Fig 1: Endothelial cell conditional knockout of MYDGF disrupts retinal vascular homeostasis in adult mice and inhibits pathological angiogenesis in OIR mice.A–G Endothelial cell conditional MYDGF knockout in adult mice impacts retinal vasculature and neuronal integrity. A Western blotting analysis of MYDGF protein levels in primary murine retinal microvascular endothelial cells (mRMECs) isolated from adult sgC control and MYDGF-eCKO (#1 and #2) mice. B Western blotting analysis of listed proteins and quantification of protein phosphorylation in mRMECs from adult sgC and MYDGF-eCKO mice. C Representative retinal vascular images and quantification of vascular branches per view from adult sgC and MYDGF-eCKO mice. D Histological analysis (trypsin digestion and PAS staining) showing acellular capillaries (red arrowheads) and quantification of their number per view in adult sgC and MYDGF-eCKO retinas. E Representative images from Evans Blue (EB) assay and quantification of retinal EB permeation in adult sgC and MYDGF-eCKO mice. F Representative images of retinal flat mounts stained with Tubb3 (green) and NeuN (magenta), and quantification of Tubb3 & NeuN positive cells per view in adult sgC and MYDGF-eCKO mice. G Representative images of retinal sections stained with RBPMS (green), and quantification of RBPMS positive cells per view in adult sgC and MYDGF-eCKO retinas. H–J Endothelial cell conditional knockout of MYDGF alleviates pathological angiogenesis in the OIR model. H, I Western blotting analysis of listed proteins in mRMECs isolated from P17 OIR sgC control and MYDGF-eCKO (#1 and #2) mice, with results quantified. J Representative images of retinal neovascular tufts and quantification of tufts area (% of total vessel) in P17 OIR sgC and MYDGF-eCKO (#1 and #2) mice. Data are mean ± SD. n = 5 independent experiments with similar outcomes. One-way ANOVA with Bonferroni’s post hoc test is applied. Age-matched 5 male adult mice were used per group. Sex was not identified during the neonatal period. Scale bars were shown. Source data are provided in the Source data file.
Fig 2: The proposed signaling pathway of this study.MYDGF promotes retinal angiogenesis and vascular growth by triggering an autocrine signaling loop that activates the Gαi1/3-Gab1 complex and the downstream Akt-mTOR cascade. Created in BioRender.Wen, B. (2026) https://BioRender.com/5cmnt0y with publication license obtained.
Fig 3: Endothelial knockdown of MYDGF inhibits neonatal mouse retinal angiogenesis.A–G UMAP visualization of 12 distinct cell subpopulations identified by scRNA-seq from mouse retinas at postnatal days P6 and P10 (GEO: GSE175895). A Shows overall cell clustering, and B displays cell distribution at P6 and P10. UMAP density plot (C) indicating MYDGF expression across cell populations, and a dot plot (D) illustrating MYDGF expression and percentage of expressing cells in various retinal cell types at P6 and P10, highlighting predominant localization to endothelial cells and pericytes. Further scRNA-seq analysis of the endothelial cell compartment showing UMAP visualization of the designated endothelial cell subtypes (Resting, Proliferative, and Tip ECs) in (E), MYDGF expression density within these subtypes in (F), and a dot plot (G) demonstrating average expression and percentage of expressing cells for MYDGF and known markers in Tip, Proliferative, and Resting ECs. H Temporal expression pattern of MYDGF during retinal vascular development: qRT-PCR analysis of MYDGF mRNA levels (top) and Western blotting analysis of MYDGF protein levels (bottom) in primary murine retinal microvascular endothelial cells (mRMECs) at P1, P3, P5, and P7. I Representative retinal flat mounts stained with IB4 from neonatal mice with retro-orbital injection endothelial-specific MYDGF knockdown (MYDGF-eKD) or scramble control shRNA (shC, in same AAV construct) at P1, assessed at P5 and P7. Images show retinal vascular area, tip cells (yellow dots), and filopodia (yellow dots). Quantifications include total retinal vascular area, tip cell number per view, and number of filopodia per view. J Western blotting analysis of listed proteins and quantified protein phosphorylation levels in primary murine retinal microvascular endothelial cells (mRMECs) from neonatal WT (no AAV injection), shC, and MYDGF-eKD mice at P7. Data are mean ± SD. n = 5 independent experiments with similar outcomes. One-way ANOVA with Bonferroni’s post hoc test is utilized. Sex was not identified during the neonatal period. Scale bars were shown. Source data are provided in the Source data file.
