Abstract: During wound healing, angiogenic capillary sprouts invade the fibrin/fibronectin-rich wound clot and within a few days organize into a microvascular network throughout the granulation tissue. As collagen accumulates in the granulation tissue to produce scar, the density of blood vessels diminishes. A dynamic interaction occurs among endothelial cells, angiogenic cytokines, such as FGF, VEGF, TGF-β, angiopoietin, and mast cell tryptase, and the extracellular matrix (ECM) environment. Specific endothelial cell ECM receptors are critical for these morphogenetic changes in blood vessels during wound repair. In particular, αvβ3, the integrin receptor for fibrin and fibronectin, appears to be required for wound angiogenesis: αvβ3 is expressed on the tips of angiogenic capillary sprouts invading the wound clot, and functional inhibitors of αvβ3 transiently inhibit granulation tissue formation. Recent investigations have shown that the wound ECM can regulate angiogenesis in part by modulating integrin receptor expression. mRNA levels of αvβ3 in human dermal microvascular endothelial cells either plated on fibronectin or overlaid by fibrin gel were higher than in cells plated on collagen or overlaid by collagen gel. Wound angiogenesis also appears to be regulated by endothelial cell interaction with the specific three-dimensional ECM environment in the wound space. In an in vitro model of human sprout angiogenesis, three-dimensional fibrin gel, simulating early wound clot, but not collagen gel, simulating late granulation tissue, supported capillary sprout formation. Understanding the molecular mechanisms that regulate wound angiogenesis, particularly how ECM modulates ECM receptor and angiogenic factor requirements, may provide new approaches for treating chronic wounds. During wound healing, angiogenic capillary sprouts invade the fibrin/fibronectin-rich wound clot and within a few days organize into a microvascular network throughout the granulation tissue. As collagen accumulates in the granulation tissue to produce scar, the density of blood vessels diminishes. A dynamic interaction occurs among endothelial cells, angiogenic cytokines, such as FGF, VEGF, TGF-β, angiopoietin, and mast cell tryptase, and the extracellular matrix (ECM) environment. Specific endothelial cell ECM receptors are critical for these morphogenetic changes in blood vessels during wound repair. In particular, αvβ3, the integrin receptor for fibrin and fibronectin, appears to be required for wound angiogenesis: αvβ3 is expressed on the tips of angiogenic capillary sprouts invading the wound clot, and functional inhibitors of αvβ3 transiently inhibit granulation tissue formation. Recent investigations have shown that the wound ECM can regulate angiogenesis in part by modulating integrin receptor expression. mRNA levels of αvβ3 in human dermal microvascular endothelial cells either plated on fibronectin or overlaid by fibrin gel were higher than in cells plated on collagen or overlaid by collagen gel. Wound angiogenesis also appears to be regulated by endothelial cell interaction with the specific three-dimensional ECM environment in the wound space. In an in vitro model of human sprout angiogenesis, three-dimensional fibrin gel, simulating early wound clot, but not collagen gel, simulating late granulation tissue, supported capillary sprout formation. Understanding the molecular mechanisms that regulate wound angiogenesis, particularly how ECM modulates ECM receptor and angiogenic factor requirements, may provide new approaches for treating chronic wounds. human dermal microvascular endothelial cells platelet-derived growth factor vascular endothelial cell growth factor Thirty-five million cutaneous wounds that require major intervention occur yearly in the U.S.A. alone. Some experts have estimated that the total number of chronic wounds exceeds 2 million and perhaps up to 5 million annually in the U.S.A. alone (1998). The social and financial tolls of chronic wounds are extremely high. The most common cause of acute wounds is thermal injury, with an estimated 2.5 million burns each year in the U.S.A. (1982). Other significant acute cutaneous wounds are caused by trauma, excision of extensive skin cancer, and medical conditions