www.mjms.usm.my © Penerbit Universiti Sains Malaysia, 2010 For permission, please email:mjms.usm@gmail.com

Abstract Background: Post-occlusive skin reactive hyperaemia (PORH) is a model used toassess microvascular reactivity. This study aims to compare PORH response among pregnanthypercholesterolaemicpatientswithageandgestationalage-matchedcontrols. Methods: This cross sectional study involved 17 hypercholesterolaemic, pregnantwomenand20pregnantcontrolsenteringtheirearlythirdtrimester.LaserDopplerfluximetry(LDF)wasusedtomeasureskinperfusion.TheprocessofPORHwasperformedbyoccludingtheupperarmwithanocclusioncuffat200mmHgfor3minutes.Skinperfusionwasrecordedbefore,during,andafterocclusionrelease.Baselineperfusion,timetoachievepeakperfusion(Tp),peakperfusionafterocclusionrelease(PORHpeak),andmaximumchangeinperfusionduetoocclusion(PORHmax)wererecorded. Results: Serumtotalcholesterol(TC)wassignificantlydifferent(P<0.001)betweenthe2groups:7.25(SEM0.18)mmol/Lforhypercholesterolaemicwomenand5.54(SEM0.15)mmol/Lforthecontrolgroup.Therewerenosignificantdifferencesintheirbaseline,PORHpeak,andPORHmax.However,Tpinthehypercholesterolaemicgroupwassignificantlyincreased(P=0.024)comparedwiththecontrolsat14.9(SEM0.6)secondsand13.1(SEM0.5)seconds,respectively. Conclusion: Pregnant hypercholesterolaemic patients showed an abnormalmicrovascular reactivity response. Tp with ischemia was significantly increased compared withnormocholesterolaemiccontrols.

Keywords:hypercholesterolaemia, laser Doppler flowmetry, microcirculation, pregnancy, reactive hyperaemia

Introduction Hypercholesterolaemia is an importantcardiovascular risk factor and a potent factorcontributingtotheprogressionofatherosclerosis(1). It is associatedwith endothelialdysfunctionand reduced endothelium-dependent responsesin the forearm and coronary circulation (2,3).Maternal hypercholesterolaemia duringpregnancy has been reported to be associatedwith enhanced fatty streak formation in humanfoetuses(4).Thehypercholesterolaemicconditionduringpregnancymayalsomodifyvascularandplacentalfunctions,contributingtocomplicationsduringpregnancyortotheoffspring.Ithasbeenshown that pregnant rats fed a diet enriched in

cholesterol showed 4-fold increase in abortions,2-fold increase in neonatal mortality, smallerlittersize,andalowerbirthweightofpups(5). Post-occlusive skin reactive hyperaemia(PORH) is the increased of blood flow aftertemporary occlusion of the arterial blood flow.Thehyperaemicresponsetoanischaemicblockisthoughttobeendothelium-dependent, involvingmyogenicresponseandreleaseofmetabolicfactorssuch as nitric oxide (NO) and prostaglandins.AssessmentofPORHbylaserDopplerfluximetry(LDF)isasimple,non-invasiveandreproduciblemethod to assess microvascular health (6,7).Besides providing information on baselineblood flow measurements, the time-courseof the cutaneous response to post-ischaemic

Original Article Abnormal Microvascular Reactivity with Hypercholesterolaemia in Pregnancy

Aida Hanum Ghulam Rasool1, Aisyah Syairah abdul Rahman1, Nor Aliza abd GhaffaR2, Nik Mohd Zaki nik mahmood2, Abd Rahim WonG3

1 Pharmacology Vascular Laboratory, School of Medical Sciences, Universiti Sains Malaysia Health Campus, 16150 Kubang Kerian, Kelantan, Malaysia

2 Department of Obstetrics and Gynaecology, School of Medical Sciences, Universiti Sains Malaysia Health Campus, 16150 Kubang Kerian, Kelantan, Malaysia

