A roadmap for therapeutic discovery in pulmonary hypertension associated with left heart failure. A scientific statement of the Heart Failure Association (HFA) of the ESC and the ESC Working Group on Pulmonary Circulation & Right Ventricular Function.

Pulmonary hypertension (PH) associated with left heart failure (LHF) (PH-LHF) is one of the most common causes of PH. It directly contributes to symptoms and reduced functional capacity and negatively affects right heart function, ultimately leading to a poor prognosis. There are no specific treatments for PH-LHF, despite the high number of drugs tested so far. This scientific document addresses the main knowledge gaps in PH-LHF with emphasis on pathophysiology and clinical trials. Key identified issues include better understanding of the role of pulmonary venous versus arteriolar remodelling, multidimensional phenotyping to recognize patient subgroups positioned to respond to different therapies, and conduct of rigorous pre-clinical studies combining small and large animal models. Advancements in these areas are expected to better inform the design of clinical trials and extend treatment options beyond those effective in pulmonary arterial hypertension. Enrichment strategies, endpoint assessments, and thorough haemodynamic studies, both at rest and during exercise, are proposed to play primary roles to optimize early-stage development of candidate therapies for PH-LHF.

GLI1+ Cells Contribute to Vascular Remodeling in Pulmonary Hypertension.

BACKGROUND: The precise origin of newly formed ACTA2+ (alpha smooth muscle actin-positive) cells appearing in nonmuscularized vessels in the context of pulmonary hypertension is still debatable although it is believed that they predominantly derive from preexisting vascular smooth muscle cells (VSMCs). METHODS: Gli1Cre-ERT2; tdTomatoflox mice were used to lineage trace GLI1+ (glioma-associated oncogene homolog 1-positive) cells in the context of pulmonary hypertension using 2 independent models of vascular remodeling and reverse remodeling: hypoxia and cigarette smoke exposure. Hemodynamic measurements, right ventricular hypertrophy assessment, flow cytometry, and histological analysis of thick lung sections followed by state-of-the-art 3-dimensional reconstruction and quantification using Imaris software were used to investigate the contribution of GLI1+ cells to neomuscularization of the pulmonary vasculature. RESULTS: The data show that GLI1+ cells are abundant around distal, nonmuscularized vessels during steady state, and this lineage contributes to around 50% of newly formed ACTA2+ cells around these normally nonmuscularized vessels. During reverse remodeling, cells derived from the GLI1+ lineage are largely cleared in parallel to the reversal of muscularization. Partial ablation of GLI1+ cells greatly prevented vascular remodeling in response to hypoxia and attenuated the increase in right ventricular systolic pressure and right heart hypertrophy. Single-cell RNA sequencing on sorted lineage-labeled GLI1+ cells revealed an Acta2high fraction of cells with pathways in cancer and MAPK signaling as potential players in reprogramming these cells during vascular remodeling. Analysis of human lung-derived material suggests that GLI1 signaling is overactivated in both group 1 and group 3 pulmonary hypertension and can promote proliferation and myogenic differentiation. CONCLUSIONS: Our data highlight GLI1+ cells as an alternative cellular source of VSMCs in pulmonary hypertension and suggest that these cells and the associated signaling pathways represent an important therapeutic target for further studies.

Restoration of Foxp3+ Regulatory T Cells by HDAC-Dependent Epigenetic Modulation Plays a Pivotal Role in Resolving Pulmonary Arterial Hypertension Pathology.

