Médea Padra, Jesper Bergwik, Anna Adler, Genevieve Marcoux, Ravi K V Bhongir, Praveen Papareddy, Arne Egesten
{"title":"Tartrate-Resistant Acid Phosphatase 5 (TRAP5) Promotes Eosinophil Migration During Allergic Asthma.","authors":"Médea Padra, Jesper Bergwik, Anna Adler, Genevieve Marcoux, Ravi K V Bhongir, Praveen Papareddy, Arne Egesten","doi":"10.1165/rcmb.2024-0304OC","DOIUrl":"https://doi.org/10.1165/rcmb.2024-0304OC","url":null,"abstract":"<p><p>Allergic asthma is characterized by type 2 inflammation and eosinophilia. Tartrate-resistant acid phosphatase 5 (TRAP5/ACP5) is a metallophosphatase expressed by alveolar macrophages that dephosphorylates osteopontin (OPN), a phosphoglycoprotein with increased expression in asthma. To investigate the role of TRAP5 during asthma, we used a murine model of ovalbumin (OVA) induced allergic airway inflammation as well as IL-33 induced airway inflammation including <i>Trap5<sup>-/-</sup></i> and wild-type (WT) mice. Histological analyses of murine lung revealed that OVA-induced inflammation induced the formation of inflammatory lesions and increased mucus production in both WT and <i>Trap5<sup>-/-</sup></i> mice. However, lower cytokine levels (including IL-5 and IL-13) were detected by multiplex immunoassay in <i>Trap5<sup>-/-</sup></i> mice after OVA-induced inflammation. Furthermore, qPCR analysis detected different gene expression profile of <i>Trap5<sup>-/-</sup></i>/OVA mice, including upregulation of <i>Il-17a</i> and downregulation of <i>Il-33</i>. Lower eosinophil numbers were measured in bronchoalveolar lavage fluid of <i>Trap5<sup>-/-</sup></i>/OVA mice using flow cytometry analysis, whereas immunofluorescence staining revealed high eosinophil number in lung tissue of both groups with OVA challenge. In the IL-33 model of type 2 inflammation, both WT and <i>Trap5<sup>-/-</sup></i> mice showed similar inflammatory responses with regard to cytokine levels and cell recruitment patterns. <i>In vitro</i>, eosinophil chemotaxis was facilitated by non-phosphorylated but not phosphorylated OPN, an effect inhibited by an α4β1 integrin inhibitor. The results suggest that TRAP5 is important in the recruitment of immune cells, including eosinophils, as well as in shaping the profile and amplification of the inflammatory response during allergic airway inflammation. Thus, TRAP5 may serve as a therapeutic target in allergic asthma. This article is open access and distributed under the terms of the Creative Commons Attribution 4.0 International License (https://creativecommons.org/licenses/by/4.0/).</p>","PeriodicalId":7655,"journal":{"name":"American Journal of Respiratory Cell and Molecular Biology","volume":" ","pages":""},"PeriodicalIF":5.9,"publicationDate":"2025-04-02","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143770942","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
{"title":"SOX17 Prevents Endothelial-Mesenchymal Transition of Pulmonary Arterial Endothelial Cells in Pulmonary Hypertension through Mediating TGF-β/Smad2/3 Signaling.","authors":"Xiaozhou Zou, Mengnan Yuan, Wei Zhou, Anqi Cai, Yili Cheng, Zibo Zhan, Yiwen Zhang, Zongfu Pan, Xiaoping Hu, Shuilian Zheng, Ting Liu, Ping Huang","doi":"10.1165/rcmb.2023-0355OC","DOIUrl":"10.1165/rcmb.2023-0355OC","url":null,"abstract":"<p><p>Endothelial-to-mesenchymal transition (EndMT) has been reported to contribute to pulmonary vascular remodeling in patients with pulmonary hypertension (PH). Our study demonstrates that SOX17, a member of the SOX (SRY-Box) transcription factor family, plays a role in regulating pulmonary arterial homeostasis through extracellular vesicles in an autocrine and paracrine manner. However, the role of SOX17 in mediating EndMT of pulmonary arterial endothelial cells (PAECs) and its intracellular mechanisms remain unclear. Here we present evidence showing that downregulation of SOX17 expression is accompanied by significant pulmonary arterial EndMT and activation of the TGF-β/Smad2/3 signaling pathway in patients with idiopathic PH and rats with PH induced by Sugen 5416/hypoxia. In primary human PAECs, canonical TGF-β (transforming growth factor-β) signaling inhibits the expression of SOX17. Overexpression of SOX17 reverses TGF-β- and hypoxia-induced EndMT. These findings suggest that SOX17 is essential for human PAECs to