Exogenous Transforming Growth Factor-β1 and Its Helminth-Derived Mimic Attenuate the Heart's Inflammatory Response to Ischemic Injury and Reduce Mature Scar Size

Figures

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  Figure 1. Circulating transforming growth factor (TGF)-β1 levels in patients with ST-elevation myocardial infarction (STEMI) at 24 hours after reperfusion positively correlate with a reduction in infarct size over 3 months. A: The median circulating level of TGF-β1 at 15 minutes and 24 hours after reperfusion in patients with STEMI. B: Infarct size measured by magnetic resonance between week 1 and 3 months after primary percutaneous intervention in patients with STEMI. C: Circulating TGF-β1 levels at 24 hours after reperfusion versus change in infarct size. Spearman's rank correlation coefficient = −0.42; P = 0.0034. ∗∗P < 0.01 (Wilcoxon two-tailed matched paired signed rank test); ∗∗∗∗P < 0.0001 (Wilcoxon matched paired samples test). %LV, percentage of the left ventricle.

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  Figure 2. Treatment with exogenous transforming growth factor (TGF)-β1 in a mouse model of transient cardiac ischemia reduces infarct size and inflammatory infiltrate 24 hours after reperfusion. A and B: Hearts were subject to 60 minutes of infarction by transient occlusion of the left anterior descending artery followed by reperfusion with or without TGF-β1 treatment (66 μg/kg). Area of infarct as a proportion of the left ventricle (LV) was measured using viable (triphenyltetrazolium chloride) staining at 24 hours after reperfusion. Infarct area of nonviable myocardium (white) is indicated. C: Immunostaining of transverse heart sections with anti-CD45 (pan-leukocyte marker) at 24 hours after reperfusion was used to measure the total area of immune infiltrate (dashed line) expressed as percentage of the LV (%LV). This was then used as a readout of tissue injury, and this area of infiltrate is absent in sham controls. D: Mean values of injured area in response to TGF-β1 treatment shown for 8 images per biological replicate, taken from transverse heart sections (200 μm apart) from the intramyocardial region of the middle LV. Data analyzed by unpaired 2-tailed t-test (data values in shams…

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  Figure 3. Exogenous transforming growth factor (TGF)-β1 treatment reduces myocardial-induced expression of major inflammatory cytokines. A–C: Quantitative PCR analysis of infarcted (60 minutes) left ventricular heart tissue at 24 hours after reperfusion showing cytokine IL-1β (A), chemokine (C-C motif) ligand 2 (CCL2) (B), and tumor necrosis factor (TNF)-α (C) compared with sham operated hearts. Gene expression data were normalized to housekeeping genes Hprt1 and Gapdh, and fold change in gene expression was calculated with respect to expression in naive hearts. Data were analyzed by one-way analysis of variance with Tukey correction for multiple comparisons. n = 5 to 6 per group. ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001, and ∗∗∗∗P < 0.0001. ns, not significant.

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  Figure 4. Transforming growth factor (TGF)-β1 treatment reduces scar size at 4 weeks after reperfusion. A: Masson's trichrome stain was used to discriminate viable muscle (pink) from collagenous scar (blue). Analysis of 17 transverse sections through each heart was used to quantify scar size as percentage of left ventricle (%LV). B: Scar size is significantly reduced in TGF-β1–treated mice compared with control mice subjected to the same cardiac injury of 60-minute transient ischemia. Data were analyed by unpaired t-test. n = 11 per group. ∗P < 0.05. Scale bars = 500 μm. LV, left ventricle; RV, right ventricle.

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  Figure 5. Treatment with exogenous Heligmosomoides polygyrus TGM (HpTGM) in a mouse model of myocardial infarction reduces infarct size, inflammatory cytokine expression, and scar size. A: Hearts were subject to 60-minute infarction by transient occlusion of the left anterior descending artery (LAD) followed by reperfusion with or without HpTGM treatment (132 μg/kg). Immunostaining of transverse heart sections with anti-CD45 (pan-leukocyte marker) at 24 hours after reperfusion was used to measure the total area of tissue injury. Mean values are shown for 24 images per biological replicate. Data were analyzed by unpaired two-tailed t-test. B: Anti-CD45 staining was used to measure the density of leukocyte accumulation in the injured region at 24 hours of reperfusion. Data were analyzed by unpaired two-tailed t-test. C–F: Quantitative PCR analysis of infarcted (60 minutes) left ventricular heart tissue at 24 hours after reperfusion shows up-regulation of cytokines chemokine (C-C motif) ligand 2 (CCL2), IL-1β, and tumor necrosis factor (TNF)-α in the left ventricle (LV) of ischemia-reperfusion (I/R) hearts from saline-treated mice compared with HpTGM-treated mice. Gene expression data were…

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  Figure 6. Cardiac protection of Heligmosomoides polygyrus TGM (HpTGM) treatment in the context of myocardial infarction is dependent on expression of transforming growth factor (TGF)-β receptor type II in endothelial cells. A and B:Tgfbr2 floxed (Tgfbr2fl/fl) control mice and endothelial specific Tgfbr2 knockout (Tgfbr2-iKOe) mice were subject to 60-minute occlusion of the left anterior descending artery followed by reperfusion with or without HpTGM treatment (132 μg/kg). Immunostaining of transverse heart sections with anti-CD45 (pan-leukocyte marker) at 24 hours after reperfusion was used to measure the total area of heart tissue injury and the density of leukocytes per field of view (FOV). Data were analyzed by one-way analysis of variance with Tukey correction (A) and Kruskal-Wallis test with Dunn correction for multiple comparisons (B). n = 7 per group. ∗P < 0.05, ∗∗P < 0.01, and ∗∗∗P < 0.001. ns, not significant.