Pulmonary Arterial Hypertension Pulmonary Artery Smooth Muscle Cells Research Articles

Abstract 15168: The Role of Mitochondrial Fission Factor (MFF) and Increased Midzone Mitochondrial Fission in Pulmonary Arterial Hypertension: Implications for Proliferation, Mitophagy, and Apoptosis

Introduction: The obstructive vasculopathy in pulmonary arterial hypertension (PAH) results from unrestricted proliferation and impaired apoptosis of pulmonary artery smooth muscle cells (PASMC). This pseudoneoplastic phenotype is partly due to increased mitotic fission i.e. the highly coordinated processes of mitochondrial fission and nuclear division. Two types of mitochondrial fission have been reported: peripheral fission, which eliminates damaged terminal portions of mitochondria via mitophagy, and midzone fission, which is linked to mitochondrial biogenesis and cellular proliferation. While all fission involves dynamin related protein 1 (Drp1), the Drp1 binding partner differs between these processes with mitochondrial fission factor (MFF) mediating midzone fission, whilst fission protein 1 (Fis1) governs peripheral fission. Here we explore the role of MFF and Fis1 in the pathogenesis of PAH. Hypothesis: Midzone fission is increased in PAH PASMC and inhibiting midzone fission by downregulating MFF will decrease cell proliferation and induce apoptosis. Methods: MitoMeter, a mitochondrial segmentation and tracking application, was adapted for automated analysis of the location of mitochondrial fission events. Immunoblots were used to assess the expression of MFF and Fis1 in control (n=4) vs. PAH (n=5) human PASMC. Flow cytometry was used to assess cell proliferation (EdU) and rates of apoptosis (Annexin V) (n=3). Results: Midzone fission, as a % of total fission events, is increased (PAH= 48.7%±6.9, Ctrl = 28.9%± 5.1p<0.05). Moreover, MFF expression is increased in PAH PASMC (p<0.05). Conversely, peripheral fission is decreased (PAH=51.2%±6.9, Ctrl = 71.0±5.1). Silencing MFF inhibited midzone fission (siCtrl=54.93.1%, siMFF = 39.37.4%), while Fis1 knockdown inhibited peripheral fission in PAH PASMC (siCtrl = 45.13.1%, siFis1 = 36.91.9%), p<0.05 each. Inhibiting MFF inhibited PAH PASMC proliferation (siCtrl =36.50.9%, siMFF = 23.50.5%, p<0.05) and induced apoptosis in PAH PASMC (siCtrl=3.90.3% versus siMFF 7.40.2%, p<0.05). Conclusion: MFF mediated midzone fission is upregulated in PAH PASMC contributing to increased proliferation, and reduced apoptosis. Inhibiting MFF may be a new therapeutic strategy in PAH.

Central role of ubiquitin-specific protease 8 in leptin signaling pathway in pulmonary arterial hypertension

Leptin receptor (ObR-b) is overexpressed in pulmonary artery smooth muscle cells (PA-SMCs) from patients with pulmonary arterial hypertension (PAH) and is implicated in both mechanisms that contribute to pulmonary vascular remodeling: hyperproliferation and inflammation. Our aim was to investigate the role of ubiquitin-specific peptidase 8 (USP8) in ObR-b overexpression in PAH. We performed in situ and in vitro experiments in human lung specimens and isolated PA-SMCs combined with 2 different in vivo models in rodents and we generated a mouse with an inducible USP8 deletion specifically in smooth muscles. Our results showed an upregulation of USP8 in the smooth muscle layer of distal pulmonary arteries from patients with PAH, and upregulation of USP8 expression in PAH PA-SMCs, compared to controls. USP8 inhibition in PAH PA-SMCs significantly blocked both ObR-b protein expression level at the cell surface as well as ObR-b-dependant intracellular signaling pathway as shown by a significant decrease in pSTAT3 expression. USP8 was required for ObR-b activation in PA-SMCs and its inhibition prevented Ob-mediated cell proliferation through STAT3 pathway. USP8 inhibition by the chemical inhibitor DUBs-IN-2 protected against the development of experimental PH in the 2 established experimental models of PH. Targeting USP8 specifically in smooth muscle cells in a transgenic mouse model also protected against the development of experimental PH. Our findings highlight the role of USP8 in ObR-b overexpression and pulmonary vascular remodeling in PAH.

Dysregulated Smooth Muscle Cell BMPR2-ARRB2 Axis Causes Pulmonary Hypertension.

