Increased cellular proliferation and differentiation leads to Increased extracellular matrix deposition

The biological plausibility of this KER is high. There is an accepted mechanistic relationship between activated myofibroblasts, and the capacity to secrete collagen (Hinz, 2016a; Hinz, 2016b; Hu & Phan, 2013).

The empirical evidence to support this KER is high. It is generally accepted knowledge that activated myofibroblasts are collagen secreting cells (Blaauboer et al., 2014; Hinz, 2016a; Li et al., 2017; For additional references see Table 1).

Mice infused subcutaneously with bleomycin showed pronounced lung fibrosis, characterised by elevated levels of TGFβ1 and collagen genes (Hoyt and Lazo, 1988). Radiation induced lung fibrosis was shown to precede high levels of TGFβ1 expression (Yi et al., 1996). Mice lacking TGFβ-receptor II showed resistance to bleomycininduced lung fibrosis (Li et al., 2011). Inhibition of fibroblast proliferation and differentiation by counteracting the activity of TGF-β attenuates bleomycin-induced lung fibrosis (Chen et al., 2013; Guan et al., 2016). Adenoviral vector-mediated gene transfer based transient overexpression of TGFb1 in lungs of mice induced progressive lung fibrosis (Bonniaud et al., 2004). Targeted inhibition of Wnt/b-catenin signalling by a small molecule drug inhibited the mesenchymal-myofibroblast transition and repressed matrix gene expression leading to attenuated lung fibrosis (Cao et al., 2018).

Dose-Response Relationship:

There are a number of in vitro and in vivo studies that indicate a dose-response relationship in this KER. At a higher dose of the stressor, an increased in fibroblast proliferation and myofibroblast differentiation leads to increases in extracellular matrix deposition.

Ma et al. (2017) studied the role of epithelial-mesenchymal transition (EMT) in cerium oxide (CeO₂) induced fibrosis. Male Sprague-Dawley rats were exposed to 0.15-7 mg/kg cerium oxide via intratracheal instillation and sacrificed at various times post-exposure. At 28 days post-exposure there was a dose-dependant increase in hydroxyproline content in lung tissue.  Mice exposed to 3.5 mg/kg showed an increase in soluble collagen levels in BALF at day 3 and day 28 and an increase in α-SMA expression levels in lung tissue with a peak at day 1 post-exposure. From CeO₂-exposed rats (3.5 mg/kg), macrophages, fibroblast, and alveolar type II (ATII) cells were isolated. Macrophages produced TGF-b1 with peaks at day 3 and 10 post-exposure. Fibroblast proliferation decreased in a dose-dependant manner, and an increase in the levels of a-SMA in fibroblasts and ATII at day 28 post-exposure. They concluded that CeO₂ exposure affects fibroblast function and induces EMT in ATII cells.

Blaauboer et al. (2014) studied the expression of elastin, type V collagen, and tenascin C during the development of lung fibrosis and the effect of myofibroblast differentiation on this expression. Female C57Bl/6 mice were exposed to a single intratracheal instillation bleomycin 30 ml (1.25 U/ml in PBS). Seven days before sacrifice, mice received 35 ml D₂O/g via intraperitoneal injection to label new collagen. Mice were sacrificed 1, 3, 4 or 5 weeks post exposure. An increase in the level of a-SMA protein level in histological staining was observed, with a peak after 2 weeks. Extracellular matrix proteins levels increased (histological staining). Elastin increased in a time-dependant manner with a peak after 4 weeks. Type V collagen and tenascin C increased after 1 week and decreased over time. They found that gene expression of elastin, type V collagen and tenascin C highly correlated to new collagen formation.  Primary normal human lung fibroblast and human fetal lung fibroblast were exposed to different concentrations of TGF-b1. The expression of ACTA, COL1A1, ELN, COL5A1 and TNC increased in a dose-response relationship after 24 h. Fibroblast cultured in elastin coatings and stimulated with 10 ng/ml TGF-b1 for 48 h, showed an increase in the levels of ACTA2, COL1A1, and ELN. The In vitro study demonstrated that fibrotic changes in the composition of the extracellular matrix have a regulatory role during fibrosis development.

