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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 hypoxia on cardiac myofibroblast invasion, migration and proliferation. Invasion assays were performed over a 48 h period in normoxia or hypoxia. Membranes were fixed, stained and cell invasion quantified by counting 10 high power (×400) fields per membrane. (A) Representative fields in normoxic and hypoxic culture. Scale bar = 100 μm. (B) Quantification of invaded cells. Data expressed as average cell number per field. ⁎P < 0.05 (n = 7). (C) For migration studies, cells were grown to confluence before creating a uniform scratch wound and incubating in collagen and normoxia/hypoxia for a further 48 h. Representative image of wound assay after 48 h, shows cell migration into denuded wound area in normoxia and hypoxia (original magnification ×40). (D). Quantification of migrated cells. Data expressed as number of cells migrated to or further than 500 μm per mm wound width. NS = not significant (n = 5). (E). Co-localisation of α-SMA (red) and vimentin (green) following 48 h of culture as seen by immunofluorescence microscopy. Nuclei stained with DAPI (blue). Scale bar = 50 μm. (F). 7-day proliferation profile of cells cultured in normoxia (solid line) and hypoxia (hashed line) in 0.5% and 10% FCS.
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fig4: Effect of hypoxia on cardiac myofibroblast invasion, migration and proliferation. Invasion assays were performed over a 48 h period in normoxia or hypoxia. Membranes were fixed, stained and cell invasion quantified by counting 10 high power (×400) fields per membrane. (A) Representative fields in normoxic and hypoxic culture. Scale bar = 100 μm. (B) Quantification of invaded cells. Data expressed as average cell number per field. ⁎P < 0.05 (n = 7). (C) For migration studies, cells were grown to confluence before creating a uniform scratch wound and incubating in collagen and normoxia/hypoxia for a further 48 h. Representative image of wound assay after 48 h, shows cell migration into denuded wound area in normoxia and hypoxia (original magnification ×40). (D). Quantification of migrated cells. Data expressed as number of cells migrated to or further than 500 μm per mm wound width. NS = not significant (n = 5). (E). Co-localisation of α-SMA (red) and vimentin (green) following 48 h of culture as seen by immunofluorescence microscopy. Nuclei stained with DAPI (blue). Scale bar = 50 μm. (F). 7-day proliferation profile of cells cultured in normoxia (solid line) and hypoxia (hashed line) in 0.5% and 10% FCS.

Mentions: Myofibroblasts cultured in the presence of Type I soluble collagen under normoxic conditions showed significant activation of MMP-2 compared with cells cultured in the absence of collagen (Fig. 1A), as evidenced by the appearance of a lower molecular weight zymogen band (62 kDa) in addition to the 72 kDa proprotein. There were no differences in secreted levels of total MMP-2 between cells cultured in the absence or presence of collagen (data not shown). After exposure to chronic hypoxia (1% O2, 48 h) collagen-induced MMP-2 activation was significantly and consistently attenuated by ∼ 50% (Figs. 1A, B). This was not the result of cell death in hypoxia, since cell numbers were similar over 7 days (Fig. 4F), confirming that differences in MMP-2 activation were not attributable to variations in cell number. We repeated these experiments using the human fibrosarcoma cell line HT-1080 in which hypoxic regulation of MMP-2 secretion and activation has previously been characterised [16]. In marked contrast to the myofibroblasts, but in agreement with this previous report, HT-1080 cells exhibited a significant increase in MMP-2 activation (∼ 200%) compared with normoxic controls (Figs. 1C, D). In further agreement with this same report, an intermediate band of MMP-2 activation (∼ 68 kDa) was observed in HT-1080 media (Fig. 1C). Thus, the effects of hypoxia observed in human cardiac myofibroblasts were markedly different from those seen in HT-1080 cells.


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 hypoxia on cardiac myofibroblast invasion, migration and proliferation. Invasion assays were performed over a 48 h period in normoxia or hypoxia. Membranes were fixed, stained and cell invasion quantified by counting 10 high power (×400) fields per membrane. (A) Representative fields in normoxic and hypoxic culture. Scale bar = 100 μm. (B) Quantification of invaded cells. Data expressed as average cell number per field. ⁎P < 0.05 (n = 7). (C) For migration studies, cells were grown to confluence before creating a uniform scratch wound and incubating in collagen and normoxia/hypoxia for a further 48 h. Representative image of wound assay after 48 h, shows cell migration into denuded wound area in normoxia and hypoxia (original magnification ×40). (D). Quantification of migrated cells. Data expressed as number of cells migrated to or further than 500 μm per mm wound width. NS = not significant (n = 5). (E). Co-localisation of α-SMA (red) and vimentin (green) following 48 h of culture as seen by immunofluorescence microscopy. Nuclei stained with DAPI (blue). Scale bar = 50 μm. (F). 7-day proliferation profile of cells cultured in normoxia (solid line) and hypoxia (hashed line) in 0.5% and 10% FCS.
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fig4: Effect of hypoxia on cardiac myofibroblast invasion, migration and proliferation. Invasion assays were performed over a 48 h period in normoxia or hypoxia. Membranes were fixed, stained and cell invasion quantified by counting 10 high power (×400) fields per membrane. (A) Representative fields in normoxic and hypoxic culture. Scale bar = 100 μm. (B) Quantification of invaded cells. Data expressed as average cell number per field. ⁎P < 0.05 (n = 7). (C) For migration studies, cells were grown to confluence before creating a uniform scratch wound and incubating in collagen and normoxia/hypoxia for a further 48 h. Representative image of wound assay after 48 h, shows cell migration into denuded wound area in normoxia and hypoxia (original magnification ×40). (D). Quantification of migrated cells. Data expressed as number of cells migrated to or further than 500 μm per mm wound width. NS = not significant (n = 5). (E). Co-localisation of α-SMA (red) and vimentin (green) following 48 h of culture as seen by immunofluorescence microscopy. Nuclei stained with DAPI (blue). Scale bar = 50 μm. (F). 7-day proliferation profile of cells cultured in normoxia (solid line) and hypoxia (hashed line) in 0.5% and 10% FCS.
Mentions: Myofibroblasts cultured in the presence of Type I soluble collagen under normoxic conditions showed significant activation of MMP-2 compared with cells cultured in the absence of collagen (Fig. 1A), as evidenced by the appearance of a lower molecular weight zymogen band (62 kDa) in addition to the 72 kDa proprotein. There were no differences in secreted levels of total MMP-2 between cells cultured in the absence or presence of collagen (data not shown). After exposure to chronic hypoxia (1% O2, 48 h) collagen-induced MMP-2 activation was significantly and consistently attenuated by ∼ 50% (Figs. 1A, B). This was not the result of cell death in hypoxia, since cell numbers were similar over 7 days (Fig. 4F), confirming that differences in MMP-2 activation were not attributable to variations in cell number. We repeated these experiments using the human fibrosarcoma cell line HT-1080 in which hypoxic regulation of MMP-2 secretion and activation has previously been characterised [16]. In marked contrast to the myofibroblasts, but in agreement with this previous report, HT-1080 cells exhibited a significant increase in MMP-2 activation (∼ 200%) compared with normoxic controls (Figs. 1C, D). In further agreement with this same report, an intermediate band of MMP-2 activation (∼ 68 kDa) was observed in HT-1080 media (Fig. 1C). Thus, the effects of hypoxia observed in human cardiac myofibroblasts were markedly different from those seen in HT-1080 cells.

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