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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

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SLIDE 1

4/20/2018 1 The Role of the Lung Microenvironment in Modulating Pulmonary Angiogenesis

Cristina M. Alvira, MD

Assistant Professor of Pediatrics Stanford University School of Medicine Stanford CHRI Tashia and John Morgridge Faculty Scholar

5

  • Cristina M. Alvira

Has documented that she has no financial relationships to disclose or Conflicts of Interest (COIs) to resolve A Significant Component of Lung Development Occurs Postnatally

Birth 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40

Gestational Weeks

2 - 10 years ?

pseudoglandular canalicular saccular alveolar

Alveolar

Burri PH, Biol Neonate, 2006

Saccular

Secondary Septation 500 million alveoli Alveolar surface area: 140 m2

Numerous Pre- and Postnatal Factors Can Disrupt Normal Lung Development

Premature Birth

Antenatal Exposures

  • Intrauterine infections
  • Intrauterine growth

restriction

Postnatal Exposures

  • Mechanical Ventilation
  • Oxidative Stress
  • Infections
  • Corticosteroids
  • Nutritional Deficits

Adapted from: Baraldi and Filipone, N Engl J Med, 2007

Gestation

Saccular Embryonic Pseudoglandular Canalicular Alveolar Birth

2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40

Gestational Weeks

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SLIDE 2

4/20/2018 2

The Immature Lung has an Increased Capacity to Regenerate after Injury

Rat Lung

Removal of left lung Compensatory growth

  • f right lung to achieve

same gas exchange surface area as with both lungs Right Left

S Surface Area Vital Capacity Groups

Age at

  • peration

(y) Number TLC % predicted for 2 lungs VC % predicted for 2 lungs RV % predicted for 2 lungs

1 0-5 9 96.4 79.1 34.2 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 5 21-25 18 78.4 64.5 38.2 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

Compensatory Lung Growth is Greatest in Young Patients after Pneumonectomy Growth of the Pulmonary Vasculature Promotes Alveolarization

Thebaud B and Abman SH, Am J Respir Crit Care Med, 2007 Angiogenic Factors Angiogenic Factor Replacement

Normal alveolar sac Alveolar sac after blocking angiogenesis Alveolar sac in BPD Restored alveolarization after angiogenic therapy

Distinct Phases of Alveolarization and Angiogenesis in the Developing Lung

Rat Human

Birth P4 Bulk Alveolarization Continued / Late Alveolarization P14 P21

Adapted from Tschanz et al, J Appl Physiol, 2014

P30 Birth P60 36w 2 Yr 3 Yr 10 Yr Microvascular Maturation Active Angiogenesis

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4/20/2018 3

The Developing Pulmonary Vasculature Remodels During Late Alveolarization

Schittney JC, Cell Tissue Res, 2017

Early Alveolar Rat Lung (P4) Adult Rat Lung (P44)

Stimuli that Impair Alveolarization Induce Premature Maturation of the Pulmonary Microvasculature

Dexamethasone treated P4 Dexamethasone treated P6

What controls the angiogenic phenotype of the pulmonary endothelial cells during development?

Roth-Kleiner, Dev Dyn, 2005

Angiogenic Function is Higher in Pulmonary Endothelial Cells from the Developing Lung

1500 1000 500 2 8 12 24

Hours

Apoptosis

*** *** *** *** Caspase 3/7 Activity

2 1 0.5 2 8 12 24

Hours

Proliferation

1.5 BrdU Incorporation

*** *** *** Early Alveolar (P6) Adult (8-10 wk)

Migration

FBS 0.2%

Area of wound covered (%)

FBS 5%

20 40 60 80 100

*** *** Early Alveolar Mice (P6) Adult Mice (10-12 week)

Primary Pulmonary Endothelial Cells

Magnet isolation

The IKK/NFkB Pathway Promotes Pulmonary Angiogenesis during Alveolarization

p50 p65 Phosphorylation and degradation

  • f IkBa

P P P P IkBa IKKa IKKb NEMO CYTOPLASM NUCLEUS Gene Expression or Repression

IKK complex NFkB

Iosef C et al, Am J Physiol Lung Cell Mol Physiol, 2012

Inactive NFkB Adult Lung

NFkB (p65) Chromatin CD31

Early Alveolar Lung (P6) Active NFkB

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4/20/2018 4

Activation of NFkB Peaks in the Pulmonary Endothelium during Early Alveolarization

