Irbesartan, an angiotensin type 1 receptor inhibitor, regulates the vascular oxidative state in patients with coronary artery disease

Subjects

Forty-seven subjects (26 men and 21 women), all with documented coronary atherosclerosis, were enrolled into the study. All patients had undergone coronary artery bypass graft and/or percutaneous transluminal coronary angioplasty at least one year before the start of the study. No patient had any subjective evidence of acute coronary syndrome in the year preceding the study. Evidence of myocardial infarction (MI) was determined by electrocardiographic findings, previous history of elevated creatine kinase-MB isoenzymes, and/or regional wall motion abnormality noted on either ventriculography or echocardiography. Left ventricular ejection fraction was determined by echocardiography. All patients were considered stable on their medical regimen and free of any acute coronary syndrome (i.e., MI or unstable angina) for at least 12 months. All subjects were on aspirin, and an equal number of subjects in each group were on nitrates, beta-blockers and HMG-CoA reductase inhibitors. No subject had taken either ACE inhibitors or AT₁receptor inhibitors for at least six months before the beginning of the study.

A survey was performed on each potential subject, and the following were excluded: current smokers, patients with an ejection fraction of <40% by echocardiography or ventriculography, diabetes with a glycosylated hemoglobin >7.5%, systolic blood pressure (BP) >150 mm Hg, renal impairment, hepatic impairment or malignancy. The protocol was approved by the research committee, and all subjects gave written informed consent.

Study design

Under a randomized, placebo-controlled protocol, subjects received either irbesartan 150 mg daily (provided by Bristol-Myers Squibb, Princeton, New Jersey) or placebo for a 12-week period. Serum samples were collected at 0, 4 and 12 weeks. Clinical characteristics and medications are denoted in Table 1. All patients tolerated irbesartan or placebo and completed the 12-week study without difficulty.

Measurement of LDL oxidation in patients with atherosclerosis

The plasma was isolated from patients at 0-, 4- and 12-week intervals, and LDL was isolated via ultracentrifugation at 39,000 rpm at 4°C. The LDL was then oxidized to ox-LDL by an in vitro assay utilizing CuSO₄, as previously described (18). The lag time indicating the susceptibility of the LDL to oxidize was measured using a spectrophotometer at 280 nm (18). Values were performed at least in triplicate.

Monocyte binding to the human CD11b receptor

The serum of the patients in either the irbesartan or placebo group was collected at 0, 4 and 12 weeks. Whole-blood immunotyping was performed in blood samples using fluorescence-activated cell sorting analysis with a monoclonal antibody to the human CD11b receptor, a protein with human binding sites and a very high binding affinity to human monocytes. The monoclonal antibody to the CD11b receptor was obtained through Pharmingen. The changes in monocyte CD11b expression were determined with flow cytometry analysis, as previously described (19).

Superoxide anion generation by neutrophils

A modification of the lucigenin-based chemiluminescence assay of NADPH oxidase activity to measure basal and postactivation levels of superoxide generation by neutrophils was used (20,21). Neutrophils were separated from venous blood using a Ficoll-Hypaque density gradient, and activation of the neutrophil respiratory burst was achieved using 1 micromole/l of N-formyl-methionyl-leucyl-phenylalanine (Sigma Chemical Corp., St. Louis, Missouri). The peak level of superoxide generated after activation was recorded in mV/5 × 10⁵neutrophils (16,20).

Measurement of lipid peroxidation in plasma

The polyunsaturated fatty acid linoleic acid (18:2) is converted into 13-hydroperoxy linoleic acid (13-HPODE) by treatment with the enzyme lipoxygenase. Previous investigations suggest that 13-HPODE may be involved in the initiating and propagating steps toward atherosclerosis (22,23). The thiobarbituric acid reactive substances (TBARS) assay was performed as an indication of lipid peroxidation in the plasma by using a colorimetric assay as previously described (24). One molecule of either malonedialdehyde or 4-hydroxyalkenal reacts with thiobarbituric acid to yield a stable chromophore (20). Spectrophotometric analysis was performed at 568 nm.

Statistical analysis

All data are presented as the mean value ± SEM. Comparisons were determined within the placebo and irbesartan groups (0, 4 and 12 weeks) and between the placebo and irbesartan groups with two-way analysis of variance (ANOVA). We determined a p value of <0.05 to be statistically significant. (Bonferroni correction applied; p values are related to ANOVA).