such as deep fungal and bacterial infections, vasculitis, scleroderma, pemphigus, toxic epidermal necrolysis to name a few. Categories of chronic wounds include arterial ulcers, diabetic ulcers, pressure ulcers, and venous ulcers. It is estimated that the prevalence of leg ulcers alone is between 0.5%-1.5% with an annual cost of nearly $1 billion (Nehls and Herrmann, 1996Nehls V. Herrmann R. The configuration of fibrin clots determines capillary morphogenesis and endothelial cell migration.Microvasc Res. 1996; 51 (10.1006/mvre.1996.0032): 347-364Crossref PubMed Scopus (136) Google Scholar). Principal goals in wound management are to achieve rapid wound closure and a functional and aesthetic scar. Over the past two decades extraordinary advances in cellular and molecular biology have greatly expanded our comprehension of the basic biologic processes involved in wound repair and tissue regeneration (Cheresh, 1987Cheresh D.A. Human endothelial cells synthesize and express an Arg-Gly-Asp-directed adhesion receptor involved in attachment to fibrinogen and von Willebrand factor.Proc Natl Acad Sci USA. 1987; 84: 6471-6475Crossref PubMed Scopus (415) Google Scholar). Ultimately these strides in basic knowledge will lead to advancements in wound care resulting in accelerated rates of ulcer and normal wound repair. Furthermore, as tumor stroma generation is similar to wound healing (Davis, 1992Davis E.D. Affinity of integrins for damaged extracellular matrix: αvβ3 binds to denatured collagen type I through RGD sites.Biochem Biophys Res Comm. 1992; 182: 1025-1031Crossref PubMed Scopus (284) Google Scholar), increased knowledge of wound repair may lead to unexpected advances in tumor therapy. Clearly today's scientific breakthroughs in molecular and cell biology will lead to tomorrow's therapeutic successes in wound care and tissue engineering (Shweiki et al., 1992Shweiki D. Itin A. Soffer D. Keshet E. Vascular endothelial growth factor induced by hypoxia may mediate hypoxia-initiated angiogenesis.Nature. 1992; 359: 843-845Crossref PubMed Scopus (4006) Google Scholar). During the early phase of cutaneous wound repair, new stroma, often called granulation tissue, begins to form approximately 4 d after injury. The name derives from the granular appearance of newly forming tissue when it is incised and visually examined. Numerous new capillaries endow the neostroma with its granular appearance. Macrophages, fibroblasts, and blood vessels move into the wound space as a unit (Heimark et al., 1986Heimark R.L. Twardzik D.R. Schwartz S.M. Inhibition of endothelial cell regeneration by type-beta transforming growth factor from platelets.Science. 1986; 233: 1078-1080Crossref PubMed Scopus (330) Google Scholar), which correlates well with the proposed biologic interdependence of these cells during tissue repair. Macrophages provide a continuing source of cytokines necessary to stimulate fibroplasia and angiogenesis, fibroblasts construct new extracellular matrix necessary to support cell ingrowth, and blood vessels carry oxygen and nutrients necessary to sustain cell metabolism. The quantity and quality of granulation tissue depends on the presence of biologic modifiers, the activity level of target cells, and the extracellular matrix environment (Jackson et al., 1995Jackson A. Tarantini F. Gamble S. Friedman S. Maciag T. The release of fibroblast growth factor-1 from NIH 3T3 cells in response to temperature involves the function of cysteine residues.J Biol Chem. 1995; 270: 33-36Crossref PubMed Scopus (94) Google Scholar;Cheresh et al., 1989Cheresh D.A. Berliner S.A. Vicente V. Ruggeri Z.M. Recognition of distinct adhesive sites on fibrinogen by related integrins on platelets and endothelial cells.Cell. 1989; 58: 945-953Abstract Full Text PDF PubMed Scopus (271) Google Scholar). Biologic modifiers include lipid mediators, metabolic products including those derived from oxygen, as well as proteins and peptides. Peptides with potent mitogenic activities are usually referred to as growth factors. Low levels of some growth factors circulate in the plasma; however, activated platelets release substantial amounts of preformed growth factors into wounded areas. Arrival of peripheral blood monocytes and their subsequent activation