Redistribution of the chromatin remodeler Brg1 directs smooth muscle–derived adventitial progenitor–to–myofibroblast differentiation and vascular fibrosis
Austin J. Jolly, Sizhao Lu, Allison M. Dubner, Keith A. Strand, Marie F. Mutryn, Aaron Pilotti-Riley, Etienne P. Danis, Raphael A. Nemenoff, Karen S. Moulton, Mark W. Majesky, Mary C.M. Weiser-Evans
Austin J. Jolly, Sizhao Lu, Allison M. Dubner, Keith A. Strand, Marie F. Mutryn, Aaron Pilotti-Riley, Etienne P. Danis, Raphael A. Nemenoff, Karen S. Moulton, Mark W. Majesky, Mary C.M. Weiser-Evans
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Research Article Vascular biology

Redistribution of the chromatin remodeler Brg1 directs smooth muscle–derived adventitial progenitor–to–myofibroblast differentiation and vascular fibrosis

Abstract

Vascular smooth muscle–derived Sca1+ adventitial progenitor (AdvSca1-SM) cells are tissue-resident, multipotent stem cells that contribute to progression of vascular remodeling and fibrosis. Upon acute vascular injury, AdvSca1-SM cells differentiate into myofibroblasts and are embedded in perivascular collagen and the extracellular matrix. While the phenotypic properties of AdvSca1-SM–derived myofibroblasts have been defined, the underlying epigenetic regulators driving the AdvSca1-SM–to–myofibroblast transition are unclear. We show that the chromatin remodeler Smarca4/Brg1 facilitates AdvSca1-SM myofibroblast differentiation. Brg1 mRNA and protein were upregulated in AdvSca1-SM cells after acute vascular injury, and pharmacological inhibition of Brg1 by the small molecule PFI-3 attenuated perivascular fibrosis and adventitial expansion. TGF-β1 stimulation of AdvSca1-SM cells in vitro reduced expression of stemness genes while inducing expression of myofibroblast genes that was associated with enhanced contractility; PFI blocked TGF-β1–induced phenotypic transition. Similarly, genetic knockdown of Brg1 in vivo reduced adventitial remodeling and fibrosis and reversed AdvSca1-SM–to–myofibroblast transition in vitro. Mechanistically, TGF-β1 promoted redistribution of Brg1 from distal intergenic sites of stemness genes and recruitment to promoter regions of myofibroblast-related genes, which was blocked by PFI-3. These data provide insight into epigenetic regulation of resident vascular progenitor cell differentiation and support that manipulating the AdvSca1-SM phenotype will provide antifibrotic clinical benefits.

Authors

Austin J. Jolly, Sizhao Lu, Allison M. Dubner, Keith A. Strand, Marie F. Mutryn, Aaron Pilotti-Riley, Etienne P. Danis, Raphael A. Nemenoff, Karen S. Moulton, Mark W. Majesky, Mary C.M. Weiser-Evans

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Figure 7

CUT&RUN assay reveals TGF-β1–mediated redistribution of Brg1 from distal enhancer sites of stem genes to promoters of myofibroblast genes, while PFI-3 blocks Brg1 occupancy on myofibroblast genes.

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CUT&RUN assay reveals TGF-β1–mediated redistribution of Brg1 from di...
Subcultured AdvSca1-SM cells isolated from SM22α-Cre-YFP mice were cultured in stem cell media or stimulated with 5 ng/mL TGF-β1 or 5 ng/mL TGF-β1 + 50μM PFI-3 (“Both”) for 72 hours. Cells were adsorbed to Concanavalin A beads, permeabilized, and incubated with 0.5 μg of anti-Brg1 or anti-H3K27Ac antibody overnight. pAG-MNase was added to the samples to facilitate chromatin digestion and release of enriched DNA bound by Brg1. Enriched DNA was submitted for library preparation and sequenced and aligned to the mouse genome. Peaks represent enriched DNA fragments associated with Brg1 binding. (A) Heatmap representing the read intensity at Brg1 peaks across the AdvSca1-SM genome. (B) Read count per million mapped reads for all conditions tested shows TGF-β1 leads to increased Brg1 peaks on 12,562 genes and decreased Brg1 peaks on 310 genes as compared with AdvSca1-SM cells in the stem condition. (C) Comparative analysis of the differential Brg1 binding from CUT&RUN versus differential gene expression from RNA-Seq comparing “Stem” versus “TGF-β” and “TGF-β” versus “Both” with the parameters of Padj < 0.05 and 2-fold change. (D) The overlapping genes that showed differential Brg1 peak intensity from CUT&RUN and differential gene expression from RNA-Seq were subject to Metascape pathway analysis. (E) Representative IGV plots of stemness-related genes (left 3 plots) and myofibroblast-related genes (right 3 plots). Stemness-related genes showed decreased Brg1 reads in distal intergenic regions when stimulated with TGF-β1. Myofibroblast-related genes showed increased Brg1 reads near promoter regions when stimulated with TGF-β1; PFI-3 blocked the recruitment of Brg1 to promoter regions of myofibroblast genes.