EGF-Like-Domain-7 Is Required for VEGF-Induced Akt/ERK Activation and Vascular Tube Formation in an Ex Vivo Angiogenesis Assay

EGFL7 is a secreted angiogenic factor, which in contrast to the well-known secreted angiogenic molecules VEGF and FGF-2, is almost exclusively expressed by endothelial cells and may act in an autocrine fashion. Prior studies have shown EGFL7 to mediate its angiogenic effects by interfering with the Notch pathway and/or via the intronic miR126. Less is known about its effects on VEGF signaling. We wanted to investigate the role of epidermal growth factor-like domain 7 (EGFL7) in VEGF-driven angiogenesis using an ex vivo Matrigel-embedded mouse eye cup assay and siRNA mediated knockdown of EGFL7 by siRNA. Our results suggested that VEGF-induced vascular tube formation was significantly impaired after siRNA downregulation of EGFL7. In addition, knockdown of EGFL7 suppressed VEGF upregulation of phospho-Akt and phospho-Erk(1/2) in endothelial cells, but did not alter VEGFR phosphorylation and neuropilin-1 protein expression or miR126 expression. Thus, in conclusion, EGFL7 is required for VEGF upregulation of the Akt/Erk (1/2) pathway during angiogenesis, and may represent a new therapeutic target in diseases of pathological neovascularization

EGF-Like-Domain-7 Is Required for VEGF-Induced Akt/ERK Activation and Vascular Tube Formation in an Ex Vivo Angiogenesis Assay

EGFL7 is a secreted angiogenic factor, which in contrast to the well-known secreted angiogenic molecules VEGF and FGF-2, is almost exclusively expressed by endothelial cells and may act in an autocrine fashion. Prior studies have shown EGFL7 to mediate its angiogenic effects by interfering with the Notch pathway and/or via the intronic miR126. Less is known about its effects on VEGF signaling. We wanted to investigate the role of epidermal growth factor-like domain 7 (EGFL7) in VEGF-driven angiogenesis using an ex vivo Matrigel-embedded mouse eye cup assay and siRNA mediated knockdown of EGFL7 by siRNA. Our results suggested that VEGF-induced vascular tube formation was significantly impaired after siRNA downregulation of EGFL7. In addition, knockdown of EGFL7 suppressed VEGF upregulation of phospho-Akt and phospho-Erk(1/2) in endothelial cells, but did not alter VEGFR phosphorylation and neuropilin-1 protein expression or miR126 expression. Thus, in conclusion, EGFL7 is required for VEGF upregulation of the Akt/Erk (1/2) pathway during angiogenesis, and may represent a new therapeutic target in diseases of pathological neovascularization

AMPK-Activated Protein Kinase Suppresses Ccr2 Expression by Inhibiting the NF-κB Pathway in RAW264.7 Macrophages

<div><p>C-C chemokine receptor 2 (Ccr2) is a key pro-inflammatory marker of classic (M1) macrophage activation. Although Ccr2 is known to be expressed both constitutively and inductively, the full regulatory mechanism of its expression remains unclear. AMP-activated protein kinase (AMPK) is not only a master regulator of energy homeostasis but also a central regulator of inflammation. In this study, we sought to assess AMPK’s role in regulating RAW264.7 macrophage Ccr2 protein levels in resting (M0) or LPS-induced M1 states. In both M0 and M1 RAW264.7 macrophages, knockdown of the AMPKα1 subunit by siRNA led to increased Ccr2 levels whereas pharmacologic (A769662) activation of AMPK, attenuated LPS-induced increases in Ccr2 expression in an AMPK dependent fashion. The increases in Ccr2 levels by AMPK downregulation were partially reversed by NF-κB inhibition whereas TNF-a inhibition had minimal effects. Our results indicate that AMPK is a negative regulator of Ccr2 expression in RAW264.7 macrophages, and that the mechanism of action of AMPK inhibition of Ccr2 is mediated, in part, through the NF-κB pathway.</p></div

AMPKα1 reduction increases Ccr2 expression in the LPS-stimulated M1 macrophages through NF-κB signaling.

<p>A, B, C and D: RAW264.7 macrophages were pretreated with IKK inhibitor (BMS345541, 0.5–2 μM) and NF-κB inhibitors (LY303511, 1–3 μM; SM7368, 4 μM) for 2 h, followed by co-treatment with 100 ng/ml of LPS and different concentration of each inhibitor for 12 h. Ccr2 expression was analyzed by flow cytometry. n = 3. ***, <i>p <</i> 0.001.</p

Proposed mechanism of regulation for AMPKα1 in Ccr2 expression in RAW264.7 macrophages.

