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Event: 1500

Key Event Title

The KE title should describe a discrete biological change that can be measured. It should generally define the biological object or process being measured and whether it is increased, decreased, or otherwise definably altered relative to a control state. For example “enzyme activity, decreased”, “hormone concentration, increased”, or “growth rate, decreased”, where the specific enzyme or hormone being measured is defined. More help

Increased, fibroblast proliferation and myofibroblast differentiation

Short name
The KE short name should be a reasonable abbreviation of the KE title and is used in labelling this object throughout the AOP-Wiki. The short name should be less than 80 characters in length. More help
Increased cellular proliferation and differentiation

Biological Context

Structured terms, selected from a drop-down menu, are used to identify the level of biological organization for each KE. Note, KEs should be defined within a particular level of biological organization. Only KERs should be used to transition from one level of organization to another. Selection of the level of biological organization defines which structured terms will be available to select when defining the Event Components (below). More help

Organ term

Further information on Event Components and Biological Context may be viewed on the attached pdf.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. More help

Key Event Components

Further information on Event Components and Biological Context may be viewed on the attached pdf.Because one of the aims of the AOP-KB is to facilitate de facto construction of AOP networks through the use of shared KE and KER elements, authors are also asked to define their KEs using a set of structured ontology terms (Event Components). In the absence of structured terms, the same KE can readily be defined using a number of synonymous titles (read by a computer as character strings). In order to make these synonymous KEs more machine-readable, KEs should also be defined by one or more “event components” consisting of a biological process, object, and action with each term originating from one of 22 biological ontologies (Ives, et al., 2017; See List). Biological process describes dynamics of the underlying biological system (e.g., receptor signalling). 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 signalling 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. 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 lung cell membrane leading to lung fibrosis KeyEvent Cataia Ives (send email) Under development: Not open for comment. Do not cite EAGMST Under Review

Stressors

This is a structured field used to identify specific agents (generally chemicals) that can trigger the KE. Stressors identified in this field will be linked to the KE in a machine-readable manner, such that, for example, a stressor search would identify this as an event the stressor can trigger. NOTE: intermediate or downstream KEs in one AOP may function as MIEs in other AOPs, meaning that stressor information may be added to the KE description, even if it is a downstream KE in the pathway currently under development.Information concerning the stressors that may trigger an MIE can be defined using a combination of structured and unstructured (free-text) fields. For example, structured fields may be used to indicate specific chemicals for which there is evidence of an interaction relevant to this MIE. By linking the KE description to a structured chemical name, it will be increasingly possible to link the MIE to other sources of chemical data and information, enhancing searchability and inter-operability among different data-sources and knowledgebases. The free-text section “Evidence for perturbation of this MIE by stressor” can be used both to identify the supporting evidence for specific stressors triggering the MIE as well as to define broad chemical categories or other properties that classify the stressors able to trigger the MIE for which specific structured terms may not exist. More help

Taxonomic Applicability

Latin or common names of a species or broader taxonomic grouping (e.g., class, order, family) can be selected from an ontology. 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 Moderate NCBI

Life Stages

The structured ontology terms for life-stage are more comprehensive than those for taxa, but may still require further description/development and explanation in the free text section. More help
Life stage Evidence
Adult High

Sex Applicability

The authors must select from one of the following: Male, female, mixed, asexual, third gender, hermaphrodite, or unspecific. More help
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. For example, the biological state being measured could be the activity of an enzyme, the expression of a gene or abundance of an mRNA transcript, the concentration of a hormone or protein, neuronal activity, heart rate, etc. The biological compartment may be a particular cell type, tissue, organ, fluid (e.g., plasma, cerebrospinal fluid), etc. The role in the biology could describe the reaction that an enzyme catalyses and the role of that reaction within a given metabolic pathway; the protein that a gene or mRNA transcript codes for and the function of that protein; the function of a hormone in a given target tissue, physiological function of an organ, etc. Careful attention should be taken to avoid reference to other KEs, KERs or AOPs. Only describe this KE as a single isolated measurable event/state. This will ensure that the KE is modular and can be used by other AOPs, thereby facilitating construction of AOP networks. More help

