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

2. Inflammation Photo Courtesy of Reynold A. Panettieri, Jr., MD Progression v Persistence

3. Aberrant Injury-Repair Responses Promote Airflow Obstruction in Asthma Lazaar and Panettieri, Am J Med, 2003

4. Asthma: A Chronic Acute Disease ? Age (years) Lung Function (FEV1) Conventional ThoughtNew Hypotheses Lazaar and Panettieri, 2003

5. Persistent Airflow Obstruction ?

6. Fixed Airway Obstruction: A phenotype of severe asthma? Decreased  2 AR responsiveness Airway remodeling: Airway hyperplasia –More cells = More contraction?

7. Decreased  2 agonist responsiveness

8. Preparation of Human Precision-Cut Lung Slices (PCLS) Healthy Human Lungs Lobe dissected and inflated with 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

9. University of Pennsylvania Obtaining a Concentration-Response Curve and EC 50 Value For relaxation studies, airways are contracted to 90% total contraction and administered relaxant agonists in the presence of the final contractile concentration.

10. University of Pennsylvania

11. Dose-Effect of Albuterol after 3, 6 & 12 hrs of Incubation on Human Small Airways to Isoproteronol 3 hours6 hours 12 hours Control 0.01  M 0.1  M 1.0  M

12. University of Pennsylvania Time Course of 1 µM Albuterol On Human Small Airways to Isoproteronol Dose-ResponseEmax values

13. 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

14. University of Pennsylvania Dex / FP added for 6 hours after Albuterol and remain together for duration of incubation Effect of POST-Incubation Steroids on Albuterol (0.1 µM; 12 hrs) Induced β-AR to Isoproteronol

15. University of Pennsylvania β-AR Desensitisation to Forskolin

16. University of Pennsylvania  2 -AR cell surface binding

17. Fixed Airway Obstruction: A phenotype of severe asthma? Decreased  2 AR responsiveness Airway remodeling: Airway hyperplasia –More cells = More contraction?

18. ASM hyperplasia: the consequences

19. Components of Airway Remodeling Lazaar and Panettieri, Am J Med, 2003

20. Mechanisms regulating ASM growth. PRO ANTI EGF, PDGF, IGF Thrombin, LPA, SPP Inflammation Matrix 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?

21. ● 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

22. Mammalian RGS family members - affymetrix gene chip ● Ross & Wilkie (2000) Annu. Rev. Biochem. 69: 795-827  ARK

23. RGS Expression in Human ASM cells Damera et al. PloSONE 2011

24. Basal EGF PDGF TNF IL-1 IL-6 RGS4 GAPDH 0 1 2 3 4 5 BasalEGFPDGFTNF IL-1IL-6 Expression over Basal CYTOKINES AND GROWTH FACTORS MODULATE RGS4 EXPRESSION Damera et al. PloSONE 2011

25. PDGF (10ng/ml) RGS4DAPIMERGE Basal

26. RGS 4 expression ASM

27. Cooper University of Pennsylvania PDGF Attenuates Carbachol-Induced Contraction in Human Small Airways Damera et al. PloSONE 2011

28. Cooper University of Pennsylvania Mediator N Control PDGF P Ach101.92 ± 0.16 1.46 ± 0.080.0160 Histamine102.80 ± 0.15 1.95 ± 0.140.0004 Thrombin10 2.12 ± 0.131.41 ± 0.090.0004 Damera et al. PloSONE 2011

29. NADPH ROS Rac ? Upstream Signaling Targets to Abrogate Airway Muscle Cell Growth Ras MEK ERK PI3K p70S6K ASM Proliferation RTKGPCR Cyclin D1

30. RGS4 PHYSICALLY INTERACTS WITH p-P85-PI3K AND Gα SUBUNIT RGS4 BASALPDGF IP : Gα RGS4 BASALPDGF IP : p-P85 BASALPDGF Gα BASALPDGF P85

31. RGS4 SILENCING IN ASM 0 20 40 60 80 100 120 0010204080160 PDGF (10 ng/ml ) SiRGS4 (n M ) - 68% TRANSFECT WITH RGS4 SiRNA % Change in Expression TREAT WITH PDGF 42h6h RT-PCR RGS4 siRGS4 0 0 10 20 40 80 160 PDGF (10ng/ml) GAPDH

32. Cooper University of Pennsylvania 0 1 2 3 4 5 6 7 BasalPDGF Cnt siRNA siRGS4 Fold change in AKT kinase activity NS P<0.005

33. Cooper University of Pennsylvania 0 10 20 30 40 50 60 70 80 90 Brdu Staining % Cnt SiSiRGS4Cnt SiSiRGS4 PDGF (10 ng/ml) P=0.044 NS P=0.0036 CNTSiRNA SiRGS4 ASM Cells Damera et al. PloSONE 2011

34. Cooper University 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. ab Damera et al. PloSONE 2011

35. Cooper University 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

36. Cooper University 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

37. A CD B

38. Cooper University of Pennsylvania GENE SUSCEPTIBILITY FOR ASTHMA IRREVERSIBLE AIRWAY OBSTRUCTION AIRWAY INJURY ALLERGEN VIRUSES RGS PROTEIN EXPRESSION ASM MASS NO ASM GROWTH GENE SUSCEPTIBILITY TO ABERRANT AIRWAY INJURY- REPAIR RESPONSE RGS PROTEIN EXPRESSION REVERSIBLE AIRWAY OBSTRUCTION ASTHMA PHENOTYPES

39. 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

40. Confocal Bronchoscopy

42. Musani et al 2010

43. Confocal Bronchoscopy

44. Pulm. Fellow Dr Ali Musani Macroscopic View Anaesthetist Confocal Screen Human Studies

45. 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 mm Musani et al 2010

46. Epithelium Cilia En Face View Smooth Muscle Basement Membrane Epithelium En Face View Musani and Sims, 2010

47. 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. 48 Bronchial Thermoplasty

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

50. 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

51. Aberrant Injury-Repair Responses Promote Airflow Obstruction in Asthma Lazaar and Panettieri, Am J Med, 2003

52. Asthma: A Chronic Acute Disease ? Age (years) Lung Function (FEV1) Conventional ThoughtNew Hypotheses Lazaar and Panettieri, 2003

53. 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