Increased, fibroblast proliferation and myofibroblast differentiation

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).

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.

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