Introduction and Objective: Our recent work reported the identification of the adaptive vasculogenic fibroblast (VF), which can generate new blood vessels during tissue repair (Pal et al., Nat Commun, 2023). While these cells are physiologic and injury-inducible, they can also be therapeutically induced by inhibiting fibroblast miR-200b using tissue nanotransfection (TNT) technology (PMIDs: 28785092, 34837085, 35819852, 36380587). In individuals with diabetes, the generation of injury-induced adaptive VF formation is blunted due to epigenetic silencing of vascular genes (PMID: 39604331).Methods: To understand the mechanisms underlying VF function and significance, in this work, we developed miR-200b-429fl/fl COL1A2creER GT(ROSA)mT/mG mice. In these mice, miR-200b regions in fibroblasts (COL1A2+ cells) are knocked out in response to tamoxifen. Tamoxifen treatment not only led to Cre excision of miR-200b-429 in these mice but also induced the constitutive expression of ROSA-driven tdTomato in fibroblasts with the onset of permanent GFP expression. To determine the potential endothelial cell contribution during VF formation, miR-200b-429fl/fl-Tie2 Cre mice were generated where endothelial miR-200b levels could be specifically depleted. To gain insight into the significance of VF in vivo, ischemic hind limb studies were performed.Results: Significant lowering of miR-200b was noted in laser capture dissected dermal fibroblast/endothelial tissue elements from the wound-edge of tamoxifen-treated mice (n=4). Tamoxifen treatment led to significantly enhanced perfusion in animals subjected to ischemic injury as determined by laser speckle perfusion imaging (Perimed Inc.) (n=8). This rescue was associated with increased abundance of GFP+CD31+ VF (n=5), which accounted for 26% of all CD31+ vascular cells in the laser captured blood vessels (n=9).Conclusion: Thus, targeted lowering of miR-200b abundance within fibroblasts resulted in substantial augmentation of VF in vivo, which was associated with improved blood flow.
K. Singh: None. C. Sen: None.
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