Ganesh Raghu, MD. Professor of Medicine and Laboratory Medicine (Adjunct) University of Washington, Seattle, WA, USA

Transcription

1 Ganesh Raghu, MD. Professor of Medicine and Laboratory Medicine (Adjunct) University of Washington, Seattle, WA, USA Director, Interstitial Lung Disease, Sarcoid and Pulmonary Fibrosis Program Medical Director, Lung Transplant Program Co-Director, Scleroderma Clinic, University of Washington Medical Center Seattle, WA, USA

2 Pulmonary Fibrosis of Unknown Cause Idiopathic Pulmonary Fibrosis Pathogenesis /pathobiology Current concepts Novel Concepts

3 Current Definition of IPF A distinct type of chronic fibrosing interstitial pneumonia of unknown cause, limited to the lungs, and associated with a pattern of UIP on HRCT scan and/or surgical lung biopsy Raghu et al : Evidence based guidelines for diagnosis and management of Idiopathic pulmonary fibrosis -ATS/ERS /JRS/ALAT Statement. Am J Respir Crit Care Med

4 Idiopathic Pulmonary Fibrosis Pathogenesis: Concepts The Normal Wound Healing Model Selman et al. Ann Intern Med 2001; 134

5 M. Selman, Ann Int Med, 2001; 134:136-51

6 Epithelial Cell Injury and Activation INJURY M. Selman, Ann Int Med, 2001; 134:136-51

7 Epithelial Cell Injury and Activation INJURY Wound clot Basement Membrane Disruption M. Selman, Ann Int Med, 2001; 134:136-51

8 Epithelial Cell Injury and Activation INJURY Wound clot Fibroblast Migration and Proliferation Basement Membrane Disruption M. Selman, Ann Int Med, 2001; 134:136-51

9 Epithelial Cell Injury and Activation INJURY Wound clot Fibroblast Migration and Proliferation Basement Membrane Disruption Myofibroblast Accumulation M. Selman, Ann Int Med, 2001; 134:136-51

10 Epithelial Cell Injury and Activation INJURY Wound clot Angiogenesis Fibroblast Migration and Proliferation Basement Membrane Disruption Myofibroblast Accumulation M. Selman, Ann Int Med, 2001; 134:136-51

11 Epithelial Cell Injury and Activation INJURY Epithelial Apoptosis Wound clot Angiogenesis Fibroblast Migration and Proliferation Basement Membrane Disruption Myofibroblast Accumulation M. Selman, Ann Int Med, 2001; 134:136-51

12 Idiopathic Pulmonary Fibrosis Disease Activity: Pulmonary Parenchyma Activity: Lung tissue Alveolar epithelial Recurrent cells Injury Fibroblast Extracellular matrix Vascular remodeling Inflammation Fibrosis Cytokines Oxidants Chemokines Coagulation factors Others Genetic predisposition

13 Idiopathic Pulmonary Fibrosis Disease Activity: Pulmonary Parenchyma Alveolar epithelial cells-injury;apoptosis Epithelial-mesenchymal transformation Mesenchymal cells (myofibroblasts, fibroblasts, fibrocytes) Genetic predisposition : Surfactant proteins Telomeres and telomerase MUC5B polymorphism

14 Individual Genetic Predispositions Pulmonary Fibrosis Extrinsic (environmental) And Other factors Idiopathic Normal Pulmonary Lung Fibrosis (IPF)

15 Familial Idiopathic Pulmonary Fibrosis (IPF) Subset of patients with IPF have history of IPF in primary -first degree biological members of their family (siblings, parents, children) Compulsive and careful history elicitation of family medical history and review of clinical data from family members will unfold families with Familial IPF and familial pulmonary fibrosis in a third of cases of otherwise sporadic cases of IPF Mageto and Raghu AJRCCM 1997(abstract,ATS)

