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Event: 1498

Key Event Title

A descriptive phrase which defines a discrete biological change that can be measured. More help

Loss of alveolar capillary membrane integrity

Short name
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Loss of alveolar capillary membrane integrity
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Biological Context

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Level of Biological Organization

Organ term

The location/biological environment in which the event takes place.The biological context describes the location/biological environment in which the event takes place.  For molecular/cellular events this would include the cellular context (if known), organ context, and species/life stage/sex for which the event is relevant. For tissue/organ events cellular context is not applicable.  For individual/population events, the organ context is not applicable.  Further information on Event Components and Biological Context may be viewed on the attached pdf. More help
Organ term

Key Event Components

The KE, as defined by a set structured ontology terms consisting of a biological process, object, and action with each term originating from one of 14 biological ontologies (Ives, et al., 2017; Biological process describes dynamics of the underlying biological system (e.g., receptor signalling).Biological process describes dynamics of the underlying biological system (e.g., receptor signaling).  The biological object is the subject of the perturbation (e.g., a specific biological receptor that is activated or inhibited). Action represents the direction of perturbation of this system (generally increased or decreased; e.g., ‘decreased’ in the case of a receptor that is inhibited to indicate a decrease in the signaling by that receptor).  Note that when editing Event Components, clicking an existing Event Component from the Suggestions menu will autopopulate these fields, along with their source ID and description.  To clear any fields before submitting the event component, use the 'Clear process,' 'Clear object,' or 'Clear action' buttons.  If a desired term does not exist, a new term request may be made via Term Requests.  Event components may not be edited; to edit an event component, remove the existing event component and create a new one using the terms that you wish to add.  Further information on Event Components and Biological Context may be viewed on the attached pdf. More help

Key Event Overview

AOPs Including This Key Event

All of the AOPs that are linked to this KE will automatically be listed in this subsection. This table can be particularly useful for derivation of AOP networks including the KE.Clicking on the name of the AOP will bring you to the individual page for that AOP. More help
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 WPHA/WNT Endorsed
Lung surfactant function inhibition leading to decreased lung function KeyEvent Brendan Ferreri-Hanberry (send email) Open for comment. Do not cite Under Development

Taxonomic Applicability

Latin or common names of a species or broader taxonomic grouping (e.g., class, order, family) that help to define the biological applicability domain of the KE.In many cases, individual species identified in these structured fields will be those for which the strongest evidence used in constructing the AOP was available in relation to this KE. More help
Term Scientific Term Evidence Link
mouse Mus musculus High NCBI
human Homo sapiens Not Specified NCBI

Life Stages

An indication of the the relevant life stage(s) for this KE. More help
Life stage Evidence
Adult High

Sex Applicability

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Term Evidence
Male High
Female Not Specified

Key Event Description

A description of the biological state being observed or measured, the biological compartment in which it is measured, and its general role in the biology should be provided. More help

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 (SP-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 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

A description of the type(s) of measurements that can be employed to evaluate the KE and the relative level of scientific confidence in those measurements.These can range from citation of specific validated test guidelines, citation of specific methods published in the peer reviewed literature, or outlines of a general protocol or approach (e.g., a protein may be measured by ELISA). Do not provide detailed protocols. More help

Proteinosis, BALF protein content:

Compromised ACM barrier integrity in vivo can be measured by measuring total protein or total albumin content in the BALF derived from experimental animals exposed to lung toxicants or in human patients suffering from lung fibrosis. In addition to albumin, the total urea in BALF 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.

Cytotoxicity assessment:

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 growth factor beta (TGF-β) (Barasova et al., 2020, Kasper et al., 2011).

Domain of Applicability

A description of the scientific basis for the indicated domains of applicability and the WoE calls (if provided).  More help


List of the literature that was cited for this KE description. More help

1. Almeida C, Nagarajan D, Tian J, Leal SW, Wheeler K, Munley M, Blackstock W, Zhao W. The role of alveolar epithelium in radiation-induced lung injury. PLoS One. 2013;8(1):e53628. doi: 10.1371/journal.pone.0053628.

