Effect of Perindopril on Large Artery Stiffness and Aortic Root Diameter in Patients With Marfan Syndrome: Title and subTitle BreakA Randomized Controlled Trial FREE

Marfan syndrome (MFS) is an autosomal dominant connective tissue disorder caused by mutations in FBN1, the gene encoding fibrillin 1. Fibrillin 1 is a key component of extracellular matrix elastic fibers and also a negative regulator of the cytokine transforming growth factor β (TGF-β), which contributes to matrix metalloproteinase (MMP) activation and extracellular matrix degeneration.¹ The main clinical manifestations of MFS involve the skeletal, ocular, and cardiovascular systems. Progressive aortic stiffening, dilatation, and rupture are the most serious complications and the most common cause of premature death. Based on a single trial² of propranolol in 32 patients with MFS and 38 controls, β-blockers are currently the standard treatment for MFS. The rationale for the use of β-blockers includes reduction in the rate of pressure change in the aortic root (ie, left ventricular ejection) and heart rate, and therefore aortic wall stress. However, β-blockers may not directly address the underlying pathobiology of aortic wall degeneration^{3 - 5} as effectively as other agents.^{6 - 9}

A number of recent studies have implicated the renin-angiotensin system in development of aortic stiffening, dilatation, and rupture in MFS through multiple mechanisms involving both the angiotensin II type 1 (AT₁) and angiotensin II type 2 (AT₂) receptors. In fibrillin 1–deficient mice, enhanced activation of and signaling by TGF-β can be blocked by the AT₁ antagonist losartan preventing aortic aneurysm.¹⁰ On the other hand, both angiotensin II and AT₂ receptor expression are increased in MFS aortas and associated with cystic medial degeneration, which contributes to aortic rupture.¹¹ Angiotensin-converting enzyme (ACE) inhibition and AT₂ receptor antagonism also significantly inhibit vascular smooth muscle cell apoptosis in cultured aortic cells from patients with MFS.¹¹ ACE inhibitors are known to reduce large artery stiffness⁶ and a small nonrandomized clinical study has also reported that ACE inhibitors increase aortic distensibility compared with β-blocker therapy in children and adolescents with MFS.¹²

Given the efficacy of ACE inhibitors to inhibit the actions of angiotensin II through both the AT₁ and AT₂ receptors, we hypothesized that such therapy would reduce arterial stiffness and aortic dilatation relative to placebo in adult patients with MFS taking standard β-blocker therapy. We also hypothesized that these aortic changes would be associated with reduction in TGF-β and MMP plasma levels, particularly MMP-2 and MMP-3, which are up-regulated by TGF-β in MFS.^{13 - 14}

Seventeen patients (mean [SD], 33 [6] years; range, 27-40 years) were recruited from the Alfred Hospital Marfan Syndrome Clinic, Melbourne, Australia, and completed the trial, which was initiated in January 2004 and completed in September 2006 (Figure 1). All patients gave written informed consent to participate in the study, which was approved by the Alfred Hospital Ethics Committee. Inclusion criteria included (1) positive strict diagnosis of MFS using Ghent criteria,¹⁵ (2) age 18 to 40 years, (3) serum creatinine level of less than 1.2 mg/dL (to convert to micromoles per liter, multiply by 88.4), (4) systolic blood pressure of 140/90 mm Hg or less (when measured on β-blocker therapy), and (5) no history of previous aortic surgery. Homocysteinuria was excluded as an alternative diagnosis. All patients were receiving long-term treatment with a β-blocker.

Computer-generated numbers specified the drug allocation sequence. A tamper-proof randomization process generated by the hospital research center randomly assigned participants to receive either perindopril (2 mg/d for 1 week, 4 mg/d for 2 weeks, and thereafter 8 mg/d for 21 weeks; Coversyl, Servier Laboratories Ltd Pty, Hawthorn Victoria, Australia) or placebo (2 mg/d equivalent for 1 week, 4 mg/d equivalent for 2 weeks, and thereafter 8 mg/d equivalent for 21 weeks) in a parallel-group, double-blind design. All patients reached the 8 mg/d dosage. Both investigators and patients were blinded to drug assignment. Furthermore, investigators did not have access to baseline data when they performed follow-up measurements and patients were not asked which treatment they thought they were receiving. No patient assigned to placebo crossed over to perindopril during the trial or vice versa. The study had 80% power to detect a 10% change in arterial stiffness parameters and a 5% change in aortic diameters with an α=.05.

