Global knockout of melanoma differentiation-associated protein 5 protects mice from chronic hypoxia/SU5416-induced pulmonary hypertension

American Journal of Physiology-Lung Cellular and Molecular Physiology · 2026-04-24

This study identifies the role of melanoma differentiation-associated protein 5 (MDA5) in the development of pulmonary hypertension and pulmonary artery muscularization. The results indicate that MDA5 is important for pulmonary artery mural cell function, and that MDA5 knockout reduces immune dysregulation by decreasing a type I interferon gene signature in immune cells, particularly monocytes and macrophages.

Endothelial <i>GATA6</i> deficiency suppresses intracellular TLR3-interferon signaling in HPAECs and promotes interferon response in HPASMCs

bioRxiv (Cold Spring Harbor Laboratory) · 2025-12-11

Abstract GATA6 is a key transcription factor crucial for maintaining endothelial cell (EC) homeostasis. The dysregulation of endothelial immune function is a central feature in diseases such as pulmonary arterial hypertension (PAH). In this study, we explored the consequences of GATA6 deficiency in human pulmonary arterial endothelial cells (HPAECs) and its impact on immune response pathways. We report that siRNA-induced GATA6 deficiency or the GATA inhibitor led to significant downregulation of interferon response genes and a marked reduction in toll-like receptor 3 ( TLR3 ) expression. GATA6 overexpression enhanced the expression of these genes, and TLR3 inhibition abrogated this response in HPAECs. Furthermore, conditioned medium (CM) from GATA6 -deficient HPAECs upregulated interferon genes in pulmonary artery smooth muscle cells ( HPASMCs ), indicating a paracrine effect. Overall, these findings highlight the critical role of GATA6 in modulating TLR3 signaling and immune responses in endothelial cells and suggest its involvement in endothelial-smooth muscle cell interactions in vascular inflammation. New and Noteworthy We show that endothelial GATA6 is required for proper activation of intracellular TLR3 -interferon signaling in HPAECs. GATA6 loss diminishes interferon pathway responses in endothelial cells while promoting an exaggerated interferon signature in adjacent smooth muscle cells via secreted factors. This work reveals a new GATA6-dependent mechanism governing endothelial–smooth muscle crosstalk in pulmonary vascular disease.

Abstract 4365999: Knockout of the cytosolic RNA receptor melanoma differentiation-associated protein 5 reduces pulmonary hypertension

Circulation · 2025-11-03

Background: Pulmonary arterial hypertension (PAH) is a severe disease affecting the pulmonary arteries, causing increased blood pressure due to lumen narrowing. Aberrant proliferation of endothelial cells (ECs) and smooth muscle cells (SMCs), along with pro-inflammatory mediators and perivascular immune cell accumulation, contributes to arterial remodeling. Hypothesis: We hypothesized that the cytosolic RNA receptor melanoma differentiation-associated protein 5 (MDA5) contributes to PAH by dysregulating pulmonary vascular cell function and immune cell response. Methods: Lung tissue sections from control and PAH patients underwent immunofluorescence staining and confocal microscopy. siRNA targeting IFIH1 (gene for MDA5) was used for gene silencing in pulmonary artery ECs. BrdU incorporation and DNA staining analyzed the cell cycle; a Matrigel assay examined network formation, and a gap closure assay measured migration . Ifih1 -/- mice were exposed to the hypoxia/SU5416 (HX/Su) protocol for 21 days. Media wall thickness was quantified in histological sections. Immunohistochemistry assessed perivascular immune cell accumulation. PCR from lung tissue showed pro-inflammatory mRNA expression. Results: In lung tissue from control patients, MDA5 immunoreactivity was widely distributed throughout the pulmonary artery wall, with the strongest expression observed in immune cells. In the remodeled pulmonary arteries of PAH patients, MDA5 immunostaining was reduced in ECs. In cultured human pulmonary artery ECs, gene silencing of IFIH1 disrupted cell cycle progression, network formation, and gap closure. Bulk RNA sequencing analysis revealed the differential expression of 2,533 genes, affecting gene ontologies such as cell death and survival, as well as cell cycle. After exposing whole-body Ifih1 -/- mice to the Hx/Su protocol, we detected reduced right ventricular systolic pressure and pulmonary artery media wall thickness compared to wild-type mice. Pro-inflammatory mediators, interferon-regulated genes, and perivascular accumulation of CD11b-positive myeloid cells were decreased in the lung tissue of Hx/Su-exposed Ifih1 -/- mice. Conclusions: Our data suggest a protective effect of whole-body MDA5 knockout in mice, which may be due to a reduction in pro-inflammatory mediators and perivascular immune cell accumulation. Whether a decrease of MDA5 in ECs promotes or protects against PH will need further investigation.

