Mark L. Ormiston

MicroRNA-138 and MicroRNA-25 Down-regulate Mitochondrial Calcium Uniporter, Causing the Pulmonary Arterial Hypertension Cancer Phenotype

Rationale: Pulmonary arterial hypertension (PAH) is an obstructive vasculopathy characterized by excessive pulmonary artery smooth muscle cell (PASMC) proliferation, migration, and apoptosis resistance. This cancer‐like phenotype is promoted by increased cytosolic calcium ([Ca2+]cyto), aerobic glycolysis, and mitochondrial fission. Objectives: To determine how changes in mitochondrial calcium uniporter (MCU) complex (MCUC) function influence mitochondrial dynamics and contribute to PAHs cancer‐like phenotype. Methods: PASMCs were isolated from patients with PAH and healthy control subjects and assessed for expression of MCUC subunits. Manipulation of the pore‐forming subunit, MCU, in PASMCs was achieved through small interfering RNA knockdown or MCU plasmid‐mediated up‐regulation, as well as through modulation of the upstream microRNAs (miRs) miR‐138 and miR‐25. In vivo, nebulized anti‐miRs were administered to rats with monocrotaline‐induced PAH. Measurements and Main Results: Impaired MCUC function, resulting from down‐regulation of MCU and up‐regulation of an inhibitory subunit, mitochondrial calcium uptake protein 1, is central to PAHs pathogenesis. MCUC dysfunction decreases intramitochondrial calcium ([Ca2+]mito), inhibiting pyruvate dehydrogenase activity and glucose oxidation, while increasing [Ca2+]cyto, promoting proliferation, migration, and fission. In PAH PASMCs, increasing MCU decreases cell migration, proliferation, and apoptosis resistance by lowering [Ca2+]cyto, raising [Ca2+]mito, and inhibiting fission. In normal PASMCs, MCUC inhibition recapitulates the PAH phenotype. In PAH, elevated miRs (notably miR‐138) down‐regulate MCU directly and also by decreasing MCUs transcriptional regulator cAMP response element‐binding protein 1. Nebulized anti‐miRs against miR‐25 and miR‐138 restore MCU expression, reduce cell proliferation, and regress established PAH in the monocrotaline model. Conclusions: These results highlight miR‐mediated MCUC dysfunction as a unifying mechanism in PAH that can be therapeutically targeted.

Identification of miR-124 as a Major Regulator of Enhanced Endothelial Cell Glycolysis in Pulmonary Arterial Hypertension via PTBP1 and PKM2

Background: Pulmonary arterial hypertension (PAH) is characterized by abnormal growth and enhanced glycolysis of pulmonary artery endothelial cells. However, the mechanisms underlying alterations in energy production have not been identified. Methods: Here, we examined the miRNA and proteomic profiles of blood outgrowth endothelial cells (BOECs) from patients with heritable PAH caused by mutations in the bone morphogenetic protein receptor type 2 (BMPR2) gene and patients with idiopathic PAH to determine mechanisms underlying abnormal endothelial glycolysis. We hypothesized that in BOECs from patients with PAH, the downregulation of microRNA-124 (miR-124), determined with a tiered systems biology approach, is responsible for increased expression of the splicing factor PTBP1 (polypyrimidine tract binding protein), resulting in alternative splicing of pyruvate kinase muscle isoforms 1 and 2 (PKM1 and 2) and consequently increased PKM2 expression. We questioned whether this alternative regulation plays a critical role in the hyperglycolytic phenotype of PAH endothelial cells. Results: Heritable PAH and idiopathic PAH BOECs recapitulated the metabolic abnormalities observed in pulmonary artery endothelial cells from patients with idiopathic PAH, confirming a switch from oxidative phosphorylation to aerobic glycolysis. Overexpression of miR-124 or siRNA silencing of PTPB1 restored normal proliferation and glycolysis in heritable PAH BOECs, corrected the dysregulation of glycolytic genes and lactate production, and partially restored mitochondrial respiration. BMPR2 knockdown in control BOECs reduced the expression of miR-124, increased PTPB1, and enhanced glycolysis. Moreover, we observed reduced miR-124, increased PTPB1 and PKM2 expression, and significant dysregulation of glycolytic genes in the rat SUGEN-hypoxia model of severe PAH, characterized by reduced BMPR2 expression and endothelial hyperproliferation, supporting the relevance of this mechanism in vivo. Conclusions: Pulmonary vascular and circulating progenitor endothelial cells isolated from patients with PAH demonstrate downregulation of miR-124, leading to the metabolic and proliferative abnormalities in PAH ECs via PTPB1 and PKM1/PKM2. Therefore, the manipulation of this miRNA or its targets could represent a novel therapeutic approach for the treatment of PAH.