Mutations in BMPR2 (bone morphogenetic protein receptor 2) are associated with familial and sporadic pulmonary arterial hypertension (PAH). The functional and molecular link between loss of BMPR2 in pulmonary artery smooth muscle cells (PASMC) and PAH pathogenesis warrants further investigation, as most investigations focus on BMPR2 in pulmonary artery endothelial cells. Our goal was to determine whether and how decreased BMPR2 is related to the abnormal phenotype of PASMC in PAH. SMC-specific Bmpr2-/- mice (BKOSMC) were created and compared to controls in room air, after 3 weeks of hypoxia as a second hit, and following 4 weeks of normoxic recovery. Echocardiography, right ventricular systolic pressure, and right ventricular hypertrophy were assessed as indices of pulmonary hypertension. Proliferation, contractility, gene and protein expression of PASMC from BKOSMC mice, human PASMC with BMPR2 reduced by small interference RNA, and PASMC from PAH patients with a BMPR2 mutation were compared to controls, to investigate the phenotype and underlying mechanism. BKOSMC mice showed reduced hypoxia-induced vasoconstriction and persistent pulmonary hypertension following recovery from hypoxia, associated with sustained muscularization of distal pulmonary arteries. PASMC from mutant compared to control mice displayed reduced contractility at baseline and in response to angiotensin II, increased proliferation and apoptosis resistance. Human PASMC with reduced BMPR2 by small interference RNA, and PASMC from PAH patients with a BMPR2 mutation showed a similar phenotype related to upregulation of pERK1/2 (phosphorylated extracellular signal related kinase 1/2)-pP38-pSMAD2/3 mediating elevation in ARRB2 (β-arrestin2), pAKT (phosphorylated protein kinase B) inactivation of GSK3-beta, CTNNB1 (β-catenin) nuclear translocation and reduction in RHOA (Ras homolog family member A) and RAC1 (Ras-related C3 botulinum toxin substrate 1). Decreasing ARRB2 in PASMC with reduced BMPR2 restored normal signaling, reversed impaired contractility and attenuated heightened proliferation and in mice with inducible loss of BMPR2 in SMC, decreasing ARRB2 prevented persistent pulmonary hypertension. Agents that neutralize the elevated ARRB2 resulting from loss of BMPR2 in PASMC could prevent or reverse the aberrant hypocontractile and hyperproliferative phenotype of these cells in PAH.

Abstract 15006: Imaging the Mitochondrial Nucleoid Body in Pulmonary Arterial Hypertension Reveals Changes in Nucleoid Body Distribution and Composition in Human Pulmonary Artery Smooth Muscle Cells

Introduction: Pulmonary arterial hypertension (PAH) exhibits excessive proliferation of pulmonary artery smooth muscle cells (PASMC). This is related to mitochondrial abnormalities, including a metabolic shift to aerobic glycolysis, and increased mitochondrial fragmentation. Mitochondrial DNA (mtDNA) encodes 13 proteins critical to mitochondrial function and is supported by multi-protein complexes called nucleoid bodies (NB) comprised of key mtDNA maintenance proteins. Previous data shows increased mtDNA damage and increased NB protein levels in PAH vs control PASMC. This seems to suggest an attempted compensatory response to mtDNA damage in PAH. We aim to identify NB composition and distribution changes in PAH. We hypothesize that NB structure and distribution throughout the mitochondrial network are dysregulated in PAH. Methods: NBs were visualized in control and PAH PASMC (n=15 per group). Cells were loaded with MitoTracker Deep Red (mitochondrial matrix dye) and visualized using stimulated emission depletion (STED) microscopy. We simultaneously imaged mtDNA and NB proteins TFAM, POLG and SSBP. NB protein distribution was quantified by comparing the rates of mtDNA-protein interactions per mitochondrial area. Results: On visualization of PASMC, mitochondrial fragmentation was evident in PAH vs control. Qualitative findings in PAH PASMC included increased heterogeneity of NB size (diameter) and spatial distribution in PAH compared to control. IF quantitation showed decreased mtDNA/cell (193±20 vs 326±28 mtDNA/cell, p<0.05) and increased SSBP (0.91±0.07 vs 0.59±0.37 SSBP/mtDNA, p<0.05) in PAH cells. These results are consistent with previous findings. Conclusion: These findings suggest a compensatory remodeling of NB in PASMC and is an important step in characterizing the NB shape, distribution, and composition in PAH. These findings will direct experiments analyzing gene and protein expression to further elucidate the role of NB in PAH.

Abstract 11484: Upregulation of Dynamin 2 Increases Mitochondrial Fission and Cell Proliferation in Pulmonary Arterial Hypertension

Introduction: The obstructive vasculopathy in pulmonary arterial hypertension (PAH) results in part from acquired mitochondrial changes in pulmonary arterial smooth muscle cells (PASMC), including increased mitotic fission. Mitotic fission, mitochondrial division which is coordinated with mitosis to equitably distribute mitochondria to daughter cells, is mediated by activation of dynamin-related protein 1 (Drp1). To complete mitochondrial fission, Drp1 requires assistance from the GTPase dynamin 2 (DNM2). Hypothesis: Pathological increase in DNM2 expression drives mitochondrial fission and proliferation of PAH PASMC. Methods: Immunoblots of human PASMC and immunohistochemistry and immunofluorescence of lung sections from human (n=5/group) and monocrotaline (MCT) rats with PAH (n=4-5/group) were used to assess the expression of DNM2. The effect of manipulating DNM2 on cell proliferation, cell cycle and apoptosis were assessed by flow cytometry. Mitochondrial fission was quantified using confocal imaging. Results: DNM2 protein expression is increased in human PAH PASMC. DNM2 is also increased in the media of small pulmonary arteries of PAH patients and MCT-PAH rats. Silencing DNM2 inhibited mitochondrial fission and reduced proliferation causing cell cycle arrest in G1/G0 phase. Silencing DNM2 in PAH PASMC also induced apoptosis. Conversely, augmenting DNM2 in normal PASMC induced mitochondrial fission and accelerated cell proliferation. miR-124-3p, a putative negative regulator of DNM2, is decreased in PAH PASMC. Augmenting miR-124-3p decreased DNM2 expression, inhibited proliferation and induced apoptosis in PAH PASMC. Conclusion: In health, DNM2 regulates mitochondrial fission whilst in disease, epigenetic upregulation of DNM2 increases mitotic fission, which drives pathologic proliferation and apoptosis resistance. The miR-124-3p-DNM2 pathway offers novel PAH therapeutic targets.

