Effects of local gene transfer of VEGF on neointima formation after balloon injury in hypercholesterolemic rabbits

Abstract

Enhancement of the generation of nitric oxide (NO) and vascular endothelial growth factor (VEGF) are suggested to prevent restenosis after angioplasty. Accordingly, we tested whether the local delivery of L-arginine (L-Arg), a substrate for NO generation and the VEGF gene, alone or in combination, can influence neointima formation in hypercholesterolemic rabbits. Balloon injury of the iliac arteries was performed in 24 New Zealand White rabbits fed a 1% cholesterol diet for 3 weeks followed by a local infusion of: (1) pSG5VEGF165 plasmid alone (1000 microg); (2) pSG5VEGF165 (1000 microg) with L-Arg (800mg); (3) L-Arg (800mg) alone; and (4) L-Arg (800 mg) with naked pSVbeta-gal plasmid (1000 microg). The animals were kept on the hypercholesterolemic diets for a further 28 days, when vessels were taken for morphometric analysis and immunocytochemistry. Endogenous rabbit VEGF concentration in the plasma increased significantly at 7 days after injury (17.06 +/- 1.57 vs 23.01 +/- 1.9 pg/ml; p < 0.02) and remained elevated for up to 28 days (28.46 +/- 5.24; p < 0.01). Injured arteries exhibited strong immunocytochemical staining for rabbit VEGF. Rabbits that received a VEGF gene transfer revealed more prominent neointima formation, whereas treatment with L-Arg was associated with significantly less intimal thickness (p < 0.05). Local transfer of the VEGF gene does not inhibit neointima formation in hypercholesterolemic rabbits. Our results suggest that VEGF gene therapy applied locally in atherosclerotic arteries may not be beneficial.

Figure 1

Expression of β-galactosidase reporter gene in rabbit iliac artery after local infusion of pSVβ-gal plasmid. Positive cells are visible as dark-stained cells (arrows). L: lumen; Adv: adventitia.

Figure 2

The effect of local delivery of VEGF plasmid on neointima formation. Morphometric analysis of I/M ratio. Delivery of pSG5VEGF₁₆₅ alone resulted in a higher I/M ratio than other treatments. *p < 0.05; ANOVA = 0.036.

Figure 3

Endogenous VEGF in rabbit plasma. Levels of rabbit VEGF slightly increased in plasma during 3 weeks of cholesterol feeding. Injury resulted in an additional significant increase of VEGF, independent of the type of treatment applied. * p < 0.02 vs value before injury; ** p < 0.01 vs value before injury.

Figure 4

Immunocytochemical demonstration of VEGF expression in the injured rabbit iliac arteries. Strong VEGF expression was observed in the vessels of hypercholesterolemic rabbits, independent of the type of treatment. VEGF was particularly strongly expressed under the endothelium (A, arrows) and at the border between neointima and media (B, arrow). IEL: internal elastica lamina. No staining was observed when the primary antibody was omitted (not shown).