3 Department of Paediatrics, School of Medical Sciences, Universiti Sains Malaysia Health Campus, 16150 Kubang Kerian, Kelantan, Malaysia

Submitted: 12Jan2010Accepted: 15Mar2010

14Malaysian J Med Sci. Oct-Dec 2010; 17(4): 14-19

Original Article |Microvascularfunctioninhypercholesterolaemicpregnancy

www.mjms.usm.my 15

reactive hyperaemia can give information onchanges occurring in the microcirculation (8).Cutaneous post-ischaemic reactive hyperaemiahasbeenused todetectmicrovascular reactivitydifferences between smokers and non-smokers(9), diabetic patients and controls, as well asbetweensubgroupsofdiabeticpatients(10).Morerecently, post-hyperaemia flux was reported tobe significantly lower, and time to achieve peakpost-ischaemic responsewas slower indiabeticscompared with controls (11). Farkas et al. havedemonstrated that hypertensive patients havea lowermaximalchange inskinbloodflowwithreactivehyperaemiacomparedwithcontrols(12). Normal pregnancy is characterized byreduced peripheral vascular resistance andblood pressure, increased cardiac output, andsubsequentincreaseofbloodflowinthesystemicandpulmonarycirculations.Brachialarteryflow-mediated dilatation was found to be increasedfrom the first trimester, reaching the highestvalue in the last trimester compared with non-pregnant values. However, no studies havedirectlycomparedmicrovascularorskinvascularreactivity using reactive hyperaemia methodbetween healthy non-pregnant and pregnantwomen. In pregnantwomen, vascular reactivity hasbeen shown tobe altered in skin vessels ofpre-eclampticpatients(13).Innormotensivepregnantsubjects, the reactive hyperaemia responsewas nearly as pronounced as the maximalvasodilatation of the vessels induced by localheating.However,inpatientswithpre-eclampsia,the reactive hyperaemia response reached wasonly half of the maximal vasodilatory capacity.Pre-eclamptic women were also shown to havelower post-occlusive reperfusion comparedwithcontrols, besides having lower basal blood flow(14). Dyslipidemia has been shown toimpair microvascular reactivity in non-pregnant subjects (7,8,15,16). The effect ofhypercholesterolaemia on the microvascularbed has not been studied in pregnant women.It is not known whether the difference inreactivity between normocholesterolaemic andhypercholesterolaemic patients persists in thepresence of altered cardiovascular, hormonal,and metabolic adaptations occurring duringpregnancy. Thus, this study aims to comparemicrovascular reactivity between pregnantwomen with hypercholesterolaemia and age-matched as well as gestational age-matchedcontrols.LDFandthePORHresponsewereusedtoassessmicrovascularreactivityinthesewomen.

Materials and Methods

Subjects

This prospective, cross sectional studyinvolved 17 pregnant hypercholesterolaemicpatientsand20ageandgestational-agedmatchedwomen.Ethical approval for the conductof thisstudywas obtained from theEthicalCommitteeof Universiti Sains Malaysia. This study wasconducted following the principles stated in theDeclaration of Helsinki; all subjects voluntarilysignedaninformedconsentform. Subjectswere recruited from theObstetricsandGynaecologicalClinic ofHospitalUniversitiSains Malaysia when they presented between25–33 weeks gestation. Pregnant women withsignificant cardiovascular diseases (such ashypertension, arrhythmia, and heart failure),diabetesmellitus,historyofpreviousandcurrentgestationaldiabetes,multiplepregnancies,foetalanomalies, and seriousmaternal illnesses (suchas liver and renal diseases) were excluded. Allsubjects underwent a modified oral glucosetolerance test (MOGTT) to exclude gestationaldiabetes. None of the subjects were taking anyvasoactivemedicationsuchasnon-steroidalanti-inflammatory drugs, corticosteroids, or lipid-lowering drugs. Women were categorized intothe hypercholesterolaemic group if their serumtotalcholesterol(TC)wasequaltoormorethan6.5mmol/L.At thischolesterol level, theriskofdevelopingcoronaryarterydiseaseissubstantiallyincreased(17).

