Epigenetic Metabolic Reprogramming of Right Ventricular Fibroblasts in Pulmonary Arterial Hypertension: A Pyruvate Dehydrogenase Kinase-Dependent Shift in Mitochondrial Metabolism Promotes Right Ventricular Fibrosis.

RATIONALE: Right ventricular (RV) fibrosis in pulmonary arterial hypertension contributes to RV failure. While RV fibrosis reflects changes in the function of resident RV fibroblasts (RVfib), these cells are understudied. OBJECTIVE: Examine the role of mitochondrial metabolism of RVfib in RV fibrosis in human and experimental pulmonary arterial hypertension. METHODS AND RESULTS: Male Sprague-Dawley rats received monocrotaline (MCT; 60 mg/kg) or saline. Drinking water containing no supplement or the PDK (pyruvate dehydrogenase kinase) inhibitor dichloroacetate was started 7 days post-MCT. At week 4, treadmill testing, echocardiography, and right heart catheterization were performed. The effects of PDK activation on mitochondrial dynamics and metabolism, RVfib proliferation, and collagen production were studied in RVfib in cell culture. Epigenetic mechanisms for persistence of the profibrotic RVfib phenotype in culture were evaluated. PDK expression was also studied in the RVfib of patients with decompensated RV failure (n=11) versus control (n=7). MCT rats developed pulmonary arterial hypertension, RV fibrosis, and RV failure. MCT-RVfib (but not left ventricular fibroblasts) displayed excess mitochondrial fission and had increased expression of PDK isoforms 1 and 3 that persisted for >5 passages in culture. PDK-mediated decreases in pyruvate dehydrogenase activity and oxygen consumption rate were reversed by dichloroacetate (in RVfib and in vivo) or siRNA targeting PDK 1 and 3 (in RVfib). These interventions restored mitochondrial superoxide and hydrogen peroxide production and inactivated HIF (hypoxia-inducible factor)-1α, which was pathologically activated in normoxic MCT-RVfib. Redox-mediated HIF-1α inactivation also decreased the expression of TGF-ß1 (transforming growth factor-beta-1) and CTGF (connective tissue growth factor), reduced fibroblast proliferation, and decreased collagen production. HIF-1α activation in MCT-RVfib reflected increased DNMT (DNA methyltransferase) 1 expression, which was associated with a decrease in its regulatory microRNA, miR-148b-3p. In MCT rats, dichloroacetate, at therapeutic levels in the RV, reduced phospho-pyruvate dehydrogenase expression, RV fibrosis, and hypertrophy and improved RV function. In patients with pulmonary arterial hypertension and RV failure, RVfib had increased PDK1 expression. CONCLUSIONS: MCT-RVfib manifest a DNMT1-HIF-1α-PDK-mediated, chamber-specific, metabolic memory that promotes collagen production and RV fibrosis. This epigenetic mitochondrial-metabolic pathway is a potential antifibrotic therapeutic target.

The molecular mechanisms of oxygen-sensing in human ductus arteriosus smooth muscle cells: A comprehensive transcriptome profile reveals a central role for mitochondria.

