1. Components and major actions of the renin angiotensin system
Content Points:
The slide lists components and major actions of the tissue renin-angiotensin and kallikrein-kinin systems. As shown, ACE is uniquely positioned to regulate the balance between pressor/profilerative effects and depressor/antiproliferative effects.
The cardiovascular actions of bradykinin are mediated through B2 receptors. By stimulating synthesis and release of nitric oxide, prostacyclin, and endothelium derived hyperpolarizing factor, bradykinin can also cause vasodilation, inhibition of platelet adhesion, and inhibition of smooth muscle cell proliferation.15
Four Ang II receptors have been identified.16 The AT1 receptor is the best characterized. The AT2 receptor may counteract the effects of the AT1 receptor, while the AT3 receptor may stimulate endothelial release of PAI-1.
The biological actions of Ang-(1-7) have only recently begun to receive attention. Like bradykinin, Ang-(1-7) produces a variety of vasodilatory, natriuretic, and antiproliferative responses to counterbalance many of the effects of Ang II.17
Ang-(1-7) is formed from Ang I by the action of several tissue-specific endopeptidases, principally neprilysin, which is located on the surface of endothelial and epithelial cells.18 It is converted to the inactive peptide Ang-(1-5) by ACE. The accumulating evidence suggests that much of the effects of Ang-(1-7) are mediated by an endothelial receptor subtype distinct from AT1 and AT2.17 AT1 receptor blockers (ARBs) do not appear to have a major effect on Ang-(1-7) activity.

2. Vasculoprotective effects of angiotensin-(1-7)
Content Points:
Substantial evidence shows that angiotensin-(1-7) [Ang (1-7)] is an important, novel hormone of the renin angiotensin system.17
Ang-(1-7) opposes the hypertensive and proliferative effects of Ang II. It also increases endothelium derived NO.
Ang-(1-7) increases the release of prostaglandins, both PG2 and PG1, from vascular smooth muscle cells, and may participate in or be the initiating factor leading to the antiproliferative effects of these substances.
Ang-(1-7) inhibits ACE, leading to increases in bradykinin.

3. ACE metabolizes Ang-(1-7) into Ang-(1-5): Effect with and without ACE inhibition
Content Points:
This study demonstrated the role of ACE as the mechanism for the metabolism of Ang-(1-7).19
In the first part of the study, which is not shown on the slide, lisinopril (alone or in combination with losartan) significantly increased the half-life of Ang-(1-7) in three strains of rats (normotensive, hypertensive, and transgenic) whereas the ARB losartan alone had no effect.
An analysis of the metabolism of Ang-(1-7) in pulmonary membranes of untreated SHR rats, a tissue with a high peptidase activity, showed that Ang-(1-7) was degraded within 15 minutes. The primary metabolite resulting from the hydrolysis of Ang-(1-7) was identified as Ang-(1-5), an inactive peptide.
As seen on the slide, the addition of the ACE inhibitor lisinopril to the membrane slowed the metabolism of Ang-(1-7) and abolished the formation of Ang-(1-5). Lisinopril increased the half-life of Ang-(1-7) approximately 15-fold (P < .01).
A similar pattern of Ang-(1-7) metabolism was found in pulmonary membranes of the normotensive rats.

4. ACE and endothelial function
Content Points:
Endothelial dysfunction is the result of the effects of ACE in two distinct and parallel systems.
In the renin angiotensin system, ACE converts Ang I to Ang II, which has multiple, well described vasoconstrictive and proliferative actions.
Increases in Ang II lead to increased production of superoxide ions via the NADH/NADPH oxidase system. The increase in oxidative stress leads to endothelial dysfunction through direct actions, and through actions that reduce NO, PG2, and endothelial hyperpolarizing factor.
In the kallikrein-kinin system, ACE acts by degrading bradykinin into active peptides. This promotes endothelial dysfunction through reductions in the vasodilating and antiproliferating substances, including NO, PG2, and endothelial hyperpolarizing factor.

5. Mechanisms for the effects of ACE inhibition on endothelial dysfunction
Content Points:
ACE inhibition exerts multiple actions that enhance endothelial function and exert anti-atherogenic effects.
The actions of ACE inhibition reduce the generation of superoxide anions, promote NO synthesis, and increase bradykinin, which leads to enhanced bioavailability of NO. This promotes increased vasodilation, decreased proliferation of smooth muscle cells, and reduced thrombosis.
ACE inhibition further reduces the availability of vascular cell adhesion molecule-1 (VCAM-I), an inflammatory molecule that plays an important role in adhesion of leukocytes to the vasculature. This leads to a decrease in vascular inflammation.

