Pulmonary arterial hypertension: molecular genetic basis and emerging treatments

YesPulmonary arterial hypertension (PAH) is a rare cardiovascular disorder caused by narrowing of blood vessels in the lung and in the absence of therapy leads to right heart failure and death. No cure for this devastating disorder is known. The major objective of the current treatments is to improve symptoms and these therapies were developed prior to the discovery that this disease has substantial genetic components. In this review, we discuss molecular genetic basis of PAH together with pathobiology, current and future therapeutic interventions

BMPR-II deficiency elicits pro-proliferative and anti-apoptotic responses through the activation of TGFbeta-TAK1-MAPK pathways in PAH

Pulmonary arterial hypertension (PAH) is a cardiovascular disorder associated with enhanced proliferation and suppressed apoptosis of pulmonary arterial smooth muscle cells (PASMCs). Heterozygous mutations in the type II receptor for bone morphogenetic protein (BMPR2) underlie the majority of the inherited and familial forms of PAH. The transforming growth factor beta (TGFbeta) pathway is activated in both human and experimental models of PAH. However, how these factors exert pro-proliferative and anti-apoptotic responses in PAH remains unclear. Using mouse primary PASMCs derived from knock-in mice, we demonstrated that BMPR-II dysfunction promotes the activation of small mothers against decapentaplegia-independent mitogen-activated protein kinase (MAPK) pathways via TGFbeta-associated kinase 1 (TAK1), resulting in a pro-proliferative and anti-apoptotic response. Inhibition of the TAK1-MAPK axis rescues abnormal proliferation and apoptosis in these cells. In both hypoxia and monocrotaline-induced PAH rat models, which display reduced levels of bmpr2 transcripts, this study further indicates that the TGFbeta-MAPK axis is activated in lungs following elevation of both expression and phosphorylation of the TAK1 protein. In ex vivo cell-based assays, TAK1 inhibits BMP-responsive reporter activity and interacts with BMPR-II receptor. In the presence of pathogenic BMPR2 mutations observed in PAH patients, this interaction is greatly reduced. Taken together, these data suggest dysfunctional BMPR-II responsiveness intensifies TGFbeta-TAK1-MAPK signalling and thus alters the ratio of apoptosis to proliferation. This axis may be a potential therapeutic target in PAH

BMPRII deficiency impairs apoptosis via the BMPRII-ALK1-BclX-mediated pathway in pulmonary arterial hypertension (PAH)

YesPulmonary Arterial Hypertension (PAH) is a devastating cardiovascular disorder characterised by the remodelling of pre-capillary pulmonary arteries. The vascular remodelling observed in PAH patients results from excessive proliferation and apoptosis resistance of pulmonary arterial smooth muscle (PASMCs) and endothelial cells (PAECs). We have previously demonstrated that mutations in the type II receptor for bone morphogenetic protein (BMPRII) underlie the majority of the familial and inherited forms of the disease. We have further demonstrated that BMPRII deficiency promotes excessive proliferation and attenuates apoptosis in PASMCs, but the underlying mechanisms remain unclear. The major objective of this study is to investigate how BMPRII deficiency impairs apoptosis in PAH. Using multidisciplinary approaches, we demonstrate that deficiency in the expression of BMPRII impairs apoptosis by modulating the alternative splicing of the apoptotic regulator, Bcl-x (B-cell lymphoma X) transcripts: a finding observed in circulating leukocytes and lungs of PAH subjects, hypoxia-induced PAH rat lungs as well as in PASMCs and PAECs. BMPRII deficiency elicits cell specific effects: promoting the expression of Bcl-xL transcripts in PASMCs whilst inhibiting it in ECs, thus exerting differential apoptotic effects in these cells. The pro-survival effect of BMPRII receptor is mediated through the activin receptor like kinase 1 (ALK1) but not the ALK3 receptor. Finally, we show that BMPRII interacts with the ALK1 receptor and pathogenic mutations in the BMPR2 gene abolish this interaction. Taken together, dysfunctional BMPRII responsiveness impairs apoptosis via the BMPRII-ALK1-Bcl-xL pathway in PAH. We suggest Bcl-xL as a potential biomarker and druggable target.This work was supported by a fellowship (awarded to MTN) from the Department of Health via the NIHR Comprehensive Biomedical Research Centre award to Guy’s & St Thomas’ NHS Foundation Trust in partnership with King’s College London, Heptagon Life Science Proof of Concept Fund (grant KCL24 to MTN), the Great Britain Sasakawa Foundation (grant B70 to MTN), the Royal Society (grant 43049 to MTN), the Medical Research Council (grant G900865 to RCT, MTN and NWM) and the University of Bradford (grants 003200, 66006/001NAS and DH005 to MTN). NS and MYB were supported by scholarships from the Commonwealth Scholarship Commission, UK and Scientific and Technological Research Council of Turkey (TUBITAK), respectively

