4.1.2.1. Characterization of the Three NO-Synthases.

NO derived from L-arginine has been involved in the responses of macrophages to inflammatory stimuli, such as bacterial lipopolysaccharides and interferon-γ, and in their ability to kill tumor cells or bacteria [643,644,986,1462,1463]. A NO-forming enzyme is present in the brain and the L-arginine-NO-cyclic-GMP pathway is activated by neurotransmitters in various neurons [516,820]. These different observations prompted the conclusion that distinct types of NO production were associated with three different subtypes of NO biosynthetic enzymes. These were cloned, characterized and termed according to the chronology of their characterization: NO-synthase I (or neuronal, nNOS, NOS-1) [144,146], NO-synthase II (or inducible, iNOS, NOS-2) [964,1781] and NO-synthase III (or endothelial, eNOS, NOS-3) [1233,1394]. In fact, the expression of the various NOS isoforms is much more diverse than their name would suggest. For instance, in the vascular wall, the three NOS can be present. nNOS is expressed not only in perivascular nerves but can also be detected in endothelial and smooth muscle cells [131,566,1186]. The expression of iNOS has been documented in all nucleated cells in the cardiovascular system. In the blood vessel wall this includes endothelial and smooth muscle cells as well as fibroblasts, leukocytes and mast cells [1187]. Actually, eNOS is expressed not only in endothelial cells but also in cardiac myocytes and platelets [57,1350]. Nevertheless, the epigenetic regulation of vascular endothelial gene expression means that the preponderant NOS isoform in healthy endothelial cells is the calcium-dependent eNOS and that eNOS is predominantly expressed in endothelial cells [1000].

The three NOS enzymes contain a reductase and an oxygenase domain. In the presence of molecular oxygen, they catalyze a five-electron oxidation of one of the guanido nitrogens of L-arginine. They all require the cofactors NADPH, FAD, FMN, heme, tetrahydrobiopterin (BH₄) and zinc in order to catalyze this reaction [145]. The conversion of L-arginine to NO by eNOS involves two independent steps. The first one is the NADPH-dependent hydroxylation of L-arginine to N^G-hydroxy-arginine, a reaction requiring calcium-calmodulin, which is accelerated by the presence of BH₄. This hydroxylation step resembles cytochrome P450 monoxygenation. The second step leading to the generation of NO with the stoichiometric formation of L-citrulline is also calcium-calmodulin-dependent, requires O₂ and NADPH and is also accelerated by BH₄ [143]. An essential feature of the three NOS, despite the ability of the reductase or the oxygenase domain to function independently, is that the NO-synthase activity requires dimerization. The heme and a tetracoordinated zinc ion held by four thiols, two from each monomer, play an essential role in the formation of the homodimer and BH₄ stabilizes the dimer once formed [38,906]. The zinc thiolate center and BH₄ are subject to attack by peroxynitrite, which disrupts and releases the zinc ion, and oxidizes the thiols. Upon thiol reduction, eNOS dimers dissociate into monomers and become uncoupled [234,1817,1819] (Figure 11).

4.1.2.2. Transcriptional and Post-Translational Regulation of eNOS in Endothelial Cells.

Since NO is a gaseous free radical with a high biological reactivity, its synthesis has to be tightly controlled in order to be delivered at the right time, the right place and in the right amount [841].

The transcription of eNOS is under epigenetic control and can be regulated by various physical, chemical and pharmacological stimuli. The endothelial cell-restricted expression of eNOS is under the control of an epigenetic mechanism of gene regulation involving DNA methylation and a specific endothelial histone code. The transcription of eNOS is enhanced, for instance, by laminar and oscillatory shear stress, hydrogen peroxide, inhibition of histone deacetylase, micro-RNA, statins and, possibly, estrogens [56,170,1000,1383]. In response to laminar flow, the transcription of eNOS is coordinated with the regulation of the expression of many other genes that not only control vascular tone but also promote an atheroprotective, anti-thrombotic and anti-inflammatory phenotype. This phenomenon involves the up-regulation of the endothelial transcription factor KLF-2 (Kruppel like factor-2) [56,548,585].

The half-life of eNOS mRNA has been reported to be as long as 10 to 35 hours, indicating that post-transcriptional regulation of the transcripts half-life could be a more rapid and efficient strategy than to regulate eNOS transcription [213]. Indeed, post-transcriptional regulation (mRNA processing and polyadenylation, mRNA export from the nucleus and mRNA degradation) is heavily controlled by various different stimuli, some of those also regulate eNOS transcription [1383].

Increasing the amount of eNOS protein can only partially predict overall enzymatic activity since eNOS is subjected to several overlapping post-translational regulatory modifications, including lipidation, phosphorylations, S-nitrosylations, acetylation and protein–protein interactions [56,347,550]. The activation of eNOS also requires the calcium-dependent binding of calmodulin [38]. Furthermore, the sub-cellular location of eNOS is critical for its state of activation and multiple levels of regulation are responsible for the trafficking of the enzyme [1144].

In endothelial cells, eNOS is predominantly located in the plasma membrane and in the Golgi. A two-step process involving the enzymatic acylation of eNOS residues is responsible for this targeting. The myristoylation of the N-terminal glycine targets eNOS to membrane structures. The subsequent reversible palmitoylation of cysteine residues Cys15 and Cys26 (according to the human eNOS sequence) then directs the enzyme specifically to caveolin-1, a protein that serves as a scaffold for the assembly of multi-protein signaling complexes. Caveolin-1 is preferentially located in caveolae, invaginations of the cell membrane and is also present in the Golgi. The association of eNOS with caveolin-1 inhibits its activity [347,439,1144].

Additionally, eNOS activity is regulated by serine, threonine and tyrosine phosphorylations. The phosphorylation of Ser1177, unphosphorylated at rest, and the coordinated dephosphorylation of Thr495, constitutively phosphorylated at rest, enhance the sensitization of eNOS to calcium–calmodulin binding. The prevention of Ser1177 phosphorylation by non-specific O-linked glycosylation of eNOS can contribute to a decreased NO formation in diabetes [56,347,455]. The phosphorylation of Tyr657 is associated with a decreased production of NO. This proline-rich tyrosine kinase 2 (PYK2)-dependent phenomenon is elicited not only in response to oxidative stress but also in response to shear stress and insulin stimulation. The former may contribute to the endothelial dysfunction occurring, for instance, in hypertension. The concomitant phosphorylation of Ser117 and Tyr657 by shear stress or insulin can allow low but regular and prolonged NO generation without being confronted with co-factor depletion (BH₄) and/or excessive peroxynitrite formation [450,948]. Additionally, dephosphorylation of Ser114, a constitutive inhibitor of eNOS activity, and phosphorylation of Ser615 and Ser633 and, possibly, Tyr81 can also enhance eNOS activity [56,76,347,455,1084].

The AMP-activated protein kinase (AMPK) is the only kinase identified that can phosphorylate eNOS on different amino acid residues, Ser1177, Ser633 and Thr495. AMPK-dependent activation of eNOS has been reported following various physical, chemical, neurohumoral and pharmacological stimuli, including shear stress, hypoxia, vascular endothelial growth factor (VEGF), adiponectin, peroxisome proliferator-activated (PPAR) receptor agonists and statins [449,455].

S-nitrosylation, the reversible covalent modification of a cysteine thiol by NO, is an additional level of post-translational control of eNOS activity. When anchored to caveolae, resting eNOS activity can be self-inhibited by S-nitrosylation of Cys94 and Cys99. Conversely, during activation, the enzyme is denitrosylated in the cytosol. Whether or not the mechanism of the inhibition by S-nitrosylation is associated with eNOS monomerization is still a matter of debate [347,922,1285].

Acetylation (or ethanoylation)/deacetylation, i.e., the introduction/removal of an acetyl functional group on lysine residues, is emerging as a significant post-translational regulatory mechanism. Histone acetyltransferase and histone deacetylase can modify the acetylation status of histone and non-histone proteins as well [550]. Sirtuin-1 (SIRT-1), a class III histone deacetylase, enhances acetylcholine- and shear stress-induced NO production by deacetylating eNOS, possibly at Lys494 and Lys504, and enhancing its binding to calmodulin [239,1011]. This mechanism is susceptible to pharmacological manipulations since aspirin enhances eNOS activation by acetylating Lys609, via a mechanism that is totally independent of cyclooxygenase inhibition [758].

Furthermore, eNOS is part of a large signaling complex that, besides calmodulin and caveolin-1, involves many other different proteins which are associated with eNOS at different stages of its trafficking. When associated with caveolin-1, this includes, for instance, the cation L-arginine transporter CAT-1 or some G-protein-coupled receptors such as the ET_B endothelin and the bradykinin B₂ receptor subtype [455,841]. The heat shock protein, Hsp90, is a chaperone protein involved in the folding of eNOS and in the heme insertion. Hsp90 increases eNOS activity by cooperatively enhancing the affinity of eNOS for calmodulin and facilitating the interaction with Akt. Endoglin, a cell membrane glycoprotein, promotes the association of eNOS with Hsp90. Furthermore, the trafficking of eNOS involves various types of proteins such as actin cytoskeleton, NOSIP (eNOS interacting protein), NOSTRIN (eNOS trafficking inducer protein), Gab1 (growth factor receptor-bound protein-associated binding protein-1), PECAM-1 (platelet–endothelial cell adhesion molecule-1) or dynamin. These various eNOS-associated proteins can be also associated with various kinases and phosphatases, and their own post-translational phosphorylations and S-nitrosylations play an important regulatory role [347,455,841,1037].

Finally, the degradation of eNOS determines its availability and must be regulated. Unfortunately, very little is known at present on these processes [841].

4.1.2.3. Bradykinin and Shear Stress as Examples of Two Different Mechanisms of eNOS Activation.

In the resting state, the myristoylated and palmitoylated eNOS is anchored to the plasma membrane, associated with caveolin-1, which inhibits its activity, and clustered with other proteins located in the caveolae. Inhibitory Ser114 and Thr495 are phosphorylated, Cyst94 and Cyst99 are S-nitrosylated and some lysine residues are acetylated.

By binding to the endothelial B₂ kinin receptor subtype, a G-protein-coupled receptor, bradykinin, promotes calcium entry and calcium release from internal stores, increasing [Ca²⁺]_i. This stimulates calmodulin binding to eNOS and the concomitant dissociation from caveolin-1. In addition, bradykinin stimulates the binding of Hsp90 and reduces that of other associated proteins such as the B₂ receptor. The production of NO is further enhanced by the phosphorylation of Ser1177 and the coordinated dephosphorylation of Thr495, mediated by the calcium/calmodulin-dependent protein kinase II (CaMKII) pathway and facilitated by the Hsp90-dependent recruitment of Akt. Phosphorylation of Ser633 and possibly Ser615, via the protein kinase A pathway, dephosphorylation of Ser114 and the denitrosylation of the two cysteins could contribute to the overall activation of eNOS [347,455,841,1084].

Shear stress stimulates NO production in two phases. A first brief transient calcium-calmodulin-dependent NO burst followed by a second sustained phase of low NO production, lasting as long as shear is imposed, which occurs at resting [Ca²⁺]_i levels. In the first phase, the rapid dissociation to caveolin and the following association with Hsp90 are again observed. Then, the protein kinase A (PKA)-dependent phosphorylation of Ser1177 is rapidly induced. In the second phase, the PKA-dependent phosphorylation of Ser633 and the PYK2-dependent phosphorylation of Tyr657, which respectively increase and reduce eNOS activity, contribute to the sustained and moderate NO generation. In endothelial cells exposed to shear stress, eNOs is predominantly localized at the level of homocellular cell junctions and is associated with PECAM-1 and the adapter Gab-1, serving as a scaffold for kinases such as PKA and phosphatases [56,126,455].

After activation, eNOS can be recycled to the Golgi or the plasma membrane caveolae and can re-associate with caveolin-1. However, eNOS can also be inactivated following depalmitoylation and redistribution to sub-cellular membrane structures, a phenomenon associated by interactions with NOSTRIN and NOSIP. Complex and not completely understood, dephosphorylations and phosphorylations as well as S-nitrosylations and denitrosylations of eNOS take place during the trafficking of the enzyme [347,841].

4.1.6.1. Availability of the Substrate L-Arginine.

When L-arginine is deficient, eNOS can generate both superoxide anions and nitric oxide leading to the detrimental production of peroxynitrite [1724]. L-arginine supplementation shows beneficial effects in pathophysiological conditions such as hypercholesterolemia and hypertension. However, since the L-arginine plasma concentration is approximately 100 μM, much above the KM of eNOS for L-arginine, it is unlikely that the circulating concentrations of this semi-essential amino acid become critical. In addition, human endothelial cells can effectively recycle L-citrulline to L-arginine and can obtain L-arginine from protein breakdown [468]. However, L-arginine could be locally submitted to competition with endogenously produced analogues such as symmetric dimethyl-L-arginine, asymmetric dimethyl-L-arginine (ADMA) and N(G)-monomethyl-L-arginine (L-NMMA). L-arginine is transported through the plasma membrane via the system Y+, a member of the family of cationic amino acid transporters, and is in competition with other amino acids such as L-lysine as well as with these analogues. Furthermore, ADMA and L-NMMA directly compete at the NOS site and are inhibitors of the enzyme [247]. The levels of ADMA represent a novel independent predictor for all causes of cardiovascular mortality. Free dimethylarginines are the products of proteolytic degradation of arginine-methylated proteins following the preferential action of protein arginine N-methyltransferase type I (PRMT-I). ADMA is mainly metabolized by dimethylarginine dimethylaminohydrolase, the DDAH-2 isoform being preponderant in the endothelial cells. Following oxidative stress or angiotensin II administration, the observed elevation in ADMA levels is associated with an increase in the enzymatic activity of PRMT and a decrease in the activity of DDAH. Silencing the DDAH-2 gene with siRNA impairs endothelium-dependent relaxation and NO production. Therefore, the inhibition of PRMT-1 and the activation and/or the enhanced expression of DDAH-2 could be beneficial in cardiovascular diseases [1179].

Additionally, endothelial cells express arginases, arginase-II being the predominant isoform, which metabolize L-arginine to L-ornithine and urea. Arginase-II competes with eNOS for the substrate and its expression and/or activity are enhanced in cardiovascular diseases, possibly following oxidative stress, a redox-sensitive element having been identified on its promoter. In vascular smooth muscle cells, the arginase-dependent formation of polyamines and L-proline may promote aberrant vessel wall remodeling and neointima formation. In animal models of cardiovascular diseases, arginase-II inhibition or gene deletion improves endothelium-dependent relaxations and vascular NO production, prevents the development of hypertension and decreases the generation of endothelial reactive oxygen species and the formation of atherosclerotic plaque [357,1327]. Arginase-II may therefore represent a promising novel therapeutic target in hypertension that could reverse vascular dysfunction.

4.1.6.2. Proper Expression and Function of eNOS.

A reduced expression of eNOS could be responsible for a decrease in NO production. However, in most situations, where endothelial dysfunction is encountered, the expression of eNOS is paradoxically increased. The increased endothelial levels of reactive oxygen species, especially hydrogen peroxide, which increase the expression of eNOS at the transcriptional and translational level, is likely to explain this paradox. An endothelial dysfunction associated with an increased expression of eNOS shows that the ability to generate NO is reduced and/or that its bioavailability is decreased. The reduction in NO generation can be attributed to the eNOS uncoupling phenomenon where the NOS itself is a source of superoxide anion and a cause of endothelial dysfunction. The decrease in NO bioavailability is associated with an oxidizing environment, i.e., superoxide anion generation from different sources, and the formation of peroxynitrite [1375].

The two independent enzymatic steps required for the eNOS-dependent conversion of L-arginine to NO necessitate the presence of BH₄. Decreased endothelial levels of this essential pteridine cofactor are not only responsible for a reduction in NO production but also for the uncoupling of eNOS. In such an uncoupled state, electrons, which normally flow from the reductase domain of one subunit to the oxygenase domain of the other subunit, are diverted to molecular oxygen rather than to L-arginine, resulting in production of superoxide anion rather than nitric oxide [1602].

BH4 is synthesized de novo from guanosine triphosphate (GTP) by the enzymes GTP-cyclohydrolase I (GTPCH-I), 6-pyruvoyl-tetrahydrobiopterin synthase and sepiapterin reductase (SR). An alternative pathway for BH4 synthesis, the so-called salvage pathway, involves the formation of sepiapterin and its subsequent reduction in dihydrobiopterin (BH2), which is further reduced by dihydrofolate reductase (DHFR) to form BH4. BH4 is very susceptible to oxidation by peroxynitrite, forming BH2 and ultimately biopterin. Following oxidation, BH4 can be regenerated either by DHFR or by dihydropteridine reductase. Increased vascular homocysteine is a potent risk factor for atherosclerosis and endothelial dysfunction, and some of this effect may be mediated by the inhibition of BH4 de novo synthesis [1064].

