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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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The fibrin-invasive activity of MT1-MMP– explants and endothelial cells. (A) Wild-type and MT1-MMP– explants display comparable neovessel outgrowth and tubulogenic activity when embedded in a 3-D gel of cross-linked fibrin (3.0 mg/ml) and cultured for 7 d with VEGF–HGF in the presence of 5% mouse serum as assessed by phase-contrast microscopy, in H&E–stained cross sections (representative vessels marked with arrowheads), or by TEM analysis (middle and bottom, respectively). (B) Isolated endothelial cells from wild-type or MT1-MMP– mice invade fibrin matrices (3.0 mg/ml) in comparable fashion in the course of a 5-d incubation period in the presence of VEGF–HGF and 20% FBS. Representative invading cells are marked with arrowheads. (C) Fibrin invasion by isolated endothelial cells from wild-type and MT1-MMP– mice are comparable, and inhibited by 1 μg/ml TIMP-2 but not 1 μg/ml TIMP-1 after a 5-d culture period under the conditions described above. Results are shown as the number of invading cells per hpf (mean ± 1 SD of 10 randomly chosen fields in two experiments).
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fig6: The fibrin-invasive activity of MT1-MMP– explants and endothelial cells. (A) Wild-type and MT1-MMP– explants display comparable neovessel outgrowth and tubulogenic activity when embedded in a 3-D gel of cross-linked fibrin (3.0 mg/ml) and cultured for 7 d with VEGF–HGF in the presence of 5% mouse serum as assessed by phase-contrast microscopy, in H&E–stained cross sections (representative vessels marked with arrowheads), or by TEM analysis (middle and bottom, respectively). (B) Isolated endothelial cells from wild-type or MT1-MMP– mice invade fibrin matrices (3.0 mg/ml) in comparable fashion in the course of a 5-d incubation period in the presence of VEGF–HGF and 20% FBS. Representative invading cells are marked with arrowheads. (C) Fibrin invasion by isolated endothelial cells from wild-type and MT1-MMP– mice are comparable, and inhibited by 1 μg/ml TIMP-2 but not 1 μg/ml TIMP-1 after a 5-d culture period under the conditions described above. Results are shown as the number of invading cells per hpf (mean ± 1 SD of 10 randomly chosen fields in two experiments).

Mentions: Consistent with the inability of MT1-MMP−/− tissue explants to mount a tubulogenic program, isolated MT1-MMP−/− endothelial cells were unable to degrade subjacent collagen (Fig. 5 B) or invade 3-D collagen gels (Fig. 5 C). A requirement for MT1-MMP during tissue-invasive activity was further confirmed by the inability of MT1-MMP– endothelial cells to penetrate an explant of collagen-rich human dermal tissue ex vivo (Fig. 5 D). Despite the fact that MT1-MMP has been posited to regulate cell adhesion or migration by proteolyzing integrins, cadherins, or surface enzymes (Gálvez et al., 2002; Seiki, 2002), MT1-MMP−/− endothelial cells did not express defects in their two-dimensional interactions with collagen, as the cells migrated across type I collagen–coated surfaces at a rate indistinguishable from that of controls (i.e., 29 ± 1 μm/24 h vs. 29 ± 1 μm/24 h; n = 3). Likewise, although MT1-MMP serves as a necessary collagenolysin in endothelial cells, fibrin-invasive and -degradative activity can be conferred by other members of the membrane-anchored MMP family, including MT3-MMP (Hotary et al., 2002). Given the ability of vessel explants as well as isolated endothelial cells to express MT3-MMP (unpublished data), morphogenesis and invasion through 3-D fibrin barriers was assessed in wild-type versus MT1-MMP−/− tissues. In contrast with their defect in collagen-invasive activity, MT1-MMP– explants mounted a tubulogenic phenotype in fibrin gels that was comparable to that mounted by wild-type controls (Fig. 6 A). Furthermore, both MT1-MMP+/+ and MT1-MMP−/− endothelial cells were able to invade fibrin gels via a TIMP-2-inhibitable process (Fig. 6, B and C). Hence, we conclude that (1) a requirement for MT1-MMP in tubulogenesis and invasion is restricted to the type I collagen matrix; and (2) although MT1-MMP has been reported to cleave a multiplicity of cell surface–associated substrates (Seiki, 2002), invasion and tubulogenesis can proceed independently of MT1-MMP in a physiologically relevant, but noncollagenous, matrix.


