Guided migration as a novel mechanism of capillary network remodeling is regulated by basic fibroblast growth factor

 To investigate mechanisms of capillary network remodeling, we developed a serum-free angiogenesis in vitro system in three-dimensional fibrin matrices which allows the study of directional growth of endothelial sprouts, anastomosis, and remodeling (’pruning’) of the primitive plexus toward more elaborated capillary trees. To follow the movements of living endothelial cells by inverse-fluorescence microscopy, we cocultured unlabeled endothelial cells with endothelial cells labeled with the carbocyanine dye 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine perchlorate (DiI). We show that elongation and retraction of neighboring capillary sprouts occurs simultaneously, resembling a tug-of-war by which endothelial cells are withdrawn from shortening sprouts to become incorporated in other sprouts nearby. For the first time, we directly demonstrate the long-suspected parallel sliding movement of endothelial cells. We show that cell migration persists within immature capillaries even after sprouts have merged to continuous capillary loops, leading to overlapping growth of opposing sprout tips. As a novel concept of capillary remodeling, we distinguish two types of endothelial cell migration: sprouting and guided migration. Sprouting is the de novo invasion of a matrix by endothelial cells, and guided migration is the locomotion of cells along preexistent capillary-like structures. We show that guided migration leads to remodeling of immature capillary networks and to the retraction of sprouts. We describe a method for quantification of sprouting versus guided migration in DiI-mosaic-labeled capillary networks, and we present evidence that endothelial cell-derived basic fibroblast growth factor serves as a chemotactic signal for other cells to migrate along a preestablished capillary-like structure. Accepted: 3 November 1997     

A microcarrier-based cocultivation system for the investigation of factors and cells involved in angiogenesis in three-dimensional fibrin matrices in vitro

Angiogenesis in situ includes coordinated interactions of various microvascular cell types, i.e., endothelial cells, pericytes and perivascular fibroblasts. To study the cellular interactions of microvascular cells in vitro, we have developed a microcarrier-based cocultivation system. The technical details of this method include seeding of endothelial cells on unstained cytodex-3 microcarriers and seeding of pericytes, fibroblasts or vascular smooth muscle cells on microcarriers which have been labeled by trypan blue staining. A mixture of both unstained and trypan blue-stained microcarriers was subsequently embedded in a three-dimensional fibrin clot. The growth characteristics of each cell type could be conveniently observed since the majority of cells left their supporting microcarriers in a horizontal direction to migrate into the transparent fibrin matrix. As differently stained microcarriers were randomly arranged in the fibrin matrix, the characteristic patterns of the microcarriers allowed location of particular points of interest at different developmental stages, facilitating the observation of cellular growth over the course of time. One further advantage of this microcarrier-based system is the possibility of reliably quantifying capillary growth by determination of average numbers of capillary-like formations per microcarrier. Thus, this model allows convenient evaluation of the effects of non-endothelial cells on angiogenesis in vitro. By using this coculture system, we demonstrate that endothelial capillary-like structures in vitro do not become stabilized by contacting vascular smooth muscle cells or pericytes during the initial stages of capillary formation.     

The basic helix-loop-helix transcription factor HESR1 regulates endothelial cell tube formation

Human endothelial cells can be induced to form capillary-like tubular networks in collagen gels. We have used this in vitro model and representational difference analysis to identify genes involved in the formation of new blood vessels. HESR1 (HEY-1/HRT-1/CHF-2/gridlock), a basic helix-loop-helix protein related to the hairy/enhancer of split/HES family, is absent in migrating and proliferating cultures of endothelial cells but is rapidly induced during capillary-like network formation. HESR1 is detectable in all adult tissues and at high levels in well vascularized organs such as heart and brain. Its expression is also enriched in aorta and purified capillaries. Overexpression of HESR1 in endothelial cells down-regulates vascular endothelial cell growth factor receptor-2 (VEGFR2) mRNA levels and blocks proliferation, migration, and network formation. Interestingly, reduction of expression of HESR1 by antisense oligonucleotides also blocks endothelial cell network formation in vitro. Finally, HESR1 expression is altered in several breast, lung, and kidney tumors. These data are consistent with a temporal model for HESR1 action where down-regulation at the initiation of new vessel budding is required to allow VEGFR2-mediated migration and proliferation, but re-expression of HESR1 is necessary for induction of tubular network formation and continued maintenance of the mature, quiescent vessel.     