Direct supplementation with BH4, or with its precursor sepiapterin, improves not only endothelial function in numerous animal models of cardiovascular diseases but also in patients with hypertension, diabetes, hypercholesterolemia or coronary diseases and in smokers. Enhancing the expression or the activity of GTPCH-I, the first and rate-limiting step of de novo BH4 synthesis, or that of DHFR, involved in BH4 regeneration, could prevent the occurrence of endothelial dysfunction. Alternative strategies include folic acid, which enhances the binding-affinity of BH4 to NOS, chemically stabilizes BH4 and, by stimulating DHFR, enhances its regeneration. Preventing peroxynitrite formation and therefore BH4 oxidation or facilitating the recycling of BH4 regeneration with vitamin C may also be considered [1365,1375].

Inhibition of eNOS causes accelerated atherosclerosis in rabbits [202] and mice [794]. When apoE-/- mice are subjected to the additional deletion of eNOS, they also display accelerated atherosclerosis and develop abdominal aortic aneurysm formation and ischemic heart disease [230,853]. These data indicate that endogenous eNOS has an anti-atherosclerotic role. However, apoE-/- mice overexpressing eNOS develop paradoxically larger atherosclerotic lesions than control apoE-/- mice [1172]. This paradox can easily be explained. In a model of vascular oxidative stress, reactive oxygen species from various sources (kindling radicals) and the formation of peroxynitrite would lead to eNOS uncoupling and the subsequent eNOS contribution to superoxide production (bonfire radicals). Therefore, the up-regulation of eNOS itself is a double-edged sword strategy. However, if the up-regulation of eNOS is associated with an increase in the availability of essential cofactors, such as BH4, the eNOS uncoupling can be prevented. Some compounds, such as AVE9488 and AVE3085, which enhance eNOS promoter activity, have demonstrated this type of coordinated activity and validated the concept that enhanced transcriptional expression of eNOS can be beneficial in cardiovascular diseases [1691].

Furthermore, the so-called pleiotropic effects of statins (3-hydroxy-3methylglutaryl-CoA reductase inhibitors), which are independent of their intended lipid-lowering effect, and the activation of SIRT-1 by resveratrol involve an increase in the expression of the transcription factor KLF-2 [573,1392], which is also activated by laminar shear stress [56]. KLF-2 is described as a master regulator of the transcription of anti-inflammatory, anti-thrombotic and vasoactive genes, including eNOS, leading to a vasoprotective phenotype [548]. Additionally, statins and polyphenols as well as other drugs such as fibrates, metformin and peroxisome proliferator-activated receptor-gamma (PPARγ) agonists activate AMP-kinase. This could lead to an increased expression of eNOS and to posttranslational modifications, such as phosphorylations, that enhance protein expression and enzymatic activity [449,856,1695].

4.1.6.3. Increasing the Levels of NO And/Or Its Bioavailability.

Besides preventing eNOS uncoupling (see above), different therapeutic strategies can be designed to restore proper NO levels. These include drugs that stimulate the release of NO from endothelial eNOS, NO donor drugs, antioxidant compounds, drugs that boost the antioxidant defense mechanisms and inhibitors of the enzyme(s) involved in the generation of reactive oxygen species.

4.1.6.3.2. Reduction of Oxidative Stress.

Besides uncoupled eNOS, potential sources of superoxide anion in the vascular wall include NAD(P)H oxidases, xanthine oxidase, cyclooxygenases, lipooxygenases, cytochrome P450 monooxygenases and the mitochondrial respiratory chain. An increased production of reactive oxygen species has been demonstrated in numerous cardiovascular diseases including in patients with essential hypertension, renovascular hypertension, malignant hypertension and preeclampsia. In essential hypertension, an increase in the levels and/or activity of vascular NAD(P)H oxidase has been identified as a predominant source of excess reactive oxygen species [1730] (Figures 11, 12 and 13). Under pathological conditions, dysregulation of reactive oxygen species generation, due to enhanced production and/or reduced antioxidant potential, results in oxidative stress, which leads to vascular dysfunction and remodeling through oxidative damage. Superoxide anion not only decreases NO bioavailability but also the peroxynitrite generated exacerbates vascular injury by producing the oxidation of sulfhydryl groups as well as the nitration and hydroxylation of aromatic groups, including tyrosine, tryptophan and guanine [431,1387].

FIGURE 13

Reactive oxygen species in the vascular wall (A) Generation and metabolism of reactive oxygen species. Superoxide anion (O_2.^-) is the product or byproduct of many cellular enzymatic reactions catalyzed by, e.g., cyclooxygenases (COX), lipooxygenases (LOX), (more...)

Thus, the scavenging of reactive oxygen species with antioxidant or its inactivation with superoxide dismutase mimetics would seem an obvious means of increasing NO bioavailability. However, if such a strategy has been successful in various animal models of hypertension, large clinical trials in hypertensive patients showed that chronic antioxidant therapy did not produce any major reduction in arterial blood pressure and did not improve the associated morbidity and mortality, with the possible exception of the long-term intake of polyphenols that are present in red wine, fruit and vegetables, i.e., the so-called beneficial effect of a Mediterranean diet [1205,1433]. The reasons underlying these failures are still not completely understood.

Direct inhibition of the generation of reactive oxygen species might be a better alternative. Over the recent years, major efforts in the synthesis of NADPH oxidase inhibitors have been made in both academia and pharmaceuticals companies. However, it is not yet clear which isoform(s) of NADPH should be inhibited, and the inhibitors available are still not selective, are not very potent and have poor pharmacokinetics properties [83].

The activation of the renin–angiotensin system and the stimulation of AT1 receptor subtype are major stimulators of NAD(P)H oxidase activation and ROS production, including human hypertension [883,1730]. Preventing the generation of reactive oxygen species, for instance, by deleting a subunit of the NADPH oxidase, induces resistance to angiotensin II-induced hypertension and markedly reduces the associated endothelial generation of superoxide anions [871]. In rat models of hypertension, ACE-I and ARB decrease the generation of superoxide anions [151,1511,1677]. They diminish the amplitude of endothelium-dependent contractions [1295] and restore the amplitude of both NO- and EDHF-mediated endothelium-dependent relaxations [571,1295]. Statins not only increase eNOS expression but also decrease the expression of NADPH subunits, thus improving the balance between NO and reactive oxygen species. These endothelial effects may contribute to the therapeutic effect exerted by these compounds, although no clinical data exist demonstrating that the reversal of endothelial dysfunction is associated with a reduction in cardiovascular events [638,899,907,1450,1488].

4.1.6.4. Soluble Guanylyl Cyclase and cGMP Production.

When NO binds to the heme moiety of soluble guanylyl cyclase, its main physiological target, the activity of the enzyme, which produces the signaling molecule cGMP, is enhanced 400-fold. Thus, regulation of nitric oxide bioactivity is not only achieved by modulation of NOS but also by affecting soluble guanylyl cyclase [398] or the half-life of cGMP, which is readily hydrolyzed by phosphodiesterases [1013].

4.1.6.4.1. Soluble Guanylyl Cyclase.

NO cannot activate the soluble guanylyl cyclase when it is heme-free [951] or when the heme moiety is oxidized (Fe³⁺), following oxidative stress [517,1457]. Pharmacological agents that interact with the soluble guanylyl cyclase and generate cGMP independently of NO have been synthesized. Based on their mechanisms of action, these compounds can be separated into two different classes, the stimulators and the activators of soluble guanylyl cyclase [1361,1456]. The former class of compounds stimulates the enzyme in an NO-independent but heme-dependent manner and, as allosteric modulators, markedly enhance NO-dependent cGMP production [398,1455]. In contrast, activators of soluble guanylyl cyclase target the soluble guanylyl cyclase when oxidized, i.e., when the heme iron is in the oxidized ferric form, or more likely when the enzyme is under the heme-free form. These compounds do not synergize with NO [1312,1363,1454,1457]. Both activators and stimulators of soluble guanylyl cyclase are potent vasodilators and inhibitors of platelet aggregation, where they synergize with the prostacyclin pathway [655,1297,1298,1454,1455,1457]. The former are currently undergoing clinical trials in acute decompensated heart failure and peripheral artery occlusive diseases, while the latter are being evaluated in pulmonary hypertension. However, the potential therapeutic indications of these new compounds include hypertension, myocardial ischemia, erectile dysfunction, atherosclerosis or thrombosis [1361,1456] (Figure 14).

FIGURE 14

Effects of stimulators and activators of soluble guanylyl cyclase. Stimulators of soluble guanylyl cyclase (sGC) stimulate the enzyme in an NO-independent but heme-dependent manner and, as allosteric modulators, markedly enhance NO-dependent cGMP production (more...)

4.1.7.1. H₂O₂ as a Vasorelaxing Agent.

H₂O₂ can induce relaxations in a number of blood vessels including human coronary and mesenteric arteries [65,98,479,624,701,997–999,1351,1545,1663,1756]. Additionally, endogenously produced H₂O₂ contributes to flow-induced relaxation and to the endothelium-dependent relaxation evoked, for instance, by acetylcholine or the endothelium-independent relaxation evoked by cromakalim [621,716,811,935,1055].

In most blood vessels, which relax in response to exogenously added H₂O₂, the relaxation is endothelium-independent. However, in some arteries, the relaxing effect of H₂O₂ can be partially endothelium-dependent, such as in the canine and porcine coronary artery. In the latter coronary artery, the endothelium-dependent relaxation elicited by H₂O₂ involves the COX-1-dependent release of PGE₂ [1317,1545]. The vasodilator effects of H₂O₂ are totally endothelium- and NO-dependent in the canine basilar artery [1756], in the rabbit and in the rat aorta [103,1757].

The endothelium-independent relaxations of arterial myocytes to H₂O₂ again involve multiple pathways and include activation of soluble guanylyl cyclase [479,481,624,693,1351] and the metabolism of arachidonic acid either through cyclooxygenases and/or lipoxygenases [64,479,622,701]. H₂O₂ also produces hyperpolarization of the vascular smooth muscle cells by activating BK_Ca [624,999,1545], K_ATP [1663], K_V [1299] and/or K_IR channels [701]. The activation of smooth muscle K_V by H₂O₂ involves redox-sensitive elements, thiol groups, on the channel itself or on associated regulatory proteins [1299]. The activation of BK_Ca by H₂O₂ is evoked by a direct action on the channel as well as by a soluble guanylyl cyclase-dependent effect [624]. In the isolated murine mesenteric artery, H₂O₂ produces an endothelium-independent relaxation providing that K_Ca channels are operational but, at the same concentrations, elicits a potent contractile response if the activity of these channels is compromised [952].

H₂O₂ also modulates the ionic channel activity of the endothelial cells which, in turn, can influence the membrane potential and thus the contractile activity of the underlying smooth muscle cells. In cultured endothelial cells of the human umbilical vein, H₂O₂ elicits both depolarization and hyperpolarization. In the micromolar range, it inhibits K_IR, while at 1 mM, it activates BK_Ca [179]. The activation of BK_Ca is due to an increase in [Ca²⁺]_i since, in concentrations higher than 100 μM, H₂O₂ activates a calcium-permeable, non-selective cation current in these cells [743]. In human aortic endothelial cells, H₂O₂ causes the release of Ca²⁺ from the endoplasmic reticulum [678].

4.1.7.2. H₂O₂ as an Endothelium-Derived Relaxing/Hyperpolarizing Agent.

Most of the evidence in favor of H₂O₂ being an endothelium-derived relaxing/hyperpolarizing factor comes from the observation that, in certain blood vessels, including human coronary arteries, the agonist- and flow-induced dilatations, studied in the presence of inhibitors of NO-synthases and cyclooxygenases, are partially or totally sensitive to catalase, the enzyme that dismutates H₂O₂ into water and oxygen. In these arteries, exogenously added H₂O₂ appears to mimic these responses, which rely on K_Ca activation [935,997–999,1055,1076,1733]. Furthermore, in murine mesenteric [999,1076] and porcine coronary arteries [998], the agonist-induced relaxations/hyperpolarizations are associated with a catalase-sensitive endothelial production of H₂O₂. Similarly, in human coronary arterioles [935,1055], flow-induced dilatation increases the production of H₂O₂ from the endothelial cell layer. In addition, without actually being released by the endothelial cells, H₂O₂ per se or substances that promote its formation can enhance endothelial responses by potentiating calcium release from endothelial stores [371,514]. In various experimental models or in arteries from patients with cardiovascular disease, H₂O₂ generation can partially compensate the decreased NO production, at least in terms of endothelium-dependent relaxations [278,279,1222].

In eNOS-knockout murine arteries, the relaxations resistant to inhibitors of NO-synthases and cyclooxygenases are sensitive to catalase and have been attributed to the NOS-3/Cu,Zn-SOD-dependent formation of H₂O₂ [999,1076]. The maintenance of these endothelium-dependent relaxations/hyperpolarizations in NOS-3 knockout mice was explained by the compensatory endothelial expression of other NOS genes. Indeed, these responses are progressively reduced as the number of the disrupted NOS genes increases (single eNOS, double nNOS/eNOS and triple nNOS/iNOS/eNOS knockouts). In parallel, the production of H₂O₂ is preserved up to the total disruption of the three NOS genes [192,1504]. Nevertheless, whether or not the decrease in the EDHF-mediated responses is directly associated with the disruption of the NOS genes or is independently associated with the severe phenotype of these mice, especially with the one observed in the triple knockout mice (hypertension, dyslipidemia, myocardial infarction and nephrogenic diabetes insipidus) remains to be fully assessed.

In these murine arteries, the NOS appear to be the sole source of H₂O₂ without the involvement of other oxidases [1504]. However, in human coronary arterioles, flow-induced endothelium-dependent dilatation is associated with H₂O₂ generated by the mitochondrial respiratory chain, while bradykinin-induced endothelium-dependent relaxation requires NADPH oxidase-derived H₂O₂ [879,938] (Figure 15).

FIGURE 15

Multiple putative pathways by which H₂O₂ can act as an endothelium-derived hyperpolarizing agent, relationship with NO. The forces exerted by the flowing blood (shear stress) and/or receptor stimulation can activate many enzymes capable of generating (more...)

Not all available data support the hypothesis that H₂O₂ is involved in endothelium-dependent responses and in particular in EDHF-mediated responses. Indeed, catalase does not inhibit such responses in the porcine, canine and rat coronary [98,491,1235,1510], human radial and mesenteric artery [205,607], murine mesenteric [388], guinea-pig carotid [552] and in rabbit ileo-femoral and mesenteric [218,716] arteries or in the mouse aorta [388]. Furthermore, H₂O₂ does not always hyperpolarize vascular smooth muscle cells [552], and its relaxing effect is not necessarily associated with hyperpolarization of the vascular smooth muscle cells and/or the opening of K⁺ channels [218].

4.1.7.3. H₂O₂ as a Vasoconstricting Agent.

In various arteries from different species, H₂O₂ also directly induces contractions [45,621,788,1296,1400,1449,1758]. In some other blood vessels, such as the rabbit aorta or the rat mesenteric artery, low concentrations of H₂O₂ (<100 μM) evoke contractions, while higher concentrations (>0.3mM) produce transient relaxations followed by sustained contractions [103,503].

Additionally, the vasoconstricting (and proliferative) actions induced by receptor stimulation, for instance, α1-adrenergic, TP and AT₁ receptors, or mechanical stimulation, for instance, stretch, involves the generation of H₂O₂ [597,1130,1142,1558,1784]. NAD(P)H is a frequent but not the exclusive source of the ROS involved in these contractile effects. In vivo in mice, the administration of either norepinephrine or angiotensin II produces a vasopressor response, which is also associated with the production of H₂O₂. In genetically modified mice overexpressing catalase, the basal arterial pressure and the basal level of H₂O₂ are similar to those of wild-type animals. However, the pressor response induced by the administration of either norepinephrine or angiotensin II is decreased in the transgenic animals and this is associated with a suppression of H₂O₂ production, indicating that endogenously produced H₂O₂ contributes to the vasopressor responses evoked by these cardiovascular hormones [1750]. Furthermore, mice with a specific endothelial overexpression of catalase show a strongly reduced steady-state concentration of vascular H₂O₂ and are hypotensive. This phenomenon is not dependent on the bioavailability of endothelial NO but is completely reversible by treatment with a catalase inhibitor, suggesting that endothelial-derived H₂O₂ exerts a tonic contractile effect on vascular smooth muscle cells [1478]. In agreement with these observations, H₂O₂ has been identified as a putative EDCF in rat arteries [505].

