P45 In vitro fibrillar collagen assay development to enable drug discovery in fibrosis
Wai Theng Mak, F Rifkhana Shah Jalan, Deyvapriya Siva, Rachel E B WatsonAbstract
Introduction and aims
The extracellular matrix (ECM) is essential for maintaining tissue architecture and function. Collagens, the most abundant ECM proteins, require tightly regulated biosynthesis for tissue homeostasis. Dysregulated collagen production contributes to fibrosis and age-related tissue degeneration, leading to impaired organ function and increased morbidity. Here, we developed 2-dimensional (2D) and 3-dimensional (3D) in vitro models to quantify collagen production to validate the effect of compounds that may modulate collagen synthesis.
Methods
Human dermal fibroblasts (FBs) were cultured until D14 postconfluence; from D7, cells received 10 ng mL−1 transforming growth factor (TGF)-β1 to increase collagen biosynthesis. On D10, cells were transferred to serum-free media containing phosphate-buffered saline (control), ethanol (vehicle), or 0.1 mmol L–1 ethyl-3,4-dihydrobenzoate (EDHB), a prolyl 4-hydroxylase inhibitor which blocks collagen biosynthesis. Collagens type I and III were assessed by quantitative polymerase chain reaction (n = 3) and Western blot (n = 3), secreted collagen via enzyme-linked immunosorbent assay (pCOL1; n = 3) and cellular and ECM-deposited proteins by immunofluorescence (n = 3; pCOL1, COL1, pCOL3, COL3). Human skin equivalents (HSEs) (3D) were developed using FBs from either healthy or hypertrophic scar skin seeded into a rat tail collagen-derived scaffold; after 2 days, matched keratinocytes were added, constructs air-lifted and cultured for a further D14. Western blots were analysed in ImageJ normalized to Ponceau staining. (Pro)collagen types I and III were assessed via immunofluorescence (n = 3); images were processed by Otsu-thresholding. In 2D cultures, integrated intensity per image was measured. In HSEs, signals were thresholded to distinguish nuclear overlapping from ECM proteins. Data were analysed using either paired Student’s t-tests or Anova, as appropriate, with significance taken at the 95% confidence level.
Results
In 2D, TGF-β1 significantly upregulated collagen (P < 0.05) while EDHB treatment significantly reduced it (P < 0.05). In HSEs, collagen levels significantly increased over time in culture and were higher in constructs derived from healthy skin.
Conclusions
This study provides methods to assess the impact of drug treatments on collagen biosynthesis.