4/20/2018 • Cristina M. Alvira The Role of the Lung Microenvironment in Has documented that she has no financial Modulating Pulmonary Angiogenesis relationships to disclose or Conflicts of Interest (COIs) to resolve Cristina M. Alvira, MD Assistant Professor of Pediatrics Stanford University School of Medicine Stanford CHRI Tashia and John Morgridge Faculty Scholar 5 Numerous Pre- and Postnatal Factors Can A Significant Component of Lung Development Disrupt Normal Lung Development Occurs Postnatally Birth Gestational Weeks Postnatal Exposures Antenatal Exposures 0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 2 - 10 years ? • Mechanical Ventilation • Intrauterine infections • Oxidative Stress • Intrauterine growth Premature Birth • Infections restriction • Corticosteroids alveolar • Nutritional Deficits saccular Gestational Birth canalicular Weeks pseudoglandular 0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 Burri PH, Biol Neonate, 2006 Gestation Secondary Alveolar Septation Saccular Embryonic Canalicular Pseudoglandular 500 million alveoli Saccular Alveolar Adapted from: Baraldi and Filipone, N Engl J Med, 2007 Alveolar surface area: 140 m 2 1
4/20/2018 The Immature Lung has an Increased Compensatory Lung Growth is Greatest in Capacity to Regenerate after Injury Young Patients after Pneumonectomy Compensatory growth Age at TLC % VC % RV % Rat Lung of right lung to achieve Groups operation Number predicted predicted predicted same gas exchange surface area as with (y) for 2 lungs for 2 lungs for 2 lungs both lungs 1 0-5 9 96.4 79.1 34.2 Removal of left lung Right Left 2 6-10 12 87.2 75.4 30.2 3 11-15 8 85.6 73.5 31.3 4 16-20 14 84.8 68.8 34.2 Vital Capacity Surface Area 5 21-25 18 78.4 64.5 38.2 S 6 26-30 19 71.2 56.8 40.0 7 31-40 18 70.4 55.9 41.4 Laros CD, et al, J Thorac Cardiovasc Surg, 1987 Distinct Phases of Alveolarization and Growth of the Pulmonary Vasculature Angiogenesis in the Developing Lung Promotes Alveolarization 36w Birth 2 Yr 3 Yr 10 Yr Human Angiogenic Factor Replacement Alveolar sac after Active Microvascular blocking angiogenesis Angiogenesis Maturation Continued / Late Bulk Alveolarization Alveolarization Normal alveolar sac Restored alveolarization after angiogenic therapy Rat Angiogenic Factors Birth P4 P14 P21 P30 P60 Alveolar sac in BPD Thebaud B and Abman SH, Am J Respir Crit Care Med, 2007 Adapted from Tschanz et al, J Appl Physiol, 2014 2
4/20/2018 Stimuli that Impair Alveolarization Induce The Developing Pulmonary Vasculature Premature Maturation of the Pulmonary Remodels During Late Alveolarization Microvasculature Roth-Kleiner, Dev Dyn, 2005 Dexamethasone Dexamethasone Early Alveolar Adult treated P4 treated P6 Rat Lung Rat Lung (P4) (P44) What controls the angiogenic phenotype of the pulmonary endothelial cells during development? Schittney JC, Cell Tissue Res, 2017 Angiogenic Function is Higher in Pulmonary The IKK/NF k B Pathway Promotes Pulmonary Endothelial Cells from the Developing Lung Angiogenesis during Alveolarization Early Alveolar Mice (P6) Magnet P P P P NUCLEUS Adult Mice isolation I k B a (10-12 week) p50 p65 Phosphorylation and degradation NF k B Gene Expression or NEMO of I k B a Repression Primary Pulmonary IKK IKK a IKK b Endothelial Cells CYTOPLASM complex Early Alveolar (P6) Adult (8-10 wk) Adult Lung Apoptosis Proliferation Migration Early Alveolar Lung (P6) 1500 2 NF k B 100 *** (p65) Area of wound covered (%) BrdU Incorporation *** Caspase 3/7 Activity 1.5 80 CD31 1000 *** *** Chromatin 60 1 *** *** 40 500 *** *** 0.5 *** 20 Active NF k B Inactive NF k B 0 Iosef C et al, Am J Physiol Lung Cell Mol Physiol, 2012 0 0 FBS FBS 2 8 12 24 2 8 12 24 0.2% 5% Hours Hours 3
