1. Endothelial Nitric Oxide in Health and Disease

386 G. M. Rubanyi

calmodulin (CaM), and requires three co-substrates including L- arginine, nicotinamide adenine dinucleotide phosphate (NADPH) and molecular oxygen.4

Three isoforms of NOS, encoded by three distinct genes on different chromosomes, have been isolated and purified. Both the neuronal (nNOSor NOS-I) and endothelial (eNOS or NOS-III) isoforms are constitutively activated and expressed upon calcium-calmodulin binding following an increase in intracellular calcium. The inducible isoform (iNOS or NOS-II) is activated by cytokines independent of calcium (calmodulin is tightly bound to NOS-II in contrast to the constitutive isoforms, probably due to the lack of an auto-inhibitory loop on NOS-II).5 All NOS isoenzymes form homodimers, and contain a heme oxygenase domain and a cytochrome P-450 reductase domain.

1.2. Physiological role of endothelial NO (“EDNO”)

Under physiological conditions endothelial NOS-III-derived NO, released by receptor activation or shear stress, freely diffuses from the endothelium towards the lumen and abluminally towards the underlying vessel wall, and plays a key role in the maintenance of vascular homeostasis.6

Endothelium-derived NO (EDNO) is a potent vasodilator, which led to its discovery as “EDRF”1 and later to its identification as NO2,3 using bioassay systems allowing the assessment of its biological half-life,7,8 and describing the characteristics of “EDRF” including its interaction with superoxide anion radical (.O2−)9 and its release by increased flow/shear stress.10

The vasodilating activity of EDNO is mediated by activation of soluble guanylate cyclase (sGC) and elevation of cGMP.3,11 NO inhibits platelet adhesion and aggregation,12 also through a cGMP-mediated pathway. NO inhibits vascular SMC proliferation13 while promoting endothelial cell growth.14,15 NO have been shown to reduce leukocyte infiltration of the endothelial barrier.16

Oxidatively modified LDL (oxLDL) is a major contributor to the pathogenesis of atherosclerosis. NO have been shown to inhibit oxidative modification of LDL.17 NO attenuates smooth muscle proliferation

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and inhibits neointima formation.18 On the other hand NO protects endothelial cells from apoptotic stimuli,19,20 which play an important role in its participation in the angiogenic process.

Endothelial NOS is localized to the caveolae of the endothelial plasma membrane21 in close proximity to key membrane receptors, ion channels and signaling molecules, positioning the NOS-III/EDNO system upstream of many regulatory pathways determining cell function and phenotype.

1.3. Endothelial NO-deficiency in cardiovascular diseases

Availability and biological activity of endothelial NO are regulated by the expression of its generating enzyme, NOS-III, as well as by the activity of the NOS-III enzyme, which is tightly controlled by co-factor and substrate availability, post-translational modifications (myristoylation, palmitoylation and phosphorylation), protein-protein interactions (e.g. caveolin and Hsp90) and subcellular localization. In addition, accumulation of endogenous NOS inhibitors (e.g. ADMA) and increased oxidative degradation of NO can also lead to diminished availability/bioactivity of endothelial NO.

“Endothelial NO-deficiency” is an early phenomenon in the progression of various cardiovascular diseases.22,23 Impaired continuous basal NO synthesis may be the first detectable evidence of endothelial dysfunction.24−26 Early signs of endothelial function are easily assessable by measuring endothelium-dependent vasoconstriction to NOS inhibitors or endothelium-dependent vasodilation in response to increased flow or receptor agonists, such as acetylcholine. Diagnostic approaches, like quantitative coronary angiography and new ultrasound/Doppler devices, are becoming mainstream tools for early detection of EDNO-deficiency in high risk patients.27

Impaired EDNO activity is associated with several cardiovascular diseases,28 including atherosclerosis, systemic and pulmonary hypertension, congestive heart failure, peripheral arterial occlusive disease as well as cardiovascular complications of diabetes. The apparent “NO-deficiency” is the net result of several different pathological processes interfering with one or more of the components regulating the

388 G. M. Rubanyi

availability and/or bioactivity of NO in the vascular wall. These processes can decrease the amount of endothelial NO at different levels, which include (a) reduced NOS-III gene expression (both at the transcription and mRNA stability level), (b) reduced activity of the NOSIII enzyme via diminished co-factor and substrate availability, or by modifications in post-translational processes, cellular localization or protein-protein interactions, and (c) reduced biological activity of NO (e.g. through oxidative inactivation).

Proof for the numerous physiological (mostly vasculoprotective) roles of endothelial NO in the cardiovascular system was provided by the development of the NOS-III-deficient (NOS-III-KO) mouse,in which NOS-III expression was genetically disrupted.29 Homozygous NOS- III-KO mice have elevated mean arterial blood pressure, consistent with the role of endothelial NO in the regulation of blood pressure and vascular tone.30 Isolated aortic rings with intact endothelium from NOS-III-KO mice do not relax to acetylcholine, which provides genetic evidence that the NOS-III gene is required for the “EDRF” activity. These mice show markedly decreased bleeding times,31 exhibit enhanced leukocyte adhesion associated with elevated surface expression of P- selection in the microcirculation32 and impaired angiogenic response.33 Myocardial ischemia and reperfusion injury were significantly exacerbated in NOS-III-KO mice.34 NOS-III deficiency also resulted in enlarged cerebral infarcts following permanent middle cerebral artery occlusion.35

1.4.Therapeutic restoration of endothelial NO production in cardiovascular diseases

Contribution of diminished endothelial NO production to the pathomechanism and the progression of different cardiovascular diseases have been demonstrated under numerous experimental and clinical conditions. Therefore drugs that can improve endothelial NO production may have significant therapeutic benefits in these pathological conditions.

Endothelial function can be restored by different classes of compounds directly or indirectly targeting regulatory mechanisms of