6. Potential benefits of suppressing cardiac ACE Content Points:
The efficacy of ACE inhibition in improving symptoms and survival in patients with heart failure has been attributed, as least in part, to suppression of cardiac ACE.20
The exact mechanisms continue to be explored, but the fact that ACE inhibitors have demonstrated greater efficacy in this setting than conventional vasodilators or antihypertensive agents implies a benefit that goes beyond preload and afterload reduction, and quite possibly includes direct positive effects on myocardial function and metabolism.
ACE inhibitors also have anti-ischemic effects, possibly resulting from direct coronary vasodilation and improvement in myocardial flow, and reduction in sympathetic stimulation and norepinephrine-mediated vasomotion.21 There may also be an effect of bradykinin on coronary tone and myocardial metabolism.
Studies have demonstrated the benefits of ACE inhibitors in postinfarction cardioprotection. These effects include reduced infarct size,22 improved myocyte energy utilization and metabolism, and reduced reperfusion arrhythmias.23, 24
Clinical investigations have shown a reduced incidence of ventricular arrhythmias in patients with severe congestive heart failure who are treated with ACE inhibition, and a decrease in left ventricular size following myocardial infarction (MI).25

7. Mechanisms of cardiac RAS activation in cardiac remodeling
Content Points:
This diagram by Dzau summarizes the mechanisms that activate the renin-angiotensin system (RAS) in the heart and lead to cardiac remodeling.
It is now known that there are two RAS, the circulatory RAS and the tissue RAS. The tissue RAS is present in cardiac tissue, vascular walls, the central nervous system, lungs, etc. It produces more than 90% of all ACE in the body. The tissue RAS exerts long-term effects on vascular structure and function.26
Increased pressure overload in the heart, associated with left ventricular hypertrophy (LVH) and hypertension, results in an adaptive ventricular response in which gene expression of ACE is increased. Resulting intracardiac activation of Ang II contributes to myocardial remodeling, to an adaptive response to overload, and ultimately to increased diastolic stiffness that is charactertic of left LVH.
Long-term treatment with ACE inhibitors blocks Ang II production and thereby limits progression of cardiac remodeling. This explains the well-documented benefits of ACE inhibition in preventing or reducing LVH.
The next slides discuss the role of the endothelium in heart failure.

8. Possible mechanisms of endothelial dysfunction in heart failure
Content Points:
A variety of mechanisms are implicated in the pathophysiology of endothelial dysfunction in patients with heart failure.27
Among these, a diminished release of NO in response to receptor or flow-mediated conditions appears to play a major role.
Reduction of L-arginine, a precursor of NO, is a potentially rate-limiting step that affects NO synthesis.
Decreased expression of NO synthase, the enzyme that generates NO from L-arginine, promotes decreased NO bioavailability.
Increased levels of Ang II stimulate the formation of oxygen-free radicals, which promotes inactivation of NO.

9. Effect of experimental heart failure on cardiac ACE activity
Content Points:
Researchers have hypothesized that local RAS may be activated in chronic disease states, such as heart failure, and that this activation may contribute to the pathophysiology of these syndromes.
In the first study to compare plasma and tissue ACE activity in the pathophysiologic state, Hirsch, Dzau, and colleagues demonstrated that compensated heart failure induced by coronary artery ligation in rats was associated with an increase in cardiac tissue ACE activity, but not with plasma or non-cardiac tissue ACE activity.28
In this seminal study, three groups of rats, including non-operated rats, sham-operated rats, and heart failure rats were examined for an average of 85 days post-operatively.
The heart failure rats exhibited a two-fold rise in right ventricular ACE activity compared with the other groups. They had a comparable rise in interventricular septal ACE activity. There was no change in plasma, pulmonary, aortic, and renal ACE activity.
Further observations by this group will be discussed on the next slide.

10. Genetic evidence that cardiac ACE correlates with experimental infarct size
Content Points:
Hirsch, Dzau, and colleagues demonstrated a positive correlation between the magnitude of left ventricular dysfunction (as indicated by histopathologic infarct size) and ACE activity in the right ventricular and in the interventricular septum of rats with heart failure. No correlation was noted between infarct size and serum or noncardiac tissue ACE levels.28
To confirm that the increase in cardiac ACE activity in rats with heart failure was attributable to increased synthesis of local ACE, right ventricular myocardium from 10 additional sham-operated and heart failure rats was analyzed to determine ACE mRNA expression.
Results revealed a two-fold increase in ACE mRNA level that also correlated with infarct size (r = 0.67, P < .05).
These data demonstrate that compensated experimental heart failure is associated with tissue-specific cardiac ACE production.
Increased cardiac ACE activity and the resultant local synthesis of Ang II in experimental heart failure may be pertinent to the beneficial effect of ACE inhibitors.
Increased cardiac ACE activity and increased local Ang II may contribute to the compensatory ventricular remodeling that is ultimately responsible for cardiac dilatation and progressive heart failure. The authors wrote that “the beneficial effects of ACE inhibitor therapy in this condition is related to the inhibition of tissue Ang II production.”

