Targeting eNOS and Beyond: Emerging Heterogeneity of the Role of Endothelial Rho Proteins in Stroke Protection

Targeting Endothelial Nitric Oxide Synthase for Stroke Protection

Role of Nitric Oxide Synthases in Stroke: Insights from Knockout Mice

An important endogenous mediator of CBF and cerebrovascular protection is endothelium-derived nitric oxide (NO), which is synthesized by endothelial NO synthase (eNOS or NOS3) through oxidative conversion of L-arginine to L-citrulline.^[5,6] NO formed by the vascular endothelium within a nanomolar range diffuses to the adjacent cells, such as vascular smooth muscle, platelets and leukocytes, activates soluble guanylate cyclase and increases intracellular cyclic guanosine monophosphate, which in turn mediates many of the beneficial effects of NO. These include vasodilation, antithrombotic, anti-inflammatory and antiproliferative effects.

In the brain, two other isoforms of NOS have been characterized. Neurons produce NO mostly by activation of neuronal NOS (nNOS or NOS1), which is constitutively expressed in these cells.^[7] Glial cells (astrocytes, microglia and macrophages) synthesize NO through inducible NOS (iNOS- or NOS2) expression by distinct stimuli.^[8] Substantial evidence suggests that NO can exert both protective and deleterious effects depending on factors such as the temporal stage after the onset of ischemic brain injury and the NOS isoform and cellular source of NO. Immediately after brain ischemia, NO release from eNOS is protective by promoting vasodilation and inhibiting microvascular aggregation and adhesion; however, after ischemia develops, NO produced by overactivation of nNOS and, later, NO release by de novo expression of iNOS contribute to brain damage.^[9]

The differential roles of NOS isoforms in stroke have been revealed by the development of knockout mice over the respective isoform for the past two decades. Following ischemia, NO levels in the brain rise several orders of magnitude, from baseline nanomolar levels to stimulated micromolar levels.^[10] nNOS- and iNOS-knockout mice have revealed that these NOS isoforms mediate this increase at early and late stages post-ischemia, since the knockout mice do not show this increase.^[11,12] When subjected to a middle cerebral artery occlusion (MCAo) model of ischemia, iNOS- and nNOS-knockout mice develop significantly smaller infarct sizes and have better neurological outcome than wild-type mice. nNOS and iNOS may contribute to neuronal toxicity in several ways, including reaction with superoxide to form peroxinitrite, activation of poly-ADP ribose synthase resulting in depletion of cellular energy stores, and direct inhibition of mitochondrial respiration.^[13–15]

By contrast, eNOS-knockout mice subjected to the MCAo model develop larger infarct sizes compared with wild-type mice.^[16] Laser Doppler flowmetry and temporal correlation mapping show that eNOS-knockout mice have significantly reduced blood flow compared with wild-type mice.^[17] This confirms that eNOS normally serves to vasodilate and preserve blood flow in the setting of ischemia; in its absence, an inability to preserve blood flow contributes to the enlarged infarct sizes observed in eNOS-knockout mice. Additionally, enhancing NO production by administration of the eNOS substrate l-arginine increases regional CBF in the ischemic territory and confers protection from stroke.^[18] This suggests that augmenting eNOS activity protects the ischemic brain, probably by maintaining sufficient CBF.

Therapeutic Strategies That Enhance eNOS for the Prevention and Treatment of Stroke

The activity of eNOS is regulated by complex, multiple mechanisms. eNOS is a constitutively expressed enzyme; however, its protein abundance can be dynamically altered by transcriptional and post-transcriptional mechanisms, and serves as a major determinant of eNOS-activity.^[19,20] In addition, eNOS enzymatic activity is post-translationally regulated by the availability of the substrate L-arginine, cofactors FAD, FMN and BH4, and intracellular Ca²⁺/calmodulin levels.^[21] Post-translational regulation of eNOS further involves phosphorylation, protein–protein interaction and intracellular localization. eNOS is phosphorylated at multiple sites, including serine 1177 and threonine 495. Importantly, eNOS serine 1177 is phosphorylated by protein kinase B/Akt, and mediates the rapid activation of eNOS and NO generation by fluid shear stress and growth factors, such as VEGF.^[22,23] eNOS is localized to caveolae by N-terminal fatty acid modifications – myristoylation and palmitoylation, as well as through interaction with heat-shock protein (hsp)90 and caveolins. While caveolin-1 and caveolin-3 bind to eNOS and inhibit its activity, hsp90 enhances eNOS activity.^[24]

Recently, various experimental modalities have shown the capacity to alleviate tissue injury in rodent models of acute ischemic stroke. Evidence shows that many of the modalities exert their effects by increasing the expression or activity, or both, of eNOS ( Box 1 ).

Expert Rev Neurother. 2009;9(8):1171-1186. © 2009  Expert Reviews Ltd.

Cite this: Targeting eNOS and Beyond: Emerging Heterogeneity of the Role of Endothelial Rho Proteins in Stroke Protection - Medscape - Aug 01, 2009.