Oligomerization-dependent regulation of motility and morphogenesis by the collagen XVIII NC1/endostatin domain

Collagen XVIII (c18) is a triple helical endothelial/epithelial basement membrane protein whose noncollagenous (NC)1 region trimerizes a COOH-terminal endostatin (ES) domain conserved in vertebrates, Caenorhabditis elegans and Drosophila. Here, the c18 NC1 domain functioned as a motility-inducing factor regulating the extracellular matrix (ECM)-dependent morphogenesis of endothelial and other cell types. This motogenic activity required ES domain oligomerization, was dependent on rac, cdc42, and mitogen-activated protein kinase, and exhibited functional distinction from the archetypal motogenic scatter factors hepatocyte growth factor and macrophage stimulatory protein. The motility-inducing and mitogen-activated protein kinase-stimulating activities of c18 NC1 were blocked by its physiologic cleavage product ES monomer, consistent with a proteolysis-dependent negative feedback mechanism. These data indicate that the collagen XVIII NC1 region encodes a motogen strictly requiring ES domain oligomerization and suggest a previously unsuspected mechanism for ECM regulation of motility and morphogenesis.     

Fluid shear stress modulates endothelial cell invasion into three-dimensional collagen matrices

Endothelial cells are subjected to biochemical and mechanical stimuli, which regulate their angiogenic potential. We determined the synergistic effects of sphingosine-1-phosphate (S1P) and fluid wall shear stress (WSS) on a previously established model of human umbilical vein endothelial cell invasion into three-dimensional collagen matrices. Collagen matrices were incorporated into a parallel-plate flow chamber to apply controlled WSS to the surface of endothelial monolayers over a period of 24 h. Cell invasion required the presence of S1P, with the effects of S1P being enhanced by shear stress to an extent comparable with S1P combined with angiogenic growth factor stimulation. The number of invading cells depended on the magnitude of shear stress, with a maximal induction at a shear stress of approximately 5 dyn/cm2, whereas the invasion distance was proportional to the magnitude of shear stress. The enhancement of invasion by 5.3 dyn/cm2 shear stress coincided with elevated phosphorylation of Akt and matrix metalloproteinase (MMP)-2 activation. Furthermore, invasion induced by the combined application of WSS and S1P was attenuated by inhibitors of MMPs (GM6001) and the phosphatidylinositol 3-kinase/Akt signaling pathway (wortmannin). These results provide evidence that shear stress is a positive modulator of S1P-induced endothelial cell invasion into collagen matrices through enhanced Akt and MMP-2 activation.     

Mesenchymal stem cell modification of endothelial matrix regulates their vascular differentiation

Mesenchymal stem cells (MSCs) respond to a variety of differentiation signal provided by their local environments. A large portion of these signals originate from the extracellular matrix (ECM). At the same time, MSCs secrete various matrix‐altering agents, including proteases, that alter ECM‐encoded differentiation signals. Here we investigated the interactions between MSC and ECM produced by endothelial cells (EC‐matrix), focusing not only on the differentiation signals provided by EC‐matrix, but also on MSC‐alteration of these signals and the resultant affects on MSC differentiation. MSCs were cultured on EC‐matrix modified in one of three distinct ways. First, MSCs cultured on native EC‐matrix underwent endothelial cell (EC) differentiation early during the culture period and smooth muscle cell (SMC) differentiation at later time points. Second, MSCs cultured on crosslinked EC‐matrix, which is resistant to MSC modification, differentiated towards an EC lineage only. Third, MSCs cultured on EC‐matrix pre‐modified by MSCs underwent SMC‐differentiation only. These MSC‐induced matrix alterations were found to deplete the factors responsible for EC‐differentiation, yet activate the SMC‐differentiation factors. In conclusion, our results demonstrate that the EC‐matrix contains factors that support MSC differentiation into both ECs and SMCs, and that these factors are modified by MSC‐secreted agents. By analyzing the framework by which EC‐matrix regulates differentiation in MSCs, we have uncovered evidence of a feedback system in which MSCs are able to alter the very matrix signals acting upon them. J. Cell. Biochem. 107: 706–713, 2009. Published 2009 Wiley‐Liss, Inc.     

