1. Beyond LDL Cholesterol: Reduction of Small Dense LDL and of Oxidized LDL With Combined Lipid Therapy
Rabih R. Azar, MD, MSc, FACC
Associate Professor of Medicine
Division of Cardiology
Hotel Dieu de France
Saint Joseph University

2. Risk of CAD according to LDL and HDL

5. IS LDL CHOLESTEROL THE “ONLY” PLAYER IN ATHEROSCLEROSIS?

6. Stages of Atherosclerosis
LUMEN
LDL
INTIMA
Here the proposed mechanism of action of Lp-PLA2 in coronary heart disease is presented in graphic form
Lp-PLA2 co-traffics in circulation with LDL-cholesterol
LDL penetrates the intima where it undergoes oxidative modification
MEDIA

7. Stages of Atherosclerosis
LUMEN
Adhesion
molecules
Oxidized LDL
Lp-PLA2
INTIMA
Oxidized LDL is the favored substrate for the enzyme Lp-PLA2
Lp-PLA2 acts on oxidatively-modified phosphatidylcholine to produce lysophosphatidylcholine (lyso-PC) and oxidized fatty acid (oxFA)
Lyso-PC triggers a host of pro-inflammatory activities, including the upregulation of adhesion molecules such as VCAM-1 and ICAM-1
Lyso-PC
OxFA
MEDIA

8. Stages of Atherosclerosis
LUMEN
Monocytes
Plaque
formation
Cytokines
Adhesion
molecules
Oxidized LDL
Lp-PLA2
Foam cell
INTIMA
The pro-inflammatory actions of lysophosphatidylcholine (lyso-PC), as well as those of oxidized fatty acid (oxFA), result in a cascade of events leading to atherosclerotic plaque formation.
The upegulation of adhesion molecules and the expression of cytokines result in the recruitment of monocytes to the intimal space
Monocytes differentiate into macrophages that engulf the oxidized LDL, developing into foam cells
Foam cells aggregate to form a fatty streak covered by a fibrous cap
Cytokines and proteases secreted by the plaque break down the collagen within the fibrous cap, making it weak and prone to rupture
Plaque rupture can result in the formation of an atherothrombotic clot that can block the artery, resulting in a coronary event
This model also suggests that Lp-PLA2 is a novel therapeutic target; if its activity could be blocked, the sequence of events leading to plaque formation and rupture could be interrupted
Macrophage
Lyso-PC
OxFA
MEDIA

9. Stages of Atherosclerosis
LUMEN
LDL
Monocytes
Plaque
formation
Cytokines
Adhesion
molecules
Oxidized LDL
Lp-PLA2
Foam cell
INTIMA
The pro-inflammatory actions of lysophosphatidylcholine (lyso-PC), as well as those of oxidized fatty acid (oxFA), result in a cascade of events leading to atherosclerotic plaque formation.
The upegulation of adhesion molecules and the expression of cytokines result in the recruitment of monocytes to the intimal space
Monocytes differentiate into macrophages that engulf the oxidized LDL, developing into foam cells
Foam cells aggregate to form a fatty streak covered by a fibrous cap
Cytokines and proteases secreted by the plaque break down the collagen within the fibrous cap, making it weak and prone to rupture
Plaque rupture can result in the formation of an atherothrombotic clot that can block the artery, resulting in a coronary event
This model also suggests that Lp-PLA2 is a novel therapeutic target; if its activity could be blocked, the sequence of events leading to plaque formation and rupture could be interrupted
Macrophage
Lyso-PC
OxFA
MEDIA

10. Risk of CAD according to LDL and HDL

11. Cholesterol distribution in CHD and non-CHD populations
In spite of major advances made in the screening, detection, and management of heart disease, a major need exists for more accurate ways to predict CV risk
Approximately 50% of individuals diagnosed with coronary artery disease do not have high blood cholesterol levels
Therefore, other factors must be involved
Framingham Heart Study — 26-year follow-up
35% of CHD occurs in people with TC considered optimal (<200mg/dL)
No CHD
CHD
Most of the focus on the prevention and treatment of coronary heart disease has been on the role of lipids, particularly cholesterol, and the identification and treatment of hypertension
It has been shown that approximately 35% of individuals diagnosed with coronary artery disease have blood cholesterol levels that are within the recommended desirable range (<200 mg/dL)
Data from the Framingham Heart Study 26-year follow-up shows that 50% of coronary heart disease cases occur in patients who have below-average total cholesterol levels (<240 mg/dL)
Factors other than traditional risk factors appear to be involved in the development of atherosclerosis
Castelli W. Atherosclerosis 1996;124(suppl):S1-S9
150
200
250
300
Total cholesterol (mg/dL)
Adapted from Castelli W. Atherosclerosis 1996

