Author + information
- Received May 28, 1998
- Revision received April 16, 1999
- Accepted June 3, 1999
- Published online September 1, 1999.
- Daniel Henrion, PhDa,* (, )
- Joelle Benessiano, PhD∗,
- Ivan Philip, MD#,
- Sandrine Vuillaumier-Barrot, PhD∗,
- Marc Iglarz, BSc∗,
- Gaetan Plantefève, MD#,
- Didier Chatel, MD§,
- Ulrich Hvass, MD§,
- Geneviève Durand, PhD∗,
- Jean-Marie Desmonts, MD#,
- Philippe Amouyel, MD∗∗ and
- Bernard I Lévy, MD, PhDa
- ↵*Reprint requests and correspondence: D. Henrion, PhD, INSERM U 141, 41 Bd de la Chapelle, 75475 Paris, Cedex 10, France
To define a link between the deletion genotype (DD) and vascular reactivity, we studied in vivo and in vitro phenylephrine (PE)-induced tone and the effect of angiotensin II (AII) at physiological (subthreshold) concentrations on PE-induced tone.
The deletion allele (D) of the angiotensin I-converting enzyme (ACE) has been associated with a higher circulating and cellular ACE activity and possibly with some cardiovascular diseases.
During cardiac surgery PE-induced contraction was studied in patients with excessive hypotension. In parallel, excess material of internal mammary artery, isolated from patients operated for bypass surgery, was mounted in an organ chamber, in vitro, for isometric vascular wall force measurement.
In patients under extracorporeal circulation, PE (25 to 150 μg) induced higher contractions in patients with the DD genotype (e.g., with PE 75 μg: 20.3 ± 2.9 vs. 11.5 ± 2.5 mm Hg/ml per min, DD vs. II/ID, n = 15 vs. 30, p < 0.03). In the mammary artery, in vitro, contractions to PE (0.1 to 100 μmol/liter) or AII (1 or 100 nmol/liter) were not affected by the genotype. Angiotensin II (10 pmol/liter) significantly potentiated PE (1 μmol/liter)-induced contraction in both groups. Potentiation of PE-induced tone by AII was significantly higher in the DD than in the II/ID group.
The DD genotype was associated with an increased reactivity to PE in vivo and potentiating effect of exogenous AII in vitro. The higher response to PE in vivo might reflect a higher potentiation by endogenous AII. These data should be considered to understand possible link(s) between cardiovascular disorders and the ACE gene polymorphism.
Angiotensin I-converting enzyme (ACE) is a key enzyme in the production of angiotensin II (AII). The ACE gene contains a polymorphism based on the presence (insertion, I) or absence (deletion, D) within an intron of a 287-base-pair nonsense DNA domain (1)resulting in three genotypes, DD, II and ID. This polymorphism is associated with differences in plasma and cellular ACE levels and may be a risk factor for some cardiovascular diseases (2–5), although this issue is still a matter of debate (6–8). The increase in ACE activity is confirmed functionally by the observation that angiotensin I is more active in contracting in vitro isolated human mammary artery segments from patients with the deletion genotype (DD) than are segments from patients with the II or ID genotypes (9). Nevertheless, physiologic (subcontractile) concentrations of angiotensin II are not directly contractile but potentiate the contractile responses of blood vessels to other agonists in vivo and in vitro (10–18).
The phenomenon of potentiation by angiotensin II has also been reported in humans (17,18). Potentiation of vascular tone by angiotensin II involves the activation of protein kinase C and a sensitization of the contractile apparatus to calcium (12–14). We hypothesized that a change in ACE activity associated with the I/D polymorphism might affect the potentiation phenomenon rather than the direct contractile effect of the peptide. We explored a possible relation between homozygosity of the ACE D allele and the AII-induced potentiation of the alpha1- adrenergic contractile response in human vessels, under in vitro and in vivo conditions. We used isolated human mammary arterial segments isolated in vitro and tested the potentiating effect of exogenous AII on phenylephrine (PE)-induced contraction. We also performed PE-induced contractions in vivo in patients under extracorporeal circulation. In these conditions, endogenous AII may exert its potentiating effect as previously shown (15,17,18).
Materials and methods
With approval by the local ethics committee and informed patient consent, we studied 45 patients with hypotensive events during extracorporeal circulation. Patients underwent elective coronary artery surgery or valve surgery and presented a hypotensive event requiring PE injections.
