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Circulation JournalOfficial Journal of the Japanese Circulation Societyhttp://www.j-circ.or.jp

tatins reduce plasma cholesterol via inhibition of 3-hydroxy-3-methylglutaryl-coenzyme-A reductase and have been shown to have a major clinical effect on

improving cardiovascular outcomes.1–4 Additional pleiotro-pic vascular effects have been described that are not attribut-able simply to a reduction in cholesterol levels but which may contribute to their cardiovascular benefits.5–7 For exam-ple, in both animal models of hypertension8 and hypertensive patients,9 statins produce a small reduction in blood pressure, which may have contributed to the beneficial effects observed in the ASCOT trial.10 Furthermore, in normocholesterolemic animals, statin therapy has been shown to protect against ischemia–reperfusion injury of the heart11 and the incidence of stroke.12 In addition, Kayikcioglu et al demonstrated an increased ischaemic threshold on exercise stress testing in patients with coronary microvascular dysfunction.13 Collec-tively, the basic and clinical data suggest that statins have beneficial cardiovascular effects, including important effects on smaller vessels.

The mechanism(s) responsible for these vascular effects may involve some of the multiple pleiotropic effects of statins, including (a) improved endothelial function, (b) inhibition of endothelin synthesis, (c) antioxidant properties or (d) antiin-flammatory effects.14,15 Several of these actions are mediated via the endothelium-derived nitric oxide (NO) pathway, because statins have been shown to increase NO bioavailabil-ity.14 Endothelium-derived NO is the primary relaxing factor in the large conduit vessels, although its production is impaired in atheromatous vessels16 and following ischemia–reperfusion injury.17 In contrast, endothelium-derived hyperpolarizing factor plays a significant role in the microvasculature in addi-tion to NO.18,19 Nevertheless there is evidence that chronic statin treatment may be beneficial in some microvascular sys-tems.13,20 Considering these observations, the role of statins in influencing constrictor responses in small vessels warrants further clarification and the responsible mechanisms identi-fied. To date, what little information there is on the direct effects of statins on small vessel reactivity21–23 relates mostly

Received September 22, 2010; revised manuscript received January 26, 2011; accepted February 28, 2011; released online April 29, 2011 Time for primary review: 20 days

Cardiology Unit, The Queen Elizabeth Hospital, Department of Medicine, The University of Adelaide, Adelaide, South Australia, AustraliaMailing address: Professor John F Beltrame, BMBS, PhD, Discipline of Medicine, The University of Adelaide, The Queen Elizabeth

Hospital, 28 Woodville Rd, Woodville South, South Australia 5011, Australia. E-mail: [email protected] doi: 10.1253/circj.CJ-10-0954All rights are reserved to the Japanese Circulation Society. For permissions, please e-mail: [email protected]

Acute Modulation of Vasoconstrictor Responses by Pravastatin in Small VesselsNader Ghaffari; Christine Ball; Jennifer A Kennedy;

Irene Stafford; John F Beltrame

Background: Statins have been shown to inhibit conduit vessel constrictor responses via the endothelial nitric oxide (NO) pathway. Clinical studies have implicated an effect in microvascular resistance vessels; however, direct effects of therapeutically relevant statin concentrations have not been examined. We examined the effect of acute pravastatin pretreatment on vasoconstrictor responsiveness of isolated rat mesenteric small vessels.

Methods and Results: Pravastatin (112 nmol/L) pretreatment for 60 min reduced both the potency and maximal constrictor responses to phenylephrine, thromboxane (U46619) and serotonin in small vessels. This effect was abol-ished by endothelial denudation, NO synthase (NOS) inhibition with N-ω-nitro-L-arginine methyl ester (L-NAME 300 μmol/L) and Akt inhibition (Akt1/2 kinase inhibitor 500 nmol/L), confirming an endothelium-dependent mechanism and implicating a NO-mediated effect via the Akt pathway. Maximal superoxide scavenging with polyethylene glycol-superoxide dismutase (PEG-SOD), 150 U/ml did not influence phenylephrine constrictor responses but potentiated pravastatin’s effect, suggesting that the statin did not increase NO bioavailability merely via an antioxidant mecha-nism. In contrast, pravastatin did not affect endothelin-1 (ET-1) constrictor responses. However, after pre-incubation with a selective endothelin-B (ETB) receptor antagonist (BQ788 3 μmol/L) pravastatin inhibited ET-1 constriction, sug-gesting that its effect is via the same mechanistic pathway as the ETB receptor.