On the morning of testing and 10 hours following the last medication dose, supine resting brachial arterial blood pressure and heart rate were measured 3 times at 3-minute intervals using an automated oscillometric blood pressure monitor (Dinamap Vital Signs Monitor 18465X, Criticon, Florida) following 15 minutes of quiet rest in a temperature-controlled room.

Arterial stiffness was assessed globally via systemic arterial compliance (carotid tonometry and Doppler velocimetry) and regionally via pulse wave velocity. Systemic arterial compliance was determined noninvasively using calculations based on the area method of Liu et al¹⁶ and a 2-element Windkessel model of the arterial system as described previously.^{16 - 17} This method has been validated to primarily assess the properties of the large vessels, including the aorta and carotid arteries.¹⁸ Pulse wave velocity is related directly to aortic stiffness and was measured centrally between the right carotid and right femoral arteries and peripherally between the right femoral and dorsalis pedis arteries, by simultaneous applanation tonometry (SPT-301; Miller Instruments, Houston, Texas).¹⁹ The experienced analyst performing the measurements (A.A.A.) was blinded to treatment allocation.

Two-dimensional, M-mode, and Doppler echocardiograms were obtained with a commercially available cardiac ultrasound system, using a 2.5-MHz transducer. Aortic root measurements were made in 2-dimensional parasternal long-axis view at end-diastole (peak of R wave on electrocardiogram) and at end-systole (T wave on electrocardiogram) at the level of the left ventricular outflow tract, sinuses of Valsalva, supra-aortic ridge, and proximal ascending aorta 1 to 2 cm above the supra-aortic ridge according to the method of Roman et al.²⁰ All measurements were made using the leading edge technique on up to 5 cycles and averaged. The severity of aortic and mitral regurgitation was graded semiquantitatively using color Doppler jet area criteria.²¹ Echocardiographic evidence of mitral valve prolapse was evaluated using established echocardiographic criteria.²² A single sonographer blinded to the clinical data and treatment assignment (K.M.D.) performed the analysis, and all echocardiograms were also assessed by 2 experienced cardiologists blinded to patient identity and treatment (A.A. and A.J.W.). Excellent comparability (and no systematic differences) in measures of aortic root diameters both in end-diastole and end-systole between readers was observed (r = 0.88-0.97). Results are expressed relative to body surface area calculated according to Roman et al.²⁰

Venous blood samples were drawn from each patient at baseline and following 24 weeks of therapy. Samples were subsequently analyzed for active and latent TGF-β using enzyme-linked immunosorbent assay (ELISA, TGF-β Emax ImmunoAssay System, Promega Corporation, Madison, Wisconsin). A fluorokine MAP-Human MMP Base Kit (R&D Systems, Minneapolis, Minnesota) was used to measure MMPs on a BioPlex platform (BioRad Laboratories Inc, Hercules, California); this assay consists of multiplexed sandwich ELISA for the quantitative measurement of MMP-2 and MMP-3. Each sample was assayed twice and averaged. The coefficient of variation between all duplicate assays was less than 5% and therefore results were averaged to obtain a single value for each sample. Unique standard curves were constructed for each bead and sample analyte concentration determined. This approach delivers the sensitivity of ELISA.

Demographic and hemodynamic indices at baseline were compared in the perindopril and placebo groups by χ² tests (categorical variables) and unpaired t tests (continuous variables). Replicates were meaned to obtain a single value for each patient at each time point (baseline and postintervention). All data are presented as mean (SEM) except age, which is mean (SD). The change in all arterial stiffness indices and echocardiographic parameters from baseline to 24 weeks was compared between the perindopril and placebo groups using an analysis of covariance model. Although groups were matched for all baseline parameters, these were included in all covariate analyses. In addition, because the change in mean arterial pressure was greater in the perindopril group compared with the placebo group, this variable was also included in covariate analyses. P values did not change when change in mean arterial pressure was included as a covariate except as otherwise noted. All statistical analyses were performed by using SPSS version 15.0 (SPSS Inc, Chicago, Illinois). P<.05 was considered to be significant. The statistician performing the analyses was blinded to treatment group.