LDF and PORH response

TheLDFDRT4 system (Moor Instruments,Axminster, UK) was used in this study to non-invasively measure the forearm skin bloodperfusion.ThisequipmentwasusedtogetherwiththeDP1T-V2skinlaserprobe(MoorInstruments,Axminster,UK).LDFofskinbloodflowisbasedon measurement of the Doppler shift principlewherein photons of laser light are scattered bymoving blood cells to produce a Doppler shifton the reflected light. The outcome is generallytermed as ‘flux’ and expressed in arbitraryperfusionunits(AU). LaserDopplermeasurementsonthepregnantwomenwerecarriedoutinthemorninginaquiteroom with room temperature maintained at 23(SD1)°C.Uponarriving,subjectslaydownforatleast 15minutes for acclimatization before theirblood pressure was taken using an automated

16 www.mjms.usm.my

Malaysian J Med Sci. Oct-Dec 2010; 17(4): 14-19

blood pressure sphygmomanometer (Omron,Japan).The rightarmof the subjectwasplacedonacushionandsupportedbyahandsupporterto reduce involuntary hand movements. Asphygmomanometer cuff (Accoson, UK) wasplacedaroundtherightupperarm1–2cmabovetheante-cubitalcrease.LaserDopplerprobeswerethenfixedonthevolarsurfaceoftherightforearmdistal to the sphygmomanometer occlusioncuff.After a stable skinperfusionwasobtained,baseline skin perfusion flux was recorded for 1minute.Forearmbloodflowwasthenperformedbyinflatingthepneumaticpressurecuffplacedonthe rightupperarm toa supra-systolicpressureof200mmHgfor3minutes.After3minutes,theocclusioncuffwasrapidlydeflatedandperfusionfluxwasrecordedforatleast2minutes.Boththeskin perfusion and temperature were measuredcontinuously before, during and after occlusionby the laserDopplerprobes.Skinperfusionwasallowedtoreturntobaseline;anintervalofatleast15minutes (or until baseline fluxwas achieved,whicheverwaslonger)wasallowedbeforearepeatoftheprocedure.Thefollowingparametersweremeasured:minimumbaselineperfusion,thetimerequiredtoreachpeakflowafterocclusionrelease(Tp), peak perfusion reached after occlusionrelease (PORHpeak), and maximum change inperfusionafterocclusioncomparedwithbaseline(PORHmax) which was calculated as PORHpeakminusminimumbaselineperfusion. Theaverageof2readingsforeachparameterwasrecorded.Asingleoperatorwasresponsibleforperformingthereactivehyperaemiaprocedurethroughout this study to reduce inter-operatorvariability.The intradayand interdaycoefficientofvariationsforPORHmaxandTpatourlaboratorywas 4.77% and 6.5%, and 8.89% and 6.87%,respectively(6). BloodsampleforthemeasurementofserumTCwastakenafteranovernightfast.SerumTCwasmeasuredattheChemicalPathologyLaboratory,Hospital Universiti Sains Malaysia, usingcommercially prepared reagents (EnzymatiqueEndpoint, Randox) on the Hitachi Model 902auto-analyser (Japan). The within and betweenruncoefficientofvariationfordeterminingserumTCatthelaboratorywaslessthan2%. StatisticalanalysiswasperformedusingtheSPSSversion12(SPSSInc.,Chicago,IL).ResultsarepresentedasmeanandSEM;P<0.05definedstatistical significance. Differences betweengroups were evaluated using the independent t testorMann–WhitneyUtest,whereapplicable.

