Advance Publication by J-STAGE

Circulation Journal Official Journal of the Japanese Circulation Society http://www. j-circ.or.jp

ORIGINAL ARTICLE
VascularMedicine

Acute Modulation of Vasoconstrictor Responses by Pravastatin in Small Vessels

Nader Ghaffari; Christine Ball; Jennifer A Kennedy; Irene Stafford; John F Beltrame

Background: Statinshavebeenshowntoinhibitconduitvesselconstrictorresponsesviatheendothelialnitric oxide(NO)pathway.Clinicalstudieshaveimplicatedaneffectinmicrovascularresistancevessels;however,direct effectsoftherapeuticallyrelevantstatinconcentrationshavenotbeenexamined.Weexaminedtheeffectofacute pravastatinpretreatmentonvasoconstrictorresponsivenessofisolatedratmesentericsmallvessels. MethodsandResults: Pravastatin (112nmol/L) pretreatment for 60min reduced both the potency and maximal constrictorresponsestophenylephrine,thromboxane(U46619)andserotonininsmallvessels.Thiseffectwasabolished by endothelial denudation, NO synthase (NOS) inhibition with N--nitro-L-arginine methyl ester (L-NAME 300 mol/L)andAktinhibition(Akt1/2kinaseinhibitor500nmol/L),confirminganendothelium-dependentmechanism andimplicatingaNO-mediatedeffectviatheAktpathway.Maximalsuperoxidescavengingwithpolyethyleneglycolsuperoxidedismutase(PEG-SOD),150U/mldidnotinfluencephenylephrineconstrictorresponsesbutpotentiated pravastatinseffect,suggestingthatthestatindidnotincreaseNObioavailabilitymerelyviaanantioxidantmechanism.Incontrast,pravastatindidnotaffectendothelin-1(ET-1)constrictorresponses.However,afterpre-incubation withaselectiveendothelin-B(ETB)receptorantagonist(BQ7883 mol/L)pravastatininhibitedET-1constriction,suggestingthatitseffectisviathesamemechanisticpathwayastheETBreceptor. Conclusions: Insmallvessels,pravastatininhibitsconstrictorresponsesbyincreasingendothelialNObioavailability 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

tatins reduce plasma cholesterol via inhibition of 3hydroxy-3-methylglutaryl-coenzyme-A reductase and have been shown to have a major clinical effect on improving cardiovascular outcomes.14 Additional pleiotropic vascular effects have been described that are not attributable simply to a reduction in cholesterol levels but which may contribute to their cardiovascular benefits.57 For example, 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 ischemiareperfusion 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 Collectively, 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) antiinflammatory 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 bioavailability.14 Endothelium-derived NO is the primary relaxing factor in the large conduit vessels, although its production is impaired in atheromatous vessels16 and following ischemiareperfusion injury.17 In contrast, endothelium-derived hyperpolarizing factor plays a significant role in the microvasculature in addition to NO.18,19 Nevertheless there is evidence that chronic statin treatment may be beneficial in some microvascular systems.13,20 Considering these observations, the role of statins in influencing constrictor responses in small vessels warrants further clarification and the responsible mechanisms identified. To date, what little information there is on the direct effects of statins on small vessel reactivity2123 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, Australia Mailing 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: john.beltrame@adelaide.edu.au ISSN-1346-9843 doi: 10.1253/circj.CJ-10-0954 All rights are reserved to the Japanese Circulation Society. For permissions, please e-mail: cj@j-circ.or.jp

Advance Publication by J-STAGE

to concentrations in excess of those considered therapeutically appropriate.2426 Accordingly, the objective of this study was to examine the direct effect of pravastatin on vasoconstrictor responses in isolated small vessels at therapeutically relevant concentrations and explore the potential mechanism mediating any observed effect.

