Limits...
Simvastatin re-couples dysfunctional endothelial nitric oxide synthase in experimental subarachnoid hemorrhage.

Sabri M, Ai J, Marsden PA, Macdonald RL - PLoS ONE (2011)

Bottom Line: SAH increased phosphorylated eNOS which was prevented by pre- or post-treatment with simvastatin.Simvastatin pre-treatment also prevented the increase in eNOS monomer formation that was associated with SAH, decreased superoxide anion radical production and increased NO.These changes were associated with decreased vasospasm, microthromboemboli and neuronal injury.

View Article: PubMed Central - PubMed

Affiliation: Division of Neurosurgery, St. Michael's Hospital, Toronto, Ontario, Canada.

ABSTRACT
Reduced endothelial nitric oxide synthase (eNOS) function has been linked to secondary complications of subarachnoid hemorrhage (SAH). We previously found that there is increased eNOS function after SAH but that it is uncoupled, leading to secondary complications such as vasospasm, microthromboembolism and neuronal apoptosis. Here we test the hypothesis that recoupling eNOS with simvastatin can prevent these complications. SAH was created in mice that were treated with vehicle or simvastatin starting 2 weeks before or 30 minutes after SAH. SAH increased phosphorylated eNOS which was prevented by pre- or post-treatment with simvastatin. Simvastatin pre-treatment also prevented the increase in eNOS monomer formation that was associated with SAH, decreased superoxide anion radical production and increased NO. These changes were associated with decreased vasospasm, microthromboemboli and neuronal injury. The data suggest that simvastatin re-couples eNOS after SAH, leading to decreased secondary complications such as vasospasm, microthromboemboli and neuronal injury.

Show MeSH

Related in: MedlinePlus

Simvastatin increases eNOS dimer/monomer ratio and NO production.A and B. Dimer/monomer ratio of eNOS protein expression for pre- and post-SAH treated groups, respectively. Pre-SAH treatment with simvastatin significantly increases dimer/monomer ratio to the naïve level (P>0.05). Post-SAH treatment also markedly increases the ratio, but to a lesser extent (P<0.05 as compared to naïve controls). Vehicle treated animals exhibit significantly lower dimer/monomer ratio than both naïve or the two simvastatin treated groups (P<0.01). C to F. Western blot quantification of separate monomer and dimer protein expression in pre- (C and D) and post-SAH (E and F) simvastatin treated animals. In pre-treated animals, both eNOS monomer and dimer expression were decreased by simvastatin to the same level as in naïve animals. They were significantly lower than that in vehicle treated animals (C and D, P<0.01 for both monomer and dimer). Similarly, in post-treated animals, both eNOS monomer and dimer expression were decreased by simvastatin as compared to that in vehicle treated animals (P<0.01 for both). However, while eNOS dimer was reduced by post-SAH treatment of simvastatin to the same level as in naïve animals (F), the eNOS monomer was still significantly higher than that in naïve controls (E, P<0.05). Representative blots of monomer and dimer protein expression are shown in H (for pre-treatment) and I (for post-treatment). Spectrometric measurement of NO shows pre-SAH treatment with simvastatin restored NO production to the level seen in naïve controls, which is significantly higher than vehicle treated (J, P<0.01). In contrast, post-SAH simvastatin treated animals shows a significantly increased NO production as compared to vehicle treated (K, P<0.05), but not to the same level as in naïve animals (K, P<0.01). All data are mean ± SD. Legend in K is for all the charts in the figure.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC3044158&req=5

pone-0017062-g002: Simvastatin increases eNOS dimer/monomer ratio and NO production.A and B. Dimer/monomer ratio of eNOS protein expression for pre- and post-SAH treated groups, respectively. Pre-SAH treatment with simvastatin significantly increases dimer/monomer ratio to the naïve level (P>0.05). Post-SAH treatment also markedly increases the ratio, but to a lesser extent (P<0.05 as compared to naïve controls). Vehicle treated animals exhibit significantly lower dimer/monomer ratio than both naïve or the two simvastatin treated groups (P<0.01). C to F. Western blot quantification of separate monomer and dimer protein expression in pre- (C and D) and post-SAH (E and F) simvastatin treated animals. In pre-treated animals, both eNOS monomer and dimer expression were decreased by simvastatin to the same level as in naïve animals. They were significantly lower than that in vehicle treated animals (C and D, P<0.01 for both monomer and dimer). Similarly, in post-treated animals, both eNOS monomer and dimer expression were decreased by simvastatin as compared to that in vehicle treated animals (P<0.01 for both). However, while eNOS dimer was reduced by post-SAH treatment of simvastatin to the same level as in naïve animals (F), the eNOS monomer was still significantly higher than that in naïve controls (E, P<0.05). Representative blots of monomer and dimer protein expression are shown in H (for pre-treatment) and I (for post-treatment). Spectrometric measurement of NO shows pre-SAH treatment with simvastatin restored NO production to the level seen in naïve controls, which is significantly higher than vehicle treated (J, P<0.01). In contrast, post-SAH simvastatin treated animals shows a significantly increased NO production as compared to vehicle treated (K, P<0.05), but not to the same level as in naïve animals (K, P<0.01). All data are mean ± SD. Legend in K is for all the charts in the figure.