Pulmonary arterial hypertension: pathogenesis and clinical management

ABSTRACT Pulmonary hypertension is defined as a resting mean pulmonary artery pressure of 25 mm Hg or above. This review deals with pulmonary arterial hypertension (PAH), a type of pulmonary hypertension that primarily affects the pulmonary vasculature. In PAH, the pulmonary vasculature is dynamically obstructed by vasoconstriction, structurally obstructed by adverse vascular remodeling, and pathologically non-compliant as a result of vascular fibrosis and stiffening. Many cell types are abnormal in PAH, including vascular cells (endothelial cells, smooth muscle cells, and fibroblasts) and inflammatory cells. Progress has been made in identifying the causes of PAH and approving new drug therapies. A cancer-like increase in cell proliferation and resistance to apoptosis reflects acquired abnormalities of mitochondrial metabolism and dynamics. Mutations in the type II bone morphogenetic protein receptor (BMPR2) gene dramatically increase the risk of developing heritable PAH. Epigenetic dysregulation of DNA methylation, histone acetylation, and microRNAs also contributes to disease pathogenesis. Aberrant bone morphogenetic protein signaling and epigenetic dysregulation in PAH promote cell proliferation in part through induction of a Warburg mitochondrial-metabolic state of uncoupled glycolysis. Complex changes in cytokines (interleukins and tumor necrosis factor), cellular immunity (T lymphocytes, natural killer cells, macrophages), and autoantibodies suggest that PAH is, in part, an autoimmune, inflammatory disease. Obstructive pulmonary vascular remodeling in PAH increases right ventricular afterload causing right ventricular hypertrophy. In some patients, maladaptive changes in the right ventricle, including ischemia and fibrosis, reduce right ventricular function and cause right ventricular failure. Patients with PAH have dyspnea, reduced exercise capacity, exertional syncope, and premature death from right ventricular failure. PAH targeted therapies (prostaglandins, phosphodiesterase-5 inhibitors, endothelin receptor antagonists, and soluble guanylate cyclase stimulators), used alone or in combination, improve functional capacity and hemodynamics and reduce hospital admissions. However, these vasodilators do not target key features of PAH pathogenesis and have not been shown to reduce mortality, which remains about 50% at five years. This review summarizes the epidemiology, pathogenesis, diagnosis, and treatment of PAH.

Epigenetic Dysregulation of the Drp1 Binding Partners MiD49 and MiD51 Increases Mitotic Mitochondrial Fission and Promotes Pulmonary Arterial Hypertension: Mechanistic and Therapeutic Implications

