1. Bradykinin and angiotensin-(1-7): Emerging allies in vascular function
Content Points:
Studies indicate that the relationship between the RAS and the KKS is more complex (and more flexible) than once believed. This program discusses new data on the interaction between these 2 systems and, in particular, the evidence suggesting that many of the beneficial effects of ACE inhibitors may be mediated by bradykinin.
There will also be a discussion of intriguing new research on the novel RAS peptide angiotensin-(1-7), hereafter referred to as Ang-(1-7).

2. Bradykinin, Ang-(1-7), and vascular health
Content Points:
Both bradykinin and Ang-(1-7) are associated with a number of favorable actions that counterbalance the unfavorable actions of Ang II. Thus, these bioactive agents have important counterregulatory roles.

3. Vasculoprotective effects of bradykinin
Content Points:
Like the RAS, the KKS is located in both plasma and tissues.1 The circulating plasma system is primarily involved in the blood clotting cascade, while the tissue system has a number of important cardiovascular effects mediated through bradykinin B1 and B2 receptors.
A local KKS has been identified in the vascular wall.2 At the vascular level, bradykinin is one of the most potent stimuli for endothelial release of tPA in vivo.3 In addition, by stimulating synthesis and release of nitric oxide (NO), prostacyclin, and endothelium-derived hyperpolarizing factor (EDHF), bradykinin can also cause vasodilation, as well as inhibition of platelet adhesion and smooth muscle cell proliferation.4

4. Bradykinin B2 receptor blockade decreases basal coronary tone
Content Points:
To investigate the role of bradykinin in vasomotor control, Groves et al infused the B2 receptor blocker icatibant into the left main coronary artery of 15 patients who were without flow-limiting coronary stenoses.5 B2 receptor blockade led to a significant reduction in coronary luminal area in proximal (P < 0.001), mid (P < 0.001), and distal (P < 0.05) segments.
The investigators concluded that tissue bradykinin is involved in mediating vasomotor responses in the human coronary circulation under basal conditions.

5. Bradykinin B1 and B2 receptor stimulation in canine coronary circulation
Content Points:
Su et al infused either bradykinin or a bradykinin B1 receptor agonist (des-Arg9-bradykinin) into canine circumflex coronary arteries.6
Both bradykinin and the B1 receptor agonist increased coronary blood flow velocity (microvessel dilation) and coronary diameter (large vessel dilation). However, the changes associated with the B1 receptor agonist, which is not degraded by ACE, were much smaller than those produced by bradykinin (P < 0.05).
The data suggest that most coronary vascular effects of bradykinin may be mediated by the B2 receptor and are significantly influenced by ACE.

6. Cardiovascular role of bradykinin as determined by knockout mice
Content Points:
Studies by Emanueli et al demonstrate that disruption of the gene for the bradykinin B2 receptor leads to hypertension, left ventricular remodeling, and functional impairment.7
Specifically, in mice that are heterozygous (B2+/-), inheriting only 1 functional gene for this receptor, blood pressure rises after approximately 4 months of age, with an associated gradual increase in left ventricular mass.
In mice that completely lack the gene (B2-/-) for this receptor, blood pressure and left ventricular mass increase sooner and more rapidly. These mice develop LV hypertrophy and decompensated heart failure.
Perivascular and reparative LV fibrosis is enhanced in both models versus B2+/+ controls that have both copies of the functional gene for this receptor.
These data imply that bradykinin is essential for the functional and structural preservation of the heart and vasculature.

7. Components and major actions of the kallikrein-kinin and renin-angiotensin systems
Content Points:
Several peptidases can hydrolyze kinins such as bradykinin; however, in vascular tissue, the principle enzyme involved is ACE.1 While ACE also converts Ang I to Ang II, bradykinin may be the preferred substrate.8
At least four sub-type Ang II receptors (designated AT1 to AT4) have been identified.9 The AT1 receptor is the best characterized and mediates the vasoconstrictive and proliferative effects of Ang II. The AT2 receptor may counteract some of the effects of the AT1 receptor, while the AT4 receptor may affect vascular integrity and stimulate endothelial release of PAI-1. Little is known at present regarding the effects of the AT3 receptor.
More recently, Ang-(1-7) has also been shown to possess biological activity and may have an important role in regulation of blood pressure and endothelial function.10 AT1 receptor blockers do not appear to have a major effect on Ang-(1-7) activity. This finding, along with other accumulating evidence (discussed in a later slide), suggests that much of the effects of Ang-(1-7) are mediated by a distinct endothelial receptor subtype, which has been designated the AT(1-7) receptor.10
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 vascular endothelial and epithelial cells11) and is converted to the inactive peptide Ang-(1-5) by ACE.
Like bradykinin, Ang-(1-7) opposes the hypertensive and proliferative actions of Ang II. In fact, as discussed in the next slide, the effects of Ang-(1-7) are mediated in part by bradykinin.