Conclusions: In small vessels, pravastatin inhibits constrictor responses by increasing endothelial NO bioavail-ability via the Akt pathway. Furthermore, ETB receptor blockade unmasks this effect in ET-1 constrictor responses.

Key Words: Endothelin-1; Phenylephrine; Pravastatin; Vasoconstriction; Wire-myograph

S

ORIGINAL ARTICLEVascular Medicine

Advance Publication by J-STAGE

to concentrations in excess of those considered therapeuti-cally appropriate.24–26 Accordingly, the objective of this study was to examine the direct effect of pravastatin on vasocon-strictor responses in isolated small vessels at therapeutically relevant concentrations and explore the potential mechanism mediating any observed effect.

MethodsMale Sprague Dawley rats (250–400 g) were killed humanely under halothane anesthesia and the mesenteric artery distal branches were carefully dissected. The protocol was approved by the institutional animal ethics committee and conformed to the Australian National Health and Medical Research Council guidelines for animal usage for experimentation.

Vascular PreparationSmall mesenteric arteries (328±6 μm in diameter) were mounted between 2 stainless steel wires (40 μm in diameter) in an automated tension myograph (Danish-Myo Technol-ogy, Denmark) to assess isometric tension.27 Vessels were bathed in Krebs-bicarbonate solution and aerated with carbo-gen (95% O2, 5% CO2) at 37°C. Constituents of the Krebs solution (mmol/L) were: NaCl (118), KH2PO4 (1.18), KCl (3.89), NaHCO3 (25), MgCl2 (1.05), CaCl2 (2.34), EDTA (0.01) and glucose (5.56) at pH 7.4. Resting vessel tension was normalized to 90% of the diameter achieved if the vessel was under an effective transmural pressure of 100 mmHg.27 After a 30-min equilibration period, vessel viability and response to a standard depolarizing stimulus were evaluated with high-potassium physiological salt solution (KPSS; 122 mmol/L KCl). After establishing a phenylephrine con-centration–response curve, endothelial integrity was assessed using acetylcholine (0.01–100 μmol/L) in vessels precon-stricted with phenylephrine to 75% of maximum. Endothe-lium was considered intact if acetylcholine relaxed the ves-sels by ≥50%. When required, endothelial denudation was achieved by passing a single hair through the lumen of the vessel and gently rubbing the endothelium.

Study ProtocolsUtilizing this in-vitro small vessel preparation, the following studies were conducted to examine the acute effects of pravas-tatin pre-incubation on constrictor responses and to assess the underlying mechanisms. A pravastatin concentration of 112 nmol/L was utilized because it approximates the therapeu-tic-equivalent plasma concentration in clinical studies.24–26

Effect of Pravastatin on Vasoconstrictor Responses Con-centration–response curves were obtained using the fol-lowing agonists: phenylephrine (0.01–100 μmol/L), throm-boxane analogue (U46619, 1 nmol/L–3 μmol/L), serotonin (5HT, 0.01–10 μmol/L), and endothelin-1 (ET-1: 1pmol/L–30 nmol/L). These concentration–response curves were then repeated following 60-min pre-incubation with pravastatin. Initial experiments indicated that a shorter incubation time of 15 min was without effect. Appropriate time controls were also undertaken and the ET-1 responses with and without pravastatin were performed on segments of the same vessel in parallel baths, due to the prolonged contractile response of this agonist.

Role of Endothelial NO on the Effect of Pravastatin To determine the influence of the endothelium on the effect of pravastatin, the phenylephrine studies just described were repeated following endothelial denudation. Loss of endothe-lial integrity was verified by loss of vasodilatory responses

to acetylcholine in phenylephrine preconstricted vessels (ie, response to acetylcholine <10%). To specifically assess the role of NO on the effect of pravastatin, the NOS inhibitor, N-ω-nitro-L-arginine methyl ester (L-NAME, 300 μmol/L) was added 30 min prior to the constrictor agents. Thus ves-sels with intact endothelium were pretreated with pravastatin alone, L-NAME alone, or pravastatin + L-NAME, and the phenylephrine/U46619 vasoconstrictor responses were reas-sessed. To evaluate the involvement of the phosphati-dylinositol-3 kinase (PI3K)/Akt pathway in the effects of pravastatin, phenylephrine responses were also performed in the presence of Akt inhibition (Akt1/2 kinase inhibitor, 500 nmol/L) following a 60-min pre-incubation. Thus ves-sels with intact endothelium were pretreated with pravastatin alone, Akt inhibitor alone, or pravastatin + Akt inhibitor, and the phenylephrine vasoconstrictor responses reassessed.