Irbesartan affects the susceptibility of LDL to oxidation in patients with coronary artery disease (CAD)

Using time-course analysis, irbesartan increased the lag time of LDL oxidation in patients, with a slight increase of 11% from placebo (Fig. 1)at four weeks of treatment (71.1 ± 8.3 s vs. 63.1 ± 5.6 s for placebo, NS). Furthermore, there was an increase of 32% in LDL lag time in the irbesartan group from placebo at 12 weeks of treatment (93.2 ± 8.4 vs. 62.7 ± 7.6 s, p < 0.05). There was no change in serum cholesterol or LDL cholesterol in any of the patients who were treated with irbesartan.

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Figure 1

Irbesartan decreases susceptibility to low-density lipoprotein (LDL) oxidation in patients with coronary artery disease (CAD). Patients with CAD were placed on placebo or irbesartan (150 mg/day) for a 12-week period. Serum samples were collected at the start of the study (0 weeks) and at 4 and 12 weeks of treatment. The LDL was isolated by centrifugation, and rate of oxidation was measured by spectrophotometric analysis as described in the Methods section. The p value, as determined by analysis of variance, was 0.027 for time, <0.0001 for group and 0.014 for the group-time interaction. Black diamond= placebo; black square= irbesartan; *p < 0.05 = difference between 0 weeks and 4 or 12 weeks within each group; #p < 0.05 = difference between placebo and irbesartan groups.

Treatment with irbesartan regulates monocyte binding in the vasculature of patients with stable CAD

Using flow cytometry analysis, treatment with irbesartan suppressed monocyte binding to CD11b at four weeks and 12 weeks of treatment as compared to placebo. Figure 2indicates that there was a significant decrease in monocyte binding capacity in the group of patients treated with irbesartan. This decreased capacity remained constant through the course of the study. On the other hand, monocyte binding capacity continued to increase in the placebo group throughout the study (from 61% binding at four weeks to 68.9% binding at 12 weeks).

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Figure 2

Irbesartan decreases monocyte binding the CD11b monoclonal antibody in patients with coronary artery disease (CAD). Patients with CAD were placed on placebo or irbesartan (150 mg/day) for a 12-week period. Serum samples were collected and red blood cells were lysed. Using flow cytometry analysis, whole-blood monocyte CD11b expression was performed. The values expressed are a percentage of expression of CD11b by circulating monocytes. The p value, as determined by analysis of variance, was <0.0001 for time, for group and for the group-time interaction. Black square= placebo; white square= irbesartan; *p < 0.05 = difference between 0 weeks and 4 or 12 weeks within each group; #p < 0.05 = difference between placebo and irbesartan groups.

Treatment with irbesartan modifies neutrophil superoxide-generating capacity in patients with stable CAD

Figure 3indicates that neutrophil superoxide generating capacity was markedly reduced in patients treated with irbesartan at both four and 12 weeks. The figure shows that treatment with irbesartan reduced neutrophil superoxide-generating capacity by 42% at 12 weeks. In contrast, the placebo group showed no significant change in neutrophil superoxide-generating capacity during the 12-week study period.

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Figure 3

Irbesartan decreases production of superoxide in the neutrophils of patients with coronary artery disease. Serum samples were collected from the patients who were placed on placebo or irbesartan (150 mg/day) for a 12-week period. Samples were collected at the start of the study (0 weeks) and at 4 and 12 weeks of treatment. The neutrophils were isolated and the peak levels of superoxide production after neutrophil respiratory burst were performed. The p value was <0.0001, as determined by analysis of variance, for time, for group and for the group-time interaction. Black diamond= placebo; black square= irbesartan; *p < 0.05 = difference between 0 and 4 or 12 weeks within each group; #p < 0.05 = difference between placebo and irbesartan groups.

Irbesartan regulates lipid peroxidation in patients with CAD

To determine whether lipid-derived free-radical production is regulated by inhibition of the AT₁receptor, the TBARS assay was utilized as a measurement of lipid peroxidation. Patients with stable CAD when treated with irbesartan showed a reduction in TBARS activity that was 33% and 36% lower than placebo (Fig. 4)at four weeks and 12 weeks (both p ≤ 0.05), respectively.

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Figure 4

Irbesartan decreases lipid peroxidation in the serum of patients with coronary artery disease. Serum samples were collected during the 12-week period of treatment at the start of the study (0 weeks) and at 4 and 12 weeks of treatment. The thiobarbituric acid reactive substances (TBARS) assay was performed, as described in the Methods section, using spectrophotometric analysis at 568 nm. The p value, as determined by analysis of variance, was 0.027 for time, 0.012 for the group and 0.004 for the group-time interaction. Black square= placebo; white square= irbesartan; *p < 0.05 = difference between 0 weeks and 4 or 12 weeks within each group; #p < 0.05 = difference between placebo and irbesartan groups.

