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.     

Capillary morphogenesis during human endothelial cell invasion of three-dimensional collagen matrices

Summary Here, we describe assay systems that utilize serum-free defined media to evaluate capillary morphogenesis during human endothelial cell (EC) invasion of three-dimensional collagen matrices. ECs invade these matrices over a 1–3-d period to form capillary tubes. Blocking antibodies to the α2β1 integrin interfere with invasion and morphogenesis while other integrin blocking antibodies do not. Interestingly, we observed increased invasion of ECs toward a population of underlying ECs undergoing morphogenesis. In addition, we have developed assays on microscope slides that display the invasion process horizontally, thereby enhancing our ability to image these events. Thus far, we have observed intracellular vacuoles that appear to regulate the formation of capillary lumens, and extensive cell processes that facilitate the interconnection of ECs during morphogenic events. These assays should enable further investigation of the morphologic steps and molecular events controlling human capillary tube formation in three-dimensional extracellular matrices.     

Immobilization of growth factors on collagen scaffolds mediated by polyanionic collagen mimetic peptides and its effect on endothelial cell morphogenesis

Angiogenesis, a morphogenic event endothelial cells (ECs) undergo in response to 3-D environmental triggers, is critical to the survival and ultimate functional capacity of engineered tissue constructs. Here we present a new collagen mimetic peptide (CMP) architecture consisting of multiple anionic charges at the peptide's N-terminus designed to attract growth factors by charge-charge interactions and bind to collagen by CMP-collagen interaction. The anionic CMPs exhibited specific binding affinity to type I collagen substrates while attracting vascular endothelial growth factors (VEGFs), which led to enhanced morphological features of ECs, indicative of tubulogenesis. The results show that these new CMPs could be used to direct proliferation and differentiation of cells in collagen scaffolds by localization and sustained delivery of growth factors and other morphogens.     

Toward single cell traction microscopy within 3D collagen matrices

Mechanical interaction between the cell and its extracellular matrix (ECM) regulates cellular behaviors, including proliferation, differentiation, adhesion, and migration. Cells require the three-dimensional (3D) architectural support of the ECM to perform physiologically realistic functions. However, current understanding of cell–ECM and cell–cell mechanical interactions is largely derived from 2D cell traction force microscopy, in which cells are cultured on a flat substrate. 3D cell traction microscopy is emerging for mapping traction fields of single animal cells embedded in either synthetic or natively derived fibrous gels. We discuss here the development of 3D cell traction microscopy, its current limitations, and perspectives on the future of this technology. Emphasis is placed on strategies for applying 3D cell traction microscopy to individual tumor cell migration within collagen gels.     

2D protrusion but not motility predicts growth factor-induced cancer cell migration in 3D collagen

Growth factor-induced migration is a critical step in the dissemination and metastasis of solid tumors. Although differences in properties characterizing cell migration on two-dimensional (2D) substrata versus within three-dimensional (3D) matrices have been noted for particular growth factor stimuli, the 2D approach remains in more common use as an efficient surrogate, especially for high-throughput experiments. We therefore were motivated to investigate which migration properties measured in various 2D assays might be reflective of 3D migratory behavioral responses. We used human triple-negative breast cancer lines stimulated by a panel of receptor tyrosine kinase ligands relevant to mammary carcinoma progression. Whereas 2D migration properties did not correlate well with 3D behavior across multiple growth factors, we found that increased membrane protrusion elicited by growth factor stimulation did relate robustly to enhanced 3D migration properties of the MDA-MB-231 and MDA-MB-157 lines. Interestingly, we observed this to be a more reliable relationship than cognate receptor expression or activation levels across these and two additional mammary tumor lines.     

