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IDIOPATHIC PULMONARY FIBROSIS, 2017:

PATHOGENESIS AND NOVEL THERAPEUTIC TARGETS

Bruce D. Uhal Department of Physiology Michigan State University

East Lansing, Michigan

Disclosures

Paid Consultantships: last date:GlaxoSmithKline 2017Actelion Pharm. Ltd 2007

Definition:• a chronic progressive and “inevitably fatal*” lung disease

Idiopathic Pulmonary Fibrosis (IPF)

NORMAL IPF

Definition:• a chronic progressive and “inevitably fatal*” lung disease

*


NORMAL IPF

Recently, pirfenidone and nintedanib, 2 antifibrotic drugs, have been proven to be effective in slowing disease progression and are now approved as treatments in the US and Europe.

Definition:• a chronic progressive and “inevitably fatal*” lung disease • distortion of the lung’s architecture with excess ECM• unknown etiology• 6.8-16cases/100,000• median survival ~3yr

Diagnostic Criteria:clinical history: exclusion of occupational exposures etc

typical HRCT pattern of Usual Interstitial Pneumonia

if HRCT non diagnostic: SLB to confirm UIP histology


NORMAL IPF


Diagnostic Criteria:-clinical history: exclusion of occupational exposures etc-typical HRCT pattern of Usual Interstitial Pneumonia



NORMAL IPF


Diagnostic Criteria:-clinical history: exclusion -typical HRCT pattern of Usual Interstitial Pneumonia:



NORMAL IPF

Subpleural & basal reticular opacitiesTraction bronchiectasisHoneycomb changes (cystic airspaces thick walls 0.3-1.0cm)





NORMAL IPF





NORMAL IPF

Injury

Alveolitis

Derangements of Parenchyma

Loss of functionalalv.-capillary units

“Honeycomb Lung”

Pathogenesis of IPF-Traditional View:

IPF viewed as an“inflammatory disease”

From: Crystal, Ferrans and Basset, Ch7.8.1 The Lung:Scientific Foundations, 1991

Traditional View

Injury

Alveolitis

Derangements of Parenchyma

Loss of functionalalv.-capillary units

“Honeycomb Lung”

Pathogenesis of IPF-Traditional View:

IPF viewed as an“inflammatory disease”

From: Crystal, Ferrans and Basset, Ch7.8.1 The Lung:Scientific Foundations, 1991

Elephant in the room:

Antiiflammatory/Immunosuppressive Approaches do not work!

Traditional View

The failure of antiinflammatory/immunosuppressive clinical trials:

AJRCCM 190:867-878, 2014

The failure of antiinflammatory/immunosuppressive clinical trials:

“An Alarming Press Release”Wells AU et al.Eur.Resp.J.39:805-6, 2012

AJRCCM 190:867-878, 2014

“Triple Therapy” prednisone azathioprene +NAC

Arguments Against the “Inflammation Hypothesis”:Selm

Selman et al. Am J RespCrit Care Med 165:1205-8, 2002

Gauldie et al. Respir Res 2002; 3: 3-11. al.

• Anti-inflammatories/immunosuppressives don’t work

• Inflammation does not correlate with stage or outcome

• Inflammation is not required for the fibrotic response

• Inflammation is not a prominent histopathologic finding

SO, what IS critical in IPF?

IPF - Pathogenesis Theory

Arguments Against the “Inflammation Hypothesis”:Selm

Selman et al. Am J RespCrit Care Med 165:1205-8, 2002

Gauldie et al. Respir Res 2002; 3: 3-11. al.