Patients operated in emergency as well as septic patients and those receiving intravenous nitroglycerin or catecholamine medication during the precardiopulmonary bypass period were excluded from the study.
Diabetes, prior myocardial infarction and congestive heart failure were defined according to medical history and specific definitions of medical conditions. In addition, hypertension was defined as an increase in blood pressure requiring medication or a blood pressure higher than 140/90 mm Hg, documented on at least three occasions during the current hospital admission.
Patient medications are listed in Table 1. Calcium channel blockers were not given on the day of the operation. Nitrates and beta-adrenergic blocking agents were administered until the morning of surgery. Patients treated with angiotensin-converting enzyme inhibitors were not included in the study.
Anesthesia and cardiopulmonary bypass (CPB) management
Patients were premedicated with intramuscular morphine (0.1 mg/kg) and oral lorazepam (2 mg) 2 h before anesthesia. A radial artery catheter and a thermodilution pulmonary arterial catheter were installed to monitor haemodynamic parameters. Anesthesia was induced with fentanyl (25 to 30 μg/kg), midazolam (0.05 to 0.1 mg/kg) and pancuronium (0.1 mg/kg). Maintenance of anesthesia was achieved by additional injections of these agents every 30 min. The total amounts of fentanyl and midazolam received before the start of the experimental protocol described next are reported in Table 2.
Nonpulsatile CPB was standardized for all patients. The extracorporeal circuit consisted of a roller pump (Cobe Stöckert, Stöckert Instrument, Rungis, France), a cardiotomy reservoir and a Sorin membrane oxygenator (Sorin Laboratories, Mirandola, Italy). Ultrafiltration was not performed during the experimental protocol. Normothermia (temperature >36.5°C) assessed by the monitoring of bladder temperature was assured during surgery and CPB. In each patient, myocardial protection was performed by cold blood cardioplegia, repeated every 30 min; a warm reperfusion before aortic unclamping was assured by the same blood cardioplegic solution, at 37°C.
Phenylephrine dose-response curve (PE-DRC)
A PE dose-response curve (DRC) was performed when the mean arterial pressure (MAP) decreased below 65 mm Hg for a pump flow ranging from 2 to 2.4 liters/min per m2. Phenylephrine DRC was generated by injection of an individual bolus of PE in the following sequence: 25, 50, 75, 100 and 150 μg every 2 min. After each injection, the peak MAP was recorded. Injections of PE were not continued when MAP reached a value of at least 85 mm Hg. During the injection of PE, pump flow was constant and no injection of other drugs was performed.
For each patient the following parameters were recorded: bladder temperature, hematocrit, arterial pH, Pco2and Po2. Patients with extreme values of hematocrit (<20% and >35%), pH (<7.4 and >7.6), Pco2and temperature were excluded.
Isolated mammary artery preparation
In a separated series of experiments, excess material of internal mammary artery was collected from patients (n = 36) undergoing bypass surgery. Arterial segments were stored in ice-cold physiologic salt solution containing penicillin (100 IU/liter) and streptomycin (100 IU/liter) and sent to the laboratory for the in vitro study. Ring segments of internal mammary artery, cleaned free of fat and connective tissues, 3 mm in length, were mounted between two stainless steel wires in 3-ml organ baths containing a physiologic salt solution of the following composition (in mmol/liter): 135.0 NaCl; 15.0 NaHCO3; 4.6 KCl; 1.5 CaCl2; 1.2 MgSO4; 11.0 glucose; 5.0 N-2-hydroxy-ethylpiperazine-N-2-ethylsulfonic acid (HEPES). The pH was 7.4 when the solution was bubbled with 95% O2and 5% CO2. A solution containing K+125 mmol/liter was prepared using 14.4 mmol/liter NaCl and 125 mmol/liter KCl. One wire was attached to a fixed support, and the second wire was connected to a movable holder supporting a tension transducer (Grass FT.03) so that isometric force measurements could be collected by a Biopac data acquisition system (Biopac MP 100, LaJolla, California) and recorded on a Macintosh Quadra 610 computer (Apple Computers, Cupertino, California) using the Acqknowledge data acquisition and analysis software (Biopac, LaJolla, California). The artery segments were allowed to recover for 30 min during which time the physiologic salt solution was replaced at 15-min intervals.