to macrophages ensures continual synthesis and release of growth factors. In addition, injured and activated parenchymal cells can synthesize and secrete growth factors. The provisional extracellular matrix also promotes granulation tissue formation. Once fibroblasts and endothelial cells express the proper integrin receptors, they invade the fibrin/fibronectin-rich clot in the wound space. New blood vessel formation is a critical component of wound healing. In the form of developing capillary sprouts, endothelial cells digest and penetrate the underlying vascular basement membrane, invade the ECM stroma, and form tube-like structures that continue to extend, branch, and create networks, pushed by endothelial cell proliferation from the rear and pulled by chemotaxis from the front. These events require a dynamic temporally and spatially regulated interaction between endothelial cells, angiogenesis factors, and surrounding ECM proteins (Cheresh et al., 1989Cheresh D.A. Berliner S.A. Vicente V. Ruggeri Z.M. Recognition of distinct adhesive sites on fibrinogen by related integrins on platelets and endothelial cells.Cell. 1989; 58: 945-953Abstract Full Text PDF PubMed Scopus (271) Google Scholar;Keck et al., 1989Keck P.J. Hauser S.D. Krivi G. Sanzo K. Warren T. Feder J. Connolly D.T. Vascular permeability factor, an endothelial cell mitogen related to PDGF.Science. 1989; 246: 1309-1313Crossref PubMed Scopus (1759) Google Scholar). The soluble factors that can stimulate angiogenesis in wound repair are gradually being elucidated (Roberts et al., 1986Roberts A.B. Sporn M.B. Assoian R.K. et al.Transforming growth factor beta: Rapid induction of fibrosis and angiogenesis in vivo and stimulation of collagen formation.Proc Natl Acad Sci (USA). 1986; 83: 4167-4171Crossref PubMed Scopus (2332) Google Scholar); however, the factors that do stimulate wound angiogenesis are less clear. Angiogenic activity can be recovered from activated macrophages as well as the epidermis and soft tissue wounds. Twelve years ago acidic fibroblast growth factor (aFGF) or basic fibroblast growth factor (bFGF) appeared to be responsible for most of these activities (Feng et al., 1999aFeng X. Clark R.A.F. Galanakis D. Tonnesen M.G. Fibrin and collagen differentially regulate human dermal microvascular endothelial cell integrins: Stabilization of αvβ3 mRNA by fibrin.J Invest Dermatol. 1999; 113 (10.1046/j.1523-1747.1999.00786.x): 913-919Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar). In the interim, other molecules have also been shown to have angiogenic activity, including vascular endothelial growth factor (VEGF) (Jerdan et al., 1991Jerdan J.A. Michels R.G. GlaSeries B.M. Extracellular matrix of newly forming vessels – an immunohistochemical study.Microvasc Res. 1991; 42: 255-265Crossref PubMed Scopus (36) Google Scholar), TGF-β (Yang and Moses, 1990Yang E.Y. Moses H.L. Transforming growth factor-β1-induced changes in cell migration, proliferation, and angiogenesis in the chicken chorioallantoic membrane.J Cell Biol. 1990; 111: 731-741Crossref PubMed Scopus (406) Google Scholar), angiogenin (Vallee and Riordan, 1997Vallee B.L. Riordan J.F. Organogenesis and angiogenin.Cell Mol Life Sci. 1997; 53: 803-815Crossref PubMed Scopus (49) Google Scholar), angiopoietin (Singer and Clark, 1999Singer A.J. Clark R.A.F. Mechanisms of disease: cutaneous wound healing.New Eng J Med. 1999; 341: 738-746Crossref PubMed Scopus (4308) Google Scholar), and human mast cell tryptase (Blair et al., 1997Blair R.J. Meng H. Marchese M.J. Ren S. Schwartz L.B. Tonnesen M.G. Gruber B.L. Human mast cells stimulate vascular tube formation. Tryptase is a novel, potent angiogenic factor.J Clin Invest. 1997; 99: 2691-2700Crossref PubMed Scopus (370) Google Scholar). aFGF and bFGF were the first members of the large FGF family to be discovered and are now designated FGF-1 and FGF-2, respectively (Abraham et al., 1996Abraham J.A. Klagsbrun M. Modulation of wound repair by members of the fibroblast growth factor family.in: Clark R.A.F. The Molecular and Cellular Biology of Wound Repair. Plenum Press, New York1996: 195-248Google Scholar). These two growth factors have potent angiogenic activity by rabbit cornea and chorioallantoic membrane assays (Feng et al., 1999aFeng X. Clark R.A.F. Galanakis D. Tonnesen M.G. Fibrin and collagen differentially regulate human dermal microvascular endothelial cell integrins: Stabilization of αvβ3 mRNA by fibrin.J Invest Dermatol. 