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Absence of SPARC results in increased cardiac rupture and dysfunction after acute myocardial infarction.

Schellings MW, Vanhoutte D, Swinnen M, Cleutjens JP, Debets J, van Leeuwen RE, d'Hooge J, Van de Werf F, Carmeliet P, Pinto YM, Sage EH, Heymans S - J. Exp. Med. (2008)

Bottom Line: The matricellular protein SPARC (secreted protein, acidic and rich in cysteine, also known as osteonectin) mediates cell-matrix interactions during wound healing and regulates the production and/or assembly of the extracellular matrix (ECM).These findings indicate that local production of SPARC is essential for maintenance of the integrity of cardiac ECM after MI.The protective effects of SPARC emphasize the potential therapeutic applications of this protein to prevent cardiac dilatation and dysfunction after MI.

View Article: PubMed Central - PubMed

Affiliation: Center for Heart Failure Research, Cardiovascular Research Institute Maastricht, University Hospital Maastricht, 6229 HX Maastricht, The Netherlands.

ABSTRACT
The matricellular protein SPARC (secreted protein, acidic and rich in cysteine, also known as osteonectin) mediates cell-matrix interactions during wound healing and regulates the production and/or assembly of the extracellular matrix (ECM). This study investigated whether SPARC functions in infarct healing and ECM maturation after myocardial infarction (MI). In comparison with wild-type (WT) mice, animals with a targeted inactivation of SPARC exhibited a fourfold increase in mortality that resulted from an increased incidence of cardiac rupture and failure after MI. SPARC- infarcts had a disorganized granulation tissue and immature collagenous ECM. In contrast, adenoviral overexpression of SPARC in WT mice improved the collagen maturation and prevented cardiac dilatation and dysfunction after MI. In cardiac fibroblasts in vitro, reduction of SPARC by short hairpin RNA attenuated transforming growth factor beta (TGF)-mediated increase of Smad2 phosphorylation, whereas addition of recombinant SPARC increased Smad2 phosphorylation concordant with increased Smad2 phosphorylation in SPARC-treated mice. Importantly, infusion of TGF-beta rescued cardiac rupture in SPARC- mice but did not significantly alter infarct healing in WT mice. These findings indicate that local production of SPARC is essential for maintenance of the integrity of cardiac ECM after MI. The protective effects of SPARC emphasize the potential therapeutic applications of this protein to prevent cardiac dilatation and dysfunction after MI.

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SPARC and TGF-β cooperate during infarct healing. (A) Representative Western blot showing increased phosphorylated and total Smad2 levels in the infarcts of AdSPARC-treated mice (n = 9; *, P < 0.05). (B) Representative Western blot showing that addition of recombinant mouse SPARC activated Smad2 signaling and augmented the Smad2-activating potential of TGF-β (n = 6; *, P < 0.05). (C) The top blot is a representative Western blot indicating that shRNA against SPARC resulted in an 80% decrease in levels of SPARC protein. The blots are representative Western blots showing that Smad2 phosphorylation in shRNA-treated cardiac fibroblasts was significantly decreased at baseline levels (left) and after 15 min of stimulation by 1 ng/ml TGF-β (right; n = 5 per group; *, P < 0.05). (D) Survival curve showing that infusion of TGF-β protected against cardiac rupture after MI in male SPARC- mice (1 out of 7 mice) in comparison with saline-treated male SPARC- mice (8 out of 12 mice; *, P < 0.05). (E, F, I, and J) Hematoxylin and eosin staining revealed striking differences between TGF-β–treated WT and SPARC- mice 7 d after MI. In WT mice, infusion of TGF-β did not significantly affect wound healing, whereas in SPARC- animals it stimulated ECM production in the infarct zone. (G, H, K, and L) Sirius Red staining and Sirius Red polarization microscopy revealed no apparent differences in collagen deposition between TGF-β1–treated SPARC- and WT mice, whereas the difference in well aligned and tightly packed (orange-red) collagen fibers in WT infarcts (H), but less mature fibers (yellow-green) in SPARC- infarcts (L), was preserved. Error bars represent the mean ± SEM. Bars: (E, G–I, K, and L) 200 μm; (F and J) 50 μm.
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fig5: SPARC and TGF-β cooperate during infarct healing. (A) Representative Western blot showing increased phosphorylated and total Smad2 levels in the infarcts of AdSPARC-treated mice (n = 9; *, P < 0.05). (B) Representative Western blot showing that addition of recombinant mouse SPARC activated Smad2 signaling and augmented the Smad2-activating potential of TGF-β (n = 6; *, P < 0.05). (C) The top blot is a representative Western blot indicating that shRNA against SPARC resulted in an 80% decrease in levels of SPARC protein. The blots are representative Western blots showing that Smad2 phosphorylation in shRNA-treated cardiac fibroblasts was significantly decreased at baseline levels (left) and after 15 min of stimulation by 1 ng/ml TGF-β (right; n = 5 per group; *, P < 0.05). (D) Survival curve showing that infusion of TGF-β protected against cardiac rupture after MI in male SPARC- mice (1 out of 7 mice) in comparison with saline-treated male SPARC- mice (8 out of 12 mice; *, P < 0.05). (E, F, I, and J) Hematoxylin and eosin staining revealed striking differences between TGF-β–treated WT and SPARC- mice 7 d after MI. In WT mice, infusion of TGF-β did not significantly affect wound healing, whereas in SPARC- animals it stimulated ECM production in the infarct zone. (G, H, K, and L) Sirius Red staining and Sirius Red polarization microscopy revealed no apparent differences in collagen deposition between TGF-β1–treated SPARC- and WT mice, whereas the difference in well aligned and tightly packed (orange-red) collagen fibers in WT infarcts (H), but less mature fibers (yellow-green) in SPARC- infarcts (L), was preserved. Error bars represent the mean ± SEM. Bars: (E, G–I, K, and L) 200 μm; (F and J) 50 μm.