Background: Mitotic fission is increased in pulmonary arterial hypertension (PAH), a hyperproliferative, apoptosis-resistant disease. The fission mediator dynamin-related protein 1 (Drp1) must complex with adaptor proteins to cause fission. Drp1-induced fission has been therapeutically targeted in experimental PAH. Here, we examine the role of 2 recently discovered, poorly understood Drp1 adapter proteins, mitochondrial dynamics protein of 49 and 51 kDa (MiD49 and MiD51), in normal vascular cells and explore their dysregulation in PAH. Methods: Immunoblots of pulmonary artery smooth muscle cells (control, n=6; PAH, n=8) and immunohistochemistry of lung sections (control, n=6; PAH, n=6) were used to assess the expression of MiD49 and MiD51. The effects of manipulating MiDs on cell proliferation, cell cycle, and apoptosis were assessed in human and rodent PAH pulmonary artery smooth muscle cells with flow cytometry. Mitochondrial fission was studied by confocal imaging. A microRNA (miR) involved in the regulation of MiD expression was identified using microarray techniques and in silico analyses. The expression of circulatory miR was assessed with quantitative reverse transcription–polymerase chain reaction in healthy volunteers (HVs) versus patients with PAH from Sheffield, UK (plasma: HV, n=29, PAH, n=27; whole blood: HV, n=11, PAH, n=14) and then confirmed in a cohort from Beijing, China (plasma: HV, n=19, PAH, n=36; whole blood: HV, n=20, PAH, n=39). This work was replicated in monocrotaline and Sugen 5416-hypoxia, preclinical PAH models. Small interfering RNAs targeting MiDs or an miR mimic were nebulized to rats with monocrotaline-induced PAH (n=4–10). Results: MiD expression is increased in PAH pulmonary artery smooth muscle cells, which accelerates Drp1-mediated mitotic fission, increases cell proliferation, and decreases apoptosis. Silencing MiDs (but not other Drp1 binding partners, fission 1 or mitochondrial fission factor) promotes mitochondrial fusion and causes G1-phase cell cycle arrest through extracellular signal-regulated kinases 1/2– and cyclin-dependent kinase 4–dependent mechanisms. Augmenting MiDs in normal cells causes fission and recapitulates the PAH phenotype. MiD upregulation results from decreased miR-34a-3p expression. Circulatory miR-34a-3p expression is decreased in both patients with PAH and preclinical models of PAH. Silencing MiDs or augmenting miR-34a-3p regresses experimental PAH. Conclusions: In health, MiDs regulate Drp1-mediated fission, whereas in disease, epigenetic upregulation of MiDs increases mitotic fission, which drives pathological proliferation and apoptosis resistance. The miR-34a-3p-MiD pathway offers new therapeutic targets for PAH.

Abnormal angiogenesis in blood outgrowth endothelial cells derived from von Willebrand disease patients

&NA; Bleeding associated with angiodysplasia is a common, often intractable complication in patients with von Willebrand disease (VWD). von Willebrand factor (VWF), the protein deficient or defective in VWD, is a negative regulator of angiogenesis, which may explain the pathologic blood vessel growth in VWD. This study explores the normal range of angiogenesis in blood outgrowth endothelial cells (BOECs) derived from healthy donors and compares this to angiogenesis in BOECs from VWD patients of all types and subtypes. BOECs were assessed for VWF and angiopoietin-2 (Ang-2) gene expression, secretion, and storage. To explore angiogenic potential, we characterized cellular proliferation, matrix protein adhesion, migration, and tubule formation. We found great angiogenic variability in VWD BOECs with respect to each of the angiogenesis parameters. However, type 1 and 3 VWD BOECs had higher Ang-2 secretion associated with impaired endothelial cell migration velocity and enhanced directionality. Type 2A and 2B BOECs were the most proliferative and multiple VWD BOECs had impaired tubule formation in Matrigel. This study highlights the angiogenic variability in BOECs derived from VWD patients. Abnormal cell proliferation, migration, and increased Ang-2 secretion are common features of VWD BOECs. Despite the many abnormalities of VWD BOECs, significant heterogeneity among individual VWD phenotypes precludes a simple description of relationship between VWD type and in vitro surrogates for angiodysplasia.

