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Chronic hypoxia inhibits MMP-2 activation and cellular invasion in human cardiac myofibroblasts.

Riches K, Morley ME, Turner NA, O'Regan DJ, Ball SG, Peers C, Porter KE - J. Mol. Cell. Cardiol. (2009)

Bottom Line: Reduced membrane expression of MT1-MMP (P<0.05) was responsible for the hypoxic reduction of MMP-2 activation, with no change in either total MMP-2 or TIMP-2.In conclusion, hypoxia reduces MMP-2 activation and subsequent invasion of human cardiac myofibroblasts by reducing membrane expression of MT1-MMP and may delay healing after MI.Regulation of these MMPs remains an attractive target for therapeutic intervention.

View Article: PubMed Central - PubMed

Affiliation: Multidisciplinary Cardiovascular Research Centre (MCRC), University of Leeds, Leeds LS2 9JT, UK.

ABSTRACT
Cardiac myofibroblasts are pivotal to adaptive remodelling after myocardial infarction (MI). These normally quiescent cells invade and proliferate as a wound healing response, facilitated by activation of matrix metalloproteinases, particularly MMP-2. Following MI these reparative events occur under chronically hypoxic conditions yet the mechanisms by which hypoxia might modulate MMP-2 activation and cardiac myofibroblast invasion have not been investigated. Human cardiac myofibroblasts cultured in collagen-supplemented medium were exposed to normoxia (20% O(2)) or hypoxia (1% O(2)) for up to 48 h. Secreted levels of total and active MMP-2 were quantified using gelatin zymography, TIMP-2 and membrane-associated MT1-MMP were quantified with ELISA, whole cell MT1-MMP by immunoblotting and immunocytochemistry and MT1-MMP mRNA with real-time RT-PCR. Cellular invasion was assessed in modified Boyden chambers and migration by scratch wound assay. In the human cardiac myofibroblast, MT1-MMP was central to MMP-2 activation and activated MMP-2 necessary for invasion, confirmed by gene silencing. MMP-2 activation was substantially attenuated by hypoxia (P<0.001), paralleled by inhibition of myofibroblast invasion (P<0.05). In contrast, migration was independent of either MT1-MMP or MMP-2. Reduced membrane expression of MT1-MMP (P<0.05) was responsible for the hypoxic reduction of MMP-2 activation, with no change in either total MMP-2 or TIMP-2. In conclusion, hypoxia reduces MMP-2 activation and subsequent invasion of human cardiac myofibroblasts by reducing membrane expression of MT1-MMP and may delay healing after MI. Regulation of these MMPs remains an attractive target for therapeutic intervention.

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Effect of MMP-2 and MT1-MMP gene silencing on MMP-2 secretion and activation, and myofibroblast function. For MMP quantification, mock-transfected and siRNA-transfected cardiac myofibroblasts were cultured in collagen for 48 h. Functional assays were performed in parallel using mock-transfected and siRNA-transfected cardiac myofibroblasts. (A) Representative zymography of conditioned media collected at 48 h. (B) Densitometric analysis of MMP-2, NS = not significant, ⁎⁎P < 0.01 (n = 5). (C) Membrane MT1-MMP expression determined by ELISA. ⁎⁎⁎P < 0.01 (n = 5). (D) Densitometric quantification of active MMP-2 relative to mock-transfected cells. ⁎⁎P < 0.01 (n = 5). (E) Quantification of invaded cells, ⁎⁎P < 0.01, ⁎⁎⁎P < 0.001, (n = 6). (F) Quantification of migrated cells. NS = not significant (n = 6).
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fig5: Effect of MMP-2 and MT1-MMP gene silencing on MMP-2 secretion and activation, and myofibroblast function. For MMP quantification, mock-transfected and siRNA-transfected cardiac myofibroblasts were cultured in collagen for 48 h. Functional assays were performed in parallel using mock-transfected and siRNA-transfected cardiac myofibroblasts. (A) Representative zymography of conditioned media collected at 48 h. (B) Densitometric analysis of MMP-2, NS = not significant, ⁎⁎P < 0.01 (n = 5). (C) Membrane MT1-MMP expression determined by ELISA. ⁎⁎⁎P < 0.01 (n = 5). (D) Densitometric quantification of active MMP-2 relative to mock-transfected cells. ⁎⁎P < 0.01 (n = 5). (E) Quantification of invaded cells, ⁎⁎P < 0.01, ⁎⁎⁎P < 0.001, (n = 6). (F) Quantification of migrated cells. NS = not significant (n = 6).

Mentions: Since we had demonstrated that hypoxia reduced invasion (but not migration), and also reduced both MT1-MMP protein levels together with MMP-2 activity, we sought to establish whether these effects were sufficient to inhibit myofibroblast invasion. Myofibroblasts transfected with siRNA targeting MT1-MMP or MMP-2 were cultured in collagen for 48 h and supernatants subjected to zymography. Total MMP-2 was selectively reduced by ∼ 50% with MMP-2 gene silencing, but was unaffected by MT1-MMP siRNA (Figs. 5A, B). Cells transfected with MT1-MMP siRNA exhibited a ∼ 50% decrease in membrane MT1-MMP expression (Fig. 5C). Importantly, selective silencing of either MT1-MMP or MMP-2 resulted in a significant reduction in active MMP-2 levels (Fig. 5D), to a level comparable with that observed in myofibroblasts during hypoxic culture.


