IKK Phosphorylation, ICAM–1, and CD11a/LFA–1 Increases Are Correlated With VEGF–Induced Blood–Retinal Barrier Breakdown and Are Blocked by Triamcinolone Acetonide in Rabbit

Abstract

Abstract: : Purpose: Intravitreal injection of VEGF–165 in rabbit induces blood–retinal barrier breakdown and retinal edema. Intravitreal triamcinolone acetonide (TAA) completely blocks these effects. The current study examined whether VEGF activates a pro–inflammatory mechanism in this model, and whether that response is blocked by TAA. Methods: VEGF–165 (500 ng) was injected intravitreally in female Dutch Belt rabbits, and retinae were obtained from 1 hr to 48 hrs after VEGF injection. Control retinae were obtained for comparison. Total protein was extracted from the vascular region of each retina and analyzed by Western blots evaluating levels of phosphorylated IKK–α (IkB Kinase–α), ICAM–1 (intercellular adhesion molecule–1), and the leukocyte integrin CD11a/LFA–1. Similar protein analyses were performed after intravitreal injection with either VEGF (48 hrs), triamcinolone acetonide (TAA: 2 mg, 4 days), both VEGF and TAA (48 hrs and 4 days, respectively), or neither (control). Retinal vascular leakage was assessed in all of these eyes by fluorescein angiography prior to obtaining the vascular retinae for protein analysis. Results: Following intravitreal injection with VEGF–165, the earliest change detected in this study was an increase in phosphorylated IKK–α (p–IKK) after 1 hr. This was followed by an additional 3–fold increase in p–IKK 48 hrs after VEGF injection. Also at 48 hrs, 55 kD soluble ICAM–1 (sICAM) and 158 kD CD11a/LFA–1 proteins dramatically increased, and these changes correlate temporally with maximal retinal vascular leakage. TAA administered intravitreally two days prior to VEGF injection completely blocked VEGF–induced increases in p–IKK, sICAM, and CD11a/LFA–1 in the retina 48 hrs after VEGF injection. This correlates with TAA's complete inhibition of VEGF–induced blood–retinal barrier breakdown in the same eyes. Conclusions: Intravitreal injection of VEGF–165 initiates a pro–inflammatory mechanism that includes early activation of endogenous retinal IKK–α at 1 hr and delayed amplification of IKK/NF–kB activation at 48 hrs. This delay correlates temporally with sICAM–1 and CD11a/LFA–1 protein increases that suggest leukocyte infiltration in the retina at the time of maximal retinal vascular leakage. The inhibition of VEGF–induced inflammatory mechanisms in the retina may be one mode by which triamcinolone acetonide blocks VEGF–induced blood–retinal barrier breakdown.

Keywords: retina • growth factors/growth factor receptors • corticosteroids