Methods
Male Sprague Dawley rats (250400 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. VascularPreparation Small mesenteric arteries (3286 in diameter) were m mounted between 2 stainless steel wires (40 in diameter) m in an automated tension myograph (Danish-Myo Technology, Denmark) to assess isometric tension.27 Vessels were bathed in Krebs-bicarbonate solution and aerated with carbogen (95% O2, 5% CO2) at 37C. 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 concentrationresponse curve, endothelial integrity was assessed using acetylcholine (0.01100 mol/L) in vessels preconstricted with phenylephrine to 75% of maximum. Endothelium was considered intact if acetylcholine relaxed the vessels by 50%. When required, endothelial denudation was achieved by passing a single hair through the lumen of the vessel and gently rubbing the endothelium. StudyProtocols Utilizing this in-vitro small vessel preparation, the following studies were conducted to examine the acute effects of pravastatin pre-incubation on constrictor responses and to assess the underlying mechanisms. A pravastatin concentration of 112 nmol/L was utilized because it approximates the therapeutic-equivalent plasma concentration in clinical studies.2426 EffectofPravastatinonVasoconstrictorResponses Concentrationresponse curves were obtained using the following agonists: phenylephrine (0.01100mol/L), thromboxane analogue (U46619, 1 nmol/L3 mol/L), serotonin (5HT, 0.0110 mol/L), and endothelin-1 (ET-1: 1pmol/L 30 nmol/L). These concentrationresponse 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. RoleofEndothelialNOontheEffectofPravastatin To determine the influence of the endothelium on the effect of pravastatin, the phenylephrine studies just described were repeated following endothelial denudation. Loss of endothelial 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--nitro-L-arginine methyl ester (L-NAME, 300mol/L) was added 30 min prior to the constrictor agents. Thus vessels with intact endothelium were pretreated with pravastatin alone, L-NAME alone, or pravastatin + L-NAME, and the phenylephrine/U46619 vasoconstrictor responses were reassessed. To evaluate the involvement of the phosphatidylinositol-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 vessels 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, concentrationresponse curves were also obtained using the endothelium-dependent vasodilator, acetylcholine (0.01 100 mol/L) before and following a 60-min pre-incubation with pravastatin (112 nmol/L). InfluenceofSuperoxideontheEffectofPravastatin Because pravastatin could potentially limit superoxide levels28,29 and thereby inhibit the degradation of NO to peroxynitrite, pravastatins effect on phenylephrine responses was assessed in the presence of maximal superoxide scavenging with polyethylene glycol-superoxide dismutase (PEG-SOD). The concentration of PEG-SOD required for maximal superoxide scavenging was determined in preliminary experiments in a cell-free system, using superoxide generation by hypoxanthinexanthine oxidase and measurement via lucigenin-derived chemiluminescence assessed using a Picolite-luminometer (Packard Instruments, CT, USA). Under these conditions, 932% (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) PEGSOD 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 mol/L).30 BQ788 was added 30 min prior to constriction with ET-1. Constrictor responses were assessed in the presence of the following pretreatments: (a) control (vehicle), (b) pravastatin 112 nmol/L, (c) BQ788 3 mol/L, and (d) pravastatin + BQ788 (112 nmol/L and 3 mol/L, respectively). DrugPreparations Acetylcholine chloride, Akt1/2 kinase inhibitor (1,3-dihydro-1-(1-((4-(6-phenyl-1H-imidazo[4,5-g]quinoxalin-7yl)phenyl)methyl)-4-piperidinyl)-2H-benzimidazol-2-one trifluoroacetate salt hydrate), 5-hydroxytryptamine creatinine sulfate complex, L-NAME, L-phenylephrine hydrochloride, PEG-SOD and 9,11-dideoxy-11 ,9-epoxy-meth ano-prostaglandinF2 (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).

Advance Publication by J-STAGE

Figure1. Effectofpre-incubationwithpravastatin(112nmol/L)onvasoconstrictorresponsesto(A)phenylephrine(PE,n=21), (B)U46619(n=6),(C)serotonin(5-HT,n=7),and(D)endothelin-1(ET,n=6)inendothelium-intactratmesentericmicrovessels. Controlsegments( )andpravastatinpretreatedsegments( ).

Table. Effect of Pravastatin on Vascular Reactivity in Rat Mesenteric Microvessels Vasoactive agent Phenylephrine Serotonin U46619 Acetylcholine Endothelin Endothelin+BQ788 Phenylephrine+PEG-SOD n 21 8 6 5 6 6 8 EC50 (Log M) Control 5.90.05 6.30.05 7.10.03 6.60.36 8.80.04 8.80.06 5.90.08 Pravastatin 5.70.06* 6.20.06* 6.80.11* 6.90.16* 8.60.09 8.50.03** 5.50.08** Emax (%KPSS) Control 1222 1246 1143 747 1115 1177 1132 Pravastatin 884* 963* 854* 863* 1115 875** 673**

*Effectofpre-incubationwithpravastatin(112nmol/L)significant,P<0.05,pairedt-test. **Effectofpre-incubationwithpravastatin(112nmol/L)plusBQ788(3mol/L)orpravastatin(112nmol/L)plusPEGSOD(150U/ml)significant,P<0.05,one-wayANOVA. nindicatesthenumberofanimalsusedandthevesselsizewasdeterminedpriortodrugadministration,usingthe Mulvanynormalizationprocedure. KPSS,high-potassiumphysiologicalsaltsolution;PEG-SOD,polyethyleneglycolsuperoxidedismutase.