In vitro rapid organization of endothelial cells into capillary-like networks is promoted by collagen matrices

We have studied the behavior of cloned capillary endothelial cells grown inside a three dimensional collagen matrix. Cell monolayers established on the surface of collagen gels were covered with a second layer of collagen. This induced the monolayers of endothelial cells to reorganize into a network of branching and anastomosing capillary-like tubes. As seen by electron microscopy, the tubes were formed by at least two cells (in transverse sections) delimiting a narrow lumen. In addition, distinct basal lamina material was present between the abluminal face of the endothelial cells and the collagen matrix. These results showed that capillary endothelial cells have the capacity to form vessel-like structures with well-oriented cell polarity in vitro. They also suggest that an appropriate topological relationship of endothelial cells with collagen matrices, similar to that occurring in vivo, has an inducive role on the expression of this potential. This culture system provides a simple in vitro model for studying the factors involved in the formation of new blood vessels (angiogenesis).     

Phorbol esters induce angiogenesis in vitro from large-vessel endothelial cells

We have shown previously that the tumor promoter phorbol myristate acetate (PMA) induces capillary endothelial cells grown on the surface of three-dimensional collagen gels to invade the underlying matrix as capillary-like tubular structures, a phenomenon mimicking angiogenic processes that occur in vivo (Montesano and Orci: Cell 42:469, 1985). To establish whether the potential to invade the extracellular matrix as capillary-like sprouts is restricted to microvascular endothelial cells or is also shared by large vessel endothelium, we have examined the response to PMA of endothelial cells isolated from the human umbilical vein and the calf pulmonary artery. The results of these experiments show that both types of macrovascular endothelial cells are able to penetrate into collagen gels as vessel-like tubes following treatment with PMA. This demonstrates that endothelial cells derived from large vessels can, in response to appropriate signals, express invasive properties thought to be associated specifically with capillary endothelial cells in vivo.     

Engineered blood and lymphatic capillaries in 3-D VEGF-fibrin-collagen matrices with interstitial flow

In vitro endothelial cell organization into capillaries is a long standing challenge of tissue engineering. We recently showed the utility of low level interstitial flow in guiding the organization of endothelial cells through a 3-D fibrin matrix-containing covalently bound vascular endothelial growth factor (VEGF). Here this synergistic phenomenon was extended to explore the effects of matrix composition on in vitro capillary morphogenesis of human blood versus lymphatic endothelial cells (BECs and LECs). Different mixtures of fibrin and collagen were used in conjunction with constant concentrations of matrix-bound VEGF and slow interstitial flow over 10 days. Interestingly, the BECs and LECs each showed a distinct preference in terms of organization for matrix composition: LECs organized the most extensively in a fibrin-only matrix, while BEC organization was optimized in the compliant collagen-containing matrices. Furthermore, the BECs and LECs produced architecturally different structures; while BECs organized in thick, branched networks containing wide lumen, the LECs were elongated into slender, overlapping networks with fine lumen. These data demonstrate the importance of the 3-D matrix composition in facilitating and coordinating BEC and LEC capillary morphogenesis, which is important for in vitro vascularization of engineered tissues.     

