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Background Pulmonary arterial hypertension (PAH) is a vascular remodeling disease characterized by enhanced proliferation of pulmonary artery smooth muscle cell (PASMC) and suppressed apoptosis. This phenotype has been associated with the upregulation of the oncoprotein survivin promoting mitochondrial membrane potential hyperpolarization (decreasing apoptosis) and the upregulation of growth factor and cytokines like PDGF, IL-6 and vasoactive agent like endothelin-1 (ET-1) promoting PASMC proliferation. Krüppel-like factor 5 (KLF5), is a zinc-finger-type transcription factor implicated in the regulation of cell differentiation, proliferation, migration and apoptosis. Recent studies have demonstrated the implication of KLF5 in tissue remodeling in cardiovascular diseases, such as atherosclerosis, restenosis, and cardiac hypertrophy. Nonetheless, the implication of KLF5 in pulmonary arterial hypertension (PAH) remains unknown. We hypothesized that KLF5 up-regulation in PAH triggers PASMC proliferation and resistance to apoptosis. Methods and results We showed that KFL5 is upregulated in both human lung biopsies and cultured human PASMC isolated from distal pulmonary arteries from PAH patients compared to controls. Using stimulation experiments, we demonstrated that PDGF, ET-1 and IL-6 trigger KLF-5 activation in control PASMC to a level similar to the one seen in PAH-PASMC. Inhibition of the STAT3 pathway abrogates KLF5 activation in PAH-PASMC. Once activated, KLF5 promotes cyclin B1 upregulation and promotes PASMC proliferation and triggers survivin expression hyperpolarizing mitochondria membrane potential decreasing PASMC ability to undergo apoptosis. Conclusion We demonstrated for the first time that KLF5 is activated in human PAH and implicated in the pro-proliferative and anti-apoptotic phenotype that characterize PAH-PASMC. We believe that our findings will open new avenues of investigation on the role of KLF5 in PAH and might lead to the identification of new therapeutic targets.

Metabolic syndrome (MetS) increases the risk of coronary artery disease, but effects of this condition on the working myocardium remain to be fully elucidated. In the present study we evaluated the consequences of diet-induced metabolic disorders on cardiac function and myocyte performance using female mice fed with Western diet. Animals maintained on regular chow were used as control (Ctrl). Mice on the Western diet (WesD) had increased body weight, impaired glucose metabolism, preserved diastolic and systolic function, but increased left ventricular (LV) mass, with respect to Ctrl animals. Moreover, WesD mice had reduced heart rate variability (HRV), indicative of altered cardiac sympathovagal balance. Myocytes from WesD mice had increased volume, enhanced cell mechanics, and faster kinetics of contraction and relaxation. Moreover, levels of cAMP and protein kinase A (PKA) activity were enhanced in WesD myocytes, and interventions aimed at stabilizing cAMP/PKA abrogated functional differences between Ctrl and WesD cells. Interestingly, in vivo β-adrenergic receptor (β-AR) blockade normalized the mechanical properties of WesD myocytes and revealed defective cardiac function in WesD mice, with respect to Ctrl. Collectively, these results indicate that metabolic disorders induced by Western diet enhance the cAMP/PKA signaling pathway, a possible adaptation required to maintain cardiac function., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

More than 10% of adults in the United States have type 2 diabetes mellitus (DM) with a 2-4 times higher prevalence of ischemic heart disease than the non-diabetics. Despite extensive research approaches to limit this life-threatening condition have proven unsuccessful, highlighting the need for understanding underlying molecular mechanisms. Long noncoding RNAs (lncRNAs), which regulate gene expression by acting as signals, decoys, guides, or scaffolds have been implicated in diverse cardiovascular conditions. However, their role in ischemic heart disease in DM remains poorly understood. We provide new insights into the lncRNA expression profile after ischemic heart disease in DM mice. We performed unbiased RNA sequencing of well-characterized type 2 DM model db/db mice or its control db/+ subjected to sham or MI surgery. Computational analysis of the RNA sequencing of these LV tissues identified several differentially expressed lncRNAs between (db/db sham vs. db/db MI) including Gm19522 and Gm8075. lncRNA Gm-19522 may regulate DNA replication via DNA protein kinases, while lncRNA Gm-8075 is associated with cancer gene dysregulation and PI3K/Akt pathways. Thus, the downregulation of lncRNAs Gm19522 and Gm8075 post-MI may serve as potential biomarkers or novel therapeutic targets to improve cardiac repair/recovery in diabetic ischemic heart disease., (© 2024. The Author(s).)

Background: Idiopathic Pulmonary fibrosis (IPF) is characterized by progressive scarring and fibrosis within the lungs. There is currently no cure for IPF; therefore, there is an urgent need to identify novel therapeutic targets that can prevent the progression of IPF. Compelling evidence indicates that the second messenger, cyclic adenosine monophosphate (cAMP), inhibits lung fibroblast proliferation and differentiation through the classical PKA pathway. However, the contribution of the e xchange p rotein directly a ctivated by c AMP 1 (Epac1) to IPF pathophysiological processes is yet to be investigated., Objective: To determine the role of the cAMP-binding protein Epac1 in the progression of IPF., Methods: We used lung samples from IPF patients or healthy controls, mouse lung samples, or lung fibroblast isolated from a preclinical mouse model of PF induced by bleomycin intratracheal injection. The effect of bleomycin (BLM) treatment was determined in Epac1 knock-out mice or wild-type littermates. Epac1 expression was modulated in vitro by using lentiviral vectors or adenoviruses. The therapeutic potential of the Epac1-selective pharmacological inhibitor, AM-001, was tested in vivo and in vitro, using a bleomycin mouse model of PF and an ex vivo precision-cut lung slices (PCLs) model of human lung fibrosis., Results: Epac1 expression was increased in the lung tissue of IPF patients, in IPF-diseased fibroblasts and in BLM-challenged mice. Furthermore, Epac1 genetic or pharmacological inhibition with AM-001 decreased normal and IPF fibroblast proliferation and the expression of profibrotic markers, αSMA, TGF-β/SMAD2/3, and interleukin-6 (IL-6)/STAT3 signaling pathways. Consistently, blocking Epac1 protected against BLM-induced lung injury and fibrosis, suggesting a therapeutic effect of Epac1 inhibition on PF pathogenesis and progression. Global gene expression profiling revealed a decrease in the key components of the profibrotic gene signature and neddylation pathway in Epac1-deficient lung fibroblasts and IPF human-derived PLCs. Mechanistically, the protective effect of Epac1 inhibition against PF development involves the inhibition of FoxO3a neddylation and its subsequent degradation by NEDD8, and in part, by limiting the proliferative capacity of lung-infiltrating monocytes., Conclusions: We demonstrated that Epac1 is an important regulator of the pathological state of fibroblasts in PF and that small molecules targeting Epac1 can serve as novel therapeutic drugs against PF.

Epigenetic mechanisms, including DNA methylation, histone modifications, and non-coding RNA, play a crucial role in the regulation of gene expression and are pivotal in biological processes like apoptosis, cell proliferation, and differentiation. SIN3a serves as a scaffold protein and facilitates interactions with transcriptional epigenetic partners and specific DNA-binding transcription factors to modulate gene expression by adding or removing epigenetic marks. However, the activation or repression of gene expression depends on the factors that interact with SIN3a, as it can recruit both transcriptional activators and repressors. The role of SIN3a has been extensively investigated in the context of cancer, including melanoma, lung, and breast cancer. Our group is interested in defining the roles of SIN3a and its partners in pulmonary vascular disease. Pulmonary arterial hypertension (PAH) is a multifactorial disease often described as a cancer-like disease and characterized by disrupted cellular metabolism, sustained vascular cell proliferation, and resistance to apoptosis. Molecularly, PAH shares many common signaling pathways with cancer cells, offering the opportunity to further consider therapeutic strategies used for cancer. As a result, many signaling pathways observed in cancer were studied in PAH and have encouraged new research studying SIN3a's role in PAH due to its impact on cancer growth. This comparison offers new therapeutic options. In this review, we delineate the SIN3a-associated epigenetic mechanisms in cancer and PAH cells and highlight their impact on cell survival and proliferation. Furthermore, we explore in detail the role of SIN3a in cancer to provide new insights into its emerging role in PAH pathogenesis.