Rationale: Immune dysregulation is a common feature of pulmonary arterial hypertension (PAH). Histone deacetylase (HDAC)-dependent transcriptional reprogramming epigenetically modulates immune homeostasis and is a novel disease-oriented approach in modern times. Objectives: To identify a novel functional link between HDAC and regulatory T cells (Tregs) in PAH, aiming to establish disease-modified biomarkers and therapeutic targets. Methods: Peripheral blood mononuclear cells were isolated from patients with idiopathic PAH (IPAH) and rodent models of pulmonary hypertension (PH): monocrotaline rats, Sugen5416-hypoxia rats, and Treg-depleted mice. HDAC inhibitor vorinostat (suberoylanilide hydroxamic acid, SAHA) was used to examine the immune modulatory effects in vivo, ex vivo, and in vitro. Measurements and Main Results: Increased HDAC expression was associated with reduced Foxp3+ Tregs and increased PD-1 (programmed cell death-1) signaling in peripheral blood mononuclear cells from patients with IPAH. SAHA differentially modified a cluster of epigenetic-sensitive genes and induced Foxp3+ Treg conversion in IPAH T cells. Rodent models recapitulated these epigenetic aberrations and T-cell dysfunction. SAHA attenuated PH phenotypes and restored FOXP3 transcription and Tregs in PH rats; interestingly, the effects were more profound in female rats. Selective depletion of CD25+ Tregs in Sugen5416-hypoxia mice neutralized the effects of SAHA. Furthermore, SAHA inhibited endothelial cytokine/chemokine release upon stimulation and subsequent immune chemotaxis. Conclusions: Our results indicated HDAC aberration was associated with Foxp3+ Treg deficiency and demonstrated an epigenetic-mediated mechanism underlying immune dysfunction in PAH. Restoration of Foxp3+ Tregs by HDAC inhibitors is a promising approach to resolve pulmonary vascular pathology, highlighting the potential benefit of developing epigenetic therapies for PAH.

Pharmacology and Rationale for Seralutinib in the Treatment of Pulmonary Arterial Hypertension.

Pulmonary arterial hypertension (PAH) is a complex disorder characterized by vascular remodeling and a consequent increase in pulmonary vascular resistance. The histologic hallmarks of PAH include plexiform and neointimal lesions of the pulmonary arterioles, which are composed of dysregulated, apoptosis-resistant endothelial cells and myofibroblasts. Platelet-derived growth factor receptors (PDGFR) α and ß, colony stimulating factor 1 receptor (CSF1R), and mast/stem cell growth factor receptor kit (c-KIT) are closely related kinases that have been implicated in PAH progression. In addition, emerging data indicate significant crosstalk between PDGF signaling and the bone morphogenetic protein receptor type 2 (BMPR2)/transforming growth factor ß (TGFß) receptor axis. This review will discuss the importance of the PDGFR-CSF1R-c-KIT signaling network in PAH pathogenesis, present evidence that the inhibition of all three nodes in this kinase network is a potential therapeutic approach for PAH, and highlight the therapeutic potential of seralutinib, currently in development for PAH, which targets these pathways.

ADPGK-AS1 long noncoding RNA switches macrophage metabolic and phenotypic state to promote lung cancer growth.

Long noncoding RNAs (lncRNAs) influence the transcription of gene networks in many cell types, but their role in tumor-associated macrophages (TAMs) is still largely unknown. We found that the lncRNA ADPGK-AS1 was substantially upregulated in artificially induced M2-like human macrophages, macrophages exposed to lung cancer cells in vitro, and TAMs from human lung cancer tissue. ADPGK-AS1 is partly located within mitochondria and binds to the mitochondrial ribosomal protein MRPL35. Overexpression of ADPGK-AS1 in macrophages upregulates the tricarboxylic acid cycle and promotes mitochondrial fission, suggesting a phenotypic switch toward an M2-like, tumor-promoting cytokine release profile. Macrophage-specific knockdown of ADPGK-AS1 induces a metabolic and phenotypic switch (as judged by cytokine profile and production of reactive oxygen species) to a pro-inflammatory tumor-suppressive M1-like state, inhibiting lung tumor growth in vitro in tumor cell-macrophage cocultures, ex vivo in human tumor precision-cut lung slices, and in vivo in mice. Silencing ADPGK-AS1 in TAMs may thus offer a novel therapeutic strategy for lung cancer.

Spatial profiling of the microenvironment reveals low intratumoral heterogeneity and STK11-associated immune evasion in therapy-naïve lung adenocarcinomas.