Induction of three-dimensional assembly and increase in apoptosis of human endothelial cells by simulated microgravity: Impact of vascular endothelial growth factor

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.     

Non-enzymatic glycation of type I collagen diminishes collagen-proteoglycan binding and weakens cell adhesion

Non-enzymatic glycation of type I collagen occurs in aging and diabetes, and may affect collagen solubility, charge, polymerization, and intermolecular interactions. Proteoglycans(1) (PGs) bind type I collagen and are proposed to regulate fibril assembly, function, and cell-collagen interactions. Moreover, on the collagen fibril a keratan sulfate (KS) PG binding region overlaps with preferred collagen glycation sites. Thus, we examined the effect of collagen modified by simple glycation on PG-collagen interactions. By affinity coelectrophoresis (ACE), we found reduced affinities of heparin and KSPGs for glycated but not normal collagen, whereas the dermatan sulfate (DS)PGs decorin and biglycan bound similarly to both, and that the affinity of heparin for normal collagen decreased with increasing pH. Circular dichroism (CD) spectroscopy revealed normal and glycated collagens to assume triple helical conformations, but heparin addition caused precipitation and decreased triple helical content-effects that were more marked with glycated collagen. A spectrophotometric assay revealed slower polymerization of glycated collagen. However, ultrastructural analyses indicated that fibrils assembled from normal and glycated collagen exhibited normal periodicity, and had similar structures and comparable diameter distributions. B-cells expressing the cell surface heparan sulfate PG syndecan-1 adhered well to normal but not glycated collagen, and endothelial cell migration was delayed on glycated collagen. We speculate that glycation diminishes the electrostatic interactions between type I collagen and PGs, and may interfere with core protein-collagen associations for KSPGs but not DSPGs. Therefore in vivo, collagen glycation may weaken PG-collagen interactions, thereby disrupting matrix integrity and cell-collagen interactions, adhesion, and migration.     

Basement membrane collagen type IV expression by human mesenchymal stem cells during adipogenic differentiation

During adipogenic differentiation human mesenchymal stem cells (hMSC) produce collagen type IV. In immunofluorescence staining differentiating hMSCs started to express collagen type IV when Oil Red O-positive fat droplets appeared intracellularly. Quantitative real time-polymerase chain reaction confirmed progressive increase of collagen type IV α1 and α2 mRNA levels over time, 18.6- and 12.2-fold by day 28, respectively, whereas the copy numbers of α3-α6 mRNAs remained rather stable and low. Type IV collagen was in confocal laser scanning microscopy seen around adipocytes, where also laminins and nidogen were found, suggesting pericellular deposition of all key components of the fully developed basement membrane. Immunofluorescence staining of matrix metalloproteinase-2 (MMP-2, 72 kD type IV collagenase, gelatinase A) and MMP-9 (92 kD type IV collagenase, gelatinase B) disclosed only faint staining of MSCs, but MMP-9 was strongly induced during adipogenesis, whereas MSC supernatants disclosed in zymography pro-MMP-2 and faint pro-MMP-9 bands, which increased over time, with partial conversion of pro-MMP-2 to its active 62 kD form. Differentiation was associated with increasing membrane type 1-MMP/MMP-14 and tissue inhibitor of metalloproteinase-2 (TIMP-2) staining, which may enable participation of type IV collagenases in basement membrane remodelling via ternary MT1-MMP/TIMP-2/MMP-2 or -9 complexes, focalizing the fully active enzyme to the cell surface. MMP-9, which increased more in immunofluorescence staining, was perhaps preferentially bound to cell surface and/or remodelling adipocyte basement membrane. These results suggest that upon MSC-adipocyte differentiation collagen type IV synthesis and remodelling become necessary when intracellular accumulation of fat necessitates a dynamically supporting and instructive, partly denatured adipogenic pericellular type IV collagen scaffold.     