Data for proteomic analysis of murine cardiomyocytic HL1 cells treated with siRNA against tissue factor

YesThis data article is related to the research article entitled Proteomics of Tissue Factor silencing in cardiomyocytic cells reveals a new role for this coagulation factor in splicing machinery control by Lento et al [1]. Tissue Factor (TF) is the key player in the coagulation cascade, but it has additional functions ranging from angiogenesis, tumor invasion and, in the heart, the maintenance of the integrity of cardiac cells. This article reports the nano-LC-MSE analysis of the cardiomyocytic HL-1 cell line proteome and describes the results obtained from a Gene Ontology analysis of those proteins affected by TF-gene silencing

Aminoglycoside-mediated promotion of translation readthrough occurs through a non-stochastic mechanism that competes with translation termination

Attempts have been made to treat nonsense-associated genetic disorders by chemical agents and hence an improved mechanistic insight into the decoding of readthrough signals is essential for the identification and characterisation of factors for the treatment of these disorders. To identify either novel compounds or genes that modulate translation readthrough, we have employed dual reporter-based high-throughput screens that use enzymatic and fluorescence activities and screened bio-active NINDS compounds (n = 1000) and siRNA (n = 288) libraries. Whilst siRNAs targeting kinases such as CSNK1G3 and NME3 negatively regulate readthrough, neither the bio-active NINDS compounds nor PTC124 promote readthrough. Of note, PTC124 has previously been shown to promote readthrough. Furthermore, the impacts of G418 on the components of eukaryotic selenocysteine incorporation machinery have also been investigated. The selenocysteine machinery decodes the stop codon UGA specifying selenocysteine in natural selenoprotein genes. We have found that the eukaryotic SelC gene promotes the selenocysteine insertion sequence (SECIS)-mediated readthrough but inhibits the readthrough activity induced by G418. We have previously reported that SECIS-mediated readthrough at UGA codons follows a non-processive mechanism. Here, we show that G418-mediated promotion of readthrough also occurs through a non-processive mechanism which competes with translation termination. Based on our observations, we suggest that proteins generated through a non-processive mechanism may be therapeutically beneficial for the resolution of nonsense-associated genetic disorders.Fellowship (awarded to MTN) from the Department of Health via the NIHR Comprehensive Biomedical Research Centre award to Guy’s & St Thomas’ NHS Foundation Trust in partnership with King’s College London, Heptagon Life Science Proof of Concept Fund (grant KCL24 to MTN), the Great Britain Sasakawa 22 Foundation (grant B70 to MTN), the Royal Society (grant 43049 to MTN) and the University of Bradford (grants 003200 and DH005 to MTN)

Resolution of coronavirus disease 2019 (COVID-19)

YesIntroduction. Coronavirus disease 2019 (COVID-19) was first detected in China in December, 2019, and declared as a pandemic by the World Health Organization (WHO) on March 11, 2020. The current management of COVID-19 is based generally on supportive therapy and treatment to prevent respiratory failure. The effective option of antiviral therapy and vaccination are currently under evaluation and development. Areas covered. A literature search was performed using PubMed between December 1, 2019–June 23, 2020. This review highlights the current state of knowledge on the viral replication and pathogenicity, diagnostic and therapeutic strategies, and management of COVID-19. This review will be of interest to scientists and clinicians and make a significant contribution toward development of vaccines and targeted therapies to contain the pandemic. Expert Opinion. The exit strategy for a path back to normal life is required, which should involve a multi-prong effort toward development of new treatment and a successful vaccine to protect public health worldwide and prevent future COVID-19 outbreaks. Therefore, the bench to bedside translational research as well as reverse translational works focusing bedside to bench is very important and would provide the foundation for the development of targeted drugs and vaccines for COVID-19 infections.Research carried out at TN laboratories are funded by the GrowMedtech, The Royal Society and University of Bradford. KH is supported by a project grant by the GrowMedtech awarded to TN. CW is funded by a Ph.D studentship

Inhibition of Overactive Transforming Growth Factor–β Signaling by Prostacyclin Analogs in Pulmonary Arterial Hypertension

