New evidence for endothelial protection




Stefano TADDEI, MD
Department of Internal Medicine
University of Pisa – Pisa, ITALY

New evidence for endothelial protection

by S. Taddei, Italy

The endothelium plays a crucial role in the regulation of vascular tone and structure through production of several substances, the most important of which is nitric oxide. Endothelial dysfunction, a condition characterized by impaired nitric oxide availability, is considered a main promoter of atherothrombosis. An important mechanism determining endothelial dysfunction is the activity of the tissue renin-angiotensin system. In patients with cardiovascular risk factors or coronary artery disease, chronic overexpression of tissue angiotensin-converting enzyme disrupts the angiotensin II/ bradykinin balance with a net result of endothelial dysfunction, mainly due to an increased production of oxidative stress and apoptosis. Imbalance between increased endothelial cell apoptosis and decrease in endothelial cell renewal from the bone marrow causes discontinuity of the endothelial layer, favoring the initiation and progression of a biochemical sequence that leads to atherosclerosis, plaque rupture, and eventually acute coronary syndromes. Thus, reversal of vascular functional and structural alterations is crucial to determine whether pharmacological treatment is having a beneficial effect. A large body of scientific evidence indicates that perindopril has a specific efficacy on both the molecular and the functional aspects of endothelial dysfunction and arterial stiffness. These effects are important for the prevention of atherosclerosis in patients with cardiovascular risk factors and of clinical events in patients with cardiovascular risk factors or established coronary artery disease.

Medicographia. 2012;34:17-24 (see French abstract on page 24)

Since the pioneering report by the 1998 Nobel Prize winner Robert Furchgott on the obligatory role of endothelium in vascular relaxation in response to acetylcholine,1 an impressive array of evidence has made it possible to state today that vascular endothelium plays a primary role in the control of vascular function and structure through the production of nitric oxide (NO).2 NO derives from the transformation of L-arginine into citrulline through the activity of the constitutive endothelial enzyme NO synthase (eNOS). NO is produced and released either basally or under the influence of agonists, such as acetylcholine, bradykinin, substance P, serotonin, and others acting on specific endothelial receptors, and by mechanical forces, such as shear stress.2 Other endothelium-derived relaxing factors include prostacyclin and the production of various endothelium-derived hyperpolarizing factors (EDHFs), which represent a compensatory vasodilating pathway triggered by reduced NO availability.3,4 In several pathological conditions, activation of endothelial cells can lead to the production and release of prostanoids (thromboxane A2 and prostaglandin H2), which are cyclooxygenase- derived endothelium-derived contracting factors (EDCFs) that counteract the relaxing activity of NO, and reactive oxygen species (ROS), which impair endothelial function by causing NO breakdown.2 ROS, mainly superoxide anion, trap NO to form substances like peroxynitrite, which are thought to alter vascular function and structure.5 NO and EDCFs not only exert an opposite effect on vascular tone, but NO also inhibits, and EDCFs activates, platelet aggregation, vascular smoothmuscle cell proliferation andmigration,monocyte adhesion, adhesion molecule expression, apoptosis, and cell regeneration, which exert an important role in the genesis of thrombosis and of the atherosclerotic plaque.6

Assessment of endothelial function in humans

_ Vascular reactivity tests
The autocrine/paracrine activity of endothelial cells makes it very difficult to investigate endothelial function in clinical research. Usually, this requires vascular reactivity studies.7 It is possible to activate or inhibit endothelial cells in several vascular regions andmeasure the changes in the vessels induced by experimental manipulation. Endothelial cells respond to agonists acting on specific receptors (acetylcholine, bradykinin…) or by increasing shear stress (flow-mediated dilation [FMD]). In addition, it is possible to block pathways involved in endothelial responses such as NOS activity (by NG-monomethyl- L-arginine [L-NMMA]), hyperpolarization of vascular smooth muscle cells (by ouabain), cyclooxygenase activity (by indomethacin), or oxidative stress (by antioxidants such as vitamin C). In describing the approach to the evaluation of endothelium-dependent mechanisms in humans, it is crucial to consider the type of vascular bed to be investigated. A distinction should be made between macrocirculation (large arteries) and microcirculation. These two vessel types are subjected to different types of regulation and therefore results obtained in large arteries cannot be extrapolated to the microcirculation. Valuable large arteries include brachial (most frequently), radial, femoral, and epicardial arteries. The microcirculation can be evaluated in peripheral muscle (usually the forearm), subcutaneous tissue, skin, and the coronary circulation.