Mentions: We asked whether the increased collagen deposition found in the infarcts of SPARC-overexpressing mice was associated with altered signaling of TGF-β, which is a known profibrotic cytokine (12). The levels of phosphorylated and total Smad2 were significantly higher in the infarcts of AdSPARC-treated WT mice (Fig. 5 A). These results were concordant with experiments in vitro, which showed that addition of recombinant SPARC increased Smad2 phosphorylation at baseline as well as after stimulation with TGF-β1 (Fig. 5 B). Inhibition of SPARC by short hairpin RNA (shRNA) resulted in an 80% decrease in levels of SPARC protein (Fig. 5 C). Although treatment with 1 ng/ml TGF-β resulted in a significant increase in the ratio of phosphorylated Smad2 (p-Smad2) to total Smad2 protein in control fibroblasts (100 ± 4% in control compared with 272 ± 30% in TGF-β–treated fibroblasts; n = 5; P < 0.05), the decrease in SPARC significantly blunted the ratio of p-Smad2/Smad2 at baseline (66 ± 8%; n = 6; P < 0.05 compared with WT fibroblasts) and after treatment with TGF-β (234 ± 14%; n = 5; P < 0.05 compared with TGF-β–treated WT fibroblasts; Fig. 5 C). We did not find significant differences in Smad2 phosphorylation between WT and SPARC- infarcts, a result which might reflect the increased presence of myofibroblasts in the SPARC- infarcts. Together, these data confirm a role for SPARC in the regulation of TGF-β signaling.


Absence of SPARC results in increased cardiac rupture and dysfunction after acute myocardial infarction.

Schellings MW, Vanhoutte D, Swinnen M, Cleutjens JP, Debets J, van Leeuwen RE, d'Hooge J, Van de Werf F, Carmeliet P, Pinto YM, Sage EH, Heymans S - J. Exp. Med. (2008)