undergo TGF-β-mediated EndMT. Mechanistically, our data demonstrate that SOX17 prevents TGF-β-induced EndMT by suppressing ROCK1 (Rho-associated kinase 1) expression through binding to the specific promoter region of ROCK1, thereby inhibiting MYPT1 (myosin phosphatase target subunit 1) and MLC (myosin light chain) phosphorylation. Furthermore, we show that Tie2-Cre rats with endothelial cell-specific overexpression of SOX17 are protected against Sugen/hypoxia-induced EndMT and subsequent pulmonary vascular remodeling. Consistent with the <i>in vitro</i> results, compared with Tie2-Cre rats treated with Sugen/hypoxia alone, rats overexpressing SOX17 exhibited reduced levels of ROCK1 as well as decreased phosphorylation levels of MYPT1 and MLC. Overall, our studies unveil a novel TGF-β/SOX17/ROCK1 pathway involved in regulating PAECs' EndMT process, and we propose the targeting of SOX17 as a potential therapeutic strategy for alleviating pulmonary vascular remodeling in PH.</p>","PeriodicalId":7655,"journal":{"name":"American Journal of Respiratory Cell and Molecular Biology","volume":" ","pages":"364-379"},"PeriodicalIF":5.9,"publicationDate":"2025-04-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142399133","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Naoko Hara, Dorota S Raclawska, Leslie E Morgan, James C NeeDell, Lucie Dao, Ayako Kato, Ana M Jaramillo, Patrick S Hume, Fernando Holguin, William J Janssen, Eszter K Vladar, Christopher M Evans
{"title":"Requirement for Fucosyltransferase 2 in Allergic Airway Hyperreactivity and Mucus Obstruction.","authors":"Naoko Hara, Dorota S Raclawska, Leslie E Morgan, James C NeeDell, Lucie Dao, Ayako Kato, Ana M Jaramillo, Patrick S Hume, Fernando Holguin, William J Janssen, Eszter K Vladar, Christopher M Evans","doi":"10.1165/rcmb.2024-0216OC","DOIUrl":"10.1165/rcmb.2024-0216OC","url":null,"abstract":"<p><p>Mucus hypersecretion is an important pathological problem in respiratory diseases. Mucus accumulates in the airways of people with asthma and contributes to airflow limitation by forming plugs that occlude airways. Current treatments have minimal effects on mucus or its chief components, the polymeric mucin glycoproteins MUC5AC and MUC5B. This treatment gap reflects a poor molecular understanding of mucins that could be used to determine how they contribute to airway obstruction. Because of the prominence of glycosylation as a defining characteristic of mucins, we investigated characteristics of mucin glycans in asthma and in a mouse model of allergic asthma. Mucin fucosylation was observed in asthma, and in healthy mice it was induced as part of a mucous metaplastic response to allergic inflammation. In allergically inflamed mouse airways, mucin fucosylation was dependent on the enzyme fucosyltransferase 2. <i>Fut2</i> gene-deficient mice were protected from asthma-like airway hyperreactivity and mucus plugging. These findings provide mechanistic and translational links between observations in human asthma and a mouse model that may help improve therapeutic targeting of airway mucus.</p>","PeriodicalId":7655,"journal":{"name":"American Journal of Respiratory Cell and Molecular Biology","volume":" ","pages":"408-417"},"PeriodicalIF":5.9,"publicationDate":"2025-04-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142493010","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Joanna Woo, Gaoyuan Cao, Nikhil Karmacharya, Jordan Lee, Justin Lee, Kingsley C Duru, Conor McClenaghan, Steven S An, Reynold A Panettieri, Joseph A Jude
{"title":"Volume-Regulated Anion Channel Complex Modulates Mechano-Electrical Signal Responses in Human Airway Smooth Muscle Shortening.","authors":"Joanna Woo, Gaoyuan Cao, Nikhil Karmacharya, Jordan Lee, Justin Lee, Kingsley C Duru, Conor McClenaghan, Steven S An, Reynold A Panettieri, Joseph A Jude","doi":"10.1165/rcmb.2024-0160OC","DOIUrl":"10.1165/rcmb.2024-0160OC","url":null,"abstract":"<p><p>LRRC8A (leucine-rich repeat containing 8A) is an obligatory constituent of the volume-regulated anion channel (VRAC) that is fundamental to a wide range of biological processes, including regulating cell size, proliferation, and migration. Here we explored the physiological role of VRAC in excitation-contraction (E-C) coupling and shortening of human airway smooth muscle (HASM). In