Rapamycin reduces elevated VEGF and HIF-1 levels in hemangioma endothelial cells.

<p><b>A:</b> Immunoblotting showing the effects of rapamycin on p70S6K phosphorylation, HIF-1α expression and VEGF-A₁₆₅ expression in normal (HDMEC) or hemangioma (EC2, EC17B, EC21A) endothelial cell lysates. <b>B and C:</b> Luminex analysis showing reduced expression of HIF-1α (B) and VEGF-A₁₆₅ (C) in hemangioma endothelial cells treated with rapamycin. Data represent mean (n = 3)±SD; *<i>P</i><0.05 compared to vehicle.</p

Schematic diagram of mTOR signaling in hemangioma endothelial cells.

<p>Low levels of VEGFR1 in hemangioma endothelial cells result in constitutive VEGFR2 signaling and cell proliferation <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0042913#pone.0042913-Jinnin1" target="_blank">[2]</a>. PI3K and its downstream kinase AKT are constitutively phosphorylated in hemangioma endothelial cells as previously described <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0042913#pone.0042913-Jinnin1" target="_blank">[2]</a>. Phosphorylation of p70S6K, a known target of PI3K/AKT signaling, promotes translation of HIF-1 mRNA into protein, which translocates to the nucleus to regulate expression of target genes such as VEGF. This causes an autocrine loop of VEGF signaling via activation of VEGFR2. The mTOR inhibitor rapamycin prevents p70S6K phosphorylation and decreases expression of HIF-1. mTOR and HIF-1 inhibition is sufficient to reduce VEGF levels and proliferation rate of hemangioma endothelial cells.</p

Elevated HIF-1 expression in hemangioma endothelial cells caused by VEGF/PI3K signaling.

<p><b>A:</b> Immunoblotting showing high expression of HIF-1α in hemangioma endothelial cells (EC2, EC17B, EC21A) compared to normal endothelial cells (HDMEC). <b>B:</b> Immunocytochemistry showing constitutive nuclear localization of HIF-1α in hemangioma endothelial cells. <b>C and D:</b> Luminex analyses demonstrating that elevated HIF-1α levels in hemangioma endothelial cells are reduced in the presence of VEGF-A₁₆₅ neutralizing antibodies (VEGF Ab) or a chemical inhibitor of PI3K (LY294002). Data represent mean (n = 3)±SD; *<i>P</i><0.01 compared to IgG or vehicle.</p

Elevated VEGF levels in hemangioma endothelial cells are HIF-1-dependent.

<p><b>A:</b> Immunoblotting showing the expression knockdown effects of HIF-1α siRNA on HIF-1α and VEGF-A₁₆₅ in normal (HDMEC) and hemangioma (EC2, EC17B, EC21A) endothelial cells. <b>B and C:</b> Luminex analysis showing suppression of HIF-1α and VEGF-A₁₆₅ protein levels in hemangioma endothelial cells by HIF-1α siRNA. Data represent mean (n = 3)±SD; *<i>P</i><0.05 compared to control siRNA.</p

Rapamycin inhibits proliferation of hemangioma endothelial cells.

<p><b>A:</b> Flow cytometry analysis of normal (HDMEC) or hemangioma (EC2, EC17B, EC21A) endothelial cell proliferation by BrdU incorporation in the presence of rapamycin. <b>B:</b> Quantification of flow cytometry assessing the effects of rapamycin on BrdU incorporation. Data represent mean (n = 3)±SD; *<i>P</i><0.01 compared to vehicle.</p

VEGF stimulates p70S6K phosphorylation and HIF-1 expression.

<p><b>A:</b> Immunoblotting showing that treatment of normal endothelial cells (HDMEC) with exogenous VEGF-A₁₆₅ increases phosphorylation of p70S6K. <b>B:</b> Immunoblotting showing increased expression of HIF-1α and VEGF-A₁₆₅ in HDMEC treated with recombinant VEGF-A₁₆₅, suggesting an autocrine loop of signaling. Rapamycin prevents these increases. <b>C:</b> Flow cytometry analysis of BrdU incorporation showing that increased proliferation of HDMEC by VEGF-A₁₆₅ is inhibited in the presence of HIF-1α siRNA. <b>D:</b> Quantification of flow cytometry analysis of BrdU incorporation. Data represent mean (n = 3)±SD; *<i>P</i><0.05.</p

HIF-1 over-expression increases VEGF-dependent proliferation of endothelial cells.

