REYNOLD A. PANETTIERI, JR., M.D. UNIVERSITY OF PENNSYLVANIA Airway Smooth Muscle: A Response Element

REYNOLD A. PANETTIERI, JR., M.D.UNIVERSITY OF PENNSYLVANIA

Airway Smooth Muscle: A Response Element.

Inflammation

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

Progression v Persistence

Aberrant Injury-Repair Aberrant Injury-Repair Responses Promote Airflow Responses Promote Airflow Obstruction in AsthmaObstruction in Asthma

Lazaar and Panettieri, Am J Med, 2003

Asthma: A Chronic Acute Disease ?

Age (years) Age (years)

Lu

ng

Fu

nc

tio

n (

FE

V1

)

Lu

ng

Fu

nc

tio

n (

FE

V1

)

Conventional Thought New Hypotheses

Lazaar and Panettieri, 2003

Persistent AirflowObstruction ?

Fixed Airway Obstruction: A phenotype of severe asthma?

• Decreased 2 AR responsiveness

• Airway remodeling: Airway hyperplasia–More cells = More contraction?

Decreased2 agonist

responsiveness

Preparation of Human Precision-Cut Lung Slices (PCLS)

Healthy Human Lungs Lobe dissected and inflatedwith low melting point agarose

Inflated lung is sectioned

Lung core is sliced(Thickness: 250 µm)

Krumdieck Tissue Slicer Sectioned lung is cored(Diameter: 8 mm)

Lung Slice Small airway located on slice

University of Pennsylvania

Obtaining a Concentration-Response Curve and EC50 Value

For relaxation studies, airways are contracted to 90% total contractionand administered relaxant agonists in the presence of the final contractileconcentration.

University of Pennsylvania

University of Pennsylvania

0

25

50

75

100

10-6 10-510-710-810-9

Control0.039

0.01M0.043

0.1M0.055

1.0M0.099

10-4

*

[ISO] M

Re

lax

ati

on

(%

)

0

25

50

75

100

10-6 10-5 10-410-710-810-9

Control0.055

0.01M0.070

0.1M0.098

1.0M0.098

***

[ISO] M

Re

lax

ati

on

(%

)

0

25

50

75

100

10-6 10-5 10-410-710-810-9

Control0.052

0.01M0.083

0.1M0.17

1.0M0.20

****

[ISO] M

Re

lax

ati

on

(%

)

Dose-Effect of Albuterol after 3, 6 & 12 hrs of Incubation onHuman Small Airways to Isoproteronol

3 hours 6 hours

12 hours

Control

0.01 M

0.1 M

1.0 M

University of Pennsylvania

0

25

50

75

100

Control3 hrs6 hrs12 hrs

10-6 10-510-710-810-9 10-4

Control0.03924

3 hrs0.09927

6 hrs0.09763

12 hrs0.1953

[ISO] M

Re

lax

ati

on

(%

)

0 3 6 120

25

50

75

100

***

* **

Time (hrs)M

ax

imu

m R

ela

xa

tio

n

Time Course of 1 µM Albuterol On Human Small Airways to Isoproteronol

Dose-Response Emax values

University of Pennsylvania

0

10

20

30

40

50

60

70

80

90

ControlAlbuterol (12 hrs, 0.1 M) Dex (1.0 M) + Albuterol (0.1 M)

FP (0.1 M + Albuterol (0.1 M)Dex (1.0 M)

10-8 10-410-610-10

Control0.053

Albuterol0.25

Dex + Alb0.030

FP + Alb0.028

Dex0.017EC50 Values (M):

[ISO] M

Re

laxa

tio

n (

%)

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 andremain for duration of incubation

University of Pennsylvania

0

25

50

75

100ControlAlbuterol 0.1 MAlbuterol + Dex 1 MAlbuterol + FP 0.1 M

10-610-8 10-4

[ISO] M

Re

lax

ati

on

(%

)

Dex / FP added for 6 hours after Albuterol andremain together for duration of incubation

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

University of Pennsylvania

0

10

20

30

40

50

60

70

ControlAlbuterol (1 M, 12 hrs)

10-610-7 10-5 10-410-8

Control0.7056

Albuterol0.9749

[Forskolin] M

Re

lax

ati

on

(%

)

β-AR Desensitisation to Forskolin

University of Pennsylvania

2-AR cell surface binding

Fixed Airway Obstruction: A phenotype of severe asthma?

• Decreased 2 AR responsiveness

• Airway remodeling: Airway hyperplasia–More cells = More contraction?

ASM hyperplasia: the consequences

Components of Airway Remodeling

Lazaar and Panettieri, Am J Med, 2003

Mechanisms regulating ASM growth.

PRO ANTI

EGF, PDGF, IGF

Thrombin, LPA, SPP

InflammationMatrix

Contractile agonists

A-Kinase

G-Kinase

Steroids

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

airway smooth muscle?

Is the a master switch?