The ductus arteriosus (DA) connects the fetal pulmonary artery and aorta, diverting placentally oxygenated blood from the developing lungs to the systemic circulation. The DA constricts in response to increases in oxygen (O2) with the first breaths, resulting in functional DA closure, with anatomic closure occurring within the first days of life. Failure of DA closure results in persistent patent ductus arteriosus (PDA), a common complication of extreme preterm birth. The DA's response to O2, though modulated by the endothelium, is intrinsic to the DA smooth muscle cells (DASMC). DA constriction is mediated by mitochondrial-derived reactive oxygen species, which increase in proportion to arterial partial pressure of oxygen (PaO2). The resulting redox changes inhibit voltage-gated potassium channels (Kv) leading to cell depolarization, calcium influx and DASMC constriction. To date, there has not been an unbiased assessment of the human DA O2-sensors using transcriptomics, nor are there known molecular mechanisms which characterize DA closure. DASMCs were isolated from DAs obtained from 10 term infants at the time of congenital heart surgery. Cells were purified by flow cytometry, negatively sorting using CD90 and CD31 to eliminate fibroblasts or endothelial cells, respectively. The purity of the DASMC population was confirmed by positive staining for α-smooth muscle actin, smoothelin B and caldesmon. Cells were grown for 96 h in hypoxia (2.5% O2) or normoxia (19% O2) and confocal imaging with Cal-520 was used to determine oxygen responsiveness. An oxygen-induced increase in intracellular calcium of 18.1% ± 4.4% and SMC constriction (-27% ± 1.5% shortening) occurred in all cell lines within five minutes. RNA sequencing of the cells grown in hypoxia and normoxia revealed significant regulation of 1344 genes (corrected p < 0.05). We examined these genes using Gene Ontology (GO). This unbiased assessment of altered gene expression indicated significant enrichment of the following GOterms: mitochondria, cellular respiration and transcription. The top regulated biologic process was generation of precursor metabolites and energy. The top regulated cellular component was mitochondrial matrix. The top regulated molecular function was transcription coactivator activity. Multiple members of the NADH-ubiquinone oxidoreductase (NDUF) family are upregulated in human DASMC (hDASMC) following normoxia. Several of our differentially regulated transcripts are encoded by genes that have been associated with genetic syndromes that have an increased incidence of PDA (Crebb binding protein and Histone Acetyltransferase P300). This first examination of the effects of O2 on human DA transcriptomics supports a putative role for mitochondria as oxygen sensors.

Postpartum alterations following inflammation in rat pregnancy: a discovery proteomic analysis.

Women with a history of preeclampsia have an increased risk of subsequent cardiovascular and metabolic disease. While aberrant inflammation during pregnancy is associated with the development of preeclampsia, whether maternal inflammation increases the risk of disease later in life is unclear. Using a rat model we determined whether aberrant inflammation in pregnancy alters the levels of plasma proteins associated with cardiovascular and metabolic disease risk in the postpartum period. Pregnant rats were administered lipopolysaccharide (LPS) or saline on gestational days 13.5-16.5 to induce inflammation. Non-pregnant controls consisted of age-matched female rats subjected to similar administration of LPS or saline. Examination of the proteomic profile of plasma collected 16 weeks after delivery or from non-pregnant controls using liquid chromatography-tandem mass spectrometry revealed 100 differentially expressed proteins. Moreover, we identified 188 proteins in pregnant rats, of which 49 were differentially expressed in saline- vs LPS-treated dams. Of the 49 proteins regulated by LPS, 28 were pregnancy specific. PANTHER classification software, DAVID database and Ingenuity Pathways analysis revealed that the differentially expressed proteins in pregnant saline vs LPS-treated rats are associated with alterations in lipid and glucose metabolism and atherosclerosis, all of which may contribute to cardiovascular and metabolic disease risk. Results from proteomic and pathway analyses were validated by immunoassay of three serum proteins selected a priori and by assessment of serum metabolites. This discovery study demonstrates that aberrant inflammation during pregnancy results in long-lasting postpartum physiological alterations known to be associated with metabolic and cardiovascular disease.

An epigenetic increase in mitochondrial fission by MiD49 and MiD51 regulates the cell cycle in cancer: Diagnostic and therapeutic implications.