Abstract 12004: Mitochondrial Fission Mediator MiD49 Regulates Proliferation via MYO1C in Pulmonary Arterial Hypertension

Introduction: Pulmonary arterial hypertension (PAH) is a lethal syndrome characterized by pulmonary vascular obstruction that is partially due to hyperproliferation of apoptosis-resistant pulmonary arterial smooth muscle cells (PASMC). This “pseudoneoplastic phenotype” of PAH PASMC is due, in part, to excessive mitochondrial fission caused by activation of dynamin related protein-1 (Drp-1) and its binding partner MiD49. The mechanism by which MiD49 regulates the proliferation of PASMC is not completely understood. Here we investigate the possibility that MiD49 promotes cellular proliferation by regulating a non-filamentaous class-1 myosin protein, MYO1C. Hypothesis: MiD49 regulates cell proliferation via a MYO1C-dependent mechanism, which is pathologically upregulated in PAH. Methods: MiD49 and MYO1C expression was compared between normal (n=4) vs PAH (n=5) human PASMC using western blot analysis. The colocalization was visualized using super-resolution confocal microscopy. The effects of manipulating MYO1C on cell proliferation and apoptosis were assessed by flowcytometry. Results: MiD49 and MYO1C are upregulated and colocalized in PAH PASMC. The expression of MYO1C positively correlated with MiD49 in PAH. Silencing MiD49 downregulated MYO1C expression whilst augmenting MiD49 upregulated MYO1C. Silencing MYO1C, however, does not affect MiD49 expression, proving that MYO1C is downstream of MiD49. The downregulation of MYO1C by MiD49 silencing is rescued by a proteasomal blockade, MG132. Downregulating MYO1C using siRNA inhibited proliferation, prevented ERK1/2 activation, and induced apoptosis in PAH PASMC. Conclusion: Increased proliferation and apoptosis resistance of PAH PASMC are in part due to the dysregulation of MiD49-MYO1C pathway. Targeting this pathway may have therapeutic implications against PAH.

Abstract 13283: Mitochondrial DNA Damage and Remodelling of Nucleoid Bodies in Pulmonary Artery Smooth Muscle Cells From Human Patients With Pulmonary Arterial Hypertension

Introduction: Pulmonary arterial hypertension (PAH) is an obstructive pulmonary vasculopathy characterized by pulmonary artery smooth muscle cell (PASMC) hyperproliferation. PAH-PASMC have mitochondrial defects, including increased fragmentation, and increased production of reactive oxygen species (ROS). Mitochondrial-derived ROS can cause DNA damage. Mitochondrial DNA (mtDNA) is surrounded by replication proteins which form nucleoid bodies (NB). Hypothesis: We hypothesize that mtDNA damage is increased and NB structure altered in human PAH PASMC. Methods & Results: We profiled mtDNA and NB composition and integrity in control and PAH PASMC (n = 5/group). mtDNA content was assessed via confocal immunofluorescence of discrete extranuclear double-stranded DNA colocalized with mitochondria. Lower mtDNA content was observed in PAH PASMC vs control (1.8±0.2 vs 3.5±0.2 mtDNAs/μm 3 mitochondria, p<0.0001). Reduced mtDNA in PAH vs control was validated by qPCR (0.69±0.07 vs 1.00±0.07 ng mtDNA/μg cellular protein, n=4-5, p=0.0182). siRNA targeting of Mitofusin2 (Mfn2) in normal PASMC to mimic the mitochondrial fragmentation of PAH, reduced total mtDNA compared to control (1.05±0.06 vs 3.5±0.2 mtDNA/μm 3 mitochondria, n=9, p<0.0001), suggesting increased fission reduces mtDNA expression. Amplification of an 8.8kb mtDNA fragment, a measure of mtDNA damage, revealed increased damage in PAH vs control (14.3±3.9 vs 28.7±2.4 pg mtDNA, n=4, p=0.02). PAH PASMC exhibited higher expression of NB proteins vs control (n=5/measurement): TFAM (+96.7±23.3%, p=0.0032), SSBP (+63.4±14.4%, p=0.0023), and POLG (+59.0±24.0%, p=0.0397). These results were confirmed via immunoblotting for TFAM (+161.9±39.3%, n=4-5, p=0.0045) . Immunofluorescence verified increased TFAM in PAH PASMC vs control. Conclusions: We report a paradoxical upregulation of NB proteins despite reduced mtDNA content and elevated mtDNA damage in PAH. We speculate there is an unsuccessful compensatory attempt to restore mtDNA levels by increasing the expression of mtDNA’s replication apparatus in PAH PASMC.

