Lin Shigang, Lin Ruoran, Zhu Pengwei, Sun Xiaotong, Qiu Chenyang, Zhang Bohuan, He Yangyan, Xu Qingbo, Zhang Hongkun
Department of Vascular Surgery, The First Affiliated Hospital, Zhejiang University School of Medicine Hangzhou, Zhejiang, China.
Department of Cardiology, The First Affiliated Hospital, Zhejiang University School of Medicine Hangzhou, Zhejiang, China.
Am J Cancer Res. 2024 Apr 15;14(4):1866-1879. doi: 10.62347/SQOC7984. eCollection 2024.
Although the formation of NETs contributes to cancer cell invasion and distant metastasis, its role in the pathological progression of limb ischemia remains unknown. This study investigated the functional significance of NETs in cell-cell crosstalk during limb ischemia. The changes of cell subsets in lower limb ischemia samples were detected by single-cell RNA sequencing. The expression of neutrophil extracellular traps (NETs) related markers in lower limb ischemia samples was detected by immunohistochemistry and Western blotting. The signaling pathway of NETs activation in fibroblasts was verified by immunofluorescence, PCR and Western blotting. Through single-cell RNA sequencing (scRNA-seq), we identified 9 distinct cell clusters, with significantly upregulated activation levels in fibroblasts and neutrophils and phenotypic transformation of smooth muscle cells (SMCs) into a proliferative state in ischemic tissue. At the same time, the interaction between fibroblasts and smooth muscle cells was significantly enhanced in ischemic tissue. NETs levels rise and fibroblast activation is induced in ischemic conditions. Mechanistically, activated fibroblasts promote smooth muscle cell proliferation through the Wnt5a pathway. In ischemic mice, inhibition of Wnt5a mitigated vascular remodeling and subsequent ischemia. These findings highlighting the role of cell-cell crosstalk in ischemia and vascular remodeling. We found that the NETs-initiated fibroblast-SMC interaction is a critical regulator of limb ischemia via Wnt5a pathway, a potential therapeutic target for the treatment.
尽管中性粒细胞胞外诱捕网（NETs）的形成有助于癌细胞侵袭和远处转移，但其在肢体缺血病理进展中的作用仍不清楚。本研究调查了NETs在肢体缺血期间细胞间串扰中的功能意义。通过单细胞RNA测序检测下肢缺血样本中细胞亚群的变化。通过免疫组织化学和蛋白质免疫印迹法检测下肢缺血样本中与中性粒细胞胞外诱捕网（NETs）相关标志物的表达。通过免疫荧光、聚合酶链反应（PCR）和蛋白质免疫印迹法验证成纤维细胞中NETs激活的信号通路。通过单细胞RNA测序（scRNA-seq），我们鉴定出9个不同的细胞簇，其中成纤维细胞和中性粒细胞的激活水平显著上调，并且缺血组织中的平滑肌细胞（SMCs）发生表型转变为增殖状态。同时，缺血组织中成纤维细胞与平滑肌细胞之间的相互作用显著增强。在缺血条件下，NETs水平升高并诱导成纤维细胞激活。机制上，激活的成纤维细胞通过Wnt5a途径促进平滑肌细胞增殖。在缺血小鼠中，抑制Wnt5a可减轻血管重塑及随后的缺血。这些发现突出了细胞间串扰在缺血和血管重塑中的作用。我们发现，NETs引发的成纤维细胞-平滑肌细胞相互作用是通过Wnt5a途径对肢体缺血起关键调节作用，是一个潜在的治疗靶点。
