Effects of basic FGF and TGF beta 1 on F-actin and ZO-1 organization during cat endothelial wound healing.
Previous studies suggest the existence of two separate and distinct mechanisms of endothelial wound healing (i.e., cell migration and cell spreading), which may be controlled by unique, injury-dependent, wound-related factors. The purpose of our study was to evaluate potential biologic mediators regulating healing of the growth arrested cat endothelium by using an ex vivo, organ-culture model. Three buttons were punched from each cornea of 11 cats with a 6-mm trephine. A 1- to 2-mm diameter endothelial scrape injury (SI) was made, and buttons were cultured in (a) serum-free media (SFM), (b) serum plus media (20% fetal calf serum), (c) SFM plus basic fibroblast growth factor (bFGF), (d) SFM plus bFGF and heparin, (e) SFM plus transforming growth factor-beta 1 (TGF beta 1), or (f) SFM plus TGF beta 1 and anti-TGF beta 1. At various times from 8-48 h after injury, buttons were stained with phalloidin and anti-ZO-1, and imaged by using laser scanning confocal microscopy. Evaluation of SI in cat corneal buttons under serum-free conditions showed maintenance of normal endothelial differentiation, indicating that the organ-culture SI model mimics in vivo SI. Addition of TGF beta 1 produced a dramatic reorganization of apical F-actin and development of stress fibers, as well as the loss of normal cell border-associated ZO-1 distribution. The effects of TGF beta 1 were blocked by the neutralizing antibodies to TGF beta 1. Addition of serum or bFGF produced much less pronounced changes in F-actin and ZO-1 distribution. These results suggest that TGF beta 1 may play a critical role in modulating the wound-healing response of the corneal endothelium.
Petroll
W M
WM
Department of Ophthalmology, University of Texas Southwestern Medical Center, Dallas 75235-9057, USA.
Jester
J V
JV
Barry-Lane
P A
PA
Cavanagh
H D
HD
D000199:Actins / Q000378:metabolism*; D000818:Animals; D001769:Blood; D002415:Cats; D016895:Culture Media, Serum-Free; D004728:Endothelium, Corneal / Q000293:injuries / Q000378:metabolism* / Q000473:pathology; D016222:Fibroblast Growth Factor 2 / Q000494:pharmacology*; D019084:Fluorescent Antibody Technique, Indirect; D008565:Membrane Proteins / Q000378:metabolism*; D018613:Microscopy, Confocal; D009924:Organ Culture Techniques; D010750:Phosphoproteins / Q000378:metabolism*; D016212:Transforming Growth Factor beta / Q000494:pharmacology*; D014945:Wound Healing / Q000502:physiology*; D062826:Zonula Occludens-1 Protein
D000199:Actins; D016895:Culture Media, Serum-Free; D008565:Membrane Proteins; D010750:Phosphoproteins; D016212:Transforming Growth Factor beta; D062826:Zonula Occludens-1 Protein; D016222:Fibroblast Growth Factor 2