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

Receptor mediated activation-termination of G protein signaling

RGS proteins modulate signaling by accelerating Gα-GTPase activity

Mammalian RGS family members - affymetrix gene chip

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

ARK

RGS Expression in Human ASM cells

Damera et al. PloSONE 2011

Basal EGF PDGF TNF IL-1 IL-6

RGS4

GAPDH

0

1

2

3

4

5

Basal EGF PDGF TNF IL-1 IL-6 E

xpre

ssio

n o

ver

Bas

al

CYTOKINES AND GROWTH FACTORS MODULATE RGS4 EXPRESSION

Damera et al. PloSONE 2011

PD

GF

(10

ng

/ml)

RGS4DAPI MERGEB

asal

RGS 4 expression ASM

CooperUniversity of Pennsylvania

0

25

50

75

100

ControlPDGF 50 ng/ml 8 hrs

10-6 10-410-510-710-8

[CCh] M

Co

ntr

acti

on

(%

)

PDGF Attenuates Carbachol-Induced Contraction in Human Small Airways

Emax values

Control PDGF0

25

50

75

100 *

Ma

x C

on

tra

cti

on

(%

)

CCh log EC50 values

Control PDGF

-1.1

-1.0

-0.9

-0.8

-0.7

-0.6

-0.5**

CC

h lo

g E

C5

0

va

lue

s ( M

)

Damera et al. PloSONE 2011

CooperUniversity of Pennsylvania

Mediator N Control PDGF 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 10 2.12 ± 0.13 1.41 ± 0.09 0.0004

Damera et al. PloSONE 2011

NADPH

ROS

Rac

?

Upstream Signaling Targets to Abrogate Airway Muscle Cell Growth

Ras

MEK

ERK

PI3K

p70S6K

ASM Proliferation

RTK GPCR

Cyclin D1

RGS4 PHYSICALLY INTERACTS WITH p-P85-PI3K AND Gα SUBUNIT

RGS4

BASAL PDGF

IP : Gα

RGS4

BASAL PDGF

IP : p-P85

BASAL PDGF

Gα

BASAL PDGF

P85

RGS4 SILENCING IN ASM

0

20

40

60

80

100

120

0 0 10 20 40 80 160

PDGF (10 ng/ml )

SiRGS4 (n M )

- 68%

TRANSFECT WITH RGS4 SiRNA

% C

han

ge

in E

xpre

ssio

n

TREAT WITH PDGF

42h 6h

RT-PCR

RGS4

siRGS4 0 0 10 20 40 80 160

PDGF (10ng/ml)

GAPDH

CooperUniversity of Pennsylvania

0

1

2

3

4

5

6

7

Basal PDGF

Cnt siRNA

siRGS4

Fo

ld c

ha

ng

e in

AK

T k

inas

e a

cti

vity

NS

P<0.005P<0.005

CooperUniversity of Pennsylvania

0

10

20

30

40

50

60

70

80

90

Brd

u S

tain

ing

%

Cnt Si SiRGS4 Cnt Si SiRGS4

PDGF (10 ng/ml)

P=0.044

NS

NS

P=0.0036

CNTSiRNA

SiRGS4

ASM Cells

Damera et al. PloSONE 2011

CooperUniversity of Pennsylvania

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.

a b

Damera et al. PloSONE 2011

CooperUniversity of Pennsylvania

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)

a

c

fe

d

b

Damera et al. PloSONE 2011

CooperUniversity of Pennsylvania

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.

a

cb

A C DB

CooperUniversity of Pennsylvania

GENE SUSCEPTIBILITY

FOR ASTHMA

IRREVERSIBLEAIRWAY

OBSTRUCTION

AIRWAY INJURY ALLERGENVIRUSES

RGS PROTEIN EXPRESSION

ASMMASS

NO ASMGROWTH

GENE SUSCEPTIBILITY TO ABERRANT

AIRWAY INJURY-REPAIR RESPONSE

RGS PROTEIN EXPRESSION

REVERSIBLEAIRWAY

OBSTRUCTION

ASTHMAPHENOTYPES

DiagnosisDiagnosis

The mainstays of determining asthma The mainstays of determining asthma severity and airway remodeling severity and airway remodeling include:include: FEV1FEV1 Symptom scoresSymptom scores Bronchial biopsiesBronchial biopsies

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

Confocal Bronchoscopy

Confocal Bronchoscopy

Confocal Bronchoscopy

Musani et al 2010

Confocal Bronchoscopy

Pulm. Fellow

Dr Ali MusaniMacroscopic View

Anaesthetist

Confocal Screen

Human Studies

Surface

Area of Regenerating Epithelium

Goblet Cell

Just Below Surface

Area of Regenerating Epithelium

Surface

Blood Vessel

Sub-Surface

Lamina Propria

Bar = 100 mM FOV = 363 mmMusani et al 2010

Epithelium

Cilia

En Face View

Smooth Muscle

Basement Membrane

Epithelium

En Face View

Musani and Sims, 2010

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

48

Bronchial Thermoplasty

.

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

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 Aberrant Injury-Repair Responses Promote Airflow Obstruction in AsthmaPromote Airflow Obstruction in Asthma

Lazaar and Panettieri, Am J Med, 2003

Asthma: A Chronic Acute Disease ?

Age (years) Age (years)

Lu

ng

Fu

nc

tio

n (

FE

V1

)

Lu

ng

Fu

nc

tio

n (

FE

V1

)

Conventional Thought New Hypotheses

Lazaar and Panettieri, 2003

Acknowledgments

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

John A. Cidlowski

NIAID

Kurt Druery

Leicester University

Chris Brightling, Yassine Amrani

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