• Anti-inflammatories/immunosuppressives don’t work

• Inflammation does not correlate with stage or outcome

• Inflammation is not required for the fibrotic response

• Inflammation is not a prominent histopathologic finding


IPF - Pathogenesis Theory


Histological Features of the IPF Lung:

F1000 Research 2016, 5(F1000 Faculty Rev):1046

Spatial heterogeneity: subpleural/paraseptal fibrosis vs. “spared” parenchyma

Temporal heterogeneity: active fibroblast foci, ECM deposition & paucity of inflammatory infiltrate vs. regions of normal lung histo


F1000 Research 2016, 5(F1000 Faculty Rev):1046

Spatial heterogeneity: subpleural/paraseptal fibrosis vs. “spared” parenchyma

Temporal heterogeneity: active fibroblast foci, ECM deposition & paucity of inflammatory infiltrate vs. regions of normal lung histo

IPF

CTL

PICROSIRIUS RED

Uhal et al. AmJPhysiol275:L1192-9, 1998


Annu Rev Pathol. 2014; 9:157-179

Key features of UIP: 1. fibroblastic foci -at the “edge” between fibrotic vs normal lung

-deposit collagens (myofibroblasts)

-Presence & number predict mortality in IPF! Nicholson et al. AJRCCM 166:173–177, 2002






Fibroblast Myofibroblast TGF-β1

collagen α−SMA






Fibroblast Myofibroblast TGF-β1

collagen α−SMA






2. “Reactive”-appearing epithelium -cuboidal

-primarily type II-like

-previously described as “hyperplastic”

Persistent and unrepaired epithelial damage (apoptosis)

Proliferation and accumulation of fibroblast/myofibroblasts

Thickening of alveolar wall by deposition of collagens I & III

Obliteration of the alveolar space.


CTL

IPF

IPFISEL

The “Hyperplastic Alveolar Epithelium” is now known to be:dividing and dying (by apoptosis) simultaneously

Uhal et al. Alveolar epithelial cell death adjacent to underlying myofibroblasts in advanced fibrotic human lung. Am. J. Physiol. 275:L1192-L1199, 1998.

Lung Fibrogenesis Theory:

Early evidence for the “Severity of Epithelial Injury Hypothesis” lung explant studies – Witschi, Haschek, Adamson, Bowden 1979-1990

BHT, O2

epith. repair

Upshot: failure of reepithelialization fibrosis in the presence or the absence of blood

ANTIFIBROTIC FUNCTIONS OF THE EPITHELIUM:

Normal Alveolar Epithelium:

BM

III

Barrier~2,000Ohm

Surfactant surface T immunomod.Antioxidant Def.

AM

Soluble Factors Growth factors (e.g. EGF, FGFs) Growth inhibitors (PGE2)

Proteases uPA(degrade fibrin) MMPs (interstit. collagenases)

ANTIFIBROTIC FUNCTIONS OF THE EPITHELIUM :

Denuded Epithelium:

BM

capillary

BarrierCollapse

AM Proteases uPA = fibrinolysis MMPsint = collagen

fibrin

Lost/damaged type II cells = Surfactant surface T immunomod. Antioxidant Def.

EGF

PGE2

+

Upshot – Epith. death releases fibroblasts from inhibitions






CTL

IPF

IPFISEL







CTL

IPF

IPFISEL


But at the same time, Fibroblasts/myofibroblasts are Resistant to apoptosis:


CTL

IPF

IPFISEL

Apoptosis in IPF Lung Biopsies:

TUNELChest 2005125:1005

ISELAm.J.Physiol. 1998275:L1192

BAX IHCChest 2005

125:1005

Little MF labeling=Resistance to apoptosis


CTL

IPF

IPFISEL

Apoptosis in IPF Lung Biopsies:

TUNELChest 2005125:1005

ISELAm.J.Physiol. 1998275:L1192

BAX IHCChest 2005

125:1005

Little MF labeling=Resistance to apoptosis

Apoptosis high in the epithelium, But low in myofibroblast foci,

= “The Apoptosis Paradox” in IPF V. Thannickal

Persistent and unrepaired epithelial damage

Epithelial-Mesenchymal Transition (EMT) blasts


Ob

literation of the alveolar space.

Mechanisms of fibrogenesis in IPF:

The “Hyperplastic Alveolar Epithelium” is now known to be:dividing and dying (by apoptosis)AND:Undergoing EMT (Epith-Mesenchymal Transition)




Oblite

ration of the alveolar space.



Type II AEC Myofibroblast TGF-β1,

mutations collagen α−SMA



Epithelial “Activation” to secrete growth factors & cytokines I & III

O

bliteration of the alveolar space.



