- 2002 -

12(R)-HYDROXYEICOSATRIENOIC ACID, A CORNEAL EPITHELIAL DERIVED EICOSANOID, STIMULATES ANGIOGENESIS VIA ERK1/2 ACTIVATION OF VEGF INDUCTION ((F. Seta, A. Mezentsev, M.W. Dunn, M. Laniado Schwartzman)) Departments of Pharmacology and Ophthalmology, New York Medical College, Valhalla, NY.

Purpose. In response to hypoxic injury, corneal epithelial cells synthesize an eicosanoid via cytochrome P450. This eicosanoid, 12(R)-hydroxyeicosatrienoic acid [12(R)-HETrE], then induces limbal vascular endothelial cells to proliferate and migrate into the cornea and begin neovascularization. We further investigate cellular mechanisms underlying its angiogenic activity using endothelial cells derived from rabbit limbal microvessels (RLMV cells). Methods. Endothelial cell cultures from rabbit limbal microvessels (RLMV) were grown until 70% confluent and then quiesced for 36 hours. The cells were treated with 12(R)-HETrE (0.1 nM) for various times. Northern or slot blot hybridization was used to determine VEGF mRNA levels and ELISA was used for measuring VEGF protein. Western blot analysis and in vitro kinase assays were used to determine MAPK activation. In vitro capillary formation assay using Matrigel was used to access functional relationship between 12(R)-HETrE and VEGF. Results. 12(R)-HETrE increased VEGF mRNA and protein in time and concentration dependent manners. Both transcriptional and posttranscriptional mechanisms accounted for the increase in VEGF mRNA. 12(R)-HETrE activated Erk1/2 as indicated by rapid increases in the level of phosphorylated Elk. 12(R)-HETrE- stimulated Erk1/2 activity as well as 12(R)-HETrE- induced VEGF expression was inhibited by the MEK selective inhibitor, PD98059. Addition of VEGF antibody to endothelial cells grown in Matrigel-coated plates inhibited 12(R)-HETrE- induced capillary formation. Conclusions. The results indicate that in microvessel endothelial cells, 12(R)-HETrE induces VEGF expression via activation Erk1/2 and that VEGF mediates at least in part the angiogenic activity of 12(R)-HETrE. Given the fact that both VEGF and 12(R)-HETrE are produced in the cornea following hypoxic injury their interaction may be an important determinant in the development of neovascularized cornea.

- 2001 -

CORNEAL EPITHELIAL VEGF AND CYP4B1 EXPRESSION IN A RABBIT MODEL OF CLOSED EYE CONTACT LENS WEAR. ((V. Mastyugin, S. Mosad, A. Bonazzi, A. Mezentsev, M.W. Dunn, M. Laniado-Schwartzman)). Departments of Pharmacology and Ophthalmology, New York Medical College, Valhalla, NY.

Purpose. The similar and overlapping activity of VEGF and the potent corneal-derived angiogenic eicosanoid 12(R)-HETrE calls for a study of the temporal relationship in the expression of these two autocoids. Since recent evidence suggests that hypoxia induces the expression of a CYP4B1 mRNA which might be involved in the conversion of arachidonic acid to 12(R)-HETrE, we determined its time-dependent expression and correlated it to that of VEGF mRNA in the rabbit model of closed eye contact lens-induced injury. Methods. Rabbit eyes were fitted with contact lenses followed by a silk suture tarsorrhaphy. The anterior surface was analyzed at 2-, 4- and 7-days by slit lamp biomicroscopy, subjective inflammatory scoring and corneal pachymetry. Corneal epithelium was scraped and CYP4B1 and VEGF mRNA levels were measured by Southern hybridization of RT-PCR products amplified from a single cornea with specific primers. Results. Corneal thickness and inflammatory scores increased in a time dependent manner in the model of closed eye contact lens induced hypoxic injury. Corneal epithelial CYP4B1 and VEGF mRNAs, as well as the production of the angiogenic eicosanoid, 12(R)-HETrE, increased in a time-dependent manner and correlated with the in situ inflammatory response. Conclusions. The present study documents the increased expression of CYP4B1 isoform in the corneal epithelium during hypoxic injury in vivo. It also demonstrates the presence of VEGF mRNA in the corneal epithelium and its increased expression in this model of hypoxic injury. All together, the results of this study raise the possibility of interaction between these autocoids, VEGF and CYP4B1-12(R)-HETrE, in mediating the neovascularization response induced by the prolonged hypoxic state brought about by closed eye contact lens wear.

THE ANGIOGENIC ACTIVITY OF 12(R)-HYDROXYEICOSATRIENOIC ACID IS MEDIATED BY VASCULAR ENDOTHELIAL GROWTH FACTOR (VEGF). ((A. Mezentsev, V. Mastyugin, A. Bonazzi, M.W. Dunn, M. Laniado Schwartzman)). Departments of Pharmacology and Ophthalmology, New York Medical College, Valhalla, NY.

Purpose. In response to hypoxic injury, corneal epithelial cells synthesize an eicosanoid via cytochrome P450. This eicosanoid, 12(R)-hydroxyeicosatrienoic acid [12(R)-HETrE], then induces limbal vascular endothelial cells to proliferate and migrate into the cornea and begin neovascularization. It is well documented that VEGF is a major mediator of angiogenesis in other tissues under hypoxic circumstances. We hypothesize that 12(R)-HETrE mediates its angiogenic actions, at least in part, through the induction of VEGF in vascular endothelial cells. Methods. Endothelial cell cultures from rabbit limbal microvessels (RLMVE) or immortalized rabbit corneal epithelial cells (RCE) were grown until 80% confluent and then quiesced for 24 hours. The cells were treated with 12(R)-HETrE (0.1nM to 10 nM) for 4-24 h. Northern Blot analysis and ELISA were used to determine VEGF mRNA and protein levels, respectively, and an in vitro capillary formation assay using Matrigel was used to assess functional relationship between 12(R)-HETrE and VEGF. Results. In RLMVE cells, 12(R)-HETrE (1 nM) time-dependently, increased VEGF mRNA. The increase in mRNA was followed by an increase in immunoreactive VEGF protein in the culture medium and this increase was concentration-dependent. 12(R)-HETrE-stimulated VEGF expression was attenuated by the PD 98059, a selective inhibitor of MEK-1. Addition of VEGF antibody to endothelial cells grown in Matrigel-coated plates inhibited 12(R)-HETrE-induced capillary formation. In RCE cells, hypoxia-induced VEGF mRNA and protein; addition of 12(R)-HETrE (1nM) increased VEGF mRNA by 5-fold. Conclusions. The ability of 12(R)-HETrE to stimulate corneal epithelial VEGF expression suggests that this eicosanoid can act within the corneal epithelium to activate and augment the angiogenic response. Moreover, the results also suggest that VEGF mediates, at least in part, the angiogenic response to 12(R)-HETrE.