renin–angiotensin–aldosterone system in brain infarction and vascular death

Renin–Angiotensin–Aldosterone System inBrain Infarction and Vascular Death

David Brenner, MD,1 Julien Labreuche,1, Bst, Odette Poirier, PhD,2 Francois Cambien, MD,2

and Pierre Amarenco, MD,1 on behalf of the GENIC Investigators

The renin–angiotensin–aldosterone system has functions that may contribute to brain infarction (BI). In 459 matched pairsof white patients and control subjects, we measured plasma angiotensin-converting enzyme (ACE) levels, seven polymor-phisms (angiotensinogen T174M and M235T, ACE I/D and 4656 2/3CT repeat [rpt], angiotensin II type 1 receptorA1166C and A153G, and aldosterone synthase CYP11B2), and evaluated 5-year poststroke mortality. Mean plasma ACElevels (�standard error) were significantly greater in patients than control subjects (37.5 � 0.9 vs 33.9 � 0.9), in patientswith lacunar stroke, and in patients with no previous vascular (cerebrovascular or cardiovascular) history. The risk for BIincreased with tertiles of plasma ACE, without an interaction with hypertension. After adjustments, the association disap-peared except among patients with cardioembolic BI and those without previous vascular events. Among the polymor-phisms, there was a weak association of BI with angiotensin II type 1 receptor 1166C, a weak protective effect withangiotensinogen 174M, and a strong association of angiotensinogen 235T with 5-year vascular mortality. These resultssuggest that renin–angiotensin–aldosterone system activity and genes contribute to cerebrovascular disease and poststrokevascular death in white patients.

Ann Neurol 2005;58:131–138

The renin–angiotensin–aldosterone system (RAAS) is im-portant for cerebrovascular research because it influencesblood pressure, vasoconstriction, thrombosis, and vesselwall damage. Renin, released from the kidney, cleaves an-giotensinogen (AGT) to angiotensin I, which then ismodified by angiotensin-converting enzyme (ACE) to an-giotensin II (ATII). ATII is an extremely potent vasocon-strictor that acts within seconds.1 ATII also stimulates al-dosterone secretion, which absorbs sodium and increasesintravascular volume.2 In addition, ATII and aldosteronepromote coagulation by increasing plasminogen-activatorinhibitor and activating platelets at sites of injury.2,3

Several studies have suggested that the RAAS contrib-utes to brain infarction (BI). The Heart Outcomes Pre-vention Evaluation (HOPE), Losartan Intervention forEndpoint Reduction in Hypertension Study (LIFE), andAcute Candesartan Cilexetil Evaluation in Stroke Survi-vors (ACCESS) studies showed that RAAS inhibition re-duced the occurrence of BI and cardiovascular disease,possibly by mechanisms other than blood pressure reduc-tion alone.2,4,5 We report the results of the Etude du pro-fil GENetique de l’Infarctus Cerebral (GENIC) study,which included more than 500 caucasion patients and

matched control subjects. We studied plasma ACE levelsafter acute BI, separated by subtypes, the frequencies ofseven RAAS polymorphisms (AGT T174M and M235T,ACE I/D and 4656 2/3CT repeat [rpt], angiotensin IItype 1 receptors [AT1R] A1166C and A153G, and aldo-sterone synthase CYP11B2 T344C), and their effect on5-year poststroke vascular death.

Patients and MethodsStudy PopulationThe Ethics Committee of Cochin Hospital (Paris, France)approved the protocol. All subjects signed informed consent.There were 510 patients recruited from 12 neurological cen-ters in France with the following criteria: clinical symptomsof a stroke, no hemorrhage on computed tomography, in-farct on magnetic resonance imaging, aged 18 to 85, bothparents were white. Patients were enrolled within 1 week af-ter the BI. Patients reporting a previous cardiovascular (myo-cardial infarction, angioplasty, coronary artery bypass sur-gery, or lower limb arterial disease) or cerebrovascular(ischemic and hemorrhagic stroke or transient ischemic at-tacks) history were eligible. There were 510 control subjectsmatched by sex, age (�5 years), and center to each patient,

From the 1Department of Neurology and Stroke Centre, BichatUniversity Hospital and Medical School, Denis Diderot University;and 2Institut National de la Sante et de la Recherche Medicale 525,Pitie-Salpetriere Medical School, Paris, France.

Received Feb 25, 2005, and in revised form May 4. Accepted forpublication May 4, 2005.

The institutions and investigators in the GENIC group is availablein the Appendix on page 136 and via the internet at http://www.ccr.jussieu.fr/GENIC/Welcome.html.

Published online Jun 27, 2005, in Wiley InterScience(www.interscience.wiley.com). DOI: 10.1002/ana.20537

Address correspondence to Dr Amarenco, Coordinating Centre forthe GENIC study, Department of Neurology and Stroke Centre,Bichat Hospital, 46, rue Henri Huchard, 75018 Paris, France.E-mail: [email protected]

© 2005 American Neurological Association 131Published by Wiley-Liss, Inc., through Wiley Subscription Services

and they were hospitalized at the same centers for nonneu-rological causes (orthopedic, 46%; ophthalmological, 12%;rheumatological, 11%; surgical, 6%; other, 25%). Controlsubjects with cardiovascular history, but not cerebrovascularhistory, were eligible. Both parents of control subjects werewhite.

