Effects of Direct Renin Inhibition on Atherosclerotic Biomarkers in Patients With Stable Coronary Artery Disease and Type 2 Diabetes Mellitus

Abstract

Objectives:

To evaluate whether the direct renin inhibitor, aliskiren, has a more favorable effect compared to amlodipine on atherosclerotic biomarkers in patients with stable coronary artery disease and diabetes currently receiving standard secondary prevention therapy.

Methods:

A total of 38 patients were randomly assigned initially to either aliskiren (150 mg daily) or amlodipine (5 mg daily) for 2 weeks after which the dose of either medication was increased to its maximum daily dose for 4 additional weeks. Baseline and 6-week blood samples were analyzed for changes from baseline and between treatment groups for vascular and intracellular cell adhesion molecule, C-reactive protein, nitric oxide, plasminogen activator inhibitor 1, 8-isoprostane, and thiobarbituric acid reactive substances.

Results:

Thirty-one patients completed the study. More of the dropouts occurred in patients receiving aliskiren. Systolic blood pressure decreased in both treatment arms with no differences between the groups being noted. Plasminogen activator inhibitor 1, nitric oxide, and C-reactive protein concentrations increased in both groups from baseline but changes from baseline or between groups were not significant. Vascular and intracellular cell adhesion molecule, thiobarbituric acid reactive substances, and isoprostane concentrations decreased in each treatment arm from baseline, but these changes were not significant and no differences were noted between the groups.

Conclusions:

Treatment with either aliskiren or amlodipine did not significantly alter surrogate biomarkers of atherosclerosis in patients with both diabetes and established cardiovascular disease already receiving appropriate secondary cardiovascular prevention therapy. The study is limited in its size and duration to see an effect.

Keywords

aliskiren amlodipine atherosclerosis biomarkers nitric oxide C-reactive protein

Introduction

Agents that attenuate the renin–angiotensin system (RAS), that is, angiotensin-converting enzyme inhibitors (ACE-Is) and angiotensin receptor blockers (ARBs), have been shown to have therapeutic benefit in a variety of cardiovascular disorders often beyond their effects on blood pressure. Use of ACE-Is, or ARBs in those considered ACE-I intolerant, is considered standard of care for secondary prevention of cardiovascular disease, by leading guidelines.^1,2 In addition to their effects on blood pressure, aldosterone, and sodium and water absorption, blockade of the RAS with these agents may alter key biomarkers of vascular inflammation, vascular endothelial function, fibrinolytic balance, or oxidative stress.³ These surrogate biomarkers are thought to play a role in the progression of atherosclerosis. Biomarkers of vascular inflammation include vascular and intracellular cell adhesion molecules (VCAM and ICAM, respectively) and C-reactive protein (CRP). Markers of endothelial function include endothelin 1 (ET-1) and the vasodilator nitric oxide (NO). In addition, plasminogen activator inhibitor 1 (PAI-1) serves as a prothrombotic marker associated with plaque proliferation and atherosclerosis progression, and both 8-isoprostane and lipid peroxidation may be markers of oxidative stress. Type 2 diabetes is a significant risk factor for coronary artery disease and as a result the 2 exist concurrently in many patients. Type 2 diabetes also affects many of the atherosclerotic biomarkers described.^4
–6

The ACE-Is produce a compensatory rise in angiotensin 1 as a result of ACE blockade that allows other enzymes (eg, chymase) to convert angiotensin 1 to angiotensin 2.⁷ This so-called ACE escape may attenuate the influence of ACE-Is despite proven benefits in clinical trials. In addition, both ACE-Is and ARBs show a compensatory increase in RAS activity.⁸ Aliskiren is a direct renin inhibitor approved for the treatment of hypertension. It is a very specific and potent inhibitor of human renin. As such, it may offer an advantage over ACE-I and ARB therapy, as it blocks the rate-limiting step of the RAS. Aliskiren appears to have additive blood pressure-lowering effects when added to ACE-I or ARB therapy.⁹ Given aliskiren’s different pharmacodynamic effects on the RAS compared to ACE-I and ARB therapy, and that angiotensin 2 levels are not fully lowered by ACE-I therapy, it is important to better understand how direct renin inhibition may influence atherosclerotic biomarkers in patients with a variety of cardiovascular disorders. The objectives of this study were to determine whether aliskiren affects atherosclerotic biomarkers in patients with stable coronary artery disease and diabetes currently receiving standard secondary prevention therapy and whether aliskiren has a more favorable effect on these markers compared to a control antihypertensive agent.