Mentions: We previously indirectly showed that SAH increased eNOS expression and O2Γ concentration in the brain, but decreased NO availability, suggesting uncoupling of eNOS [6]. We now used a direct measurement of eNOS uncoupling, namely, eNOS dimer/monomer ratio [10], [11]. SAH was associated with a significant increase in both eNOS dimer and monomer as compared to naïve controls (Fig. 2C to 2F, P< 0.01). However, the relative proportion of dimer, thus potentially functional eNOS, was significantly lower in SAH than in naïve controls (eNOS dimer/monomer ratio was 5.9±0.4 and 4.0±0.3 for SAH, 16.5±6.9 and 15.5±3.8 for naive controls in pre-treatment and post-treatment experiments, respectively, Fig. 2A and 2B, P<0.01). Treatment with simvastatin starting before SAH completely reversed the decreased ratio of dimer/monomer to naïve level (P>0.05 compared to naïve group, Fig 2A). In comparison, post-SAH treatment with simvastatin also increased the dimer/monomer ratio as compared to vehicle treatment but not to the level of naïve controls (P<0.05 compared to naïve, Fig. 2B). This was directly related to the significantly higher than normal level of monomer expression seen in the post-SAH simvastatin-treated animals (P<0.05, Fig. 2E). Nevertheless, all naive and simvastatin-treated animals had a significantly higher dimer/monomer ratio than vehicle-treated SAH (P<0.01, Fig. 2A and 2B).


Simvastatin re-couples dysfunctional endothelial nitric oxide synthase in experimental subarachnoid hemorrhage.

Sabri M, Ai J, Marsden PA, Macdonald RL - PLoS ONE (2011)

Simvastatin increases eNOS dimer/monomer ratio and NO production.A and B. Dimer/monomer ratio of eNOS protein expression for pre- and post-SAH treated groups, respectively. Pre-SAH treatment with simvastatin significantly increases dimer/monomer ratio to the naïve level (P>0.05). Post-SAH treatment also markedly increases the ratio, but to a lesser extent (P<0.05 as compared to naïve controls). Vehicle treated animals exhibit significantly lower dimer/monomer ratio than both naïve or the two simvastatin treated groups (P<0.01). C to F. Western blot quantification of separate monomer and dimer protein expression in pre- (C and D) and post-SAH (E and F) simvastatin treated animals. In pre-treated animals, both eNOS monomer and dimer expression were decreased by simvastatin to the same level as in naïve animals. They were significantly lower than that in vehicle treated animals (C and D, P<0.01 for both monomer and dimer). Similarly, in post-treated animals, both eNOS monomer and dimer expression were decreased by simvastatin as compared to that in vehicle treated animals (P<0.01 for both). However, while eNOS dimer was reduced by post-SAH treatment of simvastatin to the same level as in naïve animals (F), the eNOS monomer was still significantly higher than that in naïve controls (E, P<0.05). Representative blots of monomer and dimer protein expression are shown in H (for pre-treatment) and I (for post-treatment). Spectrometric measurement of NO shows pre-SAH treatment with simvastatin restored NO production to the level seen in naïve controls, which is significantly higher than vehicle treated (J, P<0.01). In contrast, post-SAH simvastatin treated animals shows a significantly increased NO production as compared to vehicle treated (K, P<0.05), but not to the same level as in naïve animals (K, P<0.01). All data are mean ± SD. Legend in K is for all the charts in the figure.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3044158&req=5

