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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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Retroviral reconstitution of the collagenolytic and type I collagen–invasive activity of MT1-MMP– endothelial cells. (A) Although MT1-MMP−/− endothelial cells transduced with a control cDNA (top, left) were unable to degrade a subjacent collagen film after a 7-d culture period with VEGF–HGF in the presence of 5% mouse serum, retroviral reconstitution of active MT1-MMP restores the collagenolytic activity of MT1-MMP−/− endothelial cells (top, middle). Cells transduced with a catalytically inactive MT1-MMPE/A mutant or a transmembrane-deleted, soluble form of MT1-MMP (MT1-MMPsol) do not display collagen degradative activity. Whereas the collagenolytic activity of MT1-MMP−/− endothelial cells could be rescued by expressing MT2-MMP, MT3-MMP–transduced cells did not display a collagenolytic phenotype. Bar, 50 μm. (B) Collagen-invasive activity of MT1-MMP−/− cells cultured atop a 3-D gel of type I collagen (2.2 mg/ml) for 7 d in the presence of VEGF–HGF and 20% FBS is rescued after transduction with MT1-MMP or MT2-MMP, but not EGFP, MT1-MMPsol, MT1-MMPE/A, or MT3-MMP. The inset for MT1-MMP shows the protein level of endogenous MT1-MMP in wild-type cells (lane 1), MT1-MMP– endothelial cells (lane 2), and MT1-MMP–transduced- cells (lane 3). The insets for MT2-MMP and MT3-MMP show the expression of MT2-MMP and MT3-MMP, respectively, in wild-type cells (lane 1), and MT1-MMP– endothelial cells after retroviral transduction with MT1-MMP, MT2-MMP, or MT3-MMP (lanes 2–4). (C) Immunofluorescent micrographs of 11-d-old CAM cross sections depict a dense interstitial matrix composed of types I and III collagen (top, left; bar, 50 μm). MT1-MMP+/+ or MT1-MMP−/− endothelial cells labeled with fluorescent microbeads (green) invaded 144 ± 55 μm and 53 ± 13 μm, respectively, into the CAM interstitium after a 3-d incubation (the CAM surface is marked by the black arrowheads to the right; top, middle and right, and bottom; bar, 100 μm). Retroviral gene transfer of GFP highlights the morphology of a group of invading MT1-MMP+/+ cells (MT1+/+; middle row, left and center; bar, 20 μm), which form tubular structures (left, arrows). The section of the micrograph bounded by the dashed square is enlarged (middle) and shows a group of elongated endothelial cells (arrowheads) forming a tubule with the nuclei highlighted by Hoechst staining. Morphology of GFP-labeled MT1-MMP−/− endothelial cells (MT1−/−) is shown (middle row, right). After retroviral gene transfer of MT1-MMP or MT2-MMP into MT1-MMP−/− endothelial cells, invasion increased to 147 ± 46 μm and 123 ± 34 μm, respectively. After retroviral gene transfer of MT1-MMP or MT2-MMP into MT1-MMP−/− endothelial cells, invasion increased to 147 ± 46 μm and 123 ± 34 μm, respectively (mean ± 1 SD, n = 3). The invasive activity of MT3-MMP–transduced cells was not affected relative to the MT1-MMP−/− cells (i.e., 47 ± 22 μm, n = 3).
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fig7: Retroviral reconstitution of the collagenolytic and type I collagen–invasive activity of MT1-MMP– endothelial cells. (A) Although MT1-MMP−/− endothelial cells transduced with a control cDNA (top, left) were unable to degrade a subjacent collagen film after a 7-d culture period with VEGF–HGF in the presence of 5% mouse serum, retroviral reconstitution of active MT1-MMP restores the collagenolytic activity of MT1-MMP−/− endothelial cells (top, middle). Cells transduced with a catalytically inactive MT1-MMPE/A mutant or a transmembrane-deleted, soluble form of MT1-MMP (MT1-MMPsol) do not display collagen degradative activity. Whereas the collagenolytic activity of MT1-MMP−/− endothelial cells could be rescued by expressing MT2-MMP, MT3-MMP–transduced cells did not display a collagenolytic phenotype. Bar, 50 μm. (B) Collagen-invasive activity of MT1-MMP−/− cells cultured atop a 3-D gel of type I collagen (2.2 mg/ml) for 7 d in the presence of VEGF–HGF and 20% FBS is rescued after transduction with MT1-MMP or MT2-MMP, but not EGFP, MT1-MMPsol, MT1-MMPE/A, or MT3-MMP. The inset for MT1-MMP shows the protein level of endogenous MT1-MMP in wild-type cells (lane 1), MT1-MMP– endothelial cells (lane 2), and MT1-MMP–transduced- cells (lane 3). The insets for MT2-MMP and MT3-MMP show the expression of MT2-MMP and MT3-MMP, respectively, in wild-type cells (lane 1), and MT1-MMP– endothelial cells after retroviral transduction with MT1-MMP, MT2-MMP, or MT3-MMP (lanes 2–4). (C) Immunofluorescent micrographs of 11-d-old CAM cross sections depict a dense interstitial matrix composed of types I and III collagen (top, left; bar, 50 μm). MT1-MMP+/+ or MT1-MMP−/− endothelial cells labeled with fluorescent microbeads (green) invaded 144 ± 55 μm and 53 ± 13 μm, respectively, into the CAM