Fig 1.
Simultaneous increases in proliferation and apoptosis of vascular cells in vein graft are associated with selective activation of MAPKs.
(A, B) Immunofluorescence staining with Ki67 (red) and TUNEL (green) of both non-diabetic (ND) and diabetic (D) mice shows the simultaneous proliferation and apoptosis. (C) Graph bars show Ki67 and TUNEL positive ratios. (D-I) Immunofluorescence shows activation of ERKs, JNKs and p38MAPK in non-diabetic and diabetic mouse. Scale bars, 50 μm. Arrows and stars indicate the wall thickness and lumens of the vein grafts. All the experiments were independently repeated three times and presented as mean±SEM. a above bars represented the p<0.05 compared to the non-diabetic group, n = 3 (a for p value<0.05).
Fig 2.
SS and AGEs synergistically promote simultaneous increases in proliferation and apoptosis of VSMCs.
(A-D) Immunofluorescence analysis showed Ki67 and TUNEL positive cells of VMSCs in response to AGEs and SS. Either SS or AGEs could induce simultaneous increases in proliferation (Ki-67 positive, red, arrows) and apoptosis (TUNEL positive, green, arrowheads) compared to negative controls (NC), and combined stimulation with both had a synergistic effect. Scale bars, 50 μm. (E) Graph bars showed Ki67 and TUNEL positive ratios. All the experiments were independently repeated three times and shown as mean±SEM. a above bars represented the p<0.05 compared to NC group, and b represented the p<0.05 compared to AGEs or SS group, n = 3 (a and b for p value<0.05).
Fig 3.
Combined SS and AGEs induce synergistic activation of three members of the MAPK subfamily in VSMCs.
(A) Western blot analysis showed phosphorylation and total levels of ERK1/2, JNK1/2, and p38MAPK in VSMCs in response to AGEs and SS. (B) Graph bars showed densitometry analysis of phosphorylated ERK1/2, JNK1/2, and p38MAPK normalized with total MAPKs. All the experiments were independently repeated three times and shown as mean±SEM. a above bars represented the p<0.05 compared to NC group, and b represented the p<0.05 compared to AGEs or SS group, n = 3 (a and b for p value<0.05).
Fig 4.
SS and AGEs induce selective activation of three members of the MAPK subfamily in individual VSMCs.
Immunofluorescence was used to show selective activation of ERKs, JNKs and p38MAPK in single cells induced by SS and AGEs. VSMCs were marked with SM-α-actin, and the different expression levels (arrows indicated SM-α-actin weak positive cells and arrowheads indicated SM-α-actin strong positive cells) suggested subtypes existed. (A, D, E, J) Immunofluorescence showed ERK were preferentially activated in SM-α-actin weak positive cells. (B, C, E, F, H, I, K, L) JNK and p38MAPK were preferentially activated in SM-α-actin strong positive cells. Scale bars, 20 μm.
Fig 5.
SS- and AGEs-induced simultaneous increases in proliferation and apoptosis of VSMCs are closely associated with selective activation of three members the MAPK subfamily.
The cultured quiescent VSMCs were pretreated with DMSO, ERK inhibitor PD98059 (50 μM), JNK inhibitor SP600125 (20 μM), and p38MAPK inhibitor SB202190 (20 μM) for 1 h and then treated with SS and/or AGEs for 1 h and continually cultured for 23 h. (A, E, I, M) Either SS or AGEs could induce simultaneous increases in proliferation (Ki-67 positive, red, arrows) and apoptosis (TUNEL positive, green, arrowheads), and combined stimulation with both had a synergistic effect. (B, F, J, N) PD98059 completely suppressed cell proliferation while it had no effect on apoptosis. (C, G, K, O, D, H, L, P) SP600125 and SB202190 were found to significantly decrease apoptosis without affecting the cell proliferation. Scale bars, 20 μm. (Q, R) Graph bars showed Ki67 and TUNEL positive ratios. All the experiments were independently repeated three times and shown as mean±SEM. a above bars represented the p<0.05 compared to NC group, b represented the p<0.05 compared to DMSO group and c represented the p<0.05 compared to AGEs and SS group, n = 3 (a, b and c for p value<0.05).
Fig 6.
Relationship among MAPKs, NF-κB, and Caspase-3 in VSMCs induced by SS and AGEs.
