O r i g i n a l P a p e r

Low-Dose Statin Therapy Improves EndothelialFunction in Type 2 Diabetic PatientsWith Normal Serum Total Cholesterol:A Randomized Placebo-Controlled Study

Amr Adel, MD; Zainab Abdel-Salam, MD; Wail Nammas, MD

The authors sought to explore the effect oflow-dose atorvastatin on endothelial function innormocholesterolemic patients with type 2diabetes mellitus without evidence of coronarydisease. Sixty patients with type 2 diabetesmellitus, normal serum cholesterol, and normalexercise test results were enrolled. Initialassessment was performed by measurement of thebrachial artery diameter and flow velocity both atbaseline and after induced hyperemia. Patientswere randomly assigned to receive eitheratorvastatin 10 mg daily for 4 weeks (atorvastatingroup=30 patients) or matched placebo for thesame period (placebo group=30 patients). Follow-up assessment of the brachial artery diameter andflow velocity (both baseline and hyperemic) wasperformed after 4 weeks. Initially, no significantdifference was found between the two groupsregarding brachial artery diameter or flowvelocity, both at baseline and at peak hyperemia(P>.05 for all). At follow-up, there was asignificantly higher flow velocity at baseline

(P<.05) and a significantly higher percentincrease of brachial artery diameter (from base-line to peak hyperemia) in the atorvastatin group(P<.05). In patients with type 2 diabetes mellitusand normal serum cholesterol without evidenceof coronary disease, low-dose atorvastatinimproves endothelial function. J Clin Hypertens(Greenwich). 2010;12:820–825. ª2010 Wiley

Periodicals, Inc.

There is a strong body of evidence that type 2diabetes mellitus (T2DM) is associated with

endothelial dysfunction as a result of bothdecreased production and increased oxidative inac-tivation of nitric oxide (NO) by free radicals.1

Several relevant mechanisms may operate in statesof insulin resistance and account for the decreasedbioavailability of NO and abnormal endothelialfunction. Insulin resistance is associated withelevated serum markers of inflammation such asC-reactive protein.2 Increased release of cytokinessuch as interleukin 6 and tumor necrosis factora from the adipose tissues stimulates hepaticproduction of C-reactive protein.3

Evidence-based literature indicates that statinsmay improve the bioavailability of NO by reducingoxidative stress or increasing the activity of NOsynthase.4,5 Recently, it has been proposed thatstatins may improve endothelial function bydecreasing the levels of asymmetric dimethylargi-nine (ADMA), a known competitive inhibitor ofendogenous endothelial NO synthase.6 The effect

From the Cardiology Department, Faculty of Medicine,Ain Shams University, Cairo, EgyptAddress for correspondence:Wail Nammas, MD, Cardiology Department, AinShams University Hospitals, Faculty of Medicine, AinShams University. Abbassia, Cairo, PO 11381, EgyptE-mail: wnammas@hotmail.comManuscript received June 5, 2010; revised July 12, 2010;accepted July 14, 2010

doi: 10.1111/j.1751-7176.2010.00367.x

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of statins on ADMA levels may be mediated by thedecrease in oxidized low-density lipoprotein levels.Similarly, the ability of statins to depress cytokineexpression (especially tumor necrosis factor a)might indirectly decrease ADMA levels.6

In a prospective, randomized, double-blind,placebo-controlled study, we sought to explore theeffect of low-dose atorvastatin on endothelial func-tion, as reflected by flow-mediated dilatation (FMD)of the brachial artery in patients with T2DM andnormal serum cholesterol levels without evidence ofcoronary artery disease.

METHODSPatient SelectionWe enrolled 60 patients with T2DM referred toour diabetes outpatient clinic during the periodfrom October 2007 to July 2008 for routine fol-low-up. Patients were considered eligible for inclu-sion if they had normal total serum cholesterollevels (<5.4 mmol ⁄L) and a normal exercise elec-trocardiographic (ECG) test result. Patients withabnormal serum triglycerides and ⁄or high-densitylipoprotein cholesterol levels were also included.We excluded patients with known ischemic heartdisease and cerebrovascular or peripheral vasculardisease and those with contraindication to statins(eg, history of chronic liver impairment or skeletalmyopathy). Before inclusion, informed writtenconsent was obtained from each patient after fullexplanation of the study protocol, and the studyprotocol was reviewed and approved by our localinstitutional human research committee as it con-forms to the ethical guidelines of the 1975 Declara-tion of Helsinki, as revised in 2002.

