The endothelium executes important anticoagulant and anti-inflammatory functions. However, endothelial dysfunction in disease primarily entails impaired endothelium-dependent dilator responses to acetylcholine(ACh) and bradykinin. Endothelial Nitric Oxide Synthase(eNOS)-derived NO and cycloxygenase(COX)-derived prostacyclin(PGI2) mediate two well-defined endothelium-dependent relaxation pathways. Agonist- or shear stress-induced endothelium-dependent hyperpolarisation (EDH) was not defined as a distinct third pathway until 1988, when VSMC relaxation and hyperpolarisation that persisted in the presence of COX and NOS inhibition was reported.4 Over thirty years later, three mechanisms of EDH have been proposed, although the underlying molecular constituents remain elusive, with different pathways exhibiting dominance in different vascular beds, species and experimental conditions. Furthermore, the physiological contribution of endothelium-dependent hyperpolarisation (EDH) to vascular tone and its role in pathophysiological states is uncertain. This review evaluates current knowledge of the EDH response in coronary vessels, in physiological and pathophysiological states, and highlights mechanistic components that represent promising therapeutic targets for restoration of endothelium-dependent dilation and, therefore, endothelial function in disease.
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Figure.1: 3 pathways of endothelium-dependent vasorelaxation
The first experiment describing EDH was carried out in 1979 in guinea-pig coronary arteries.5 Later studies reported a shift in endothelium-dependent vasodilation from NO to an EDH pathway as coronary vessel size decreases, suggesting EDH might regulate coronary flow.6,7 The most consistent reports of EDH in coronary vasculature investigate arterioles, although, such experiments are challenging because of difficulties in mounting microvasculature onto myographs and in the identification of 'functional resistance vessels', as disease may shift resistance to smaller vessels, for example, in epicardial stenoses.8
To maintain blood flow, the coronary microcirculation is subject to stringent autoregulation according to transmural pressure and metabolic factors. Myogenic and EDH responses represent diametrically opposing pathways that antagonistically regulate BKCa channels to control vascular tone and coronary perfusion.9 Since hypertension increases myogenic responses and commonly leads to atherosclerosis, coronary artery disease, myocardial infarction and cardiomyopathies, the study of coronary arterioles to establish mechanistic components that may present promising therapeutic targets to augment EDH may confer useful coronary protection.
Ex vivo experiments with coronary vessels from animal models should produce reproducible reports of EDH in physiology or pathophysiology, since laboratory animals have less variation in risk factors. In healthy coronary vessels, basal NO synthesis suppresses EDH,10 whereas in coronary arterioles of Spontaneously Hypertensive Rats(SHRs) when NO and PGI2 pathways are impaired, EDH is augmented.11 These studies suggest the EDH pathway integrity is preserved, and can compensate for impaired NO-mediated vasodilation in endothelial dysfunction, indicating EDH may represent a useful therapeutic target. However, contradictory experiments in SHR coronary arterioles report decreased EDH.12 If impaired EDH contributes to disease progression, enhancing EDH therapeutically may also produce benefit. Since SHRs used in these studies exhibit similar age, gender and arterial blood pressure distributions, this conundrum highlights the importance of mimicking in vivo environments in experiments.13 For example, one group measured tension of isolated arteriolar rings,12 whilst the other recorded intact pressurised arteriole diameter.11,13
Since chronic progressive cardiovascular disease is not accurately represented in animal models, studies of human coronary arterioles(HCA) with underlying pathology are valuable. HCAs are readily obtained from right atrial appendages, a frequently discarded tissue in surgery. Ex vivo experiments with HCAs from patients undergoing cardiopulmonary bypass procedures show significant reduction in EDH-mediated vasodilation with age.14 Impaired EDH may, therefore, contribute to increasing blood pressure with age. Some groups assume the physiological role of EDH cannot be accurately studied in humans even if HCAs are not explicitly diseased, as results are influenced by many risk factors, including underlying chronic cardiovascular disease, aging, hyperlipidaemia or smoking.14 However, critics argue that whereas ECs in resistance vessels become dysfunctional with aging, young arterioles relevant to EDH investigations can be used to evaluate the physiological role of EDH because they are spared in patients with evidence of hypertension or atherosclerosis, where endothelial dysfunction is limited to larger coronary vessels.8,15 Furthermore, studies of explicitly diseased HCAs are useful since inherent endothelium dysfunction causes endothelium-dependent vasodilation to be almost exclusively EDH-mediated, eliminating the confounding influence of NO in investigations.REF!