The space radiation (IR) environment contains high charge and energy (HZE) nuclei emitted from galactic cosmic rays with the ability to overcome current shielding strategies, posing increased IR-induced cardiovascular disease risks for astronauts on prolonged space missions. Little is known about the effect of 5-ion simplified galactic cosmic ray simulation (simGCRsim) exposure on left ventricular (LV) function. Three-month-old, age-matched male Apolipoprotein E (ApoE) null mice were irradiated with 137 Cs gamma (γ; 100, 200, and 400 cGy) and simGCRsim (50, 100, 150 cGy all at 500 MeV/nucleon (n)). LV function was assessed using transthoracic echocardiography at early/acute (14 and 28 days) and late/degenerative (365, 440, and 660 days) times post-irradiation. As early as 14 and 28-days post IR, LV systolic function was reduced in both IR groups across all doses. At 14 days post-IR, 150 cGy simGCRsim-IR mice had decreased diastolic wall strain (DWS), suggesting increased myocardial stiffness. This was also observed later in 100 cGy γ-IR mice at 28 days. At later stages, a significant decrease in LV systolic function was observed in the 400 cGy γ-IR mice. Otherwise, there was no difference in the LV systolic function or structure at the remaining time points across the IR groups. We evaluated the expression of genes involved in hemodynamic stress, cardiac remodeling, inflammation, and calcium handling in LVs harvested 28 days post-IR. At 28 days post-IR, there is increased expression of Bnp and Ncx in both IR groups at the lowest doses, suggesting impaired function contributes to hemodynamic stress and altered calcium handling. The expression of Gals3 and β-Mhc were increased in simGCRsim and γ-IR mice respectively, suggesting there may be IR-specific cardiac remodeling. IR groups were modeled to calculate the Relative Biological Effectiveness (RBE) and Radiation Effects Ratio (RER). No lower threshold was determined using the observed dose-response curves. These findings do not exclude the possibility of the existence of a lower IR threshold or the presence of IR-induced cardiovascular disease (CVD) when combined with additional space travel stressors, e.g., microgravity., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Brojakowska, Jackson, Bisserier, Khlgatian, Jagana, Eskandari, Grano, Blattnig, Zhang, Fish, Chepurko, Chepurko, Gillespie, Dai, Kumar Rai, Garikipati, Hadri, Kishore and Goukassian.)

Doxorubicin (Dox) is an effective anticancer molecule, but its clinical efficacy is limited by strong cardiotoxic side effects. Lysosomal dysfunction has recently been proposed as a new mechanism of Dox-induced cardiomyopathy. However, to date, there is a paucity of therapeutic approaches capable of restoring lysosomal acidification and function in the heart. Methods: We designed novel poly(lactic-co-glycolic acid) (PLGA)-grafted silica nanoparticles (NPs) and investigated their therapeutic potential in the primary prevention of Dox cardiotoxicity in cardiomyocytes and mice. Results: We showed that NPs-PLGA internalized rapidly in cardiomyocytes and accumulated inside the lysosomes. Mechanistically, NPs-PLGA restored lysosomal acidification in the presence of doxorubicin or bafilomycin A1, thereby improving lysosomal function and autophagic flux. Importantly, NPs-PLGA mitigated Dox-related mitochondrial dysfunction and oxidative stress, two main mechanisms of cardiotoxicity. In vivo, inhalation of NPs-PLGA led to effective and rapid targeting of the myocardium, which prevented Dox-induced adverse remodeling and cardiac dysfunction in mice. Conclusion: Our findings demonstrate a pivotal role for lysosomal dysfunction in Dox-induced cardiomyopathy and highlight for the first time that pulmonary-driven NPs-PLGA administration is a promising strategy against anthracycline cardiotoxicity., Competing Interests: Competing Interests: The authors have declared that no competing interest exists., (© The author(s).)

Pulmonary arterial hypertension (PAH) is a multifactorial disease associated with the remodeling of pulmonary blood vessels. If left unaddressed, PAH can lead to right heart failure and even death. Multiple biological processes, such as smooth muscle proliferation, endothelial dysfunction, inflammation, and resistance to apoptosis, are associated with PAH. Increasing evidence suggests that epigenetic factors play an important role in PAH by regulating the chromatin structure and altering the expression of critical genes. For example, aberrant DNA methylation and histone modifications such as histone acetylation and methylation have been observed in patients with PAH and are linked to vascular remodeling and pulmonary vascular dysfunction. In this review article, we provide a comprehensive overview of the role of key epigenetic targets in PAH pathogenesis, including DNA methyltransferase (DNMT), ten-eleven translocation enzymes (TET), switch-independent 3A (SIN3A), enhancer of zeste homolog 2 (EZH2), histone deacetylase (HDAC), and bromodomain-containing protein 4 (BRD4). Finally, we discuss the potential of multi-omics integration to better understand the molecular signature and profile of PAH patients and how this approach can help identify personalized treatment approaches., (© 2023. The Author(s).)

The lifetime effects of space irradiation (IR) on left ventricular (LV) function are unknown. The cardiac effects induced by space-type IR, specifically 5-ion simplified galactic cosmic ray simulation (simGCRsim), are yet to be discovered. Three-month-old, age-matched, male C57BL/6J mice were irradiated with 137 Cs gamma (γ; 100, 200 cGy) and simGCRsim (50 and 100 cGy). LV function was assessed via transthoracic echocardiography at 14 and 28 days (early), and at 365, 440, and 660 (late) days post IR. We measured the endothelial function marker brain natriuretic peptide in plasma at three late timepoints. We assessed the mRNA expression of the genes involved in cardiac remodeling, fibrosis, inflammation, and calcium handling in LVs harvested at 660 days post IR. All IR groups had impaired global LV systolic function at 14, 28, and 365 days. At 660 days, 50 cGy simGCRsim-IR mice exhibited preserved LV systolic function with altered LV size and mass. At this timepoint, the simGCRsim-IR mice had elevated levels of cardiac fibrosis, inflammation, and hypertrophy markers Tgfβ1 , Mcp1 , Mmp9 , and βmhc , suggesting that space-type IR may induce the cardiac remodeling processes that are commonly associated with diastolic dysfunction. IR groups showing statistical significance were modeled to calculate the Relative Biological Effectiveness (RBE) and Radiation Effects Ratio (RER). The observed dose-response shape did not indicate a lower threshold at these IR doses. A single full-body IR at doses of 100-200 cGy for γ-IR, and 50-100 cGy for simGCRsim-IR decreases the global LV systolic function in WT mice as early as 14 and 28 days after exposure, and at 660 days post IR. Interestingly, there is an intermediate time point (365 days) where the impairment in LV function is observed. These findings do not exclude the possibility of increased acute or degenerative cardiovascular disease risks at lower doses of space-type IR, and/or when combined with other space travel-associated stressors such as microgravity.

With planned deep space and commercial spaceflights, gaps remain to address health risks in astronauts. Multiple studies have shown associations between clonal expansion of hematopoietic cells with hematopoietic malignancies and cardiometabolic disease. This expansion of clones in the absence of overt hematopoietic disorders is termed clonal hematopoiesis (CH) of indeterminate potential (CHIP). Using deep, error-corrected, targeted DNA sequencing we assayed for somatic mutations in CH-driver genes in peripheral blood mononuclear cells isolated from de-identified blood samples collected from 14 astronauts who flew Shuttle missions between 1998-2001. We identified 34 nonsynonymous mutations of relatively low variant allele fraction in 17 CH-driver genes, with the most prevalent mutations in TP53 and DNMT3A. The presence of these small clones in the blood of relatively young astronaut cohort warrants further retrospective and prospective investigation of their clinical relevance and potential application in monitoring astronaut's health., (© 2022. The Author(s).)

