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Airway Smooth Muscle: A Response Element. Reynold a. panettieri, Jr., M.D. UNIVERSITY OF PENNSYLVANIA. Inflammation. Progression v Persistence. Photo Courtesy of Reynold A. Panettieri, Jr., MD. Aberrant Injury-Repair Responses Promote Airflow Obstruction in Asthma.

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Reynold a panettieri jr m d university of pennsylvania

Airway Smooth Muscle: A Response Element.

Reynold a. panettieri, Jr., M.D.UNIVERSITY OF PENNSYLVANIA


Inflammation

Inflammation

Progression v

Persistence

Photo Courtesy of Reynold A. Panettieri, Jr., MD


Aberrant injury repair responses promote airflow obstruction in asthma

Aberrant Injury-Repair Responses Promote Airflow Obstruction in Asthma

Lazaar and Panettieri, Am J Med, 2003


Asthma a chronic acute disease

Asthma: A Chronic Acute Disease ?

Conventional Thought

New Hypotheses

Lung Function (FEV1)

Lung Function (FEV1)

Age (years)

Age (years)

Lazaar and Panettieri, 2003


Reynold a panettieri jr m d university of pennsylvania

Persistent Airflow

Obstruction ?


Fixed airway obstruction a phenotype of severe asthma

Fixed Airway Obstruction: A phenotype of severe asthma?

  • Decreased b2 AR responsiveness

  • Airway remodeling: Airway hyperplasia

    • More cells = More contraction?


Reynold a panettieri jr m d university of pennsylvania

Decreased

b2 agonist responsiveness


Reynold a panettieri jr m d university of pennsylvania

Preparation of Human Precision-Cut Lung Slices (PCLS)

Inflated lung is

sectioned

Lobe dissected and inflated

with low melting point agarose

Healthy Human Lungs

Lung core is sliced

(Thickness: 250 µm)

Sectioned lung is cored

(Diameter: 8 mm)

Krumdieck Tissue Slicer

Lung Slice

Small airway located on slice


Reynold a panettieri jr m d university of pennsylvania

Obtaining a Concentration-Response Curve and EC50 Value

For relaxation studies, airways are contracted to 90% total contraction

and administered relaxant agonists in the presence of the final contractile

concentration.


Reynold a panettieri jr m d university of pennsylvania

Dose-Effect of Albuterol after 3, 6 & 12 hrs of Incubation on

Human Small Airways to Isoproteronol

3 hours

6 hours

Control

m

0.01

M

12 hours

m

0.1

M

m

1.0

M


Reynold a panettieri jr m d university of pennsylvania

Time Course of 1 µM Albuterol On

Human Small Airways to Isoproteronol

Dose-Response

Emax values


Reynold a panettieri jr m d university of pennsylvania

Effect of PRE-Incubation of Steroids on Albuterol (0.1 µM; 12 hrs) Induced β-AR to Isoproteronol

Dex / FP added 1 hour before Albuterol and

remain for duration of incubation


Reynold a panettieri jr m d university of pennsylvania

Effect of POST-Incubation Steroids on Albuterol (0.1 µM; 12 hrs) Induced β-AR to Isoproteronol

Dex / FP added for 6 hours after Albuterol and

remain together for duration of incubation


Reynold a panettieri jr m d university of pennsylvania

β-AR Desensitisation to Forskolin


Reynold a panettieri jr m d university of pennsylvania

b2-AR cell surface binding


Fixed airway obstruction a phenotype of severe asthma1

Fixed Airway Obstruction: A phenotype of severe asthma?

  • Decreased b2 AR responsiveness

  • Airway remodeling: Airway hyperplasia

    • More cells = More contraction?


Reynold a panettieri jr m d university of pennsylvania

ASM hyperplasia: the consequences


Components of airway remodeling

Components of Airway Remodeling

Lazaar and Panettieri, Am J Med, 2003


Mechanisms regulating asm growth

ANTI

PRO

Mechanisms regulating ASM growth.

What is/are the mechanism(s) that regulate contractile and proliferative phenotypes of airway smooth muscle?

Is the a master switch?