Type II AEC Myofibroblast TGF-β1,

mutations collagen α−SMA

MECHANISMS IF FIBROGENESIS IN IPF:

surviving “REACTIVE” Epithelium: (dying/proliferating??)

BM

capillary

Cytokines IL-1,6,8,10,11 GMCSF CCL2 CXCL12

Growth Factors TGF-β1 FGF’s, CTGF PDGF’s, IGF’s, TGF-α ET-1

+

MECHANISMS IF FIBROGENESIS IN IPF:

surviving “REACTIVE” Epithelium: (dying/proliferating??)

BM

capillary

Cytokines IL-1,6,8,10,11 GMCSF CCL2 CXCL12

Growth Factors TGF-β1 FGF’s, CTGF PDGF’s, IGF’s, TGF-α ET-1

+

recruit circulatingfibrocytes




Entry & engraftment of circulating fibrocytes of t

he alveolar space.


The “Hyperplastic Alveolar Epithelium” is now known to be:-dividing and dying (by apoptosis)-undergoing EMT-secreting GFs/cytokines

Circulating fibrocytes




Entry & engraftment of circulating fibrocytes

Genetic predispositions the alveolar space.


The “Hyperplastic Alveolar Epithelium” is now known to be:-dividing and dying (by apoptosis)-undergoing EMT-secreting GFs/cytokines

Circulating fibrocytes

Genetic Mutations Predisposing to Pulmonary Fibrosis (IPF-like)

Wolters P et al. AnnRev Pathol 9:157-179, 2014

Surfactant Protein C / A

Mucin 5B

Telomerase

Genetic Mutations Predisposing to Pulmonary Fibrosis (IPF-like)

Wolters P et al. AnnRev Pathol 9:157-179, 2014

Surfactant Protein C / A

Mucin 5B(elevated MUC5B protein)

-relation to IPF unclear

Telomerase(shortened telomeres)-increased apoptosis?

SP-C BRICHOS DOMAIN MUTATIONS:

Discovery:

Pedigree:Black = IPFSquare = males# = DNA seq

Proband Biospies

SP-C G100S mutation causes familial pulmonary fibrosis in Japanese kindred. Ono et al. ERJ 38:861-869, 2011.


Cause disruption of protein folding and packaging into LBs

SP-C:

Made only by Type IIepithelial Cells

ESSENTIALfor normallung function

Lamellar Bodies:Fibrogenic non-fibrogenic

100S

SP-C BRICHOS DOMAIN MUTATIONS:Mechanisms of Toxicity:

1.UPR (Unfolded Protein Response) & ER Stress

2.Apoptosis of type II AECs & loss of antifibrotic functions

a

*Other activators of ER Stress: viruses, hyperoxia, hypoxia etc.3. EMT (Epithelial-Mesenchymal Transition)

(X-box BP-1)C/EBP-homol.PActivating TFs

Chaperones:

CHOP ATF-4,-6

Ubiquitin Proteasome System

Ono et al. ERJ 38:861-869, 2011 Lawson et al. AJP 294L1119, 2008Mulugeta et al. AJRCMB 32:521-530, 2005

Zhong et al. AJRCMB 45(3):498-509, 2011




a



Chaperones:

CHOP ATF-4,-6






a



Chaperones:

CHOP ATF-4,-6



Zhong et al. AJRCMB 45(3):498-509, 2011

Type II cell Myofibroblast ER Stress

collagen α−SMA


Implications on Lung Fibrosis Pathogenesis:

1.SP-C is expressed only by type II alveolar epithelial cells

2.Fibrogenic SP-C mutants induce AEC apoptosis

3.Fibrogenic SP-C mutants also induce EMT4.“Activated” epithelia recruit circulating fibrocytes

5.The above strongly suggest that Epithelial Injury initiates and likely helps propogate fibrosis progression







The “Witschi Hypothesis” ~ 1979a.k.a.“Severity of Epithelial Injury” theory

AJP:Lung 305:L906-L911, 2013








AJP:Lung 305:L906-L911, 2013








AJP:Lung 305:L906-L911, 2013

Pathophysiologic Stages of Idiopathic Pulmonary Fibrosis (IPF)

Summary of IPF Pathogenesis:

Epithelial Pathologic Cells: Mesenchymal Cells

telomere length?