Fibroblast morphogenesis on 3D collagen matrices: The balance between cell clustering and cell migration

Fibroblast clusters have been observed in tissues under a variety of circumstances: in fibrosis and scar, in the formation of hair follicle dermal papilla, and as part of the general process of mesenchymal condensation that takes place during development. Cell clustering has been shown to depend on features of the extracellular matrix, growth factor environment, and mechanisms to stabilize cell–cell interactions. In vitro studies have shown that increasing the potential for cell–cell adhesion relative to cell–substrate adhesion promotes cell clustering. Experimental models to study fibroblast clustering have utilized centrifugation, hanging drops, and substrata with poorly adhesive, soft and mechanically unstable properties. In this review, we summarize work on a new, highly tractable, cell clustering research model in which human fibroblasts are incubated on the surfaces of collagen matrices. Fibroblast clustering occurs under procontractile growth factor conditions (e.g., serum or the serum lipid agonist lysophosphatidic acid) but not under promigratory growth factor conditions (e.g., platelet-derived growth factor) and can be reversed by switching growth factor environments. Cell contraction plays a dual role in clustering to bring cells closer together and to stimulate cells to organize fibronectin into a fibrillar matrix. Binding of fibroblasts to a shared fibronectin fibrillar matrix stabilizes clusters, and fragmentation of the fibrillar matrix occurs when growth factor conditions are switched to promote cell dispersal.     

Establishment and characterization of a novel human myoepithelial cell line and matrix-producing xenograft from a parotid basal cell adenocarcinoma

Summary Myoepithelial cells exert important paracrine effects on epithelial morphogenesis and mitogenesis through direct cell-cell interactions and through synthesis of a basement membrane extracellular matrix. To study these effects further, this study established the first immortalized human myoepithelial cell line, HMS-1, and transplantable xenograft, HMS-X, from the rare parotid basal cell adenocarcinoma. The cell line exhibited a fully differentiated myoepithelial phenotype and the xenograft exhibited the rare property of accumulating an abundant extracellular matrix composed of both basement membrane and nonbasement membrane components with the latter predominating. With HMS-1 as a feeder layer, dramatic and specific induction of epithelial morphogenesis (sheroid formation) occurred with selected normal epithelial and primary carcinoma target cells. HMS-1 and HMS-X provide distinct advantages over the conventional murine matrices in existence. They will be invaluable in future studies of human tumor-myoepithelial and matrix interactions important for tumor cell growth, invasion, and metastasis.     

Induction of apoptotic cell death in vascular endothelial cells cultured in three-dimensional collagen lattice

Endothelial cells derived from fetal bovine aorta (BAECs) undergo apoptosis in three-dimensional (3-D) type I collagen lattice in the absence of specific angiogenic factor. In the presence of angiogenic factor, BAECs survive and form a capillary-like tube structure in 3-D culture. In the present study we elucidate the mechanisms of BAECs apoptosis or survival and tube formation in 3-D culture. When BAECs embedded in collagen lattice were cultured with angiogenic factor (fibroblast growth factor-2 (FGF-2) or 4beta-phorbol 12-myristate 13-acetate (PMA)) in the presence of PD98059, a specific inhibitor of mitogen-activated protein kinase kinase, BAECs did not form tube structures and underwent apoptosis in collagen lattice. Function-blocking antibody against alphavbeta3 integrin also inhibited tube formation and induced apoptosis in 3-D culture in the presence of angiogenic factors. Exposure of BAECs to FGF-2 and PMA had no effect on the alphavbeta3 integrin expression but induced the activation of alphavbeta3 integrin. PD98059 attenuated alphavbeta3 integrin activation in response to angiogenic factor. KB-R8301, a hydroxamic acid-based matrix metalloproteinase (MMP) inhibitor, prevented apoptotic cell death in the absence of angiogenic factor in 3-D culture and enhanced capillary-like tube formation in the presence of angiogenic factor, which was not inhibited by the anti-alphavbeta3 integrin antibody. The results suggest that angiogenic factor-induced alphavbeta3 integrin activation through the MEK-ERK pathway regulates the BAEC fate between apoptosis and angiogenesis in collagen lattice. MMP derived from BAECs seems to play a key role in the release of cryptic ligands for alphavbeta3 integrin from intact collagen.     

