GLI1+ cells contribute to vascular remodeling in hypoxia- and cigarette smokeinduced pulmonary hypertension

The precise origin of newly formed alpha smooth muscle actin-positive (ACTA2+) cells appearing in non-muscularized vessels in the context of pulmonary hypertension (PH) is still debatable, although it is believed that they predominantly derive from pre-existing vascular smooth muscle cells (VSMCs). Here, Gli1Cre-ERT2; tdTomatoflox mice were used to lineage-trace GLI1+ cells in the context of PH using two independent models of vascular remodeling (VR) and reverse remodeling (RR): Hypoxia and cigarette-smoke exposure (SE). Hemodynamic measurements, right ventricular hypertrophy assessment, flow cytometry, and histological analysis of thick lung sections followed by state-of-the-art 3D reconstruction and quantification using Imaris software were used to investigate the contribution of GLI1+ cells to neo-muscularization of the pulmonary vasculature. The data show that GLI1+ cells are abundant around distal, nonmuscularized vessels during steady state, and that this lineage contributes to around 50% of newly formed ACTA2+ cell around these normally non-muscularized vessels. During RR, cells derived from the GLI1+ lineage are largely cleared in parallel to the reversal of muscularization. Partial ablation of GLI1+ cells greatly prevented VR in response to hypoxia and attenuated the increase in RVSP and right heart hypertrophy. Single-cell RNA sequencing (scRNA-seq) on sorted lineage-labeled GLI1+ cells revealed an Acta2high fraction of cells with pathways in cancer and MAPK signaling as potential players in reprogramming these cells during VR. Analysis of human lungderived material suggests that GLI1 signaling is overactivated in both Group 1 and Group 3 PH and can promote proliferation and myogenic differentiation. Our data highlight GLI1+ cells as an alternative cellular source of VSMCs in PH and suggest that these cells and the associated signaling pathways represent an important therapeutic target for further studies. human Pulmonary Artery Smooth Muscle Cells (hPASMCs) treated or not with GLI antagonist (GANT61), 20uM, 24 hours

肺动脉高压（pulmonary hypertension, PH）背景下非肌化血管中出现的新生α平滑肌肌动蛋白阳性（alpha smooth muscle actin-positive, ACTA2+）细胞的确切来源仍存在争议，尽管学界普遍认为其主要源自已存在的血管平滑肌细胞（vascular smooth muscle cells, VSMCs）。本研究采用Gli1Cre-ERT2; tdTomatoflox小鼠，通过两种独立的血管重构（vascular remodeling, VR）与逆向重构（reverse remodeling, RR）模型——缺氧模型与香烟烟雾暴露（cigarette-smoke exposure, SE）模型，对肺动脉高压背景下的GLI1阳性（GLI1+）细胞进行谱系示踪。通过血流动力学检测、右心室肥厚评估、流式细胞术、厚肺组织切片组织学分析，结合Imaris软件开展前沿三维重建与定量分析，探究GLI1+细胞对肺血管新生肌化的贡献。研究数据显示，稳态下GLI1+细胞大量富集于远端非肌化血管周围，且该谱系细胞约占此类正常非肌化血管周围新生ACTA2+细胞的50%。在逆向重构过程中，源自GLI1+谱系的细胞会随肌化逆转被大量清除。部分敲除GLI1+细胞可显著阻断缺氧诱导的血管重构，并缓解右心室收缩压（right ventricular systolic pressure, RVSP）升高与右心肥厚。对分选得到的谱系标记GLI1+细胞进行单细胞RNA测序（single-cell RNA sequencing, scRNA-seq）分析，发现存在高表达ACTA2的细胞亚群，其癌症通路与丝裂原活化蛋白激酶（mitogen-activated protein kinase, MAPK）信号通路或为血管重构过程中细胞重编程的潜在调控通路。对人类肺组织样本的分析表明，1型与3型肺动脉高压中GLI1信号均存在过度激活，且可促进细胞增殖与肌源性分化。本研究证实GLI1+细胞是肺动脉高压中血管平滑肌细胞的另一重要细胞来源，并提示此类细胞及其相关信号通路可作为后续研究的关键治疗靶点。本研究还使用了经20μM GLI拮抗剂（GANT61）处理24小时，以及未接受处理的人类肺动脉平滑肌细胞（human Pulmonary Artery Smooth Muscle Cells, hPASMCs）。