Methods and Materials

Patients and Study Design

The Human Subjects Institutional Review Board at Texas Tech University Health Sciences Center approved the study, and all the patients provided written informed consent. This was a prospective, randomized, open-label trial. Patients were recruited between April 2008 and March 2011. Inclusion criteria for consent and randomization include men and women of age 18 to 85 years with a documented history of both type 2 diabetes mellitus and stable coronary artery disease (CAD). Stable CAD was defined as a history of coronary artery bypass graft, percutaneous intervention (coronary angioplasty or stent placement), or myocardial infarction at least 3 months or more in the past. Additional inclusion criteria included a hemoglobin A1c of ≤9.5% in the previous 3 months, concurrent ACE-I or ARB therapy (at a dose ≥50% of the maximum daily dose), concurrent antiplatelet and statin therapy, baseline blood pressure >100/75 mm Hg, and a body mass index 25 to 35 kg/m². Patients were excluded if they were currently receiving therapy with a calcium channel blocker, existing peripheral edema, hyperkalemia, serum creatinine >2.0 mg/dL, a documented history of cirrhosis or liver function tests 2 times the upper limit of normal, documented history of serious rheumatologic disorder, or concurrent use of corticosteroids.

Intervention

Eligible patients were randomly assigned and stratified based on the baseline blood pressure (>140/90 mm Hg or ≤140/90 mg Hg) to receive 1 of the 2 treatments: aliskerin 150 mg daily or amlodipine 5 mg daily for 2 weeks. If at that time the patient’s blood pressure was ≥100/75 mm Hg, the dosage of the agent was doubled and maintained at that dose for an additional 4 weeks. The dosage was not increased for the remainder of the 6-week study for those whose blood pressure was <100/75 mm Hg or who were experiencing signs or symptoms of hypotension. For those patients who had an increase in dosage at the 2-week interval and then experienced any hypotensive symptoms, their dose was reduced to the starting dose for the remainder of the study. Blood pressures were obtained by a single investigator to determine study eligibility and during the study period. Blood pressure was obtained manually using standard techniques using a sphygmomanometer with an appropriate cuff size for an individual patient.

Blood Collection Procedures

Blood samples for measuring the biologic markers were obtained before treatment and 6 weeks after treatment. All the samples were collected after an overnight fast in citrate, heparin, and EDTA collection tubes, between 0700 and 0900 hours to minimize confounding diurnal variations. Following collection, the samples were centrifuged at 760g for 15 minutes. The plasma was then transferred to cryogenic vials, labeled accordingly, and frozen immediately at −90°C before being analyzed.

Analytical Procedures

The PAI-1 antigen, ICAM-1, and VCAM-1 were determined through quantitative enzyme-linked immunosorbent assay (ELISA) techniques. The PAI-1 antigen was determined with a Hyphen Biomed kit (Neuville-sur-Oise, France). Both ICAM-1 and VCAM-1 were measured with assays from R&D Systems Inc (Minneapolis, Minnesota). In these assays, the respective samples and standards were added to the microwell plates coated with assay’s primary antibodies, which recognize specific antigen. Then, enzyme-conjugated secondary antibodies were applied, and the reactions were visualized by adding the enzyme-specific substrate, whose absorbance was detected using a Bio Rad 3550-UV Microplate Reader (Bio Rad Laboratories, Hercules, California), at wavelengths recommended by the assay protocol. The standard diluted solution gave the calibrated concentration of the standard curve, which was then used to interpolate the patient’s plasma concentration of the respective biologic marker. A commercially available NO assay (Cayman Chemical, Ann Arbor, Michigan) was used to determine total NO with the Griess reagent after the enzymatic conversion of nitrate to nitrite by the enzyme nitrate reductase. Total 8-isoprostane (free and esterified) was measured by ELISA (Cayman Chemical) in sample extracts obtained by ethyl acetate extraction according to a method published earlier.¹⁰ Lipid peroxidation was estimated with thiobarbituric acid reactive substances assay (TBARS) method as reported previously.¹¹ Serum CRP was measured by turbidometric method using Sigma CRP kit (Sigma Diagnostics, Inc, St Louis, Missouri).

