Hypertension. Endothelium-dependent contractions, associated with endothelial dysfunctions, were observed first in the isolated aorta of spontaneously hypertensive rats (SHR; Luscher and Vanhoutte, 1986). They have been extensively characterized thereafter in that blood vessel. In the arteries of this hypertensive model, the generation of a diffusible EDCF opposes the relaxing effect of NO. Endothelium-dependent contractions are positively correlated with the severity of hypertension and the aging process, are delayed in female SHR and also occur in aging normotensive Wistar–Kyoto control rats (WKY; Félétou et al., 2009).
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 (i) abnormal calcium handling, (ii) an increased expression of COX-1, (iii) the associated enhanced production of reactive oxygen species, (iv) a major increase in prostacyclin synthase expression, (v) the enhanced release of prostacyclin, thromboxane A2, and possibly PGH2. In vascular smooth muscle cells, they include (i) an exacerbated response of the TP receptor to prostacyclin and PGH2, (ii) a deficient IP receptor function and (iii) an early dysfunction in the adenylyl cyclase pathway (Félétou et al., 2009; 2010a,b).
When compared with WKY aorta and in response to receptor-mediated stimuli (acetylcholine), the amplitude of the endothelium-dependent contractions and the increase in intracellular calcium ([Ca2+]i) in SHR endothelial cells 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 (Gluais et al., 2005; 2006; Tang et al., 2007). 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, the activation of the calcium-independent phospholipase A2 (iPLA2) allows the store-operated calcium channels (SOC)–dependent influx of extracellular calcium and the subsequent activation of cPLA2. It mediates the initial part of the signalling cascade leading to endothelium-dependent contractions of the SHR aorta in response to acetylcholine. Substances, such as calcium ionophores, that bypass the cell membrane receptors causes an increase in [Ca2+]i, and a direct activation of cPLA2 (Wong et al., 2010b). Therefore, the iPLA2 pathway associated with calcium mobilization is defective in SHR endothelial cells (Figure 4).
Figure 4. Calcium signalling and the COX-1 production of endothelium-derived contracting factors (EDCF). Acetylcholine (ACh) activates muscarinic receptors (M) on the endothelial cell membrane and triggers the release of calcium from intracellular stores. The resulting calcium depletion process displaces the inhibitory calmodulin (CaM) from iPLA2. Activated iPLA2 produces lysophospholipids (LysoPL), which in turn open store-operated calcium channels (SOCs) leading to the influx of extracellular calcium into the endothelial cells. This large influx of calcium ions then activates cPLA2, which catalyses the production of arachidonic acids (AA). The later is then metabolized by cyclooxygenase-1 (COX-1) to prostanoids. cPLA2, calcium dependent phospholipase A2; EC, endothelial cells; iPLA2, calcium independent phospholipase A2 (modified from Wong and Vanhoutte, 2010).
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The subsequent steps involve the activation of COX and the production of reactive oxygen species along with that of prostanoids. Aortic endothelial cells express preferentially COX-1 versus COX-2 (Kawka et al., 2007; Tang and Vanhoutte, 2008). In SHR endothelial cells, the mRNA and protein expression of COX-1 are enhanced when compared with that of WKY, and in the two strains, both are augmented by aging (Ge et al., 1995; Tang and Vanhoutte, 2008). Conversely, in SHR, the decrease expression of COX-1 produced by a chronic treatment with vitamin D reduces the endothelium-dependent contractions (Wong et al., 2010a). In response to acetylcholine, endothelium-dependent contractions and the associated generation of PGs are blocked consistently by selective inhibitors of COX-1 and partially inhibited, although to various extent depending on the experimental conditions, by selective inhibitors of COX-2 (Ge et al., 1995; Yang et al., 2003a; Gluais et al., 2005; 2006). However, if the endothelium-dependent contractions and the release of PGs by A 23187 are also fully blocked by COX-1 inhibitors, these responses are less sensitive to COX-2 inhibition (Figure 5). 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 (Morita, 2002). 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 thromboxane A2 (Mitchell et al., 2006; Warner et al., 2006). 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 (Tang et al., 2005).
Figure 5. Effects of inhibitors of COX-1 and COX-2 on endothelium-dependent contractions and prostaglandins production in SRH aortic rings. Top panels: effects of acetylcholine. Lower panels: effects of A 23187. The effects of A23187 are less sensitive to the COX-2 inhibitor, NS 398, than those produced by acetylcholine. Data are shown as means ± SEM. The asterisk indicates a statistically significant effect of a COX inhibitor. The sharp sign indicates that the response in presence of the COX-1 inhibitor, SC 560, is significantly different from the response observed in the presence of NS 398. COX, cyclooxygenase; SHR, spontaneously hypertensive rat.
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Additionally, COX is also involved in the endothelial generation of reactive oxygen species, a key factor in the generation of endothelium-dependent contractions (Yang et al., 2002; Tang et al., 2007). Reactive oxygen species decrease NO bioavailability (Gryglewski et al., 1986; Rubanyi and Vanhoutte, 1986) and, as a positive feedback loop, the formation of hydroperoxides further activates COX (Morita, 2002). In addition, since reactive oxygen species diffuse towards the vascular smooth muscle cells, they can stimulate COX in these cells and produce more contractile prostanoids.
The generated PGs diffuse towards the vascular smooth muscle cells and directly activate TP receptors (Luscher and Vanhoutte, 1986; Auch-Schwelk et al., 1990; Yang et al., 2003a). In the rat aorta, the five major PGs and 8-isoprostane produce contractions that predominantly involve TP receptor activation (Figure 2). However, the involvement of PGD2 and 8-isoprostane in endothelium-dependent contractions can be ruled out since their generation is not affected by acetylcholine (Gluais et al., 2005).
