Chen Xiaoyun, Li Fajiu, Ma Guofeng, Qiang Haifeng, Chen Maohe, Chen Shi, Huang Yedong, Lai Xingyue, Lin Qinghuang, Deng Chaosheng
The School of Clinical Medicine, Fujian Medical University, Department of Respiratory and Critical Care Medicine, Fujian Provincial Geriatric Hospital, Fuzhou, China.
Department of Pulmonary and Critical Care Medicine, The Sixth Hospital of Wuhan, Affiliated Hospital of Jianghan University, Wuhan, China.
Front Immunol. 2025 Nov 17;16:1681485. doi: 10.3389/fimmu.2025.1681485. eCollection 2025.
Venous thrombus fibrosis contributes to post-thrombotic syndrome (PTS) and chronic thromboembolic pulmonary hypertension (CTEPH). M2 macrophages promote fibrosis via TGF-β1 secretion. This study investigates whether sphingosine kinase 1 (SPHK1) promotes thrombus fibrosis by regulating M2 macrophage polarization.
Histological staining and immunofluorescence (IF) were performed on thrombus tissues from patients with acute thrombosis and CTEPH. Single-cell RNA sequencing (scRNA-seq) was used to characterize immune cell heterogeneity and to identify SPHK1 expression within macrophage subsets. , a rat model of thrombus was established via inferior vena cava (IVC) ligation, and the SPHK1 inhibitor PF543 was administered to evaluate its effects on fibrosis and macrophage polarization. , bone marrow-derived macrophages (BMDMs) were subjected to M2 polarization and co-cultured with fibroblasts to assess the TGF-β1-dependent fibroblast activation.
Histological analysis revealed significantly increased ECM deposition and macrophage infiltration in CTEPH thrombi compared to acute thrombi. Masson staining demonstrated extensive collagen fiber accumulation in CTEPH samples. Immunofluorescence analysis of fibrotic thrombi from a rat inferior vena cava (IVC) ligation model showed strong co-expression of SPHK1 and CD68, indicating the presence of SPHK1-expressing macrophages in thrombus remodeling. scRNA-seq analysis further revealed high SPHK1 expression in M2 macrophage subsets, particularly in the MARCO-1 cluster, and its expression was closely correlated with TGF-β1 secretion. , PF543 treatment significantly reduced collagen deposition, TGF-β1 expression, and M2 macrophage polarization in thrombus tissue. , SPHK1 knockdown markedly suppressed the expression of TGF-β1, Arg1, CD36, and FASN in BMDMs, indicating an inhibition of pro-fibrotic macrophage function. Co-culture experiments further confirmed that M2 macrophages activated fibroblasts via a TGF-β1-dependent mechanism.
This study demonstrates that SPHK1 promotes M2 macrophage polarization and drives TGF-β1-dependent thrombus fibrosis, underscoring its critical role in the progression of CTEPH. Pharmacological inhibition of SPHK1 by PF543 effectively attenuates fibrotic remodeling and suppresses M2 macrophage polarization, suggesting that SPHK1 may serve as a promising therapeutic target for the treatment of chronic thrombus-associated fibrosis.
静脉血栓纤维化会导致血栓后综合征（PTS）和慢性血栓栓塞性肺动脉高压（CTEPH）。M2巨噬细胞通过分泌转化生长因子-β1（TGF-β1）促进纤维化。本研究旨在探讨鞘氨醇激酶1（SPHK1）是否通过调节M2巨噬细胞极化来促进血栓纤维化。
对急性血栓形成患者和CTEPH患者的血栓组织进行组织学染色和免疫荧光（IF）检测。采用单细胞RNA测序（scRNA-seq）来表征免疫细胞异质性，并确定巨噬细胞亚群中SPHK1的表达。通过下腔静脉（IVC）结扎建立大鼠血栓模型，并给予SPHK1抑制剂PF543以评估其对纤维化和巨噬细胞极化的影响。对骨髓来源的巨噬细胞（BMDMs）进行M2极化处理，并与成纤维细胞共培养，以评估TGF-β1依赖性成纤维细胞活化情况。
组织学分析显示，与急性血栓相比，CTEPH血栓中的细胞外基质（ECM）沉积和巨噬细胞浸润显著增加。Masson染色显示CTEPH样本中有广泛的胶原纤维堆积。对大鼠下腔静脉（IVC）结扎模型的纤维化血栓进行免疫荧光分析，结果显示SPHK1与CD68强烈共表达，表明在血栓重塑过程中存在表达SPHK1的巨噬细胞。scRNA-seq分析进一步揭示了M2巨噬细胞亚群中SPHK1的高表达，特别是在MARCO-1簇中，且其表达与TGF-β1分泌密切相关。PF543处理显著减少了血栓组织中的胶原沉积、TGF-β1表达和M2巨噬细胞极化。SPHK1基因敲低显著抑制了BMDMs中TGF-β1、精氨酸酶1（Arg1）、CD36和脂肪酸合酶（FASN）的表达，表示对促纤维化巨噬细胞功能有抑制作用。共培养实验进一步证实，M2巨噬细胞通过TGF-β1依赖性机制激活成纤维细胞。
本研究表明，SPHK1促进M2巨噬细胞极化并驱动TGF-β1依赖性血栓纤维化，突显了其在CTEPH进展中的关键作用。PF543对SPHK1的药理学抑制有效减轻了纤维化重塑并抑制了M2巨噬细胞极化，表明SPHK1可能是治疗慢性血栓相关纤维化的一个有前景的治疗靶点。