Data Collection, Risk Factor Definition,and InvestigationsDemographic characteristics, risk factors, and definitionshave been reported previously.6 Electrocardiogram, extracra-nial duplex, transcranial Doppler, and carotid ultrasonogra-phy studies were performed on all subjects. One centralizedlaboratory performed all blood work. Plasma ACE concen-trations were determined by spectrophotometry using N-[3-(2-furyl)acryloyl]-L-phenylalanylglycylglycine (FAPGG) as asubstrate (Trinity Biotech, Oxfordshire, United Kingdom).The laboratory was blinded to the case–control status of thesubjects. The genotyping protocol is available at the Gen-eCanvas Web site (http://www.genecanvas.org).

Stroke Subtype ClassificationThe patients were classified into BI subtypes by two neurol-ogists (P.A., F. C.) by reviewing imaging studies, case reportforms, and clinical reports. Details have been published pre-viously and are available on the GENIC Web site (http://www.ccr.jussieu.fr/GENIC/SMSub.html).6

Follow-upFollow-up included a 10-day visit during hospitalization or ondischarge (median, 11 days; range, 3–40 days), a 6-monthvisit with a local neurologist (median, 192 days; range, 94–565 days), and a home visit by a research nurse (median, 2.7days; range, 1.4–4.0 years). Patients or families were called at5 years (median, 5.3 years; range, 4.0–6.6 years) to assessmortality. If contact could not be made, we searched primarycare records or the Registrar of Births, Marriages and Deathsfrom the city of birth (or city of Nantes for births outsideFrance). We used the Institut National de la Sante et de laRecherche Medicale (INSERM) registry of death certificates toclassify cases into vascular death, which included all cardiovas-cular and cerebrovascular causes.

Statistical AnalysisStatistical analysis was based on 459 pairs of patients and con-trols for whom genotypes and plasma ACE levels were avail-able. Comparison of mean plasma ACE levels between pa-tients and controls was performed using analysis of covarianceadjusted on matching variables (ie, age, sex, and center), ACEinhibitor use, and blood sample delay. The linearity of theplasma ACE/BI relation was tested using conditional logisticregression for matched sets adjusted on ACE inhibitor use andblood sample delay. After categorization of plasma ACE intotertiles, the odds ratios (ORs) and 95% confidence intervals(CIs) of BI for the upper two tertiles relative to the lowesttertile were calculated. Because ORs for BI increased with ter-tiles of plasma ACE, we also computed OR per 1 standarddeviation (SD) increase of plasma ACE.

Allelic frequencies were calculated by gene counting, andthe Hardy–Weinberg equilibrium was tested in control sub-

jects with the �2 test. We compared genotype distributionsbetween patients and control subjects using the �2 log likeli-hood statistic of conditional logistic regression for matchedsets. Because there was a trend for significant effect for theAGT T174M and AT1R A1166C polymorphisms, we calcu-lated ORs and 95% CIs for BI assuming a dominant effect ofthese alleles. The log likelihood of the dominant model wasnot significantly different from the codominant genetic model.

Genotype analysis was performed for the whole study groupand repeated after stratifying for main BI subtypes (ie, athero-thrombotic strokes, lacunar strokes, cardioembolic strokes, andstrokes of unknown cause). For each subtype, cases were com-pared with their matched control subjects. The analysis wasalso performed for subjects free of previous vascular (cardio-vascular and cerebrovascular) history. Matched case–controlcomparisons were adjusted for cerebrovascular risk factors (hy-pertension, diabetes, tobacco use, cholesterol, and cardiovascu-lar history). The homogeneity of ORs for patients with andwithout history of hypertension was tested by introducing thecorresponding interaction term into models. We also investi-gated the relation between plasma ACE and RAAS polymor-phisms on vascular mortality at 5-year follow-up for patients.We estimated and compared vascular mortality rates accordingto tertiles of plasma ACE and RAAS polymorphisms using theKaplan–Meier method and the log-rank test, respectively. Pa-tients who died of causes other than vascular were censored atthe time of death. Cox proportional hazard models were usedto estimate the relative risk for vascular death before and afteradjustment on baseline handicap level (Rankin Scale � 2), BIsubtypes, and vascular risk factors. The assumptions of pro-portional hazards were checked by introducing time-dependent variables into models.

Statistical testing was done with a two-tailed � level of 0.05.Data were analyzed using the SAS package (SAS, Cary, NC).

ResultsGeneral characteristics of subjects are presented in Ta-ble 1. Patients had a greater prevalence of cardiovascu-lar risk factors and reported previous cardiovascularevents more often than control subjects. The frequen-cies of BI subtypes were atherothrombotic, 23% (n �105); lacunar, 21% (n � 96), cardioembolic, 16%(n � 73); undetermined (multiple coexisting causes),13% (n � 58); dissections, 2% (n � 11); rare causes;2% (n � 9); unknown, 23% (n � 107).

Matched Case–Control ComparisonsPLASMA ANGIOTENSIN-CONVERTING ENZYME LEVELS.