_ Circulating markers of endothelial function
Use of circulating markers of endothelial function was recently introduced.7,8 These include direct products of endothelial cells that change when the endothelium is activated, such as measures of NO biology, inflammatory cytokines, adhesion molecules, as well as markers of endothelial damage and repair.

A promising method for assessing endothelial function by means of circulating markers is to determine the rate of endothelial cell death and regeneration. Imbalance between endothelial apoptosis (death) and renewal from the bone marrow (life) causes discontinuity of the endothelial layer, favoring the initiation and progression of a biochemical sequence that leads to atherosclerosis, plaque rupture, and eventually acute coronary syndromes. Circulating endothelial apoptotic cells or endothelial progenitor cells (CEPCs) can be counted, providing a novel and exciting means of monitoring the determinants of endothelial injury and repair.9 The expression of surface markers on CEPCs can be measured, but because a wide range of hematopoietic progenitor cells have the potential to adopt an endothelial phenotype, the specificity of these measurements is debated.10 Other methods to characterize CEPC biology include quantification of the potential to differentiate into an endothelial cell phenotype, as well as determination of functional characteristics, which include migration toward a chemical stimulus, adhesion, formation of vascular tubules, and the ability toattenuate ischemia in animalmodels.11

Clinical significance of endothelial dysfunction

In patients with cardiovascular risk factors, endothelial dysfunction is considered as a common mechanism deeply affecting vascular function and structure, including vasomotor function and promotion of atherosclerosis and thrombosis, thereby contributing to cardiovascular events. This is supported by mounting evidence showing the association of endothelial dysfunction with markers of vascular damage and with cardiovascular events, both in patients with essential hypertension and, more generally speaking, in patients with atherosclerotic disease. A recent meta-analysis by Lerman and Zeiher evaluated the available longitudinal studies relating to the prognostic impact of endothelial dysfunction.12 This metaanalysis included around 2500 patients with atherosclerotic coronary disease or at high cardiovascular risk. The outcomes, evaluated over a wide follow-up range (from 1 to 92 months) included major cardiovascular events. The authors observed that endothelial dysfunction, evaluated either in the coronary territory or in the peripheral circulation, significantly predicted cardiovascular events, independently of traditional cardiovascular risk factors.12 These studies suggest that endothelial dysfunction is prognostically relevant in high-risk patients with coronary artery disease (CAD).

Treatment of endothelial dysfunction

Since impaired endothelium-dependent vasodilation promotes atherosclerosis and cardiovascular events in patients with cardiovascular risk factors, eg, essential hypertension, improving impaired endothelial function is an important treatment target. However, blood pressure lowering with antihypertensive therapy is not sufficient per se to reverse endothelial dysfunction.13 Thus, antihypertensive compounds, beyond their ability to reduce blood pressure, need to have additional specific properties to restore endothelial function. Diuretics or β-blockers show little evidence of being able to restore endothelial dysfunction in patients with hypertension or CAD. In contrast, renin-angiotensin system (RAS) blockers and calcium antagonists are strongly associated with a beneficial effect on endothelial dysfunction in patients with hypertension or CAD.14 However, this beneficial effect differs depending on which drug class or vascular district is considered.14 Calcium antagonists