Following this recovery period, a 2- to 4-g preload, resulting in optimal stretch (determined for each ring segment), was applied to the artery segments, which were allowed to equilibrate for an additional 90 min. Only mammary artery segments responding to PE (10 μmol/liter) by a contraction higher than 5 mN and to acetylcholine (1 μmol/liter) by a dilation higher than 50% were considered as valid and could be included in the study. In addition, a patient was included only if three segments of mammary artery were valid simultaneously.
Experimental protocols on isolated arteries
At least four arterial segments were isolated from each sample of mammary artery, and concentration-response curves to PE were performed, preceded by the determination of the maximum response to K+(125 mmol/liter) and by the determination of the dilator capacity of acetylcholine (detailed in the next section). At the end of the experiment, ring segments of mammary arteries were blotted dry and left overnight in a glass tube. Dry weight was then determined.
In each arterial segment three concentration-response curves to PE were obtained by cumulative addition of PE (0.1 to 10 μmol/liter) to the bath solution. The third concentration-response curve to PE was made after pretreatment of the tissues with AII (10 pmol/liter) for 10 min (12,13). Control segments did not receive AII but instead the solvent was added (physiologic salt solution). Thus, two groups were obtained: AII and control (or solvent; Fig. 1). Changes in response to PE after addition of AII (10 pmol/liter) were estimated by comparing the third to the second concentration-response curve to PE (Fig. 1). Hence, for each concentration of PE the difference due to the addition of AII or the solvent was calculated as the change in tension attributable to AII to the solvent (12,13). In two other segments of mammary artery the direct contractile effect of AII (1 or 100 nmol/liter) was tested.
The response to acetylcholine (10 μmol/liter) was assayed in each arterial segment after precontraction of the arterial segments with PE (1 μmol/liter). Data are expressed as % dilation of PE-induced preconstriction.
Determination of ACE genotypes
The ACE gene contains a polymorphism based on the presence (insertion:I) or absence (deletion:D) in intron 16 of a 287-base-pair (bp) nonsense DNA domain, resulting in three different genotypes: DD and II homozygotes and ID heterozygotes (1).
Genomic DNA was prepared from frozen peripheral blood with standard procedures (19)and the D and I alleles were identified, as previously described, on the basis of polymerase chain reaction (PCR) amplification of the respective fragments of the ACE gene (1). The PCR products were separated by agarose gel electrophoresis, and DNA was visualized by ethidium bromide staining. Subjects were then classified, according to the absence or the presence of the 287-bp insertion in intron 16 of the ACE gene, as DD, II or heterozygous for insertion/deletion ID. Reanalysis of the genotypes, performed with dimethylsulfoxide added to the reaction, confirmed the original determination.
In addition, possible mistyping of ID heterozygotes was controlled according to Shanmugam et al. (20). Each sample with a DD genotype was subjected to a second independent PCR amplification with a primer pair that recognizes an insertion-specific sequence. Other conditions were identical to the first PCR. No ID genotype was identified as DD in the present study.
Results are expressed as mean value ± SEM. Comparisons between groups were made using a one-factor analysis of variance (ANOVA) followed by a Dunnett’s test when significant or by a two-factor ANOVA for repeated measures to compare the concentration-response curves to PE. A probability level of p < 0.05 was considered significant. Pre- and intraoperative characteristics of the two groups of patients were compared using the chi-square test.
In the laboratory in which the in vitro study was conducted as well as in the operating room, where the in vivo study was performed, the patient’s genotype was not known. Analysis of the effect of the genotypes on the vascular reactivity in vivo or in vitro was performed at the end of the protocol.
Drugs were purchased from Sigma (L’Isle D’Abeau Chesnes, France).
Except for hypotensive events, surgery was uneventful. There was no complication due to the experimental protocol performed and there was no operative mortality. Patients’ preoperative characteristics are shown in Table 1. Both groups were similar as regard to age, gender, body area, left ventricular function and the performed surgery. In addition, there was no significant difference in pharmacologic therapy or in medical conditions between the two groups (Table 1). Table 2summarizes patients’ intraoperative characteristics. Both groups were similar with regard to dose of anesthetic agent. Arterial blood gas analysis (pH, Pco2and Po2), bladder temperature and hematocrit were similar in the two groups.
During the study 468 patients underwent extracorporeal circulation (37% with the DD genotype, 53% with the ID genotype and 10% with the II genotype) and 45 patients with a hypotensive event were included in the study. In these patients the repartition of the genotypes was not significantly different from the entire population (33% with the DD genotype, 60% with the ID genotype and 7% with the II genotype).