11. Neointima formation: Correlation with blood pressure and residual serum and tissue ACE after ACE-I
Content Points:
Rakugi and colleagues demonstrated a relationship between vascular ACE inhibition (and therefore ACE inhibitor dose) and vascular remodeling (neointima formation) after balloon injury.29
Rats were treated with increasing doses of an ACE inhibitor (quinapril: .05, 5.0, and 50 mg/kg/day). Blood pressure, serum ACE and tissue ACE activity, and neointimal area were measured.
Inhibition of serum ACE and tissue ACE activity and neointima formation were dose-related. However, the dose that reduced blood pressure and inhibited plasma ACE (5.0 mg/kg/day) was significantly lower than the dose that suppressed neointima formation (50 mg/kg/day).
The degree of neotintima formation was more closely correlated with tissue ACE inhibition than with blood pressure or serum ACE inhibition.
The investigators concluded that 1) tissue ACE inhibition was important to prevent vascular remodeling and 2) the dose response results indicate the relative difficulty in inhibiting tissue ACE activity and the potential need for higher doses to inhibit vascular remodeling.

12. ACE inhibitors and fibrinolysis
Content Points:
The tissue RAS and circulating RAS work with the fibrinolytic system to maintain a smoothly functioning cardiovascular system.30
Bradykinin and Ang II compete at the level of vasoconstriction. Ang II is a potent vasoconstrictor and bradykinin is a potent vasodilator.
Bradykinin is probably the most potent stimulus for the production and secretion of tissue plasminogen activator (t-PA). In contrast, Ang II is an extremely powerful stimulus for the production and secretion of plasminogen activator inhibitor 1 (PAI-I). These two peptides compete at the vascular interface to determine net fibrinolytic balance.
The action of ACE inhibitors to elevate bradykinin and decrease Ang II would be expected to promote higher levels of t-PA and decrease PAI-I levels, thereby enhancing fibrinolysis.

13. Vasculoprotective effects of tissue ACE inhibition
Content Points:
The prevention of cardiovascular disease by ACE inhibitors involves complex mechanisms.31
Locally generated Ang II may contribute to the secondary structural changes seen in cardiovascular disorders, such as cardiac remodeling, CAD, and atherosclerosis. Therefore, inhibition of Ang II formation by ACE inhibitors, particularly at the tissue level, may provide valuable effects that protect the heart and vasculature.
Through their ability to reduce Ang II and increase NO formation, ACE inhibitors influence smooth muscle cell and cardiac myocyte growth, as well as matrix synthesis.
In addition, MI may be prevented by reducing those factors that are involved in the ischemic syndrome, including the formation of t-PA and PAI-1, which affect fibrinolysis. ACE inhibition also reduces inflammation and monocyte adhesion.

14. Cardiovascular role of bradykinin as determined by knockout mice
Content Points:
Activation of B2 receptors by kinins may have cardioprotective effects relative to myocardial ischemia and heart failure. In this study, Emanueli and colleagues studied the bradykinin B2 receptor (BK B2) gene in knockout mice, to determine whether their absence affects cardiac structure and function.32
They examined changes in blood pressure, heart rate, and heart structure in BK B2 receptor gene knockout (B2–/–) mice, and compared them with heterozygous (B2+/–) and wild type (B2+/+) mice.
The blood pressure of B2-/- mice, which was still normal at 50 days of age, gradually increased as they matured, reaching a plateau at 6 months (136 ± 3 versus 109 ± 1 mm Hg in B2+/+ (P < .01).
At 40 days, heart rate was higher in B2-/-and B2+/- mice than in B2+/+ mice (P < .01), but the left ventricular (LV) weight and chamber volume were similar among the groups.
Thereafter, the LV growth rate accelerated in B2-/- and B2+/- mice, leading at 360 days to a LV weight-to-body weight ratio that was 9% and 17% higher, respectively, than that of B2+/+ mice.
In B2-/- mice, LV hypertrophy was associated with a marked chamber dilatation (42% larger than that of B2+/+ mice), a significant elevation in LV end-diastolic pressure, and reparative fibrosis.
In summary, the disruption of the BK B2 receptor leads to hypertension, LV remodeling, and functional impairment, implying that kinins are essential for the functional and structural preservation of the heart.
A partial deficiency of the BK B2 receptor causes hypertension and LV hypertrophy. Total deficiency induces decompensated hypertrophy and heart failure similar to hypertrophic hypertensive cardiomyopathy seen in humans.