YesHeterozygous loss of function mutations in the type II bone morphogenetic protein receptor (BMPR-II), a member of the transforming growth factor (TGF-β) receptor family, underlie the majority of familial cases of pulmonary arterial hypertension (PAH). The TGF-β1 pathway is activated in PAH and inhibitors of TGF-β1 signaling prevent the development and progression of PAH in experimental models. However, the effect of currently utilized therapies on the TGF-β pathway is not known. Prostacyclin analogues remain the first line of treatment for clinical PAH. We hypothesized that these agents effectively decrease the activity of the TGF-β1 pathway. Beraprost sodium (BPS), a prostacyclin analogue selectively inhibits proliferation in a dose-dependent manner in mouse primary pulmonary arterial smooth muscle cells (PASMCs) harbouring a pathogenic BMPR2 nonsense mutation in both the presence and absence of TGF-β1 stimulation. This study demonstrates that this agent inhibits TGF-β1–induced SMAD-dependent and -independent signaling via a PKA dependent pathway by reducing the phosphorylation of SMADs 2 and 3 and p38MAPK proteins. Finally, in a monocrotaline (MCT)-induced rat model of PAH, which is associated with increased TGF-β signaling, this study confirms that treprostinil (TPS), a stable prostacyclin analogue, inhibits the TGF-β pathway by reducing SMAD3 phosphorylation. Taken together, these data suggest that prostacyclin analogues inhibit dysregulated TGF-β signaling in vitro and in vivo and reduce BMPR-II-mediated proliferation defects in mutant mice PASMCs.The authors acknowledge financial support from the British Heart Foundation, United Kingdom (Programme Grant 1-2004-357 to R.C.T. and N.W.M.), a Heptagon Life Science Proof of Concept Fund (grants KCL24 and KCL25 to M.T.N. and R.C.T., respectively), and the Great Britain Sasakawa Foundation (grant B70 to M.T.N.

Recurrent mutations in the U2AF1 splicing factor in myelodysplastic syndromes

Myelodysplastic syndromes (MDS) are hematopoietic stem cell disorders that often progress to chemotherapy-resistant secondary acute myeloid leukemia (sAML). We used whole-genome sequencing to perform an unbiased comprehensive screen to discover the somatic mutations in a sample from an individual with sAML and genotyped the loci containing these mutations in the matched MDS sample. Here we show that a missense mutation affecting the serine at codon 34 (Ser34) in U2AF1 was recurrently present in 13 out of 150 (8.7%) subjects with de novo MDS, and we found suggestive evidence of an increased risk of progression to sAML associated with this mutation. U2AF1 is a U2 auxiliary factor protein that recognizes the AG splice acceptor dinucleotide at the 3' end of introns, and the alterations in U2AF1 are located in highly conserved zinc fingers of this protein. Mutant U2AF1 promotes enhanced splicing and exon skipping in reporter assays in vitro. This previously unidentified, recurrent mutation in U2AF1 implicates altered pre-mRNA splicing as a potential mechanism for MDS pathogenesis

Proteomics of tissue factor silencing in cardiomyocytic cells reveals a new role for this coagulation factor in splicing machinery control

YesIt has long been known that Tissue Factor (TF) plays a role in blood coagulation and has a direct thrombotic action that is closely related to cardiovascular risk, but it is becoming increasingly clear that it has a much wider range of biological functions that range from inflammation to immunity. It is also involved in maintaining heart haemostasis and structure, and the observation that it is down-regulated in the myocardium of patients with dilated cardiomyopathy suggests that it influences cell-to-cell contact stability and contractility, and thus contributes to cardiac dysfunction. However, the molecular mechanisms underlying these coagulation-independent functions have not yet been fully elucidated. In order to analyse the influence of TF on the cardiomyocitic proteome, we used functional biochemical approaches incorporating label-free quantitative proteomics and gene silencing, and found that this provided a powerful means of identifying a new role for TF in regulating splicing machinery together with the expression of several proteins of the spliceosome, and mRNA metabolism with a considerable impact on cell viability

A double reporter assay for detecting changes in the ratio of spliced and unspliced mRNA in mammalian cells

Current methods for measuring the efficiency of splicing in mammalian cells rely on either direct analysis of the RNA, which does not lend itself to rapid assays, or on single reporter functions that are subject to numerous intrinsic variables. If two protein activities are encoded within a single reading frame but on separate exons, with an intervening sequence containing termination codons, then the expression of the second activity is dependent on removal of the intervening sequence by pre-mRNA splicing. Thus, the ratio of the activities encoded by exon 2 to exon 1 reflects the ratio of expression from spliced mRNA to the total expression of spliced and unspliced RNA. This provides a rapid and convenient assay for the effects on splicing efficiency of trans-acting factors or of alterations in the sequences of the intron and surrounding exon sequences