Fig 2: Retinal neovascularization and higher expression of AQP1 and COX-2 in the Ins2Akita mice. a, b Upper panels: Hematoxylin/eosin-stained retinal cross-sections from sex- and age-matched (1 year-old) C57BL/6 non-diabetic control mice (a) and D2.B6-Ins2 Akita (b). Magnification: 60x. Lower panels: immunohistochemistry of CD31-stained retinal cross-sections from D2.B6-Ins2 Akita (a) and sex- and age-matched (1 year-old) C57BL/6 non-diabetic control mice (b), showing higher CD31 immunoreactivity in the D2.B6-Ins2 Akita mice. Magnification: 40x and 100x. c The extent of angiogenesis was determined by measuring total vessels (arterioles and capillaries) density in light microscopy sections. N = 2 eyes per animal, with each experimental group consisting of n = 7 mice. The bar graph represents for each value the mean ± S.D. from 3 separate experiments. **, P < 0.01 vs C57BL/6 control mice. d, e: Western analysis of COX-2 and AQP1 expression in retinas from D2.B6-Ins2 Akita mouse and from sex- and age-matched (1 year-old) C57BL/6 non-diabetic control mice, with β-actin serving as a loading control. N = 2 eyes/group, with each group consisting of n = 7 mice. The bar graph represents for each value the mean ± S.D. from three separate experiments. **, P < 0.01 vs C57BL/6 control mice

Fig 3: The involvement of AQP1 in COX-2 expression in the Ins2Akita and C57BL/6 mice. a Western analysis of COX-2 and AQP1 expression in retinas from from D2.B6-Ins2 Akita and sex- and age-matched (1 year-old) C57BL/6 non-diabetic control mice, after the intravitreal injection of saline (sham), or siRNA against scrambled sequence (siRNA-scr) or siRNA against AQP1 (siRNA-AQP1), with β-actin serving as a loading control. b n = 2 eyes per animal, with each experimental group consisting of n = 7 mice. The bar graph represents the means ± S.D. from three separate experiments. **, P < 0.01 vs C57BL/6 control mice °°, P < 0.01 vs sham and siRNA-scr. Glyco AQP1 represents the glycosylated form of AQP1 able to insert itself in the plasma membrane

Fig 4: The involvement of AQP1, COX-2 and TonEBP/NFAT5 in glucose-induced in vitro angiogenic activity. A, C, D The effect of gene silencing to AQP1 or to TonEBP/NFAT5 on high glucose- and high mannitol-induced angiogenic activity of endothelial cells. B The effect of the COX-2 inhibitor NS-398 on high glucose- and high mannitol-induced angiogenic activity of endothelial cells. E The effect of TonEBP/NFAT5 gene silencing on high glucose- and high mannitol-induced endothelial cell migration. Serum-starved human aortic endothelial cells (HAECs) were transfected with Cy5-coniugated-siRNA to AQP1 or with FITC-coniugated-siRNA to TonEBP/NFTA5 and/or siRNA with scrambled sequence for 24 h (D), and then treated with stimuli for 24 h with high glucose or high mannitol. At the end of treatments, transfection efficiency was checked by observing the plates under fluorescence microscopy (D), while TonEBP/NFAT5 gene knockdown efficiency was detected at the protein level by Western analysis (D). At the end of incubations cells were enzymatically harvested, plated on Matrigel-coated 96 well-plates for the in vitro angiogenesis assay (A–C, E) or cell migration assay (F), and further incubated with stimuli for 24 h. In parallel experiments (B), serum starved HAECs were treated with high glucose or high mannitol (both at 25 mmol/L) in the presence or absence of 5 µg/mL NS-938 (NS) for 48 h and, after enzymatic detachment, plated on Matrigel-coated 96 well-plates, and further incubated with stimuli for 24 h. After incubation, images to document angiogenic activity were taken and analyzed as indicated in Methods. Upper panels (A–E): representative images. Lower panels (C, F): quantitation of angiogenic activity (network area, C) and for cell migration (F). *, P < 0.05 mannitol- or glucose-treated vs control HAECs; §, P < 0.05 vs without AQP1-siRNA or without TonEBP/NFAT-siRNA; # P < 0.05 vs without NS-938. RFU: relative fluorescence units

Fig 5: The involvement of AQP1 in retinal neovascularization in the Ins2Akita and C57BL/6 mice. Immunofluorescence staining of retinal cross-sections for α-smooth muscle actin (ASMA) and 4′,6-diamidino-2-phenylindole (DAPI) for nuclei from D2.B6-Ins2 Akita (G-N) and sex- and age-matched (1 year-old) C57BL/6 non-diabetic control mice (a–f), after intravitreal injection of saline (sham, panels a, b g, h), or siRNA with scrambled sequence (siRNA-scr, panels c, d, i, l) or siRNA against AQP1 (siRNA-AQP1, panels e, f, n, m). Magnification: 10x