Fig 4: Gab1 acts downstream of Gαi1/3 to mediate MYDGF-induced Akt/mTOR activation and angiogenesis.A–D Gαi1/3 are critical upstream regulators of MYDGF-induced Gab1 phosphorylation. A Western blotting analysis of MYDGF-induced Gab1 phosphorylation (p-Gab1) in WT, Gαi1 KO, Gαi3 KO, and Gαi1/3 DKO mouse embryonic fibroblasts (MEFs). Total Gab1 levels were shown. B Western blotting analysis of MYDGF-induced p-Gab1 in shC-MEFs and Gαi1/3 shRNA-expressing MEFs. Total Gab1 levels were shown. C Western blotting analysis of MYDGF-induced p-Gab1 in Gαi1/3 DKO MEFs, or reconstituted with OE-Gαi1 or OE-Gαi3. Total Gab1 and GAPDH levels were shown. D Western blotting analysis of MYDGF-induced p-Gab1 in WT-MEFs and MEFs co-overexpressing Gαi1 and Gαi3 (OE-Gαi1/3). Total Gab1 levels were shown. E, F Gab1 mediates MYDGF-induced Akt/mTOR signaling activation and interacts with Gαi1/3. E Western blotting analysis of MYDGF-induced phosphorylation of Akt (p-Akt), GSK3α/β (p-GSK3α/β), S6K (p-S6K), and S6 (p-S6) in WT-MEFs and Gab1 KO-MEFs. Total protein levels were shown. F Co-immunoprecipitation (Co-IP) experiments in hRMECs demonstrating MYDGF (25 ng/mL)-induced formation of Gαi1/3-Gab1 signaling complexes. Inputs were also shown. G–K Gab1 is essential for MYDGF-induced pro-angiogenic cellular responses in hRMECs. G Western blotting analysis of MYDGF-induced p-Akt and p-S6K in stable Gab1 knockdown (shGab1-hRMECs), CRISPR/Cas9-mediated Gab1 knockout (koGab1-hRMECs) and shC+sgC control hRMECs treated with MYDGF (25 ng/mL, 15 min), total proteins and Gab1 expression were shown. H Western blotting analysis confirming Gab1 overexpression and showing MYDGF (25 ng/mL, 15 min)-induced p-Gab1, p-Akt, and p-S6K in vector control (Vec) and oeGab1 hRMECs. Total protein levels, Gαi1, and Gαi3 expression were also shown. I Cell proliferation (EdU-positive cells), J cell migration (Transwell assay) and K tube formation in Vec and oeGab1 hRMECs treated with MYDGF (25 ng/mL, for designated hours) were shown. Data are mean ± SD. n = 5 independent experiments with similar outcomes. Two-tailed Student’s t-test (B, D, I–K) and one-way ANOVA with Bonferroni’s post hoc test (A, C, G). Scale bars were shown. Source data are provided in the Source data file.
Fig 5: Gαi1 and Gαi3 are essential for MYDGF-induced signaling and pro-angiogenic responses.A–F Gαi1/3 are critical for MYDGF-induced Akt-mTOR signaling and pro-angiogenic functions in human retinal microvascular endothelial cells (hRMECs). A Western blot of Gαi1/3 expression and Akt/S6K phosphorylation in shC and shGαi1/3 hRMECs after 15 min stimulation with 25 ng/mL MYDGF; total protein levels are shown. B Representative images and quantification of EdU proliferation, Transwell migration, and tube formation in shC and shGαi1/3 hRMECs stimulated with 25 ng/mL MYDGF. C CRISPR/Cas9-mediated Gαi1/3 knockout was verified by Western blot; Akt/S6K phosphorylation was examined in koC and koGαi1/3 hRMECs following 15 min MYDGF (25 ng/mL) stimulation. D Gαi1/3 overexpression was confirmed by western blot; Akt/S6K phosphorylation was detected in WT and OE-Gαi1/3 hRMECs after 15 min MYDGF treatment. E Proliferation, migration, and tube formation were assessed and quantified in WT and OE-Gαi1/3 hRMECs with 25 ng/mL MYDGF stimulation. F hRMECs were treated with 25 ng/mL MYDGF from 15 min (15’) to 24 h (24 h); Gαi1 and Gαi3 protein levels were analyzed by western blot and quantified. G–I Endothelial Gαi1/3 knockdown inhibits MYDGF-induced retinal angiogenesis in vivo. G Representative retinal flat mounts stained with IB4 and quantification of neovascular tufts area (% total area) from OIR mice with intravitreal injection of shC or endothelial-specific Gαi1/3 shRNA-AAV (Gαi1/3-eKD) at P3, followed by intravitreal MYDGF 50 pg injection at P12, with assessment at P17. H Representative retinal flat mounts stained with IB4 from neonatal mice with intravitreal injection of shC or Gαi1/3-eKD at P1, or with MYDGF 50 pg treatment, assessed at P5. Quantifications include total retinal vascular area, tip cell number per view, and number of filopodia per view. I Western blotting analysis of listed proteins and quantified phosphorylation levels in primary mRMECs from P5 neonatal mice treated as in (H). Data are mean ± SD. n = 5 independent experiments with similar outcomes. Two-tailed Student’s t-test (E, D) and one-way ANOVA with Bonferroni’s post hoc test (A–C, F–I). Sex was not identified during the neonatal period. Scale bars were shown. Source data are provided in the Source data file.
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