preferentially to native type IV (basement membrane) collagen over other collagens. A fragment of laminin (Mr 600 000) containing the three short chains (Mr 200 000) but lacking the long chain (Mr 400 000) showed the same affinity for type IV collagen as the intact protein. The heparan sulfate proteoglycan binds well to laminin and to type IV collagen. These studies show that laminin, type IV collagen and heparan sulfate proteoglycan interact with each other. Such interactions in situ may determine the structure of basement membranes.     

Interactions of basement membrane components

The binding of laminin, type IV collagen, and heparan sulfate proteoglycan to each other was assessed. Laminin binds preferentially to native type IV (basement membrane) collagen over other collagens. A fragment of laminin (M_r 600 000) containing the three short chains (M_r 200 000) but lacking the long chain M_r 400 000) showed the same affinity for type IV collagen as the intact protein. The heparan sulfate proteoglycan binds well to laminin and to type IV collagen. These studies show that laminin, type IV collagen and heparan sulfate proteoglycan interact with each other. Such interactions in situ may determine the structure of basement membranes.     

Glandular-like morphogenesis of the human submandibular tumor cell line A253 on basement membrane components

We have studied the interaction of a human tumor cell line, A253, derived from a submandibular gland carcinoma with a differentiation promoting reconstituted basement membrane extract, Matrigel. When cultured on plastic, these cells maintain a flat, cobblestone, epithelial morphology. On Matrigel, A253 cells initially form a honeycomb network of cords of cells which subsequently thickens. With time, these cords of cells become discontinuous and blunted, whereupon multilobular clusters of cells develop. These clusters possess a lumen with polarized, PAS(+) cells containing numerous desmosomes and an abundance of glycogen. Culture of the cells on laminin, the most abundant protein found in Matrigel, also induces this morphologic differentiation. Using synthetic laminin-derived peptides, the biologically active IKVAV-containing site of laminin was most active in attachment assays, as well as in inhibiting glandular-like morphogenesis when added to the media of cells cultured on Matrigel. Antibodies to the cell surface 67- and 32-kDa laminin binding proteins partially inhibited the glandular-like morphogenesis, suggesting that multiple interactions with laminin are likely required for the differentiation process. Our data demonstrate that A253 cells can undergo glandular-like morphogenesis on basement membrane and that laminin appears to be the major initiating factor.     

In vitro studies of membrane protein folding.

The study of membrane protein folding is a new and challenging research field. Consequently, there are few direct studies on the in vitro folding of membrane proteins. This review covers work aimed at understanding folding mechanisms and the intermolecular forces that drive the folding of integral membrane proteins. We discuss the kinetic and thermodynamic studies that have been undertaken. Our review also draws on closely related research, mainly from purification studies of functional membrane proteins, and gives an overview of some of the successful methods. A brief survey is also given of the large body of mutagenesis and fragment work on membrane proteins, as this too has relevance to the folding problem. It is noticeable that the choice of solubilizing detergents and lipids can determine the success of the method, and indeed it appears that particular lipid properties can be used to control the rate and efficiency of folding. This has important ramifications for much in vitro folding work in that it aids our understanding of how to obtain and handle folded, functional protein. With this in mind, we also cover some relevant properties of model, lipid-bilayer systems.     

Cryoinjury in endothelial cell monolayers

Developing successful cryopreservation strategies for corneas have proven to be more difficult than anticipated, because of the resulting loss of viability and detachment of endothelial cells from Descemet's membrane following cryopreservation of corneas. The objectives of this study are to develop a more detailed understanding of cryoinjury in human corneal endothelial cell (HCEC) monolayers and to examine the effects of storage temperature, cryoprotectant type and concentration, and cooling/warming rates on HCEC monolayers. Monolayers of endothelial cells attached to collagen-coated glass, immersed in an experimental solution (with and without cryoprotectant) were cooled at 1 degrees C/min to various temperatures (-5 to -40 degrees C), then thawed directly or cooled rapidly to -196 or to -80 degrees C before thawing. Cryoprotectants used were dimethyl sulfoxide and propylene glycol in concentrations of 1 and 2M. Monolayers were assessed for membrane integrity and detachment using SYTO/ethidium bromide fluorescent stain. The presence of cryoprotectants resulted in high recovery of membrane integrity and low monolayer detachment in monolayers thawed directly from temperatures down to -40 degrees C. In contrast, there was excessive detachment and loss of membrane integrity in monolayers cooled to -196 degrees C compared to monolayers cooled to -80 degrees C. Also, increasing cryoprotectant concentrations did not improve recovery of the monolayers. The higher recovery and lower detachment after storage at -80 degrees C compared to storage at -196 degrees C suggest that storage temperatures for corneas should be re-evaluated.     

