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Event: 1500

Key Event Title

A descriptive phrase which defines a discrete biological change that can be measured. 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. More help
Increased cellular proliferation and differentiation
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Biological Context

Structured terms, selected from a drop-down menu, are used to identify the level of biological organization for each KE. More help
Level of Biological Organization
Tissue

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

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; https://aopwiki.org/info_pages/2/info_linked_pages/7#List). 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

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 Moderate NCBI

Life Stages

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

Sex Applicability

An indication of the the relevant sex for this KE. 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. 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 extracellular matrix (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 Alpha smooth muscle actin (α-SMA) 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 (EMT) (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 C-C motif chemokine receptor (CCR)2, C-X-C chemokine receptor (CXCR)4 and CCR7 in addition to a characteristic pattern of biomarkers, including collagen I and III, CD34, CD43 and CD45 (Abe et al., 2001; Bucala et al., 1994; Chesney et al., 1998). 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 C-X-C motif chemokine ligand (CXCL)12 (which is the only chemokine known to bind to CXCR4) (Phillips 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.

Literature evidence for its perturbation in the context of pulmonary fibrosis:

Idiopathic pulmonary fibrosis is characterised by progressive fibroblast and myofibroblast proliferation and excessive deposition of ECM (Kuhn and McDonald., 1991). High levels of α -SMA protein and increased number of α-SMA positive cells were observed in mouse lungs treated with multi-walled carbon nanotubes (MWCNTs) as early as day 1 post-exposure (Dong and Ma, 2016). Fibrotic lesions observed in mice treated with asbestos show proliferating fibroblasts and collagen deposition. The same study also demonstrated that bronchoalveolar lavage fluid 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 α-SMA expressing myofibroblasts (Vyalov et al., 1993). Several in vitro studies have shown fibroblast proliferation following carbon nanotube treatment (Hussain et al., 2014; Wang et al., 2010a; Wang et al., 2010b).

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

Immunohistochemistry (routinely used and recommended):

Proliferation of fibroblasts and activation of myofibroblasts is normally detected using individual antibodies against vimentin, procollagen 1 and α-SMA, specific markers of fibroblasts and myofibroblasts (Zhang, 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 semiquantitative, 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 mm) 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 α-SMA 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 cell surface antigen (Thy-1), cyclooxygenase-2 (COX2) and caveolin-1 (CAV1) are used as markers of homogeneous population of fibroblasts. Increased expression of Transforming growth factor beta (TGF-β) and α-SMA is used as markers of differentiated myofibroblasts. Transcription factor SMAD family member 3 (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 EMT (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 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 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 (Azad et al., 2013; Vietti et al., 2013) to the same material.

Advanced co-culture models (myofibroblast differentiation):

Co-culture models that mimic the alveolar capillary membrane (such as those listed for Event 1496 & Event 1498) can be used to assess myofibroblast differentiation in response to pro-fibrotic stressors using immunofluorescent staining for α-SMA. More complex in vitro microfluidic lung-on-a-chip models (such as the one listed for Event 1497) can be used to assess myofibroblast differentiation in the same stead. These provide a more realistic exposure model as opposed to a submerged monoculture of fibroblasts, however they require a higher degree of technical skill and advanced fabrication which may not be suitable for all labs.

Domain of Applicability

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

References

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

1. Abe R, Donnelly SC, Peng T, Bucala R, Metz CN. Peripheral blood fibrocytes: differentiation pathway and migration to wound sites. J Immunol. 2001 Jun 15;166(12):7556-62. doi: 10.4049/jimmunol.166.12.7556. 

2. Azad N, Iyer AK, Wang L, Liu Y, Lu Y, Rojanasakul Y. Reactive oxygen species-mediated p38 MAPK regulates carbon nanotube-induced fibrogenic and angiogenic responses. Nanotoxicology. 2013 Mar;7(2):157-68. doi: 10.3109/17435390.2011.647929.

3. Bucala R, Spiegel LA, Chesney J, Hogan M, Cerami A. Circulating fibrocytes define a new leukocyte subpopulation that mediates tissue repair. Mol Med. 1994 Nov;1(1):71-81.

4. Chesney J, Metz C, Stavitsky AB, Bacher M, Bucala R. Regulated production of type I collagen and inflammatory cytokines by peripheral blood fibrocytes. J Immunol. 1998 Jan 1;160(1):419-25.

5. Dong J, Ma Q. Myofibroblasts and lung fibrosis induced by carbon nanotube exposure. Part Fibre Toxicol. 2016 Nov 4;13(1):60. doi: 10.1186/s12989-016-0172-2.  