HASM cells, pharmacological inhibition of VRAC with DCPIB (4-[2-butyl-6,7-dichloro-2-cyclopentyl-indan-1-on-5-yl]) (0.1-10 μM) markedly attenuated swell-activated Cl<sup>-</sup> conductance, and contractile agonist (histamine or carbachol)-induced cellular stiffening as measured by single-cell patch-clamp and optical magnetic twisting cytometry, respectively. In addition, HASM cells treated with DCPIB or transfected with LRRC8A-targeting siRNA showed reduced agonist-induced phosphorylation of protein kinase B (i.e., AKT), paxillin, MYPT1, and myosin light chain. Consistent with the changes of these E-C coupling effectors, DCPIB appreciably decreased agonist-induced small airways narrowing in human precision-cut lung slices. Taken together, our findings shed new light on the mechanistic link between HASM shortening and regulatory volume decrease via LRRC8A, revealing a previously unrecognized nodal point for modulation of E-C coupling and acute airway constriction.</p>","PeriodicalId":7655,"journal":{"name":"American Journal of Respiratory Cell and Molecular Biology","volume":" ","pages":"418-428"},"PeriodicalIF":5.9,"publicationDate":"2025-04-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142520727","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
{"title":"Can ENaC \"TIP\" the Scales to Reduce Endothelial Reactive Oxygen Species and Vascular Leak during Pneumococcal Lung Injury?","authors":"Alison W Ha, Eleftheria Letsiou, Steven M Dudek","doi":"10.1165/rcmb.2024-0486ED","DOIUrl":"10.1165/rcmb.2024-0486ED","url":null,"abstract":"","PeriodicalId":7655,"journal":{"name":"American Journal of Respiratory Cell and Molecular Biology","volume":" ","pages":"349-351"},"PeriodicalIF":5.9,"publicationDate":"2025-04-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142602771","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Yanyan Xing, Yeseul Nho, Katy Lawson, Yuyan Zhu, Alexandra E Ellison, Margaret Y Chang, William Hancock, Liang Han
{"title":"MrgprC11<sup>+</sup> Jugular Neurons Control Airway Hyperresponsiveness in Allergic Airway Inflammation.","authors":"Yanyan Xing, Yeseul Nho, Katy Lawson, Yuyan Zhu, Alexandra E Ellison, Margaret Y Chang, William Hancock, Liang Han","doi":"10.1165/rcmb.2024-0153OC","DOIUrl":"10.1165/rcmb.2024-0153OC","url":null,"abstract":"<p><p>The lung is densely innervated by sensory nerves, the majority of which are derived from the vagal sensory neurons. Vagal ganglia consist of two different ganglia, termed <i>nodose</i> and <i>jugular</i> ganglia, with distinct embryonic origins, innervation patterns, and physiological functions in the periphery. Because nodose neurons constitute the majority of the vagal ganglia, our understanding of the function of jugular nerves in the lung is very limited. This study aims to investigate the role of MrgprC11<sup>+</sup> jugular sensory neurons in a mouse allergic asthma model. Our previous study has shown that MrgprC11<sup>+</sup> jugular neurons mediate cholinergic bronchoconstriction. In this study, we found that, in addition to MrgprC11, several other Mrgpr family members, including MrgprA3, MrgprB4, and MrgprD, are also specifically expressed in the jugular sensory neurons. MrgprC11<sup>+</sup> jugular neurons exhibit dense innervation in the respiratory tract, including the larynx, trachea, proximal bronchus, and distal bronchus. We also found that receptors for IL-4 and oncostatin M, two critical cytokines promoting allergic airway inflammation, are mainly expressed in jugular sensory neurons. Both IL-4 and oncostatin M can sensitize the neuronal responses of MrgprC11<sup>+</sup> jugular neurons. Moreover, ablation of MrgprC11<sup>+</sup> neurons significantly inhibited airway hyperresponsiveness in the asthmatic lung, demonstrating the critical role of MrgprC11<sup>+</sup> neurons in controlling airway constriction. Our results emphasize the critical role of jugular sensory neurons in respiratory diseases and present MrgprC11<sup>+</sup> neurons as a potential therapeutic target for treating airway hyperresponsiveness.</p>","PeriodicalId":7655,"journal":{"name":"American Journal of Respiratory Cell and Molecular Biology","volume":" ","pages":"393-407"},"PeriodicalIF":5.9,"publicationDate":"2025-04-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142455964","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Maritza J Romero, Qian Yue, Won Mo Ahn, Jürg Hamacher, Yusra Zaidi, Stephen Haigh, Supriya Sridhar, Joyce Gonzales, Martina Hudel, Yuqing Huo, Alexander D Verin, Betty S Pace, Brian K Stansfield, Mazharul Maishan, Enid R Neptune, Perenlei Enkhbaatar, Yunchao Su, Trinad Chakraborty, Graydon Gonsalvez, Edith Hummler, William B Davis, Vladimir Y Bogdanov, David J R Fulton, Gabor Csanyi, Michael A Matthay, Douglas C Eaton, Rudolf Lucas