Interommatidial cells build a tensile collagen network during Drosophila retinal morphogenesis

1. Download : Download high-res image (188KB)
2. Download : Download full-size image

     

Double-layered collagen gel hemisphere for cell invasion assay: successful visualization and quantification of cell invasion activity

Although various methods for collagen gel-based cell invasion assays have been described, there continues to be a need for a simpler and more objective assay. Here, we describe an easy-to-prepare double-layered collagen gel hemisphere (DL-CGH) system that satisfies these requirements, and we demonstrate the advantages of this new system for visualizing cell movements during invasion. DL-CGH consists of a central core collagen layer surrounded by an outer cover collagen layer. A droplet of collagen I solution (containing cells to be examined) naturally forms a small hemisphere on the bottom of the culture dish. After this central core layer gels, a second droplet is placed atop the first gel, encapsulating it completely. The hemisphere is submerged in the medium and cultured. The invasive activity of cells that infiltrate from the inner to the outer layer can be evaluated optically. Using this in vitro system, we measured the inhibitory effect of E-cadherin expression on cancer cell invasion. DL-CGH also allowed visualization of interactions between invading cancer cells and the stroma. Cancer cells, which lack the proteases required for direct entrance into the three-dimensional collagen matrix, were seen to slip like amoebas through matrix gaps generated by the pericellular proteolytic activity of fibroblasts. [Supplementary materials are available for this article. Go to the publisher's online edition of Cell Communication and Adhesion for the following free supplemental resources: Movies 1-3; 4a and b].     

Modeled gravitational unloading induced downregulation of endothelin-1 in human endothelial cells

Many space missions have shown that prolonged space flights may increase the risk of cardiovascular problems. Using a three-dimensional clinostat, we investigated human endothelial EA.hy926 cells up to 10 days under conditions of simulated microgravity (microg) to distinguish transient from long-term effects of microg and 1g. Maximum expression of all selected genes occurred after 10 min of clinorotation. Gene expression (osteopontin, Fas, TGF-beta(1)) declined to slightly upregulated levels or rose again (caspase-3) after the fourth day of clinorotation. Caspase-3, Bax, and Bcl-2 protein content was enhanced for 10 days of microgravity. In addition, long-term accumulation of collagen type I and III and alterations of the cytoskeletal alpha- and beta-tubulins and F-actin were detectable. A significantly reduced release of soluble factors in simulated microgravity was measured for brain-derived neurotrophic factor, tissue factor, vascular endothelial growth factor (VEGF), and interestingly for endothelin-1, which is important in keeping cardiovascular balances. The gene expression of endothelin-1 was suppressed under microg conditions at days 7 and 10. Alterations of the vascular endothelium together with a decreased release of endothelin-1 may entail post-flight health hazards for astronauts.     

Controlling the mechanical properties of three-dimensional matrices via non-enzymatic collagen glycation

The mechanical properties of the extracellular matrix play an important role in maintaining cellular function and overall tissue homeostasis. Recently, a number of hydrogel systems have been developed to investigate the role of matrix mechanics in mediating cell behavior within three-dimensional environments. However, many of the techniques used to modify the stiffness of the matrix also alter properties that are important to cellular function including matrix density, porosity and binding site frequency, or rely on amorphous synthetic materials. In a recent publication, we described the fabrication, characterization and utilization of collagen gels that have been non-enzymatically glycated in their unpolymerized form to produce matrices of varying stiffness. Using these scaffolds, we showed that the mechanical properties of the resulting collagen gels could be increased 3-fold without significantly altering the collagen fiber architecture. Using these matrices, we found that endothelial cell spreading and outgrowth from multi-cellular spheroids changes as a function of the stiffness of the matrix. Our results demonstrate that non-enzymatic collagen glycation is a tractable technique that can be used to study the role of 3D stiffness in mediating cellular function. This commentary will review some of the current methods that are being used to modulate matrix mechanics and discuss how our recent work using non-enzymatic collagen glycation can contribute to this field.     