Fibroblasts are non-hematopoietic, non-epithelial and non-endothelial cells. In steady state conditions, they are distributed throughout the mesenchyme. During the wound healing process, fibroblasts are rapidly recruited from mesenchymal cells or in case of exaggerated repair, and they can also be derived from fibrocytes in the bone marrow. They are not terminally differentiated. They synthesise structural proteins (fibrous collagen, elastin), adhesive proteins (laminin and fibronectins) and ground substance (glycosaminoglycans – hyaluronan and glycoproteins) proteins of the ECM that provide structural support to tissue architecture and function. Fibroblasts play an important role in ECM maintenance and turnover, wound healing, inflammation and angiogenesis. They provide structural integrity to the newly formed wound. Fibroblasts with a-smooth muscle actin expression are called myofibroblasts. It is thought that differentiating fibroblasts residing in the lung are the primary source of myofibroblast (CD45- Col I+ α-SMA+) cells (Hashimoto et al., 2001; Serini and Gabbiani, 1999). Myofibroblasts can also originate from epithelial-mesenchymal transition (Kim et al., 2006). The other sources of fibroblasts include fibrocytes that likely originate in the bone marrow and migrate to the site of injury upon cytokine signaling. Fibrocytes are capable of differentiating into fibroblasts or myofibroblasts, and comprise less than 1% of the circulating pool of leukocytes and express chemokines CCR2, CXCR4 and CCR7 in addition to a characteristic pattern of biomarkers, including collagen I and III, CD34, CD43 and CD45 (Bucala, 1994; Chesney et al., 1998; Abe et al., 2001).  In bleomycin induced lung fibrosis model, human CD34+ CD45+ collagen I CXCR4+ cells (fibrocytes) are shown to migrate to the lungs in response to both bleomycin and CXCL12 (which is the only chemokine known to bind to CXCR4) (Phillip et al., 2004). Myofibroblasts exhibit features of both fibroblasts and smooth muscle cells. The myofibroblasts synthesise and deposit ECM components that eventually replace the provisional ECM. Because of their contractile properties, they play a major role in contraction and closure of the wound tissue. Apart from secreting ECM components, myofibroblasts also secrete proteolytic enzymes such as metalloproteinases and their inhibitors tissue inhibitor of metalloproteinases, which play a role in the final phase of the wound healing which is scar formation phase or tissue remodelling.

Evidence for its perturbation

IPF is characterised by progressive fibroblast and myofibroblast proliferation and excessive deposition of extracellular matrix (Kuhn et al., 1991). High levels of a-SMA protein and increased number of a-SMA positive cells were observed in mouse lungs treated with MWCNTs as early as day 1 post-exposure (Dong et al., 2015). Fibrotic lesions observed in mice treated with asbestos show proliferating fibroblasts and collagen deposition. The same study also demonstrated that BALF supernatant derived from asbestos exposed lungs was sufficient to stimulate fibroblast proliferation in vitro (Lemaire et al., 1986). Fibrotic foci developed in rat lungs following exposure to bleomycin show a-SMA expressing myofibroblasts (Vyalov et al., 1993). Several in vitro studies have shown fibroblast proliferation following CNT treatment (Wang et al., 2010; Wang et al., 2010; Hussain et al., 2014).

How It Is Measured or Detected

One of the primary considerations in evaluating AOPs is the relevance and reliability of the methods with which the KEs can be measured. The aim of this section of the KE description is not to provide detailed protocols, but rather to capture, in a sentence or two, per method, the type(s) of measurements that can be employed to evaluate the KE and the relative level of scientific confidence in those measurements. Methods that can be used to detect or measure the biological state represented in the KE should be briefly described and/or cited. 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).Key considerations regarding scientific confidence in the measurement approach include whether the assay is fit for purpose, whether it provides a direct or indirect measure of the biological state in question, whether it is repeatable and reproducible, and the extent to which it is accepted in the scientific and/or regulatory community. Information can be obtained from the OECD Test Guidelines website and the EURL ECVAM Database Service on Alternative Methods to Animal Experimentation (DB-ALM). ?

