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Key Event Title
|Level of Biological Organization|
Key Event Components
|collagen biosynthetic process||collagen||increased|
Key Event Overview
AOPs Including This Key Event
|AOP Name||Role of event in AOP||Point of Contact||Author Status||OECD Status|
|Protein Alkylation to Liver Fibrosis||KeyEvent||Brigitte Landesmann (send email)||Open for citation & comment||WPHA/WNT Endorsed|
|Latent TGFbeta1 activation leads to pulmonary fibrosis||KeyEvent||Marvin Martens (send email)||Under development: Not open for comment. Do not cite|
|lysosomal uptake induced liver fibrosis||KeyEvent||Marina Kuburic (send email)||Under development: Not open for comment. Do not cite||EAGMST Under Review|
|Binding to ACE2 leads to lung fibrosis||KeyEvent||Young Jun Kim (send email)||Open for comment. Do not cite||Under Development|
|AT1R, lung fibrosis||KeyEvent||Young Jun Kim (send email)||Under development: Not open for comment. Do not cite||Under Development|
|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 Do not cite||EAGMST Under Review|
|All life stages|
Key Event Description
Collagen is mostly found in fibrous tissues such as tendons, ligaments and skin. It is also abundant in corneas, cartilage, bones, blood vessels, the gut, intervertebral discs, and the dentin in teeth. In muscle tissue, it serves as a major component of the endomysium. Collagen is the main structural protein in the extracellular space in the various connective tissues, making up from 25% to 35% of the whole-body protein content. In normal tissues, collagen provides strength, integrity, and structure. When tissues are disrupted following injury, collagen is needed to repair the defect. If too much collagen is deposited, normal anatomical structure is lost, function is compromised, and fibrosis results.
The fibroblast is the most common collagen producing cell.Collagen-producing cells may also arise from the process of transition of differentiated epithelial cells into mesenchymal cells (EMT). This has been observed e.g. during renal fibrosis (transformation of tubular epithelial cells into fibroblasts) and in liver injury (transdifferentiation of hepatocytes and cholangiocytes into fibroblasts) (Henderson and Iredale, 2007).
There are close to 20 different types of collagen found with the predominant form being type I collagen. This fibrillar form of collagen represents over 90 percent of our total collagen and is composed of three very long protein chains which are wrapped around each other to form a triple helical structure called a collagen monomer. Collagen is produced initially as a larger precursor molecule called procollagen. As the procollagen is secreted from the cell, procollagen proteinases remove the extension peptides from the ends of the molecule. The processed molecule is referred to as collagen and is involved in fiber formation. In the extracellular spaces the triple helical collagen molecules line up and begin to form fibrils and then fibers. Formation of stable crosslinks within and between the molecules is promoted by the enzyme lysyl oxidase and gives the collagen fibers tremendous strength (Diegelmann,2001). The overall amount of collagen deposited by fibroblasts is a regulated balance between collagen synthesis and collagen catabolism. Disturbance of this balance leads to changes in the amount and composition of collagen. Changes in the composition of the extracellular matrix initiate positive feedback pathways that increase collagen production.
Normally, collagen in connective tissues has a slow turn over; degradating enzymes are collagenases, belonging to the family of matrix metalloproteinases (MMPs).Other cells that can synthesize and release collagenase are macrophages, neutrophils, osteoclasts, and tumor cells (Di Lullo et al., 2001; Prockop and Kivirikko, 1995; Miller and Gay, 1987;Kivirikko and Risteli, 1976).
How It Is Measured or Detected
Determination of the amount of collagen produced in vitro can be done in a variety of ways ranging from simple colorimetric assays to elaborate chromatographic procedures using radioactive and non-radioactive material. What most of these procedures have in common is the need to destroy the cell layer to obtain solubilized collagen from the pericellular matrix. Rishikof et al describe several methods to assess the in vitro production of type I collagen: Western immunoblotting of intact alpha1(I) collagen using antibodies directed to alpha1(I) collagen amino and carboxyl propeptides, the measurement of alpha1(I) collagen mRNA levels using real-time polymerase chain reaction, and methods to determine the transcriptional regulation of alpha1(I) collagen using a nuclear run-on assay (Rishikof et al., (2005) .
Histological staining with stains such as Masson Trichrome, Picro-sirius red are used to identify the tissue/cellular distribution of collagen, which can be quantified using morphometric analysis both in vivo and in vitro. The assays are routinely used and are quantitative.
Sircol Collagen Assay for collagen quantification:
The Serius dye has been used for many decades to detect collagen in histology samples. The Serius Red F3BA selectively binds to collagen and the signal can be read at 540 nm (Chen & Raghunath, 2009; Nikota et al., 2017).
Hydroxyproline is a non-proteinogenic amino acid formed by the prolyl-4-hydroxylase. Hydroxyproline is only found in collagen and thus, it serves as a direct measure of the amount of collagen present in cells or tissues. Colorimetric methods are readily available and have been extensively used to quantify collagen using this assay (Chen & Raghunath, 2009; Nikota et al., 2017).