Numerous mechanisms can explain the contractile responses produced by endogenous and exogenous H₂O₂ and include activation of P2 purinergic receptors [1400], production of COX-derivatives, possibly thromboxane A₂ and the activation of TP receptors [504,509,1744], stimulation of ERK-MAP kinase [502,1540] and PKC [597], inhibition of potassium channels, for instance, K_V [263,1038] and BK_Ca by altering cysteine-mediated calcium sensing [1518], activation of chloride channels [578] and interplay with calcium signaling at multiple levels [1564].

In addition, H₂O₂ can impair endothelium-dependent vasodilatations. Thus, H₂O₂ promotes proline-rich tyrosine kinase-2 (Pyk2)-dependent phosphorylation of eNOS Tyr657, preventing NO generation [948], and directly inhibits cytochrome P450 epoxygenase and the production of vasodilatory EETs [881]. Furthermore, in endothelial cells of the porcine renal artery, patched in situ, H₂O₂ inhibits the NO-dependent activation of BK_Ca and the associated endothelial hyperpolarization [138]. Finally, H₂O₂ reduces the activity of IK_Ca [183] and may therefore prevent EDHF-mediated responses.

These apparently controversial results are still difficult to explain. Nevertheless, in different arteries, endothelium-derived H₂O₂ can evoke the hyperpolarization and/or the relaxation of smooth muscle cells and can compensate a decrease in NO bioavailability [1412]. Additionally, this peroxide appears to play an important role in flow-induced vasodilatation [935]. In vivo, this reactive oxygen species could play an important role in coronary autoregulation [1733] and a cardioprotective role during ischemia–reperfusion injury [1732].

4.2.1.1. The HO–CO Pathway.

The predominant biological source of CO is from the degradation of heme (hemoglobin, myoglobin, cytochrome P450, etc.) by heme-oxygenase (HO), which generates equimolar quantities of biliverdin, free ferrous iron and carbon monoxide. Biliverdin is rapidly converted to bilirubin, while free iron is promptly sequestered by ferritin [355]. These bile pigments are not simple by-products of heme catabolism, they have antioxidant properties which protect the vascular cells from oxidative stress. Three isoforms of heme oxygenase have been described: HO-1 (or heat shock protein 32, inducible isoform), HO-2 (constitutive isoform) and HO-3, which is closely related to HO-2 but is nearly devoid of catalytic activity [238,1643]. HO-1 is ubiquitously expressed in response to a variety of stimuli including its own substrate, heme, and several oxidants. HO-2 is also ubiquitously expressed with notably high levels in brain and testes. Both HO-1 and HO-2 are expressed in vascular smooth muscle and endothelial cells [355,1644].

In many pathophysiological situations, the HO-CO pathway compensates for the decrease in NO bioavailability [1709]. CO is a potent vasodilator in most, but not all, vascular beds. The biological activity of CO is generally associated with the modulation of heme-dependent proteins and although it has been postulated that CO could also bind to targets that lack a heme moiety, direct evidence for these effects is still missing [1083,1709]. The mechanisms of CO-induced vasodilatation can involve the stimulation of soluble guanylate cyclase [1647], the inhibition of cytochrome P450 [1300] and/or the activation of various populations of K⁺ channels [1709].

4.2.1.2. CO as a Vasodilator.

The involvement of guanylyl cyclase activation in CO-induced vascular relaxation was recognized early. However, this gaseous mediator is many times less potent than NO and is able to activate the purified guanylyl cyclase only in the high micromolar range [495,765]. Nevertheless, in canine cerebral arteries, cyclic-GMP-dependent activation of BK_Ca has been reported [840]. Additionally, CO can activate BK_Ca in a cGMP-independent manner. The inhibition of cytochrome P450 by CO suppresses the synthesis of 20-HETE and lifts the inhibition of BK_Ca produced by this cytochrome P450 derivative [262,1300]. Furthermore, CO can directly increase the open probability of BK_Ca [1648,1649] (Figure 16).

FIGURE 16

Carbon monoxide (CO) as an endogenous vasodilator. In response to stimuli, such as oxidant stress, carbon monoxide is produced by heme oxygenase (HO) in both endothelial and vascular smooth muscle cells. Endothelium-derived carbon monoxide can diffuse (more...)

The direct action of CO on BK_Ca differs from that of NO on this population of potassium channels [1708]. CO increases the apparent calcium sensitivity of the α subunit, enhancing the probability of eliciting STOCs in response to calcium sparks [730,1721]. The molecular mechanism could involve the heme group that binds with high affinity to the α subunit of BK_Ca and inhibits its activity [730,1721]. Indeed, BK_Ca has recently been recognized as a hemoprotein since its activity is regulated by free heme [672,731,1519,1682]. CO can activate BK_Ca channels by binding to the channel-bound heme and altering the interaction between the heme and the heme-binding domain. Furthermore, HO and BK_Ca channels are colocalized in the membrane [1682]. HO activation of BK_Ca may not only be associated with the production of CO but also with the metabolism of the membrane-associated heme, the very substrate required for the generation of CO. Thus, it is conceivable that compartmentalization of the CO generator (HO), CO receptor (heme) and its downstream target (BK_Ca channel) may regulate cellular excitability through more than one local signaling pathway [731]. Additionally, CO and CO-donors can activate smooth muscle K_ATP [467] and/or K_V [63].

4.2.1.3. Cardiovascular Effects of Endogenous CO Production.

Endogenously produced CO attenuates the sensitivity to vasconstrictors and to pressure-induced vasoconstriction in various vascular beds [764,894,1647,1793].

Angiogenic factors, such as VEGF, mediate some of their effects through the induction of HO-1 [350]. Nevertheless, HO-1 knockout mice are viable and normotensive but have an abnormal postnatal lung development. When subjected to stress or injury, these genetically deficient animals show exacerbated cardiovascular responses [238,1812]. For instance, HO-1 gene disruption accelerates the formation of arterial thrombosis and enhances oxidative damage to the endothelial cell [1568]. Induction of HO-1 protects against atherosclerotic disease in part by promoting reendothelialization. In HO-1-deficient mice, the generation of endothelial progenitor cells from bone marrow is significantly impaired [1704]. In contrast, overexpression of HO-1 plays a protective role in hypoperfusion and ischemia–reperfusion injury [238,1812]. The pharmacological induction of HO-1 expression by hemin or cobalt protoporphyrin administration produces long-lasting normalization of arterial blood pressure in SHR and reduces the elevation of blood pressure in angiotensinII hypertensive rats, respectively [1458,1646]. However, in the latter model, the lowering of arterial blood pressure was not associated with an improvement in endothelial function [1458].

HO-2 knockout mice are also normotensive but their endothelial cells show an activated phenotype characterized by increases in inflammatory, oxidative and angiogenic factors. In these animals, stroke damage in response to injuries is accentuated, indicating that HO-2 plays an endogenous neuroprotective role in the brain [91,341]. In vivo, during myocardial ischemia, HO-2 activation can evoke a cGMP-mediated vasodilation, but only as a back-up mechanism, which is activated when the other pathways of vasodilation are exhausted [1128].

An endothelial production of CO, contributing to endothelium-dependent relaxations in response to neurohumoral substances, has been demonstrated only in a limited number of arteries [62,1785]. As HO-1 is induced in response to a variety of stresses and especially oxidant stress, the endothelial and even more so the smooth muscle cell release of CO, since the latter is a more important source of CO production in the vascular wall [1644], could contribute to the regulation of vascular tone under pathological conditions such as atherosclerosis [713].

4.2.1.4. CO Contribution to Endothelial Dysfunction.

However, CO also contributes to endothelial dysfunction. Indeed, this gas, by binding to its prosthetic heme, acts as a tonic inhibitor of eNOS and, in resistance arteries, can produce vasoconstriction and impair flow-induced dilatation [356,750,751]. Furthermore, CO induces contractions by stimulating the production of ROS, generated by various enzymes (including NO-synthase, NADPH oxidase, xanthine oxidase and complex IV of the mitochondrial chain), and the subsequent activation of the TP receptors [870]. In transgenic mice overexpressing HO-1, specifically in vascular smooth muscle cells, the systemic arterial blood pressure is elevated when compared to the wild-type controls [1467].

Nevertheless, carbon monoxide releasing molecules have vasodilator, anti-ischemic and anti-inflammatory effects and may present some therapeutic value in cardiovascular diseases [1082,1580].

4.2.2.1. The L-Cysteine–H₂S Pathways.

Hydrogen sulfide (H₂S), like NO and CO, is a toxic gas. It is colorless with a strong smell of rotten eggs and is produced not only by bacteria but also by mammalian cells, including vascular cells [674,1644,1803]. Two main enzymes are predominantly responsible for the production of H₂S, cystathionine β-synthase and cystathionine γ-lyase, and both use L-cysteine as substrate. A third pathway, recently identified, involves the production of H₂S in the mitochondria, still from L-cysteine, via the combined action of 3-mercaptopyruvate sulfurtransferase and cysteine aminotransferase. In the cardiovascular system, the production of H₂S is generally associated with the activation of cystathionine γ-lyase, while cystathionine β-synthase is the predominant enzyme involved in the nervous system. The major sources of H₂S in the blood are likely to be the vascular smooth muscle and the red blood cells [1482,1644,1652]. However, in the endothelial cells, both the cystathionine γ-lyase and the 3-mercaptopyruvate sulfurtransferase–cysteine aminotransferase pathways are expressed and produce H₂S [1407,1749] (Figure 17).

FIGURE 17

H₂S and SO₂ biosynthetic pathways and vasodilator action. In the cardiovascular system, the production of H₂S is generally associated with the activation of cystathionine γ-lyase, while cystathionine β-synthase is the predominant enzyme (more...)

4.2.2.2. H₂S as a Vasoactive Mediator.

The predominant effect of H₂S is to produce vascular smooth muscle hyperpolarization and relaxation. H₂S activates K_ATP channels by causing the S-sulfhydration of specific cystein residues of the SUR subunit protein of the K_ATP channel complex [1645,1803]. Additionally, H₂S could stimulate the metabolism of arachidonic acid by activating phospholipase A₂ [306] and acts as a non-specific inhibitor of phosphodiesterases, including PDE5, which enhance the smooth muscle cell levels of cAMP and cGMP. This latter effect could again be attributed to cystein sulfhydration or to the binding of this compound to the coordinated zinc atom, which is essential for the enzymatic activity of phosphodiesterase. Furthermore, H₂S, in a cGMP-dependent or -independent manner, could activate other populations of potassium channels such as BK_Ca and K_V [27,222,1436,1823]. An interesting property of H₂S is its ability to act synergically with NO. Indeed, low concentrations of H₂S greatly enhance the relaxation induced by NO [674]. Finally, in some blood vessels, H₂S is an endothelium-dependent relaxing agent, possibly in part because it increases endothelial [Ca²⁺]_i, by modulating calcium entry and calcium handling [75,306,1436].

Conversely, H₂S can also be a vasoconstricting substance. This gaseous mediator can reduce NO bioavailability by forming with NO a nitrosothiol compound [25,1675], inhibit eNOS by preventing its interaction with BH₄ [848,849], and produce a general down-regulation of the L-arginine-NO pathway not only by decreasing eNOS activity but also by reducing eNOS transcript abundance and L-arginine transport [536]. Additionally, H₂S generates vasoconstricting prostaglandins [306] and impairs the cAMP-dependent relaxing pathway in vascular smooth muscle cells [921].

4.2.2.3. Cardiovascular Effects of H₂S.

H₂S is not only a vasodilator but also has anti-inflammatory and anti-oxidant properties, produces inhibition of vascular smooth muscle cell proliferation, has pro-angiogenic effects, depresses cardiac function and decreases arterial blood pressure [1482,1483,1644,1652]. Furthermore, microvascular endothelial cells and hepatocytes can release cystathionine γ-lyase and cystathionine β-synthase in the circulation. These transsulfuration enzymes generate H₂S from homocysteine and protect the endothelium from oxidative stress [81].

Mice with deletion of the cystathionine γ-lyase are hypertensive and, in the mesenteric artery, the endothelium-dependent relaxations are virtually abolished [1749]. In this artery, H₂S is produced and released by the endothelial cells in a calcium-dependent manner following neurohumoral stimulation, and evokes relaxation and hyperpolarization of vascular smooth muscle cells by activating K_ATP channels [1749]. Thus, these results suggest that H₂S is an endothelium-derived relaxing and hyperpolarizing factor. However, the precise role of this mediator needs to be further substantiated. In most studies, the endothelium-dependent relaxations of the murine mesenteric artery involve NO release and EDHF-mediated responses, which are not necessarily associated with the activation of K_ATP channel [430]. The disappearance of both the NO- and EDHF-mediated component of the endothelium-dependent relaxation in cystathionine γ-lyase knockout mice is unexplained at present but could be attributed to the increase in homocysteine levels [313,1244], and/or to the disappearance of the facilitating role of H₂S on NO-mediated relaxations [674].

H₂S donors are currently being synthesized and have therapeutic potential in cardiovascular diseases associated with inflammatory processes such as reperfusion injury, circulatory shock, atherosclerosis, diabetes, pulmonary hypertension, ischemia–reperfusion injury and possibly hypertension [1652].

4.3.2.1. Prostaglandin D₂ and DP Receptors.

PGD₂ is a major prostaglandin in the central nervous system and in immune cells. It can also be synthesized in the vascular wall as both types of PGD-synthases are expressed in endothelial cells, where they are up-regulated in response to an increase in fluid shear stress [1485]. However, PGD₂ is mainly involved in the regulation of sleep and in allergic responses [1444].

Two distinct types of PGD-synthases have been identified, the lipophilic ligand-carrier protein (lipocalin)-type enzyme and the hematopoietic enzyme. PGD₂ is dehydrated to produce the J series of prostaglandin (PGJ₂ analogues). PGJ₂ acts as a ligand for the peroxisome proliferator-activated receptor-gamma (PPARγ), a transcription factor of the nuclear hormone receptor superfamily, which plays a predominant role in the differentiation of adipocytes [1578]. PPARγ is also expressed in endothelial and smooth muscle cells [722,886] and is involved in the regulation of vascular function and blood pressure, in addition to its well-recognized role in metabolism. Thiazolidinediones, PPARγ agonists, prevent proliferation and migration of smooth muscle and inhibit the interaction between leukocytes and endothelial cells [1629]. They lower blood pressure and have protective vascular effects. In contrast, loss-of-function dominant-negative mutations in human PPARγ cause insulin resistance and severe early onset hypertension [803].

Two PGD₂ receptors, DP1 and DP2, have been identified, the latter being also known as CRTH2, a member of the chemokine receptor family [649]. Activation of DP1 increases blood flow and vascular permeability [1444]. For instance, flushing, an unwanted secondary effect of some drugs, such as nicotinic acid, involves in part the COX-1-dependent PGD₂ production by Langherans cells and the subsequent activation of skin microvascular DP1 receptors [354]. PGD₂ can produce not only endothelium- and NO-dependent relaxations [3] but also direct smooth muscle contractions, the latter effect involving activation of TP receptors [553]. PGD₂ is also produced by COX-1 in platelets, where it can elevate cAMP and inhibit their aggregation [382].

4.3.2.2. Prostaglandin E₂ and EP Receptors.

Prostaglandin E₂ (PGE₂), the most abundant prostaglandin in the human body, is involved in multiple physiological effects including gastric secretion and motility, bone formation, pain, inflammation, female reproductive function, body temperature, sleep–wake activity, and the regulation of kidney functions and blood pressure (glomerular filtration rate, tubular salt and water transport, renin release, renal blood flow and vascular tone). The diverse effects of PGE₂ (for instance, this prostaglandin can produce both relaxation and contraction of vascular smooth muscle) can be attributed to the existence of four receptor subtypes, namely, EP1, EP2, EP3 (of which three splice variants exist in the mouse and at least eight in the human) and EP4, which are coupled to different signaling pathways [620,895,1190,1470]. In microvascular endothelial cells, PGE₂ is the predominant COX product [539].