16 Familial Idiopathic Pulmonary Fibrosis (IPF) Ascertained diagnosis of IPF in at least 2 members of primary biological family* Clinically indistinguishable from sporadic IPF Tends to affect relatively younger patients Approximately one of every 50 patients with IPF has an affected first-degree family member** Inheritance: autosomal dominant with incomplete/variable penetrance * Raghu G, Hert R: Genetic predisposition and inherited interstitial lung disease. Sem Respir Med 14(5): , **Marshall et al, Thorax 2000; 55:143-6

17 Familial Pulmonary Fibrosis % of cases in UK % of cases in Finland % of patients referred for lung transplantation Clinically identical to sporadic IPF may manifest at a relatively younger age predominant, smokers

18 Idiopathic Pulmonary Fibrosis Genetic Predisposition : Genetic predisposition : Surfactant proteins Telomeres and telomerase MUC5B polymorphism

19 Idiopathic Pulmonary Fibrosis Surfactant Protein A and B Genetic Variants on Potential Markers to Identify Subgroups of Patients at Risk* 84 unrelated IPF patients Associations between IPF and genetic polymorphic variants of surfactant proteins, SP- A1, SP-A2, SP-B, SP-C and SP-D One SP-A1 (6A 4 ) allele and single nucleotide polymorphisms (SPNs) that characterize the 6A 4 allele, and one SP-B (B1580_C) higher frequency in IPF (P<0.01) * Selman M. et al. Human Genetics 2003; 113:542-50

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21 Idiopathic Interstitial Pneumonia (IPF): Genetic Mutations in Surfactant Protein C (SFTPC) in IPF and Nonspecific Interstitial Pneumonia (NSIP) Single nucleotide polymorphisms (SNPs) found by genetic sequencing of the SFTPC gene Uncommon in sporadic cases of IPF + NSIP 13/135 patients with PF (9.6%, 95% CI ) SFTPC mutations: May have implication for familial ILD (esp. in children) Clearly defined genetic association remain elusive Molecular genetic studies: Ongoing in number of centers Lawson et al. Thorax 2004; 59:977-80

22 Idiopathic Pulmonary Fibrosis Genetic Predisposition : Genetic predisposition : Surfactant proteins Telomeres and telomerase MUC5B polymorphism

23 Telomeres DNA-protein structures that protect chromosome ends Shorten successively with each cell division Short telomeres: activate DNA damage response cell death/permanent cell cycle arrest Telomere shortening implicated in degenerative age-related disease D Adda di Faggana et al. Nature 2003 Harley et al. Nature 1990; Lee et al. Nature 1998

24 Telomerase Important for telomere replication and stabilization against its shortening htert: highly expressed only in specific germ line cells, proliferative stem cells of renewal tissues, cancer cells Mutations in either htert or htr lead to decrease in telomerase dose accelerated telomere shortening organ failure Harley et al. 1990;, Feng et al. 1995, Nakamura et al. 1997; Lee et al ; Cong et al 2002; D Adda di Faggana et al. Nature 2003

25 Telomerase Maintains Chromosomal Ends

26 Model for Telomerase Dysfunction Inherited Set Point Number of Replications Genetic Mutations: TERT TERC DKC1 Others Telomere Shortening Environmental Influences (Tobacco, Antioxidants,etc) Cell cycle arrest Progenitor cell senescence Clinical Disease

27 Telomere Dynamics in Humans Telomeres progressively shorten with cell division and age. Telomerase expression restricted to stem cells in humans.

28 Familial Idiopathic Pulmonary Fibrosis (IPF)

29 Familial Pulmonary Fibrosis 56 year old Caucasian-Asian male with progressive dyspnea and nagging nonproductive cough. PMH: Osteopenia Social Hx: Past smoker, 22 pk-years Family Hx: Brother recently diagnosed with IPF. Father died at age 56 of pulmonary fibrosis. Jonathan Weissler, MD