To confirm the endothelial-dependent effect of pravastatin, concentration–response curves were also obtained using the endothelium-dependent vasodilator, acetylcholine (0.01–100 μmol/L) before and following a 60-min pre-incubation with pravastatin (112 nmol/L).

Influence of Superoxide on the Effect of Pravastatin Be-cause pravastatin could potentially limit superoxide levels28,29 and thereby inhibit the degradation of NO to peroxynitrite, pravastatin’s effect on phenylephrine responses was assessed in the presence of maximal superoxide scavenging with poly-ethylene glycol-superoxide dismutase (PEG-SOD). The con-centration of PEG-SOD required for maximal superoxide scavenging was determined in preliminary experiments in a cell-free system, using superoxide generation by hypoxan-thine–xanthine oxidase and measurement via lucigenin-derived chemiluminescence assessed using a Picolite-luminometer (Packard Instruments, CT, USA). Under these conditions, 93±2% (n=5) of the superoxide generated was scavenged at a concentration of 150 U/ml PEG-SOD. This concentration was then used in subsequent experiments with an additional 30 min prior to the constrictor agent. Phenylephrine constrictor responses were repeated with the following pretreatments: (a) control (vehicle only), (b) pravastatin 112 nmol/L, (c) PEG-SOD 150 U/ml, and (d) pravastatin + PEG-SOD (112 nmol/L and 150 U/ml, respectively).

Endothelin-B Receptor and Pravastatin Effect on ET-1 Vasoconstriction Because the net ET-1 constrictor response is influenced by endothelin-ETB receptors that stimulate NO production, the pravastatin response was also assessed in the presence of selective endothelin-ETB receptor blockade with BQ788 (3 μmol/L).30 BQ788 was added 30 min prior to con-striction with ET-1. Constrictor responses were assessed in the presence of the following pretreatments: (a) control (vehi-cle), (b) pravastatin 112 nmol/L, (c) BQ788 3 μmol/L, and (d) pravastatin + BQ788 (112 nmol/L and 3 μmol/L, respectively).

Drug PreparationsAcetylcholine chloride, Akt1/2 kinase inhibitor (1,3-dihy-dro-1-(1-((4-(6-phenyl-1H-imidazo[4,5-g]quinoxalin-7-yl)phenyl)methyl)-4-piperidinyl)-2H-benzimidazol-2-one trifluoroacetate salt hydrate), 5-hydroxytryptamine creati-nine sulfate complex, L-NAME, L-phenylephrine hydro-chloride, PEG-SOD and 9,11-dideoxy-11α,9α-epoxy-meth-ano-prostaglandinF2α (U46619) were purchased from Sigma (St Louis, MO, USA). ET-1 acetate salt was purchased from AUSPEP (Parkville, Victoria, Australia), BQ788 from BACHEM (Bubendorf, Switzerland), and pravastatin sodium salt from Calbiochem (Merck, Victoria, Australia).


Statistical AnalysisContractile responses are expressed as a percentage of the KPSS response. Results are presented as mean ± SEM. The Emax and EC50 values were derived from concentration–response curves constructed by non-linear curve fitting (Graph Pad Prism version 4.0 Software). Comparisons between con-trol and pravastatin pretreated vessels were performed with paired Student’s t-tests for both Emax and log EC50 values. Comparisons among 3 or more treatment groups was under-taken by 1-way analysis of variance (ANOVA) with Bonfer-

roni’s multiple comparison post-hoc-test. A critical value of P<0.05 was adopted for statistical significance. n refers to the number of animals studied.

ResultsEffect of Pravastatin on Vasoconstrictor ResponsesThe 60-min pre-incubation with a therapeutically relevant concentration of pravastatin (112 nmol/L) inhibited maximal constriction (Emax) to phenylephrine by 34±4%, to U46619

Figure 1. Effect of pre-incubation with pravastatin (112 nmol/L) on vasoconstrictor responses to (A) phenylephrine (PE, n=21), (B) U46619 (n=6), (C) serotonin (5-HT, n=7), and (D) endothelin-1 (ET, n=6) in endothelium-intact rat mesenteric microvessels. Control segments (□) and pravastatin pretreated segments (■).

