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MT1-MMP-dependent neovessel formation within the confines of the three-dimensional extracellular matrix.

Chun TH, Sabeh F, Ota I, Murphy H, McDonagh KT, Holmbeck K, Birkedal-Hansen H, Allen ED, Weiss SJ - J. Cell Biol. (2004)

Bottom Line: Extracellular matrix-degradative enzymes, including the matrix metalloproteinases (MMPs) MMP-2 and MMP-9, are thought to play key roles in angiogenesis by binding to docking sites on the cell surface after activation by plasmin- and/or membrane-type (MT) 1-MMP-dependent processes.Unexpectedly, neither MMP-2, MMP-9, their cognate cell-surface receptors (i.e., beta3 integrin and CD44), nor plasminogen are essential for collagenolytic activity, endothelial cell invasion, or neovessel formation.Instead, the membrane-anchored MMP, MT1-MMP, confers endothelial cells with the ability to express invasive and tubulogenic activity in a collagen-rich milieu, in vitro or in vivo, where it plays an indispensable role in driving neovessel formation.

View Article: PubMed Central - PubMed

Affiliation: Division of Molecular Medicine and Genetics, Department of Internal Medicine, University of Michigan, Ann Arbor, MI 48109, USA.

ABSTRACT
During angiogenesis, endothelial cells initiate a tissue-invasive program within an interstitial matrix comprised largely of type I collagen. Extracellular matrix-degradative enzymes, including the matrix metalloproteinases (MMPs) MMP-2 and MMP-9, are thought to play key roles in angiogenesis by binding to docking sites on the cell surface after activation by plasmin- and/or membrane-type (MT) 1-MMP-dependent processes. To identify proteinases critical to neovessel formation, an ex vivo model of angiogenesis has been established wherein tissue explants from gene-targeted mice are embedded within a three-dimensional, type I collagen matrix. Unexpectedly, neither MMP-2, MMP-9, their cognate cell-surface receptors (i.e., beta3 integrin and CD44), nor plasminogen are essential for collagenolytic activity, endothelial cell invasion, or neovessel formation. Instead, the membrane-anchored MMP, MT1-MMP, confers endothelial cells with the ability to express invasive and tubulogenic activity in a collagen-rich milieu, in vitro or in vivo, where it plays an indispensable role in driving neovessel formation.

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ECM remodeling during ex vivo neovessel formation. (A) Neovessel formation from mouse aortic ring explants. After culturing for 2 d in a 3-D type I collagen gel (2.2 mg/ml) in the presence of VEGF and HGF (50 ng/ml each) and 20% FBS, vessel wall–associated cells initiate migration from matrix-embedded aortic rings (a; bar, 500 μm). The asterisk indicates the position of the aortic explant and the inset displays a scanning electron micrograph of the type I collagen matrix that highlights the dense packing of the surrounding fibrillar gel (bar, 5 μm). Patent tubular structures form by day 5 (b) with PECAM-1–positive endothelial cells (c, green) comprising the wall of the formed neovessels (c). Cell nuclei are stained blue with DAPI (c; bar, 100 μm). By phase-contrast microscopy, the newly formed tubules (margins of a vessel are bordered by arrowheads) allow the passive outflow of red blood cells that remain in the aortic rings after isolation (d; bar, 50 μm). Cross sections of sprouting neovessels display tubular structures of varying diameters after a 7-d culture period (e; bar, 100 μm), whereas transmission electron micrographs show that endothelial cells line a patent lumen (f, asterisk) that is surrounded by pericyte-like accessory cells (arrowheads; bar, 5 μm). (B) Type I collagen degradation products surrounding egressing neovessels, as detected with antibodies directed against collagen neoepitopes generated after the proteolysis of collagen into three-quarter and one-quarter fragments, are shown on the left (green). Type I collagen denaturation products after proteolysis are shown in the middle (red). Coincident with the generation of type I collagen cleavage production, the endothelial cells deposit a matrix of type IV collagen (right; green). Cell nuclei are stained blue with DAPI. (C) Neovessel formation is blocked in the presence of 5 μm BB-94 (neovessel length decreases from 1,079 ± 20 μm to 271 ± 9 μm, n = 3), but proceeds in an unaffected manner from aortic rings isolated from plg−/− mice and cultured in plg−/− serum after a 7-d incubation period.
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fig1: ECM remodeling during ex vivo neovessel formation. (A) Neovessel formation from mouse aortic ring explants. After culturing for 2 d in a 3-D type I collagen gel (2.2 mg/ml) in the presence of VEGF and HGF (50 ng/ml each) and 20% FBS, vessel wall–associated cells initiate migration from matrix-embedded aortic rings (a; bar, 500 μm). The asterisk indicates the position of the aortic explant and the inset displays a scanning electron micrograph of the type I collagen matrix that highlights the dense packing of the surrounding fibrillar gel (bar, 5 μm). Patent tubular structures form by day 5 (b) with PECAM-1–positive endothelial cells (c, green) comprising the wall of the formed neovessels (c). Cell nuclei are stained blue with DAPI (c; bar, 100 μm). By phase-contrast microscopy, the newly formed tubules (margins of a vessel are bordered by arrowheads) allow the passive outflow of red blood cells that remain in the aortic rings after isolation (d; bar, 50 μm). Cross sections of sprouting neovessels display tubular structures of varying diameters after a 7-d culture period (e; bar, 100 μm), whereas transmission electron micrographs show that endothelial cells line a patent lumen (f, asterisk) that is surrounded by pericyte-like accessory cells (arrowheads; bar, 5 μm). (B) Type I collagen degradation products surrounding egressing neovessels, as detected with antibodies directed against collagen neoepitopes generated after the proteolysis of collagen into three-quarter and one-quarter fragments, are shown on the left (green). Type I collagen denaturation products after proteolysis are shown in the middle (red). Coincident with the generation of type I collagen cleavage production, the endothelial cells deposit a matrix of type IV collagen (right; green). Cell nuclei are stained blue with DAPI. (C) Neovessel formation is blocked in the presence of 5 μm BB-94 (neovessel length decreases from 1,079 ± 20 μm to 271 ± 9 μm, n = 3), but proceeds in an unaffected manner from aortic rings isolated from plg−/− mice and cultured in plg−/− serum after a 7-d incubation period.