Excessive proliferation and apoptosis-resistance are hallmarks of cancer. Increased dynamin-related protein 1 (Drp1)-mediated mitochondrial fission is one of the mediators of this phenotype. Mitochondrial fission that accompanies the nuclear division is called mitotic fission and occurs when activated Drp1 binds partner proteins on the outer mitochondrial membrane. We examine the role of Drp1-binding partners, mitochondrial dynamics protein of 49 and 51 kDa (MiD49 and MiD51), as drivers of cell proliferation and apoptosis-resistance in non-small cell lung cancer (NSCLC) and invasive breast carcinoma (IBC). We also evaluate whether inhibiting MiDs can be therapeutically exploited to regress cancer. We show that MiD levels are pathologically elevated in NSCLC and IBC by an epigenetic mechanism (decreased microRNA-34a-3p expression). MiDs silencing causes cell cycle arrest through (a) increased expression of cell cycle inhibitors, p27Kip1 and p21Waf1 , (b) inhibition of Drp1, and (c) inhibition of the Akt-mTOR-p70S6K pathway. Silencing MiDs leads to mitochondrial fusion, cell cycle arrest, increased apoptosis, and tumor regression in a xenotransplant NSCLC model. There are positive correlations between MiD expression and tumor size and grade in breast cancer patients and inverse correlations with survival in NSCLC patients. The microRNA-34a-3p-MiDs axis is important to cancer pathogenesis and constitutes a new therapeutic target.

Physiological and Transcriptomic Changes in the Hypothalamic-Neurohypophysial System after 24 h of Furosemide-Induced Sodium Depletion.

BACKGROUND/AIMS: Furosemide is a loop diuretic widely used in clinical practice for the treatment of oedema and hypertension. The aim of this study was to determine physiological and molecular changes in the hypothalamic-neurohypophysial system as a consequence of furosemide-induced sodium depletion. METHODS: Male rats were sodium depleted by acute furosemide injection (10 and 30 mg/kg) followed by access to low sodium diet and distilled water for 24 h. The renal and behavioural consequences were evaluated, while blood and brains were collected to evaluate the neuroendocrine and gene expression responses. RESULTS: Furosemide treatment acutely increases urinary sodium and water excretion. After 24 h, water and food intake were reduced, while plasma angiotensin II and corticosterone were increased. After hypertonic saline presentation, sodium-depleted rats showed higher preference for salt. Interrogation using RNA sequencing revealed the expression of 94 genes significantly altered in the hypothalamic paraventricular nucleus (PVN) of sodium-depleted rats (31 upregulated and 63 downregulated). Out of 9 genes chosen, 5 were validated by quantitative PCR in the PVN (upregulated: Ephx2, Ndnf and Vwf; downregulated: Caprin2 and Opn3). The same genes were also assessed in the supraoptic nucleus (SON, upregulated: Tnnt1, Mis18a, Nr1d1 and Dbp; downregulated: Caprin2 and Opn3). As a result of these plastic transcriptome changes, vasopressin expression was decreased in PVN and SON, whilst vasopressin and oxytocin levels were reduced in plasma. CONCLUSIONS: We thus have identified novel genes that might regulate vasopressin gene expression in the hypothalamus controlling the magnocellular neurons secretory response to body sodium depletion and consequently hypotonic stress.

Clinical value of non-coding RNAs in cardiovascular, pulmonary, and muscle diseases.

Although a majority of the mammalian genome is transcribed to RNA, mounting evidence indicates that only a minor proportion of these transcriptional products are actually translated into proteins. Since the discovery of the first non-coding RNA (ncRNA) in the 1980s, the field has gone on to recognize ncRNAs as important molecular regulators of RNA activity and protein function, knowledge of which has stimulated the expansion of a scientific field that quests to understand the role of ncRNAs in cellular physiology, tissue homeostasis, and human disease. Although our knowledge of these molecules has significantly improved over the years, we have limited understanding of their precise functions, protein interacting partners, and tissue-specific activities. Adding to this complexity, it remains unknown exactly how many ncRNAs there are in existence. The increased use of high-throughput transcriptomics techniques has rapidly expanded the list of ncRNAs, which now includes classical ncRNAs (e.g., ribosomal RNAs and transfer RNAs), microRNAs, and long ncRNAs. In addition, splicing by-products of protein-coding genes and ncRNAs, so-called circular RNAs, are now being investigated. Because there is substantial heterogeneity in the functions of ncRNAs, we have summarized the present state of knowledge regarding the functions of ncRNAs in heart, lungs, and skeletal muscle. This review highlights the pathophysiologic relevance of these ncRNAs in the context of human cardiovascular, pulmonary, and muscle diseases.