Immunohistochemistry (routinely used and recommended)

Proliferation of fibroblasts and activation of myofibroblasts is normally detected using individual antibodies against vimentin, procollagen 1 and alpha-smooth muscle actin, specific markers of fibroblasts and myofibroblasts (Kai, 1994). It is recommended to use more than one marker to confirm the activation of fibroblasts. The species-specific antibodies for all the markers are commercially available and the technique works in both in vitro and in vivo models as well as in human specimens. Immunohistochemistry is performed using immunoperoxidase technique. Formalin fixed and paraffin embedded lung sections are sliced in 3-5µm thin slices and reacted with diluted H2O2 for 10 min to block the endogenous peroxidase activity. The slices are then incubated with appropriate dilutions of primary antibody against the individual markers followed by incubation with the secondary antibody that is biotinylated. The slices are incubated for additional 30 minutes for avidin-biotin amplification and reacted with substrate 3’3’ diaminobenzidine before visualising the cells under the light microscope. Although only semi-quantitative, morphometric analysis of the lung slices can be conducted to quantify the total number of cells expressing the markers against the control lung sections where expression of specific markers is expected to be low or nil. For the morphometric analysis, using ocular grids, images of 20-25 non-overlapping squares (0.25 mm2) from 2-3 random lung section are taken under 20x magnification. Minimum of three animals per treatment group are assessed. Some researchers include only those cells that are positive for both procollagen I and alpha smooth muscle markers.

The limitation of the technique is that the antibodies have to be of high quality and specific. Background noise due to non-specific reactions can yield false-positive results.

In vitro, expression of type-1 collagen, Thy-1, cyclooxygenase-2 and vaeolin-1 are used as markers of homogeneous population of fibroblasts. Increased expression of TGF-b and a-smooth muscle actin is used as markers of differentiated myofibroblasts. Transcription factor Smad3 is the other marker measured in vitro to assess the fibroblast proliferation and differentiation. Several in vitro studies using lung epithelial cells (e.g. A549 cells) have shown that asbestos induces markers of epithelial-mesenchymal transition (Tamminen et al., 2012), which is mediated by the activation of TGF-β-p-Smad2 (Kim et al., 2006).

Hydrogels

Hydrogels are water-swollen crosslinked polymer networks. They are used to mimic the original extracellular matrix (ECM). Hydrogels consist of collagen, fibrin, hyaluronic acid or synthetic materials such as polyacrylamide enriched with ECM proteins, etc. Hydrogels can be prepared to express inherent biological signals, mechanical properties (e.g., modulus) and biochemical properties (e.g., proteins) of the ECM. Fibroblasts are usually cultured in fibrin and type-1 collagen that represent the matrix of the wound healing. Thus, the well-constructed hydrogel can be used to assess cell proliferation, activation and matrix synthesis as reflective of fibroblast activation. For naturally derived hydrogen scaffolds, cells derived directly from animal or human tissues can be used (Smithmyer et al., 2014).

Fibroblast proliferation assay

Several primary and immortalised fibroblast types can be used for the assay. Proliferation assays such as water-soluble tetrazolium salts (WST)-1 and propidium iodide (PI) staining of cells have been used to show dose-dependent increase in MWCNT-induced increase in fibroblast proliferation that is in alignment with in vivo mouse fibrogenic response (Vietti et al., 2013; Azad et al., 2013) to the same material.

Domain of Applicability

This free text section should be used to elaborate on the scientific basis for the indicated domains of applicability and the WoE calls (if provided). While structured terms may be selected to define the taxonomic, life stage and sex applicability (see structured applicability terms, above) of the KE, the structured terms may not adequately reflect or capture the overall biological applicability domain (particularly with regard to taxa). Likewise, the structured terms do not provide an explanation or rationale for the selection. The free-text section on evidence for taxonomic, life stage, and sex applicability can be used to elaborate on why the specific structured terms were selected, and provide supporting references and background information.  More help