A novel angiogenesis model using interstitial cells derived from skeletal muscle

A novel angiogenesis model using co-culture of endothelial and interstitial cells was developed, in which bovine capillary endothelial cells (BCEC) formed capillary-like structure on the monolayer of interstitial cells (like smooth muscle cells) isolated from rat skeletal muscle. The capillary formation of BCEC occurred even under serum-free conditions. Insulin stimulated the capillary growth under serum-free condition, but not BCEC growth in the single culture system. These results suggested that the insulin effect on the capillary growth was brought about indirectly through the interstitial cells, and that this co-culture system may be useful for the study of angiogenesis (especially in skeletal muscle).     

Induction of angiogenesis in vitro by vanadate, an inhibitor of phosphotyrosine phosphatases

We have previously shown that capillary endothelial cells grown on the surface of three-dimensional collagen gels can be induced to invade the underlying fibrillar matrix and to form capillary-like tubular structures in response to tumor-promoting phorbol esters or the angiogenic agent fibroblast growth factor (FGF). Since both phorbol esters and FGF stimulate phosphorylation of tyrosine residues, we treated endothelial cells with vanadate, an inhibitor of phosphotyrosine-specific phosphatases, to determine whether this agent could induce the expression of an angiogenic phenotype in these cells. We show here that vanadate stimulates endothelial cells to invade collagen matrices and to organize into characteristic tubules resembling those induced by FGF or phorbol esters. We have further observed that vanadate concomitantly stimulates endothelial cells to produce plasminogen activators (PAs), proteolytic enzymes which are induced by phorbol esters and FGF, and which have been implicated in the neovascular response; this stimulation can be accounted for by an increase in the levels of urokinase-type PA and tissue type PA mRNA. These results suggest a role for tyrosine phosphorylation in the regulation of the angiogenic phenotype in capillary endothelial cells.     

In vitro angiogenesis in fibrin matrices containing fibronectin or hyaluronic acid.

Angiogenesis involved numerous interactions between extracellular matrix and endothelial cells which may exhibit changes in actin filament distribution. Using an in vitro model, capillary endothelial cells were grown in fibrin matrix containing fibronectin or hyaluronic acid. Actin filament distribution, nucleus localization and cell morphology were observed. Preliminary study showed the formation of tube-, branche- and capillary-like structures within fibrin. In the presence of both fibrin and fibronectin, cells with actin filament stress fibers were more spreading than those in fibrin. In the presence of hyaluronic acid, tubes were limited in extension into the fibrin. In addition, the study of co-localization of nucleus and actin filaments showed different cell behaviours. Migratory cells seem to arrange in parallel to each other and a capillary-like structure may be formed at the proximal extremity of this cell pattern.     

CRIM1 is involved in endothelial cell capillary formation in vitro and is expressed in blood vessels in vivo

In endothelial cells that form capillary-like structures in vitro a variety of genes is upregulated as we have demonstrated previously. In addition to well known genes, we also identified genes never described in endothelial cells before. Here, we report the further characterization of one selected gene called cysteine-rich motor neuron 1 (CRIM1). CRIM1 is strongly upregulated in endothelial cells during tube formation and is expressed by a variety of adherent growing cell lines whereas cell lines grown in suspension do not express CRIM1. By using antisense technology we were able to inhibit CRIM1 expression and demonstrate impaired formation of capillary-like structures in vitro in transfected endothelial cells. Furthermore, we show that CRIM1 is a glycosylated type I transmembrane protein, that accumulates at sites of close cell-to-cell contact upon stimulation. Finally, we found CRIM1 protein to be expressed by endothelial cells of the inner lining of blood vessels in vivo. Taken together our results imply a possible role of CRIM1 in capillary formation and maintainance during angiogenesis.     