Sex-specific differences in cardiovascular and metabolic hormones with integrated signalling in the paraventricular nucleus of the hypothalamus.

NEW FINDINGS: What is the topic of this review? We describe roles of crucial signalling molecules in the paraventricular nucleus of the hypothalamus and highlight recent data suggesting sex-specific changes in the expression of crucial signalling molecules and their receptors, which may underlie sex differences in both cardiovascular and metabolic function. What advances does it highlight? This review highlights the integrative capacity of the paraventricular nucleus in mediating cardiovascular and metabolic effects by integrating information from multiple signalling molecules. It also proposes that these signalling molecules have sex-specific differential gene expression, indicating the importance of considering these differences in our ongoing search to understand the female-male differences in the regulation of crucial autonomic systems. Many traditional cardiovascular hormones have been implicated in metabolic function. Conversely, many hormones traditionally involved in metabolic regulation have an effect on cardiovascular function. Many of these signalling molecules exert such effects through specific actions in the paraventricular nucleus, an integrative autonomic control centre located in the hypothalamus. Here, we focus on four cardiovascular/metabolic peptide hormones that signal within the paraventricular nucleus, namely angiotensin II, orexin, adiponectin and nesfatin-1. Each of these hormones has specific electrophysiological effects on paraventricular nucleus neurons that can be related to its physiological actions. In addition, we introduce preliminary transcriptomic data indicating that the genes for some of these hormones and their receptors have sex-specific differential expression.

A comparison of physiological and transcriptome responses to water deprivation and salt loading in the rat supraoptic nucleus.

Salt loading (SL) and water deprivation (WD) are experimental challenges that are often used to study the osmotic circuitry of the brain. Central to this circuit is the supraoptic nucleus (SON) of the hypothalamus, which is responsible for the biosynthesis of the hormones, arginine vasopressin (AVP) and oxytocin (OXT), and their transport to terminals that reside in the posterior lobe of the pituitary. On osmotic challenge evoked by a change in blood volume or osmolality, the SON undergoes a function-related plasticity that creates an environment that allows for an appropriate hormone response. Here, we have described the impact of SL and WD compared with euhydrated (EU) controls in terms of drinking and eating behavior, body weight, and recorded physiological data including circulating hormone data and plasma and urine osmolality. We have also used microarrays to profile the transcriptome of the SON following SL and remined data from the SON that describes the transcriptome response to WD. From a list of 2,783 commonly regulated transcripts, we selected 20 genes for validation by qPCR. All of the 9 genes that have already been described as expressed or regulated in the SON by osmotic stimuli were confirmed in our models. Of the 11 novel genes, 5 were successfully validated while 6 were false discoveries.

Inhibition of Egr1 expression underlies the anti-mitogenic effects of cAMP in vascular smooth muscle cells.

AIMS: Cyclic AMP inhibits vascular smooth muscle cell (VSMC) proliferation which is important in the aetiology of numerous vascular diseases. The anti-mitogenic properties of cAMP in VSMC are dependent on activation of protein kinase A (PKA) and exchange protein activated by cAMP (EPAC), but the mechanisms are unclear. METHODS AND RESULTS: Selective agonists of PKA and EPAC synergistically inhibited Egr1 expression, which was essential for VSMC proliferation. Forskolin, adenosine, A2B receptor agonist BAY60-6583 and Cicaprost also inhibited Egr1 expression in VSMC but not in endothelial cells. Inhibition of Egr1 by cAMP was independent of cAMP response element binding protein (CREB) activity but dependent on inhibition of serum response element (SRE) activity. SRF binding to the Egr1 promoter was not modulated by cAMP stimulation. However, Egr1 expression was dependent on the SRF co-factors Elk1 and 4 but independent of MAL. Inhibition of SRE-dependent Egr1 expression was due to synergistic inhibition of Rac1 activity by PKA and EPAC, resulting in rapid cytoskeleton remodelling and nuclear export of ERK1/2. This was associated with de-phosphorylation of the SRF co-factor Elk1. CONCLUSION: cAMP inhibits VSMC proliferation by rapidly inhibiting Egr1 expression. This occurs, at least in part, via inhibition of Rac1 activity leading to rapid actin-cytoskeleton remodelling, nuclear export of ERK1/2, impaired Elk1-phosphorylation and inhibition of SRE activity. This identifies one of the earliest mechanisms underlying the anti-mitogenic effects of cAMP in VSMC but not in endothelial cells, making it an attractive target for selective inhibition of VSMC proliferation.

