Retinal VEGF-A Overexpression Is Not Sufficient to Induce Lymphangiogenesis Regardless of VEGF-C Upregulation and Lyve1(+) Macrophage Infiltration

PURPOSE: No lymphatic vessels have been identified in the retina. This study investigated whether pathological VEGF-A–overexpressing diabetic retina causes lymphangiogenesis. METHODS: Three genetic mouse models of diabetic retinopathy (DR) (Akita [Ins2(+/−)], Kimba [vegfa(+/+)], and Akimba [Akita × Kimba] mice) were used. Retinas were examined by fundus photography, fluorescence angiography (FA), and immunostaining to detect lymphangiogenesis or angiogenesis. Lyve1-GFP (Lyve1(EGFP/Cre)) mice were used to examine Lyve1-expressing cells by immunostaining. Lymphatic-related factors were investigated in mouse retina and vitreous fluid from proliferative diabetic retinopathy (PDR) patients by RT-PCR and ELISA, respectively. Aged Kimba and Akimba mice were used to examine the retinal phenotype at the late phase of VEGF overexpression. RESULTS: FA and immunostaining showed retinal neovascularization in Kimba and Akimba mice but not wild-type and Akita mice. Immunohistochemistry showed that lymphangiogenesis was not present in the retinas of Akita, Kimba, or Akimba mice despite the significant upregulation of lymphatic-related factors (Lyve1, podoplanin, VEGF-A, VEGF-C, VEGF-D, VEGFR2, and VEGFR3) in the retinas of Kimba and Akimba mice by RT-PCR (P < 0.005). Furthermore, lymphangiogenesis was not present in aged Kimba or Akimba mice. Significantly increased numbers of Lyve1-positive cells present in the retinas of Kimba and Akimba mice, especially in the peripheral areas, were CD11b positive, indicating a macrophage population (P < 0.005). VEGF-C in PDR vitreous with vitreous hemorrhage (VH) was higher than in PDR without VH or a macular hole. CONCLUSIONS: Retinal VEGF-A overexpression did not cause typical lymphangiogenesis despite upregulated lymphatic-related factors and significant Lyve1-positive macrophage infiltration.