Ex vivo experiments striving to define the nature of the EDH-response and vasodilation to ACh, bradykinin or flow in precontracted vessels, essentially mimic the approach outlined in figure.2. Endothelium-dependency is ascertained by observing the corollary of mechanical or detergent endothelial denudation, and an EDH-response defined by residual hyperpolarisation in the presence of COX- and NOS-blockade. High concentrations of COX- and NOS-inhibitors or the use of eNOS-/- and COX-1-/- double knock-out mice16 are common approaches, although they negate to eliminate NO derived from plasma proteins, 5-nitrosothiols or preformed stores in VSMCs.17,18 The best studies additionally use NO-scavengers and guanylate cyclase inhibitors to abolish the NO pathway17, although critics object that too many pharmacological agents perturb the system under investigation.13 Protocols should also show that procedures used to abrogate the EDH do not reduce vasodilation via PGI2 or NO disruption by confirming pathway integrity by demonstrating relaxation to iloprost and glyceryl trinitrate. The clearest experiments measure membrane potentials and KCa channel conductances to explicitly define EDH responses.7 However, the difficulties of electrophysiological approaches in the microcirculation prompts groups to rely on diameter or tension measurements and the sensitivity of the vasodilation to BKca channel inhibitors (iberiotoxin) or high K+ concentrations (to clamp membrane potential) to characterise the EDH component, since VSMC BKCa K+ conductance is critical to EDH.7
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Figure.2: Experimental procedure to investigate EDH mechanisms ex vivo.
In vivo experiments are critical in demonstrating the contribution of EDH to vascular tone in order to ascertain whether this mechanism is worthwhile exploiting therapeutically. Contribution of EDH to global haemodynamics has been investigated in SKCa, IKCa or BKCa-deficient mice, which exhibit significantly raised arterial blood pressure, indicating EDH may be a useful therapeutic target in hypertension.19-22 However, in vivo investigation of EDH mechanisms in coronary arterioles is difficult, since cardiac action potentials preclude accurate recording of arteriolar membrane potentials and KCa channel conductances necessary to unequivocally define EDH.7 This is only achieved by sensitivity of vasodilation to iberiotoxin and high K+ concentrations. However, changes in vessel diameter remain difficult to measure by intravital microscopy because of cardiac motion7. Two groups report EDH to contribute about one third of ACh-induced vasodilation in canine coronary arterioles in vivo.6,7 However, in vivo experiments involving anaesthesia should be interpreted cautiously, since volatile and intra-venous general anaesthetics have been reported to disrupt the EDH pathway, in particular CYP450 activity23 and IKCa-activation.24 Moreover, this emphasises the importance of stringent cardiovascular control during surgery of patients with impaired NO bioavailability in endothelial dysfunction.
Figure.3: EDH-mediated VSMC relaxation
Whilst understanding of the EDH mechanism in coronary vessels remains sparse, and, therefore, potential therapeutic targets are difficult to identify, all EDH-mediated pathways begin with EC hyperpolarisation. Hyperpolarisation spreads from ECs to VSMCs via several pathways leading to vasodilation (outlined in figure.3). The primary trigger of EDH is Ca2+ entry to ECs, particularly via non-selective cation channels, TRPV4. Indeed, genetic deficits of TRPV4 in mice dampens ACh-induced EC hyperpolarisations and arteriolar dilations.25 Enhancing EC Ca2+ entry may represent a powerful therapeutic target since it initiates EDH. Indeed TRPV4 agonists augment EC Ca2+ and endothelium-dependent relaxation, although they are unlikely to be a useful therapeutically since they are reported to cause circulatory collapse in three species through disrupted endothelial morphology.26
The role of K+ conductance in EDH was established from the earliest investigation of ACh-induced EDH, which reported a reversal potential of ~-70mV, a value approximate to the Nernst potential for K+.5 Further work shows hyperpolarisation amplitude inversely correlates with extracellular K+concentration and is completely eliminated at concentrations >25mM.4 Moreover, ACh-induced EDH involves K+efflux since Cerenkov counting detected 86Rb efflux from preloaded arteries.4
The requirement for K+conductance together with EC Ca2+-dependency prompted investigation of calcium-dependent K+channels (KÂCa). There are eight KCa channels in the human genome, classified according to conductances and mode of gating.27 Coexistence of three groups of channels is not evolutionary functional redundancy, since they exhibit distinct subcellular localisation within the vasculature and perform different roles (table.1).
