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Key Event Title
Loss of alveolar capillary membrane integrity
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 lung cell membrane leading to lung fibrosis||KeyEvent||Sabina Halappanavar (send email)||Under development: Not open for comment. Do not cite||EAGMST Under Review|
|Lung surfactant function inhibition leading to decreased lung function||KeyEvent||Jorid Birkelund Sørli (send email)||Open for comment. Do not cite||Under Development|
Key Event Description
Loss of alveolar capillary membrane integrity
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 (Fakuda et al., 1987; Shwarz 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 D are few of the markers of decreasing lung compliance arising from the lost integrity of ACM (Johnson and Matthay, 2010).
KE3 associative event 1 - Chronic inflammation
In the presence of continuous stimulus (e.g., presence of biopersistent toxic fibres such as asbestos, MWCNTs) or following repeated stimulus (e.g., repeated exposure to silica or coal dust), the ensuing cell injury fuels the inflammatory mechanisms leading to accumulation of immune cells, prolonged inflammation and aggravated tissue damage. This sustained and perpetuated immunological response is termed as chronic inflammation. During this phase, active inflammation, tissue injury and destruction, and tissue repair processes proceed in tandem. Thus, the causative substance must contain unique physico-chemical properties that grant the material biopersistance in the pulmonary environment or the pulmonary system has to be repeatedly exposed to the same substance that perpetuates the tissue injury leading to loss of ACM. Although, increases in number of neutrophils are observed during chronic inflammation, mononuclear phagocytes (circulating monocytes, tissue macrophages) and lymphoid cells mark this phase. The macrophages, components of mononuclear phagocyte system, are the predominant cells in chronic inflammation. Activated macrophages release a variety of cytokines, chemokines, growth factors, which when uncontrolled, lead to extensive tissue injury, cellular death and necrosis, the other characteristics of chronic inflammation, leading to ACM loss. The other types of inflammatory cells found in chronic inflammation include eosinophils in allergen induced lung fibrosis, lymphocytes and epithelial cells.
KE3 associative event 2 - Oxidative stress
Superoxide anion (O2-) and the hydroxyl radical (OH) are the common ROS found in the biological systems that are unstable due to unpaired electrons. As such, all tissues including lung have efficient antioxidant system to counteract the ROS induced oxidation. The antioxidant enzymes including superoxide dismutase, catalase and glutathione peroxidase act directly to inactivate ROS and associated reactions. In addition, phase II detoxifying enzymes, including glutathione-S-transferase, NADPH quinone oxidoreductase and glutamate-cysteine ligase catalytic act as indirect antioxidant enzymes. However, when the balance between oxidant and antioxidants is tipped towards oxidants, oxidative stress occurs. ROS, when interacted with biomolecules, initiate their oxidation. During the process, proteins, DNA and lipids are oxidized. Oxidative stress modulates the cellular signalling processes and contributes to oxidative stress-induced tissue injury. It also plays a role in the tissue injury caused by inflammatory processes in lungs. The infiltrating neutrophils and macrophages generate superoxide anion which is converted to hydrogen peroxide by superoxide dismutase enzyme. OH is formed by a secondary reaction in the presence of Fe2+. ROS can also be produced by NADPH oxidase present in phagocytes. The other enzyme that contributes to ROS synthesis during inflammatory processes is the myeloperoxidase from neutrophils. In a self-perpetuating loop, inflammatory cells generate oxidative stress leading to increased airspace epithelial permeability, increased cell death and increased expression of pro-inflammatory genes, all of which lead to secretion of inflammatory cytokines/chemokines leading to prolonged and chronic inflammation.
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 pro-fibrotic drug bleomycin destroys structural architecture of the 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 expression (Almeida et al., 2013; Gabazza et al., 2004).
Epithelium and basement membrane injury is a prerequisite for the development of fibrotic lesion (Brody et al., 1981). 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 IL-1 (Kolb et al., 2001), TNFa (Sime et al., 1998), T-helper type 2 cytokine IL-13 or exposure to specific proteinases initiate ACM injury, significant loss of the epithelium and endothelium of the ACM resulting in loss of the barrier. In patients diagnosed with idiopathic pulmonary fibrosis, both type 1 pneumocyte and endothelial cell injury with the ACM barrier loss is observed.
Bleomycin and silica exposure generate persistent inflammation and lung damage (Thrall, 1995; Chau, 2005). Exposure to SWCNTs induces persistent inflammation, granuloma and diffuse intestinal fibrosis in mice after pharyngeal aspiration (Shevedova, 2005). MWCNTs act as allergens and induce lung infiltration of eosinophils and cause airway hypersensitivity (Beamer, 2013). Inhaled particles induce chronic inflammation (Hamilton, 2008; Thakur, 2008; Ernst, 2002). Increased numbers of alveolar macrophages, neutrophils and eosinophils are observed in the BALF of patients suffering from IPF and chronic inflammation is associated with decreased survival (Parra, 2007; Schwartz 1991; Yasuoka, 1985).
The BALF of patients diagnosed with interstitial diseases contains increased levels of 8-isoprostane (Psathakis, 2006) and carbonyl-modified proteins (Lenz, 1996), markers of oxidative modification of lipids and proteins. In vivo, increased ROS levels in rodents (Ghio, 1998) and enzymatic production of nitric oxide in rat alveolar macrophages is observed after asbestos exposure (Quinlan, 1998). Some nanoparticles induce oxidative stress that contributes to cellular toxicity (Shi, 2012). NADPH oxidase derived ROS is a critical determinant of the pulmonary response to SWCNTs in mice (Shvedova, 2008). Oxidative lipidomics analysis of the lungs of CNT-exposed mice showed, phospholipid oxidation (Tyurina, 2011). ROS synthesis is suggested to be important for inflammosome activation involving NLR-related protein 3 complex, activated caspase-1 and IL-1b, which is observed following exposure to a variety of pro-fibrotic stimuli including, asbestos and crystalline silica (Dostert, 2008; Cassel, 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, 2008).
How It Is Measured or Detected
Proteinosis, BAL fluid protein content
The compromised ACM 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).
Cel 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, 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.
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 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 NMs 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 RT-PCR or ELISA assays for tight junction proteins, cell adhesion molecules and inflammatory mediators such as IFNg, IL-10, and IL-13.
Domain of Applicability
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- Gabazza, E., Kasper, M., Ohta, K., Keane, M., D'Alessandro-Gabazza, C., Fujimoto, H., Nishii, Y., Nakahara, H., Takagi, T., Menon, A., Adachi, Y., Suzuki, K. and Taguchi, O. (2004). Decreased expression of aquaporin-5 in bleomycin-induced lung fibrosis in the mouse. Pathology International, 54(10), pp.774-780.
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