In patients with hypertension, a wide range of calcium antagonists— including amlodipine, isradipine, lacidipine, lercanidipine, nifedipine, verapamil, and diltiazem—have been shown to improve endothelial function, suggesting that all these agents share a class effect. Schiffrin and Deng15 found that nifedipine GITS (gastrointestinal therapeutic system) normalized endothelial function as well as the structure of gluteal subcutaneous small arteries. Conversely, use of atenolol in similar patients resulted in equally well-controlled blood pressure, but abnormal endothelial function and thicker small arteries. Taddei et al reported a decrease in circulating plasma lipoperoxides and isoprostanes, an increase in plasma antioxidant capacity, and an improved forearm vasodilator response to acetylcholine in a study in 15 hypertensive patients vs 15 healthy subjects after 3 months of nifedipine treatment.16

Investigators have also examined the effects of calciumantagonists on endothelium-mediated vasodilation in patients with CAD. ENCORE I (Evaluation of Nifedipine and Cerivastatin On Recovery of coronary Endothelial function–I) showed a significant reduction in acetylcholine-induced vasoconstriction in patients with CAD receiving nifedipine.17 However, results are not universally positive, and calcium antagonists failed to improve endothelial function, assessed by measurement of brachial artery FMD, in hypertensive patients18 or patients with CAD.19

Figure 1
Figure 1. Effect of antihypertensive drugs on conduit artery endothelial
function.

In hypertensive patients, reduced endothelial function, assessed as brachial
artery flow-mediated dilation, is improved by chronic treatment with perindopril,
whereas other antihypertensive drugs have no effect.
After reference 8: Versari et al. Curr Pharm Des. 2007;13:1811-1824. © 2007,
Bentham Science Publishers.

RAS inhibitors: are ACE inhibitors and ARBs equally effective in reversing endothelial dysfunction?

The possibility that angiotensin-converting enzyme (ACE) inhibitors may improve endothelial function has been raised following reports in the literature from experimental studies showing that the RAS—and in particular angiotensin II— plays a dramatic role in inhibiting NO production and activity, mainly by inducing ROS generation.20 However, this hypothesis on the role of angiotensin II is not the only explanation for the efficacy of ACE inhibitors regarding endothelial function, since the literature suggests a significant superiority of this drug class vs angiotensin II type 1 (AT1) receptor antagonists, at least in certain clinical situations.

The BANFF study (Brachial Artery Normalization of Forearm Function) compared the effect of drugs belonging to different classes on endothelial function by assessing brachial artery FMD in patients with CAD.19 It was found that while the ACE inhibitor quinapril was able to improve FMD, the AT1 receptor antagonist losartan failed to elicit any significant improvement in endothelial function.

An important confirmation of these findings derives from a study that enrolled a large population (n=168) of essential hypertensive patients (Figure 1) and compared the effects ofthe principal classes of antihypertensive drugs on peripheral conduit artery endothelial dysfunction (by measuring brachial artery FMD) in essential hypertension.18 The drugs compared in the study were the ACE inhibitor perindopril, the AT1 receptor antagonist telmisartan, the -β-blockers atenolol and nebivolol (the latter expected to be effective on endotheliumdependent vasodilation), and the calcium antagonists amlodipine and nifedipine. The only compound able to improve endothelial function was perindopril, while the other drugs, including the AT1 receptor antagonist telmisartan, failed to increase endothelium-dependent vasodilation. In addition to perindopril’s specific ability to improve FMD in the peripheral circulation, its action was also confirmed in coronary epicardial arteries in patients with essential hypertension. In these patients, acute endovenous administration of perindoprilat restored normal vascular response to endothelial stimuli, thereby determining an increase in coronary flow.21

Under other experimental conditions, AT1 receptor antagonists can improve endothelial function—an effect shared by the ACE inhibitors. However, analysis of the literature indicates that inhibition of converting-enzyme activity has a greater efficacy than blockade of the AT1 receptor.14

Table I
Table I. Evidence-based vascular protection with perindopril

Endothelial and vascular dysfunction: is there a specific role for perindopril?