Potential antioxidant mechanisms seen with the blockade of the angiotensin II type 1 receptor

The binding of angiotensin II to the AT₁receptor is responsible for most of the peripheral and central effects of angiotensin II on BP, osmoregulation and cell growth, and thus for the effect of the renin-angiotensin system in cardiovascular pathology (25,26). Drugs that block the renin-angiotensin system have been shown to provide benefits beyond BP control because of their inhibition of angiotensin II (27). Recent interest has focused on the role of angiotensin II acting at the AT₁receptor in the vasculature, specifically in the processes of atherosclerosis (26).

In the current study, we determined that irbesartan reduces the susceptibility of LDL to oxidize in patients with CAD. These patients had LDL cholesterol values that were treated with lipid-lowering agents, and treatment with irbesartan did not alter their LDL cholesterol values during the entire course of the study. It has been reported previously that saralasin, an AT₁receptor blocker, inhibits angiotensin II macrophage-mediated oxidation of LDL (17). Additionally, our findings indicate that treatment with irbesartan reduces the adhesion of monocytes in whole blood. Therefore, it may be that cells such as monocytes are inactivated in their ability to induce mechanisms of inflammation that are significant in atherogenesis.

The study also showed that treatment of CAD patients with irbesartan reduced the capacity of neutrophils to make superoxide anion. The NAD(P)H oxidase system in endothelial cells, smooth muscle cells and phagocytes is an important source of reactive oxygen species in cardiovascular disease (20,28), and this activity may be regulated by angiotensin II operating primarily through the AT₁receptor. It has been shown that angiotensin II mediates vascular superoxide production via the NAD(P)H oxidase system (29,30)and that cardiovascular diseases exhibit increased levels of superoxide in the vasculature (16,20,31). Our findings suggest that inhibition of the AT₁receptor regulates the production of reactive oxygen species that may in turn modulate the oxidative state in the vasculature. These findings suggest that the reduction of reactive oxygen species such as superoxide anion by irbesartan may be significant in decreasing irbesartan’s ability to modify LDL to an oxidized form. This decrease in turn may reduce the uptake of LDL by inflammatory cells via the scavenger receptor or other receptors that would subsequently form an atherogenic foam cell seen in the pathogenesis of atherosclerosis. Furthermore, these inhibitory mechanisms would then reduce the ability of inflammatory cells such as monocytes to adhere to the vessel wall and thereby reduce the formation of an atherosclerotic plaque. These potential antioxidant mechanisms may be specific to irbesartan and its chemical structure and may not necessarily be an effect of this class of antihypertensive compounds.

Our findings with the TBARS assay indicate that irbesartan reduced the oxidative stress responsible for lipid peroxidation. It has been observed that oxidative stress may contribute to endothelial dysfunction in cardiovascular diseases such as atherosclerosis by reducing NO synthase activity and by reacting with NO to form peroxynitrite (20,32). Recent studies in the aorta suggest that angiotensin II interacts with the bradykinin/NO system by an AT₂receptor-dependent mechanism (33,34). Recent studies indicate that the AT₂receptor mediates antiproliferative and potentially antioxidative effects (35–37), and it is conceivable that many if not all of the antioxidant effects by irbesartan may be modulated by upregulation of AT₂receptor activity. Further investigations must be undertaken to assess the validity of these potential mechanisms of action.

Study limitations

Our findings are primarily confined to patients with known CAD, and it is not clear whether treatment with irbesartan would be beneficial in the primary prevention of CAD. Unlike AT₁receptor inhibitors such as losartan or valsartan, the pharmacological properties of irbesartan are that the compound blocks 100% of the AT₁receptor and has insurmountable properties (38). This difference indicates that the properties seen with irbesartan should not be considered a class effect of the AT₁receptor antagonists and that further investigations with the other AT₁receptor inhibitors need to be performed. Also, the TBARS assay is not an accurate indicator of the oxidative state in the vasculature, but it is an indirect measurement of lipid peroxidation, and has been utilized with reliable results in previous investigations (20,24). Finally, our results have been observed in only 12 weeks of therapy. A large group study of long-term therapy with an AT₁receptor inhibitor, similar to previous reported results with ACE inhibitors (39), is required to determine whether these agents would be beneficial in secondary prevention of CAD.

Conclusions

Because inflammatory and pro-oxidant mechanisms are significant in the pathogenesis of atherosclerosis, the findings in the present study suggest that AT₁receptor inhibitors such as irbesartan may have benefits in treatment of atherosclerosis. We have previously shown that irbesartan reduces markers of inflammation in the serum of patients with early CAD (16). The present findings extend these observations to indicate that antioxidant-sensitive mechanisms may be responsible for reducing these inflammatory markers independent of BP reduction.