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What is fibrosis?

- deposition of extracellular matrix
- part of wound healing
- role in development of organs
- when aberrant is fibrosis
- imbalance of synthesis and degradation


What are chronic obstructive lung diseases?

- asthma (airway remodelling)
-- collagen of ECM being laid down beneath the basement membrane that thickens between the basement membrane and the muscle (lamina propria)
-- also get excessive matrix being laid down between the muscle and the alveoli
-- great functional effects on the airway

- chronic obstructive pulmonary disease (COPD)
-- emphysema
-- bronchitis
-- remodelling around the airways and parenchyma

- fibrosing diseases of the lung are a category separate from chronic obstructive lung diseases


What are fibrosing diseases of the lung?

idiopathic interstitial pneumonias
- idiopathic pulmonary fibrosis (IPF) most common


What other diseases have a similar pattern of pathology (non-idiopathic)?

Lung fibrosis as part of rheumatic-type diseases (collagen/vascular diseases)
- scleroderma (systemic fibrosis)
- rheumatoid arthritis
- mixed connective tissue disease
- polymyositis/dermatomyositis
- systemic lupus erythematosus

Lung fibrosis triggered by medical treatments
- antibiotics (e.g. nitrofurantoin)
- antiarrhythmics (e.g. amiodarone)
- anti-inflammatory drugs
- anticonvulsants
- chemotherapy agents (e.g. bleomycin)
- radiotherapy
- oxygen

Lung fibrosis caused by inhaled dust particles
- inorganic dusts (asbestosis, silicosis [coal worker's pneumoconiosis], talc pneumonia)
- organic dusts (extrinsic allergic alveolitis)

Lung fibrosis in association with systemic diseases of unknown origin
- sarcoidosis
- amyloidosis
- Niemann-Pick/Gaucher's disease
- Hermansky-Pudlak syndrome

Lung fibrosis of unknown origin
- idiopathic pulmonary fibrosis (IPF)
- non-specific interstitial pneumonia (NSIP)


What is idiopathic pulmonary fibrosis (IPF)?

- chronic, progressive, irreversible, usually lethal lung disease of unknown cause
- median age at diagnosis 66 years (range 55 - 75) i.e. disease of the elderly
- limited to the lungs
- histological and radiological pattern known as 'usual interstitial pneumonia'


What is the epidemiology/what are the risk factors for IPF?

- rising incidence of 4.6 - 16.3 per 100,000
- prevalence of 13 – 20 cases per 100,000
- predominance in men (1.5-1.7:1)
- risk factors:
-- cigarette smoking
-- exposure to metal and wood dust
-- genetic transmission .5 - 3.7% or higher autosomal dominant (familial IPF)


What are the clinical phenotypes? What is the prognosis for IPF?

Survival 2.5-3.5 years after diagnosis
- symptoms are cough and progressive dyspnea

Heterogeneous natural history
- stable or slowly progressive course
- accelerated variant (male cigarette smokers)
- acute exacerbations (will speed up progression)

- asymptomatic for years beforehand
- can have a rapid course of disease, or a slow progressive course
- can be a bit sped up by the presence of emphysema


How is IPF diagnosed?

- high resolution computed tomography (HRCT)
- patchy, subpleural reticular opacities and honeycombing with basal predominance (heterogeneous gaps in the tissue)
- septa between the lobes are thickened

surgical biopsy, histopathology
- fibroblastic foci
- not as much inflammation as in other lung diseases
- some oedema

- the histopathologic hallmark and chief diagnostic criteria is a heterogenous appearance at low magnification in which areas of fibrosis with scarring and honeycomb alternate with areas of less affected or normal parenchyma


What are the major criteria for diagnosis?

1. exclusion of other known causes of interstitial lung disease
2. abnormal lung function tests - restriction/impaired gas exchange
3. abnormalities of chest radiographs HRCT


What are the minor criteria for diagnosis?

