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