1. Interactions of lipids and RAS: Mechanisms for risk reduction
Content Points:
Accumulating data from recent experimental and clinical studies suggest that the pathways by which Ang II and LDL-C lead to atherosclerosis may frequently overlap.
The first part of this slide program covers experimental data on potential molecular mechanisms. An understanding of these basic data is necessary to approach the clinical findings covered in the second part of this program.

2. Section 1: Interactions of lipids and RAS: Potential molecular mechanisms
Effect of ACE inhibition on blood pressure and atherosclerosis progression in apo E deficient mice
Content Points:
Hayek et al examined the relation between blood pressure reduction and atherosclerosis progression in apolipoprotein E (apo E) deficient mice.1 This model develops severe hypercholesterolemia on a low-fat, low-cholesterol diet.
As shown, high-dose ACE inhibitor (ACEI, fosinopril 25 mg/kg/day) reduced mean blood pressure from 93 mm Hg before treatment to 70 mm Hg at 12 weeks (P < versus placebo). The average lesion area at 12 weeks was also significantly lower in ACEI-treated mice (P < versus placebo).

3. Effect of ACE inhibition on LDL oxidation in apo E deficient mice
Content Points:
Hayek et al also examined the effect of high-dose ACEI on LDL oxidation as measured by levels of thiobarbituric acid reactive substance (TBARS) or by formation of conjugated dienes.1 As shown, the ACEI-treated mice demonstrated a 90% reduction in TBARS versus the placebo group (P < 0.001). ACE inhibition also suppressed formation of conjugated dienes.
These findings suggest that Ang II promotes LDL oxidation.

4. Effect of ACE inhibition, AT1 receptor blockade, and hydralazine on LDL oxidation
Content Points:
Hayek et al conducted a second study with a low dose of ACEI (fosinopril 5 mg/kg/day) or AT1 receptor blocker (AT1 RB, losartan 5 mg/kg/day) that did not lower blood pressure.1 Control animals were given either placebo or a dose of hydralazine that lowered blood pressure comparably to the higher dose of ACEI.
LDL oxidation as measured by levels of TBARS or by formation of conjugated dienes was suppressed by low-dose ACEI, somewhat suppressed by AT1 RB, and unaffected by placebo or hydralazine.
Thus, Ang II increases oxidation of LDL and this action is mediated by AT1 receptors. This suggests that the antiatherosclerotic effects of ACEIs may be due at least in part to direct inhibition of LDL oxidation and other Ang II actions in the vascular wall.

5. Ang II and atherosclerosis in apo E deficient mice
Content Points:
Keidar et al also examined the atherogenic effects of Ang II in apo E deficient mice, but in this case administered Ang II via intraperitoneal injection (0.1 mL of 10-7 M).2
Mice that received Ang II once daily for 30 days demonstrated increased lesion area versus controls (P < 0.001), confirming the pro-atherosclerotic effects of Ang II.

6. Effect of Ang II and ACE inhibition on cholesterol biosynthesis
Content Points:
Using macrophages harvested from the peritoneum after injection of Ang II, Keidar et al were also able to demonstrate that Ang II stimulated macrophage cholesterol biosynthesis (P < 0.01 versus control).2
In mice that received ACEI (fosinopril 25 mg/kg/day), a reduction in cholesterol biosynthesis of approximately 70% was observed (P < 0.01 versus control).

7. Role of AT1 receptor in cholesterol biosynthesis
Content Points:
In an accompanying in vitro study, Keidar et al looked at the effects of AT1 and AT2 receptor blockade on cholesterol synthesis in cultured macrophages harvested from the peritoneal fluid of BALB/c mice.2 Macrophages were pre-incubated for 1 hour with an AT1 RB (losartan) or the investigational AT2 RB PD prior to addition of Ang II and a further 18 hours of incubation.
Control macrophages demonstrated stimulation of cholesterol synthesis by Ang II. This effect was inhibited by an AT1 RB but not by an AT2 RB.