Data Analysis

Data were evaluated with the Excel statistics add-on package Analyze-it v 2.07 1997-2007 (Analyze-it Software, Ltd, Leeds, England, United Kingdom). The continuous data were evaluated with the Shapiro-Wilk test for normality and all were parametric; therefore, all central tendencies are presented as means ± standard deviations. Paired and unpaired student t tests were utilized as appropriate. The α level of significance was set at .05.

Results

A total of 38 patients consented to be enrolled and subsequently randomized in this project. Of these, 31 completed the required intervention. The majority (6 of 7) of the patients who dropped out of the study were in the aliskiren treatment arm. In all, 4 were due to suspected adverse effects (elevated BP, dizziness, general malaise), 1 was due to a protocol violation, and 1 was lost to follow-up. One patient in the amlodipine treatment arm dropped out due to dizziness and elevated blood pressure. The baseline characteristics for each arm are provided in Table 1. Patients were in their middle 60s, primarily male, overweight, and had only mildly elevated hemoglobin A1c values. There were more African Americans in the amlodipine arm, and the baseline blood pressure was similar between the 2 groups.

Table 1.

Baseline Patient Characteristics.^a

	Aliskiren (n = 16)	Amlodipine (n = 15)
Age, years	68 (10)	63 (16)
Gender, n (%)
Male	14 (87.5)	10 (66.7)
Female	2 (12.5)	5 (33.3)
Ethnicity, n (%)
Caucasian	7 (44)	7 (47)
Hispanic	9 (56)	6 (40)
African American	0 (0)	2 (13)
BMI, kg/m²	29.9 (3.6)	29.7 (7.4)
A1c (%)	7.2 (1.1)	7.3 (1.0)
Duration of diabetes diagnosis, year	9.8 (7.5)	11.2 (11.6)
Tobacco smoker, n (%)	1 (6)	2 (13)
Cardiac condition, n (%)
Myocardial infarction	6 (37)	3 (20)
PCI	7 (44)	5 (33)
Bypass surgery	2 (13)	5 (33)
Documented stenosis	1 (6)	2 (13)
Systolic blood pressure, mm Hg	138 (21)	138 (19)
Diastolic blood pressure, mm Hg	75 (8)	77 (7)

Abbreviations: BMI, body mass index; A1c, hemoglobin A1c; PCI, percutaneous intervention; n, number.

^aApplicable continuous variables depicted as mean (standard deviation).

Both treatment groups had a statistically significant reduction in systolic blood pressure after 6 weeks of treatment (Table 2). Patients in the amlodipine group showed a significant reduction in diastolic blood pressure, while the decrease was not significant in the aliskiren group. No differences in blood pressure changes were noted between the groups upon the study completion. The concentration of PAI-1, NO, and CRP increased in both the groups from baseline but changes from baseline or between groups were not significant. The VCAM, ICAM, TBARS, and isoprostane concentrations decreased in each treatment arm from baseline but these changes were not significant, and no differences were noted between the groups.

Table 2.

Changes in Blood Pressures and Biomarkers.