Endothelial ADAR1 Deficit Induces the NOCT-IRF7 Axis in Pulmonary Hypertension

Circulation Research · 2025-12-04 · 4 citations

BACKGROUND: Early apoptosis of pulmonary artery endothelial cells (PAECs) is a driver of vascular remodeling and pulmonary hypertension (PH), but its regulation is poorly defined. Adenosine deaminase acting on RNA 1 (ADAR1, gene name ADAR ) is an RNA editing enzyme that converts adenosine to inosine (A-to-I) in RNA transcripts and participates in RNA metabolism. While deficiency in ADAR1-mediated RNA editing stimulates cellular innate immunity signaling and can promote apoptosis, the exact ADAR1 RNA editing targets and downstream mechanisms regulating PAEC survival are unknown. We sought to define the functions and targets of ADAR1-dependent RNA editing that control pulmonary endothelial pathophenotypes in PH. METHODS: ADAR1 or Nocturnin (NOCT) expression and A-to-I RNA editing levels were evaluated in human PAH lungs by immunofluorescent staining and single cell RNA sequencing, respectively. Mice carrying a human missense ADAR mutation and genetic deletion of Noct with interleukin-6 (il6) transgene were studied in chronic hypoxia-induced PH in vivo models. RESULTS: ADAR1 expression was downregulated in the pulmonary vascular endothelium and in lung tissue of human and mouse PH. Global A-to-I RNA editing was decreased in lungs from PAH patients and hypoxic PH mice. In vitro, hypoxia, a PH trigger, downregulated ADAR1 in PAECs. Circadian gene NOCT was identified as a direct ADAR1 target which carries two active A-to-I RNA editing sites in the 3’UTR. In human PAH lungs, NOCT editing levels were reduced, while NOCT protein level increased. Correspondingly, in vitro, ADAR silencing increased NOCT mRNA levels, thus inducing dsRNA-MDA5 sensing interferon signaling and PAEC apoptosis. Importantly, silencing of NOCT reversed these changes. Forced NOCT expression phenocopied the effect of ADAR1 knockdown, upregulating interferon signaling molecules and increasing apoptosis. Chronically hypoxic PH mice carrying human ADAR mutation displayed worsened PH. Forced adeno-associated virus (AAV) expression of Adar improved monocrotaline-induced PH in rats. Genetic deletion of Noct mitigated PH in hypoxic il6-expressing transgenic PH mice, emphasizing the crucial role of NOCT in PH pathogenesis. CONCLUSIONS: Hypoxia-induced ADAR1 deficiency upregulates NOCT expression to induce PAEC interferon signaling activation, PAEC apoptosis, and PH. This study provides impetus to target the ADAR1-NOCT axis for more effective diagnostics and therapeutics for PH.

Lactate dehydrogenase-induced DNA Topoisomerase 1 is a novel regulator of smooth muscle cell proliferation and remodeling in pulmonary arterial hypertension