StatisticalAnalysis Contractile 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 control and pravastatin pretreated vessels were performed with paired Students t-tests for both Emax and log EC50 values. Comparisons among 3 or more treatment groups was undertaken by 1-way analysis of variance (ANOVA) with Bonfer-

ronis 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.

Results
EffectofPravastatinonVasoconstrictorResponses The 60-min pre-incubation with a therapeutically relevant concentration of pravastatin (112 nmol/L) inhibited maximal constriction (Emax) to phenylephrine by 344%, to U46619

Advance Publication by J-STAGE

Figure3. (A) Effect of pre-incubation with pravastatin (112nmol/L)onvasoconstrictorresponsestophenylephrine (PE, n=6), in the absence and presence of Akt inhibitor (500nmol/L)inendothelium-intactratmesentericmicrovessels. Control segments ( ), pravastatin pretreated segments (), Akt inhibitor pretreated segments () and Akt inhibitor+pravastatinpretreatedsegments().(B)Effectof pre-incubation with pravastatin (112nmol/L) on vasoconstrictor responses to phenylephrine (PE, n=6), in the absence and presence of PEG-SOD (150U/ml) in endothelium-intact rat mesenteric microvessels. Control segments ( ), pravastatin pretreated segments (), PEG-SOD pretreatedsegments()andPEG-SOD+pravastatinpretreated segments (). PEG-SOD, polyethylene glycol-superoxidedismutase.

Figure2. Effectofpre-incubationwithpravastatin(112nmol/L) onvasoconstrictorresponsesto(A)phenylephrine(PE,n=6) inendothelium-denudedratmesentericmicrovessels(endothelium-denuded segments () and endothelium-denuded segments with pravastatin pretreatment ()), (B) PE (n=7) and(C)U46619(n=6)inendothelium-intactratmesenteric microvessels following nitric oxide synthase inhibition with L-NAME (300 mol/L). L-NAME pretreated segments () andL-NAME+pravastatinpretreatedsegments().L-NAME, N--nitro-L-argininemethylester.

by 304%, and to 5HT by 286% 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 approximately 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 1242% and 1222% in control and pravastatin pretreated vessels, respectively (P=0.14, n=7). Furthermore, phenylephrine EC50 concentrations (log M) were 5.90.08 in the control segments and 5.80.26 (P=0.23, n=7) in those pretreated for only 15 min with pravastatin. In contrast to the other constrictor agents, both the Emax

Advance Publication by J-STAGE

Figure4. Effectofpre-incubationwithpravastatin(112nmol/L) on endothelium dependent relaxation responses to acetylcholine(ACh,n=8)inendothelium-intactratmesentericmicrovessels.Controlsegments( )andpravastatinpretreatedsegments().