Table.1: Grouping and properties of KCa channels. Most significant channels contributing to EDH shown in red.
Gating of channel
Bind Ca2+ directly
Gated by calmodulin
-inactivates at negative membrane potentials
-active even at negative membrane potentials
Distribution in vascular context
At sites of MEGJs28
At interface between adjacent ECs28
Associates with Ca2+-sensing receptors29
Associates with TRPV430
Common target for diffusible EDHFs
Repolarisation of precontracted VSMCs31.
EDH of VSMC31.
For many years, progress regarding which KCachannels mediate the EDH-response was hindered by use of non-selective channel blockers (table.2-shaded). Whilst the involvement of SKCa channels was inferred from abrogated hyperpolarisations in the presence of apamin (selective SKCa-blocker), the role of IKCa channels is confounded in the early literature by use of charybdotoxin, since nanomolar concentrations block both BKCa and IKCa.17,32 However, the contribution of IKCa to EDH was later confirmed by the development of IKCa-specific TRAM-34/39.29,33 Often studies still compromise between potent, specific venom-derived toxins, which are costly at high amounts necessary in vivo, and inexpensive organic molecules with lower selectivity.32 The importance of KCa channels in controlling blood pressure in vivo is evident in mice deficient in both or either SK3 or IKCa, which exhibit impaired EDH correlating with increased vascular tone and raised arterial blood pressure.19-21 In an SK3-inducible transgenic mouse, normotensive arterial blood pressure is restored by SK3 overexpression.19
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Table.2: Relative potencies of drugs for different channels 32 are elucidated by comparison of IC50 (half maximal inhibitory concentration) values. Shaded: poorly selective agents.
Small organic molecules
100nM-300Î¼M depending on expression of Î±-subunits in species35
Since SKCa and IKCa channels function early in the EDH pathway, they represent promising therapeutic targets to significantly augment EDH responses. Recently, a new IKCa-opener, SKA-31, was demonstrated to augment ACh-induced EDH-mediated dilations in murine carotid arteries and decrease arterial blood pressure in wild-type and angiotensin-II-infused hypertensive mice.37 NS309, which activates both IKCa and SKCa channels, demonstrated enhanced ACh-induced hyperpolarisations in guinea-pig carotid arteries.38 However, its short plasma-half-life and off-target inhibition of L-type Ca2+ channels may render it unsuitable for therapeutic use.27,39 Furthermore, the abundance of IKCa channels on epithelial, immune and neoplastic cells, and SKCa in the CNS may cause side-effects in targeted therapeutics, and it is possible that drugs may exhibit tachyphylaxis if channels become downregulated27.
BKCa channels are only sparsely detected on ECs. Immunolabelling and reverse-transcription qPCR techniques show abundant BKCa expression in coronary VSMCs.40 Studies in coronary arterioles demonstrating EDH-sensitivity to tetraethylammonium at concentrations <3mM,8 or to more specific agents such as iberiotoxin(table.2) justifiably attribute the effect to blockade of BKCa channels.40 Many proposed diffusible endothelium-derived hyperpolarising factors (EDHFs) are postulated to activate VSMC BKCa channels.
The physiological significance of BKCa function in control of vasomotion is apparent from enhanced myogenic tone and raised blood pressure in BKCa-deficient mice.22 This is confirmed by epidemiological studies that report gain- or loss-of-function single nucleotide polymorphisms (SNPs) in BKCa-coding genes to lower or increase the occurrence of hypertension, myocardial infarction and stroke, respectively, within a population.27,41 Indeed, increased BKCa conductance is reported in the SHR, correlating with raised BKCa Î±-subunit expression in coronary VSMCs.42
Since many EDH pathways converge to VSMC BKCa, this channel represents an attractive potential therapeutic target. Indeed, BKCa-openers not only suppress myogenic constriction, but significantly enhance EDH in rat coronary vessels, suggesting that BKCaÂ-openers may be useful to 'reduce coronary risk in hypertension'.9 However, poor efficacy and selectivity, leading to side-effects in vivo have caused most clinical trials of BKCa-openers to be terminated.43 BKCa-activators may produce promising therapeutics if an effective technique to selectively target specific cells is developed. Interestingly, BKCa ß-subunit transcript expression declines with ageing in rat coronary vessels44, which may contribute to progressive endothelial dysfunction and cardiovascular disease with age, precluding benefit from BKca-activating therapeutics. Conceivably, therapeutics enhancing BKCa expression may be useful, which may contribute to reduced pulmonary hypertension in rats treated with the clinically-prescribed steroid, dehydroepiandrosterone.45
There are three distinct pathways proposed by which ECs communicate hyperpolarisation to VSMCs (figure 4).
