Inactivation of AXL in Cardiac Fibroblasts Alleviates Right Ventricular Remodeling in Pulmonary Hypertension

Abstract

Right ventricular (RV) adaptation critically determines survival in pulmonary hypertension (PH). Since cardiac fibroblasts (FBs) are crucial mediators of cardiac fibrosis, we aim to uncover the mechanism underlying their activation during RV remodeling. Using single-nucleus RNA sequencing (snRNA-seq) across three rodent PH models-hypobaric hypoxia and Sugen 5416/hypoxia in mice, and monocrotaline in rats-we identified elevated Axl expression in RV FBs under PH. AXL upregulation was consistently observed in RV FBs from both human PH patients and animal models. Cardiac FB-specific Axl overexpression exacerbated RV remodeling in both PH and pulmonary artery banding (PAB) models, whereas Axl knockdown in FBs alleviated this remodeling. Functionally, AXL promoted FB proliferation, migration, and extracellular matrix synthesis via the PI3K-AKT pathway, facilitating nuclear translocation of NFIC, which in turn promoted the transcription of targeted genes such as COL1A1. Inhibiting PI3K or administering R428 mitigated AXL-driven RV remodeling in PH, and R428 also ameliorated remodeling in PAB mice. In conclusion, AXL signals the PI3K-AKT pathway to license nuclear translocation of NFIC, thereby dictating the transcription of fibrotic genes in FBs and driving RV remodeling. These findings reveal novel insights into RV pathophysiology and highlight AXL as a potential therapeutic target for PH-induced RV remodeling.

Keywords: AXL; pulmonary hypertension; right ventricular remodeling; single‐nucleus RNA sequencing; transcription factor nuclear factor I C.

Figure 1

Elevated AXL expression levels in RV FBs of human and rodent PH. A) The experimental scheme involved harvesting RV tissues from PH mice under hypobaric hypoxia (HH) conditions and control mice in ambient air (NN) for the single‐nuclei RNA sequencing. B) RV free wall thickness (RVFWT) in PH mice (n = 5) and the control group (n = 8). C, D) Representative images of WGA staining (C) and the quantification of RV cardiomyocytes (D) in PH mice compared to the control group (n = 5/group); Scale bar: 50 µm. E) UMAP visualization of cell clustering across RV samples from both groups. F) Increased ligand‐receptor signaling in PH RV, with cardiomyocytes and FBs serving as signal‐sending (source) cells, and FBs acting as signal‐receiving (target) cells. G) Trajectory analysis of FBs showing gene signatures according to the group and cell state. H) Gene expression patterns in FBs of two transition states due to PH development were visualized in a heatmap and categorized into four clusters (C1‐C4) based on functional gene ontology. I) Axl expression intensity across trajectory states. J) Representative images of double immunofluorescent staining against AXL and Vimentin and quantification of AXL in FBs of RV tissues from HH or NN group (n = 6/ group); Scale bar: 50 µm. K) The proportion of AXL‐expressing FBs in RVs of the HH group (n = 10) or NN group (n = 9) by flow cytometry. L) AXL protein levels in the RVs of PH mice and the control group (n = 5/group). M) Representative images of double immunofluorescent staining against AXL and Vimentin and quantification of AXL in RV tissues from human PAH patients or controls; Scale bar: 20 µm. Data represent mean ± SEM. ^* P < 0.05, ^** P < 0.01, ^*** P < 0.001, ^**** P < 0.0001 compared to corresponding controls, as analyzed by unpaired t test.

Figure 2

Axl overexpression in cardiac FBs aggravates RV remodeling in PH. A) Experimental scheme illustrating the treatment of mice with AAV9‐Postn‐hAXL (to overexpress AXL in cardiac FBs) or AAV9‐Postn‐Ctrl, followed by HH or NN conditions for four weeks (G1‐G4 groups as indicated). B–E) Right ventricular systolic pressure (RVSP; n = 9‐11/group) (B); Fulton index (n = 4‐8/group) (C); right ventricular free wall thickness (RVFWT; n = 10‐12/group) (D) and tricuspid annular plane systolic excursion (TAPSE; n = 10‐12/group) (E) in PH or control mice receiving AAV9‐Postn‐hAXL or AAV9‐Postn‐Ctrl. F, G) The transcriptional level of Nppa (F) and Nppb (G) in RV tissues from PH or control mice receiving AAV9‐Postn‐hAXL or AAV9‐Postn‐Ctrl (n = 5‐6/group). H–M) Representative HE staining (H) and assessment (I) of right ventricular size measured by right ventricular hypertrophy fraction; WGA staining (J) and cardiomyocyte size (K); IHC staining for collagen I (L) and the abundance of collagen deposition (M) measured by collagen I‐positive area; Masson staining for fibrosis N) and the quantification O) of fibrotic area in RV tissues from PH or control mice receiving AAV9‐Postn‐hAXL or AAV9‐Postn‐Ctrl, respectively. n = 4‐6/group; Scale bar: 50 µm. Data represent mean ± SEM. ^* P < 0.05, ^** P < 0.01, ^*** P < 0.001, ^**** P < 0.0001 compared to the indicated group, as analyzed by One‐way ANOVA test as appropriate.

