Temporal expression and activation of matrix metalloproteinases-2, -9, and membrane type 1 - Matrix metalloproteinase following acute hindlimb ischemia

Bart E. Muhs, George Plitas, Yara Delgado, Ioana Ianus, Jason P. Shaw, Mark A. Adelman, Patrick Lamparello, Peter Shamamian, Paul Gagne

Research output: Contribution to journalArticle

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Abstract

Objective. Matrix metalloproteinase (MMP) activity is essential for remodeling of ischemic tissue. The murine hindlimb ischemia model exhibits tissue remodeling including revascularization in part due to angiogenesis. MMP-2 and -9 are type IV collagenases necessary for basement membrane degradation as a part of extracellular matrix remodeling and angiogenesis. Polymorphonuclear leukocytes (PMNs) contain MMP-9, and in the presence of membrane type 1 (MT1)-MMP, are able to activate proMMP-2 in vitro. Activation of MMP-2 and -9 may be essential in ischemic limbs both for tissue remodeling and revascularization via angiogenesis. We hypothesized that MMP-2 and -9 would be activated following acute hindlimb ischemia (HI), and this activation would be temporally related to PMN infiltration. Design of study. HI was achieved by unilateral femoral artery ligation in 20 FVB/N mice. Five mice underwent sham operation without hindlimb ischemia. Gastrocnemius muscle was harvested from both hindlimbs at 1, 3, 14, and 30 days following ligation and assayed for MMP-2, -9 (gelatin zymography), and MT1-MMP (Western blotting). MMP-2 and -9 expression and activation were analyzed by gelatin zymography and quantified by densitometry with NIH Image Analysis software. Neutrophils per high power field were counted. The results were expressed as a ratio of ischemic to nonischemic limbs and compared at each time point using ANOVA. Results. Zymographic analysis revealed a 212% increase in active MMP-2 3 days postligation (P < .05). Active MMP-9 reached its maximum level (800% over baseline) on postoperative day 3 and continued to be elevated on day 14 (737% over baseline) (P < .05). The increase in active MMP-2 and -9 levels paralleled PMN infiltration that also peaked 3 days postligation (1184% over baseline) (P < .05). PMN count, MMP-2, and -9 all returned to baseline levels by postoperative Day 30. MT1-MMP was present in tissue samples from all time points as confirmed by Western blot. Conclusions. Limb ischemia causes an early activation of MMP-2 and -9 in temporal relation to PMN infiltration. HI may prime PMNs, leading to their sequestration in ischemic tissue. Primed PMNs, along with constitutively expressed MT1-MMP, may activate MMPs-2 and -9 and enable tissue remodeling essential for limb revascularization and angiogenesis.

Original languageEnglish (US)
Pages (from-to)8-15
Number of pages8
JournalJournal of Surgical Research
Volume111
Issue number1
DOIs
StatePublished - May 1 2003
Externally publishedYes

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Matrix Metalloproteinase 14
Matrix Metalloproteinase 2
Matrix Metalloproteinase 9
Hindlimb
Ischemia
Extremities
Gelatin
Matrix Metalloproteinases
Ligation
Neutrophils
Western Blotting
Matrix Metalloproteinase 3
Densitometry
Collagenases
Femoral Artery
Basement Membrane
Extracellular Matrix
Analysis of Variance
Skeletal Muscle
Software

Keywords

  • Acute ischemia
  • Hindlimb
  • Matrix metalloproteinase
  • MMP
  • MMP-2
  • MMP-9
  • MT1-MMP

ASJC Scopus subject areas

  • Surgery

Cite this

Temporal expression and activation of matrix metalloproteinases-2, -9, and membrane type 1 - Matrix metalloproteinase following acute hindlimb ischemia. / Muhs, Bart E.; Plitas, George; Delgado, Yara; Ianus, Ioana; Shaw, Jason P.; Adelman, Mark A.; Lamparello, Patrick; Shamamian, Peter; Gagne, Paul.

In: Journal of Surgical Research, Vol. 111, No. 1, 01.05.2003, p. 8-15.