Homeobox B3 promotes capillary morphogenesis and angiogenesis

Endothelial cells (EC) express several members of the Homeobox (Hox) gene family, suggesting a role for these morphoregulatory mediators during angiogenesis. We have previously established that Hox D3 is required for expression of integrin alphavbeta3 and urokinase plasminogen activator (uPA), which contribute to EC adhesion, invasion, and migration during angiogenesis. We now report that the paralogous gene, Hox B3, influences angiogenic behavior in a manner that is distinct from Hox D3. Antisense against Hox B3 impaired capillary morphogenesis of dermal microvascular EC cultured on basement membrane extracellular matrices. Although levels of Hox D3-dependent genes were maintained in these cells, levels of the ephrin A1 ligand were markedly attenuated. Capillary morphogenesis could be restored, however, by addition of recombinant ephrin A1/Fc fusion proteins. To test the impact of Hox B3 on angiogenesis in vivo, we constitutively expressed Hox B3 in the chick chorioallantoic membrane using avian retroviruses that resulted in an increase in vascular density and angiogenesis. Thus, while Hox D3 promotes the invasive or migratory behavior of EC, Hox B3 is required for the subsequent capillary morphogenesis of these new vascular sprouts and, together, these results support the hypothesis that paralogous Hox genes perform complementary functions within a particular tissue type.     

Matrix loading: assembly of extracellular matrix collagen fibrils during embryogenesis

Nothing in biology stimulates the imagination like the development of a single fertilized egg into a newborn child. Consequently, a major focus of biomedical research is aimed at understanding cell differentiation, proliferation, and specialization during child health and human development. However, the fact that the increase in size and shape of the growing embryo has as much to do with the extracellular matrix (ECM) as with the cells themselves, is largely overlooked. Cells in developing tissues are surrounded by a fiber-composite ECM that transmits mechanical stimuli, maintains the shape of developing tissues, and functions as a scaffold for cell migration and attachment. The major structural element of the ECM is the collagen fibril. The fibrils, which are indeterminate in length, are arranged in different tissues in exquisite supramolecular architectures, including parallel bundles, orthogonal lamellae, and concentric weaves. This article reviews our current understanding of the synthesis and assembly of collagen fibrils, and discusses challenging questions about how cells assemble an organized ECM during embryogenesis.     

Contraction of collagen by human fibroblasts and keratinocytes

Summary In the process of wound healing keratinocytes and fibroblasts play an important role, keratinocytes in the re-epithelization process and fibroblasts in the process of wound contraction. We have studied the role of human keratinocytes and fibroblasts in the rearrangement of collagen in a collagen lattice model system. Our results revealed that keratinocytes as well as fibroblasts rearrange the collagen lattice; this occurs in a cell number and collagen concentration dependent manner. The optimal gel contraction is obtained in the presence of keratinocytes on the top of and of fibroblasts in the collagen lattice, the situation most closely approaching the in vivo situation. Between the two types of cells, differences in morphologic behavior were observed: when incorporated into the gel the keratinocytes retained their spherical shape throughout the whole culture period, but fibroblasts became elongated and formed extensions. Our data suggest that not only fibroblasts but also keratinocytes may be involved in the process of wound contraction. This work was supported by the Koningin Wilhelmina Fonds (Netherlands Cancer Foundation, grant 84-10).     

