Abstract 489: ROCK2, but not ROCK1, Mediates NO-dependent Neuroprotective Effects Following Transient Focal Cerebral Ischemia
Background: Previous studies have shown that Rho kinase (ROCK) inhibitors protect against cerebrovascular injury through upregulation of endothelial nitric oxide synthase (eNOS). Because ROCK inhibitors cannot distinguish between ROCK1 and ROCK2, the isoform specific role of ROCKs in neuroprotection is not known. Using mice with target deletion of ROCK1 or ROCK2 alleles, we investigated whether ROCK1 and/or ROCK2 mediates changes in eNOS expression and contributes to neuroprotection.
Methods and Results: ROCK1−/ − and ROCK2−/ −mice were generated and found to be lethal embryonically and postnatally. However, haploinsufficient ROCK1+/− and ROCK2+/− mice are phenotypically normal and have half of the level of ROCK1 and ROCK2 protein compared to that of WT mice, respectively. There were no compensatory changes of ROCK2 in ROCK1+/− mice or of ROCK1 in ROCK2+/− mice. Furthermore, no differences in hemodynamic parameters such as BP, heart rate, and body weight were observed between WT, ROCK1+/−, and ROCK2+/− mice. We subjected these 10-wk old male mice to transient intraluminal filament occlusion of the middle cerebral artery (MCAO) for 2 hrs followed by 22 hrs of reperfusion. Twenty-four hrs after MCAO, cerebral infarct size was substantially reduced in ROCK2+/− (48.9 ± 7.7 mm3, P<0.05), but not ROCK1+/− mice (73.8 ± 7.3 mm3, P=NS), compared to that of WT mice (80.0 ± 8.9 mm3; n=8–12). This correlated with a 29% decrease in neurological deficit score in ROCK2+/− (P<0.05), but not ROCK1+/− mice (P=NS), compared to that of WT mice. In brains and primary cultures of endothelial cells, the expression and Ser1177 phosphorylation of eNOS were 3-fold higher in ROCK2+/− mice and 1.7-fold higher in ROCK1+/− mice compared to those in WT mice (P<0.05 for both). Indeed, the half-life of eNOS mRNA was 55 hrs in ROCK2+/− mice, 31 hrs in ROCK1+/−mice, and 20 hrs in WT mice (P<0.05 between all groups).
Conclusions: These findings indicate that eNOS mRNA stability and expression are increased to a greater extent in ROCK2+/−mice compared to that of ROCK1+/− or WT mice, and suggest that inhibition of ROCK2 may contribute to the primary benefits of ROCK inhibitors in stroke protection. These results suggest that ROCK2 could be a therapeutic target in ischemic stroke.