1999; 113 (10.1046/j.1523-1747.1999.00786.x): 913-919Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar). Neither FGF-1 nor FGF-2, however, have a transmembrane sequence and therefore cannot be secreted. Nevertheless, at least some forms of cell injury can cause FGF-1 release (Hunt, 1980Hunt T.K. Wound Healing and Wound Infection: Theory and Surgical Practice. Appleton-Century-Crofts, New York1980Google Scholar). Perhaps these two factors are released from disrupted parenchymal cells at a wound site resulting in the initial stimulus for angiogenesis. Although TGF-β promotes angiogenesis in vivo (Risau, 1997Risau W. Mechanisms of angiogenesis.Nature. 1997; 386: 671-674Crossref PubMed Scopus (4614) Google Scholar;Yang and Moses, 1990Yang E.Y. Moses H.L. Transforming growth factor-β1-induced changes in cell migration, proliferation, and angiogenesis in the chicken chorioallantoic membrane.J Cell Biol. 1990; 111: 731-741Crossref PubMed Scopus (406) Google Scholar), it inhibits the growth and proliferation of endothelial cell monolayers in vitro (Baird and Durkin, 1986Baird A. Durkin T. Inhibition of endothelial cell proliferation by type-beta transforming growth factor: interactions with acidic and basic fibroblast growth factors.Biochem Biophys Res Commun. 1986; 138: 476-482Crossref PubMed Scopus (229) Google Scholar;Feng et al., 1999bFeng X. Clark R.A.F. Galanakis D. Tonnesen M.G. Fibrin, but not collagen, 3-dimensional matrix supports sprout angiogenesis of human dermal microvascular endothelial cells.Am J Pathol. 1999Google Scholar;Frater-Schroder et al., 1986Frater-Schroder M. Muller G. Birchmeirer W. Bohlem P. Transforming growth factor-beta inhibits endothelial cell proliferation.Biochem Biophys Res Commun. 1986; 137: 295-302Crossref PubMed Scopus (227) Google Scholar). This apparent discrepancy between in vivo and in vitro activities may be attributable, in part, to the capacity of TGF-β in vivo to recruit and stimulate macrophages that then produce other active angiogenesis factors (Weisman et al., 1988Weisman D.M. Polverini P.J. Kamp D.W. Leibovich S.J. Transforming growth factor-beta (TGF-β) is chemotactic for human monocytes and induces their expression of angiogenic activity.Biochem Biophys Res Comm. 1988; 157: 793-800Crossref PubMed Scopus (181) Google Scholar). An alternative, but not preclusive, explanation is that TGF-β is a growth inhibitor for cultured endothelial cell monolayers, but a mitogen for cultured endothelial cells that have formed capillary-like tubes (Horton et al., 1985Horton M.A. Lewis D. McNulty K. Pringle J.A.S. Chambers T.J. Monoclonal antibodies to osteoclastomas (giant cell bone tumors): Definition of osteoclast specific antigens.Cancer Res. 1985; 45: 5663-5669PubMed Google Scholar). In fact, the types of TGF-β receptors on endothelial cells are altered when cultured endothelial cells form tubes (Ruoslahti, 1991Ruoslahti E. Integrins.J Clin Invest. 1991; 87: 1-5Crossref PubMed Scopus (1441) Google Scholar). Likewise cultured monolayer endothelial cells make PDGF-BB but have no receptor (PDGFR-β) for this ligand. In contrast, once the cultured cells form tubes, they express PDGFR-β and respond to the ligand that they no longer produce (Battegay et al., 1994Battegay E.F. Rupp J. Iruela-Arispe L. Sage E.H. Pech M. PDGF-BB modulates endothelial proliferation and angiogenesis in vitro via PDGF β-receptors.J Cell Biol. 1994; 125: 917-928Crossref PubMed Scopus (311) Google Scholar). VEGF, a member of the PDGF family of growth factors, has potent angiogenesis, as well as vasopermeability, activity which led to its initial designation as vasopermeability factor (VPF) (Detmar et al., 1997Detmar M. Brown L.F. Berse B. Jackman R.W. Elicker B.M. Dvorak H.F. Claffey K.P. Hypoxia regulates the expression of vascular permeability factor/vascular endothelial growth factor (VPF/VEGF) and its receptors in human skin.J Invest Dermatol. 1997; 108: 263-268Crossref PubMed Scopus (225) Google Scholar). This factor is produced in large quantities by the epidermis during wound healing (Brown et al., 1992Brown L.F. Yeo K.