Mean (�standard error) plasma ACE was significantlyincreased in cases (37.5 � 0.9) compared with controlsubjects (33.9 � 0.9). According to BI subtypes, the dif-ference in plasma ACE levels was significant only forlacunar strokes (Table 2), without heterogeneity (p forinteraction � 0.10). Analysis restricted to subjects with-out vascular history showed similar results, except thesignificant association between plasma ACE and cardio-embolic stroke (see Table 2). After categorizing ACElevels into tertiles, the risk for BI increased gradually

132 Annals of Neurology Vol 58 No 1 July 2005

with increasing plasma ACE (Fig 1), both in overall pa-tients and subjects without vascular history (p fortrend � 0.0015). Therefore, we also computed the ORsper 1 SD increase in ACE levels (see Table 2). The as-sociation between ACE levels and established cerebrovas-cular risk factors were studied separately in patients andcontrol subjects. There were no significant associationsbetween plasma ACE and hypertension, diabetes, andcardiovascular history in either patients or control sub-jects. A significant correlation between ACE levels andtotal cholesterol was found both in control subjects(Pearson’s correlation coefficient � 0.13; p � 0.001)and patients (p � 0.004). Smokers had greater plasmaACE levels than nonsmokers, both in patients and con-trol subjects (p � 0.07). After adjusting for cerebrovas-cular risk factors, the relation between ACE levels andBI was no longer significant in all subjects (OR, 1.16;95% CI, 0.98–1.36), but it was borderline significant in

subjects without vascular history (OR, 1.24; 95% CI,1.00–1.53). Among BI subtypes, cardioembolic strokeremained significantly associated with plasma ACE onlyin subjects without vascular history (OR, 2.36; 95% CI,1.09–5.11). There was no interaction with a history ofhypertension (p � 0.72). ACE levels increased graduallywith the number of ACE D and 4566 2CT rpt alleles,both in patients and control subjects (Fig 2).

GENETIC POLYMORPHISMS OF THE RENIN–ANGIOTENSIN–

ALDOSTERONE SYSTEM AND BRAIN INFARCTION. Allelicfrequencies of all polymorphisms were in the range ofpreviously published studies.7,8 The frequencies did notdeviate from Hardy–Weinberg equilibrium, except forAT1R A1166C (p � 0.008). Genotype distributionswere not significantly different between patients andcontrol subjects; however, two polymorphisms had bor-derline associations with BI (Table 3). The frequency of

Table 2. Association between Plasma ACE Levels and Brain Infarction

Stroke Type Cases Controls pa OR (95% CI)b OR (95% CI)b,c pb,c

All subjects (N � 459 pairs)All stroke 37.5 (0.9) 33.9 (0.9) 0.0003 1.29 (1.12–1.49) 1.16 (0.98–1.36) 0.078Atherothrombotic strokes 35.5 (2.4) 32.4 (2.4) 0.14 1.25 (0.93–1.69) 1.15 (0.74–1.76) 0.53Lacunar strokes 40.0 (1.8) 32.7 (1.8) 0.0003 1.76 (1.20–2.60) 1.31 (0.86–1.97) 0.20Cardioembolic strokes 40.1 (2.5) 35.9 (2.7) 0.14 1.28 (0.92–1.79) 1.43 (0.93–2.21) 0.10Strokes of unknown cause 36.4 (2.1) 35.8 (2.2) 0.76 1.05 (0.74–1.47) 0.88 (0.59–1.33) 0.56

Cases and matched controls free of previous vascular history (N � 263 pairs)All stroke 36.4 (1.2) 32.5 (1.2) 0.0024 1.36 (1.12–1.66) 1.24 (1.00–1.53) 0.05Atherothrombotic strokes 35.5 (3.3) 30.6 (3.2) 0.094 1.41 (0.93–2.13) 1.17 (0.68–2.00) 0.58Lacunar strokes 42.1 (2.3) 34.5 (2.5) 0.003 1.66 (1.06–2.60) 1.41 (0.85–2.34) 0.18Cardioembolic strokes 39.2 (3.5) 31.4 (3.8) 0.02 2.00 (1.06–3.79) 2.36 (1.09–5.11) 0.03Strokes of unknown cause 30.0 (2.8) 31.6 (2.9) 0.50 0.93 (0.59–1.45) 0.87 (0.53–1.42) 0.57

Values are adjusted mean plasma ACE levels (SE).aANCOVA adjusted on matching variables (ie, age, sex, and center); ACE inhibitor use and blood sampling delay.bORs associated with an increase in plasma ACE levels of 1 standard deviation (15.4U/L); conditional logistic regression for matched setsadjusted on ACE inhibitor use and blood sampling delay.cAdditional adjustment for other risk factors (ie, hypertension, diabetes, total cholesterol, current smoking, and cardiovascular history).

ACE � angiotensin-converting enzyme; OR � odds ratio; CI � confidence interval; ANCOVA � analysis of covariance.