SPARC and TGF-β cooperate during infarct healing. (A) Representative Western blot showing increased phosphorylated and total Smad2 levels in the infarcts of AdSPARC-treated mice (n = 9; *, P < 0.05). (B) Representative Western blot showing that addition of recombinant mouse SPARC activated Smad2 signaling and augmented the Smad2-activating potential of TGF-β (n = 6; *, P < 0.05). (C) The top blot is a representative Western blot indicating that shRNA against SPARC resulted in an 80% decrease in levels of SPARC protein. The blots are representative Western blots showing that Smad2 phosphorylation in shRNA-treated cardiac fibroblasts was significantly decreased at baseline levels (left) and after 15 min of stimulation by 1 ng/ml TGF-β (right; n = 5 per group; *, P < 0.05). (D) Survival curve showing that infusion of TGF-β protected against cardiac rupture after MI in male SPARC- mice (1 out of 7 mice) in comparison with saline-treated male SPARC- mice (8 out of 12 mice; *, P < 0.05). (E, F, I, and J) Hematoxylin and eosin staining revealed striking differences between TGF-β–treated WT and SPARC- mice 7 d after MI. In WT mice, infusion of TGF-β did not significantly affect wound healing, whereas in SPARC- animals it stimulated ECM production in the infarct zone. (G, H, K, and L) Sirius Red staining and Sirius Red polarization microscopy revealed no apparent differences in collagen deposition between TGF-β1–treated SPARC- and WT mice, whereas the difference in well aligned and tightly packed (orange-red) collagen fibers in WT infarcts (H), but less mature fibers (yellow-green) in SPARC- infarcts (L), was preserved. Error bars represent the mean ± SEM. Bars: (E, G–I, K, and L) 200 μm; (F and J) 50 μm.
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fig5: SPARC and TGF-β cooperate during infarct healing. (A) Representative Western blot showing increased phosphorylated and total Smad2 levels in the infarcts of AdSPARC-treated mice (n = 9; *, P < 0.05). (B) Representative Western blot showing that addition of recombinant mouse SPARC activated Smad2 signaling and augmented the Smad2-activating potential of TGF-β (n = 6; *, P < 0.05). (C) The top blot is a representative Western blot indicating that shRNA against SPARC resulted in an 80% decrease in levels of SPARC protein. The blots are representative Western blots showing that Smad2 phosphorylation in shRNA-treated cardiac fibroblasts was significantly decreased at baseline levels (left) and after 15 min of stimulation by 1 ng/ml TGF-β (right; n = 5 per group; *, P < 0.05). (D) Survival curve showing that infusion of TGF-β protected against cardiac rupture after MI in male SPARC- mice (1 out of 7 mice) in comparison with saline-treated male SPARC- mice (8 out of 12 mice; *, P < 0.05). (E, F, I, and J) Hematoxylin and eosin staining revealed striking differences between TGF-β–treated WT and SPARC- mice 7 d after MI. In WT mice, infusion of TGF-β did not significantly affect wound healing, whereas in SPARC- animals it stimulated ECM production in the infarct zone. (G, H, K, and L) Sirius Red staining and Sirius Red polarization microscopy revealed no apparent differences in collagen deposition between TGF-β1–treated SPARC- and WT mice, whereas the difference in well aligned and tightly packed (orange-red) collagen fibers in WT infarcts (H), but less mature fibers (yellow-green) in SPARC- infarcts (L), was preserved. Error bars represent the mean ± SEM. Bars: (E, G–I, K, and L) 200 μm; (F and J) 50 μm.
Mentions: We asked whether the increased collagen deposition found in the infarcts of SPARC-overexpressing mice was associated with altered signaling of TGF-β, which is a known profibrotic cytokine (12). The levels of phosphorylated and total Smad2 were significantly higher in the infarcts of AdSPARC-treated WT mice (Fig. 5 A). These results were concordant with experiments in vitro, which showed that addition of recombinant SPARC increased Smad2 phosphorylation at baseline as well as after stimulation with TGF-β1 (Fig. 5 B). Inhibition of SPARC by short hairpin RNA (shRNA) resulted in an 80% decrease in levels of SPARC protein (Fig. 5 C). Although treatment with 1 ng/ml TGF-β resulted in a significant increase in the ratio of phosphorylated Smad2 (p-Smad2) to total Smad2 protein in control fibroblasts (100 ± 4% in control compared with 272 ± 30% in TGF-β–treated fibroblasts; n = 5; P < 0.05), the decrease in SPARC significantly blunted the ratio of p-Smad2/Smad2 at baseline (66 ± 8%; n = 6; P < 0.05 compared with WT fibroblasts) and after treatment with TGF-β (234 ± 14%; n = 5; P < 0.05 compared with TGF-β–treated WT fibroblasts; Fig. 5 C). We did not find significant differences in Smad2 phosphorylation between WT and SPARC- infarcts, a result which might reflect the increased presence of myofibroblasts in the SPARC- infarcts. Together, these data confirm a role for SPARC in the regulation of TGF-β signaling.

Bottom Line: The matricellular protein SPARC (secreted protein, acidic and rich in cysteine, also known as osteonectin) mediates cell-matrix interactions during wound healing and regulates the production and/or assembly of the extracellular matrix (ECM).These findings indicate that local production of SPARC is essential for maintenance of the integrity of cardiac ECM after MI.The protective effects of SPARC emphasize the potential therapeutic applications of this protein to prevent cardiac dilatation and dysfunction after MI.

View Article: PubMed Central - PubMed

Affiliation: Center for Heart Failure Research, Cardiovascular Research Institute Maastricht, University Hospital Maastricht, 6229 HX Maastricht, The Netherlands.

ABSTRACT
The matricellular protein SPARC (secreted protein, acidic and rich in cysteine, also known as osteonectin) mediates cell-matrix interactions during wound healing and regulates the production and/or assembly of the extracellular matrix (ECM). This study investigated whether SPARC functions in infarct healing and ECM maturation after myocardial infarction (MI). In comparison with wild-type (WT) mice, animals with a targeted inactivation of SPARC exhibited a fourfold increase in mortality that resulted from an increased incidence of cardiac rupture and failure after MI. SPARC- infarcts had a disorganized granulation tissue and immature collagenous ECM. In contrast, adenoviral overexpression of SPARC in WT mice improved the collagen maturation and prevented cardiac dilatation and dysfunction after MI. In cardiac fibroblasts in vitro, reduction of SPARC by short hairpin RNA attenuated transforming growth factor beta (TGF)-mediated increase of Smad2 phosphorylation, whereas addition of recombinant SPARC increased Smad2 phosphorylation concordant with increased Smad2 phosphorylation in SPARC-treated mice. Importantly, infusion of TGF-beta rescued cardiac rupture in SPARC- mice but did not significantly alter infarct healing in WT mice. These findings indicate that local production of SPARC is essential for maintenance of the integrity of cardiac ECM after MI. The protective effects of SPARC emphasize the potential therapeutic applications of this protein to prevent cardiac dilatation and dysfunction after MI.

Show MeSH
Related in: MedlinePlus