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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 nonessential role of MMP-2 and MMP-9 in regulating the collagenolytic and collagen-invasive activities of microvascular endothelial cells. (A) Littermate control, MMP-2−/−, or MMP-9−/− endothelial cells focally degrade a subjacent film (∼100 μg) of fluorescently labeled type I collagen after a 7-d culture period in the presence of VEGF–HGF and 5% autologous wild-type or knockout sera. Areas of collagenolytic activity expressed by MMP-2+/+, MMP-2−/−, MMP-9+/+, and MMP-9−/− correspond precisely with sites subjacent to overlying endothelial cells as shown in merged images of MMP-2–, phalloidin-stained cells (top, right). Collagenolytic activity by control littermate and  cells is completely blocked by 3 μg/ml TIMP-2 (bottom, right). Bar, 50 μm. (B) Invasion of 3-D collagen gels (2.2 mg/ml) by microvascular endothelial cells isolated from wild-type (MMP-2+/+/C57BL6 and MMP-9+/+/129SvEv), MMP-2–, or MMP-9– mice are shown after a 5-d incubation period with VEGF–HGF in the presence of 5% autologous serum. Endothelial cell invasion was insensitive to 3 μg/ml TIMP-1, but inhibited completely by 3 μg/ml TIMP-2. Bar, 50 μm. (C) The invasive activity of isolated wild-type, MMP-2−/−, or MMP-9−/− endothelial cells was quantified after a 5-d incubation period in the absence or presence of BB-94, TIMP-1, TIMP-2, or Ala-TIMP-2 mutant. Results are shown as the mean number of invading cells per high powered field (hpf) in two experiments.
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fig4: A nonessential role of MMP-2 and MMP-9 in regulating the collagenolytic and collagen-invasive activities of microvascular endothelial cells. (A) Littermate control, MMP-2−/−, or MMP-9−/− endothelial cells focally degrade a subjacent film (∼100 μg) of fluorescently labeled type I collagen after a 7-d culture period in the presence of VEGF–HGF and 5% autologous wild-type or knockout sera. Areas of collagenolytic activity expressed by MMP-2+/+, MMP-2−/−, MMP-9+/+, and MMP-9−/− correspond precisely with sites subjacent to overlying endothelial cells as shown in merged images of MMP-2–, phalloidin-stained cells (top, right). Collagenolytic activity by control littermate and cells is completely blocked by 3 μg/ml TIMP-2 (bottom, right). Bar, 50 μm. (B) Invasion of 3-D collagen gels (2.2 mg/ml) by microvascular endothelial cells isolated from wild-type (MMP-2+/+/C57BL6 and MMP-9+/+/129SvEv), MMP-2–, or MMP-9– mice are shown after a 5-d incubation period with VEGF–HGF in the presence of 5% autologous serum. Endothelial cell invasion was insensitive to 3 μg/ml TIMP-1, but inhibited completely by 3 μg/ml TIMP-2. Bar, 50 μm. (C) The invasive activity of isolated wild-type, MMP-2−/−, or MMP-9−/− endothelial cells was quantified after a 5-d incubation period in the absence or presence of BB-94, TIMP-1, TIMP-2, or Ala-TIMP-2 mutant. Results are shown as the mean number of invading cells per high powered field (hpf) in two experiments.

Mentions: The tubulogenic response mounted by tissue explants involves multiple cell types (e.g., Nicosia and Madri, 1987) that might conceivably mask or compensate for an endothelial cell–specific defect in collagenolytic or invasive activity. Hence, microvascular endothelial cells were isolated from MMP-2 and MMP-9– mice, and their ability to degrade a subjacent bed of type I collagen fibrils or invade 3-D type I collagen gels was assessed. As shown in Fig. 4 A, wild-type endothelial cells stimulated with a VEGF–hepatocyte growth factor (HGF) mixture proteolyzed a subjacent film of rhodamine-labeled collagen in an area confined to the boundaries of the overlying cell by a process sensitive to either tissue inhibitor of MP (TIMP) 2 or BB-94 (not depicted). Of note, subjacent collagenolytic activity was not affected by targeting either MMP-2 or MMP-9 (Fig. 4 A). Furthermore, consistent with the collagen-degradative phenotypes displayed by either population of -endothelial cells, MMP-2 or MMP-9 knockout cells retained the wild-type capability to invade 3-D collagen gels (Fig. 4, B and C). Invasion, like collagen degradation, was also blocked completely by both BB-94 and wild-type TIMP-2, but was not affected by TIMP-1, a potent inhibitor of MMP-2 and MMP-9 (Hotary et al., 2003; Fig. 4 C). Although TIMP-2 can affect cell function independently of its ability to block MMP activity (Seo et al., 2003), endothelial cell invasion was not inhibited by an Ala–TIMP-2 mutant devoid of anti-MMP activity (Fig. 4 C).