OBJECTIVE: Intratumoral heterogeneity was found to be a significant factor causing resistance to lung cancer therapies, including immune checkpoint blockade. Lesser is known about spatial heterogeneity of the tumor microenvironment (TME) and its association with genetic properties of the tumor, which is of particular interest in the therapy-naïve setting. MATERIALS AND METHODS: We performed multi-region sampling (2-4 samples per tumor; total of 55 samples) from a cohort of 19 untreated stage IA-IIIB lung adenocarcinomas (n = 11 KRAS mutant, n = 1 ERBB2 mutant, n = 7 KRAS wildtype). For each sample the expression of 770 immunooncology-related genes was analyzed using the nCounter platform, while the mutational status was determined by hybrid capture-based next-generation sequencing (NGS) using a large panel covering more than 500 genes. RESULTS: Global unsupervised analyses revealed clustering of the samples into two groups corresponding to a 'hot' or 'cold' immunologic tumor contexture based on the abundance of immune cell infiltrates. All analyzed specific immune cell signatures (ICsig) showed a significantly higher intertumoral than intratumoral heterogeneity (p < 0.02), as most of the analyzed cases (14/19) showed a very homogenous spatial immune cell profile. PD-L1 exhibited a significantly higher intertumoral than intratumoral heterogeneity (p = 1.03e-13). We found a specific association with 'cold' TME for STK11 (11/14, p < 0.07), but not KRAS, TP53, LRP1B, MTOR, U2AF1 co-mutations, and validated this finding using The Cancer Genome Atlas (TCGA) data. CONCLUSION: Early-stage lung adenocarcinomas show considerable intertumoral, but limited intratumoral heterogeneity, which is clinically highly relevant as assessment before neoadjuvant treatment is based on small biopsies. STK11 mutations are specifically associated with a 'cold' TME, which could affect the efficacy of perioperative immunotherapy.

The HDAC2-SP1 Axis Orchestrates Protumor Macrophage Polarization.

Tumor-associated macrophages (TAM), including antitumor M1-like TAMs and protumor M2-like TAMs, are transcriptionally dynamic innate immune cells with diverse roles in lung cancer development. Epigenetic regulators are key in controlling macrophage fate in the heterogeneous tumor microenvironment. Here, we demonstrate that the spatial proximity of HDAC2-overexpressing M2-like TAMs to tumor cells significantly correlates with poor overall survival of lung cancer patients. Suppression of HDAC2 in TAMs altered macrophage phenotype, migration, and signaling pathways related to interleukins, chemokines, cytokines, and T-cell activation. In coculture systems of TAMs and cancer cells, suppressing HDAC2 in TAMs resulted in reduced proliferation and migration, increased apoptosis of cancer cell lines and primary lung cancer cells, and attenuated endothelial cell tube formation. HDAC2 regulated the M2-like TAM phenotype via acetylation of histone H3 and transcription factor SP1. Myeloid cell-specific deletion of Hdac2 and pharmacologic inhibition of class I HDACs in four different murine lung cancer models induced the switch from M2-like to M1-like TAMs, altered infiltration of CD4+ and CD8+ T cells, and reduced tumor growth and angiogenesis. TAM-specific HDAC2 expression may provide a biomarker for lung cancer stratification and a target for developing improved therapeutic approaches. SIGNIFICANCE: HDAC2 inhibition reverses the protumor phenotype of macrophages mediated by epigenetic modulation induced by the HDAC2-SP1 axis, indicating a therapeutic option to modify the immunosuppressive tumor microenvironment.

Targeting Wnt-ß-Catenin-FOSL Signaling Ameliorates Right Ventricular Remodeling.

BACKGROUND: The ability of the right ventricle (RV) to adapt to an increased pressure afterload determines survival in patients with pulmonary arterial hypertension. At present, there are no specific treatments available to prevent RV failure, except for heart/lung transplantation. The wingless/int-1 (Wnt) signaling pathway plays an important role in the development of the RV and may also be implicated in adult cardiac remodeling. METHODS: Molecular, biochemical, and pharmacological approaches were used both in vitro and in vivo to investigate the role of Wnt signaling in RV remodeling. RESULTS: Wnt/ß-catenin signaling molecules are upregulated in RV of patients with pulmonary arterial hypertension and animal models of RV overload (pulmonary artery banding-induced and monocrotaline rat models). Activation of Wnt/ß-catenin signaling leads to RV remodeling via transcriptional activation of FOSL1 and FOSL2 (FOS proto-oncogene [FOS] like 1/2, AP-1 [activator protein 1] transcription factor subunit). Immunohistochemical analysis of pulmonary artery banding -exposed BAT-Gal (ß-catenin-activated transgene driving expression of nuclear ß-galactosidase) reporter mice RVs exhibited an increase in ß-catenin expression compared with their respective controls. Genetic inhibition of ß-catenin, FOSL1/2, or WNT3A stimulation of RV fibroblasts significantly reduced collagen synthesis and other remodeling genes. Importantly, pharmacological inhibition of Wnt signaling using inhibitor of PORCN (porcupine O-acyltransferase), LGKK-974 attenuated fibrosis and cardiac hypertrophy leading to improvement in RV function in both, pulmonary artery banding - and monocrotaline-induced RV overload. CONCLUSIONS: Wnt- ß-Catenin-FOSL signaling is centrally involved in the hypertrophic RV response to increased afterload, offering novel targets for therapeutic interference with RV failure in pulmonary hypertension.