Judge et al. (2015) determined that the lactate dehydrogenase-A (LDHA) enzyme was upregulated in radiation and that lactate is required for radiation-induced myofibroblast differentiation. In lung biopsies obtained from patients who received thoracic radiation for cancer treatment, an overexpression of LDHA and a-SMA by immunostaining was seen, as well as the accumulation of collagen fibers. Mice C57BL/6mice were exposed to 5 Gy total body plus 10 Gy thoracic radiation. They found that LDHA overexpressed in lungs at 26 week, and LDHA mRNA increased over time at 16-26 weeks (post-radiation). Primary human lung fibroblasts were exposed to 3, and 7 Gy. At the highest dose 5 days post-radiation, an increase in the levels of LDHA protein expression, extracellular acidification, lactate levels in supernatants, a-SMA protein expression, soluble collagen I, Col1A1, and Col3A1 mRNA levels, and TGF-b1 bioactivity was seen. LDHA siRNA and an LDH inhibitor, inhibits radiation-induced myofibroblast differentiation.

Lia et al. (2018) studied whether copper oxide nanoparticles (CuO NPs) could induce epithelial cell injury, pulmonary inflammation, and fibrosis in C57BL/6 mice. Animals were nasally instilled with 1, 2.5, 5, and 10 mg/kg of CuO NPs, and responses were evaluated at 7, 14, and 28 days post-exposure. In a dose-dependent manner, authors found increased mRNA levels of proinflammatory genes such as CCL-2, CCL-3, IL-4, IL-10, IFN-α, and TGF-β1 in lung tissue. Cell apoptosis was also increased in a dose-dependant manner at 1, 2.5, and 5 mg/Kg. Also, the increase of TGF-B1 in BALF at day 14 and the increase of α-SMA at day 28 in lung tissue followed a dose-response relation ship at 2.5 and 5 mg/Kg. After 28 days of exposure, there was an increase in collagen-I and hydroxyproline content at 2.5 and 5 mg/Kg.

Temporal Relationship:

In vitro and in vivo studies highlight the temporal relationship between the two KEs in this KER.

Osterholzer et al. (2013) evaluated local inflammation and fibrosis after a targeted epithelial insult. Wild type (WT) C57BL/6 and transgenic mice expressing the diphtheria toxin receptor were intraperitoneally injected with diphtheria toxin (DT) once daily for 14 days at a dose of 100 mg/kg in 100 ml of PBS. Observations were evaluated at various days post DT initiation. At day 7 and 14, an accumulation of exudate macrophages and Ly-6C^high monocytes was observed. The immunophenotype of ExM and Ly-6C^high monocytes at day 14 showed an expression of arginase, iNOs, IL-13, TGF-b, CD45+, Col1+, and CCR4. Chemokine-receptor-2 deficient mice did not show an accumulation of inflammatory cells and fibrosis. Finally, at day 21, lung collagen deposition was evident, as measured by hydroxyproline content. 

Fang et al. (2018) studied the endothelial-mesenchymal transition characterized by the loss of endothelial specific markers (Cdh5, PECAM1), the acquisition of the mesenchymal markers (Col1A1, Acta 2), and the expression of a-SMA and Collagen I and III. Stock TEK-GFP 287 Sato/JNiu Tie2-GFP mice were administered with 0.5 g/Kg SiO₂ instilled intratracheally in one dose. After 28 days of treatment GFP were localized with a-SMA/Acta2 and the amount of Sirius red (collagen I and III) increased.  Mouse microvascular lung cells (MML1) were exposed to 50 mg/cm² for 0, 6, 12, 24, and 48 h. An increase in the level of mesenchymal markers, a decrease in the level of endothelial markers, and an increase in cell proliferation and migration were observed after 12 h in a time-dependant manner. The exposure to SiO₂ increased the expression of circHECTD1 (a circular RNA which regulates the SiO₂-induced endothelial mesenchymal transition) after 1, 3 and 24 h of exposure, and decrease the expression of HECTD1 12, 24 and 48 h post-exposure.