Results

Meanageandgestationalageforthesubjectswere32.8 (SEM1.1)years (ranged21–45years)and28.4(SEM0.3)weeks(ranged25–33weeks),respectively. Baseline characteristics for thecontrols and hypercholesterolaemic patients areshowninTable1.ApartfromtheserumTCvalues,therewerenosignificantdifferencesbetweenthecontrols and hypercholesterolaemic subjects intheir age, gestational age, bloodpressure, bloodcountvalues,bodymassindex,andfastingplasmaglucose values. There was also no significantdifference in their skin temperature during thePORHprocedure. Arterial occlusion induced by the occlusioncuff around the upper arm and inflated to 200mmHg for 3minutes resulted in a significantlylower perfusion recording compared withbaseline. After occlusion release, the blood flowincreased rapidly to apeak value approximately5-foldcomparedwithbaseline;buttheperfusiondecreasedagaintowardsbaselinesoonafter. BothgroupsshowedsignificantincreasesinPORHpeak comparedwith baseline (P <0.001 inboth groups). Baseline perfusion, Tp, PORHpeak,andPORHmaxforthenormocholesterolaemicandhypercholesterolaemicgroupsareshowninTable2.Therewerenosignificantdifferencesbetweenthe 2 groups in baseline perfusion, PORHpeak,and PORHmax. There was however, significantdifference between the 2 groups for Tp; thehypercholesterolaemicgroupshowedsignificantlylongerTpcomparedwiththecontrols. LinearregressionanalysiswasperformedtoassesstherelationshipbetweenserumTCandTp.Therewasapositive significant correlation (P=0.015,r=0.4)betweenthe2parameters;higherserumTCvalueswereassociatedwithlongerTp.

Discussion

We found that the presence ofhypercholesterolaemia in pregnancy prolongedtheTpwiththeprocessofPORHcomparedwiththe age and gestational age-matched controls.Microvascular reactivity among non-pregnant,hypercholesterolaemic subjects has beenreported in the literature (7,8,16,18).Binggeli etal. reported that post-ischaemic skin bloodflowmeasured using the LDFwasmarkedly reducedin hypercholesterolaemic patients comparedwith healthy controls and statin improved thepost-ischaemic hyperaemia in the subjectsafter an average of 6 months of treatment (7).

Original Article |Microvascularfunctioninhypercholesterolaemicpregnancy

www.mjms.usm.my 17

Table1:Demographicandlaboratorycharacteristicsofcontrolsandhypercholesterolaemicpatients

Controlsn=20

Hypercholesterolaemicpatientsn=17

Age(years) 32.60(1.29) 33.00(1.91)Bodymassindex(kg/m2) 27.13(0.92) 26.75(1.38)Gestationalage(weeks) 28.15(0.49) 28.71(0.49)Fastingplasmaglucose(mmol/L) 4.03(0.14) 4.23(0.14)Serumtotalcholesterol(mmol/L) 5.54(0.15) 7.25(0.18)a

Systolicbloodpressure(mmHg) 103.6(2.1) 108.2(2.9)Diastolicbloodpressure(mmHg) 63.8(1.9) 66.9(2.3)Bloodhematocrit(109/L) 33.98(0.56) 34.7(0.49)Glycosylatedhemoglobin(%) 5.10(0.15) 5.01(0.18)Skintemperatureduringreactivehyperaemia(°C) 28.6(0.23) 28.6(0.21)

Dataareexpressedinmean(SEM). aP<0.001indicateshighlysignificantdifferenceinserumtotalcholesterol forhypercholesterolaemicpatientsincomparisonwithcontrols(independent t test).

Table2:Microvascularperfusionvaluesinnormocholesterolaemicandhypercholesterolaemicpregnantpatientsduringpost-occlusivereactivehyperaemia

Controlsn=20

Hypercholesterolaemicpatientsn=17

Tp(seconds) 13.1(0.5) 14.9(0.6)a

Baselineperfusion(AU) 10.3(1.3)b 11.0(1.9)b

PORHpeak (AU) 49.1(3.7) 51.1(4.7)

PORHmax (AU) 38.8(3.3) 40.1(4.2)

Dataareexpressedinmean(SEM).aP=0.024indicatessignificantdifferenceinTpforhypercholesterolaemicpatientsincomparisonwithcontrols(independentttest),whereasbP <0.001indicateshighlysignificantdifferenceinPORHpeakincomparisonwithbaselineforbothgroups(pairedttest).Abbreviation:AU=arbitraryperfusionunits,PORHmax=maximumchange inperfusionafterocclusioncomparedwithbaseline,PORHpeak=peakperfusionreachedafterocclusionrelease,Tp=timerequiredtoreachpeakflowafterocclusionrelease.