Table. Effect of Pravastatin on Vascular Reactivity in Rat Mesenteric Microvessels

Vasoactive agent nEC50 (Log M) Emax (%KPSS)

Control Pravastatin Control Pravastatin

Phenylephrine 21 –5.9±0.05 –5.7±0.06*　 122±2 88±4*　

Serotonin 8 –6.3±0.05 –6.2±0.06*　 124±6 96±3*　

U46619 6 –7.1±0.03 –6.8±0.11*　 114±3 85±4*　

Acetylcholine 5 –6.6±0.36 –6.9±0.16*　 74±7 86±3*　

Endothelin 6 –8.8±0.04 –8.6±0.09　　 111±5 111±5　　　　

Endothelin + BQ788 6 –8.8±0.06 –8.5±0.03** 117±7 87±5**

Phenylephrine + PEG-SOD 8 –5.9±0.08 –5.5±0.08** 113±2 67±3**

*Effect of pre-incubation with pravastatin (112 nmol/L) significant, P<0.05, paired t-test.**Effect of pre-incubation with pravastatin (112 nmol/L) plus BQ788 (3μ mol/L) or pravastatin (112 nmol/L) plus PEG-SOD (150 U/ml) significant, P<0.05, one-way ANOVA.n indicates the number of animals used and the vessel size was determined prior to drug administration, using the Mulvany normalization procedure.KPSS, high-potassium physiological salt solution; PEG-SOD, polyethylene glycol superoxide dismutase.


by 30±4%, and to 5HT by 28±6% in rat mesenteric vessels with intact endothelium (Figure 1, Table). Furthermore, pravastatin shifted the constrictor response curves to the right, reducing the potency of phenylephrine by approxi-mately 1.7-fold, U46619 by approximately 2.2-fold and 5HT by approximately 1.2-fold (Figure 1, Table).

However, pravastatin had no effect on vasoconstrictor responses to phenylephrine when a shorter pre-incubation of 15 min was used. Emax responses to phenylephrine were 124±2% and 122±2% in control and pravastatin pretreated vessels, respectively (P=0.14, n=7). Furthermore, phenyleph-rine EC50 concentrations (log M) were −5.9±0.08 in the con-trol segments and −5.8±0.26 (P=0.23, n=7) in those pretreated for only 15 min with pravastatin.

In contrast to the other constrictor agents, both the Emax

Figure 2. Effect of pre-incubation with pravastatin (112 nmol/L) on vasoconstrictor responses to (A) phenylephrine (PE, n=6) in endothelium-denuded rat mesenteric microvessels (endo-thelium-denuded segments (□) and endothelium-denuded segments with pravastatin pretreatment (■)), (B) PE (n=7) and (C) U46619 (n=6) in endothelium-intact rat mesenteric microvessels following nitric oxide synthase inhibition with L-NAME (300 μmol/L). L-NAME pretreated segments (□) and L-NAME+pravastatin pretreated segments (■). L-NAME, N-ω-nitro-L-arginine methyl ester.

Figure 3. (A) Effect of pre-incubation with pravastatin (112 nmol/L) on vasoconstrictor responses to phenylephrine (PE, n=6), in the absence and presence of Akt inhibitor (500 nmol/L) in endothelium-intact rat mesenteric microves-sels. Control segments (□), pravastatin pretreated seg-ments (■), Akt inhibitor pretreated segments (○) and Akt inhibitor+pravastatin pretreated segments (●). (B) Effect of pre-incubation with pravastatin (112 nmol/L) on vasocon-strictor responses to phenylephrine (PE, n=6), in the ab-sence and presence of PEG-SOD (150 U/ml) in endotheli-um-intact rat mesenteric microvessels. Control segments (□), pravastatin pretreated segments (■), PEG-SOD pre-treated segments (○) and PEG-SOD + pravastatin pretreat-ed segments (●). PEG-SOD, polyethylene glycol-superox-ide dismutase.


and EC50 responses to ET-1 were unchanged by 60-min pre-incubation with pravastatin (Figure 1, Table).

