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. 2021 Mar 26;12(4):327.
doi: 10.1038/s41419-021-03603-0.

Trajectory modeling of endothelial-to-mesenchymal transition reveals galectin-3 as a mediator in pulmonary fibrosis

Wangyue Jia #  1   2   3 , Zhaoyan Wang #  1   2   3 , Ceshu Gao #  4 , Jian Wu  5 , Qiong Wu  6   7
Affiliations

Trajectory modeling of endothelial-to-mesenchymal transition reveals galectin-3 as a mediator in pulmonary fibrosis

Wangyue Jia et al. Cell Death Dis. .

Abstract

The endothelial-to-mesenchymal transition (EndMT) is an important source of fibrotic cells in idiopathic pulmonary fibrosis (IPF). However, how endothelial cells (ECs) are activated and how EndMT impact IPF remain largely elusive. Here, we use unsupervised pseudotemporal analysis to recognize the heterogeneity of ECs and reconstruct EndMT trajectory of bleomycin (BLM)-treated Tie2creER/+;Rosa26tdTomato/+ IPF mice. Genes like C3ar1 and Lgals3 (protein name galectin-3) are highly correlated with the transitional pseudotime, whose expression is gradually upregulated during the fate switch of ECs from quiescence to activation in fibrosis. Inhibition of galectin-3 via siRNA or protein antagonists in mice could alleviate the pathogenesis of IPF and the transition of ECs. With the stimulation of human pulmonary microvascular endothelial cells (HPMECs) by recombinant proteins and/or siRNAs for galectin-3 in vitro, β-catenin/GSK3β signaling and its upstream regulator AKT are perturbed, which indicates they mediate the EndMT progress. These results suggest that EndMT is essential to IPF process and provide potential therapeutic targets for vascular remodeling.

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Conflict of interest statement

The authors declare no competing interests.