DefinitionsThe presence of diabetes mellitus was defined asfasting plasma glucose �126 mg ⁄dL and ⁄or 2-hourpost-load glucose �200 mg ⁄dL or specific antidia-betic drug therapy. Dyslipidemia was defined asserum triglycerides >150 mg ⁄dL and ⁄or high-density lipoprotein cholesterol <40 mg ⁄dL in menand <50 mg ⁄dL in women. Normal exercise ECGtest result was defined as attainment of >85%of age-predicted maximal heart rate, without anginaor other symptoms suggestive of myocardialischemia, in the absence of ischemic ST-segmentdeviation.

Initial Assessment of Endothelium-DependentFMDPatients were instructed to discontinue all vasoac-tive medications for 4 times the half-life and to

refrain from cigarette smoking for at least 6 hoursbefore assessment. Assessment was performed ina quiet dark room with a constant temperature(24�C), barometric pressure (760 mb), and humid-ity (50%). Patients were evaluated after a 12-hourfasting period, in the supine position, with the armcomfortable and supported. Resting blood pressurewas measured with a sphygmomanometer cuff.Imaging of the brachial artery in the dominant armwas performed using a General Electric Vivid 7 Procardiac ultrasound machine (General Electric, Hor-ten, Norway). After 15 minutes of relaxation, a7.5-MHz linear array probe was used to obtaingood-quality images of the brachial artery in thelongitudinal plane, above the antecubital fossa. Thebaseline brachial artery diameter was measuredat rest from media-adventitia interface to media-adventitia interface. The baseline blood flow veloc-ity was measured from the pulsed-wave Dopplersignal obtained with the sample volume at mid-artery level. Hyperemic response was induced byinflating the sphygmomanometer cuff (wrappedaround the distal forearm) to a pressure 50 mm Hgabove the measured systolic pressure for 5 minutes.After release of the cuff, the hyperemic blood flowvelocity was measured as before within 15 secondsafter cuff release. The hyperemic brachial arterydiameter was then measured as before, approxi-mately 60 seconds after cuff release or 45 to60 seconds after the peak hyperemic flow velocity.7

FMD was calculated as the percent increase of bra-chial artery diameter from baseline to peak hyper-emic measurement. The percent increase of bloodflow velocity from baseline to peak hyperemic mea-surement was also calculated. Assessment was per-formed offline by a single experienced operator(Z.A.), blinded to individual group assignment aswell as to the chronologic order of assessment (ini-tial or follow-up).

Pharmacologic InterventionAfter enrollment and initial assessment of endo-thelium-dependent FMD, patients were randomlyassigned, on an individual basis, to receive eitheratorvastatin calcium 10 mg daily orally for 4 weeks(atorvastatin group=30 patients) or matchedplacebo for the same period (placebo group=30patients). They remained in the same allocationthroughout the study period. Standard antidiabeticmedications were allowed and remained unchangedduring the study period. None of the patientsregularly consumed multivitamin ⁄mineral tablets orrelevant amounts of food particularly rich inantioxidants.

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Follow-Up Assessment of Endothelium-Dependent FMDAssessment of the brachial artery diameter andblood flow velocity (both baseline and hyperemic)was performed after 4 weeks, as before.

Statistical AnalysisAll continuous variables were presented asmean � standard deviation, if they were normallydistributed. Data were tested for normal distribu-tion using the Kolmogorov–Smirnov test. Categoricvariables were described with absolute and rela-tive (percentage) frequencies. Comparisons betweenthe two individual groups were performed usingthe unpaired t test for continuous variables and thePearson chi-square test for categoric variables. Base-line data were compared with follow-up datawithin the same group using the paired t test forcontinuous variables. All tests were 2-sided and aprobability value of P<.05 was considered statisti-cally significant. Analyses were performed withSPSS version 12.0 statistical package (SPSS Inc,Chicago, IL).

RESULTSBaseline Clinical CharacteristicsA total of 60 patients with T2DM and normalserum cholesterol levels were enrolled in the currentstudy, which comprised 30 patients randomlyassigned to receive atorvastatin (atorvastatin group=30 patients) and 30 others randomly assigned toreceive matched placebo (placebo group=30patients). Table I shows the baseline clinical charac-teristics of the whole series, as well as the two indi-vidual groups. The mean age of the study cohortwas 38.3�10.5 years, 30 (50%) being men. Thetwo individual groups were statistically matchedregarding age, sex, duration of DM, hypertension,smoking, and dyslipidemia.

Initial Brachial Artery MeasurementsTable II shows initial brachial artery measurements(both baseline and hyperemic) of the two individualgroups. No statistically significant difference wasfound between the two groups regarding brachialartery diameter or flow velocity, both at baselineand at peak hyperemia (P>.05 for all). However,there was a trend toward a higher percent increaseof brachial artery diameter (from baseline to peakhyperemia) in the placebo group compared withthe atorvastatin group (P=.095).