Table 1. General Characteristics of Brain Infarction Cases and Controls

Characteristic Cases (n � 459) Controls (n � 459)

Age, yr, median (range) 69 (20–86) 68 (20–89)Male sex, % (n) 61.4 (282/459) 61.4 (282/459)BMI, kg/m2, mean (SD) 25.5 (4.4) 25.7 (4.8)History of hypertension, %(n) 51.6 (236/457) 36.0 (164/456)a

History of diabetes, % (n) 19.0 (87/457) 11.8 (54/459)b

Total cholesterol, mg/dl, mean (SD) 202 (43) 182 (43)a

Current smokers, % (n) 28.6 (131/458) 20.5 (94/459)b

Cardiovascular history, % (n) 21.8 (99/455) 11.6 (53/456)a

Previous stroke history, % (n) 20.9 (96/459) —

Mac Nemar and paired t tests were used to compare proportions and continuous variables, respectively. (Medians of age were compared withWilcoxon test.)ap � 0.001; bp � 0.01.

BMI � body mass index; SD � standard deviation.

Brenner et al: RAAS in Brain Infarction and Vascular Death 133

AGT 174M was greater in control subjects (0.144 vs0.107; p � 0.052), and the frequency of AT1R 1166Cwas greater in patients (0.300 vs 0.268; p � 0.07).

ANGIOTENSINOGEN T174M POLYMORPHISM. The ORof BI associated with of at least one copy of AGT 174Mwas 0.71 (95% CI, 0.52–0.99; p � 0.04), thus suggest-ing a weak protective effect against BI. This relation wasnot modified by a history of hypertension (p � 0.20).After adjusting for cerebrovascular risk factors, the ORwas of similar magnitude but no longer significant(0.72; 95% CI, 0.50–1.04; p � 0.08). Similar resultswere observed in the analysis restricted to subjects with-out vascular history (adjusted OR, 0.74; 95% CI, 0.46–1.18; p � 0.21). When stratifying BI into subtypes, wefound no significant association (data not shown).

ANGIOTENSIN RECEPTOR A1166C POLYMORPHISM. TheOR for all BI subtypes associated with the presence ofat least one copy of AT1R 1166C was significant (1.33;95% CI, 1.02–1.74; p � 0.037) and was not modifiedby a history of hypertension (p � 0.16). When strat-ifying BI into subtypes (Table 4), there was a signifi-cant effect for 1166C only for atherothrombotic

strokes (OR, 1.82; 95% CI, 1.07–3.11). In multivari-ate analysis, including cerebrovascular risk factors, theOR was not significant in the whole group (1.28; 95%CI, 0.94–1.73; p � 0.12) and for each BI subtype.Sensitivity analysis, restricted to subjects without vas-cular history, showed similar results (see Table 4).

Case Follow-upAfter a median follow-up of 5.3 (range, 1.5–6.6) yearsfor 459 patients, 152 patients died and 19 were lost tofollow-up after the third examination. Fatal vascularevents, including all cardiovascular and cerebrovascularcauses, occurred in 86 cases.

PLASMA ANGIOTENSIN-CONVERTING ENZYME LEVELS

AND VASCULAR DEATH. There was no significant dif-ference in survival of patients with respect to tertiles ofACE levels (log-rank test: p � 0.46). The adjusted haz-ard ratio for vascular death per 1 SD increase in ACElevels was 0.96 (95% CI, 0.76–1.20; p � 0.72).

GENETIC POLYMORPHISMS OF THE RENIN–ANGIOTENSIN–

ALDOSTERONE SYSTEM AND VASCULAR DEATH. A signif-icant difference in poststroke survival was observed with

Fig 1. Odds ratio of brain infarction associated with tertiles of plasma angiotensin-converting enzyme (ACE) levels.

Fig 2. Mean plasma angiotensin-converting enzyme (ACE) levels in cases and controls according to angiotensin converting enzymepolymorphisms. Standard errors and p values for trend are indicated.


the AGT M235T polymorphism (Fig 3). The hazard ra-tio for vascular death associated with at least one copy ofthe AGT 235T allele was 2.33 (95% CI, 1.38–3.92; p �0.001). This association remained significant in multivar-iate analysis, including potential confounding factors (haz-ard ratio, 3.34; 95% CI, 1.36–4.02; p � 0.002). Therewas no significant association between vascular mortalityand other RAAS polymorphisms, assuming recessive ordominant genetic models (log-rank test: p � 0.10).

DiscussionIn this large cohort of European adults, these resultssuggest an association between plasma ACE and BI,with an increased risk for BI with greater plasma ACElevels. There were also strong associations between theACE D and ACE 4656 2CT rpt alleles and increasedplasma ACE levels. Among the genotypes, there was aweak association of the AT1R 1166C allele with BI, aweak protective effect of the AGT 174M allele against

Table 3. Distribution of Genotypes for Polymorphisms of Renin-Angiotensin- Aldosterone System in Brain InfarctionCases and Controls

Polymorphism Genotypes Second Allele (%) pa,b

ACE 4656 2/3CT rpt 22 23 33Cases 32.5 (149) 46.2 (212) 21.3 (98) 44.4 0.60/0.35Controls 29.4 (135) 49.0 (225) 21.6 (99) 46.1

ACE I/D II ID DDCases 20.5 (94) 46.2 (212) 33.3 (153) 56.4 0.72/0.40Controls 18.7 (86) 48.8 (224) 32.5 (149) 56.9

AGT T174M TT TM MMCases 80.2 (368) 18.3 (84) 1.5 (7) 10.7 0.05/0.10Controls 74.5 (342) 22.2 (102) 3.3 (15) 14.4