8. ACE and Ang II in the aorta following a high-cholesterol diet (6 months)
Content Points:
The role of AT1 receptors in mediating Ang II pro-atherosclerotic effects was demonstrated in another model by Miyazaki et al, who examined the effects of an ACEI (trandolapril) and an AT1 RB (HR 720) on atherosclerosis progression in Cynomolgus monkeys fed a high-cholesterol diet for 6 months.3
Plasma renin and ACE activities showed no difference between the normal and high-cholesterol diet groups. However, ACE activity and Ang II concentration were significantly increased in the aorta of monkeys fed the high-cholesterol diet (P < 0.05). The ACEI significantly reduced ACE activity and Ang II concentration in the aorta.

9. Antiatherosclerotic effect of ACE inhibition and AT1 receptor blockade
Content Points:
Miyazaki et al then showed that an ACEI and an AT1 RB reduced formation of atherosclerotic lesions in animals fed a high-cholesterol diet (P < 0.05).3

10. Superoxide release in hyperlipidemic rabbit aortas: Effects of endothelium removal
Content Points:
Warnholtz et al studied superoxide production in the aortas of rabbits fed a diet containing 0.5% cholesterol.4 In their first study, they looked at the effects of endothelium removal on vascular superoxide production in control and Watanabe rabbits (hypercholesterolemia secondary to an LDL-receptor defect).
As shown on the slide, rates of superoxide production were increased approximately 2-fold in aortic segments from Watanabe rabbits compared with controls (P < 0.05). This increase was abolished by removal of the endothelium from the Watanabe segments (P < 0.05 versus segments with endothelium).
NADH oxidase activity was significantly increased in Watanabe rabbits versus controls, while NADPH activity was similar in the 2 groups. Endothelial removal decreased NADH oxidase activity (not shown on slide).
These findings suggest that hypercholesterolemia is associated with increased superoxide production secondary to activation of vascular NADH oxidase.

11. Effect of AT1 receptor blockade on superoxide production and NADH oxidase activity
Content Points:
Warnholtz et al then measured the effects of an AT1 RB (Bay ) on superoxide production and NADH oxidase activity in aortas from rabbits fed a normal diet (controls) and cholesterol-fed rabbits.4
AT1 receptor blockade reduced superoxide production and inhibited NADH oxidase activity in cholesterol-fed animals.
The investigators concluded that in hypercholesterolemic animals, NADH oxidase represents a major vascular source of superoxide and that increased vascular levels of Ang II may cause increased NADH oxidase activity.

12. Potential interaction of LOX-1 and Ang II in endothelial cells
Content Points:
As we have seen, LDL-mediated AT1 receptor upregulation leads to production of ox-LDL.
Ox-LDL, in turn, upregulates its own receptor (LOX-1), which leads to activation of mitogen-activated protein kinase (MAPK) and nuclear factor-kB (NF-kB).5 NF-kB is an important transcription factor in the expression of genes for many cytokines, enzymes, and adhesion molecules.6 Ox-LDL also increases expression of monocyte chemoattractant protein-1 (MCP-1), a factor that plays a crucial role in monocyte adhesion to endothelial cells.7
It has been hypothesized that the adverse effects of ox-LDL on the vascular wall may be mediated by LOX-1.5,7

13. Section 2: Interactions of lipids and RAS: Clinical perspectives
Prevalence of dyslipidemia by blood pressure stage
Content Points:
The association of hypertension with hyperlipidemia has been noted in several population studies. However, these studies used older, less-rigorous definitions than are currently recommended. Recently, Lloyd-Jones et al evaluated 4962 subjects from the Framingham Heart Offspring Study and cross classified them according to the Sixth Report of the Joint National Committee on the Prevention, Detection, Evaluation, and Treatment of High Blood Pressure (JNC VI).8 Data were collected from subjects examined between 1990 and 1995.
The prevalence of dyslipidemia (defined as total cholesterol >240 mg/dL, HDL-C <35 mg/dL, or currently receiving lipid-lowering therapy) increased with increasing blood pressure in men and women. On average, over 40% of men and 33% of women with blood pressure >145/>90 mm Hg were also dyslipidemic.
These data demonstrate that hypertension and hypercholesterolemia are frequently associated, even when current rigorous definitions are used. The data also suggest that individuals with hypertension may be more likely to become dyslipidemic over time.