inhibition with L-NAME had no significant effect on either Emax (1173% vs. 1294%, P=0.059; n=7) or EC50 values (log M: 5.80.08 vs. 6.10.08, P=0.12; n=7). However, in the presence of NOS inhibition, pravastatin pretreatment did not alter the phenylephrine Emax (1294% vs. 1225%, P=0.3; n=7) or EC50 value compared with controls (log M: 5.80.08 vs. 6.10.07; P=0.099, n=7) (Figure 2B). A similar abolition of pravastatins effect on the U46619 constrictor responses occurred in the presence of L-NAME (Figure 2C). These findings support an endothelial NO mechanism being responsible for pravastatins inhibition of constrictor responses. Compared with controls, Akt inhibition did not affect phenylephrines constrictor potency (EC50, log M: 5.90.06 vs. 5.50.2, P>0.05; n=6) or maximal constrictor response (Emax: 1295% vs. 1235%, P>0.05; n=6). However, Akt inhibition abolished the inhibitory effect of pravastatin on the phenylephrine responses (Figure 3A), with Emax (1276%, P>0.05, n=6) and EC50 values (log M: 5.40.14, P>0.05; n=6) similar to those for the controls, suggesting pravastatins 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 pravastatins effect on acetylcholine-induced relaxation (Figure 4). Acetylcholine relaxation responses in endothelium-intact vessels were enhanced by pravastatin pretreatment, with a 124% increase in Emax (P=0.028, n=5) and a reduction in EC50 of approximately 2.1fold (Figure 4, Table). InfluenceofSuperoxideontheEffectofPravastatin In rat mesenteric small arteries preconstricted with phenylephrine, PEG-SOD pretreatment alone did not affect constrictor responsiveness, as neither the Emax (1132%) nor the EC50 value (log M: 5.90.08) was significantly different to the control (1202%, 6.10.07; n=8). However, when PEG-SOD was co-incubated with pravastatin, the extent of inhibition of the phenylephrine concentrationresponse curve (approximately 3.9-fold decrease in potency and 533% 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 336% 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 198% 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 pravastatins inhibition of constrictor responses is not mediated by a reduction in superoxide concentrations because maximal superoxide scavenging by PEG-SOD did not abolish pravastatins effect but enhanced it, suggesting an independent synergistic effect. Endothelin-BReceptorBlockadeandPravastatinEffect onET-1 ET-1 constrictor responses were not influenced by pravastatin (112 nmol/L) pretreatment or treatment with BQ788 (3 mol/L) alone (P>0.05, 1-way ANOVA). However, pretreatment with pravastatin in the presence of BQ788 inhibited the ET-1 Emax response by 303% and reduced the potency of ET-1 by approximately 2-fold (Figure 5, Table). Thus, blocking endothelin-ETB receptors prior to stimulation by ET-1 unmasked pravastatins inhibition of this constrictor response.

Figure5. Effectofpre-incubationwithpravastatin(112nmol/L) on vasoconstrictor responses to endothelin-1 (n=6) in the absenceandpresenceofBQ788(3 mol/L)inendothelium intact rat mesenteric microvessels. Control segments ( ), pravastatin pretreated segments (), BQ788 pretreated segments () and BQ788+pravastatin pretreated segments().

and EC50 responses to ET-1 were unchanged by 60-min preincubation with pravastatin (Figure 1, Table). RoleofEndothelialNOontheEffectofPravastatin Removal of endothelium in mesenteric vessels increased the maximum contractile response to phenylephrine (1275% vs. 1165% 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: 5.70.12 vs. 5.80.06 in vessels with intact endothelium, P=0.12, n=6). However, endothelial denudation abolished pravastatins inhibition of phenylephrine responses (Figure 2A), with pravastatin and controls having similar Emax values (1368% vs. 1275%, P>0.05) and EC50 values (log M: 5.80.05 vs. 5.70.12, P>0.05; n=6). Compared with the control phenylephrine responses, NOS

