CD34⁺ PI16⁺ fibroblast progenitors aggravate neointimal lesions of allograft arteries via CCL11/CCR3-PI3K/AKT pathway

Rationale: Transplant-accelerated arteriosclerosis is a common complication that limits the long-term survival of organ transplant recipients. While previous studies have indicated the involvement of CD34⁺ stem/progenitor cells (SPCs) in this process, their heterogeneity and potential adverse effects remains incompletely understood. Methods: To investigate the role of CD34⁺ SPCs in transplant arteriosclerosis, we used various genetically modified mouse models, including BALB/c, C57BL/6J, CD34-CreER^T2, Rosa26-tdTomato, Rosa26-iDTR, CD34-Dre, PI16-CreER^T2, and CAG-LSL-RSR-tdTomato-2A-DTR mice. Single-cell RNA sequencing (scRNA-seq), chemokine antibody microarrays, ELISA assays, and immunohistochemistry were employed to identify fibroblast progenitors and their interactions with smooth muscle cells. Furthermore, in vivo and in vitro experiments targeting the CCL11/CCR3-PI3K/AKT signaling pathway were conducted to assess its role in the pathogenesis of transplant arteriosclerosis. Results: Single-cell RNA-seq and genetic lineage tracing revealed a subpopulation of fibroblast progenitors, characterized by high CD34 and PI16 expression, which differentiated into a distinct chemotactic fibroblast subset. Proteomic and scRNA analysis revealed that this CD34⁺ PI16^- subgroup released CCL11 (Eotaxin-1), which promoted intimal hyperplasia through the paracrine activation of smooth muscle cells. Binding of CCL11 to its receptor CCR3 activated the PI3K/AKT signaling pathway in smooth muscle cells, driving their proliferation and migration. In vivo, overexpression of CCL11 promoted neointimal hyperplasia, while neutralizing CCL11 or inhibiting CCR3 alleviated neointimal formation. Conclusions: These findings identified CD34⁺ PI16⁺ fibroblast progenitors that differentiate into specific chemotactic fibroblasts, releasing chemokines pivotal for neointima formation, suggesting a therapeutic strategy targeting their chemotactic activity.