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

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The Year in Review

Basic Science 2017

11th International Conference on Neonatal and Childhood Pulmonary Vascular Disease April 19-21, 2018

Stephen L. Archer, MD

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

Methodology

PubMed Literature Review: Search Terms “Pulmonary Hypertension” and “2017” Manual review of 3600 papers Excluded clinical papers and case reports and articles in languages not in English ~50 basic science papers

Topics

• IPSC-EC reveal compensatory mechanisms protecting against BMPR2

mutations in familial PAH

• Targeting the Right ventricle

– Disorders of the T-tubules and junctophilin 2 – Restoring T-tubule function and RV function using colchicine

• 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)

Why don’t BMPR2 carriers ALWAYS get disease?

• In families affected by PAH, a BMPR2 mutation has a

disease penetrance of only 27%

– greater in females, 42% than in males, 14%

– Larkin EK et al. Longitudinal analysis casts doubt on the presence of genetic anticipation in heritable pulmonary arterial hypertension. AJRCCM 186: 892-896, 2012.

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Inducible Progenitor Cells

• Dr. Shinya Yaminaka

Yamanaka Factors

ALL PAH PATIENTS

15% familial 70% fPAH (+) BMPR2 mutation 20% of IPAH also carry BMPR2 mutation idiopathic/associated PAH Only 20% BMPR2 carriers within families develop clinical symptoms

fPAH FAMILY WITH BMPR2 Mutation

Why are the purple people protected?

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

FAMILY 1 FAMILY 2 FAMILY 3

Metrics in IPSC differentiated into endothelial cells (11 people from 3 families)

CELL SURVIVAL CELL Adhesion MORPHOLOGY GENOTYPE Compensatory Pathways

EC BMPR2 GENOTYPE SIMILAR IN UMC AND FPAH iPSC-ECs

Wild-type Mutant ddPCR emulsion (20K)

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IMPAIRED CELL ADHESION AND SURVIVAL of iPSC-ECs in FPAH patient vs UMC subject

Cell adhesion (variety of substrates) Elevated caspase activity (before/after hypox)

Adhesion Impairment > In FPAH iPSC-ECs Apoptosis marker > in FPAH iPSCs

Upregulation of noncanonical pP38 signaling pathway in unaffected family members maintains cell adhesion and survival

ADHESION BMP +/-

LRP1 – regulates the BMP ligand- receptor complex & promotes ITGB1 maturation and transport to cell 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)

More enhancers + fewer inhibitors of BMPR2 pathway in unaffected BMPR2 carriers Family-specific reasons for improved downstream pP38 signaling

Co-transfection of FPAH cells with LRP, ITGB1, MKK6 in FPAH iPSC-ECs Compensatory pP38 Signaling and Cell Adhesion in UMCs Are Related to BMP Regulators

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Genome Editing of the BMPR2 Mutation in FPAH iPSCEC Improves Adhesion

Cell adhesion is rescued with BMPR2 repair

Tube formation still impaired with correction Cell migration still impaired with correction Correction + BMP4 stimulation rescued tube formation and migration Driving the BMPR2 pathway with BMP4 stimulation rescues tube formation and migration Baculoviral IAP repeat containing 3 (BIRC3) RNAseq identifies a decrease in BIRC3 (maintains EC survival) in FPAH (vs UMCs & control iPSC-Ecs)

• BIR3 K/O mimics FPAH

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Conclusions

Unaffected BMPR2 Mutation Carriers Have Different Compensatory Signaling Mechanisms and Gene Expression

Gu et al, Cell Stem Cell, April 2017

The Right Ventricle

Junctophilin-2 Misregulation Through Microtubule Remodeling Contributes to RV Dysfunction in PAH

Compensated RV Decompensated RV

Normal RV PAH RV Microtubules T-tubules

Increased tubulin and decreased junctophilin2 in RVH Benefits of depolymerizng agent, colchicine

b-Tubulin Junctophilin2

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Colchicine Partially Prevents T-tubule Remodeling and Improves RV Function in MCT Rats

Conclusions

Normal RV PAH RV Colchicine Microtubule remodeling Colchicine in PAH RV

Restored junctophilin-2 to the t-tubules Improved t-tubule architecture Enhanced RV function

Microtubule remodeling causes junctophiiln-2 misregulation

Epigenetics Driving Cell Proliferation and Apoptosis Resistance

• PASMC:

– SOD2, MCU

• Endothelial cell &

fibroblast: – PKM2

Courtesy of Zdenko Herceg, PhD, Lyon France

MCU at the intersection of Mitochondrial Dynamics & Metabolism

20mm Fragmented Mitochondria Warburg Metabolism Hong et al Am J Respir Crit Care Med 2017;195:515–529.

