Despite advances in treatment, the prognosis for patients with pulmonary arterial hypertension (PAH) remains dismal, highlighting the need for further therapeutic advances. By using RNA sequencing on pulmonary artery smooth muscle cells (PASMCs), functional enrichment, and connectivity map analyses, we identify Aurora kinase B (AURKB) as a candidate therapeutic target. We show that AURKB inhibition blocks cell cycle progression and reverses the gene signature of PAH-PASMCs. We also report that PAH-PASMCs that escape apoptosis acquire a senescence-associated secretory phenotype. In vivo, AURKB inhibition using barasertib improves hemodynamics in two preclinical models of established PAH by attenuating pulmonary vascular remodeling. A therapeutic effect is also observed in human precision-cut lung slices. Finally, we demonstrate that the combination of barasertib with a p21 attenuator is more effective in reducing vascular remodeling than either drug alone. These findings provide insight into strategies for therapeutic manipulation.

Die Genetik und Pathobiolgie wurde beim 7. Weltsymposium für pulmonale Hypertonie in den Task Forces 2 und 3 behandelt. Die Genetik-Taskforce fokussierte sich zusätzlich auf präzisionsmedizinische Ansätze und die Pathobiologie-Arbeitsgruppe konzentrierte sich stark auf neue Omics-Technologien. Daher werden im Folgenden nicht nur die aktuellen Wissensstände zur Genetik, genetischen Untersuchungsverfahren und molekularen pathophysiologischen Änderungen zusammengefasst, sondern durch Kommentare eingeordnet und ergänzt. Abschließend werden die Wichtigkeit von nationalen und internationalen Biobanken und Kohorten sowie die aktive Einbindung von Patient*innen und Familien hervorgehoben.

Die pulmonalarterielle Hypertonie (PAH) ist eine komplexe vaskuläre Erkrankung, die durch eine ausgeprägte pathobiologische und genetische Heterogenität gekennzeichnet ist. Im Rahmen der 7. Weltkonferenz für pulmonale Hypertonie (7. WSPH) wurden neue Erkenntnisse zu zentralen Mechanismen wie Gefäßumbauprozesse, epigenetischen Modifikationen, metabolischen Dysregulationen und Ionenkanalveränderungen präsentiert. Fortschritte in der Genetik, Transkriptomik, Epigenetik und Multiomics-Integration ermöglichen tiefere Einblicke in molekulare Prozesse und eröffnen neue therapeutische Perspektiven. Zunehmend wird deutlich, dass sich diese pathobiologischen Prozesse nicht isoliert betrachten lassen, sondern eng mit genetischen Prädispositionen und individuellen Expositionen gegenüber Risikofaktoren verwoben sind. Die Integration moderner molekularer Analysen in die pathophysiologische Forschung schafft die Grundlage für ein umfassenderes Verständnis der Krankheitsentstehung und -progression. Insbesondere seltene genetische Varianten in Genen wie BMPR2, TBX4 oder SOX17 liefern wichtige Hinweise auf die individuelle Krankheitsanfälligkeit und untermauern das Potenzial personalisierter Therapieansätze. Dieses Review fasst die aktuellen pathobiologischen und genetischen Konzepte zusammen und diskutiert zukünftige Forschungsprioritäten sowie die Notwendigkeit internationaler, diverser Patientenkohorten.