8. Vasculoprotective effects of Ang-(1-7)
Content Points:
Ang-(1-7) has the following beneficial physiologic effects:
– Increases NO release12-14
– Releases vasodilatory prostaglandins (PGE2 and PGI2)15-19
– Increases bradykinin levels, since Ang-(1-7) is also an inhibitor of ACE as well as being a substrate 12,14,20,21

9. Ang-(1-7) levels are significantly reduced in hypertension
Content Points:
Ferrario et al demonstrated that untreated hypertensive individuals have significantly lower urinary levels of Ang-(1-7) than normotensive individuals.22 As shown on the slide, the Ang-(1-7) urinary excretion rate (corrected for urinary excretion of creatinine) was lower in hypertensives than normotensives (P < 0.01). Furthermore, both urinary Ang-(1-7) levels and age were independent predictors of systolic blood pressure.
Findings suggest that this peptide may serve as a marker of RAS activity and perhaps may serve as a measure of the therapeutic effectiveness of antihypertensive drugs.

10. Differing effects of Ang-(1-7) and Ang II on VSMC growth
Content Points:
Studies by Freeman et al in cultured vascular smooth muscle cells (VSMC) demonstrated that Ang-(1-7) and Ang II have opposite effects on VSMC growth.23 As shown, labeled thymidine uptake was reduced by Ang-(1-7) versus control, indicating reduced VSMC proliferation. In contrast, incorporation of thymidine was stimulated by increasing concentrations of Ang II.
The investigators also conducted studies to identify the receptor that mediated these effects on VSMC growth. These data are summarized on the next slide.

11. Ang-(1-7) inhibits VSMC growth through activation of a non-AT1, non-AT2, D-Ala-Ang-(1-7)-sensitive receptor
Content Points:
Thymidine uptake was reduced versus control to a similar degree by Ang-(1-7) plus the AT1 antagonist L-158,809 or the AT2 antagonist PD , indicating that these compounds had no effect on Ang-(1-7) antiproliferative effects.23
However, thymidine uptake was not significantly different from control with Ang-(1-7) plus the angiotensin analog [Sar1, Thr8] Ang II or the Ang-(1-7) analog [D-Ala7]Ang-(1-7), indicating that these compounds abolish Ang-(1-7) antiproliferative effects.
These data suggest that Ang-(1-7) suppresses VSMC growth through activation of a non-AT1, non-AT2, D-Ala-Ang-(1-7)-sensitive receptor. This receptor has been designated the AT(1-7) receptor.10

12. Ang-(1-7) receptor blockade elevates blood pressure of salt-depleted hypertensive rats
Content Points:
Dietary salt depletion stimulates the activity of the RAS and potentiates the antihypertensive action of angiotensin converting enzyme inhibitors and angiotensin II receptor antagonists.
IV administration of a selective AT(1-7) receptor antagonist [(D-Ala)-Ang-(1-7)] to conscious spontaneously hypertensive rats (SHR) and transgenic hypertensive [mRen-2]27 rats maintained on a reduced dietary intake of salt for 12 days produced a dose-dependent increase in arterial pressure.24 The AT(1-7) receptor antagonist had no effect on hypertensive animals maintained on a normal salt intake.
These findings illustrate an endogenous role of Ang-(1-7) in modulating the pressor actions of increased Ang II in response to dietary salt intake.

13. ACE inhibition increases NO formation via bradykinin
Content Points:
In isolated dog coronary microvessels, captopril (an ACE inhibitor with low tissue specificity), enalaprilat (tissue specificity similar to captopril), and ramiprilat (relatively high tissue specificity) caused increases in NO production of 66%, 83%, and 97% (when given alone), respectively.25 These changes were blocked by addition of the B2 receptor blocker icatibant, by an inhibitor of endothelial NO synthase (L-NAME), and by inhibitors of bradykinin synthesis (TI, DCIC, aprotinin).
These changes in NO synthesis were accompanied by reductions in myocardial oxygen consumption (not shown on the slide). The changes in myocardial oxygen consumption were also blocked by B2 receptor blockade.
The investigators concluded that there is a local KKS in coronary microvessels, and that inhibition of kinin degradation with tissue ACE inhibition increases endothelium-derived NO formation, which reduces myocardial oxygen consumption.

