Simvastatin increases notch signaling activity and promotes arteriogenesis after stroke - PubMed (original) (raw)

Simvastatin increases notch signaling activity and promotes arteriogenesis after stroke

Alex Zacharek et al. Stroke. 2009 Jan.

Abstract

Background and purpose: Notch signaling activity regulates arteriogenesis. Presenilin 1 (PS1) mediates Notch signaling activity via cleavage of Notch, liberating Notch intracellular domain (NICD). We tested the hypothesis that simvastatin enhances arteriogenesis after stroke by increasing PS1 activation of the Notch signaling pathway.

Methods: Rats were subjected to middle cerebral artery occlusion (MCAo) and treated with or without simvastatin (1 mg/kg) starting 24 hours after stroke and daily for 7 days; they were euthanized 14 days after stroke. Immunostaining, Western blot, and real-time polymerase chain reaction assays were performed.

Results: Simvastatin significantly increased arterial diameter, density, and vascular smooth muscle cell proliferation, and upregulated PS1, Notch1, and NICD expression in the ischemic border tissue and in the cerebral arteries compared with MCAo control rats, respectively. However, simvastatin did not increase arteriogenesis, PS1, and NICD expression in sham control animals. To investigate the mechanisms of simvastatin-induced arteriogenesis, primary cerebral artery cultures were used. Rats were subjected to MCAo and treated with or without simvastatin daily for 7 days. The cerebral arteries derived from these stroke rats were cultured in matrigel and treated with or without a gamma40-secretase inhibitor II, which blocks Notch signaling activity, inhibiting NICD production. Arterial cell migration was measured. simvastatin treatment significantly increased arterial cell migration compared to control MCAo artery, whereas inhibition of Notch signaling activity by the gamma40-secretase inhibitor II significantly attenuated simvastatin-induced arterial cell migration.

Conclusions: These data indicate that simvastatin increases arteriogenesis after stroke, and that simvastatin upregulation of PS1 expression and Notch signaling activity may facilitate an increase in arteriogenesis.

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Figures

Figure 1

Figure 1

Designated boxes identify areas in the ischemic border where immunostaining was analyzed.

Figure 2

Figure 2

Simvastatin treatment of stroke increases arteriogenesis and PS1 and NICD expression in the ischemic brain. A, _α_-SMA immunostaining in MCAo control and simvastatin-treated rats, and arterial density, diameter, and perimeter quantitative data in MCAo or sham-operated rats treated with or without simvastatin (n=8/group). B, Double immunostaining BrdU (FITC, green)/_α_-SMA (Cy5, red) and quantitative data in MCAo or sham-operated rats treated with or without simvastatin (n=8/group). NICD (C) and PS1 (D) immunostaining MCAo control and simvastatin-treated rats and quantitative data. *P<0.05 compared with MCAo control (n=8/group). Scale bar B and C=50 _μ_m; A and D=100 _μ_m.

Figure 3

Figure 3

Simvastatin regulates Notch1 and PS1 gene and protein expression in the ischemic brain and cerebral artery measured by Western blot and real-time PCR. Western blot assay is shown. PS1, Notch1, and NICD protein expression in sham-operated rats brain tissue (A), MCAo rat ischemic brain tissue (B), and ischemic brain cerebral artery (C). D to F, Quantitative data of Western blot analysis. G, Gene expression in the ischemic brain tissue treated with or without simvastatin measured by real-time PCR (n=5/group).

Figure 4

Figure 4

Cerebral artery migration assay. A, Sham-operated nontreatment for control. B, Sham-operated simvastatin-treated artery. C, MCAo nontreatment for control. D, MCAo simvastatin-treated artery. E, MCAo simvastatin-treated artery plus _γ_-secretase inhibitor. F, Migration quantitative analysis (n=6/group). Scale bar A=100 _μ_m.

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