Endothelial nitric oxide (NO) and its pathophysiologic regulation.

Regulation of NO and NO-mediated effects can occur through multiple mechanisms, including targeting of eNOS, availability of factors that regulate NOS activity or through modulation of protein-protein interactions of NOS with its partners, most importantly, hsp90. The complex response of endothelial cells to pathophysiological stimuli among different vascular beds or among different animal models of complex vascular disorders, such as diabetes and sepsis is a reflection of the complexity of NO regulation in endothelial cells. Although the role of tetrahydrobiopterin, reactive oxygen species, and hsp90, among many others, in regulating NO biological activity are well established, a more thorough understanding of their role is required to translate these findings into successful clinical therapies

Reduced bioavailability of NO is considered as one of the most important factors associated with vascular disease. It is unclear however whether this is a cause or a result of endothelial dysfunction. There are a number of factors which affect the production of NO and the ability of NO to reach or diffuse to its cellular targets. An important aspect of NOS function is the availability of substrates and cofactors. It is highly unlikely that L-arginine can become a rate-limiting factor since the K m of eNOS is approximately 2.9μM ( Pollock et al., 1991 ), while intra-cellular levels of L-arginine are 100 fold higher both in culture cells and in vivo ( Arnal et al., 1999 ; Baydoun et al., 1990 ). Studies conducted in vitro and in vivo however suggest that L-arginine can influence NO production. L-arginine supplementation partially reverses the impairment of endothelium-dependent vasodilation in response to acetylcholine in hypercholesterolemic patients and animal models ( Cooke et al., 1992 ; Cooke and Tsao, 1994 ). This unexpected response to l-arginine inspite of a large intracellular excess of l-arginine has been termed “the arginine paradox” ( Bode-Boger et al., 2007 ). The synthesis of NO from L-arginine can be blocked pharmacologically by a variety of arginine analogues. In the cardiovascular system, these inhibitors of NOS can induce vasoconstriction, thrombus formation and atherogenesis ( Nava et al., 1995 ).Two of these inhibitors, NG-mono-methyl-L-arginine (L-NMMA) and asymmetrical dimethylarginine (ADMA) are naturally occurring compounds that circulate in plasma ( Vallance and Leiper, 2004 ). The levels of AMDA are regulated by a dynamic process. It is synthesized by the methylation of arginine within proteins, released by proteolysis, and metabolized to citrulline by the enzymes dimethylarginine dimethylaminohydrolase (DDAH)-1 and -2 ( Leiper et al., 1999 ). A novel functional mutation of DDAH-1 carries a significantly elevated risk for cardiovascular disease and a tendency to develop hypertension ( Valkonen et al., 2005 ). Increased plasma AMDA levels have been described in a number of vascular disorders including hypercholesterolaemia, hypertension and is a strong predictor of the risk for acute coronary events ( Vallance and Leiper, 2004 ). Tetrahydrobiopterin (BH 4 ), an essential co-factor for NOS, has profound effects on NOS function, including stabilizing its dimeric structure and facilitating and enhancing binding of l-arginine ( Cosentino and Luscher, 1999 ). Reduced bioavailability of BH 4 results in uncoupling of NOS, leading to superoxide (O2 .