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 nonessential role of MMP-2 and MMP-9 in regulating the collagenolytic and collagen-invasive activities of microvascular endothelial cells. (A) Littermate control, MMP-2−/−, or MMP-9−/− endothelial cells focally degrade a subjacent film (∼100 μg) of fluorescently labeled type I collagen after a 7-d culture period in the presence of VEGF–HGF and 5% autologous wild-type or knockout sera. Areas of collagenolytic activity expressed by MMP-2+/+, MMP-2−/−, MMP-9+/+, and MMP-9−/− correspond precisely with sites subjacent to overlying endothelial cells as shown in merged images of MMP-2–, phalloidin-stained cells (top, right). Collagenolytic activity by control littermate and  cells is completely blocked by 3 μg/ml TIMP-2 (bottom, right). Bar, 50 μm. (B) Invasion of 3-D collagen gels (2.2 mg/ml) by microvascular endothelial cells isolated from wild-type (MMP-2+/+/C57BL6 and MMP-9+/+/129SvEv), MMP-2–, or MMP-9– mice are shown after a 5-d incubation period with VEGF–HGF in the presence of 5% autologous serum. Endothelial cell invasion was insensitive to 3 μg/ml TIMP-1, but inhibited completely by 3 μg/ml TIMP-2. Bar, 50 μm. (C) The invasive activity of isolated wild-type, MMP-2−/−, or MMP-9−/− endothelial cells was quantified after a 5-d incubation period in the absence or presence of BB-94, TIMP-1, TIMP-2, or Ala-TIMP-2 mutant. Results are shown as the mean number of invading cells per high powered field (hpf) in two experiments.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2172577&req=5

fig4: A nonessential role of MMP-2 and MMP-9 in regulating the collagenolytic and collagen-invasive activities of microvascular endothelial cells. (A) Littermate control, MMP-2−/−, or MMP-9−/− endothelial cells focally degrade a subjacent film (∼100 μg) of fluorescently labeled type I collagen after a 7-d culture period in the presence of VEGF–HGF and 5% autologous wild-type or knockout sera. Areas of collagenolytic activity expressed by MMP-2+/+, MMP-2−/−, MMP-9+/+, and MMP-9−/− correspond precisely with sites subjacent to overlying endothelial cells as shown in merged images of MMP-2–, phalloidin-stained cells (top, right). Collagenolytic activity by control littermate and cells is completely blocked by 3 μg/ml TIMP-2 (bottom, right). Bar, 50 μm. (B) Invasion of 3-D collagen gels (2.2 mg/ml) by microvascular endothelial cells isolated from wild-type (MMP-2+/+/C57BL6 and MMP-9+/+/129SvEv), MMP-2–, or MMP-9– mice are shown after a 5-d incubation period with VEGF–HGF in the presence of 5% autologous serum. Endothelial cell invasion was insensitive to 3 μg/ml TIMP-1, but inhibited completely by 3 μg/ml TIMP-2. Bar, 50 μm. (C) The invasive activity of isolated wild-type, MMP-2−/−, or MMP-9−/− endothelial cells was quantified after a 5-d incubation period in the absence or presence of BB-94, TIMP-1, TIMP-2, or Ala-TIMP-2 mutant. Results are shown as the mean number of invading cells per high powered field (hpf) in two experiments.
Mentions: The tubulogenic response mounted by tissue explants involves multiple cell types (e.g., Nicosia and Madri, 1987) that might conceivably mask or compensate for an endothelial cell–specific defect in collagenolytic or invasive activity. Hence, microvascular endothelial cells were isolated from MMP-2 and MMP-9– mice, and their ability to degrade a subjacent bed of type I collagen fibrils or invade 3-D type I collagen gels was assessed. As shown in Fig. 4 A, wild-type endothelial cells stimulated with a VEGF–hepatocyte growth factor (HGF) mixture proteolyzed a subjacent film of rhodamine-labeled collagen in an area confined to the boundaries of the overlying cell by a process sensitive to either tissue inhibitor of MP (TIMP) 2 or BB-94 (not depicted). Of note, subjacent collagenolytic activity was not affected by targeting either MMP-2 or MMP-9 (Fig. 4 A). Furthermore, consistent with the collagen-degradative phenotypes displayed by either population of -endothelial cells, MMP-2 or MMP-9 knockout cells retained the wild-type capability to invade 3-D collagen gels (Fig. 4, B and C). Invasion, like collagen degradation, was also blocked completely by both BB-94 and wild-type TIMP-2, but was not affected by TIMP-1, a potent inhibitor of MMP-2 and MMP-9 (Hotary et al., 2003; Fig. 4 C). Although TIMP-2 can affect cell function independently of its ability to block MMP activity (Seo et al., 2003), endothelial cell invasion was not inhibited by an Ala–TIMP-2 mutant devoid of anti-MMP activity (Fig. 4 C).

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