Methodology The Year in Review PubMed Literature Review: Basic - PowerPoint PPT Presentation

4/21/2018 Methodology The Year in Review PubMed Literature Review: Basic Science 2017 Search Terms “Pulmonary Hypertension” and “2017” 11th International Conference on Neonatal and Manual review of 3600 papers Childhood Pulmonary Vascular Disease April 19-21, 2018 Excluded clinical papers and case reports and articles in languages not in English Stephen L. Archer, MD ~50 basic science papers Professor and Head, Department of Medicine Queen’s University Tier 1 Canada Research Chair Mitochondrial Dynamics Slides derived from selected papers provided Other articles in bibliography Topics Why don’t BMPR2 carriers ALWAYS get disease? • In families affected by PAH, a BMPR2 mutation has a • IPSC-EC reveal compensatory mechanisms protecting against BMPR2 mutations in familial PAH disease penetrance of only 27% • Targeting the Right ventricle – greater in females, 42% than in males, 14% – Disorders of the T-tubules and junctophilin 2 – Larkin EK et al . Longitudinal analysis casts doubt on the presence of genetic anticipation in – Restoring T-tubule function and RV function using colchicine heritable pulmonary arterial hypertension. AJRCCM 186: 892-896, 2012. • Epigenetic drivers of metabolism promote PAH’s cancer-like, Warburg, phenotype and drives proliferation and apoptosis resistance in PASMC, endothelial cells and fibroblasts – Mitochondrial calcium uniporter (MCU) downregulation in PASMC – Pyruvate Kinase M2 upregulation in EC and fibroblasts • Platelets: Mitochondria and TAFI – Is CTEPH a consequence of unresolved PE or is it due to the upregulation of an antifibrinolytic factor (TAFI) that promotes inflammation, vascular leak, metabolic remodeling of PAECs and cell proliferation? – Platelets mitochondria-a glycolytic shift in PAH may serve as a biomarker • sFlt1 and BPD (in handout) 1

4/21/2018 Inducible Progenitor Cells fPAH FAMILY WITH ALL PAH PATIENTS Dr. Shinya Yaminaka BMPR2 Mutation Yamanaka Factors Only 20% BMPR2 carriers within families develop clinical symptoms Why are the purple people 15% familial protected? idiopathic/associated PAH 70% fPAH (+) BMPR2 mutation 20% of IPAH also carry BMPR2 mutation GOAL: FIND PROTECTIVE MODIFIERS OF THE BMPR2 MUTATION IN UNAFFECTED MUTATION CONTROLS ( UMC) THAT ARE RESPONSIBLE FOR NORMAL CELLULAR FUNCTION THAT MIGHT INFORM PROSPECTIVE PAH TREATMENT STRATEGIES EC BMPR2 GENOTYPE SIMILAR IN UMC AND Metrics in IPSC differentiated into endothelial cells (11 people from 3 families) FPAH iPSC-ECs GENOTYPE MORPHOLOGY FAMILY 1 Wild-type Mutant Compensatory Pathways FAMILY 2 CELL Adhesion FAMILY 3 CELL SURVIVAL ddPCR emulsion (20K) 2

4/21/2018 Upregulation of noncanonical pP38 signaling pathway in IMPAIRED CELL ADHESION AND SURVIVAL of iPSC-ECs unaffected family members maintains cell adhesion and survival in FPAH patient vs UMC subject BMP +/- Cell adhesion Elevated caspase activity (variety of substrates) (before/after hypox) Adhesion Impairment Apoptosis marker > In FPAH iPSC-ECs > in FPAH iPSCs ADHESION Compensatory pP38 Signaling and Cell Adhesion in More enhancers + fewer inhibitors of BMPR2 pathway in unaffected BMPR2 carriers Family-specific reasons for improved downstream pP38 signaling UMCs Are Related to BMP Regulators LRP1 – regulates the BMP ligand- receptor complex & promotes ITGB1 Co-transfection of FPAH cells with maturation and transport to cell LRP, ITGB1, MKK6 in FPAH iPSC-ECs membrane (Family 1 and 2) CAV1 – Stabilizes BMPR2 increasing downstream pSMAD1/5-1D1 signaling (Family 2) GREM1 – BMPR2 inhibitor (interacts with BMP ligands) (family 1 and 2) FKBP1A – BMPR2 inhibitor (binds to type I receptors) (family 1 and 2) 3

4/21/2018 Genome Editing of the BMPR2 Mutation in FPAH iPSCEC Improves Adhesion Cell adhesion is rescued with BMPR2 repair Driving the BMPR2 pathway with BMP4 stimulation Baculoviral IAP repeat containing 3 (BIRC3) rescues tube formation and migration RNAseq identifies a decrease in BIRC3 (maintains EC survival) in Tube formation still FPAH (vs UMCs & control iPSC-Ecs) impaired with correction Cell migration still impaired with correction Correction + BMP4 stimulation rescued tube formation • BIR3 K/O mimics FPAH and migration 4

4/21/2018 Conclusions The Right Ventricle Unaffected BMPR2 Mutation Carriers Have Different Compensatory Signaling Mechanisms and Gene Expression Gu et al, Cell Stem Cell, April 2017 Junctophilin-2 Misregulation Through Microtubule Increased tubulin and decreased junctophilin2 in RVH Remodeling Contributes to RV Dysfunction in PAH Benefits of depolymerizng agent, colchicine b -Tubulin Microtubules Normal RV Compensated RV Junctophilin2 PAH RV T-tubules Decompensated RV 5

