Modulation of corneal fibroblast contractility within fibrillar collagen matrices.
OBJECTIVE
To investigate the migratory and contractile behavior of isolated human corneal fibroblasts in fibrillar collagen matrices.


METHODS
A telomerase-infected, extended-lifespan human corneal fibroblast cell line (HTK) was transfected by using a vector for enhanced green fluorescent protein (GFP)-alpha-actinin. Cells were plated at low density on top of or within 100-microm-thick fibrillar collagen lattices. After 18 hours to 7 days, time-lapse imaging was performed. At each 1- to 3-minute interval, GFP and Nomarski differential interference contrast (DIC) images were acquired in rapid succession. Serum-containing (S+) medium was used initially for perfusion. After 2 hours, perfusion was switched to either serum-free (S-) or S+ medium containing the Rho-kinase inhibitor Y-27632 for 1 to 2 hours. Finally, perfusion was changed back to S+ medium for 1 hour.


RESULTS
Two to 4 days after plating, many cells underwent spontaneous contraction and/or relaxation in S+ medium. A decrease in the distance between consecutive alpha-actinin-dense bodies along stress fibers was measured during contraction, and focal adhesion and matrix displacements correlated significantly. Removal of serum or inhibition of Rho-kinase induced cell body elongation and relaxation of matrix stress, as confirmed using finite element modeling. Rapid formation and extension of pseudopodia and filopodia were also observed, and transient tractional forces were generated by these extending processes.


CONCLUSIONS
Cultured human corneal fibroblasts can undergo rapid changes in the subcellular pattern of force generation that are mediated, in part, by Rho-kinase. Sarcomeric shortening of stress fibers in contracting corneal fibroblasts is also demonstrated for the first time.
Department of Ophthalmology, Southwestern Medical Center, Dallas, Texas 75390-9057, USA.
Mridula
M
Vishwanath
Lisha
L
Ma
Carol A
CA
Otey
James V
JV
Jester
W Matthew
WM
Petroll
D000185:Actinin / Q000378:metabolism; D000577:Amides / Q000494:pharmacology; D001696:Biomechanical Phenomena; D015153:Blotting, Western; D002460:Cell Line; D002465:Cell Movement / Q000502:physiology*; D003319:Corneal Stroma / Q000166:cytology / Q000502:physiology*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D024022:Fibrillar Collagens / Q000378:metabolism*; D005347:Fibroblasts / Q000502:physiology*; D049452:Green Fluorescent Proteins; D006801:Humans; D007091:Image Processing, Computer-Assisted; D047908:Intracellular Signaling Peptides and Proteins; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism; D017346:Protein Serine-Threonine Kinases / Q000037:antagonists & inhibitors; D011725:Pyridines / Q000494:pharmacology; D022502:Stress Fibers / Q000378:metabolism; D014162:Transfection; D014945:Wound Healing; D054460:rho-Associated Kinases
D000577:Amides; D004791:Enzyme Inhibitors; D024022:Fibrillar Collagens; D047908:Intracellular Signaling Peptides and Proteins; D008164:Luminescent Proteins; D011725:Pyridines; D000185:Actinin; C108830:Y 27632; D049452:Green Fluorescent Proteins; D017346:Protein Serine-Threonine Kinases; D054460:rho-Associated Kinases