Discussion
The major findings of this study relate to the direct vascular

Advance Publication by J-STAGE

effects of pravastatin and include (1) attenuation of constrictor responses mediated via the -adrenergic, serotonergic, and thromboxane pathways at a clinically relevant statin concentration; (2) the effects of pravastatin being endotheliumdependent and inhibited by both NOS and Akt inhibitors but not maximal superoxide scavenging with PEG-SOD, suggesting an endothelial NO mechanism mediated via the Akt pathway that did not involve reduced NO degradation by superoxide; and (3) ET-1 mediated constrictor responses being only attenuated by pravastatin in the presence of a specific endothelin-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 vessels of a statin at a clinically relevant concentration. EffectofStatinsonSmallVessels Direct 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 demonstrated that pravastatin pretreatment had anti-ischemic effects in cardiac syndrome-X patients who have normal angiography 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 resistance vessels. The demonstration that statins can modulate constrictor responses provides an explanation for these clinical observations. In particular, this studys results suggest that sympathetic nerve ( -adrenergic) and plateletthrombus (serotonergicthromboxane) mediated constriction could be attenuated by pravastatin but not endothelin-mediated constriction. MechanismofPravastatinsEffectsonResponses inSmallVessels The 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 mechanisms include either (1) increased NO production by (a) stabilizing 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 signaling,34 and/or (d) inhibition of caveolin, which inhibits endothelial NOS activation35 or (2) increased NO bioavailability 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 superoxide 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 enhancement of relaxation of rat aorta to acetylcholine after atorvastatin treatment was eliminated in the presence of SOD, suggesting that reduction in superoxide was the major mechanism for the enhanced relaxation response to acetylcholine.38 Similar protective effects against oxidative stress have been suggested 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 pravastatins attenuation of constrictor responses is abolished following endothelial 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 promote 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 proposed mechanism via the PI3K/Akt signaling pathway, we also used the PI3K/Akt inhibitor (LY294002). However LY294002 (10mol/L) reduced the phenylephrine vasoconstrictor 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 pravastatins antioxidant effects. Firstly, in a cell-free system with hypoxanthinexanthine oxidase generated superoxide measured by lucigenin-derived chemiluminescence, the clinically relevant concentration of pravastatin had no scavenging effect (data not shown). This result is consistent with a previous study43 demonstrating superoxide-scavenging by fluvastatin but not pravastatin at low concentrations, but contrasts with another study in which pravastatin exhibited a superoxide-scavenging effect at very high concentrations.44 Our second 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 superoxide 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 alternative explanation for the increased inhibition of vasoconstrictor 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 phenylephrine responses in the absence of pravastatin. As documented by previous investigators, NO typically has a lesser role in regulating vascular tone in resistance vessels, 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 mesenteric arteries (400500 than in resistance mesenteric m) arteries (150250 47 Consistent with their findings, our m). study demonstrated that the physiologic role of NO in regulating 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 pravastatins effect by L-NAME indicates that NO plays a significant role in modifying vascular tone after acute exposure to a statin. Thus pravastatin

Advance Publication by J-STAGE

appears to have a similar potentiating effect on NO bioavailability in small vessels to that observed in large vessels, and this effect appears to be mediated through enhanced NO production via the PI3K/Akt pathway. Compared with the study by Takamura et al,47 we used vessels that were intermediate in size (3286 m), 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 (3537 m m; n=14) and those <300 (2693 n=7), observing a simim m; lar reduction in the Emax between the 2 groups (353% vs. 317% reduction, respectively, P=0.60). This was confirmed by examining the relationship between vessel size and maximal contraction to phenylephrine in control and pravastatintreated 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-intercepts 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 progressive gradient in responses in vessels between 236 and 381 in diameter and NOS inhibition eliminated the pravasm tatin effect irrespective of vessel size. In addition to the above endothelium-dependent mechanisms, 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-dependent calcium channels and intracellular calcium stores.48 These direct vascular smooth muscle effects vary with different statins. Simvastatin,48,49 atorvastatin48 and fluvastatin23 have all been shown to directly effect vascular smooth muscle cells by inhibiting calcium mobilization. This acute effect is mediated via opening of vascular smooth muscle cell voltage-dependent (Kv) potassium channels.23 In contrast, 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. PravastatinandET-1ConstrictorResponsesinSmallVessels Unlike the other vasoconstrictors, ET-1 constrictor responses were not affected by pravastatin. However, ET-1 stimulates both endothelin-ETA and endothelin-ETB receptors, including 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 receptorendothelin-ETB receptor-mediated constriction, and endothelin-ETB receptor-mediated relaxation. Pravastatin treatment alone does not appear to provide any incremental NO effect in the presence of maximal endothelin-ETB receptor stimulation and thus on NO release. However, selective endothelin-ETB receptor blockade (with BQ788) restores pravastatins 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 identified an interaction between pravastatins vascular effects and endothelin-ETB receptor blockade. From our observations, it would be anticipated that a non-selective endothelin-ETA endothelin-ETB receptor blocker would enhance pravastatins inhibition of constrictor responses, whereas pravastatin would have no effect in the presence of a selective endothelin-ETA receptor blocker because of the unopposed endothelin-ETB receptor effects on NO. Whether this impairment of pravastatins inhibition of ET-1 constrictor responses in the presence of selective endothelin-ETA blockers translates into a clinically significant difference is open to speculation. In particular, does endothelin-ETB receptor stimulation by ET-1 impair any of pravastatins clinically documented (NO-mediated) protective effects? Alternatively, does the use of pravastatin with a non-selective endothelin-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 current study. StudyLimitations The findings of this study are limited by the use of rat mesenteric vessels rather than human tissue. Furthermore, we examined in-vitro vessels from healthy rats, and the effects of acute statin treatment observed cannot necessarily be extrapolated 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 endothelial function and therefore influencing the pravastatin response. A second limitation is the use of a single concentration 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 hyperpolarizing factor on the response to pravastatin in smaller vessels cannot be excluded.