During spaceflight, astronauts are exposed to various physiological and psychological stressors that have been associated with adverse health effects. Therefore, there is an unmet need to develop novel diagnostic tools to predict early alterations in astronauts' health. Small nucleolar RNA (snoRNA) is a type of short non-coding RNA (60-300 nucleotides) known to guide 2'-O-methylation (Nm) or pseudouridine (ψ) of ribosomal RNA (rRNA), small nuclear RNA (snRNA), or messenger RNA (mRNA). Emerging evidence suggests that dysregulated snoRNAs may be key players in regulating fundamental cellular mechanisms and in the pathogenesis of cancer, heart, and neurological disease. Therefore, we sought to determine whether the spaceflight-induced snoRNA changes in astronaut's peripheral blood (PB) plasma extracellular vesicles (PB-EV) and peripheral blood mononuclear cells (PBMCs). Using unbiased small RNA sequencing (sRNAseq), we evaluated changes in PB-EV snoRNA content isolated from astronauts ( n = 5/group) who underwent median 12-day long Shuttle missions between 1998 and 2001. Using stringent cutoff (fold change > 2 or log 2 -fold change >1, FDR < 0.05), we detected 21 down-and 9-up-regulated snoRNAs in PB-EVs 3 days after return (R + 3) compared to 10 days before launch (L-10). qPCR validation revealed that SNORA74A was significantly down-regulated at R + 3 compared to L-10. We next determined snoRNA expression levels in astronauts' PBMCs at R + 3 and L-10 ( n = 6/group). qPCR analysis further confirmed a significant increase in SNORA19 and SNORA47 in astronauts' PBMCs at R + 3 compared to L-10. Notably, many downregulated snoRNA-guided rRNA modifications, including four Nms and five ψs. Our findings revealed that spaceflight induced changes in PB-EV and PBMCs snoRNA expression, thus suggesting snoRNAs may serve as potential novel biomarkers for monitoring astronauts' health., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Rai, Rajan, Bisserier, Brojakowska, Sebastian, Evans, Coleman, Mills, Arakelyan, Uchida, Hadri, Goukassian and Garikipati.)

There are unique stressors in the spaceflight environment. Exposure to such stressors may be associated with adverse effects on astronauts' health, including increased cancer and cardiovascular disease risks. Small extracellular vesicles (sEVs, i.e., exosomes) play a vital role in intercellular communication and regulate various biological processes contributing to their role in disease pathogenesis. To assess whether spaceflight alters sEVs transcriptome profile, sEVs were isolated from the blood plasma of 3 astronauts at two different time points: 10 days before launch (L-10) and 3 days after return (R+3) from the Shuttle mission. AC16 cells (human cardiomyocyte cell line) were treated with L-10 and R+3 astronauts-derived exosomes for 24 h. Total RNA was isolated and analyzed for gene expression profiling using Affymetrix microarrays. Enrichment analysis was performed using Enrichr. Transcription factor (TF) enrichment analysis using the ENCODE/ChEA Consensus TF database identified gene sets related to the polycomb repressive complex 2 (PRC2) and Vitamin D receptor (VDR) in AC16 cells treated with R+3 compared to cells treated with L-10 astronauts-derived exosomes. Further analysis of the histone modifications using datasets from the Roadmap Epigenomics Project confirmed enrichment in gene sets related to the H3K27me3 repressive mark. Interestingly, analysis of previously published H3K27me3-chromatin immunoprecipitation sequencing (ChIP-Seq) ENCODE datasets showed enrichment of H3K27me3 in the VDR promoter. Collectively, our results suggest that astronaut-derived sEVs may epigenetically repress the expression of the VDR in human adult cardiomyocytes by promoting the activation of the PRC2 complex and H3K27me3 levels., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Bisserier, Brojakowska, Saffran, Rai, Lee, Coleman, Sebastian, Evans, Mills, Addya, Arakelyan, Garikipati, Hadri and Goukassian.)

Over the past decades, a better understanding of the genetic and molecular alterations underlying several respiratory diseases has encouraged the development of new therapeutic strategies. Gene therapy offers new therapeutic alternatives for inherited and acquired diseases by delivering exogenous genetic materials into cells or tissues to restore physiological protein expression and/or activity. In this review, we review (1) different types of viral and non-viral vectors as well as gene-editing techniques; and (2) the application of gene therapy for the treatment of respiratory diseases and disorders, including pulmonary arterial hypertension, idiopathic pulmonary fibrosis, cystic fibrosis, asthma, alpha-1 antitrypsin deficiency, chronic obstructive pulmonary disease, non-small-cell lung cancer, and COVID-19. Further, we also provide specific examples of lung-targeted therapies and discuss the major limitations of gene therapy.

During spaceflight, astronauts are exposed to multiple unique environmental factors, particularly microgravity and ionizing radiation, that can cause a range of harmful health consequences. Over the past decades, increasing evidence demonstrates that the space environment can induce changes in gene expression and RNA processing. Long non-coding RNA (lncRNA) represent an emerging area of focus in molecular biology as they modulate chromatin structure and function, the transcription of neighboring genes, and affect RNA splicing, stability, and translation. They have been implicated in cancer development and associated with diverse cardiovascular conditions and associated risk factors. However, their role on astronauts' health after spaceflight remains poorly understood. In this perspective article, we provide new insights into the potential role of exosomal lncRNA after spaceflight. We analyzed the transcriptional profile of exosomes isolated from peripheral blood plasma of three astronauts who flew on various Shuttle missions between 1998-2001 by RNA-sequencing. Computational analysis of the transcriptome of these exosomes identified 27 differentially expressed lncRNAs with a Log 2 fold change, with molecular, cellular, and clinical implications., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Bisserier, Saffran, Brojakowska, Sebastian, Evans, Coleman, Walsh, Mills, Garikipati, Arakelyan, Hadri and Goukassian.)

Background: Electrophysiological (EP) properties have been studied mainly in the monocrotaline model of pulmonary arterial hypertension (PAH). Findings are confounded by major extrapulmonary toxicities, which preclude the ability to draw definitive conclusions regarding the role of PAH per se in EP remodeling., Objective: The purpose of this study was to investigate the EP substrate and arrhythmic vulnerability of a new model of PAH that avoids extracardiopulmonary toxicities., Methods: Sprague-Dawley rats underwent left pneumonectomy (Pn) followed by injection of the vascular endothelial growth factor inhibitor Sugen-5416 (Su/Pn). Five weeks later, cardiac magnetic resonance imaging was performed in vivo, optical action potential (AP) mapping ex vivo, and molecular analyses in vitro., Results: Su/Pn rats exhibited right ventricular (RV) hypertrophy and were highly prone to pacing-induced ventricular tachycardia/fibrillation (VT/VF). Underlying this susceptibility was disproportionate RV-sided prolongation of AP duration, which promoted formation of right-sided AP alternans at physiological rates. While propagation was impaired at all rates in Su/Pn rats, the extent of conduction slowing was most severe immediately before the emergence of interventricular lines of block and onset of VT/VF. Measurement of the cardiac wavelength revealed a decrease in Su/Pn relative to control. Nav1.5 and total connexin 43 expression was not altered, while connexin 43 phosphorylation was decreased in PAH. Col1a1 and Col3a1 transcripts were upregulated coinciding with myocardial fibrosis. Once generated, VT/VF was sustained by multiple reentrant circuits with a lower frequency of RV activation due to wavebreak formation., Conclusion: In this pure model of PAH, we document RV-predominant remodeling that promotes multiwavelet reentry underlying VT. The Su/Pn model represents a severe form of PAH that allows the study of EP properties without the confounding influence of extrapulmonary toxicity., (Copyright © 2021 Heart Rhythm Society. Published by Elsevier Inc. All rights reserved.)