Role of Endothelial NO on the Effect of PravastatinRemoval of endothelium in mesenteric vessels increased the maximum contractile response to phenylephrine (127±5% vs. 116±5% in vessels with intact endothelium, P=0.007, n=6) as previously seen in larger vessels,31 but did not affect the potency of phenylephrine (EC50 values, log M: −5.7±0.12 vs. −5.8±0.06 in vessels with intact endothelium, P=0.12, n=6). However, endothelial denudation abolished pravastatin’s inhibition of phenylephrine responses (Figure 2A), with pravastatin and controls having similar Emax values (136±8% vs. 127±5%, P>0.05) and EC50 values (log M: −5.8±0.05 vs. −5.7±0.12, P>0.05; n=6).

Compared with the control phenylephrine responses, NOS

inhibition with L-NAME had no significant effect on either Emax (117±3% vs. 129±4%, P=0.059; n=7) or EC50 values (log M: −5.8±0.08 vs. −6.1±0.08, P=0.12; n=7). However, in the presence of NOS inhibition, pravastatin pretreatment did not alter the phenylephrine Emax (129±4% vs. 122±5%, P=0.3; n=7) or EC50 value compared with controls (log M: −5.8±0.08 vs. −6.1±0.07; P=0.099, n=7) (Figure 2B). A similar aboli-tion of pravastatin’s effect on the U46619 constrictor responses occurred in the presence of L-NAME (Figure 2C). These findings support an endothelial NO mechanism being respon-sible for pravastatin’s inhibition of constrictor responses.

Compared with controls, Akt inhibition did not affect phenylephrine’s constrictor potency (EC50, log M: −5.9±0.06 vs. −5.5±0.2, P>0.05; n=6) or maximal constrictor response (Emax: 129±5% vs. 123±5%, P>0.05; n=6). However, Akt inhibition abolished the inhibitory effect of pravastatin on the phenylephrine responses (Figure 3A), with Emax (127±6%, P>0.05, n=6) and EC50 values (log M: −5.4±0.14, P>0.05; n=6) similar to those for the controls, suggesting pravastatin’s effects were mediated via the Akt pathway.

These documented endothelium-dependent NO and Akt pathway mediated pravastatin effects on phenylephrine responses is further supported by pravastatin’s effect on ace-tylcholine-induced relaxation (Figure 4). Acetylcholine relax-ation responses in endothelium-intact vessels were enhanced by pravastatin pretreatment, with a 12±4% increase in Emax (P=0.028, n=5) and a reduction in EC50 of approximately 2.1-fold (Figure 4, Table).

Influence of Superoxide on the Effect of PravastatinIn rat mesenteric small arteries preconstricted with phenyleph-rine, PEG-SOD pretreatment alone did not affect constrictor responsiveness, as neither the Emax (113±2%) nor the EC50 value (log M: −5.9±0.08) was significantly different to the control (120±2%, −6.1±0.07; n=8). However, when PEG-SOD was co-incubated with pravastatin, the extent of inhibition of the phenylephrine concentration–response curve (approxi-mately 3.9-fold decrease in potency and 53±3% change in maximal constriction; P<0.05, n=8, Figure 3B) was greater than the effect with pravastatin alone (approximately 1.7-fold reduction in potency and 33±6% change in Emax; P<0.05, n=8 compared with control). Thus, in the presence of PEG-SOD the addition of pravastatin reduced maximal contraction a further 19±8% and reduced the potency a further 2.3-fold, over and above the effect of pravastatin alone (P<0.05, n=8). These results imply that pravastatin’s inhibition of constric-tor responses is not mediated by a reduction in superoxide concentrations because maximal superoxide scavenging by PEG-SOD did not abolish pravastatin’s effect but enhanced it, suggesting an independent synergistic effect.

Endothelin-B Receptor Blockade and Pravastatin Effect on ET-1ET-1 constrictor responses were not influenced by pravastatin (112 nmol/L) pretreatment or treatment with BQ788 (3 μmol/L) alone (P>0.05, 1-way ANOVA). However, pretreatment with pravastatin in the presence of BQ788 inhibited the ET-1 Emax response by 30±3% and reduced the potency of ET-1 by approximately 2-fold (Figure 5, Table). Thus, blocking endo-thelin-ETB receptors prior to stimulation by ET-1 unmasked pravastatin’s inhibition of this constrictor response.

DiscussionThe major findings of this study relate to the direct vascular

Figure 4. Effect of pre-incubation with pravastatin (112 nmol/L) on endothelium dependent relaxation responses to acetyl-choline (ACh, n=8) in endothelium-intact rat mesenteric mi-crovessels. Control segments (□) and pravastatin pretreat-ed segments (■).

