Table 1_SPHK1-mediated M2 macrophage polarization drives TGF-β1-dependent thrombus fibrosis.docx

Background and objectiveVenous 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.
MethodsHistological 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. In vivo, 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. In vitro, bone marrow-derived macrophages (BMDMs) were subjected to M2 polarization and co-cultured with fibroblasts to assess the TGF-β1-dependent fibroblast activation.
ResultsHistological 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. In vivo, PF543 treatment significantly reduced collagen deposition, TGF-β1 expression, and M2 macrophage polarization in thrombus tissue. In vitro, 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.
ConclusionThis 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.

背景与目的
静脉血栓纤维化可诱发血栓后综合征（post-thrombotic syndrome, PTS）与慢性血栓栓塞性肺动脉高压（chronic thromboembolic pulmonary hypertension, CTEPH）。M2型巨噬细胞可通过分泌转化生长因子-β1（transforming growth factor-β1, TGF-β1）促进纤维化进程。本研究旨在探讨鞘氨醇激酶1（sphingosine kinase 1, SPHK1）是否通过调控M2巨噬细胞极化进而促进血栓纤维化。

方法
对急性血栓患者与CTEPH患者的血栓组织进行组织学染色与免疫荧光（immunofluorescence, IF）检测。采用单细胞RNA测序（single-cell RNA sequencing, scRNA-seq）分析免疫细胞异质性，并鉴定巨噬细胞亚群中SPHK1的表达情况。体内实验方面，通过下腔静脉（inferior vena cava, IVC）结扎构建大鼠血栓模型，给予SPHK1抑制剂PF543，以评估其对纤维化进程与巨噬细胞极化的影响。体外实验中，分离骨髓来源巨噬细胞（bone marrow-derived macrophages, BMDMs）并诱导其发生M2极化，随后与成纤维细胞共培养，以探究TGF-β1依赖的成纤维细胞活化机制。

结果
组织学分析显示，相较于急性血栓，CTEPH患者的血栓组织中细胞外基质（extracellular matrix, ECM）沉积与巨噬细胞浸润均显著增加。Masson染色结果证实CTEPH样本中存在大量胶原纤维蓄积。对大鼠下腔静脉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存在强烈共表达，提示血栓重塑过程中存在表达SPHK1的巨噬细胞。单细胞RNA测序分析进一步揭示，M2巨噬细胞亚群（尤其是MARCO-1细胞簇）中SPHK1表达水平较高，且其表达与TGF-β1分泌密切相关。体内实验中，PF543处理可显著降低血栓组织中的胶原沉积、TGF-β1表达水平与M2巨噬细胞极化程度。体外实验中，敲低SPHK1可显著抑制BMDMs中转化生长因子-β1（TGF-β1）、精氨酸酶1（arginase 1, Arg1）、分化簇36（cluster of differentiation 36, CD36）及脂肪酸合成酶（fatty acid synthase, FASN）的表达，表明其可削弱促纤维化巨噬细胞的功能。共培养实验进一步证实，M2巨噬细胞可通过TGF-β1依赖的途径活化成纤维细胞。

结论
本研究证实，SPHK1可促进M2巨噬细胞极化并驱动TGF-β1依赖的血栓纤维化过程，明确了其在CTEPH进展中的关键作用。通过PF543对SPHK1进行药理学抑制，可有效减轻纤维化重塑并抑制M2巨噬细胞极化，提示SPHK1或可作为慢性血栓相关性纤维化的潜在治疗靶点。