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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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A required role for MT1-MMP in neovessel formation, endothelial cell invasion, and collagenolytic activity. (A) Aortic vessel explants isolated from MT1-MMP– mice display defective capillary sprouting (middle) compared with wild-type tissue, after a 7-d culture period in type I collagen gels in the presence of VEGF–HGF and 20% FBS (left) or 5% autologous serum (not depicted). The co-culture of wild-type and MT1-MMP– aortic rings (left and right, respectively, of the right panel) does not affect the sprouting behavior of wild-type or MT1-MMP– explants. (B) Phalloidin-stained endothelial cells (green) circumscribe zones of collagen degradation generated by wild-type, but not MT1-MMP−/−, cells cultured atop a type I collagen film in the presence of VEGF–HGF in 20% FBS for 7 d. Bar, 10 μm. Insets show a low magnification image of multiple sites of collagenolysis relative to the complete absence of degradation observed when MT1-MMP– endothelial cells are cultured atop fluorescently labeled collagen gels (bar, 50 μm). (C) In contrast with wild-type endothelial cells, MT1-MMP−/− cells do not invade 3-D type I collagen gels (2.2 mg/ml), as assessed by phase-contrast microscopy (bar, 50 μm), in H&E–stained cross sections (bar, 50 μm), or by TEM analysis (bar, 5 μm) after a 7-d culture period with VEGF–HGF in the presence of 20% FBS. Arrowheads and asterisk indicate the positions of invading MT1-MMP+/+ endothelial cells. (D) An acellular explant of type I/III collagen–rich human dermis stained with Sirius red is infiltrated by GFP-labeled control endothelial cells, but not by MT1-MMP– endothelial cells. The double-headed arrow marks the boundary of the explant tissue beneath the monolayer of seeded endothelial cells. Bar, 100 μm.
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fig5: A required role for MT1-MMP in neovessel formation, endothelial cell invasion, and collagenolytic activity. (A) Aortic vessel explants isolated from MT1-MMP– mice display defective capillary sprouting (middle) compared with wild-type tissue, after a 7-d culture period in type I collagen gels in the presence of VEGF–HGF and 20% FBS (left) or 5% autologous serum (not depicted). The co-culture of wild-type and MT1-MMP– aortic rings (left and right, respectively, of the right panel) does not affect the sprouting behavior of wild-type or MT1-MMP– explants. (B) Phalloidin-stained endothelial cells (green) circumscribe zones of collagen degradation generated by wild-type, but not MT1-MMP−/−, cells cultured atop a type I collagen film in the presence of VEGF–HGF in 20% FBS for 7 d. Bar, 10 μm. Insets show a low magnification image of multiple sites of collagenolysis relative to the complete absence of degradation observed when MT1-MMP– endothelial cells are cultured atop fluorescently labeled collagen gels (bar, 50 μm). (C) In contrast with wild-type endothelial cells, MT1-MMP−/− cells do not invade 3-D type I collagen gels (2.2 mg/ml), as assessed by phase-contrast microscopy (bar, 50 μm), in H&E–stained cross sections (bar, 50 μm), or by TEM analysis (bar, 5 μm) after a 7-d culture period with VEGF–HGF in the presence of 20% FBS. Arrowheads and asterisk indicate the positions of invading MT1-MMP+/+ endothelial cells. (D) An acellular explant of type I/III collagen–rich human dermis stained with Sirius red is infiltrated by GFP-labeled control endothelial cells, but not by MT1-MMP– endothelial cells. The double-headed arrow marks the boundary of the explant tissue beneath the monolayer of seeded endothelial cells. Bar, 100 μm.

Mentions: In contrast with wild-type explants, tissues isolated from MT1-MMP−/− mice are completely unable to generate neovessels during a 7-d culture period (Fig. 5 A). Co-cultures of MT1-MMP– explants with wild-type aorta rings demonstrate that soluble inhibitors of capillary formation are not released from the knockout tissues and that wild-type tissues do not generate soluble factors that are able to rescue the phenotype (Fig. 5 A). Similar, if not identical, results are obtained when explants of lung, myocardium, or skin are recovered from MT1-MMP– mice and tested ex vivo (unpublished data). Furthermore, although neovessel formation by control aortic explants resulted in the release of 6.2 ± 1.1 μg hydroxyproline, MT1-MMP−/− explants released only 1.2 ± 0.6 μg hydroxyproline in the course of a 7-d culture period. In the presence of TIMP-2, collagenolysis by wild-type and MT1-MMP−/− explants was inhibited completely (0.4 ± 0.3 μg and 0 ± 0 μg hydroxyproline released, respectively; n = 3). Though MT1-MMP has been posited to regulate cell function by activating latent TGFβ or generating denatured collagen products that mediate integrin signaling (Heissig et al., 2003), neither the addition of active TGFβ nor that of proteolyzed collagen affected the MT1-MMP−/− phenotype (unpublished data).