The perindopril and placebo groups were similar in age, body mass index, body surface area, and other cardiovascular risk factors (Table 1). The presence of mild aortic regurgitation, mitral valve prolapse, and mitral valve regurgitation was comparable between the 2 groups. The type and dose of β-blocker did not vary between groups. In the perindopril group, 5 patients were taking 200 mg/d of atenolol, 3 patients were taking 100 mg/d of atenolol, 1 patient was taking 240 mg/d, and 1 patient was taking 200 mg/d of propranolol. In the placebo group, 3 patients were taking 200 mg/d of atenolol, 2 patients were taking 100 mg/d of atenolol, 1 patient was taking 240 mg/d, and 1 patient was taking 160 mg/d of propranolol. None of the above doses were changed during the trial. One patient in the perindopril group and 2 patients in the placebo group were taking lipid-lowering therapy. None of the patients were taking any other cardiovascular-active drugs. No adverse events were reported throughout the trial.

Perindopril therapy increased mean systemic arterial compliance by 0.2 (SEM, 0.1) mL/mm Hg and was significantly different from placebo (–0.01 [0.03] mL/mm Hg, P = .004) (Table 2 and Figure 2). Consistent with these findings, perindopril also reduced central pulse wave velocity (–1.6 [0.2] m/s for perindopril vs 0.4 [0.1] m/s for placebo, P < .001) and peripheral pulse wave velocity (–2.2 [0.2] m/s for perindopril vs 0.2 [0.1] m/s for placebo, P < .001) (Table 2 and Figure 2).

In young healthy individuals, central systolic and pulse pressure are normally lower than the corresponding brachial pressure; however, due to the increase in arterial stiffness in MFS,²³ this differential was not evident. Relative to placebo, perindopril significantly reduced both carotid and brachial systolic blood pressure as well as brachial diastolic blood pressure; however, these changes were small (reduction of 1-4 mm Hg from baseline, P < .001 vs placebo) (Table 2). Although brachial pulse pressure was unaffected by the perindopril intervention, carotid pulse pressure was reduced compared with placebo (P = .03). The reduction in mean arterial pressure was small but significant (mean [SEM], –1 [1] mm Hg for perindopril vs 1 [1] mm Hg for placebo; P = .004). Importantly, the observed changes in arterial stiffness parameters remained significant (P = .001-.006) when mean arterial pressure was included as a covariate. There was no significant difference in resting heart rate between the 2 groups (Table 2).

At the initial examination, all echocardiographic variables were comparable between the 2 groups (Table 3). Aortic root diameters during systole increased during the placebo intervention, and this increase was significantly attenuated in the perindopril group for all diameters (Table 3 and Figure 3). During diastole, aortic root diameters increased marginally in the placebo group, while perindopril therapy led to a reduction (1.2-3.0 mm/m²) in end-diastolic diameters compared with the placebo group (Table 3 and Figure 3). The differences in changes in aortic diameters remained significant (P < .001) when mean arterial pressure was included as a covariate.

Perindopril reduced total MMP-2 protein levels (mean [SEM], 157 [14] ng/mL at baseline to 135 [14] ng/mL at 24 weeks; decrease of 22 [6] ng/mL in the perindopril group vs increase of 8 [3] ng/mL in the placebo group, P < .001) and total MMP-3 proteins levels (18 [1] ng/mL at baseline to 13 [1] ng/mL at 24 weeks; decrease of 5 [1] ng/mL in the perindopril group vs increase of 2 [1] ng/mL in the placebo group, P < .001) (Figure 4). Perindopril also reduced latent TGF-β levels (59 [6] ng/mL at baseline to 45 [3] ng/mL at 24 weeks; decrease of 14 (4.5) ng/mL), which was significantly different from placebo (51 [8] ng/mL at baseline to 54 [7] ng/mL at 24 weeks; increase of 2 [2.3] ng/mL, P = .01) (Figure 5). Active TGF-β levels were also reduced following perindopril therapy (46 [2] ng/mL at baseline to 42 [1] ng/mL at 24 weeks; decrease of 4 [1] ng/mL in the perindopril group vs increase of 3 [1] ng/mL in the placebo group, P = .02) (Figure 5).