Three distinct PGE-synthases, responsible for the synthesis of PGE₂, have been identified and characterized, one cytosolic, cPGES, and two membrane-bound, mPGES-1 and mPGES-2 [851]. cPGES is a constitutive enzyme expressed ubiquitously and in abundance in many tissues and cell types. This cytosolic form is preferentially associated with COX-1, suggesting that this isoform contributes physiologically to the production of PGE₂ [1091,1190], and its function is thought to overlap that of COX-1. Knockout mice studies demonstrate that the deletion of cPGES is lethal [609].

mPGES-1 is localized in perinuclear membranes and shows significant homology with the superfamily of membrane-associated proteins involved in eicosanoid and glutathione metabolism (MAPEG), which includes 5-lipoxygenase-activating protein (FLAP) and leukotriene C4 synthase [734]. In the vascular wall, both the endothelium and the vascular smooth muscle can express this isoform. mPGES-1 is up-regulated in response to stimuli that induce COX-2 expression and is associated preferentially with COX-2 [1091]. Cells from mice deficient in mPGES-1 display impaired LPS-stimulated PGE₂ generation but the basal formation, which is cPGES-dependent, is preserved [609]. The deletion of the mPGES-1 gene does not affect arterial blood pressure or thrombogenesis but delays atherogenesis and prevents angiotensin-II-induced oxidative stress and aortic aneurysm formation [243,1639,1640]. Conversely, mPGES-1-deficient mice show adverse left ventricular remodeling after myocardial infarction [299] and can, depending on the model, develop severe hypertension when subjected to a high-salt diet [472,744].

mPGES-2 is a constitutive, Golgi membrane-associated protein that can be coupled to both COX-1 and COX-2 [851]. The real significance of its contribution in PGE₂ production under physiological or pathophysiological conditions is questionable since knockout of this enzyme does not result in a specific phenotype or a reduction in PGE₂ generation [609].

Although PGE₂ is the common ligand of the four receptor subtypes, the amino acid homology among these receptors is limited. The stimulation of the EP1 receptor is predominantly coupled with an increase in intracellular calcium concentration (calcium channel gating and phospholipase C-dependent IP₃ formation), that of EP2 and EP4 with an increase in cAMP (activation of adenylyl cyclase) and that of EP3 with a decrease in cAMP (inhibition of adenylyl cyclase) [895,1470]. In the rat aorta, the mRNAs of the four receptor subtypes are expressed and in various rat arteries EP1 activation is associated with contraction [117,1512]. In addition, PGE₂ can also produce contraction by activating TP receptors [553]. In contrast, the activation of EP2 and EP4 receptors produces vasodilatation, a phenomenon which also contributes to skin flushing, an unwanted side-effect of the drugs briefly described above [608]. PGE₂ activates platelets via EP3 and possibly EP1 receptor stimulation, but at elevated concentrations can inhibit their aggregation by stimulating the IP receptor [399].

The deletion of the various EP receptor genes shows that in the kidney, PGE₂ stimulates renin release via the stimulation of EP2 and EP4 receptors but that the four subtypes are involved in the control of renal vascular tone, with EP1 and EP3 increasing and EP2 and EP4 decreasing it [822,1378]. EP1-deficient mice have a urine concentration defect due to a decrease in vasopressin release resulting in hypotension. EP1 receptor deletion reduces the elevated blood pressure and cardiac hypertrophy induced by angiotensin-II [594,1444]. Paradoxically in EP1 knockout males, but not in females, the depressor response to exogenous administration of PGE₂ is reduced. However, in both genders, EP2 receptors contribute to the decrease in blood pressure produced by PGE₂ [52] and EP2 knockout mice develop salt-sensitive hypertension [965]. EP3 receptor-deficient mice have a bleeding tendency and are resistant to thromboembolism [1780]. In male mice, EP3 receptor activation opposes the vasodepressor response to PGE₂, while in female mice, EP4 receptors also contribute to the depressor action of PGE₂ [52]. Endothelium- and NO-dependent vasodilatation to PGE₂ involves EP4 activation [677]. The germline disruption of the EP4 gene delays the closure of the ductus arteriosus [1119] and enhances infarct size in the heart following ischemia–reperfusion [1726] and specific deletion of EP4 in cardiomyocytes exacerbates the decline in cardiac function observed after myocardial infarction [1444]. Additionally, EP4 receptor activation has been associated with atherosclerotic plaque progression and destabilization [254,1507].

4.3.2.3. Prostaglandin F_2α and FP Receptors.

Prostaglandin F₂ isoforms are synthesized not only from PGH₂, by the membrane-associated 9,11-endoperoxide reductase but also from PGD₂ and PGE₂ by cytosolic PGD₂ 11-ketoreductase and PGE₂ 9-ketoreductase (or in the endometrium by the 20α-hydroxysteroid dehydrogenase), respectively [632,1660].

Prostaglandin F_2α interacts with its preferential receptor, the FP receptor, which generally generates an increase in the intracellular concentration of calcium. Prostaglandin F_2α is required in the female reproductive system for normal parturition. In the kidney, this prostaglandin is involved in water absorption, causing natriuresis and diuresis and, in the eye, is involved in the regulation of intraocular pressure. Prostaglandin F_2α is produced in the vascular wall, including by the endothelial cells. It is also a potent vasoconstrictor as it stimulates not only its preferential FP receptor but also TP receptors and could be involved in the development of cardiac hypertrophy [433,553,620,755,1444,1698]. Additionally, FP receptor can be expressed in endothelial cells and induce endothelium and NO-dependent relaxations when stimulated [229]. Deletion of FP receptors reduces arterial blood pressure and delays atherogenesis in hyperlipidemic mice [1779].

4.3.2.4. Prostaglandin I₂ and IP Receptors.

Prostacyclin is an unstable substance formed by prostacyclin synthase, a member of the cytochrome P450 superfamily (in the human CYP8A1) of enzymes. In endothelial cells, prostacyclin synthase is highly expressed [1515] and the enzyme has been reported to be closely associated with COX-1 [796] or COX-2 [1136]. Prostacyclin synthase is also expressed in vascular smooth muscle cells, heart, lung, kidneys, gastric mucosa and intestinal epithelial cells, brain, macrophages, oviducts and embryonic cells [382,1707].

Prostacyclin is a potent platelet inhibitor, preventing their aggregation and their adhesion to the endothelial cell surface and an endothelium-derived vasodilator [1068,1069,1071,1269,1270]. However, this prostaglandin has numerous other physiological functions. Prostacyclin is involved in overall renal function by regulating renal blood flow, glomerular filtration rate and renin release. In addition, prostacyclin is involved in the development, transport and implantation of the embryo, in pain tolerance, in gastric acid secretion and in intracellular signaling by interacting with nuclear receptors and regulating gene transcription [1443,1444,1688,1707] (Figures 19 and 20).

FIGURE 19

Multiple functions of the prostacyclin IP receptor. The IP receptor belongs to the G-protein-coupled seven transmembrane domain family [1461].

FIGURE 20

Prostacyclin as a vasorelaxing and an anti-aggregating agent. Upper panel: In rat, isolated aortic rings contracted with phenylephrine, prostacyclin produces a concentration-dependent relaxation, which is potentiated in the presence of the specific TP (more...)

The deletion of prostacyclin synthase generates hypertensive mice with thickening and sclerosis of the arterial wall and with kidney infarction associated with interstitial fibrosis and nephrosclerosis [1443,1707]. Conversely, overexpression of prostacyclin synthase, alone or in association with COX-1, prevents injury-induced intimal hyperplasia, pulmonary hypertension and vascular remodeling and protects brain tissue from ischemia–reperfusion injury [1707]. Prostacyclin synthase is among the most sensitive targets of peroxynitrite and is inactivated by concentrations as low as 50 nM [1364,1818,1819]. When PGI₂ synthase is inactivated, the excess PGH₂ is shunted toward other metabolic pathways leading to a variety of products that are, in general, deleterious to vascular function [433,553].

Prostacyclin is the preferential ligand of IP receptors (Figures 19 and 20). Although several types of G-proteins are likely to be coupled to the IP receptor, in platelets and to a lesser extent in vascular smooth muscle cells, the Gs-adenylyl-cyclase-cyclic adenosine monophosphate (cAMP)-PKA pathway is a preponderant signaling system [255,1443,1688,1689]. The genetic deletion of IP receptors is associated with increased injury-induced restenosis [242], thrombotic events [1093], atherosclerosis [383,823] and reperfusion injury [1725]. Some of the negative effects observed with IP receptor deletion, including restenosis and enhanced platelet activation, can be abrogated by coincidental TP receptor deletion, indicating that prostacyclin regulates the cardiovascular effects of thromboxane A2, and vice-versa [440] (Figure 20). A cross-talk between the two prostanoid-binding sites may involve the heterodimerization of these two receptors. TP receptor agonists stimulating these heterodimers would produce accumulation of cAMP. This and regulation of receptor endocytosis and trafficking would explain some of the braking effects of IP receptors on the cellular effects of TP receptor activation [1685,1686]. In humans, a defect in prostacyclin receptor signaling, as observed in patients with a dysfunctional IP receptor mutation, leads to accelerated atherothrombosis [46]. The accelerated cardiovascular disease associated with this IP^R212C mutation is observed in individuals carrying only one copy of the variant allele and can thus be attributed to a dominant negative action not only when dimerized with the wild-type IP receptor but also through dimerization with the TP receptor since the activation of such dimers can no longer lead to cAMP production [692].

4.3.2.4.1. Prostacyclin and Vascular Function.

Since inhibitors of cyclooxygenases abolish the basal and stimulated generation of PGI₂, and potent and specific antagonists of the IP receptor block the vasodilator responses that it elicits [553,562], the contribution of PGI₂ in endothelium-dependent responses can be assessed. This prostaglandin plays a role in flow-mediated vasodilatation [348,834], but its contribution to acute endothelium-dependent relaxations in response to neurohumoral mediators is often considered as minimal because cyclooxygenase inhibitors, in particular indomethacin, do not affect these responses. However, the role of prostacyclin as an endogenous mediator contributing to endothelium-derived relaxation has generally been overlooked since in some vascular beds, a major vasodilator effect of COX derivatives can only be observed when the other pathways leading to endothelium-dependent responses have been inhibited [272,1830]. The contribution of PGI₂ to endothelium-dependent responses is increased in eNOS knockout mice [211,1473]. In coronary arteries from insulin-resistant obese Zucker rats or in the mesenteric vascular bed of mice with streptozotocin-induced diabetes, endothelial dysfunction is prevented by a complementary up-regulation of COX-2 expression and activity [1094,1336]. Similarly, in humans with diabetes and cardiovascular diseases, COX-2-derived prostacyclin can play a compensatory role for the decreased NO bioavailability [157,1025,1484]. This increased contribution of COX-2-derived prostacyclin when NO bioavailability is reduced may explain some of the detrimental cardiovascular effects associated with COX-2 inhibitors [36].

The relaxation to PGI₂, or its synthetic analogues, is often associated with the concomitant hyperpolarization of the smooth muscle cells and can involve the opening of K_ATP [274,1194], K_V [914], K_IR [1165], BK_Ca [256,1370] and/or two-pore domain (K_2P) K⁺ channel family [1156]. The activation of K⁺ channels following IP receptor stimulation relies on cAMP-dependent and -independent pathways. The latter pathway could be associated with the direct activation of the K⁺ channel by Gs protein [1735]. The contribution of the hyperpolarizing mechanism in the relaxation process can be very significant in some tissues [724,1371] (Figure 21).

FIGURE 21

Prostacyclin as a hyperpolarizing and a vasoconstricting factor. Upper panel: In the guinea pig isolated carotid artery, prostacyclin (PGI₂) and its stable analogue, iloprost, produce vascular smooth muscle hyperpolarization. The K_ATP blocker, glibenclamide (more...)

Therefore, in numerous vascular beds, PGI₂ can act not only as an endothelium-derived relaxing factor but also as an endothelium-derived hyperpolarizing substance [430,1194]. Additionally, prostacyclin is also a potent anti-proliferative agent in vascular smooth muscle cells, and it reduces oxidant stress and prevents cellular adhesion to the vascular wall [382].

However, prostacyclin can also evoke depolarization, e.g., in the guinea-pig carotid artery [274], and contractile responses in various vascular beds including human coronary and umbilical arteries [127,297,904,963,1234,1683]. These depolarizations and/or contractions could be attributed in some instances to the release of endothelium-derived contracting factors [9] but are generally associated with the direct activation of thromboxane receptors by elevated concentrations of prostacyclin, showing that prostacyclin can contribute to endothelium-dependent contractions [274,419,433,1683,1804] (Figure 21). This latter phenomenon will be developed in a subsequent chapter.

Specific and potent agonists and antagonists of the IP receptors have been synthesized. IP receptor antagonists could be of interest for the treatment of pain, inflammation and overactive bladder, providing that cardiovascular side effects can be avoided [755]. IP receptor agonists are prescribed for pulmonary hypertension but are currently administered intravenously or by inhalation and are associated with serious side effects. The recent synthesis of orally active and long-acting IP receptor agonists may prove beneficial not only in pulmonary hypertension but possibly also in atherosclerosis obliterans [440,863].

4.3.2.5. Thromboxane A₂ and TP Receptors.

The existence of an intermediate compound in the conversion of prostaglandin G₂ into thromboxane B₂ was first demonstrated in platelets [606]. This unstable compound, termed thromboxane A₂, has a short half-life (approximately 30 seconds at 37°C and pH 7.4). In the cardiovascular system, thromboxane A₂ is not only predominantly derived from platelet COX-1 [493] but can also be produced by COX-2, for instance, in macrophages. In the vascular wall, both the endothelial cells and the smooth muscle cells synthesize thromboxane A₂ [434]. Thromboxane A₂ is enzymatically produced from PGH₂ as a substrate by a specific synthase, the thromboxane synthase, which also belongs to the cytochrome P450 superfamily (in the human, CYP5) [1771]. This enzyme catalyzes two distinct reactions, the formation of thromboxane A₂ and that of 12-hydroxyheptadecatrienoic acid plus malondialdehyde. The human gene encoding for thromboxane synthase is a single copy gene, and the protein is widely expressed in mammalian tissues including platelets, vascular smooth muscle and to a lesser extent endothelial cells [1401,1417,1515,1638]. Thromboxane A₂ elicits diverse physiological/pathophysiological reactions, including direct platelet aggregation and amplification of the responses to other aggregating agents, as well as contraction and proliferation of vascular smooth muscle. Additionally, thromboxane A₂ is involved in allergies, modulation of acquired immunity, atherogenesis, neovascularization and metastasis of cancer cells [1098] (Figure 22).

FIGURE 22

Multiple functions of the thromboxane/endoperoxide TP receptor. The TP receptor belongs to the G-protein-coupled seven transmembrane domain family. In human and some other species, but not in rodents, two isoforms, which differ only in their C-terminal (more...)

Thromboxane A₂ is the preferential ligand of the TP receptor, which was the first receptor of the prostaglandin/endoperoxide pathway to be cloned [651]. The transduction signal associated with the activation of TP receptors mainly involves two types of G-proteins, the Gq (Gq, G11 G15, G16) and the G13 (G12, G13) families, resulting in the activation of phospholipase C and RhoGEF, respectively (Figure 7). Additionally, TP receptors can also be coupled to Gh (phospholipase C) as well as Gi and Gs (adenylyl cyclase)-coupling proteins [1098]. In humans, but not in rodents, two alternatively spliced isoforms of TP receptors, TPα and TPβ, have been described, which differ only in their C-terminal portions (Figure 22). Both isoforms are coupled to phospholipase C activation but TPα stimulates adenylyl cyclase whereas TPβ inhibits it, at least in transfected cells [652]. Furthermore, TP receptors can be engaged in cross-talks with receptor tyrosine kinases, such as the EGF receptor, to induce cell proliferation and differentiation [1098] and with the IP receptor (see above). PGH₂ is another potent agonist of TP receptor and higher concentrations of other prostaglandins, isoprostanes and hydroxyeicosatetraenoic acids (HETEs, generated by lipoxygenases and cytochrome P450 monoxygenases or formed by non-enzymatic lipid peroxidation in endothelial cells and leukocytes) can also activate it with various ranges of potency [419,433]. By contrast, epoxyeicosatrienoic acids (EETs), which act as endothelium-derived hyperpolarizing factors in some vascular beds [430], and their dihydro-derivatives (DiHETs), are endogenous antagonists of TP receptors [84].

Reactive oxygen species enhance the stability and increase the density of functional TP receptors at the cell membrane [1583,1684] and, in endothelial cells, the activation of TP receptors inhibits NO production [930]. The generation of deleterious eicosanoids, the post-transcriptional stabilization of TP receptors and the decreased production of NO are reactive oxygen species-dependent feed-forward loops further altering the unbalance between relaxing/anti-thrombosis and contracting/pro-thrombosis pathways. Taken in conjunction, this experimental evidence indicates that TP receptors are likely to play a pivotal role in cardiovascular diseases [419,433].