Antagonism of orexin receptors significantly lowers blood pressure in spontaneously hypertensive rats.

In normal rats, central administration of orexin or exposure to certain forms of stress can induce significant increases in blood pressure and sympathetic nerve activity, which can be blocked by orexin receptor antagonists. The resting blood pressure is, however, unaffected by such antagonists, but is significantly lower in rodents with total loss of orexin, such as prepro-orexin knockout mice and orexin neuron-ablated orexin/ataxin-3 transgenic rats. We hypothesize that orexin is involved in the pathophysiology and maintenance of high blood pressure in the spontaneously hypertensive rat (SHR), a model of primary hypertension. To test this hypothesis, we measured orexin-A mRNA expression in the rostral ventrolateral medulla and antagonized both orexin receptors using an orally administered potent dual orexin receptor antagonist, almorexant, in SHRs and normotensive Wistar-Kyoto rats. In SHRs, there was a strong trend towards an increased orexin-A mRNA expression in the rostral ventrolateral medulla, and blocking orexin receptors markedly lowered blood pressure (from 182/152 ± 5/6 to 149/119 ± 9/8 mmHg; P < 0.001), heart rate (P < 0.001), sympathetic vasomotor tone (P < 0.001) and the noradrenaline levels in cerebrospinal fluid and plasma (P < 0.002). The significant antihypertensive effects of almorexant were observed in wakefulness and non-rapid eye movement sleep during both dark and light phases of the diurnal cycle only in SHRs. Blocking orexin receptors had no effect on blood pressure and sympathetic tone in normotensive Wistar-Kyoto rats. Our study links the orexin system to the pathogenesis of high blood pressure in SHRs and suggests that modulation of the orexin system could be a potential target in treating some forms of hypertension.

Medium-chain Acyl-COA dehydrogenase deficiency: Pathogenesis, diagnosis, and treatment.

INTRODUCTION: Medium-Chain Acyl-CoA Dehydrogenase Deficiency (MCADD) is the most common inherited metabolic disorder of ß-oxidation. Patients with MCADD present with hypoketotic hypoglycemia, which may quickly progress to lethargy, coma, and death. Prognosis for MCADD patients is highly promising once a diagnosis has been established, though management strategies may vary depending on the severity of illness and the presence of comorbidities. METHODS AND RESULTS: Given the rapid developments in the world of gene therapy and implementation of newborn screening for inherited metabolic disorders, the provision of concise and contemporary knowledge of MCADD is essential for clinicians to effectively manage patients. Thus, this review aims to consolidate current information for physicians on the pathogenesis, diagnostic tools, and treatment options for MCADD patients. CONCLUSION: MCADD is a commonly inherited metabolic disease with serious implications for health outcomes, particularly in children, that may be successfully managed with proper intervention.

Using omics to breathe new life into our understanding of the ductus arteriosus oxygen response.