Abstract 12932: An Epigenetic Upregulation of Dynamin 2, a Regulator of Mitochondrial Fission, Promotes Human and Experimental Pulmonary Arterial Hypertension

Introduction: Increase in mitotic fission in pulmonary arterial smooth muscle cells (PASMC) contributes to the obstructive vasculopathy in pulmonary arterial hypertension (PAH). PAH’s fissogenic phenotype is due, in part, to excessive mitochondrial fission caused by activation of dynamin related protein-1 (Drp1). Recent observation suggests that to complete mitochondrial fission, Drp1 requires assistance from the GTPase dynamin 2 (DNM2). In this study we explore the role of DNM2 in the pathogenesis of PAH. Hypothesis: In PAH, increased expression of DNM2 increases mitochondrial fission and proliferation of PASMC. Methods: Immunoblot was used to quantify the expression of DNM2 in control (n=4) and PAH PASMC (n=5). Immunofluorescence was used to quantify the expression of DNM2 in the pulmonary arteries of monocrotaline (MCT) vs control rats. Flow cytometry was used to assess the effects of manipulating the expression of DNM2 on cell proliferation, cell cycle progression and rates of unstimulated apoptosis. Mitochondrial network structure was assessed using confocal imaging of cells infected with Adv-TOM20 mNeon green. Results: The expression of DNM2 was increased in human PAH PASMC vs control PASMC. Increase in the expression of DNM2 expression was also observed in the pulmonary arteries of MCT-PAH rats. Silencing DNM2 inhibited mitochondrial fission in PAH PASMC. Furthermore, silencing DNM2 inhibited proliferation in PAH PASMC by blocking the cell cycle at G1/G0. Conversely, augmenting DNM2 in normal PASMC induced mitochondrial fission and accelerated cell proliferation, recapitulating the PAH phenotype. In silico analysis identified miR-124-3p as a putative negative regulator of DNM2. Augmenting miR-124-3p decreased DNM2 expression, inhibited proliferation and induced apoptosis in PAH PASMC. Conclusion: DNM2 promotes mitochondrial fission and regulates cell proliferation in human PAH PASMC. Decreased miR-124-3p expression may contribute to upregulation of DNM2 in PAH. This work highlights the importance of DNM2 as partner with Drp1 in regulation of mitochondrial fission and shows that like Drp1, DNM2 is increased in human PAH. DNM2 may constitute new therapeutic target for PAH.

Abstract 13094: Pharmacokinetics and Therapeutic Efficacy of Drpitor1a, a Novel Dynamin-Related Protein 1 Inhibitor, in a Preclinical Model of Pulmonary Arterial Hypertension

Introduction: Increased dynamin-related protein 1 (Drp1)-mediated mitochondrial fission contributes to the pathogenesis of pulmonary arterial hypertension (PAH). We investigated the pharmacokinetic characteristics of Drpitor1a, a novel, small molecule, ellipticine Drp1 inhibitor (prepared in-house), and tested its therapeutic efficacy in a pilot study using the rat monocrotaline (MCT)-PAH model. Methods: A single dose of Drpitor1a (5mg/kg) was administered IV or PO to male and female rats (n=3/sex). Blood was collected at 0, 5 and 30 minutes, and at 1, 2, 4, 6, 8 and 24 hours post drug administration. Plasma, lung and right ventricle (RV) were collected and tissue concentration of Drpitor1a was measured using HPLC coupled with mass spectrometry. The effects of Drpitor1a on mitochondrial fission and cell proliferation were assessed in human PAH pulmonary artery smooth muscle cells (PASMC). In an MCT-PAH model, a catheter was implanted in the left jugular vein on day 14 post-MCT injection. Drpitor1a (1mg/kg) was administered by IV every 48 hours from day 17 to day 27. On day 28, RV function and pulmonary hemodynamics were assessed. Results: Drpitor1a’s half-life varies by sex (9.9±5.3 hours and 24.3±21.4 hours in male and female rats respectively). Oral bioavailability of Drpitor1a was similar in males (21.1%) and females (19.7%). Lung concentration of Drpitor1a was similar between males and females (0.441±0.015 nM/mg and 0.444±0.581 nM/mg, respectively). In the RV, Drpitor1a level was higher in females (0.102±0.053 nM/mg) than males (0.055±0.015 nM/mg). Drpitor1a inhibited mitochondrial fission and cell proliferation in human PAH PASMC. In addition, Drpitor1a regressed pulmonary artery medial thickening (25.5% vs 34.9%), inhibited RV hypertrophy (Fulton index: 0.33 vs 0.39) and improved RV function (TAPSE: 2.2mm vs 1.8mm) in female MCT-PAH rats. Conclusion: Drpitor1a is a potential therapeutic agent for preclinical PAH and may be more effective in females.

Regulation of Type I cytokine receptors as a target for pulmonary arterial hypertension treatment?

In pulmonary arterial hypertension (PAH), the current therapies do not target pulmonary vascular remodeling (PVR), the main cause of the disease. Type I cytokine receptors (TypeIR) are known to be overexpressed in PAH pulmonary artery smooth muscle cells (PA-SMCs) and involved in PVR. To assess whether the regulation of TypeIR intracellular trafficking is altered in PAH, leading to TypeIR overexpression and PVR. We studied TypeIR trafficking through the main regulatory proteins ubiquitin-specific protease 8 (USP8) and ring finger protein (RNF) 41. In PAH patients and in controls, we performed in situ immunostaining in lung samples and western blotting in primary cultures of PA-SMCs isolated from human tissues. We transfected human PA-SMCs with siRNA against USP8 and RNF41 in vitro . In two experimental models, the monocrotaline- and chronic hypoxia-induced PAH, we performed hemodynamic measurements followed by in situ immunostaining and western blotting analysis in lung tissues. We showed an increased USP8 and decreased RNF41 expression in PAH lungs compared to controls, and a similar shift in USP8/RNF41 ratio at the protein level in PAH PA-SMCs compared to controls ( n = 10). We modulated USP8/RNF41 ratio with siRNAs and restored TypeIR expression and its signalling ( n = 5). Consistent with our human in situ and in vitro results, we confirmed the altered USP8/RNF41 ratio in the two in vivo models ( n = 8 and n = 5). TypeIR intracellular trafficking regulation is altered in PAH patients compared to controls, due to a shift in USP8/RNF41 ratio. Restoring USP8/RNF41 ratio in PA-SMCs may represent a potential target in PAH.

