Macrophage-produced VEGFC is induced by efferocytosis to ameliorate cardiac injury and inflammation
Kristofor E. Glinton, … , Guillermo Oliver, Edward B. Thorp
Kristofor E. Glinton, … , Guillermo Oliver, Edward B. Thorp
Published March 10, 2022
Citation Information: J Clin Invest. 2022;132(9):e140685. https://doi.org/10.1172/JCI140685.
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Research Article Inflammation Vascular biology

Macrophage-produced VEGFC is induced by efferocytosis to ameliorate cardiac injury and inflammation

Abstract

Clearance of dying cells by efferocytosis is necessary for cardiac repair after myocardial infarction (MI). Recent reports have suggested a protective role for vascular endothelial growth factor C (VEGFC) during acute cardiac lymphangiogenesis after MI. Here, we report that defective efferocytosis by macrophages after experimental MI led to a reduction in cardiac lymphangiogenesis and Vegfc expression. Cell-intrinsic evidence for efferocytic induction of Vegfc was revealed after adding apoptotic cells to cultured primary macrophages, which subsequently triggered Vegfc transcription and VEGFC secretion. Similarly, cardiac macrophages elevated Vegfc expression levels after MI, and mice deficient for myeloid Vegfc exhibited impaired ventricular contractility, adverse tissue remodeling, and reduced lymphangiogenesis. These results were observed in mouse models of permanent coronary occlusion and clinically relevant ischemia and reperfusion. Interestingly, myeloid Vegfc deficiency also led to increases in acute infarct size, prior to the amplitude of the acute cardiac lymphangiogenesis response. RNA-Seq and cardiac flow cytometry revealed that myeloid Vegfc deficiency was also characterized by a defective inflammatory response, and macrophage-produced VEGFC was directly effective at suppressing proinflammatory macrophage activation. Taken together, our findings indicate that cardiac macrophages promote healing through the promotion of myocardial lymphangiogenesis and the suppression of inflammatory cytokines.

Authors

Kristofor E. Glinton, Wanshu Ma, Connor Lantz, Lubov S. Grigoryeva, Matthew DeBerge, Xiaolei Liu, Maria Febbraio, Mark Kahn, Guillermo Oliver, Edward B. Thorp

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Figure 2

Vegfc is induced in macrophages during efferocytosis.

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Vegfc is induced in macrophages during efferocytosis. (A) Photomicrogra...
(A) Photomicrograph depicts primary BMDMs (red) cocultivated with fluorescently labeled (green) ACs. Original magnification, ×40. Bar graph shows the quantification of gene expression at the indicated time points after AC cocultivation. n = 3–5. *P < 0.03 and **P < 0.003, by 1-way ANOVA with Tukey’s test. (B) Macrophage gene expression after treatment with either ACs or LPS (100 ng/mL). n = 6 per group. **P < 0.006. (C) Representative protein immunoblots of VEGFC, 6 hours after efferocytosis and densitometric analysis. n = 6 pooled from 2 independent experiments. **P < 0.005. (D) VEGFC ELISA of macrophage supernatant 9 hours after treatment with ACs versus control. n = 6. *P < 0.01. (E) BMDMs from Cd36+/+ and Cd36–/– animals were cocultured for 3 hours with ACs. After sequential washes, cells were imaged on an Olympus fluorescence microscope, and the percentage of efferocytosis was calculated from 10 random fields per replicate. Scale bar: 20 μm. Data are representative of 2 independent experiments with n = 3 wells per group. **P < 0.01. (F) BMDMs from Cd36+/+ and Cd3–/– animals was assessed for gene expression before and after treatment with ACs. n = 3–8 per group. ***P < 0.0005 and ****P < 0.0001. (G) Macrophage Vegfc gene expression after treatment with either ACs or with ACs plus etomoxir (ETO). n = 3–4 wells per group. *P < 0.01. (H) Quantification of gene expression in macrophages treated with ACs versus ACs plus cytochalasin D (CytoD). n = 3 per group. *P < 0.02. (I) Inhibition of STAT6 with AS1517499 blocked efferocytic Vegfc induction. *P < 0.01. (J) To assess STAT6 phosphorylation, macrophages were cultured as above, and lysates were prepared in RIPA buffer and then assessed by Western blotting. n = 6 pooled samples from 2 independent experiments. *P < 0.05. Data are presented as the mean ± SEM. Statistical significance was determined by 2-way ANOVA followed by Tukey’s test (B and F–J) and 2-tailed, unpaired t test (C–E). Ctrl, control.