6. Hashimoto S, Gon Y, Takeshita I, Maruoka S, Horie T. IL-4 and IL-13 induce myofibroblastic phenotype of human lung fibroblasts through c-Jun NH2-terminal kinase-dependent pathway. J Allergy Clin Immunol. 2001 Jun;107(6):1001-8. doi: 10.1067/mai.2001.114702.

7. Hussain S, Sangtian S, Anderson SM, Snyder RJ, Marshburn JD, Rice AB, Bonner JC, Garantziotis S. Inflammasome activation in airway epithelial cells after multi-walled carbon nanotube exposure mediates a profibrotic response in lung fibroblasts. Part Fibre Toxicol. 2014 Jun 10;11:28. doi: 10.1186/1743-8977-11-28. 

8. Kim KK, Kugler MC, Wolters PJ, Robillard L, Galvez MG, Brumwell AN, Sheppard D, Chapman HA. Alveolar epithelial cell mesenchymal transition develops in vivo during pulmonary fibrosis and is regulated by the extracellular matrix. Proc Natl Acad Sci U S A. 2006 Aug 29;103(35):13180-5. doi: 10.1073/pnas.0605669103.

9. Kuhn C, McDonald JA. The roles of the myofibroblast in idiopathic pulmonary fibrosis. Ultrastructural and immunohistochemical features of sites of active extracellular matrix synthesis. Am J Pathol. 1991 May;138(5):1257-65. 

10. Lemaire I, Beaudoin H, Massé S, Grondin C. Alveolar macrophage stimulation of lung fibroblast growth in asbestos-induced pulmonary fibrosis. Am J Pathol. 1986 Feb;122(2):205-11.

11. Phillips RJ, Burdick MD, Hong K, Lutz MA, Murray LA, Xue YY, Belperio JA, Keane MP, Strieter RM. Circulating fibrocytes traffic to the lungs in response to CXCL12 and mediate fibrosis. J Clin Invest. 2004 Aug;114(3):438-46. doi: 10.1172/JCI20997. 

12. Serini G, Gabbiani G. Mechanisms of myofibroblast activity and phenotypic modulation. Exp Cell Res. 1999 Aug 1;250(2):273-83. doi: 10.1006/excr.1999.4543.

13. Smithmyer ME, Sawicki LA, Kloxin AM. Hydrogel scaffolds as in vitro models to study fibroblast activation in wound healing and disease. Biomater Sci. 2014 May 1;2(5):634-650. doi: 10.1039/C3BM60319A. 

14. Tamminen JA, Myllärniemi M, Hyytiäinen M, Keski-Oja J, Koli K. Asbestos exposure induces alveolar epithelial cell plasticity through MAPK/Erk signaling. J Cell Biochem. 2012 Jul;113(7):2234-47. doi: 10.1002/jcb.24094.

15. Vietti G, Ibouraadaten S, Palmai-Pallag M, Yakoub Y, Bailly C, Fenoglio I, Marbaix E, Lison D, van den Brule S. Towards predicting the lung fibrogenic activity of nanomaterials: experimental validation of an in vitro fibroblast proliferation assay. Part Fibre Toxicol. 2013 Oct 10;10:52. doi: 10.1186/1743-8977-10-52. 

16. Vyalov SL, Gabbiani G, Kapanci Y. Rat alveolar myofibroblasts acquire alpha-smooth muscle actin expression during bleomycin-induced pulmonary fibrosis. Am J Pathol. 1993 Dec;143(6):1754-65. 

17. Wang L, Mercer RR, Rojanasakul Y, Qiu A, Lu Y, Scabilloni JF, Wu N, Castranova V. Direct fibrogenic effects of dispersed single-walled carbon nanotubes on human lung fibroblasts. J Toxicol Environ Health A. 2010a;73(5):410-22. doi: 10.1080/15287390903486550. 

18. Wang X, Xia T, Ntim SA, Ji Z, George S, Meng H, Zhang H, Castranova V, Mitra S, Nel AE. Quantitative techniques for assessing and controlling the dispersion and biological effects of multiwalled carbon nanotubes in mammalian tissue culture cells. ACS Nano. 2010b Dec 28;4(12):7241-52. doi: 10.1021/nn102112b. 

19. Zhang K, Rekhter MD, Gordon D, Phan SH. Myofibroblasts and their role in lung collagen gene expression during pulmonary fibrosis. A combined immunohistochemical and in situ hybridization study. Am J Pathol. 1994 Jul;145(1):114-25.