Mice genetically deficient in TP receptors are normotensive but have abnormal vascular responses to thromboxane A₂ and show a tendency to bleeding [1550]. The deletion of TP receptors decreases vascular proliferation and platelet activation in response to intimal lesions [242], delays atherogenesis in apoE^-/- mice [823], prevents angiotensin-II- and L-NAME-induced hypertension and the associated cardiac hypertrophy but reduces the extent of kidney injury only in the former hypertensive model [471,473]. TP receptor knockout mice are also protected against various lipopolysaccharide-induced responses such as the increase in iNOS expression [1734], acute renal failure [116] and inflammatory tachycardia [1508]. The phenotype of thromboxane A₂ synthase knockout mice is much less pronounced [1776], most likely because thromboxane A₂ is only one of the endogenous agonists of TP receptor and also because the deletion of this enzyme may redirect the arachidonic cascade toward less pathogenic synthases.

Metabolically stable agonists of TP receptors with high-affinity and potency, such as U46619, have been synthesized and numerous synthetic TP receptor antagonists have been designed, including SQ-29,548, ifetroban (BMS180291) or S 18886 (terutroban). One of them, seratrodast (AA-2414), is used for the treatment of asthma [755,1098,1430].

4.3.2.6. Endothelium-Dependent Contractions.

Very soon after the seminal discovery of Fuchgott and Zawadzki in 1980 [496] concerning the obligatory role of the endothelium in the relaxation of isolated arteries to acetylcholine, it was observed that in veins, the endothelial cells could cause contractions rather than relaxations of the surrounding vascular smooth muscle cells [305]. These responses were attributed to the production of a diffusible factor(s), termed “endothelium-derived contracting factor” (EDCF) and were shown to depend upon cyclooxygenase activation [1045]. Soon thereafter, cyclooxygenase- and endothelium-dependent contractions were also reported in arteries of different species in response to various agonists, substances that increase endothelial [Ca²⁺]_i in a receptor-independent manner (calcium ionophores, thapsigargin, cyclopiazonic acid) and physical stimuli such as stretch [175,700,789,790,1129,1150,1418,1513,1746,1809]. These endothelium-dependent contractions were observed in healthy blood vessels, suggesting that they play a physiological role in the endothelium-dependent regulation of vascular tone.

However, endothelium-dependent contractions are also frequently associated with cardiovascular disease in both animals and humans [1599]. EDCF-mediated increases in tension associated with endothelial dysfunction were first observed in SHR [963] and then in other models of hypertension, in diabetic animals, in estrogen-deprived female rats, as well as in humans with essential hypertension and diabetes [419,431,433,1599].

4.3.2.6.1. SHR Aorta: The Archetypal Model.

Endothelium-dependent contractions have been extensively characterized in the isolated aorta of the SHR. In this blood vessel, the endothelium-dependent relaxations are impaired [940,963] and are associated with the generation of a diffusible EDCF that opposes the relaxing effect of nitric oxide with no or little alteration in its production [661,1745] (Figure 23). These endothelium-dependent contractions are positively correlated with the severity of hypertension and the aging process [720,824,825], are delayed in female SHR [574,795] and also occur in aging normotensive Wistar–Kyoto rats (WKY) [824,825]. The generation of EDCF is observed in response to the activation of endothelial G-protein-coupled receptors such as muscarinic M3 and purinergic receptors [133,825,1067,1746] and of tyrosine kinase receptors [931], as well as in response to receptor-independent stimuli, for instance, the calcium ionophore, A 23187 [1514,1746]. EDCF contributes to the contractile responses of endothelin [1489,1490] and in the presence of an inhibitor of NOS, a tonic generation of EDCF is observed in SHR aorta and in that of aging WKY [1].

FIGURE 23

Endothelium-dependent contractions and EDCF release. Upper panel: Endothelial dysfunction and endothelium-dependent contractions in SHR aorta. In contracted rings of spontaneously hypertensive rats (SHR) and in the congenic normotensive strain, the Wistar–Kyoto (more...)

In SHR, inhibitors of COX abolish the endothelium-dependent contractions and fully restore the impaired endothelium-dependent relaxations, indicating that there is no or little alteration in NO production [963], a conclusion strengthened by perfusion–superfusion bioassay studies [661]. Further bioassay studies using layered “sandwich” preparations demonstrated that endothelium-dependent contractions to ACh involve the endothelial release of diffusible contractile COX derivatives which oppose the relaxing effect of NO [1745] (Figure 23).

In SHR aorta, endothelium-dependent contractions are associated with multiple dysfunctions in both the endothelial and the smooth muscle cells. In the endothelial cells, they include: 1) abnormal calcium handling, 2) an increased expression of COX-1, 3) the associated enhanced production of reactive oxygen species, 4) a major increase in prostacyclin synthase expression, 5) the enhanced release of prostacyclin, thromboxane A₂, and possibly PGH₂. In vascular smooth muscle cells, they include: 1) an exacerbated response of the TP receptor to prostacyclin and PGH₂, 2) a deficient IP receptor function and 3) an early dysfunction in the adenylyl cyclase pathway [419,433,434].

4.3.2.6.1.1. Abnormal Calcium Handling.

When compared to WKY aorta and in response to receptor mediated stimuli (acetylcholine), the increase in intracellular calcium ([Ca2+]i) in SHR endothelial cells and the amplitude of the endothelium-dependent contractions are exacerbated, while in response to receptor-independent stimuli (calcium ionophore, A 23187), the maximal amplitude of the endothelium-dependent contractions and the changes in [Ca2+]i in both strains are similar [553,554,1514]. Any event leading to an increase in endothelial [Ca2+]i activates the calcium-dependent phospholipase A2 (cPLA2) and provokes the mobilization of arachidonic acid. However, in response to receptor-dependent stimuli, such as acetylcholine, the activation of the calcium-independent phospholipase A2 (iPLA2) allows the store-operated calcium channel (SOC)-dependent influx of extracellular calcium and the subsequent activation of cPLA2. Substances, such as calcium ionophores, which bypass the cell membrane receptors, cause an increase in [Ca2+]i, and a direct activation of cPLA2 [1696]. Therefore, the iPLA2 pathway associated with calcium mobilization is defective in SHR endothelial cells.

4.3.2.6.1.2. Cyclooxygenases.

The subsequent steps involve the activation of cyclooxygenase and the production of reactive oxygen species along with that of prostanoids. Aortic endothelial cells express preferentially COX-1 versus COX-2 [796,1515]. In SHR endothelial cells, the mRNA and protein expression of COX-1 are enhanced when compared to that of WKY and, in the two strains, both are augmented by aging [528,1515]. In response to acetylcholine, endothelium-dependent contractions and the associated generation of prostaglandins are blocked consistently by specific inhibitors of COX-1 and partially inhibited, to a varying degree, depending on the experimental conditions, by specific inhibitors of COX-2 [528,553,1745] (Figure 23). However, if the endothelium-dependent contractions and the release of prostaglandins by A 23187 are also fully blocked by COX-1 inhibitors, these responses are less sensitive to COX-2 inhibition. This could possibly be explained by the fact that low concentrations of arachidonic acid are preferentially oxygenated by COX-2, while higher ones are preferentially metabolized by COX-1 [1080]. Alternatively, the effects observed with the COX-2 inhibitors could nevertheless be attributed to COX-1 inhibition. Indeed, the ability of COX-2 inhibitors to inhibit COX-1 depends obviously not only on the degree of selectivity of any given inhibitor but also on other factors such as substrate availability, endogenous lipid peroxide levels and plasma protein concentration, explaining why COX-2 inhibitors are systematically more potent in preventing the endothelial production of PGI₂ than the platelet production of tromboxane A₂ [1052,1658]. In agreement with a preponderant role for COX-1 in endothelium-dependent contractions, these responses are abolished in aortae taken from COX-1 knockout mice while they are maintained in aortic rings of COX-2 knockout animals [1513].

However, in addition to COX-1, COX-2 also can generate vasoconstrictor prostanoids in the SHR endothelial cells. In both WKY and SHR endothelial cells, not only the induction of COX-2 in the aorta but also in resistance arteries is accelerated by ageing and can be associated with the generation of endothelium-derived contractile prostanoids [33,107,642,1406,1619]. For instance, in WKY rats, the impairment of the aortic endothelium-dependent relaxations, observed after chronic treatment with fenofibrate, has been attributed to the endothelial release of COX-2-derived PGE₂ acting on smooth muscle TP receptors [108]. In L-NAME-hypertensive rats, the up-regulation of COX-2 enhances the release of EDCFs [1258]. Additionally, the release of COX-2-derived PGF_2α and 8-isoprostane augments α-adrenoceptor-induced contractions in SHR arteries [33] and COX-2 also mediates vasoconstrictions in response to tert-butyl hydroperoxide, a product of lipid peroxidation [508].

Additionally, COXs are also involved in the endothelial generation of reactive oxygen species, a key factor in the generation of endothelium-dependent contractions [1514,1744]. Reactive oxygen species decrease NO bioavailability [590,1316] and, as a positive feedback loop, the formation of hydroperoxides further activates COXs [1080]. In addition, since reactive oxygen species diffuse toward the vascular smooth muscle cells, they can stimulate COXs in these cells and produce more contractile prostanoids. Furthermore, oxidative stress enhances the response to TP receptor stimulation [1747]. Finally, the oxidative stress can produce isoprostanes [1081], which could theoretically act as EDCFs and contribute to the endothelial dysfunction [1619].

4.3.2.6.1.3. TP Receptor Activation.

Endothelium-dependent contractions are blocked by TP receptor antagonists, indicating that the generated prostaglandins diffuse toward the vascular smooth muscle cells and directly activate these receptors [51,963,1745] (Figure 23). In the rat aorta, the five major prostaglandins and 8-isoprostane produce contractions that predominantly involve TP receptor activation. However, the involvement of PGD2 and 8-isoprostane in endothelium-dependent contractions can be ruled out since their generation is not affected by acetylcholine [553]. In SHR aortic endothelial cells, the expression of thromboxane synthase is enhanced when compared to that in WKY endothelium [1515]. In response to ATP or the calcium ionophore A 23187, endothelium-dependent contractions are associated with an increased generation of thromboxane A2 and are partially inhibited by dazoxiben, a specific inhibitor of thromboxane synthase that abrogates the production of thromboxane A2 [554,555]. By contrast, acetylcholine produces only a minor dazoxiben-sensitive increase in thromboxane A2 production, and the endothelium-dependent contractions that it evokes are not affected by the presence of the thromboxane synthase inhibitor, indicating that thromboxane A2 is only one of the EDCFs that can be released from SHR aortic endothelial cells [528,553–555,787,825] (Figure 24).

FIGURE 24

Thromboxane A₂ involvement in endothelium-dependent contractions. (A) The calcium ionophore, A23187, produces a significantly larger endothelial release of thromboxane B₂, the stable metabolite of thromboxane A₂, than acetylcholine (ACh). (B) The endothelial (more...)

4.3.2.6.1.3.1. PROSTACYCLIN INVOLVEMENT.

Paradoxically, prostacyclin is likely to be a major EDCF in SHR aorta. This conclusion is based on the following observations [553–555]:

1. In the SHR aorta, prostacyclin is a contracting but not a relaxing factor [1282] (Figure 21). The absence of relaxation in response to prostacyclin is attributed to an early (as young as 12 weeks old) dysfunction of the IP-receptors of vascular smooth muscle. This dysfunction is tissue-specific since the platelet response to prostacyclin (or its analogues) is unaffected or even enhanced [35,562]. In order to explain this specific smooth muscle cell dysfunction, a decrease in the aortic expression of IP receptors [1138] and an early impairment of adenylyl cyclase signaling have been evoked [35,993]. However, these two hypotheses can only, at best, partially explain the total disappearance of IP-receptor-mediated relaxations in SHR aorta. Indeed, the decreased expression of the IP receptor was not confirmed in later experiments [1515] and when compared to WKY, the relaxations to prostacyclin in SHR aorta are much more severely affected than those produced by other agents that stimulate adenylyl cyclase, such as isoproterenol and forskolin [562]. A potential additional/alternative hypothesis, which requires proper validation, could be the oxidative damage of the IP receptor itself, which contains redox-sensitive cysteines that play an essential role in determining its structure, addressing and function [1461].
2. Prostacyclin is a more potent contracting agent in SHR than in WKY.
3. The contractions evoked by prostacyclin mimic the endothelium-dependent contractions produced by acetylcholine both in terms of duration and amplitude.
4. Endothelium-dependent contractions and prostacyclin-induced contractions both involve activation of TP receptors (Figures 21 and 23).
5. Prostacyclin is by far the most abundant prostaglandin released from the endothelial cells in response not only to receptor-dependent stimuli but also to calcium ionophores. This may come as a surprise since prostacyclin synthase is rapidly nitrosylated and inactivated by peroxynitrite [1364,1818,1819]. However, in the SHR aorta, the massive increase in the expression of prostacyclin synthase [1515] may compensate the loss of activity due to peroxynitrite-dependent tyrosine nitration (Figure 25).
6. The release of prostacyclin is nearly two times greater in SHR than in WKY (Figure 25).
7. The time course of the release of prostacyclin is compatible with the time course of the observed endothelium-dependent contractions.
8. The release of prostacyclin correlates with the amplitude of the endothelium-dependent contractions over the full concentration range of ACh in both WKY and SHR (Figure 25).
9. The endothelium-dependent contractions and the release of prostacyclin are affected similarly by COX inhibitors.
10. The expression of PGIS is by far the most abundant of the prostaglandin synthases expressed in the rat aortic endothelial cells and PGIS and COX-1 co-segregate [796].
11. The inhibition of prostacyclin synthesis enhances the ACh-induced endothelium-dependent contractions. Paradoxically, this latter observation also supports the hypothesis that prostacyclin contributes to endothelium-dependent contractions since the inhibition of PGIS may enhance PGH₂ spillover, a more potent TP receptor agonist than prostacyclin itself [553,1282].

FIGURE 25

Prostacyclin involvement in endothelium-dependent contractions. Upper panel: Basal and acetylcholine-dependent release of 6-keto-prostaglandin F_1α (6-keto PGF_1α), the stable metabolite of prostacyclin (PGI₂), and thromboxane B₂ (TxB₂), (more...)

Prostacyclin has also been identified as a major contributing factor accounting for the endothelial dysfunction observed in the aorta and mesenteric artery of WKY and SHR treated with aldosterone [107,1719]. Thus, although, as a rule, prostacyclin is a vasodilator and an anti-aggregating agent, depending on the circumstances, it can also act as an EDCF.

4.3.2.6.1.3.2. OTHER PROSTAGLANDINS.

Any level of prostacyclin synthase inactivation would theoretically lead to an excess of free PGH₂. Since PGH₂ is the second most potent agonist at TP receptors and is more effective in activating TP receptors in vascular smooth muscle from SHR than that of WKY, the endoperoxide is also a suitable candidate as EDCF [528,553,787]. Finally, the shunting of PGH₂ metabolism toward other metabolic pathways can lead to a variety of products, including PGE₂ and/or PGF_2α. Therefore, thromboxane A₂, PGH₂, PGI₂, PGE₂ and PGF_2α can all theoretically act as EDCF and contribute to endothelial dysfunction [419,433,553] (Figure 26).

FIGURE 26

Mechanisms of endothelium-dependent contractions in WKY and SHR aortic rings. M: muscarinic receptor; AA: arachidonic acid; PLA₂: phospholipase A₂; eNOS: endothelial nitric oxide synthase; NO^.: nitric oxide, L-arg: L-arginine; O₂^.-: superoxide anion; (more...)

The EDCF- and TP-mediated responses, first observed in the aorta of the SHR, are not ubiquitous in SHR arteries, but have been reported in other vascular territories such as the mesenteric, skeletal muscle and renal vascular beds [434]. In these peripheral arteries the endothelial dysfunction additionally includes a marked attenuation of the EDHF-mediated component of the endothelium-dependent relaxations which in turn can favor the development of endothelium-dependent contractions.

4.3.2.6.7. Endothelium-Dependent Contractions in Humans.

Endothelial dysfunction was first reported in human coronary arteries of atherosclerotic patients [956]. Since then, endothelial dysfunction has been associated not only with hypertension or atherosclerosis but also with physiological and pathophysiological processes, including aging, heart and renal failure, coronary syndrome, microalbuminuria, dialysis, thrombosis, intravascular coagulation, preeclampsia, Type I and Type II diabetes, impaired glucose tolerance, insulin resistance, hyperglycemia, obesity, postprandial lipemia, hypercholesterolemia, hyperhomocysteinemia, elevated asymmetric dimethylarginine plasma levels, inflammation, vasculitis, infections, sepsis, rheumatoid arthritis, periodontitis, trauma, transplantation, low birth weight, postmenopause in women, mental stress, sleep apnea syndrome, smoking, nitrate tolerance, glucocorticoids and so on [431]. In the human, reactive oxygen species are elevated in many cardiovascular diseases. An increased production of reactive oxygen species, a decreased antioxidant activity, and a reduced ability to scavenge oxygen-derived free radicals all contribute to oxidative stress [883,1559]. In a number of these conditions, the endothelial dysfunction involves the activation of TP receptors.

