Impaired infarct healing in atherosclerotic mice with Ly-6C(hi) monocytosis

Abstract

Objectives: The aim of this study was to test whether blood monocytosis in mice with atherosclerosis affects infarct healing.

Background: Monocytes are cellular protagonists of tissue repair, and their specific subtypes regulate the healing program after myocardial infarction (MI). Inflammatory Ly-6C(hi) monocytes dominate on Day 1 to Day 4 and digest damaged tissue; reparative Ly-6C(lo) monocytes dominate on Day 5 to Day 10 and promote angiogenesis and scar formation. However, the monocyte repertoire is disturbed in atherosclerotic mice: Ly-6C(hi) monocytes expand selectively, which might disrupt the resolution of inflammation.

Methods: Ex vivo analysis of infarcts included flow cytometric monocyte enumeration, immunoactive staining, and quantitative polymerase chain reaction. To relate inflammatory activity to left ventricular remodeling, we used a combination of noninvasive fluorescence molecular tomography (FMT-CT) and physiologic imaging (magnetic resonance imaging).

Results: Five-day-old infarcts showed >10x more Ly-6C(hi) monocytes in atherosclerotic (apoE(-/-)) mice compared with wild-type mice. The injured tissue in apoE(-/-) mice also showed a more pronounced inflammatory gene expression profile (e.g., increased tumor necrosis factor-alpha and myeloperoxidase and decreased transforming growth factor-beta) and a higher abundance of proteases, which are associated with the activity of Ly-6C(hi) monocytes. The FMT-CT on Day 5 after MI showed higher proteolysis and phagocytosis in infarcts of atherosclerotic mice. Serial magnetic resonance imaging showed accelerated deterioration of ejection fraction between Day 1 and Day 21 after MI in apoE(-/-). Finally, we could recapitulate these features in wild-type mice with artificially induced Ly-6C(hi) monocytosis.

Conclusions: Ly-6C(hi) monocytosis disturbs resolution of inflammation in murine infarcts and consequently enhances left ventricular remodeling. These findings position monocyte subsets as potential therapeutic targets to augment tissue repair after infarction and to prevent post-MI heart failure.

Figure 1

Experimental protocol for ex (A, C) and in vivo (B, D) studies. MI: myocardial infarction induced by coronary ligation, FRI: ex vivo Fluorescence Reflectance Imaging, FACS: Fluorescence Activated Cell Sorting, PCR: Polymerase Chain Reaction-based gen expression profiling, IHC: Immunohistochemistry, DE MRI: delayed enhancement MRI for infarct size measurement.

Figure 2

(A) Original flow cytometry dot plots of blood and infarct tissue in wild type and apoE^-/- mice 5 days after coronary ligation. (B) Enumeration of cells shows significantly increased blood and tissue levels for inflammatory Ly-6C^hi monocytes in apoE^-/- mice.

Figure 3

(A) Immunoreactive staining and quantitation for the presence of the imaging target protease Cathepsin B and F4/80 positive macrophages. Furthermore, presence of MMP2, MMP9, VEGF and CD31 was profiled. Magnification 200×, the scale bar denotes 100 μm. (B) Quantitative RT-PCR: higher expression of MAC-3 and CD68 corroborate in vivo and ex vivo findings of increased cell recruitment into the infarct of apoE^-/-. Transcription of MPO and TNF-α is higher in apoE^-/- mice, whereas TGF-β is reduced. As a control, we determined apoE, which was undetectable in apoE^-/- mice. *p<0.05, **p<0.001.

Figure 4

Image fusion. (A) Multimodal imaging cartridge. (B-E) FMT-CT phantom imaging. (B-C) Coronal CT and FMT-CT. (D) Maximum intensity projection of fused FMT-CT. The well inside the phantom is filled with fluorochrome VT-680 and in addition contains iodine CT contrast. (E) Correlation between CT and FMT-CT dimensions shows good agreement of fused data. (F-K) CT, FMT and FMT-CT in a mouse with MI injected with CLIO. Arrows denote fiducial landmarks used for fusion.

Figure 5

(A) FMT-CT 5 days after MI. Arrows denote apical infarct, arrowhead denotes calcification, presumably in an atherosclerotic lesion. On 3D Prosense and CLIO FMT-CT images, arrows point towards infarct signal. Arrowheads denote fluorescence signal in the carotid artery. (B) Ex vivo fluorescence reflectance imaging corroborates in vivo FMT findings. TBR: target to background ratio. *p<0.05.

Figure 6

Serial cardiac MRI. Initial assessment of infarct size by delayed enhancement after injection of Gd-DTPA (arrows) showed similar values in both groups. Over time, ventricular dilatation was enhanced in apoE^-/- mice, which resulted in significantly worse EF on day 21 after MI. *p<0.05.

Figure 7

(A) Flow cytometry on day 5 and serial imaging in wild type mice in which blood monocytosis was induced by LPS. Blood monocytosis led to sustained monocyte presence in the infarct on day 5, increased protease and phagocytic signal on FMT and accelerated left ventricular dilation. (B) ApoE^-/- mice in which neutrophils were depleted with injections of anti-Ly-6G antibody show similar inflammation in the infarct by FMT-CT and remodeling by MRI. *p<0.05.