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

ABSTRACT: 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.

ORGANISM(S): Homo sapiens

PROVIDER: GSE253415 | GEO | 2024/05/14

REPOSITORIES: GEO

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Project description:Repair-supportive mesenchymal cells (RSMCs) have been recently reported in the context of naphthalene (NA)-induced airway injury and regeneration. These cells transiently express smooth muscle actin (Acta2) and are enriched with platelet-derived growth factor receptor alpha (Pdgfra) and fibroblast growth factor 10 (Fgf10) expression. Genetic deletion of Ctnnb1 (gene coding for beta catenin) or Fgf10 in these cells using the Acta2-Cre-ERT2 driver line after injury (defined as NA-Tam condition; Tam refers to tamoxifen) led to impaired repair of the airway epithelium. In this study, we demonstrate that RSMCs are mostly captured using the Acta2-Cre-ERT2 driver when labeling occurs after (NA-Tam condition) rather than before injury (Tam-NA condition), and that their expansion occurs mostly between days 3 and 7 following NA treatment. Previous studies have shown that lineage-traced peribronchial GLI1+ cells are transiently amplified after NA injury. Here, we report that Gli1 expression is enriched in RSMCs. Using lineage tracing with Gli1Cre-ERT2 mice combined with genetic inactivation of Fgf10, we show that GLI1+ cells with Fgf10 deletion fail to amplify around the injured airways, thus resulting in impaired airway epithelial repair. Interestingly, Fgf10 expression is not upregulated in GLI1+ cells following NA treatment, suggesting that epithelial repair is mostly due to the increased number of Fgf10-expressing GLI1+ cells. Co-culture of SCGB1A1+ cells with GLI1+ cells isolated from non-injured or injured lungs showed that GLI1+ cells from these two conditions are similarly capable of supporting bronchiolar organoid (or bronchiolosphere) formation. Single-cell RNA sequencing on sorted lineage-labeled cells showed that the RSMC signature resembles that of alveolar fibroblasts. Altogether, our study provides strong evidence for the involvement of mesenchymal progenitors in airway epithelial regeneration and highlights the critical role played by Fgf10-expressing GLI1+ cells in this context.

Project description:Left heart disease (LHD) frequently causes lung vascular remodelling and pulmonary hypertension (PH). Yet, pharmacological treatment for PH in LHD is lacking and its pathophysiological basis remains obscure. We aimed to identify candidate mechanisms of PH in LHD and to test their relevance and therapeutic potential. In rats, LHD was induced by supracoronary aortic banding. Whole genome microarray analyses were performed, candidate genes were confirmed by RT-PCR and Western blots and functional relevance was tested in vivo by genetic and pharmacological strategies. In lungs of LHD rats, mast cell activation was the most prominently upregulated gene ontology cluster. Mast cell gene upregulation was confirmed at RNA and protein levels and remodelled vessels showed perivascular mast cell accumulations. In LHD rats treated with the mast cell stabiliser ketotifen, or in mast cell deficient Ws/Ws rats, PH and vascular remodelling were largely attenuated. Both strategies also reduced PH and vascular remodelling in monocrotaline-induced pulmonary arterial hypertension, suggesting that the role of mast cells extends to noncardiogenic PH. In PH of different aetiologies, mast cells accumulate around pulmonary blood vessels and contribute to vascular remodelling and PH. Mast cells and mast cell-derived mediators may present promising targets for the treatment of PH. Whole rat genome microarray analyses were performed in lung homogenates of three rats with established PH following supracoronary aortic banding and in three sham-operated controls. Out of a total of 28,000 analysed genes, differential expression defined as 2-fold change with p<0.05 was evident for 120 genes. Of these, 76 were upregulated and 44 downregulated in aortic banding compared with control lungs gene. Enrichment analysis revealed regulation of mast cell specific genes - 13 out of 20 genes were significantly upregulated in aortic banding compared with control lungs. To test for a putative functional role of mast cells in lung vascular remodelling and PH in LHD, we applied a pharmacological approach by treatment of aortic banding rats with the mast cell stabiliser ketotifen. Microarray analysis then compared rats that were treated with untreated.

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

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