Fig 1: VEGFD Reduces Formation of Empty Sleeves Triggered by Intravitreal NMDA Injection(A) Representative images of retinal whole mounts at d7 after intravitreal injections of NMDA or PBS from mice injected either with rAAV-LacZ or rAAV-VEGFD as indicated. Vessels were immunolabeled against collagen IV (Coll-IV, green) and using isolectin B4 (IB4, red). Scale bar, 30 μm. (B) Representative images of retinal whole mounts at d7 after intravitreal injections of PBS, NMDA, rVEGFD, or rVEGFD+NMDA. Vessels were immunolabeled against collagen IV (Coll-IV, green) and using isolectin B4 (IB4, red). Scale bar, 30 μm. (C) Quantification of empty sleeves at d7 after intravitreal injections, injected as indicated in (A). Two-way ANOVA, Bonferroni’s post hoc test. n = 6. rAAV-LacZ+PBS versus rAAV-LacZ+NMDA, p = 0.0008; rAAV-VEGFD+PBS versus rAAV-VEGFD+NMDA, p = 0.6565; rAAV-LacZ+PBS versus rAAV-VEGFD+PBS, p = 0.2950; rAAV-LacZ+NMDA versus rAAV-VEGFD+NMDA, p = 0.1484. (D) Quantification of empty sleeves at d7 after intravitreal injections, injected as indicated in (A). Number of empty sleeves in the NMDA-treated eye was normalized to the PBS control eye from the same animal. n = 6. (E) Quantification of empty sleeves at d1 after intravitreal injections with PBS, NMDA, rVEGFD, or rVEGFD+NMDA. Two-way ANOVA, Bonferroni’s post hoc test. n = 6. PBS versus NMDA (sham), p = 0.0003; PBS versus NMDA (rVEGFD), p = 0.5067; sham versus rVEGFD (PBS), p > 0.9999; sham versus rVEGFD (NMDA), p = 0.0022. (F) Quantification of empty sleeves at d7 after intravitreal injections, injected as indicated in (B). Two-way ANOVA, Bonferroni’s post hoc test. n = 6. PBS versus NMDA (sham), p = 0.0001; PBS versus NMDA (rVEGFD), p = 0.1673; sham versus rVEGFD (PBS), p = 0.8438; sham versus rVEGFD (NMDA), p = 0.0329. (G) Quantification of empty sleeves at d7 after intravitreal injections, injected as indicated in (B). Number of empty sleeves in the NMDA-treated eye was normalized to the PBS control eye from the same animal. n = 6. (H) Quantification of empty sleeves at d1 after intravitreal injections of PBS, NMDA, rGFP, or rGFP+NMDA. Two-way ANOVA, Bonferroni’s post hoc test. n = 6. PBS versus NMDA (sham), p = 0.0019; PBS versus NMDA (rGFP), p = 0.0004; sham versus rGFP (PBS), p > 0.9999; sham versus rGFP (NMDA), p = 0.6279. (I) Quantification of empty sleeves at d7 after intravitreal injections, injected as indicated in (H). Two-way ANOVA, Bonferroni’s post hoc test. n = 6. PBS versus NMDA (sham), p = 0.0143; PBS versus NMDA (rGFP), p = 0.0050; sham versus rGFP (PBS), p = 0.5439; sham versus rGFP (NMDA), p = 0.1376. (J) Quantification of empty sleeves at d7 after intravitreal injections, injected as indicated in (H). Number of empty sleeves in the NMDA-treated eye was normalized to the PBS control eye from the same animal. n = 6. Graphs represent mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001. See also Figure S1.
Fig 2: NMDA-Triggered Excitotoxicity Reduces VEGFD Expression in RGCs(A) qRT-PCR analysis of vegfd, vegfc, vegfa, flt4, flt1, and kdr expression in retinal homogenates after intravitreal injection of NMDA. Unpaired t test. n = 6. vegfd, p = 0.0085; vegfc, p = 0.2052; vegfa, p = 0.8524; flt4, p = 0.9414; flt1, p = 0.6074; kdr, p = 0.2016. (B) Representative images of human retinal whole mounts. Retinas were immunolabeled with antibodies against Brn3a (red) and VEGFD (green). Scale bars, 10 μm. Negative controls of human retinas were immunolabeled only with secondary antibodies. (C) Representative bands of VEGFD, VEGFA, and β-actin cDNA products following qRT-PCR analysis of human retinal extracts obtained from two donors (subject 1, subject 2). (D) Quantification of VEGFD protein levels in cells in the ganglion cell layer of retinas of mice injected as indicated. Unpaired t test. n = 3. PBS versus NMDA, p = 0.0192. (E) Representative images of mouse sagittal retina sections at d7 after intravitreal injections of NMDA or PBS. Retinas were immunolabeled for VEGFD (green). Nuclei were labeled with Hoechst (blue). GCL, ganglion cell layer. Scale bar, 20 μm. (F) qRT-PCR analysis of vegfd, vegfc, vegfa, and flt4 expression in brain ECs (b.END3) 24 h after 20 μM NMDA treatment. Unpaired t test. n = 3. vegfd, p = 0.1744; vegfc, p = 0.7598; vegfa, p = 0.8998; flt4, p = 0.4457. (G) qRT-PCR analysis of vegfd, vegfc, vegfa, and flt4 expression in primary astrocytes 24 h after 20 μM NMDA treatment. Unpaired t test. n = 3. vegfd, p = 0.8716; vegfc, p = 0.4918; vegfa, p = 0.9902; flt4, p = 0.9331. Bars represent mean ± SEM. Dots represent single values. **p < 0.01.
