INVITED REVIEW ARTICLE
Endothelial Dysfunction in Resistance Arteries of Hypertensive Humans: Old and New Conspirators Agostino Virdis, MD and Stefano Taddei, MD
(See Editorial: A Tireless Giant in Vascular Research by Aimin Xu and Yu Huang. Journal of Cardiovascular Pharmacology, 2016 67:5;359–360)
Abstract: A large body of homogeneous reports documented that endothelial dysfunction, which characterizes human essential hypertension is largely dependent on an impaired endothelial nitric oxide availability and an increased production of endothelium-derived contracting factors. These factors include endothelin, prostanoids, and reactive oxygen species (ROS). In particular, it was evidenced that acute intraarterial administration of indomethacin, a nonselective cyclooxygenase (COX)-inhibitor, and ascorbic acid, an antioxidant, normalized the blunted endothelial dysfunction by restoring nitric oxide availability at the level of peripheral microcirculation, thus demonstrating that COX-derived endothelium-derived contracting factors are one of the major sources of ROS. Recent studies put in evidence new lights on the mechanisms involved in endothelial dysfunction in human hypertension, identified as “new conspirators.” In particular, functional and immunohistochemical experiments with selective COX inhibitors identified the isoform COX-2 as the main source of intravascular ROS generation in isolated small vessels from essential hypertensive patients. In addition, important vascular protective properties by human ghrelin have been demonstrated in human hypertension, in terms that its systemic reduction is involved in the pathophysiology of endothelial dysfunction, while a normalization of its levels may restore vascular homeostasis. Key Words: cyclooxygenase, endothelium, ghrelin, oxidative stress (J Cardiovasc Pharmacol Ô 2016;67:451–457)
INTRODUCTION There is no doubt that endothelium represents the fundamental homeostatic tissue for the regulation of the vascular tone and structure, which is physiologically characterized by a balance between substances with vasodilating and antithrombogenic properties, and substances with vasoconstricting and prothrombotic activities. Under healthy conditions, the most characterized compound is nitric oxide (NO), which exerts its cardiovascular protective role by relaxing Received for publication September 23, 2015; accepted January 5, 2016. From the Department of Clinical and Experimental Medicine, University of Pisa, Pisa, Italy. The authors report no conflicts of interest. Reprints: Stefano Taddei, MD, Department of Clinical and Experimental Medicine, University of Pisa, Via Roma 67, 56100 Pisa, Italy (e-mail: [email protected]). Copyright © 2016 Wolters Kluwer Health, Inc. All rights reserved.
J Cardiovasc Pharmacol ä Volume 67, Number 6, June 2016
media-smooth muscle cells, preventing leukocyte adhesion and migration into the arterial wall, muscle cell proliferation, platelet adhesion and aggregation, and adhesion molecule expression. Under disease conditions, including essential hypertension, the endothelium loses its protective role, becoming a proatherosclerotic structure.1 The loss of the normal endothelial function is referred to as “endothelial dysfunction,” characterized by impaired NO bioavailability. This can be determined by either a reduced production of NO by endothelial nitric oxide synthase (eNOS) or, more frequently, an increased breakdown by reactive oxygen species (ROS). Under such conditions, endothelial cells produce substances and mediators with vasoconstricting and prothrombotic activities, called endothelium-derived contracting factors (EDCFs). These include endothelin-1 (ET-1), prostanoids such as thromboxane A2 or prostaglandin H2, and ROS.2,3 Accordingly, both NO deficiency and activation of EDCFs are implicated in the pathogenesis of atherosclerosis and thrombosis.4 The aim of this review was to give a brief overview of the known mechanisms involved in the pathogenesis of endothelial dysfunction in the microcirculation of hypertensive patients. In particular, the recognized classical EDCFs, referred as “old conspirators,” together with the so-called “new conspirators,” according to the most recent discoveries in peripheral circulation of hypertensive patients, will be discussed.
HOW TO ASSESS ENDOTHELIAL FUNCTION IN HUMANS Compounds and substances released by endothelial cells exert a vascular autocrine/paracrine activity. For these reasons, the mechanisms regulating the endothelial physiology differ in various organs and tissues and, within the same vascular bed, largely vary according to vessel size.5 Usually, endothelial function in humans is indirectly assessed by vascular reactivity tests, by stimulation with specific external mechanical and pharmacological stimuli.5 It is possible to activate or inhibit endothelium-dependent pathways in several vascular regions and measure the vessel diameter changes induced by experimental perturbation. Specific agonists operating through specific receptors or increasing shear stress may activate endothelium-dependent pathways. In addition, it is possible to block pathways involved in endothelial responses such as NOS activity by NG-mono-methyl-L-arginine (LNMMA), hyperpolarization of vascular smooth muscle cells (by ouabain or sulfaphenazole), cyclooxygenase (COX) activity (by indomethacin or specific COX isoforms inhibitors), or oxidative stress (by antioxidant compounds).6 When www.jcvp.org |
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approaching to the evaluation of endothelium-dependent mechanisms in humans, it is crucial to distinguish between macrocirculation and microcirculation. These 2 vascular districts are differently regulated and therefore results obtained in large arteries cannot be extrapolated to microvascular regions. The next session will focus on the evaluation of endothelial function in microcirculation, whereas for assessment of endothelial function in large arteries, not discussed here, the reader is referred to excellent Guidelines on this topic.7
production, and a generation of COX-derived EDCFs.9,10 Unfortunately, its invasive nature limits the use in patients with advanced disease and precludes repeated testing. Many years ago, venous plethysmography was introduced to assess peripheral microcirculation by evaluation of forearm blood flow changes. An increased or decreased forearm blood flow changes by venous plethysmography is a highly reliable index of vasodilation or vasoconstriction, respectively. This approach was used thereafter for studying local endothelium-dependent vasodilation by the intraarterial infusion of endothelial agonists and antagonists (Fig. 1).6,11 Of note, this technique allows a great possibility to investigate several fine biochemical mechanisms accounting for endothelial dysfunction in humans, mainly by intraarterial infusion of compounds able to inhibit different pathways involved in endothelial function. Thus, the NO pathway may be specifically inhibited by L-arginine analogues such as L-NMMA, ROS by scavengers such as ascorbic acid and, finally, EDCFs by COX inhibitors.12 By this technique, a strict relationship between endothelial dysfunction and cardiovascular outcome was demonstrated.13 However, its invasiveness may limit the number of patients enrolled and the possibility to repeat testing frequently. Subcutaneous microcirculation can be studied using the Halpern–Mulvany myograph system, an in vitro ex vivo technique.14,15 This technique allows to assess functional characteristics of isolated resistance arterioles (lumen diameter 150–300 mm), taken from subcutaneous tissue obtained by skin biopsies. Once cleaned of adherent connective tissue, vessels are investigated with the “wire myograph” or the
Assessment of Microcirculation Most of the literature that evaluated endothelial function in human microcirculation is obtained in functionally isolated vascular districts such as the coronary circulation, or the peripheral muscle (usually the forearm), subcutaneous, or skin tissue. The coronary microcirculation can be evaluated by measuring coronary blood flow with Doppler flow wire and quantitative angiography during the intracoronary infusion of various substances, which can influence either endothelium-dependent or endothelium-independent vasodilation. Responses to endothelial agonists, including acetylcholine and substance P, have been measured, while nitroglycerin is usually used to assess endotheliumindependent vasodilation. In addition, intracoronary infusion of L-NMMA has defined the contribution of NO to these vasomotor responses.8 This experimental model has shown the presence of endothelial dysfunction in essential hypertensive patients with angiographically normal coronary arteries, an alteration caused by a reduction in agonist-induced NO
FIGURE 1. Schematic representation of the perfused forearm technique to evaluate endothelial function in human peripheral microcirculation. The brachial artery is cannulated for drug infusion at systemically ineffective rates, intraarterial blood pressure (BP) and Heart rate (HR) monitoring. Forearm blood flow is measured by strain-gauge venous plethysmography.