Figure 3

Knockdown of Axl in cardiac FBs ameliorates RV Remodeling in PH. A) Experimental scheme depicting the four‐week exposure of Axl ^flox/flox mice to HH or normoxic air (NN) after administration of either AAV9‐Postn‐Cre (to generate Axl ^ΔFB mice) or AAV9‐Postn‐Ctrl (control mice). The four experimental groups (G1‐G4) are indicated. B–E) RVSP (n = 8‐10/group) (B); Fulton index (n = 4‐6/group) (C); RVFWT (n = 9‐10/group) (D) and TAPSE (n = 9‐10/group) (E) in Axl ^ΔFB mice or controls under HH or NN conditions. F, G) The transcriptional level of Nppa (F) and Nppb (G) in RV tissues from G1 to G4 groups (n = 4‐6/group). H–M) Representative HE staining (H) and quantification (I) of right ventricular size measured by right ventricular hypertrophy fraction; WGA staining (J) and cardiomyocyte size by cardiomyocyte score (K); and IHC staining for collagen I (L) and the abundance of collagen deposition (M) measured by collagen I‐positive area; Masson staining for fibrosis N) and the quantification O) of fibrotic area in RV tissues from Axl ^ΔFB mice or controls under HH or NN conditions, respectively. n = 5/group; Scale bar: 50 µm. Data represent mean ± SEM. ^* P < 0.05, ^** P < 0.01, ^*** P < 0.001, **** P < 0.0001 compared to the indicated group, as analyzed by One‐way ANOVA test.

Figure 4

The impact of AXL expression on FB Function. A) The enrichment pathways of upregulated genes in Axl‐positive FBs compared to Axl‐negative FBs. BP, biological process; MF, molecular function. B–D) The GSEA analysis indicated enriched gene profiling related to cellular response to growth factor stimulus (B), cell migration (C), and ECM structural constituent (D) in Axl‐positive FBs. E–G) The differences in growth score (E), migration score (F), and ECM score (G) between Axl‐positive and Axl‐negative FBs. H) The effect of Axl silencing on FB proliferation in response to hypoxia for 24 h, measured by EdU assay (distributed in four independent experiments). I) Correlation analysis reveals a significant positive relationship between Axl expression and collagen‐related genes in FBs. J–M) The effect of AXL inhibitor R428 on proliferation (J), migration (K, L), and synthesis of collagen COL1A1 (M) of FBs infected with AdAxl or AdCtrl in the presence or absence of R428 under hypoxic conditions (distributed in three independent experiments). Data represent mean ± SEM. ^** P < 0.01, ^*** P < 0.001 compared to indicated group, as analyzed by One‐way ANOVA test.

Figure 5

Suppression of NFIC rescues FB activation in response to AXL upregulation. A) The differentially expressed transcription factors (TFs) in FBs from PH RV relative to control RVs. The letter “g” is used as the abbreviation for genes regulated by transcription factors, and the number represents the number of genes. B) The combinatorial patterns of expressed TFs based on the connection specificity index (CSI) using SCENIC. C) Predicted TFs regulating the genes in a positive relationship with the Axl expression levels in FBs. D) The effect of Nfic knockdown on the proliferation of FBs infected with AdAxl or AdCtrl under hypoxic conditions, measured by EdU incorporation assay (distributed in six independent experiments). E) Representative images and quantification of scratch assays showed decreased migratory ability in FBs with Axl overexpression upon Nfic suppression (distributed in three independent experiments). F) The effect of Nfic knockdown on the synthesis of collagen COL1A1 in FBs infected with AdAxl or AdCtrl under hypoxic conditions, as measured by ELISA (distributed in three independent experiments). G, H) Representative blotting image (G) and quantification (H) of NFIC protein levels in cytosolic and nuclear fractions from FBs infected with either AdAxl or AdCtrl under hypoxic or normoxic conditions (distributed in four independent experiments). Protein levels were normalized to GAPDH in the cytosol and histone H3 in the nucleus. I) Venn diagram illustrating 18 genes commonly regulated by Nfic, identified via ENCODE and TRUST databases, with Col1a1 featured as a key target. J) The identification of NFIC's binding to the Col1a1 promoter in FBs with Axl overexpression in response to hypoxia by CHIP‐PCR (distributed in three independent experiments). The bands from the Input sample serve as the positive control for the ChIP‐PCR. Data represent mean ± SEM. ^* P < 0.05, ^** P < 0.01, ^*** P < 0.001, ^**** P < 0.0001 compared to the indicated group, as analyzed by One‐way ANOVA test.