Pulmonary arterial hypertension (PAH) is a severe and progressive cardiopulmonary disease characterized by pathological remodeling of the resistance pulmonary arteries (PA), ultimately leading to right ventricular (RV) failure and death. Animal models have been particularly useful for unraveling the pathogenesis of PAH by providing incisive experimental strategies that were impossible in human studies. Over the past decade, gene therapy has been making considerable progress as an alternative strategy for treating PAH disease. Animal models mimicking PAH disease are essential at preclinical stages for assessing the therapeutic potential of gene therapy and determining genome viral vectors transduction, safety, dosage, and localization of transgene expression. The most commonly used PAH rat models in gene therapy studies are the monocrotaline (MCT), the chronic hypoxia-Sugen 5416, and the pneumonectomy (PNT)-MCT models. Here, we provide detailed protocols for creating these preclinical rodent models of PAH commonly used to assess the efficiency of lung gene therapy in PAH., (© 2022. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Background Space travel-associated stressors such as microgravity or radiation exposure have been reported in astronauts after short- and long-duration missions aboard the International Space Station. Despite risk mitigation strategies, adverse health effects remain a concern. Thus, there is a need to develop new diagnostic tools to facilitate early detection of physiological stress. Methods and Results We measured the levels of circulating cell-free mitochondrial DNA in blood plasma of 14 astronauts 10 days before launch, the day of landing, and 3 days after return. Our results revealed a significant increase of cell-free mitochondrial DNA in the plasma on the day of landing and 3 days after return with vast ~2 to 355-fold interastronaut variability. In addition, gene expression analysis of peripheral blood mononuclear cells revealed a significant increase in markers of inflammation, oxidative stress, and DNA damage. Conclusions Our study suggests that cell-free mitochondrial DNA abundance might be a biomarker of stress or immune response related to microgravity, radiation, and other environmental factors during space flight.

Pulmonary arterial hypertension (PAH) is a devastating lung disease characterized by the progressive obstruction of the distal pulmonary arteries (PA). Structural and functional alteration of pulmonary artery smooth muscle cells (PASMC) and endothelial cells (PAEC) contributes to PA wall remodeling and vascular resistance, which may lead to maladaptive right ventricular (RV) failure and, ultimately, death. Here, we found that decreased expression of sarcoplasmic/endoplasmic reticulum Ca 2+ ATPase 2a (SERCA2a) in the lung samples of PAH patients was associated with the down-regulation of bone morphogenetic protein receptor type 2 (BMPR2) and the activation of signal transducer and activator of transcription 3 (STAT3). Our results showed that the antiproliferative properties of SERCA2a are mediated through the STAT3/BMPR2 pathway. At the molecular level, transcriptome analysis of PASMCs co-overexpressing SERCA2a and BMPR2 identified STAT3 amongst the most highly regulated transcription factors. Using a specific siRNA and a potent pharmacological STAT3 inhibitor (STAT3i, HJC0152), we found that SERCA2a potentiated BMPR2 expression by repressing STAT3 activity in PASMCs and PAECs. In vivo, we used a validated and efficient model of severe PAH induced by unilateral left pneumonectomy combined with monocrotaline (PNT/MCT) to further evaluate the therapeutic potential of single and combination therapies using adeno-associated virus (AAV) technology and a STAT3i. We found that intratracheal delivery of AAV1 encoding SERCA2 or BMPR2 alone or STAT3i was sufficient to reduce the mean PA pressure and vascular remodeling while improving RV systolic pressures, RV ejection fraction, and cardiac remodeling. Interestingly, we found that combined therapy of AAV1.hSERCA2a with AAV1.hBMPR2 or STAT3i enhanced the beneficial effects of SERCA2a. Finally, we used cardiac magnetic resonance imaging to measure RV function and found that therapies using AAV1.hSERCA2a alone or combined with STAT3i significantly inhibited RV structural and functional changes in PNT/MCT-induced PAH. In conclusion, our study demonstrated that combination therapies using SERCA2a gene transfer with a STAT3 inhibitor could represent a new promising therapeutic alternative to inhibit PAH and to restore BMPR2 expression by limiting STAT3 activity.

With the continued rise of the global incidence of COVID-19 infection and emergent second wave, the need to understand characteristics that impact susceptibility to infection, clinical severity, and outcomes remains vital. The objective of this study was to assess modifying effects of demographic factors on COVID-19 testing status and outcomes in a large, diverse single health system cohort. The Mount Sinai Health System de-identified COVID-19 database contained records of 39,539 patients entering the health system from 02/28/2020 to 06/08/2020 with 7,032 laboratory-confirmed cases. The prevalence of qRT-PCR nasopharyngeal swabs (χ2 = 665.7, p<0.0001) and case rates (χ2 = 445.3, p<0.0001) are highest in Hispanics and Black or African Americans. The likelihood of admission and/or presentation to an intensive care unit (ICU) versus non-ICU inpatient unit, emergency department, and outpatient services, which reflects the severity of the clinical course, was also modified by race and ethnicity. Females were less likely to be tested [Relative Risk(RR) = 1.121, p<0.0001], and males had a higher case prevalence (RR = 1.224, p<0.001). Compared to other major ethnic groups, Whites experienced a higher prevalence of mortality (p<0.05). Males experienced a higher risk of mortality (RR = 1.180, p = 0.0012) at relatively younger ages (70.58±11.75) compared to females (73.02±11.46) (p = 0.0004). There was an increased severity of disease in older patient populations of both sexes. Although Hispanic and Black or African American race was associated with higher testing prevalence and positive testing rates, the only disparity with respect to mortality was a higher prevalence in Whites., Competing Interests: We would like to indicate the authors have declared that no competing interests exist.

Background: Epigenetic mechanisms are critical in the pathogenesis of pulmonary arterial hypertension (PAH). Previous studies have suggested that hypermethylation of the BMPR2 (bone morphogenetic protein receptor type 2) promoter is associated with BMPR2 downregulation and progression of PAH. Here, we investigated for the first time the role of SIN3a (switch-independent 3a), a transcriptional regulator, in the epigenetic mechanisms underlying hypermethylation of BMPR2 in the pathogenesis of PAH., Methods: We used lung samples from PAH patients and non-PAH controls, preclinical mouse and rat PAH models, and human pulmonary arterial smooth muscle cells. Expression of SIN3a was modulated using a lentiviral vector or a siRNA in vitro and a specific adeno-associated virus serotype 1 or a lentivirus encoding for human SIN3a in vivo., Results: SIN3a is a known transcriptional regulator; however, its role in cardiovascular diseases, especially PAH, is unknown. It is interesting that we detected a dysregulation of SIN3 expression in patients and in rodent models, which is strongly associated with decreased BMPR2 expression. SIN3a is known to regulate epigenetic changes. Therefore, we tested its role in the regulation of BMPR2 and found that BMPR2 is regulated by SIN3a. It is interesting that SIN3a overexpression inhibited human pulmonary arterial smooth muscle cells proliferation and upregulated BMPR2 expression by preventing the methylation of the BMPR2 promoter region. RNA-sequencing analysis suggested that SIN3a downregulated the expression of DNA and histone methyltransferases such as DNMT1 (DNA methyltransferase 1) and EZH2 (enhancer of zeste 2 polycomb repressive complex 2) while promoting the expression of the DNA demethylase TET1 (ten-eleven translocation methylcytosine dioxygenase 1). Mechanistically, SIN3a promoted BMPR2 expression by decreasing CTCF (CCCTC-binding factor) binding to the BMPR2 promoter. Last, we identified intratracheal delivery of adeno-associated virus serotype human SIN3a to be a beneficial therapeutic approach in PAH by attenuating pulmonary vascular and right ventricle remodeling, decreasing right ventricle systolic pressure and mean pulmonary arterial pressure, and restoring BMPR2 expression in rodent models of PAH., Conclusions: All together, our study unveiled the protective and beneficial role of SIN3a in pulmonary hypertension. We also identified a novel and distinct molecular mechanism by which SIN3a regulates BMPR2 in human pulmonary arterial smooth muscle cells. Our study also identified lung-targeted SIN3a gene therapy using adeno-associated virus serotype 1 as a new promising therapeutic strategy for treating patients with PAH.

With the continuing rise of SARS-CoV2 infection globally and the emergence of various waves in different countries, understanding characteristics of susceptibility to infection, clinical severity, and outcomes remain vital. In this retrospective study, data was extracted for 39,539 patients from the de-identified Mount Sinai Health System COVID-19 database. We assessed the risk of mortality based on the presence of comorbidities and organ-specific sequelae in 7,032 CoV2 positive (+) patients. Prevalence of cardiovascular and metabolic comorbidities was high among SARS-CoV2+ individuals. Diabetes, obesity, coronary artery disease, hypertension, atrial fibrillation, and heart failure all increased overall mortality risk, while asthma did not. Ethnicity modified the risk of mortality associated with these comorbidities. With regards to secondary complications in the setting of infection, individuals with acute kidney injury and acute myocardial injury showed an increase in mortality risk. Cerebral infarcts and acute venous thromboembolic events were not associated with increased risk of mortality. Biomarkers for cardiovascular injury, coagulation, and inflammation were compared between deceased and survived individuals. We found that cardiac and coagulation biomarkers were elevated and fell beyond normal range more often in deceased patients. Several, but not all, inflammatory markers evaluated were increased in deceased patients. In summary, we identified comorbidities and sequelae along with peripheral blood biomarkers that were associated with elevated clinical severity and poor outcomes in COVID-19 patients. Overall, these findings detail the granularity of previously reported factors which may impact susceptibility, clinical severity, and mortality during the course of COVID-19 disease., Competing Interests: The authors have declared that no competing interests exist.