Abstract Background: Therapies targeting immune checkpoint have significantly transformed the treatment of several cancers, including lung cancer. However, only 20% of lung cancer patients show a response to immunotherapies. KRAS and EGFR mutations are significant factors in the development and progression of lung cancer and its response to immunotherapy. Hence, it's vital to understand intra-tumoral heterogeneity, the makeup of the Tumor Microenvironment (TME), and the cellular interactions in tumors with KRAS and EGFR mutations to uncover new insights. Methods: Lung cancer tissues from 200 patients were collected, focusing on KRAS and EGFR mutation statuses, specifically KRAS+/EGFR-, KRAS-/EGFR+, and KRAS-/EGFR-. For each patient, two samples were taken, resulting in a total of 400 tissue microarray (TMA) cores. These samples were imaged at single-cell resolution using the PhenoCycler®-Fusion system, which employed a 42-plex antibody panel targeting immune, tumor, proliferation, and apoptosis markers. The first step in the analysis was cell segmentation, performed using a fine-tuned deep learning model. This process successfully segmented a total of 2.16 million cells across the entire collection of samples. Following this, the protein expressions for each cell were calculated, and unsupervised clustering was performed, resulting in 36 distinct clusters, that were manually annotated into 14 cell phenotypes. The proportions of these cell phenotypes were then quantified for each of the 400 TMA cores and compared across the three mutation groups. The analysis also included the spatial proximity and cellular neighborhoods in relation to these mutation groups. Results: Quantitative analysis focusing on the relative abundance of cell types revealed that the ratio of tumor cells to immune cells was significantly lower in KRAS-/EGFR- tumors compared to those with KRAS+/EGFR- and KRAS-/EGFR+ mutations. Furthermore, the percentages of Regulatory T cells (Tregs) and M1 Macrophages were significantly higher in the KRAS+/EGFR- group compared to the KRAS-/EGFR+ group. Spatial proximity analysis indicated that in the KRAS-/EGFR+ group, M2 Macrophages were significantly closer to Cytotoxic T cells and Helper T cells compared to the distances observed in the other two groups. Moreover, neighborhood analysis identified 20 distinct cellular neighborhoods, each defined by specific cell-cell interactions. COX hazard analysis based on these cellular neighborhoods demonstrated notable variations among the three mutation groups, which are linked to differences in patient outcomes. Conclusions: Through single-cell spatial phenotyping, this work has shown that the spatial immune landscape of lung tumors is influenced by KRAS and EGFR mutation statuses. This presents a novel opportunity to enhance our understanding of the spatial structure of lung cancer and to identify more effective therapeutic targets. Citation Format: Rajender Nandigama, Bassem Ben Cheikh, Ning Ma, Jochen Wilhelm, Laura Klotz, Florian Eichhorn, Mark Kriegsmann, Marek Bartkuhn, Jamal Nabhanizadeh, Sascha Seidel, Thorsten Stiewe, Albrecht Stenzinger, Andreas Weigert, Mario Looso, Friedrich Grimminger, Werner Seeger, Soni Savai Pullamsetti, Niyati Jhaveri, Hauke Winter, Rajkumar Savai. Single-cell spatial landscape of the mutation-specific human lung tumor immune microenvironment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5508.

In recent years, major advances have been made in the understanding of the cellular and molecular mechanisms driving pulmonary vascular remodelling in various forms of pulmonary hypertension, including pulmonary arterial hypertension, pulmonary hypertension associated with left heart disease, pulmonary hypertension associated with chronic lung disease and hypoxia, and chronic thromboembolic pulmonary hypertension. However, the survival rates for these different forms of pulmonary hypertension remain unsatisfactory, underscoring the crucial need to more effectively translate innovative scientific knowledge into healthcare interventions. In these proceedings of the 7th World Symposium on Pulmonary Hypertension, we delve into recent developments in the field of pathology and pathophysiology, prioritising them while questioning their relevance to different subsets of pulmonary hypertension. In addition, we explore how the latest omics and other technological advances can help us better and more rapidly understand the myriad basic mechanisms contributing to the initiation and progression of pulmonary vascular remodelling. Finally, we discuss strategies aimed at improving patient care, optimising drug development, and providing essential support to advance research in this field.

Abstract Background: Patients with lung cancer often have a high prevalence of severe comorbidities, primarily due to the significant correlation with cigarette smoking and the aging process. Dysregulations of the immune response are the cause of chronic inflammatory diseases such as chronic obstructive pulmonary disease (COPD), pulmonary arterial hypertension (PAH), pulmonary fibrosis (PF) and lung cancer. In addition, the lung microenvironment in these chronic inflammatory diseases has not yet been sufficiently investigated. Therefore, we aimed to investigate the spatio-temporal localization and cellular distribution in the lung microenvironment and to compare lung cancer with the concomitant diseases of lung cancer. Methods: FFPE lung sections, each 3µm in thickness, were collected from patients with diagnoses of lung cancer, COPD, PAH, PF, and from healthy donors, with three samples taken for each category. These sections were subsequently imaged using the PhenoCycler®-Fusion system, utilizing a comprehensive panel of 45 antibodies. including immune, epithelial, vascular, proliferation and apoptosis markers. The first step of the analysis workflow consists of cell segmentation using a fine-tuned deep learning model leading to a total of 5.2 million cells across the whole dataset. Protein expressions were then calculated from the segmented cells, and unsupervised clustering was performed based on the normalized expression values. The resulting clusters were manually annotated into 16 cell phenotypes based on their protein expression patterns as displayed on a hierarchical clustering heatmap. The percentages of cell phenotypes were then calculated for each sample and compared between disease groups. Furthermore, the spatial proximity and cellular neighborhood analyses were performed to study the spatial organization of the different cell phenotypes in the tissues. Results: Quantitative assessment revealed unique characteristics specific to each disease, identified by the varying abundance of different immune cell subtypes. Additionally, when comparing lungs from healthy donors with those affected by lung diseases, notable differences were observed in the spatial arrangement of immune cells and their proximity to lung blood vessels. Conclusions: This study presents the first architectural map of lung cancer, COPD, PAH, and PF linking various immune cell types to their spatial location within the tissue. Such in-depth spatial analyses will aid in comprehending the spatial and temporal interplay within the lung's microenvironment in diverse lung conditions, as well as in developing targeted therapies for specific cell types. Citation Format: Rajender Nandigama Nandigama, Bassem Ben Cheikh Cheikh, Jamal Nabhanizadeh, Friedrich Grimminger, Werner Seeger, Niyati Jhaveri, Soni Savai Pullamsetti, Rajkumar Savai. High-resolution spatial atlas reveals insight into spatial landscape of lung cancer and chronic lung diseases [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5507.

