Vildagliptin Reduces Stenosis of Injured Carotid Artery in Diabetic Mouse Through Inhibiting Vascular Smooth Muscle Cell Proliferation via ER Stress/NF-κB Pathway

Abstract

Dipeptidyl peptidase-4 (DPP-4) inhibitors are novel anti-hyperglycemic drugs for type 2 diabetes. It has been reported that DDP-4 inhibitor could exert pleiotropic effects on cardiovascular system. This study was to explore the effect and mechanism of vildagliptin on the stenosis of injured carotid artery in diabetic mouse. Twenty six-week-old male db/db mice (BKS) were randomized into vildagliptin treated and vehicle control groups. Ligation injury was first performed in left carotid arteries of all diabetic mice, then oral vildagliptin or equal amount of PBS was correspondingly administered to the mice from the next day to ligation injury for 4 weeks. Effects on proliferation were detected via histological and morphometric analysis. Endoplasmic reticulum (ER) stress and nuclear factor kappa B (NF-κB) markers were determined by immunoblot analysis. After 4 weeks of vildagliptin delivery, it was observed that the intimal area and neointimal thickness of the ligated carotid arteries were significantly reduced as compared to the control group. In vivo, vildagliptin suppressed the expressions of PCNA and α-SMA, phospho-p65, phospho-IKKα/β, GRP78 and CHOP, as well as IRE-1 in vascular smooth muscle cells (VSMCs). In vitro, the proliferation and hypertrophy of VSMCs were significantly inhibited by blocking the IRE-1 pathway, and the inhibition of phospho-IRE-1 expression down-regulated the expression of phospho-IKKα/β in VSMCs. Vildagliptin reduced the stenosis of injured carotid arteries in diabetic mice, and this effect was achieved via inhibiting the activation of ER stress/NF-κB pathway.

Keywords: VSMCs proliferation; phospho-IKKα/β; phospho-IRE-1; phospho-p65; vildagliptin.

FIGURE 1

Non-fasting blood glucose (A) and body weight (B) of mice before (baseline) and after 1, 2, 3, and 4 weeks of treatment.

FIGURE 2

Vildagliptin inhibited ligation injury-induced neointimal hyperplasia. (A) Representative hematoxylin and eosin staining of carotid arteries. (B). Quantifications of lumen area. ^∗P < 0.05 vs. sham mice, ^#P < 0.05 vs. injured mice (n = 3). (C) Representative immunohistochemical staining of α-SMA, CD31, and PCNA in carotid arteries. (D) Representative immunofluorescence staining of α-SMA and PCNA in injured arteries treated with or without vildagliptin.

FIGURE 3

Vildagliptin inhibited ligation injury-induced proliferation and hypertrophy in VSMCs. (A) Representative expressions of PCNA, Cyclin D and CDK2 (indices of proliferation) analyzed by Western-blot. (B) Representative expression of α-SMA (an index of hypertrophy) analyzed by Western-blot. (C). Quantification data of PCNA, Cyclin D1, CDK2, and α-SMA were determined by calculating the ratio of the intensity of the signal for the protein of interest to that of the normalization control. GAPDH served as the loading control. The value from the sham carotid artery treated with PBS was considered as 100% (control). Bars represented the ratio of the quantitative data from experimental groups to that from the control group. ^∗P < 0.05 vs. sham, ^#P < 0.05 vs. injured carotid artery (n = 3).

FIGURE 4

Vildagliptin inhibited arterial injury-induced activation of NF-κB pathway. (A) Representative expression of phospho-IKKα/β and phospho-p65 (markers of NF-κB activation) analyzed by Western-blot. (B) Representative expressions of TNF-α and IL-1β (downstream of NF-κB) analyzed by Western-blot. (C) Quantification data of p-p65 and phospho-IKKα/β were determined by calculating the ratio of the intensity of the signal for the protein of interest to that of the p65 and IKKα/β separately. Quantification data of TNF-α and IL-1β were determined by the ratio of signal for the protein of interest to that of the normalization control. GAPDH served as the loading control. The value from the sham carotid artery treated with PBS was considered as 100% (control). Bars represented the ratio of the quantitative data from experimental groups to that from the control group. ^∗P < 0.05 vs. sham, ^#P < 0.05 vs. injured carotid artery (n = 3).

FIGURE 5

Vildagliptin inhibited ligation injury-induced activation of IRE-1 pathway. (A) Representative expressions of CHOP and GRP78 (markers of ER stress) analyzed by Western-blot. (B) Representative expressions of phospho-eIF2α, phospho-IRE1 and ATF-6 (markers of three activation pathways in ER stress, respectively) analyzed by Western-blot. (C) Quantification data of phospho-eIF2α and phospho-IRE1 were determined by calculating the ratio of the intensity of the signal for the protein of interest to that of the eIF2α and IRE1 separately. Quantification data of CHOP, GRP78, and ATF-6 were determined by the ratio of signal for the protein of interest to that of the normalization control. GAPDH served as the loading control. The value from the sham carotid artery treated with PBS was considered as 100% (control). Bars represented the ratio of the quantitative data from experimental groups to that from the control group. ^∗P < 0.05 vs. sham, ^#P < 0.05 vs. injured carotid artery (n = 3).

FIGURE 6

NF-κB and ER stress involved in high glucose induced proliferation and hypertrophy in VSMCs. (A) Enhanced proliferation in a time-dependent manner by CCK-8 assay. ^∗P < 0.05 vs. normal glucose treated cells, ^#P < 0.05 vs. high glucose treated cells (n = 5). (B) Representative Brdu staining of A7r5 cells in normal glucose, high glucose, high glucose treated with taurine, and high glucose treated with ACHP by EdU incorporation assay. (C) Quantifications of Brdu positive cells to hoechst positive cells ratio (n = 5). (D) The mean values of cell capacitances in cells in normal glucose, high glucose, high glucose treated with taurine, and high glucose treated with ACHP conditions. ^∗∗P < 0.05 (n = 5).

FIGURE 7

IRE-1 pathway involved in high glucose induced NF-κB activation. (A) Representative expressions of PCNA (an index of proliferation), α-SMA (an index of hypertrophy), phospho-IRE1 (marker of activation in ER stress IRE-1 pathway), phospho-p65 (marker of NF-κB activation) in A7r5 cells in normal glucose, high glucose, high glucose treated with taurine, and high glucose treated with ACHP conditions by Western-blot assay. (B) Quantification data of phospho-IRE1 and phospho-IKKα/β were determined by calculating the ratio of the intensity of the signal for the protein of interest to that of the IRE1 and IKKβ separately. Quantification data of α-SMA and PCNA were determined by the ratio of signal for the protein of interest to that of the normalization control. GAPDH served as the loading control. The value from the normal glucose treated cells was considered as 100% (control). Bars represented the ratio of the quantitative data from experimental groups to that from the control group. ^∗P < 0.05 vs. normal glucose treated cells, ^#P < 0.05 vs. high glucose treated cells (n = 3).