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Endothelial Dysfunction in Human Hypertension
“pressurized myograph.” Briefly, the first technique implies that 2 wires are threaded through the vessel, while in the pressurized system the artery is slipped into 2 glass microcannulae and exposed to a constant pressure.15,16 Whichever myograph approach is used, in this vascular district a reduced endothelial function in essential hypertensive patients was clearly documented.17,18 As an in vitro technique, such methodology allows exploration of several pathways and testing of many compounds nonapplicable in vivo, although the prognostic value of endothelial dysfunction in isolated small vessels from hypertensive patients is still under debate.19
and stimuli applied. Infusion of acetylcholine on SHR aorta evokes a large production of ROS within endothelial cells. This effect is prevented by indomethacin, thus COX appears as a main source of ROS in such conditions. In turn, ROS can amplify the EDCF-mediated effect, either by triggering EDCF-mediated responses or indirectly by reducing the availability of NO, thus favoring the occurrence of EDCF response.26
ENDOTHELIAL DYSFUNCTION IN HYPERTENSION: THE OLD CONSPIRATORS Experimental studies evidenced that endothelium induces contractile responses to endothelial agonists. Several agents induce contraction through the activation of various biochemical pathways leading to the production and secretion of different EDCFs in different animal species and vascular districts. Although relaxing factors play a crucial effect in the regulation of circulatory vasomotion, a large body of evidence supports the concept that also contracting factors have a significant role, which becomes particularly important in several pathological conditions, including essential hypertension.20
Human EDCFs The principal EDCF is ET-1, generated by the vascular endothelium, which acts through specific ETA and ETB receptors. ETA receptors are located on smooth muscle cells and promote growth and contraction. ETB receptors are located on both endothelial and smooth muscle cells, with opposite effects. Activation of smooth muscle cell ETB evokes contraction, whereas activation of endothelial ETB induces relaxation.21,22 The overall biological effect of these activated receptors on vasculature derives from the balance between their protective or deleterious effects, and this delicate equilibrium likely explains why this physiological substance may shift to a pathological role in cardiovascular disease, including essential hypertension.23 By using the isolated forearm technique, we previously observed that intraarterial infusion of TAK-044, an ETA/ETB receptor antagonist, caused an increased vasodilation among hypertensive patients compared with healthy subjects. Moreover, vasoconstriction to L-NMMA was decreased in hypertensive patients compared with controls. In addition, vasodilation to TAK-044 and vasoconstriction to L-NMMA showed an inverse correlation.24 These findings allowed to conclude that in essential hypertensive patients, endogenous ET-1 shows greater vasoconstrictor activity. Beyond ET-1, the main EDCFs are represented by endoperoxides, deriving from the metabolism of arachidonic acid by COX activity into the unstable intermediate PGH2, which, in turn, is converted by an array of downstream enzymes to form a range of bioactive prostanoids, including thromboxane A2 or prostacyclin.2,25 The role of ROS in endothelium-dependent contractions largely depends on the different experimental models Copyright © 2016 Wolters Kluwer Health, Inc. All rights reserved.
The Source of EDCFs Several bioassay studies conducted in different animal vascular districts documented that the endothelium-dependent contractions to arachidonic acid or acetylcholine were abolished by the COX inhibitor indomethacin, thus demonstrating that the metabolism of arachidonic acid through endothelial COX is deeply involved in the genesis of endothelium-dependent contractions.26 Of importance, COX-derived EDCFs diffuse to the underlying vascular smooth muscle cells and through the activation of specific receptors (thromboxane prostanoid receptors) induce contraction.2 Accordingly, most COX-mediated EDCFs effects are inhibited by smooth muscle cell thromboxane prostanoid– receptor antagonists.27,28 The metabolism of oxygen by cells generates ROS. In healthy conditions, the rate and magnitude of oxidant formation is counterbalanced by the rate of oxidant elimination. An imbalance between prooxidants and antioxidants results in oxidative stress, which is the pathogenic outcome of oxidant overproduction. Vascular ROS derive primarily from NAD(P)H oxidase. Other important sources include COX, dysfunctional eNOS (uncoupled NOS), and xanthine oxidase.29 Most of the available data on EDCFs in humans have been obtained in essential hypertensive patients. As already mentioned, human hypertension is associated with a reduced NO availability.1,6 The first experiments assessing the role of EDCFs on endothelial dysfunction in the forearm microcirculation of essential hypertensive patients demonstrated that intraarterial administration of the COX inhibitor indomethacin improved the vasodilation to acetylcholine and restored the inhibitory effect of L-NMMA on that response, indicating that COX generates substances that reduce the availability of NO.30,31 Moreover, intraarterial infusion of the ROS scavenger ascorbic acid evoked similar effect as indomethacin in these patients, with no further potentiation when the 2 compounds were coinfused.32 In conjunction, these findings represent the demonstration that the COX pathway is a source of ROS in essential hypertension. Of note, COX-inhibition failed to affect the acetylcholine-induced relaxation in the forearm microcirculation of patients with secondary forms of hypertension,30 thus indicating that production of EDCFs is not a consequence of a mere blood pressure increase, but is genetically related to essential hypertension. When does the EDCFs generation occur during the lifespan of a hypertensive patient? Important knowledge on this topic were provided from vascular studies on the cross-relation between the hypertensive progress and ageing. The increasing age is the most powerful determinants of endothelial dysfunction and is accompanied by a progressive www.jcvp.org |
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FIGURE 2. Schematic diagram showing the hypothetic involvement of COX-2 in endothelial dysfunction. Once activated by acetylcholine, COX-2 is able to transform arachidonic acid (AA) into prostaglandin H2 (PGH2) and then prostanoids, which in turn activate the contracting thromboxane-prostanoid receptors. Simultaneously, COX-2 is a source of ROS, with the consequent reduced NO availability.
worsening of NO availability in resistance circulation.33–36 The main mechanism responsible for age-related endothelial dysfunction in the peripheral microcirculation is a primary defect in the L-arginine–NO pathway. After the age of 60 years, along with a further impairment of the L-arginine– NO pathway, COX-dependent EDCF production becomes evident and significant.37 Such age-related endothelial dysfunction is anticipated by hypertension, which therefore represents a condition of premature vascular ageing. Thus, although not effective in younger hypertensive patients (,30 years), in adult patients (31–45 years) indomethacin begins to show some effect, and in the older patients (46– 60 and .60 years) COX-derived oxidative stress generation becomes the main determinant of endothelial dysfunction.37 In conclusion, this study evidenced that ageing is an important factor altering endothelium-dependent vasodilation and that the mechanisms involved include a defect in the L-arginine–NO pathway and production of COX-dependent EDCFs. However, whereas in normotensive subjects, ageing mainly affects the formation of NO and EDCF production characterizes only old age, the presence of hypertension causes an earlier onset of altered L-arginine–NO pathway and also earlier formation of vasoconstrictor prostanoids.
pioneering investigations have left undetermined the question of which COX isoenzymes is effectively involved.