12. Prevalence of major risk factors in men with CHD
Traditional risk factors are a useful first step in determining who could be at risk for a coronary event
Exposure to one or more CHD risk factors is also highly prevalent in individuals who do not develop clinical CHD
Less than 10% of patients have 3 or 4 major risk factors
Secondary testing can be used to further stratify individuals for CHD risk
4 major risk factors
3 major risk factors
0 major risk factors
2 major risk factors
62.4% to 1 major risk factor
In a meta-analysis compiling data from 14 clinical studies, it was determined that the majority of patients with CHD had 0 to 1 of the major modifiable risk factors associated with CHD
Conversely, very few of these individuals had 3 or 4 of the major modifiable risk factors
A similar analysis determined that the vast majority of clinical trial patients who did not develop CHD also had at least one major risk factor above desirable levels
Assessment of the major modifiable risk factors is a critical first step in identifying individuals with elevated CHD risk, but does not always provide the necessary diagnostic and prognostic information to determine the clinical management strategy for every individual patient
Further testing may be useful in determining the absolute risk of an individual with a single major risk factor
Khot, et al. JAMA. 2003;290:
1 major risk factor
N=87,869
4 Major modifiable risk factors: hypertension, smoking, hypercholesterolemia, diabetes
Khot, et al. JAMA. 2003

17. Similar LDL Levels Do NOT Mean Similar Risk
Phenotype B
LDL = 81
Phenotype A

18. Small Dense LDL
LDL particles are heterogeneous in size, density and composition. Individual can be classified according to their predominant LDL size into:
Phenotype A: large particle size > 26.3 nm in diameter
Phenotype B: Small particle size < 25.8 nm
Phenotype I: Intermediate particle size, nm
LDL phenotype B is in part genetically determined
LDL phenotype B is also influenced by acquired conditions such as:
Obesity
Type 2 DM
Metabolic syndrome

19. Mechanism of Increased Atherogenicity
Direct Mechanisms:
Enhanced oxidative susceptibility
Reduced clearance by LDL receptors in the liver with increased LDL receptor-independent binding in the arterial wall
Endothelial dysfunction
Indirect Mechanisms:
Inverse relationship with HDL-C
Marker for accumulation of atherogenic triglyceride remnant particles
Insulin resistance

21. Inverse Relationship Between Small Dense LDL and HDL-C

22. The “Good” and “Bad” LDL
Large LDL subclasses 1 and 2 are the “good or normal” LDL that are responsible for the transport of cholesterol throughout the body
Small LDL subclasses 3 through 7 are “bad or abnormal” that are easily oxidized and promote cardiovascular disease

23. Role of Small Dense LDL in Predicting Ischemic Heart Disease: The Quebec Cardiovascular Study
2034 men; all initially free of IHD
Followed for 5 years
108 first IHD recorded
Polyacrylamide gradient gel electrophoresis was used to measure small dense LDL
Circulation 2001, 104:2295-9

24. The Quebec Cardiovascular Study: Risk Factors for IHD
Variable
IHD free
IHD Cases p
Age
<0.001
BMI
0.07
Systolic BP
< 0.001
Type 2 DM
4.4%
14.8%
0.003
Cholesterol, mmol/L
HDL, mmol/L
0.002
Cholesterol/HDL ratio
TG, mmol/L
Apo B, mg/L
Lp(a), mg/dL
0.01

25. Small Dense LDL (<255Å) is the Best Predictor of Ishemic Heart Disease (IHD) in a Multivariate Model
RRs of IHD according to baseline LDL, apoB, and TG and small dense LDL (proportion of small dense LDL above or below median of 39.6%). RR were adjusted for age, BMI, BP, DM, medication use at baseline and family history.
Circulation 2001, 104:2295-9

26. The Quebec Cardiovascular Study RESULTS:
Among all lipid parameters, small dense LDL (< 255 Å) showed the strongest association with the risk of IHD (RR in men = 4.6; p < 0.001)
This was independent of all nonlipid risk factors and of LDL cholesterol, HDL, TG and Lp(a)

27. LDL particle number and risk of future cardiovascular disease in the Framingham Offspring Study (J Clin Lipidol 2007;1:583-92)

30. Stages of Atherosclerosis
LUMEN
Monocytes
Plaque
formation
Cytokines
Adhesion
molecules
Lp-PLA2
Foam cell
Oxidized LDL
INTIMA
The pro-inflammatory actions of lysophosphatidylcholine (lyso-PC), as well as those of oxidized fatty acid (oxFA), result in a cascade of events leading to atherosclerotic plaque formation.
The upegulation of adhesion molecules and the expression of cytokines result in the recruitment of monocytes to the intimal space
Monocytes differentiate into macrophages that engulf the oxidized LDL, developing into foam cells
Foam cells aggregate to form a fatty streak covered by a fibrous cap
Cytokines and proteases secreted by the plaque break down the collagen within the fibrous cap, making it weak and prone to rupture
Plaque rupture can result in the formation of an atherothrombotic clot that can block the artery, resulting in a coronary event
This model also suggests that Lp-PLA2 is a novel therapeutic target; if its activity could be blocked, the sequence of events leading to plaque formation and rupture could be interrupted
Macrophage
Lyso-PC
OxFA
MEDIA