Figure 5. Effect of pre-incubation with pravastatin (112 nmol/L) on vasoconstrictor responses to endothelin-1 (n=6) in the absence and presence of BQ788 (3 μmol/L) in endothelium-intact rat mesenteric microvessels. Control segments (□), pravastatin pretreated segments (■), BQ788 pretreated segments (○) and BQ788 + pravastatin pretreated seg-ments (●).


effects of pravastatin and include (1) attenuation of constric-tor responses mediated via the α-adrenergic, serotonergic, and thromboxane pathways at a clinically relevant statin con-centration; (2) the effects of pravastatin being endothelium-dependent and inhibited by both NOS and Akt inhibitors but not maximal superoxide scavenging with PEG-SOD, suggest-ing an endothelial NO mechanism mediated via the Akt path-way that did not involve reduced NO degradation by superox-ide; and (3) ET-1 mediated constrictor responses being only attenuated by pravastatin in the presence of a specific endo-thelin-ETB receptor blocker. Although some of these findings have previously been documented in large conduit vessels, this is the first study examining the direct effect in small ves-sels of a statin at a clinically relevant concentration.

Effect of Statins on Small VesselsDirect modulation of vascular responses by statins has been postulated to explain some of their clinical benefits. Although some of these effects (eg, plaque stabilization) can be explained by their effects on large conduit vessels, other responses cannot. For example, Kayikcioglu et al demon-strated that pravastatin pretreatment had anti-ischemic effects in cardiac syndrome-X patients who have normal angiogra-phy and evidence of coronary microvascular dysfunction.17 Statins also have mild blood pressure lowering properties that can also be accounted for by their effects on the resis-tance vessels. The demonstration that statins can modulate constrictor responses provides an explanation for these clini-cal observations. In particular, this study’s results suggest that sympathetic nerve (α-adrenergic) and platelet–thrombus (serotonergic–thromboxane) mediated constriction could be attenuated by pravastatin but not endothelin-mediated con-striction.

Mechanism of Pravastatin’s Effects on Responses in Small VesselsThe previous studies examining the mechanisms responsible for statin-associated modulation of large vessel constrictor responses have described both endothelium-dependent and -independent mechanisms. The endothelium-dependent mech-anisms include either (1) increased NO production by (a) sta-bilizing endothelial NOS mRNA and therefore its expression,32 (b) activating endothelial NOS by phosphorylating its serine 1,177 residue,33 (c) endothelial NOS activation via Akt sig-naling,34 and/or (d) inhibition of caveolin, which inhibits endothelial NOS activation35 or (2) increased NO bioavail-ability by prevention of its degradation by superoxide. For example, 18 h exposure to atorvastatin or pravastatin not only enhanced endothelial NOS activity but also inhibited super-oxide generation in rat aorta, an effect attributed to inhibition of isoprenylation of p21 Rac,15,36,37 which is a critical step in the assembly of NADPH oxidase.38 Moreover, the enhance-ment of relaxation of rat aorta to acetylcholine after atorvas-tatin treatment was eliminated in the presence of SOD, sug-gesting that reduction in superoxide was the major mechanism for the enhanced relaxation response to acetylcholine.38 Simi-lar protective effects against oxidative stress have been sug-gested for both acute29 and chronic in-vivo39 and in-vitro40 protection by statins against low-density lipoprotein-induced endothelial dysfunction in rat aorta.

In this study we demonstrated that pravastatin’s attenua-tion of constrictor responses is abolished following endo-thelial denudation and NOS inhibition, implicating an endothelial NO mechanism in small vessels. This increased NO bioavailability by pravastatin appears to be a result of

increased production via the PI3K/Akt pathway, because the effect of pravastatin was abolished in the presence of an Akt-inhibitor. Statins have been shown to rapidly pro-mote the activation of Akt in endothelial cells, leading to eNOS phosphorylation and increased NO production.34 At low (clinically relevant) statin concentrations, this is achieved via endothelial Ras with subsequent activation of Akt.34,41 However, high statin concentrations are toxic to endothelial cells,34 presumably because of inhibition of protein prenylation.32 In an attempt to support the pro-posed mechanism via the PI3K/Akt signaling pathway, we also used the PI3K/Akt inhibitor (LY294002). However LY294002 (10 μmol/L) reduced the phenylephrine vaso-constrictor responses by approximately 40% (data not shown), preventing interpretation of its effects on the statin response. This PI3K inhibitory effect of LY294002 on the phenylephrine responses is consistent with results from a recent study in mouse cerebral arteries.42