In vivo vascular reactivity
In patients under extracorporeal circulation, cumulative doses of PE induced a dose-dependent vasoconstrictor response, as reflected by a dose-dependent increase in blood pressure (Fig. 2). The contractile responses to PE were significantly higher in patients with the DD genotype (Fig. 2).
In vitro vascular reactivity
Thirty-six patients were included in the study of the vascular reactivity of the mammary artery in vitro (12 with the DD genotype, 22 with the ID genotype and 2 with the II genotype). Repartition of the three genotypes in this population was not significantly different from repartition of the genotypes in the population excluded according to the selection criteria given in the Methods section (18 with the DD genotype, 30 with the ID genotype and 5 with the II genotype).
In human mammary artery ring segments, the contractile response to K+(125 mmol/liter) was similar in the different experimental groups (Table 3). Acetylcholine (10 μmol/liter) induced a dilation of PE (1 μmol/liter)-preconstricted rings in both groups. Angiotensin II (1 and 100 pmol/liter) produced a significant contraction of isolated mammary arteries (Table 3). No significant difference was found between the two groups (Table 3).
Phenylephrine (0.1 to 100 μmol/liter) induced a concentration-dependent contraction of mammary artery ring segments. The genotype had no significant influence on this response to PE (Fig. 3).
Angiotensin II (10 pmol/liter) produced no contraction by itself, but induced a significant potentiation of PE (0.1 to 100 μmol/liter)-induced tone (Fig. 3).
Angiotensin II (10-pmol/liter)-induced potentiation of PE (0.1 to 100 μmol/liter)-induced tone, as appreciated by the difference between the DRC to PE before and after addition of AII, was significantly higher in mammary artery ring segments from patients with the DD genotype than in patients with the II or ID genotypes (Fig. 3).
Higher concentrations of AII (1 and 100 nmol/liter) induced a significant contraction in mammary arteries from patients with the DD or the II or ID genotypes (Table 3). The genotype had no significant effect on AII (1 and 100 nmol/liter)-induced direct contractions.
Phenylephrine had a higher contractile effect in patients with the DD genotype, in the process of an extracorporeal circulation. In addition, potentiation of PE-induced tone by subthreshold concentrations of AII in the mammary artery in vitro was higher in patients with the DD genotype. This study provides functional evidence that the deletion genotype (DD) for angiotensin-converting enzyme is associated with an increased vascular reactivity in vivo and in vitro, although some care should be taken when considering the results of the in vivo study, because it was performed in patients under deep anesthesia and under extracorporeal circulation.
Potentiating effect of angiotensin II
Previous studies have demonstrated that subcontractile concentrations of AII potentiate the contractile response to sympathetic nerve stimulation (10,17,21,22), norepinephrine (11–13,23), clonidine (14), thrombin (14), histamine (12), potassium-induced depolarization (10)and caffeine (12). Furthermore, we have recently reported that endogenous AII acts in vivo to potentiate PE-induced tone (15). In humans, the phenomenon of potentiation has also been described (17,18). Potentiation of agonist-induced tone by AII has a low calcium requirement and depends on protein kinase C activation, suggesting a sensitization of the contractile apparatus to calcium (12–14). We found in human mammary artery segments a potentiation by AII of the whole range of concentrations of PE tested. This potentiation affected preferentially the low concentrations of PE, which is in agreement with our previous observations (11,13). The concentrations of AII used in the present study are compatible with physiologic concentrations of the peptides (24). Thus, at physiologic concentrations, AII participates to the maintenance of a basal constrictor tone in blood vessels, most probably by its potentiating effect (15,17). This peptide is part of a larger puzzle, combined with other vasoactive substances such as neuropeptide Y (25), endothelin-1 (26)and serotonin (27), which also amplify the contractile responses of blood vessels to contractile agonists.