UnlikethestudybyBinggelietal.,nodifferencein peak hyperaemic response was seen in ourstudy. Two possible reasons may explain thisdifference; firstly, themean age of our subjectswas 33 years compared with theirs, whichwas 42 years. It is possible that older subjectsexperience more vascular function abnormalitywith hypercholesterolaemia compared with theyoungeragegroup.Theolderagegroupmayalsohave been exposed to a longer duration of highlipidlevels.Secondly,oursubjectswereallfemaleandpregnant,whereasintheirstudy,17outof19subjectsweremalesandtherewerenopregnantwomen. Most of their subjects were also onother drugs,mostly cardiovascular-related, such

as acetyl salicylic acid, antihypertensives, anddiuretics, indicating that some of these patientsmay already had some vascular/endothelialdysfunctionpresent.InthestudybyBinggelietal.,Tpachievedbyeachgroupwasnotreported.Stulcetal.haveobservedthatmicrovascularreactivityin hypercholesterolaemic patients withoutcoronary artery disease was not different fromcontrols.Microvascular reactivity was, however,reduced in hypercholesterolaemic patientswith coronary artery disease (16). Patients withhypertriglyceridemiawerereportedbyTuretal.toshowcutaneousmicrocirculatory changes in theforearm.Untreatedhypertriglyceridemicpatientshave significantly lower peak flow compared

18 www.mjms.usm.my

Malaysian J Med Sci. Oct-Dec 2010; 17(4): 14-19

withcontrolsandpatientstreatedwiththelipid-lowering drug, bezafibrate (8). The hyperaemicreactionwasalsofasterinthebezafibrate-treatedgroup comparedwith the other 2 groups.Apartfrom our current study, there are no studiesassessing the effect ofhypercholesterolaemiaonmicrovascularreactivityinpregnancy. Haak et al. reported that Tp during PORHwas shorter in hyperlipidaemic patients aftertreatment with the statin fluvastatin; this wasassociated with a 22.8% reduction in serumTC level (18).Gomes et al. reported thatType 1diabetic patients took longer to reach peak fluxduringhyperaemiaresponsecomparedwithage-matched controls (19). The mean age of theirsubjects was 33 years. Similar to our study, nodifferenceinPORHmaxwasobservedbetweenthe2groups(19).Similarly,Turetal.haddemonstratedthatindiabeticpatients,PORHpeakwaslowerthannon-diabetic controls; diabetics also had longerTpcomparedwithcontrols(10). Thereareafewpossiblemechanismswherebyhypercholesterolaemia contributes to prolongedTp.Functionaloracombinationoffunctionalwithstructuralvascularchangesmightproducethesemicrovascular abnormalities. Firstly, elevatedserum TC may increase blood viscosity andincrease the Tp. Secondly, theremay be failureofpromptvasodilatationbysmoothmusclecellsinresponsetoischemiainhypercholesterolaemicsubjects.During reactive hyperaemia, ischaemicstimulation forces the endothelium to releasevasodilating substances such as NO andprostaglandins.Innormalsubjects,theresponsetoocclusionandreleasewouldbefastandefficient,but in hypercholesterolaemia, the endotheliummay take a longer time to respond, causing alonger time for reperfusion.Thismaybedue toimpairmentinthediffusionofNOintothesmoothmuscleofthevesselwall.Hypercholesterolaemiahas been reported to impair endothelium-dependent relaxation in the microcirculationand macrocirculation in experimental animalsand humans (20,21). This may be due toreduced synthesis of endothelium-derivedrelaxing factors, or altered membrane receptorcoupling mechanisms that affected the releaseof these factors. Since NO normally interfereswith the action and synthesis of endothelin (anendogenous vasoconstrictor), the lack of NOactivity may favour the vascular expression ofendothelin. Thirdly, hypercholesterolaemia maycause a reduction in vasodilating substancesproduced by the endothelium by creating aconditionofoxidative stress.Free radicals, suchassuperoxideanion,havebeenknowntoscavenge