To ascertain if increased NO bioavailability was related to reduced degradation via superoxide we examined pravas-tatin’s antioxidant effects. Firstly, in a cell-free system with hypoxanthine–xanthine oxidase generated superoxide mea-sured by lucigenin-derived chemiluminescence, the clini-cally relevant concentration of pravastatin had no scavenging effect (data not shown). This result is consistent with a previ-ous study43 demonstrating superoxide-scavenging by fluvas-tatin but not pravastatin at low concentrations, but contrasts with another study in which pravastatin exhibited a superox-ide-scavenging effect at very high concentrations.44 Our sec-ond approach involved combining PEG-SOD (at maximal superoxide scavenging concentrations) with the clinically relevant concentration of pravastatin in the small arteries. The increased effect of PEG-SOD + pravastatin suggests that the latter does not mediate its vasomotor effect via superox-ide scavenging but via an alternate mechanism such as those described above.

In addition to NO, nitroxyl may also be partially derived from NOS45 and it is possible that some of the acute effects of pravastatin observed in the present study involve increased nitroxyl production. However, the effects of nitroxyl would not be expected to be enhanced by PEG-SOD, because the nitroxyl concentration is not limited by superoxide. An alter-native explanation for the increased inhibition of vasocon-strictor tone when PEG-SOD was combined with pravastatin includes the possibility that PEG-SOD increased the tissue concentration of hydrogen peroxide, which has been described as a hyperpolarizing factor in mouse mesenteric vessels.19 However, at odds with these explanations is the observation that PEG-SOD alone had no significant effect on phenyleph-rine responses in the absence of pravastatin.

As documented by previous investigators, NO typically has a lesser role in regulating vascular tone in resistance ves-sels, where endothelium-dependent hyperpolarizing factor plays a greater role than in conduit vessels.46 Specifically, the study by Takamura et al showed that the contribution of NO to shear stress-induced relaxation is greater in rat large mes-enteric arteries (400–500 μm) than in resistance mesenteric arteries (150–250 μm).47 Consistent with their findings, our study demonstrated that the physiologic role of NO in regu-lating basal vascular tone in small vessels is minimal, because endothelial NOS inhibition by L-NAME did not affect the vasoconstrictor responses to phenylephrine and U46619. In contrast, the inhibition of pravastatin’s effect by L-NAME indicates that NO plays a significant role in modifying vas-cular tone after acute exposure to a statin. Thus pravastatin


appears to have a similar potentiating effect on NO bioavail-ability in small vessels to that observed in large vessels, and this effect appears to be mediated through enhanced NO pro-duction via the PI3K/Akt pathway.

Compared with the study by Takamura et al,47 we used vessels that were intermediate in size (328±6 μm), which raises the possibility that if smaller vessels were utilised then a lesser effect would have been observed with pravastatin, given that the pravastatin effect was eliminated by endothelial NOS inhibition with L-NAME. We therefore performed a post-hoc subanalysis comparing the effect of pravastatin on the phenylephrine responses in vessels ≥300 μm (353±7 μm; n=14) and those <300 μm (269±3 μm; n=7), observing a simi-lar reduction in the Emax between the 2 groups (35±3% vs. 31±7% reduction, respectively, P=0.60). This was confirmed by examining the relationship between vessel size and maxi-mal contraction to phenylephrine in control and pravastatin-treated vessels using linear regression analysis. The slopes obtained (−0.018 control vs. −0.107 pravastatin-treated) did not significantly deviate from zero, nor were they significantly different from each other (F1,38 =0.93, P=0.34). The axis-inter-cepts at zero size (127.4 control vs. 122.7 pravastatin-treated) were significantly different (F1,39 =69.9, P<0.0001), indicating the lines are parallel with distinct intercepts. Although we can not exclude a change in this relationship had smaller vessels been examined, there does not appear to be a significant pro-gressive gradient in responses in vessels between 236 and 381 μm in diameter and NOS inhibition eliminated the pravas-tatin effect irrespective of vessel size.

