3/11/2017 History: Role of Hypoxia Inducible Factor-1 α in Pulmonary Vascular Disease I am convinced that the disease is caused by the increasing mass of inner vascular tissue during its development….to connective tissue…caused “ inflammation ” to characterize a process with by inflammation. However, I contest the term David N. Cornfield, M.D. nothing whatsoever to do with inflammation. Anne T. and Robert M. Bass Professor of Pulmonary Medicine Director-Center for Excellence in Pulmonary Biology Division Director- Pulmonary, Asthma, and Sleep Medicine Professor Julius Klob, M.D., 1839-1879 Stanford University Medical School regarded as the first to describe primary pulmonary hypertension Pulmonary hypertension: No effective treatment or cure Hypoxia-inducible factor-1 α is regulated by oxygen 7 year median survival No cure or reliably effective therapies Despite substantial therapeutic pipeline ($), patient specific Tx Two complementary mechanisms: Complicates many childhood diseases- 1. Degradation High O 2 BPD, Primary, CDH, CHD, Immunologic, hypoxia-related pulmonary 2. Inactivation parenchymal), portal pulmonary HTN, thrombotic, sickle cell, HIV- related, schistosomiasis Ubiquitin Degradation pVHL Might cell specific expression of hypoxia-inducible Proly-hydroxylases Ubiquitylation O OH OH PHD 1,2,3 factors modulate normal and abnormal PVR? Hormonal regulation Energy Metabolism P402 P564 HIF-1 α bHLH PAS TAD-N TAD-C Epo, leptin anaerobic Angiogenesis VEGF, VEGFR Growth, Apoptosis Cell Migration Vasomotor Tone HIF-1, 2, 3 CXCR4, c-Met ET-1, eNOS, Transcriptional Control Matrix, Barrier Function Transport Viral 1
3/11/2017 Hypoxia-inducible factor-1 α is regulated by How is HIF-1 α regulated by oxygen? oxygen Two complementary mechanisms: Two complementary mechanisms: 1. Degradation 1. Degradation High O 2 High O 2 2. Inactivation 2. Inactivation Ubiquitin Degradation pVHL Ubiquitylation Proly-hydroxylases O OH OH OH PHD 1,2,3 Asparagyl-hydroxylase FIH-1 P402 P564 N803 HIF-1 α bHLH PAS TAD-N TAD-C bHLH PAS TAD-N TAD-C HIF-1 α p300 Does cell specific expression of HIF-1 α modulate lung development? Altering HIF-1 α expression-implications HIF-1 α in pulmonary hypertension- for vascular tone and development state of the art HIF +/+ Fijalkowska, I. et al. Am. J. Pathol. 2010. Yu AY, et al. Am. J. Physiol. 1998;275:L818-L826 HIF -/- Yu AY, et al. J. Clin. Invest. 1999;103: 691-696 Control PAH Tuder RM, et al. J. Pathol. 2001;195:367-374 Marsboom G, et al. Circ. Res 2012;110:1484-1497 Iyer, et al., Genes&Dev, 1998 2
3/11/2017 SMC specific deletion of HIF-1 α increases pulmonary SM22-driven deletion of HIF-1 α increases myosin light arterial pressure and increases the response to hypoxia chain phosphorylation in SMC, not muscularization Kim, et al., Circ Res, 2013 Kim, et al., Circ Res, 2013 MHC-driven deletion of HIF-1 α in SMC decreased HIF-2 α in pulmonary endothelial cells modulates PVR pulmonary arterial pressure, but not vascular remodeling and vascular remodeling Tan, et al. JBC, 2013 Adult mice 4 month old mice Ball, et al., AJRCCM, 2014 Cowburn et al. PNAS 2016. Tan, et al. JBC, 2013 3
3/11/2017 Demographic Data from control and PH Patients- In PA SMC from PH patients, HIF-1 α expression is Pulmonary Hypertension Breakthrough Initiative decreased, pMLC is increased Patient ID# Disease Gender Age Race/Ethnicity Control Control PASMC PASMC-C1 HT, IH, stroke Female 60 Caucasian IPAH Control IPAH PASMC-C2 IH Male Caucasian 25 4 6 PASMC-C3 1 2 3 4 1 2 3 4 HT, IH Caucasian *** Male 25 3.5 (relative expression) (relative expression) 5 HIF-1 α / β -actin PASMC-C4 IH HIF-1 α Female 49 Caucasian 100kD 3 pMLC / MLC 4 PASMC-C5 MI 2.5 Female 57 Caucasian 50kD β -actin PASMC-C6 Stroke 2 3 Male 49 Caucasian 37kD ** 1.5 2 IPAH pMLC 20kD 1 1 0.5 MLC PASMC-P1 IPAH Male 40 Hispanic 20kD 0 0 PASMC-P2 IPAH Female 32 Caucasian PASMC-P3 Caucasian IPAH Male 51 PASMC-P4 IPAH Female 27 Caucasian PASMC-P5 IPAH Female 40 Caucasian PASMC-P6 Hispanic IPAH Female 28 Barnes, E..A., et al., FASEB, 2017 In PA SMC from PH patients, HIF-1 α expression and In pulmonary artery endothelial cells from PAH nuclear translocation are decreased patients, HIF-1 α and HIF-2 α expression are increased Control IPAH Control IPAH PAEC 3.5 1.6 * HIF-1 α Nuclei Overlay *** 1.4 Control IPAH 3.0 (relative expression) (relative expression) HIF-1 α / β -actin HIF-2 α / β -actin 1.2 2.5 1 2 3 4 1 2 3 4 Control 1.0 2.0 HIF-1 α 100kD 0.8 1.5 0.6 100kD HIF-2 α HIF-1 α 1.0 0.4 50kD 0.5 0.2 β -actin 37kD 0 0 IPAH + Normoxic expression of HIF-1 α Barnes, E..A., et al., FASEB, 2017 Barnes, E..A., et al., FASEB, 2017 Cornfield Lab 4