4/21/2018 Colchicine Partially Prevents T-tubule Remodeling and Improves Conclusions RV Function in MCT Rats Colchicine in PAH RV Normal RV PAH RV Microtubule Colchicine remodeling Restored junctophilin-2 to the t-tubules Microtubule remodeling causes Improved t-tubule architecture junctophiiln-2 misregulation Enhanced RV function Epigenetics Driving Cell Proliferation MCU at the intersection of Mitochondrial Dynamics & Metabolism and Apoptosis Resistance Hong et al Am J Respir Crit Care Med 2017;195:515–529. Control PAH Fragmented Mitochondria • PASMC: – SOD2, MCU • Endothelial cell & fibroblast: – PKM2 Warburg Metabolism 20 m m Courtesy of Zdenko Herceg, PhD, Lyon France 6

4/21/2018 MCU is Decreased in Human PAH miR-138-5p &25/ MCU/Ca2+/ Mitochondria Pathway A B Control PAH STED MCU + Complex 1 Control PASMC 100 µ Hong, Z et al Am J Respir Crit Care Med. 2016 Sep 20 PAH PASMC Control ** PAH Impaired MCUC Function Increases Cytosolic Ca 2+ and Depresses Intramitohondrial Ca 2+ in PAH MCU EMRE MICU1 MICU2 MCUb Nebulized anti-miR therapy reverses MCT-PAH MCU Downregulation Lowers intramitochondrial Calicum, Initiates Fission and Warburg Metabolism in PAH Monocrotaline Treatment Treadmill 100mg/kg sc Initiation Hemodynamics A) Ca 2+ ** mito 10 days Therapy (Nebulization twice weekly) 14days Control PAH Antagomir-138, Antagomomir-25 or Vehicle B) Fission PAH Control plasmid MCU plasmid si-Control si-MCU B A C) Metabolism miR-25 miR-138 PAH si-Control si-MCU Control * * * Control PASMC si- si- Control MCU Control PAH Control PAH MCU plasmid plasmid Control Hong, Z et al Am J Respir Crit Care Med. 2016 Sep 20 7

4/21/2018 Conclusions Epigenetic Activation of Activation of Glycolysis Promotes Proliferation of Fibroblasts and Mitochondrial Calcium Transport: A Potentially Prominent, Therapeutically Targetable Contributor to Pulmonary Arterial Hypertension Progression Endothelial Cells….another route to Warburg Gupte and Wolin Editorial. American Journal of Respiratory and Critical Care Medicine Volume 195 Number 4 | February 15 2017 miR-124 and aerobic glycolysis: Role of miR-124/ PTBP1/PKM pathway miR-124 over-expression reverses the dysregulation observed in BMPR2 pyruvate kinase M2 Loss of miR-124 increases PTBP1 expression driving a mutant BOECs of the target PTBP1 and of enzymes involved in glycolysis proglycolytic pyruvate kinase splice variant, PKM2 over-expression of miR-124 in BOECs Glucose Glucose miR-124 PTBP1 PKM2 Biosynthesis Biosynthesis PTBP1 PEP PEP AAAAA 8 9 11 8 10 11 PKM1 PKM2 Pyruvate Pyruvate TCA cycle TCA cycle 8 9 10 11 Lactate Lactate Pyruvate Kinase M Low Glycolysis High Glycolysis The mimic-mediated over-expression of miR-124 in BOECs isolated from • Binding of the heterogeneous nuclear ribonucleoprotein, polypyrimidine tract-binding protein (PTBP1) to the splice sites flanking exon 9 in PKM transcripts results in exon 9 exclusion HPAH patients significantly reverse the up-regulation of PTBP1, PKM2 and exon 10 inclusion, generating PKM2. and LDHA, suggesting a reduction of glycolysis • PKM2 converts PEP to pyruvate less efficiently than PKM1, leading to the accumulation of glycolytic metabolites for anabolic metabolism (Efficiencies indicated by thickness of arrows) Caruso et al Circulation 2017 Caruso et al Circulation 2017 8

4/21/2018 miR-124 and PTBP1 manipulation reverse the Proliferation rate (and glycolytic phenotype) hyperproliferation observed in HPAH BOECs in PAH BOEC-EC Corrected by: N=4 MIMIC-miR124 siPTBP1 siBMPR2 • BMPR2 mutant BOECs treated with a mimic-124 or a siPTBP1 show a •BMPR2 mutant BOECs present an expected glycolysis rate comparable with control cells; hyperproliferative phenotype vs control BOECs; • The up-regulation of miR-124 and the down-regulation of PTBP1 can •The up-regulation of miR-124 and the down-regulation of reverse the increased glycolysis induced by the down-regulation of PTBP1 can reverse this increased hyperproliferation. BMPR2 in control BOECs. Caruso et al Circulation 2017 Caruso et al Circulation 2017 The Levels of PKM2 /PKM1 is Increased in PH-Fibs and is accompanied by PKM2 Knockdown and Promoting PKM2 Tetramer formation by decreased MPC and SIRT3 Expression TTEP-46 in PH-Fibs (human and bovine) Rescues Metabolic Reprogramming and Decreases Cell Proliferation Human Bovine Zhang H, et al., Circulation, 2017; 2468 Zhang H, et al., Circulation, 2017; 2468 9