{"title":"Endothelial ENaC-α Restrains Oxidative Stress in Lung Capillaries in Murine Pneumococcal Pneumonia-associated Acute Lung Injury.","authors":"Maritza J Romero, Qian Yue, Won Mo Ahn, Jürg Hamacher, Yusra Zaidi, Stephen Haigh, Supriya Sridhar, Joyce Gonzales, Martina Hudel, Yuqing Huo, Alexander D Verin, Betty S Pace, Brian K Stansfield, Mazharul Maishan, Enid R Neptune, Perenlei Enkhbaatar, Yunchao Su, Trinad Chakraborty, Graydon Gonsalvez, Edith Hummler, William B Davis, Vladimir Y Bogdanov, David J R Fulton, Gabor Csanyi, Michael A Matthay, Douglas C Eaton, Rudolf Lucas","doi":"10.1165/rcmb.2023-0440OC","DOIUrl":"10.1165/rcmb.2023-0440OC","url":null,"abstract":"<p><p>Infection of lung endothelial cells with pneumococci activates the superoxide-generating enzyme NOX2 (nicotinamide adenine dinucleotide phosphate hydrogen [NADPH] oxidase 2), involving the pneumococcal virulence factor PLY (pneumolysin). Excessive NOX2 activity disturbs capillary barriers, but its global inhibition can impair bactericidal phagocyte activity during pneumococcal pneumonia. Depletion of the α subunit of ENaC (epithelial sodium channel) in pulmonary endothelial cells increases expression and PMA-induced activity of NOX2. Direct ENaC activation by TIP peptide improves capillary barrier function-measured by electrical cell substrate impedance sensing in endothelial monolayers and by Evans blue dye incorporation in mouse lungs-after infection with pneumococci. PLY-induced hyperpermeability in human lung microvascular endothelial cell monolayers is abrogated by both NOX2 inhibitor gp91dstat and TIP peptide. Endothelial NOX2 expression is assessed by increased surface membrane presence of phosphorylated p47<sup>phox</sup> subunit (Western blotting) <i>in vitro</i> and by colocalization of CD31 and gp91<sup>phox</sup> in mouse lung slices using DuoLink, whereas NOX2-generated superoxide is measured by chemiluminescence. TIP peptide blunts PMA-induced NOX2 activity in cells expressing ENaC-α, but not in neutrophils, which lack ENaC. Conditional endothelial ENaC-α knockout (enENaC-α knockout) mice develop increased capillary leak upon intratracheal instillation with PLY or pneumococci, compared with wild-type animals. TIP peptide diminishes capillary leak in <i>Streptococcus pneumoniae</i>-infected wild-type mice, without significantly increasing lung bacterial load. Lung slices from <i>S. pneumoniae</i>-infected enENaC-α knockout mice have significantly increased endothelial NOX2 expression, compared with infected cyclization recombination mice. In conclusion, enENaC may represent a novel therapeutic target to reduce NOX2-mediated oxidative stress and capillary leak in acute respiratory distress syndrome, without impairing host defense.</p>","PeriodicalId":7655,"journal":{"name":"American Journal of Respiratory Cell and Molecular Biology","volume":" ","pages":"429-440"},"PeriodicalIF":5.9,"publicationDate":"2025-04-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142455963","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Abdul S Qadir, Sukanta Das, Swathi Nedunchezian, Kaori Masuhara, Tushar J Desai, Jalees Rehman, Preetish Kadur Murthy, Yoshikazu Tsukasaki, Lijian Shao, Asrar B Malik