A Potential Role for Exosomal Translationally Controlled Tumor Protein Export in Vascular Remodeling in Pulmonary Arterial Hypertension

Abstract Pulmonary arterial hypertension (PAH) is characterized by increased proliferation and resistance to apoptosis of pulmonary vascular cells. Increased expression of translationally controlled tumor protein (TCTP), a prosurvival and antiapoptotic mediator, has recently been demonstrated in patients with heritable PAH; however, its role in the pathobiology of PAH remains unclear. Silencing of TCTP in blood outgrowth endothelial cells (BOECs) isolated from control subjects led to significant changes in morphology, cytoskeletal organization, increased apoptosis, and decreased directionality during migration. Because TCTP is also localized in extracellular vesicles, we isolated BOEC‐derived extracellular vesicles (exosomes and microparticles) by sequential ultracentrifugation. BOECs isolated from patients harboring BMPR2 mutations released more exosomes than those derived from control subjects in proapoptotic conditions. Furthermore, TCTP expression was significantly higher in exosomes than in microparticles, indicating that TCTP is mainly exported via exosomes. Coculture assays demonstrated that exosomes transferred TCTP from ECs to pulmonary artery smooth muscle cells, suggesting a role for endothelial‐derived TCTP in conferring proliferation and apoptotic resistance. In an experimental model of PAH, rats treated with monocrotaline demonstrated increased concentrations of TCTP in the lung and plasma. Consistent with this finding, we observed increased circulating TCTP levels in patients with idiopathic PAH compared with control subjects. Therefore, our data suggest an important role for TCTP in regulating the critical vascular cell phenotypes that have been implicated in the pathobiology of PAH. In addition, this research implicates TCTP as a potential biomarker for the onset and development of PAH.

A sex-specific reconstitution bias in the competitive CD45.1/CD45.2 congenic bone marrow transplant model

Allelic variants of the pan-haematopoietic cell marker CD45, identified as CD45.1 and CD45.2, have been established as a marker system to track haematopoietic cells following congenic mouse bone marrow transplants. Despite the frequent use of this model for studying the impact of genetic modifications on relative differentiation potential, it is now evident that a bias exists in CD45.1 versus CD45.2 cell reconstitution. While this bias has been demonstrated by reduced reconstitution potential in B cells of CD45.1 origin, differences in the development of other lymphocytes, as well as the impact of sex on this bias, remain uncertain. We performed bone marrow transplants with wild-type CD45.1 and CD45.2 donor cells, and characterised haematopoietic cell reconstitution in dual-expressing CD45.1/2 host mice. We report an increase in CD45.2 reconstitution in the bone marrow that persists in the spleen, thymus and blood. Through the use of CD45.1/2 hosts, we demonstrate the intrinsic bias towards CD45.2 reconstitution is independent of an immunogenic response to the CD45.1 epitope. Furthermore, we identify a sex-specific difference in reconstitution efficiencies, with female mice exhibiting a greater bias towards CD45.2 reconstitution than males. This work sheds new light on the limitations of the CD45.1/CD45.2 congenic system for tracking lymphocyte development.

Immune regulation of systemic hypertension, pulmonary arterial hypertension, and preeclampsia: shared disease mechanisms and translational opportunities

Systemic hypertension, preeclampsia, and pulmonary arterial hypertension (PAH) are diseases of high blood pressure in the systemic or pulmonary circulation. Beyond the well-defined contribution of more traditional pathophysiological mechanisms, such as changes in the renin-angiotensin-aldosterone system, to the development of these hypertensive disorders, there is substantial clinical evidence supporting an important role for inflammation and immunity in the pathogenesis of each of these three conditions. Over the last decade, work in small animal models, bearing targeted deficiencies in specific cytokines or immune cell subsets, has begun to clarify the immune-mediated mechanisms that drive changes in vascular structure and tone in hypertensive disease. By summarizing the clinical and experimental evidence supporting a contribution of the immune system to systemic hypertension, preeclampsia, and PAH, the current review highlights the cellular and molecular pathways that are common to all three hypertensive disorders. These mechanisms are centered on an imbalance in CD4+ helper T cell populations, defined by excessive Th17 responses and impaired Treg activity, as well as the excessive activation or impairment of additional immune cell types, including macrophages, dendritic cells, CD8+ T cells, B cells, and natural killer cells. The identification of common immune mechanisms in systemic hypertension, preeclampsia, and PAH raises the possibility of new therapeutic strategies that target the immune component of hypertension across multiple disorders.