14. Cholesterol lowering reduces blood pressure response to mental stress
Content Points:
Sung et al examined the blood pressure response to a standard mental arithmetic test in 37 healthy normotensive subjects with hypercholesterolemia (mean total cholesterol 263 mg/dL) and 33 normotensive subjects with normal cholesterol levels.9 None of the hypercholesterolemic group were receiving lipid-lowering therapy prior to study entry.
In the first part of the study, the blood pressure response to the arithmetic test was determined. The blood pressure response during the arithmetic test was significantly higher in the hypercholesterolemic group compared with the normocholesterolemic group ( versus mm Hg, respectively, P < 0.05).
In the second part, the hypercholesterolemic group was divided into 2 subgroups that received either 6 weeks of lovastatin or 6 weeks of placebo in a double-blind, crossover design. There were 26 evaluable patients in this part of the study.
As shown on the slide, statin treatment resulted in significant reductions from baseline in total cholesterol and LDL-C (P = for both comparisons). Compared with placebo, statin treatment was associated with lower mean systolic blood pressure prior to ( versus mm Hg, P = 0.07) and during the arithmetic test ( versus mm Hg, P < 0.05).
Diastolic blood pressure changes were not significantly correlated with lipid lowering.
These findings demonstrate that individuals with hypercholesterolemia have exaggerated systolic blood pressure responses to mental stress and that lipid lowering improves the systolic response to stress.

15. Combined and distinct vascular effects of ACE inhibitor and statin: Effect on blood pressure
Content Points:
Nazzaro et al examined the effects of lipid lowering on blood pressure in a study of 30 subjects with coexisting hypertension and hypercholesterolemia.10 Subjects received placebo for 4 weeks and then were divided into 2 groups:
. First group (N = 15): mean blood pressure 167/97 mm Hg, mean total cholesterol 260 mg/dL; received a statin (simvastatin 10 mg) for 14 weeks
. Second group (N = 15): mean blood pressure 164/100 mm Hg, mean total cholesterol 259 mg/dL; received an ACEI (enalapril 20 mg) for 14 weeks
After the monotherapy phase, each group received both medications for an additional 14 weeks. Blood pressure was measured during stressful stimuli (Stroop color test plus the cold pressor forehead test).
As expected, enalapril lowered blood pressure. Interestingly, however, simvastatin also lowered blood pressure (although to a lesser extent) and the combination of both medications achieved greater blood pressure reduction than either alone.

16. Combined and distinct vascular effects of ACE inhibitor and statin: Total regional (forearm) hemodynamic reactivity
Content Points:
Nazzaro et al also measured postischemic forearm blood flow and minimal vascular resistance to evaluate the effects of mental stress on vasodilatative capacity and vascular structure, respectively.10
These parameters demonstrated the same trends as blood pressure: both monotherapies improved these parameters, but combination therapy was associated with a superior improvement than either monotherapy.
This finding indicated that the underlying mechanism involved effects of ACEI and statin on the vascular wall.

17. Hypercholesterolemia-induced AT1 receptor regulation
Content Points:
Nickenig et al assessed the blood pressure response to Ang II infusion in normocholesterolemic (mean total cholesterol 181 mg/dL) and hypercholesterolemic men (mean total cholesterol 294 mg/dL).11
As expected, Ang II caused an increase in blood pressure in both groups. However, the blood pressure response was exaggerated in the hypercholesterolemic group and this response could be blunted by LDL-C lowering with statins (atorvastatin, simvastatin).
The investigators also measured platelet AT1 receptor density and found that there was a linear relationship between AT1 receptor expression and LDL-C concentration. They concluded that hypercholesterolemia induces AT1 receptor overexpression and, thereby, enhances response to Ang II.

