Liam A. Hurst

Mitochondrial DNA Damage Can Promote Atherosclerosis Independently of Reactive Oxygen Species Through Effects on Smooth Muscle Cells and Monocytes and Correlates With Higher-Risk Plaques in Humans

Background— Mitochondrial DNA (mtDNA) damage occurs in both circulating cells and the vessel wall in human atherosclerosis. However, it is unclear whether mtDNA damage directly promotes atherogenesis or is a consequence of tissue damage, which cell types are involved, and whether its effects are mediated only through reactive oxygen species. Methods and Results— mtDNA damage occurred early in the vessel wall in apolipoprotein E–null (ApoE−/−) mice, before significant atherosclerosis developed. mtDNA defects were also identified in circulating monocytes and liver and were associated with mitochondrial dysfunction. To determine whether mtDNA damage directly promotes atherosclerosis, we studied ApoE−/− mice deficient for mitochondrial polymerase-&ggr; proofreading activity (polG−/−/ApoE−/−). polG−/−/ApoE−/− mice showed extensive mtDNA damage and defects in oxidative phosphorylation but no increase in reactive oxygen species. polG−/−/ApoE−/− mice showed increased atherosclerosis, associated with impaired proliferation and apoptosis of vascular smooth muscle cells, and hyperlipidemia. Transplantation with polG−/−/ApoE−/− bone marrow increased the features of plaque vulnerability, and polG−/−/ApoE−/− monocytes showed increased apoptosis and inflammatory cytokine release. To examine mtDNA damage in human atherosclerosis, we assessed mtDNA adducts in plaques and in leukocytes from patients who had undergone virtual histology intravascular ultrasound characterization of coronary plaques. Human atherosclerotic plaques showed increased mtDNA damage compared with normal vessels; in contrast, leukocyte mtDNA damage was associated with higher-risk plaques but not plaque burden. Conclusions— We show that mtDNA damage in vessel wall and circulating cells is widespread and causative and indicates higher risk in atherosclerosis. Protection against mtDNA damage and improvement of mitochondrial function are potential areas for new therapeutics.

TNFα drives pulmonary arterial hypertension by suppressing the BMP type-II receptor and altering NOTCH signalling

Heterozygous germ-line mutations in the bone morphogenetic protein type-II receptor (BMPR-II) gene underlie heritable pulmonary arterial hypertension (HPAH). Although inflammation promotes PAH, the mechanisms by which inflammation and BMPR-II dysfunction conspire to cause disease remain unknown. Here we identify that tumour necrosis factor-α (TNFα) selectively reduces BMPR-II transcription and mediates post-translational BMPR-II cleavage via the sheddases, ADAM10 and ADAM17 in pulmonary artery smooth muscle cells (PASMCs). TNFα-mediated suppression of BMPR-II subverts BMP signalling, leading to BMP6-mediated PASMC proliferation via preferential activation of an ALK2/ACTR-IIA signalling axis. Furthermore, TNFα, via SRC family kinases, increases pro-proliferative NOTCH2 signalling in HPAH PASMCs with reduced BMPR-II expression. We confirm this signalling switch in rodent models of PAH and demonstrate that anti-TNFα immunotherapy reverses disease progression, restoring normal BMP/NOTCH signalling. Collectively, these findings identify mechanisms by which BMP and TNFα signalling contribute to disease, and suggest a tractable approach for therapeutic intervention in PAH.

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.

T1 TNFα mediates ectodomain shedding of BMPR-II: a mechanism for inflammation as a trigger for pulmonary arterial hypertension

