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Key Event Title
Loss of alveolar capillary membrane integrity
|Level of Biological Organization|
Key Event Components
Key Event Overview
AOPs Including This Key Event
|AOP Name||Role of event in AOP||Point of Contact||Author Status||OECD Status|
|Substance interaction with the pulmonary cell membrane leading to pulmonary fibrosis||KeyEvent||Cataia Ives (send email)||Under development: Not open for comment. Do not cite||EAGMST Under Review|
|Lung surfactant function inhibition leading to decreased lung function||KeyEvent||Brendan Ferreri-Hanberry (send email)||Open for comment. Do not cite||Under Development|
Key Event Description
The alveolar-capillary membrane (ACM) is the gas exchange surface of the lungs that is only ~0.3µm thick and is the largest surface area within the lung that separates the interior of the body from the environment. It is comprised of the microvascular endothelium, interstitium, and alveolar epithelium. As a consequence of its anatomical position, and the large surface area, it is the first point of contact for any inhaled pathogen, particles or toxic substances. Thus, ACM is subjected to injury constantly and rapidly repaired following the external insults without formation of fibrosis or scar tissue. The extent of ACM injury or how rapidly its integrity is restored is a pivotal determinant of whether the lung restores its normal functioning following an injury or is replaced by fibrotic lesion or scar tissue (Fukuda et al., 1987; Schwarz et al., 2001). Significant loss of endothelium and epithelium of the ACM results in loss of the barrier and membrane integrity. Increased membrane permeability leading to efflux of protein-rich fluid into the peribronchovascular interstitium and the distal airspaces of the lung, disruption of normal fluid transport via downregulated Na+ channels or malfunctioning Na+/K+ATPase pumps, loss of surfactant production, increased expression of epithelial or endothelial cell markers such as Intercellular adhesion molecule-1 (ICAM-1) or decreased expression of surfactant protein D are few of the markers of decreasing lung compliance arising from the lost integrity of ACM (Johnson and Matthay, 2010).
Literature evidence for its perturbation:
Bleomycin exposure causes alveolar barrier dysfunction (Miyoshi et al., 2013). Cigarette smoke impairs tight junction proteins and leads to altered permeability of the epithelial barrier (Schamberger et al., 2014). Exposure to bleomycin destroys the structural architecture of tight junctions, increases permeability, epithelial death and loss of specialised repair proteins such as claudins. Thoracic radiation and bleomycin induced lung injury results in decreased expression of E-cadherin and Aquaporin-5 (AQP5) expression (Almeida et al., 2013; Gabazza et al., 2004).
Repeated exposure to or biopersistent toxic substances, pathogens or lung irritants initiate non-resolving inflammation and ACM injury (Costabel et al., 2012). Chronic inflammation mediated by overexpression of cytokines such as Interleukine (IL)-1 (Kolb et al., 2001), Tumor necrosis factor alpha (TNF-α) (Sime et al., 1998), T helper type 2 cytokine IL-13 or exposure to specific proteinases initiate ACM injury, leading to significant loss of the epithelium and endothelium of the ACM resulting loss of barrier integrity. In patients diagnosed with idiopathic pulmonary fibrosis (IPF), both type 1 pneumocyte & endothelial cell injury with ACM barrier loss is observed.
Bleomycin and silica exposure generate persistent inflammation and lung damage (Chua et al., 2005; Thrall and Scaliso, 1995). Exposure to single-walled carbon nanotubes (SWCNTs) induces persistent inflammation, granuloma formation and diffuse intestinal fibrosis in mice after pharyngeal aspiration (Shvedova et al., 2005). Multi-walled carbon nanotubes act as allergens and induce lung infiltration of eosinophils and cause airway hypersensitivity (Beamer et al., 2013). Inhaled particles induce chronic inflammation (Ernst et al., 2002; Hamilton et al., 2008; Thakur et al., 2008). Increased numbers of alveolar macrophages, neutrophils and eosinophils are observed in the bronchoalveolar lavage fluid (BALF) of patients suffering from IPF and chronic inflammation is associated with decreased survival (Parra et al., 2007; Schwartz et al., 1991; Yasuoka et al., 1985).