Endothelium-dependent vasodilatations to physical (shear stress) and/or pharmacological (e.g., acetylcholine, bradykinin, substance P) stimuli are impaired in the forearm, coronary and renal vasculature as well as in various microcirculatory beds of patients with essential or secondary hypertension [926,1185,1492,1495,1498,1499,1501]. In patients with essential hypertension, but not in those with secondary hypertension (primary aldosteronism or renovascular hypertension), non-specific COX inhibitors partially restore the reduced acetylcholine-induced forearm vasodilatation, suggesting in the former the release of EDCF [1492,1495,1499]. In primary hypertensive patients, selective inhibition of COX-1 partially reverses the impairment of vasodilator responses to acetylcholine, while the selective inhibition of COX-2, which does not produce adverse effects in the forearm of healthy subjects [1611], further reduces the increase in forearm blood flow produced by the muscarinic agonist [157]. These results indicate that COX-1-derived vasoconstrictor prostaglandins contribute to the endothelial dysfunction and that the production of vasodilator prostaglandins by COX-2 is of minor importance in subjects with normal endothelial function, but become relatively more important in hypertensive patients with endothelial dysfunction, presumably playing a beneficial compensatory role. Vitamin C normalizes the impaired endothelium-dependent vasodilatation in the forearm and the coronary circulation in patients with essential hypertension, confirming that the generation of reactive oxygen species contributes to the endothelial dysfunction [1446,1493,1612]. Similarly, during the aging process, progressive endothelial impairment has been demonstrated both in conduit arteries and at the microcirculatory level, in the forearm as well as in the coronary circulation [1498,1499,1787]. In normotensive subjects up to the age of 60 years, the main mechanism responsible for the progressive endothelial dysfunction is a primary defect in the L-arginine–NO pathway, without evidence of a substantial contribution of EDCF [1499]. However, in subjects older than 60 years, the infusion of indomethacin potentiates the vasodilatation to acetylcholine, suggesting that EDCF contributes to the dysfunction [1304]. The production of COX-dependent factors is associated with a further and parallel impairment in the L-arginine–NO pathway [1499]. In essential hypertensive patients on the other hand, the contribution of COX-derived vasoconstrictor substances is already detectable in the age group of 31–45 years, and augments in parallel with increasing age [1499]. This finding certainly supports the conclusion that the production of COX-derived EDCF is a characteristic of the aging blood vessel wall, with essential hypertension merely causing an earlier onset and an acceleration of this endothelial alteration [1600].

In patients with coronary artery disease, the impaired acetylcholine- and flow-induced forearm vasodilatation is restored by the administration of a TP receptor antagonist [85]. The fact that the patients in this latter study were already treated with aspirin suggests that COX-2 activity, rather than COX-1, could be the main source of the vasoconstrictor prostanoids involved in this endothelial dysfunction. Indeed, in patients with severe coronary artery disease, COX-2 inhibition improved flow-mediated dilatation [240]. Additionally, bone morphogenic protein 4 (BMP4) and COX-2 are elevated in the renal arteries of hypertensive patients. By increasing NADPH oxidase-derived reactive oxygen species, BMP4 up-regulates COX-2 resulting in the impairment of endothelium-dependent relaxations and appearance of endothelium-dependent contractions. The BMP4 antagonist, noggin, a selective COX-2 inhibitor, or a TP receptor antagonist normalize the relaxations and/or abolish endothelium-dependent contractions [1304,1699].

In addition, women subjected to ovariectomy/hysterectomy, and therefore to acute estrogen deprivation, show a decrease in the forearm vasodilator response to acetylcholine. In healthy adult women, the vasodilatation is unaffected by COX-inhibitors, but after ovariectomy, indomethacin enhances the response. In these subjects, estrogen replacement therapy restores the proper acetylcholine-induced vasodilatation [1225]. This suggests that estrogen protects endothelial function by preventing the formation of COX-derived EDCFs. COX inhibition also improves the endothelial dysfunction observed in patients with atherosclerosis [687] or with congestive heart failure [792] and in impotent diabetic patients, the reduced endothelium-dependent relaxation of the corpus cavernosum involves the activation of TP receptors [40].

These results indicate that, as has been observed in animal models, COX-1, COX-2 or both isoforms can contribute to endothelial dysfunction. Since in most cases, the activation of TP receptors is the common downstream effector, specific antagonists of this receptor could be of therapeutic interest in the treatment of cardiovascular disorders (Figure 27).

FIGURE 27

Physiological and pathophysiological effects of IP and TP receptor activation. COX: cyclooxygenases; LOX: lipoxygenases; P450: cytochrome P450 monooxygenases; ROS: reactive oxygen species; PGS: prostaglandin synthases; PGIS: prostacyclin synthase; TXS: (more...)

4.3.3.1. 5-Lipoxygenases.

5-Lipoxygenase converts arachidonic acid to 5(S)-hydoperoxy-6-trans-8,11,14-cis-eicosatetraenoic acid (5-HPETE) and then further hydratation of 5-HPETE leads to the formation of the allylic epoxide leukotriene A4 (LTA₄), which can in turn be converted to the dihydroxic acid LTB₄ or the cysteinyl leukotrienes LTC₄, LTD₄ and LTE₄ [1268]. LTB₄ and cysteinyl leukotrienes interact with G-coupled, seven transmembrane-spanning domain receptors, BLT1 and BLT2 for the former and CysLT1 and CystLT2 for the latter. 5-Lipoxygenase is a calcium-sensitive enzyme, which needs to be translocated to the nuclear membrane, and to a lesser extent to other organelles, in order to show leukotriene synthesis activity. A 5-lipoxygenase-activating protein (FLAP), once thought to be required for the anchoring of the 5-lipoxygenase to the cell membrane, functions as a scaffold protein and is essential for the activation of the enzyme [1267,1268] (Figure 28). Leukotrienes are involved not only in acute inflammation and asthma but also in the generation of cardiovascular diseases including atherosclerosis, abdominal aortic aneurysm and myocardial infarction and reperfusion injury [1230]. 5-Lipoxygenase knockout mice and rats treated with a FLAP inhibitor develop less right ventricular hypertrophy and less pulmonary hypertension under chronic hypoxic conditions than wild-type animals, suggesting that in rodents, the enzyme also contributes to the generation of chronic pulmonary hypertension [492,1620].

4.3.3.2. 12-Lipoxygenases.

12S-lipoxygenase introduces molecular oxygen into arachidonic acid to form 12S-hydroperoxy-5,8,10,14-eicosatetraenoic acid (12S-HPETE). There are three isoforms of 12S-lipoxygenases named after the cells in which they were first identified: platelet, leukocyte and epidermis [1772]. In the human, only the platelet type 12S-lipoxygenase is expressed. The production of 12S-HPETE and the expression of platelet-type 12-lipoxygenase are increased in hypertension [568]. There is a marked up-regulation of 12-lipoxygenase mRNA in the wall of arteries injured by a balloon catheter, which affects mainly smooth muscle and infiltrating inflammatory cells [1108]. Leukocyte-type 12S-lipoxygenase (rat, mouse, pig) shows higher identity with the human and rabbit 15-lipoxygenase-type I than with the other 12-lipoxygenase enzymes, and they are therefore called 12/15-lipoxygenases [1772].

4.3.3.3. 15-Lipoxygenases.

15-Lipoxygenases are lipid-peroxidizing enzymes that catalyze the stereo-selective introduction of molecular oxygen at carbon 15 of arachidonic acid to form 15S hydroperoxy-5,8,11,13-eicosatetraenoic acid (15S-HPETE). Subsequently, 15S-HPETE becomes the substrate for the synthesis of more complex bioactive lipids such as lipoxins. Two types of 15-lipoxygenases have been cloned in the human. Type I, or reticulocyte-type 15-lipoxygenase, as mentioned above, shares a highly phylogenic relatedness with the rat, murine and porcine leukocyte-type 12-lipoxygenase. Type II, or epidermis-type 15-lipoxygenase, is related to the murine 8-lipoxygenase, which shares only a low degree of homology with type I [855]. Type I is expressed ubiquitously, while the pattern of expression of type II appears to be more restricted (skin, prostate, lung and cornea). Type I has a broad substrate specificity which includes linoleic acid with the introduction of oxygen at carbon 13 giving rise to 13-HODE (13-hydroperoxyoctadecadienoic acid), an inhibitor of platelet adhesion to the endothelial cell surface [1591] (Figures 2 and 28). Liberation of arachidonic acid from the phospholipid stores is not an absolute prerequisite for the enzymatic activity of type I 15-lipoxygenase. This isoform may oxygenate polyenoic fatty acid esterified in the membrane and subsequently hydro(pero)xy derivatives can be released by the phospholipases [207,854]. NO may act as a reversible inhibitor of 15-lipoxygenase and can be effectively consumed during 15-lipoxygenase-catalyzed oxidation of polyenoic fatty acids, suggesting that this enzyme could be considered as a regulator of cellular NO metabolism [668,1141]. The biological and pathophysiological roles of 15-lipoxygenases are multiple and include cell differentiation and maturation, inflammation, hypereactivity of smooth muscle (e.g., asthma), carcinogenesis, apoptosis and atherosclerosis [855]. Mouse knock-out for the leukocyte-type 12-lipoxygenase, the enzyme which is most closely related to human platelet type 15-lipoxygenase, when crossed with other strains susceptible to develop atherosclerosis (apolipoprotein E deficient, low-density lipoprotein deficient), shows a 50% reduction in aortic atherosclerotic lesions [492].

4.3.3.4. Lipoxygenases Metabolites as Endothelium-Derived Vasoactive Factors.

Under physiological and/or pathophysiological conditions, endothelial cells express various lipoxygenases including 5-lipoxygenase, platelet-type 12-lipoxygenase, 12/15 lipoxygenase (rat, mouse, pig) and 15-lipoxygenase type I [593,805,891,1801]. These enzymes metabolize arachidonic acid into vasoactive derivatives (relaxing and contracting substances).

4.3.3.4.1. Endothelium-Dependent Relaxations and Hyperpolarizations.

Following the demonstration of the contribution of metabolites of arachidonic acid to certain endothelium-dependent relaxations [305], the involvement of a short-lived metabolite of arachidonic acid produced through either the cytochrome P450 monooxygenase or the lipoxygenase pathway was proposed to explain certain EDHF-mediated responses [839,1224,1318]. In several arteries, endothelium-dependent responses are blocked by inhibitors of phospholipase A2 [7,629,688,1318], and depending on the species and/or the arteries, cytochrome P450- or lipoxygenase-derived products play a predominant role. Thus, in rabbit aorta and mesenteric arteries as well as various arteries from rodents, dog, pig, and human, the lipoxygenases-dependent metabolization of arachidonic acid can be the predominant pathway(s) involved in the synthesis of endothelium-derived relaxing factors in response to arachidonic acid, neurohumoral substances (e.g., acetylcholine, thrombin, arachidonic acid) or oxidative stress. Some of the derivatives can be considered as endothelium-derived hyperpolarizing factors since they are released by endothelial cells and produce hyperpolarization of the underlying vascular smooth muscle cells [216,523].

4.3.3.4.1.1. 12-Lipoxygenase Metabolites.

In coronary arteries of the rat, the endothelium-dependent vasodilatation, resistant to COX and NOS inhibitors, in response to protease-activated receptor-2 involves such a lipoxygenase-derived eicosanoid [1022]. In the basilar artery of the same species, the arachidonic acid-induced vasodilatation is attributed to the release of 12-(S)-HETE and the subsequent activation of BK_Ca on the smooth muscle [406]. Similarly, in porcine coronary microvessels, and to a much lesser extent in large conduit coronary arteries, 12-(S)-HETE, released by the endothelial cells in response to oxidative stress, acts as a potent relaxing and hyperpolarizing factor by activating smooth muscle BK_Ca [1814]. In these coronary arteries the relaxing and hyperpolarizing effects of H₂O₂ involve the lipoxygenase-dependent activation of BK_Ca [64] (Figures 28 and 29).

FIGURE 29

Endothelium-derived vasoactive metabolites of arachidonic acid produced by 12- and 15-lipoxygenase and potential physiopathological mechanisms of regulation. The 15-lipoxygenase pathway has been predominantly characterized in rabbit arteries, the vasoconstrictor (more...)

4.3.3.4.1.2. 15-Lipoxygenase Metabolites.

The best characterization of a lipoxygenase derivative as an endothelium-derived hyperpolarizing factor was obtained in rabbit and murine arteries. This NO- and PGI₂-independent component of the endothelium-dependent relaxation relies on the activation of smooth muscle potassium channels [1219,1220,1517,1790]. Upon exposure to acetylcholine, arachidonic acid, mobilized by phospholipase A₂, is metabolized sequentially by the 15-lipoxygenase type I and hydroperoxide isomerase into an hydroxyepoxyeicosatrienoic acid (HEETA), which is hydrolyzed by a soluble epoxide hydrolase (sEH) to trihydroxyeicosatrienoic acid (11,12,15-THETA) and 11,14,15-THETA, the former possessing relaxing and hyperpolarizing properties while the latter is inactive [187,215,1221]. In the smooth muscle cells of the rabbit aorta, 11,12,15-THETA produces relaxation by activating an apamin-sensitive but charybdotoxin-insensitive K_Ca of “small” conductance (24 pS) [526] while, in the mesenteric artery, BK_Ca are involved [1790] (Figures 28 and 29). The synthesis of analogs showed that, in order to observe potassium channel activation and vascular relaxation, specific structures and proper stereochemical configurations were required, suggesting that these compounds interact with a specific binding site(s) or receptor(s) [519]. Furthermore, in rabbit aorta and mesenteric arteries, the combination of 15-lipoxygenase and cytochrome P450 2J2 can convert arachidonic acid into two distinct HEETA metabolites, which are hydrolyzed by soluble epoxide hydrolase into an acid-stable endothelium-derived hyperpolarizing factor, 13-hydroxy-trans-14,15-epoxy-eicosatrienoic acid (13,14,15-THETA) [217].

In rabbits, the endothelial overexpression of 15-lipoxygenase-1 enhances acetylcholine-induced endothelium-dependent relaxation in vitro and acetylcholine induces hypotension in vivo [12]. Similarly, chronic hypoxia enhances expression of 15-lipoxygenase-1 and the generation of 11,12,15-THETA and increases endothelium-dependent relaxations [15]. Conversely, the age-related reduction in the NO and prostaglandin-independent relaxations and the hypotension observed in response to acetylcholine are associated with a reduction in vascular 15-lipoxygenase-1 expression and in THETA and HEETA synthesis [13]. These data suggest that, at least in rabbits, endothelial-derived lipoxygenase metabolites of arachidonic acid contribute to the in vivo regulation of vascular tone under physiological and pathophysiological conditions (Figure 29).

However, this pathway has no or little effect in the regulation of blood pressure, since in normotensive animals, pharmacological inhibition of lipoxygenase, and either the genetic deletion or overexpression of 15-lipoxygenase-1 does not affect heart rate or blood pressure [216]. In fact, in angiotensin II-hypertensive animals, lipoxygenase inhibitors lower arterial blood pressure due to a diminished production of vasoconstrictor HETE(s) [216]. Furthermore, in placental tissue of preeclamptic women, enhanced production of 5-HETE, 12-HETE and 15-HETE metabolites has been observed, suggesting either a compensatory or a pathophysiological role of these eicosanoids in pregnancy-induced hypertension [1204]. Finally, the 12/15-LO inhibitor, baicalein, and its derivatives have favorable effects in animal models of ischemic stroke [216]. Much further work is therefore required to determine whether or not 15-lipoxygenase plays a beneficial role in the regulation of cardiovascular homeostatis or conversely is involved in vascular diseases.

Although there is a strong case for arguing that lipoxygenase derivatives act as endothelium-derived mediators in some arteries, most of the EDHF-mediated responses do not involve an arachidonic acid metabolite from this pathway. Evidence based on the effects observed with inhibitors of lipoxygenases must be taken with caution since they are far from specific. For instance, they can affect guanylyl cyclase as well as ionic channels including potassium channels or voltage-dependent calcium channels [257,1262,1738]. Nevertheless, in guinea-pig carotid and coronary arteries [272,369,1131,1262], rat uterine artery [486], porcine coronary artery [1235,1664] and human resistance arteries [174], the involvement of a lipoxygenase-derivative in the genesis of endothelium-dependent relaxations and hyperpolarizations is extremely unlikely.

