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

1. Airway Smooth Muscle: A Response Element. Reynold a. panettieri, Jr., M.D.UNIVERSITY OF PENNSYLVANIA

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

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

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

5. Persistent Airflow Obstruction ?

6. Fixed Airway Obstruction: A phenotype of severe asthma? • Decreased b2 AR responsiveness • Airway remodeling: Airway hyperplasia • More cells = More contraction?

7. Decreased b2 agonist responsiveness

8. 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

9. 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.

11. 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

12. Time Course of 1 µM Albuterol On Human Small Airways to Isoproteronol Dose-Response Emax values

13. 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. 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

15. β-AR Desensitisation to Forskolin

16. b2-AR cell surface binding

17. Fixed Airway Obstruction: A phenotype of severe asthma? • Decreased b2 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. 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

21. 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

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

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

24. 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

25. DAPI RGS4 MERGE Basal PDGF (10ng/ml)

26. RGS 4 expression ASM

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

28. 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 102.12 ± 0.13 1.41 ± 0.09 0.0004 Damera et al. PloSONE 2011

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

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

31. 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

32. 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

33. 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

34. 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

35. 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

36. 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.

37. C D A B

38. 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

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

41. Confocal Bronchoscopy

42. Confocal Bronchoscopy Musani et al 2010

43. Confocal Bronchoscopy

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

45. 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

46. Basement Membrane Cilia En Face View En Face View Smooth Muscle Epithelium Epithelium 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. 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