interstitium after a 3-d incubation (the CAM surface is marked by the black arrowheads to the right; top, middle and right, and bottom; bar, 100 μm). Retroviral gene transfer of GFP highlights the morphology of a group of invading MT1-MMP+/+ cells (MT1+/+; middle row, left and center; bar, 20 μm), which form tubular structures (left, arrows). The section of the micrograph bounded by the dashed square is enlarged (middle) and shows a group of elongated endothelial cells (arrowheads) forming a tubule with the nuclei highlighted by Hoechst staining. Morphology of GFP-labeled MT1-MMP−/− endothelial cells (MT1−/−) is shown (middle row, right). After retroviral gene transfer of MT1-MMP or MT2-MMP into MT1-MMP−/− endothelial cells, invasion increased to 147 ± 46 μm and 123 ± 34 μm, respectively. After retroviral gene transfer of MT1-MMP or MT2-MMP into MT1-MMP−/− endothelial cells, invasion increased to 147 ± 46 μm and 123 ± 34 μm, respectively (mean ± 1 SD, n = 3). The invasive activity of MT3-MMP–transduced cells was not affected relative to the MT1-MMP−/− cells (i.e., 47 ± 22 μm, n = 3).

Mentions: To define the structural features that underlie the ability of MT1-MMP to confer endothelial cells with collagen-degradative and -invasive activity, MT1-MMP– endothelial cells were transduced with wild-type MT1-MMP or MT1-MMP variants harboring either an inactivating point mutation in the catalytic domain or a deletion of the transmembrane domain (Hotary et al., 2003). Although full-length MT1-MMP bestowed MT1-MMP−/− endothelial cells with both collagenolytic and invasive activity, catalytically inactive MT1-MMP did not rescue the phenotype (Fig. 7, A and B). Similarly, transduction of MT1-MMP– cells with a catalytically active, but soluble, form of MT1-MMP failed to confer collagenolytic or invasive activity, presumably because the recipient cell loses its ability to focus proteolytic activity to the subjacent compartment (Fig. 7, A and B).


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)

Retroviral reconstitution of the collagenolytic and type I collagen–invasive activity of MT1-MMP– endothelial cells. (A) Although MT1-MMP−/− endothelial cells transduced with a control cDNA (top, left) were unable to degrade a subjacent collagen film after a 7-d culture period with VEGF–HGF in the presence of 5% mouse serum, retroviral reconstitution of active MT1-MMP restores the collagenolytic activity of MT1-MMP−/− endothelial cells (top, middle). Cells transduced with a catalytically inactive MT1-MMPE/A mutant or a transmembrane-deleted, soluble form of MT1-MMP (MT1-MMPsol) do not display collagen degradative activity. Whereas the collagenolytic activity of MT1-MMP−/− endothelial cells could be rescued by expressing MT2-MMP, MT3-MMP–transduced cells did not display a collagenolytic phenotype. Bar, 50 μm. (B) Collagen-invasive activity of MT1-MMP−/− cells cultured atop a 3-D gel of type I collagen (2.2 mg/ml) for 7 d in the presence of VEGF–HGF and 20% FBS is rescued after transduction with MT1-MMP or MT2-MMP, but not EGFP, MT1-MMPsol, MT1-MMPE/A, or MT3-MMP. The inset for MT1-MMP shows the protein level of endogenous MT1-MMP in wild-type cells (lane 1), MT1-MMP– endothelial cells (lane 2), and MT1-MMP–transduced- cells (lane 3). The insets for MT2-MMP and MT3-MMP show the expression of MT2-MMP and MT3-MMP, respectively, in wild-type cells (lane 1), and MT1-MMP– endothelial cells after retroviral transduction with MT1-MMP, MT2-MMP, or MT3-MMP (lanes 2–4). (C) Immunofluorescent micrographs of 11-d-old CAM cross sections depict a dense interstitial matrix composed of types I and III collagen (top, left; bar, 50 μm). MT1-MMP+/+ or MT1-MMP−/− endothelial cells labeled with fluorescent microbeads (green) invaded 144 ± 55 μm and 53 ± 13 μm, respectively, into the CAM interstitium after a 3-d incubation (the CAM surface is marked by the black arrowheads to the right; top, middle and right, and bottom; bar, 100 μm). Retroviral gene transfer of GFP highlights the morphology of a group of invading MT1-MMP+/+ cells (MT1+/+; middle row, left and center; bar, 20 μm), which form tubular structures (left, arrows). The section of the micrograph bounded by the dashed square is enlarged (middle) and shows a group of elongated endothelial cells (arrowheads) forming a tubule with the nuclei highlighted by Hoechst staining. Morphology of GFP-labeled MT1-MMP−/− endothelial cells (MT1−/−) is shown (middle row, right). After retroviral gene transfer of MT1-MMP or MT2-MMP into MT1-MMP−/− endothelial cells, invasion increased to 147 ± 46 μm and 123 ± 34 μm, respectively. After retroviral gene transfer of MT1-MMP or MT2-MMP into MT1-MMP−/− endothelial cells, invasion increased to 147 ± 46 μm and 123 ± 34 μm, respectively (mean ± 1 SD, n = 3). The invasive activity of MT3-MMP–transduced cells was not affected relative to the MT1-MMP−/− cells (i.e., 47 ± 22 μm, n = 3).