bioRxiv (Cold Spring Harbor Laboratory) · 2025-11-12

Pulmonary arterial hypertension (PAH) manifests by increased proliferation and survival of pulmonary vascular cells in small pulmonary arteries (PAs), PA remodeling and unresolved increase of PA pressure. PA smooth muscle cells (PASMCs) in PAH undergo metabolic shift to glycolysis resulting in over-production of lactate, hyper-proliferation, and apoptosis resistance, but the mechanisms are not completely understood. By using lung tissues and pulmonary vascular cells from PAH and non-diseased human lungs, unbiased proteomics, network analysis, and gain-and-loss of function approaches, we here report that up-regulation of lactate dehydrogenase A (LDHA)-lactate axis promotes PASMC-specific over-lactylation and consequent over-accumulation of DNA topoisomerase 1 (TOP1) in small remodeled PAs from PAH lungs, leading to the up-regulation of Akt-mechanistic target of rapamycin 1 (mTORC1) signaling, hyper-proliferation, and reduced apoptosis. Smooth muscle-specific LDHA knockdown prevented, and Ldha inhibitor oxamate reversed SU5416/hypoxia-induced TOP1 accumulation, pulmonary vascular remodeling, and pulmonary hypertension (PH) in mice. Pharmacological inhibition of TOP1 with indotecan suppressed Akt-mTORC1, decreased proliferation, induced apoptosis in human PAH, but not control PASMCs, and reversed PA remodeling, PH, and RV dysfunction in rats. Collectively, these data provide a novel mechanistic link from LDHA-driven lactate over-production through lactylation and overaccumulation of TOP1, to the up-regulation of Akt-mTORC1, hyper-proliferation and apoptosis resistance of PASMCs, pulmonary vascular remodeling, and PH, and identify TOP1 as a new potentially attractive molecular target for the remodeling-focused therapeutic intervention. Take-home message: LDHA-lactate-induced over-lactylation and overaccumulation of Topoisomerase 1 (TOP1) promotes pulmonary artery smooth muscle cell hyper-proliferation, remodeling, and pulmonary arterial hypertension, which are reversed by TOP1 inhibitor indotecan.

Noncanonical HIPPO-MST1/2 Promotes Hyperproliferation of Pulmonary Vascular Cells through CDC20

American Journal of Respiratory Cell and Molecular Biology · 2025-09-05 · 1 citations

Novel Insights Into Right Ventricular Dysfunction in Pulmonary Arterial Hypertension: The Role of Mitochondrial Dysfunction and Cell Cycle Arrest

American Journal of Respiratory and Critical Care Medicine · 2025-05-01

Abstract Rationale: Pulmonary arterial hypertension (PAH) is a progressive and fatal disease characterized by pulmonary artery remodeling. The subsequently increased right ventricle (RV) afterload leads to RV hypertrophy and dysfunction, which are the main causes of death in PAH patients. However, despite the critical role of RV function in patient prognosis, no existing therapies effectively prevent RV dysfunction. This study utilized a novel rat model of severe mitochondrial dysfunction (MD) induced by a point mutation in the NFU1 protein (NFU1G206C). These rats develop a spontaneous PAH phenotype with severe obliterative small pulmonary artery disease, increased RV pressure, RV hypertrophy, and RV dysfunction. However, the molecular players that connect MD with RV dysfunction remain unclear. Methods: RV functional in 10-week-old NFU1G206C and control wild-type (WT) rats was investigated using pressure-volume loops. The RV transcriptome profile was assessed by 10X single-cell RNA sequencing (scRNA-seq). Additionally, we employed pulmonary artery banding (PAB) as a model of isolated RV pressure overload to validate key findings in NFU1G206C RVs. Results: NFU1G206C rats exhibited a Group 1 PAH phenotype with increased RV afterload, decreased RV ejection fraction, stroke volume, cardiac output, and RV-PA uncoupling. The scRNA-seq profiling of NFU1G206C vs. WT RV cardiomyocytes (CMs) revealed 35 differentially expressed genes (DEGs, fold change ≥5) identified as transcriptional factors or signaling pathways regulating angiogenesis or responsible for cytoskeletal reorganization, activation of proteasome degradation, metabolic adaptation, and antioxidant defense. While these genes reflected the compensatory mechanisms to RV stress, the potential contributors to RV maladaptation were identified as cell cycle inhibitors Gadd45g, Ypel3, and Nr1d1. The assessment of protein levels confirmed a significant increase, 1.00 ± 0.39 vs 2.92 ± 0.94 (p&amp;lt;0.0001), 1.00 ± 0.70 vs 3.36 ± 1.10 (p&amp;lt;0.001) and 1.00 ± 0.99 vs 6.38 ±3.08 (p&amp;lt;0.01) respectively in the expression of cell cycle regulators in the RV tissues of NFU1G206C compared to WTs. A similar upregulation of Gadd45g and Ypel3 was observed in RVs of PAB rats and human subjects with decompensated RV function. Conclusion: The sustained G2/M phase arrest is a recognized contributor to cardiac hypertrophy, yet its precise regulators in RV remain unknown. This study, for the first time, reports the significant overexpression of G2/M progression inhibitors in the dysfunctional RVs from novel and established preclinical models of RV stress and decompensated PAH patient RVs. It also implicates the critical role of MD as a key driver of CM cell cycle dysregulation and RV dysfunction development.