Mentions: To determine whether neovessel formation is linked to a collagen remodeling process, mouse tissue explants were embedded in a 3-D type I collagen gel, stimulated with a growth factor cocktail, and monitored for vessel outgrowth as well as collagenolysis. After the egress of fibroblast-like cells during the first two days of ex vivo culture (Fig. 1 A, a), capillary sprouts emerge and coalesce to form an anastomosing network of PECAM-1–positive vessels surrounded by pericyte-like cells at day 7 (Fig. 1 A, b–f). Coincident with the expression of the tubulogenic program, type I collagen degradation products appear in the pericellular environment, as detected with antibodies specific for either (1) the COOH-terminal neopeptide of the three-quarter fragment of cleaved type I collagen or (2) denatured collagen products that have lost their triple-helical integrity (Fig. 1 B). Though the formation of capillary-like structures is associated with type I collagenolytic activity, the degradative phenotype is coordinated with the neodeposition of type IV collagen (Fig. 1 B) and laminin (not depicted) as maturing neovessels initiate basement membrane synthesis (Nicosia and Madri, 1987).


MT1-MMP-dependent neovessel formation within the confines of the three-dimensional extracellular matrix.

Chun TH, Sabeh F, Ota I, Murphy H, McDonagh KT, Holmbeck K, Birkedal-Hansen H, Allen ED, Weiss SJ - J. Cell Biol. (2004)

ECM remodeling during ex vivo neovessel formation. (A) Neovessel formation from mouse aortic ring explants. After culturing for 2 d in a 3-D type I collagen gel (2.2 mg/ml) in the presence of VEGF and HGF (50 ng/ml each) and 20% FBS, vessel wall–associated cells initiate migration from matrix-embedded aortic rings (a; bar, 500 μm). The asterisk indicates the position of the aortic explant and the inset displays a scanning electron micrograph of the type I collagen matrix that highlights the dense packing of the surrounding fibrillar gel (bar, 5 μm). Patent tubular structures form by day 5 (b) with PECAM-1–positive endothelial cells (c, green) comprising the wall of the formed neovessels (c). Cell nuclei are stained blue with DAPI (c; bar, 100 μm). By phase-contrast microscopy, the newly formed tubules (margins of a vessel are bordered by arrowheads) allow the passive outflow of red blood cells that remain in the aortic rings after isolation (d; bar, 50 μm). Cross sections of sprouting neovessels display tubular structures of varying diameters after a 7-d culture period (e; bar, 100 μm), whereas transmission electron micrographs show that endothelial cells line a patent lumen (f, asterisk) that is surrounded by pericyte-like accessory cells (arrowheads; bar, 5 μm). (B) Type I collagen degradation products surrounding egressing neovessels, as detected with antibodies directed against collagen neoepitopes generated after the proteolysis of collagen into three-quarter and one-quarter fragments, are shown on the left (green). Type I collagen denaturation products after proteolysis are shown in the middle (red). Coincident with the generation of type I collagen cleavage production, the endothelial cells deposit a matrix of type IV collagen (right; green). Cell nuclei are stained blue with DAPI. (C) Neovessel formation is blocked in the presence of 5 μm BB-94 (neovessel length decreases from 1,079 ± 20 μm to 271 ± 9 μm, n = 3), but proceeds in an unaffected manner from aortic rings isolated from plg−/− mice and cultured in plg−/− serum after a 7-d incubation period.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2172577&req=5