The cultured quiescent VSMCs were pretreated with DMSO, NF-κB inhibitor PDTC (20 μM) and Caspase-3 inhibitor Z-DEVD-FMK (20 μM), ERK inhibitor PD98059 (50 μM), JNK inhibitor SP600125 (20 μM) and p38 inhibitor SB202190 (20 μM) for 1 h and then treated with AGEs or SS as indicated above. (A) Western Blot analysis showed cells pretreated with PDTC and Z-DEVD-FMK had no effect on phosphorylation of MAPKs. (B) Graph bars showed densitometry analysis of MAPKs activation levels normalized with total MAPKs. (C) Western Blot analysis showed cells pretreated with PDTC suppressed the activation of both NF-κB/p65 (Ser536), and Z-DEVD-FMK also inhibited the Caspase-3 activation while had no effect on NF-κB/p65 (Ser536) phosphorylation of VSMCs. (D) Graph bars showed densitometry analysis of NF-κB/p65 (Ser536) activation levels after PDTC and Z-DEVD-FMK pretreatment normalized with NF-κB. (E) Graph bars showed densitometry analysis of Caspase-3 activation levels after PDTC and Z-DEVD-FMK pretreatment normalized with β-actin. (F) Western Blot analysis showed PD98059 significantly inhibited phosphorylation of NF-κB/p65 (Ser536), while it had no influence on Caspase-3 activation. (G) Graph bars showed densitometry analysis of NF-κB/p65 (Ser536) activation levels after PD98059 pretreatment normalized with NF-κB. (H) Graph bars showed densitometry analysis of Caspase-3 activation levels after PD98059 pretreatment normalized with β-actin. (I) Western Blot analysis showed NF-κB/p65 (Ser536) and Caspase-3 activation could be suppressed by both SP600125 and SB202190. (J) Graph bars showed densitometry analysis of NF-κB/p65 (Ser536) activation levels after SP600125 and SB202190 pretreatment normalized with NF-κB. (K) Graph bars showed densitometry analysis of Caspase-3 activation levels after SP600125 and SB202190 pretreatment normalized with β-actin. All the experiments were independently repeated three times and shown as mean±SEM. a above bars represented the p<0.05 compared to NC group, b represented the p<0.05 compared to AGEs and SS group and c represented the p<0.05 compared to DMSO group, n = 3 (a, b and c for p value<0.05).
Fig 7.
RAGE mediates simultaneous increases of proliferation and apoptosis of VSMCs.
The cultured VSMCs were transfected with siRNA-RAGE (SiR), siRNA-control (SiC) and Lipofectamine 2000 (LIP) for 24 h and then serum-starved for additional 48 h. Cells were treated with AGEs and/or SS for 1 h and cultured for additional 23 h. (A, D, G, J) Immunofluorescence showed either AGEs or SS could increase the cell proliferation (red, arrows) and apoptosis (green, arrowheads), and the combination had a synergistic effect. (B, E, H, K) Immunofluorescence showed SiC had no effect on AGEs and SS induced increases of cell proliferation and apoptosis. (C, F, I, L) Immunofluorescence showed SiR significantly suppressed both proliferation and apoptosis of VSMCs in response to AGEs and SS. Scale bars, 20 μm. (M, N) Graph bars showed Ki67 and TUNEL positive ratios. All the experiments were independently repeated three times and shown as mean±SEM. a above bars represented the p<0.05 compared to NC group, b represented the p<0.05 compared to DMSO group and c represented the p<0.05 compared to AGEs and SS group, n = 3 (a, b and c for p value<0.05).
Fig 8.
Potential signal pathway in the simultaneous increases of proliferation and apoptosis of VSMCs induced by SS and AGEs via RAGE/MAPK selective activation.
Rapidly increased blood pressure triggers increased SS on the vein grafts. SS non-specifically activates RAGE and its downstream signal molecules, including selective activation of ERK/NF-κB and JNK/p38MAPK/NF-κB/Caspase-3 signaling, which leads to simultaneous increases in proliferation (Ki-67 expression) and apoptosis (TUNEL positive) of VSMCs. Hyperglycemia-induced numerous AGEs deposits on the vascular wall directly and specifically interacts with RAGE to further amplify the SS-initiated intracellular signaling molecules, which lead to synergistic increases in proliferation and apoptosis of VSMCs. Blocking RAGE and its downstream molecules might inhibit selective activation of MAPKs, leading to simultaneous decreases in the proliferation and apoptosis of VSMCs.