endothelium-derivedrelaxingfactor(22).Lastly,structuralchangesinthebloodvessel,suchaswallthickening,mayalsoreducethespeedofresponsetoischaemia. The primary limitation of this study inthat only the serum TC value was available,while the detailed lipoprotein results werenot. However, the results from this study willhopefullyformthebasisformoredetailedstudyin this area. Another limitation of this study isthat the duration of hypercholesterolaemia inthese patients is unknown; some women maybe more relaxed in their dietary habits duringpregnancy.However,thestrengthofthisstudyisthat,apart fromtheserumTCvalues, therewaslittledifference in characteristicsof the controlsandthehypercholesterolaemicsubjects,whereinbothgroupswere screened for impairedglucosetolerance,theywerenotonanyvasoactivedrugs,none of the hypercholesterolaemic subjects hadeverbeentreatedwithlipidloweringagents,andneithergrouphadanycardiovasculardiseases.

Conclusion

We conclude that hypercholesterolaemiain pregnancy affects microvascular reactivityby increasing the time needed to achieve peakperfusion after temporary arterial occlusion.Moredetailedstudyinthispopulationcorrelatingdetailed lipid profile, microvascular reactivity,placenta blood flow, and pregnancy outcome issuggested.

Acknowledgement

We thank Universiti Sains Malaysia forproviding financial assistance to conduct thisstudy(grantno:304/PPSP/6131501).

Authors’ Contributions

Conception and design, obtaining of funding,criticalrevisionandfinalapprovalofthearticle:AHGR,NAAG,NMZNM,ARWProvisionofpatients:AHGR,NAAG,NMZNMCollection and assembly of data, analysis andinterpretation of the data, administrative,technical,orlogisticsupport:AHGR,ASARDraftingofthearticle:AHGR,ASAR,ARW

Original Article |Microvascularfunctioninhypercholesterolaemicpregnancy

www.mjms.usm.my 19

Correspondence

AssociateProfessorDrAidaHanumGhulamRasoolMBBS(Flinders),PhDClinicalPharmacology(USM)SchoolofMedicalSciencesUniversitiSainsMalaysiaHealthCampus16150KubangKerianKelantan,MalaysiaTel:+609-7676123/6143Fax:+609-7653370Email:aida@kb.usm.my

References

1. Gould AL, Rossouw JE, Santanello NC, HeyeseJF, Furberg CD. Cholesterol reduction yieldsclinical benefit: Impact of statin trials. Circulation. 1998;97(10):946–952.

2. ChowienczykPJ,WattsGF,CockcroffJR,RitterJM.Impaired endothelium-dependent vasodilatation offorearmresistancevessels inhypercholesterolaemia.Lancet.1992;340(8833):1430–1432.

3. Perrault LP,Mahlberg F, Breugnot C, Bidouard JP,VilleneuveN,VilaineJP,etal.Hypercholesterolaemicincreases coronary endothelial dysfunction, lipidcontent, and accelerated atherosclerosis afterheart transplant. Arterioscler Thromb Vasc Biol. 2000;20(3):728–736.

4. NapoliC,D’ArmientoFP,ManciniFP,PostiglioneA,WitztumJL,PalumboG,etal.Fattystreakformationoccursinhumanfetalaortasandisgreatlyenhancedbymaternalhypercholesterolemia.Intimalaccumulationof lowdensity lipoprotein and its oxidationprecedemonocyte recruitment into early atheroscleroticlesions.J Clin Invest.1997;100(11):2680–2690.

5. Marseille-Tremblay C, Ethier-Chiasson M, ForestJC, Giguère Y, Masse A, Mounier C, et al. Impactof maternal circulating cholesterol and gestationaldiabetesmellitusonlipidmetabolisminhumantermplacenta.Mol Reprod Dev.2008;75(6):1054–1062.