ALDH1A3 Coordinates Metabolism With Gene Regulation in Pulmonary Arterial Hypertension.

Metabolic alterations provide substrates that influence chromatin structure to regulate gene expression that determines cell function in health and disease. Heightened proliferation of smooth muscle cells (SMC) leading to the formation of a neointima is a feature of pulmonary arterial hypertension (PAH) and systemic vascular disease. Increased glycolysis is linked to the proliferative phenotype of these SMC. RNA sequencing was applied to pulmonary arterial SMC (PASMC) from PAH patients with and without a BMPR2 (bone morphogenetic receptor 2) mutation versus control PASMC to uncover genes required for their heightened proliferation and glycolytic metabolism. Assessment of differentially expressed genes established metabolism as a major pathway, and the most highly upregulated metabolic gene in PAH PASMC was aldehyde dehydrogenase family 1 member 3 (ALDH1A3), an enzyme previously linked to glycolysis and proliferation in cancer cells and systemic vascular SMC. We determined if these functions are ALDH1A3-dependent in PAH PASMC, and if ALDH1A3 is required for the development of pulmonary hypertension in a transgenic mouse. Nuclear localization of ALDH1A3 in PAH PASMC led us to determine whether and how this enzyme coordinately regulates gene expression and metabolism in PAH PASMC. ALDH1A3 mRNA and protein were increased in PAH versus control PASMC, and ALDH1A3 was required for their highly proliferative and glycolytic properties. Mice with Aldh1a3 deleted in SMC did not develop hypoxia-induced pulmonary arterial muscularization or pulmonary hypertension. Nuclear ALDH1A3 converted acetaldehyde to acetate to produce acetyl coenzyme A to acetylate H3K27, marking active enhancers. This allowed for chromatin modification at NFYA (nuclear transcription factor Y subunit α) binding sites via the acetyltransferase KAT2B (lysine acetyltransferase 2B) and permitted NFY-mediated transcription of cell cycle and metabolic genes that is required for ALDH1A3-dependent proliferation and glycolysis. Loss of BMPR2 in PAH SMC with or without a mutation upregulated ALDH1A3, and transcription of NFYA and ALDH1A3 in PAH PASMC was β-catenin dependent. Our studies have uncovered a metabolic-transcriptional axis explaining how dividing cells use ALDH1A3 to coordinate their energy needs with the epigenetic and transcriptional regulation of genes required for SMC proliferation. They suggest that selectively disrupting the pivotal role of ALDH1A3 in PAH SMC, but not endothelial cells, is an important therapeutic consideration.

Serum and pulmonary uric acid in pulmonary arterial hypertension

Previous studies have suggested an association between uric acid (UA) and the severity of pulmonary arterial hypertension (PAH), but it is unknown whether UA contributes to disease pathogenesis.The aim of this study was to determine the prognostic value of circulating UA in the era of current management of PAH and to investigate the role of UA in pulmonary vascular remodelling.Serum UA levels were determined in idiopathic, heritable or anorexigen PAH at baseline and first re-evaluation in the French Pulmonary Hypertension Network. We studied protein levels of xanthine oxidase (XO) and the voltage-driven urate transporter 1 (URATv1) in lungs of control and PAH patients and of monocrotaline (MCT) and Sugen/hypoxia (SuHx) rats. Functional studies were performed using human pulmonary artery smooth muscle cells (PA-SMCs) and two animal models of pulmonary hypertension (PH).High serum UA levels at first follow-up, but not at baseline, were associated with a poor prognosis. Both the generating enzyme XO and URATv1 were upregulated in the wall of remodelled pulmonary arteries in idiopathic PAH patients and MCT and SuHx rats. High UA concentrations promoted a mild increase in cell growth in idiopathic PAH PA-SMCs, but not in control PA-SMCs. Consistent with these observations, oxonic acid-induced hyperuricaemia did not aggravate MCT-induced PH in rats. Finally, chronic treatment of MCT and SuHx rats with benzbromarone mildly attenuated pulmonary vascular remodelling.UA levels in idiopathic PAH patients were associated with an impaired clinical and haemodynamic profile and might be used as a non-invasive indicator of clinical prognosis during follow-up. Our findings also indicate that UA metabolism is disturbed in remodelled pulmonary vascular walls in both experimental and human PAH.