	Change From Baseline Aliskiren Group Mean (95% CI)	Change From Baseline Amlodipine Group Mean (95% CI)	Difference Between Groups at the End of the Study (Aliskiren– Amlodipine) Mean (95%CI)
Systolic blood pressure, mm Hg	−14.9 (0.3-29.4) P = .04	−12.3 (1.9-22.7) P = .02	3.1 (−8.2-14.3) P = .58
Diastolic blood pressure, mm Hg	−3.1 (−1.5-7.8) P = .17	−6.5 (1.1-11.8) P = .02	−1.5 (−8.5-5.4) P = .65
PAI-1, ng/mL	0.65 (−7.33-8.62) P = .86	3.82 (−5.16-12.81) P = .37	−3.17 (−14.63-8.28) P = .57
NO, µmol/L)	2.66 (−7.45-12.76) P = .58	7.49 (−1.43-16.41) P = .09	−4.83 (−17.65-7.99) P = .44
CRP, mg/L	0.0397 (−0.2310-0.3104) P = 76	0.1032 (−0.1974-0.4038) P = 47	0.0635 (−0.3225-0.4495) P = .74
VCAM, ng/mL	−2.4 (−41.1-36.3) P = .90	−45.9 (−112.8-21.0) P = .16	43.53 (−29.19-116.25) P = .23
ICAM, ng/mL	−4.9 (−17.2-7.4) P = .41	−10.3 (−31.2-10.6) P = .31	5.44 (−17.40-28.28) P = .63
TBARS, (%)	−3.6 (−24.2-17.1) P = .72	−3.3 (−50.6-44.0) P = .88	0.27 (−47.93-48.46) P = .99
Isoprostane, pg/mL	−10.3 (−169.5-148.9) P = .89	−36.5 (−178.5-105.5) P = .59	26.18 (−176.81-229.16) P = .79

Abbreviations: CI, confidence interval; CRP, C-reactive protein; ICAM, intracellular cell adhesion molecule; NO, nitric oxide; PAI-1, plasminogen activator inhibitor 1; TBARS, thiobarbituric acid reactive substances assay; VCAM, vascular cell adhesion molecule.

Discussion

Given the residual risk for cardiovascular events despite treatment with ACE-Is or ARBs, other research has focused on RAS blockade through the combination of ACE-I and ARBs or with direct renin inhibition. Although ACE-I/ARB combinations have resulted in the reduction of albuminuria and hypertension, clinical trials suggest a lack of benefit on cardiovascular outcomes, and the combination may increase the risk for nephropathy and mortality.^12,13 Optimal RAS suppression is difficult to achieve with ACE-Is or ARBs alone or in combination, because both activate compensatory feedback mechanisms that result in increased plasma renin activity.¹⁴ The renin inhibitor, aliskiren, has been introduced as a novel antihypertensive drug that blocks the upstream and rate-limiting step in the RAS.¹⁵ The agent’s ability to improve blood pressure has been well documented.¹⁶ Aliskiren has also been shown to decrease the development, progression, and vulnerability of atherosclerosis plaques in rodent models.^17,18 Direct renin inhibition may have a favorable action on endothelial function through the downregulation of adhesion molecules, decrease in chemotactic molecules and inflammation, improved vasodilation, and enhanced anticoagulant properties.^19
–21 These effects may be equivalent to the reduction achieved with ARBs or ACE-Is and may not be dependent on blood pressure control.

The combination of aliskiren with an ARB or ACEI provides a synergistic reduction in blood pressure.^22,23 The potential benefit can be explained in part by inhibition of the compensatory rise of renin activity.⁹ There is little data on the effect of a direct renin inhibitor in combination with an ACE-I or ARB on endothelial dysfunction in humans. Aliskiren, in combination with valsartan, has been shown to minimize the compensatory rise in plasma renin, angiotensin, and angiotensin 2 levels induced by ARBs.¹⁴ In patients with a history of systolic dysfunction or ventricular hypertrophy, the combination of aliskiren to either an ACE-I or an ARB has shown to reduce ventricular mass index and neurohumoral effects but does not appear to affect end-systolic/diastolic volume or ejection fraction.^24
–26 Aliskiren, when added to an ARB in patients with diabetes and existing proteinuria, reduces urinary albumin excretion better than an ARB alone.^27
–29