AGT M235T MM MT TTCases 35.7 (164) 48.2 (221) 16.1 (74) 40.2 0.90/0.89Controls 34.4 (158) 48.6 (223) 17.0 (78) 41.3

AT1R A1166C AA AC CCCases 49.2 (226) 41.6 (191) 9.2 (42) 30.0 0.07/0.28Controls 56.0 (257) 34.4 (158) 9.6 (44) 26.8

AT1R A153G AA AG GGCases 67.5 (310) 29.0 (133) 3.5 (16) 18.0 0.91/1.00Controls 68.2 (313) 27.9 (128) 3.9 (18) 17.9

CYP11B2 T344C TT TC CCCases 31.4 (144) 48.6 (223) 20.0 (92) 44.3 0.55/0.85Controls 31.2 (143) 51.4 (236) 17.4 (80) 43.1

aUsing-2 log-likelihood statistic of conditional logistic regression for matched sets.bAnalyses restricted to cases and matched controls both free of vascular history.

Table 4. Association between AT1R A1166C and Brain Infarction according to Main Causative Subtypes

Stroke Type AA AC CC

SecondAllele(%) OR (95% CI) p OR (95% CI) OR (95% CI)a,b

Atherothrombotic strokesCases (n � 105) 49.5 (52) 42.9 (45) 7.6 (8) 29.0 1.82 (1.07–3.11) 0.03 1.65 (0.83–3.26) 1.31 (0.50–3.44)Control (n � 105) 65.7 (69) 29.5 (31) 4.8 (5) 19.5

Lacunar strokesCases (n � 96) 54.2 (52) 38.5 (37) 7.3 (7) 26.6 0.97 (0.51–1.85) 0.93 0.92 (0.41–2.06) 0.62 (0.23–1.71)Control (n � 96) 54.2 (52) 33.3 (32) 12.5 (12) 29.2

Cardioembolic strokesCases (n � 73) 53.4 (39) 41.1 (30) 5.5 (4) 26.0 1.16 (0.58–2.29) 0.67 1.24 (0.57–2.70) 0.96 (0.36–2.55)Control (n � 73) 56.1 (41) 32.9 (24) 11.0 (8) 27.4

Strokes of unknown causeCases (n � 107) 43.9 (47) 42.1 (45) 14.0 (15) 35.0 1.48 (0.82–2.67) 0.19 1.47 (0.76–2.84) 1.45 (0.64–3.29)Control (n � 107) 51.4 (55) 36.4 (39) 12.2 (13) 30.4

ORs are calculated assuming a dominant model, using conditional logistic regression for matched sets.aAdjustments for cerebrovascular risk factors (ie, history of hypertension and diabetes, current smoking, total cholesterol, and cardiovascularhistory).bAnalyses restricted to cases and matched controls both free of vascular history.OR � odds ratio; CI � confidence interval.


BI, and the AGT 235T allele was strongly predictive of5-year poststroke vascular death (see Fig 3).

Plasma Angiotensin-Converting Enzyme LevelsACE levels were significantly increased in the wholegroup of patients, in patients with no previous vascularhistory (cardiovascular or cerebrovascular), and in pa-tients with lacunar stroke. However, after adjusting forACE inhibitor use, hypertension, diabetes, smoking,and cholesterol, these associations disappeared, butthey remained significant for cardioembolic BI in pa-tients with no vascular history (see Table 2). We un-expectedly found a strong correlation between plasmaACE and cholesterol levels that explained the disap-pearance of the univariate association between lacunarBI and plasma ACE because lacunar BI was associatedwith plasma cholesterol in our study.

No other study has reported increased plasma ACElevels with BI, although some reports have claimed thatincreased plasma ACE level is associated with othercardiovascular diseases. Cambien and colleagues9 re-ported that myocardial infarction patients had in-creased plasma ACE levels, although they reported asmaller difference (30.8 � 0.5 vs 29.5 � 0.4).Bonithon-Kopp and colleagues10 reported increasedplasma ACE levels in subjects with carotid wall thick-ening with an OR of 2.29 (95% CI, 1.16–4.52; p �0.02) per 1 SD increase of plasma ACE. In addition,there are reports that increased plasma ACE level is as-sociated with other vascular events.11,12

Increased RAAS activity could contribute to BIthrough its effect on blood pressure, vasospasm, bloodcoagulation, and vessel wall injury. However, increasedplasma ACE level alone is not necessarily a marker ofRAAS activity. Studies have reported that plasma ACElevels can be increased during chronic ACE inhibitortherapy, presumably because of stimulation of the feed-back mechanism.13,14 In addition, angiotensin receptor

dysfunction could result in increased plasma ACE lev-els without an increase in RAAS activity.

One drawback to our study is that we cannot ex-clude that the plasma ACE level is acutely increasedafter BI, because we did not measure the plasma ACElevel at follow-up. However, several arguments suggestthat greater plasma ACE levels were not secondary tothe acute event. Both patients and control subjects hadsimilar subdistributions of ACE levels for ACE I/Dand ACE 4656 2/3CT rpt genotypes, thus suggestingplasma ACE is a function of these polymorphisms, notthe acute event. In addition, there are no reports ofalterations of plasma ACE levels after BI.