Conclusion
Pravastatin at a clinically relevant concentration inhibits vasoconstrictor responses in small vessels via an endothelium-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 receptormediated NO release. These findings have important clinical implications and suggest that pravastatin may have additional benefits in addition to lowering cholesterol.
Acknowledgments
The 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 Foundation/The University of Adelaide Scholarships. John Beltrame is a South Australian Cardiovascular Research Development Program Fellow.

References
1. Thavendiranathan P, Bagai A, Brookhart MA, Choudhry NK.

Advance Publication by J-STAGE

2.

3.

4.

5. 6.

7. 8.

9.

10.

11. 12.

13. 14. 15. 16. 17. 18. 19. 20.

21.

22.

23.

Primary prevention of cardiovascular diseases with statin therapy: A meta-analysis of randomized controlled trials. ArchInternMed 2006; 166: 2307 2313. Kinoshita T, Asai T, Nishimura O, Hiramatsu N, Suzuki T, Kambara A, et al. Statin for prevention of atrial fibrillation after off-pump coronary artery bypass grafting in Japanese patients. CircJ 2010; 74: 1846 1851. Tani S, Nagao K, Anazawa T, Kawamata H, Furuya S, Takahashi H, et al. Coronary plaque regression and lifestyle modification in patients treated with pravastatin: Assessment mainly by daily aerobic exercise and an increase in the serum level of high-density lipoprotein cholesterol. CircJ2010; 74: 954 961. Sano K, Nakamura T, Hirano M, Kitta Y, Kobayashi T, Fujioka D, et al. Comparative study of bezafibrate and pravastatin in patients with coronary artery disease and high levels of remnant lipoprotein. CircJ 2010; 74: 1644 1650. Goldstein JL, Brown MS. Regulation of the mevalonate pathway. Nature 1990; 343: 425 430. Pedersen TR, Kjekshus J, Berg K, Haghfelt T, Faergeman O, Faergeman G, et al. Randomised trial of cholesterol lowering in 4444 patients with coronary heart disease: The Scandinavian Simvastatin Survival Study (4S): 1994. AtherosclerSuppl 2004; 5: 81 87. Levine GN, Keaney JF Jr, Vita JA. Cholesterol reduction in cardiovascular disease: Clinical benefits and possible mechanisms. NEnglJMed1995; 332: 512 521. Wassmann S, Laufs U, Baumer AT, Muller K, Ahlbory K, Linz W, et al. HMG-CoA reductase inhibitors improve endothelial dysfunction in normocholesterolemic hypertension via reduced production of reactive oxygen species. Hypertension 2001; 37: 1450 1457. Iwakura K, Ito H, Kawano S, Okamura A, Kurotobi T, Date M, et al. Chronic pre-treatment of statins is associated with the reduction of the no-reflow phenomenon in the patients with reperfused acute myocardial infarction. EurHeartJ 2006; 27: 534 539. Sever PS, Dahlof B, Poulter NR, Wedel H, Beevers G, Caulfield M, et al. Prevention of coronary and stroke events with atorvastatin in hypertensive patients who have average or lower-than-average cholesterol concentrations, in the Anglo-Scandinavian Cardiac Outcomes TrialLipid Lowering Arm (ASCOT-LLA): A multicentre randomised controlled trial. Lancet 2003; 361: 1149 1158. Lefer AM, Campbell B, Shin YK, Scalia R, Hayward R, Lefer DJ. Simvastatin preserves the ischemic-reperfused myocardium in normocholesterolemic rat hearts. Circulation 1999; 100: 178 184. Endres M, Laufs U, Huang Z, Nakamura T, Huang P, Moskowitz MA, et al. Stroke protection by 3-hydroxy-3-methylglutaryl (HMG)CoA reductase inhibitors mediated by endothelial nitric oxide synthase. ProcNatlAcadSciUSA 1998; 95: 8880 8885. Kayikcioglu M, Payzin S, Yavuzgil O, Kultursay H, Can LH, Soydan I. Benefits of statin treatment in cardiac syndrome-X1. EurHeartJ 2003; 24: 1999 2005. Bonetti PO, Lerman LO, Napoli C, Lerman A. Statin effects beyond lipid lowering: Are they clinically relevant? EurHeartJ 2003; 24: 225 248. Zhou Q, Liao JK. Pleiotropic effects of statins: Basic research and clinical perspectives. CircJ 2010; 74: 818 826. Osborne JA, Siegman MJ, Sedar AW, Mooers SU, Lefer AM. Lack of endothelium-dependent relaxation in coronary resistance arteries of cholesterol-fed rabbits. AmJPhysiol 1989; 256: C591 C597. Lefer AM, Lefer DJ. The role of nitric oxide and cell adhesion molecules on the microcirculation in ischaemia-reperfusion. CardiovascRes 1996; 32: 743 751. Garland CJ, Plane F, Kemp BK, Cocks TM. Endothelium-dependent hyperpolarization: A role in the control of vascular tone. Trends PharmacolSci1995; 16: 23 30. Shimokawa H, Yasuda S. Myocardial ischemia: Current concepts and future perspectives. JCardiol 2008; 52: 67 78. Trinkl A, Vosko MR, Wunderlich N, Dichgans M, Hamann GF. Pravastatin reduces microvascular basal lamina damage following focal cerebral ischemia and reperfusion. EurJNeurosci 2006; 24: 520 526. Alvarez De Sotomayor M, Herrera MD, Marhuenda E, Andriantsitohaina R. Characterization of endothelial factors involved in the vasodilatory effect of simvastatin in aorta and small mesenteric artery of the rat. BrJPharmacol2000; 131: 1179 1187. Fatehi-Hassanabad Z, Imen-Shahidi M, Fatehi M, Farrokhfall K, Parsaeei H. The beneficial in vitro effects of lovastatin and chelerythrine on relaxatory response to acetylcholine in the perfused mesentric bed isolated from diabetic rats. EurJPharmacol 2006; 535: 228 233. Mukai Y, Shimokawa H, Matoba T, Hiroki J, Kunihiro I, Fujiki

