Sotatercept Reverses SIN3a Deficiency-Driven PAH by Reprogramming BMPR2/TGF-β-HIF-1α Signaling Pathways

Figures

• 

  Figure 1.. SIN3a overexpression induces global transcriptional programs in PASMCs. (A) Quantitative RT-PCR and immunoblot analysis confirming efficient SIN3a overexpression at the mRNA and protein levels in human proliferating failed donor FD-PASMCs following adenoviral transduction either with an empty vector (Ad-Control) or an adenoviral vector encoding human SIN3a (Ad-SIN3a) for 48 hours. (B) RNA sequencing workflow and analysis in SIN3a-overexpressing PASMCs and controls. (C) Heatmap of the top 5,000 DEGs illustrates a broad reprogramming of gene expression, consistent with SIN3a’s role as a master transcriptional regulator. (D) GAGE pathway enrichment analysis of differentially expressed genes reveals significant enrichment of pathways associated with TGF-β signaling, epithelial–mesenchymal transition (EMT), hypoxia, and ROS stress responses in SIN3a-overexpressing PASMCs. Functional interaction network of enriched pathways reveals SIN3a-driven upregulation (green) and downregulation (red) of signaling cascades, prominently involving extracellular matrix (ECM) dynamics and cellular stress responses. (E) WikiPathways enrichment identifies diverse regulatory modules following SIN3a…

• 

  Figure 2.. SIN3a modulates hypoxia- and TGF-β-induced signaling pathways in PASMCs. (A) (A) qRT-PCR analysis of SIN3a mRNA expression in PASMCs cultured under normoxic conditions or exposed to hypoxia (1% O2) and/or TGF-β1 stimulation. (B-C) qRT-PCR analysis of TGFB1 and BMPR2 mRNA expression under hypoxic and/or TGF-β1 conditions in the presence or absence of SIN3a overexpression. (D) ENCODE ChIP-seq analysis demonstrating HIF-1α binding at promoter regions of TGF-β signaling components, including TGFBR1 and TGFB1. (E) qRT-PCR analysis of HIF-1α mRNA expression under hypoxia and/or TGF-β1 stimulation, with or without SIN3a overexpression. (F) Representative immunoblot analysis of HIF-1α and BMPR2 protein expression under identical experimental conditions. (G) Immunofluorescence staining showing nuclear localization of HIF-1α and phosphorylation of SMAD1/5/9 under hypoxic and TGF-β1 conditions, demonstrating attenuation of hypoxia-induced nuclear translocation by SIN3a. (H-I) qRT-PCR analysis of COL1A1, COL3A1, NOX4, and NFE2L2 mRNA expression in PASMCs exposed to hypoxia (1% O2) and TGF-β1, with or without SIN3a overexpression. (J) Reactive oxygen species detection in PASMCs cultured under…

• 

  Figure 3.. Smooth muscle-specific SIN3a deficiency exacerbates RV function and remodeling in SuHx-induced PH. (A) SIN3a mRNA expression in aortic tissue from tamoxifen-induced SMMHC-CreERT2 SIN3afl/fl mice (SIN3aSMC−/−) mice and littermates SIN3afl/fl control mice. (B) Representative co-immunofluorescence staining of SIN3a (red) and α-smooth muscle actin (α-SMA; green) in aortic sections of SIN3aSMC−/− mice. (C) Right heart catheterization analysis showing right ventricular systolic pressure (RVSP; left panel) in SIN3aSMC−/− KO and littermate control mice under normoxic and Sugen/Hypoxia (SuHx) conditions for 21 days. RV hypertrophy was quantified using the Fulton index (RV/LV+S), right panel. (D) Quantification of cardiomyocyte cross area assessed by wheat germ agglutinin (WGA) staining in SIN3aSMC−/− KO and littermate control mice under normoxic and SuHx conditions. (E) qRT-PCR analysis of pro-hypertrophic gene markers (ANP, BNP, BMHC) in RV from SIN3a-deficient mice compared to littermates under normoxic and SuHx conditions. (F) Representative Masson’s trichrome staining of RV sections of SIN3aSMC−/− KO mice and littermate control mice under normoxic and SuHx conditions. (G)…