2. Barosova H, Maione AG, Septiadi D, Sharma M, Haeni L, Balog S, O'Connell O, Jackson GR, Brown D, Clippinger AJ, Hayden P, Petri-Fink A, Stone V, Rothen-Rutishauser B. Use of EpiAlveolar Lung Model to Predict Fibrotic Potential of Multiwalled Carbon Nanotubes. ACS Nano. 2020 Apr 28;14(4):3941-3956. doi: 10.1021/acsnano.9b06860. 

3. Beamer CA, Girtsman TA, Seaver BP, Finsaas KJ, Migliaccio CT, Perry VK, Rottman JB, Smith DE, Holian A. IL-33 mediates multi-walled carbon nanotube (MWCNT)-induced airway hyper-reactivity via the mobilization of innate helper cells in the lung. Nanotoxicology. 2013 Sep;7(6):1070-81. doi: 10.3109/17435390.2012.702230. 

4. Cassel SL, Eisenbarth SC, Iyer SS, Sadler JJ, Colegio OR, Tephly LA, Carter AB, Rothman PB, Flavell RA, Sutterwala FS. The Nalp3 inflammasome is essential for the development of silicosis. Proc Natl Acad Sci U S A. 2008 Jul 1;105(26):9035-40. doi: 10.1073/pnas.0803933105.

5. Chua F, Gauldie J, Laurent GJ. Pulmonary fibrosis: searching for model answers. Am J Respir Cell Mol Biol. 2005 Jul;33(1):9-13. doi: 10.1165/rcmb.2005-0062TR.

6. Costabel U, Bonella F, Guzman J. Chronic hypersensitivity pneumonitis. Clin Chest Med. 2012 Mar;33(1):151-63. doi: 10.1016/j.ccm.2011.12.004.

7. Dostert C, Pétrilli V, Van Bruggen R, Steele C, Mossman BT, Tschopp J. Innate immune activation through Nalp3 inflammasome sensing of asbestos and silica. Science. 2008 May 2;320(5876):674-7. doi: 10.1126/science.1156995.

8. Ernst H, Rittinghausen S, Bartsch W, Creutzenberg O, Dasenbrock C, Görlitz BD, Hecht M, Kairies U, Muhle H, Müller M, Heinrich U, Pott F. Pulmonary inflammation in rats after intratracheal instillation of quartz, amorphous SiO2, carbon black, and coal dust and the influence of poly-2-vinylpyridine-N-oxide (PVNO). Exp Toxicol Pathol. 2002 Aug;54(2):109-26. doi: 10.1078/0940-2993-00241. 

9. Fukuda Y, Ishizaki M, Masuda Y, Kimura G, Kawanami O, Masugi Y. The role of intraalveolar fibrosis in the process of pulmonary structural remodeling in patients with diffuse alveolar damage. Am J Pathol. 1987 Jan;126(1):171-82.

10. Gabazza EC, Kasper M, Ohta K, Keane M, D'Alessandro-Gabazza C, Fujimoto H, Nishii Y, Nakahara H, Takagi T, Menon AG, Adachi Y, Suzuki K, Taguchi O. Decreased expression of aquaporin-5 in bleomycin-induced lung fibrosis in the mouse. Pathol Int. 2004 Oct;54(10):774-80. doi: 10.1111/j.1440-1827.2004.01754.x.

11. Ghio AJ, Kadiiska MB, Xiang QH, Mason RP. In vivo evidence of free radical formation after asbestos instillation: an ESR spin trapping investigation. Free Radic Biol Med. 1998 Jan 1;24(1):11-7. doi: 10.1016/s0891-5849(97)00063-4. 