- ) and H 2 O 2 production ( Stroes et al., 1998 ). Endothelial cells isolated from diabetic rats have reduced BH 4 levels and reduced NO production ( Meininger et al., 2000 ). Depletion of BH 4 and increased endothelial superoxide production in diabetic wild-type mice with deficient endothelial function, is prevented by maintenance of BH 4 levels in GCH-Tg diabetic mice, over-expressing GTP-cyclohydrolase, the rate-limiting enzyme for BH 4 biosynthesis ( Alp et al., 2003 ). Similarly, in spontaneously hypertensive rats, BH 4 supplementation improves endothelial dysfunction ( Heitzer et al., 2000 ; Hong et al., 2001 ). Oral administration of BH 4 shows promise for the treatment of oxidative stress-induced disorders, such as the metabolic syndrome ( Wang et al., 2007 )and improves endothelium dependent vascular relaxation after 10 weeks of high-cholesterol diet ( Hattori et al., 2007 ). Thus, BH 4 represents a therapeutically relevant tool to modulate NOS function in different diseases that are characterized by reduced NOS activity or NO synthesis. All mammalian cells including endothelial cells generate superoxide anions (O 2 .- ), which are inactivated mainly by superoxide dismutases (SOD) ( Rubbo et al., 1996 ). The foremost mechanism for the loss of bioavailable NO is thought to be due to its interaction with superoxide. If levels of superoxide increase significantly, NO outcompetes SOD for O 2 .- , a reaction which is diffusion-limited for NO and approximately six times faster than dismutation of (O 2 .- ) by SOD ( Beckman and Koppenol, 1996 ). This reaction has the triple effect of scavenging NO, reducing its bioavailability and producing a potent oxidant, peroxynitrite (ONOO - ). Once formed, peroxynitrite can chemically modify amino acids, nucleic acids and thiol containing proteins and peptides ( Koppenol et al., 1992 ). At physiological pH of 7.4, 20% of peroxynitrite is protonated to form peroxynitrous acid (ONOOH), which decomposes to form nitrogen dioxide radical (NO 2 . )and hydroxyl radical (OH . )( Beckman and Koppenol, 1996 ). The NO 2 . attacks phenol groups to produce nitrophenols ( Ischiropoulos et al., 1992 ). In biological systems this leads to modification of tyrosine residues to produce 3-nitrotyrosine. The formation of 3-nitrotyrosine can be thought of as a stable biological marker for the formation of peroxynitrite, and is elevated in a number of cardiovascular diseases ( Greenacre and Ischiropoulos, 2001 ; Peluffo and Radi, 2007 ). Endothelial cells constantly produce low levels of O 2 .- , which are significantly increased when the cells become activated ( Matsubara and Ziff, 1986 ). Many vascular diseases are associated with increased superoxide formation. The enzymatic origin of O 2 .- may vary in different types of disease and could potentially involve NAD(P)H oxidases, xanthine oxidase, lipoxygenase and NOS. However both animal and human studies suggest that the primary enzymes responsible for O 2 .- production in the vasculature are the NAD(P)H oxidases ( Clempus and Griendling, 2006 ; Ferder et al., 2006 ; Inoguchi and Nawata, 2005 ; Schulman et al., 2006 ). Hence, a dysfunctional endothelium can contribute to the reduced bioavailability of NO by releasing O 2 .- . Heat shock protein 90 (hsp90), an abundant molecular chaperone (constituting almost 1∼2% of total cytosolic protein) is highly conserved from prokaryotes to eukaryotes, and is involved in the folding, stability and maturation of numerous client proteins including nitric oxide synthases ( Richter and Buchner, 2001 ). The hsp90 chaperone machinery is in a constant flux between two conformations. The ADP bound hsp90, which corresponds to an “open” conformation, binds to its client proteins with the assistance of different co-chaperones. Replacement of ADP by ATP results in transient association of the N-terminal domains giving rise to a “closed” structural conformation, which now effectively clamps the client protein and aids in its proper folding, stabilization and maturation ( Chadli et al., 2000 ). Hsp90 inhibitors such as geldanamycin and radicicol, interact with the “N-terminal ATP binding site” of hsp90 and result in destabilization and degradation of the client proteins ( Prodromou and Pearl, 2003 ). Geldanamycin-bound hsp90 resembles the chaperone’s ADP-bound “open” conformation, and this results in the recruitment of other hsp90-interacting proteins such as E3 ubiquitin ligases (e.g. CHIP) which interact with hsp90 and promote ubiquitylation and subsequent proteasomal degradation of client proteins ( Marcu et al., 2000 ).