Follow-Up Brachial Artery MeasurementsSimilarly, Table III shows brachial artery measure-ments (both baseline and hyperemic) of the twoindividual groups at 4 weeks of follow-up. Therewas a significantly higher percent increase ofbrachial artery diameter (from baseline to peakhyperemia) in the atorvastatin group as comparedwith the placebo group (P<.05). Moreover, therewas a significantly higher flow velocity at baseline(P<.05) and a trend toward a higher flow velocity

Table I. Baseline Clinical Characteristics of the Entire Cohort and Both Study Groups

Cohort (N=60) Statin Group (n=30) Placebo Group (n=30) P Valuea

Age, y 38.3�10.5 38.7�11 37.9�9.9 >.05

Men 30 (50) 14 (46.7) 16 (53.3) >.05Duration of diabetes mellitus, y 3.6�8.3 3.0�5.4 3.9�9.5 >.05Hypertension 48 (80) 25 (83.3) 23 (76.7) >.05

Smoking 25 (41.7) 13 (43.3) 12 (40) >.05Dyslipidemiab 20 (33.3) 9 (30) 11 (36.7) >.05Total serum cholesterol, mmol ⁄ L 4.8�0.3 4.7�0.3 4.9�0.2 >.05

Continuous variables are presented as mean � standard deviation, while categoric variables are presented as numbers

(percentage). aComparison between statin group and placebo group. bAbnormal serum triglycerides and ⁄ or high-densitylipoprotein cholesterol levels.

Table II. Initial Brachial Artery Measurements in Both

Study Groups at Baseline and at Peak Hyperemia

Statin

Group

(n=30)

Placebo

Group

(n=30) P Value

Brachial artery diameter

Baseline, mm 4.03�0.64 3.89�0.66 .661Hyperemic, mm 4.43�0.68 4.39�0.73 .902Percent increase, % 7.7 13.2 .095

Brachial artery flow velocity

Baseline, cm ⁄ s 66.7�23.2 62.8�26.4 .734Hyperemic, cm ⁄ s 71.5�29.2 69.4�27.2 .670Percent increase, % 12.2 7.5 .367

Continuous variables are presented as mean � standarddeviation.

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at peak hyperemia (P=.091) in the atorvastatingroup as compared with the placebo group. Yet,no statistically significant difference was foundbetween the two groups regarding the brachialartery diameter (both at baseline and at peakhyperemia) or the percent increase of flow velocity(from baseline to peak hyperemia) (P>.05 for all).

The drug was well-tolerated by all patients, withno major side effects during the 4-week treatmentperiod. Moreover, no patients reported any clinicalevents during this period.

DISCUSSIONThe current study demonstrated that low-doseatorvastatin (10 mg daily) induced an appreciableimprovement of endothelial function in normocho-lesterolemic patients with T2DM, as reflected byimprovement of brachial artery FMD. FMD (thepercent increase of brachial artery diameter frombaseline to peak hyperemia) has increased in theatorvastatin group, from 7.7% initially to 18.3%at 4-week follow-up (P<.05). In comparison, whilethe FMD was initially higher in the placebo group(as compared with the atorvastatin group), itremained almost the same at 4-week follow-up(P>.05). Furthermore, while brachial artery flowvelocities (both baseline and hyperemic) were ini-tially similar between the two individual groups(P>.05), both values were higher in the atorvasta-tin group at 4-week follow-up (compared with theplacebo group).

Endothelial Dysfunction in T2DMHyperglycemia is associated with reduced bioavail-ability of NO as a result of decreased synthesis byendothelial NO synthase enzyme and increased

consumption by oxygen-derived free radicals.1 Arecently proposed pathophysiologic mechanismpotentially contributing to endothelial dysfunctionin patients with T2DM is mediated by the increasein ADMA levels. By inhibiting endothelial NO syn-thase, ADMA hampers the regeneration of depletedNO in the vessel wall.6,8 Moreover, the exposureto hyperglycemia induces a nonenzymatic glycationof proteins and lipids with ensuing formation ofadvanced glycation end-products, which are pre-valent in the vasculature of diabetic patients.9

Advanced glycation end-products decrease the bio-availability of NO both by reducing endothelialNO synthase activity and by depleting the alreadypresent NO.10

Effect of Statins on Endothelial FunctionStatins likely improve endothelial function byincreasing NO synthase enzyme activity, reducingoxidative stress, and decreasing levels of ADMA.4–6