12. Halappanavar S, van den Brule S, Nymark P, Gaté L, Seidel C, Valentino S, Zhernovkov V, Høgh Danielsen P, De Vizcaya A, Wolff H, Stöger T, Boyadziev A, Poulsen SS, Sørli JB, Vogel U. Adverse outcome pathways as a tool for the design of testing strategies to support the safety assessment of emerging advanced materials at the nanoscale. Part Fibre Toxicol. 2020 May 25;17(1):16. doi: 10.1186/s12989-020-00344-4.

13. Hamilton RF Jr, Thakur SA, Holian A. Silica binding and toxicity in alveolar macrophages. Free Radic Biol Med. 2008 Apr 1;44(7):1246-58. doi: 10.1016/j.freeradbiomed.2007.12.027.

14. Johnson ER, Matthay MA. Acute lung injury: epidemiology, pathogenesis, and treatment. J Aerosol Med Pulm Drug Deliv. 2010 Aug;23(4):243-52. doi: 10.1089/jamp.2009.0775.

15. Kasper J, Hermanns MI, Bantz C, Maskos M, Stauber R, Pohl C, Unger RE, Kirkpatrick JC. Inflammatory and cytotoxic responses of an alveolar-capillary coculture model to silica nanoparticles: comparison with conventional monocultures. Part Fibre Toxicol. 2011 Jan 27;8(1):6. doi: 10.1186/1743-8977-8-6. 

16. Kolb M, Margetts PJ, Anthony DC, Pitossi F, Gauldie J. Transient expression of IL-1beta induces acute lung injury and chronic repair leading to pulmonary fibrosis. J Clin Invest. 2001 Jun;107(12):1529-36. doi: 10.1172/JCI12568.

17. Lenz AG, Costabel U, Maier KL. Oxidized BAL fluid proteins in patients with interstitial lung diseases. Eur Respir J. 1996 Feb;9(2):307-12. doi: 10.1183/09031936.96.09020307. 

18. Miyoshi K, Yanagi S, Kawahara K, Nishio M, Tsubouchi H, Imazu Y, Koshida R, Matsumoto N, Taguchi A, Yamashita S, Suzuki A, Nakazato M. Epithelial Pten controls acute lung injury and fibrosis by regulating alveolar epithelial cell integrity. Am J Respir Crit Care Med. 2013 Feb 1;187(3):262-75. doi: 10.1164/rccm.201205-0851OC. 

19. Parra ER, Kairalla RA, Ribeiro de Carvalho CR, Eher E, Capelozzi VL. Inflammatory cell phenotyping of the pulmonary interstitium in idiopathic interstitial pneumonia. Respiration. 2007;74(2):159-69. doi: 10.1159/000097133.

20. Psathakis K, Mermigkis D, Papatheodorou G, Loukides S, Panagou P, Polychronopoulos V, Siafakas NM, Bouros D. Exhaled markers of oxidative stress in idiopathic pulmonary fibrosis. Eur J Clin Invest. 2006 May;36(5):362-7. doi: 10.1111/j.1365-2362.2006.01636.x.

21. Quinlan TR, BeruBe KA, Hacker MP, Taatjes DJ, Timblin CR, Goldberg J, Kimberley P, O'Shaughnessy P, Hemenway D, Torino J, Jimenez LA, Mossman BT. Mechanisms of asbestos-induced nitric oxide production by rat alveolar macrophages in inhalation and in vitro models. Free Radic Biol Med. 1998 Mar 15;24(5):778-88. doi: 10.1016/s0891-5849(97)00357-2.

22. Schamberger AC, Mise N, Jia J, Genoyer E, Yildirim AÖ, Meiners S, Eickelberg O. Cigarette smoke-induced disruption of bronchial epithelial tight junctions is prevented by transforming growth factor-β. Am J Respir Cell Mol Biol. 2014 Jun;50(6):1040-52. doi: 10.1165/rcmb.2013-0090OC. 

