Quantitative assessment of local collagen matrix remodeling in 3-D culture: the role of Rho kinase.
The purpose of this study was to quantitatively assess the role of Rho kinase in modulating the pattern and amount of local cell-induced collagen matrix remodeling. Human corneal fibroblasts were plated inside 100-microm thick fibrillar collagen matrices and cultured for 24 h in media with or without the Rho kinase inhibitor Y-27632. Cells were then fixed and stained with phalloidin. Fluorescent (for f-actin) and reflected light (for collagen fibrils) 3-D optical section images were acquired using laser confocal microscopy. Fourier transform analysis was used to assess collagen fibril alignment, and 3-D cell morphology and local collagen density were measured using MetaMorph. Culture in serum-containing media induced significant global matrix contraction, which was inhibited by blocking Rho kinase (p<0.001). Fibroblasts generally had a bipolar morphology and intracellular stress fibers. Collagen fibrils were compacted and aligned parallel to stress fibers and pseudopodia. When Rho kinase was inhibited, cells had a more cortical f-actin distribution and dendritic morphology. Both local collagen fibril density and alignment were significantly reduced (p<0.01). Overall, the data suggests that Rho kinase-dependent contractile force generation leads to co-alignment of cells and collagen fibrils along the plane of greatest resistance, and that this process contributes to global matrix contraction.
Kim
Areum
A
Graduate Program in Biomedical Engineering, University of Texas Southwestern Medical Center, Dallas, TX, USA.
Lakshman
Neema
N
Petroll
W Matthew
WM
D000199:Actins / Q000378:metabolism; D000577:Amides / Q000494:pharmacology; D018929:Cell Culture Techniques; D048430:Cell Shape; D002478:Cells, Cultured; D003094:Collagen / Q000378:metabolism*; D003315:Cornea / Q000166:cytology; D005109:Extracellular Matrix / Q000737:chemistry / Q000378:metabolism*; D005347:Fibroblasts / Q000166:cytology / Q000378:metabolism*; D005583:Fourier Analysis; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins / Q000378:metabolism*; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*; D011725:Pyridines / Q000494:pharmacology; D054460:rho-Associated Kinases
D000199:Actins; D000577:Amides; D047908:Intracellular Signaling Peptides and Proteins; D011725:Pyridines; C108830:Y 27632; D003094:Collagen; D017346:Protein Serine-Threonine Kinases; D054460:rho-Associated Kinases