14. Effect of selective or combined blockade of ACE and AT1 receptors on Ang-(1-7)
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Chronic ACE inhibition augments plasma concentrations of Ang-(1-7) in hypertensive subjects26 and animals with genetic forms of high blood pressure.27,28
Yamada et al administered a 15-minute infusion of Ang-(1-7) to 21 spontaneously hypertensive rats (SHR), 18 Sprague-Dawley (SD) rats, and 36 mRen-2 transgenic (TG+) rats (ie no renin).27
Lisinopril, losartan, or both agents in combination significantly increased plasma levels of Ang-(1-7) when compared with corresponding control (vehicle-treated) rats (P < 0.01). Baseline plasma Ang-(1-7) levels were higher, however, in TG+ rats given lisinopril than in similarly treated SD rats.
Among strains, the highest values of Ang-(1-7) were observed in TG+. Within each strain, however, the highest concentrations of Ang-(1-7) were present in rats treated with lisinopril either alone or in combination with losartan. Compared with control rats, the final plasma concentrations of Ang-(1-7) in lisinopril-treated rats increased by 68% in SD (P < 0.02), 76% in SHR (P < ), and 55% (P < 0.005) in TG+.
These data show that ACE is the predominant enzyme involved in the metabolism of Ang-(1-7).

15. Ang-(1-7) contributes to antihypertensive effects of ACE inhibitors and ARBs
Content Points:
Iyer et al studied the effects of IV infusions of a specific, monoclonal Ang-(1-7) antibody (Ab) on the blood pressure of SHR chronically treated with combined oral lisinopril and losartan.28 Infusion of the Ang-(1-7) Ab caused partial and significant reversal of the fall in arterial pressure obtained by chronic inhibition of the formation and activity of Ang II. The specificity of the response was shown by the demonstration that co-infusion of the non-selective Ang II receptor antagonist ([Sar1, Thr.8]-Ang II) had no further effect on the blood pressure of these rats.
This finding indicates that Ang-(1-7) contributes to the antihypertensive mechanisms stimulated by blockade of ACE and AT1 receptors. These data further suggest that Ang-(1-7) may be the primary mechanism leading to increased tissue bradykinin and nitric oxide activity, the final signaling pathways determining the beneficial antihypertensive effects of RAS blockade.

16. ACE inhibition increases vasodilation by a bradykinin mechanism in response to flow-mediated dilation
Content Points:
Hornig et al studied the vascular effects of IV quinaprilat (a tissue-avid ACE inhibitor) with or without B2 receptor blockade in 10 healthy volunteers.29 The ACE inhibitor increased endothelium-dependent vasodilation, while coinfusion of quinaprilat and a B2 receptor blocker resulted in a decrease.
This finding indicates that tissue bradykinin is involved in the vascular effects of ACE inhibitors. Furthermore, when the B2 receptor blocker was infused under basal conditions, some endothelium-dependent vasodilation was lost, indicating that local bradykinin activity is responsible for a component of physiologic flow regulation.

17. Effect of ACE inhibition on FMD in patients with CHF
Content Points:
Further insight into the role of tissue bradykinin in flow regulation was provided by another study conducted by Hornig et al, comparing the effects of IV quinaprilat and enalaprilat in 30 patients with heart failure.30 The doses used were equipotent with regard to plasma Ang II blockade as evidenced by BP. Quinaprilat increased endothelium-dependent dilation from 6.9 ± 0.6% to 10.2 ± 0.6% (P < 0.01 versus baseline), while enalaprilat had no effect on dilation even when given in ascending doses.
The investigators concluded that ACE inhibitors with more potent tissue effects (like quinaprilat) enhance endothelial release of NO and EDHF via increased bioavailability of local bradykinin.

18. Bradykinin contributes to ACE inhibitor antihypertensive effects
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Gainer et al studied the effects of ACE inhibition, AT1 receptor blockade, and B2 receptor blockade in 20 individuals with normal blood pressure and 7 hypertensive patients.31 All study participants were salt depleted to ensure maximal stimulation of the RAS and KKS. Each participant received a single dose of the following: oral captopril 25 mg plus IV saline; oral captopril plus IV B2 receptor blockade; oral losartan 75 mg plus IV saline; and oral placebo plus IV saline.
B2 receptor blockade attenuated the blood pressure lowering effect of the ACE inhibitor by 53%. The blood pressure level achieved with captopril plus a B2 receptor blocker was similar to that achieved with AT1 blockade alone. This effect was independent of the presence or absence of hypertension and was observed in both black and white subjects (not shown on slide).
Investigators concluded that bradykinin contributes to the antihypertensive effect of ACE inhibitors. The residual vasodilator effect observed after the combined treatment with ACE inhibition and B2 receptor blockade implicates a contribution of Ang-(1-7) to the overall antihypertensive effect of captopril.

