Stroke protection by 3-hydroxy-3-methylglutaryl (HMG)-CoA reductase inhibitors mediated by endothelial nitric oxide synthase

Abstract

The treatment of ischemic strokes is limited to prophylactic agents that block the coagulation cascade. Here, we show that cholesterol-lowering agents, 3-hydroxy-3-methylglutaryl (HMG)-CoA reductase inhibitors, protect against cerebral injury by a previously unidentified mechanism involving the selective up-regulation of endothelial NO synthase (eNOS). Prophylactic treatment with HMG-CoA reductase inhibitors augments cerebral blood flow, reduces cerebral infarct size, and improves neurological function in normocholesterolemic mice. The up-regulation of eNOS by HMG-CoA reductase inhibitors is not associated with changes in serum cholesterol levels, but is reversed by cotreatment with L-mevalonate and by the downstream isoprenoid, geranylgeranyl pyrophosphate and not by farnesyl pyrophosphate. The blood flow and neuroprotective effects of HMG-CoA reductase inhibitors are completely absent in eNOS-deficient mice, indicating that enhanced eNOS activity by HMG-CoA reductase inhibitors is the predominant if not the only mechanism by which these agents protect against cerebral injury. Our results suggest that HMG-CoA reductase inhibitors provide a prophylactic treatment strategy for increasing blood flow and reducing brain injury during cerebral ischemia.

Figure 1

(A) The effects of Sim (0.2, 2.0 or 20 mg/kg s.c. daily for 14 days) on cerebral infarct volume after 2 h of filamentous MCA occlusion and 22 h of reperfusion compared with vehicle-injected 129/SV mice. Cerebral infarction volume was determined quantitatively by TTC staining as described (31). Smaller infarct sizes were confirmed with an indirect method that corrects for brain swelling (31). (n = 9–18 per group). ∗, P < 0.05; ∗∗, P < 0.01. (B) Neurological sensory-motor deficits were significantly reduced in Sim-treated 129/SV mice compared with controls at 24 h. Deficits were evaluated blindly by an observer using an established rating system ranging from 0 (no deficit) to 3 (severe) (31). (n = 9–18 per group). ∗, P < 0.05.

Figure 2

Effects of Sim on absolute CBF during focal cerebral ischemia in anesthetized and ventilated wild-type 129/SV mice. Mice were injected s.c. with saline (n = 10) or Sim 20 mg/kg (n = 10) for 14 days. Absolute CBF was determined 30 min after filamentous occlusion of the left MCA in the ischemic tissue (ipsilateral) and corresponding tissue from the contralateral hemisphere by using an indicator fractionation technique (32). ∗, P < 0.05.

Figure 3

The Ca²⁺-dependent NOS catalytic activity was measured in aortas from wild-type 129/SV mice treated with Sim (0.2, 2.0 or 20 mg/kg s.c. for 14 days) compared with vehicle and determined by the conversion of [¹⁴C]arginine to [¹⁴C]citrulline in vitro. ∗, P < 0.05; ∗∗, P < 0.01.

Figure 4

Semiquantitative reverse transcription–PCR showing the effects of Sim on eNOS mRNA expression in aortas (a) and brains (b) of 129/SV mice after 14 days of treatment. Specificity was determined by the lack of eNOS mRNA in eNOS-deficient mice (KO). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA expression did not change with treatment and was used as an internal standard (n = 5 per group).

Figure 5

Immunoblots (20 μg protein/lane) showing the concentration-dependent effects of Sim and Lov on eNOS protein levels in human endothelial cells in the presence and absence of l-mevalonate (200 μM) (17, 18) (a) and in the presence and absence of farnesyl pyrophosphate (FPP, 5 μM) and geranylgeranyl pyrophosphate (GGPP, 5 μM) (b).

Figure 6

Effects of Sim (Sim, 20 mg/kg daily for 14 days) on (A) absolute CBF in eNOS-deficient mice (eNOS^−/−) and (B) cerebral infarct volume and (C ) neurological deficits in eNOS-deficient mice (eNOS^−/−) after MCA occlusion/reperfusion (n = 10 per group).