24. 25. 26.

27. 28.

29. 30.

31.

32. 33.

34.

35.

36.

37. 38.

39. 40.

41. 42.

43.

44. 45.

T, et al. Acute vasodilator effects of HMG-CoA reductase inhibitors: Involvement of PI3-kinase/Akt pathway and Kv channels. J CardiovascPharmacol 2003; 42: 118 124. Corsini A, Bellosta S, Baetta R, Fumagalli R, Paoletti R, Bernini F. New insights into the pharmacodynamic and pharmacokinetic properties of statins. PharmacolTher 1999; 84: 413 428. Hatanaka T. Clinical pharmacokinetics of pravastatin: Mechanisms of pharmacokinetic events. ClinPharmacokinet 2000; 39: 397 412. Pentikainen PJ, Saraheimo M, Schwartz JI, Amin RD, Schwartz MS, Brunner-Ferber F, et al. Comparative pharmacokinetics of lovastatin, simvastatin and pravastatin in humans. JClinPharmacol 1992; 32: 136 140. Mulvany MJ, Halpern W. Contractile properties of small arterial resistance vessels in spontaneously hypertensive and normotensive rats. CircRes 1977; 41: 19 26. Yamamoto E, Yamashita T, Tanaka T, Kataoka K, Tokutomi Y, Lai ZF, et al. Pravastatin enhances beneficial effects of olmesartan on vascular injury of salt-sensitive hypertensive rats, via pleiotropic effects. ArteriosclerThrombVascBiol 2007; 27: 556 563. Deng HF, Xiong Y. Effect of pravastatin on impaired endotheliumdependent relaxation induced by lysophosphatidylcholine in rat aorta. ActaPharmacolSin 2005; 26: 92 98. Tirapelli CR, Casolari DA, Yogi A, Montezano AC, Tostes RC, Legros E, et al. Functional characterization and expression of endothelin receptors in rat carotid artery: Involvement of nitric oxide, a vasodilator prostanoid and the opening of K+ channels in ETBinduced relaxation. BrJPharmacol 2005; 146: 903 912. de la Lande IS, Siebert TE, Bennett CL, Stafford I, Horowitz JD. Influence of the endothelium on ex vivo tolerance and metabolism of glyceryl trinitrate in rat aorta. EurJPharmacol 2004; 486: 201 207. Laufs U, La Fata V, Plutzky J, Liao JK. Upregulation of endothelial nitric oxide synthase by HMG CoA reductase inhibitors. Circulation 1998; 97: 1129 1135. Scotland RS, Morales-Ruiz M, Chen Y, Yu J, Rudic RD, Fulton D, et al. Functional reconstitution of endothelial nitric oxide synthase reveals the importance of serine 1179 in endothelium-dependent vasomotion. CircRes 2002; 90: 904 910. Kureishi Y, Luo Z, Shiojima I, Bialik A, Fulton D, Lefer DJ, et al. The HMG-CoA reductase inhibitor simvastatin activates the protein kinase Akt and promotes angiogenesis in normocholesterolemic animals. NatMed2000; 6: 1004 1010. Feron O, Dessy C, Desager JP, Balligand JL. Hydroxy-methylglutaryl coenzyme A reductase inhibition promotes endothelial nitric oxide synthase activation through a decrease in caveolin abundance. Circulation 2001; 103: 113 118. Rashid M, Tawara