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)

A required role for MT1-MMP in neovessel formation, endothelial cell invasion, and collagenolytic activity. (A) Aortic vessel explants isolated from MT1-MMP– mice display defective capillary sprouting (middle) compared with wild-type tissue, after a 7-d culture period in type I collagen gels in the presence of VEGF–HGF and 20% FBS (left) or 5% autologous serum (not depicted). The co-culture of wild-type and MT1-MMP– aortic rings (left and right, respectively, of the right panel) does not affect the sprouting behavior of wild-type or MT1-MMP– explants. (B) Phalloidin-stained endothelial cells (green) circumscribe zones of collagen degradation generated by wild-type, but not MT1-MMP−/−, cells cultured atop a type I collagen film in the presence of VEGF–HGF in 20% FBS for 7 d. Bar, 10 μm. Insets show a low magnification image of multiple sites of collagenolysis relative to the complete absence of degradation observed when MT1-MMP– endothelial cells are cultured atop fluorescently labeled collagen gels (bar, 50 μm). (C) In contrast with wild-type endothelial cells, MT1-MMP−/− cells do not invade 3-D type I collagen gels (2.2 mg/ml), as assessed by phase-contrast microscopy (bar, 50 μm), in H&E–stained cross sections (bar, 50 μm), or by TEM analysis (bar, 5 μm) after a 7-d culture period with VEGF–HGF in the presence of 20% FBS. Arrowheads and asterisk indicate the positions of invading MT1-MMP+/+ endothelial cells. (D) An acellular explant of type I/III collagen–rich human dermis stained with Sirius red is infiltrated by GFP-labeled control endothelial cells, but not by MT1-MMP– endothelial cells. The double-headed arrow marks the boundary of the explant tissue beneath the monolayer of seeded endothelial cells. Bar, 100 μm.
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Related In: Results  -  Collection

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fig5: A required role for MT1-MMP in neovessel formation, endothelial cell invasion, and collagenolytic activity. (A) Aortic vessel explants isolated from MT1-MMP– mice display defective capillary sprouting (middle) compared with wild-type tissue, after a 7-d culture period in type I collagen gels in the presence of VEGF–HGF and 20% FBS (left) or 5% autologous serum (not depicted). The co-culture of wild-type and MT1-MMP– aortic rings (left and right, respectively, of the right panel) does not affect the sprouting behavior of wild-type or MT1-MMP– explants. (B) Phalloidin-stained endothelial cells (green) circumscribe zones of collagen degradation generated by wild-type, but not MT1-MMP−/−, cells cultured atop a type I collagen film in the presence of VEGF–HGF in 20% FBS for 7 d. Bar, 10 μm. Insets show a low magnification image of multiple sites of collagenolysis relative to the complete absence of degradation observed when MT1-MMP– endothelial cells are cultured atop fluorescently labeled collagen gels (bar, 50 μm). (C) In contrast with wild-type endothelial cells, MT1-MMP−/− cells do not invade 3-D type I collagen gels (2.2 mg/ml), as assessed by phase-contrast microscopy (bar, 50 μm), in H&E–stained cross sections (bar, 50 μm), or by TEM analysis (bar, 5 μm) after a 7-d culture period with VEGF–HGF in the presence of 20% FBS. Arrowheads and asterisk indicate the positions of invading MT1-MMP+/+ endothelial cells. (D) An acellular explant of type I/III collagen–rich human dermis stained with Sirius red is infiltrated by GFP-labeled control endothelial cells, but not by MT1-MMP– endothelial cells. The double-headed arrow marks the boundary of the explant tissue beneath the monolayer of seeded endothelial cells. Bar, 100 μm.
Mentions: In contrast with wild-type explants, tissues isolated from MT1-MMP−/− mice are completely unable to generate neovessels during a 7-d culture period (Fig. 5 A). Co-cultures of MT1-MMP– explants with wild-type aorta rings demonstrate that soluble inhibitors of capillary formation are not released from the knockout tissues and that wild-type tissues do not generate soluble factors that are able to rescue the phenotype (Fig. 5 A). Similar, if not identical, results are obtained when explants of lung, myocardium, or skin are recovered from MT1-MMP– mice and tested ex vivo (unpublished data). Furthermore, although neovessel formation by control aortic explants resulted in the release of 6.2 ± 1.1 μg hydroxyproline, MT1-MMP−/− explants released only 1.2 ± 0.6 μg hydroxyproline in the course of a 7-d culture period. In the presence of TIMP-2, collagenolysis by wild-type and MT1-MMP−/− explants was inhibited completely (0.4 ± 0.3 μg and 0 ± 0 μg hydroxyproline released, respectively; n = 3). Though MT1-MMP has been posited to regulate cell function by activating latent TGFβ or generating denatured collagen products that mediate integrin signaling (Heissig et al., 2003), neither the addition of active TGFβ nor that of proteolyzed collagen affected the MT1-MMP−/− phenotype (unpublished data).

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