Glycation of fibronectin inhibits VEGF‐induced angiogenesis by uncoupling VEGF receptor‐2‐c‐Src crosstalk

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  FIGURE 1. Characterization of glycation of FN by MGO. FN (1 mg/mL) was incubated with MGO (0, 0.1, 1, 10 and 50 mM) at 37°C for 7 days. A, The samples were separated by SDS‐PAGE, and FN was detected with immunoblotting. B, AGEs were also immunoblotted on the same blots after stripping. C, The fluorescence intensity of MGO‐FN (50 mM MGO) was measured at 370/440 nm in the fractions. Results represent the mean ± SD for triplicate determinations. **P < .01

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  FIGURE 2. Glycation of FN inhibits VEGF signalling and VEGF‐induced angiogenesis. A, MGO‐FN inhibits VEGF‐induced activation of VEGFR‐2. HUVECs were cultured on FN or MGO‐FN and stimulated with VEGF (50 ng/mL) or vehicle control for 10 minutes. Phosphorylation (p) of VEGFR‐2, Akt and ERK1/2, and total VEGFR‐2, Akt and ERK1/2 were analysed by Western blotting in total cell lysates. Representative images of three independent experiments and densitometric analysis of phosphorylated VEGFR‐2, Akt and ERK1/2 normalized to total VEGFR‐2, Akt and ERK1/2 are shown. All data shown are mean ± SD for triplicate experiments and are expressed as fold changes. **P < .01. B, MGO‐FN inhibits VEGF‐induced cell migration. HUVECs were added to the upper chambers containing FN or MGO‐FN‐coated porous filters and then exposed to VEGF (50 ng/mL) or vehicle control. After 24 hours, the cells were fixed, stained with crystal violet and the cells that had migrated to the lower chambers were counted. Representative images of cell migration are shown. Scale bar is 200 μm. Quantitative assessment of triplicate cell migration experiments was performed. All data shown are mean ± SD and are expressed as % of control. **P…

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  FIGURE 3. Glycation of FN inhibits VEGF‐induced VEGFR‐2‐c‐Src interaction. HUVECs were cultured on FN or MGO‐FN and stimulated with VEGF (50 ng/mL) or vehicle control for 10 minutes. A, Cell lysates were prepared and immunoprecipitated with anti‐VEGFR‐2 antibody and protein A/G plus agarose beads. B, Cell lysates were immunoprecipitated with anti‐c‐Src antibody and protein A/G plus agarose beads. Then, captured proteins were analysed by Western blotting with anti‐c‐Src and anti‐VEGFR‐2 antibodies as indicated. Representative images of three independent experiments and densitometric analysis are shown. All data shown are mean ± SD for triplicate experiments and are expressed as fold changes. **P < .01

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  FIGURE 4. Glycated FN directly binds to RAGE. A, 96‐well plates were coated with FN or MGO‐FN at 4°C overnight. After washing and blocking, recombinant RAGE (5 μg/mL) was added and incubated at 37°C for 1 hour. Bound RAGE was detected by sequential addition of biotinylated anti‐RAGE antibody, streptavidin‐HRP conjugate and HRP substrate and measurement of OD450. Data shown are mean ± SD for triplicate experiments. B, HUVECs were seeded onto FN or MGO‐FN for 0, 15, 30, 60 and 120 minutes. Phosphorylation (p) of NF‐κB and total NF‐κB were analysed by Western blotting in total cell lysates. Representative images of three independent experiments and densitometric analysis of phosphorylated NF‐κB normalized to total NF‐κB are shown. All data shown are mean ± SD for triplicate experiments and are expressed as fold changes. **P < .01

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  FIGURE 5. A pool of c‐Src is sequestered by RAGE upon glycated FN stimulation. A and B, Glycation of FN induces RAGE‐c‐Src complex formation. MGO‐FN induces RAGE‐c‐Src interaction. HUVECs were cultured on FN or MGO‐FN and stimulated with VEGF (50 ng/mL) or vehicle control for 10 minutes. Cell lysates were prepared and immunoprecipitated with anti‐RAGE or anti‐c‐Src antibodies and protein A/G plus agarose beads. Then, captured proteins were analysed by Western blotting with anti‐c‐Src and anti‐RAGE antibodies as indicated. Representative images of three independent experiments and densitometric analysis are shown. All data shown are mean ± SD for triplicate experiments and are expressed as fold changes. **P < .01. C, The blockade of RAGE restores activation of VEGF signalling. HUVECs were pre‐treated with anti‐RAGE antibody (10 μg/mL) or control IgG for 30 minutes. Then, cells were seeded onto MGO‐FN‐coated 6‐well plates, followed by stimulation with VEGF (50 ng/mL) or vehicle control for 10 minutes. Phosphorylation (p) of VEGFR‐2, Akt and ERK1/2, and total VEGFR‐2, Akt and ERK1/2 were analysed by Western blotting in total cell lysates. Representative images of three independent experiments…

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  FIGURE 6. Glycation of FN inhibits VEGF‐induced angiogenesis in Matrigel plug assay. Matrigel supplemented with FN, FN and VEGF, MGO‐FN or MGO‐FN and VEGF was injected subcutaneously in mice for 14 days. Then, the Matrigel plugs were harvested, fixed and sectioned. A, The plugs were stained with haematoxylin and eosin, and the representative images are shown. B, The endothelial cells were labelled by immunochemical staining of CD31, and the representative images of CD31‐positive microvessels (arrows) in Matrigel plugs are shown. Scale bar is 100 μm. Quantitative assessment of microvessel formation was performed, and all data shown are mean ± SD. ***P < .001

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  FIGURE 7. Schematic diagrams representing the molecular mechanism underlying the glycation of FN‐regulated VEGF signalling and VEGF‐induced angiogenesis. A, Under the physiology condition, the binding of FN to VEGF enhances VEGF signalling and VEGF‐induced angiogenesis and the recruitment of c‐Src to VEGFR‐2 is critical for VEGFR‐2 phosphorylation and the downstream angiogenic signalling activation. B, Glycation of FN directly binds to RAGE and results in the interaction of RAGE to c‐Src, thereby preventing the binding of c‐Src to VEGFR‐2 and the consequent activation of VEGF‐VEGFR‐2 signalling pathway that leads to angiogenesis