Expression of cell adhesion molecules and vascular endothelial growth factor in experimental choroidal neovascularisation in the rat

Abstract

Aims-To investigate the longevity and reproducibility of choroidal neovascularisation (CNV) induced by krypton laser photocoagulation in the rat. The presence of cell adhesion molecules (CAMs) and vascular endothelial growth factor (VEGF) during the development of CNV was also studied. Methods-67 pigmented rats underwent retinal photocoagulation by krypton laser. The eyes were examined by either single or serial fluorescein angiography at 3 days, 1, 2-3, 4-5, 7-8, and 12 weeks post photocoagulation. The expression of CAMs (ICAM-1, E-selectin, and CD44) and VEGF post photocoagulation was studied by immunohistochemistry. Results-CNV related fluorescein leakage appeared in 46.4% of 766 laser spots delivered to the 58 eyes that were tested at 2-3 weeks post treatment. The ratio of hyperfluorescent laser sites did not change significantly at 8 weeks post laser. The number of leaky spots was independent of the total number of lesions delivered to each eye (at 2-3 weeks post laser 10-15 spots/eye: 44% and 25-30 spots/eye: 49%; t=0.7673; p=0.3903). Nine eyes were followed by serial angiography between 2 and 12 weeks. The laser spots with fluorescein leakage at 2 weeks (51.5%) remained leaky at 12 weeks (51.5%). Histopathologically, macrophage accumulation peaked at 5 days and CNV was firstly observed at 1 week post photocoagulation. ICAM-1, E-selectin, CD44, and VEGF were maximally induced at 3-5 days post laser photocoagulation, and were localised to RPE, choroidal vascular endothelial, and inflammatory cells. VEGF was also detected in intravascular leucocytes at the sites of laser lesions. Conclusions-These studies demonstrated that krypton laser photocoagulation can be successfully used to produce lesions similar to those of human CNV. The response induced remained present for an extended period of time (12 weeks), thus oVering a potential model to screen candidate CNV inhibitory agents. In addition, it is proposed that the expression of ICAM-1, E-selectin, CD44, and VEGF before new vessel formation might be linked to the initiation of CNV.

Potential long-term inhibition of ocular neovascularisation by recombinant adeno-associated virus-mediated secretion gene therapy

“…In the choroidal NV study, the presence of infiltrating cells and other inflammatory cytokines and molecules have also been observed at the site of laser photocoagulation. 21,22 It is probable that injection with AAV-CMV.sflt similarly suppressed the activity of VEGF as a stimulator of inflammation and angiogenesis in this model.…”

“…28,36 It is widely believed that VEGF up-regulation also plays a major part in the development of ocular NV. [6][7][8][9][20][21][22] Several studies have further shown that inhibition of VEGF activity by administration of VEGF-specific kinase inhibitors, VEGF antibodies, VEGF receptors and VEGF antisense oligonucleotides successfully suppressed the development of retinal and choroidal NV. [37][38][39][40] In particular, Honda et al 41 showed that expression of adenovirus-mediated sFlt-1 suppressed choroidal NV.…”

“…At 5 weeks post-laser photocoagulation, approximately 60% of the 103 laser spots delivered to AAV-CMV.gfp-injected eyes demonstrated leakage (Table 2, Figure 5f), consistent with our previous study. 22 Rats treated with AAV-CMV.sflt showed leakage in 41% of the 106 laser spots delivered indicating a 19% reduction in percentage of leaky lesions (P = 0.002, Table 2; Figure 5e). This was also reflected in the mean score per eye, which was 22% lower in AAV-CMV.sflt-treated eyes (P = 0.001, Table 2), indicating less leakage on the whole in these treated eyes.…”

“…However, one of the difficulties of ocular anti-angiogenic therapies is that several types of cells such as retinal pigment epithelial (RPE) cells, Muller cells, ganglion cells, the inner nuclear layer, macrophages and choroidal vascular endothelial cells have all been identified as potential contributors to VEGF upregulation. [20][21][22] Some of these target cells are difficult to access, transduce or undesirable to disturb, especially within the macula, the central part of the retina responsible for 80% of human vision. 23 To fulfil the specific requirements of ocular anti-angiogenic therapy, we explored the use of secretion gene therapy (SGT).…”

“…Both models have been shown to induce VEGF expression resulting in extensive blood vessel growth in the normally avascular cornea and subretinal space, respectively. 22,26,27 Based upon the proven efficacy of soluble VEGF receptor 1, sFlt-1, as a potent anti-angiogenic agent, [28][29][30] we selected sFlt-1 as our therapeutic gene and investigated the efficacy of AAV-mediated delivery and expression of this protein as SGT against global corneal NV and choroidal NV.…”