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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 ? in Asthma

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 in Asthma

Obstruction ?


Fixed airway obstruction a phenotype of severe asthma
Fixed Airway Obstruction: A phenotype of severe asthma? in Asthma

  • Decreased b2 AR responsiveness

  • Airway remodeling: Airway hyperplasia

    • More cells = More contraction?


Reynold a panettieri jr m d university of pennsylvania

Decreased in Asthma

b2 agonist responsiveness


Reynold a panettieri jr m d university of pennsylvania

Preparation of Human Precision-Cut Lung Slices (PCLS) in Asthma

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 EC in Asthma50 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 onµM 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 onµM; 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 onµM; 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 on-AR Desensitisation to Forskolin


Reynold a panettieri jr m d university of pennsylvania

b on2-AR cell surface binding


Fixed airway obstruction a phenotype of severe asthma1
Fixed Airway Obstruction: A phenotype of severe asthma? on

  • Decreased b2 AR responsiveness

  • Airway remodeling: Airway hyperplasia

    • More cells = More contraction?



Components of airway remodeling
Components of Airway Remodeling on

Lazaar and Panettieri, Am J Med, 2003


Mechanisms regulating asm growth

ANTI on

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 signaling

bARK

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


Reynold a panettieri jr m d university of pennsylvania

RGS Expression in signaling

Human ASM cells

Damera et al. PloSONE 2011


Reynold a panettieri jr m d university of pennsylvania

5 signaling

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 signaling

RGS4

MERGE

Basal

PDGF (10ng/ml)



Reynold a panettieri jr m d university of pennsylvania

PDGF Attenuates Carbachol-Induced signaling

Contraction in Human Small Airways

Damera et al. PloSONE 2011


Reynold a panettieri jr m d university of pennsylvania

Mediator N Control signalingPDGF P

Ach 10 1.92 ± 0.16 1.46 ± 0.08 0.0160

Histamine 10 2.80 ± 0.15 1.95 ± 0.14 0.0004

Thrombin 102.12 ± 0.13 1.41 ± 0.09 0.0004

Damera et al. PloSONE 2011


Reynold a panettieri jr m d university of pennsylvania

Cyclin D1 signaling

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 signaling

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 signaling

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 signaling

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 signaling

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 signaling

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 signaling

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 signaling

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 signaling

D

A

B


Reynold a panettieri jr m d university of pennsylvania

GENE SUSCEPTIBILITY signaling

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 signaling

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 bronchoscopy2
Confocal Bronchoscopy signaling

Musani et al 2010



Reynold a panettieri jr m d university of pennsylvania

Human Studies signaling

Macroscopic View

Dr Ali Musani

Pulm. Fellow

Confocal Screen

Anaesthetist


Reynold a panettieri jr m d university of pennsylvania

Blood Vessel signaling

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 signaling

Cilia

En Face

View

En Face

View

Smooth Muscle

Epithelium

Epithelium

Musani and Sims, 2010


Confocal bronchoscopy4
Confocal Bronchoscopy signaling

  • 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

. signaling

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 ? in Asthma

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 in Asthma

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