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Endogenous thrombospondin 1 protects the pressure-overloaded myocardium by modulating fibroblast phenotype and matrix metabolism

Hypertension (2011) 58(5) 902-911
DOI: 10.1161/HYPERTENSIONAHA.111.175323
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Abstract

The matricellular protein thrombospondin (TSP) 1 is induced after tissue injury and may regulate reparative responses by activating transforming growth factor-β, by suppressing angiogenesis and by modulating inflammation and matrix metabolism. We hypothesized that endogenous TSP-1 may be involved in the pathogenesis of cardiac remodeling in the pressure-overloaded heart. Myocardial TSP-1 expression was increased in a mouse model of pressure overload because of transverse aortic constriction. TSP-1 -/- mice exhibited increased early hypertrophy and enhanced late dilation in response to pressure overload. Pressure-overloaded TSP-1 null mice had intense degenerative cardiomyocyte changes, exhibiting more extensive sarcomeric loss and sarcolemmal disruption when compared with wild-type hearts. Accentuated hypertrophy and cardiomyocyte injury in TSP-1 -/- hearts was accompanied by increased myofibroblast density. However, despite a 2-fold higher infiltration of the cardiac interstitium with myofibroblasts, pressureoverloaded TSP-1 null hearts did not exhibit significantly increased collagen content when compared with wild-type hearts. The disproportionately low collagen content in TSP-1 null hearts was attributed to infiltration with abundant, but functionally defective, fibroblasts that exhibited impaired myofibroblast differentiation and reduced collagen expression in comparison with wild-type fibroblasts. Impaired myofibroblast activation in TSP-1 null hearts was associated with reduced Smad2 phosphorylation reflecting defective transforming growth factor-β signaling. Moreover, TSP-1 null hearts had increased myocardial matrix metalloproteinase 3 expression and enhanced matrix metalloproteinase 9 activation after pressure overload. TSP-1 upregulation in the pressure-overloaded heart critically regulates fibroblast phenotype and matrix remodeling by activating transforming growth factor-β signaling and by promoting matrix preservation, thus preventing chamber dilation. © 2011 American Heart Association, Inc.