4.3.4.1. Bis-Allylic Oxidation (Lipoxygenase-Like Reactions).

The products of bis-allylic oxidation are structurally similar to those of lipoxygenases with no evidence that hydroperoxide intermediates are formed. These reactions are catalyzed by cytochrome P450 of the 1A, 3A, 2C and 4F families [1300]. They generate any of the six regio-isomeric hydroxyeicosatetraenoic acids (5-, 8-, 9-, 11-, 12-, and 15-HETE). 12R-HETE is the predominant enantiomer generated by cytochrome P450-catalyzed reactions and is a powerful and enantioselective inhibitor of Na⁺/K⁺-ATPase [190,1377].

4.3.4.2. Arachidonic Acid ω/ω Hydroxylase.The ω/ω-1 hydroxylation of arachidonic acid is catalyzed mainly by the cytochrome P450s of the 4A, 4B and 4F families, yielding 16-, 17-, 18-, 19- or 20-HETE.

Cytochrome 4A is expressed in blood vessels, mostly in the smooth muscle cells, and is responsible for the synthesis of 20-HETE [793,1300], an endogenous blocker of BK_Ca and a potent vasoconstrictor, especially in small arteries [611,1816].

4.3.4.3. Olefin Epoxidation (Arachidonic Acid Epoxygenase).

This reaction, producing the four epoxyeicosatrienoic acids (5,6-; 8,9-; 11,12-; 14,15-EET), is catalyzed by numerous cytochrome P450s, namely, the 1A, 1B, 2B, 2C, 2D, 2E, 2J, 3A and 4A families. Human endothelial cells express the 2C8, 2C9, 2J2, 3A and 2B1 families that all produce EETs. EETs are potent vasodilators. They increase the open probability of BK_Ca in vascular smooth muscle cells, produce hyperpolarization and a subsequent relaxation [186,533]. Only a very limited number of observations suggest that vascular smooth muscle cells can produce EETs [1300]. EETs are converted by epoxide hydrolases into dihydroxyeicosatetraenoic acids (DiHETE). DiHETEs are, in general, less potent vasodilators than the corresponding EETs [707] with some exceptions, such as the coronary circulation where EETS and DiHETE can be equipotent [882,1160].

Cytochrome P450 monooxygenases can catalyze only one or several type of reactions. For instance, CYP 4A1 catalyzes only the ω/ω-1 hydroxylation while CYP 4A2 and CYP4A3 catalyze both ω/ω-1 hydroxylation and epoxygenation [190,1300]. Additionally, the metabolism of arachidonic acid by cytochrome P450 is a significant source of oxygen-derived reactive species and therefore can contribute to oxidative stress. For instance, in endothelial cells of the porcine coronary artery, CYP2C9, which produces vasodilator EETs, also produces superoxide anions [460].

4.3.4.4. EETs

4.3.4.4.1. EETs and Vascular Smooth Muscle Relaxation and Hyperpolarization.

One of the earliest reports demonstrating the vasodilator action of EETs concerned the intestinal microcirculation [1253]. In fact, EETs directly relax many blood vessels and especially the coronary arteries from many species, including humans [186,369,451,490,533,575,1057,1160]. In some cases, EETs produce endothelium-dependent and NO-dependent relaxation of blood vessels [637].

EETs hyperpolarize arterial smooth muscle cells [186,369,451] and increase the open-state probability of BK_Ca [186,451,532,533,623,679,914], without affecting their unitary conductance or their calcium sensitivity [485]. The mechanism by which EETs activate BK_Ca in vascular smooth muscle cells is not completely understood. Although in some cell types, EETs can interact directly with BK_Ca to enhance the mean open time of this channel [352], as has been previously shown with other fatty acids [1164], they do not directly activate these channels in vascular smooth muscle cells. EETs are likely to interact with a G-protein-coupled “receptor,” which remains to be identified, to activate ADP-ribosyltransferase. This enzyme catalyzes the ADP-ribosylation of G_sα, promoting the phosphorylation and the activation of the Slo1 α subunit of BK_Ca [912,913]. The observation that some EET analogues act in a stereo-specific manner either as inhibitors of the action of EETs (i.e., “antagonists”), such as 14,15-epoxyeicosa-5(Z)-enoic acid (14,15-EEZE) and 14,15-epoxyeicosa-5(Z)-enoic-methylsulfonylimide (14,15-EEZE-mSI), or as EETS mimetics (i.e., “agonists”), such as 14,15-epoxyeicosatrienoyl-sulfonamides, supports the existence of such receptors [155,235,522,1755]. However, if the presence of such a receptor(s)/high-affinity binding site(s) has been shown in guinea pig mononuclear cells [1697], its existence in vascular cells remains to be demonstrated. In smooth muscle cells of rat small mesenteric arteries, EETs activate K_ATP and not BK_Ca. This again is dependent on the ADP-ribosylation of G_sα but, in this artery, involves the stimulation of adenylyl cyclase and the subsequent cyclic-AMP-dependent activation of PKA. PKA in turn phosphorylates and activates vascular K_ATP channels, producing membrane hyperpolarization and vasodilatation [1763,1764] (Figure 31).

FIGURE 31

Epoxyeicosatrienoic acids (EETs) induced relaxation and hyperpolarization of vascular smooth muscle cells. Some neurohumoral mediators, such as bradykinin, and shear stress activate the metabolism of arachidonic acid (AA) via cytochrome P450 monooxygenase (more...)

Additionally, EETs can also activate BK_Ca in much more indirect manners. Indeed, 11,12-EET can increase the activity of heme-oxygenase (HO) and the subsequent generation of carbon monoxide (CO) activates BK_Ca [1328]. Furthermore, EETs can promote Ca²⁺ entry into smooth muscle cells by activating cationic channels such as the vanilloid transient receptor potential channel (TRPV4). The Ca²⁺ influx through TRPV4 preferentially stimulates ryanodine receptors located on the sarcoplasmic reticulum, increasing the frequency of unitary Ca²⁺ release events (Ca²⁺ sparks). The EET-induced Ca²⁺ sparks activate nearby sarcolemmal BK_Ca and increase the frequency of spontaneous transient outward currents (STOCs). This EET-dependent activation of the Ca²⁺-signaling complex (TRPV4-ryanodine receptors-BK_Ca) hyperpolarizes and relaxes vascular smooth muscle cells [363] (Figure 31).

4.3.4.4.6. EETs: Therapeutic Potential.

Since EETs are potent vasodilators, possess anti-inflammatory properties and appear in general cardioprotective, this arachidonic acid pathway is likely to contain therapeutic targets (Figure 32).

FIGURE 32

Cytochrome P450 pathway and potential therapeutic targets in cardiovascular diseases. CYP: cytochrome P450; DHETE: dihydroxyeicosatetraenoic acids; EET: epoxyeicosatrienoic acids; HETE: hydroxyeicosatetraenoic acids; sEH: soluble epoxide hydrolase.

The most promising one is the inhibition of soluble epoxide hydrolase, the enzyme that metabolized the EETs in the less active corresponding diols (dihydroxyeicosatrienoic acids; DHETs) [706]. The genetic polymorphisms of cytochrome P450 2C8, 2C9 and 2J enzymes have been associated with hypertension, myocardial infarction and cardiovascular disease and those involving the soluble epoxide hydrolase gene (EPHX2) with coronary artery disease, ischemic stroke and insulin resistance [527,592,704,1148,1815]. Potent and selective inhibitors of this enzyme have been designed and are currently undergoing clinical trials for the treatment of hypertension. The additional anti-inflammatory and cardioprotective properties of soluble epoxide hydrolase inhibitors also make them attractive in patients with heart failure, cardiac arrhythmias, ischemic stroke, atherosclerosis and in the treatment of end-organ damage patients with cardiometabolic syndrome and/or hypertension [704,706].

Metabolically stable analogues of EETs have been designed in order to act as putative agonists of the EET “receptors.” In various animal models of cardiovascular disease and metabolic syndrome, they demonstrate antihypertensive, anti-inflammatory and cardioprotective effects. Similarly, cardioprotective and antidiabetic effects were observed by overexpressing cytochrome P450 2J [587,704,706,1396,1731]. Additionally, existing therapies such as statins, calcium channel blockers and fibrates are potent inducers of cytochrome P450 epoxygenases and the resulting increase in EET generation may contribute to their beneficial therapeutic effects [89].

4.3.4.5. 20-HETE.

In the vascular wall, while endothelial cells preferentially express cytochrome P450 epoxygenase and are the predominant source of EETs, the vascular smooth muscle cells preferentially express cytochrome P450 hydrolase, CYP4A and CYP4F isoforms, and are the predominant source of 20-HETE [1060].

4.4.1.1. Anandamide as an Endothelium-Derived Relaxing and Hyperpolarizing Factor.

In isolated and perfused mesenteric and coronary arterial beds of the rat, anandamide induces a dilatation which mimics the endothelium-dependent vasodilation that could be observed in the presence of inhibitors of NO-synthases and cyclooxygenases [1277]. However, further analysis in isolated blood vessels from various species demonstrated beyond any doubt that anandamide did not fulfill the criteria to be classified as an endothelium-derived hyperpolarizing factor [212,1227,1672,1828]. For instance, in the isolated mesenteric artery of the rat, the acetylcholine-induced endothelium-dependent hyperpolarization involves the activation of both IK_Ca and SK_Ca and is reproducible over time, while the endothelium-independent hyperpolarization elicited by anandamide is tachyphylactic and involves the activation of K_ATP [212]. Furthermore, EDHF-mediated responses are not inhibited by pharmacologically relevant concentrations of the CB₁ receptor antagonist [212,1227,1248,1828]. Finally, some of the effects of anandamide are endothelium-dependent, i.e., anandamide, depending on the vascular bed, releases NO, prostacyclin and induces EDHF-mediated responses or conversely inhibits these responses [212,221,390,462,1248,1828].

4.4.1.2. Physiological and Pathophysiological Cardiovascular Effects of CB 1 and CB2 Receptor Activation.

Nevertheless, anandamide and some of the other endocannabinoids, besides their actions in the central and peripheral nervous system, are likely to play an important role in the regulation of the cardiovascular system [648,1275,1278]. In vivo administration of cannabinoids evokes hypotension, bradycardia, and depressed cardiac contractility. Both in isolated blood vessels and in anesthetized animals, endogenous and exogenous cannabinoids usually have vasodilator properties. In vitro, the vasodilatory effect of endocannabinoids and synthetic analogues is tissue- and species-specific and could result from the activation of CB1 receptor, the endothelial “abnormal” cannabinoid receptor and/or TRPV1 channels, and involves NO-mediated or NO-independent mechanisms [1278]. Under numerous pathological conditions (hemorrhagic, endotoxic and septic shock, advanced liver cirrhosis and heart failure), the endocannabinoid system may become over-activated and contribute to hypotension/cardiodepression through the activation of cardiovascular CB1 receptors [1174,1175]. Endocannabinoid-mediated CB1 signaling is also involved in the pathogenesis of atherosclerosis. If, on the one hand, endocannabinoids released from endothelial cells, macrophages or platelets reduce hypertension, on the other hand, they induce CB1-dependent platelet and macrophage activation, endothelial generation of reactive oxygen species, endothelial dysfunction and death as well as smooth muscle cell migration and proliferation [1272,1273,1276,1469]. The endocannabinoid system is also involved in the regulation of food intake and obesity-related metabolic disorders, including alterations in lipid profile and glucose homeostasis [394,996]. Clinical trials have shown the pharmacological CB1 blockade to have multiple therapeutic benefits in obesity and associated cardiometabolic disorders. However, the increased incidence of anxiety and depression in treated patients has halted these clinical trials [754,1354].

Activation of CB2 receptors attenuates endothelial inflammatory response, chemotaxis and adhesion of inflammatory cells to the activated endothelium and the consequent release of a variety of proinflammatory mediators, key processes involved both in the initiation and progression of atherosclerosis and restenosis, and in the mediation of reperfusion-induced tissue damage. CB2 receptor activation also diminishes smooth muscle cell proliferation. The potent anti-inflammatory and anti-mitogenic effects of CB2 receptors could be of interest in ischemia–reperfusion injuries and in atherosclerosis [1174,1175].

4.5.2.1. Endothelins.

A turning point in the quest to identify endothelium-derived contracting factors was the discovery that, besides superoxide anions, endoperoxides and thromboxane A₂, endothelial cells in culture synthesize and secrete a vasoconstrictor peptide [544,645,1313]. A gene coding for this peptide was then cloned and the product of this gene, endothelin-1 (ET-1), a peptide of 21-amino acid residues, was identified as one of the most potent known vasoconstrictors [1741]. Soon after, two other closely related peptides, ET-2 and ET-3, coded by two different genes, were characterized [708]. Nevertheless, ET-1, an autocrine, paracrine and endocrine factor, is the most abundant and important endothelin isoform produced by endothelial cells. However, endothelins are also produced by a wide range of tissues, and indeed cells other than the endothelial cells are likely to be involved in the major role that these peptides play in development and/or physiology [859,1355] (Figure 33).

FIGURE 33

The endothelins. ET: endothelin; ECE: endothelin-converting enzyme.

4.5.2.1.2. Pathophysiological Action of Endothelins

4.5.2.1.2.1. Endothelin Receptors.

Endothelins interact with two G-protein-coupled receptors termed ET_A and ET_B (Figure 33). ET-1 is the preferential ligand for the ET_A receptor subtype, while the three peptides show a similar affinity toward the ET_B receptor subtype [43,1334]. The ET_A receptor is widely distributed and can be expressed in virtually every cell type of the body, but is especially predominant in vascular smooth muscle cells. The distribution of the ET_B receptor is restricted to the cardiovascular and pulmonary systems, neurons, bone, pancreas and kidneys, but its expression is elevated in endothelial cells and renal tubules [828,1662].

ET-1, beyond its function as a vasoactive peptide, also plays a crucial role in the atherogenic process by enhancing mitogenesis, inducing extracellular matrix formation and contributing to the development of inflammation within the vessel wall [1355]. Both ET_A and ET_B receptors are localized on vascular smooth muscle cells where they induce their vasoconstrictor, proliferative and hypertrophic action, but in arteries, the ET_A receptor is the predominant vasoconstrictor receptor [1315] (Figure 34).

FIGURE 34

Endothelin-1 and regulation of vascular tone. Endothelin-1 (ET-1) is a vasoconstrictor predominantly by activating the endothelin A receptor subtype (ETA). It can also produce vasodilatation by stimulating the endothelin ETB receptor subtype (ETB) and (more...)

The contractions elicited by ET_A receptor activation are unusual when compared to those produced by most other agonists as they are slowly developing and long lasting even after washing out the peptide. The almost irreversible binding of the peptide to this receptor and its persistent association even after internalization are likely to explain, at least in part, this prolonged signaling [828]. The signal transduction following ET_A receptor stimulation involves several G-protein-dependent and -independent pathways, including phospholipase C, phospholipase A₂, adenylate cyclase, Rho kinase, transactivation of receptor tyrosine kinases, beta-arrestin and mitogen activated protein kinase cascades. The G-protein-dependent activation of phospholipase C and phospholipase A (predominantly Gq/11 and G12/13) leads to the usual cascade of events, i.e., on the one hand, the formation of IP₃ and diacylglycerol and the resulting activation of protein kinase C, and on the other hand, the metabolism of arachidonic acid. The G-protein-independent pathway involves a direct interaction with β-arrestin, leading to the src/extracellular signal-regulated kinase/mitogen-activated protein kinase pathway (src/ERK/MAPK). These pathways contribute to the increase in [Ca²⁺]_i, linked to the facilitation of calcium influx and calcium mobilization, and the changes in calcium sensitivity, both being essential for ET-1-induced contraction (and proliferation) of vascular smooth muscle cells [719,828,1315,1551].

In the vascular wall, the ET_B receptor is predominantly expressed in the endothelial cells and, beyond its role as a clearance receptor, its activation is associated with an increase in [Ca²⁺]_i and leads to the release of the potent vasodilators and anti-proliferative factors, NO and prostacyclin [307,1551]. Both in vivo and in vitro, these endothelial effects of ET-1 counterbalance the effects of ET_A stimulation on the vascular smooth muscle cells. However, in numerous cardiovascular diseases, this response is impaired and is associated with endothelial dysfunction, leaving the ET_A-dependent responses unimpeded [1551]. In vascular smooth muscle cells, the activation of ET_B receptors produces vasoconstriction, the signaling pathway being similar to that described for the ET_A receptor (Figure 34).

However, besides the endothelial cells, in the renal collecting duct, the ET_B receptor is highly expressed and plays a major role in regulating sodium excretion and therefore arterial blood pressure [828].

Additionally, the ET_A receptor can form heterodimers with the ET_B receptor and possibly with other receptors, suggesting that the effects of ET-1 may be much more complex than those predicted so far [1551,1662].