Fabrication of endothelialized tube in collagen gel as starting point for self-developing capillary-like network to construct three-dimensional organs in vitro

A possible strategy for creating three-dimensional (3D) tissue-engineered organs in vitro with similar volumes to the primary organs is to develop a capillary network throughout the constructs to provide sufficient oxygenation and nutrition to the cells composing them. Here, we propose a novel approach for the creation of a capillary-like network in vitro, based on the spontaneous tube-forming activity of vascular endothelial cells (ECs) in collagen gel. We fabricated a linear tube of 500 microm in diameter, the inner surface of which was filled with bovine carotid artery vascular endothelial cells (BECs), in type I collagen gel as a starting point for the formation of a capillary-like network. The BECs exposed to a medium containing vascular endothelial growth factor (VEGF) migrated into the ambient gel around the tube. After 2 weeks of VEGF exposure, the distance of the migration into the ambient gel in the radial direction of the tube reached approximately 800 microm. Cross-sections of capillary-like structures composed of the migrating BECs, with a lumen-like interior space, were observed in slices of the gel around the tube stained with hematoxylin-eosin (H&E). These results demonstrate that this approach using a pre-established tube, which is composed of ECs, as a starting point for a self-developing capillary-like network is potentially useful for constructing 3D organs in vitro.     

Tissue engineering of blood vessels with endothelial cells differentiated from mouse embryonic stem cells

Endothelial cells (TEC3 cells) derived from mouse embryonic stem (ES) cells were used as seed cells to construct blood vessels. Tissue engineered blood vessels were made by seeding 8 X 106 smooth muscle cells (SMCs) ob-tained from rabbit arteries onto a sheet of nonwoven polyglycolic acid (PGA) fibers, which was used as a biode-gradable polymer scaffold. After being cultured in DMEM medium for 7 days in vitro, SMCs grew well on the PGA fibers, and the cell-PGA sheet was then wrapped around a silicon tube, and implanted subcutaneously into nude mice. After 6～8 weeks, the silicon tube was replaced with another silicon tube in smaller diameter, and then the TEC3 cells (endothelial cells differentiated from mouse ES cells) were injected inside the engineered vessel tube as the test group. In the control group only culture medium was injected. Five days later, the engineered vessels were harvested for gross observation, histological and immunohistochemical analysis. The preliminary results demonstrated that the SMC-PGA construct could form a tubular structure in 6-8 weeks and PGA fibers were completely degraded. Histological and immunohistochemical analysis of the newly formed tissue revealed a typical blood vessel structure, including a lining of endothelial cells (ECs) on the lumimal surface and the presence of SMC and collagen in the wall. No EC lining was found in the tubes of control group. Therefore, the ECs differentiated from mouse ES cells can serve as seed cells for endothelium lining in tissue engineered blood vessels.     

Assessing the permeability of engineered capillary networks in a 3D culture

Many pathologies are characterized by poor blood vessel growth and reduced nutrient delivery to the surrounding tissue, introducing a need for tissue engineered blood vessels. Our lab has developed a 3D co-culture method to grow interconnected networks of pericyte-invested capillaries, which can anastamose with host vasculature following implantation to restore blood flow to ischemic tissues. However, if the engineered vessels contain endothelial cells (ECs) that are misaligned or contain wide junctional gaps, they may function improperly and behave more like the pathologic vessels that nourish tumors. The purpose of this study was to test the resistance to permeability of these networks in vitro, grown with different stromal cell types, as a metric of vessel functionality. A fluorescent dextran tracer was used to visualize transport across the endothelium and the pixel intensity was quantified using a customized MATLAB algorithm. In fibroblast-EC co-cultures, the dextran tracer easily penetrated through the vessel wall and permeability was high through the first 5 days of culture, indicative of vessel immaturity. Beyond day 5, dextran accumulated at the periphery of the vessel, with very little transported across the endothelium. Quantitatively, permeability dropped from initial levels of 61% to 39% after 7 days, and to 7% after 2 weeks. When ECs were co-cultured with bone marrow-derived mesenchymal stem cells (MSCs) or adipose-derived stem cells (AdSCs), much tighter control of permeability was achieved. Relative to the EC-fibroblast co-cultures, permeabilities were reduced 41% for the EC-MSC co-cultures and 50% for the EC-AdSC co-cultures after 3 days of culture. By day 14, these permeabilities decreased by 68% and 77% over the EC-fibroblast cultures. Co-cultures containing stem cells exhibit elevated VE-cadherin levels and more prominent EC-EC junctional complexes when compared to cultures containing fibroblasts. These data suggest the stromal cell identity influences the functionality and physiologic relevance of engineered capillary networks.     