The first confirmed case of novel Coronavirus Disease 2019 (COVID-19) in the United States was reported on January 20, 2020. As of November 24, 2020, close to 12.2 million cases of COVID-19 was confirmed in the US, with over 255,958 deaths. The rapid transmission of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), its unusual and divergent presentation has strengthened the status of COVID-19 as a major public health threat. In this review, we aim to 1- discuss the epidemiological data from various COVID-19 patient cohorts around the world and the USA as well the associated risk factors; 2- summarize the pathophysiology of SARS-CoV-2 infection and the underlying molecular mechanisms for the respiratory and cardiovascular manifestations; 3- highlight the potential treatments and vaccines as well as current clinical trials for COVID-19., (Copyright © 2020. Published by Elsevier Ltd.)

Pulmonary arterial hypertension (PAH) is a rare and chronic lung disease characterized by progressive occlusion of the small pulmonary arteries, which is associated with structural and functional alteration of the smooth muscle cells and endothelial cells within the pulmonary vasculature. Excessive vascular remodeling is, in part, responsible for high pulmonary vascular resistance and the mean pulmonary arterial pressure, increasing the transpulmonary gradient and the right ventricular "pressure overload", which may result in right ventricular (RV) dysfunction and failure. Current technological advances in multi-omics approaches, high-throughput sequencing, and computational methods have provided valuable tools in molecular profiling and led to the identification of numerous genetic variants in PAH patients. In this review, we summarized the pathogenesis, classification, and current treatments of the PAH disease. Additionally, we outlined the latest next-generation sequencing technologies and the consequences of common genetic variants underlying PAH susceptibility and disease progression. Finally, we discuss the importance of molecular genetic testing for precision medicine in PAH and the future of genomic medicines, including gene-editing technologies and gene therapies, as emerging alternative approaches to overcome genetic disorders in PAH.

Compared to low doses of gamma irradiation (γ-IR), high-charge-and-energy (HZE) particle IR may have different biological response thresholds in cardiac tissue at lower doses, and these effects may be IR type and dose dependent. Three- to four-month-old female CB6F1/Hsd mice were exposed once to one of four different doses of the following types of radiation: γ-IR 137 Cs (40-160 cGy, 0.662 MeV), 14 Si-IR (4-32 cGy, 260 MeV/n), or 22 Ti-IR (3-26 cGy, 1 GeV/n). At 16 months post-exposure, animals were sacrificed and hearts were harvested and archived as part of the NASA Space Radiation Tissue Sharing Forum. These heart tissue samples were used in our study for RNA isolation and microarray hybridization. Functional annotation of twofold up/down differentially expressed genes (DEGs) and bioinformatics analyses revealed the following: (i) there were no clear lower IR thresholds for HZE- or γ-IR; (ii) there were 12 common DEGs across all 3 IR types; (iii) these 12 overlapping genes predicted various degrees of cardiovascular, pulmonary, and metabolic diseases, cancer, and aging; and (iv) these 12 genes revealed an exclusive non-linear DEG pattern in 14 Si- and 22 Ti-IR-exposed hearts, whereas two-thirds of γ-IR-exposed hearts revealed a linear pattern of DEGs. Thus, our study may provide experimental evidence of excess relative risk (ERR) quantification of low/very low doses of full-body space-type IR-associated degenerative disease development.

Rationale: Metabolic syndrome (MetS) is a cluster of interrelated risk factors for cardiovascular diseases and atherosclerosis. Circulating levels of large extracellular vesicles (lEVs), submicrometer-sized vesicles released from plasma membrane, from MetS patients were shown to induce endothelial dysfunction, but their role in early stage of atherosclerosis and on vascular smooth muscle cells (SMC) remain to be fully elucidated., Objective: To determine the mechanisms by which lEVs lead to the progression of atherosclerosis in the setting of MetS., Methods and Results: Proteomic analysis revealed that the small GTPase, Rap1 was overexpressed in lEVs from MetS patients compared with those from non-MetS subjects. Rap1 was in GTP-associated active state in both types of lEVs, and Rap1-lEVs levels correlated with increased cardiovascular risks, including stenosis. MetS-lEVs, but not non-MetS-lEVs, increased Rap1-dependent endothelial cell permeability. MetS-lEVs significantly promoted migration and proliferation of human aortic SMC and increased expression of proinflammatory molecules and activation of ERK (extracellular signal-regulated kinase) 5/p38 pathways. Neutralization of Rap1 by specific antibody or pharmacological inhibition of Rap1 completely prevented the effects of lEVs from MetS patients. High-fat diet-fed ApoE -/- mice displayed an increased expression of Rap1 both in aortas and circulating lEVs. lEVs accumulated in plaque atherosclerotic lesions depending on the progression of atherosclerosis. lEVs from high-fat diet-fed ApoE -/- mice, but not those from mice fed with a standard diet, enhanced SMC proliferation. Human atherosclerotic lesions were enriched in lEVs expressing Rap1., Conclusions: These data demonstrate that Rap1 carried by MetS-lEVs participates in the enhanced SMC proliferation, migration, proinflammatory profile, and activation of ERK5/p38 pathways leading to vascular inflammation and remodeling, and atherosclerosis. These results highlight that Rap1 carried by MetS-lEVs may be a novel determinant of diagnostic value for cardiometabolic risk factors and suggest Rap1 as a promising therapeutic target against the development of atherosclerosis. Graphical Abstract: A graphical abstract is available for this article.

Aims: Cyclic adenosine monophosphate (cAMP) is the predominant intracellular second messenger that transduces signals from Gs-coupled receptors. Intriguingly, there is evidence from various cell types that an extracellular cAMP pathway is active in the extracellular space. Herein, we investigated the role of extracellular cAMP in the lung and examined whether it may act on pulmonary vascular cell proliferation and pulmonary vasculature remodelling in the pathogenesis of pulmonary hypertension (PH)., Methods and Results: The expression of cyclic AMP-metabolizing enzymes was increased in lungs from patients with PH as well as in rats treated with monocrotaline and mice exposed to Sugen/hypoxia. We report that inhibition of the endogenous extracellular cAMP pathway exacerbated Sugen/hypoxia-induced lung remodelling. We found that application of extracellular cAMP induced an increase in intracellular cAMP levels and inhibited proliferation and migration of pulmonary vascular cells in vitro. Extracellular cAMP infusion in two in vivo PH models prevented and reversed pulmonary and cardiac remodelling associated with PH. Using protein expression analysis along with luciferase assays, we found that extracellular cAMP acts via the A2R/PKA/CREB/p53/Cyclin D1 pathway., Conclusions: Taken together, our data reveal the presence of an extracellular cAMP pathway in pulmonary arteries that attempts to protect the lung during PH, and suggest targeting of the extracellular cAMP signalling pathway to limit pulmonary vascular remodelling and PH., (Published on behalf of the European Society of Cardiology. All rights reserved. © The Author(s) 2019. For permissions, please email: journals.permissions@oup.com.)