-T. Berse B. Yeo T.-K. Senger D.R. Dvorak H.F. Van De Water L. Expression of vascular permeability factor (vascular endothelial growth factor) by epidermal keratinocytes during wound healing.J Exp Med. 1992; 176: 1375-1379Crossref PubMed Scopus (749) Google Scholar). Low oxygen tension, as occurs in tissue hypoxia, is a major inducer of this growth factor (Sankar et al., 1996Sankar S. Mahooti-Brooks N. Bensen L. McCarthy T.L. Centrella M. Madri J.A. Modulation of transforming growth factor β receptor levels on microvascular endothelial cells during in vitro angiogenesis.J Clin Invest. 1996; 97: 1436-1446Crossref PubMed Scopus (153) Google Scholar;Clark et al., 1996Clark R.A.F. Tonnesen M.G. Gailit J. Cheresh D.A. Transient functional expression of αvβ3 on vascular cells during wound repair.Am J Path. 1996; 148: 1407-1421PubMed Google Scholar) and its receptor (Brogi et al., 1996Brogi O. Schatteman G. Wu T. Kim E.A. Varticovski L. Keyt B. Isner J.M. Hypoxia-induced paracrine regulation of vascular endothelial growth factor receptor expression.J Clin Invest. 1996; 97: 469-476Crossref PubMed Scopus (335) Google Scholar). Thus, cell disruption and hypoxia, hallmarks of tissue injury, appear to be strong initial inducers of potent angiogenesis factors at the wound site. Recent data suggest that bFGF may set the stage for angiogenesis during the first 3 d of wound repair, whereas VEGF may be critical for angiogenesis during granulation tissue formation from day 4 through 7 (Nehls and Drenckhahn, 1995Nehls V. Drenckhahn D. A novel, microcarrier-based in vitro assay for rapid and reliable quantiifcation of three-dimensional cell migration and angiogenesis.Microvasc Res. 1995; 50 (10.1006/mvre.1995.1061): 311-322Crossref PubMed Scopus (200) Google Scholar). Several additional members of the VEGF family have been found recently (VEGF-B, VEGF-C, and VEGF-D) (Veikkola and Alitalo, 1999Veikkola T. Alitalo K. VEGFs receptors and angiogenesis [In Process Citation].Semin Cancer Biol. 1999; 9 (10.1006/scbi.1998.0091): 211-220Crossref PubMed Scopus (433) Google Scholar). Although their general role in angiogenesis processes is quickly being elucidated, their specific function in wound angiogenesis is not yet clear. The angiopoietins have recently joined the members of the VEGF family as the only known growth factors largely specific for vascular endothelium. The angiopoietins include a naturally occurring agonist, angiopoietin-1, as well as a naturally occurring antagonist, angiopoietin-2, both of which act by means of the Tie2 receptor. Two new angiopoietins, angiopoietin-3 in mouse and angiopoietin-4 in human, have recently been identified but their function in angiogenesis is unknown (Valenzuela et al., 1999Valenzuela D.M. Griffiths J.A. Rojas J. et al.Angiopoietins 3 and 4: diverging gene counterparts in mice and humans.Proc Natl Acad Sci USA. 1999; 96: 1904-1909Crossref PubMed Scopus (388) Google Scholar). Neither bind the Tie2 receptor. Recently one of us collaborated in research that demonstrated that mast cell tryptase is an additional angiogenesis factor (Blair et al., 1997Blair R.J. Meng H. Marchese M.J. Ren S. Schwartz L.B. Tonnesen M.G. Gruber B.L. Human mast cells stimulate vascular tube formation. Tryptase is a novel, potent angiogenic factor.J Clin Invest. 1997; 99: 2691-2700Crossref PubMed Scopus (370) Google Scholar). The frequent presence of mast cells near capillary sprouting sites suggests an association between mast cells and angiogenesis. Coculture of human mast cells (HMC) with human dermal microvascular endothelial cells (HDMEC) led to a dose-dependent increase in the network area of vascular tube growth. Moreover, the extent of neovascularization was enhanced greatly when HMC were degranulated in the presence of HDMEC. Further examination using antagonists to various mast cell products revealed a diminished response (73%-88% decrease) in the area of vascular tube formation if specific inhibitors of tryptase were present. Tryptase (3 microg per ml) directly added to HDMEC caused a significant augmentation of capillary growth, which was suppressed by specific tryptase inhibitors. Tryptase also directly induced cell proliferation of HDMEC in a dose-dependent