Non-canonical HIPPO-MST1/2 promotes hyper-proliferation of pulmonary vascular cells through CDC20

bioRxiv (Cold Spring Harbor Laboratory) · 2025-04-04 · 1 citations

HIPPO components mammalian Ste20-like protein kinases 1 and 2 (MST1/2) are well described growth suppressors. However, in pulmonary arterial hypertension (PAH), MST1/2 switch their roles and become pro-proliferative and pro-survival molecules, supporting hyper-proliferation of pulmonary artery (PA) smooth muscle cells (PASMCs) and adventitial fibroblasts (PAAFs), remodeling of small PAs, and pulmonary hypertension. Here, we report that MST1/2 promotes hyper-proliferation and apoptosis resistance of human PAH PASMCs and PAAFs by up-regulating cell division cycle protein 20 (CDC20), establishing novel link between HIPPO-MST1/2 and cell cycle regulation in PAH. Authors Contributions: conception and design of the work (EAG, SSP, TVK); acquisition, analysis, and interpretation of data (TD, IZ, LJ, SOO, AP, TA, DL, DG, JRG, PJW, HD, TK); drafting and editing the manuscript (EAG, SSP, TVK, JRG, PJW).

Actin Cytoskeletal Remodeling Mediates SUN1/ARPC5 Protein Accumulation to Drive Nuclear Architectural Alteration; A Novel Signaling Axis in Pulmonary Hypertension

American Journal of Respiratory and Critical Care Medicine · 2025-05-01

Abstract Rationale Pulmonary hypertension (PH) is a deadly disease characterized by hyperproliferation and reduced apoptotic ability of pulmonary vascular cells. Due to limited treatment options, unravelling novel drug able signaling axis to be modulated as a therapeutic strategy is of utmost interest.The nuclear cellular compartment is a key organelle driving numerous biological processes, it is therefore important that the nuclear structural integrity is maintained to ensure its optimal functioning. Unfortunately, the nuclear structural integrity could be compromised or altered as a result of cellular stress, or mechanosensing. This has been a concept in various pathological conditions but not yet elucidated in the context of pulmonary hypertension. Thus, the aim of this study is to unravel how actin cytoskeleton can alter nuclear structure, and drive the phenotype observed in pulmonary hypertension towards novel therapeutic approach. Methods Using confocal microscopy, we profiled possible alterations in cytoskeleton and nuclear structures in IPAH patients, Hypoxic PCLS and Jasplakinolide-stimulated pulmonary arterial smooth muscle cells. In addition, we employed siRNA mediated knockdown strategy, pharmacological inhibition, gene and protein expression profiling (western blotting, immunofluorescence and qPCR) and bulk RNA-sequencing to identify key drivers and pathways involved the observed phenotype.Also, chromatin accessibility assay was used to investigate the enrichment of the signaling molecule of the predicted pathway upon actin disruption and our patients’ cohort. Finally, we studied the regulation of SUN1/ARPC5 and the effect of XMU-MP-1 (Hippo inhibitor) using Monocrotaline-induced PH rat model. Result: Our results show that Jasplakinolide induced the accumulation of SUN1 protein which correlates with increased actin related protein complex 5 (ARPC5), Loss of nuclear circularity, volume and nuclear shrinking. This same phenotype was observed in IPAH, Hypoxic PCLS and MCT-induced PH when compared with respective control groups.RNA-seq and chromatin accessibility assay inferred dysregulation of Hippo signaling and altered enrichment of Hippo molecules upon actin maneuver. Moreover, siRNA KD of SUN1/ARPC5 axis ameliorated the phenotype observed upon actin polymerization, IPAH phenotype and dysregulates hippo pathway.In vivo data using the Hippo signaling inhibitor (XMU-MP-1) attenuated PH-like phenotype, ablated the SUN1/ARPC5 axis and reduced hemodynamic alterations upon treatment.Interestingly, MCT-rats treated with XMU-MP-1 showed reduced expression of epigenetic players PCAF, and TBP upon treatment which correlates with reduced expression of key PAH signature genes. Conclusion: our data suggest actin mediated nuclear architectural alteration as a key signature driving PH and can be modulated via ARPC5/SUN1/Hippo signaling axis. Thus, a potential therapeutic option in PH.