1. > 50 years
2. insidious onset of otherwise unexplained dyspnea on exertion
3. duration of illness >3 months
4. bibasilar inspiratory crackles


What is the gross lung appearance in IPF?

- pleural surface has a bosselated or cobblestone appearance
- these correspond to airspace enlargement and fibrotic retraction
- this is termed gross honeycombing


What are the microscopic features of IPF?

Usual interstitial pneumonia
- patchy fibrotic reaction
- fibrosis prominent in peripheral secondary pulmonary lobule
- central portion of lobule spared
- temporal and spacial heterogeniety

fibrotic foci
- epithelial surface of cuboidal cells

paucity of inflammation

lack of uniform involvement

acute exacerbations of usual interstitial pneumonia includes diffuse alveolar damage
- type II pneumocyte hyperplasia (gone from representing a small percentage to a large percentage)
- edematous alveolar septa
- hyaline membranes (sign of epithelial damage)
- distal airway squamous metaplasia
- thrombi in small pulmonary arteries


Why do we need to understand the pathobiology of IPF?

- essential if effective therapies are to be developed in the future


How do we study IPF?

- biopsies from patients
-- from dead patients only
-- dead, fixed tissue
-- observational
-- histopathology

- cell lines
-- not really typical of what's typical of the disease
-- different microenvironment

- primary cell culture from patients samples
-- retain phenotype seen in person

- animal models


What is the main animal model of IPF?

- bleomycin model
- observation: side effect of cancer drug bleomycin is pulmonary fibrosis
- administration to mice leads to disease model
-- lacks human chronicity
-- mouse lifespan and anatomy
-- used to test potential therapies
-- free radicals/DNA damage (bit different to humans)

- sheep model under development


How do people think about patholobiology?

come up with a paradigm that gives you something to work with


What is the paradigm for pathobiology of IPF?

- has gone from inflammation to aberrant wound healing

- rather than starting with inflammation causing fibrosis
- disease is actually starting at the epithelial cell
- alveolar epithelial cells are somewhat vulnerable to injury, endogenous vs exogenous?
- epithelial cells release various factors that cause fibrosis (activation of fibroblasts to become myofibroblasts)


What clue did familial IPF give to indicate the role of alveolar epithelium?

- genetics - familial IPF
- all genes are expressed in alveolar epithelial cells
- IPF have short telomeres even without TERT/TERC mutation - why?
- mutant allele leads to increased MUC5B

- MUC5B: gene that codes for mucin (protein component of mucus) (35%)
- TERT/TERC: related to telomere length, telomeres important for cell senescence and ageing (3%)
- SPA/C: surfactant protein (1%) (1%)

changes in these epithelial genes are found in disease, so maybe this is disease has an epithelial origin


What is ER stress?

- imbalance between protein synthesis and ER ability to make proteins
- markers of UPR are up in alveolar type II cells in IPD
- virus infection can lead to UPR, other triggers need to be identified

- Stress > sensor > unfolded protein response (UPR) > terminal UPR > apoptosis

- epithelial cells in IPF had ER stress

- in the model environmental insults cause misfolding of proteins in the cell > UPR > apoptosis and/or > fibrosis


How is TGFβ activation important to IPF?

- TGFβ is the key fibrotic cytokine
- Inactive TGFβ is secreted bound to latency associated peptide (LAP)
- during fibrosis avB6-integrin binds LAP
- epithelial cell contracts and latent TGFβ is released and becomes activated
- outcomes: epithelial cell proliferation, myofibroblast differentiation, epithelial - mesenchymal transition (EMT)


What is epithelial-mesenchymal transition?

- seen in development, cancer and fibrosis
- in vitro observations
- demonstrated by immunohistochemistry
-- colocalization of type II cell marker TTF1 with αSMA, N-cadherin, calponin 1 mesenchymal markers
-- controversy – do epithelial cells acquire sufficient mesenchymal markers to be classified as fibroblasts? cells are meant to stay differentiated.
-- what is the contribution to the fibrotic process?