The major novel finding of our study was that perindopril therapy for 24 weeks reduced aortic diameters relative to placebo in both systole and diastole in patients with MFS taking standard β-blocker therapy. In systole, perindopril reduced the progression of aortic dilatation observed in the placebo group. However, in diastole, perindopril actually reduced aortic diameters below baseline levels by an average of between 1.2 and 3.0 mm/m². Aortic root dilatation and associated aortic regurgitation, dissection, and rupture is the major life-threatening complication of MFS. Prophylactic repair of the aorta with a composite valve and graft is effective therapy once the ascending aorta becomes widely dilated.²⁴ The current indication for surgery is an aortic root diameter of 50 mm.²⁵ However, even after surgery, approximately 10% of patients develop complications of the residual aorta.²⁴ In addition, some patients will have a major complication (ie, dissection or rupture), even though the aortic root dimension is less than 50 mm.²⁵ Postoperatively, the patient is required to remain on life-long warfarin treatment and has the risk of prosthetic valve endocarditis. Thus, there is a great deal of interest in medical therapy for MFS, which protects the aorta and prevents or delays surgery. The current use of β-blockers as standard therapy may not directly address the underlying pathobiology of aortic wall degeneration.^{3 - 5}

In MFS, there is good evidence that angiotensin II is increased¹¹ and that signaling through both the AT₁¹⁰ and AT₂¹¹ receptor pathways contributes to aortic degeneration. Specifically, in a mouse MFS model, the AT₁ receptor antagonist losartan reduced aortic growth rate and prevented elastic fiber degeneration, presumably through hemodynamic actions and effects on TGF-β signaling.¹⁰ Angiotensin II also stimulates Smad-2 dependent signaling in vascular smooth muscle cells and vessel wall fibrosis in a mouse model by an AT₁ receptor-dependent but TGF-β–independent mechanism.²⁶ In addition, AT₂ receptor mechanisms are associated with cystic medial degeneration in MFS and contribute to aortic rupture.¹¹ As ACE inhibitors reduce angiotensin II production, they act via both AT₁ and AT₂ dependent pathways (Figure 6). The effect of inhibiting both pathways in the context of MFS was unknown²⁷ before the current study.

Our data indicate that adjunct therapy with the ACE inhibitor perindopril reduced large artery stiffness and aortic diameter in patients with MFS taking standard β-blocker therapy. This likely occurred by reducing signaling through both the AT₁ and AT₂ receptors (Figure 6). The observed reduction in TGF-β and MMP-2 and MMP-3 are probably secondary to reduced AT₁ receptor signaling.¹⁰ Reduction in AT₂ receptor signaling may provide additional benefit through protection from cystic medial degeneration.¹¹ Based on our data and previous literature, we therefore suggest that ACE inhibition will provide greater efficacy in aortic root protection in MFS than specific blockade of either of the angiotensin II receptors alone.

Whether our findings represent an effect specific to perindopril or a more broad class effect cannot be determined. ACE inhibitors vary in lipophilicity, tissue binding, duration of action, and metabolism, which may contribute to variable efficacy with regard to arterial actions.²⁸ An extensive literature exists regarding the beneficial effects of perindopril on large artery properties^{29 - 31} ; however, whether perindopril has greater efficacy than other ACE inhibitors due to its relative lipophilicity or other properties will require direct comparisons.

This study was limited because of its small sample size and relatively short duration. However, it provides a valid basis for further investigation in a larger clinical trial.

In conclusion, therapy with perindopril reduced both aortic stiffness and aortic root diameter in patients with MFS taking standard β-blocker therapy. These findings warrant further investigation in a larger, longer-term clinical trial.