In SHR aortic endothelial cells, the expression of thromboxane synthase is enhanced when compared with that in WKY endothelium (Tang and Vanhoutte, 2008). In response to ATP or the calcium ionophore A 23187, this is associated with an increase generation of thromboxane A2, and the endothelium-dependent contractions are partially inhibited by dazoxiben, a selective inhibitor of thromboxane synthase that abrogates the production of thromboxane A2 (Gluais et al., 2006; 2007). 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 (Koga et al., 1989; Kato et al., 1990; Ge et al., 1995; Gluais et al., 2005; 2006; 2007).
Paradoxically, prostacyclin is likely to be a major EDCF in SHR aorta. In SHR endothelial cells, prostacyclin is by far the most abundant PG released in response not only to receptor-dependent stimuli but also to calcium ionophores (Gluais et al., 2005; 2006; 2007). This may come as a surprise since prostacyclin synthase is rapidly nitrosylated and inactivated by peroxynitrite (Zou et al., 2002a,b; Schmidt et al., 2003). However, in the SHR aorta, the massive increase in the expression of prostacyclin synthase (Tang and Vanhoutte, 2008) may compensate the loss of activity due to peroxynitrite-dependent tyrosine nitration. Furthermore, in that preparation, prostacyclin does not produce relaxations but evokes TP receptor–dependent contractions (Rapoport and Williams, 1996; Gluais et al., 2005; Figure 2). In fact, prostacyclin, like PGH2, is also more potent in producing contraction in SHR than in WKY aortae (Ge et al., 1995; Gluais et al., 2005). 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 (Anand-Srivastava, 1993; Gomez et al., 2008). In order to explain this specific smooth muscle cell dysfunction, a decrease in the aortic expression of IP receptors (Numaguchi et al., 1999) and an early impairment of adenylyl cyclase signalling have been evoked (Anand-Srivastava, 1988; Masuzawa et al., 1989). However, these two hypotheses can only, at best, partially explain the total disappearance of IP receptor–mediated relaxations in SHR aorta. Indeed, the decrease expression of the IP receptor has not been confirmed in latter experiments (Tang and Vanhoutte, 2008), and when compared with 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 (Gomez et al., 2008). A potential additional/alternative hypothesis, which requires proper validation, could be the oxidative damage of the IP receptor itself, which contain redox-sensitive cysteines that play an essential role in determining its structure, addressing and function (Stitham et al., 2006).
Prostacyclin has also been identified as a major contributing factor accounting for the endothelial dysfunction in the aorta and mesenteric artery of WKY and SHR treated with aldosterone (Blanco-Rivero et al., 2005; Xavier et al., 2008). Thus, although as a rule prostacyclin is a vasodilator and an anti-aggregating agent, depending on the circumstances, the prostanoid can also act as an EDCF.
Any levels 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 in that of WKY, the endoperoxide is also a suitable candidate as EDCF (Kato et al., 1990; Ge et al., 1995; Gluais et al., 2005; Figure 2). Finally, the shunting of PGH2 metabolism towards other metabolic pathways can lead to a variety of products, including PGE2 and/or PGF2α, which also produce contractions by activating TP receptors (Figure 2). Therefore, thromboxane A2, PGH2, PGI2, PGE2 and PGF2α can all act theoretically as EDCF (Gluais et al., 2005; Félétou et al., 2010a,b).
In addition, in the SHR aorta, PGE2-mediated relaxations are impaired, which could contribute to the observed endothelial dysfunction (Tang et al., 2008) and, in the femoral artery of diabetic rats, activation of the EP1 receptor contributes to the endothelium-dependent contractions (Shi et al., 2007).
Furthermore, some alterations at the level of the TP receptors should also be considered. Hydrogen peroxide prevents the translocation and degradation of TP receptors, increasing their density at the cell membrane and TP activation enhances TP stability through a reactive oxygen species–dependent post-transcriptional mechanism (Valentin et al., 2004; Wilson et al., 2009). This may explain the enhanced TP receptor–dependent contractions in response to PGH2, prostacyclin and exogenously generated reactive oxygen species observed in SHR aorta (Auch-Schwelk et al., 1989; Ge et al., 1995; Yang et al., 2002; 2003b, Gluais et al., 2005; García-Redondo et al., 2009). In addition, TP receptors are also expressed in endothelial cells and their stimulation induces the Rho kinase–dependent inhibition of NO production (Liu et al., 2009). Conversely, the isoform α of the human TP receptor is negatively and independently regulated by either NO or prostacyclin, following the phosphorylation of serine residues by protein kinase G and A respectively (Reid and Kinsella, 2003). Additionally, NO can inhibit the activity of thromboxane synthase (Wade and Fitzpatrick, 1997), indicating that a decrease in NO bioavailability may facilitate the TP receptor–dependent signalling pathway. Finally, 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 (Félétou et al., 2009). In these peripheral arteries, the endothelial dysfunction additionally includes a marked attenuation of the EDHF-mediated component of the endothelium-dependent relaxations (Félétou and Vanhoutte, 2006b). TP receptor stimulation induces a loss in the activity of endothelial small conductance calcium-activated potassium channels (Crane and Garland, 2004; McNeish and Garland, 2007), an essential component of EDHF-mediated responses (Félétou and Vanhoutte, 2006b). Conversely, the impairment of EDHF-mediated responses can favour the development of endothelium-dependent contractions (Michel et al., 2008).