Intercellular gap formation induced by thrombin in confluent cultured bovine retinal pigment epithelial cells

Abstract

Purpose: Thrombin is formed at the site of intraocular hemorrhage and may be important in the development of progressive retinal damage. The authors observed that thrombin-treated bovine retinal pigment epithelial (RPE) cell cultures develop intercellular gaps and initiated this study to examine in detail the effects of thrombin on RPE cell morphology, adhesion, and cytoskeleton.

Methods: Confluent cultures of bovine RPE cells were incubated for various times (0 to 24 hours) with alpha-thrombin (0.1 to 100 U/ml) or enzymatically inactive thrombin. Intercellular gaps were quantitated by light microscopy in ten representative fields (magnification X400) as number of gaps per field (gaps/f). RPE cytoskeleton was studied using immunofluorescent staining for vinculin and F-actin. The mechanism of thrombin-induced RPE cell gap formation was studied by preincubation with specific drugs, including a protein kinase inhibitor (staurosporine), protein kinase C inhibitors (H-7 and calphostin C), cyclic adenosine monophosphate (cAMP) inducer (forskolin), and cytoskeleton-disrupting agents (cytochalasin B or colchicine).

Results: Intercellular gaps (20 to 80 microns in diameter) were markedly increased in number in thrombin-treated cultures in a dose-dependent and time-dependent manner and were associated with an alteration in the distribution of F-actin and vinculin. Whereas control cultures showed 3.3 +/- 2.4 gaps/f, incubation with 8 U/ml of alpha-thrombin for 3 hours resulted in 44.8 +/- 15.3 gaps/f. These changes were most prominent shortly after the 3-hour coincubation, but the cultures did return to their original confluent state within 24 hours. Cultures treated with an enzymatically inactive thrombin showed fewer intercellular gaps than those treated with enzymatically active thrombin but had significantly more intercellular gaps than control cultures. Thrombin-induced intercellular gap formation was blocked by preincubation with forskolin (14.6 +/- 7.1 gaps/f), staurosporine (10.2 +/- 5.0 gaps/f), or H-7 (24.5 +/- 9.8 gaps/f).

Conclusions: Exposure to an enzymatically active thrombin results in formation of intercellular gaps between cultured RPE cells. Inhibition of this phenomenon by protein kinase inhibitors and by a cAMP inducer suggests that this effect is mediated, at least in part, through protein kinase C- and cAMP-dependent pathways. Thrombin generation associated with intraocular hemorrhage may thus result in direct damage to the RPE monolayer, possibly via the same pathway(s).