Chronic hypoxia inhibits MMP-2 activation and cellular invasion in human cardiac myofibroblasts.

Riches K, Morley ME, Turner NA, O'Regan DJ, Ball SG, Peers C, Porter KE - J. Mol. Cell. Cardiol. (2009)

Effect of MMP-2 and MT1-MMP gene silencing on MMP-2 secretion and activation, and myofibroblast function. For MMP quantification, mock-transfected and siRNA-transfected cardiac myofibroblasts were cultured in collagen for 48 h. Functional assays were performed in parallel using mock-transfected and siRNA-transfected cardiac myofibroblasts. (A) Representative zymography of conditioned media collected at 48 h. (B) Densitometric analysis of MMP-2, NS = not significant, ⁎⁎P < 0.01 (n = 5). (C) Membrane MT1-MMP expression determined by ELISA. ⁎⁎⁎P < 0.01 (n = 5). (D) Densitometric quantification of active MMP-2 relative to mock-transfected cells. ⁎⁎P < 0.01 (n = 5). (E) Quantification of invaded cells, ⁎⁎P < 0.01, ⁎⁎⁎P < 0.001, (n = 6). (F) Quantification of migrated cells. NS = not significant (n = 6).
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Show All Figures
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fig5: Effect of MMP-2 and MT1-MMP gene silencing on MMP-2 secretion and activation, and myofibroblast function. For MMP quantification, mock-transfected and siRNA-transfected cardiac myofibroblasts were cultured in collagen for 48 h. Functional assays were performed in parallel using mock-transfected and siRNA-transfected cardiac myofibroblasts. (A) Representative zymography of conditioned media collected at 48 h. (B) Densitometric analysis of MMP-2, NS = not significant, ⁎⁎P < 0.01 (n = 5). (C) Membrane MT1-MMP expression determined by ELISA. ⁎⁎⁎P < 0.01 (n = 5). (D) Densitometric quantification of active MMP-2 relative to mock-transfected cells. ⁎⁎P < 0.01 (n = 5). (E) Quantification of invaded cells, ⁎⁎P < 0.01, ⁎⁎⁎P < 0.001, (n = 6). (F) Quantification of migrated cells. NS = not significant (n = 6).
Mentions: Since we had demonstrated that hypoxia reduced invasion (but not migration), and also reduced both MT1-MMP protein levels together with MMP-2 activity, we sought to establish whether these effects were sufficient to inhibit myofibroblast invasion. Myofibroblasts transfected with siRNA targeting MT1-MMP or MMP-2 were cultured in collagen for 48 h and supernatants subjected to zymography. Total MMP-2 was selectively reduced by ∼ 50% with MMP-2 gene silencing, but was unaffected by MT1-MMP siRNA (Figs. 5A, B). Cells transfected with MT1-MMP siRNA exhibited a ∼ 50% decrease in membrane MT1-MMP expression (Fig. 5C). Importantly, selective silencing of either MT1-MMP or MMP-2 resulted in a significant reduction in active MMP-2 levels (Fig. 5D), to a level comparable with that observed in myofibroblasts during hypoxic culture.

Bottom Line: Reduced membrane expression of MT1-MMP (P<0.05) was responsible for the hypoxic reduction of MMP-2 activation, with no change in either total MMP-2 or TIMP-2.In conclusion, hypoxia reduces MMP-2 activation and subsequent invasion of human cardiac myofibroblasts by reducing membrane expression of MT1-MMP and may delay healing after MI.Regulation of these MMPs remains an attractive target for therapeutic intervention.

View Article: PubMed Central - PubMed

Affiliation: Multidisciplinary Cardiovascular Research Centre (MCRC), University of Leeds, Leeds LS2 9JT, UK.

ABSTRACT
Cardiac myofibroblasts are pivotal to adaptive remodelling after myocardial infarction (MI). These normally quiescent cells invade and proliferate as a wound healing response, facilitated by activation of matrix metalloproteinases, particularly MMP-2. Following MI these reparative events occur under chronically hypoxic conditions yet the mechanisms by which hypoxia might modulate MMP-2 activation and cardiac myofibroblast invasion have not been investigated. Human cardiac myofibroblasts cultured in collagen-supplemented medium were exposed to normoxia (20% O(2)) or hypoxia (1% O(2)) for up to 48 h. Secreted levels of total and active MMP-2 were quantified using gelatin zymography, TIMP-2 and membrane-associated MT1-MMP were quantified with ELISA, whole cell MT1-MMP by immunoblotting and immunocytochemistry and MT1-MMP mRNA with real-time RT-PCR. Cellular invasion was assessed in modified Boyden chambers and migration by scratch wound assay. In the human cardiac myofibroblast, MT1-MMP was central to MMP-2 activation and activated MMP-2 necessary for invasion, confirmed by gene silencing. MMP-2 activation was substantially attenuated by hypoxia (P<0.001), paralleled by inhibition of myofibroblast invasion (P<0.05). In contrast, migration was independent of either MT1-MMP or MMP-2. Reduced membrane expression of MT1-MMP (P<0.05) was responsible for the hypoxic reduction of MMP-2 activation, with no change in either total MMP-2 or TIMP-2. In conclusion, hypoxia reduces MMP-2 activation and subsequent invasion of human cardiac myofibroblasts by reducing membrane expression of MT1-MMP and may delay healing after MI. Regulation of these MMPs remains an attractive target for therapeutic intervention.

Show MeSH
Related in: MedlinePlus