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Fibroblast-dependent differentiation of human microvascular endothelial cells into capillary-like 3-dimensional networks¹

OMAIDA C. VELAZQUEZ^*,, RUTHANNE SNYDER^*,, ZHAO-JUN LIU^*,, RONALD M. FAIRMAN and MEENHARD HERLYN^*2

^* The Wistar Institute and
Department of Surgery, University of Pennsylvania, Pennsylvania, USA

²Correspondence: The Wistar Institute, 3601 Spruce St., Philadelphia, PA 19104, USA. E-mail: herlynm{at}wistar.upenn.edu

SPECIFIC AIMS

Investigations on the mechanism of angiogenesis require an efficient in vitro model that closely mimics the physiological in vivo processes of endothelial cell-extracellular matrix (ECM) interactions and endothelial-stromal cell communications. A new in vitro model was established that uses collagen type I as a matrix for endothelial cell invasion and differentiation, and integrates human fibroblasts to understand their critical role in vessel morphogenesis.

PRINCIPAL FINDINGS

1. Establishment and characterization of a novel in vitro 3-dimensional angiogenesis model
To establish an angiogenesis model that reflects endothelial cell detachment, invasion, migration, proliferation, and vacuole and tube formation, human microvascular endothelial cells (HMVECs) were cultured on collagen type I-coated dishes to 80% confluency, then overlaid with acellular collagen prepared in M199 medium supplemented with heparin, vitamin C, endothelial cell growth serum, and 10% FBS. After polymerization, the collagen was overlaid with a second collagen layer containing 5 x 10⁵ cells/mL primary human dermal fibroblasts. Endothelial cell growth medium was changed every 48 h. Labeling of the endothelial cells with DiI and subsequent in situ observation of the reconstruct by inverted fluorescence microscopy showed that detachment of the endothelial cells from the substrate and migration into the acellular collagen layer began within 4 h. After 24 h, the endothelial cells had migrated through the acellular collagen into the fibroblast-containing collagen layer and were found there throughout with increasing density over time. Double staining for the proliferation marker Ki67 and endothelial-specific marker von Willebrand factor (vWF) demonstrated that endothelial cells proliferated in the matrix even after 5 days. The endothelial cells came into close contact with fibroblasts and began forming vacuoles within 2 days and aligned into cords that structured into branching networks within 3–5 days. Capillary networks formed by day 4 to 5 and increased through day 11. Endothelial differentiation into true capillary-like morphology was confirmed by electron microscopy (Fig. 1 A). The outlines of branching tubular structures with true lumen could be visualized by staining cross sections for endothelial cell-specific PECAM-1 (Fig. 1B-D ). The lumen of the tubular structures stained positive for vWF on whole mount preparations (Fig. 1E, F ).

View larger version (39K): [in this window] [in a new window]   Figure 1. Characterization of fibroblast-induced differentiation of HMVECs into capillary-like networks. A). Electron micrograph of cross section of endothelial cell in collagen demonstrating capillary-like differentiation. ECN, endothelial cell nucleus; L, lumen of capillary formed by differentiated endothelial cell; FF, fibroblast adjacent and in close contact (white arrows) to endothelial cell. Insert shows high magnification of area indicated by black arrow showing endothelial cell-cell junction, indicating true capillary differentiation of the endothelial cell. The two white arrows point to the close cell-cell contact between the endothelial cell membrane and the fibroblast membrane. B–H). Endothelial cells from monolayers having migrated through collagen into a second layer of collagen with or without fibroblasts. B). PECAM-1 staining of longitudinal section after 5 days showing mature, capillary-like morphology. Counterstained fibroblast nuclei (negative for PECAM-1, blue arrowheads in inserts) are observed in close proximity to the endothelial annular structures (black arrows). x5, inserts x40. C, D) Cross sections of gel after 11 days stained for PECAM-1 expression. x10. E, F) Projection views of whole mounts of collagen gels with invading endothelial cells forming hollow tubular structures stained for vWF within the luminal surface of the tubes (red arrows). vWF-negative fibroblast nuclei stained with Hoechst dye are seen in close proximity to endothelial cells forming a tubule (E, insert). F) Insert shows a tubular structure on oblique cross section, x10, inserts x20. G, H) HMVECs’ migration into collagen in the absence of fibroblasts and staining for PECAM-1 (G) and vWF (H). There was greatly diminished migration of endothelial cells into collagen and they formed only immature disconnected cords. x10.