19. Regression of LVH with ACE inhibitors (rat model): Role of bradykinin
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Linz et al induced hypertension and left-ventricular hypertrophy (LVH) in rats by aortic banding.32 The tissue ACE inhibitor ramipril, given at a dose of 1 mg/kg/day for 6 weeks, prevented the increase in blood pressure (not shown) and development of LVH. A lower dose of 10 mg/kg/day for 6 weeks did not prevent the increase in blood pressure and had no effect on plasma ACE activity but prevented LVH.
Coadministration of a B2 receptor blocker abolished the antihypertensive and antihypertrophic effects of the ACE inhibitor at both doses, indicating that these effects are bradykinin-mediated.

20. Bradykinin and antihypertrophic effects of ACE inhibition: Evidence from a canine direct shock model
Content Points:
McDonald et al used direct current shock to produce LV necrosis and subsequent dysfunction in dogs.33 Twenty-four hours after direct current shock, dogs were assigned to a control group, a group receiving ramipril 10 mg bid, and a group receiving ramipril 10 mg bid plus an IV B2 receptor blocker. The increase in LV mass in the control group was similar to that observed in the ACE inhibitor plus B2 receptor blocker group (0.73 g/kg versus 0.75 g/kg).
In contrast, there was a net decrease in LV mass in the ramipril alone group (–0.48 g/kg, P = versus control and P = versus the group receiving concomitant B2 receptor blocker), providing further support for the role of tissue bradykinin in the antihypertrophic effects of ACE inhibition.
In a separate study in the same model, an AT1 receptor blocker failed to prevent remodeling.34

21. Reduction in infarct size with ACE inhibition: Involvement of bradykinin
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Hoshida et al induced myocardial infarction via coronary occlusion in rabbits fed either normal- or high-cholesterol diets for 10 weeks.35 Vascular ACE activity increased with cholesterol feeding. ACE activity was also greater in ischemic myocardium than in nonischemic myocardium.
As shown on the slide, treatment with quinapril 3 mg/kg/day (+) ameliorated the severity of ischemic injury in both the normal and cholesterol-fed groups. Both the B2 receptor blockade and NO synthase inhibition abolished these effects of quinapril.
Treatment with quinapril also significantly reduced the ACE activity in ischemic and nonischemic myocardium (not shown on slide).
In a separate study in the same model, AT1 receptor blockade failed to prevent remodeling.36

22. Comparative effects of ACE inhibition and AT1 receptor blockade on fibrinolytic balance
Content Points:
Bradykinin is a potent inducer of tPA synthesis (via a B2 receptor-mediated mechanism),3,37 while there are data suggesting that Ang II increases expression of PAI-1 through the AT4 receptor.38
Brown et al compared the effects of ACE inhibition with quinapril and AT1 receptor blockade with losartan on fibrinolytic balance in 25 normotensive subjects who were salt-depleted to ensure stimulation of the RAS and KKS.39 ACE inhibition decreased PAI-1 antigen and PAI-1 activity (P = 0.03 and P = 0.018, respectively).
Since tPA frequently circulates as a complex with PAI-1, a slight decrease in circulating tPA level might have been expected. However, tPA levels with ACE inhibition were unchanged, which suggested a net increase in tPA synthesis.
Thus, ACE inhibition may favorably affect fibrinolytic balance via decreased PAI-1 and increased tPA.
AT1 receptor blockade had no effect on PAI-1 but was associated with significantly decreased tPA levels (P = 0.03), implying an undesirable change in fibrinolytic balance.

23. Role of bradykinin in risk reduction: Implications for clinical practice
Content Points:
In summary, many vasculoprotective effects of tissue ACE inhibitors are either partially or fully suppressed by blockade of B2 receptors.
Vasodilator actions of bradykinin following inhibition of ACE may be produced, in part, by increased levels of Ang-(1-7).
Increased tissue bradykinin appears to be an important beneficial mechanism of the action of ACE inhibitors. In fact, data suggest that this may be a more important beneficial mechanism than reduction in circulating Ang II production.