Activated macrophages secrete TIMP1 into the ECM to inhibit matrix metalloproteinases and this could promote cell proliferation and inhibit fibroblast apoptosis through CD63/integrin b1 ERK signaling. Dong et al. (2017) characterized TiMP1 expression after multiwalled carbon nanotube (MWCNT) exposure. Male C57BL/6J WT and B6.129S4-Timp1tm1Pds/J (Timp1 KO) mice were administered with MWCNT at 40 mg per mouse by pharyngeal aspiration. Lungs were harvested at 1, 3, 7- and 14-days post-exposure. TIMP1 mRNA and protein levels increased in lung, BALF and serum at day 1, and then decreased over time. A similar behavior was observed for FN1, FSP, Ki67 and PCNA expression with a peak at 7 and 14 days post exposure. Collagen deposition was observed at 1 day post exposure with a peak at day 7. At day 7 they also observed an increase in the expression of Hsp47, vimentin, a-SMA, PDGFR-b, and genes involved in cell cycle regulation (Bub1b, Capg, Cenpa, Kif2c, Kif22, Mcm5, Plk1 and Tuba 6). TIMP1 KO mice displayed reduced responses. The formation of TIMP1/CD63/integrin b1 complex on the cell surface lead to an activation of the Erk1/3 pathway.

Hu et al. (2015) studied the effects of conditional mesenchymal-specific deletion of Notch1 on pulmonary fibrosis. A conditional knockout of Notch1 (CKO) in collagen I-expressing mesenchymal cells was generated (Notch1fl/fl, Col1a2-cre-ER(T)+/0). Col1a2-cre-ER(T)+/0 with WT Notch1 mice and CKO were given daily intra peritoneal injections of tamoxifen for 8 days to induce mesenchymal cell-specific expression of the Cre-ER(T) recombinase and the removal of the floxed Notch1 (Notch1 CKO Mice). Control mice and Notch1 CKO were injected endotracheally with 2 U/Kg Bleomycin. Mice were sacrificed after 7, 14, and 21 days. Jagged1 and Notch1 protein expression increased with a peak at 7- and 14-days post-exposure. After 14 days of the treatment, an increase in the levels of mRNA and protein of a-SMA and Col1 was seen, as well as an increase in the percentage of a-SMA+ lung fibroblasts. 28 days post-exposure there was an increase in the content of hydroxyproline in lungs. CKO mice showed a significant attenuation of collagen deposition and myofibroblast differentiation.

Li et al. (2017) evaluated whether low-dose cadmium exposure induces peribronchiolar fibrosis through site-specific phosphorylation of vimentin. C57BL/6 mice were exposed to 0.009 or 0.018 mg/kg cadmium chloride (CdCl₂) via non-surgical intratracheal instillation in saline every other day for eight weeks. On weeks 1, 2, 4, and 8, mice were sacrificed, and lungs were removed for histology. At week 4, the expression of α-SMA, collagen-I, and picro-sirius red increased. Also, subepithelial thickness and airway resistance increased at this time point. Collagen content was also raised in a time-dependant manner. In a parallel experiment, primary human fibroblasts were incubated with CdCl₂ at 5, 10, and 20 μM for 3 h and then allowed to recover for 3, 24, 48, and 72 h. α-SMA protein expression and soluble collagen increased in a dose-dependent manner; meanwhile, α-SMA, fibronectin, and collagen-I increased in a time-dependant manner. These results demonstrated that cadmium induces myofibroblast differentiation and extracellular matrix deposition around small airways.

Several studies have shown that inhibition of TGF-β involved in fibroblast activation and collagen deposition results in attenuated fibrotic response in lungs; however, results are inconsistent. More studies are required to support the quantitative KER.