To date, perindopril is the only compound with documented efficacy on several parameters reflecting vascular dysfunction or structural alterations in the different segments of the arterial tree (Table I).18,21-28 Several hypotheses can be raised to explain perindopril’s specific efficacy.

One such hypothesis concerns the tissue affinity of ACE inhibitors. It is now well documented that vascular homeostasis is regulated by tissue RAS and, therefore, that vascular absorption is crucial for drug efficacy on endothelial function or arterial structural alterations.29 Perindopril is the ACE inhibitor with the greatest documented vascular absorption in comparison with other drugs of the same class, including quinapril, ramipril, enalapril, fosinopril, and captopril.30 This specific characteristic may therefore be one of the possible explanations for perindopril’s unique effects on vascular function and structure.

Another important mechanism, too often forgotten, is the reduction in bradykinin (BK) breakdown determined by ACE blockade.29 BK is an important autacoid with significant effects on endothelium. In addition to the classic stimulation of the NO pathway,31 BK can also release EDHFs,32 which represent an important compensatory mechanism when NO availability is reduced as observed in presence of cardiovascular risk factors, including hypertension.432 The ability of BK to stimulate EDHF production in presence of reduced NO availability is not shared by other classic endothelial activators, including acetylcholine.32

The ability of ACE inhibitors to increase BK tissue concentration by blocking its degradation could be an important mechanism for vascular protection, especially in the presence of cardiovascular risk factors or CAD, which cause endothelial dysfunction by NO inactivation. In these patients, chronic overexpression of tissue ACE disrupts the angiotensin II/BK balance, with increased and decreased tissue levels of angiotensin II and BK, respectively, the net result being endothelial dysfunction. The latter is characterized by augmentation of oxidative stress and inflammation with the consequent impairment of NO availability and increase in endothelial damage and apoptosis.23 Apoptosis is an emerging concept in cardiovascular medicine. Thus, endothelium undergoes a life and death cycle, characterized by a programmed cell suicide (apoptosis) associated with subsequent regeneration.23 Imbalance between endothelial apoptosis and regeneration is now considered a promoter of atherosclerosis.33

However, the effect of the different ACE inhibitors on angiotensin II/BK balance and the rate of apoptosis varies markedly. ACE has two different catalytic domains, one cleaving angiotensin I, the other inactivating BK.34 When the ACE inhibitors enalaprilat, perindoprilat, quinaprilat, ramiprilat, and trandolaprilat were tested to compare their binding affinity for the two ACE domains, two major findings emerged. First, all ACE inhibitors have a greater affinity for the BK than for the angiotensin I binding site, supporting the concept that these compounds are primarily inhibitors of BK degradation rather than inhibitors of angiotensin II production.34 Second, perindoprilat has the highest selectivity for the BK binding site while trandolaprilat and enalaprilat had the lowest one (Figure 2).18,34

The effect of the aforementioned ACE inhibitors (enalapril, perindopril, quinapril, ramipril, and trandolapril) on the rate of endothelial apoptosis was assessed in vivo in rats. Apopto- sis was induced by endotoxic shock with Escherichia coli lipopolysaccharides (LPS).35 Chronic in vivo administration of ACE inhibitors to rats at equihypotensive dosages resulted in a significant reduction in rate of LPS-induced apoptosis with perindopril (P<0.001) and a nonsignificant reduction with the other ACE inhibitors. The order of potency of the ACE inhibitors tested was perindopril > ramipril >> quinapril = trandolapril = enalapril.35

Figure 2
Figure 2. Comparative affinity of ACE inhibitors for bradykinin vs
angiotensin I binding sites.