Supporting the results of the current study, severalvalidated peer-reviewed articles in the literaturedemonstrated that statins improved endothelial dys-function in patients with T2DM by a mechanismunrelated to the lipid-lowering effect.11–13 In con-trast, other studies reported no or insignificantimprovement of FMD in T2DM patients treatedwith statins.14–16 This discrepancy of results amongthe different studies can be attributed—at least inpart—to the divergence in the methodology of mea-surement of FMD. For instance, defining lumendiameter as the distance between intima-lumeninterfaces—rather than media-adventitia inter-faces—would decrease reference lumen diametersand augment the calculated FMD. In addition,inducing hyperemia by inflating the sphygmoma-nometer cuff around the upper arm instead of theforearm would again result in higher FMD. Finally,the timing of measurement of the hyperemic bra-chial artery diameter would also be critical. Record-ing measurements every 15 seconds after deflationof the cuff would be more representative than sim-ply taking a single measurement 1 minute later.Inconsistent findings would also reflect the hetero-geneous nature of the underlying disease process,the lack of uniformity in patient selection and studyprotocols among different studies, and the interplayof various genetic and environmental factors relatedto the diverse ethnical and cultural backgrounds ofthe studied populations.

Clinical ImplicationsTwo recent mega trials, namely the CollaborativeAtorvastatin Diabetes Study (CARDS) and the

Table III. Follow-Up Brachial Artery Measurements in

Both Study Groups at Baseline and at Peak Hyperemia

Statin

Group

(n=30)

Placebo

Group

(n=30) P Value

Brachial artery diameter

Baseline, mm 3.87�0.74 3.79�0.46 .884Hyperemic, mm 4.49�0.69 4.33�0.62 .405Percent increase, % 18.3 13.2 .015a

Brachial artery flow velocity

Baseline, cm ⁄ s 70.6�18.2 58.2�20.8 .033a

Hyperemic, cm ⁄ s 74.5�20.3 63.8�20.7 .091Percent increase, % 10.9 10.1 .724

Continuous variables are presented as mean � standarddeviation. aSignificant P value.

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Anglo-Scandinavian Cardiac Outcomes Trial–Lipid-Lowering Arm (ASCOT-LLA), reported thattreatment with atorvastatin reduced major cardio-vascular events in patients with T2DM.17,18 Impor-tantly, the benefits of atorvastatin use were notrelated to initial serum cholesterol levels, and thesedata add further credence to the theory that allpatients with type 2 diabetes may benefit from sta-tin therapy. Nevertheless, although these data sug-gest that treatment with low-dose atorvastatin(10 mg ⁄d) is sufficient to reduce cardiovascular riskin patients with T2DM and normal serum choles-terol levels, this paradigm is not widely acknowl-edged.19 We conducted the current study inpatients with T2DM and normal serum cholesterollevels, excluding those with known ischemic heartdisease and those with equivocal or ischemicresponse to exercise stress ECG test. The observa-tion, in the current study, that a low-dose atorvast-atin regimen improves endothelial dysfunction (asreflected by the improvement of FMD) innormocholesterolemic diabetic patients without evi-dence of coronary artery disease may provideinsight into the underlying mechanism of reductionof cardiovascular risk in this patient category, pre-viously reported in the literature,17,18 and warrantfurther reconsideration of such regimens in patientswith T2DM. Based on the available body of evi-dence, statin therapy should now be routinely con-sidered for all diabetic patients at sufficiently highrisk for major cardiovascular events, irrespective oftheir initial cholesterol concentrations.

LIMITATIONSOur findings are based on a single-center study witha relatively small sample size of the cohort, a factthat makes it difficult to generalize our results to allpatients with T2DM and normal serum cholesterollevels. Multicenter studies using the same protocoland examining a larger number of patients areneeded. Moreover, the presence of ‘‘silent’’ ischemicheart disease cannot be completely ruled out in thisseries of diabetic patients. Furthermore, the sensitiv-ity of exercise stress ECG testing might be less thanideal in identifying patients with ischemic heartdisease. More sophisticated stress myocardial perfu-sion imaging studies or stress echocardiographywould have better isolated patients with ischemicheart disease.

CONCLUSIONSIn patients with T2DM and normal serum choles-terol levels without evidence of coronary artery

disease, low-dose atorvastatin improves endothelialfunction, as reflected by FMD in the brachialartery.

Acknowledgements and disclosures: The authors deeply appre-ciate the faithful efforts and sincere collaboration of allmembers of the staff of the echocardiography laboratory inAin Shams University Hospital who rigorously participated inthe production of this work. No authors have any conflict ofinterest to declare.

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