Vascular COX-2 Isoform
As described in the previous section, studies conducted in human hypertension documented that COX pathway actively interferes with NO availability, and represents a source of ROS in the peripheral microcirculation of patients with essential hypertension. Until now, COX represents the unique pathway identified by acting as a source of ROS in human hypertension. However, these
Two different isoforms of COX are known to exist.38 In most tissues, COX-1 is regarded as constitutively expressed to produce physiological prostanoids, whereas COX-2 is often induced by a number of stimuli, including inflammation or growth factors.39 Nevertheless, COX-2 is also expressed constitutively in several organs. In particular, within the vasculature, endothelial and vascular smooth muscle cells do express both isoforms, with COX-1 being usually expressed at a higher extent than COX-2.39 Recently, we investigated which COX isoform contributes to ROS generation in human hypertension. Thus, in isolated small resistance arteries from essential hypertensive patients, the role of COX-1 and COX2, their cross-talks with NO and ROS, as well as whether other pathways may participate as adjunctive sources of ROS was assessed. In brief, it was found that in small vessels from hypertensive patients, the blunted vascular response to acetylcholine, while not modified by the COX-1 inhibitor SC560, was significantly improved by the selective COX-2 inhibitor Dup-697, which also partly restored the inhibitory effect of L-NAME on acetylcholine.40 In addition, an augmented COX-2 protein presence in vessels from these patients was documented. Furthermore, immunohistochemistry indicated a marked upregulation of COX-2 mainly in the vascular media layer.40 Of note, the fluorescent dihydroethidium technique revealed that in these patients the intravascular superoxide excess was dramatically reduced by incubation with the selective COX-2 inhibitor, and moderately blunted by the antioxidant apocynin. These data provide the first evidence that, in small arteries isolated from essential hypertensive patients, an overexpression and increased activity of COX-2
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ENDOTHELIAL DYSFUNCTION IN HYPERTENSION: THE NEW CONSPIRATOR
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Endothelial Dysfunction in Human Hypertension
plasma and stomach: acylated ghrelin, which represents the biologically active form, and desacyl-ghrelin, which although not totally biologically inactive, seems not to exert endocrine activities.43 At the beginning, ghrelin was identified as an endogenous ligand for the orphan receptor (GHS-R) and reported to exert a growth hormone-releasing activity, and to participate in the regulation of energy balance, food intake, and reducing fat utilization.44 Although basically a gastric hormone, adjunctive growth hormone-independent properties have been recently attributed to ghrelin. Among others, a variety of cardiovascular properties, including vascular effects, are ascribable to this peptide.
Vascular Effects of Ghrelin
FIGURE 3. Representative dihydroethidium staining (upper) and quantitative analysis of the red signal (lower) in small arteries from controls and essential hypertensive (EH) patients at baseline (vehicle) or after incubation with ghrelin, gp91 ds-tat, or both. *P , 0.001 versus other groups; †P , 0.05 versus control. Modified from Virdis et al.51 Adaptations are themselves works protected by copyright. So in order to publish this adaptation, authorization must be obtained both from the owner of the copyright in the original work and from the owner of copyright in the translation or adaptation.
occur, playing a major role in reducing NO availability. COX-2 represents a major source of ROS generation in essential hypertension (Fig. 2). In this context, NAD(P)H oxidase participates also, although with a minor role, in promoting superoxide generation in these patients.41 COX-2 as a main source of ROS generation likely represents a new conspirator of endothelial dysfunction or, in other words, a new source of old conspirators (ROS) and in these terms such isoform might open future therapeutic options.
Human Ghrelin Ghrelin is a peptide recently discovered from human stomachs.42 Two major forms of ghrelin are present in the Copyright © 2016 Wolters Kluwer Health, Inc. All rights reserved.
Although mainly generated and released by the gastric cells, growing evidence revealed the presence of ghrelin and its receptor, growth hormone secretagogues 1a (GHS-R 1a) at the level of the cardiovascular system. In particular, expression of ghrelin has been demonstrated in endothelial cells of human arteries and veins and in secretory vesicles of cultured endothelial cells,45 whereas GHS-R 1a has been stained within endothelial cells and vascular smooth muscle cells from different vascular beds.46 Accordingly, both animal and human reports demonstrated an active effect of this peptide on the cardiovascular system, including the endothelial function. In vitro studies documented that ghrelin acutely stimulated increased production of NO in bovine aortic endothelial cells and in human aortic endothelial cells in a timeand dose-dependent manner, using a signaling pathway that involves GHS-R 1a and increased expression of eNOS.47 Ghrelin also demonstrated an antioxidant property, an activity dependent on the activation of cellular signaling pathways leading to inhibition of enzymes involved in ROS generation, including NAD(P)H oxidase,48 thus suggesting a novel and promising mechanism of vascular protection. The first evidence of the vascular effects of ghrelin in in vivo human studies comes from patients with metabolic syndrome. In the forearm microcirculation of these patients, intraarterial infusion of human ghrelin was able to reverse endothelial dysfunction by increasing NO availability,49 and to exert a beneficial effect on NO and ET-1 imbalance,50 thus contributing to restore a balance between endothelium-derived contracting and vasodilator forces. More recently, we investigated the impact of exogenous ghrelin on endothelial dysfunction in human hypertension. To this purpose, we assessed whether exogenous ghrelin may ameliorate endothelial dysfunction in the forearm resistance circulation of patients with essential hypertension, focusing on the involvement of NO availability and ROS. Second, in isolated small arteries from essential hypertensive patients, we studied the abrogation of intravascular ROS generation and NAD(P)H oxidase pathway as possible mechanisms exerted by ghrelin. The main results indicated that acute ghrelin infusion improved endothelium-dependent vasodilation and restored the inhibition by L-NMMA on response to acetylcholine in the forearm resistance arterioles of essential hypertensive patients, thus representing the first demonstration in the peripheral microcirculation of hypertensive patients of the ability of ghrelin to restore endothelial NO www.jcvp.org |
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availability.51 The antioxidant property of ghrelin was also assessed in our study, both in the forearm resistance circulation and in isolated small vessels. In the first vascular district, acute ghrelin infusion was able to induce a great reduction of markers of oxidative stress in our hypertensive population. In isolated small arteries from hypertensive patients, the dihydroethidium staining showed that ghrelin dramatically reduced the enhanced superoxide generation. An identical result was obtained on incubation with the NAD(P)H oxidase inhibitor gp91ds-tat, and no further superoxide reduction was seen when ghrelin and gp91 were simultaneously incubated (Fig. 3). The possibility that ghrelin prevented vascular ROS generation by downregulating NAD(P)H oxidase was demonstrated by the Western blot analysis.51 Taken together these findings demonstrate, for the first time in 2 different microvascular districts of hypertensive patients, that the beneficial activity of ghrelin is related to its antioxidant property, an effect obtained through a marked inhibition of NAD(P)H oxidase activation, leading to an amelioration of NO activity. These data highlight the role of ghrelin as a physiological mediator of the vascular system: a systemic reduction of this peptide is involved in the pathophysiology of endothelial dysfunction, whereas a normalization of its levels may restore vascular homeostasis.