Figure.4: Three pathways proposed to mediate EDH
The first mechanism involves direct heterocellular communication through MEGJs, which provide conduits through which hyperpolarising current or small EDHFs flow from ECs to VSMCs.46 This efficient EDH pathway has proved difficult to investigate because of a shortage of selective agents to block connexins.32,47 Furthermore, no MEGJs have been reported in HCA, and, therefore, this pathway is not discussed further in this review.
Electrophysiological experiments suggested a K+-mediated pathway in which endothelial SKCa- and IKCa-derived K+ions (5-15mM) accumulate in the interstitial space and activate VSMC inward-rectifying K+ channels (KIR) and electrogenic Na+/K+ATPases to elicit hyperpolarisation.48 However, experiments in bovine coronary vasculature with identical K+ concentrations produce divergent results regarding the sensitivity of BK-induced EDH to ouabain.13,49,50 Discrepancies may result from different degrees of depolarisation in vessel precontraction,13 different exogenous K+ and ouabain incubation times prior to EDH measurement, or by off-target suppression of the NO-pathway by ouabain.50 Since this evidence is confounding and the K+hypothesis has not been investigated in HCA, it is not discussed further in this review.
The third EDH pathway involves release of a diffusible EDHF by the EC to act on targets in VSMCs. Many potential transmitters have been uncovered including epoxyeicosatrienoic acids(EETs), H2O2, CO, H2S, lipoxygenase metabolites, and C-type natriuretic peptide in various vascular beds.51 NO and PGI2 have also been demonstrated to mediate EDH in coronary vessels, although these pathways involve KATP channel conductance, and challenge the definition that EDH is a NO- and PGI2-independent pathway.52 Diffusible EDHFs should satisfy four criteria53 (Table.3). Evidence suggests eicosatrienoic acids (EETs) and H2O2 are dominant EDHFs in HCA8,54, and thus represent the most amenable pathways to be targeted therapeutically.
Table.3: Criteria to define EDHFs 53
EDHF activity is reduced if synthesis of the proposed EDHF is inhibited
EDHF bioactivity is mimicked by exogenous application of proposed EDHF
No other vasodilator accounts for the relaxation
The proposed EDHF is synthesised in ECs (4a) and acts on VSMCs (4b)
EETs are derived from arachidonic acid by cytochrome P450 epoxygenases (CYP2C and CYP2J) in ECs. Criterion-1 is satisfied by targeting of ECs with antisense oligonucleotides directed against CYP2C to preclude EET synthesis, which abrogates bradykinin-induced EDH and vasodilation in porcine coronary arteries.55 Furthermore, earlier studies using non-specific clotrimazole have been confirmed with the specific CYP inhibitor, miconazole, which precludes EDH in canine coronary arterioles in vivo.7,56 Criteria-2&3 are satisfied by experiments in which exogenous application of EETs elicits concentration-dependent vasodilations of preconstricted bovine coronary artery rings in the presence of L-NNA and indomethacin.57 Subsequent application of the synthetic EET antagonist, 14,15-EEZE, completely abolishes bradykinin-induced EDH and vasodilation.57 Criterion-4 is satisfied by in tandem double organ-chamber bioassay performed with L-NNA and diclofenac, in which a clotrimazole-sensitive bradykinin-induced diffusible factor, released into the superfusate from donor porcine coronary ECs, elicits hyperpolarization and relaxation of downstream VSMCs.56 Patch clamp experiments show exogenous application of EETs indirectly promotes the open-probability of VSMC BKCa channels.58 A recent review also suggests that EETs may augment EDH by activation of EC TRP channels to augment intracellular Ca2+ and hyperpolarisation which may then be communicated via MEGJs to VSMCs.59 However, the lack of MEGJs in HCA precludes this mechanism being of therapeutic interest for human coronary vasculature.