Extracellular matrix components of the mouse thymic microenvironment. II. In vitro modulation of basement membrane proteins by interferon-gamma: relationship with thymic epithelial cell proliferation

We studied the effects of recombinant interferon-gamma (IFN-gamma) on some aspects of the physiology of two murine thymic epithelial cell (TEC) lines. Besides the expected induction of MHC class II antigens, this lymphokine was able to modulate the extracellular matrix (ECM) expression by growing TEC, as well as modulate their adhesion and proliferation patterns. As regards the influence of rIFN-gamma on ECM expression, we observed that when applied in very low doses, it promoted an increase in the amounts of basement membrane proteins, mainly fibronectin. In contrast, relatively high doses of this lymphokine (10(1) to 10(2) IU/ml) induced the opposite effect. Interestingly, both the stimulatory and the blocking effects of IFN-gamma on ECM expression were paralleled by equivalent modulation of cell proliferation, in both mouse and rat TEC lines. It should be pointed out that all these effects could be significantly abrogated by an anti-IFN-gamma monoclonal antibody. Searching for a putative mechanism that could be involved in the modulation of TEC proliferation by IFN-gamma, we observed a clear-cut positive correlation between cell adhesion and proliferation of TEC growing onto ECM-containing substrata produced following IFN-gamma treatment. The bulk of the data presented herein suggests that IFN-gamma may play a relevant role in TEC physiology and ontogeny, not only by inducing MHC class II antigen expression but also by regulating TEC growth via the control of extracellular matrix production by these cells.     

In vitro growth and differentiation of human kidney tubular cells on a basement membrane substrate

Summary Kidney cortical tubular cells, mainly proximal tubular cells, isolated from human kidney and grown either on a basement membrane substrate in chemically defined medium or on plastic in serum-supplemented medium, had substantial proliferative potential and could be propagated for more than 10 generations or 8 passages before senescence. Basement membrane produced on a plastic substrate by the HR-9 endodermal cell line could replace serum supplementation in promoting tubular cell growth. Tubular cells grown on an HR-9 basement membrane substrate exhibited stable epithelial morphology over an extended period of time; in the presence of 5% serum they differentiated into organized structures such as hemicysts and cell cords. Cells grown on plastic failed to differentiate and gradually degenerated. Tubular cells on HR-9 basement membrane were characterized by densely packed microvilli, abundant rough endoplasmic reticulum and free polysomes, basal cell membrane interdigitations, a well-developed endocytotic apparatus, and conspicuous junctional complexes—all features of the proximal tubular cell. Compared with cells on plastic substrate, there were higher levels of the brush border enzymes γ-glutamyl transpeptidase,l-leucine aminopeptidase, and alkaline phosphatase in cells maintained on an HR-9 basement membrane substrate, further supporting the conclusion that a basement membrane substrate promoted differentiation of tubular cells. These data and morphological observations indicate that a basement membrane substrate can promote growth and both functional and morphologic differentiation of human kidney tubular cells. This work was supported by the Veterans Administration.     

Effects of endothelial basement membrane on neutrophil adhesion and migration

The influence of the sub-endothelial basement membrane (BM) on the adhesion and migration of leukocytes is not well-defined. We therefore investigated the behaviour of human neutrophils on purified BM proteins and on BM deposited by short- or long-term cultures of endothelial cells (EC). The adhesion, but not migration velocities, of neutrophils activated with interleukin-8 was dependent on the coating concentrations of purified collagen, laminin or fibronectin. In contrast, adhesion was similar on matrices deposited by 3-day or 20-day cultures of EC, but neutrophils migrated more slowly on the distinct BM that formed over 20 days. In addition, while adhesion on all surfaces was greatly reduced when neutrophils were treated with antibody against β₂-integrins, antibody against β₁-integrins only inhibited adhesion to the 20-day BM. Thus, the native BM has distinct effects on integrin usage and migration by neutrophils, which are not reproduced by purified proteins or matrix deposited early during endothelial culture.     

Mechanical effects on endothelial cell morphology: In vitro assessment

Summary Endothelial cells are subjected to fluid mechanical forces which accompany blood flow. These cells become elongated and orient their long axes parallel to the direction of shear stress when the cultured cells are subjected to flow in an in vitro circulatory system. When the substrate is compliant and cyclically deformed, to simulate effects of pressure in the vasculature, the cells elongate an orient perpendicular to the axis of deformation. Cell shape changes are reflected in the alignment of microtubule networks. The systems described provide tools for assessing the individual roles of shear stress, pressure, and mechanical strain on vascular cell structure and function. This work was partially supported by grants HL 17437, HL 18072, and HL 23016 from the National Institutes of Health, Bethesda, MD, and grant C-938 from the Robert A. Welch Foundation.     

Protein kinase C regulates endothelial cell tube formation on basement membrane matrix, Matrigel.