30 Familial Pulmonary Fibrosis Spirometry FVC 4.41L (93%) FEV1 3.21L (84%) Ratio 73% DLco 60%

31 Familial Pulmonary Fibrosis

32 Familial Pulmonary Fibrosis No clinical response to immunosuppression.

33 Familial Pulmonary Fibrosis

34 Familial Pulmonary Fibrosis Ganesh Raghu, MD

35 TRF Analysis of Telomere Length

36 Telomere Lengths of Familial PF Kindreds Jenni Cronkhite, PhD

37 Idiopathic Pulmonary Fibrosis (Familial) Mutations in Telomerase: summary of findings* 46 families with FIPF Whole genomic, single nucleotide polymorphism (SNP) linkage Linkage to map the disease gene in 2 of the largest families to chromosome 5 F11 & F31: IPF = 5, PF = 5, Pulm disease = 6 Both families: linkage to chromosome 5p15 with LOD score of 2.8 Mutations in TERT & TERC Heterozygous carriers of all of the mutations in TERT or TERC: Shorter telomeres than age-matched family members without the mutations CONCLUSION :Mutations in TERT or TERC (telomere shortening): Increase susceptibility to IPF * Tsakiri et al PNAS 2007; 104:7552-7

38 Familial Idiopathic Pulmonary Fibrosis (IPF)

39 Familial Idiopathic Pulmonary Fibrosis (IPF)

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41 Idiopathic Pulmonary Fibrosis and Short Telomeres : SUMMARY A subset of IPF (~15% of familial IPF) Telomerase deficiancy without physical manifestations of Dyskeratosis congenita (DKC) (DKC: a severe form of a syndrome of telomere shortening characterized by abnormal skin manifestations and premature death due to bone marrow failure and ILD. Dokal 2000; Br J Hematol; Dokal 2001, Lancet 358, S27) In familial IPF with telomerase mutations, anemia may be mild and hidden

42 Idiopathic Pulmonary Fibrosis and Short Telomeres SUMMARY (CONTD) A subset of IPF (~15% of familial IPF) Pulmonary phenotype: Incomplete penetrant Telomere shortening Cigarette smoking (Morla, ERJ 2006) Oxidative damage (von Zglinicki, 2002) Since telomerase protein expression in cells with proliferative potential, IPF? Loss or senescence of a lung cell population to respond to repetitive injuries over time? Premature aging of lung? Influenced by environmental factors Extrinsic (cigarette smoking)? Intrinsic (?? Micro aspiration)

43 FPF: A Disease of Telomere Shortening Not enough Telomerase Time Environment Long telomeres Short telomeres

44 Idiopathic Pulmonary Fibrosis Genetic Predisposition : Genetic predisposition : Surfactant proteins Telomeres and telomerase MUC5B polymorphism

45 Siebold et al. NEJM 2011; 364: (April 21, 2011)

46 Immunohistochemical Staining of MUC5B in Lung Tissue from Subjects with Idiopathic Pulmonary Fibrosis and Controls. Immunohistochemical staining showed MUC5B distribution in the cytoplasm of the secretory columnar cells of the bronchi and larger proximal bronchioles in a specimen of lung tissue from a control subject Siebold et al. NEJM 2011; 364: (April 21, 2011)

47 Immunohistochemical Staining of MUC5B in Lung Tissue from Subjects with Idiopathic Pulmonary Fibrosis and Controls. In subjects with idiopathic pulmonary fibrosis, regions of dense accumulation of MUC5B were observed in areas of microscopical honeycombing and involved patchy staining of the metaplastic epithelia lining the honeycomb cysts Siebold et al. NEJM 2011; 364: (April 21, 2011)

48 Immunohistochemical Staining of MUC5B in Lung Tissue from Subjects with Idiopathic Pulmonary Fibrosis and Controls. Accumulation was also observed in the mucous plugs within the cysts Siebold et al. NEJM 2011; 364: (April 21, 2011)

49 MUC5B Expression in 33 Subjects with Idiopathic Pulmonary Fibrosis (IPF) and 47 Healthy Controls, Stratified According to MUC5B Promoter Single Nucleotide Polymorphism (SNP) (rs ) Genotype and Smoking Status. Panel A shows the distribution of MUC5B expression among subjects with wild-type or heterozygous rs genotypes of MUC5B according to the presence or absence of IPF. Siebold et al. NEJM 2011; 364: (April 21, 2011)