Biocompatible scaffolds based on collagen and oxidized dextran for endothelial cell survival and function in tissue engineering

Angiogenesis is a vital step in tissue regeneration. Hence, the current study aimed to prepare oxidized dextran (Odex)/collagen (Col)-hydrogels with laminin (LMN), as an angiogenic extracellular matrix (ECM) component, for promoting human umbilical vein endothelial cell (HUVEC) proliferation and function. Odex/Col scaffolds were constructed at various concentrations and temperatures. Using oscillatory rheometry, scanning electron microscopy (SEM), and cell viability testing, the scaffolds were characterized, and then HUVEC proliferation and function was compared with or without LMN. The gelation time could be modified by altering the Odex/Col mass ratio as well as the temperature. SEM showed that Odex/Col hydrogels had a more regular three-dimensional (3D) porous structure than the Col hydrogels. Moreover, HUVECs grew faster in the Col scaffold (12 mg/mL), whereas the Odex (30 mg/mL)/Col (6 mg/mL) scaffold exhibited the lowest apoptosis index. Furthermore, the expression level of vascular endothelial growth factor (VEGF) mRNA in the group without LMN was higher than that with LMN, and the Odex (30 mg/mL)/Col (6 mg/mL) scaffold without LMN had the highest VEGF protein secretion, allowing the cells to survive and function effectively. Odex/Col scaffolds, with or without LMN, are proposed as a tissue engineering construct to improve HUVEC survival and function for angiogenesis.     

Inhibition of endothelial cell morphogenetic interactions in vitro by alpha- and beta-xylosides

Summary Bovine aortic endothelial cells retain the ability to undergo histotypic morphogenetic interactions in vitro as evidenced by a) the reversible expression of a sprouting cell phenotype and b) the patterned self-association of these sprouting cells into three-dimensional meshworks and tubule-like structures. These morphogenetic events are inhibited by xylosides in a dose-dependent manner. Two types of beta-xylosides (p-nitrophenyl-beta-d-xylopyranoside and 4-methylumbelliferyl-beta-d-xylopyranoside) and one alpha-xyloside (p-nitrophenyl-alpha-d-xylopyranoside) were tested. Beta-xylosides are well characterized acceptors of glycosaminoglycan chains, whereas alpha-xylosides do not function in this capacity and have been extensively used as negative controls when studying the effects of beta-xylosides. Both alpha-and beta-xylosides inhibited endothelial morphogenetic interactions. This inhibition was slowly reversed during the 6- to 7-d period following removal of the xyloside. Inhibition of morphogenetic interactions by xylosides occurred at concentrations (0.5 to 2.0 mM) that had no demonstrable effects on cell proliferation, migration, or adhesion to 2-D plastic or collagen substrata. The xylosides seemed to inhibit cell spreading on a 3-D environment, they also inhibited the incorporation of [³H]-proline and Na₂ ³⁵SO₄ into the extracellular matrix deposited by the cells, suggesting that the inhibition of morphogenesis may be related to the inhibition of matrix deposition. Endothelial morphogenetic interactions were not inhibited by the extracellular matrix or by the conditioned medium produced by cells cultured in the presence of xylosides.     

Optimization of culture conditions for human corneal endothelial cells

Summary Long-term cultivation of human corneal endothelial cells (HCEC) was optimized with respect to different components of the culture system: 25 different nutrient media, different sera, 6 mitogens and various substrates were tested in their ability to influence clonal growth and morphology of HCEC. F99, a 1∶1 mixture of the two media M199 and Ham’s F12, was the most effective basal medium in promoting clonal growth of HCEC. Among various sera, human serum and fetal bovine serum showed optimal growth promoting activities in combination with F99, whereas newborn bovine serum (NBS) was by far superior for the development of a typically corneal endothelial morphology. Crude fibroblast growth factor (FGF), or alternatively endothelial cell growth supplement, was absolutely essential for clonal growth of HCEC at low serum concentrations, for example 5% NBS. Formation of a monolayer with a morphology similar to corneal endothelium in vivo was observed only on culture dishes coated with basal membrane components such as collagen type IV, laminin, or fibronectin. The most pronounced effect on morphologic appearance was obtained by culturing the cells on the extracellular matrix (ECM) produced by bovine corneal endothelial cells. Moreover, ECM could substitute for crude FGF in clonal growth assays.