4. Lerman A, Zeiher AM. Endothelial function: cardiac events. Circulation. 2005;111:363–368. 5. Deanfield J, Donald A, Ferri C, et al. Endothelial function and dysfunction. Part i: methodological issues for assessment in the different vascular beds: a statement by the working group on endothelin and endothelial factors of the european society of hypertension. J Hypertens. 2005;23:7–17. 6. Virdis A, Ghiadoni L, Versari D, et al. Endothelial function assessment in complicated hypertension. Curr Pharm Des. 2008;14: 1761–1770. 7. Corretti MC, Anderson TJ, Benjamin EJ, et al. Guidelines for the ultrasound assessment of endothelial-dependent flow-mediated vasodilation of the brachial artery: a report of the international brachial artery reactivity task force. J Am Coll Cardiol. 2002;39:257–265. 8. Goodhart DM, Anderson TJ. Role of nitric oxide in coronary arterial vasomotion and the influence of coronary atherosclerosis and its risks. Am J Cardiol. 1998;82:1034–1039. 9. Egashira K, Suzuki S, Hirooka Y, et al. Impaired endothelium-dependent vasodilation of large epicardial and resistance coronary arteries in patients with essential hypertension. Different responses to acetylcholine and substance p. Hypertension. 1995;25:201–206. 10. Marzilli M, Taddei S, Virdis A, et al. Endothelial function and coronary microcirculaiton in essential hypertension. J Hypertens. 1998;16 (suppl 8):S59–S63. 11. Virdis A, Neves MF, Duranti E, et al. Microvascular endothelial dysfunction in obesity and hypertension. Curr Pharm Des. 2013;19: 2382–2389. 12. Versari D, Daghini E, Virdis A, et al. Endothelium-dependent contractions and endothelial dysfunction in human hypertension. Br J Pharmacol. 2009;157:527–536. 13. Heitzer T, Schlinzig T, Krohn K, et al. Endothelial dysfunction, oxidative stress, and risk of cardiovascular events in patients with coronary artery disease. Circulation. 2001;104:2673–2678. 14. Mulvany MJ, Baumbach GL, Aalkjaer C, et al. Vascular remodeling. Hypertension. 1996;28:505–506. 15. Virdis A, Savoia C, Grassi G, et al. Evaluation of microvascular structure in humans: a ’state-of-the-art’ document of the working group on macrovascular and microvascular alterations of the Italian society of arterial hypertension. J Hypertens. 2014;32:2120–2129. 16. Virdis A, Colucci R, Fornai M, et al. Cyclooxygenase-2 inhibition improves vascular endothelial dysfunction in a rat model of endotoxic shock: role of inducible nitric-oxide synthase and oxidative stress. J Pharmacol Exp Ther. 2005;312:945–953. 17. Rizzoni D, Porteri E, Castellano M, et al. Endothelial dysfunction in hypertension is independent from the etiology and from vascular structure. Hypertension. 1998;31:335–341. 18. Endemann DH, Pu Q, De Ciuceis C, et al. Persistent remodeling of resistance arteries in type 2 diabetic patients on antihypertensive treatment. Hypertension. 2004;43:399–404. 19. Rizzoni D, Porteri E, De Ciuceis C, et al. Lack of prognostic role of endothelial dysfunction in subcutaneous small resistance arteries of hypertensive patients. J Hypertens. 2006;24:867–873. 20. Luscher TF. Endogenous and exogenous nitrates and their role in myocardial ischaemia. Br J Clin Pharmacol. 1992;34(suppl 1):29S–35S. 21. Cardillo C, Kilcoyne CM, Cannon RO, et al. Interactions between nitric oxide and endothelin in the regulation of vascular tone of human resistance vessels in vivo. Hypertension. 2000;35:1237–1241. 22. Dhaun N, Goddard J, Kohan DE, et al. Role of endothelin-1 in clinical hypertension: 20 years on. Hypertension. 2008;52:452–459. 23. Schiffrin EL. State-of-the-art lecture. Role of endothelin-1 in hypertension. Hypertension. 1999;34:876–881. 24. Taddei S, Virdis A, Ghiadoni L, et al. Vasoconstriction to endogenous endothelin-1 is increased in the peripheral circulation of patients with essential hypertension. Circulation. 1999;100:1680–1683. 25. Vanhoutte PM. Endothelium and control of vascular function. State of the art lecture. Hypertension. 1989;13:658–667. 26. Vanhoutte PM, Tang EH. Endothelium-dependent contractions: when a good guy turns bad! J Physiol. 2008;586:5295–5304. 27. Yang D, Feletou M, Boulanger CM, et al. Oxygen-derived free radicals mediate endothelium-dependent contractions to acetylcholine in aortas from spontaneously hypertensive rats. Br J Pharmacol. 2002; 136:104–110.
CONCLUSIONS Homogeneous literature clearly documented that in essential hypertensive patients, an impaired vascular NO availability in the peripheral circulation occurs. A large body of evidence from the past 2 decades indicates an increased production of EDCFs, which include COXderived prostanoids and ROS, as the most important determinants for the impaired agonist-stimulated vasodilation, and NO breakdown. More recent reports, while confirming the role of EDCFs and ROS, were able to put in evidence new lights on mechanisms involved in endothelial dysfunction in hypertensive patients, identified as “new conspirator.” In particular, COX-2 is the isoform identified as a main source of intravascular ROS generation in peripheral microcirculation. In addition, important vascular protective properties by ghrelin have been demonstrated, in terms that its systemic reduction is involved in the pathophysiology of endothelial dysfunction, whereas a normalization of its levels may restore vascular homeostasis. When considering the importance of a reduced NO availability in the pathogenesis of atherosclerotic disease, it is evident that the discovery of protective effects of ghrelin toward the vasculature is opening up many new research perspectives, thus highlighting the ghrelin system as a promising candidate for cardiovascular drug discovery. REFERENCES 1. Flammer AJ, Anderson T, Celermajer DS, et al. The assessment of endothelial function: from research into clinical practice. Circulation. 2012;126:753–767. 2. Vanhoutte PM, Feletou M, Taddei S. Endothelium-dependent contractions in hypertension. Br J Pharmacol. 2005;144:449–458. 3. Virdis A, Ghiadoni L, Taddei S. Human endothelial dysfunction: Edcfs. Pflugers Arch. 2010;459:1015–1023.