Genotypes Affecting Plasma Angiotensin-ConvertingEnzyme LevelsWe found strong linear relations (p � 0.0001) be-tween the ACE D and 4656 2CT rpt alleles andplasma ACE levels, which are consistent with previousreports.15,16 Control subjects with the ACE ID andDD genotypes had increased ACE levels 19% and38%, respectively, over the II genotype. Control sub-jects with the ACE 23CT rpt and 22CT rpt genotypeshad increased ACE levels 22% and 38%, respectively,over 33CT rpt. However, we found no direct associa-tion with the ACE I/D polymorphism and BI. It ispossible that the associations with ACE D and BIshown in other studies were actually caused by in-creased plasma ACE levels.

Angiotensin Receptor A1166C PolymorphismThe results suggest that the AT1R A1166C polymor-phism is associated with all BI subtypes grouped to-gether and with atherothrombotic BI. The associationsdisappeared after adjusting for risk factors (cholesterol,diabetes, hypertension, smoking, and cardiovasculardisease history). AT1R is a G-protein receptor that me-diates vasoconstriction on binding of ATII. The

Fig 3. Vascular survival of brain infarction cases according to AGT M235T genotype.


A1166C variant is a single-nucleotide polymorphismfrom adenine to cytosine in the 3� untranslated regionof the gene and has been shown previously to be asso-ciated with vascular disease.

Previous reports have found associations with the1166C allele in ischemic stroke, lacunar stroke, hyper-tension, cardiac hypertrophy, coronary heart disease,myocardial infarction, angina, and diabetic nephropa-thy.17–24 It is currently debated whether this polymor-phism contributes directly to hypertension, which, inturn, leads to these other vascular events. More re-search is needed to understand how the A1166C poly-morphism affects receptor structure and function.

Angiotensinogen T174M PolymorphismThe AGT T174M polymorphism represents a pointmutation of threonine to methionine in exon 2 of theAGT gene. There was a weak protective trend of the Mallele, when grouping all subtypes together, but the as-sociation disappeared after separating cases into sub-types. In contrast to these results, many studies found anassociation of the 174M allele with vascular disease, suchas coronary atherosclerosis and hypertension.24–26 How-ever, there are also many reports that found no associa-tion of this polymorphism and vascular disease.27,28 In arecent metaanalysis of the AGT gene, there was no con-sistent association of the AGT T174M polymorphismand disease, but a nonsignificant trend for decreasedstroke risk with the 174M allele in men matched forage, smoking, hypertension, and hypercholesterolemia.27

In the GENIC cohort, only 22 of 918 patients werehomozygous for 174M, so we cannot exclude that ourfinding was because of chance. Further research isneeded to elucidate the effect of this polymorphism.

Angiotensinogen M235T PolymorphismThe AGT M235T polymorphism is a mutation of me-thionine to theonine at position 235 of the gene,changing a nonpolar amino acid to a polar amino acid,and thus possibly altering the structure of AGT.27

Some studies have reported this single-nucleotide poly-morphism is associated with increased plasma AGTlevels, hypertension, ischemic heart disease, and isch-emic stroke.29–31 The T allele was strongly associatedwith greater rates of 5-year vascular death, includingischemic stroke, hemorrhagic stroke, myocardial infarc-tion, sudden cardiac death, congestive heart failure,and other cerebrovascular and cardiovascular causes.These results suggest that this polymorphism has someeffect on vascular pathology.

Other Renin–Angiotensin–AldosteroneSystem PolymorphismsThere were no associations with BI and the other poly-morphisms (ACE I/D and 4656 2/3CTrpt, AT1RA153G, and CYP11B2). A strength of our study is that it

was restricted to caucasian subjects. The genotypes may bemore or less prevalent in different ethnic populations.

In conclusion, the association between the RAASand BI is complex, with our results showing greaterplasma ACE levels associated with BI, the AT1R1166C allele weakly associated with BI, the AGT174M allele possibly protecting against BI, and theAGT 235T associated with a greater 5-year vascularmortality rate in caucasian patients.