Human umbilical vein endothelial cells differentiate within 12 h to form capillary-like networks of tube structures when the cells are plated on Matrigel, a mixture of basement membrane proteins. Nothing is known about the intracellular signaling events involved in this differentiation. As a first step to define the process, we investigated the possible role of protein kinase C activation by beta-phorbol 12-myristate 13-acetate (PMA) in regulating the formation of the tube structures. In this model, PMA increased tube formation several-fold in a dose-dependent manner with half-maximum stimulation of tube formation at approximately 5 nM PMA. In the absence of serum, essentially little or no tubes were formed on Matrigel unless PMA was added to the medium. Only active phorbol analogs increased tube formation, while the protein kinase C inhibitor, H-7, blocked tube formation. The protein kinase C activators and inhibitors were effective only when added at or just after plating of the cells and did not affect already formed tubes. This study suggests that protein kinase C is involved in the early events of in vitro endothelial cell tube formation on Matrigel.     

Haemocytes secrete basement membrane components in embryonic locusts

Several monoclonal antibodies raised against a glycoprotein-enriched fraction of adult muscle membranes of Locusta migratoria selectively stain particles within haemocytes and basement membrane in developing locust embryos. Haemocytes containing immunoreactive particles are found associated with areas where basement membrane is being laid down. The underlying ectoderm does not show immunoreactivity. We conclude that haemocytes contribute to basement membrane formation in embryonic locusts.     

Recombinant vascular basement membrane derived multifunctional peptide blocks endothelial cell angiogenesis and neovascularization

Angiogenesis is an innovative target in the therapy of cancer and other diseases, but the effects of anti‐angiogenic drugs have been rather modest in clinical trials. We have developed a small peptide, recombinant vascular basement membrane derived multifunctional peptide (rVBMDMP), which significantly inhibits endothelial cells in vitro. Here we test the mechanisms of rVBMDMP in angiogenesis balance in assays of tubule formation, colony formation, and apoptosis in HUVE‐12 endothelial cells. We also analyzed the differential expression of phosphorylation proteins and related genes in a protein phosphorylation chip and extracellular matrix adhesion molecule cDNA microarray, and validated changes with Western blot or real‐time quantitative PCR, respectively. rVBMDMP dose‐dependently inhibited colony formation, induced apoptosis, and inhibited in vitro tubule formation. rVBMDMP increased the phosphorylation of 88 signal proteins, including caspase‐3, death receptor 3, 4, and 5, and integrin αV, β1, and β3, and down‐regulated 41 signal proteins, including EGFR, pEGFR, VEGFR‐1, and survivin versus control. rVBMDMP upregulated 14 genes, including collagen 4, 7, and 27, and down‐regulated 21 genes, including integrin αVβ3, MMP10, and MMP12. Our study suggests that rVBMDMP inhibits angiogenesis and may be a viable drug candidate in anti‐angiogenesis and anticancer therapies. J. Cell. Biochem. 111: 453–460, 2010. © 2010 Wiley‐Liss, Inc.     

Collective cell motion in endothelial monolayers

Collective cell motility is an important aspect of several developmental and pathophysiological processes. Despite its importance, the mechanisms that allow cells to be both motile and adhere to one another are poorly understood. In this study we establish statistical properties of the random streaming behavior of endothelial monolayer cultures. To understand the reported empirical findings, we expand the widely used cellular Potts model to include active cell motility. For spontaneous directed motility we assume a positive feedback between cell displacements and cell polarity. The resulting model is studied with computer simulations and is shown to exhibit behavior compatible with experimental findings. In particular, in monolayer cultures both the speed and persistence of cell motion decreases, transient cell chains move together as groups and velocity correlations extend over several cell diameters. As active cell motility is ubiquitous both in vitro and in vivo, our model is expected to be a generally applicable representation of cellular behavior.     

Expression and adhesive properties of basement membrane proteins in cerebral capillary endothelial cell cultures

Previous studies have indicated the importance of basement membrane components both for cellular differentiation in general and for the barrier properties of cerebral microvascular endothelial cells in particular. Therefore, we have examined the expression of basement membrane proteins in primary capillary endothelial cell cultures from adult porcine brain. By indirect immunofluorescence, we could detect type IV collagen, fibronectin, and laminin both in vivo (basal lamina of cerebral capillaries) and in vitro (primary culture of cerebral capillary endothelial cells). In culture, these proteins were secreted at the subcellular matrix. Moreover, the interaction between basement membrane constituents and cerebral capillary endothelial cells was studied in adhesion assays. Type IV collagen, fibronectin, and laminin proved to be good adhesive substrata for these cells. Although the number of adherent cells did not differ significantly between the individual proteins, spreading on fibronectin was more pronounced than on type IV collagen or laminin. Our results suggest that type IV collagen, fibronectin, and laminin are not only major components of the cerebral microvascular basal lamina, but also assemble into a protein network, which resembles basement membrane, in cerebral capillary endothelial cell cultures.