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Deficiency of Smooth Muscle ADAR1 RNA Editing Exacerbates Vascular Remodeling and Pulmonary Hypertension [pul43_RNA-seq]

ABSTRACT: Adenosine deaminase acting on RNA 1 (ADAR1) catalyzes the conversion of adenosine (A) to inosine (I) in double-stranded RNA (dsRNA), which is critical to prevent auto-inflammatory responses mediated by activation of the type I interferon (IFN) signaling. Here, we define the role of ADAR1-dependent RNA editing in interferon-β(IFNβ) activation and pulmonary artery smooth muscle cell (PASMC) remodeling in pulmonary arterial hypertension (PAH), a devastating disease leading to right heart failure and premature death. Methods: RNA editing levels were analyzed in human pulmonary artery smooth muscle cells (PASMCs) from idiopathic PAH (IPAH) patients versus healthy controls. A conditional transgenic line ADAR1SMC-KO was generated by knocking out ADAR1 selectively in α-smooth muscle actin-positive cells, followed by hypoxic exposure to induce PH. Results: PASMCs from IPAH patients displayed decreased levels of ADAR1 mRNA and protein, accompanied by reduced A-to-I editing compared to healthy PASMCs. ADAR1 knockdown in PASMCs upregulated MDA5, PKR, IFN-β and IFN-Stimulated Genes (ISGs). Compared with controls in vivo, hypoxic ADAR1SMC-KO mice developed severe PH, as evidenced by excessive vascular remodeling in distal arterioles and increased vascular leakage resulting in elevated right ventricular systolic pressure and right ventricular hypertrophy. Mechanistically, IFNβ signaling in ADAR1SMC-KO lungs induced the recruitment of inflammatory cells, including macrophages, which in turn enhanced PASMC proliferation and muscularization. Correspondingly, pharmacological treatment with PKR kinase inhibitor Imoxin decreased IFNβ thus attenuating the hypoxia-induced PH phenotype of ADAR1SMC-KO mice. Conclusions: ADAR1-dependent A-to-I RNA editing tempers IFN signaling in PASMCs. Pathologic reduction of ADAR1 in SMCs increased IFNβ activity, macrophage recruitment, and worsened PH, thus demonstrating a direct detrimental role of vascular innate immune responses in PH. Targeting PKR could be the new therapeutic strategy aimed at treating PAH.

ORGANISM(S): Homo sapiens

PROVIDER: GSE228133 | GEO | 2025/09/03

REPOSITORIES: GEO

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Project description:Adenosine deaminase acting on RNA 1 (ADAR1) catalyzes the conversion of adenosine (A) to inosine (I) in double-stranded RNA (dsRNA), which is critical to prevent auto-inflammatory responses mediated by activation of the type I interferon (IFN) signaling. Here, we define the role of ADAR1-dependent RNA editing in interferon-β(IFNβ) activation and pulmonary artery smooth muscle cell (PASMC) remodeling in pulmonary arterial hypertension (PAH), a devastating disease leading to right heart failure and premature death. Methods: RNA editing levels were analyzed in human pulmonary artery smooth muscle cells (PASMCs) from idiopathic PAH (IPAH) patients versus healthy controls. A conditional transgenic line ADAR1SMC-KO was generated by knocking out ADAR1 selectively in α-smooth muscle actin-positive cells, followed by hypoxic exposure to induce PH. Results: PASMCs from IPAH patients displayed decreased levels of ADAR1 mRNA and protein, accompanied by reduced A-to-I editing compared to healthy PASMCs. ADAR1 knockdown in PASMCs upregulated MDA5, PKR, IFN-β and IFN-Stimulated Genes (ISGs). Compared with controls in vivo, hypoxic ADAR1SMC-KO mice developed severe PH, as evidenced by excessive vascular remodeling in distal arterioles and increased vascular leakage resulting in elevated right ventricular systolic pressure and right ventricular hypertrophy. Mechanistically, IFNβ signaling in ADAR1SMC-KO lungs induced the recruitment of inflammatory cells, including macrophages, which in turn enhanced PASMC proliferation and muscularization. Correspondingly, pharmacological treatment with PKR kinase inhibitor Imoxin decreased IFNβ thus attenuating the hypoxia-induced PH phenotype of ADAR1SMC-KO mice. Conclusions: ADAR1-dependent A-to-I RNA editing tempers IFN signaling in PASMCs. Pathologic reduction of ADAR1 in SMCs increased IFNβ activity, macrophage recruitment, and worsened PH, thus demonstrating a direct detrimental role of vascular innate immune responses in PH. Targeting PKR could be the new therapeutic strategy aimed at treating PAH.

Project description:Sensing of double-stranded RNA (dsRNA) is an important component of innate immunity. Proteins like PKR and MDA5 recognize dsRNA and activate various pathways to fight viral infection. In addition to viral dsRNA, many endogenous RNAs containing double-stranded regions can be misrecognized as immunogenic. The RNA editing enzyme ADAR1, specifically its p150 isoform, has been shown to suppress activation of PKR and MDA5 through its A-to-I editing activity. While the cytoplasmic ADAR1-p150 isoform has been well established within this role, the functions of the nuclear ADAR1-p110 isoform are less understood. To address this knowledge gap, we utilized proximity labeling by APEX2 to identify putative ADAR1-p110 interacting proteins. We identified 110 proteins across three breast cancer cell lines that either interact with p110 or are within close proximity. Many of these proteins have known roles in RNA metabolism. Nine of the identified proteins belong to the DEAD or DEAH box families of RNA helicases, including DHX9. DHX9 is overexpressed in breast cancer, with expression closely correlated with that of p110. By co-immunoprecipitation we confirmed that p110 interacts with DHX9. Knockdown of DHX9 in several triple-negative breast cancer cell lines caused cell death and activation of the dsRNA sensor PKR. In two cell lines that are refractory to knockdown of ADAR1, the combined knockdown of DHX9 and ADAR1 caused a substantial increase in PKR activity, where knockdown of either alone had no effect. Knockdown of DHX9 and ADAR1 caused activation of the type I IFN pathway, RNase L and NF-KB signaling. Activation of these proteins and pathways was not seen with individual knockdown of ADAR1 or DHX9. Activation of PKR following combined knockdown of ADAR1 and DHX9 could be rescued by expression of p110, p150, DHX9, and catalytically inactive DHX9. Additionally PKR activation could be rescued by the dsRBM of DHX9, revealing an important role for dsRBMs in suppressing PKR activation. Together these results reveal an important role for DHX9 in suppressing dsRNA sensing by multiple pathways.

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Deficiency of Smooth Muscle ADAR1 RNA Editing Exacerbates Vascular Remodeling and Pulmonary Hypertension [pul43_RNA-seq]

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