Glycation of Tie-2 Inhibits Angiopoietin-1 Signaling Activation and Angiopoietin-1-Induced Angiogenesis

Figures

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  Figure 1. Tie-2 is glycated in HFD–fed mice. (a) The aortae derived from ND-fed C57BL/6J mice were isolated, fixed, embedded in paraffin, and sectioned. The Tie-2 and AGEs were then immunohistochemically stained in two serial aortic sections. Distance bars, 50 μm. Representative images are shown. n = 4. (b) The aortae derived from HFD-fed C57BL/6J mice were isolated, fixed, embedded in paraffin, and sectioned. The Tie-2 and AGEs were then immunohistochemically stained in two serial aortic sections. The solid black rectangles show the colocalization of Tie-2 and AGEs. Distance bars, 50 μm. Representative images are shown. n = 4. (c) Quantitative assessment of AGE expression was performed. (d) Quantitative assessment of Tie-2 expression was performed. *** p < 0.001. Error bar represents the standard deviation and p value was generated by t test.

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  Figure 2. Tie-2 is glycated in MGO–treated endothelial cells. (a) HUVECs were exposed to MGO (100 μM) or vehicle control for 24 h. The cell lysates were immunoprecipitated with anti-Tie-2 antibody and the captured proteins were analyzed by Western blotting with anti-AGE and anti-Tie2 antibodies. Representative images of three independent experiments are shown. (b) HUVECs were stimulated with MGO (0, 50, 100 and 200 μM) for 24 h and total expression of Tie-2 was detected using Western blotting. Representative images of three independent experiments are shown and densitometric analysis of Tie-2 normalized to GAPDH was performed. (c) HUVECs were stimulated with MGO (100 μM) or vehicle for 24 h and the cell membrane proteins were separated. The Tie-2 expression at the cell membrane was determined with Western blotting and representative images of three independent experiments are shown. The densitometric analysis of Tie-2 normalized to Na+-K+-ATPase was carried out. Error bar represents the standard deviation and p value was generated by t test.

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  Figure 3. MGO inhibits Ang-1 signaling activation and Ang-1–induced angiogenesis. (a) HUVECs were exposed to MGO (100 μM) or vehicle control for 24 h, followed by stimulation with Ang-1 (200 ng/mL) for 15 min. Phosphorylation (p) of Tie-2 and Akt and total Tie-2 and Akt were detected by Western blotting. Representative images of three independent experiments are shown and densitometric analysis of phosphorylated Tie-2 and Akt normalized to total Tie-2 and Akt was performed. (b) HUVECs cultured in six-well plates were exposed to MGO (100 μM) or vehicle control for 24 h. The cells were then detached and added to the upper chambers containing porous filters, followed by stimulation with Ang-1 (200 ng/mL) or vehicle control. After 24 h, the cells were fixed, stained, and the cells that had migrated to the lower chambers were counted. Representative images of three independent experiments are shown and quantitative assessment was performed. (c) HUVECs cultured in six-well plates were exposed to MGO (100 μM) or vehicle control for 24 h. The cells were then detached and seeded onto Matrigel in the presence of Ang-1 (200 ng/mL) or vehicle control for 18 h. Representative images of three…

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  Figure 4. AG prevents MGO–induced Ang-1/Tie-2 signaling dysfunction. (a) HUVECs were pre-treated with AG (100 μM) or vehicle control for 1 h and then exposed to MGO (100 μM) for 24 h, followed by stimulation with Ang-1 (200 ng/mL) or vehicle control for 15 min. Phosphorylation (p) of Tie-2 and Akt and total Tie-2 and Akt were detected by Western blotting. Representative images of three independent experiments are shown and densitometric analysis of phosphorylated Tie-2 and Akt normalized to total Tie-2 and Akt was performed. (b) HUVECs cultured in six-well plates were pre-treated with AG (100 μM) or vehicle control for 1 h, followed by exposure to MGO (100 μM) for 24 h. The cells were then detached and added to the upper chambers containing porous filters, followed by stimulation with Ang-1 (200 ng/mL) or vehicle control. After 24 h, the cells were fixed, stained, and the cells that had migrated to the lower chambers were counted. Representative images of three independent experiments are shown and quantitative assessment was performed. Distance bars, 200 μm. (c) HUVECs cultured in six-well plates were pre-treated with AG (100 μM) or vehicle control for 1 h, followed by exposure to MGO…

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  Figure 5. Glycation of Tie-2 inhibits Ang-1–induced vessel outgrowth ex vivo. The aortae derived from ND-fed C57BL/6J mice were isolated, treated with AG (100 μM) or vehicle control for 1 h, followed by exposure to MGO (100 μM) for 24 h. (a) The aortae were then fixed, embedded in paraffin, and sectioned. The Tie-2 and AGEs were immunohistochemically stained in two serial aortic sections. The solid black rectangles show the colocalization of Tie-2 and AGEs. Distance bars, 50 μm. Representative images are shown. n ≥ 6 per group. (b) The aortic rings were embedded into collagen I gel and stimulated with Ang-1 (200 ng/mL) or vehicle control for 14 d. Representative images are shown. n = 8 per group. Quantitative analysis of microvessel sprouts from the aortic rings was carried out. ** p < 0.01, *** p < 0.001. Error bar represents the standard deviation and p value was generated by one-way ANOVA followed by post hoc comparison.

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  Figure 6. The molecular mechanism underlying Tie-2 glycation–regulated Ang-1 signaling and Ang-1–induced angiogenesis. (a) Under the physiology condition, the binding of Ang-1 to Tie-2 induces Tie-2 and Akt activation, causing endothelial cell migration and tube formation, consequently leading to angiogenesis. (b) However, hyperglycemia or high MGO in diabetes mellitus induces AGEs–Tie-2 adduct formation (Tie-2 glycation), which inhibits Ang-1 signaling activation and Ang-1-induced angiogenesis.