pone-0017062-g002: Simvastatin increases eNOS dimer/monomer ratio and NO production.A and B. Dimer/monomer ratio of eNOS protein expression for pre- and post-SAH treated groups, respectively. Pre-SAH treatment with simvastatin significantly increases dimer/monomer ratio to the naïve level (P>0.05). Post-SAH treatment also markedly increases the ratio, but to a lesser extent (P<0.05 as compared to naïve controls). Vehicle treated animals exhibit significantly lower dimer/monomer ratio than both naïve or the two simvastatin treated groups (P<0.01). C to F. Western blot quantification of separate monomer and dimer protein expression in pre- (C and D) and post-SAH (E and F) simvastatin treated animals. In pre-treated animals, both eNOS monomer and dimer expression were decreased by simvastatin to the same level as in naïve animals. They were significantly lower than that in vehicle treated animals (C and D, P<0.01 for both monomer and dimer). Similarly, in post-treated animals, both eNOS monomer and dimer expression were decreased by simvastatin as compared to that in vehicle treated animals (P<0.01 for both). However, while eNOS dimer was reduced by post-SAH treatment of simvastatin to the same level as in naïve animals (F), the eNOS monomer was still significantly higher than that in naïve controls (E, P<0.05). Representative blots of monomer and dimer protein expression are shown in H (for pre-treatment) and I (for post-treatment). Spectrometric measurement of NO shows pre-SAH treatment with simvastatin restored NO production to the level seen in naïve controls, which is significantly higher than vehicle treated (J, P<0.01). In contrast, post-SAH simvastatin treated animals shows a significantly increased NO production as compared to vehicle treated (K, P<0.05), but not to the same level as in naïve animals (K, P<0.01). All data are mean ± SD. Legend in K is for all the charts in the figure.
Mentions: We previously indirectly showed that SAH increased eNOS expression and O2Γ concentration in the brain, but decreased NO availability, suggesting uncoupling of eNOS [6]. We now used a direct measurement of eNOS uncoupling, namely, eNOS dimer/monomer ratio [10], [11]. SAH was associated with a significant increase in both eNOS dimer and monomer as compared to naïve controls (Fig. 2C to 2F, P< 0.01). However, the relative proportion of dimer, thus potentially functional eNOS, was significantly lower in SAH than in naïve controls (eNOS dimer/monomer ratio was 5.9±0.4 and 4.0±0.3 for SAH, 16.5±6.9 and 15.5±3.8 for naive controls in pre-treatment and post-treatment experiments, respectively, Fig. 2A and 2B, P<0.01). Treatment with simvastatin starting before SAH completely reversed the decreased ratio of dimer/monomer to naïve level (P>0.05 compared to naïve group, Fig 2A). In comparison, post-SAH treatment with simvastatin also increased the dimer/monomer ratio as compared to vehicle treatment but not to the level of naïve controls (P<0.05 compared to naïve, Fig. 2B). This was directly related to the significantly higher than normal level of monomer expression seen in the post-SAH simvastatin-treated animals (P<0.05, Fig. 2E). Nevertheless, all naive and simvastatin-treated animals had a significantly higher dimer/monomer ratio than vehicle-treated SAH (P<0.01, Fig. 2A and 2B).

Bottom Line: SAH increased phosphorylated eNOS which was prevented by pre- or post-treatment with simvastatin.Simvastatin pre-treatment also prevented the increase in eNOS monomer formation that was associated with SAH, decreased superoxide anion radical production and increased NO.These changes were associated with decreased vasospasm, microthromboemboli and neuronal injury.

View Article: PubMed Central - PubMed

Affiliation: Division of Neurosurgery, St. Michael's Hospital, Toronto, Ontario, Canada.

ABSTRACT
Reduced endothelial nitric oxide synthase (eNOS) function has been linked to secondary complications of subarachnoid hemorrhage (SAH). We previously found that there is increased eNOS function after SAH but that it is uncoupled, leading to secondary complications such as vasospasm, microthromboembolism and neuronal apoptosis. Here we test the hypothesis that recoupling eNOS with simvastatin can prevent these complications. SAH was created in mice that were treated with vehicle or simvastatin starting 2 weeks before or 30 minutes after SAH. SAH increased phosphorylated eNOS which was prevented by pre- or post-treatment with simvastatin. Simvastatin pre-treatment also prevented the increase in eNOS monomer formation that was associated with SAH, decreased superoxide anion radical production and increased NO. These changes were associated with decreased vasospasm, microthromboemboli and neuronal injury. The data suggest that simvastatin re-couples eNOS after SAH, leading to decreased secondary complications such as vasospasm, microthromboemboli and neuronal injury.

Show MeSH
Related in: MedlinePlus