In addition to the above endothelium-dependent mecha-nisms, endothelium-independent mechanisms have also been identified for the vascular effect of statins via direct effects on vascular smooth muscle cells. These involve statin inhibition of calcium mobilization both via voltage-depen-dent calcium channels and intracellular calcium stores.48 These direct vascular smooth muscle effects vary with dif-ferent statins. Simvastatin,48,49 atorvastatin48 and fluvastatin23 have all been shown to directly effect vascular smooth mus-cle cells by inhibiting calcium mobilization. This acute effect is mediated via opening of vascular smooth muscle cell voltage-dependent (Kv) potassium channels.23 In con-trast, the hydrophilic statin, pravastatin, was chosen for this study because it does not directly alter vascular smooth muscle cell calcium mobilization,23,48 but appears to operate via endothelium-dependent mechanisms, consistent with a complete loss of effect after endothelium denudation in the present study.

Pravastatin and ET-1 Constrictor Responses in Small VesselsUnlike the other vasoconstrictors, ET-1 constrictor responses were not affected by pravastatin. However, ET-1 stimulates both endothelin-ETA and endothelin-ETB receptors, includ-ing endothelin-ETB receptors on the endothelium, resulting in the release of endothelium-derived NO. Hence the net constrictor effect of ET-1 is determined by a balance between endothelin-ETA receptor–endothelin-ETB receptor-mediated constriction, and endothelin-ETB receptor-mediated relax-ation. Pravastatin treatment alone does not appear to provide any incremental NO effect in the presence of maximal endo-thelin-ETB receptor stimulation and thus on NO release. However, selective endothelin-ETB receptor blockade (with BQ788) restores pravastatin’s ability to inhibit the contractile response. This may suggest that pravastatin-mediated NO release and endothelin-ETB receptor-mediated NO release share a common pathway.

More interestingly, this study for the first time has identi-fied an interaction between pravastatin’s vascular effects and endothelin-ETB receptor blockade. From our observa-tions, it would be anticipated that a non-selective endothe-lin-ETA – endothelin-ETB receptor blocker would enhance pravastatin’s inhibition of constrictor responses, whereas pravastatin would have no effect in the presence of a selec-tive endothelin-ETA receptor blocker because of the unop-posed endothelin-ETB receptor effects on NO. Whether this impairment of pravastatin’s inhibition of ET-1 constrictor responses in the presence of selective endothelin-ETA block-ers translates into a clinically significant difference is open to speculation. In particular, does endothelin-ETB receptor stimulation by ET-1 impair any of pravastatin’s clinically documented (NO-mediated) protective effects? Alterna-tively, does the use of pravastatin with a non-selective endo-thelin-ETA – endothelin-ETB receptor blocker enhance the vascular benefits of these agents in the treatment of such conditions as pulmonary hypertension? These questions require further investigations beyond the scope of the cur-rent study.

Study LimitationsThe findings of this study are limited by the use of rat mesen-teric vessels rather than human tissue. Furthermore, we exam-ined in-vitro vessels from healthy rats, and the effects of acute statin treatment observed cannot necessarily be extrap-olated to vessels displaying endothelial dysfunction in-vivo. However, a human small vessel model is the focus of our future studies exploring the role of disorders affecting endo-thelial function and therefore influencing the pravastatin response. A second limitation is the use of a single concentra-tion of pravastatin. Accordingly, we may not have achieved the maximal possible acute effect of pravastatin. However, we wished to examine a concentration that was relevant to clinical use. This study also did not address the effect of pravastatin on endothelium-derived hyperpolarizing factor. Thus, although NO is clearly the major mechanism whereby pravastatin exerts its vasomotor effect in the small vessels studied, the influence of endothelium-derived hyperpolariz-ing factor on the response to pravastatin in smaller vessels cannot be excluded.

ConclusionPravastatin at a clinically relevant concentration inhibits vasoconstrictor responses in small vessels via an endothe-lium-dependent NO mechanism. The mechanism whereby pravastatin increases NO bioavailability in the small vessels is likely to be via increased NO production via endothelial NOS involving the Akt pathway. This appears to share a common pathway with endothelial endothelin-ETB receptor-mediated NO release. These findings have important clinical implications and suggest that pravastatin may have addi-tional benefits in addition to lowering cholesterol.

AcknowledgmentsThe studies were in part supported by a National Heart Foundation of Australia Grant-in-Aid (G 05A 2080). Nader Ghaffari and Christine Ball are supported by The Queen Elizabeth Hospital Research Founda-tion/The University of Adelaide Scholarships. John Beltrame is a South Australian Cardiovascular Research Development Program Fellow.

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