Inhibitory effects of RAGE-aptamer on development of monocrotaline-induced pulmonary arterial hypertension in rats

BackgroundThe receptor for advanced glycation end products (RAGE), a transmembrane receptor belonging to the immunoglobulin superfamily, is overexpressed in pulmonary artery smooth muscle cells (PASMCs) in patients with pulmonary arterial hypertension (PAH) and is implicated in the etiology of PAH. Recently, we reported that RAGE-aptamer, a short and single-stranded DNA directed against RAGE, inhibited an inappropriate increase in cultured PASMCs in PAH. The aim of this study was to determine the efficacy of RAGE-aptamer in monocrotaline-induced PAH in rats. Methods and ResultsRats were assigned to either an untreated control group, a group that received continuous subcutaneous administration of RAGE-aptamer immediately after monocrotaline injection, or a group that received control-aptamer immediately after monocrotaline injection. All rats survived 21 days after injection of monocrotaline and control-aptamer or RAGE-aptamer. Injection of monocrotaline with continuous subcutaneous delivery of control-aptamer resulted in higher right ventricular systolic pressure compared with controls. This increase was attenuated by continuous subcutaneous delivery of RAGE-aptamer. The proportion of small pulmonary arteries with full muscularization was greater in the monocrotaline and control-aptamer group than in the control group. Continuous subcutaneous delivery of RAGE-aptamer significantly reduced the percentage of small pulmonary arteries with full muscularization. ConclusionsContinuous subcutaneous delivery of RAGE-aptamer suppresses development of monocrotaline-induced PAH in rats. Inhibition of RAGE ameliorates muscularization of small pulmonary arteries. Treatment with RAGE-aptamer might be a new therapeutic option for PAH.

Abstract 13575: Lack of Both Mitochondrial Proteins Sirt3 and Ucp2 in Mice Recapitulates Many Critical Features of Human Pulmonary Arterial Hypertension (PAH), Including Inflammatory Plexogenic Lesions, Supporting the Metabolic Theory of PAH

Loss-of function SNPs for Sirt3 (a mitochondrial deacetylase) and Ucp2 (an atypical uncoupling protein enabling mitochondrial calcium entry) have been associated with insulin resistance and obesity in humans; and patients with PAH have insulin resistance without being obese. In a cohort of PAH patients (n=60) we found these SNPs often both in the same patient in a homozygous or heterozygous manner and their presence correlated positively with the degree of PAH upon referral, the presence of type II diabetes and outcomes (death, transplantation). We generated and studied double KO mice for Sirt3 and Ucp2 using closed-chest right heart catheterization and echocardiography and found increasing severity of pulmonary hypertension (PHT) in Sirt3 +/- -Ucp2 +/ - , Sirt3 -/- -Ucp2 +/ - , Sirt3 +/- -Ucp2 -/- and Sirt3 -/- -Ucp2 -/- , associated with decreasing cardiac output and increasing right ventricular hypertrophy, dilatation and dysfunction (TAPSE), compared to wild-type (WT) mice. The LVEDP in all mice was normal. There was increasing severity of vascular remodeling with increasing levels of CD4 + cell infiltration, while Sirt3 -/- -Ucp2 +/- , Sirt3 +/- -Ucp2 -/- and Sirt3 -/- -Ucp2 -/- mice also developed frequent plexogenic lesions. In vivo and in vitro pulmonary artery smooth muscle cells (PASMC) expressed higher levels of Ki67, compared to WT mice. In vitro, the Sirt3 -/- -Ucp2 +/- , Sirt3 +/- -Ucp2 -/- and Sirt3 -/- -Ucp2 -/- PASMC exhibited more apoptosis-resistance, expressed higher nuclear levels of proliferative transcription factors (HIF1, NFATc2), exhibited decreased respiration and higher levels of glycolysis, similarly to previous reports of animal and human PAH PASMC. The Sirt3 -/- -Ucp2 -/- , but not the WT mice, also developed in vivo evidence of insulin resistance. Our work supports the metabolic theory of PAH and shows that these mice exhibit spontaneous severe PHT (without environmental or chemical triggers) in a gene dose-response dependent manner that mimics human PAH. No other mouse model of PHT has shown frequent and predictable plexogenic lesions. Our study is relevant to the PAH patients that carry loss-of-function SNPs of mitochondrial proteins and offers a new mouse model of PAH, with more features of human PAH than previous mice models.

Pulmonary Artery Smooth Muscle Cell Bone Morphogenetic Protein 2 Promotes Pulmonary Endothelial Cell Migration and Angiogenesis