Ex vivo precision cut tissue slices
Precision cut tissue slices mimic the whole organ response and allow histological assessment, an endpoint of interest in regulatory decision making. While this technique uses animals, the number of animals required to conduct a dose-response study can be reduced to 1/4th of what will be used in whole animal exposure studies (Rahman et al., 2020).
Domain of Applicability
Humans: Bataller and Brenner, 2005; Decaris et al., 2015.
Mice: Dalton et al., 2009; Leung et al., 2008; Nan et al., 2013.
Rats: Hamdy and El-Demerdash, 2012; Li, Li et al., 2012; Natajaran et al., 2006; Luckey and Petersen, 2001.
- Henderson, N.C. and J.P. Iredale (2007), Liver fibrosis: cellular mechanisms of progression and resolution, Clin Sci (Lond), vol. 112, no. 5, pp. 265-280.
- Diegelmann, R.F. (2001), Collagen Metabolism, Wounds, vol. 13, no. 5, available at www.medscape.com/viewarticle/423231 (accessed on 20 Jamuary 2016).
- Di Lullo, G.A. et al. (2001), Mapping the Ligand-binding Sites and Disease-associated Mutations on the Most Abundant Protein in the Human, Type I Collagen, J. Biol. Chem, vol. 277, no. 6, pp. 4223–4231.
- Prockop, D.J. and K.I. Kivirikko (1995), Collagens: molecular biology, diseases, and potentials for therapy, Annu Rev Biochem, vol. 64, pp. 403-434.
- Miller, E.J. and S. Gay (1987), The collagens: an overview and update, Methods Enzymol, vol. 144, pp. 3-41.
- Kivirikko, K.I. and L. Risteli (1976), Biosynthesis of collagen and its alterations in pathological states, Med Biol, vol. 54, no. 3, pp. 159-186.
- Rishikof, D.C. et al. (2005), Methods for measuring type I collagen synthesis in vitro, Methods, Mol Med, vol. 117, pp.129-140.
- Bataller, R. and D.A. Brenner (2005), Liver Fibrosis, J.Clin. Invest, vol. 115, no. 2, pp. 209-218.
- Decaris, M.L. et al. (2015), Turnover rates of hepatic collagen and circulating collagen- associated proteins in humans with chronic liver disease, PLoS One, vol. 10, no. 4, e0123311.
- Dalton, S.R. et al. (2009), Carbon tetrachloride-induced liver damage in asialoglycoprotein receptor-deficient mice, Biochem Pharmacol, vol. 77, no. 7, pp. 1283-1290.
- Leung, T.M. et al. (2008), Endothelial nitric oxide synthase is a critical factor in experimental liver fibrosis, Int J Exp Pathol, vol. 89, no. 4, pp. 241-250.
- Nan, Y.M. et al. (2013), Activation of peroxisome proliferator activated receptor alpha ameliorates ethanol mediated liver fibrosis in mice, Lipids in Health and Disease, vol. 12, p. 11.
- Hamdy, N. and E. El-Demerdash. (2012), New therapeutic aspect for carvedilol: antifibrotic effects of carvedilol in chronic carbon tetrachloride-induced liver damage, Toxicol Appl Pharmacol, vol. 261, no. 3, pp. 292-299.
- Li, Li et al. (2012), Establishment of a standardized liver fibrosis model with different pathological stages in rats, Gastroenterol Res Pract; vol. 2012, Article ID 560345.Natajaran, S.K. et al. (2006), Oxidative stress in the development of liver cirrhosis: a comparison of two different experimental models, J Gastroenterol Hepatol, vol. 21, no. 6, pp. 947-957.
- Natarajan SK, Thomas S, Ramamoorthy P, et al. Oxidative stress in the development of liver cirrhosis: a comparison of two different experimental models. J Gastroenterol Hepatol. 2006;21:947–957. doi: 10.1111/j.1440-1746.2006.04231.x
- Luckey, S.W., and D.R. Petersen (2001), Activation of Kupffer cells during the course of carbon tetrachloride-induced liver injury and fibrosis in rats, Exp Mol Pathol, vol. 71, no. 3, pp. 226-240.
Nikota, J., Banville, A., Goodwin, L., Wu, D., Williams, A., Yauk, C., Wallin, H., Vogel, U. and Halappanavar, S. (2017). Stat-6 signaling pathway and not Interleukin-1 mediates multi-walled carbon nanotube-induced lung fibrosis in mice: insights from an adverse outcome pathway framework. Particle and Fibre Toxicology, 14(1).
Rahman, L., Williams, A., Gelda, K., Nikota, J., Wu, D., Vogel, U., & Halappanavar, S. (2020). 21st Century Tools for Nanotoxicology: Transcriptomic Biomarker Panel and Precision-Cut Lung Slice Organ Mimic System for the Assessment of Nanomaterial-Induced Lung Fibrosis. Small, 16(36), e2000272.
Chen, C. and Raghunath, M. (2009). Focus on collagen: in vitro systems to study fibrogenesis and antifibrosis _ state of the art. Fibrogenesis & Tissue Repair, 2(1).