Fibrocytes boost tumor-supportive phenotypic switches in the lung cancer niche via the endothelin system.

Fibrocytes are bone marrow-derived monocytic cells implicated in wound healing. Here, we identify their role in lung cancer progression/ metastasis. Selective manipulation of fibrocytes in mouse lung tumor models documents the central role of fibrocytes in boosting niche features and enhancing metastasis. Importantly, lung cancer patients show increased number of circulating fibrocytes and marked fibrocyte accumulation in the cancer niche. Using double and triple co-culture systems with human lung cancer cells, fibrocytes, macrophages and endothelial cells, we substantiate the central features of cancer-supporting niche: enhanced cancer cell proliferation and migration, macrophage activation, augmented endothelial cell sprouting and fibrocyte maturation. Upregulation of endothelin and its receptors are noted, and dual endothelin receptor blockade suppresses all cancer-supportive phenotypic alterations via acting on fibrocyte interaction with the cancer niche. We thus provide evidence for a crucial role of fibrocytes in lung cancer progression and metastasis, suggesting targets for treatment strategies.

Epigenetic reactivation of transcriptional programs orchestrating fetal lung development in human pulmonary hypertension.

Phenotypic alterations in resident vascular cells contribute to the vascular remodeling process in diseases such as pulmonary (arterial) hypertension [P(A)H]. How the molecular interplay between transcriptional coactivators, transcription factors (TFs), and chromatin state alterations facilitate the maintenance of persistently activated cellular phenotypes that consequently aggravate vascular remodeling processes in PAH remains poorly explored. RNA sequencing (RNA-seq) in pulmonary artery fibroblasts (FBs) from adult human PAH and control lungs revealed 2460 differentially transcribed genes. Chromatin immunoprecipitation sequencing (ChIP-seq) revealed extensive differential distribution of transcriptionally accessible chromatin signatures, with 4152 active enhancers altered in PAH-FBs. Integrative analysis of RNA-seq and ChIP-seq data revealed that the transcriptional signatures for lung morphogenesis were epigenetically derepressed in PAH-FBs, including coexpression of T-box TF 4 (TBX4), TBX5, and SRY-box TF 9 (SOX9), which are involved in the early stages of lung development. These TFs were expressed in mouse fetuses and then repressed postnatally but were maintained in persistent PH of the newborn and reexpressed in adult PAH. Silencing of TBX4, TBX5, SOX9, or E1A-associated protein P300 (EP300) by RNA interference or small-molecule compounds regressed PAH phenotypes and mesenchymal signatures in arterial FBs and smooth muscle cells. Pharmacological inhibition of the P300/CREB-binding protein complex reduced the remodeling of distal pulmonary vessels, improved hemodynamics, and reversed established PAH in three rodent models in vivo, as well as reduced vascular remodeling in precision-cut tissue slices from human PAH lungs ex vivo. Epigenetic reactivation of TFs associated with lung development therefore underlies PAH pathogenesis, offering therapeutic opportunities.

Picturing of the Lung Tumor Cellular Composition by Multispectral Flow Cytometry.

The lung tumor microenvironment plays a critical role in the tumorigenesis and metastasis of lung cancer, resulting from the crosstalk between cancer cells and microenvironmental cells. Therefore, comprehensive identification and characterization of cell populations in the complex lung structure is crucial for development of novel targeted anti-cancer therapies. Here, a hierarchical clustering approach with multispectral flow cytometry was established to delineate the cellular landscape of murine lungs under steady-state and cancer conditions. Fluorochromes were used multiple times to be able to measure 24 cell surface markers with only 13 detectors, yielding a broad picture for whole-lung phenotyping. Primary and metastatic murine lung tumor models were included to detect major cell populations in the lung, and to identify alterations to the distribution patterns in these models. In the primary tumor models, major altered populations included CD324+ epithelial cells, alveolar macrophages, dendritic cells, and blood and lymph endothelial cells. The number of fibroblasts, vascular smooth muscle cells, monocytes (Ly6C+ and Ly6C-) and neutrophils were elevated in metastatic models of lung cancer. Thus, the proposed clustering approach is a promising method to resolve cell populations from complex organs in detail even with basic flow cytometers.