2. Validation of the model by demonstrating the mitogenic and motogenic effects of VEGF and Ang-1
Vascular endothelial growth factor (VEGF) and angiopoietin-1 (Ang-1) are two well-known angiogenic factors with mitogenic and/or motogenic properties. We used these two factors to validate the model for studies on angiogenesis. Reducing FBS to 1% completely inhibited the capillary formation while not inducing significant endothelial cell apoptosis. Capillary-like differentiation could be rescued by infection of fibroblasts with recombinant adenoviral vectors encoding either VEGF or Ang-1. Transduction of VEGF or Ang-1 into fibroblasts stimulated endothelial cell migration, whereas VEGF also increased endothelial cell proliferation under reduced conditions. Both VEGF and Ang-1 overexpressed in fibroblasts could restore capillary-like differentiation of HMVECs but only partially. Instead of mature capillary-like complexes, VEGF induced disconnected branching cords with few true lumina whereas Ang-1 enhanced formation of more mature endothelial networks, which showed true lumina but were not interconnected and were sparse in numbers. Addition of neutralizing Ab to VEGF or blocking Ab to VEGF-R2 (KDR) significantly inhibited vessel formation. Thus, VEGF and Ang-1 showed reproducible angiogenic effects in this new in vitro model and validated its applicability for angiogenesis study.

3. Formation of capillary-like structure is fibroblast dependent
Development of capillary networks in this model was fibroblast dependent. In the absence of fibroblasts, few HMVEC invaded the first collagen layer and proliferated within 24–48 h with rudimentary alignment into immature disconnected cords. There was progressive cell death after 48 h with no maturation of the tubular networks (Fig. 1G, H ). The presence of fibroblasts in the top collagen layer, however, greatly increased migration of the endothelial cells into the top layer housing the fibroblasts and decreased endothelial apoptosis. Fibroblasts did not stimulate proliferation, but rather impelled differentiation toward capillary-like structures.

The mechanism of how fibroblasts modulate endothelial cell differentiation into the capillary phenotype is unclear. It may be through direct fibroblast-endothelial cell contact and indirectly through secreted soluble factors. Some cells lining the lumina of the capillary-like networks stained positive for the pericyte marker smooth muscle actin, suggesting some fibroblasts may differentiate into pericytes and thereby stabilize the capillary-like structures. Induction of mature capillary-like networks by fibroblasts was inhibited by separating the fibroblast-containing layer from the endothelial cell monolayer with membranes that restricted cellular migration by the pore size (0.4 µm) while allowing exchange of soluble factors. When 0.4 µm membranes prevented close or direct fibroblast-endothelial cell interactions, endothelial cells migrated into the acellular collagen layer but formed only a few scattered immature networks, likely supported by fibroblast-derived soluble factors. Membranes with 12 µm pore size, on the other hand, allowed transmigration and cell-cell contact, and stimulated formation of mature capillary networks, suggesting that direct cell-cell contact between fibroblasts and endothelial cells is essential for capillary network formation. Close associations between endothelial cells and fibroblasts were confirmed by electron microscopy (Fig. 1A ), and could be seen in whole mount preparations in which the endothelial cells were stained for vWF and unstained fibroblasts were identified by Hoechst nuclear dye (Fig. 1E , insert). During formation of capillary-like structures, the fibroblasts remained in close proximity to the endothelial cells and over time organized concentrically around the forming vessels, supposedly stabilizing lumen formation (Fig. 1B, C ).

To study whether tumor cells could substitute for fibroblasts, fibroblasts within the second collagen layer were replaced with seven melanoma cell lines derived from radial and vertical growth phase primary and metastatic melanomas. None could induce migration of the endothelial cells into the collagen or capillary-like differentiation, implicating a unique and critical role for fibroblasts in the control of endothelial cell differentiation.

