A Reservoir of Fibroblasts Promotes Recovery from Limb Ischemia

ABSTRACT: Background: The angiogenic response to ischemia restores perfusion so as to preserve tissue. A role for mesenchymal-to-endothelial transition in the angiogenic response is controversial. This study is to determine if resident fibroblasts contribute to angiogenesis. Methods: We utilized the murine model of hindlimb ischemia, and in vivo matrigel plug assay together with lineage tracing studies and single cell RNA-sequencing (scRNA-seq) to examine the transcriptional and functional changes in fibroblasts in response to ischemia. Results: Lineage tracing using Fsp1-Cre: R26R-EYFP mice revealed the emergence within the ischemic hindlimb of a small subset of YFP+ CD144+ CD11b- fibroblasts (E*cells) that expressed endothelial cell (EC) genes. Subcutaneous administration of matrigel in Fsp1-Cre: R26R-EYFP mice generated a plug that became vascularized within 5 days. Isolation of YFP+ CD11b- cells from the plug revealed a small subset of YFP+ CD144+ CD11b- E*cells which expressed EC genes. Pharmacological or genetic suppression of innate immune signaling reduced vascularity of the matrigel plug and abrogated the generation of these E*cells. These studies were repeated using human fibroblasts, with FACS analysis revealing that a small percentage of human fibroblasts that were induced to express EC markers in the matrigel plug assay. Pharmacological suppression or genetic knockout of inflammatory signaling abolished the generation of E*cells, impaired perfusion recovery and increased tissue injury after femoral artery ligation. To further characterize these E*cells, we performed scRNA-seq and revealed eight discrete clusters of cells expressing characteristic fibroblast genes, of which two clusters (C5 and C8) also expressed some EC genes. Ischemia of the hindlimb induced expansion of clusters C5 and C8. The C8 cells did not express CD144, nor did they form networks in Matrigel, but did generate angiogenic cytokines. The C5 fibroblasts most resembled E* cells in their expression of CD144 and their ability to form EC-like networks in Matrigel. Conclusion: Together, these studies indicate the presence of subsets of tissue fibroblasts which seem poised to contribute to the angiogenic response. The expansion of these subsets with ischemia is dependent upon activation of innate immune signaling, and contributes to recovery of perfusion and preservation of ischemic tissue.

ORGANISM(S): Mus musculus

PROVIDER: GSE150383 | GEO | 2020/08/08

REPOSITORIES: GEO