fashion (2 pM-2 nM). These results are consistent with the concept that mast cells act at sites of new vessel formation by secreting tryptase, which then functions as a potent and previously unrecognized angiogenesis factor. The ECM of a healing wound undergoes rapid changes as the fibrin clot is replaced by fibronectin and hyaluronan and subsequently by types I and III collagen (Cheresh et al., 1989Cheresh D.A. Berliner S.A. Vicente V. Ruggeri Z.M. Recognition of distinct adhesive sites on fibrinogen by related integrins on platelets and endothelial cells.Cell. 1989; 58: 945-953Abstract Full Text PDF PubMed Scopus (271) Google Scholar). These transitions from fibrin-rich provisional matrix to a second-order provisional matrix to a collagenous scar are highly orchestrated and tightly regulated both spatially and temporally. As fibroblasts invade the fibrin clot it is lysed and fibronectin and hyaluronan are deposited, forming early granulation tissue. This process initially occurs in the periphery of the clot and later more centrally as the granulation tissue grows into the wound space. At any given time, the ECM at the wound margin differs qualitatively and quantitatively from the ECM situated centrally. Orchestration and regulation of the rapid new tissue development observed in wound healing indubitably depends not only on the cells and cytokines present but also on the ECM microenvironment. The complex interaction and feedback control of cells/cytokines/matrix has been termed ‘‘dynamic reciprocity’' (Bissell et al., 1982Bissell M.J. Hall H.G. Parry G. How does the extracellular matrix direct gene expression?.J Theor Biol. 1982; 99: 31-68Crossref PubMed Scopus (1031) Google Scholar). For example, previous studies from our laboratory have demonstrated that three-dimensional ECM proteins regulate ECM receptor expression of normal human dermal fibroblasts (Xu and Clark, 1996Xu J. Clark R.A.F. Extracellular matrix alters PDGF regulation of fibroblast integrins.J Cell Biol. 1996; 132: 239-249Crossref PubMed Scopus (182) Google Scholar). Thus, ECM proteins control fibroblast expression of ECM receptors, which regulate fibroblast interaction with and alteration of the ECM. Therefore, besides the growth factors and chemotactic factors, an appropriate ECM is also necessary for wound angiogenesis (Keck et al., 1989Keck P.J. Hauser S.D. Krivi G. Sanzo K. Warren T. Feder J. Connolly D.T. Vascular permeability factor, an endothelial cell mitogen related to PDGF.Science. 1989; 246: 1309-1313Crossref PubMed Scopus (1759) Google Scholar). Dilated and hypertrophied blood vessels adjacent to the wound transiently (from 3 to 5 d after injury) deposit increased amounts of fibronectin within their vascular walls (Clark, 1996aClark R.A.F. The Molecular and Cellular Biology of Wound Repair. Plenum Press, New York1996Google Scholar,Clark et al., 1982bClark R.A.F. Lanigan J.M. DellaPelle P. Manseau E. Dvorak H.F. Colvin R.B. Fibronectin and fibrin (ogen) provide a provisional matrix for epidermal cell migration during wound reepithelialization.J Invest Dermatol. 1982; 79: 264-269Abstract Full Text PDF PubMed Scopus (490) Google Scholar), whereas fibrin and fibronectin leak from the blood into the perivascular stroma (Clark, unpublished data). At day 4 post-injury capillary sprouts emanate from these ‘‘mother’' vessels and invade the wound clot (Magnatti et al., 1989Magnatti P. Tsuboi R. Robbins E. Rifkin D.B. In vitro angiogenesis on the human amniotic membrane: requirement for basic fibroblast growth factor-induced proteinases.J Cell Biol. 1989; 108: 671-682Crossref PubMed Scopus (363) Google Scholar). Remarkably, neovascular invasion of the wound fibrin clot precedes fibroblast invasion and lysis of the clot (Figure 1) (Clark et al., 1995Clark R.A.F. Nielsen L.D. Welch M.P. McPherson J.M. Collagen matrices attenuate the collagen synthetic response of cultured fibroblasts to TGF-β.J Cell Sci. 1995; 108: 1251-1261PubMed Google Scholar;Shweiki et al., 1992Shweiki D. Itin A. Soffer D. Keshet E. Vascular endothelial growth factor induced by hypoxia may mediate hypoxia-initiated angiogenesis.Nature. 