Sepsis and septic shock are associated with overproduction of nitric oxide primarily through iNOS ( Titheradge, 1999 ). The endothelium is a key player in initiating, perpetuating, and modulating the host response to infection. Role of eNOS in the pathophysiology of sepsis has recently gained controversy due to findings that indicated eNOS as a pro-inflammatory candidate in inflammatory disease conditions. Chronic eNOS over-expression in the endothelium of transgenic mice resulted in resistance to LPS-induced hypotension, lung injury, and death ( Yamashita et al., 2000 ) whereas, in another study, tissue (heart, liver, lungs, aorta ) iNOS expression was greatly reduced in eNOS knockout mice after LPS injection with an improved hemodynamic profile. This and other in vitro ( Connelly et al., 2003 ) and in vivo ( Bucci et al., 2005 ) studies highlight a pro-inflammatory role of eNOS in inflammatory diseases. We have shown that pretreating mice with hsp90 inhibitors markedly improves survival and lung function following a lethal dose of LPS and is associated with reduced lung injury and formation of hsp90-iNOS complexes and NO metabolites ( Chatterjee et al., 2007 ). Our preliminary experiments also revealed reduced pulmonary eNOS expression in septic mice, pretreated with hsp90 inhibitors ( unpublished data). Therefore, in our model, hsp90 appears to exert a pro-inflammatory role through its association with eNOS and iNOS. How hsp90 regulates endothelial NO post sepsis remains an interesting topic to be investigated.

Endothelial cells have a constitutive expression of eNOS and like other constitutively expressed proteins, the eNOS promoter lacks the typical TATA box. Instead, it has other multiple cis -regulatory DNA sequences like SP-1, GATA, activator protein-1, activator protein-2, nuclear factor-1, sheer stress response elements and sterol-regulatory elements ( Marsden et al., 1993 ). The presence of these consensus sites is consistent with evidence showing that levels of eNOS transcripts are elevated by sheer stress ( Davis et al., 2001 ; Woodman et al., 2005 ), exercise ( Sessa et al., 1994 ; Yang et al., 2002 ) and hypoxia ( Le Cras et al., 1996 ). Regulation of eNOS transcription by estrogens is still a matter of debate ( Arnal et al., 1996 ; Kleinert et al., 1998 ), however, estradiol relaxes rat aortic segments via endothelium-dependent and -independent mechanisms involving the NO-cGMP signaling system ( Abou-Mohamed et al., 2003 ). Both lipopolysaccharide ( Arriero et al., 2000 ) and tumor necrosis factor-α ( Yoshizumi et al., 1993 ) decrease eNOS gene expression by reducing the stability of eNOS mRNAs. Basal human eNOS transcription is controlled by two regulatory regions, the positive regulatory domains I and II (PRD I, PRD II). These regulatory domains bind various types of transcription and trans -acting factors and regulate eNOS transcription by complex cis and trans interactions ( Searles, 2006 ). Moreover, these regions also contain differentially methylated nucleotides that restrict eNOS transcription largely in vascular endothelial cells ( Chan et al., 2004 ). The constitutively expressed eNOS mRNA is about 4052 nucleotide long and has a half-life of 10-35 hours. Therefore, synthesis of the encoded proteins is likely to persist long after gene expression has been repressed. Thus, altering the half-life of stable transcripts may be the most rapid and efficient means of modulating steady-state mRNA levels and gene expression. Posttranscriptional control of eNOS-mRNA is largely mediated by cis -acting RNA elements located in 3′ — mRNA untranslated regions (UTRs). Bovine eNOS-mRNA is stabilized by deletion of a 45-nt located at the origin of the bovine 3′-UTR ( Searles et al., 1999 ). A CU-rich 158-nucleotide sequence, located in the medial portion of human 3′-UTR is important for regulating eNOS-mRNA stability ( Lai et al., 2003 ). Another mechanism for post-transcriptional regulation of eNOS has been proposed, based on the evidence of an antisense mRNA (sONE) that is derived from a transcription unit (NOS3AS) on the opposite DNA strand from which the human eNOS ( NOS3 ) mRNA is transcribed at human chromosome 7q36 ( Robb et al., 2004 ). The mRNA for sONE can be detected in a variety of cell types, both in vivo and in vitro , but not in vascular endothelial cells. Suppression of sONE leads to over expression of eNOS whereas over-expression of sONE in human endothelial cells leads to decreased eNOS expression ( Robb et al., 2004 ). These findings suggest a model for cell-specific expression of eNOS that involves a functional interaction between the eNOS and NOS3AS genes at the posttranscriptional level. Further studies are needed to determine how sONE expression is regulated in endothelial cells and its role in eNOS expression under various physiological and pathophysiological conditions.