DIRECT STIMULATION OF LIMBAL MICROVESSEL ENDOTHELIAL-CELL PROLIFERATION AND CAPILLARY FORMATION IN-VITRO BY A CORNEAL-DERIVED EICOSANOID

12(R)-Hydroxyeicosatrienoic acid (12(R)-HETrE), a corneal epithelial d erived inflammatory eicosanoid, elicits blood vessel growth into the a vascular cornea in the classical corneal micropocket bioassay. Wing an in vivo simulated angiogenesis assay, and 12(R)-HETrE as the angiogen ic stimulus, we isolated a homogeneous population of rabbit limbal mic rovessel endothelial cells, the target for angiogenic factors in the a nterior surface of ocular tissues, and analyzed the mitogenic and angi ogenic potential of this eicosanoid. 12(R)-HETrE stereospecifically in creased cell number by similar to 45%, an effect comparable to that of basic fibroblast growth factor (0.6 nmol/L 10 ng/ml). This potent mit ogenic response was maximal at 0.1 nmol/L. An additive effect (similar to 90% above control) on cell proliferation was observed when 12(R)-H ETrE (0.1 nmol/L) and basic fibroblast growth factor (0.6 nmol/L) were added to quiescent cultures of rabbit limbal microvessel endothelial cells, We also show that 12(R)-HETrE, but not 12(S)-HETrE, induces cul tured rabbit limbal microvessel endothelial cells to organize themselv es as a network of branching cords reminiscent of capillaries, This ef fect was evident within 48 hours, maximal by 5 days of culture, and pa ralleled the effect observed with basic fibroblast growth factor, This study describes a novel method for testing site-directed angiogenesis in vitro and further strengthens the angiogenic properties of 12(R)-H ETrE by demonstrating a direct effect on limbal microvessel endothelia l cells.