The phosphorylation state of eNOS modulates vascular reactivity and outcome of cerebral ischemia in vivo

Abstract

NO plays critical roles in vascular function. We show that modulation of the eNOS serine 1179 (S1179) phosphorylation site affects vascular reactivity and determines stroke size in vivo. Transgenic mice expressing only a phosphomimetic (S1179D) form of eNOS show greater vascular reactivity, develop less severe strokes, and have improved cerebral blood flow in a middle cerebral artery occlusion model than mice expressing an unphosphorylatable (S1179A) form. These results provide a molecular mechanism by which multiple diverse cardiovascular risks, such as diabetes and obesity, may be centrally integrated by eNOS phosphorylation in vivo to influence blood flow and cardiovascular disease. They also demonstrate the in vivo relevance of posttranslational modification of eNOS in vascular function.

Figure 1. Generation of S1179A and S1179D eNOS transgenic mice.

(A) Transgenic construct. A 1.6-kb fragment of the human eNOS promoter drives the bovine eNOS cDNA carrying the S1179D or S1179A mutation, an HA tag, and the SV40 polyadenylation site. (B) Genotyping of transgenic and native eNOS genes by PCR. Primers E1 and E2 (see Methods) span an intron, so they amplify an 800-bp product from the endogenous WT eNOS gene but a 280-bp product from the S1179A and S1179D transgenes. Mice bred onto the homozygous eNOS KO background do not show the 800-bp product. (C) Western blot analysis of heart protein (60 μg) of WT, S1179D transgenic/eNOS KO (S1179D/eNOS KO), and S1179A transgenic/eNOS KO (S1179A/eNOS KO) mice, using antibody directed against total eNOS.

Figure 2. Subcellular localization of WT and transgenic eNOS.

(A) En face immunostaining of the common carotid arteries using eNOS antibody shows localization of eNOS in perinuclear Golgi that are aligned along the long axes of the cells in the endothelium. VE-cadherin (VE-cad) staining shows the outlines of the endothelial cell membranes. (B) En face immunostaining of the common carotid arteries using anti-HA shows perinuclear staining in the Golgi apparatus in a pattern similar to that of eNOS immunostaining in WT mice. Expression levels of the transgene are comparable in the S1179A/eNOS KO mice and S1179D/eNOS KO mice. Original magnification, ×600.

Figure 3. Interactions between caveolin-1 (Cav-1) and eNOS are unaffected in mice carrying transgenes.

En face immunostaining of the common carotid arteries using eNOS antibody and caveolin-1 antibody shows that interactions between eNOS and caveolin-1 are unaffected in S1179D/eNOS KO mice and S1179A/eNOS KO mice. Original magnification, ×600.

Figure 4. Effect of S1179D and S1179A mutations on vascular reactivity.

(A) Left common carotid arteries were constricted with phenylephrine (10^–5 M) and then subjected to increasing doses of ACh from 1 × 10^–9 to 1 × 10^–5 M. S1179A/eNOS KO mice, n = 8; S1179D/eNOS KO mice, n = 8; eNOS KO mice, n = 6; and WT mice, n = 8. *P < 0.01 by 1-way ANOVA; ^†P < 0.05 by 1-way ANOVA. (B) Left common carotid arteries were treated with l-NAME (3 × 10^–4 M) for 30 minutes, constricted with phenylephrine (10^–5 M), and then subjected to increasing doses of SNP from 1 × 10^–9 to 1 × 10^–5 M. Data are expressed as mean ± SEM.

Figure 5. Effect of S1179A and S1179D eNOS mutations on cerebral infarct size.

Mice were subjected to the filament model of MCA occlusion for 1 hour, followed by 23 hours of reperfusion. The brains were cut into 2-mm coronal sections and stained using 2,3,5-triphenyltetrazolium chloride. (A) Rostral to caudal distribution of infarct areas (mm²) in coronal sections. Abscissa shows the distance (mm) from the rostral surface of the brain. n = 7 mice for each group. *P < 0.05 versus corresponding sections from S1179A/eNOS KO mice; ^†P < 0.05 versus corresponding sections from eNOS KO mice using 1-way ANOVA. (B) Infarct volumes were determined by integrating the infarct areas in each section over the entire brain, using the indirect method, which corrects for edema. *P < 0.05 versus infarct volume from S1179A/eNOS and from eNOS KO mice using ANOVA. Neurologic scores for each group of mice are as follows: eNOS KO, 2.5; S1179A/eNOS KO, 2.67; S1179D/eNOS KO, 1.57; WT, 1.86. Kruskal-Wallis 1-way ANOVA on ranks showed a statistically significant difference between eNOS KO and S1179A/eNOS KO mice and WT and S1179D/eNOS KO mice (P < 0.05).

Figure 6. Effect of S1179A and S1179D eNOS mutations on cerebral blood flow.

(A) Laser speckle contrast images in mice subjected to MCA occlusion. Superimposed areas (blue) indicate regions with ≤30% residual blood flow. Imaging field dimensions are 6 × 8 mm. Thresholded images were recorded after 60 minutes of ischemia. (B) Composite graph showing the area of cortex with ≤20% (black) or 21–30% (blue) residual blood flow compared with preischemic baseline. *P < 0.05, comparing the area with ≤20% of residual blood flow of eNOS KO and S1179A/eNOS KO mice versus WT and S1179D/eNOS KO mice. CBF, cerebral blood flow.