VEGFR-3 in adult angiogenesis.

Vascular endothelial growth factor receptor 3 (VEGFR-3, Flt-4), the receptor for vascular endothelial growth factors (VEGFs) C and D, is expressed on lymphatic endothelium and may play a role in lymphangiogenesis. In embryonic life, VEGFR-3 is essential for blood vessel development. The purpose of this study was to investigate whether VEGFR-3 is also involved in blood vessel angiogenesis in the adult. This was studied in human tissues showing angiogenesis and in a model of VEGF-A-induced iris neovascularization in the monkey eye, by the use of immunohistochemistry at the light and electron microscopic level. VEGFR-3 was expressed on endothelium of proliferating blood vessels in tumours. In granulation tissue, staining was observed in the proliferative superficial zone in plump blood vessel sprouts, in the intermediate zone in blood vessels and long lymphatic sprouts, and in the deeper fibrous zone in large lymphatics, in a pattern demonstrating that lymphangiogenesis follows behind blood vessel angiogenesis in granulation tissue formation. At the ultrastructural level, VEGFR-3 was localized in the cytoplasm and on the cell membrane of endothelial cells of sprouting blood vessels and sprouting lymphatics. In monkey eyes injected with VEGF-A, blood vessel sprouts on the anterior iris surface and pre-existing blood vessels in the iris expressed VEGFR-3. In conclusion, these results support a role for VEGFR-3 and its ligands VEGF-C and/or VEGF-D in cell-to-cell signalling in adult blood vessel angiogenesis. The expression of VEGFR-3 in VEGF-A-induced iris neovascularization and in pre-existing blood vessels exposed to VEGF-A suggests that this receptor and possibly its ligands are recruited in VEGF-A-driven angiogenesis.

Endothelial cell hypertrophy induced by vascular endothelial growth factor in the retina: new insights into the pathogenesis of capillary nonperfusion.

OBJECTIVE: To investigate the mechanism leading to capillary nonperfusion of the retina in a monkey model of vascular endothelial growth factor A (VEGF)-induced retinopathy in which capillary closure occurs in a late stage after VEGF treatment. METHODS: Two monkeys received 4 intravitreous injections of 0.5 microg of VEGF in one eye and of phosphate-buffered saline in the other eye and were killed at day 9. After perfusion and enucleation, retinal samples were snap frozen for immunohistochemical analysis with the panendothelial cell marker CD31 or were fixed for morphometric analysis at the light and electron microscopic level. RESULTS: At the light microscopic level, all capillaries in the retina of VEGF-injected eyes displayed hypertrophic walls with narrow lumina. In a quantitative analysis of the deep capillary plexus in the inner nuclear layer, VEGF-injected eyes had a significant 5- to 7-fold decrease in total capillary luminal volume. CD31 staining showed that this decrease was not accompanied by a change in the number of capillaries. Electron microscopy revealed that the luminal volume of individual capillaries of the inner nuclear layer of VEGF-injected eyes was significantly decreased due to a 2-fold hypertrophy of the endothelial cells. CONCLUSIONS: Luminal narrowing caused by endothelial cell hypertrophy occurs in the deep retinal capillary plexus in VEGF-induced retinopathy in monkeys. This suggests a causal role of endothelial cell hypertrophy in the pathogenesis of VEGF-induced retinal capillary closure. A similar mechanism may operate in retinal conditions in humans associated with ischemia and VEGF overexpression. CLINICAL RELEVANCE: Capillary nonperfusion occurs in diabetic retinopathy and other ischemic diseases associated with overexpression of VEGF. In addition, VEGF-induced endothelial cell hypertrophy may be causative for capillary closure in these diseases.