Background: Adenosine deaminase acting on RNA 1 (ADAR1) catalyzes the conversion of adenosine (A) to inosine (I) in double-stranded RNA, 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 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: Total RNAs from IPAH PASMCs (N=3) and healthy control (N=3) were deep sequenced by RNA-seq and determined the fraction of reads with a ‘G’ nucleotide editing sites using RADAR database. A transgenic line ADAR1 SMC-KO was generated by knocking down ADAR1 selectively in αSMA+ cells, followed by 3 weeks of hypoxia. PKR inhibitor Imoxin was used to treat ADAR1 SMC-KO . Results: SMC layers of human IPAH tissues expressed reduced levels of ADAR1. Moreover, IPAH PASMCs displayed decreased mRNA and protein levels of ADAR1, along with reduced editing sites compared to healthy PASMCs (Fig.1A). The knockdown of ADAR1 in PASMCs resulted in the activation of the MDA5-PKR-IFNβ signaling pathway. Compared with controls in vivo , hypoxic ADAR1 SMC-KO mice developed severe PH, as evidenced by excessive vascular remodeling in distal arterioles (Fig.1B) and increased vascular leakage resulting in elevated right ventricular systolic pressure (37 vs 23 mmHg) and right ventricular hypertrophy by Fulton Index (46 vs. control 20%) (Fig.1C). The immunofluorescence staining showed higher expression of IFNβ and increased recruitment of inflammatory immune cells. Imoxin treatment prevented PH & RVH. Conclusions: The deficiency of ADAR1 leads to the activation of IFNβ thus contributing to the pathological processes observed in PAH. Inhibiting PKR activity could attenuate the aberrant IFNβ activation and potentially serve as a new therapeutic strategy to treat PAH.

Introduction: Early apoptosis of pulmonary artery endothelial cells (PAECs) is a key driver of vascular remodeling and pulmonary hypertension (PH), but its regulation is poorly defined. Adenosine deaminase acting on RNA 1 (ADAR1) is an RNA editing enzyme that converts adenosine to inosine (A-to-I) in RNA transcripts. We found that ADAR1 expression was reduced in PH endothelium. While reduced RNA editing stimulates aberrant cytosolic innate immunity and can drive apoptosis, the exact RNA editing targets and downstream mechanisms regulating pulmonary endothelial survival are unknown. Hypothesis: Deficiency in ADAR1-mediated RNA editing promotes pulmonary endothelial pathophenotypes and drives PH. Methods & Results: ADAR1 expression was downregulated in the pulmonary vascular endothelium and in lung tissues of human and mouse PH. Global A-to-I editing was decreased in the whole lungs and PBMCs from PAH patients. In vitro , hypoxia, a known PH trigger, downregulated ADAR1 in PAECs via upregulation of the ADAR1-targeting microRNA, miR-17. By RNA sequencing of PAECs after ADAR1 knockdown, we identified the circadian gene Nocturnin ( NOCT ) as a direct ADAR1 target that carries two active A-to-I RNA editing sites in the 3’UTR. By single cell RNA sequencing of human PAH lungs, NOCT editing levels were reduced, while NOCT transcript levels increased. Correspondingly, in vitro , ADAR1 silencing increased NOCT mRNA levels, thus activating an innate immune response and PAEC apoptosis. Forced NOCT expression phenocopied the effect of ADAR1 deficiency, inducing RNA sensing innate immunity signaling pathway and increasing apoptosis. In vivo , in mice chronically exposed to hypoxia, administration of the ADAR1 inhibitor 8-Azaadenosine (Aza) resulted in worsened indices of PH. Notably, genetic deletion of NOCT mitigated PH induced by Aza in hypoxic IL6 transgenic mice, emphasizing the crucial role of NOCT in ADAR1-mediated pathogenesis. Conclusions: Hypoxia-induced ADAR1 deficiency promotes NOCT mRNA dysregulation to induce PAEC innate immunity, PAEC apoptosis, and PH. This study provides impetus to target the ADAR1-NOCT axis for more effective diagnostics and therapeutics for PH.