Research output: Contribution to journalArticle

Muhs, Bart E. ; Plitas, George ; Delgado, Yara ; Ianus, Ioana ; Shaw, Jason P. ; Adelman, Mark A. ; Lamparello, Patrick ; Shamamian, Peter ; Gagne, Paul. / Temporal expression and activation of matrix metalloproteinases-2, -9, and membrane type 1 - Matrix metalloproteinase following acute hindlimb ischemia. In: Journal of Surgical Research. 2003 ; Vol. 111, No. 1. pp. 8-15.
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title = "Temporal expression and activation of matrix metalloproteinases-2, -9, and membrane type 1 - Matrix metalloproteinase following acute hindlimb ischemia",
abstract = "Objective. Matrix metalloproteinase (MMP) activity is essential for remodeling of ischemic tissue. The murine hindlimb ischemia model exhibits tissue remodeling including revascularization in part due to angiogenesis. MMP-2 and -9 are type IV collagenases necessary for basement membrane degradation as a part of extracellular matrix remodeling and angiogenesis. Polymorphonuclear leukocytes (PMNs) contain MMP-9, and in the presence of membrane type 1 (MT1)-MMP, are able to activate proMMP-2 in vitro. Activation of MMP-2 and -9 may be essential in ischemic limbs both for tissue remodeling and revascularization via angiogenesis. We hypothesized that MMP-2 and -9 would be activated following acute hindlimb ischemia (HI), and this activation would be temporally related to PMN infiltration. Design of study. HI was achieved by unilateral femoral artery ligation in 20 FVB/N mice. Five mice underwent sham operation without hindlimb ischemia. Gastrocnemius muscle was harvested from both hindlimbs at 1, 3, 14, and 30 days following ligation and assayed for MMP-2, -9 (gelatin zymography), and MT1-MMP (Western blotting). MMP-2 and -9 expression and activation were analyzed by gelatin zymography and quantified by densitometry with NIH Image Analysis software. Neutrophils per high power field were counted. The results were expressed as a ratio of ischemic to nonischemic limbs and compared at each time point using ANOVA. Results. Zymographic analysis revealed a 212{\%} increase in active MMP-2 3 days postligation (P < .05). Active MMP-9 reached its maximum level (800{\%} over baseline) on postoperative day 3 and continued to be elevated on day 14 (737{\%} over baseline) (P < .05). The increase in active MMP-2 and -9 levels paralleled PMN infiltration that also peaked 3 days postligation (1184{\%} over baseline) (P < .05). PMN count, MMP-2, and -9 all returned to baseline levels by postoperative Day 30. MT1-MMP was present in tissue samples from all time points as confirmed by Western blot. Conclusions. Limb ischemia causes an early activation of MMP-2 and -9 in temporal relation to PMN infiltration. HI may prime PMNs, leading to their sequestration in ischemic tissue. Primed PMNs, along with constitutively expressed MT1-MMP, may activate MMPs-2 and -9 and enable tissue remodeling essential for limb revascularization and angiogenesis.",
keywords = "Acute ischemia, Hindlimb, Matrix metalloproteinase, MMP, MMP-2, MMP-9, MT1-MMP",
author = "Muhs, {Bart E.} and George Plitas and Yara Delgado and Ioana Ianus and Shaw, {Jason P.} and Adelman, {Mark A.} and Patrick Lamparello and Peter Shamamian and Paul Gagne",
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T1 - Temporal expression and activation of matrix metalloproteinases-2, -9, and membrane type 1 - Matrix metalloproteinase following acute hindlimb ischemia

AU - Muhs, Bart E.

AU - Plitas, George

AU - Delgado, Yara

AU - Ianus, Ioana

AU - Shaw, Jason P.

AU - Adelman, Mark A.