Fig 3: Recombinant VEGFD Prevents Decrease of Pattern Electroretinography Amplitudes after NMDA-Triggered Damage to RGCs(A) Representative images of retinal whole mounts at d7 after intravitreal injections of PBS, NMDA, rVEGFD, or rVEGFD+NMDA. RGCs were immunolabeled against Brn3a. Scale bar, 20 μm. (B) Amplitude of the second harmonic of the fast Fourier transformation plotted against spatial frequency of stimulation. Asterisks indicate statistical significance between NMDA- and rVEGFD+NMDA-treated animals. Two-way ANOVA, Bonferroni’s post hoc test. n = 4. PBS versus NMDA (0.05 cyc/deg), p = 0.6877; PBS versus NMDA (0.10 cyc/deg), p = 0.0076; PBS versus NMDA (0.20 cyc/deg), p = 0.0964; PBS versus NMDA (noise), p = 0.8182; PBS versus rVEGFD (0.05 cyc/deg), p = 0.9669; PBS versus rVEGFD (0.10 cyc/deg), p = 0.9140; PBS versus rVEGFD (0.20 cyc/deg), p = 0.5811; PBS versus rVEGFD (noise), p = 0.9509; rVEGFD versus rVEGFD+NMDA (0.05 cyc/deg), p = 0.4433; rVEGFD versus rVEGFD+NMDA (0.10 cyc/deg), p = 0.9976; rVEGFD versus rVEGFD+NMDA (0.20 cyc/deg), p = 0.8797; rVEGFD versus rVEGFD+NMDA (noise), p = 0.9980; NMDA versus rVEGFD+NMDA (0.05 cyc/deg), p = 0.0144; NMDA versus rVEGFD+NMDA (0.10 cyc/deg), p = 0.0296; NMDA versus rVEGFD+NMDA (0.20 cyc/deg), p = 0.2776; NMDA versus rVEGFD+NMDA (noise), p = 0.9980. (C) Representative pattern electroretinography waveforms recorded from mice injected as indicated in (A). Bars represent mean ± SEM. Dots represent single values. *p < 0.05, **p < 0.01.
Fig 4: VEGFD Protection against NMDA-Triggered Excitotoxic Retinal Damage Is Mediated by Neuronal VEGFR3 Expression(A) Representative confocal scans of retinal whole mounts at d7 after intravitreal injections of PBS, NMDA, rVEGFD, or rVEGFD+NMDA from mice intravitreally injected either with rAAV-shSCR (control) or rAAV-shVEGFR3. RGCs were immunolabeled using Brn3a. Scale bar, 20 μm. (B) Quantification of RGCs injected as indicated in (A). Two-way ANOVA, Bonferroni’s post hoc test. n = 8. rAAV-shSCR+PBS versus rAAV-shSCR+NMDA, p = 0.0067; rAAV-shSCR+PBS versus rAAV-shSCR+rVEGFD, p = 0.9583; rAAV-shSCR+PBS versus rAAV-shSCR+rVEGFD+NMDA, p = 0.4943; shVEGFR3+PBS versus rAAV-shVEGFR3+NMDA, p = 0.0046; rAAV-shVEGFR3+PBS versus rAAV-shVEGFR3+rVEGFD, p = 0.7359; rAAV-shVEGFR3+PBS versus rAAV-shVEGFR3+rVEGFD+NMDA, p = 0.0017. (C) Quantification of RGCs at d7 after intravitreal injections, injected as indicated in (A), in rAAV-shSCR-injected eyes. RGC numbers in the NMDA-treated eye were normalized to the PBS control eye from the same animal. n = 8. (D) Quantification of RGCs at d7 after intravitreal injections injected as indicated in (A), in rAAV-shVEGFR3-injected eyes. RGC numbers in the NMDA-treated eye were normalized to the PBS control eye from the same animal. n = 8. (E) Representative confocal scans of retinal whole mounts at d7 after intravitreal injections of PBS, NMDA, rVEGFD, or rVEGFD+NMDA from mice injected either with rAAV-shSCR or rAAV-shVEGFR3 as indicated. Vessels were immunolabeled using collagen IV (Coll-IV, green) and isolectin B4 (IB4, red). Scale bar, 30 μm. (F) Quantification of empty sleeves injected as indicated in (E). Two-way ANOVA, Bonferroni’s post hoc test. n = 6. rAAV-shSCR+PBS versus rAAV-shSCR+NMDA, p = 0.0018; rAAV-shSCR+PBS versus rAAV-shSCR+rVEGFD, p = 0.5622; rAAV-shSCR+PBS versus rAAV-shSCR+rVEGFD+NMDA, p = 0.1073; rAAV-shVEGFR3+PBS versus rAAV-shVEGFR3+NMDA, p = 0.0009; rAAV-shVEGFR3+PBS versus rAAV-shVEGFR3+rVEGFD, p = 0.2623; rAAV-shVEGFR3+PBS versus rAAV-shVEGFR3+rVEGFD+NMDA, p = 0.0001. (G) Quantification of empty sleeves at d7 after intravitreal injections, injected as indicated in (E), in rAAV-shSCR-injected eyes. Number of empty sleeves in the NMDA-treated eye was normalized to the PBS control eye from the same animal. n = 8. (H) Quantification of empty sleeves at d7 after intravitreal injections, injected as indicated in (E), in rAAV-shVEGFR3-injected eyes. Number of empty sleeves in the NMDA-treated eye was normalized to the PBS control eye from the same animal. n = 8. (I) Schema of neuronal-mediated VEGFD protection against excitotoxic retinal damage. Upper row, left panel: NMDA triggers RGC and EC degeneration in rAAV-shSCR-injected mice by direct action on RGCs (red arrows). RGCs and ECs express VEGFR3 (VEGFR3+). Lower row, left panel: VEGFD prevents RGCs and ECs from NMDA-triggered degeneration in rAAV-shSCR-injected mice. RGCs and ECs express VEGFR3 (VEGFR3+). VEGFD can act on VEGFR3 in both RGCs and ECs (green arrows). Upper row, right panel: NMDA triggers RGC and EC degeneration in rAAV-shVEGFR3-injected mice by direct action on RGCs (red arrows). RGCs do not express VEGFR3 (VEGFR3−); ECs express VEGFR3 (VEGFR3+). Lower row, right panel: In rAAV-shVEGFR3-injected mice, VEGFD does not prevent RGCs and ECs from NMDA-triggered degeneration. RGCs do not express VEGFR3 (VEGFR3−); ECs express VEGFR3 (VEGFR3+). VEGFD cannot act on VEGFR3 in RGCs, but on VEGFR3 in ECs (green arrows). Graphs represent mean ± SEM. **p < 0.01, ***p < 0.001, ****p<0.0001 See also Figure S1.
Fig 5: VEGFD Overexpression in RGCs Protects Them from NMDA-Mediated Damage(A) Representative images of retinal whole mounts at d7 after intravitreal injections of NMDA or PBS from mice injected either with rAAV-LacZ or rAAV-VEGFD as indicated. RGCs were immunolabeled against Brn3a (green). Scale bar, 15 μm. (B) Representative images of sagittal optic nerve sections injected as indicated. Stressed axons were immunolabeled using SMI-32 (green). Nuclei were labeled using Hoechst (blue). Scale bar, 50 μm. (C) Quantification of RGCs injected as indicated. Two-way ANOVA, Bonferroni’s post hoc test. n = 9. rAAV-LacZ+PBS versus rAAV-LacZ+NMDA, p < 0.0001; rAAV-VEGFD+PBS versus rAAV-VEGFD+NMDA, p = 0.5035; rAAV-LacZ+PBS versus rAAV-VEGFD+PBS, p > 0.9999; rAAV-LacZ+NMDA versus rAAV-VEGFD+NMDA, p = 0.0006. (D) Quantification of RGCs injected as indicated. RGC numbers in the NMDA-treated eye were normalized to the PBS control eye from the same animal. n = 9. (E) Quantification of SMI-32+ clusters in optic nerves from eyes injected as indicated. Two-way ANOVA, Bonferroni’s post hoc test. n = 6. rAAV-LacZ+PBS versus rAAV-LacZ+NMDA, p < 0.0001; rAAV-VEGFD+PBS versus rAAV-VEGFD+NMDA, p = 0.7794; rAAV-LacZ+PBS versus rAAV-VEGFD+PBS, p > 0.9999; rAAV-LacZ+NMDA versus rAAV-VEGFD+NMDA, p < 0.0001. (F) Quantification of SMI-32+ clusters in optic nerves from eyes injected as indicated. SMI-32+ clusters in the NMDA-treated eye were normalized to the PBS control eye from the same animal. n = 6. Bars represent mean ± SEM. Dots represent single values. ***p < 0.001, ****p < 0.0001. See also Figure S1.
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