Matrix density alters zyxin phosphorylation,which limits peripheral process formation and extension in endothelial cells invading 3D collagen matrices

This study was designed to determine the optimal conditions required for known pro-angiogenic stimuli to elicit successful endothelial sprouting responses. We used an established, quantifiable model of endothelial cell (EC) sprout initiation where ECs were tested for invasion in low (1 mg/mL) and high density (5 mg/mL) 3D collagen matrices. Sphingosine 1-phosphate (S1P) alone, or S1P combined with stromal derived factor-1α (SDF) and phorbol ester (TPA), elicited robust sprouting responses. The ability of these factors to stimulate sprouting was more effective in higher density collagen matrices. S1P stimulation resulted in a significant increase in invasion distance, and with the exception of treatment groups containing phorbol ester, invasion distance was longer in 1 mg/mL compared to 5 mg/mL collagen matrices. Closer examination of cell morphology revealed that increasing matrix density and supplementing with SDF and TPA enhanced the formation of multicellular structures more closely resembling capillaries. TPA enhanced the frequency and size of lumen formation and correlated with a robust increase in phosphorylation of p42/p44 Erk kinase, while S1P and SDF did not. Also, a higher number of significantly longer extended processes formed in 5 mg/mL compared to 1 mg/mL collagen matrices. Because collagen matrices at higher density have been reported to be stiffer, we tested for changes in the mechanosensitive protein, zyxin. Interestingly, zyxin phosphorylation levels inversely correlated with matrix density, while levels of total zyxin did not change significantly. Immunofluorescence and localization studies revealed that total zyxin was distributed evenly throughout invading structures, while phosphorylated zyxin was slightly more intense in extended peripheral processes. Silencing zyxin expression increased extended process length and number of processes, while increasing zyxin levels decreased extended process length. Altogether these data indicate that ECs integrate signals from multiple exogenous factors, including changes in matrix density, to accomplish successful sprouting responses. We show here for the first time that zyxin limited the formation and extension of fine peripheral processes used by ECs for matrix interrogation, providing a molecular explanation for altered EC responses to high and low density collagen matrices.     

ADAM17 co-purifies with TIMP-3 and modulates endothelial invasion responses in three-dimensional collagen matrices

In this study, we investigated potential mechanisms through which the known anti-angiogenic factor, tissue inhibitor of metalloproteinase-3 (TIMP-3) blocks angiogenesis. As a strategy to identify TIMP-3 binding proteins, we used tandem affinity purification, employing recombinant adenoviruses constructed to deliver TIMP-3 fused to C-terminal S and His tags (TIMP-3-S-His) or TIMP-1-S-His control to endothelial cells prior to extraction. Western blotting of final eluates revealed robust binding of A Disintegrin and Metalloproteinase (ADAM) 17 and a slight association of ADAM15 to TIMP-3, but not TIMP-1 control. To confirm a functional requirement for ADAM15 and 17 in mediating angiogenic events, a model of endothelial cell invasion was utilized. Silencing of ADAM17, but not ADAM15, expression using small interfering RNA (siRNA) interfered with invasion, resulting in decreased density of invading cells and decreased invasion distance. Stable EC lines expressing short hairpin RNA directed to ADAM17 were similarly inhibited. To confirm these results, dominant negative mutants (ΔMPs) of ADAM10, ADAM15 or ADAM17 were delivered using recombinant lentiviruses. Expression of ADAM17 ΔMP, but not ADAM10 or ADAM15 ΔMP, decreased invasion density and distance. Further, time-lapse analyses revealed ADAM17 ΔMP cells exhibited far greater numbers of protruding sprouts compared to control, suggesting an inability of extended processes to retract properly. Immunofluorescence analyses revealed ADAM17 localized to bifurcations in invading sprouts. These data jointly indicate a role for ADAM17 in modulating endothelial sprouting events during angiogenesis.     