• 

  Figure 4.. Loss of SIN3a promotes inflammation, oxidative stress, and vascular remodeling in SuHx-induced PH. (A) Representative co-immunofluorescence staining of α-smooth muscle actin (α-SMA; green) and SIN3a (red) in lung sections from littermate control and SIN3aSMC−/− mice under normoxic and SuHx conditions. (B) Quantification of pulmonary arterial medial wall thickness and vascular remodeling in SIN3aSMC−/− mice littermate control and SIN3aSMC−/− mice under normoxic and SuHx conditions, as assessed by hematoxylin and eosin staining. (C-D) qRT-PCR analysis of inflammatory (TNFA) and profibrotic markers (TGFB, COL1A1, and COL3A1) in lungs from littermates and SIN3aSMC−/− mice under to normoxic and SuHx conditions. (E) qRT-PCR analysis of oxidative stress–related genes (SOD2, NOX4, NFE2L2) in lungs from controls littermates and SIN3aSMC−/− mice under normoxic and SuHx conditions. (F-G) qRT-PCR analysis of BMPR2 and HIF1A transcript levels in lungs of littermates and SIN3aSMC−/− mice under normoxic and SuHx conditions. (H) Representative immunoblot analysis of BMPR2, phospho-SMAD2/3, and phospho-SMAD1/5/9 protein levels in lungs from SIN3aSMC−/− lungs. (I-J) Densitometric quantification of…

• 

  Figure 5.. Global transcriptomic profiling of PAH-PASMCs following SIN3a overexpression and combined SIN3a with Sotatercept treatment. (A) Principal component analysis (PCA) of RNA-seq data showing distinct clustering of control, SIN3a-overexpressing, and SIN3a + sotatercept-treated PAH-PASMCs. (B) Heatmap of significantly differentially expressed genes (DEGs) across experimental conditions. (C) Unsupervised hierarchical clustering illustrating treatment-specific transcriptional signatures and regulation of genes associated with proliferative signaling, cellular homeostasis, stress response, and TGF-β signaling. (D) Gene set enrichment analysis (GSEA) reveals enrichment for hypoxia- and oxidative stress-related pathways. (E) Differential expression of BMPR2 downstream target genes ID1, ID2, and ID3 following SIN3a and SIN3a + Sotatercept treatment. (F-G) Gene Ontology (GO) enrichment analyses highlighting biological processes related to vascular remodeling, ECM organization, hypoxia response, oxidative stress, and PASMC proliferation. (H) FPKM-normalized expression levels of ID1, ID2, and ID3 across control, SIN3a-overexpressing, and SIN3a + Sotatercept-treated PAH-PASMCs.

• 

  Figure 6.. Sotatercept attenuates PAH in SIN3aSMC−/− mice by restoring BMPR2/TGF-β signaling balance and suppressing HIF-1α. (A) Schematic overview of the experimental design used to evaluate the therapeutic efficacy of Sotatercept in the SuHx-induced PAH conditional SIN3aSMC−/− KO mouse model. Tissues were harvested 35 days after initiation of treatment for molecular and histological analyses. (B) RVSP (left panel) and RV hypertrophy assessed by the Fulton Index (RV/[LV+S]; right panel) in vehicle- and Sotatercept-treated SIN3aSMC KO mice. (C). Representative right ventricular sections stained with fluorescent WGA to assess cardiomyocyte cross-sectional area, with corresponding quantitative analysis shown to the right. (D). qRT-PCR analysis of hypertrophic marker gene expression (ANP, BNP, BMHC) in RV tissue from the indicated groups. (E) Representative Masson’s trichrome-stained RV sections from vehicle- and Sotatercept-treated SIN3aSMC−/− KO mice. (F). qRT-PCR analysis of profibrotic gene expression (TGFB, COL1A1, COL3A1) in RV tissue from the indicated groups. (G). Representative hematoxylin and eosin (H&E)-stained lung sections from the indicated mice, with quantification of pulmonary…

• 

  Figure 7.. Restoration of BMPR2 and Suppression of TGF-β/HIF-1α Signaling by Sotatercept in SIN3aSMC−/− KO mice. (A) qRT-PCR analysis of ID1, ID2, and ID3 mRNA expression in lungs from SIN3aSMC−/− KO lungs treated with Sotatercept compared to vehicle-treated controls. (B) qRT-PCR analysis of HIF1α and BMPR2 mRNA expression in lungs from SIN3aSMC−/− KO lungs treated with Sotatercept compared to vehicle-treated controls. (C-D) Representative immunoblot analysis (C) and corresponding densitometric quantification (D) of SIN3a, BMPR2, SMAD signaling components, and HIF-1α protein levels in lungs from vehicle- and Sotatercept- treated SIN3aSMC−/− KO mice. (E) Schematic model illustrating the proposed mechanism by which SIN3a loss of function promotes PAH features and how Sotatercept treatment counteracts SIN3a deficiency. Sotatercept represses TGF-β signaling, HIF-1α expression, pro-inflammatory and oxidative stress pathways, while restoring BMPR2 signaling and pathway balance. Restoration of BMPR2/TGF-β signaling by SIN3a and Sotatercept limits oxidative stress, inflammation, and extracellular matrix remodeling through inhibition of HIF-1α–dependent pathways. Schematic Created with BioRender.com.