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Endothelial Dysfunction in Resistance Arteries of Hypertensive Humans: Old and New Conspirators Agostino Virdis, MD and Stefano Taddei, MD
(See Editorial: A Tireless Giant in Vascular Research by Aimin Xu and Yu Huang. Journal of Cardiovascular Pharmacology, 2016 67:5;359–360)
Abstract: A large body of homogeneous reports documented that endothelial dysfunction, which characterizes human essential hypertension is largely dependent on an impaired endothelial nitric oxide availability and an increased production of endothelium-derived contracting factors. These factors include endothelin, prostanoids, and reactive oxygen species (ROS). In particular, it was evidenced that acute intraarterial administration of indomethacin, a nonselective cyclooxygenase (COX)-inhibitor, and ascorbic acid, an antioxidant, normalized the blunted endothelial dysfunction by restoring nitric oxide availability at the level of peripheral microcirculation, thus demonstrating that COX-derived endothelium-derived contracting factors are one of the major sources of ROS. Recent studies put in evidence new lights on the mechanisms involved in endothelial dysfunction in human hypertension, identified as “new conspirators.” In particular, functional and immunohistochemical experiments with selective COX inhibitors identified the isoform COX-2 as the main source of intravascular ROS generation in isolated small vessels from essential hypertensive patients. In addition, important vascular protective properties by human ghrelin have been demonstrated in human hypertension, in terms that its systemic reduction is involved in the pathophysiology of endothelial dysfunction, while a normalization of its levels may restore vascular homeostasis. Key Words: cyclooxygenase, endothelium, ghrelin, oxidative stress (J Cardiovasc Pharmacol Ô 2016;67:451–457)
INTRODUCTION There is no doubt that endothelium represents the fundamental homeostatic tissue for the regulation of the vascular tone and structure, which is physiologically characterized by a balance between substances with vasodilating and antithrombogenic properties, and substances with vasoconstricting and prothrombotic activities. Under healthy conditions, the most characterized compound is nitric oxide (NO), which exerts its cardiovascular protective role by relaxing Received for publication September 23, 2015; accepted January 5, 2016. From the Department of Clinical and Experimental Medicine, University of Pisa, Pisa, Italy. The authors report no conflicts of interest. Reprints: Stefano Taddei, MD, Department of Clinical and Experimental Medicine, University of Pisa, Via Roma 67, 56100 Pisa, Italy (e-mail: [email protected]). Copyright © 2016 Wolters Kluwer Health, Inc. All rights reserved.
J Cardiovasc Pharmacol ä Volume 67, Number 6, June 2016
media-smooth muscle cells, preventing leukocyte adhesion and migration into the arterial wall, muscle cell proliferation, platelet adhesion and aggregation, and adhesion molecule expression. Under disease conditions, including essential hypertension, the endothelium loses its protective role, becoming a proatherosclerotic structure.1 The loss of the normal endothelial function is referred to as “endothelial dysfunction,” characterized by impaired NO bioavailability. This can be determined by either a reduced production of NO by endothelial nitric oxide synthase (eNOS) or, more frequently, an increased breakdown by reactive oxygen species (ROS). Under such conditions, endothelial cells produce substances and mediators with vasoconstricting and prothrombotic activities, called endothelium-derived contracting factors (EDCFs). These include endothelin-1 (ET-1), prostanoids such as thromboxane A2 or prostaglandin H2, and ROS.2,3 Accordingly, both NO deficiency and activation of EDCFs are implicated in the pathogenesis of atherosclerosis and thrombosis.4 The aim of this review was to give a brief overview of the known mechanisms involved in the pathogenesis of endothelial dysfunction in the microcirculation of hypertensive patients. In particular, the recognized classical EDCFs, referred as “old conspirators,” together with the so-called “new conspirators,” according to the most recent discoveries in peripheral circulation of hypertensive patients, will be discussed.
HOW TO ASSESS ENDOTHELIAL FUNCTION IN HUMANS Compounds and substances released by endothelial cells exert a vascular autocrine/paracrine activity. For these reasons, the mechanisms regulating the endothelial physiology differ in various organs and tissues and, within the same vascular bed, largely vary according to vessel size.5 Usually, endothelial function in humans is indirectly assessed by vascular reactivity tests, by stimulation with specific external mechanical and pharmacological stimuli.5 It is possible to activate or inhibit endothelium-dependent pathways in several vascular regions and measure the vessel diameter changes induced by experimental perturbation. Specific agonists operating through specific receptors or increasing shear stress may activate endothelium-dependent pathways. In addition, it is possible to block pathways involved in endothelial responses such as NOS activity by NG-mono-methyl-L-arginine (LNMMA), hyperpolarization of vascular smooth muscle cells (by ouabain or sulfaphenazole), cyclooxygenase (COX) activity (by indomethacin or specific COX isoforms inhibitors), or oxidative stress (by antioxidant compounds).6 When www.jcvp.org |
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approaching to the evaluation of endothelium-dependent mechanisms in humans, it is crucial to distinguish between macrocirculation and microcirculation. These 2 vascular districts are differently regulated and therefore results obtained in large arteries cannot be extrapolated to microvascular regions. The next session will focus on the evaluation of endothelial function in microcirculation, whereas for assessment of endothelial function in large arteries, not discussed here, the reader is referred to excellent Guidelines on this topic.7
production, and a generation of COX-derived EDCFs.9,10 Unfortunately, its invasive nature limits the use in patients with advanced disease and precludes repeated testing. Many years ago, venous plethysmography was introduced to assess peripheral microcirculation by evaluation of forearm blood flow changes. An increased or decreased forearm blood flow changes by venous plethysmography is a highly reliable index of vasodilation or vasoconstriction, respectively. This approach was used thereafter for studying local endothelium-dependent vasodilation by the intraarterial infusion of endothelial agonists and antagonists (Fig. 1).6,11 Of note, this technique allows a great possibility to investigate several fine biochemical mechanisms accounting for endothelial dysfunction in humans, mainly by intraarterial infusion of compounds able to inhibit different pathways involved in endothelial function. Thus, the NO pathway may be specifically inhibited by L-arginine analogues such as L-NMMA, ROS by scavengers such as ascorbic acid and, finally, EDCFs by COX inhibitors.12 By this technique, a strict relationship between endothelial dysfunction and cardiovascular outcome was demonstrated.13 However, its invasiveness may limit the number of patients enrolled and the possibility to repeat testing frequently. Subcutaneous microcirculation can be studied using the Halpern–Mulvany myograph system, an in vitro ex vivo technique.14,15 This technique allows to assess functional characteristics of isolated resistance arterioles (lumen diameter 150–300 mm), taken from subcutaneous tissue obtained by skin biopsies. Once cleaned of adherent connective tissue, vessels are investigated with the “wire myograph” or the
Assessment of Microcirculation Most of the literature that evaluated endothelial function in human microcirculation is obtained in functionally isolated vascular districts such as the coronary circulation, or the peripheral muscle (usually the forearm), subcutaneous, or skin tissue. The coronary microcirculation can be evaluated by measuring coronary blood flow with Doppler flow wire and quantitative angiography during the intracoronary infusion of various substances, which can influence either endothelium-dependent or endothelium-independent vasodilation. Responses to endothelial agonists, including acetylcholine and substance P, have been measured, while nitroglycerin is usually used to assess endotheliumindependent vasodilation. In addition, intracoronary infusion of L-NMMA has defined the contribution of NO to these vasomotor responses.8 This experimental model has shown the presence of endothelial dysfunction in essential hypertensive patients with angiographically normal coronary arteries, an alteration caused by a reduction in agonist-induced NO
FIGURE 1. Schematic representation of the perfused forearm technique to evaluate endothelial function in human peripheral microcirculation. The brachial artery is cannulated for drug infusion at systemically ineffective rates, intraarterial blood pressure (BP) and Heart rate (HR) monitoring. Forearm blood flow is measured by strain-gauge venous plethysmography.