{"title":"Physiological Modeling of the Vascularized Human Lung Organoid.","authors":"Abdul S Qadir, Sukanta Das, Swathi Nedunchezian, Kaori Masuhara, Tushar J Desai, Jalees Rehman, Preetish Kadur Murthy, Yoshikazu Tsukasaki, Lijian Shao, Asrar B Malik","doi":"10.1165/rcmb.2024-0413MA","DOIUrl":"10.1165/rcmb.2024-0413MA","url":null,"abstract":"<p><p>Human lung organoids (hLOs) derived from induced pluripotent stem cells (iPSCs) are of great interest, as they inform lung development, such as differentiation of lung epithelial subtypes in the distal alveolar unit. An unaddressed question is whether introducing endothelial cells (ECs) and vascularization provides a better representation of hLOs. Here we describe a method in which vessels become integrated with hLOs. hLOs were generated by combining human iPSC-derived lung progenitor cells (LPs) with ECs at varying LP:EC ratios. At the optimal combination of both cells, we observed vessel infiltration of hLOs compared to without ECs. Red blood cells were seen in hLOs implanted into kidney capsules of NOD/SCID mice. Both human and mouse ECs conjoined to form chimeric vessels in hLOs. The vascularized hLOs showed alveolar type II epithelial (ATII) cells and ATI cells, although there was no difference in 1:1 ATII/ATI ratio. We observed primitive airway sacs with alveolar epithelial cells lining the lumen of vascularized hLOs. Electron microscopy revealed surfactant production in ATII cells of vascularized hLOs in contrast to absence of vessels. The vascularized hLOs also mounted a robust inflammatory response characterized by influx of mouse neutrophils after challenging mice with LPS. Thus, interactions of ECs with LPs generated vascularized hLOs that induced ATII and ATI differentiation, although not reaching to the ratio of 1:9 seen in mature human lungs. hLOs also showed the LPS induced inflammatory response upon transplantation into recipient mice. Our results show the potential of vascularized hLOs for studying human lung development and inflammatory lung injury.</p>","PeriodicalId":7655,"journal":{"name":"American Journal of Respiratory Cell and Molecular Biology","volume":" ","pages":"354-363"},"PeriodicalIF":5.9,"publicationDate":"2025-04-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142602779","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Adam L Fellows, Chien-Nien Chen, Chongyang Xie, Nayana Iyer, Lukas Schmidt, Xiaoke Yin, Luke A Yates, Manuel Mayr, Andrew Cowburn, Lan Zhao, Beata Wojciak-Stothard
{"title":"ARF6 as a Novel Activator of HIF-2α in Pulmonary Arterial Hypertension.","authors":"Adam L Fellows, Chien-Nien Chen, Chongyang Xie, Nayana Iyer, Lukas Schmidt, Xiaoke Yin, Luke A Yates, Manuel Mayr, Andrew Cowburn, Lan Zhao, Beata Wojciak-Stothard","doi":"10.1165/rcmb.2024-0149OC","DOIUrl":"10.1165/rcmb.2024-0149OC","url":null,"abstract":"<p><p>ARF6 (ADP-ribosylation factor 6), a GTPase associated with cancer metastasis, is activated in the lung endothelium in pulmonary arterial hypertension (PAH). To identify ARF6-regulated pathways relevant to PAH, we performed a state-of-the-art proteomic analysis of human pulmonary artery endothelial cells (HPAECs) overexpressing the wild-type, constitutively active, fast-cycling, and dominant-negative mutants of ARF6. The analysis revealed a novel link of ARF6 with HIF (hypoxia-inducible factor), in addition to endocytotic vesicle trafficking, cell proliferation, angiogenesis, oxidative stress, and lipid metabolism. Active ARF6 markedly increased expression and activity of HIF-2, critical in PAH, with HIF-1 relatively unaffected. Hypoxic ARF6 activation was a prerequisite for HIF-2 activation and HIF-dependent gene expression in HPAECs, PAH blood-derived late-outgrowth endothelial colony-forming cells, and hypoxic mouse lungs <i>in vivo</i>. A novel ARF6 inhibitor, chlortetracycline (CTC), reduced hypoxia-induced HIF-2 activation, proliferation, and angiogenesis in HPAECs and reduced HIF-2 expression in lung and heart tissues of hypoxic mice. PAH endothelial colony-forming cells showed elevated expression and activity of ARF6 and HIF2, which was attenuated by CTC, and oral CTC attenuated development of pulmonary hypertension in chronically hypoxic mice. We identify EGFR (epidermal growth factor receptor) as a direct interactor of ARF6 and EGFR signaling as a crucial mechanism linking ARF6 and HIF activation. In conclusion, we are the first to demonstrate a key role of ARF6 in the regulation of HIF-2α activation <i>in vitro</i> and <i>in vivo</i> and show that HIF-2α, a master regulator of vascular remodeling in PAH, can be targeted by a clinically approved antibiotic CTC.</p>","PeriodicalId":7655,"journal":{"name":"American Journal of Respiratory Cell and Molecular Biology","volume":" ","pages":"380-392"},"PeriodicalIF":5.9,"publicationDate":"2025-04-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142646690","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}