Pulmonary arterial hypertension (PAH) is a progressive and fatal lung disease of multifactorial etiology. Most of the available drugs and FDA-approved therapies for treating pulmonary hypertension attempt to overcome the imbalance between vasoactive and vasodilator mediators, and restore the endothelial cell function. Traditional medications for treating PAH include the prostacyclin analogs and receptor agonists, phosphodiesterase 5 inhibitors, endothelin-receptor antagonists, and cGMP activators. While the current FDA-approved drugs showed improvements in quality of life and hemodynamic parameters, they have shown only very limited beneficial effects on survival and disease progression. None of them offers a cure against PAH, and the median survival rate remains less than three years from diagnosis. Extensive research efforts have led to the emergence of innovative therapeutic approaches in the area of PAH. In this review, we provide an overview of the current FDA-approved therapies in PAH and discuss the associated clinical trials and reported-side effects. As recent studies have led to the emergence of innovative therapeutic approaches in the area of PAH, we also focus on the latest promising therapies in preclinical studies such as stem cell-based therapies, gene transfer, and epigenetic therapies., Competing Interests: The authors have declared that no conflict of interest exists., (© 2020 Bisserier et al. Published by IMR press.)

Inhibition of pulmonary fibrosis (PF) by restoring sarco/endoplasmic reticulum calcium ATPase 2a isoform (SERCA2a) expression using targeted gene therapy may be a potentially powerful new treatment approach for PF. Here, we found that SERCA2a expression was significantly decreased in lung samples from patients with PF and in the bleomycin (BLM) mouse model of PF. In the BLM-induced PF model, intratracheal aerosolized adeno-associated virus serotype 1 (AAV1) encoding for human SERCA2a (AAV1.hSERCA2a) reduces lung fibrosis and associated vascular remodeling. SERCA2a gene therapy also decreases right ventricular pressure and hypertrophy in both prevention and curative protocols. In vitro, we observed that SERCA2a overexpression inhibits fibroblast proliferation, migration, and fibroblast-to-myofibroblast transition induced by transforming growth factor β (TGF-β1). Thus, pro-fibrotic gene expression is prevented by blocking nuclear factor κB (NF-κB)/interleukin-6 (IL-6)-induced signal transducer and activator of transcription 3 (STAT3) activation. This effect is signaled toward an inhibitory mechanism of small mother against decapentaplegic (SMAD)/TGF-β signaling through the repression of OTU deubiquitinase, ubiquitin aldehyde binding 1 (OTUB1) and Forkhead box M1 (FOXM1). Interestingly, this cross-inhibition leads to an increase of SKI and SnoN expression, an auto-inhibitory feedback loop of TGF-β signaling. Collectively, our results demonstrate that SERCA2a gene transfer attenuates bleomycin (BLM)-induced PF by blocking the STAT3/FOXM1 pathway and promoting the SNON/SKI Axis. Thus, SERCA2a gene therapy may be a potential therapeutic target for PF., (Copyright © 2019 The American Society of Gene and Cell Therapy. Published by Elsevier Inc. All rights reserved.)

Idiopathic pulmonary fibrosis (IPF) is an interstitial lung disease characterized by an accumulation of scar tissue within the lungs and the common presence of usual interstitial pneumonia. Unfortunately, only a few FDA-approved therapeutic options are currently available for the treatment of IPF and IPF remains associated with poor prognosis. Therefore, the identification of new pharmacological targets and strategies are critical for the treatment of IPF. This commentary aims to further discuss the role of sarcoplasmic reticulum Ca 2+ -ATPase 2a and its downstream signaling in IPF. Finally, this commentary offers new insights and perspectives regarding the therapeutic potential of AAV-mediated SERCA2A gene therapy as an emerging therapy for respiratory diseases., Competing Interests: Competing Interests The authors declare that they have no competing interests.

Pulmonary arterial hypertension (PAH) is a multifactorial cardiopulmonary disease characterized by an elevation of pulmonary artery pressure (PAP) and pulmonary vascular resistance (PVR), which can lead to right ventricular (RV) failure, multi-organ dysfunction, and ultimately to premature death. Despite the advances in molecular biology, the mechanisms underlying pulmonary hypertension (PH) remain unclear. Nowadays, there is no curative treatment for treating PH. Therefore, it is crucial to identify novel, specific therapeutic targets and to offer more effective treatments against the progression of PH. Increasing amounts of evidence suggest that epigenetic modification may play a critical role in the pathogenesis of PAH. In the presented paper, we provide an overview of the epigenetic mechanisms specifically, DNA methylation, histone acetylation, histone methylation, and ncRNAs. As the recent identification of new pharmacological drugs targeting these epigenetic mechanisms has opened new therapeutic avenues, we also discuss the importance of epigenetic-based therapies in the context of PH., Competing Interests: Declaration of interest The authors declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of this review.

In this protocol, we detail the correct procedural steps and necessary precautions to successfully perform a left pneumonectomy and induce PAH in rats with the additional administration of monocrotaline (MCT) or SU5416 (Sugen). We also compare these two models to other PAH models commonly used in research. In the last few years, the focus of animal PAH models has moved towards studying the mechanism of angioproliferation of plexiform lesions, in which the role of increased pulmonary blood flow is considered as an important trigger in the development of severe pulmonary vascular remodeling. One of the most promising rodent models of increased pulmonary flow is the unilateral left pneumonectomy combined with a "second hit" of MCT or Sugen. The removal of the left lung leads to increased and turbulent pulmonary blood flow and vascular remodeling. Currently, there is no detailed procedure of the pneumonectomy surgery in rats. This article details a step-by-step protocol of the pneumonectomy surgical procedure and post-operative care in male Sprague-Dawley rats. Briefly, the animal is anesthetized and the chest is opened. Once the left pulmonary artery, pulmonary vein, and bronchus are visualized, they are ligated and the left lung is removed. The chest then closed and the animal recovered. Blood is forced to circulate only on the right lung. This increased vascular pressure leads to a progressive remodeling and occlusion of small pulmonary arteries. The second hit of MCT or Sugen is used one week post-surgery to induce endothelial dysfunction. The combination of increased blood flow in the lung and endothelial dysfunction produces severe PAH. The primary limitation of this procedure is that it requires general surgical skills.

Background: Pulmonary arterial hypertension (PAH) results in right ventricular (RV) failure, electro-mechanical dysfunction and heightened risk of sudden cardiac death (SCD), although exact mechanisms and predisposing factors remain unclear. Because impaired chronotropic response to exercise is a strong predictor of early mortality in patients with PAH, we hypothesized that progressive elevation in heart rate can unmask ventricular tachyarrhythmias (VT) in a rodent model of monocrotaline (MCT)-induced PAH. We further hypothesized that intra-tracheal gene delivery of aerosolized AAV1.SERCA2a (AAV1.S2a), an approach which improves pulmonary vascular remodeling in PAH, can suppress VT in this model., Objective: To determine the efficacy of pulmonary AAV1.S2a in reversing electrophysiological (EP) remodeling and suppressing VT in PAH., Methods: Male rats received subcutaneous injection of MCT (60 mg/kg) leading to advanced PAH. Three weeks following MCT, rats underwent intra-tracheal delivery of aerosolized AAV1.S2a (MCT + S2a, N = 8) or saline (MCT, N = 9). Age-matched rats served as controls (CTRL, N = 7). The EP substrate and risk of VT were determined using high-resolution optical action potential (AP) mapping ex vivo. The expression levels of key ion channel subunits, fibrosis markers and hypertrophy indices were measured by RT-PCR and histochemical analyses., Results: Over 80% of MCT but none of the CTRL hearts were prone to sustained VT by rapid pacing (P < .01). Aerosolized gene delivery of AAV1.S2a to the lung suppressed the incidence of VT to <15% (P < .05). Investigation of the EP substrate revealed marked prolongation of AP duration (APD), increased APD heterogeneity, a reversal in the trans-epicardial APD gradient, and marked conduction slowing in untreated MCT compared to CTRL hearts. These myocardial EP changes coincided with major remodeling in the expression of K and Ca channel subunits, decreased expression of Cx43 and increased expression of pro-fibrotic and pro-hypertrophic markers. Intra-tracheal gene delivery of aerosolized AAV1 carrying S2a but not luciferase resulted in selective upregulation of the human isoform of SERCA2a in the lung but not the heart. This pulmonary intervention, in turn, ameliorated MCT-induced APD prolongation, reversed spatial APD heterogeneity, normalized myocardial conduction, and suppressed the incidence of pacing-induced VT. Comparison of the minimal conduction velocity (CV) generated at the fastest pacing rate before onset of VT or at the end of the protocol revealed significantly lower values in untreated compared to AAV1.S2a treated PAH and CTRL hearts. Reversal of EP remodeling by pulmonary AAV1.S2a gene delivery was accompanied by restored expression of key ion channel transcripts. Restored expression of Cx43 and collagen but not the pore-forming Na channel subunit Nav1.5 likely ameliorated VT by improving CV at rapid rates in PAH., Conclusion: Aerosolized AAV1.S2a gene delivery selectively to the lungs ameliorates myocardial EP remodeling and VT susceptibility at rapid heart rates. Our findings highlight for the first time the utility of a non-cardiac gene therapy approach for arrhythmia suppression., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Resistance to targeted therapies has become increasingly prevalent. We noted that resistance to different targeted therapies occurs by largely common mechanisms. In this study, we used this information for identifying the mechanisms of resistance to enhancer of zeste homolog 2 (EZH2) inhibitors in diffuse large B-cell lymphoma (DLBCL) harboring EZH2 mutations. We discovered that EZH2 inhibitor-resistant DLBCL cells showed activation of the insulin-like growth factor 1 receptor (IGF-1R), MEK, and phosphoinositide-3-kinase (PI3K) pathways. Constitutive activation of IGF-1R, MEK, or PI3K pathways was sufficient to confer resistance to EZH2 inhibitors in DLBCL. The activation of the PI3K/AKT and MAPK pathways decreased TNFSF10 and BAD expression through a FOXO3-dependent mechanism, which was required for the antitumor effects of EZH2i GSK126. We also identified multiple acquired mutations in EZH2 inhibitor-resistant DLBCL cell lines. These mutations independently conferred resistance to EZH2 inhibitors. Mechanistically, cellular thermal shift assays revealed that the acquired EZH2 mutations that confer resistance to EZH2 inhibitors prevent EZH2 inhibitor binding to the EZH2 mutants. Notably, EZH2 inhibitor GSK126- and EPZ-6438-resistant DLBCL cells remained sensitive to the EZH2 inhibitor UNC1999 and embryonic ectoderm development protein inhibitor EED226, which provides an opportunity to treat DLBCLs that are resistant to these drugs. Collectively, our results underpin the importance for developing a unified approach for forestalling drug resistance by prospectively considering lessons learned from the use of different targeted therapeutic agents., (© 2018 by The American Society of Hematology.)

