Quantitative assessment of collagen assembly by live cells

Abstract

Changes in supramolecular assembly of matrix, specifically collagen, have important functional consequences, especially for tissues requiring high mechanical strength. Thus modulation of collagen assembly could be used as a therapeutic intervention or to control the development of tissue-engineered constructs containing natural matrix. Quantitative methods that monitor such effects currently are lacking. Using live cultured cells, we developed a convenient way either to visualize by fluorescence microscopy or to measure directly, using a high throughput fluorescence assay, the supramolecular assembly of FITC-labeled collagen monomers. The wide applicability of this assay was confirmed by testing the assay using two major collagen sources, rat tail and bovine skin, and vascular smooth muscle cells from two different origins, mouse aorta and human saphenous vein. We further determined that treatments that interfere with the function of the cytoskeleton modulate collagen assembly. Use of positive and negative regulators of lysyl-oxidase indicated that while the assay does not require active production of endogenous collagen, it can be used to monitor the incorporation of such de novo synthesized collagen into labeled fibrils. Thus we have designed a novel quantitative assay that can monitor assembly of exogenous and endogenous collagen by live cells and reveal the effects of various interventions upon this process.

Matrix Metalloproteinase-2 and −9 Differentially Regulate Smooth Muscle Cell Migration and Cell-Mediated Collagen Organization

“…To gain mechanistic insight into this process, we used a novel assay that allows quantification of cell-mediated supramolecular assembly of collagen monomers. 15 After adding subgelling concentration of collagen monomers to cultured MMP-2 KO, MMP-9 KO, and WT SMC, levels of fluorescence detectable after 24 hours were significantly different (8. …”

“…Collagen assembly was measured using a novel assay with FITC-labeled (Fluos labeling kit, Roche) rat-tail collagen (Becton Dickenson), which we previously described in detail. 15 Briefly, 10 to 100 g/mL FITC-labeled collagen (subgelling concentrations) in serum-free DMEM was added to quiesced, confluent SMC seeded in 96-well tissue culture plates. After washing collagen monomers unbound after 24 hours, SMC monolayers and associated assembled collagen were fixed.…”

High resolution imaging of collagen organisation and synthesis using a versatile collagen specific probe

“…However, with confocal reflection microscopy alone it is not possible to distinguish between different constituents, whereas with multiple fluorescent labels in combination with laser scanning microscopy this is possible. Collagen assembly by cells has previously been quantified using FITC labelled rat-tail collagen (Johnson and Galis, 2003) to monitor the remodelling and structure of exogenous collagen. The study presented here is the first to look at fluorescently enhanced endogenous collagen.…”

The expression of MMP‐1 and MMP‐9 is up‐regulated by smooth muscle cells after their cross‐talk with macrophages in high glucose conditions

“…In a first step, the collagen Type I obtained from rat tail (powder from Sigma) was labelled with Texas Red as previously described . Briefly, collagen was solubilized in 2 mmol/L HCl to a final concentration of 10 mg/mL on a shaker at room temperature.…”