Figure 6

Inhibition of the PI3K‐AKT pathway alleviates FB activation in response to AXL. A) The analysis of enriched pathways for DEGs revealed 21 common pathways between Axl‐positive and Axl‐negative FBs, as well as between PH and control FBs. B) Major differential pathways (as in A) were visualized in a bar plot. C) EdU incorporation assay showed cell proliferation increased under hypoxia in Axl‐overexpressing FBs, and this effect was reversed by a PI3K‐Akt inhibitor (LY294002) (distributed in three independent experiments). D) The scratch assay demonstrated Axl overexpression in FBs under hypoxia enhanced cell migration, an effect that could be counteracted by PI3K inhibition (distributed in three independent experiments). E) COL1A1 expression at the protein level was elevated in Axl‐overexpressing FBs, and this increase was blunted by PI3K inhibition (distributed in three independent experiments). F, G) Representative blotting image (F) and quantification (G) of NFIC protein levels in cytosolic and nuclear fractions from FBs infected with either AdAxl or AdCtrl under hypoxic conditions in the presence or absence of PI3K inhibitor (distributed in three independent experiments). Protein levels were normalized to GAPDH in the cytosol and histone H3 in the nucleus. H) AKT phosphorylation in RVs from either AAV9‐Postn‐hAXL or AAV9‐Postn‐Ctrl recipients under HH conditions (n = 5/group). I) AKT phosphorylation in RVs from either Axl ^ΔFB mice or control mice under HH conditions (n = 5/group). Data represent mean ± SEM. ^* P < 0.05, ^** P < 0.01, ^*** P < 0.001, ^**** P < 0.0001 compared to indicated group, as analyzed by One‐way ANOVA test; ns indicates not significant.

Figure 7

AXL‐exacerbated RV remodeling in PH is rescued by R428 or LY294002. A) Experimental scheme outlining the treatment protocol for mice that received either AAV9‐Postn‐hAXL or AAV9‐Postn‐Ctrl, followed by administration of LY294002 (a PI3K inhibitor) or R428 (an AXL inhibitor) every other day for two weeks, starting two weeks after HH exposure (G1‐G4 groups as indicated). B–D) RVSP (n = 5‐9/group) (B); RVFWT (n = 5‐9/group) (C) and TAPSE (n = 5‐9/group) (D) in mice receiving either AAV9‐Postn‐hAXL or AAV9‐Postn‐Ctrl, followed by administration of LY294002 or R428 under HH conditions. E, F) The transcriptional level of Nppa (E) and Nppb (F) in RV tissues from G1 to G4 groups (n = 5‐9/group). G, H) Representative HE staining images (G) and quantification analysis (H) revealed PI3K blockade or R428 mitigated RV hypertrophy fraction in AAV9‐Postn‐hAXL recipients exposed to HH conditions (n = 5–8/group). I, J) Representative WGA staining images (I) and quantification (J) demonstrated a reduced cardiomyocyte size in AAV9‐Postn‐hAXL recipients exposed to HH conditions (n = 5–8/group). K, L) Representative IHC staining images (K) and quantification (L) showed a reduction in Collagen I‐positive areas in RV tissue of mice receiving AAV9‐Postn‐hAXL under HH conditions (n = 5–8/group). M) Synopsis of proposed mechanism: In the context of PH, excessive AXL signaling activates the PI3K‐AKT pathway, facilitating the nuclear translocation of NFIC. This process promotes the transcription of targeted genes that enhance FB proliferation, migration, and collagen synthesis, contributing to RV remodeling. This remodeling can be mitigated by blocking the PI3K pathway with LY294002 and using R428 (Created in BioRender. Yan, Y. (2025) https://BioRender.com/s34e956). Data represent mean ± SEM. ^* P < 0.05, ^** P < 0.01, ^*** P < 0.001, ^**** P < 0.0001 compared to indicated group, as analyzed by One‐way ANOVA test; Scale bar: 50 µm.