The BALF of patients diagnosed with interstitial diseases contains increased levels of 8-isoprostane (Psathakis et al., 2006) and carbonyl-modified proteins (Lenz et al., 1996), markers of oxidative modification of lipids and proteins. In vivo, increased reactive oxygen species (ROS) levels in rodents (Ghio et al., 1998) and enzymatic production of nitric oxide in rat alveolar macrophages is observed after asbestos exposure (Quinlan et al., 1998). Some nanoparticles induce oxidative stress that contributes to cellular toxicity (Shi et al., 2012). Nicotinamide adenine dinucleotide phosphate (NADPH) oxidase derived ROS is a critical determinant of the pulmonary response to SWCNTs in mice (Shvedova et al., 2008). Oxidative lipidomics analysis of the lungs of carbon nanotube (CNT)-exposed mice showed, phospholipid oxidation (Tyurina et al., 2011). ROS synthesis is suggested to be important for inflammosome activation involving NLR-related protein 3 complex, activated caspase-1 and IL-1β, which is observed following exposure to a variety of pro-inflammatory stimuli including, asbestos and crystalline silica (Cassel et al., 2008; Dostert et al., 2008) and long needle-like CNTs. In the case of asbestos, frustrated phagocytosis triggered ROS synthesis leads to inflammosome activation, which is associated with asbestos induced pathology (Dostert et al., 2008).
How It Is Measured or Detected
Proteinosis, BAL fluid protein content:
Compromised ACM barrier integrity in vivo can be measured by measuring total protein or total albumin content in the BAL fluid derived from experimental animals exposed to lung toxicants or in human patients suffering from lung fibrosis. In addition to albumin, the total urea in BAL fluid is also a good indicator of the ACM integrity loss (Schmekel et al., 1992).
Cell type considerations:
ACM loss is a tissue level event. In vitro, assays with human cells are desired; however, the use of cells derived from experimental animals including alveolar macrophages, dendritic cells, epithelial cells, and neutrophils are routinely used. Primary cells are preferred over immortalised cell types that are in culture for a long period of time. In vitro, studies often assess the altered expression of pro-inflammatory mediators, increased ROS synthesis or oxidative stress and cytotoxicity events, an interplay between these three biological events occurring following exposure to stressors, is suggested to induce cell injury, which is reflective of tissue injury or loss of ACM (Halappanavar et al., 2019) in vivo.
Cellular viability or cytotoxicity assays are the most commonly used endpoints to assess the leaky or compromised cell membrane. The most commonly employed method is the trypan blue exclusion assay – a dye exclusion assay where cells with intact membrane do not permit entry of the dye into cells and thus remain clear, whereas the dye diffuses into cells with damaged membrane turning them to blue colour. Other high throughput assays that use fluorescent DNA stains such as ethidium bromide or propidium iodide can also be used and cells that have incorporated the dye can be scored using flow cytometry.
Lactate dehydrogenase (LDH) release assay is a very sensitive cytotoxicity assay that measures the amount of LDH released in the media following membrane injury. The assay is based on measuring the reduction of nicotinamide adenine dinucleotide (NAD) and conversion of a tetrazolium dye that is measured at a wavelength of 490 nm.
The Calcein AM assay depends on the hydrolysis of calcein AM ( a non-fluorescent hydrophobic compound that permeates live cells by simple diffusion) by non-specific intracellular esterases resulting in production of calcein, a hydrophilic and strongly fluorescent compound that is readily released into the cell culture media by the damaged cells.
Although the above mentioned assays work for almost all chemicals, insoluble substances such as nanomaterials can confound the assay by inhibiting the enzyme activity or interfering with the absorbance reading. Thus, care must be taken to include appropriate controls in the assays.
Transepithelial/transendothelial electrical resistance (TEER):
TEER is an accepted quantitative technique that measures the integrity of tight junctions in cell culture models of endothelial and epithelial cell monolayers. They are based on measuring ohmic resistance or measuring impedance across a wide range of frequencies.
The other methods include targeted reverse transcription polymerase chain reaction (RT-PCR) or enzyme-linked immunosorbent assays (ELISA) for tight junction proteins, cell adhesion molecules and inflammatory mediators such as Interferon gamma (IFN-γ), IL-10, and IL-13. Advanced in vitro co-culture models, like the EpiAlveolar model system, and other similar systems present an intact capillary membrane that can be used to assess loss in the membrane integrity (via TEER) after exposure to pro-fibrotic stressors like crystalline silica and Transforming growht factor beta (TGF-β) (Barasova et al., 2020, Kasper et al., 2011).
Domain of Applicability
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