Figures

Fig. 1
Fig. 1. Verification of EndMT in a mouse model of pulmonary fibrosis.
A Schematic diagram of the construction of the IPF model in Tie2creER/+;Rosa26tdTomato/+ mice based on a C57bl/6 genetic background. B Immunofluorescence imaging of ECs (CD31, red) and fibroblasts (COL1, green) in the BLM/control groups. The region in blue represents nucleus. Asterisks indicate the co-localization of ECs and fibroblasts. Scale bar = 13 μm. C Comparative analysis of the relative proportions of EC-derived cells (Tie2+) and ECs (Tie2+CD31+) in the BLM/control groups presented as means ± SD, n = 2. x-axis: different mice derived from C57BL/6. Tie2-positive cells represent all cells of endothelial origin; Tie2 + CD31 + cells represent resident ECs that remain endothelium-feature supplemented with BLM or saline solution. y-axis: the percentage of subpopulations of Tie2-positive and Tie2CD31-positive cells. n = 2. D Gating strategy for calculating the fraction that underwent EndMT (Tie2+Vim+) cultured in vitro for 72 h after isolation from lungs by PBS perfusion. Specifically, cells gated by rectangles in black are subpopulation of interest.
Fig. 2
Fig. 2. Transcriptional heterogeneity of ECs during EndMT in IPF at single-cell level.
A Sing-cell transcriptome sequencing (scRNA-seq) using the Fluidigm C1 platform. Lung cells from the BLM and control groups were collected and sorted by FACS to harvest Tie2+ cells for scRNA-seq. B UMAP visualization of single-cell transcriptomes in the BLM and control groups. Left panel: colored by origin of cells (BLM or control). Right panel: colored according to the unsupervised clustering of cells. C Cluster-wide expression of EndMT-related markers/regulators screened among differentially expressed genes of high significance (p < 0.01) based on their GO-terms. D Expression levels of candidate regulators in whole lung samples from the BLM (day 7) and control groups according to RT-PCR. The data are presented as means ± SD, n = 3. *p < 0.05, **p < 0.01, ***p < 0.001.
Fig. 3
Fig. 3. Pseudotemporal trajectory analysis of ECs revealing factors related to the EndMT process.
A Merged pseudotime analysis of both groups revealing the transitional trajectory of ECs using Monocle3. Hollow circles: start/end nodes; black circles: branching nodes; black curve: pseudotemporal trajectory. B Expression of genes highly correlated with the pseudotemporal trajectory representing the EndMT process. C3ar1 complementary component 3a receptor 1, Lgals3 galectin-3, S100A4 S100 calcium binding protein A4, Fn1 fibronectin 1, Icam-1 intercellular adhesion molecule-1, Col4a1 collagen type IV alpha 1 chain, Tgfbi transforming growth factor beta induced, Hif1a hypoxia inducible factor 1 subunit alpha. C Expression levels of candidate regulators in whole-lung samples from the BLM and control groups according to RT-PCR. CD31: platelet and endothelial cell adhesion molecule 1, α-SMA: actin, alpha 2, smooth muscle, aorta. The data presented as the means ± SD, n = 3. *p < 0.05, **p < 0.01, ***p < 0.001.
Fig. 4
Fig. 4. Alleviation of pulmonary fibrosis by targeting C3ar1 and galectin-3 in vivo.
A Schematic diagram showing the functional exploration of C3ar1 and galectin-3 in IPF using lentivirus and galectin-3 interference (shRNA group), respectively. Alternatively, antagonists were used to block the binding affinity of C3a and galectin-3 (antagonist group). B, C Body weight and survival of IPF mice with galectin-3 shRNA lentivirus (B) (LV C3ar1 shRNA and LV galectin-3 shRNA, n = 5) or antagonists (C) (SB290157: antagonist of C3ar1; GB1107: antagonist of GB1107, n = 6). D, E HE and Masson’s trichrome staining of mouse lung sections following lentivirus or antagonist treatments. Scale bar = 500 μm (global view) or 50 μm (detailed view). F Semiquantitative morphological index/scoring of lung sections. The grade ranges from 1 (normal) to 8 (complete fibrosis). n = 30 for each group; *p < 0.05, **p < 0.01, ***p < 0.001.
Fig. 5
Fig. 5. Galectin-3 promotes the EndMT process in vitro.
A Schematic diagram showing the exploration of the potential of galectin-3 in inducing EndMT in vitro using HPMECs. B Morphological changes of HPMECs under different conditions. Complete medium (control): DMEM containing 10% FBS; starvation medium (S): DMEM containing 1% FBS; T: supplemented with 10 ng/ml TGF-β; G: supplemented with 1 μg/ml galectin-3. Scale bar = 50 μm. C Quantitative analysis of cell length of 30 randomly selected HPMECs from B using ImageJ software. D CCK-8 assay for determining the cytotoxicity of galectin-3 for ECs at days 1, 2, 4, and 7. E Expression levels of the fibroblast-related gene α-SMA in different experimental groups according to RT-PCR. Error bars represent standard deviations, n = 3. *p < 0.05, **p < 0.01, ***p < 0.001.
Fig. 6
Fig. 6. Inhibition of galectin-3 downregulates the AKT/GSK3β/β-catenin pathway in IPF mice.
Western blot analysis of galectin-3 and AKT/GSK3β/β-catenin pathway proteins in lung tissue lysates. Control: saline for intratracheal injection. BLM + DMSO: positive control using BLM for pulmonary fibrosis model construction and intraperitoneal injection of vehicle (DMSO). BLM + GB1107: intraperitoneal injection of GB1107 to inhibit galectin-3 expression, n = 5 per group. Protein targets are A galectin-3; B β-catenin; pY654 β-catenin; C AKT, pS473 AKT; D pS9 GSK3β; *p < 0.05, **p < 0.01, ***p < 0.001.
Fig. 7
Fig. 7. Inhibition of galectin-3 expression ameliorates the EndMT process by downregulating the AKT/β-catenin pathway in HPMECs.
A Schematic showing the knockdown of galectin-3 expression with siRNA, and its impact on the transition of HPMECs. HPMECs were first cultured in complete medium for 24 h, and then transfected with siRNA targeting galectin-3 for 6 h. After treatment with the siRNA, the complete medium was replaced with starvation medium (DMEM containing 1% FBS), followed by further cultivation for 48 h. B The knockdown efficiency of galectin-3 siRNA according to RT-PCR. siNC: negative control. C–G HPMECs were stimulated with TGF-β (10 ng/ml, S + T) or galectin-3 (1 μg/ml, S + G) and interfered with galectin-3 siRNA (S + T + galectin-3 siRNA and S + G + galectin-3 siRNA, respectively) for 48 h. Analysis of the expression level analysis of the target genes galectin-3, β-catenin, and SMAD2, as well as the fibroblast-related genes α-SMA and S100A in different experimental groups using RT-PCR. Error bars represent standard deviations, n = 3. H HPMECs lysate was analyzed for CD31, α-SMA, and AKT/β-catenin signaling pathway regulators, total AKT and pS473 AKT, total-β-catenin and pY654 β-catenin, PI3K and GSK3β by Western blotting. *p < 0.05, **p < 0.01, ***p < 0.001.
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