Fig 2: VEGFR3 mediates retinal neovascularization in ligand dependent and independent manner.a Upper panel: western blot analysis of control and the indicated time periods of VEGFA (40 ng/ml)-treated HRMVECs for phospho (Y705) and total STAT3 levels. Middle and bottom panels: The effect of siControl, siVEGFR2 and siVEGFR3 (100 nM) on VEGFA (40 ng/ml)-induced STAT3 phosphorylation. The blots were sequentially reprobed for total STAT3, VEGFR2 or VEGFR3 levels to show the loading control and the specificity and efficacy of siRNAs on their target molecules. b Upper two panels: Eyes from normoxic and 24 h (i.e., at P13) of hypoxic WT mice pups that were injected intravitreally with siControl, siVEGFR2 (0.5 μg/0.5 μl/eye) or sVEGFR2 (0.05 μg/0.5 μl/eye) at P11 and P12 were enucleated, retinas were isolated, protein extracts were prepared and analyzed by western blotting for phospho and total STAT3 levels using their specific antibodies. Lower two panels: Eyes from normoxic and 24 h (i.e., at P13) of hypoxic WT mice pups that were injected intravitreally with siControl, siVEGFR3 (0.5 μg/0.5 μl/eye) or sVEGFR3 (0.05 μg/0.5 μl/eye) at P11 and P12 were enucleated, retinas were isolated, protein extracts were prepared and analyzed by western blotting for phospho and total STAT3 levels using their specific antibodies. c Mice pups were injected intravitreally with vehicle or 0.05 μg/0.5 μl/eye of soluble VEGFR3 at P11, P12 and P13 and normoxic and 72 h of hypoxic eyes were enucleated, fixed, sections were made and coimmunostained for CD31 and Ki67. d All the conditions were the same as in (c) except that eyes were enucleated at 120 h of hypoxia (i.e., at P17), fixed, retinas were isolated, stained with isolectin B4, flat mounts were made and examined for filopodia at 40× magnification (scale bar, 50 μm). Bar graph shows quantitative analysis of the number of filopodia/unit vessel length. e All the conditions were the same as in (d) except that the flat mounts were examined for retinal vascularization. Retinal vascularization is shown in the first column at 2.5× magnification (scale bar, 500 μm). Neovascularization is highlighted in red in the second column. The third column shows the selected rectangular areas of the images in the first column at 10× magnification (scale bar, 200 μm). f,g Retinal neovascularization (f) and avascular area (g) were determined as described in “Materials and Methods.” The values are presented as mean±SD. *p < 0.01 vs normoxia; **p < 0.01 vs control + hypoxia.

Fig 3: Depletion of VEGFR2 levels blunts retinal neovascularization.a Upper panel: Equal amount of protein from control and the indicated time periods of VEGFA-treated HRMVECs were analyzed for phospho and total VEGFR1, 2 and 3 levels. Lower panel: The cell extracts were analyzed for VEGFR2 and VEGFR3 complex formation. b HRMVECs were transfected with siControl or siVEGFR2 (100 nM), quiesced, treated with and without VEGFA (40 ng/ml) for 10 min (for pPKCθ) or 120 min (for JunB and VEGFR3) and cell extracts were prepared and analyzed by western blotting for the indicated proteins. c Retinas from normoxic and 24 h (i.e., at P13) of hypoxic WT mice pups that received siControl or siVEGFR2 (1 μg/0.5 μl/eye) by intravitreal injections at P10 and P11 were isolated, extracts were prepared and analyzed by western blotting for the indicated proteins. d Eyes from normoxic and 72 h (i.e., at P15) of hypoxic WT mice pups that received siControl or siVEGFR2 (1 μg/0.5 μl/eye) by intravitreal injections at P11, P12 and P13 were enucleated, fixed, sections were made and coimmunostained for CD31 and VEGFR2 (left panel) and CD31 and VEGFR3 (right panel). e Eyes from normoxic and 24 h (i.e., at P13) of hypoxic WT mice pups that were injected intravitreally with vehicle or 0.05 μg/0.5 μl/eye of soluble VEGFR2 at P11 and P12 were enucleated, retinas were isolated, protein extracts were prepared and analyzed by western blotting for the indicated proteins using their specific antibodies. f Eyes from normoxic and 24 h of hypoxic mice pups that were injected intravitreally with siControl or siVEGFR2 (0.5 μg/0.5 μl/eye) at P11 and P12 were enucleated, retinas were isolated, protein extracts were prepared and analyzed by western blotting for VEGFR3 levels using its specific antibodies and the blot was normalized for β-tubulin. g All the conditions were the same as in (d) except that sections were coimmunostained for CD31 and Ki67. The bar graph shows quantification of proliferating ECs per section. h All the conditions were the same as in (d) except that eyes were enucleated at P17, fixed, retinas were isolated, stained with isolectin B4, flat mounts were made and examined for filopodia at 40× magnification (scale bar, 50 μm). Bar graph shows quantification of the number of filopodia/unit vessel length. i All the conditions were the same as in (h) except that the flat mounts were examined for retinal vascularization. Retinal vascularization is shown in the first column at 2.5× magnification (scale bar, 500 μm). Neovascularization is highlighted in red in the second column. The third column shows the selected rectangular areas of the images in the first column at 10× magnification (scale bar, 200 μm). j,k Retinal neovascularization (j) and avascular area (k) were determined as described in “Materials and Methods.” The values are presented as mean ± SD. *p < 0.01 vs normoxia; **p < 0.01 vs siControl + hypoxia.