Identification of novel angiogenesis inhibitors

Vascular endothelial growth factor (VEGF) is a key stimulant of angiogenesis, which is the process of generating new capillary blood vessels. Inhibition of the vascular endothelial growth factor receptor (VEGFR) kinase is known to result in blockage of angiogenesis. A pharmacophore was developed based on the binding of ATP to the hinge region of the kinase domain of VEGFR and a database search of 18,000 compounds was conducted. Selected hits were assessed for their ability to limit the induction of web-like network of capillary tubes by the human umbilical vascular endothelial cells. Two compounds (1 and 4) showed good inhibitory ability to prevent sprouting and closed polygon formation of the tubular networks, promising them to be lead compounds. Compound 4 showed 60% inhibition at 0.05 microM.     

Cell patterning using magnetite nanoparticles and magnetic force

Technologies for fabricating functional tissue architectures by patterning cells precisely are highly desirable for tissue engineering. Although several cell patterning methods such as microcontact printing and lithography have been developed, these methods require specialized surfaces to be used as substrates, the fabrication of which is time consuming. In the present study, we demonstrated a simple and rapid cell patterning technique, using magnetite nanoparticles and magnetic force, which enables us to allocate cells on arbitrary surfaces. Magnetite cationic liposomes (MCLs) developed in our previous study were used to magnetically label the target cells. When steel plates placed on a magnet were positioned under a cell culture surface, the magnetically labeled cells lined on the surface where the steel plate was positioned. Patterned lines of single cells were achieved by adjusting the number of cells seeded, and complex cell patterns (curved, parallel, or crossing patterns) were successfully fabricated. Since cell patterning using magnetic force may not limit the property of culture surfaces, human umbilical vein endothelial cells (HUVECs) were patterned on Matrigel, thereby forming patterned capillaries. These results suggest that the novel cell patterning methodology, which uses MCLs, is a promising approach for tissue engineering and studying cell-cell interactions in vitro.     

Fabrication of artificial endothelialized tubes with predetermined three-dimensional configuration from flexible cell-enclosing alginate fibers

One possible strategy for creating three-dimensional (3D) tissue-engineered organs in vitro is to develop a vasculature for sufficient transport of oxygen and nutrients within these constructs. Here, we describe a novel technique to fabricate endothelialized tubes with predetermined 3D configuration, as a starting point for self-developing capillary-like networks in vitro. Calcium-alginate hydrogel fibers of ca. 250 and 500 mum in diameter, enclosing bovine carotid artery vascular endothelial cells (BECs), were used as templates for endothelialized tubes. Fibers were prepared by extruding a 2% (w/v) sodium alginate solution containing BECs into a 100 mM calcium chloride solution flowing in the same direction. Fibers were embedded in type I collagen gels and enzymatically degraded by alginate lyase, resulting in channels with predetermined 3D configuration filled with a BEC suspension. Cells attached to and covered the surfaces of the channels. Exposing the cells to medium containing basic fibroblast growth factor resulted in their migration into the ambient collagen gel and self-assembly into capillary-like structures. These results demonstrate that using artificial endothelialized tubes with predetermined 3D configuration, as a starting point for a self-developing capillary-like network, could be potentially useful for constructing 3D tissue-engineered organs.     