A-Kinase

EGF, PDGF, IGF

G-Kinase

Thrombin, LPA, SPP

Steroids

Matrix

Inflammation

Contractile agonists


Receptor mediated activation termination of g protein signaling

Receptor mediated activation-termination of G protein signaling

RGS proteins modulate signaling

by accelerating Gα-GTPase activity

● Siderowski & Willard (2005) Int. J. Biol. Sci. 1: 51-66


Mammalian rgs family members affymetrix gene chip

Mammalian RGS family members - affymetrix gene chip

bARK

● Ross & Wilkie (2000) Annu. Rev. Biochem. 69: 795-827


Reynold a panettieri jr m d university of pennsylvania

RGS Expression in

Human ASM cells

Damera et al. PloSONE 2011


Reynold a panettieri jr m d university of pennsylvania

5

4

3

Expression over Basal

RGS4

Basal EGF PDGF TNF IL-1 IL-6

GAPDH

2

1

0

IL-1

IL-6

Basal

EGF

PDGF

TNF

CYTOKINES AND GROWTH FACTORS MODULATE RGS4 EXPRESSION

Damera et al. PloSONE 2011


Reynold a panettieri jr m d university of pennsylvania

DAPI

RGS4

MERGE

Basal

PDGF (10ng/ml)


Reynold a panettieri jr m d university of pennsylvania

RGS 4 expression ASM


Reynold a panettieri jr m d university of pennsylvania

PDGF Attenuates Carbachol-Induced

Contraction in Human Small Airways

Damera et al. PloSONE 2011


Reynold a panettieri jr m d university of pennsylvania

Mediator N Control PDGF P

Ach101.92 ± 0.161.46 ± 0.080.0160

Histamine102.80 ± 0.151.95 ± 0.140.0004

Thrombin102.12 ± 0.131.41 ± 0.090.0004

Damera et al. PloSONE 2011


Reynold a panettieri jr m d university of pennsylvania

Cyclin D1

Upstream Signaling Targets to Abrogate Airway Muscle Cell Growth

RTK

GPCR

Ras

PI3K

Rac

MEK

p70S6K

NADPH

ERK

ROS

?

ASM Proliferation


Reynold a panettieri jr m d university of pennsylvania

BASAL

PDGF

BASAL

PDGF

P85

IP : p-P85

IP : Gα

BASAL

PDGF

BASAL

PDGF

RGS4

RGS4

RGS4 PHYSICALLY INTERACTS WITH

p-P85-PI3K AND Gα SUBUNIT


Reynold a panettieri jr m d university of pennsylvania

120

100

80

RGS4

GAPDH

60

0 0 10 20 40 80 160

siRGS4

- 68%

40

PDGF (10ng/ml)

20

0

SiRGS4 (n M )

0

0

10

20

40

80

160

PDGF (10 ng/ml )

TREAT WITH PDGF

RT-PCR

42h

6h

RGS4 SILENCING IN ASM

% Change in Expression

TRANSFECT WITH RGS4 SiRNA


Reynold a panettieri jr m d university of pennsylvania

P<0.005

P<0.005

7

Cnt siRNA

6

siRGS4

5

4

Fold change in AKT kinase activity

3

NS

2

1

0

Basal

PDGF


Reynold a panettieri jr m d university of pennsylvania

P=0.0036

90

ASM Cells

NS

80

CNTSiRNA

SiRGS4

70

60

50

Brdu Staining %

P=0.044

40

30

NS

20

10

0

Cnt Si

SiRGS4

Cnt Si

SiRGS4

PDGF (10 ng/ml)

Damera et al. PloSONE 2011


Reynold a panettieri jr m d university of pennsylvania

a

b

Bronchial biospy from a) a severe asthmatic and b) healthy control stained for a-smooth muscle actin (x200) illustrating that in severe asthma the airway smooth muscle (ASM)-bundle represents an increased percentage of the cross-sectional area of the biopsy, as we have previously described (Siddiqui et al JACI 2008), and is closer to the epithelium.

Damera et al. PloSONE 2011


Reynold a panettieri jr m d university of pennsylvania

a

b

c

d

e

f

a) Bronchial biospy from a severe asthmatic stained with goat immunoglobulin (negative control) (x100) and b) the same subject illustarting the ASM-bundle (x400). c) a-smooth muscle actin staining of the ASM-bundle in a severe asthmatic and d) a healthy control (x200). d) and e) are sequential sections from the same subjects stained for RGS4 illustrating a cluster of positive cells in the severe asthmatic and negative staining in the healthy control (x200)

Damera et al. PloSONE 2011


Reynold a panettieri jr m d university of pennsylvania

a

b

c

a) Bronchial biospy from a different severe asthmatic stained for RGS4 illustrating positive cells within the lamina propria and clustered at the edge of the ASM-bundle (x100). b) and c) are the insets at higher power (x400) illustrating RGS4+ cells within and adjacent to the ASM-bundle.