50 MUC5B Expression in 33 Subjects with Idiopathic Pulmonary Fibrosis (IPF) and 47 Healthy Controls, Stratified According to MUC5B Promoter Single Nucleotide Polymorphism (SNP) (rs ) Genotype and Smoking Status. Panel B shows MUC5B expression among all controls, controls who smoked, and controls who did not smoke, according tors genotype Siebold et al. NEJM 2011; 364: (April 21, 2011)

51 IPF: Molecular Pathogenesis Genetic Insults (SP-A2, SP-C) Recurrent Insults over time Extrinsic (environmental)? Intrinsic (micro aspiration) IPF Epith Cell Injuryrespo nse X Proliferation EMT Fibrotic Response TGFb MMPs Fibrotic Resolution Regeneration from stem cells TERT-dependent

52 Gene Expression Profiles Distinguish Idiopathic Pulmonary Fibrosis (IPF) from Hypersensitivity Pneumonitis (HP) Gene expression patterns : enriched for genes that are functionally associated with inflammation,t-cell activation and immune responses in HP. With tissue remodeling,epithelial and myofibroblast activities of fibrosis in IPF. Selman M et al. AJRCCM 2006; 173:188-98

53 Regulation of Lung Fibrosis Epithelial Injury Alveolar Epithelial cells Fibroblast Myofibroblast formation Matrix Synthesis and Deposition

54 Idiopathic pulmonary fibrosis and abnormal acid Gastroesophagial Reflux* *AJRCCM 1998; 158: *Raghu et al : ERJ 2006

55 Gastroesophageal Reflux and Idiopathic Pulmonary Fibrosis Hypothesis Etiology of IPF Genetic predisposition (putative PF gene/s) Lung: recurrent insults (variable intensity and extent) by gastric acid (GER)

56 Abnormal Gastroesophageal Reflux and Idiopathic Pulmonary Fibrosis/Acute Exacerbation Raghu Hypothesis : In a genetically predisposed adult to manifest IPF/preexisting IPF Provocative observations Future directions - studies required Cause/effect Clarify mechanisms Genetic predisposition Identify preclinical stages of IPF and/or acute exacerbation Clinical trials

57 IPF : Regulation of Lung Fibrosis Epithelial Injury Acid/gastric juice?? Alveolar Epithelial cells Fibroblast TGFb CTGF ET-1 PDGF Myofibroblast formation and proliferation Matrix Synthesis and Deposition

58 Pulmonary Fibrosis of Unknown Etiology Conceptual Pathogenesis of Usual Interstitial Pneumonia (UIP) Genetic predisposition factors Born with normal lung Growing ages Adult, Lung exposed to fibrogenic stimuli Environment (domestic, occupation) Drugs Cigarette smoke Cell Modulating Factors Proinflammatory Matrix Profibrotic cytokines Autoimmune Infection (viral?) Recurrent, chronic aspiration Others? Recurrent insult/injury x yrs, Epithelium, EMT Alveolar epithelial cells Injury Cell Clinical manifestation Basement membrane

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60 Growth of Pulmonary Fibroblast-like Cells Mean + SEM

61 % Cycling cells Differential Proliferation of Fibroblasts Cultured from Normal and Fibrotic Human Lungs Normal adult lung, n=6 ILD, Alveolitis bx, n=4 ILD, dense fibrosis, n= Alone + 10% FBS + Mitogen s Raghu G, et al. Am Rev Resp Disease. 1988;138: PDGF only - PDGF + Other factors

62 Fibroblast Recruitment and Activation in IPF Raghu et al UpTo Date 2010 Genetically Predisposed Person Alveolar epithelial cells Injury Bone marrow derived Basement membrane TGFb EMT Injured epithelial cells Chemokines Recruitment Bone marrow derived circulating fibrocytes TGFb Interstitial Fibroblast (resident) Fibrotic Foci (Myofibroblasts/Mesenchymal cells activation, proliferation)

63 Where do the Fibroblasts/Myofibroblasts seen in Pulmonary Fibrosis come from? Resident fibroblasts Tissue injury induces activation of a resident fibroblast to proliferate and express constituents of the ECM. Epithelial-mesenchymal transformation Tissue injury induces epithelial-mesenchymal transformation into the fibroblast/myofibroblast that subsequently contributes to fibroproliferation. Circulating fibrocytes Marrow-derived stem cells, migrate to sites of tissue injury, differentiate into fibroblasts/myofibroblasts, and contribute to fibroproliferation.

