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Key Event Title
Increased, fibroblast proliferation and myofibroblast differentiation
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 Do not cite||EAGMST Under Review|
Key Event Description
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
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 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
- 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.
- 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.
- 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.
- 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.
- 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.
- 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.
- 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.
- 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.
- 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.
- 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.
- Serini, G. and Gabbiani, G. (1999). Mechanisms of Myofibroblast Activity and Phenotypic Modulation. Experimental Cell Research, 250(2), pp.273-283.
- 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.
- 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.
- 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.
- 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.
- 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.
- 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.
- Zhang K. (1994). Myofibroblasts and Their Role in Lung Collagen Gene Expression during Pulmonary Fibrosis. American Journal of Pathology, Vol. 145, No. 1.