AU - Lamparello, Patrick

AU - Shamamian, Peter

AU - Gagne, Paul

PY - 2003/5/1

Y1 - 2003/5/1

N2 - Objective. Matrix metalloproteinase (MMP) activity is essential for remodeling of ischemic tissue. The murine hindlimb ischemia model exhibits tissue remodeling including revascularization in part due to angiogenesis. MMP-2 and -9 are type IV collagenases necessary for basement membrane degradation as a part of extracellular matrix remodeling and angiogenesis. Polymorphonuclear leukocytes (PMNs) contain MMP-9, and in the presence of membrane type 1 (MT1)-MMP, are able to activate proMMP-2 in vitro. Activation of MMP-2 and -9 may be essential in ischemic limbs both for tissue remodeling and revascularization via angiogenesis. We hypothesized that MMP-2 and -9 would be activated following acute hindlimb ischemia (HI), and this activation would be temporally related to PMN infiltration. Design of study. HI was achieved by unilateral femoral artery ligation in 20 FVB/N mice. Five mice underwent sham operation without hindlimb ischemia. Gastrocnemius muscle was harvested from both hindlimbs at 1, 3, 14, and 30 days following ligation and assayed for MMP-2, -9 (gelatin zymography), and MT1-MMP (Western blotting). MMP-2 and -9 expression and activation were analyzed by gelatin zymography and quantified by densitometry with NIH Image Analysis software. Neutrophils per high power field were counted. The results were expressed as a ratio of ischemic to nonischemic limbs and compared at each time point using ANOVA. Results. Zymographic analysis revealed a 212% increase in active MMP-2 3 days postligation (P < .05). Active MMP-9 reached its maximum level (800% over baseline) on postoperative day 3 and continued to be elevated on day 14 (737% over baseline) (P < .05). The increase in active MMP-2 and -9 levels paralleled PMN infiltration that also peaked 3 days postligation (1184% over baseline) (P < .05). PMN count, MMP-2, and -9 all returned to baseline levels by postoperative Day 30. MT1-MMP was present in tissue samples from all time points as confirmed by Western blot. Conclusions. Limb ischemia causes an early activation of MMP-2 and -9 in temporal relation to PMN infiltration. HI may prime PMNs, leading to their sequestration in ischemic tissue. Primed PMNs, along with constitutively expressed MT1-MMP, may activate MMPs-2 and -9 and enable tissue remodeling essential for limb revascularization and angiogenesis.

AB - Objective. Matrix metalloproteinase (MMP) activity is essential for remodeling of ischemic tissue. The murine hindlimb ischemia model exhibits tissue remodeling including revascularization in part due to angiogenesis. MMP-2 and -9 are type IV collagenases necessary for basement membrane degradation as a part of extracellular matrix remodeling and angiogenesis. Polymorphonuclear leukocytes (PMNs) contain MMP-9, and in the presence of membrane type 1 (MT1)-MMP, are able to activate proMMP-2 in vitro. Activation of MMP-2 and -9 may be essential in ischemic limbs both for tissue remodeling and revascularization via angiogenesis. We hypothesized that MMP-2 and -9 would be activated following acute hindlimb ischemia (HI), and this activation would be temporally related to PMN infiltration. Design of study. HI was achieved by unilateral femoral artery ligation in 20 FVB/N mice. Five mice underwent sham operation without hindlimb ischemia. Gastrocnemius muscle was harvested from both hindlimbs at 1, 3, 14, and 30 days following ligation and assayed for MMP-2, -9 (gelatin zymography), and MT1-MMP (Western blotting). MMP-2 and -9 expression and activation were analyzed by gelatin zymography and quantified by densitometry with NIH Image Analysis software. Neutrophils per high power field were counted. The results were expressed as a ratio of ischemic to nonischemic limbs and compared at each time point using ANOVA. Results. Zymographic analysis revealed a 212% increase in active MMP-2 3 days postligation (P < .05). Active MMP-9 reached its maximum level (800% over baseline) on postoperative day 3 and continued to be elevated on day 14 (737% over baseline) (P < .05). The increase in active MMP-2 and -9 levels paralleled PMN infiltration that also peaked 3 days postligation (1184% over baseline) (P < .05). PMN count, MMP-2, and -9 all returned to baseline levels by postoperative Day 30. MT1-MMP was present in tissue samples from all time points as confirmed by Western blot. Conclusions. Limb ischemia causes an early activation of MMP-2 and -9 in temporal relation to PMN infiltration. HI may prime PMNs, leading to their sequestration in ischemic tissue. Primed PMNs, along with constitutively expressed MT1-MMP, may activate MMPs-2 and -9 and enable tissue remodeling essential for limb revascularization and angiogenesis.

KW - Acute ischemia

KW - Hindlimb

KW - Matrix metalloproteinase

KW - MMP

KW - MMP-2

KW - MMP-9

KW - MT1-MMP

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