Endothelial cell polarity and extracellular matrix composition require functional ATP6AP2 during developmental and pathological angiogenesis

Figures

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  Figure 1. Defective retinal angiogenesis in EC-specific Atp6ap2-KO mice. (A) Strategy for EC-specific deletion of Atp6ap2 in postnatal mice (Atp6ap2 induced endothelial cell KO; Atp6ap2iECKO) by tamoxifen administration at P1–P3 for early induction or at P5–P7 for late induction. Murine retinas were analyzed at indicated ages. (B) qPCR analysis of isolated lung ECs (iLECs) show Atp6ap2 expression levels in control and Atp6ap2iECKO mice at P7 (n = 3, triplicates for each sample). (C) Western blot analysis of iLECs from control and Atp6ap2iECKO mice at P7. (D) Densitometric quantification of ATP6AP2 levels in C (n = 3). (E–H) Whole-mount isolectin-IB4–stained (IB4-stained) retinas (dotted circles represent outgrowth in the control retina) (E) and quantification of radial outgrowth (F), vascular density (G), and branch points in control and Atp6ap2iECKO mice at P7 (n =7) (H). Scale bars: 1,000 µm. (I–L) Whole-mount IB4-stained retinas and closeup images of IB4+ vessels in the superficial plexus and deep plexus (I) with quantification of radial outgrowth (n = 4) (J), vascular density in the superficial plexus (n = 4) (K), and vascular density in the deep plexus (n = 4) (L) in control and…

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  Figure 2. ATP6AP2 is critical for sprouting angiogenesis. (A) Images of IB4+ vessels from P7 control and Atp6ap2iECKO retinas, with white arrowheads indicating sprouts at the vascular front. Scale bars: 100 µm. (B) Quantification of the number of sprouts in control and Atp6ap2iECKO mice at P7 (n = 4). (C) Images of IB4+ vessels at high magnification showing filopodia in sprouts (white asterisks) at the vascular front . Scale bars: 10 µm. (D) Quantification of the number of filopodia per sprout in control and Atp6ap2iECKO mice at P7 (n = 4). (E) Western blot analysis of ATP6AP2 and β-actin in control-siRNA and Atp6ap2 siRNA–treated TeloHAECs. (F) Densitometric quantification of ATP6AP2 levels in E normalized to β-actin in TeloHAECs following siRNA treatments (n = 3). (G) qPCR analysis of control-siRNA and Atp6ap2 siRNA–treated TeloHAECs for Atp6ap2 mRNA levels normalized to GAPDH transcripts (n = 3; triplicates for each sample). (H) Representative images of TeloHAEC sprouting bead assays embedded in 3D fibrinogen gel at 120 hours following control and Atp6ap2-siRNA treatments. scale bars: 100 µm. Black arrowheads indicate vessel bifurcations. (I) Quantification of the number of sprouts per…

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  Figure 3. ATP6AP2 regulates endothelial cell migration and polarity in vivo and in vitro. (A) Representative images of ERG+ nuclei of ECs at the vascular front in P7 control and Atp6ap2iECKO retinas. Scale bars: 25 µm. (B) Quantification of nuclear ellipticity in control and Atp6ap2iECKO mice at P7 (n = 4). (C) Scratch wound assays performed on TeloHAEC monolayers following control and Atp6ap2 siRNA treatments. Images at 0 and 40 hours following the scratch. Scale bars: 75 µm. (D) Quantification of the wound closure and rate of migration in control and Atp6ap2 siRNA–treated TeloHAECs at 40 hours (n = 12). (E) Images of fluorescently labeled IB4+ vessels, ERG+ nuclei in ECs, and Golgi Matrix Protein GM130+ Golgi apparatuses at the vascular front with their respective insets (white dashed-line boxed) highlighting tip cells in control and Atp6ap2iECKO mice at P7. Scale bars: 50 µm and 15 μm (insets). White arrowheads denote the Golgi apparatus position in respect to the nucleus. White arrows indicate direction of the retinal vasculature outgrowth. (F) Images of DAPI+ nuclei, GM130+ Golgi apparatuses, and F-actin+ cytoskeleton of indicated TeloHAECs at 12 hours after initiating cell migration…