18. TREND: Effects of ACE inhibition in endothelial response according to LDL-C level
Content Points:
The Trial on Reversing Endothelial Function (TREND) was conducted in 129 patients with CAD and preserved left ventricular function.12 Coronary artery diameter responses to stepwise infusions of acetylcholine (10-6 and 10-4 mol/L) were measured by quantitative coronary angiography. The main finding of this trial was that 6 months of an ACEI (quinapril, which has a high affinity for tissue ACE) significantly prevented constriction in coronary artery segments relative to placebo.
In a subsequent subgroup analysis, the acetylcholine response was classified according to mean LDL-C level.13 As shown, the ACEI had greater efficacy in improving endothelial function in the group with LDL-C >130 mg/dL than in the group with LDL-C <130 mg/dL. In addition, placebo patients with LDL-C >130 mg/dL demonstrated a trend towards worsening endothelial function.

19. QUIET: Effect of quinapril on CAD progression according to LDL-C level
Content Points:
The Quinapril Ischemic Events Trial (QUIET) was conducted in 1750 post-PCI patients who received ACEI (quinapril) or placebo.14 This trial was designed with 2 components: a 3-year clinical end point trial and a 3-year angiographic follow-up study of a subset of the clinical trial patients. The primary angiographic end point was angiographic progression or nonprogression of atherosclerosis.
While the primary angiographic end point was neutral, there was a different finding when the results were classified according to LDL-C level.
As shown on the slide, in patients with LDL-C <130 mg/dL, quinapril had only a slight effect on progression of disease.15 However, in patients with LDL-C >130 mg/dL, there was significantly less progression in the ACEI group (P = 0.08).
Thus, the rapid disease progression seen in placebo patients with higher LDL-C levels did not occur in ACEI-treated patients. As in the TREND study, the ACEI appeared to have greater efficacy in patients with higher LDL-C levels.

20. ACE genotype determines LDL-C response to statin
Content Points:
The previous slides presented data suggesting that lipids can affect blood pressure and other RAS actions. The data shown on this slide suggest that, in turn, the RAS may affect responses to lipid-lowering agents.
The Lipoprotein and Coronary Atherosclerosis Study (LCAS) was conducted in 429 patients with CAD and at least 1 lesion of 30% to 75% diameter stenosis.16 Subjects were randomized to statin (fluvastatin) or placebo for 2.5 years and the primary end point was change in minimum lumen diameter as assessed by quantitative coronary angiography.
Marian et al studied response to statin therapy according to ACE insertion/deletion (I/D) phenotype in the LCAS population.16 As shown on the slide, subjects with DD, ID, or II phenotypes achieved reductions of 31%, 25%, and 21%, respectively. There was a significant genotype-by-treatment interaction (P = 0.005).
A similar result was obtained for reduction in total cholesterol (not shown on slide). Subjects with DD phenotype also had a higher rate of regression and a lower rate of progression than subjects with the other 2 phenotypes.

21. Hypertension and hypercholesterolemia: Interactions and potential mechanisms
Content Points:
In summary, hypertension and hypercholesterolemia, the major risk factors for atherosclerotic disease, are frequently associated and data from clinical studies suggest the existence of lipoprotein-neurohormonal interactions that may adversely affect vascular structure and reactivity.
Data from preclinical studies suggest that the vascular RAS may be involved via LDL-mediated AT1 receptor upregulation, which leads to production of ox-LDL. Adverse effects of ox-LDL on the vascular wall may be mediated by the LOX-1 receptor.
These findings extend our understanding of the interplay among risk factors to synergistically increase cardiovascular risk and of the antiatherosclerotic effects of local ACE inhibition to reduce cardiovascular risk.