Slide 1

In the corneal epithelium, cytochrome P450, primarily CYP 4B1, metabolizes arachidonic acid to two major 12-hydroxyeicosanoids, 12-HETE and 12-HETrE, with stereospecificity that favors the R isomers. Both R-isomers are bioactive and their biological effects point them as inflammatory mediators: 12(R)-HETE is a potent inhibitor of Na, K-ATPase activity and 12(R)-HETrE is a powerful vasodilator, chemotactic, and angiogenic factor. Based on their biological activities we postulated that these eicosanoids mediate ocular surface inflammation.

Slide 2

We hypothesize that injury to the cornea induces the expression and activity of a cytochrome P450, that we already identified as CYP4B1, leading to increased production of 12-HETE and 12-HETrE, which through their action on the corneal endothelium and the vascular endothelium of the adjacent limbal vessels mediate the inflammatory response to the injurious insult including edema, vasodilation and neovascularization.

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This hypothesis is supported by numerous studies showing that READ THE SLIDE

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The goal of the present study was: READ THE SLIDE

Slide 5

The experimental design was the following: New Zeeland White Male Rabbits were transfected with pIRES2 vector carrying EGFP or EGFP plus CYP4B1. We dipped a needle into the plasmid solution and repeatedly inserted it into the limbus over 360 degrees. This followed by topical application of the remaining plasmid solution. The development of the inflammatory response was monitored by slit lamp microscopy for up to 7 days after the transfection. Some rabbits were sacrificed in 4 days after the transfection. Corneas were dissected in four pieces and placed on Matrigel for ex vivo angiogenic activity assay. In parallel, medium was collected for measurements of 12-HETrE by GC/MS analysis.

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After transfection with the EGFP and 4B1 the ocular surface becomes progressively inflamed over a 7-day period. This inflammatory response was characterized by dilation of limbal vessels and beginning of neovascularization. In contrast, the eyes transfected with EGFP only showed a little if any limbal dilation or neovascularization sprouting.

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Using fluorescent microscopy we clearly showed that our method of transfection effectively transduced EGFP expression into the cornea. Moreover, EGFP expression was primarily associated with the corneal epithelial cell layer and was sustained in the cornea for 6 days after the transfection.

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The EGFP fluorescence was also evident in corneas from eyes transfected with EGFP and 4B1.

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Corneal explants taken from eyes transfected with EGFP and 4B1 demonstrated a significantly higher angiogenic activity compared to corneal explants from the sham transfected eyes and from the eyes transfected with the control EGFP plasmid. We also observed there a massive capillary-like network.

Slide 10
To examine the endothelial origin of pseudocapillary cells, we performed a binding assay with lectin I from Grifonia simpliconica labeled with rhodamine. As seen in this slide, rhodamine conjugated lectin I specifically bound to pseudocapillary cells indicating that the capillary network is composed of endothelial cells.

Slide 11

Previously, we established that CYP4B1 is an enzyme that is induced in the corneal epithelium in response to injury. We also linked cytochrome P450 arachidonic acid metabolism to the production of the angiogenic eicosanoid, 12(R)-HETrE. In addition to significant inflammatory response in vivo, CYP4B1 transfection markedly increased the angiogenic activity of corneal-limbal explants. As seen in this slide, angiogenic activity measured as total capillary length was about 3 fold higher in explants obtained after transfection with 4B1 than the control explants. Moreover, transfection with CYP4B1 also resulted in a marked production of 12(R)-HETrE by the corneal explants. In fact, 12(R)-HETrE level in samples from 4B1 transfected corneas significantly exceeded the levels of 12(R)-HETrE in the controls suggesting a relationship between the angiogenic activity and the production of 12-HETrE.

Slide 12

In summary; READ THE SLIDE or SAY THIS: Transfection of the cornea with EGFP or EGFP-4B1 plasmids resulted in expression of EGFP in corneal epithelial cells. In the eyes transfected with plasmid containing the 4B1, transfection was accompanied by increased inflammation in vivo, as well as 3-fold increase in capillary growth ex vivo and neovessel sprouting composed of endothelial cells. This was associated with increased levels of 12(R)-HETrE, the angiogenic eicosanoid formed by CYP4B1. The results implicate the corneal CYP4B1 as a component of the inflammatory cascade initiated by the injury of the ocular surface.