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  Figure 4. Loss of Atp6ap2 in ECs results in misregulation of angiogenesis, directed cell migration, and extracellular matrix–related genes. (A) Outline of the workflow used to collect iLECs from control and Atp6ap2iECKO mice at P7 (n = 3) for RNA-Seq analyses. (B) MA plot of differentially expressed genes between Atp6ap2iECKO and control iLECs. Red dots, upregulated genes (823 genes); blue dots, downregulated genes (1,053 genes). (C) Summary of Atp6ap2iECKO-upregulated and -downregulated EC-gene distribution among organ EC–specific mRNAs. (D) Top GO biological process terms enriched in up- or downregulated genes in Atp6ap2iECKO iLECs (FDR ≤ 0.05). (E–G) Representative clustered heatmaps of gene count Z scores for angiogenesis (E), cell migration (F), and extracellular matrix–related genes (G) that are differentially expressed upon loss of Atp6ap2 in ECs. Columns represent individual biological replicates. (H) Images of P7 control and Atp6ap2iECKO retinas immunofluorescently labeled for IB4 and collagen IV (COLIV). Note reduced levels of COLIV in Atp6ap2 mutants. Scale bars: 50 μm. (I–L) qPCR analysis of isolated retinal ECs (iRECs) shows Atp6ap2, α-parvin, Col3a1, and Lama2 expression…

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  Figure 5. ATP6AP2 knockdown in ECs results in impaired ERK1/2 signaling and downregulation of extracellular matrix proteins. (A) Western blot analysis of collagen III, laminin, and β-actin from control and Atp6ap2iECKO iLECs at P7 (n = 3). (B and C) Densitometric quantification of collagen III (B) and laminin (C) levels in control and Atp6ap2iECKO iLECs from A. (D) Western blot analyses for the indicated proteins in prorenin-stimulated TeloHAECs at indicated time points following control and Atp6ap2 siRNA treatment. Note that the β-actin blots correspond to the same protein samples represented directly above them. For instance, the β-actin blot at the bottom corresponds to the samples used to assess ATP6AP2. (E–G) Densitometric quantifications of pERK1/2 (E), collagen III (F), laminin (G) levels in control and Atp6ap2 siRNA–treated cells at indicated time points following prorenin stimulation (n = 3 for each time point). Data are shown as mean ± SD; 2-tailed unpaired t test. ***P < 0.001, ****P < 0.0001.

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  Figure 6. ATP6AP2 is required for proper revascularization during pathological angiogenesis in the OIR mouse model. (A) Schematic showing the timeline of the OIR protocol with hyperoxia phase from P7 to P12, tamoxifen administration from P12 to P14, and retina analysis at P17. (B) Whole-mount IB4 stained retinas showing the avascular area (yellow) and neovascular tuft (NVT) area (red) in control OIR and Atp6ap2iECKO OIR mice at P17. Scale bars: 1,000 µm. (C and D) Quantification of the avascular area and NVT area in control OIR (n = 8) and Atp6ap2iECKO OIR mice (n = 9) at P17. (E) Images of IB4+ vessels, ERG+ nuclei of ECs, and GM130+ Golgi apparatuses at the neovascularization edge in control OIR and Atp6ap2iECKO OIR P17 retinas. Scale bars: 50 µm. The respective insets (white dashed-line boxes) show magnified views of tip cells. White arrowheads indicate the Golgi apparatus position in respect to the nucleus of tip ECs. White arrows indicate direction of revascularization toward the avascular area. Data are shown as mean ± SD; 2-tailed unpaired t test. ****P < 0.0001.