The study compares different ACE inhibitors in terms of selectivity for bradykinin
vs angiotensin I binding sites. Perindopril has the highest selectivity for bradykinin
vs angiotensin I binding sites, and enalapril has the lowest. The resulting greater
endogenous concentrations of bradykinin may explain, at least in part, the beneficial
vascular effects of perindopril.
Abbreviations: ACE, angiotensin-converting enzyme; Ang I, angiotensin I;
BK, bradykinin.
After reference 34: Ceconi et al. Eur J Pharmacol. 2007;577:1-6. © 2007,
Elsevier B. V.

Taken together, these findings provide a likely explanation for the primary role of perindopril in reversing endothelial dysfunction, as further supported by the results obtained in humans in vivo in the PERTINENT study (PERindopril— Thrombosis, InflammatioN, Endothelial dysfunction and Neurohormonal activation Trial),22 a substudy of the EUROPA program (EUropean trial on Reduction Of cardiac events with Perindopril in stable Artery coronary disease).36 In PERTINENT, the authors measured von Willebrand factor (vWf) as a marker of endothelial damage. It is worth noting that elevated vWf at baseline was an independent predictor of cardiovascular events in EUROPA, confirming the relevance of endothelial alterations in determining cardiovascular prognosis. One-year treatment with perindopril was able to reduce vWf. In addition, plasma levels of angiotensin II, BK, tumor necrosis factor–α (TNF-α), nitrite/nitrate, and endothelial function at the cellular level by determining protein expression/activity of eNOS and the rate of apoptosis were also measured at baseline and after 1 year of treatment with either perindopril or placebo. At baseline, CAD patients showed decreased BK and increased angiotensin II plasma concentrations, respectively, compared with healthy controls. However, perindopril caused a significant reduction in levels of angiotensin II and an increase in BK, therefore normalizing the BK/angiotensin II ratio. In addition, perindopril also decreased TNF-α and increased nitrite nitrate (P<0.05 for all). Finally, perindopril upregulated the protein expression/activity ratio of eNOS by 19%/27% (P<0.05) and reduced the rate of apoptosis by 31% (P<0.05) (Figure 3).22

Taken together these results demonstrate: (i) an excess of angiotensin II and TNF-α (proapoptotic substances by increasing oxidative stress) and a reduction in BK (an antiapoptotic substance) cause endothelial dysfunction and increase apoptosis; (ii) treatment with perindopril restores a normal balance between angiotensin II and BK and reduces inflammation (TNF-α), thereby reversing endothelial dysfunction and preventing apoptosis.

Figure 3
Figure 3. Results from the PERTINENT study.

Rate of apoptosis in human umbilical vein endothelial cells
incubated with in serum from healthy controls or in serum
collected from PERTINENT patients at baseline and after
1 year. Perindopril significantly reduces the rate of
apoptosis vs placebo.22
Abbreviations: CAD, coronary artery disease;
PERTINENT, PERindopril—Thrombosis, InflammatioN,
Endothelial dysfunction and Neurohormonal activation Trial.

As previously stated, while apoptosis is a marker of endothelial cell death,23 CEPCs are an important marker of endothelial cell regeneration,9 a crucial mechanism to maintain the integrity of the endothelial layer. Thus, to completely prevent endothelial dysfunction, optimal treatment should not only inhibit apoptosis, but also stimulate CEPC production. In experimental conditions, perindopril was shown to increase the number of CEPCs in the spontaneously hypertensive rat(SHR) with hindlimb ischemia, while losartan has no effect on the number of CEPCs.23,37 Perindopril also reverses structural alterations, as shown in the DAPHNET study (Diabetes Artery Perindopril Hypertension Normalization Excess sTiffness),25 which found a direct blood pressure–independent effect of perindopril on arterial stiffness. The study enrolled hypertensive patients with type 2 diabetes whose blood pressure values were normalized by perindopril 4 mg daily. Patients were then randomized to continue on the 4-mg daily dose or increase to 8 mg daily while carotid distensibility was measured at baseline and after 7 months of treatment. It was found that, despite similar blood pressure control, only perindopril 8 mg daily reduced carotid stiffness, suggesting a specificeffect on vessel structure (Figure 4). This study again underlines that mere blood pressure control is not the only parameter to take into account to determine the efficacy of antihypertensive treatment, and that more specific organ protection can be obtained with higher doses, which should always be used in high-risk patients. With regard to aortic structural changes, a comparative study26 showed that perindopril decreased aortic pulse wave velocity, an established marker of aortic stiffness; this effect was also seen with the comparator dihydropyridine calcium-channel blocker, but not with the diuretic or the β-blocker. Finally, in a large population of hypertensive patients, perindopril had a positive effect on aortic elastic properties associated with an attenuation of inflammatory status assessed by the measurement of C-reactive protein, interleukin 1α and 1β and TNF-α.38

