Focus on collagen: in vitro systems to study fibrogenesis and antifibrosis _ state of the art

Abstract

Fibrosis represents a major global disease burden, yet a potent antifibrotic compound is still not in sight. Part of the explanation for this situation is the difficulties that both academic laboratories and research and development departments in the pharmaceutical industry have been facing in re-enacting the fibrotic process in vitro for screening procedures prior to animal testing. Effective in vitro characterization of antifibrotic compounds has been hampered by cell culture settings that are lacking crucial cofactors or are not holistic representations of the biosynthetic and depositional pathway leading to the formation of an insoluble pericellular collagen matrix. In order to appreciate the task which in vitro screening of antifibrotics is up against, we will first review the fibrotic process by categorizing it into events that are upstream of collagen biosynthesis and the actual biosynthetic and depositional cascade of collagen I. We point out oversights such as the omission of vitamin C, a vital cofactor for the production of stable procollagen molecules, as well as the little known in vitro tardy procollagen processing by collagen C-proteinase/BMP-1, another reason for minimal collagen deposition in cell culture. We review current methods of cell culture and collagen quantitation vis-à-vis the high content options and requirements for normalization against cell number for meaningful data retrieval. Only when collagen has formed a fibrillar matrix that becomes cross-linked, invested with ligands, and can be remodelled and resorbed, the complete picture of fibrogenesis can be reflected in vitro. We show here how this can be achieved. A well thought-out in vitro fibrogenesis system represents the missing link between brute force chemical library screens and rational animal experimentation, thus providing both cost-effectiveness and streamlined procedures towards the development of better antifibrotic drugs.

Control of lung vascular permeability and endotoxin-induced pulmonary oedema by changes in extracellular matrix mechanics

“…LOX inhibition also can render collagen more susceptible to degradation 50 ; however, we could not detect any change in collagen protein levels or degradation (measured by quantitating hydroxyproline levels) in BAPN-treated mice. On the other hand, past studies have shown that even a partial inhibition of collagen crosslinking results in changes in aortic wall stiffness 51,52 .…”

Fibroblast-specific inhibition of TGF-β1 signaling attenuates lung and tumor fibrosis

“…Fibroblasts were cultured on a 10-cm dish until confluent. The medium was then changed to DMEM containing 5% FBS, 100 μM l-ascorbic acid with 500 kDa dextran sulfate at 100 μg/ml, and 50 ng/ml recombinant human LOXL2 for 7 days (36,37). The cell layer was extracted with 0.5 M acetic acid and 0.1 mg/ml pepsin overnight at 4°C.…”

“…in monomeric collagens then undergo a series of spontaneous condensation reactions that result in the formation of intra-and intermolecular covalent collagen cross-links (35). We first established an assay of collagen cross-linking induced by recombinant LOXL2 (36,37) and found that corilagin and all other trihydroxyphenolics tested prevented cross-linking (IC 50 = 10 nM; Supplemental Figure 3, A-C) whereas an inhibitor of TGF-β1 signaling (SB431542) and an antioxidant (N-acetylcysteine) had no effect ( Figure 3B). LOXL2 enzymatic activity toward a model substrate was assessed monitoring H 2 O 2 release.…”

Activation of Prostaglandin FP and EP2 Receptors Differently Modulates Myofibroblast Transition in a Model of Adult Primary Human Trabecular Meshwork Cells

“…For the detection of collagen deposition, L-ascorbic acid 2-phosphate (Sigma-Aldrich Corp.) was added to the TM cell medium to allow collagen maturation. 40 Ninety-six hours after cell treatment with the various compounds tested, TM cells were fixed either with ice-cold methanol for the detection of collagen deposition or fibronectin, or with 4% paraformaldehyde for the detection of actin stress fibers, a-SMA, and vinculin. The cells were treated with 0.1% Triton X-100 and 3% normal goat serum (NGS) for 1 hour and then incubated overnight at þ48C with relevant antibodies in a 1% NGS solution.…”