1. 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

2. 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 BX A1)

3. 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

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

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

6. 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
↑ incidence/prevalence with age (66 = average age at diagnosis)
↓ survival with advancing age

7. 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

8. 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.

9. 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

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

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

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

13. 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

14. 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

15. 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

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

17. 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)

18. Severity of Fibrosis

19. Severity of Fibrosis

20. Resolution of Fibrosisns

21. 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

22. 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?