Background Mutations in BMP type II receptor (BMPR-II) account for 70% of heritable pulmonary arterial hypertension (PAH) cases, but low penetrance (~20%) in mutation carriers implies a ‘second hit’ is required for disease initiation. Inflammation has been implicated, yet the molecular mechanisms by which it influences pathology are unclear. Tumour necrosis factor alpha (TNFα) has been reported to influence BMPR-II expression, yet the molecular interplay remains unclear. Methods Human pulmonary arterial smooth muscle cells (PASMCs) were stimulated with TNFα (1ng/mL). Biochemical (immunoprecipitation, western blotting), molecular biology (quantitative PCR, plasmid DNA transfection, site-directed mutagenesis, RNA interference) and pharmacological (metalloprotease, proteasome and lysosome inhibitors) approaches were used to assess the impact of TNFα on BMPR-II and BMP signalling pathways. To assess this in vivo, two animal models of PAH were utilised: a mouse model overexpressing TNFα specifically in the lung as well as a moncrotaline induced PAH (MCT-PAH) rat model. Results TNFα stimulation reduced BMPR-II mRNA and protein expression, leading to loss of BMP signalling, as evidenced by abrogated Smad 1/5 and ID1 activation. Notably, a low molecular weight form of BMPR-II accumulated in PASMC lysates following prolonged TNFα exposure: identified as a C-terminal cleavage product of BMPR-II. Furthermore, the N-terminal ectodomain of BMPR-II could be immunoprecipitated from PASMC conditioned media and was quantified by ELISA. TNFα increased expression of two A Disintegrin and Metalloproteinase Domain-containing proteins (ADAMs); ADAM10/17. Pharmacological blockade and RNA interference revealed both proteases were capable of BMPR-II cleavage. Mutation of the putative cleavage site restored BMP signalling. The cleaved ectodomain acted as a ligand trap, sequestering BMP ligands and inhibiting their signalling capacity. Proliferation assays revealed loss of BMP2/4 induced PASMC anti-proliferation in the presence of BMPR-II ectodomain. Finally, both animal models revealed reduced BMPR-II and c-terminal cleavage product in lung tissue - highlighting this event occurs in vivo. Conclusions We identified a novel mechanism by which TNFα impairs BMP signalling and promotes PASMC proliferation in the lung vasculature. TNFa may provide the critical link between inflammation and disease initiation in PAH. Our in vivo observations highlight TNFα as a potential therapeutic target in PAH.

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

S40 TNFα Reduces BMPR-II Expression But Enhances BMP6 Signalling Via ActR-IIa in Pulmonary Arterial Smooth Muscle Cells

Mutations in the bone morphogenetic protein type-II receptor (BMPR-II) underlie ∼70% of heritable pulmonary arterial hypertension (PAH) cases. However, the low penetrance in mutation carriers implies a ‘second hit’ is required for disease onset. A possible trigger is inflammation. Circulating tumour necrosis factor alpha (TNFα) is raised in PAH patients and TNFα can alter smooth muscle cell proliferation. Thus, we examined the effect of TNFα on BMP signalling. Human pulmonary arterial smooth muscle cells (PASMCs) were derived from patients with BMPR-II mutations and from disease-free controls. Quantitative polymerase chain reaction (qPCR) and western blotting revealed that 24 hours of TNFα stimulation reduced the expression of BMP2 and BMPR-II but increased BMP6 expression. TNFa reduced BMP2-mediated signalling, as expected. In contrast, BMP6 treatment in the presence of TNFα increased smad1/5 activation and upregulated ID1 expression, implying that BMP6 can signal independently of BMPR-II Furthermore, BMPR-II mutant PASMCs proliferated in response to BMP6 and TNFα. Quantitative PCR revealed increased ActR-IIa expression following TNFα stimulation and siRNA knockdown of ActR-IIa abrogated this enhanced proliferative response. In summary, TNFα increases BMP6 expression, which in the absence of functional BMPR-II, can bind to receptor complexes involving ActR-IIa to accelerate PASMC proliferation. The potential contribution of this novel mechanism to vascular remodelling in the systemic and pulmonary circulation requires further study.

Response to Letter Regarding Article, “Mitochondrial DNA Damage Can Promote Atherosclerosis Independently of Reactive Oxygen Species Through Effects on Smooth Muscle Cells and Monocytes and Correlates With Higher-Risk Plaques in Humans”

We welcome the opportunity to respond to comments from Drs Stocker and Maghzal on our article.1 Increased reactive oxygen species (ROS) occur in human atherosclerosis in many cell types, and multiple experimental manipulations (predominantly in mice) suggest that ROS promote atherosclerosis. Because mitochondria are an important source of ROS that could be amplified by mitochondrial DNA (mtDNA) damage, the concept has arisen that mtDNA damage promotes atherosclerosis by elevating ROS. However, the polymerase-γ proofreading activity (polG) mouse model of mtDNA damage has been extensively characterized using multiple methods and does not show increased ROS by the age atherosclerosis was studied. Our use of the mitochondria-targeted mass spectrometry probe MitoB (Figure 2C) supported this interpretation. We also showed no difference in …