AppendixThe Genic investigators are affiliated with the Saint-AntoineHospital, Pierre and Marie Curie University, Paris: Pierre Am-arenco, M.D., Gina Lutz, M.D., Pierre-Jean Touboul, M.D.,Jean-Luc Gerard, M.D., Valerie Adraı, M.D., Sophie Dereadt(CIC), M.D., Laurent Tennez (CIC), M.D. the Clinical Inves-tigation Center personnel; Jean Minjoz Hospital, Centre Hos-pitalier et Universitaire de Besancon, Besancon: Fabrice Vuillier,M.D., Marie-Helene Snidaro, M.D.,Laurent Tatu, M.D., Thi-erry Moulin, M.D., Jean-Pierre Weissert, M.D., Francoise Cat-tain, M.D; Centre Hospitalier Ge�ne�ral, Meaux: Francois Che-�dru, M.D., Alain Ameri, M.D., Jean-Francois Lefort, M.D.,Laurent Marcy, M.D., Philippe Chantereau, M.D., ChantalKaci, Francois Thuillier; Centre Hospitalier et Universitaire deLille, Lille: Christian Lucas, M.D., Ghislaine Deklunder, M.D.,Ph.D., Corinne Gautier, M.D., Marc Gobert, M.D., Alexan-drine Prevost;Centre Hospitalier et Universitaire de Grenoble,Grenoble: Assia Jaillard, M.D., Marc Hommel, M.D., BernardBertrand, M.D., Patrick Carpentier, M.D.;Centre Hospitalier etUniversitaire d’Amiens, Amiens: Sandrine Canaple, M.D.,Marie-Pierre Guillaumont, M.D., Alain Rosa, M.D.; Sainte-Anne Hospital, Paris V University, Paris: Isabelle An, M.D., CArquizan, M.D., Mathieu Zuber, M.D., A Dilouya, M.D.,Jean-Louis Mas, M.D.; Pasteur Hospital, Nice: Haıel Alchaar,M.D., Marie-Helene Mahagne, M.D., Maurice Chatel, M.D.;La Timone Hospital, Marseille: Loic Milandre, M.D., Jean-Pierre Schildberg, M.D.;Hopital Central, Strasbourg: Gilles Ro-dier, M.D., Francis Vuillemet, M.D., Jean-Marie Warter, M.D.;Delafontaine Hospital, Saint-Denis: Thomas De Broucker,M.D., Pierre Rozier, M.D., Leonie Chartier; Cote de NacreUniversity Hospital, Cean: Serge Iglesias, M.D., Fausto Viader,M.D.;Central Blood Sample Analysis, Laboratoire de BiochimieB and Unite� de Recherche Associee of the Centre National dela Recherche Scientifique, Saint-Antoine Hospital, Paris: FatiDriss, M.D., Gilbert Bereziat, M.D.; DNA Banking and Geno-typing, Service Commun N°7, Institut National de la Sante etde la Recherche Medicale: Francois Cambien, M.D.; StatisticalCenter, Unite 360 Institut National de la Sante et de la Recher-che Medicale: Alexis Elbaz, M.D., Marion Gautier-Bertrand;Central Reading Panels: magnetic resonance imaging (gradingof white mater lucencies and atrophy): Didier Leys, M.D.,Philip Scheltens, M.D., Ph.D., Florence Pasquier, M.D., Ph.D.;intima-media thickness measurements of the common carotidarteries: Cornelia Koller, M.D., Pierre-Jean Touboul, M.D.; 12-lead electrocardiogram reading: Stephane Berroir, M.D.; strokeetiology and MRI ischemic lesion classifications: Pierre Am-arenco, M.D., Francois Chedru, M.D.; Data Monitoring andCoordinating Center, Department of Neurology, Saint-AntoineHospital: Pierre Amarenco, M.D., Homma Madrakian, ValerieLebretton, Chantal Nouharet


This study was supported by Grants-in-Aid from the Fondation CNPpour la Sante, Caisse Nationale d’Assurance Maladie des TravailleursSalaries (3AM001, P.A.), Institut National de la Sante et de la Recher-che Medicale (INSERM, P.A.), Programme Hospitalier de RechercheClinique of the French Ministry of Health (AOA9402, P.A.), andSanofi-Synthelabo and Bristol-Myers Squibb Laboratories; P.A. by In-stitut National de la Sante et de la Recherche Medicale and AssistancePublique-Hopitaux de Paris at the Clinical Investigation Centre ofSaint-Antoine University Hospital; by SOS-ATTAQUE CEREBRALEAssociation (J.L.); and by the William M. Feinberg Memorial Fellow-ship, Sarver Heart Center (University of Arizona; D.B.).

Assistance Publique Hopitaux of Paris held legal responsibility forthis study (P930902, P.A.).

References1. Reid I. The renin-angiotensin system: physiology, pathophysiology,

and pharmacology. Adv Physiol Educ 1998;20:S236–S245.2. Gorelick P. New horizons for stroke prevention: PROGRESS

and HOPE. Lancet Neurol 2002;1:149–156.3. Weber KT. Aldosterone in congestive heart failure. N Engl

J Med 2001;345:1689–1697.4. The Heart Outcome Prevention Evaluation Study Investigators.

Effects of an angiotensin-converting enzyme inhibitor, ramipril,on cardiovascular events in high-risk patients. N Engl J Med2000;342:145–153.

5. PROGRESS Collaborative Group. Randomised trial of aperindopril-based blood-pressure-lowering regimen among6105 individuals with previous stroke or transient ischaemic at-tack. Lancet 2001;358:1033–1041.

6. Touboul P, Elbaz A, Cornelia K, et al. Common carotid arteryintima-media thickness and brain infarction. Circulation 2000;102:313–318.

7. Rubattu S, Di Angelantonio E, Stanzione R, et al. Gene poly-morphisms of the renin-angiotensin-aldosterone system and therisk of ischemic stroke: a role of the A1166C/AT1 gene variant.Hypertension 2004;22:2129–2134.

8. Sethi AG, Tybjaerg-Hansen A, Gronholdt ML, et al. Angio-tensinogen mutations and risk for ischemic heart disease, myo-cardial infarction, and ischemic cerebrovascular disease. Sixcase-control studies from the Copenhagen City Heart Study.Ann Intern Med 2001;134:941–954.

9. Cambien F, Costerousse O, Tiret L, et al. Plasma level andgene polymorphism of angiotensin-converting enzyme in rela-tion to myocardial infarction. Circulation 1994;90:669–676.