1992; 359: 843-845Crossref PubMed Scopus (4006) Google Scholar). At day 4 the spatial distance between these two tissue cell invasion zones is approximately 100 μm. Thus, the capillary tips of angiogenic blood vessels are surrounded by plasma-derived fibrin and fibronectin, not wound fibroblast-derived ECM composed of fibronectin and hyaluronan. As the wound granulation tissue matures during the second week after injury, the neostroma accumulates increasing amounts of types I and III collagen (Welch et al., 1990Welch M.P. Odland G.F. Clark R.A.F. Temporal relationships of F-actin bundle formation, collagen and fibronectin matrix assembly, and fibronectin receptor expression to wound contraction.J Cell Biol. 1990; 110: 133-145Crossref PubMed Scopus (272) Google Scholar;Clark et al., 1982aClark R.A.F. DellaPelle P. Manseau E. Lanigan J.M. Dvorak H.F. Colvin R.B. Blood vessel fibronectin increases in conjunction with endothelial cell proliferation and capillary ingrowth during wound healing.J Invest Dermatol. 1982; 79: 269-276Abstract Full Text PDF PubMed Scopus (150) Google Scholar). The density of blood vessels present in the granulation tissue bed diminishes as collagen accumulates (unpublished data). Such delineation of the precise ECM present around wound blood vessels and at the tip of capillary sprouts is necessary for constructing meaningful investigations of the dynamic interactions between endothelial cells and the surrounding ECM milieu during wound angiogenesis. Using a microcarrier-based angiogenesis assay,McClain et al., 1996McClain S.A. Simon M. Jones E. et al.Mesenchymal cell activation is the rate limiting step of granulation tissue induction.Am J Path. 1996; 149: 1257-1270PubMed Google Scholar) demonstrated that fibrin structure played an important role in bovine pulmonary artery endothelial cell migration and capillary morphogenesis. They showed that the degree of rigidity of fibrin gel strongly influenced tube formation by bovine endothelial cells in response to bFGF or VEGF. They did not, however, compare fibrin with collagen gels.Swerlick et al., 1993Swerlick R.A. Brown E.J. Xu Y. Lee K.H. Manos S. Lawley T.J. Expression and modulation of the vitronectin receptor on human dermal microvascular endothelial cells.J Invest Dermatol. 1993; 99: 715-722Crossref Scopus (56) Google Scholar reported that addition of fibrin into type I collagen gel significantly increased the length of the tubular structures formed by monolayer bovine capillary endothelial cells cultured on the gel by about 180% compared with type I collagen alone. This assay, however, appears to more closely simulate vasculogenesis as occurs during embryogenesis rather than sprout angiogenesis as occurs in wound healing (Phillips and Dover, 1991Phillips T.J. Dover J.S. Leg ulcers.J Am Acad Dermatol. 1991; 25: 965-987Abstract Full Text PDF PubMed Scopus (185) Google Scholar). We have established an in vitro system of human microvascular sprout angiogenesis by modifying the original assay described by Nehls (Matsuyama et al., 1989Matsuyama T. Yamada A. Kay J. Yamada K.M. Akiyama S.K. Schlossman S.F. Morimoto C. Activation of CD4 cells by fibronectin and anti-CD3 antibody: a synergistic effect mediated by the VLA-5 fibronectin receptor complex.J Exp Med. 1989; 117: 1133-1148Crossref Scopus (243) Google Scholar). HDMEC are cultured on microcarrier beads and embedded in a three-dimensional extracellular matrix (3-D ECM) (Enenstein et al., 1992Enenstein J. Waleh N.S. Kramer R.H. Basic FGF and TGF-β differentially modulate integrin expression of human microvascular endothelial cells.Exp Cell Res. 1992; 203: 499-503Crossref PubMed Scopus (150) Google Scholar). When the ECM was a fibrin gel and an angiogenesis factor, such as VEGF or bFGF, was added to the culture construct, capillary-like sprouts developed within 24 h and capillary networks developed by 5 d. Such an in vitro environment, in fact, recapitulates angiogenesis invading a wound clot. The presence of lumina in these sprouts was confirmed by confocal microscopy. If a collagen gel was used for the 3-D ECM instead of fibrin, VEGF and bFGF induced endothelial cells to invade the matrix as individual cells, without formation of tubes. If fibrin, however, was added to the collagen matrix, capillary-like tubes sprouted from the beads. The fibrin/collagen 3-D ECM in vitro environment, in fact, simulates tumor