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fig7: Retroviral reconstitution of the collagenolytic and type I collagen–invasive activity of MT1-MMP– endothelial cells. (A) Although MT1-MMP−/− endothelial cells transduced with a control cDNA (top, left) were unable to degrade a subjacent collagen film after a 7-d culture period with VEGF–HGF in the presence of 5% mouse serum, retroviral reconstitution of active MT1-MMP restores the collagenolytic activity of MT1-MMP−/− endothelial cells (top, middle). Cells transduced with a catalytically inactive MT1-MMPE/A mutant or a transmembrane-deleted, soluble form of MT1-MMP (MT1-MMPsol) do not display collagen degradative activity. Whereas the collagenolytic activity of MT1-MMP−/− endothelial cells could be rescued by expressing MT2-MMP, MT3-MMP–transduced cells did not display a collagenolytic phenotype. Bar, 50 μm. (B) Collagen-invasive activity of MT1-MMP−/− cells cultured atop a 3-D gel of type I collagen (2.2 mg/ml) for 7 d in the presence of VEGF–HGF and 20% FBS is rescued after transduction with MT1-MMP or MT2-MMP, but not EGFP, MT1-MMPsol, MT1-MMPE/A, or MT3-MMP. The inset for MT1-MMP shows the protein level of endogenous MT1-MMP in wild-type cells (lane 1), MT1-MMP– endothelial cells (lane 2), and MT1-MMP–transduced- cells (lane 3). The insets for MT2-MMP and MT3-MMP show the expression of MT2-MMP and MT3-MMP, respectively, in wild-type cells (lane 1), and MT1-MMP– endothelial cells after retroviral transduction with MT1-MMP, MT2-MMP, or MT3-MMP (lanes 2–4). (C) Immunofluorescent micrographs of 11-d-old CAM cross sections depict a dense interstitial matrix composed of types I and III collagen (top, left; bar, 50 μm). MT1-MMP+/+ or MT1-MMP−/− endothelial cells labeled with fluorescent microbeads (green) invaded 144 ± 55 μm and 53 ± 13 μm, respectively, into the CAM interstitium after a 3-d incubation (the CAM surface is marked by the black arrowheads to the right; top, middle and right, and bottom; bar, 100 μm). Retroviral gene transfer of GFP highlights the morphology of a group of invading MT1-MMP+/+ cells (MT1+/+; middle row, left and center; bar, 20 μm), which form tubular structures (left, arrows). The section of the micrograph bounded by the dashed square is enlarged (middle) and shows a group of elongated endothelial cells (arrowheads) forming a tubule with the nuclei highlighted by Hoechst staining. Morphology of GFP-labeled MT1-MMP−/− endothelial cells (MT1−/−) is shown (middle row, right). After retroviral gene transfer of MT1-MMP or MT2-MMP into MT1-MMP−/− endothelial cells, invasion increased to 147 ± 46 μm and 123 ± 34 μm, respectively. After retroviral gene transfer of MT1-MMP or MT2-MMP into MT1-MMP−/− endothelial cells, invasion increased to 147 ± 46 μm and 123 ± 34 μm, respectively (mean ± 1 SD, n = 3). The invasive activity of MT3-MMP–transduced cells was not affected relative to the MT1-MMP−/− cells (i.e., 47 ± 22 μm, n = 3).
Mentions: To define the structural features that underlie the ability of MT1-MMP to confer endothelial cells with collagen-degradative and -invasive activity, MT1-MMP– endothelial cells were transduced with wild-type MT1-MMP or MT1-MMP variants harboring either an inactivating point mutation in the catalytic domain or a deletion of the transmembrane domain (Hotary et al., 2003). Although full-length MT1-MMP bestowed MT1-MMP−/− endothelial cells with both collagenolytic and invasive activity, catalytically inactive MT1-MMP did not rescue the phenotype (Fig. 7, A and B). Similarly, transduction of MT1-MMP– cells with a catalytically active, but soluble, form of MT1-MMP failed to confer collagenolytic or invasive activity, presumably because the recipient cell loses its ability to focus proteolytic activity to the subjacent compartment (Fig. 7, A and B).

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