Inhibition of apoptotic signalling in spermine-treated vascular smooth muscle cells by a novel glutathione precursor

Abstract

CKD (chronic kidney disease) is a public health problem, mediated by haemodynamic and non-haemodynamic events including oxidative stress. We investigated the effect of two GSH (glutathione) precursors, NAC (N-acetylcysteine) and cystine as the physiological carrier of cysteine in GSH with added selenomethionine (F1) in preventing spermine (uraemic toxin)-induced apoptosis in cultured human aortic VSMC (vascular smooth muscle cells). VSMCs exposed to spermine (15 microM) with or without antioxidants (doses 50, 100, 200 and 500 microg/ml) were assessed for apoptosis, JNK (c-Jun-NH2-terminal kinase) activation and iNOS (inducible nitric oxide synthase) induction and activation of intrinsic pathway signalling. Spermine exposure resulted in activation of JNK and iNOS induction and apoptosis. NAC and F1 (dose range 50-500 microg/ml) attenuated spermine-induced acceleration of VSMC apoptosis but only F1 (at 200 and 500 microg/ml) maintained spermine-induced apoptosis at control levels. Spermine-induced JNK activation was prevented by 200 microg/ml of both NAC and F1, while iNOS induction was blocked only by F1. Notably, the adverse effects of spermine on BAX/BCL-2 ratio, cytochrome c release and caspase activation was fully attenuated by F1. In conclusion, F1 was more effective than NAC in preventing spermine-induced apoptosis and downstream changes in related signal transduction pathways in VSMCs. Further studies are needed to examine the effect of these compounds in preventing CKD-associated vascular disease.

Fig. 1

DAPI (I–IV) and TUNEL (V–VIII) show effective suppression of spermine-induced VSMC apoptosis by antioxidant treatments. Compared to control, where no apoptosis is detected, spermine results in activation of VSMC apoptosis (arrow) and which could be effectively prevented by concomitant treatments with both NAC and F1. Nuclei are visualized by DAPI staining. Scale bar = 50 μm.

Fig. 2

Effect of graded doses (50–500 μg/ml) of antioxidants on spermine-induced VSMC apoptosis after 24 h of treatment. Co-treatment with 200 or 500 μg/ml F1 but not NAC fully (P<0.001) suppressed spermine-induced VSMC apoptosis. Values are mean ± SEM. Means with unlike superscripts differ significantly.

Fig. 3

Activation of JNK, as measured by Titer Enzyme EIA Kit (A) and by western blotting (B). (A) JNK activation, as evidenced by a significant (P<0.05) increase in phospho-JNK levels, is detected in VSMC lysates 24 h after exposure to spermine (sp). Co-treatments with both NAC and F1 significantly (P<0.05) prevents spermine-induced activation of JNK. Values are mean ± SEM. Means with unlike superscripts differ significantly. (B) Representative western blots of cell lysates from control (C), spermine (Sp), spermine + NAC, and spermine + F1 groups show effective suppression of spermine-induced activation of JNK by both antioxidants, while induction of iNOS by spermine is only blocked by F1. GAPDH in the immunoblot is shown as a loading control. (C) Immunocytochemical changes in the iNOS expression in VSMCs in various treatment groups. Compared with control, an increase in iNOS immunoreactivity (arrow) is noted in these cells after spermine treatment. Concomitant treatments with F1 but not NAC prevent such spermine-induced increase in iNOS expression in these cells. Scale bar = 50 μm. (D) Computerized densitometric analysis shows a significant increase in iNOS immunoreactivity in VSMCs after spermine treatment compared to that of controls. Co-treatment with F1 significantly (P<0.001) prevents such spermine-induced increase in iNOS expression. Values are mean ± SEM. Means with unlike superscripts differ significantly.

Fig. 3

Activation of JNK, as measured by Titer Enzyme EIA Kit (A) and by western blotting (B). (A) JNK activation, as evidenced by a significant (P<0.05) increase in phospho-JNK levels, is detected in VSMC lysates 24 h after exposure to spermine (sp). Co-treatments with both NAC and F1 significantly (P<0.05) prevents spermine-induced activation of JNK. Values are mean ± SEM. Means with unlike superscripts differ significantly. (B) Representative western blots of cell lysates from control (C), spermine (Sp), spermine + NAC, and spermine + F1 groups show effective suppression of spermine-induced activation of JNK by both antioxidants, while induction of iNOS by spermine is only blocked by F1. GAPDH in the immunoblot is shown as a loading control. (C) Immunocytochemical changes in the iNOS expression in VSMCs in various treatment groups. Compared with control, an increase in iNOS immunoreactivity (arrow) is noted in these cells after spermine treatment. Concomitant treatments with F1 but not NAC prevent such spermine-induced increase in iNOS expression in these cells. Scale bar = 50 μm. (D) Computerized densitometric analysis shows a significant increase in iNOS immunoreactivity in VSMCs after spermine treatment compared to that of controls. Co-treatment with F1 significantly (P<0.001) prevents such spermine-induced increase in iNOS expression. Values are mean ± SEM. Means with unlike superscripts differ significantly.

Fig. 4

(A) Western blot analysis of BAX and BCL-2 in VSMC lysates from control and various treatments groups show no obvious alterations of BAX levels, whereas BCL-2 levels appear to be markedly decreased after spermine treatment. Addition of both NAC and F1 effectively restored spermine-induced decrease in BCL-2 expression. GAPDH in the immunoblot is shown as a loading control. (B) Immunocytochemical changes in the expression of BCL-2 in VSMCs in various treatment groups. Exposure of VSMC to spermine results in a decrease in BCL-2 immunostaining after exposure to spermine. Addition of F1 to spermine fully restored such spermine-induced decrease in BCL-2 expression in these cells. Scale bar = 50 μm. (C) Computerized densitometric analysis shows a significant decrease in BCL-2 immunoreactivity in VSMCs after spermine treatment compared to that of controls. Co-treatments with F1 but not NAC significantly (P<0.05) restores such spermine-induced depletion in BCL-2 expression. Values are mean ± SEM. Means with unlike superscripts differ significantly.

Fig. 5

Visualization of cytochrome c release after activation of apoptosis and its prevention by antioxidants by immunocytochemistry. Control VSMCs exhibit a strong punctate perinuclear staining of cytosolic cytochrome (asterisk) indicative of its mitochondrial localization. After apoptosis induction, these cells show mostly diffuse cytoplasmic staining, which is consistent with its translocation from mitochondria to cytoplasm. Many apoptotic cells, identified by cell shrinkage and chromatin condensation, also display cytochrome c immunoreactivity in the nuclei (arrow). Concomitant administration of F1 in particular, is able to prevent such spermine-induced cytochrome c release. Scale bar = 25 μm.

Fig. 6

(A) Activation of caspase 9, as evidenced by immunocytochemical staining of active caspase 9 in VSMCs after spermine treatment, which could be effectively prevented by either NAC or F1. Compared with control, where little or no expression of active caspase 9 is detected, a marked increase in active caspase 9 immunoreactivity is noted in these cells 24 h after spermine treatment. Co-treatment with either NAC or F1 effectively prevents such spermine-induced activation of caspase 9. Scale bar = 50 μm. (B) Western blot analysis shows that spermine-induced VSMC apoptosis is associated with activation of caspase 9 and caspase 3. Notably, co-treatment with F1 but not NAC fully prevents spermine-induced activation of caspase 9 and caspase 3 in these cells.