The ductus arteriosus (DA) connects the aorta to the pulmonary artery (PA), directing placentally oxygenated blood away from the developing lungs. High pulmonary vascular resistance and low systemic vascular resistance facilitate shunting of blood in utero from the pulmonary to the systemic circulation through the widely patent DA, thereby optimizing fetal oxygen (O2) delivery. With the transition from fetal (hypoxia) to neonatal (normoxia) oxygen conditions, the DA constricts while the PA dilates. This process often fails in prematurity, promoting congenital heart disease. Impaired O2-responsivness in the DA promotes persistent ductus arteriosus (PDA), the most common form of congenital heart disease. Knowledge of DA oxygen sensing has greatly advanced in the past few decades, however we still lack a complete understanding of the sensing mechanism. The genomic revolution of the past two decades has facilitated unprecedented discovery in every biological system. This review will demonstrate how multiomic integration of data generated from the DA can breathe new life into our understanding of the DA's oxygen response.

A transcriptome analysis of basal and stimulated VWF release from endothelial cells derived from patients with type 1 VWD.

Type 1 von Willebrand disease (VWD) is associated with a reduction in qualitatively normal von Willebrand factor (VWF). Current diagnostic guidelines only take into consideration the contribution of basal VWF levels, despite a lack of correlation with bleeding severity. Defects in stimulated VWF release, which occurs after hemostatic challenge, may contribute to bleeding in type 1 VWD, but the pathogenic mechanisms are poorly defined. In this study, a layered multiomic approach including messenger RNA (mRNA) and microRNA (miRNA) sequencing was used to evaluate transcriptome-wide differences between type 1 VWD- and control-derived endothelial colony forming cells (ECFCs) during basal and stimulated VWF release. ECFCs from 8 patients with type 1 VWD and 4 other patients were included in this study as controls. VWF protein analysis revealed heterogenous responses to stimulation among type 1 VWD and control ECFCs. During basal VWF release, 64 mRNAs and 7 miRNAs were differentially regulated between type 1 VWD and control ECFCs, and 65 putatively pathogenic miRNA-mRNA interactions were identified. During stimulated VWF release, 190 mRNAs and 5 mRNAs were differentially regulated between type 1 VWD and control ECFCs, and 110 putatively pathogenic miRNA-mRNA interactions were identified. Five gene ontology terms including coagulation, regulation of cell shape, and regulation of cell signaling were also differentially regulated in type 1 VWD ECFCs during stimulated release. To our knowledge, we have shown for the first time that transcriptome-wide differences exist between type 1 VWD and control ECFCs. These differences may contribute to bleeding in type 1 VWD, and further investigation may reveal novel biomarkers and therapeutic targets.

The mechanistic and structural role of von Willebrand factor in endotoxemia-enhanced deep vein thrombosis in mice.

BACKGROUND: Although the concept of immunothrombosis has established a link between inflammation and thrombosis, the role of inflammation in the pathogenesis of deep vein thrombosis remains to be fully elucidated. Further, although various constituents of venous thrombi have been identified, their localizations and cellular and molecular interactions are yet to be combined in a single, multiplexed analysis. OBJECTIVES: The objective of this study was to investigate the role of the von Willebrand factor (VWF) in inflammation-associated venous thrombosis. We also performed a proof-of-concept study of imaging mass cytometry to quantitatively and simultaneously analyze the localizations and interactions of 10 venous thrombus constituents. METHODS: We combined the murine inferior vena cava stenosis model of deep vein thrombosis with the lipopolysaccharide model of endotoxemia. We also performed a proof-of-concept study of imaging mass cytometry to assess the feasibility of this approach in analyzing the structural composition of thrombi. RESULTS: We found that lipopolysaccharide-treated mice had significantly higher incidences of venous thrombosis, an effect that was mitigated when VWF was inhibited using inhibitory αVWF antibodies. Our detailed structural analysis also showed that most thrombus components are localized in the white thrombus regardless of endotoxemia. Moreover, although endotoxemia modulated the relative representation and interactions of VWF with other thrombus constituents, the scaffolding network, comprised VWF, fibrin, and neutrophil extracellular traps, remained largely unaffected. CONCLUSIONS: We observe a key role for VWF in the pathogenesis of inflammation-associated venous thrombosis while providing a more comprehensive insight into the molecular interactions that constitute the architecture of venous thrombi.