From Wolters P et al. AnnRev Pathol 9:157-179, 2014

type II cell myofibroblast

Investigational Therapies for IPF, 2015From Ahluwalia et al. AJRCCM 190:867-868, 2014

Targets: Epithelial InjuryDrug: NAC

Matrix Accumulation

Immune LOXL2 mAB

ActivationIL-13 mABs

Vascular Leak

Fibroblast recruitment, Myofibroblast invasion, proliferation evolution

tyr kinase inhib,, antifibrotic. Integrin αvβ6mABLysoPA-R antagonist

TARGETING THE EPITHELIUM IN IPF:

Inducers of Apoptosis in Lung Epithelial CellsInducer Mediators . Chemotherapy, amiodarone caspases, Bcl-2 family, AT1RFas ligand caspases, Bcl-fam, AT1RTNF-α caspases, Bcl-fam, AT1RTGF-β1 caspases, EGR-1SP-C mutations ER stress, UPR, ATIR Eur Respir Rev 17: 109, 138–143, 2008 al Strategy – block or inhibit expressionCaspase inhibitors* Am.J.Physiol. 279:L143, 2000 HGF* Pediatric Res.56:336, 2004

p21 overexpression* Am.J.Physiol. 286:L727, 2004

AT1 receptor blockers* Am. J. Pathol. 163:2523, 2003 ACE-2/ANG1-7/mas activation * Am.J.Physiol. 279:L143, 2000

* In vivo confirmation (mice)

Blockers of Epithelial Apoptosis & Collagen Deposition

TARGETING THE EPITHELIUM IN IPF: Manipulating apoptosis/EMT as potential therapy:-promising candidates:

a) HGF - inhibits TNF-induced epithelial apoptosispromotes epithelial proliferation & repairinduces myofibroblast apoptosis

b) AT1R antagonistsinhibit alveolar epithelial apoptosisinhibit fibroblast proliferationinhibit collagen deposition

c) ACE-2*/ANG1-7/mas activation*blocks AEC apoptosis *inhibits fibroblast proliferation*inhibits collagen deposition *GSK pipeline

ACKNOWLEDGEMENTS

Collaborators:Dr. M. Molina-Molina BarcelonaDr. A. Xaubet Barcelona

Dr. Karen Friderici Michigan State

Dr. Yuehua Cui Michigan State

Dr. Xiaopeng Li U. Iowa

Dr. Amal Abdul-Hafez Michigan State

Funding: NIH, Am.Heart Assoc., Am.Physiol. Soc., IDIBAPS, SEPAR, SOCAP-FUCAP

Factors that Confer Resistance to Apoptosis in Lung Myofibroblasts/fibroblastsMolecule Basic Mechanism Mediators . IL-6 reduces FasL induction Stat3, Bcl-2

TGF-β1 blocks IL-1β induction iNOS, Bcl-2

Thrombin* blocks FasL induction PAR-1, PKC-ε , p21

IGF-1 blocks GF-withdrawal Akt, ERK phospho.

FIZZ1 blocks TNF-α induction ERK-1/2 phospo.(a.k.a. RELM-α, resistin-like molecule α)

Anti-α2β1* ligation of integrin PI3K, Akt, PKB integrin

Potential strategy- attempt blockade *human cells

Roles of ACE-2 in Lung Fibrosis

Angiotensinogen ANGI ANGII ACEchymase

ANG1-7

ACE-2

APOPTOSIS,FIBROSIS

ACE-2 SHOULD BE PROTECTIVE - IT IS!

AND, ACE-2 ISREDUCED

IN IPF

Am J Physiol 295(1):L178-L185, 2008

Tools to study ACE-2:Competitive inhibitor (DX600) ACE-2 siRNAs (mouse lung)

Mechanisms of Protection by ACE-2/ANG1-7:

Alveolar epithelial cells: fibroblasts, leukocytes: