Improved heart function follows enhanced inflammatory cell recruitment and angiogenesis in 11betaHSD1-deficient mice post-MI

Abstract

Aims: Mice unable to locally regenerate corticosterone due to deficiency of 11β-hydroxysteroid dehydrogenase type 1 (11βHSD1) have enhanced angiogenesis during acute myocardial infarct healing. The present study investigates the hypotheses that in these mice (i) inflammation and angiogenic signalling are promoted and (ii) longer-term remodelling and function are improved.

Methods and results: Myocardial infarction (MI) was induced by coronary artery ligation in 11βHSD1(-/-) and wild-type (C57BL/6) mice. Studies were terminated 2, 4, 7, and 28 days post-surgery. Increased vessel density (CD31 immunoreactivity) on the infarct border was confirmed 7 days after MI in 11βHSD1(-/-) hearts (P < 0.05) and was accompanied by improved ejection fraction (ultrasound) compared with C57BL/6. During wound healing, recruitment of neutrophils (at 2 days after MI) and macrophages (from 4 days after MI) and expression of monocyte-chemoattractant protein-1 was increased in 11βHSD1(-/-) compared with C57BL/6 hearts (P < 0.05). Recruitment of alternatively activated YM1-positive macrophages was particularly enhanced in the period preceding increased vessel density and was accompanied by increased expression of pro-angiogenic IL-8. By 28 days post-MI, when the infarct scar had matured, higher vessel density was maintained in 11βHSD1(-/-) hearts and vessels were smooth-muscle coated. Infarct scars were thicker (P < 0.001) in 11βHSD1(-/-) compared with C57BL/6 hearts and ejection fraction was higher (P < 0.05).

Conclusion: Increased vessel density in healing infarcts of mice deficient in 11(-/-)HSD1 follows recruitment of pro-reparative macrophages and increased pro-angiogenic signalling. Mature infarcts show less thinning and cardiac function is improved relative to wild-type mice, suggesting that 11βHSD1 may be a novel therapeutic target after MI.

Figure 1

Neutrophil infiltration during infarct healing. (A and B) Neutrophils were identified in the left ventricular myocardium (LV) by their distinctive multi-lobed nuclei in haematoxylin and eosin-stained sections, counted and expressed as number per 10 μm². (B) Tiled heart 2 days post-infarction showing neutrophil invading infarct border. IA, infarct area; RV, right ventricle. Arrows point to neutrophils. n = 8, C57BL/6 sham; n = 12, C57BL/6 MI; n = 4, 11βHSD1^−/− sham; n = 6, 11βHSD1^−/− MI. ^##P < 0.01, ^###P < 0.001 (versus matched sham), **P < 0.01 (C57BL/6 versus 11βHSD1^−/−). Scale bar, 10 μm.

Figure 2

Macrophage accumulation during infarct healing. (A, D, E) mac 2 immunohistochemistry was used to detect macrophage infiltration and was quantified as percent staining of the infarct border (IB). (B) Monocyte chemoattractant protein-1 (MCP-1) mRNA levels in heart tissue normalized to GAPDH housekeeping gene. (C, F) YM1-positive staining in heart tissue showing alternatively activated macrophages and quantified as percent staining of the infarct border. YM1 staining was absent from the hearts of mice that underwent sham operation (C). n = 8, C57BL/6 sham; n = 12, C57BL/6 MI; n = 4, 11βHSD1^−/− sham; n = 6, 11βHSD1^−/− MI; for RT–PCR n = 6 per group. ^##P < 0.01, ^###P < 0.001 (sham versus MI). *P < 0.05, **P < 0.01, ***P < 0.001 (C57BL/6 versus 11βHSD1^−/−). Scale bar, 10 μm.

Figure 3

Vessel density and cell proliferation during infarct healing. (A) CD31-positive vessels <200 μm in diameter in the LV, expressed per 400 μm². (B) Nuclei positive for BrdU incorporation in the LV, expressed per mm². (C) Representative sections of infarct border at 7 days after infarction, arrows point to CD31-positive vessels. (D) Interleukin 8 (IL-8) and vascular endothelial growth factor α (VEGFα) mRNA expression levels in heart tissue normalized to GAPDH housekeeping gene at 7 days after MI. n = 8, C57BL/6 sham; n = 12, C57BL/6 MI; n = 4, 11βHSD1^−/− sham; n = 6, 11βHSD1^−/− MI; for RT–PCR n = 6 per group. ^#P < 0.05, ^###P < 0.001 (sham versus MI). *P < 0.05, **P < 0.01 (C57BL/6 versus 11βHSD1^−/−). Scale bar, 10 μm.

Figure 4

Heart function during and after infarct healing. Ejection fraction was calculated from the LV end-diastolic area (LVEDA) and LV end-systolic area (LVESA) and expressed as a percentage. Data are presented as mean ± SEM, lighter columns C57BL/6, darker columns 11βHSD1^−/−. (for C57BL/6, n = 12 MI during early healing at 2, 4, and 7 days after MI, n = 10 for MI at day 28; for 11βHSD1^−/− n = 6 MI day 2, 4, and 7, and  n = 9 for MI day 28). *P < 0.05, **P < 0.01 C57BL/6 versus 11βHSD1^−/−.

Figure 5

Blood vessel density and pericyte coverage 28 days after MI. (A) CD31 and (B) α smooth muscle actin (αSMA smooth muscle cells) positive vessels <200 μm in diameter counted in sequential sections from the LV, expressed per 400 μm². (C) Representative sections showing typical double immuno-staining for CD31 (brown) and αSMA (blue) on the infarct border of C57BL/6 (C57BL6) and 11βHSD1^−/− (11HSD1−/−) hearts . Filled arrows point to pericyte, smooth muscle coated vessels, open arrows point to pericyte poor, smooth muscle negative vessels. n = 10, C57BL/6 MI; n = 9, 11βHSD1^−/− MI. *P < 0.05, ***P < 0.001. Scale bar, 10 μm.

Figure 6

Fibrosis and scar formation 28 days after MI. (A) Collagen deposition measured from Picrosirius Red stained sections and expressed as percent staining of the LV in C57BL/6 (C57BL6, light columns) and 11βHSD1^−/− (11HSD1−/−, dark columns) hearts . (B–E) Scar dimensions and infarct lengths were assessed from Masson's Trichrome stained sections (B). (C) Scar area expressed as the percentage of the LV. (D) Scar thickness was averaged from three points taken across the scar. (E) Epicardial infarct length expressed as a percentage of the epicardial LV length. n = 10, C57BL/6 MI; n = 9, 11βHSD1^−/− MI for Picrosirius Red and Masson's Trichrome staining. ***P < 0.001 (C57BL/6 versus 11βHSD1^−/−). Scale bar, 10 μm.