Smooth muscle–derived progenitor cell myofibroblast differentiation through KLF4 downregulation promotes arterial remodeling and fibrosis
Sizhao Lu, Austin J. Jolly, Keith A. Strand, Allison M. Dubner, Marie F. Mutryn, Karen S. Moulton, Raphael A. Nemenoff, Mark W. Majesky, Mary C.M. Weiser-Evans
Sizhao Lu, Austin J. Jolly, Keith A. Strand, Allison M. Dubner, Marie F. Mutryn, Karen S. Moulton, Raphael A. Nemenoff, Mark W. Majesky, Mary C.M. Weiser-Evans
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Research Article Stem cells Vascular biology

Smooth muscle–derived progenitor cell myofibroblast differentiation through KLF4 downregulation promotes arterial remodeling and fibrosis

Abstract

Resident vascular adventitial SCA1+ progenitor (AdvSca1) cells are essential in vascular development and injury. However, the heterogeneity of AdvSca1 cells presents a unique challenge in understanding signaling pathways orchestrating their behavior in homeostasis and injury responses. Using smooth muscle cell (SMC) lineage-tracing models, we identified a subpopulation of AdvSca1 cells (AdvSca1-SM) originating from mature SMCs that undergo reprogramming in situ and exhibit a multipotent phenotype. Here we employed lineage tracing and RNA-sequencing to define the signaling pathways regulating SMC-to-AdvSca1-SM cell reprogramming and AdvSca1-SM progenitor cell phenotype. Unbiased hierarchical clustering revealed that genes related to hedgehog/WNT/beta-catenin signaling were significantly enriched in AdvSca1-SM cells, emphasizing the importance of this signaling axis in the reprogramming event. Leveraging AdvSca1-SM–specific expression of GLI-Kruppel family member GLI1 (Gli1), we generated Gli1-CreERT2-ROSA26-YFP reporter mice to selectively track AdvSca1-SM cells. We demonstrated that physiologically relevant vascular injury or AdvSca1-SM cell–specific Kruppel-like factor 4 (Klf4) depletion facilitated the proliferation and differentiation of AdvSca1-SM cells to a profibrotic myofibroblast phenotype rather than macrophages. Surprisingly, AdvSca1-SM cells selectively contributed to adventitial remodeling and fibrosis but little to neointima formation. Together, these findings strongly support therapeutics aimed at preserving the AdvSca1-SM cell phenotype as a viable antifibrotic approach.

Authors

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

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Figure 5

AdvSca1-SM cells adopt a myofibroblast, and only rarely macrophage, phenotype in response to vascular injury.

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AdvSca1-SM cells adopt a myofibroblast, and only rarely macrophage, phen...
Gli1-CreERT-YFP mice were subjected to carotid arterial injury, and uninjured right and injured left carotids were harvested at 3 days, 7 days, and 3 weeks postinjury; fixed; and embedded in OCT. (A) Arterial sections were immunofluorescently stained for YFP (green), and in situ hybridization was used to detect periostin transcripts (red). Representative images from N = 3 at each time point. Note strong induction of periostin in AdvSca1-SM cells in response to injury. (B and C) Arterial sections were immunofluorescently stained for YFP (green) and CD68 (B; red) or MAC2 (C; red). Representative images from N = 3 per time point. Note that AdvSca1-SM cells do not coexpress either macrophage marker. Scale bars for panels A–C: 50 μm. M, arterial media; A, arterial adventitia; NI, neointima. Dashed lines indicate the external and internal elastic laminae. (D) Single-cell suspensions were isolated from uninjured and 7-day postinjured carotid arteries; cells were stained for SCA1, YFP, CD11c, MHCII, LY6G, CD11b, CD64, αSMA, and Aqua VI and analyzed by flow cytometry. Total macrophages (left graph) and YFP+ AdvSca1-SM cell-derived macrophages (middle right) were quantified as percentage of all live cells. Median expression of αSMA (middle left) and CD64 (right) by the YFP+ cell population was expressed as absolute value. Each point represents an individual carotid artery; N = 3. Data represent mean ± SEM; paired Student’s t tests (2 tailed); *P < 0.05; **P < 0.01.