References

List of the literature that was cited for this KE description. Ideally, the list of references, should conform, to the extent possible, with the OECD Style Guide (https://www.oecd.org/about/publishing/OECD-Style-Guide-Third-Edition.pdf) (OECD, 2015). More help
  1. Abe, R., Donnelly, S., Peng, T., Bucala, R. and Metz, C. (2001). Peripheral Blood Fibrocytes: Differentiation Pathway and Migration to Wound Sites. The Journal of Immunology, 166(12), pp.7556-7562.
  2. Azad N, Iyer A.K.V., Lu Y., Wang L., Rojanasakul Y. (2013). P38/MAPK Regulates Single-walled Carbon Nanotube-Induced Fibroblast Proliferation and Collagen Production. Nanotoxicology, 7(2), 157–168.
  3. Bucala, R., Spiegel, L., Chesney, J., Hogan, M. and Cerami, A. (1994). Circulating Fibrocytes Define a New Leukocyte Subpopulation That Mediates Tissue Repair. Molecular Medicine, 1(1), pp.71-81.
  4. Chesney J, Metz C, Stavitsky A-B, et al. (1998) Regulated production of type I collagen and inflammatory cytokines by peripheral blood fibrocytes. J Immunol 160:419–425.
  5. Dong, J., Porter, D., Batteli, L., Wolfarth, M., Richardson, D. and Ma, Q. (2014). Pathologic and molecular profiling of rapid-onset fibrosis and inflammation induced by multi-walled carbon nanotubes. Archives of Toxicology, 89(4), pp.621-633.
  6. Hashimoto, S., Gon, Y., Takeshita, I., Maruoka, S. and Horie, T. (2001). IL-4 and IL-13 induce myofibroblastic phenotype of human lung fibroblasts through c-Jun NH2-terminal kinase–dependent pathway. Journal of Allergy and Clinical Immunology, 107(6), pp.1001-1008.
  7. Hussain, S., Sangtian, S., Anderson, S., Snyder, R., Marshburn, J., Rice, A., Bonner, J. and Garantziotis, S. (2014). Inflammasome activation in airway epithelial cells after multi-walled carbon nanotube exposure mediates a profibrotic response in lung fibroblasts. Particle and Fibre Toxicology, 11(1), p.28.
  8. Kim, K., Kugler, M., Wolters, P., Robillard, L., Galvez, M., Brumwell, A., Sheppard, D. and Chapman, H. (2006). Alveolar epithelial cell mesenchymal transition develops in vivo during pulmonary fibrosis and is regulated by the extracellular matrix. Proceedings of the National Academy of Sciences, 103(35), pp.13180-13185.
  9. Lemaire I, Beaudoin H, Massé S, Grondin C. (1986). Alveolar macrophage stimulation of lung fibroblast growth in asbestos-induced pulmonary fibrosis. Am J Pathol. 122(2):205–211.
  10. Phillips, R., Burdick, M., Hong, K., Lutz, M., Murray, L., Xue, Y., Belperio, J., Keane, M. and Strieter, R. (2004). Circulating fibrocytes traffic to the lungs in response to CXCL12 and mediate fibrosis. Journal of Clinical Investigation, 114(3), pp.438-446.
  11. Serini, G. and Gabbiani, G. (1999). Mechanisms of Myofibroblast Activity and Phenotypic Modulation. Experimental Cell Research, 250(2), pp.273-283.
  12. Smithmyer, M., Sawicki, L. and Kloxin, A. (2014). Hydrogel scaffolds asin vitromodels to study fibroblast activation in wound healing and disease. Biomater. Sci., 2(5), pp.634-650.
  13. Tamminen, J., Myllärniemi, M., Hyytiäinen, M., Keski-Oja, J. and Koli, K. (2012). Asbestos exposure induces alveolar epithelial cell plasticity through MAPK/Erk signaling. Journal of Cellular Biochemistry, 113(7), pp.2234-2247.
  14. Vietti, G., Ibouraadaten, S., Palmai-Pallag, M., Yakoub, Y., Bailly, C., Fenoglio, I., Marbaix, E., Lison, D. and van den Brule, S. (2013). Towards predicting the lung fibrogenic activity of nanomaterials: experimental validation of an in vitro fibroblast proliferation assay. Particle and Fibre Toxicology, 10(1), p.52.
  15. Vyalov SL, Gabbiani G, Kapanci Y. (1993). Rat alveolar myofibroblasts acquire alpha-smooth muscle actin expression during bleomycin-induced pulmonary fibrosis. Am J Pathol. 143(6):1754–1765.
  16. Wang, L., Mercer, R., Rojanasakul, Y., Qiu, A., Lu, Y., Scabilloni, J., Wu, N. and Castranova, V. (2010). Direct Fibrogenic Effects of Dispersed Single-Walled Carbon Nanotubes on Human Lung Fibroblasts. Journal of Toxicology and Environmental Health, Part A, 73(5-6), pp.410-422.
  17. Wang, X., Xia, T., Ntim, S., Ji, Z., George, S., Meng, H., Zhang, H., Castranova, V., Mitra, S. and Nel, A. (2010). Quantitative Techniques for Assessing and Controlling the Dispersion and Biological Effects of Multiwalled Carbon Nanotubes in Mammalian Tissue Culture Cells. ACS Nano, 4(12), pp.7241-7252.
  18. Zhang K. (1994). Myofibroblasts and Their Role in Lung Collagen Gene Expression during Pulmonary Fibrosis. American Journal of Pathology, Vol. 145, No. 1.