Propagation of viruses on micropatterned host cells

We have developed a technique to characterize the in vitro propagation of viruses. Microcontact printing was used to generate linear arrays of alkanethiols on gold surfaces, which served as substrates for the patterned culture of baby hamster kidney (BHK-21) cells. Vesicular stomatitis virus (VSV) was added to unpatterned cell reservoirs adjacent to the patterned cells and incubated, setting in motion a continuously advancing viral infection into the patterned cells. At different incubation times, multiple arrays were chemically fixed to stop the viral propagation. Viral propagation distances into the patterned cells were determined by indirect immunofluorescent labeling and visualization of the VSV surface glycoprotein (G). The infection spread at approximately 50 microm/h in the 140-microm lines. Moreover, different temporal stages of the infection process were simultaneously visualized along individual lines. These stages included initiation of infection, based on G protein expression; cell-cell fusion, based on virus-induced clustering of cell nuclei; and cytoskeletal degradation, based on localized release of cells from the surface. This work sets a foundation for parallel, high-throughput characterization of viral and cellular processes.     

Angiogenic activity of porcine granulosa cells co-cultured with endothelial cells in a microcarrier-based three-dimensional fibrin gel.

To verify the possible role played by pig granulosa cells in the ovarian angiogenic process, we have developed a reliable in vitro system which allows the evaluation of endothelial sprouting and capillary growth in three-dimensional matrices. Granulosa cells collected from porcine follicles of different size were co-cultured with porcine aortic endothelial cells (PAEC) in a microcarrier-based fibrin gel system; after 2 and 5 days of co-culture, we determined the number and length of all endothelial sprouts; moreover, these parameters were quantified only in capillary-like structures, which were defined as continuous multicellular sprouts at least 200 microm long. In granulosa cells- PAEC co-cultures we observed an increase of angiogenic activity as compared to controls (PAEC alone). Granulosa cells from follicles of different size regulate angiogenesis differently: cells from the small follicle group significantly enhanced endothelial sprouting, while those from the large follicle group favoured mainly capillary elongation. Our observations seem therefore to suggest that the development and growth of thecal vascular bed is controlled by paracrine factors of granulosa cell origin that may induce the formation of a primitive capillary plexus during the early phases of antral follicle growth, which will be remodelled in more advanced phases of follicular development.     

Identification of a subpopulation of human renal microvascular endothelial cells with capacity to form capillary-like cord and tube structures

Summary Endothelial specialization is a prominent feature within distinct capillary beds of organs such as mammalian kidney, yet immunological markers for functionally distinct subpopulations of cultured endothelial cells from tissue sources such as kidney have not been available. We developed a simple and reproducible isolation and culture procedure to recover human renal microvascular endothelial cells (HRMEC) from the cortex of unused donor kidneys. This procedure yields highly purified preparations of cells that display endothelial markers that include Factor VIII antigen, acetyl-LDL receptors, and determinants that bind Ulex europaeus lectin. HRMEC assemble into capillary-like cord and tube structures when plated on the surface of basement membrane-like matrix (BMM) in media containing phorbol myristate acetate. To further define subpopulations of HRMEC, we generated a panel of monoclonal antibodies and screened for those recognizing cell surface determinants. One monoclonal antibody recovered from this screen recognized a cell surface protein expressed on a subpopulation of HRMEC that we have designated PEC-1 (pioneer endothelial cell antigen-1). Cells expressing PEC-1 extended long, interconnecting filopodial processes in response to phorbol myristate acetate and assembled into capillary-like structures when plated on BMM. Anti-PEC-1 immunoprecipitated proteins of 25 and 27 kDa. Magnetic bead separation of PEC-1 (+) cells selected cells that assemble into capillary-like cord and tube structures. The remaining PEC-1 (−) HRMEC population formed matrix adherent patches. In the kidney, the PEC-1 determinant is expressed on a small subpopulation of microvascular glomerular cells and is prominently expressed on the apical membrane of proximal tubule cells. The PEC-1 determinant discriminates among subpopulations of HRMEC, identifying a subpopulation that contributes to assembly of capillary-like structures.