In-Silico Design of a Novel Tridecapeptide Targeting Spike Protein of SARS-CoV-2 Variants of Concern.

Several mutations in severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) have increased the transmission and mortality rate of coronavirus disease-19 (COVID-19) across the globe. Although many vaccines have been developed, a large proportion of the global population remains at high risk of infection. The current study aims to develop an antiviral peptide capable of inhibiting the interaction of SARS-CoV-2 spike protein and its six major variants with the host cell angiotensin-converting enzyme 2 (ACE2) receptor. An in-silico approach was employed to design a therapeutic peptide inhibitor against the receptor-binding domain (RBD) of the spike (S) protein of SARS-CoV-2 and its variants (B.1.1.7, B.1.351, P.1, B.1.617.1, B.1.617.2 and B.1.617.3). The binding specificity and affinity of our designed peptide inhibitor Mod13AApi (YADKYQKQYKDAY) with wild-type S-RBD and its six variants was confirmed by molecular docking using the HPEPDOCK tool, whereas complex stability was determined by the MD simulation study. The physicochemical and ADMET (absorption, distribution, metabolism, excretion, and toxicity) properties of inhibitory peptides were determined using the ExPASy tool and pkCSM server. The docking results and its properties from our in-silico analysis present the Mod13AApi, a promising peptide for the rapid development of anti-coronavirus peptide-based antiviral therapy. Blockage of the binding of the spike protein of SARS-CoV-2 variants with ACE2 in the presence of the therapeutic peptide may prevent deadly SARS-CoV-2 variants entry into host cells. Therefore, the designed inhibitory peptide can be utilized as a promising therapeutic strategy to combat COVID-19, as evident from this in-silico study.

Disrupted PI3K subunit p110α signaling protects against pulmonary hypertension and reverses established disease in rodents.

Enhanced signaling via RTKs in pulmonary hypertension (PH) impedes current treatment options because it perpetuates proliferation and apoptosis resistance of pulmonary arterial smooth muscle cells (PASMCs). Here, we demonstrated hyperphosphorylation of multiple RTKs in diseased human vessels and increased activation of their common downstream effector phosphatidylinositol 3'-kinase (PI3K), which thus emerged as an attractive therapeutic target. Systematic characterization of class IA catalytic PI3K isoforms identified p110α as the key regulator of pathogenic signaling pathways and PASMC responses (proliferation, migration, survival) downstream of multiple RTKs. Smooth muscle cell-specific genetic ablation or pharmacological inhibition of p110α prevented onset and progression of pulmonary hypertension (PH) as well as right heart hypertrophy in vivo and even reversed established vascular remodeling and PH in various animal models. These effects were attributable to both inhibition of vascular proliferation and induction of apoptosis. Since this pathway is abundantly activated in human disease, p110α represents a central target in PH.

Hidden Treasures: Macrophage Long Non-Coding RNAs in Lung Cancer Progression.

Ever since RNA sequencing of whole genomes and transcriptomes became available, numerous RNA transcripts without having the classic function of encoding proteins have been discovered. Long non-coding RNAs (lncRNAs) with a length greater than 200 nucleotides were considered as "junk" in the beginning, but it has increasingly become clear that lncRNAs have crucial roles in regulating a variety of cellular mechanisms and are often deregulated in several diseases, such as cancer. Lung cancer is the leading cause of cancer-related deaths and has a survival rate of less than 10%. Immune cells infiltrating the tumor microenvironment (TME) have been shown to have a great effect on tumor development with macrophages being the major cell type within the TME. Macrophages can inherit an inflammatory M1 or an anti-inflammatory M2 phenotype. Tumor-associated macrophages, which are predominantly polarized to M2, favor tumor growth, angiogenesis, and metastasis. In this review, we aimed to describe the complex roles and functions of lncRNAs in macrophages and their influence on lung cancer development and progression through the TME.