Figures

  • Figure 1. Thrombospondin (TSP) 1 upregulation in the pressure-overloaded heart. A, Quantitative RT-PCR demonstrated that TSP-1 mRNA was markedly induced in the pressure-overloaded myocardium after 3 to 7 days of transverse aortic constriction (TAC; *P 0.01 vs sham; *P 0.05 vs sham). B and C, Immunohistochemical localization of TSP-1 protein in the myocardium after 7 (B) and 28 days (C) of TAC. TSP-1 was predominantly localized in the perivascular and interstitial space (arrows). Sections were counterstained with eosin. D, KaplanMeier curves demonstrating survival rates of wild-type (WT) and TSP-1 null mice undergoing pressure overload protocols. TSP-1 null mice and WT animals showed comparable mortality after 28 days of TAC (P value not significant) but had lower survival rates during the first 3 days of pressure overload (P 0.05).
  • Table 1. Echocardiographic Analysis of Systolic Function and Chamber Dimensions
  • Figure 2. Effects of thrombospondin (TSP) 1 loss on cardiomyocyte injury and apoptosis in the pressure-overloaded myocardium. A through D, Hematoxylin-eosin staining shows histopathologic alterations after 7 days of transverse aortic constriction (TAC) in wild-type (WT; A and C) and TSP-1 null (B and D) hearts. Low ( 100; A and B) and high ( 400; C and D) magnification images demonstrate that TSP-1 null hearts (B and D) exhibited more intense alterations in cardiomyocyte morphology (including sarcomeric loss, hypereosinophilia, and contraction band necrosis; arrows) than WT hearts. E through H, Apoptosis of cardiomyocytes and noncardiomyocytes was quantitatively assessed using TUNEL staining (green, arrows), combined with wheat germ agglutinin lectin histochemistry that outlines the surface of cardiomyocytes (red). The density of apoptotic cardiomyocytes (G, arrow) and interstitial cells (H, arrows) was quantitatively assessed in WT (E) and TSP-1 / (F) hearts after 3 to 28 days of TAC. 4 ,6- Diamidino-2-phenylindole (DAPI) staining (blue) was used to label all of the nuclei in the murine myocardium. I and J, The majority of apoptotic cells were noncardiomyocytes; TUNEL cardiomyocytes were rare. TSP-1 / mice had lower early cardiomyocyte apoptosis after 3 days of TAC but exhibited a significantly higher apoptotic cardiomyocyte density after 7 days (*P 0.05 vs WT; I). The number of apoptotic noncardiomyocytes was comparable between groups (J; ˆ P 0.05, ˆˆP 0.01 vs sham).
  • Figure 3. Although thrombospondin (TSP) 1 null pressure-overloaded hearts are infiltrated with abundant fibroblasts, collagen content is not significantly increased. A through C, Myofibroblasts were identified in the pressure-overloaded myocardium as spindle-shaped cells expressing -smooth muscle actin (SMA) located outside the vascular media (arrows). Quantitative analysis demonstrated that myofibroblast density was significantly higher in TSP-1 / hearts after 3 to 28 days of transverse aortic constriction (TAC; **P 0.01 vs wild-type [WT]). Representative images showing -SMA staining to identify myofibroblasts in WT (B) and TSP-1 null (C) hearts after 7 days of pressure overload are shown. D through F, Cardiac pressure overload was associated with increased total and insoluble collagen content (measured with a hydroxyproline biochemical assay) and a higher ratio of insoluble:total collagen (ˆˆP 0.01 vs sham). Despite a markedly increased myofibroblast density, collagen content was not significantly higher in TSP-1 null hearts after 7 days of pressure overload (D). The amount of insoluble collagen (E) and the ratio of insoluble:total collagen (F) were comparable between TSP-1 / and WT animals. Sirius red staining showed that, whereas pressure-overloaded WT hearts had interstitial deposition of dense collagen (G, arrows), TSP-1 / animals had more extensive cardiomyocyte loss, associated with replacement by a matrix network composed of loose collagen (H, arrows).
  • Table 2. Cytokine mRNA Expression in the Pressure-Overloaded Heart
  • Figure 4. Thrombospondin (TSP) 1 / and wild-type (WT) mice show comparable infiltration of the pressure-overloaded heart with macrophages and exhibit no significant difference in microvascular density. A and B, Mac2 immunohistochemistry was used to identify macrophages in WT (A) and TSP-1 null (B) pressure-overloaded hearts. C, Quantitative analysis showed infiltration of the pressureoverloaded myocardium with Mac2 cells after 3 to 7 days of transverse aortic constriction (TAC; ˆˆP 0.01 vs sham). The density of Mac2 macrophages was comparable between WT and TSP-1 null animals at all of the time points examined. D, Quantitative PCR demonstrated that cardiac mRNA expression of the CC chemokine monocyte chemoattractant protein (MCP) 1, a potent mononuclear cell chemoattractant, was comparable between TSP-1 null and WT hearts. E and F, Vascular endothelial cells were identified in the mouse myocardium using staining for CD31. Microvascular density was compared between WT (E) and TSP-1 / (F) animals. No statistically significant difference was observed in subendocardial (G, endo) and subepicardial (H, epi) microvascular density between WT and knockout hearts after 7 to 28 days of TAC (P value not significant).
  • Figure 5. Thrombospondin (TSP) 1 null mice exhibit impaired activation of transforming growth factor (TGF)- /Smad2 signaling. A, Quantitative PCR demonstrated that TGF- 1 mRNA was upregulated in pressure-overloaded hearts after 3 days of transverse aortic constriction (TAC; ˆP 0.05 vs sham). However, TGF- 1 mRNA (A) and protein (B) levels were comparable between TSP-1 null and wildtype (WT) animals. C, D, Activation of the Smad2/3 pathway was assessed using Western blotting for p-Smad2. TSP-1 null hearts exhibited significantly lower Smad2 phosphorylation after 7 days of transverse aortic constriction (TAC) than WT hearts (*P 0.05).
  • Figure 6. Thrombospondin (TSP) 1 null hearts have increased matrix metalloproteinase (MMP) 3 expression and enhanced MMP-9 activity after 7 days of pressure overload. A through D, Quantitative analysis of Western blotting showed that MMP-3 protein levels were significantly higher in pressure-overloaded TSP-1 / hearts (**P 0.01 vs corresponding wild-type [WT]); in contrast MMP-2, MMP-9, and tissue inhibitor of metalloproteinase (TIMP) 1 levels were comparable between groups (ˆP 0.05 vs sham). E, F, Zymography showed significantly increased MMP-2 and MMP-9 activity in the pressure-overloaded myocardium after 7 days of TAC (ˆP 0.05 vs sham). Active MMP-9 levels (aMMP-9) were significantly higher in TSP-1 hearts (C; *P 0.05 vs WT), whereas MMP-2 activity (aMMP-2) was comparable between WT and / hearts (B). G, A representative image of a zymogram comparing MMP activity in the pressure-overloaded myocardium between WT ( / ) and TSP-1 / mice after 7 days of TAC. Bands corresponding with active MMP-2 and active MMP-9 are identified.

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Xia, Y., Dobaczewski, M., Gonzalez-Quesada, C., Chen, W., Biernacka, A., Li, N., … Frangogiannis, N. G. (2011). Endogenous thrombospondin 1 protects the pressure-overloaded myocardium by modulating fibroblast phenotype and matrix metabolism. Hypertension, 58(5), 902–911. https://doi.org/10.1161/HYPERTENSIONAHA.111.175323

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