S84 Identification of MIR-124a as a major regulator of enhanced endothelial cell glycolysis in pulmonary arterial hypertension

Introduction Pulmonary arterial hypertension (PAH) is a rare desease characterised by profound vascular abnormalities in the peripheral arteries of the lung, leading to a progressive increase in pulmonary vascular resistance, right heart failure and death. The disease exists in several forms including a heritable form (HPAH) caused primarily by mutations in bone morphogenetic protein receptor type 2 (BMPR2) and an idiopathic form (IPAH). Endothelial cell (EC) dysfunction is considered a critical initiating factor in the pathobiology of PAH, manifested by increased susceptibility to apoptosis, heightened permeability and enhanced endothelial proliferation. Substantial changes in bioenergetics of ECs, including higher rates of glycolysis, have been reported in PAH patients. However, the mechanisms underlying alterations in energy production have not been identified. Methods We measured glycolysis in blood outgrowth endothelial cells (BOECs) from HPAH patients carrying mutations in BMPR2 and IPAH patients to confirm the metabolic abnormalities previously. We also employed an unbiased genome-wide microarray and proteomic screening approach to detect miRNAs and proteins dysregulated in the same groups to determine the mechanisms underlying abnormal endothelial glycolysis. Results HPAH and IPAH BOECs recapitulated the metabolic phenotype previously observed in PAECs. These alterations were found to be associated with the downregulation of miR-124 and the upregulation of its known target, splicing factor polypyrimidine-tract-binding protein (PTBP1). We also demonstrated that increased PTBP1 promotes the switching in expression of two forms of pyruvate kinase, PKM1 and PKM2, resulting in an increase of aerobic glycolysis, consequently increasing cell proliferation (mechanism schematized in Figure 1). Overexpression of miR-124, or siRNA silencing of PTPB1, restoring normal expression levels of PKM2, also restored normal proliferation and glycolysis in HPAH BOECs. Finally, we observed reduced miR-124 and increased PTPB1 and PKM2 expression in a well-established rat model of PAH, characterised by endothelial proliferation, supporting the presence of this mechanism in vivo. Conclusions Loss of function of BMPR2 results in the downregulation of miR-124 and consequently in the glycolytic abnormalities reported in PAH ECs. Therefore, the manipulation of this miRNA, or its targets, could represent a novel and effective strategy to achieve clinical benefits in the treatment of PAH. Abstract S84 Figure 1

S67 Characterisation of the endothelial outgrowth cell

The existence of cells circulating in the peripheral blood with the capacity of an endothelial progenitor cell (EPC) remains controversial. This is best exemplified by the original cell posited by Asahara and colleagues to be an EPC in 1997,1 which has now been clearly phenotyped as a monocyte with dendritic features.2 There remains a viable candidate known as the endothelial colony-forming cell (ECFC) or endothelial outgrowth cell (OEC), named because it cannot be seen in ex vivo culture for at least 10 days. These cells proliferate in culture, form a cobblestone monocellular layer, and form networks in ex vivo assays. Given the lack of understanding of previous candidate cells we sought to better characterise the EOC. Here we present data from electron microscopy studies, immunohistochemistry, ligand stimulation studies, and mRNA microarrays that demonstrate the EOC is a true endothelial cell. Furthermore we demonstrate that these cells can potentially be used as endothelial surrogates in patients with pulmonary hypertension due to a mutation in the bone morphogenetic protein type II receptor (BMPRII). Cells taken from the peripheral blood of patients have a deficiency in intracellular signalling in the BMPRII pathway on stimulation with BMP9. This can be demonstrated by reduced phosphoSmad 1/5 protein on western blotting, and downstream with reduced Id1 gene induction by qPCR. Therefore regardless of its origin and biologically intended function, the EOC is an endothelial cell, and has the capacity to act as an easily derivable endothelial surrogate from patients in whom vascular tissue is not normally obtainable.