Figure 4
Figure 4. Results from the DAPHNET study: dose-dependent effects
of perindopril on carotid artery function in diabetic patients.

In hypertensive patients with type 2 diabetes, long-term administration of perindopril
at adequate dose improves carotid structure and function, independently of
blood pressure reduction.
Abbreviations: DAPHNET, Diabetes Artery Perindopril Hypertension Normalization
Excess sTiffness [study].
After reference 25: Tropeano AI et al. Hypertension. 2006;48:80-86. © 2006,
American Heart Association, Inc.

The improvement in functional and structural vascular alterations with perindopril contributes to preventing atherosclerosis, as shown by the PERSPECTIVE study (PERindopril’S Prospective Effect on Coronary aTherosclerosis by IntraVascular ultrasound Evaluation),28 another substudy of the EUROPA program. This study evaluated the progression of coronary atherosclerosis by intracoronary ultrasound in coronary segments containing noncalcified or calcified plaques in 118 CAD patients enrolled in EUROPA study. A post hoc analysis assessed the effect of perindopril vs placebo on progression/ regression of atherosclerosis based on the degree of calcification. Findings were as follows: (i) coronary plaques with no or little calcium (0%-25%) regressed on perindopril, but did not change on placebo (–0.33±1.74 vs –0.03±1.66, respectively; P=0.04); (ii) plaques with moderate calcium content (group 25%-50%) did not change; and (iii) plaques with high calcium content (group 5%-100%) progressed similarly. The important conclusion of the study is that noncalcified plaques may be amenable to regression with perindopril treatment. PERTINENT is the only study to have evidenced the ability of an ACE inhibitor to prevent progression of atherosclerosis in the coronary circulation in vivo.

Conclusions

Endothelium plays a central role in the maintenance of vascular homeostasis. Tests have been developed to study endothelial function in humans and assess its changes in relation with subclinical and clinical target organ damage as well as the effect of treatment. Endothelial dysfunction and structural vascular alterations are of particular clinical relevance since they promote atherosclerosis and thrombosis, which are typical complications of hypertension and other cardiovascular risk factors, and thereby play a key role in the occurrence of clinical events.

Not all cardiovascular drugs are able to reverse endothelial and structural dysfunction. Analysis of the literature clearly indicates that perindopril has a unique profile of action, characterized by its effect on both the molecular and functional aspects of endothelial dysfunction and arterial stiffness. This effect has important implications for the prevention of atherosclerosis in patients with cardiovascular risk factors and of clinical events in patients with established CAD. A recentmetaanalysis of EUROPA,36 ADVANCE (Action in Diabetes and Vascular disease: PreterAx and DiamicroN Modified-Release Controlled Evaluation),39 and PROGRESS (Perindopril pROtection aGainst REcurrent Stroke Study)40 provides strong evidence in favor of consistent cardiovascular protection with a perindopril- based regimen, by improving survival and reducing the risk of major cardiovascular events.41 These specific vascular properties of perindopril may account, at least in part, for the documented superiority of this compound in the prevention of cardiovascular disease. _

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Keywords: ACE inhibitor; angiotensin II; bradykinin; cardiovascular risk factor; coronary artery disease; endothelium, hypertension; large artery, microcirculation; nitric oxide; perindopril