Why are external stressors considered important to IPF?

- mice KO for the genetic changes listed earlier do not develop spontaneous pulmonary fibrosis
- may be a role for environmental insult
-- gastrointestinal reflux - microaspiration
-- treatment with proton pump inhibitors slows disease progression


What is the role of the mesenchyme in IPF?

- likely that more than just epithelium and EMT is involved in pathogenesis
- there is an interaction between dysregulated epithelium and fibroblasts (professional collagen secreting cells of the lung)


What are fibrocytes?

- precursor cells for fibroblasts, go through circulation
- bone marrow derived CD34, CD45
- evolved to home to sites of tissue injury
- increased percentage of circulating fibrocytes is associated with exacerbation
- >5% of circulating leukocytes - poor prognosis
- recruitment: alveolar epithelium in IPF may express CXCL12 which binds the CXCR4 chemokine receptor on fibrocytes


How can fibroblasts become myofibroblasts?

- TGFβ causes differentiation to myofibroblast
- this is a transient cell type and needs ongoing activation (can take advantage by using anti TGFβ drugs)
- fibroblasts in IPF can also be resistant to apoptosis and acquire an invasive phenotype
- these changes are durable (cells cultured from patients retain phenotype)
- resist apoptosis when exposed to FAS ligand
- prosurvival genes of FoxO3a


What are the three ways you can get myofibroblasts?

- epithelial cells --> EMT --> fibroblasts --> myofibroblast transition --> myofibroblasts
- fibrocytes --> fibrocyte to fibroblast differentiation --> myofibroblast transition
- fibroblasts --> myofibroblast transition --> myofibroblasts

- probablt all of these are happening


What is IPF's similarity with cancer?

- not monoclonal but
- relentless progression
- invasiveness
- foci of fibroblasts are like tumorlets


What is epithelial-mesenchymal crosstalk?

- epithelial cells and fibroblasts are in close apposition
- epithelial cells secrete TGFβ, integrin avβ6 to activate it, also secrete PDGF - promotes fibroblast proliferation, wnts – stimulate collagen production by fibroblasts

i.e. epithelial cells secrete things that get the fibroblasts going, but also the mesenchyme will interact with the ECM (laid down aberrant ECM) that by itself will drive changes in the fibroblasts, and the epithelial cells
- put down different layers of ECM on cell culture dishes that contain proteins that are only contained in disease, they cause a change in the epithelial cells/fibroblasts that are grown on top of them


How macrophages contribute to IPF?

- may make most of active TGFβ (controversial)
- TSP-1 secreted by macrophages activates latent TGFβ
- CCL18 expressed by M2 macrophages and correlates with severity

- Inflammatory M1 macrophage: makes a number of inflammatory things that may be involved in fibrosis, e.g. TNFalpha, MMPs (break down collagen but that allows the tissue to turn over and make more)
- profibrotic M2 macrophage: makes TGFβ etc, helps drive production of collagen etc


Why do we get propagation of lung fibrosis?

- still don't know why IPF has such a relentless progressive clinical course
- epithelial microscopic injury hypothesis:
-- not much clinical or histopathological evidence
-- although, can be seen in acute exacerbations – evidence: hyaline membranes, alveolar filling with proteinaceous material

- ECM includes molecules that signal mesenchymal cells to secrete more ECM
- feed forward autoamplified loop of matrix remodelling
- epigenetics: tobacco smoke, air pollution, ageing causing epigenetic changes
-- DNA methylation
-- microRNAs decreased


What are therapeutic strategies in IPF?

- prednisone/azathiprine/N-acetyl cysteine (NAC)
-- slower decline in lung function
-- further studies showed increase death with the combination therapy so now just looking at NAC (antioxidant)

- interferon γ - antifibrotic
- endothelin receptor antagonists
- etanercept - anti-TNFα
- pirfenidone - anti-inflamm/anti-oxidant/anti-fibrotic - mixed resutls
- only proven treatment is lung transplant