6. Yvonne-TeeGB,RasoolAH,HalimAS,RahmanAR.Reproducibilityofdifferent laserDopplerfluximetryparameters of postocclusive reactive hyperemia inhumanforearmskin. J Pharmacol Toxicol Methods. 2005;52(2):286–292.

7. Binggeli C, Spieker LE, Corti R, Sudano I,Stojanovic V, Hayoz D, et al. Statins enhancepostischemic hyperaemia in the skin circulation ofhypercholesterolemic patients. J Am Coll Cardiol. 2003;42(1):71–77.

8. TurE,PolitiY,RubinsteinA.Cutaneousbloodflowabnormalities in hypertriglyceridemia. J Invest Dermatol.1994;103(4):597–600.

9. Tur E, Yosipovitch G, Oren-Vulfs S. Chronic andacuteeffectsofcigarettesmokingonskinbloodflow.Angiology.1992;43(4):328–335.

10. Tur E, Yosipovitch G, Bar-On Y. Skin reactivehyperemia in diabetic patients. A study by laserDoppler flowmetry. Diabetes Care. 1991;14(11):958–962.

11. JafferU,AslamM,StandfieldN.Impairedhyperaemicandrhythmicvasomotorresponseintype1diabetesmellitus patients: A predictor of early peripheralvascular disease. Eur J Vasc Endovasc Surg.2008;35(5):603–606.

12. FarkasK,KolossváryE,JáraiZ,NemcsikJ,FarsangC. Non-invasive assessment of microvascularendothelial function by laser Doppler flowmetry inpatientswithessentialhypertension.Atherosclerosis. 2004;173(1):97–102.

13. BeinderE, SchlembachD. Skin flux during reactivehyperemia and local hyperthermia in patients withpreeclampsia.Obstet Gynecol.2001;98(3):313–318.

14. Stark MJ, Dierkx L, Clifton VL, Wright IM.Alterationsinthematernalperipheralmicrovascularresponseinpregnanciescomplicatedbypreeclampsiaand the impactof fetal sex.J Soc Gynecol Investig. 2006;13(8):573–578.

15. KhanF,LitchfieldSJ,StonebridgePA,BelchJJ.Lipid-lowering and skin vascular responses in patientswith hypercholesterolaemia and peripheral arterialobstructivedisease.Vasc Med.1999;4(4):233–238.

16. Stulc T, Kasalová Z, PráznýM,VrablikM, Skrha J,Ceska R. Microvascular reactivity in patients withhypercholesterolaemia: Effect of lipid loweringtreatment.Physiol Res.2003;52(4):439–445.

17. Martin MJ, Browner WS, Hulley SB, Kuller LH,WentworthD.Serumcholesterol,bloodpressure,andmortality:Implicationsfromacohortof361662men.Lancet.1986;328(8513):933–936.

18. HaakE,AbletshauserC,WeberS,GoedickeC,MartinN,HermannsN,etal.Fluvastatin therapy improvesmicrocirculation in patients with hyperlipidaemia.Atherosclerosis.2001;155(2):395–401.

19. Gomes MB, Matheus AS, Tibiriçá E. Evaluationof microvascular endothelial function in patientswith type 1 diabetes using laser-Doppler perfusionmonitoring: Which method to choose? Microvasc Res.2008;76(2):132–133.

20. Vanhoutte PM. Hypercholesterolaemia,atherosclerosis and release of endothelium-derivedrelaxingfactorbyaggregatingplatelets.Eur Heart J.1991;12(SupplE):S25–S32.

21. ShimokawaH,VanhouttePM.Impairedendothelium-dependent relaxation to aggregating platelets andrelated vasoactive substances in porcine coronaryarteriesinhypercholesterolemiaandatherosclerosis.Circ Res.1989;64(5):900–914.

22. RubanyiGM,Vanhoutte PM. Superoxide anion andhyperoxia inactivate endothelial derived relaxingfactor.Am J Physiol.1986;250(5Pt2):S822–S827.