Reynold a panettieri jr m d university of pennsylvania

C

D

A

B


Reynold a panettieri jr m d university of pennsylvania

GENE SUSCEPTIBILITY

TO ABERRANTAIRWAY INJURY-REPAIR RESPONSE

IRREVERSIBLE

AIRWAY

OBSTRUCTION

ASM

MASS

RGS PROTEIN EXPRESSION

GENE SUSCEPTIBILITY FOR ASTHMA

ASTHMA

PHENOTYPES

RGS PROTEIN EXPRESSION

NO ASM

GROWTH

REVERSIBLE

AIRWAY

OBSTRUCTION

AIRWAY INJURY ALLERGEN

VIRUSES


Diagnosis

Diagnosis

The mainstays of determining asthma severity and airway remodeling include:

  • FEV1

  • Symptom scores

  • Bronchial biopsies

    Other noninvasive measures may be useful for serial monitoring in severe asthma


Confocal bronchoscopy

Confocal Bronchoscopy


Confocal bronchoscopy1

Confocal Bronchoscopy


Confocal bronchoscopy2

Confocal Bronchoscopy

Musani et al 2010


Confocal bronchoscopy3

Confocal Bronchoscopy


Reynold a panettieri jr m d university of pennsylvania

Human Studies

Macroscopic View

Dr Ali Musani

Pulm. Fellow

Confocal Screen

Anaesthetist


Reynold a panettieri jr m d university of pennsylvania

Blood Vessel

Lamina Propria

Sub-Surface

Area of Regenerating Epithelium

Area of Regenerating Epithelium

Surface

Surface

Goblet Cell

Just Below Surface

Musani et al 2010

FOV = 363 mm

Bar= 100 mM


Reynold a panettieri jr m d university of pennsylvania

Basement Membrane

Cilia

En Face

View

En Face

View

Smooth Muscle

Epithelium

Epithelium

Musani and Sims, 2010


Confocal bronchoscopy4

Confocal Bronchoscopy

  • Confocal bronchoscopy is feasible in large mammals.

  • First studies could easily characterize: goblet cell number and size, epithelial cell number, sub-basement membrane thickness, bronchial vessel size and number.

  • Assess ASM mass likely with refinement.

  • Phase 1 human studies completed


Reynold a panettieri jr m d university of pennsylvania

Bronchial Thermoplasty


Reynold a panettieri jr m d university of pennsylvania

.

GILDEA T R et al. Cleveland Clinic Journal of Medicine 2011;78:477-485


Reynold a panettieri jr m d university of pennsylvania

The AIR2 trial: Effect of bronchial thermoplasty and sham thermoplasty on health care utilization Health care utilization in the 12 months after real or sham thermoplasty.

GILDEA T R et al. Cleveland Clinic Journal of Medicine 2011;78:477-485


Aberrant injury repair responses promote airflow obstruction in asthma1

Aberrant Injury-Repair Responses Promote Airflow Obstruction in Asthma

Lazaar and Panettieri, Am J Med, 2003


Asthma a chronic acute disease1

Asthma: A Chronic Acute Disease ?

Conventional Thought

New Hypotheses

Lung Function (FEV1)

Lung Function (FEV1)

Age (years)

Age (years)

Lazaar and Panettieri, 2003


Reynold a panettieri jr m d university of pennsylvania

Acknowledgments

University of Pennsylvania Medical Center, Philadelphia Airways Biology Initiative

Pulmonary, Allergy and Critical Care Division

Omar Tlibi, Vera Krysmkaya, Angela Haczku

Elena Goncharova, Dimtri Gonchorova

Bill Jester, Homer Fogel, Heng Jiang

Gautum Damera, Phil Cooper, Audreesh Banergee

Laboratory of Signal Transduction, National Institute of Environmental Health Science, NC

John A. Cidlowski

NIAID

Kurt Druery

Leicester University

Chris Brightling, Yassine Amrani


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