15. ACE inhibition increases vasodilation by a bradykinin mechanism in response to flow-mediated dilation
Content Points:
In this work, Hornig and Drexler demonstrated that ACE inhibition enhances flow-mediated, endothelium-dependent dilation by a bradykinin-dependent mechanism.33 They used high-resolution ultrasound and Doppler to measure radial artery diameter and blood flow in 10 healthy volunteers.
The vascular effects of the ACE inhibitor quinaprilat, the selective bradykinin B2 receptor antagonist icatibant, and their combination were determined at rest and during reactive hyperemia following wrist occlusion. During reactive hyperemia, increased flow causes endothelium-dependent dilation. They also infused sodium nitroprusside, which causes endothelium-independent vasodilation.
At rest, there were no changes in arterial diameter with either quinapril, icatibant, or their combination, but changes occurred during reactive hyperemia, as shown in the graph.
During reactive hyperemia, quinaprilat significantly increased FMD (P < .001 compared with control). In contrast, icatibant significantly reduced radial artery diameter (P < .01 compared with control).
Co-infusion of icatibant reduced FMD (P < .001 compared with control and with quinaprilat). The reduction was similar to the reduction with icatibant alone.
Infusion of sodium nitroprusside significantly increased arterial diameter (P < .001).
The observation indicates ACE inhibition involves a bradykinin-dependent mechanisms and that accumulation of endogenous bradykinin is involved in the vascular effects of ACE inhibitors.

16. Effects of ACE-I vs other antihypertensive agents on endothelial function in hypertensive patients
Content Points:
Endothelial dysfunction is a component of essential hypertension. This study by Higashi and coworkers compared the effect of different antihypertensive agents, calcium antagonists, ACE inhibitors, b-blockers, and diuretic agents on endothelial function.34
Forearm blood flow was measured in 296 patients with essential hypertension at baseline and during reactive hyperemia, which is an index of endothelial function. Mean age of the patients was 48 years.
The forearm blood flow during reactive hyperemia in hypertensive patients was significantly less than in normotensive control subjects.
The group that was treated with an ACE inhibitor had a significantly greater maximal forearm blood flow response from reactive hyperemia than the groups treated with calcium antagonists, b-blockers, diuretic agents, or nothing (P < .05). Reductions in blood pressure were similar with each of the antihypertensive agents used.
Thus reduction in blood pressure with antihypertensive therapy may not always lead to improved endothelial function in patients with essential hypertension. Although all of these agents were effective in reducing blood pressure, only ACE inhibitors improved endothelial function.
The next slide discusses a second part of the study to determine the possible mechanisms involved.

17. ACE inhibitors and endothelial function: Evidence for the role of NO and bradykinin
Content Points:
In a second part of the study, Higashi and colleagues measured the effect of the various antihypertensive agents on NO and prostaglandin release.34 Forearm blood flow response to reactive hyperemia was measured after the patients were given L-NMMA, which inhibits NO synthase, and indomethacin, which inhibits prostaglandin synthesis.
Inhibition of NO synthase with L-NMMA ended the enhancement in reactive hyperemia in the patients treated with ACE inhibitors, suggesting the NO plays a major role in this process.
Thus, ACE inhibitors improve endothelial function in patients with essential hypertension through increases in NO production. ACE inhibitors decrease bradykinin degradation, leading to increases in NO release.
Inhibition of prostaglandin synthesis with indomethacin did not change reactive hyperemia. Thus, although the decrease in bradykinin degradation by ACE inhibitors has been shown to increase prostaglandins, this observation suggests that prostaglandins do not contribute to the increase in endothelial vasodilation with ACE inhibition.

18. ACE inhibitors upregulate b -adrenergic receptors on cardiac myocytes by a bradykinin mechanism
Content Points:
Yonemochi and colleagues conducted this study to determine whether bradykinin upregulation of b-adrenergic receptors (b-AR) contributes to the improvement in cardiac function attributable to ACE inhibition.
Incubation of cultured rat cardiac myocytes for 24 hours with the ACE inhibitor captopril increased b-AR density by 35%.
There was also an increase in spontaneous beating frequency of the myocytes in response to isoprotenerol. Radioimmunoassay confirmed that bradykinin levels were 1.4-fold higher in the medium containing captopril than in untreated medium.
To determine the mechanism involved, as seen on the left side of the slide, Yonemochi and colleagues also incubated the cells for 24 hours with an AT1 receptor blocker (CV-11974) and Ang I. In contrast to the effect seen with captopril, these agents had no effect on b-AR density.
They then incubated the cells with captopril and HOE 140, a bradykinin B2 receptor antagonist, which abolished the effect of captopril on b-AR upregulation in a dose-dependent manner, as seen on the right side of the slide.
The protein kinase C activator staurosporine also inhibited the captopril-induced increase in b-ARs density (not shown here).
These findings suggest that bradykinin upregulation of b-ARs contributes to the improvement in cardiac function induced by ACE inhibition through an effect that is mediated by stimulation of B2 receptors and protein kinase C activation.
These observations provide some insight into the different roles of ACE inhibition and AT1 receptor blockade in heart failure.