Targeting EETs represents another therapeutic approach to enhance EDH in disease. One study reports oxidised LDL, a contributor to atherosclerotic progression, to reduce CYP450 expression and suppress EET-mediated EDH in HCA.60 Moreover, the addition of 11,12-EETs to hyperkalaemic preservation solutions of porcine transplanted hearts improved EDH in grafted coronary vessels.61 Although the mechanism is unclear, the Ca2+channel blocker, Nifedipine, used clinically in hypertension, also augments CYP2C expression and EET synthesis in porcine coronary ECs, which may confer additional coronary protection in hypertension.62 However, CYP450-activating therapeutics may be undesirable, since induction of CYP450 produces reactive oxygen species(ROS). Therefore restraining EET metabolism by suppressing soluble epoxide hydrolase (sEH) may be a more appropriate therapeutic approach. Indeed, administration of sEH inhibitors restored ACh-induced vasodilation in DOCA-salt hypertensive mice without effect on sodium nitroprusside-induced vasodilation. This amelioration of endothelial dysfunction restrained the increasing blood pressure.63 sEH inhibition may be therapeutically useful in humans since infusion of isolated HCA with sEH inhibitors significantly improves dilation mediated by 14,15-EET.64 A double-blinded, placebo-controlled phase-IIa clinical trial is currently underway for an sEH-inhibitor, including glucose-intolerant pre-diabetic patients with moderate hypertension. [i] However, critics are concerned that long-term use of sEH inhibitors may augment other mechanisms to metabolise EETs, producing side-effects and tachyphylaxis.65 Furthermore, excess EETs increase the risk of cancer by promoting angiogenesis and suppressing apoptosis.65
In HCA H2O2 has also been suggested as an EDHF.54 Moreover, H2O2 has been proposed to interact with CYP2C to restrain bradykinin-induced EET synthesis in HCA, implicating H2O2 as the dominant EDHF. However, following H2O2 degradation by catalase, EETs become the principal EDHF in HCA.66 This may account for inconsistent reports of H2O2 or EETs as the primary EDHF in HCA published by the same group8,54.
Like EETs, H2O2 fulfils the criteria for an EDHF in coronary arterioles. Criterion-1 is satisfied by observing bradykinin-induced HCA dichlorodihydrofluorescein (H2O2) histofluorescence in ECs and demonstrating sensitivity of EDH to apocynin (NADPH-inhibitor), indicating NADPH-derived H2O2 as an EDHF.67 Criteria-2&3 are satisfied by the catalase-sensitivity of flow-induced EDH in the presence of COX- and NOS-blockade and the dose-dependent VSMC hyperpolarisation with application of exogenous H2O2.54 The release of H2O2 from HCA as a diffusible EDHF is shown by in tandem bioassay experiments, satisfying criterion-4.66 Patch clamp studies show H2O2 increases VSMC BKCa conductance in porcine coronary arterioles.68
Critics suggest H2O2 may not exert significant EDH in vivo since plasma antioxidants scavenge reactive oxygen species (ROS), although studies of canine coronary vessels in vivo demonstrate H2O2 contributes significantly to EDH and vasodilation in coronary autoregulation, and in compensation for NO in ischaemia-reperfusion injury.6,69 Nevertheless, augmentation of H2O2 is unlikely to present novel therapeutics for endothelial dysfunction in cardiovascular disease, since the pro-mitogenic, pro-adhesive and pro-aggregatory properties of H2O2 contribute to platelet dysfunction and the development of atherosclerosis.70
In conclusion, in vivo experiments and epidemiological studies of SNPs suggest that restoration of endothelial dysfunction by improvement of EDH protects against and ameliorates cardiovascular disease. Patients unresponsive to conventional pharmacological interventions targeting the NO pathway may benefit from augmentation of EDH. Ex vivo studies of EDH in animal and human coronary arterioles have revealed several mechanistic components that represent potential therapeutic targets. Whilst TRP agonists, CYP2C inducers and H2O2 are unlikely candidates, SKCa,IKCa and BKCa activators and sEH inhibitors may represent promising therapeutics to help combat the cardiovascular disease epidemic.