4/20/2018 Inhibiting NF k B Disrupts Angiogenic Function in Activation of NF k B Peaks in the Pulmonary Early Alveolar Lung Endothelial Cells Endothelium during Early Alveolarization Apoptosis Proliferation Control 1750 BAY (2.5 m M) 1.4 *** *** BAY (5 m M ) NF k B Activation in Primary Pulmonary Endothelial Cells (PEC) *** 1500 1.2 1250 Active BrdU § *** Caspase 3/7 Incorporation 1.0 1000 750 0.8 500 0.6 250 Late Saccular Early Alveolar Late Alveolar Adult 0 0 Con BAY PEC PEC PEC PEC 2 4 6 8 24 24h Time (hours) NF k B Migration In Vitro Angiogenesis Nucleus IKK- a IKK- b Control Control IKK- a IKK- b siRNA siRNA siRNA siRNA siRNA siRNA 100 5 * 4 80 *** Total tube ** Moderate Peak Minimal No % scratch 3 60 *** length per area 2 Activation Activation Activation Activation 40 field covered (10 4 m m) 1 20 0 Control IKK- a IKK- b 0 IKK- b Control IKK- a siRNA siRNA siRNA siRNA siRNA siRNA Iosef C et al, Am J Physiol Lung Cell Mol Physiol, 2012 Secreted Factors from Early Alveolar Lung can Systemic NF k B Inhibition and Endothelial-Specific Deletion of IKK b Impair Alveolarization Activate Angiogenic Function in Adult PEC Normoxia Early Alveolar Lung Early alveolar lung Secreted Inactive NF k B Active NF k B factors in lung conditioned Adult PEC Adult PEC media (LCM) FBS Early Alveolar Late Alveolar Adult Adult Lung Hyperoxia 0.2% LCM (P6) LCM (P16) LCM Migration 100 NF k B (p65) 80 Adult PEC 60 150 Area of *** *** scratch covered *** 40 100 Total intensity PBS BAY 11-7082 IKK b +/+ IKK b -/- (%) nuclear staining vs. * 20 * 50 nuclear area Endothelial-Specific Systemic Pharmacologic 0 Deletion of IKK b 0 NF k B Inhibition EA- FBS FBS Adult FBS EA- LA- Adult- LCM 0.2% 5% LCM 0.2% LCM LCM LCM 4
4/20/2018 Proteomic Analysis of Secreted Factors in Proteomic Screening of Putative Angiogenic Lung Conditioned Media Using 2D-DIGE Factors Secreted by the Early Alveolar Lung Adult LCM Adult LCM Late Alveolar-LCM vs. vs. vs. Early Alveolar-LCM Late Alveolar-LCM Early Alveolar-LCM Early Alveolar Lung Adult Lung kD Proteins Increased in Early Alveolar Lung Appear Red or Orange Proteins Equally Expressed Size Appear Yellow Only Red spots Only Red spots Red or Orange Proteins Increased in Adult Lung selected selected spots selected Appear Green pH Isoelectric point Transforming Growth Factor– b Induced Protein TGFBI is Highly Expressed by the Early (TGFBI) is Uniquely Expressed by the Early Alveolar Lung Alveolar Lung Lung conditioned media Lung tissue P6 P16 P30 Early Alveolar vs. Adult TGFBI > < TGFBI EA-LCM MA-LCM Adult LCM < b -Actin 1 Relative Expression Intensity (pixel 10 3 ) Total Integrated 30 Early TGFBI *** vs. b -actin Alveolar 20 0.5 ** 10 0 EA-LCM LA-LCM Adult LCM *** 0 P6 P16 P30 Lung tissue in situ TGFBI Adult CD31 Early TGFBI Alveolar TGFBI vs. Adult Early Alveolar Adult Early Alveolar Lung Lung Lung (High Mag) 5
4/20/2018 Neutralizing TGFBI Limits the Ability of the TGFBI-Mediated Pulmonary Endothelial Migration is IKK/NF k B Dependent Early Alveolar Secreted Factors to Promote Angiogenic Function NF k B p65 NF k B Activation Migration DAPI Adult 100 PEC CD31 *** *** 80 Starvation TGFBI *** FBS EA-LCM + Rat EA-LCM + 0.2% IgG Anti-TGFBI Ab Area of * 60 wound IKK b +/+ PEC 80 100 covered (%) IKK b -/- PEC *** 80 Total intensity * 40 60 § nuclear staining % scratch 60 vs. * area nucelar area 40 40 20 covered 20 20 0 0 EA-LCM FBS EA-LCM FBS FBS 5% Rat anti- 0.2% Rat anti- 0.2% 0 IgG TGFBI Ab IgG TGFBI Ab Starv TGFBI EGM Future Directions Conclusions How Do Alterations of the Lung Microenvironment During Acute Injury • Growth of the pulmonary vascular by angiogenesis is essential for alveolarization Alter the Angiogenic Phenotype of the Pulmonary Endothelium? • The angiogenic phenotype of pulmonary endothelial cells is developmentally regulated; NF k B is a key pathway promoting angiogenesis in the early alveolar pulmonary endothelium Immature Adult Injury Control Early Alveolar Lung Adult Lung Adult Injury Maturation TGFBI TGFBI TGFBI TGFBI Immature Control Adult Early Alveolar Active NF k B Inactive NF k B Pulmonary Pulmonary Premature loss of Premature induction of Endothelium Endothelium Angiogenesis Quiescence developmentally developmentally regulated pro-angiogenic regulated anti- factors angiogenic factors • Unique factors present in microenvironment of the early alveolar lung have the ability to activate NF k B and enhance angiogenic function of adult pulmonary endothelial cells. 6
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