Fig 4: Changes in axon transport are accompanied by increased expression of phosphorylated p38 MAPK and downstream Hsp27, but not Tau. (a) Immunolocalization of phosphorylated p38 MAPK (p-p38 MAPK; magenta), in retinas from the 48-h post-CTB injection transport studies, revealed increased expression in eyes treated with sVEGFR-2 (right panels), in comparison with IgG1 controls (left panels). Original magnification= × 10; green=CTB, blue=DAPI. Higher magnification ( × 40) images (left middle and right) showing elevated p-p38 MAPK as well as nuclear localization in some cells (arrows) in sVEGFR-2 treated retinas. (b) Differences in expression of phosphorylated Hsp27 were also observed between IgG1 (left panels) and sVEGFR-2 (left panels) treatment. Phosphorylated Hsp27 (green) was strongly upregulated following sVEGFR-2 administration, and colocalized with GFAP (red),41 indicating astrocyte expression. Blue=DAPI. Left and right middle panels – original magnification= × 10; left middle and right panels – original magnification= × 40. (c) In contrast, no changes were detected in phosphorylated Tau levels between IgG1 (left panels) and sVEGFR-2 (right panels). Red=p-Tau, green=CTB, blue=DAPI. Original magnifications= × 10 (left and right middle) and × 40 (left middle and right)

Fig 5: VEGFR3 mediates VEGFA-induced angiogenic events in HRMVECs.a Western blot analysis of control and the indicated time periods of VEGFA (40 ng/ml)-treated HRMVECs for VEGFR3 and β-tubulin levels. b,c The effect of siControl, siPKCθ and siJunB (100 nM) on VEGFA (40 ng/ml)-induced (2 h) VEGFR3 levels. The blots were sequentially reprobed for PKCθ and β-tubulin levels or JunB and β-tubulin levels to show the specificity and efficacy of siRNA on its target and off target molecules. d Upper panel: Retinal ECs were isolated from WT and PKCθ−/− mice and tested for the effect of VEGFA on PKCθ phosphorylation and JunB and VEGFR3 expression. Lower panel: Retinal ECs from PKCθ−/− mice were transfected with empty vector or JunB expression vector and two days later cell extracts were prepared and analyzed by western blotting for JunB, VEGFR3 and β-tubulin levels. e Upper panel: western blot analysis of VEGFR2, VEGFR3 and β-tubulin levels to show the specificity and efficacy of siControl and siVEGFR3 (100 nM) in HRMVECs. Bottom panel: The effect of siControl, siVEGFR3 and MAZ51 (5 μM) on VEGFA (40 ng/ml)-induced HRMVEC migration. f–h All the conditions were same as in (e) except that cells were treated with and without VEGFA (40 ng/ml) and DNA synthesis (f), sprouting (g) or tube formation (h) were measured. The bar graphs represent quantitative analysis of three independent experiments. The values are presented as mean ± SD. *p < 0.01 vs vehicle control or siControl; **p < 0.01 vs siControl + VEGFA. Scale bars in (g) and (h) are 50 and 200 μm, respectively.