Fibroblast polarization over the myocardial infarction time continuum shifts roles from inflammation to angiogenesis

Alan J Mouton,Alan J Mouton,Kristine Y Deleon-Pennell,Kristine Y Deleon-Pennell,Kristine Y Deleon-Pennell,Michael R Garrett,Michael J Daseke,Michael J Daseke,Tom C Freeman,Tom C Freeman,Yonggang Ma,Yonggang Ma,Elizabeth R Flynn,Elizabeth R Flynn,Osvaldo J Rivera Gonzalez,Merry L Lindsey,Merry L Lindsey,Merry L Lindsey

doi:10.1007/s00395-019-0715-4

Abstract

Cardiac fibroblasts are the major producers of extracellular matrix (ECM) to form infarct scar. We hypothesized that fibroblasts undergo a spectrum of phenotype states over the course of myocardial infarction (MI) from early onset to scar formation. Fibroblasts were isolated from the infarct region of C57BL/6J male mice (3–6 months old, n = 60) at days 0 (no MI control) and 1, 3, or 7 after MI. Whole transcriptome analysis was performed by RNA-sequencing. Of the genes sequenced, 3371 were differentially expressed after MI. Enrichment analysis revealed that MI day 1 fibroblasts displayed pro-inflammatory, leukocyte-recruiting, pro-survival, and anti-migratory phenotype through Tnfrsf9 and CD137 signaling. MI day 3 fibroblasts had a proliferative, pro-fibrotic, and pro-angiogenic profile with elevated Il4ra signaling. MI day 7 fibroblasts showed an anti-angiogenic homeostatic-like myofibroblast profile and with a step-wise increase in Acta2 expression. MI day 7 fibroblasts relied on Pik3r3 signaling to mediate Tgfb1 effects and Fgfr2 to regulate PI3K signaling. In vitro, the day 3 MI fibroblast secretome stimulated angiogenesis, while day 7 MI fibroblast secretome repressed angiogenesis through Thbs1 signaling. Our results reveal novel mechanisms for fibroblasts in expressing pro-inflammatory molecules and regulating angiogenesis following MI.

Highlights

Myocardial infarction (MI) occurs when coronary blood flow to the left ventricle (LV) is blocked for a sufficient duration to result in tissue necrosis
By mass spectrometry analysis of the day 0 and MI day 7 fibroblast secretomes, collagen I alpha 1 and 2 chains, secreted protein acidic and rich in cysteine (SPARC), and lysyl oxidase were significantly increased in the day 7 MI fibroblast secretome, while periostin showed no change (Online Resource 2b)
Our results indicate the fibroblast activation marker collagens and collagen cross-linking enzymes are regulated at the protein level in MI cardiac fibroblasts

Summary

Introduction

Myocardial infarction (MI) occurs when coronary blood flow to the left ventricle (LV) is blocked for a sufficient duration to result in tissue necrosis. The cardiac wound healing response involves robust inflammation, extracellular matrix. While the cardiac fibroblast serves a baseline homeostasis role or transforms to an activated myofibroblast in response to injury, intermediary phenotypes exist, and fibroblasts display a range of phenotypes in response to individual in vitro stimuli [1, 61]. Cardiac fibroblasts stimulated with lipopolysaccharide show increased pro-inflammatory cytokine production and decreased collagen synthesis [37, 59]. Fibroblasts stimulated with transforming growth factor (TGF)β1 increase collagen synthesis and differentiate to a myofibroblast phenotype [62, 65]

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