<p><b>A:</b> Immunoblotting showing increased HIF-1α and VEGF-A₁₆₅ levels in HDMEC containing the pcDNA3-HIF-1α expression plasmid. <b>B:</b> Flow cytometry analysis of BrdU incorporation showing increased proliferation of cells with the pcDNA3-HIF-1α plasmid, which is prevented by VEGF neutralizing antibodies. Non-specific IgG and pcDNA3 vector were used as negative controls. <b>C:</b> Quantification of flow cytometry analysis of BrdU incorporation. Data represent mean (n = 3)±SD; *<i>P</i><0.01.</p

Inhibiting HIF-1 expression decreases proliferation of hemangioma endothelial cells.

<p><b>A:</b> Flow cytometry analysis of BrdU incorporation in normal (HDMEC) and hemangioma (EC2, EC17B, EC21A) endothelial cells transfected with control or HIF-1α siRNA duplexes. <b>B:</b> Quantification of BrdU incorporation showing decreased proliferation of hemangioma endothelial cells transfected with HIF-1α siRNA. Data represent mean (n = 3)±SD; *<i>P</i><0.05 compared to control siRNA.</p

Loss of <i>Lrp5</i> causes retinal hypovascularization and neovascularization.

<p>(A) ColIV whole mount IF staining showing retinal vessels of <i>Lrp5</i>^<i>-/-</i> and control mice at P9 and P30. Quantification of vascular sprout numbers at P5 shown at right. (B) Adult <i>Lrp5</i>^<i>-/-</i> retinas showing persistent hyaloid vessels (black arrows, CD31 IHC staining), aneurysms (open arrow, CD31 IHC staining), neovascular overgrowth (white arrows, fibronectin IF staining) and lack of IPL and OPL vascular development (lower panels, green: FITC-Dextran perfusion). (C) FITC-Dextran perfusion (green) showing retinal vascular leakage (white arrows) in adult <i>Lrp5</i>^<i>-/-</i> mice compared to control. Scale bars = 100μm. (D) EM analysis of endothelium of <i>Lrp5</i>^<i>-/-</i> and control retinas at P5, P8 and P30. Arrows point to area of endothelial fenestration. Scale bars = 500nm. (E) Total amounts of VEGF protein in retinas of <i>Lrp5</i>^<i>-/-</i> and control mice at P5, P8 and P30. Each ELISA was done in duplicate and normalized to total retinal protein amount. <i>n</i> = 9, 5, 12 for controls (at P5, P8, P30) and 9, 8, 8 for <i>Lrp5</i>^<i>-/-</i> (at P5, P8, P30). * <i>p</i><0.05, ** <i>p</i><0.01. Data are represented as means ± SD. Ctrl, control.</p

Endothelium-derived <i>Lrp6</i> is dispensable for retinal vascular development.

<p>Whole mount IF staining of ColIV (red) and FITC-Dextran perfusion showing (A) Retinal vasculature of <i>Tie2-Cre;Lrp5</i>^<i>fl/+</i><i>;Lrp6</i>^<i>fl/+</i> CKO mice. (B) Retinal vasculature of <i>Tie2-Cre;Lrp5</i>^<i>fl/+</i><i>;Lrp6</i>^<i>fl/fl</i> CKO mice. (C) Retinal vasculature of <i>Tie2-Cre;Lrp5</i>^<i>fl/fl</i> CKO mice. (D) Retinal vasculature of <i>Tie2-Cre;Lrp5</i>^<i>fl/fl</i><i>;Lrp6</i>^<i>fl/+</i> CKO mice. All mice are 8 weeks of age. Scale bars = 100nm.</p