Rationale: Although the second messenger cyclic AMP (cAMP) is physiologically beneficial in the heart, it largely contributes to cardiac disease progression when dysregulated. Current evidence suggests that cAMP is produced within mitochondria. However, mitochondrial cAMP signaling and its involvement in cardiac pathophysiology are far from being understood., Objective: To investigate the role of MitEpac1 (mitochondrial exchange protein directly activated by cAMP 1) in ischemia/reperfusion injury., Methods and Results: We show that Epac1 (exchange protein directly activated by cAMP 1) genetic ablation ( Epac1 -/- ) protects against experimental myocardial ischemia/reperfusion injury with reduced infarct size and cardiomyocyte apoptosis. As observed in vivo, Epac1 inhibition prevents hypoxia/reoxygenation-induced adult cardiomyocyte apoptosis. Interestingly, a deleted form of Epac1 in its mitochondrial-targeting sequence protects against hypoxia/reoxygenation-induced cell death. Mechanistically, Epac1 favors Ca 2+ exchange between the endoplasmic reticulum and the mitochondrion, by increasing interaction with a macromolecular complex composed of the VDAC1 (voltage-dependent anion channel 1), the GRP75 (chaperone glucose-regulated protein 75), and the IP3R1 (inositol-1,4,5-triphosphate receptor 1), leading to mitochondrial Ca 2+ overload and opening of the mitochondrial permeability transition pore. In addition, our findings demonstrate that MitEpac1 inhibits isocitrate dehydrogenase 2 via the mitochondrial recruitment of CaMKII (Ca 2+ /calmodulin-dependent protein kinase II), which decreases nicotinamide adenine dinucleotide phosphate hydrogen synthesis, thereby, reducing the antioxidant capabilities of the cardiomyocyte., Conclusions: Our results reveal the existence, within mitochondria, of different cAMP-Epac1 microdomains that control myocardial cell death. In addition, our findings suggest Epac1 as a promising target for the treatment of ischemia-induced myocardial damage., (© 2017 American Heart Association, Inc.)

Mutations in the NRAS oncogene are present in up to 20% of melanoma. Here, we show that interferon alpha-inducible protein 6 (IFI6) is necessary for NRASQ61K-induced transformation and melanoma growth. IFI6 was transcriptionally upregulated by NRASQ61K, and knockdown of IFI6 resulted in DNA replication stress due to dysregulated DNA replication via E2F2. This stress consequentially inhibited cellular transformation and melanoma growth via senescence or apoptosis induction depending on the RB and p53 pathway status of the cells. NRAS-mutant melanoma were significantly more resistant to the cytotoxic effects of DNA replication stress-inducing drugs, and knockdown of IFI6 increased sensitivity to these drugs. Pharmacological inhibition of IFI6 expression by the MEK inhibitor trametinib, when combined with DNA replication stress-inducing drugs, blocked NRAS-mutant melanoma growth. Collectively, we demonstrate that IFI6, via E2F2 regulates DNA replication and melanoma development and growth, and this pathway can be pharmacologically targeted to inhibit NRAS-mutant melanoma., Competing Interests: The authors declare that no competing interests exist.

Physical exercise or hypertension requires that the heart increases its hemodynamic work. However, this adaptation is based on distinct cardiac remodelling according to the physiological or pathological origin of the stress. As shown here with two examples, understanding the molecular events leading to cardiac remodeling may offer new opportunities for the development of therapies for heart failure. The recently described Epac1 protein is an effector of the second messenger cAMP. Following a pathological stress, the cAMP-binding protein Epac1 induces cardiac hypertrophy and fibrosis as well as alteration of calcium cycling suggesting that Epac1 pharmacological inhibition may be of therapeutic value. Furthermore, the protein carabin is an important regulator of several effectors of pathological cardiac remodelling. Experimental manipulation of carabin expression profoundly alters the development of heart failure., (© 2015 médecine/sciences – Inserm.)

Background: Cardiac hypertrophy is an early hallmark during the clinical course of heart failure and is regulated by various signaling pathways. However, the molecular mechanisms that negatively regulate these signal transduction pathways remain poorly understood., Methods and Results: Here, we characterized Carabin, a protein expressed in cardiomyocytes that was downregulated in cardiac hypertrophy and human heart failure. Four weeks after transverse aortic constriction, Carabin-deficient (Carabin(-/-)) mice developed exaggerated cardiac hypertrophy and displayed a strong decrease in fractional shortening (14.6±1.6% versus 27.6±1.4% in wild type plus transverse aortic constriction mice; P<0.0001). Conversely, compensation of Carabin loss through a cardiotropic adeno-associated viral vector encoding Carabin prevented transverse aortic constriction-induced cardiac hypertrophy with preserved fractional shortening (39.9±1.2% versus 25.9±2.6% in control plus transverse aortic constriction mice; P<0.0001). Carabin also conferred protection against adrenergic receptor-induced hypertrophy in isolated cardiomyocytes. Mechanistically, Carabin carries out a tripartite suppressive function. Indeed, Carabin, through its calcineurin-interacting site and Ras/Rab GTPase-activating protein domain, functions as an endogenous inhibitor of calcineurin and Ras/extracellular signal-regulated kinase prohypertrophic signaling. Moreover, Carabin reduced Ca(2+)/calmodulin-dependent protein kinase II activation and prevented nuclear export of histone deacetylase 4 after adrenergic stimulation or myocardial pressure overload. Finally, we showed that Carabin Ras-GTPase-activating protein domain and calcineurin-interacting domain were both involved in the antihypertrophic action of Carabin., Conclusions: Our study identifies Carabin as a negative regulator of key prohypertrophic signaling molecules, calcineurin, Ras, and Ca(2+)/calmodulin-dependent protein kinase II and implicates Carabin in the development of cardiac hypertrophy and failure., (© 2014 American Heart Association, Inc.)