10. Bonithon-Kopp C, Ducimetiere P, Touboul P, et al. Plasmaangiotensin-converting enzyme activity and carotid wall thick-ening. Circulation 1994;89:952–954.

11. Ribichini F, Ferrero V, Matullo G, et al. Association study ofthe I/D polymorphism and plasma angiotensin-converting en-zyme (ACE) as risk factors for stent restenosis. Clin Sci 2004;107:381–389.

12. Nagi D, Foy C, Mohamed-Ali V, et al. Angiotensin-1-converting enzyme (ACE) gene polymorphism, plasma ACElevels, and their association with the metabolic syndrome andelectrocardiographic coronary artery disease in Pima Indians.Metabolism 1998;47:622–626.

13. Sassano P, Chatellier G, Billaud E, et al. Treatment of mild tomoderate hypertension with or without the converting enzymeinhibitor Enalapril. Am J Med 1988;83:227–235.

14. Larochelle P, Genest J, Juchel O, et al. Effect of Captopril onblood pressure, plasma renin activity and angiotensin I. CanMed Assoc J 1979;121:309–316.

15. Tiret L, Rigat B, Visvikis S, et al. Evidence, from combinedsegregation and linkage analysis, that a variant of the angioten-sin I-converting enzyme (ACE) gene controls plasma ACE lev-els. Am J Hum Genet 1992;51:197–205.

16. Paillard F, Chansel D, Brand E, et al. Genotype-phenotype re-lationships for the renin-angiotensin-aldosterone system in anormal population. Hypertension 1999;34:423–429.

17. Takami S, Imai Y, Katsuya T, et al. Gene polymorphism of therenin-angiotensin system associates with risk for lacunar infarc-tion. The Ohasama study. Am J Hypertens 2000;13:121–127.

18. Canavy I, Henry M, Morange P, et al. Genetic polymorphismsand coronary artery disease in the south of France. ThrombHaemost 2000;83:212–216.

19. Benetos A, Gautier S, Ricard S, et al. Influence of angiotensin-converting enzyme and angiotensin II type 1 receptor genepolymorphisms on aortic stiffness in normotensive and hyper-tensive patients. Circulation 1996;94:698–703.

20. Hindorff L, Heckbert S, Tracy R, et al. Angiotensin II type 1receptor polymorphisms in the cardiovascular health study: re-lation to blood pressure, ethnicity, and cardiovascular events.Am J Hypertens 2002;15:1050–1056.

21. Reich H, Duncan J, Weinstein J, et al. Interactions betweengender and the angiotensin type 1 receptor gene polymorphism.Kidney Int 2003;63:1443–1449.

22. Henskens L, Spiering W, Stoffers H, et al. Effects of the ACEI/D and AT1R polymorphisms on blood pressure in a healthynormotensive primary care population: first results of the Hip-pocrates study. J Hypertens 2003;21:81–86.

23. Lajemi M, Labat C, Gautier S, et al. Angiotensin II type 1receptor 153A/G and 1166A/C gene polymorphisms and in-crease in aortic stiffness with age in hypertensive subjects. J Hy-pertens 2001;19:407–413.

24. Rubattu S, Di Angelantonio E, Stanzione R, et al. Gene poly-morphisms of the renin-angiotensin-aldosterone system and therisk of ischemic stroke: a role of the A1166C/AT1 gene variant.J Hypertens 2004;22:2129–2134.

25. Takashima Y, Kokaze A, Matsunaga N, et al. Relations ofblood pressure to angiotensinogen gene T174M polymorphismand alcohol intake. J Physiol Anthropol Appl Human Sci 2003;22:187–194.

26. Agachan B, Isbir T, Yilmaz H, Akoglu E. Angiotensin convert-ing enzyme I/D, angiotensin T174M-M235T and angiotensinII type 1 receptor A1166C gene polymorphisms in Turkish hy-pertensive patients. Exp Mol Med 2003;35:545–549.

27. Sehti A, Tybjaerg-Hansen A, Gronholdt M, et al. Angio-tensinogen mutations and risk for ischemic heart disease, myo-cardial infarction, and ischemic cerebrovascular disease. Sixcase-control studies from the Copenhagen City Heart Study.Ann Intern Med 2001;134:941–954.

28. Tiret L, Blanc H, Ruidavents J, et al. Gene polymorphisms ofthe renin-angiotensin system in relation to hypertension andparental history of myocardial infarction and stroke: the PE-GASE study. J Hypertens 1998;16:37–44.

29. Jeunemaitre X, Soubrier F, Koteletsev Y, et al. Molecular basisof human hypertension: role of angiotensinogen. Cell 1992;71:169–180.

30. Katsuya T, Koike G, Yee T, et al. Association of angiotensino-gen gene T235 variant with increased risk of coronary heartdisease. Lancet 1995;345:1600–1603.

31. Nakata T, Kutsuya T, Rakugi H, et al. Polymorphism of an-giotensin converting enzyme, angiotensinogen, and apolipopro-tein E genes in a Japanese population with cerebrovascular dis-ease. Hypertension 1997;10:1391–1395.


renin–angiotensin–aldosterone system in brain infarction and vascular death

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