64 Genetics and Genomics of human pulmonary fibrosis and idiopathic pulmonary fibrosis IPF : complex disorder (heterogeneity-clinical and genetic; complex trait) Importance of recognition of clinical phenotypes, clinical manifestations Predisposition factors : genetic, developmental, environmental Suscebtability gene/s : cause, or compensate mutations Molecular genetic Approaches : associations- linkage disequilibrium/analyses, genome wide, systems biology, etc Microarray methods in Biomarkers activity (gene expressions in tissue, peripheral blood) Ascertainment of accurate diagnosis in familial aggregates and sib-pairs by thorough screening of pedigrees and clinical evaluation of all affected and apparently unaffected/ healthy members.

65 Genetic Predisposition of of Human Pulmonary fibrosis Fibrosis Fibrosis Healthy

66 Pulmonary Fibrosis of Unknown Cause Idiopathic Pulmonary Fibrosis Pathogenesis /pathobiology Current concepts Novel Concepts

67 Idiopathic Pulmonary Fibrosis The Fibroblast Foci Characteristic histologic feature of UIP Leading edge of a complex reticulum that is highly interconnected and extends from pleura into the underlying parenchyma Cool et al. AJRCCM 2006; 174:654-8

68 Familial Idiopathic Pulmonary Fibrosis (IPF)

69 Idiopathic Pulmonary Fibrosis : Pathogenesis - Links to Cancer: Concepts* * Vancheri C, Failla M, Crimi N and Raghu G. Eur Respir J :

70 Idiopathic Pulmonary Fibrosis : Pathogenesis - Links to Cancer: Concepts* * Vancheri C, Failla M, Crimi N and Raghu G. Eur Respir J :

71 Idiopathic Pulmonary Fibrosis : Pathogenesis - Links to Cancer: Concepts* * Vancheri C, Failla M, Crimi N and Raghu G. Eur Respir J :

72 Idiopathic Pulmonary Fibrosis : Pathogenesis - Links to Cancer: Concepts* Pathogenic Similarities Between Idiopathic Pulmonary Fibrosis and Cancer Genetic alterations Tumor suppressor gene mutations Telomere shortening Epigenetic alterations Hypermethylation of the Thy-1 promoter region Uncontrolled proliferation Self-sufficiency in growth signals Insensitivity to growth inhibitory signals Evasion of apoptosis Altered cell-to-cell communications * Vancheri C, Failla M, Crimi N and Raghu G. Eur Respir J :

73 Idiopathic Pulmonary Fibrosis : Pathogenesis - Links to Cancer: Concepts* Pathogenic Similarities Between Idiopathic Pulmonary Fibrosis and Cancer Tissue invasion Myofibroblasts recruitment and differentiation Myofibroblast infiltrative ability Expression invasive molecules Signal transduction pathways Activation Wnt/b-catenin pathway Activation phosphoinositide 3-kinase/phosphatase and tensin homologue-akt pathway * Vancheri C, Failla M, Crimi N and Raghu G. Eur Respir J :

74 IPF :Therapeutic Targets Epithelial cell injury activation apoptosis Fibroblast migration proliferation myofibroblast accumulation Aberrent angiogenesis (?) Cancer biology approach?

75 Pulmonary fibrosis of unknown cause: identification of susceptibility factors - Implications for Therapeutics: Vision Prevention Utility of Genetic Screening Maintenance of alveolar type II progenitor cells (stem cells) Drugs that target the molecular pathway leading to pulmonary fibrosis Stem cell transplant

76 Genetics and Genomics of Lung Disease in Man Healthy Precise Identification of genetic/inherent factors and avoidance of extrinsic environmental factors attributable to disease