Effects of collagen density on cardiac fibroblast behavior and gene expression

Interactions between cells and the extracellular matrix (ECM) play essential roles in modulating cell behavior during development and disease. The myocardial ECM is composed predominantly of interstitial collagen type I and type III. The composition, organization, and accumulation of these collagens are altered concurrent with cardiovascular development and disease. Changes in these parameters are thought to play significant roles in myocardial function. While a number of studies have examined how changes in the ECM affect myocardial function as a whole, much less is known regarding the response at the cellular level to changes in the collagenous ECM. Experiments were carried out to determine the effects of alterations in collagen density and ECM stiffness on the behavior of isolated heart fibroblasts. In vitro bioassays were performed to measure the effects of changes in collagen concentration (0.75-1.25 mg/ml) on adhesion, migration, spreading, and gene expression by heart fibroblasts. Increased density of collagen in 3-dimensional gels resulted in more efficient adhesion, spreading, and migration by heart fibroblasts. These experiments indicated that the density of the collagen matrix has a significant impact on fibroblast function. These studies begin to elucidate the effects of ECM density at the cellular level in the myocardium.     

B16/F10 tumors in aged 3D collagen in vitro simulate tumor growth and gene expression in aged mice in vivo

Although the incidence of cancer rises with age, tumor growth is often slowed in older hosts. The B16/F10 melanoma cell line is commonly used in murine models of age-related tumor growth suppression. We wished to determine if the growth pattern and gene expression of B16/10 tumors grown in aged mice could be simulated in 3D collagen matrices derived from aged mice. Outcome measures were tumor size in vitro and gene expression of the key growth regulatory molecules: growth hormone receptor (GHR), IL-10Rβ, IL-4Rα, and IL-6. B16/F10 tumors were grown in 20–25-mo-old C57/BL6 male mice. Tumor sizes ranged from 30 to 4,910 mg in vivo. Tumors from a subset of mice were removed after euthanasia, and equivalent amounts of each tumor were placed in aged 3D collagen and grown for 5 d. Tumor sizes in aged 3D collagen correlated highly with their original tumor size in vivo. Gene expression changes noted in vivo were also maintained during tumor growth in aged 3D collagen in vitro. The relative expression of GHR was increased, IL-10Rβ was unchanged, and IL-4Rα and IL-6 were decreased in the larger tumors relative to the smaller tumors in vitro, in a pattern similar to that noted in vivo. We propose that 3D matrices from aged mice provide an in vitro model of tumor growth that correlates highly with tumor size and expression of key regulatory molecules in vivo.     

T3 affects expression of collagen I and collagen cross-linking in bone cell cultures

Thyroid hormones (T3, T4) have a broad range of effects on bone, however, its role in determining the quality of bone matrix is poorly understood. In-vitro, the immortalized mouse osteoblast-like cell line MC3T3-E1 forms a tissue like structure, consisting of several cell layers, whose formation is affected by T3 significantly. In this culture system, we investigated the effects of T3 on cell multiplication, collagen synthesis, expression of genes related to the collagen cross-linking process and on the formation of cross-links.T3 compared to controls modulated cell multiplication, up-regulated collagen synthesis time and dose dependently, and stimulated protein synthesis. T3 increased mRNA expressions of procollagen-lysine-1,2-oxoglutarate 5-dioxygenase 2 (Plod2) and of lysyloxidase (Lox), both genes involved in post-translational modification of collagen. Moreover, it stimulated mRNA expression of bone morphogenetic protein 1 (Bmp1), the processing enzyme of the lysyloxidase-precursor and of procollagen. An increase in the collagen cross-link-ratio Pyr/deDHLNL indicates, that T3 modulated cross-link maturation in the MC3T3-E1 culture system. These results demonstrate that T3 directly regulates collagen synthesis and collagen cross-linking by up-regulating gene expression of the specific cross-link related enzymes, and underlines the importance of a well-balanced concentration of thyroid hormones for maintenance of bone quality.     

Endothelial cell protrusion and migration in three-dimensional collagen matrices

Many cells display dramatically different morphologies when migrating in 3D matrices vs. on planar substrata. How these differences arise and the implications they have on cell migration are not well understood. To address these issues, we examined the locomotive structure and behavior of bovine aortic endothelial cells (ECs) either inside 3D collagen gels or on 2D surfaces. Using time-lapse imaging, immunofluorescence, and confocal microscopy, we identified key morphological differences between ECs in 3D collagen gels vs. on 2D substrata, and also demonstrated important functional similarities. In 3D matrices, ECs formed cylindrical branching pseudopodia, while on 2D substrata they formed wide flat lamellae. Three distinct cytoplasmic zones were identified in both conditions: (i) a small, F-actin-rich, rapidly moving peripheral zone, (ii) a larger, more stable, intermediate zone characterized by abundant microtubules and small organelles, and (iii) a locomotively inert central zone rich in microtubules, and containing the larger organelles. There were few differences between 2D and 3D cells in the content and behavior of their peripheral and central zones, whereas major differences were seen in the shape and types of movements displayed by the intermediate zone, which appeared critical in distributing cell-matrix adhesions and directing cytoplasmic flow. This morphological and functional delineation of cytoplasmic zones provides a conceptual framework for understanding differences in the behavior of cells in 3D and 2D environments, and indicates that cytoskeletal structure and dynamics in the relatively uncharacterized intermediate zone may be particularly important in cell motility in general.     

Control of endothelial cell gene expression by flow

The vessel wall is constantly subjected to, and affected by, the stresses resulting from the hemodynamic stimuli of transmural pressure and flow. At the interface between blood and the vessel wall, the endothelial cell plays a crucial role in controlling vessel structure and function in response to changes in hemodynamic conditions. Using bovine aortic endothelium monolayers, we show that fluid shear stress causes simultaneous differential regulation of endothelial-derived products. We also report that the downregulation of endothelin-1 mRNA by flow is a reversible process, and through the use of uncharged dextran supplementation demonstrate it to be shear stress-rather than shear rate-dependent. Recent work on the effect of fluid shear stress on endothelial cell gene expression of a number of potent endothelial products is reviewed, including vasoactive substances, autocrine and paracrine growth factors, thrombosis/fibrinolysis modulators, chemotactic factors, surface receptors and immediate-early genes. The encountered patterns of gene expression responses are classified into three categories: a transient increase with return to baseline (type I), a sustained increase (type II) and a biphasic response consisting of an early transient increase of varying extent followed by a pronounced and sustained decrease (type III). The importance of the dynamic character of the flow stimulus and the magnitude dependence of the response are presented. Potential molecular mechanisms of shear-induced gene regulation, including putative shear stress response elements (SSRE), are discussed. These results suggest exquisite modulation of endothelial cell phenotype by local fluid shear stress and may offer insight into the mechanism of flow-dependent vascular remodeling and the observed propensity of atherosclerosis formation around bifurcations and areas of low shear stress.     

Collagen fibril flow and tissue translocation coupled to fibroblast migration in 3D collagen matrices

In nested collagen matrices, human fibroblasts migrate from cell-containing dermal equivalents into surrounding cell-free outer matrices. Time-lapse microscopy showed that in addition to cell migration, collagen fibril flow occurred in the outer matrix toward the interface with the dermal equivalent. Features of this flow suggested that it depends on the same cell motile machinery that normally results in cell migration. Collagen fibril flow was capable of producing large-scale tissue translocation as shown by closure of a approximately 1-mm gap between paired dermal equivalents in floating, nested collagen matrices. Our findings demonstrate that when fibroblasts interact with collagen matrices, tractional force exerted by the cells can couple to matrix translocation as well as to cell migration.     

Effects of basic fibroblast growth factor on human microvessel endothelial cell migration on collagen I correlates inversely with adhesion and is cell density dependent

Angiogenesis, new vessel growth from existing vessels, is critical to tissue development and healing. Much is known about the molecular and cellular elements of angiogenesis, such as the effects of growth factors and matrix molecules on proliferation and migration. However, it is not clear how these elements are coordinated to produce specific microvascular beds. To address this, the effects of basic fibroblast growth factor (bFGF) on β1 integrin-mediated adhesion relative to migration in human microvessel endothelial cells (HMVEC) was examined. Using two assays of migration that differ in the density of cells being examined, bFGF stimulated single cell migration and reduced cell migration from a confluent monolayer on collagen I. Adhesion to collagen I of HMVEC treated at low density (2−4 × 10⁴ cells/cm²) with bFGF for 22 h was reduced, while bFGF increased cell adhesion of HMVEC treated at high density (6−8 × 10⁴ cells/cm²). Adhesion of both bFGF-treated and untreated HMVEC was mediated by the β1 integrin matrix receptor. Basic FGF treatment did not significantly alter surface expression of the β1 integrin subunit. Reduction in bFGF-mediated adhesion correlated with delayed cell spreading and altered organization of β1 integrin into substrate contacts. Thus, integrin-mediated cell adhesion in microvessel endothelial cells is sensitive to regulation by a growth factor. Furthermore, the nature of the response to this signal depends on another cell regulator, cell density. In addition, modulation of cell adhesion by a growth factor may be a central regulatory feature in controlling endothelial cell migration. © 1996 Wiley-Liss, Inc.     

急性低氧和间断低氧习服对HepG2细胞内血管内皮细胞生长因子基因表达的影响

目的:观察急性低氧和间断低氧习服对人HepG2细胞内血管内皮细胞生长因子(VEGF)及转录因子低氧诱导因子-1α(HIF-1α)的mRNA和蛋白含量的影响及其可能的生物学意义.方法:HepG2细胞随机分为常氧对照组,急性低氧组和间断低氧习服组.采用Northern blot和Western blot分别检测不同组别HepG2细胞内VEGF和HIF-1α mRNA表达和蛋白含量的变化.结果:急性低氧诱导HepG2细胞内VEGF和HIF-1α基因的转录,增加两种蛋白在细胞内的含量.间断低氧习服组的细胞内VEGF和HIF-1α的mRNA含量分别为常氧对照组细胞的(108.6±17.7)%和(116.74±19.8)%,与常氧对照组相比无显著差异(P＞0.05);而其蛋白表达的含量分别为对照组细胞的1.4和2.7倍,都明显低于急性低氧组细胞内两种蛋白的含量(P＜0.05).结论:HepG2细胞达到低氧习服状态后,抑制急性低氧对HepG2细胞内VEGF基因表达的促进作用,其中HIF-1α可能起着重要的调节作用.     

Heregulin and retinoids synergistically induce branching morphogenesis of breast cancer cells cultivated in 3D collagen gels

C-erbB and retinoid receptor signaling control mammary epithelial cell proliferation, differentiation, and morphology. Here, we examined the morphogenetic activities of c-erbB specific ligands such as heregulin and of retinoids on non-malignant (primary, MTSV1-7) and malignant (T47D, SKBR-3) human mammary epithelial cells (HMEC) cultivated in 3D collagen type I gels. These cells are positive for both c-erbB and retinoid receptors. Non-malignant primary HMEC spontaneously formed branched structures in collagen, whereas SV40 large T antigen-immortalized non-tumorigenic MTSV1-7 spontaneously formed balls and required heregulin or retinoid X receptor alpha-selective retinoid Ro 25-7386 for branching, which was further stimulated by combination of both types of agents. In malignant cells, heregulin alone induced ball formation and cooperated either with Ro 25-7386 (T47D) or with retinoic acid receptor alpha-selective AM580 (SKBR-3) for branching morphogenesis, which was accompanied by changes in the subcellular distribution of alpha(2)beta(1)-integrin and E-cadherin, and by down-regulation of c-erbB-2, -3, or -4. Heregulin and/or retinoids correspondingly increased the integrin-dependent adhesion of malignant cells to type I collagen. Our data demonstrate cooperative signaling of c-erbB and retinoid receptor pathways at the levels of morphogenesis and immunophenotypic differentiation.