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“pressurized myograph.” Briefly, the first technique implies that 2 wires are threaded through the vessel, while in the pressurized system the artery is slipped into 2 glass microcannulae and exposed to a constant pressure.15,16 Whichever myograph approach is used, in this vascular district a reduced endothelial function in essential hypertensive patients was clearly documented.17,18 As an in vitro technique, such methodology allows exploration of several pathways and testing of many compounds nonapplicable in vivo, although the prognostic value of endothelial dysfunction in isolated small vessels from hypertensive patients is still under debate.19
and stimuli applied. Infusion of acetylcholine on SHR aorta evokes a large production of ROS within endothelial cells. This effect is prevented by indomethacin, thus COX appears as a main source of ROS in such conditions. In turn, ROS can amplify the EDCF-mediated effect, either by triggering EDCF-mediated responses or indirectly by reducing the availability of NO, thus favoring the occurrence of EDCF response.26
ENDOTHELIAL DYSFUNCTION IN HYPERTENSION: THE OLD CONSPIRATORS Experimental studies evidenced that endothelium induces contractile responses to endothelial agonists. Several agents induce contraction through the activation of various biochemical pathways leading to the production and secretion of different EDCFs in different animal species and vascular districts. Although relaxing factors play a crucial effect in the regulation of circulatory vasomotion, a large body of evidence supports the concept that also contracting factors have a significant role, which becomes particularly important in several pathological conditions, including essential hypertension.20
Human EDCFs The principal EDCF is ET-1, generated by the vascular endothelium, which acts through specific ETA and ETB receptors. ETA receptors are located on smooth muscle cells and promote growth and contraction. ETB receptors are located on both endothelial and smooth muscle cells, with opposite effects. Activation of smooth muscle cell ETB evokes contraction, whereas activation of endothelial ETB induces relaxation.21,22 The overall biological effect of these activated receptors on vasculature derives from the balance between their protective or deleterious effects, and this delicate equilibrium likely explains why this physiological substance may shift to a pathological role in cardiovascular disease, including essential hypertension.23 By using the isolated forearm technique, we previously observed that intraarterial infusion of TAK-044, an ETA/ETB receptor antagonist, caused an increased vasodilation among hypertensive patients compared with healthy subjects. Moreover, vasoconstriction to L-NMMA was decreased in hypertensive patients compared with controls. In addition, vasodilation to TAK-044 and vasoconstriction to L-NMMA showed an inverse correlation.24 These findings allowed to conclude that in essential hypertensive patients, endogenous ET-1 shows greater vasoconstrictor activity. Beyond ET-1, the main EDCFs are represented by endoperoxides, deriving from the metabolism of arachidonic acid by COX activity into the unstable intermediate PGH2, which, in turn, is converted by an array of downstream enzymes to form a range of bioactive prostanoids, including thromboxane A2 or prostacyclin.2,25 The role of ROS in endothelium-dependent contractions largely depends on the different experimental models Copyright © 2016 Wolters Kluwer Health, Inc. All rights reserved.
The Source of EDCFs Several bioassay studies conducted in different animal vascular districts documented that the endothelium-dependent contractions to arachidonic acid or acetylcholine were abolished by the COX inhibitor indomethacin, thus demonstrating that the metabolism of arachidonic acid through endothelial COX is deeply involved in the genesis of endothelium-dependent contractions.26 Of importance, COX-derived EDCFs diffuse to the underlying vascular smooth muscle cells and through the activation of specific receptors (thromboxane prostanoid receptors) induce contraction.2 Accordingly, most COX-mediated EDCFs effects are inhibited by smooth muscle cell thromboxane prostanoid– receptor antagonists.27,28 The metabolism of oxygen by cells generates ROS. In healthy conditions, the rate and magnitude of oxidant formation is counterbalanced by the rate of oxidant elimination. An imbalance between prooxidants and antioxidants results in oxidative stress, which is the pathogenic outcome of oxidant overproduction. Vascular ROS derive primarily from NAD(P)H oxidase. Other important sources include COX, dysfunctional eNOS (uncoupled NOS), and xanthine oxidase.29 Most of the available data on EDCFs in humans have been obtained in essential hypertensive patients. As already mentioned, human hypertension is associated with a reduced NO availability.1,6 The first experiments assessing the role of EDCFs on endothelial dysfunction in the forearm microcirculation of essential hypertensive patients demonstrated that intraarterial administration of the COX inhibitor indomethacin improved the vasodilation to acetylcholine and restored the inhibitory effect of L-NMMA on that response, indicating that COX generates substances that reduce the availability of NO.30,31 Moreover, intraarterial infusion of the ROS scavenger ascorbic acid evoked similar effect as indomethacin in these patients, with no further potentiation when the 2 compounds were coinfused.32 In conjunction, these findings represent the demonstration that the COX pathway is a source of ROS in essential hypertension. Of note, COX-inhibition failed to affect the acetylcholine-induced relaxation in the forearm microcirculation of patients with secondary forms of hypertension,30 thus indicating that production of EDCFs is not a consequence of a mere blood pressure increase, but is genetically related to essential hypertension. When does the EDCFs generation occur during the lifespan of a hypertensive patient? Important knowledge on this topic were provided from vascular studies on the cross-relation between the hypertensive progress and ageing. The increasing age is the most powerful determinants of endothelial dysfunction and is accompanied by a progressive www.jcvp.org |
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FIGURE 2. Schematic diagram showing the hypothetic involvement of COX-2 in endothelial dysfunction. Once activated by acetylcholine, COX-2 is able to transform arachidonic acid (AA) into prostaglandin H2 (PGH2) and then prostanoids, which in turn activate the contracting thromboxane-prostanoid receptors. Simultaneously, COX-2 is a source of ROS, with the consequent reduced NO availability.
worsening of NO availability in resistance circulation.33–36 The main mechanism responsible for age-related endothelial dysfunction in the peripheral microcirculation is a primary defect in the L-arginine–NO pathway. After the age of 60 years, along with a further impairment of the L-arginine– NO pathway, COX-dependent EDCF production becomes evident and significant.37 Such age-related endothelial dysfunction is anticipated by hypertension, which therefore represents a condition of premature vascular ageing. Thus, although not effective in younger hypertensive patients (,30 years), in adult patients (31–45 years) indomethacin begins to show some effect, and in the older patients (46– 60 and .60 years) COX-derived oxidative stress generation becomes the main determinant of endothelial dysfunction.37 In conclusion, this study evidenced that ageing is an important factor altering endothelium-dependent vasodilation and that the mechanisms involved include a defect in the L-arginine–NO pathway and production of COX-dependent EDCFs. However, whereas in normotensive subjects, ageing mainly affects the formation of NO and EDCF production characterizes only old age, the presence of hypertension causes an earlier onset of altered L-arginine–NO pathway and also earlier formation of vasoconstrictor prostanoids.
pioneering investigations have left undetermined the question of which COX isoenzymes is effectively involved.
