Impaired Myofibroblast Dedifferentiation Contributes to Non-Resolving Fibrosis in Aging Louise Hecker, PhD Assistant Professor of Medicine Division of Pulmonary, Allergy, Critical Care & Sleep Southern Arizona VA Health Cara System (SAVAHCS) Pulmonary Fibrosis Foundation November 14, 2015

Acknowledgements Joe G. N. “Skip” Garcia Yoon-Joo Shin Sunmi Palumbo Ken Knox Victor Thannickal Naomi Logsdon Deepali Kurundkar Funding: This work was supported by the Veterans Health Administration (CDA2: 1 IK2 BX001477-01A1)

Pathogenesis? Physiological Fibrosis: Pathological Fibrosis Tissue damage Inflammation Fibroblast proliferation & migration Myofibroblast differentiation, contraction, & ECM deposition Myofibroblast apoptosis Re-epithelialization Physiological Fibrosis: Pathogenesis? Pathological Fibrosis Continued myofibroblast activation/accumulation Excessive matrix deposition Impaired re-epithelialization Dedifferentiation

The Myofibroblast: a terminally differentiated cell type? Proliferative Differentiated TGF-β1

Myofibroblasts are not terminally differentiated cells: they retain the capacity to dedifferentiate Hecker et. al., Exp Cell Res, 2011

IPF is a disease of aging Incidence Prevalence 120 300 100 250 Male Male 80 Female 200 Female Per Hundred Thousand Per Hundred Thousand 60 150 40 100 20 50 45-54 55-64 65-74 75+ 45-54 55-64 65-74 75+ Raghu G, et al. Am J Respir Crit Care Med. 2006. ↑ incidence/prevalence with age (66 = average age at diagnosis) ↓ survival with advancing age

Myofibroblast senescence in the IPF lung p16 ki67 IPF Fibroblastic Focus Hecker et. al., Sci Trans Med, 2014 The effects of aging/senescence in the capacity for myofibroblast dedifferentiation is largely unknown

Hypothesis: In the context of aging/senescence, myofibroblasts demonstrate a diminished capacity for dedifferentiation, leading to alterations in their subsequent apoptotic fate and ultimately impaired fibrosis resolution.

Western Immunoblotting Senescent myofibroblasts demonstrate an impaired capacity for dedifferentiation TGFb1 48h Serum Deprived 5 days 16h +/- FBS (20% vs. 0%) Day 0: Western Immunoblotting

Impaired dedifferentiation capacity in senescent myofibroblasts is associated with an apoptosis-resistant phenotype Staurosporine

IPF lung myofibroblasts demonstrate impaired dedifferentiation and apoptosis resistance aSMA GAPDH IPF FBS: _ + Day 0 Day 5

Elevated MyoD expression in IPF myofibroblasts aSMA GAPDH IPF FBS: _ + Day 0 Day 5 MyoD Tubulin MyoD IPF Control

MyoD is upregulated in the lungs of aged mice with persistent fibrosis Hecker et. al., Sci Trans Med, 2014 ctrl 3w 2m Young Aged MyoD β-actin

Therapeutic targeting of MyoD in the lungs of aged mice with established fibrosis restores the capacity for fibrosis resolution TGFβ: __ + MyoD NT siRNA: αSMA GAPDH mu fb Drug administration Age-dependent persistent fibrosis * Control 3 weeks NT- siRNA MyoD- 6 weeks Trichrome NT-siRNA MyoD-siRNA

Summary: Senescent and IPF myofibroblasts demonstrate an impaired capacity for dedifferentiation Myofibroblast dedifferentiation was associated with greater susceptibility to apoptosis. Whereas, senescent and IPF myofibroblasts, that fail to undergo dedifferentiation, confer an apoptosis-resistant phenotype. Knockdown of MyoD in the lungs of aged mice with established fibrosis led to a reversal of fibrosis. These studies support the concept that the capacity for myofibroblast dedifferentiation may be critical to normal physiologic versus pathologic responses to tissue injury. Strategies to induce myofibroblast dedifferentiation and/or apoptosis may be highly relevant cellular phenotypes for therapeutic targeting

Thank you! Lhecker@email.arizona.edu Funding: This work was supported by the Veterans Health Administration (CDA2: 1 IK2 BX001477-01A1)

Aged mice are deficient in their ability to resolve fibrosis Peak Fibrosis Injury Resolution (young) Time: 3 weeks 2 months 4 months Persistent Fibrosis (aged)

Severity of Fibrosis

Severity of Fibrosis

Resolution of Fibrosis ns

Why does fibrosis become pathological? Tissue Repair is a Complex Biological Process Influenced by Various Factors Age Environment Genetics Life expectancy is increasing Growing elderly population Increased incidence of fibrotic diseases

Physiological healing Fibrosis: The formation of fibrous “scar tissue” as a reparative or reactive process “Normal” Physiological healing “Abnormal” Pathological disease (Reversible) (Persistent/Progressive) Why Does Fibrosis Become Pathological?