HIV and Schistosoma infections have been individually associated with pulmonary vascular disease. Co-infection with these pathogens is very common in tropical areas, with an estimate of six million people co-infected worldwide. However, the effects of HIV and Schistosoma co-exposure on the pulmonary vasculature and its impact on the development of pulmonary vascular disease are largely unknown. Here, we have approached these questions by using a non-infectious animal model based on lung embolization of Schistosoma mansoni eggs in HIV-1 transgenic (HIV) mice. Schistosome-exposed HIV mice but not wild-type (Wt) counterparts showed augmented pulmonary arterial pressure associated with markedly suppressed endothelial-dependent vasodilation, increased endothelial remodeling and vessel obliterations, formation of plexiform-like lesions and a higher degree of perivascular fibrosis. In contrast, medial wall muscularization was similarly increased in both types of mice. Moreover, HIV mice displayed an impaired immune response to parasite eggs in the lung, as suggested by decreased pulmonary leukocyte infiltration, small-sized granulomas, and augmented residual egg burden. Notably, vascular changes in co-exposed mice were associated with increased expression of proinflammatory and profibrotic cytokines, including IFN-γ and IL-17A in CD4+ and γδ T cells and IL-13 in myeloid cells. Collectively, our study shows for the first time that combined pulmonary persistence of HIV proteins and Schistosoma eggs, as it may occur in co-infected people, alters the cytokine landscape and targets the vascular endothelium for aggravated pulmonary vascular pathology. Furthermore, it provides an experimental model for the understanding of pulmonary vascular disease associated with HIV and Schistosoma co-morbidity.

Rationale: Supplementary oxygen leads to bronchopulmonary dysplasia (BPD) in preterm infants, a disease characterized by lung growth arrest and matrix remodeling. We demonstrated that hyperoxia reduces Krüppel-like factor 4 (Klf4), a key transcription factor in cell biology. Since myofibroblasts are crucial in lung injury and remodeling, we investigated the functional role of Klf4 in the pathogenesis of BPD and in the homeostasis of lung fibroblasts. Methods: (1) Newborn C57BL/6N mice were exposed to 85% O2 (HYX) or 21% O2 (NOX) until postnatal day 28 (P28). (2) Primary neonatal murine lung fibroblasts (pnF) were transfected with a Klf4 overexpressing plasmid or siRNA technique for overexpression (OE) and knockdown of Klf4 (del), respectively, and exposed to HYX or NOX. (3) BioID (Proximity-dependent Biotin Identification) and CHIP-Seq were performed. Results: (1) Reduced Klf4 expression in lungs after HYX was associated with an activation of TGFβ signaling and an increase of myofibroblasts, promoting thereby matrix remodeling. (2) TGFβ inhibited Klf4 expression in cultured pnF. Klf4del decreased migration, induced proliferation and increased mRNA expression of matrix markers (Ctgf, Col1a1 & Col4a1) and Pdgfra (myofibroblasts). In contrast, Klf4OE increased adhesion, but reduced proliferation, apoptosis, migration and expression of fibrotic genes in pnF. (3) Finally, BioID and CHIP-Seq identified a novel Klf4 interactome that regulates FoxO1 transcriptionally and its activation via acetylation. Conclusion: Our data unveil a novel Klf4-FoxO1 axis in lung fibroblasts that could promote matrix remodeling and lung growth arrest in lungs with BPD.

Cardiac valve disease can lead to severe cardiac dysfunction and is thus a frequent cause of morbidity and mortality. Its main treatment is valve replacement, which is currently greatly limited by the poor recellularization and tissue formation potential of the implanted valves. As we still lack suitable animal models to identify modulators of these processes, here we used the adult zebrafish and found that, upon valve decellularization, they initiate a striking regenerative program that leads to the formation of new functional valves. After injury, endothelial and kidney marrow-derived cells undergo cell cycle re-entry and differentiate into new extracellular matrix-secreting valve cells. The Transforming Growth Factor beta (TGFβ) signaling pathway promotes this process by enhancing progenitor cell proliferation as well as valve cell differentiation. These findings reveal a key role for TGFβ signaling in valve regeneration and also establish the zebrafish as a model to identify and test factors promoting valve recellularization and growth.