S, Fukumoto Y, Seto M, Yano K, Shimokawa H. Importance of Rac1 signaling pathway inhibition in the pleiotropic effects of HMG-CoA reductase inhibitors. CircJ 2009; 73: 361 370. Rikitake Y, Hirata K. Inhibition of RhoA or Rac1? Mechanism of cholesterol-independent beneficial effects of statins. CircJ 2009; 73: 231 232. Wagner AH, Kohler T, Ruckschloss U, Just I, Hecker M. Improvement of nitric oxide-dependent vasodilatation by HMG-CoA reductase inhibitors through attenuation of endothelial superoxide anion formation. ArteriosclerThrombVascBiol 2000; 20: 61 69. Ma FX, Liu LY, Xiong XM. Protective effects of lovastatin on vascular endothelium injured by low density lipoprotein. Acta PharmacolSin 2003; 24: 1027 1032. Yasunari K, Maeda K, Minami M, Yoshikawa J. HMG-CoA reductase inhibitors prevent migration of human coronary smooth muscle cells through suppression of increase in oxidative stress. ArteriosclerThrombVascBiol 2001; 21: 937 942. Urbich C, Dernbach E, Zeiher AM, Dimmeler S. Double-edged role of statins in angiogenesis signaling. CircRes 2002; 90: 737 744. Drouin A, Thorin E. Flow-induced dilation is mediated by Aktdependent activation of endothelial nitric oxide synthase-derived hydrogen peroxide in mouse cerebral arteries. Stroke 2009; 40: 1827 1833. Yamamoto A, Hoshi K, Ichihara K. Fluvastatin, an inhibitor of 3hydroxy-3-methylglutaryl-CoA reductase, scavenges free radicals and inhibits lipid peroxidation in rat liver microsomes. EurJPharmacol 1998; 361: 143 149. Kassan M, Montero MJ, Sevilla MA. In vitro antioxidant activity of pravastatin provides vascular protection. EurJPharmacol 2010; 630: 107 111. Schmidt HH, Hofmann H, Schindler U, Shutenko ZS, Cunningham DD, Feelisch M. No .NO from NO synthase. ProcNatlAcadSci USA 1996; 93: 14492 14497.

Advance Publication by J-STAGE

46. Shimokawa H, Yasutake H, Fujii K, Owada MK, Nakaike R, Fukumoto Y, et al. The importance of the hyperpolarizing mechanism increases as the vessel size decreases in endothelium-dependent relaxations in rat mesenteric circulation. J Cardiovasc Pharmacol 1996; 28: 703 711. 47. Takamura Y, Shimokawa H, Zhao H, Igarashi H, Egashira K, Takeshita A. Important role of endothelium-derived hyperpolarizing factor in shear stress-induced endothelium-dependent relaxations in the rat mesenteric artery. JCardiovascPharmacol 1999; 34: 381

387. 48. Tesfamariam B, Frohlich BH, Gregg RE. Differential effects of pravastatin, simvastatin, and atorvastatin on Ca2+ release and vascular reactivity. JCardiovascPharmacol 1999; 34: 95 101. 49. Alvarez de Sotomayor M, Perez-Guerrero C, Herrera MD, Marhuenda E. Effect of simvastatin on vascular smooth muscle responsiveness: Involvement of Ca(2+) homeostasis. Eur J Pharmacol 2001; 415: 217 224.