fig1: ECM remodeling during ex vivo neovessel formation. (A) Neovessel formation from mouse aortic ring explants. After culturing for 2 d in a 3-D type I collagen gel (2.2 mg/ml) in the presence of VEGF and HGF (50 ng/ml each) and 20% FBS, vessel wall–associated cells initiate migration from matrix-embedded aortic rings (a; bar, 500 μm). The asterisk indicates the position of the aortic explant and the inset displays a scanning electron micrograph of the type I collagen matrix that highlights the dense packing of the surrounding fibrillar gel (bar, 5 μm). Patent tubular structures form by day 5 (b) with PECAM-1–positive endothelial cells (c, green) comprising the wall of the formed neovessels (c). Cell nuclei are stained blue with DAPI (c; bar, 100 μm). By phase-contrast microscopy, the newly formed tubules (margins of a vessel are bordered by arrowheads) allow the passive outflow of red blood cells that remain in the aortic rings after isolation (d; bar, 50 μm). Cross sections of sprouting neovessels display tubular structures of varying diameters after a 7-d culture period (e; bar, 100 μm), whereas transmission electron micrographs show that endothelial cells line a patent lumen (f, asterisk) that is surrounded by pericyte-like accessory cells (arrowheads; bar, 5 μm). (B) Type I collagen degradation products surrounding egressing neovessels, as detected with antibodies directed against collagen neoepitopes generated after the proteolysis of collagen into three-quarter and one-quarter fragments, are shown on the left (green). Type I collagen denaturation products after proteolysis are shown in the middle (red). Coincident with the generation of type I collagen cleavage production, the endothelial cells deposit a matrix of type IV collagen (right; green). Cell nuclei are stained blue with DAPI. (C) Neovessel formation is blocked in the presence of 5 μm BB-94 (neovessel length decreases from 1,079 ± 20 μm to 271 ± 9 μm, n = 3), but proceeds in an unaffected manner from aortic rings isolated from plg−/− mice and cultured in plg−/− serum after a 7-d incubation period.
Mentions: To determine whether neovessel formation is linked to a collagen remodeling process, mouse tissue explants were embedded in a 3-D type I collagen gel, stimulated with a growth factor cocktail, and monitored for vessel outgrowth as well as collagenolysis. After the egress of fibroblast-like cells during the first two days of ex vivo culture (Fig. 1 A, a), capillary sprouts emerge and coalesce to form an anastomosing network of PECAM-1–positive vessels surrounded by pericyte-like cells at day 7 (Fig. 1 A, b–f). Coincident with the expression of the tubulogenic program, type I collagen degradation products appear in the pericellular environment, as detected with antibodies specific for either (1) the COOH-terminal neopeptide of the three-quarter fragment of cleaved type I collagen or (2) denatured collagen products that have lost their triple-helical integrity (Fig. 1 B). Though the formation of capillary-like structures is associated with type I collagenolytic activity, the degradative phenotype is coordinated with the neodeposition of type IV collagen (Fig. 1 B) and laminin (not depicted) as maturing neovessels initiate basement membrane synthesis (Nicosia and Madri, 1987).

Bottom Line: Extracellular matrix-degradative enzymes, including the matrix metalloproteinases (MMPs) MMP-2 and MMP-9, are thought to play key roles in angiogenesis by binding to docking sites on the cell surface after activation by plasmin- and/or membrane-type (MT) 1-MMP-dependent processes.Unexpectedly, neither MMP-2, MMP-9, their cognate cell-surface receptors (i.e., beta3 integrin and CD44), nor plasminogen are essential for collagenolytic activity, endothelial cell invasion, or neovessel formation.Instead, the membrane-anchored MMP, MT1-MMP, confers endothelial cells with the ability to express invasive and tubulogenic activity in a collagen-rich milieu, in vitro or in vivo, where it plays an indispensable role in driving neovessel formation.

View Article: PubMed Central - PubMed

Affiliation: Division of Molecular Medicine and Genetics, Department of Internal Medicine, University of Michigan, Ann Arbor, MI 48109, USA.

ABSTRACT
During angiogenesis, endothelial cells initiate a tissue-invasive program within an interstitial matrix comprised largely of type I collagen. Extracellular matrix-degradative enzymes, including the matrix metalloproteinases (MMPs) MMP-2 and MMP-9, are thought to play key roles in angiogenesis by binding to docking sites on the cell surface after activation by plasmin- and/or membrane-type (MT) 1-MMP-dependent processes. To identify proteinases critical to neovessel formation, an ex vivo model of angiogenesis has been established wherein tissue explants from gene-targeted mice are embedded within a three-dimensional, type I collagen matrix. Unexpectedly, neither MMP-2, MMP-9, their cognate cell-surface receptors (i.e., beta3 integrin and CD44), nor plasminogen are essential for collagenolytic activity, endothelial cell invasion, or neovessel formation. Instead, the membrane-anchored MMP, MT1-MMP, confers endothelial cells with the ability to express invasive and tubulogenic activity in a collagen-rich milieu, in vitro or in vivo, where it plays an indispensable role in driving neovessel formation.

Show MeSH
Related in: MedlinePlus