Endothelial nitric oxide synthase transgenic models of endothelial dysfunction

Abstract

Endothelial production of nitric oxide is critical to the regulation of vascular responses, including vascular tone and regional blood flow, leukocyte-endothelial interactions, platelet adhesion and aggregation, and vascular smooth muscle cell proliferation. A relative deficiency in the amount of bioavailable vascular NO results in endothelial dysfunction, with conditions that are conducive to the development of atherosclerosis: thrombosis, inflammation, neointimal proliferation, and vasoconstriction. This review focuses on mouse models of endothelial dysfunction caused by direct genetic modification of the endothelial nitric oxide synthase (eNOS) gene. We first describe the cardiovascular phenotypes of eNOS knockout mice, which are a model of total eNOS gene deficiency and thus the ultimate model of endothelial dysfunction. We then describe S1177A and S1177D eNOS mutant mice as mouse models with altered eNOS phosphorylation and therefore varying degrees of endothelial dysfunction. These include transgenic mice that carry the eNOS S1177A and S1177D transgenes, as well as knockin mice in which the endogenous eNOS gene has been mutated to carry the S1177A and S1177D mutations. Together, eNOS knockout mice and eNOS S1177 mutant mice are useful tools to study the effects of total genetic deficiency of eNOS as well as varying degrees of endothelial dysfunction caused by eNOS S1177 phosphorylation.

Fig. 1

Biological roles of endothelial NO: inhibition of platelets aggregation, modulation of leukocyte–endothelial interactions, modulation of smooth muscle cell proliferation, and maintenance of vascular tone. eNOS is activated by physiological and metabolic stimuli, shear stress, and receptor-dependent agonists. S1177 phosphorylation site is an important regulatory mechanism for eNOS activity. eNOS catalytic activity depends on the intracellular localization to caveolae and the protein–protein interactions with caveolins and hsp-90. NO activates cGMP, inducing PKG-dependent relaxation of smooth muscle cells and increasing luminal diameter and blood flow. P phosphorylation; S1177 serine 1177, Hsp-90 heat shock protein-90, PKG serine–threonine-specific protein kinase G

Fig. 2

Effect of eNOS mutations on CBF deficit. a Laser speckle contrast images in mice subjected to middle cerebral artery occlusion. Blue areas indicate regions with ≤30% residual blood flow after 60 min of ischemia. b The area of cortex with≤20% (black) or 21–30% (blue) residual blood flow as compared with preischemic baseline. *P<0.05, comparing the area with ≤20% of residual blood flow of eNOS knockout and S1177A transgenic mice with WT and S1177D transgenic mice

Fig. 3

a Temporal correlation mapping image shows blood flow after focal cerebral ischemia in wild-type mouse. Normal hemodynamic status is shown as green. Regions with abnormal CBF are represented with a red-to-black sliding color scale. The core appears black, and the penumbra is the reddish rim surrounding the core. An extensive hemodynamic penumbra exist in wild-type mice 30 min after focal ischemia. b The hemodynamic deficit in eNOS knockout mice after focal ischemia is more severe. The core is larger, and the penumbra is correspondingly smaller compared with wild-type mice

Fig. 4

Subcellular localization of eNOS S1177 mutation in transgenic mice. a En face immunostaining of carotid arteries shows localization of eNOS in perinuclear Golgi. VE-cadherin (VE-cad) staining shows the outlines of the endothelial membranes. b En face immunostaining of carotid arteries shows perinuclear location of the transgene hemagglutinin tag in the perinuclear Golgi in a pattern similar to eNOS immunostaining in wild-type mice. Original magnification ×600

Fig. 5

Effect of S1177 mutations on cerebral infarct volume. Mice were subjected to 1 h of focal brain ischemia followed by reperfusion for 23 h. The brains were cut into coronal sections and stained using 2,3,5-triphenyltetrazolium chloride. a Abscissa shows the distance (millimeter) from the rostral surface of the brain. For each group, n=7 mice. *P<0.05 versus S1177A transgenic mice, ^†P<0.05 versus eNOS KO mice. b Infarct volumes were determined by the indirect method, which corrects for edema. *P<0.05 versus infarct volume from S1177A mutant and from eNOS knockout mice