Quantitative assessment of collagen assembly by live cells.
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.
Johnson
Chad
C
Division of Cardiology, Emory University School of Medicine, 1639 Pierce Drive, Atlanta, Georgia 30322, USA.
Galis
Zorina S
ZS
D000818:Animals; D001011:Aorta / Q000166:cytology; D002417:Cattle; D002478:Cells, Cultured; D003094:Collagen / Q000096:biosynthesis*; D005109:Extracellular Matrix / Q000737:chemistry / Q000648:ultrastructure; D016650:Fluorescein-5-isothiocyanate; D006651:Histocytochemistry / Q000379:methods*; D006801:Humans; D007700:Kinetics; D051379:Mice; D008856:Microscopy, Fluorescence; D009131:Muscle, Smooth, Vascular / Q000166:cytology*; D051381:Rats; D012501:Saphenous Vein / Q000166:cytology
D003094:Collagen; D016650:Fluorescein-5-isothiocyanate