BACKGROUNDPulmonary Arterial Hypertension (PAH) is an incurable disease and remains a significant cause of morbidity and mortality in children and adults. In PAH, pulmonary arteries narrow in diameter which increases blood flow resistance in the lungs. Eventually the increase in pulmonary blood pressure causes irreversible damage to the heart. There are a variety of reasons why the pulmonary arteries become obstructed, known and unknown; yet, the molecular mechanisms driving this disease remain to be elucidated. Approximately 1000 new cases of PAH are diagnosed each year in the U.S. alone. The average survival rate for patients diagnosed with PAH is 7 years. Therefore, any insight into the molecular mechanisms that promote PAH progression may represent new strategies to treat patients with PAH.OBJECTIVEUp to 20% of PAH patients with no family history of PAH carry a bone morphogenetic protein receptor 2 (BMPR2) mutation, whereas in patients with a family history of PAH up to 82% carry a BMPR2 mutation. Therefore, it appears extremely likely that BMPR2 loss of function promotes the dysfunctional angio‐proliferative phenotype of PAH. In fact, it has recently been shown that in endothelial cells (EC) deficient in BMPR2 exposed to exogenous bone morphogenetic protein 2 (BMP2), a ligand for BMPR2, are migratory compared to control cells. To determine if the loss of BMPR2 promotes the PAEC dysfunctional angiogenesis observed in patients with PAH, we used cells from control and PAH patients to determine the role of pulmonary artery smooth muscle cell (PASMC) secreted BMP2 in promoting the migration of pulmonary artery endothelial cells (PAEC) and angiogenesis.METHODSPASMC and PAEC from control and PAH patients were examined for the expression of BMPR2 and BMP2. Cell migration, cell invasion, and angiogenetic assays were performed using PASMC and PAEC from control and PAH patients to assess differences in migration, invasion, and angiogenesis.RESULTSPAEC from PAH patients have a significant reduction in the expression of BMPR2, consistent with published reports. PASMC from PAH secrete BMP2, while BMP2 is undetectable in control cells. Co‐culturing PAH PASMC with PAH PAEC promotes PAEC migration. This migration is further enhanced in the presence of exogenous BMP2. PAH PASMC co‐cultured with PAH PAEC promote the development of PAEC‐derived angiogenetic masses. This dysfunctional angiogenetic response is further amplified in the presence of exogenous BMP2. In contrast, control PAEC exposed to control PASMC respond normally and do not form disorganized masses of vessels. Control PAEC exposed to exogenous BMP2 further promotes a normal angiogenetic response.CONCLUSIONSPASMC from PAH patients secrete BMP2. BMP2 exposure promotes the migration of PAH PAEC and a dysfunctional angiogenetic response. In contrast, BMP2 promotes a normal angiogenetic response when added to co‐cultures of control PASMC and PAEC. These data suggest that in PAH, angiogenesis becomes dysfunctional in part due to PAEC exposure to BMP2; therefore, inhibitors of BMP2 may represent a therapeutic strategy to pursue in the treatment of PAH.

Berberine alleviates pulmonary hypertension through Trx1 and β-catenin signaling pathways in pulmonary artery smooth muscle cells

Pulmonary arterial hypertension (PAH) is closely associated with profound vascular remodeling, especially pulmonary arterial medial hypertrophy and muscularization, due to aberrant proliferation of pulmonary artery smooth muscle cells (PASMCs). Berberine, a drug commonly used to treat inflammation, may be a novel therapeutic option for PAH by improving pulmonary artery remodeling. The present study investigated whether berberine affected Trx1/β-catenin expression and/or activity and whether it could reduce the development of pulmonary hypertension in an experimental rat model and proliferation in human PASMCs (HPASMCs). The results showed that increased proliferation in hypoxia-induced healthy PASMCs or PAH PASMCs was associated with a significant increase in Trx1 and β-catenin expression. Treatment with the Trx1-specific inhibitor PX-12 significantly reduced pulmonary arterial pressure and vascular remodeling, as well as improved in vivo cardiac function and right ventricular hypertrophy, in Su/Hox-induced PAH rats. Berberine reversed right ventricular systolic pressure and right ventricular hypertrophy and decreased pulmonary vascular remodeling in the rats. Furthermore, berberine had an antiproliferative effect on hypoxia-induced HPASMC proliferation in a manner likely mediated by inhibiting Trx1 and its target gene β-catenin expression. Our work will help elucidate novel strategies for PAH treatment involving the traditional Chinese medicine berberine, and Trx1/β-catenin may be a promising therapeutic target.

Therapeutic effect of pirfenidone in the sugen/hypoxia rat model of severe pulmonary hypertension.

Heightened pulmonary artery smooth muscle cell (PA-SMC) proliferation and migration and dynamic remodeling of the extracellular matrix are hallmark pathogenic features of pulmonary arterial hypertension (PAH). Pirfenidone (PFD) is an orally bioavailable pyridone derivative with antifibrotic, antiinflammatory, and antioxidative properties currently used in the treatment of idiopathic pulmonary fibrosis. We therefore evaluated the efficacy of curative treatments with PFD in the sugen/hypoxia (SuHx) rat model of severe pulmonary hypertension. Treatment with PFD (30 mg/kg per day by mouth 3 times a day for 3 wk) started 5 wk after sugen injection partially reversed established pulmonary hypertension, reducing total pulmonary vascular resistance and remodeling. Consistent with these observations, we found that continued PFD treatment decreases PA-SMC proliferation and levels of extracellular matrix deposition in lungs and right ventricles in SuHx rats. Importantly, PFD attenuated the proproliferative and promigratory potentials of cultured PA-SMCs from patients with idiopathic PAH and their capacity to produce extracellular matrix components. Finally, we found that PFD dose dependently enhanced forkhead box O1 protein levels and its nuclear translocation in cultured idiopathic PAH PA-SMCs and in PFD-treated SuHx rats. PFD appears to be a potential therapy for PAH worthy of investigation and evaluation for clinical use in conjunction with current PAH treatments.-Poble, P.-B., Phan, C., Quatremare, T., Bordenave, J., Thuillet, R., Cumont, A., Huertas, A., Tu, L., Dorfmüller, P., Humbert, M., Ghigna, M.-R., Savale, L., Guignabert, C. Therapeutic effect of pirfenidone in the sugen/hypoxia rat model of severe pulmonary hypertension.