4.5.2.1.2.2. Lessons from Genetically Modified Animals.

The total deletion of the endothelin-1 gene is lethal due to craniofacial deformity [859], and the phenotype of the ET_A receptor knockout mice is very similar [260]. The disruption of murine ET-3 or the ET_B receptor gene produces a phenotype of aganglionic megacolon and pigmentary disorders that are similar to those observed in Hirschsprung's disease, characterized by an absence of enteric ganglia in the distal colon and a failure of innervation in the gastrointestinal tract [673,1256]. As mentioned earlier, mice deleted for ECE-1, which catalyzes the proteolytic activation of big-ET-1 to ET-1 as well as big-ET-3 to ET-3, show the combined phenotype of ET_A or ET-1 knockout mice and the phenotype of ET_B or ET-3 knockout mice [1742]. Heterozygous knockout mice for ECE-1 develop normally but show an impaired ventilatory response to hypoxia [1290], suggesting that critical levels of endothelins are required for normal foetal development.

Therefore, the extremely severe phenotype of homozygous mice knockout for any component of the endothelin-ET_A system does not allow proper appraisal of the role of ET-1 in cardiovascular physiology and pathophysiology. The arterial blood pressure of heterozygous knockout mice for the ET_A receptor is normal, while the heterozygous knockout mice for the ET_B receptor are hypertensive [102]. Hypertension in these latter mice was initially attributed to a decrease in ET-1 clearance, but specific deletion of the ET_B receptor in endothelial cells produced normotensive mice [55]. Since knocking out ET-1 specifically in the cells of the collecting duct causes hypertension and sodium retention [19], ET-1 generated in the kidney may promote natriuresis and diuresis and contribute to the lowering of blood pressure, by activating ET_B receptors located most likely on the inner medullary collecting duct cells [827]. Transgenic mice overexpressing ET-1 are normotensive with renal pathology leading to renal fibrosis and fatal kidney disease [656]. Thus, these genetic murine models emphasize the importance of ET-1 in the kidney. Additionally, ET-1 may regulate tubular sodium delivery by controlling blood flow, i.e., vasoconstriction in the afferent arterioles and vasodilatation in the efferent arterioles [828].

Mice with specific conditional deletion of the ET-1 gene in the endothelial cells are hypotensive and provide direct evidence for a paracrine vasoregulatory pathway mediated by endothelial cell-derived ET-1 acting on the vascular smooth muscle ET_A receptor [814]. Conversely, transgenic mice overexpressing human ET-1 gene selectively in the endothelium exhibit vascular injury in the absence of blood pressure elevation. This overexpression results in early changes in gene expression associated with enhanced lipid biosynthesis, leading to an acceleration in the progression of atherosclerosis [1426].

4.5.2.1.2.3. Cardiovascular Diseases.

In endothelial cells, ET-1 is stored in Weibel–Palade bodies and in storage vesicles, suggesting that the peptide could be released on demand [615,1325]. Constitutive release of ET-1 maintains basal tone and blood pressure as it has been shown in genetically modified mice [814] and contributes to basal vascular tone in normotensive human subjects [626]. However, attempts to link the peptide to the normal control of moment-to-moment changes in vascular tone were not very conclusive. Nevertheless, it has been recently demonstrated that an acute de novo endothelial synthesis and release of ET-1 in response to thrombin could occur in aortic rings of aging rats and contribute to the contractile response of the underlying smooth muscle cells [557].

A causal or early role of ET-1 in pathologies, such as hypertension, is not obvious [1109,1597]. If in various models of hypertension (such as salt-sensitive hypertension or hypertension associated with insulin resistance and genetic forms of hypertension) endothelin-1 may participate in blood pressure elevation and vascular growth, the endothelial damage caused by a rise in blood pressure may activate the expression of endothelin-1 in blood vessels and in the heart. Thus, the involvement of the endothelin system may be secondary to hypertension rather than primary [1356]. ET-1 could possibly contribute to the late stages of vascular and possibly other diseases [58,69,114,1399]. The availability of endothelin-antagonists has confirmed that endothelin-1 may progressively increase with age [1546,1593] and in diseases such as pulmonary hypertension [114,311,500]. In the latter case, besides the direct vasoconstrictor effect of the peptide [66], the trophic and mitogenic effect of endothelin-1 must play a key role [294]. The production of this peptide is therefore a sign of pathology and, under normal circumstances, it does not appear to play a preponderant role in vascular homeostasis [1598]. Once initiated, the production of ET-1 not only progresses linearly with time (at least in cultured endothelial cells) but can also be up-regulated by a number of factors believed to be involved in vascular disease [298,695,1359]. Thus, under normal physiological conditions, the production and/or the action of endothelins have to be kept under control by a counteracting system [1598].

4.5.2.2. Angiotensin II.

The renin–angiotensin system (RAS) has traditionally been viewed as a circulating system. Kidney-derived renin cleaves liver-derived angiotensinogen to form angiotensin I in circulating blood. Subsequently, angiotensin I is converted into angiotensin II, the main effector peptide of the RAS, by angiotensin-converting enzyme (ACE) predominantly located at the luminal side of the endothelium. Angiotensin II exerts its effects by activating angiotensin II receptors, of which at least two types have been described, AT₁ and AT₂. However, nearly 25 years ago, it was suggested that, besides the circulating RAS, angiotensin II could be produced locally by a vascular RAS [292,359].

In isolated arteries, a renin substrate, a tetradecapeptide mimetic of angiotensinogen and angiotensin I could be metabolized to form angiotensin II, via renin-dependent and -independent pathways [385,416,426,864]. However, whether or not endothelial cells do express renin remains controversial since renin binds to the vascular wall and can be internalized. Additionally, tissue angiotensin II generation may occur not only on the endothelial cell surface but also within the cells. So angiotensin II-derived from the vasculature could be involved in the autocrine and paracrine control of vascular resistance and the compliance of large conduit arteries. Tissular generation of angiotensin II may also occur in the heart, brain, pancreas and adipose tissue [292,459].

It is now known that the RAS is a much more complex system that overlaps other systems (for instance, the bradykinin/kallikrein system) and includes other enzymatic pathways (ACE-2, NEP, PEP, trypsin, kallikrein, cathepsin, tPA, aminopeptidases), leading to the generation of various other angiotensin peptides (pro-angiotensin 12, angiotensin 1–9, 1–7, 1–5, angiotensin III, angiotensin IV), some acting at other receptor sites (MAS). Furthermore, ACE and the (pro)renin receptor have been identified as membrane receptors able to activate an intracellular signaling cascade [167,458,1063,1118]. The ACE-2-dependent production of angiotensin 1–7 plays a significant role in the regulation of heart and kidney function and acts as a physiological regulator of the classic renin–angiotensin system by counteracting its detrimental effects (Figure 35).

FIGURE 35

Renin–angiotensin and bradykinin–kallikrein systems. The “original” renin–angiotensin system is shown in dark gray while the recently identified additional pathways are shown in pale gray. Additionally, the interconnections (more...)

Activation of the AT1 receptor subtype causes renal and systemic vasoconstriction, vascular smooth muscle proliferation and promotes tubular sodium reabsorption directly at the proximal tubule and indirectly via the action of aldosterone at the distal nephron. These combined effects allow the maintenance of extracellular fluid volume in a context of fluid loss and dehydration, but favor the development of hypertension under pathophysiological conditions. Additionally, as mentioned earlier, the stimulation of the renin–angiotensin system is associated with the generation of oxidative stress, mainly through the activation of NAD(P)H oxidase (Figure 13). ROS impairs NO and EDHF-mediated relaxation and enhances COX-dependent contractions causing endothelial dysfunction. Additionally, the generation of ROS, in a positive feedback loop, activates the RAS, leading to a vicious circle exacerbating the end-organ damage and atherosclerosis associated with hypertension [400,431,883,1730].

In conclusion, the renin–angiotensin–aldosterone system plays a predominant role in the control of cardiovascular homeostasis. Extremely useful therapeutic targets have been identified within the classic RAS system, i.e., renin, ACE, AT1 receptor, aldosterone receptor, and the relevance of the new targets recently identified in this expended system is currently being assessed [265,497,966,1199,1596].

4.5.2.3. Urotensin-II and Urotensin-II-Related Peptide.

Urotensin-II and urotensin-II-related peptide are two closely related peptides of 11 and 8 amino acids, respectively, issued from two different genes. They share the same receptor, a G-protein-coupled receptor identified as the orphan receptor GPR14 [34]. Urotensin-II is widely expressed within the cardiovascular system, including by the endothelial cells [1324]. Urotensin-II is one of the most, if not the most, potent vasoconstricting peptide identified to date, but it also produces endothelium- and NO-dependent relaxations. Besides, this peptide promotes vascular smooth muscle cell proliferation, produces inotropic and hypertrophic effect in cardiac muscle, regulates renal vascular tone and glomerular filtration and shows effects outside the cardiovascular system including inhibition of insulin release and regulation of food intake [1307].

The expression of urotensin-II is increased in various cardiovascular diseases, including hypertension, preeclampsia, coronary and renal diseases, heart failure, and type II diabetes [1811]. However, the stimuli leading to the release of urotensin-II have not been properly identified, and whether or not it plays a detrimental or a beneficial role (or both) is not yet completely understood [1324].

4.5.2.4. Neuropeptide Y.

Neuropeptide Y (NPY) is a 36-amino acid peptide belonging to a family which, in mammals, also includes peptide YY (PYY) and pancreatic peptide (PP). They interact, with a variable degree of affinity with 4 to 5 G-protein-coupled receptors, termed NPY Y₁, NPY Y₂, NPY Y₄, NPY Y₅ and NPY y6, the latter being functionally expressed only in the mouse and rabbit. They are involved in a variety of physiological functions such as the regulation of appetite, circadian rhythm, anxiety, gastrointestinal motility and cardiovascular system, including heart rate, regional blood flow, vascular smooth muscle cell growth and angiogenesis [349,420,421,423,878]. The NPY activity is modulated by the endothelial ectoenzyme dipeptidyl peptidase IV (DPPIV/cd26), which inactivates the NPY Y1-agonistic activity of NPY but generates the active fragment NPY(3–36), agonist of both NPY Y2 and Y5 receptors [415,1824].

NPY is widely expressed, especially in the brain, the adrenal medulla and peripheral sympathetic nerves where it plays an important role in modulating the contribution of the sympathetic nervous system to the control of vascular tone. NPY is also expressed in the vasculature including in endothelial and endocardial cells, and can act as an autocrine and paracrine mediator [2,726,1425]. In HUVEC, this expression is modulated by inflammatory mediators such as LPS, TNFα or interferon γ, suggesting that NPY could link sympathetic nerves, blood vessels and the immune system [1424,1824].

NPY Y1 receptors are expressed in vascular smooth muscles and potentiate the contractile action of norepinephrine and ATP. This receptor is also responsible for smooth muscle growth and proliferation and could be involved in neointima formation. The NPY Y2 receptor is expressed in sympathetic nerves terminals and modulates the pool of co-transmitters released at the neuroeffector junction. Therefore, NPY plays a dual role as a modulator of sympathetic co-transmission, on the one hand, it facilitates vascular smooth muscle reactivity and, on the other hand, it modulates the presynaptic release of neurotransmitters. Additionally, the angiogenic effect of NPY, as shown by endothelial cell adhesion, migration and proliferation, as well as vessel sprouting and capillary tube formation, is mainly attributed to NPY Y2 and possibly also to Y1 and Y5 receptor stimulation [962,1237,1825].

However, so far, there is little evidence to show that endothelial release of NPY contributes to the moment-to-moment regulation of vascular tone.

4.5.2.5. CNP.

The natriuretic peptide family includes four distinct peptides, atrial natriuretic peptide (ANP), brain natriuretic peptide (BNP), C-type natriuretic peptide (CNP) and urodilatin, which are involved in the control of body fluid homeostasis and blood pressure. CNP, a 22-amino acid peptide, was originally isolated from the porcine brain. In contrast to ANP and BNP that are synthesized predominantly in the heart, CNP is produced in the central nervous system but has also been detected in peripheral endothelial cells [835,1099]. Endothelial cells express the transcript of the CNP receptor gene, synthesize and constitutively release CNP. The gene expression and release of CNP are regulated by various cytokines [1111,1460,1468].

CNP evokes relaxation and hyperpolarization of arterial and venous smooth muscle cells, and these vasodilator effects are generally attributed to the activation of natriuretic peptide receptor B subtype (NPR-B) on the smooth muscle, followed by the stimulation of particulate guanylate cyclase, leading to accumulation of cGMP and the subsequent opening of BK_Ca and K_ATP channels [669,1303,1468]. In porcine coronary arteries and canine femoral veins, CNP induces the accumulation of cyclic-GMP and opens BK_Ca, and this peptide has therefore been proposed as a potential endothelium-derived hyperpolarizing factor [60]. Furthermore, in the rat mesenteric artery, it has been suggested that CNP, when released by the endothelial cells, would activate the smooth muscle NPR-C receptor subtype and that the following cGMP-independent activation of a G-protein regulated inward-rectifier potassium channel (GIRK) would provoke the hyperpolarization of the smooth muscle cells [214,1616]. However, in various vascular tissues, this hypothesis has not been confirmed [68,336,903] and in mice deficient for the NPR-C gene, EDHF-mediated responses are not altered [1021].

CNP is therefore unlikely to act as an endothelium-derived relaxing/hyperpolarizing substance or to contribute to moment-to-moment endothelium-dependent regulation of vascular tone. Nevertheless, this peptide plays a key role in preventing smooth muscle proliferation, leukocyte recruitment and platelet reactivity. As such, CNP is likely to exert an anti-atherogenic influence on the blood vessel walls [17,961,1345].

4.5.2.6. Adrenomedullin, CGRP, VIP, PACAP.

Calcitonin gene-related peptides (αCGRP and βCGRP) and adrenomedullin are extremely potent vasodilator peptides belonging to a peptide family that also includes calcitonin and amylin [136,815,1245]. These peptides activate a family of CGRP receptors consisting of a calcitonin receptor-like protein linked to one of the three receptor activity-modifying proteins (RAMPs) which are required for the functional activity of the receptor [136,1245].

Adrenomedullin was originally identified in the extracts of human pheochromocytoma tissue but is expressed constitutively in various other cells including epithelium and cardiac myocytes, fibroblasts, vascular smooth muscle and especially endothelial cells [813,815,1471]. The vasodilator effects of adrenomedullin are predominantly endothelium-dependent, the endothelium-independent relaxations being explained by the increased formation of cAMP. Thus, adrenomedullin is a potent endothelium-dependent vasodilator but is unlikely per se to be a relevant endothelium-derived relaxing factor. Besides, adrenomedullin has a wide range of biological actions such as reduction of oxidative stress and inhibition of endothelial cell apoptosis and could be of potential therapeutic interest in arteriosclerosis. Additionally, adrenomedullin possesses angiogenetic properties [786].

CGRP is expressed mainly in neurons and endocrine cells [136], but can also be expressed in endothelial cells where the peptide is stored in Weibel–Palade bodies [1171,1705]. CGRP is predominantly an endothelium-independent relaxing substance and, in most blood vessels, the cAMP-dependent relaxations are associated with the opening of K_ATP and, in some instance, BK_Ca channels. Similarly, endogenous CGRP, released by transmural nerve stimulation or by capsaicin analogues, evokes endothelium-independent relaxations which involve activation of K_ATP [136]. CGRP receptor antagonists do not inhibit endothelium-dependent responses in any of the studies conducted to date. However, in the hepatic artery of the rat [664] and possibly also in the mesenteric artery of the same species 1382], acetylcholine releases CGRP from sensory nerves producing an endothelium-independent relaxation and hyperpolarization. Thus, CGRP should be considered as a nerve-derived relaxing/hyperpolarizing factor [664].

Vasoactive intestinal peptide (VIP) and pituitary adenylyl cyclase-activating polypeptide (PACAP) belong to the superfamily of structurally related peptides which includes glucagon, glucagon-like peptides, secretin and growth hormone-releasing factor. VIP produces not only endothelium-dependent relaxations by releasing NO and/or prostacyclin but also endothelium-independent relaxations by stimulating adenylyl cyclase and activating K_ATP, BK_Ca or K_V [636]. PACAP is predominantly an endothelium-independent vasodilator, also via the activation of adenylyl cyclase [1603]. However, whereas VIP and PACAP are well established as neuroendocrine hormones and neurotransmitters, there are only a few anecdotal reports of the expression of these peptides in endothelial cells [826,875]. Therefore, neither VIP nor PACAP can qualify as endothelium-derived mediators. Nevertheless, as both peptides are expressed in nerve endings surrounding arteries and veins, they can be considered, in a similar manner to CGRP, as nerve-derived relaxing/hyperpolarizing factors.