Adult PEC No Activation Late Alveolar PEC Minimal Activation Nucleus NFkB Late Saccular PEC Early Alveolar PEC

NFkB Activation in Primary Pulmonary Endothelial Cells (PEC)

Moderate Activation Peak Activation

Inhibiting NFkB Disrupts Angiogenic Function in Early Alveolar Lung Endothelial Cells

Active Caspase 3/7 Time (hours)

2 4 6 8 24 250 500 750 1000 1250 1500 1750

*** *** ***

§

Control BAY (2.5 mM) BAY (5 mM )

Apoptosis

BrdU Incorporation Con BAY 24h

***

0.6 0.8 1.0 1.2 1.4

Proliferation

Iosef C et al, Am J Physiol Lung Cell Mol Physiol, 2012

***

% scratch area covered

20 40 60 80 100

IKK-a siRNA Control siRNA IKK-b siRNA

*** Migration

IKK-a siRNA Control siRNA IKK-b siRNA IKK-a siRNA

*

1 2 3 4 5

Control siRNA

**

IKK-b siRNA Total tube length per field (104 mm) IKK-a siRNA Control

siRNA

IKK-b siRNA

In Vitro Angiogenesis

Systemic NFkB Inhibition and Endothelial-Specific Deletion of IKKb Impair Alveolarization

Early Alveolar Lung PBS BAY 11-7082 Adult Lung

Systemic Pharmacologic NFkB Inhibition Endothelial-Specific Deletion of IKKb

IKKb +/+ Normoxia Hyperoxia IKKb -/-

Secreted Factors from Early Alveolar Lung can Activate Angiogenic Function in Adult PEC

Early alveolar lung

Inactive NFkB

FBS 5%

***

Adult LCM

*

EA- LCM

***

20 40 60 80 100

FBS 0.2%

Area of scratch covered (%)

Migration

50 100 150

EA- LCM FBS 0.2%

Total intensity nuclear staining vs. nuclear area

***

LA- LCM

*

Late Alveolar LCM (P16)

Adult- LCM

Adult LCM Adult PEC

Active NFkB

Adult PEC

Secreted factors in lung conditioned media (LCM)

NFkB (p65) FBS 0.2% Early Alveolar LCM (P6) Adult PEC

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4/20/2018 5

Proteomic Analysis of Secreted Factors in Lung Conditioned Media Using 2D-DIGE

Early Alveolar Lung Adult Lung

Isoelectric point pH kD Size

Proteins Increased in Early Alveolar Lung Appear Red or Orange Proteins Equally Expressed Appear Yellow Proteins Increased in Adult Lung Appear Green

Proteomic Screening of Putative Angiogenic Factors Secreted by the Early Alveolar Lung

Adult LCM vs. Early Alveolar-LCM Adult LCM vs. Late Alveolar-LCM Late Alveolar-LCM vs. Early Alveolar-LCM

Only Red spots selected Only Red spots selected Red or Orange spots selected

Early Alveolar vs. Adult

Adult Early Alveolar Early Alveolar vs. Adult

TGFBI TGFBI TGFBI

Transforming Growth Factor–b Induced Protein (TGFBI) is Uniquely Expressed by the Early Alveolar Lung

EA-LCM MA-LCM Adult LCM Total Integrated Intensity (pixel 103)

10 20 30

***

TGFBI >

Lung conditioned media

Early Alveolar Lung (High Mag)

TGFBI

CD31

Early Alveolar Lung Adult Lung

Lung tissue in situ

< TGFBI

P6 P16 P30

< b-Actin

Lung tissue

P6 P16 P30

*** **

EA-LCM LA-LCM Adult LCM

TGFBI is Highly Expressed by the Early Alveolar Lung

1 0.5

Relative Expression

  • vs. b-actin
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SLIDE 6

4/20/2018 6

FBS 0.2% EA-LCM + Rat IgG EA-LCM + Anti-TGFBI Ab 20 40 60 80 100

***

Total intensity nuclear staining vs. nucelar area

* NFkB Activation

Adult PEC FBS 0.2% EA-LCM Rat IgG anti- TGFBI Ab

***

EA-LCM Rat IgG

Migration

20 Area of wound covered (%) 100 40 60 80

***

FBS 0.2% FBS 5%

Neutralizing TGFBI Limits the Ability of the Early Alveolar Secreted Factors to Promote Angiogenic Function