<p>AMPKα1 negatively regulates Ccr2 expression by inhibiting NF-κB pathway in both M0 and M1 macrophages.</p

AMPKα1 negatively regulates Ccr2 expression in the M0 and the LPS-stimulated M1 macrophages.

<p>A: Reduced AMPKα1 protein levels in macrophages treated with AMPKα1 siRNA RAW264.7 were confirmed by Western blotting of whole cell lysates. β-actin was probed as an internal control. B: Whole cell lysates of RAW264.7 macrophages treated with either control or AMPKα1 siRNA were examined by Western blotting to confirm compensation by AMPKα2. Tissue lysates prepared from mouse retina were used as a positive control for expression of AMPKα2 protein. β-actin was probed as an internal control. C: Ccr2 expression on RAW264.7 macrophages was analyzed by flow cytometry. RAW264.7 macrophages were stimulated with 10–1000 ng/ml of LPS for 12 h. D: Flow cytometry analysis of Ccr2 expression on RAW264.7 macrophages treated with either control or AMPKα1 siRNA. RAW264.7 macrophages were stimulated with 100 ng/ml of LPS for 12 h to induce the M1 state. n = 3. ***, <i>p <</i> 0.001.</p

VEGF-induced tube formation is EGFL7 dependent.

<p><i>A,</i> Mouse eye cups of each group were treated with EGFL7 or control siRNA after embedding them in Matrigel. Samples were cultured in VEGF (25 ng/ml) containing medium. At 3 and 5 days after knockdown of EGFL7, the tube length of neovascular from samples was evaluated by immunofluorescence using CD31 antibody. Bar equals 1000 μm. <i>B, ANOVA</i> Statistical analysis performed to evaluate the area of tube length. (n = 6) <i>*, P<0.01. **, P<0.05.</i> NS, not significant. C. At 3, 5 and 7 days after knockdown of EGFL7, endothelial cells were collected using anti-mouse CD31 antibody-coated magnetic beads (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0091849#pone.0091849.s004" target="_blank">Figure S4</a>). The amounts of EGFL7 in isolated cells were examined by Western blotting.</p

Pharmacological activation of AMPK counter-regulates Ccr2 expression in the LPS-stimulated M1 macrophages.

<p>A: RAW264.7 macrophages treated with either control or AMPKα1 siRNA were additionally treated with 25–100 μM of the AMPK activator, A769662. The phosphorylation of AMPKα (p-AMPKα) after A769662 treatment was examined by Western blotting. β-actin was probed as an internal control. B: RAW264.7 macrophages were pretreated with 25–100 μM A769662 for 2 h, followed by co-treatment with 100 ng/ml of LPS and each different concentration of A769662 for 12 h. Dimethyl sulfoxide (DMSO) was used as a control. Ccr2 expression was analyzed by flow cytometry. C: RAW264.7 macrophages treated with either control or AMPKα1 siRNA were pretreated with 50 μM A769662 for 2 h, followed by co-treatment with 100 ng/ml of LPS and 50 μM A769662 for 12 h. DMSO was used as a control. Ccr2 expression was analyzed by flow cytometry. n = 3. *, <i>p <</i> 0.05; ***, <i>p <</i> 0.001.</p

EGFL7 knockdown does not influence VEGFR2 phosphorylation or neuropilin 1 expression.

<p><i>A,</i> Mouse eye cups of each group were treated with EGFL7 or control siRNA after embedding them in Matrigel. Samples were cultured in VEGF (25 ng/ml) containing medium. At 3 days after knockdown of EGFL7, endothelial cells were collected using anti-mouse CD31 antibody-coated magnetic beads (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0091849#pone.0091849.s004" target="_blank">Figure S4</a>). The amounts of p-VEGFR2, neuropilin 1 and EGFL7 were examined by Western blotting. <i>B,</i> Densitometry of p-VEGFR2 in panel A. <i>C,</i> Densitometry of neuropilin 1 in panel A. <i>D,</i> Densitometry of EGFL7 in panel A. <i>ANOVA</i> Statistical analysis performed. (n = 3) <i>*, P<0.01. **, P<0.05.</i> NS, not significant. <i>*, P<0.01.</i> NS, not significant.</p