Depression and anxiety following acute myocardial infarction in women.

Cardiovascular disease is the leading global cause of mortality, with ischemic heart disease causing the majority of cardiovascular deaths. Despite this, diagnostic delay commonly occurs in women experiencing acute myocardial infarction (AMI) who have a higher associated in-hospital mortality. Several studies have demonstrated that women are significantly more likely than men to experience depression and anxiety following AMI which is linked with increased morbidity, rehospitalization, and mortality, as well as decreased quality of life. Thus, it is imperative that future work aims to understand the factors that put women at higher risk for depression and anxiety following AMI, informing prevention and intervention. This narrative review will summarize the current literature on the association between AMI and mental health in women, including the impact on morbidity, mortality, and quality of life.

The comprehensive transcriptome of human ductus arteriosus smooth muscle cells (hDASMC).

The Ductus Arteriosus (DA) is a fetal vessel that connects the aorta to the pulmonary artery ensuring that placental oxygenated blood is diverted from the lungs to the systemic circulation. Following exposure to oxygen (O2), in the first few days of life, the DA responds with a functional closure that is followed by anatomical closure. Here, we study human DA smooth muscle cells (DASMC) taken from 10 term infants during congenital heart surgery. Purification of these cells using flow cytometry ensured a pure population of DASMCs, which we confirmed as responsive to O2. An oxygen-induced increase in intracellular calcium of 18.1%±4.4% and SMC constriction (-27%±1.5% shortening) occurred in all cell lines within five minutes. These cells were maintained in either hypoxia (2.5% O2), mimicking in utero conditions or in normoxia (19% O2) mimicking neonate conditions. We then used 3' RNAsequencing to identify the transcriptome of DASMCs in each condition [1]. In this paper, we present the full differentially regulated gene list from this experiment.

The transcriptome of the medullary area postrema: the thirsty rat, the hungry rat and the hypertensive rat.

The area postrema (AP) is a sensory circumventricular organ characterized by extensive fenestrated vasculature and neurons which are capable of detecting circulating signals of osmotic, cardiovascular, immune and metabolic status. The AP can communicate these messages via efferent projections to brainstem and hypothalamic structures that are able to orchestrate an appropriate response. We have used microarrays to profile the transcriptome of the AP in the Sprague-Dawley (SD) and Wistar-Kyoto rat and present here a comprehensive catalogue of gene expression, focusing specifically on the population of ion channels, receptors and G protein-coupled receptors expressed in this sensory tissue; of the G protein-coupled receptors expressed in the rat AP, we identified ∼36% that are orphans, having no established ligand. We have also looked at the ways in which the AP transcriptome responds to the physiological stressors of 72 h dehydration (DSD) and 48 h fasting (FSD) and have performed microarrays in these conditions. Comparison between the DSD and SD or between FSD and SD revealed only a modest number of AP genes that are regulated by these homeostatic challenges. The expression levels of a much larger number of genes are altered in the spontaneously hypertensive rat AP compared with the normotensive Wistar-Kyoto control rat, however. Finally, analysis of these 'hypertension-related' elements revealed genes that are involved in the regulation of both blood pressure and immune function and as such are excellent targets for further study.

Osmoregulation of the transcriptome of the hypothalamic supraoptic nucleus: A resource for the community.

The hypothalamic supraoptic nucleus (SON) is a core osmoregulatory control centre that deciphers information about the metabolic state of the organism and orchestrates appropriate homeostatic (endocrine) and allostatic (behavioural) responses. We have used RNA sequencing to describe the polyadenylated transcriptome of the SON of the male Wistar Han rat. These data have been mined to generate comprehensive catalogues of functional classes of genes (enzymes, transcription factors, endogenous peptides, G protein coupled receptors, transporters, catalytic receptors, channels and other pharmacological targets) expressed in this nucleus in the euhydrated state, and that together form the basal substrate for its physiological interactions. We have gone on to show that fluid deprivation for 3 days (dehydration) results in changes in the expression levels of 2247 RNA transcripts, which have similarly been functionally catalogued, and further mined to describe enriched gene categories and putative regulatory networks (Regulons) that may have physiological importance in SON function related plasticity. We hope that the revelation of these genes, pathways and networks, most of which have no characterised roles in the SON, will encourage the neuroendocrine community to pursue new investigations into the new 'known-unknowns' reported in the present study.