Vascular COX-2 Isoform
As described in the previous section, studies conducted in human hypertension documented that COX pathway actively interferes with NO availability, and represents a source of ROS in the peripheral microcirculation of patients with essential hypertension. Until now, COX represents the unique pathway identified by acting as a source of ROS in human hypertension. However, these
Two different isoforms of COX are known to exist.38 In most tissues, COX-1 is regarded as constitutively expressed to produce physiological prostanoids, whereas COX-2 is often induced by a number of stimuli, including inflammation or growth factors.39 Nevertheless, COX-2 is also expressed constitutively in several organs. In particular, within the vasculature, endothelial and vascular smooth muscle cells do express both isoforms, with COX-1 being usually expressed at a higher extent than COX-2.39 Recently, we investigated which COX isoform contributes to ROS generation in human hypertension. Thus, in isolated small resistance arteries from essential hypertensive patients, the role of COX-1 and COX2, their cross-talks with NO and ROS, as well as whether other pathways may participate as adjunctive sources of ROS was assessed. In brief, it was found that in small vessels from hypertensive patients, the blunted vascular response to acetylcholine, while not modified by the COX-1 inhibitor SC560, was significantly improved by the selective COX-2 inhibitor Dup-697, which also partly restored the inhibitory effect of L-NAME on acetylcholine.40 In addition, an augmented COX-2 protein presence in vessels from these patients was documented. Furthermore, immunohistochemistry indicated a marked upregulation of COX-2 mainly in the vascular media layer.40 Of note, the fluorescent dihydroethidium technique revealed that in these patients the intravascular superoxide excess was dramatically reduced by incubation with the selective COX-2 inhibitor, and moderately blunted by the antioxidant apocynin. These data provide the first evidence that, in small arteries isolated from essential hypertensive patients, an overexpression and increased activity of COX-2
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ENDOTHELIAL DYSFUNCTION IN HYPERTENSION: THE NEW CONSPIRATOR
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plasma and stomach: acylated ghrelin, which represents the biologically active form, and desacyl-ghrelin, which although not totally biologically inactive, seems not to exert endocrine activities.43 At the beginning, ghrelin was identified as an endogenous ligand for the orphan receptor (GHS-R) and reported to exert a growth hormone-releasing activity, and to participate in the regulation of energy balance, food intake, and reducing fat utilization.44 Although basically a gastric hormone, adjunctive growth hormone-independent properties have been recently attributed to ghrelin. Among others, a variety of cardiovascular properties, including vascular effects, are ascribable to this peptide.
Vascular Effects of Ghrelin
FIGURE 3. Representative dihydroethidium staining (upper) and quantitative analysis of the red signal (lower) in small arteries from controls and essential hypertensive (EH) patients at baseline (vehicle) or after incubation with ghrelin, gp91 ds-tat, or both. *P , 0.001 versus other groups; †P , 0.05 versus control. Modified from Virdis et al.51 Adaptations are themselves works protected by copyright. So in order to publish this adaptation, authorization must be obtained both from the owner of the copyright in the original work and from the owner of copyright in the translation or adaptation.
occur, playing a major role in reducing NO availability. COX-2 represents a major source of ROS generation in essential hypertension (Fig. 2). In this context, NAD(P)H oxidase participates also, although with a minor role, in promoting superoxide generation in these patients.41 COX-2 as a main source of ROS generation likely represents a new conspirator of endothelial dysfunction or, in other words, a new source of old conspirators (ROS) and in these terms such isoform might open future therapeutic options.
Human Ghrelin Ghrelin is a peptide recently discovered from human stomachs.42 Two major forms of ghrelin are present in the Copyright © 2016 Wolters Kluwer Health, Inc. All rights reserved.
Although mainly generated and released by the gastric cells, growing evidence revealed the presence of ghrelin and its receptor, growth hormone secretagogues 1a (GHS-R 1a) at the level of the cardiovascular system. In particular, expression of ghrelin has been demonstrated in endothelial cells of human arteries and veins and in secretory vesicles of cultured endothelial cells,45 whereas GHS-R 1a has been stained within endothelial cells and vascular smooth muscle cells from different vascular beds.46 Accordingly, both animal and human reports demonstrated an active effect of this peptide on the cardiovascular system, including the endothelial function. In vitro studies documented that ghrelin acutely stimulated increased production of NO in bovine aortic endothelial cells and in human aortic endothelial cells in a timeand dose-dependent manner, using a signaling pathway that involves GHS-R 1a and increased expression of eNOS.47 Ghrelin also demonstrated an antioxidant property, an activity dependent on the activation of cellular signaling pathways leading to inhibition of enzymes involved in ROS generation, including NAD(P)H oxidase,48 thus suggesting a novel and promising mechanism of vascular protection. The first evidence of the vascular effects of ghrelin in in vivo human studies comes from patients with metabolic syndrome. In the forearm microcirculation of these patients, intraarterial infusion of human ghrelin was able to reverse endothelial dysfunction by increasing NO availability,49 and to exert a beneficial effect on NO and ET-1 imbalance,50 thus contributing to restore a balance between endothelium-derived contracting and vasodilator forces. More recently, we investigated the impact of exogenous ghrelin on endothelial dysfunction in human hypertension. To this purpose, we assessed whether exogenous ghrelin may ameliorate endothelial dysfunction in the forearm resistance circulation of patients with essential hypertension, focusing on the involvement of NO availability and ROS. Second, in isolated small arteries from essential hypertensive patients, we studied the abrogation of intravascular ROS generation and NAD(P)H oxidase pathway as possible mechanisms exerted by ghrelin. The main results indicated that acute ghrelin infusion improved endothelium-dependent vasodilation and restored the inhibition by L-NMMA on response to acetylcholine in the forearm resistance arterioles of essential hypertensive patients, thus representing the first demonstration in the peripheral microcirculation of hypertensive patients of the ability of ghrelin to restore endothelial NO www.jcvp.org |
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availability.51 The antioxidant property of ghrelin was also assessed in our study, both in the forearm resistance circulation and in isolated small vessels. In the first vascular district, acute ghrelin infusion was able to induce a great reduction of markers of oxidative stress in our hypertensive population. In isolated small arteries from hypertensive patients, the dihydroethidium staining showed that ghrelin dramatically reduced the enhanced superoxide generation. An identical result was obtained on incubation with the NAD(P)H oxidase inhibitor gp91ds-tat, and no further superoxide reduction was seen when ghrelin and gp91 were simultaneously incubated (Fig. 3). The possibility that ghrelin prevented vascular ROS generation by downregulating NAD(P)H oxidase was demonstrated by the Western blot analysis.51 Taken together these findings demonstrate, for the first time in 2 different microvascular districts of hypertensive patients, that the beneficial activity of ghrelin is related to its antioxidant property, an effect obtained through a marked inhibition of NAD(P)H oxidase activation, leading to an amelioration of NO activity. These data highlight the role of ghrelin as a physiological mediator of the vascular system: a systemic reduction of this peptide is involved in the pathophysiology of endothelial dysfunction, whereas a normalization of its levels may restore vascular homeostasis.