The DD genotype and reactivity to PE and AII
The higher amplifying effect of angiotensin II, as seen in patients with the DD genotype, might induce an increased responsiveness to other vasoconstrictor agents, which might lead to a higher frequency of deleterious vasospasm. Such an increase in AII potentiation would explain the fact that PE-induced contraction was increased in vivo and not in vitro. In vitro, endogenous AII has been washed out, whereas in vivo, endogenous angiotensin II is present when PE is injected. In vivo PE-induced tone is potentiated by endogenous AII. Thus, the in vivo conditions are equivalent to the conditions in which a subthreshold concentration of exogenous angiotensin II is added to the bath of a mammary artery in vitro before performing a concentration-response curve to PE. Only in conditions where AII (endogenous in vivo and exogenous in vitro) can exert its potentiating effect was an increased vascular reactivity observed in relation to the DD genotype. Indeed, the DD genotype has been linked to a higher risk of myocardial infarction (2,28)and a higher risk of cardiovascular events after infarction (28). Moreover, a correlation was found between stroke history and the DD genotype (29), and the DD genotype is prevalent in patients presenting severe pulmonary hypertension (30). Nevertheless, the association between the risk of myocardial infarction or ischemic heart disease was not found by other investigators (8), and the exact role of the DD genotype in cardiovascular disorders remains to be established (8).
Both plasma (31,32)and cardiac (33)angiotensin-converting enzyme (ACE) activity are increased in patients with the DD genotype, yet when assessed by the dermal response to bradykinin the ACE activity could not be corelated to the presence of the deletion (34). In internal mammary arteries isolated from patients with the DD genotype the pressor response to angiotensin I, but not to AII, is increased (35), which is compatible with the present study where we found no effect of the genotype on direct contractions to AII (1 and 100 nmol/liter). From the increased contractile effect of angiotensin I in mammary artery segments from patients with the DD genotype (35), we can assume that, at least in this vessel, the tissue-converting enzyme activity is increased. In addition, we found that the direct contractile responses to PE and potassium in vitro were not changed by the DD genotype. We can thus assume that the contractile apparatus responsiveness was not directly affected by the DD genotype. Hence, a possible mechanism that could link the DD genotype with a higher-converting enzyme activity to the higher-potentiating effect of AII could be that, locally, a higher conversion of AI into angiotensin II would sensitize the contractile apparatus to a further potentiating effect of AII. A possible candidate might be a non-calcium-sensitive protein kinase C involved in the vascular potentiating effect of AII at low concentrations (12,13).
DD genotype and nitric oxide (NO) release
Our results are at variance with a previous study in which phenylephrine-induced contraction was found to be lower in patients with the DD genotype as it was more inhibited by basal NO release, although NO-dependent dilation stimulated by an agonist was lower in these patients (9). The main differences between the two studies are the repartition of genotypes (less II genotype in the present study, 6% vs. 29%) and acetylcholine-induced dilation was much lower (35% to 45% (9)than in the present study (76% to 83%). Thus, there might be a difference in the population studied, but there was also a large difference in the vascular reactivity of the arteries used.
Number of patients included in the study
Finally, it should also be noted that in the present study the co-dominant effect of the ACE genotypes could not be analyzed owing to the small number of patients with the II genotype.
We conclude that the DD genotype for ACE was associated with an increased AII-induced potentiation of vascular tone in vitro and probably also in vivo. This observation provides a possible mechanism to be considered to understand the link between cardiovascular disorders and the DD genotype.
☆ This work was funded by INSERM, Paris, France.
- angiotensin I-converting enzyme
- angiotensin II
- cardiopulmonary bypass
- deletion genotype
- dose-response curve
- mean arterial pressure
- Received May 28, 1998.
- Revision received April 16, 1999.
- Accepted June 3, 1999.
- American College of Cardiology
- Rigat B,
- Hubert C,
- Alhenc-Gelas F,
- Cambien F,
- Corvol P,
- Soubrier F
- Buikema H,
- Pinto Y.M,
- Rooks G,
- Grandjean J.G,
- Schunkert H,
- van Gilst W.H
- Henrion D,
- Laher I,
- Laporte R,
- Bevan J.A
- Henrion D,
- Benessiano J,
- Lévy B.I
- Lyons D,
- Webster J,
- Benjamin N
- Weber M.A,
- Purdy R.E,
- Stupecky G.L,
- Prins B.A
- Duckles S.P
- Vanhoutte P.M,
- Verbeuren T.J,
- Webb R.C
- Simon D,
- Romestand B,
- Huang H,
- et al.
- Henrion D,
- Laher I
- Van Nueten J.M
- Dakik H.A,
- Mahmarian J.J,
- Verani M.S,
- Farmer J.A,
- Zhao G,
- Marian A.J
- Margaglione M,
- Celentano E,
- Grandone E,
- et al.
- Lachurié M.L,
- Azizi M,
- Guyene T.T,
- Alhenc-Gelas F,
- Ménard J
- Danser A.H,
- Schalekamp M.A,
- Bax W.A,
- et al.