Abstract 17192: ALDH1A3 Induces NFY to Coordinate Transcription of Cell Cycle and Metabolic Genes Necessary for Smooth Muscle Proliferation in Pulmonary Hypertension

Background: In developing disease modifying therapies for pulmonary arterial hypertension (PAH), it is important to understand the molecular basis for hyperproliferation of pulmonary arterial smooth muscle cells (PASMC) that progressively occlude the vessel lumen. Previously we presented studies using H3K27 ChIP Seq and RNA Seq in PAH vs. control PASMC, and showed that an increase in ALDH1A3 is responsible for both heightened proliferation and H3K27ac enhancer marks in cell cycle and metabolic enzyme genes. Methods and Results: We now present molecular and metabolic studies and experiments in transgenic mice that delineate the mechanism by which ALDH1A3 plays a critical role in PAH PASMC proliferation. Promoter binding motif analysis of sites that are ALDH1A3 dependent in PAH PA SMC identified NFY family members as putative transcription factors. We confirmed their role in increasing three energy producing metabolic enzymes PKM2, DLD and IDH1 and two cell cycle genes, CCNA2 and CDC20. We verified that their mRNA transcripts and protein levels are elevated in PAH PASMC and dependent on increased ALDH1A3 and NFY(n=3, p<0.05) The accumulation of acetyl-CoA is increased in PAH PASMC with siControl vs. siALDH1A3. The nuclear localization of increased PAH PASMC ALDH1A3 indicates that generation of acetyl CoA from acetylaldehde could acetylate histones at H3K27 target sites marked by NFY binding. From non-targeted metabolomic analyses by LC/MS, we found ALDH1A3 dependent metabolites in the glycolytic and pentose phosphate pathways in PAH PASMC, consistent with the functions of PKM2, DLD and IDH1. We then showed that ALDH1A3 is induced by hypoxia, and required for SMC proliferation and pulmonary hypertension in mice exposed to three weeks of chronic hypoxia (10% oxygen). Gender matched transgenic mice with SMC deletion of Aldh1a3 had reduced pulmonary hypertension when compared to littermate controls, assessed by right ventricular systolic pressure (n=8, p<0.05). Conclusions: Our studies reveal an ALDH1A3-dependent coordination of histone acetylation at NFY transcription factor binding sites of key metabolic enzyme and cell cycle genes in highly proliferative PAH PASMC from PAH patients, that is critical in the development of pulmonary hypertension.

A1511 Identification of CERB as a master transcription factor in monocrotaline–induced pulmonary arterial hypertension

Objectives: A large number of differential expressed genes have been identified in the pathogenesis of pulmonary arterial hypertension (PAH) by microarray/transcriptome sequencing (RNA-seq), several transcription factors might be responsible for transcription of these differential expressed genes. The purpose of this study was to identify master regulator transcription factors in the pathogenesis of PAH. Methods: RNA-seq was performed on rats subjected to 1, 2, 3 and 4 weeks of monocrotaline (MCT) treatment. Differentially expressed genes were identified by a stand of |MCT vs Ctr log2Fold Change|≥1 and p≤0.05, master regulator transcription factors were enrichment by HOMER analysis and CREB target genes were identified by CREB target gene database. Phospho-CREB and CREB protein in rat lungs and isolated pulmonary arterial smooth muscle cells (PASMCs) were determined by western blot. Results: Differential expression of 280, 1342, 1649 and 3155 genes were identified by RNA-seq on week 1, 2, 3 and 4, respectively. HOMER analysis identified CREB as the most significant enrichment transcription factor in a cluster of sustained upregulated genes (Fig.1A). In addition, CRE-containing genes were selectively differential expressed on week 4 in rat induced by MCT (Fig.1B). In subsequent validation experiment, a sustained increase in phospho-CREB and CREB expression was observed in both total PAH lung homogenates and isolated rat PAH PASMCs by western blot. Conclusion: CREB was a master transcription factor in the pathogenesis of PAH induced by MCT.

Sirtuin 1 regulates pulmonary artery smooth muscle cell proliferation: role in pulmonary arterial hypertension.

Energy metabolism shift from oxidative phosphorylation toward glycolysis in pulmonary artery smooth muscle cells (PASMCs) is suggested to be involved in their hyperproliferation in pulmonary arterial hypertension (PAH). Here, we studied the role of the deacetylase sirtuin1 (SIRT1) in energy metabolism regulation in PASMCs via various pathways including activation of peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α), master regulator of mitochondrial biogenesis. Contents of PGC-1α and its downstream targets as well as markers of mitochondrial mass (voltage-dependent anion channel and citrate synthase) were diminished in human PAH PASMCs. These cells and platelet-derived growth factor-stimulated rat PASMCs demonstrated a shift in cellular acetylated/deacetylated state, as evidenced by the increase of the acetylated forms of SIRT1 targets: histone H1 and Forkhead box protein O1. Rat and human PASMC proliferation was potentiated by SIRT1 pharmacological inhibition or specific downregulation via short-interfering RNA. Moreover, after chronic hypoxia exposure, SIRT1 inducible knock out mice displayed a more intense vascular remodeling compared with their control littermates, which was associated with an increase in right ventricle pressure and hypertrophy. SIRT1 activator Stac-3 decreased the acetylation of histone H1 and Forkhead box protein O1 and strongly inhibited rat and human PASMC proliferation without affecting cell mortality. This effect was associated with the activation of mitochondrial biogenesis evidenced by higher expression of mitochondrial markers and downstream targets of PGC-1α. Altered acetylation/deacetylation balance as the result of SIRT1 inactivation is involved in the pathogenesis of PAH, and this enzyme could be a promising therapeutic target for PAH treatment.