The present study found no additional improvement in surrogate biomarkers of atherosclerosis, with the addition of the direct renin inhibitor aliskiren to an ACE-I or ARB and what is considered standard secondary prevention therapy in patients with both diabetes and cardiovascular disease. The results are consistent with a recently published study that also showed no improvement in the effects of aliskiren combined with an ACE-I or ARB on fibrinolytic system biomarkers including PAI-1, fibrinogen, and d-dimer.²³ Our study hypothesis was the combination of aliskiren with an ACE-I or ARB would have a favorable effect on the selected biomarkers (PAI-1, NO, ET-1, CRP, VCAM, ICAM, TBARS, and isoprostane) of atherosclerosis. These biomarkers were chosen as surrogates of endothelial dysfunction, anticoagulation properties, oxidative stress, and inflammation because of their potential roles in the pathogenesis of atherosclerosis. These biomarkers have been extensively evaluated and validated in the previous trials as markers of endothelial dysfunction or atherosclerosis and may correlate with the clinical outcome.^30

–34 The calcium channel blocker amylodipine was used as a control to rule out potential changes related to a decrease in shear stress and blood pressure. Amlodipine has not been associated with significant changes in the fibrinolytic system or makers of inflammation and atherosclerosis.^35
–37

There are several limitations of our study and possible explanations for the lack of benefit observed. Combination therapy may involve the reduction in other vasoprotective agents in the RAS with the higher blockade from combination therapy.^38,39 However, the role of these agents has not been fully explored, and the effect of renin inhibitors on these agents has not been studied. The combination therapy can also result in a significant increase in plasma prorenin and renin concentration that may be associated with adverse cardiovascular effects through different pathways including prorenin/renin receptors.⁴⁰ Aliskiren does not inhibit prorenin/renin interaction with receptors nor affect the expression of receptors.^41
–43 The lack of an effect on the biomarkers assessed when aliskiren was added to an ACE-I or ARB could be due to adequate baseline RAS blockade with the patients' existing therapies. Each of the biomarkers studied showed significant variation in both baseline and follow-up analyses, making comparisons between groups or changes from baseline difficult. Some of this variation could be due to the differences in the evaluated baseline characteristics such as tobacco use, gender, or ethnicity. Other factors not evaluated such as statin dose, renal function, variations in glycemic control, or use of medications potentially affecting the biomarkers during the study cannot be ruled out. Future studies that include larger number of patients and longer duration follow-up may provide a more firm answer and permit for subgroup analysis. Our patients were already on optimal medical therapy, including an ACE-I or ARB, with good blood pressure control and no recent ischemic events and the expected benefit may require a larger study population. However, one of the previously mentioned studies investigating the potential benefit of adding aliskiren to an ACE-I or ARB in patients with diabetes and renal impairment was terminated prematurely due an increased risk for hypotension and hyperkalemia and a higher incidence of stroke or death, worsening renal function, while showing no improvements in cardiovascular outcomes.²⁹ Due to these more recent concerns with the combination of aliskiren therapy with ACE-Is or ARBs, further study in a population similar to that in our study may not be appropriate.

Conclusion

This study showed that the combination therapy with aliskerin with either an ACE-I or an ARB results in a significant and synergistic reduction in blood pressure. However, this reduction in pressure was not significantly better than a comparator antihypertensive, and no significant difference was seen on the various biomarkers of atherosclerosis. This project suggests there is little potential benefit in improving markers of atherosclerosis, with the combination of aliskerin and ACE-I or ARB therapy. The combination of aliskiren and ACE-Is or ARBs is still an area of research to explore, though recent clinical studies suggest caution is warranted particularly in those with diabetes and moderate renal impairment.

Footnotes

Authors’ Note

All work was conducted at the Texas Tech University Health Sciences Center in Lubbock, TX, USA.

Acknowledgment

The authors wish to acknowledge Elsayed Abo-Salem, MD, for his contributions in laboratory assessment of patient samples during this project.

Declaration of Conflicting Interests

The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding

The author(s) disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: This project was funded by an internal grant from the Texas Tech University Health Sciences Center School of Medicine. This research received no specific grant from any funding.

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