CONCLUSIONS

Earlier research has demonstrated the ability of malignant cells to induce angiogenesis in vivo and in vitro. This study is the first to demonstrate that normal human fibroblasts induce differentiation of microvascular endothelial cells into 3-dimensional capillary-like structures under conditions lacking tumor promoters (Fig. 2 ). The effects of fibroblasts are remarkably all-or-none, thus indicating that fibroblasts are essential for the formation of capillary-like structures. Fibroblasts are an integral component of all tissues; they contribute to their architecture by producing matrix proteins that serve as scaffolding for various organ structures, including the vasculature. Fibroblasts are also a rich source of growth factors for self-stimulation and activation of other cell types in the microenvironment. Activated fibroblasts produce angiogenic growth factors, including VEGF, FGF, or PDGF. In this model, fibroblasts likely function as supporting cells through both cell-cell contact with endothelial cells and secretion of soluble factors. Although vertical growth phase or metastatic melanoma cells are known to secret VEGF, the failure of these melanoma cells to induce migration of the endothelial cells into the collagen suggested that close or direct fibroblast-endothelial specific cell-cell interactions are required.

View larger version (86K): [in this window] [in a new window]   Figure 2. Schematic diagram of in vitro model of human angiogenesis in fibroblast-constricted collagen. When HMVECs are grown on collagen type I-coated plates to an 80% confluent monolayer, the cells are overlaid with the first collagen layer (acellular layer), followed by a second collagen layer that contains fibroblasts (A). Endothelial cells begin within 4 h to detach, migrate into the first collagen layer, and are found by day 1 in the second layer (B), where they are stimulated by soluble factors and establish direct contact with the fibroblasts, finally forming a network of branching tubular capillary-like structures by day 5 (C).

Rat brain astrocytes can induce microvascular endothelial cells to form capillary-like structures in vitro through direct or close cell-cell contact. Bovine microvascular endothelial cells are induced to form capillary-like structures by Swiss 3T3 mouse embryo fibroblasts via a paracrine mechanism. In our system, although direct cell-cell contact is required, fibroblast-mediated effects were not completely dependent on this mechanism because angiogenesis could be enhanced by VEGF and also be partially blocked with KDR blocking or VEGF neutralizing Ab. Earlier work demonstrated that fibroblasts are an important source of angiogenic factors. In our model, embedded fibroblasts were activated by collagen but we could not find detectable levels of secreted VEGF in medium collected daily. This could be due to low secretion levels or because secreted VEGF was bound to VEGF receptors or to matrix proteins. When we evaluated cross sections of the gels by anti-VEGF immunohistochemistry, increased VEGF staining was noted throughout the fibroblast-containing collagen gels but not the collagen-only controls, suggesting that low levels of VEGF are secreted and trapped within the collagen matrix. Alternatively, fibroblasts produce other soluble factors because anti-VEGF or anti-KDR Abs could block only 40–50% of the fibroblast-induced angiogenic effects, and adenoviral-mediated overexpression of VEGF under reduced serum conditions only stimulated endothelial cell migration and proliferation but did not induce mature capillary-like networks.

Our model contains three major components—fibroblasts, endothelial cells, and type I collagen—and thus is suitable to study cell-cell contacts and cell-ECM interactions. Development of capillary-like structures in our system not only required fibroblasts but also collagen, which may function as a scaffolding for cells and storage for growth factors. Only the very initial phase of the angiogenic cascade—1) detachment from the basal matrix and 2) initial invasion into overlaying collagen—is independent of fibroblasts and apparently induced only by the matrix. Endothelial cells could invade into collagen layer without fibroblasts and align into polarized cords, but the structure showed little maturation, suggesting that cooperation between fibroblasts and endothelial cells is essential for mature vessel formation.

FOOTNOTES

¹ To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.01-1011fje; to cite this article, use FASEB J. (June 7, 2002) 10.1096/fj.01-1011fje

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