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)

The fibrin-invasive activity of MT1-MMP– explants and endothelial cells. (A) Wild-type and MT1-MMP– explants display comparable neovessel outgrowth and tubulogenic activity when embedded in a 3-D gel of cross-linked fibrin (3.0 mg/ml) and cultured for 7 d with VEGF–HGF in the presence of 5% mouse serum as assessed by phase-contrast microscopy, in H&E–stained cross sections (representative vessels marked with arrowheads), or by TEM analysis (middle and bottom, respectively). (B) Isolated endothelial cells from wild-type or MT1-MMP– mice invade fibrin matrices (3.0 mg/ml) in comparable fashion in the course of a 5-d incubation period in the presence of VEGF–HGF and 20% FBS. Representative invading cells are marked with arrowheads. (C) Fibrin invasion by isolated endothelial cells from wild-type and MT1-MMP– mice are comparable, and inhibited by 1 μg/ml TIMP-2 but not 1 μg/ml TIMP-1 after a 5-d culture period under the conditions described above. Results are shown as the number of invading cells per hpf (mean ± 1 SD of 10 randomly chosen fields in two experiments).
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2172577&req=5

fig6: The fibrin-invasive activity of MT1-MMP– explants and endothelial cells. (A) Wild-type and MT1-MMP– explants display comparable neovessel outgrowth and tubulogenic activity when embedded in a 3-D gel of cross-linked fibrin (3.0 mg/ml) and cultured for 7 d with VEGF–HGF in the presence of 5% mouse serum as assessed by phase-contrast microscopy, in H&E–stained cross sections (representative vessels marked with arrowheads), or by TEM analysis (middle and bottom, respectively). (B) Isolated endothelial cells from wild-type or MT1-MMP– mice invade fibrin matrices (3.0 mg/ml) in comparable fashion in the course of a 5-d incubation period in the presence of VEGF–HGF and 20% FBS. Representative invading cells are marked with arrowheads. (C) Fibrin invasion by isolated endothelial cells from wild-type and MT1-MMP– mice are comparable, and inhibited by 1 μg/ml TIMP-2 but not 1 μg/ml TIMP-1 after a 5-d culture period under the conditions described above. Results are shown as the number of invading cells per hpf (mean ± 1 SD of 10 randomly chosen fields in two experiments).
Mentions: Consistent with the inability of MT1-MMP−/− tissue explants to mount a tubulogenic program, isolated MT1-MMP−/− endothelial cells were unable to degrade subjacent collagen (Fig. 5 B) or invade 3-D collagen gels (Fig. 5 C). A requirement for MT1-MMP during tissue-invasive activity was further confirmed by the inability of MT1-MMP– endothelial cells to penetrate an explant of collagen-rich human dermal tissue ex vivo (Fig. 5 D). Despite the fact that MT1-MMP has been posited to regulate cell adhesion or migration by proteolyzing integrins, cadherins, or surface enzymes (Gálvez et al., 2002; Seiki, 2002), MT1-MMP−/− endothelial cells did not express defects in their two-dimensional interactions with collagen, as the cells migrated across type I collagen–coated surfaces at a rate indistinguishable from that of controls (i.e., 29 ± 1 μm/24 h vs. 29 ± 1 μm/24 h; n = 3). Likewise, although MT1-MMP serves as a necessary collagenolysin in endothelial cells, fibrin-invasive and -degradative activity can be conferred by other members of the membrane-anchored MMP family, including MT3-MMP (Hotary et al., 2002). Given the ability of vessel explants as well as isolated endothelial cells to express MT3-MMP (unpublished data), morphogenesis and invasion through 3-D fibrin barriers was assessed in wild-type versus MT1-MMP−/− tissues. In contrast with their defect in collagen-invasive activity, MT1-MMP– explants mounted a tubulogenic phenotype in fibrin gels that was comparable to that mounted by wild-type controls (Fig. 6 A). Furthermore, both MT1-MMP+/+ and MT1-MMP−/− endothelial cells were able to invade fibrin gels via a TIMP-2-inhibitable process (Fig. 6, B and C). Hence, we conclude that (1) a requirement for MT1-MMP in tubulogenesis and invasion is restricted to the type I collagen matrix; and (2) although MT1-MMP has been reported to cleave a multiplicity of cell surface–associated substrates (Seiki, 2002), invasion and tubulogenesis can proceed independently of MT1-MMP in a physiologically relevant, but noncollagenous, matrix.

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