API

Event: 1500

Key Event Title

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Increased, fibroblast proliferation and myofibroblast differentiation

Short name

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Increased cellular proliferation and differentiation

Biological Context

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Level of Biological Organization
Tissue


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Key Event Components

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Process Object Action

Key Event Overview


AOPs Including This Key Event

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Sex Applicability

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Key Event Description

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Fibroblasts are non-hematopoietic, non-epithelial and non-endothelial cells. In steady state conditions, they are distributed throughout the mesenchyme (Phan SH, 2008). 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. Fibroblasts are the workhorse of the connective tissue that joins and holds other tissues together in the body. 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. In the context of lung fibrosis, fibroblasts play an important role in ECM maintenance and turnover, wound healing, inflammation and angiogenesis (Kendall RT, 2014). 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 et al 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 (CXCL12 binds 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[SH1] . 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.

Epithelial-mesenchymal transition is a process by which epithelial cells lose their original phenotype, acquire fibroblast-like properties, and display reduced cell adhesion (which is crucial for the detachment of epithelial cells prior to migration to the site of injury) and increased motility (Lekkerkerker et al 2012). During this process, epithelial cells also lose typical markers such as E-cadherin and zona occludens-1 (ZO-1) and acquire mesenchymal markers such as fibroblast-specific protein-1, vimentin and α-SMA (Grunert et al 2003; Zeisberg and Neilson 2009). 

 [SH1]Darby Clinical, Cosmetic and Investigational Dermatology 2014:7 301–311


How It Is Measured or Detected

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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 (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 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 (Megan 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 to the same material (Vietti 2013; Azad 2013)


Domain of Applicability

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References

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  1. Phan SH. Biology of Fibroblasts and Myofibroblasts. Proceedings of the American Thoracic Society. 2008;5(3):334-337. doi:10.1513/pats.200708-146DR.
  2. Kendall RT, Feghali-Bostwick CA. Fibroblasts in fibrosis: novel roles and mediators. Frontiers in Pharmacology. 2014;5:123. doi:10.3389/fphar.2014.00123.
  3. Shu Hashimoto, Yasuhiro Gon, Ikuko Takeshita, Shuichiro Maruoka, and Takashi Horie. 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 , Volume 107 , Issue 6 , 1001 – 1008.
  4. Serini G, Gabbiani G. Mechanisms of myofibroblast activity and phenotypic modulation. Exp Cell Res. 1999 Aug 1;250(2):273–283.
  5. 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 USA 2006;103:13180–13185Kim et al 2006. Proc. Natl. Acad. Sci. USA 103: 13180-13185.
  6. 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.
  7. 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.
  8. Riichiro Abe, Seamas C. Donnelly, Tina Peng, Richard Bucala and Christine N. Metz. J Peripheral Blood Fibrocytes: Differentiation Pathway and Migration to Wound Sites Immunol  June 15, 2001,  166  (12)  7556-7562.
  9. N. Lekkerkerker, Annemarie; Aarbiou, Jamil; van Es, Thomas; A.J. Janssen, Richard. Cellular Players in Lung Fibrosis Current Pharmaceutical Design, Volume 18, Number 27, September 2012, pp. 4093-4102(10).
  10. Grünert S, Jechlinger M, Beug H. Diverse cellular and molecular mechanisms contribute to epithelial plasticity and metastasis. 2003. Nat. Rev. Mol. Cell Biol. 4: 657-665.
  11. Zeisberg M, Neilson EG. Biomarkers for epithelial-mesenchymal transitions. The Journal of Clinical Investigation. 2009;119(6):1429-1437.
  12. Kai Zhang, Mark D. Rekhter, David Gordon,and Sem H. Phan. Myofibroblasts and Their Role in Lung Collagen Gene Expression during Pulmonary Fibrosis A Combined Immunohistochemical and in Situ Hybridization Study. American Journal of Pathology, Vol. 145, No. 1, July 1994
  13. Tamminen, J. A., Myllärniemi, M., Hyytiäinen, M., Keski-Oja, J. and Koli, K. (2012), Asbestos exposure induces alveolar epithelial cell plasticity through MAPK/Erk signaling. J. Cell. Biochem., 113: 2234–2247.
  14. Kim KK, Kugler MC, Wolters PJ, et al. 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 of the United States of America. 2006;103(35):13180-13185.
  15. Megan E. Smithmyer, Lisa A. Sawicki and April M. Kloxin. Hydrogel scaffolds as in vitro models to study fibroblast activation in wound healing and disease Biomater. Sci., 2014, 2, 634.
  16. Vietti G, Ibouraadaten S, Palmai-Pallag M, et al. Towards predicting the lung fibrogenic activity of nanomaterials: experimental validation of an in vitro fibroblast proliferation assay. Particle and Fibre Toxicology. 2013;10:52. doi:10.1186/1743-8977-10-52.
  17. Azad N, Iyer A.K.V., Lu Y., Wang L., Rojanasakul Y. P38/MAPK Regulates Single-walled Carbon Nanotube-Induced Fibroblast Proliferation and Collagen Production. Nanotoxicology (2013),7(2), 157–168.