23. Schmekel B, Bos JA, Khan AR, Wohlfart B, Lachmann B, Wollmer P. Integrity of the alveolar-capillary barrier and alveolar surfactant system in smokers. Thorax. 1992 Aug;47(8):603-8. doi: 10.1136/thx.47.8.603. 

24. Schwartz DA, Helmers RA, Dayton CS, Merchant RK, Hunninghake GW. Determinants of bronchoalveolar lavage cellularity in idiopathic pulmonary fibrosis. J Appl Physiol (1985). 1991 Nov;71(5):1688-93. doi: 10.1152/jappl.1991.71.5.1688. 

25. Schwarz MA. Acute lung injury: cellular mechanisms and derangements. Paediatr Respir Rev. 2001 Mar;2(1):3-9. doi: 10.1053/prrv.2000.0095.

26. Shi J, Karlsson HL, Johansson K, Gogvadze V, Xiao L, Li J, Burks T, Garcia-Bennett A, Uheida A, Muhammed M, Mathur S, Morgenstern R, Kagan VE, Fadeel B. Microsomal glutathione transferase 1 protects against toxicity induced by silica nanoparticles but not by zinc oxide nanoparticles. ACS Nano. 2012 Mar 27;6(3):1925-38. doi: 10.1021/nn2021056. 

27. Shvedova AA, Kisin ER, Mercer R, Murray AR, Johnson VJ, Potapovich AI, Tyurina YY, Gorelik O, Arepalli S, Schwegler-Berry D, Hubbs AF, Antonini J, Evans DE, Ku BK, Ramsey D, Maynard A, Kagan VE, Castranova V, Baron P. Unusual inflammatory and fibrogenic pulmonary responses to single-walled carbon nanotubes in mice. Am J Physiol Lung Cell Mol Physiol. 2005 Nov;289(5):L698-708. doi: 10.1152/ajplung.00084.2005. 

28. Shvedova AA, Kisin ER, Murray AR, Kommineni C, Castranova V, Fadeel B, Kagan VE. Increased accumulation of neutrophils and decreased fibrosis in the lung of NADPH oxidase-deficient C57BL/6 mice exposed to carbon nanotubes. Toxicol Appl Pharmacol. 2008 Sep 1;231(2):235-40. doi: 10.1016/j.taap.2008.04.018. 

29. Sime PJ, Marr RA, Gauldie D, Xing Z, Hewlett BR, Graham FL, Gauldie J. Transfer of tumor necrosis factor-alpha to rat lung induces severe pulmonary inflammation and patchy interstitial fibrogenesis with induction of transforming growth factor-beta1 and myofibroblasts. Am J Pathol. 1998 Sep;153(3):825-32. doi: 10.1016/s0002-9440(10)65624-6. 

30. Thakur SA, Hamilton RF Jr, Holian A. Role of scavenger receptor a family in lung inflammation from exposure to environmental particles. J Immunotoxicol. 2008 Apr;5(2):151-7. doi: 10.1080/15476910802085863.

31. Thrall, R. S., & Scaliso, P. J. (1995). Bleomycin. Pulmonary Fibrosis. Edited by SH Phan, RS Thrall.

31. Tyurina YY, Kisin ER, Murray A, Tyurin VA, Kapralova VI, Sparvero LJ, Amoscato AA, Samhan-Arias AK, Swedin L, Lahesmaa R, Fadeel B, Shvedova AA, Kagan VE. Global phospholipidomics analysis reveals selective pulmonary peroxidation profiles upon inhalation of single-walled carbon nanotubes. ACS Nano. 2011 Sep 27;5(9):7342-53. doi: 10.1021/nn202201j. 

32. Yasuoka S, Nakayama T, Kawano T, Ogushi F, Doi H, Hayashi H, Tsubura E. Comparison of cell profiles of bronchial and bronchoalveolar lavage fluids between normal subjects and patients with idiopathic pulmonary fibrosis. Tohoku J Exp Med. 1985 May;146(1):33-45. doi: 10.1620/tjem.146.33.