*

anti- TGFBI Ab

***

TGFBI-Mediated Pulmonary Endothelial Migration is IKK/NFkB Dependent

Starvation TGFBI NFkB p65 DAPI CD31

40 80 20 60

% scratch area covered

EGM Starv

IKKb +/+ PEC IKKb -/- PEC §

TGFBI

*

Conclusions

Early Alveolar Lung Inactive NFkB Quiescence Active NFkB Angiogenesis Adult Lung

Adult Pulmonary Endothelium Early Alveolar Pulmonary Endothelium TGFBI TGFBI TGFBI TGFBI

Maturation

  • Growth of the pulmonary vascular by angiogenesis is essential for alveolarization
  • The angiogenic phenotype of pulmonary endothelial cells is developmentally

regulated; NFkB is a key pathway promoting angiogenesis in the early alveolar pulmonary endothelium

  • Unique factors present in microenvironment of the early alveolar lung have the

ability to activate NFkB and enhance angiogenic function of adult pulmonary endothelial cells.

Future Directions

Immature Control Adult Injury Adult Control Immature Injury Premature loss of developmentally regulated pro-angiogenic factors Premature induction of developmentally regulated anti- angiogenic factors

How Do Alterations of the Lung Microenvironment During Acute Injury Alter the Angiogenic Phenotype of the Pulmonary Endothelium?

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SLIDE 7

4/20/2018 7

Acknowledgments

The Center for Excellence in Pulmonary Biology

Cristiana Iosef Yanli Hou

The Alvira Lab

Katie Concepcion Kavitha Thiagarajan

Cristiana Funding for this work was provided by the Department of Pediatrics, the Children’s Health Research Institute at Stanford TIP Grant and Morgridge Faculty Scholar Award, the American Heart Association Fellow to Faculty Transition Award, Beginning Grant in Aid and the NIH/NHLBI 1P30: HL101315-01 and R01 HL122918.

Marlene Rabinovitch David Cornfield Richard Bland

Mentors

Elizabeth Foley Min Liu Vinicio de Jesus Perez Sarah Heilshorn Shailaja Rao

Collaborators

Simon Conway Phyllis Dennery

Min Shailaja

Gray Umbach

Stanford University Indiana University University of Cologne

Miguel Alejandre Alcazar

University of Colorado

Blair Dodson Steven Abman

Duke University

Mary Sunday Sha Fu Racquel Domingo- Gonzalez FBS 0.2% 21% O2 40% O2 75% O2 85% O2 Early Alveolar Lung Conditioned Media

Scratch area covered (%) 20 40 60

***

§§§

# # # # # #

***P<0.001 vs FBS 0.02%, §§§P<0.001 vs. FBS 0.02% and EA- LCM 21% O2, and ### P<0.001 vs. EA-LCM 21% and 40% O2

Hyperoxia exposure blunts the pro-migratory effects of the early alveolar lung conditioned media

The IKK/NFkB dependent is suppressed in PAEC derived from a sheep model of IUGR

Control IUGR

Unpublished data from RB Dodson et al

Recombinant TGFBI Promotes Chemotactic Migration of Pulmonary Endothelial Cells

Microfluidic Chemotaxis Assays

Cell chamber Source Sink

Negative Control (Starvation media)

n=226 p=0.156

y axis (mm)

100 200 300

  • 100
  • 200
  • 300
  • 200

200

x axis (mm) y axis (counts) x axis (counts)

Early alveolar lung conditioned media

n=200 p=0.000383

y axis (mm)

100 200 300

  • 100
  • 200
  • 300
  • 200

200

x axis (mm) y axis (counts) x axis (counts)

Starv + TGFBI (10mg/mL)

n=185 p=0.000486

y axis (mm)

100 200 300

  • 100
  • 200
  • 300
  • 200

200

x axis (mm) y axis (counts) x axis (counts)

Positive Control (VEGF: 100 ng/ml)

n=190 p=0.0000789

y axis (mm)

100 200 300

  • 100
  • 200
  • 300
  • 200

200

x axis (mm) y axis (counts) x axis (counts)

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SLIDE 8

4/20/2018 8

Acute Injury Acute Injury

Future Directions

Immature Control Adult Injury Adult Control Immature Injury Premature loss of developmentally regulated pro- angiogenic factors Premature induction

  • f developmentally

regulated anti- angiogenic factors Putative protective factors that enhance repair Putative detrimental factors that impair repair Immature Injury Immature Control Adult Injury Adult Control

Repair Repair