4. Lerman A, Zeiher AM. Endothelial function: cardiac events. Circulation. 2005;111:363–368. 5. Deanfield J, Donald A, Ferri C, et al. Endothelial function and dysfunction. Part i: methodological issues for assessment in the different vascular beds: a statement by the working group on endothelin and endothelial factors of the european society of hypertension. J Hypertens. 2005;23:7–17. 6. Virdis A, Ghiadoni L, Versari D, et al. Endothelial function assessment in complicated hypertension. Curr Pharm Des. 2008;14: 1761–1770. 7. Corretti MC, Anderson TJ, Benjamin EJ, et al. Guidelines for the ultrasound assessment of endothelial-dependent flow-mediated vasodilation of the brachial artery: a report of the international brachial artery reactivity task force. J Am Coll Cardiol. 2002;39:257–265. 8. Goodhart DM, Anderson TJ. Role of nitric oxide in coronary arterial vasomotion and the influence of coronary atherosclerosis and its risks. Am J Cardiol. 1998;82:1034–1039. 9. Egashira K, Suzuki S, Hirooka Y, et al. Impaired endothelium-dependent vasodilation of large epicardial and resistance coronary arteries in patients with essential hypertension. Different responses to acetylcholine and substance p. Hypertension. 1995;25:201–206. 10. Marzilli M, Taddei S, Virdis A, et al. Endothelial function and coronary microcirculaiton in essential hypertension. J Hypertens. 1998;16 (suppl 8):S59–S63. 11. Virdis A, Neves MF, Duranti E, et al. Microvascular endothelial dysfunction in obesity and hypertension. Curr Pharm Des. 2013;19: 2382–2389. 12. Versari D, Daghini E, Virdis A, et al. Endothelium-dependent contractions and endothelial dysfunction in human hypertension. Br J Pharmacol. 2009;157:527–536. 13. Heitzer T, Schlinzig T, Krohn K, et al. Endothelial dysfunction, oxidative stress, and risk of cardiovascular events in patients with coronary artery disease. Circulation. 2001;104:2673–2678. 14. Mulvany MJ, Baumbach GL, Aalkjaer C, et al. Vascular remodeling. Hypertension. 1996;28:505–506. 15. Virdis A, Savoia C, Grassi G, et al. Evaluation of microvascular structure in humans: a ’state-of-the-art’ document of the working group on macrovascular and microvascular alterations of the Italian society of arterial hypertension. J Hypertens. 2014;32:2120–2129. 16. Virdis A, Colucci R, Fornai M, et al. Cyclooxygenase-2 inhibition improves vascular endothelial dysfunction in a rat model of endotoxic shock: role of inducible nitric-oxide synthase and oxidative stress. J Pharmacol Exp Ther. 2005;312:945–953. 17. Rizzoni D, Porteri E, Castellano M, et al. Endothelial dysfunction in hypertension is independent from the etiology and from vascular structure. Hypertension. 1998;31:335–341. 18. Endemann DH, Pu Q, De Ciuceis C, et al. Persistent remodeling of resistance arteries in type 2 diabetic patients on antihypertensive treatment. Hypertension. 2004;43:399–404. 19. Rizzoni D, Porteri E, De Ciuceis C, et al. Lack of prognostic role of endothelial dysfunction in subcutaneous small resistance arteries of hypertensive patients. J Hypertens. 2006;24:867–873. 20. Luscher TF. Endogenous and exogenous nitrates and their role in myocardial ischaemia. Br J Clin Pharmacol. 1992;34(suppl 1):29S–35S. 21. Cardillo C, Kilcoyne CM, Cannon RO, et al. Interactions between nitric oxide and endothelin in the regulation of vascular tone of human resistance vessels in vivo. Hypertension. 2000;35:1237–1241. 22. Dhaun N, Goddard J, Kohan DE, et al. Role of endothelin-1 in clinical hypertension: 20 years on. Hypertension. 2008;52:452–459. 23. Schiffrin EL. State-of-the-art lecture. Role of endothelin-1 in hypertension. Hypertension. 1999;34:876–881. 24. Taddei S, Virdis A, Ghiadoni L, et al. Vasoconstriction to endogenous endothelin-1 is increased in the peripheral circulation of patients with essential hypertension. Circulation. 1999;100:1680–1683. 25. Vanhoutte PM. Endothelium and control of vascular function. State of the art lecture. Hypertension. 1989;13:658–667. 26. Vanhoutte PM, Tang EH. Endothelium-dependent contractions: when a good guy turns bad! J Physiol. 2008;586:5295–5304. 27. Yang D, Feletou M, Boulanger CM, et al. Oxygen-derived free radicals mediate endothelium-dependent contractions to acetylcholine in aortas from spontaneously hypertensive rats. Br J Pharmacol. 2002; 136:104–110.
CONCLUSIONS Homogeneous literature clearly documented that in essential hypertensive patients, an impaired vascular NO availability in the peripheral circulation occurs. A large body of evidence from the past 2 decades indicates an increased production of EDCFs, which include COXderived prostanoids and ROS, as the most important determinants for the impaired agonist-stimulated vasodilation, and NO breakdown. More recent reports, while confirming the role of EDCFs and ROS, were able to put in evidence new lights on mechanisms involved in endothelial dysfunction in hypertensive patients, identified as “new conspirator.” In particular, COX-2 is the isoform identified as a main source of intravascular ROS generation in peripheral microcirculation. In addition, important vascular protective properties by ghrelin have been demonstrated, in terms that its systemic reduction is involved in the pathophysiology of endothelial dysfunction, whereas a normalization of its levels may restore vascular homeostasis. When considering the importance of a reduced NO availability in the pathogenesis of atherosclerotic disease, it is evident that the discovery of protective effects of ghrelin toward the vasculature is opening up many new research perspectives, thus highlighting the ghrelin system as a promising candidate for cardiovascular drug discovery. REFERENCES 1. Flammer AJ, Anderson T, Celermajer DS, et al. The assessment of endothelial function: from research into clinical practice. Circulation. 2012;126:753–767. 2. Vanhoutte PM, Feletou M, Taddei S. Endothelium-dependent contractions in hypertension. Br J Pharmacol. 2005;144:449–458. 3. Virdis A, Ghiadoni L, Taddei S. Human endothelial dysfunction: Edcfs. Pflugers Arch. 2010;459:1015–1023.
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40. Virdis A, Bacca A, Colucci R, et al. Endothelial dysfunction in small arteries of essential hypertensive patients: role of cyclooxygenase-2 in oxidative stress generation. Hypertension. 2013;62:337–344. 41. Vanhoutte PM. One or two, does it matter as long as the arterial wall is coxygenated? Hypertension. 2013;62:244–246. 42. Kojima M, Hosoda H, Date Y, et al. Ghrelin is a growth-hormonereleasing acylated peptide from stomach. Nature. 1999;402:656–660. 43. Otto B, Spranger J, Benoit SC, et al. The many faces of ghrelin: new perspectives for nutrition research? Br J Nutr. 2005;93:765–771. 44. Tschop M, Smiley DL, Heiman ML. Ghrelin induces adiposity in rodents. Nature. 2000;407:908–913. 45. Kleinz MJ, Maguire JJ, Skepper JN, et al. Functional and immunocytochemical evidence for a role of ghrelin and des-octanoyl ghrelin in the regulation of vascular tone in man. Cardiovasc Res. 2006;69: 227–235. 46. Iglesias MJ, Pineiro R, Blanco M, et al. Growth hormone releasing peptide (ghrelin) is synthesized and secreted by cardiomyocytes. Cardiovasc Res. 2004;62:481–488. 47. Iantorno M, Chen H, Kim JA, et al. Ghrelin has novel vascular actions that mimic pi 3-kinase-dependent actions of insulin to stimulate production of no from endothelial cells. Am J Physiol Endocrinol Metab. 2007; 292:E756–E764. 48. Kawczynska-Drozdz A, Olszanecki R, Jawien J, et al. Ghrelin inhibits vascular superoxide production in spontaneously hypertensive rats. Am J Hypertens. 2006;19:764–767. 49. Tesauro M, Schinzari F, Iantorno M, et al. Ghrelin improves endothelial function in patients with metabolic syndrome. Circulation. 2005;112: 2986–2992. 50. Tesauro M, Schinzari F, Rovella V, et al. Ghrelin restores the endothelin 1/nitric oxide balance in patients with obesity-related metabolic syndrome. Hypertension. 2009;54:995–1000. 51. Virdis A, Duranti E, Colucci R, et al. Ghrelin restores nitric oxide availability in resistance circulation of essential hypertensive patients: role of nad(p)h oxidase. Eur Heart J. 2015;36:3023–3030.
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