Innate immune memory is associated with increased disease-free survival in bladder cancer patients treated with bacillus Calmette-Guérin.

INTRODUCTION: While studies suggest that innate immune memory acquired by circulating monocytes may mediate the benefit of bacillus Calmette-Guérin (BCG) in the treatment of patients with high-risk non-muscle-invasive bladder cancer (NMIBC), prospective studies are lacking. Innate immune memory is defined by enhanced release of pro-inflammatory cytokines by innate immune cells following a secondary challenge with pattern recognition receptor (PRR) ligands. METHODS: Peripheral blood monocytes isolated from 33 patients with intermediate- or high-risk NMIBC before and after two or five induction BCG instillations were stimulated with the PRR ligand lipopolysaccharide (LPS). Inflammatory cytokine levels in the culture medium were measured. Extent of innate immune memory acquisition was determined by dividing the levels of cytokines released after BCG instillation by the levels released prior to BCG therapy. RESULTS: Monocytes secreted variable levels of TNFα, IL-1ß, IL-6, IFNγ, IL-12, and IL-10. Compared with patients with recurrences, the post-BCG:pre-BCG ratio of IL-12 in monocyte cultures from patients without recurrences after five BCG instillations was significantly increased. Patients with no innate immune memory (based on IL-12 ratios) had significantly shorter time to recurrence than patients with innate immune memory (p<0.001). Eighty-four percent (16/19) of patients with innate immune memory vs. only 22% (2/9) of patients without memory had disease-free survival of over 500 days. CONCLUSIONS: Results demonstrate a potential link between BCG-induced innate immune memory peripherally and local anti-tumor responses. Further validation will increase our understanding of the mode of action of BCG and, therefore, will be used to enhance its effectiveness.

The MNK1/2-eIF4E Axis Supports Immune Suppression and Metastasis in Postpartum Breast Cancer.

Breast cancer diagnosed within 10 years following childbirth is defined as postpartum breast cancer (PPBC) and is highly metastatic. Interactions between immune cells and other stromal cells within the involuting mammary gland are fundamental in facilitating an aggressive tumor phenotype. The MNK1/2-eIF4E axis promotes translation of prometastatic mRNAs in tumor cells, but its role in modulating the function of nontumor cells in the PPBC microenvironment has not been explored. Here, we used a combination of in vivo PPBC models and in vitro assays to study the effects of inactivation of the MNK1/2-eIF4E axis on the protumor function of select cells of the tumor microenvironment. PPBC mice deficient for phospho-eIF4E (eIF4ES209A) were protected against lung metastasis and exhibited differences in the tumor and lung immune microenvironment compared with wild-type mice. Moreover, the expression of fibroblast-derived IL33, an alarmin known to induce invasion, was repressed upon MNK1/2-eIF4E axis inhibition. Imaging mass cytometry on PPBC and non-PPBC patient samples indicated that human PPBC contains phospho-eIF4E high-expressing tumor cells and CD8+ T cells displaying markers of an activated dysfunctional phenotype. Finally, inhibition of MNK1/2 combined with anti-PD-1 therapy blocked lung metastasis of PPBC. These findings implicate the involvement of the MNK1/2-eIF4E axis during PPBC metastasis and suggest a promising immunomodulatory route to enhance the efficacy of immunotherapy by blocking phospho-eIF4E. SIGNIFICANCE: This study investigates the MNK1/2-eIF4E signaling axis in tumor and stromal cells in metastatic breast cancer and reveals that MNK1/2 inhibition suppresses metastasis and sensitizes tumors to anti-PD-1 immunotherapy.