Control PAH

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Impaired MCUC Function Increases Cytosolic Ca2+ and Depresses Intramitohondrial Ca2+ in PAH miR-138-5p &25/ MCU/Ca2+/ Mitochondria Pathway

Control PAH

B A 100 µ

Control PASMC PAH PASMC

STED

MCU + Complex 1

MCU is Decreased in Human PAH

MCU EMRE MICU1 MICU2 MCUb ** Control PAH Hong, Z et al Am J Respir Crit Care Med. 2016 Sep 20

MCU Downregulation Lowers intramitochondrial Calicum, Initiates Fission and Warburg Metabolism in PAH

si-Control si-MCU

A

Control plasmid MCU plasmid

B

PAH B) Fission

** Control PAH

A) Ca2+

mito

si- Control

*

Control

si- MCU

*

PAH

Control plasmid MCU plasmid

si-Control si-MCU

Control PASMC

C) Metabolism

Therapy (Nebulization twice weekly) 14days 10 days Monocrotaline 100mg/kg sc Treatment Initiation Antagomir-138, Antagomomir-25 or Vehicle Treadmill Hemodynamics

Nebulized anti-miR therapy reverses MCT-PAH

miR-138 * miR-25

Control PAH Control PAH

Hong, Z et al Am J Respir Crit Care Med. 2016 Sep 20

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Mitochondrial Calcium Transport: A Potentially Prominent, Therapeutically Targetable Contributor to Pulmonary Arterial Hypertension Progression Gupte and Wolin Editorial. American Journal of Respiratory and Critical Care Medicine Volume 195 Number 4 | February 15 2017

Conclusions

Epigenetic Activation of Activation of Glycolysis Promotes Proliferation of Fibroblasts and Endothelial Cells….another route to Warburg

miR-124 and aerobic glycolysis: Role of pyruvate kinase M2

AAAAA

miR-124 PTBP1 8 9 10 11 Glucose PEP Biosynthesis Pyruvate TCA cycle Lactate 8 10 11 PKM2 Glucose PEP Biosynthesis Pyruvate TCA cycle Lactate PKM1 8 11 9 Pyruvate Kinase M High Glycolysis Low Glycolysis

• 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 and exon 10 inclusion, generating PKM2.

• 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)

Loss of miR-124 increases PTBP1 expression driving a proglycolytic pyruvate kinase splice variant, PKM2

Caruso et al Circulation 2017

miR-124/ PTBP1/PKM pathway

miR-124 over-expression reverses the dysregulation observed in BMPR2 mutant BOECs of the target PTBP1 and of enzymes involved in glycolysis

The mimic-mediated over-expression of miR-124 in BOECs isolated from HPAH patients significantly reverse the up-regulation of PTBP1, PKM2 and LDHA, suggesting a reduction of glycolysis Caruso et al Circulation 2017

• ver-expression of miR-124 in BOECs

PTBP1 PKM2

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miR-124 and PTBP1 manipulation reverse the hyperproliferation observed in HPAH BOECs

• BMPR2 mutant BOECs present an expected

hyperproliferative phenotype vs control BOECs;

• The up-regulation of miR-124 and the down-regulation of

PTBP1 can reverse this increased hyperproliferation.

N=4

Caruso et al Circulation 2017

Proliferation rate (and glycolytic phenotype) in PAH BOEC-EC Corrected by:

• BMPR2 mutant BOECs treated with a mimic-124 or a siPTBP1 show a

glycolysis rate comparable with control cells;

• The up-regulation of miR-124 and the down-regulation of PTBP1 can

reverse the increased glycolysis induced by the down-regulation of BMPR2 in control BOECs.

Caruso et al Circulation 2017 MIMIC-miR124 siPTBP1 siBMPR2

The Levels of PKM2 /PKM1 is Increased in PH-Fibs and is accompanied by decreased MPC and SIRT3 Expression

Human Bovine

Zhang H, et al., Circulation, 2017; 2468

PKM2 Knockdown and Promoting PKM2 Tetramer formation by TTEP-46 in PH-Fibs (human and bovine) Rescues Metabolic Reprogramming and Decreases Cell Proliferation

Zhang H, et al., Circulation, 2017; 2468

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MiR-124 overexpression or PTBP1 silencing decreases the PKM2/PKM1 ratio and regulates the metabolic phenotype in PH-Fibs

Zhang H, et al., Circulation, 2017; 2468

Restoring PKM2/PKM1 balance reverses metabolic programming in PH

Zhang H, et al., Circulation, 2017; 2468

PAH Therapy

Platelets and TAFI

Biomarkers to Therapy

Increased Glycolysis in Platelet Mitochondria in PAH

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Platelets from PAH Patients are Glycolytic Increased Mitochondrial Reserve Capacity Predicts Disease Severity

Conclusions

• In PAH platelets have increased glycolysis
• Increased Glycolysis/OCR is c/w Warburg Metabolism
• bserved in pulmonary vascular cells and RV myocytes
• Association of increased RV SWI, a measure of RV

workload and contractility, with mitochondrial energetics in these well-compensated patients suggests an adaptive role for enhanced mitochondrial capacity.