Aims: Stimulation of β-adrenergic receptors (β-AR) increases cAMP production and contributes to the pathogenesis of cardiac hypertrophy and failure through poorly understood mechanisms. We previously demonstrated that Exchange protein directly activated by cAMP 1 (Epac1)-induced hypertrophy in primary cardiomyocytes. Among the mechanisms triggered by cardiac stress, autophagy has been highlighted as a protective or harmful response. Here, we investigate whether Epac1 promotes cardiac autophagy and how altered autophagy has an impact on Epac1-induced cardiomyocyte hypertrophy., Methods and Results: We reported that direct stimulation of Epac1 with the agonist, Sp-8-(4-chlorophenylthio)-2'-O-methyl-cAMP (Sp-8-pCPT) promoted autophagy activation in neonatal cardiomyocytes. Stimulation of β-AR with isoprenaline (ISO) mimicked the effect of Epac1 on autophagy markers. Conversely, the induction of autophagy flux following ISO treatment was prevented in cardiomyocytes pre-treated with a selective inhibitor of Epac1, CE3F4. Importantly, we found that Epac1 deletion in mice protected against β-AR-induced cardiac remodelling and prevented the induction of autophagy. The signalling mechanisms underlying Epac1-induced autophagy involved a Ca(2+)/calmodulin-dependent kinase kinase β (CaMKKβ)/AMP-dependent protein kinase (AMPK) pathway. Finally, we provided evidence that pharmacological inhibition of autophagy using 3-methyladenine (3-MA) or down-regulation of autophagy-related protein 5 (Atg5) significantly potentiated Epac1-promoted cardiomyocyte hypertrophy., Conclusion: Altogether, these findings demonstrate that autophagy is an adaptive response to antagonize Epac1-promoted cardiomyocyte hypertrophy., (Published on behalf of the European Society of Cardiology. All rights reserved. © The Author 2014. For permissions please email: journals.permissions@oup.com.)

Epacs (exchange proteins directly activated by cAMP) act as guanine-nucleotide-exchange factors for the Ras-like small G-proteins Rap1 and Rap2, and are now recognized as incontrovertible factors leading to complex and diversified cAMP signalling pathways. Given the critical role of cAMP in the regulation of cardiac function, several studies have investigated the functional role of Epacs in the heart, providing evidence that Epacs modulate intracellular Ca2+ and are involved in several cardiac pathologies such as cardiac hypertrophy and arrhythmia. The present review summarizes recent data on the Epac signalling pathway and its role in cardiac pathophysiology. We also discuss recent advances in the discovery of novel pharmacological modulators of Epacs that were identified by high-throughput screening and their therapeutic potential for the treatment of cardiac disorders.

Background: Pulmonary arterial hypertension (PAH) is a vasculopathy characterized by enhanced pulmonary artery smooth muscle cell (PASMC) proliferation and suppressed apoptosis. This results in both increase in pulmonary arterial pressure and pulmonary vascular resistance. Recent studies have shown the implication of the signal transducer and activator of transcription 3 (STAT3)/bone morphogenetic protein receptor 2 (BMPR2)/peroxisome proliferator-activated receptor gamma (PPARγ) in PAH. STAT3 activation induces BMPR2 downregulation, decreasing PPARγ, which both contribute to the proproliferative and antiapoptotic phenotype seen in PAH. In chondrocytes, activation of this axis has been attributed to the advanced glycation end-products receptor (RAGE). As RAGE is one of the most upregulated proteins in PAH patients' lungs and a strong STAT3 activator, we hypothesized that by activating STAT3, RAGE induces BMPR2 and PPARγ downregulation, promoting PAH-PASMC proliferation and resistance to apoptosis., Methods and Results: In vitro, using PASMCs isolated from PAH and healthy patients, we demonstrated that RAGE is overexpressed in PAH-PASMC (6-fold increase), thus inducing STAT3 activation (from 10% to 40% positive cells) and decrease in BMPR2 and PPARγ levels (>50% decrease). Pharmacological activation of RAGE in control cells by S100A4 recapitulates the PAH phenotype (increasing RAGE by 6-fold, thus activating STAT3 and decreasing BMPR2 and PPARγ). In both conditions, this phenotype is totally reversed on RAGE inhibition. In vivo, RAGE inhibition in monocrotaline- and Sugen-induced PAH demonstrates therapeutic effects characterized by PA pressure and right ventricular hypertrophy decrease (control rats have an mPAP around 15 mm Hg, PAH rats have an mPAP >40 mm Hg, and with RAGE inhibition, mPAP decreases to 20 and 28 mm Hg, respectively, in MCT and Sugen models). This was associated with significant improvement in lung perfusion and vascular remodeling due to decrease in proliferation (>50% decrease) and BMPR2/PPARγ axis restoration (increased by ≥60%)., Conclusion: We have demonstrated the implications of RAGE in PAH etiology. Thus, RAGE constitutes a new attractive therapeutic target for PAH.

The cAMP-binding protein Epac is a therapeutic target for the treatment of various diseases such as cardiac hypertrophy and tumor invasion. This points out the importance to develop Epac inhibitors to better understand the involvement of these cAMP sensors in physiology and pathophysiology. Here, we have developed a functional fluorescence-based high-throughput assay with a Z' value around 0.7 for screening Epac-specific antagonists. We identified an Epac1 inhibitor compound named CE3F4 that blocked Epac1 guanine nucleotide exchange activity toward its effector Rap1 both in cell-free systems and in intact cells. CE3F4 is a tetrahydroquinoline analog that fails to influence protein kinase A holoenzyme activity. CE3F4 inhibited neither the interaction of Rap1 with Epac1 nor directly the GDP exchange on Rap1. The kinetics of inhibition by CE3F4 indicated that this compound did not compete for binding of agonists to Epac1 and suggested an uncompetitive inhibition mechanism with respect to Epac1 agonists. A structure-activity study showed that the formyl group on position 1 and the bromine atom on position 5 of the tetrahydroquinoline skeleton were important for CE3F4 to exert its inhibitory activity. Finally, CE3F4 inhibited Rap1 activation in living cultured cells, following Epac activation by either 8-(4-chlorophenylthio)-2'-O-methyl-cAMP, an Epac-selective agonist, or isoprenaline, a non-selective β-adrenergic receptor agonist. Our study shows that CE3F4 and related compounds may serve as a basis for the development of new therapeutic drugs.

Pulmonary arterial hypertension (PAH) is an obstructive vasculopathy characterized by enhanced pulmonary artery smooth muscle cell (PASMC) proliferation and suppressed apoptosis. This phenotype is sustained by the activation of the Src/signal transducer and activator of transcription 3 (STAT3) axis, maintained by a positive feedback loop involving miR-204 and followed by an aberrant expression/activation of its downstream targets such as Pim1 and nuclear factor of activated T-cells (NFATc2). Dehydroepiandrosterone (DHEA) is a steroid hormone shown to reverse vascular remodeling in systemic vessels. Since STAT3 has been described as modulated by DHEA, we hypothesized that DHEA reverses human pulmonary hypertension by inhibiting Src/STAT3 constitutive activation. Using PASMCs isolated from patients with PAH (n = 3), we demonstrated that DHEA decreases both Src and STAT3 activation (Western blot and nuclear translocation assay), resulting in a significant reduction of Pim1, NFATc2 expression/activation (quantitative RT-PCR and Western blot), as well as Survivin and upregulation of bone morphogenetic protein receptor 2 (BMPR2) and miR-204. Src/STAT3 axis inhibition by DHEA is associated with 1) mitochondrial membrane potential (tetramethylrhodamine methyl-ester perchlorate; n = 150; P < 0.05) depolarization increasing apoptosis by 25% (terminal deoxynucleotidyl transferase dUTP-mediated nick-end labeling; n = 150; P < 0.05); and 2) decreased intracellular Ca(2+) concentration (fluo-3 AM; n = 150; P < 0.05) and proliferation by 30% (PCNA). Finally, in vivo similarly to STAT3 inhibition DHEA improves experimental PAH (monocrotaline rats) by decreasing mean PA pressure and right ventricle hypertrophy. These effects were associated with the inhibition of Src, STAT3, Pim1, NFATc2, and Survivin and the upregulation of BMPR2 and miR-204. We demonstrated that DHEA reverses pulmonary hypertension in part by inhibiting the Src/STAT3.