Liraglutide Inhibits Endothelial-to-Mesenchymal Transition and Attenuates Neointima Formation after Endovascular Injury in Streptozotocin-Induced Diabetic Mice

Figures

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  Figure 1. Liraglutide promotes endothelium recovery and reduces neointima formation in wire-injured carotid arteries of diabetic mice. Streptozotocin (STZ)-induced diabetic C57BL/6 mice were subjected to a wire-induced carotid artery injury with or without liraglutide treatment. (a) The percentage of the area of re-endothelisation was determined after the systemic injection of Evans blue at day one and day five (n = 3 per group). The stained blue area corresponds to the area with poor re-endothelialisation. The black arrowheads point at the area without blue staining area, which indicated re-endothelialised cells. Data are shown as mean ± SEM. * P < 0.05 analyzed by Student’s t-test. (b) The right common carotid arteries were collected on day 28 post-injury for haematoxylin and eosin (H and E) staining to evaluate the severity of neointima formation between groups. Data are shown as the mean ± SD (all groups, n = 10 per group), * P < 0.05 analyzed by Student’s t-test. IEL, internal elastic lamina; EEL, external elastic lamina; L, lumen; I, intima; M, media; A, adventitia.

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  Figure 2. Endothelia-derived cells contribute to neointima formation through EndMT. (a) Schematic representation of the induction of endothelial-specific GFP labelling after tamoxifen injection in TekCreERT2/DRG mice. (b) Representative images of immunofluorescence staining for the protein expression of GFP (green) and α-SMA (red) in the common carotid artery. Nuclei were counterstained with 4′,6-diamidino-2-phenylindole·2HCl (DAPI) (blue). The enlarged field (yellow square) represents the GFP expression in the endothelial layer of the carotid artery. (c) STZ-induced diabetic TekCreERT2/DRG mice were subjected to a wire-induced injury or a wire injury plus liraglutide treatment (n = 4 in each group). The common carotid arteries were collected on day 28. Representative images show the immunofluorescence staining for GFP (green), α-SMA (red), and nuclei (blue) in wire-injured common carotid arteries. The enlarged field (yellow square) represents the co-localization of GFP expression in α-SMA-positive cells (white arrowheads) in the neointima. (d) Quantification of the number of GFP+SMA+ cells in the neointima area between groups. (e) Quantification of the neointima to media ratio between…

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  Figure 3. High glucose induces mesenchymal-like phenotypes in human umbilical venous endothelial cells (HUVECs). HUVECs were cultured in a control medium with a normal glucose level (NG, 5.5 mM of glucose), mannitol (M, 5.5 mM of glucose + 24.5 mM of mannitol), or high glucose (HG, 30 mM of glucose). (a) Representative bright-field images show the morphological changes in cells after 96 h of culture at different conditions. Scale bar represents 200 μm. The expression of VE-cadherin (green) and SM22α (red) in each group was analyzed using immunofluorescence staining. Nuclei were counterstained with DAPI (blue). HUVECs cultured in a HG medium showed a higher SM22α expression and a lower VE-cadherin expression compared to those in NG- or mannitol-treated groups. Scale bar represents 50 μm. (b) qRT-PCR analysis of the mRNA levels of CD31, SM22α, vimentin, and Snail in NG-, mannitol-, and HG-treated HUVECs. Data are shown as the mean ± SD from three independent experiments. * P < 0.05. The statistical significance was assessed by Kruskal–Wallis test.

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  Figure 4. High glucose promotes EndMT in HUVECs. (a) Representative images of the western blot analysis of p-Smad2 protein levels. Total Smad2/3 served as the loading control. The quantification of protein levels is presented as mean ± SD from three independent experiments. (b) Representative images of the western blot analysis of CD31, SM22α, vimentin, and Snail protein levels in HUVECs treated with different conditions. (c) Quantification of the relative fold increase is shown as the mean ± SD from three independent experiments. * P < 0.05. The statistical significance was assessed by Kruskal–Wallis test.

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  Figure 5. Liraglutide attenuates high glucose- and IL-1β-induced TGF-β signaling in HUVECs. HUVECs were cultured in the conditions indicated for 24 h. Cells were harvested for western blot analyses of the protein levels of (a) TGF-β and Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) as an internal control, (b) p-Smad2 and total Smad2/3 as an internal control, and (c) p-AMPK and total AMPK as an internal control. Quantification of the relative fold increase is shown as the mean ± SD from three independent experiments. * P < 0.05 compared to saline control. # P < 0.05 compared to HG+IL-1β group. The statistical significance was assessed by Kruskal–Wallis test.

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  Figure 6. Liraglutide inhibits EndMT in HUVECs via the AMPK pathway. HUVECs were cultured in the following four conditions for 72 h: (1) Saline; (2) HG (30 mM glucose) plus IL-1β; (3) HG plus IL-1β and Liraglutide; (4) Liraglutide and Compound C. Cell lysates were collected for western blot analyses. (a) Western blot analysis of the protein levels of p-AMPKα and p-Smad2. Total AMPKα and Smad2/3 were used as loading controls for p-AMPK and p-Smad2, respectively. (b) Western blot analysis of the protein levels of CD31, SM22α, vimentin, and Snail in HUVECs treated with different conditions. Β-actin was used as the loading control. (c) Quantification of the relative fold increase is shown as the mean ± SD from three independent experiments. * P < 0.05. The statistical significance was assessed by Kruskal–Wallis test.

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  Figure 7. Liraglutide activates AMPK and enhances endothelial recovery in vivo. (a) STZ-induced diabetic C57BL/6 mice were subjected to a wire-induced carotid artery injury with or without liraglutide treatment. The expression of α-SMA (green) and p-AMPK (red) in the carotid arteries was analyzed by immunofluorescence staining. The endothelium was labelled by isolectin B4 (IB4, grey). Nuclei were stained with DAPI. (b) Number of p-AMPK+IB4+ endothelial cells per high-power field (HPF). (c) Percentage of endothelial coverage of the vascular lumen. Data are shown as mean ± SD, from three independent experiments. * P < 0.05. The statistical significance was assessed by Kruskal–Wallis test.

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  Figure 8. Liraglutide attenuates diabetes-associated neointima formation and pathogenic EndMT via the AMPK pathway. (a) The proposed underlying mechanisms of the therapeutic effects of liraglutide on the attenuation of a diabetes-associated vascular injury. Upon diabetic conditions such as hyperglycaemia in combination with pro-inflammatory stimulation by IL-1β, endothelial cells undergo the pathogenic EndMT process with increased mesenchymal marker expression (vimentin and SM22α). Liraglutide, a GLP-1 agonist, activates downstream AMPK signaling via GLP-1R and attenuates diabetic insult-induced EndMT in endothelial cells. (b) In vivo, endothelium-derived smooth muscle-like cells contribute to the progression of neointima formation after a wire-induced carotid artery injury. Reduced neointima formation in diabetic mice upon liraglutide treatment may be partially attributed to the reduced EndMT process.