• This presents a mechanistic link between altered

myeloid function and metabolic dysregulation in PAH

CTEPH and TAFI

• Plasma, thrombin-activatable fibrinolysis inhibitor (TAFI) (AKA

carboxypeptidase B2, Cpb2), significantly elevated in CTEPH patients vs PAH patients or controls

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TAFI

Is CTEPH caused by unresolved PE vs TAFI activation?

Increased TAFI (Cpb2) in CTEPH patients (plasma & PAs)

Transgenic ovexpression of TAFI-Promotes Hypoxic PH

Transgenic TAFI overexpression causes PA-specific endothelial dysfunction

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PPARa activator (WY14643) Reduces TAFI Activation and Regresses PAH

Conclusions

• TAFI upregulation in hypoxia is specific to PA (aorta unaffected)- explaining CTEPH’s tissue specificity
• TAFI increase vascular leak and cytokine expression
• PPARa agonists (WY14643) reduce hypoxic PH in trangenic mice
• Pregnancies complicated by preeclampsia(PE) and chorioamnionitis (CA) increase the

risk for bronchopulmonary dysplasia (BPD).

• The biological mechanisms linking the prenatal factors with BPD are uncertain.
• Levels of sFlt-1 (soluble fms-like tyrosine kinase 1), an endogenous VEGF antagonist is

increased in amniotic fluid and maternal blood of PE and CA

• Hypothesis: Fetal exposure to sFlt-1 decreases lung growth, lung function and causes

pulmonary hypertension during infancy.

• Corollary: Inhibiting sFlt-1 could be an effective therapeutic strategy against BPD
• Conclusion: anti-sFlt-1mAb improves lung structure and function in 2 rat models of BPD

and early AB therapy may be a novel preventative strategy

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Study Design

Fig.1 Beneficial effects of prenatal intraamniotic monoclonal antibody treatment on lung structure and right ventricular hypertrophy in experimental preeclampsia

• Fig. 2

Beneficial effects of prenatal maternal uterine artery monoclonal anti-Flt antibody treatment in experimental preeclamsia Fig.3

• Fig. 4

Effects of intraamniotic sFlt-1 exposure and uterine artery monoclonal antibody treatment on lung function as assessed on 14-day-old rats.

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Effects of intraamniotic sFlt-1 exposure on lung VEGF and eNOS protein expression at birth

• Fig. 5

Effects of postnatal monoclonal antibody therapy after intraamniotic sFlt-1 exposure.

• Fig. 6

Fig.7 Effects of prenatal intraamniotic sFlt-1 and intraamniotic endotoxin on sFlt-1 content in newborn rats at birth

• Fig. 8

Effects of prenatal intraamniotic monoclonal antibody treatment on lung structure and right ventricular hypertrophy RVH after endotoxin exposure

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BIRC3 siRNA resulted in increased activation of caspase

Caspase activity: Reflects vulnerability to apoptosis in response to either serum withdrawal or reoxygenation after hypoxia”

FAMILY 1 FAMILY 2 FAMILY 3

Induced pluripotent stem cell-derived Ecs (iPSC-ECs): Taken from skin fibroblasts

Vs. Vs. UMC fPAH Gender matched control

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

miR-124/ PTBP1/PKM pathway

• Epigenetic upregulation of a heterogeneous nuclear ribonucleoprotein, polypyrimidine

tract-binding protein (PTBP1). PTBP1 binds to the flanking sequences of exon 9, thus inhibiting formation o the PKM1 isoform. The resulting increase in the PKM2/PKM1 ratio favours uncoupled glycolysis.

• Pro-glycolytic change in pyruvate production drives proliferation of endothelial cells &

fibroblasts in PAH.

hnRNP proteins control metabolic switch between oxidative phosphorylation &aerobic glycolysis by regulating the PKM alternative splicing.

PKM2 converts PEP to pyruvate less efficiently than PKM1

Colchicine Depolymerizes Microtubules and Corrects Junctophilin-2 Mislocalization in the MCT RV

From Peng et al., 2014 PBS MCT MCT-Colch b-tubulin

Microtubules Junctophilin

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Pyruvate Kinase: Part of the Warburg Metabolic Shift in PAH

Uncoupled Glycolysis Drives a Cancer-Like Phenotype

Stephen L. Archer, Circulation. 2017;136:2486-2490 https://doi.org/10.1161/CIRCULATIONAHA.117.031655