3/11/2017 In PA SMC from PH patients, prolyl hydroxylase In PA SMC from PH patients, HIF-1 α expression is activity is increased decreased, pMLC is increased Control IPAH N H N H §§§ 14 Normoxia 12 Hypoxia HIF-1 α (relative expression) 100kD HIF-1 α / β -actin Control IPAH Control IPAH 10 50kD PHD1 8 6 PHD2 50kD 4 *** 37kD PHD3 2 50kD 0 pMLC β -actin 37kD Vinculin Control IPAH pMLC Control §§§ 20 0.9 IPAH 18 * DMSO ** 18 16 0.7 (relative expression) DMOG 16 HIF-1 α / β -actin 12 14 Iron (ng/mL) Ascorbate §§§ (ng/mL) 0.5 12 10 § 10 8 8 0.3 6 6 §§ 4 4 0.1 ** *** 2 2 0 0 0 Fe(II) Fe(III) Control IPAH Barnes, E..A., et al., FASEB, 2017 Barnes, E..A., et al., FASEB, 2017 In PA SMC from PH patients, myosin light chain In PA SMC from PH patients, contractility is kinase activity and pMLC are increased increased which is mimicked by loss of HIF-1 α *** 120 Control IPAH siNTC No SMC siHIF-1 α ML7: - - 120 *** *** 100 No SMC Surface Area (% of siNTC) 4.0 150kD Control MLCK 100 80 *** (relative expression) (% of no SMC) Surface Area IPAH 100kD * Control 80 60 3.0 pMLC / MLC HIF-1 α *** 100kD § 60 40 2.0 50kD §§ 40 20 β -actin 37kD IPAH 20 0 1.0 pMLC 20kD 0 0 MLC 20kD ML7: - - §§§ 250 Control IPAH *** Control Control 2.5 IPAH * Control 16 Focal Adhesions 200 siNTC ** 3 F-actin IPAH 14 siHIF-1 α (per cell) Vinculin 150 §§§ Active MLCK *** 2.5 Active MLCK 12 10 2 100 (ng) (ng) IPAH 8 1.5 50 6 1 4 0 0.5 2 1h 18h Barnes, E..A., et al., FASEB, 2017 0 0 Barnes, E..A., et al., FASEB, 2017 5
3/11/2017 Prolyl hydroxylase activity modifies contractility Pulmonary vascular cell specific HIF-1-3 α and pMLC expression cells from control patients and PH patients * 10 *** *** - - (relative expression) DMOG PASMC PAEC Fibroblasts 8 HIF-1 α / β -actin HIF-1 α 100kD Asc Control IPAH Control IPAH Control IPAH 6 50kD β -actin +++ + + ++ HIF-1 α N.D. N.D. 37kD 4 + + pMLC 20kD HIF-2 α + ++ N.D. N.D. Non- 2 detectable - - +++ + MLC HIF-3 α - - 20kD 0 ** ** *** ** 4.0 120 (relative expression) 100 3.5 (% of no SMC) pMLC / MLC Surface Area 2.5 80 2.0 60 40 1.5 20 0.5 0 0 - DMOG Asc Alveolarization HIF likely has cell, temporal and isoform specific effects Burri PH, Biol Neonate, 2006 Saccular Alveolar Secondary Septation Low Tone Pulmonary Artery SMC: High Tone Pulmonary Artery SMC: “New BPD” impaired secondary septation and capillary formation, “arrested development” -HIF-1 α present in normoxia -HIF-1 α decreased in normoxia -Prolyl hydroxylase activity + -Prolyl hydroxylase activity ++++ • Risk inversely proportional to weight -MLC kinase constrained -MLC kinase elevated • 85% of infants 700g, 30% between 700 - MLC- P - MLC P and 1000 g. -[Ca 2+ ] I limited (K Ca subunit) -[Ca 2+ ] i increased (K Ca subunit) •2/3 of affected infants with only minimal -Decreased contractile state -Increased contractile state supplemental O 2 or MV • 10,000 new BPD cases annually PA endothelial cells and SMC hypoxia-inducible factor form and function are distinct • Indeterminate but significant PH Courtesy of C. Alvira, MD 6
3/11/2017 Hypoxia-induced HIF-1 α protein stabilization is more durable in fetal than adult PA SMC Hypothesis: SM-22 α HIF-1 α expression modulates lung development ? ** ** * * (relative expression) * HIF-1 α /tubulin * Does expression of HIF-1 α in SM22 α cells * contribute to lung development? On C57B/6 Mice, HIF-1 α deleted using an SM-22 α promoter Adult Fetal PA SMC PA SMC Tissue Specific, deletion confirmed, no increased neonatal mortality * p<0.05, vs. normoxia ** p<0.01, vs. normoxia Alveolarization is Compromised in 8 day old Length of α -SMC actin positive arteries is and adult SM22 α -HIF-1 α -/- Mice decreased in SM-22 α HIF-1 α -/- mice. SM22 α -HIF-1 α +/+ SM22 α -HIF-1 α -/- 14 HIF-1 α +/+ SM22 α -HIF-1 α +/+ SM22 α -HIF-1 α -/- §§§ 12 HIF-1 α -/- Radial Alveolar Count §§ §§§ v v 10 ** §§ b b a ** 8 3d a v 6 v 4 b 2 b a a 0 3d 8d Adult v 8d α -SMA 60 Mean Linear Intercept (mm) 50 § ** §§ 40 ** 30 §§ §§ Adult 20 10 0 Pulmonary artery Pulmonary artery 3d 8d Adult 7
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