DEXAMETHASONE SYNCHRONIZES ASTHMATIC RESPIRATORY EPITHELIUM REPAIR IN VITRO

Sabah F Iqbal, MD 1,2, Angela S Benton, BAS 1, Alan M Watson, PhD, 1,2 Mary C Rose, PhD 1,2, Robert J Freishtat, MD, MPH 1,2

1Children’s National Medical Center, Washington, DC2George Washington University, Washington, DC

Disclosure Statement The authors have documented that they

have nothing to disclose.

Background: Asthma Increasing prevalence and severity

14.7 million annual lost school days* 196,000 hospital admissions** 730,000 ED visits**

*National Health Interview Survey, 2002**National Hospital Medical Care Survey, 2002

1980 2002

35/1000 83/1,000*

The role of glucocorticoids (GC)

Inflammation

Remodeling

?

Circadian Rhythm

Hypothesis

• Injured asthmatic respiratory epithelium is characterized by asynchronous regeneration and will resynchronize in the presence of glucocorticoids

Normal Cell Cycle

Experimental Design: ALI

Primary differentiated HBE cells at an air-liquid interface (ALI) were used (Mattek, Ashland, MA)

‘Gold standard’ model in airway epithelial research as it mimics the human airway

Heterogeneous mixture of ciliated, goblet and basal cells

Mucus layer

Stimuli

Goblet cell Ciliate

d cellCollage

n

Experimental Design:Timeline

-26 -24 -2 0 22 24 46 48

+/- DEX

Mechanical Wound

Photo

+/- DEX

+/- DEX

+/- DEX

Asthma - DEX

Normal - DEX

Normal + DEX

Asthma + DEX

0 48 hours

Bright –field microscopy (16X)

1mm

Experimental analysis Flow cytometry

BrdU (bromodeoxyuridine) Cellular proliferation/mitosis

7-AAD (7-amino-actinomycin-D) DNA label/cell cycle phase

Normal Cell Cycle

G2

G1

Cell cycle analysis by flow cytometry

Summary: Proliferating normal cells were more

likely to be found in G1 of the cell cycle Normal cell cycling

Proliferating asthmatic cells were more evenly distributed among G1 and S Consistent with asynchronous cell cycling

DEX, given at doses analogous to endogenous steroids, re-synchronized the cell cycle

Discussion Chronotherapeutic trials (Pincus et al, 1997)

Single late-afternoon dose of GC improves lung function, symptoms, and inflammation

Rethinking glucocorticoids

Inflammation

Remodeling

?

Future directions DEX at pharmacologic doses may

correct for the asynchronous processes and allow normal rapid healing

Use nasal epithelium cells from pediatric patients with asthma and in controls

Acknowledgements: K12 Mentoring Committee:

Mary Rose Ph.D. Robert Freishtat M.D. Eric Hoffman Ph.D. Anamaris Colberg-Poley Ph.D. Stephen Teach M.D.

Angela Benton Alan Watson Ph.D. Teresa Hawley Ph.D. Funded by the National Institutes of Health -

National Heart, Lung, and Blood Institute grant number K12HL090020