Non-canonical CDC20-securin Signaling Drives Pulmonary Vascular Cell Hyper-proliferation, Remodeling, and Pulmonary Hypertension

American Journal of Respiratory and Critical Care Medicine · 2025-05-01

Abstract Background: Pulmonary Arterial Hypertension (PAH) is a progressive disease defined by a mean pulmonary arterial pressure exceeding 20 mmHg at rest. PAH is characterized by pulmonary vascular remodeling caused by increased proliferation and survival of resident PA cells. Cell Division Cycle 20 (CDC20) promotes cell cycle progression via activating the anaphase-promoting complex (APC/C) ubiquitin ligase and consequent ubiquitination and degradation of securin and cyclin B. The role of CDC20 in PAH is not known. Goal: To investigate the role of CDC20 in PAH. Methods: RNA sequencing, immunoblotting, immunohistochemistry, proliferation and apoptosis assays, SU5416/Hypoxia pulmonary hypertension (PH) rodent models. Results: CDC20 was overexpressed in distal pulmonary arteries (PAs) from human PAH and rat SU546/hypoxia PH lungs and in human PAH PA smooth muscle cells (PASMCs) and adventitial fibroblasts (PAAFs), but not endothelial cells, compared to controls. This was associated with increased proliferation and deficiency of pro-apoptotic protein Bcl-2 interacting mediator of cell death (BIM). Knockdown of CDC20 reduced proliferation, up-regulated BIM, and induced apoptosis in human PAH PASMCs and PAAFs, while the pan-APC/C inhibitor proTAME had no effect, suggesting APC/C-independent mechanism of CDC20 action. Interestingly, overexpression of CDC20 in PAH PASMCs and PAAFs was accompanied by over-accumulation of canonical CDC20-APC/C substrate securin. Immunocytochemical and immunoprecipitation experiments revealed that securin co-localizes and directly interacts with CDC20.CDC20 knockdown or treatment with apcin (blocks CDC20 interaction with its substrates, including securin) reduced securin protein levels. To test potential mechanisms of CDC20 up-regulation in PAH, we treated non-diseased PASMCs and PAAFs with pro-PH factors, including CCL2, PDGF-BB, 10 ng/ml TGFβ, and TNFα. Interestingly, only TNFα induced accumulation of CDC20 and securin in control cells. Furthermore, significantly higher levels of TNFα were detected in media conditioned by PAH PASMCs (PAH PASMC-CM), but not PAAFs. PAH PASMC-CM induced hyper-proliferation and CDC20 over-accumulation in non-diseased PASMCs and PAAFs. CM from PAH PASMCs transfected with siRNA CDC20 was unable to induce PASMC proliferation, suggesting that CDC20 over-accumulation is self-supported via TNFα feed-forward loop. Treatment with CDC20-specific degrader PROTAC CP5V effectively reduced CDC20 and securin protein content, inhibited proliferation, restored BIM, and induced apoptosis in PAH PASMCs and PAAFs, without affecting control cells. CP5V treatment reversed SuHx-induced PA remodeling, PH, and right ventricular hypertrophy in male and female mice. Conclusions: CDC20 promotes PASMC and PAAF hyper-proliferation and survival, remodeling, and PH through a non-canonical up-regulation of Securin. Targeting CDC20 may represent a promising strategy to treat PAH.