31. Lipid entry into the vascular wall Content points:
After penetrating the arterial wall, LDL adheres to proteoglycans, which entrap it and increase its susceptibility to oxidation by lipoxygenases (LO), myeloperoxidase (MPO), and inducible nitric oxide synthase (iNOS).1
Scavenger receptors such as CD36 and SRA take up oxLDL, resulting in foam cell formation by macrophages.
VLDL is modified by lipoprotein lipase (LPL) to form remnants that are trapped by proteoglycans, oxidized, and taken up by macrophages.
1 Li AC, Glass CK. The macrophage foam cell as a target for therapeutic intervention. Nat Med ;8:

33. Pro-atherogenic effects of oxidized LDL
Oxidized LDL is degraded at a faster rate than native LDL by macrophages leading to lipid accumulation.
Oxidized LDL is chemotactic to monocytes, smooth muscle cells, and T lymphocytes, and induces T-cell activation and monocyte differentiation.
Oxidized LDL inhibits macrophage motility, potentially trapping macrophages in the artery.
Components of oxidized LDL are cytotoxic to cells.
Oxidized LDL inhibits endothelium-dependent relaxation factor.
Oxidized LDL enhances monocyte adhesion to endothelium.
Oxidized LDL induces the expression of monocyte chemotactic protein-1 and granulocyte-macrophage colony stimulating factors.
Oxidized LDL inhibits the migration of endothelial cells.
Oxidized LDL induces the expression of adhesion molecules on the endothelium.Components of oxidized LDL induces interleukin-1 synthesis and secretion by macrophages

41. Refining cardiac risk assessment in asymptomatic patients
Initial risk assessment and physical examination
Low risk 35%
Intermediate risk 40%
High risk 25%
No major risk factors and low Framingham risk score
At least one major risk factor or family history
Established CHD or CHD risk equivalent
Initial risk assessment and physical examination are the first steps in assessing absolute coronary risk
Once this is done, a risk level should be determined for the individual patient and they should be counseled on their risk status
Low-risk patients (35% of the population) should be provided reassurance of their low risk status and can avoid further risk assessment for about 5 years
High-risk patients (25% of population) are candidates for intensive risk factor intervention and aggressive goals are recommended
High-risk patients can have established CHD, other clinical forms of atherosclerosis, type 2 diabetes, or are older adults with multiple CHD factors
According to the NCEP ATP III guidelines, such patients should have all of their CHD risk factors treated to reduce the risk of CHD and total cardiovascular disease
Intermediate-risk patients (40% of population) do not qualify for the most intensive risk factor interventions; however, they may benefit from non-invasive testing for further risk assessment
These individuals should be counseled about their intermediate CHD risk and offered general dietary and lifestyle modifications in order to reduce their risk. Some of these patients may be eligible to receive cholesterol-lowering drugs
Further non-invasive testing can be useful in determining the most appropriate clinical strategy moving forward
Greenland P, et al. Circulation. 2001;104:
Further testing required to determine appropriate clinical direction
5-year follow up
Intensive risk intervention
CRP; Lp-PLA2; Small dense LDL; Ox LDL
Greenland P, et al. Circ. 2001

42. Effect of Ezetimibe/Atorvastatin Combination on Oxidized LDL-Cholesterol in Patients With CAD or CAD Equivallent
Rabih Azar, Georges Badaoui, Antoine Sarkis, Mireille Azar, Hermine Aydanian, Serge Harb, Guy Achkouty and Roland Kassab
Will be presented on March 14 at the Meeting of the American College of Cardiology in Atlanta, USA
In press: J Am Coll Cardiol March 2010 (abstract)
In press: Am J Cardiol July 2010 (manuscript)
Sponsored by Pharmaline

43. RATIONAL
Ox-LDL is a more potent predictor of cardiovascular risk than standard lipid parameters
The majority of clinical trials have tested the efficacy of lipid lowering therapy against standard lipid parameters
Rare trials: statins decrease ox-LDL
Ezetimibe allows potent reductions of LDL when added to statin therapy
The effect of ezetimibe on ox-LDL has however, never been studied
This effect is important to investigate given the controversy surrounding ezetimibe’s use

44. RATIONAL
Ox-LDL and small dense LDL are more potent predictors of cardiovascular risk than standard lipid parameters
The majority of clinical trials have tested the efficacy of lipid lowering therapy against standard lipid parameters
Few studies have shown that statins decrease ox-LDL. The effect of ezetimibe on ox-LDL is however, unknown
This effect is important to investigate given the controversy surrounding ezetimibe’s use

45. OBJECTIVE
TO EVALUATE THE EFFECT OF ATORVASTATIN 40 mg and of ATORVASTATIN 40 mg + EZETIMIBE ON OX-LDL CHOLESTEROL

46. Prospective, randomized, double-blind, placebo-controlled trial
Effect of Ezetimibe/Atorvastatin Combination on Oxidized LDL-Cholesterol in Patients With CAD or CAD Equivallent
Prospective, randomized, double-blind, placebo-controlled trial
Inclusion criteria:
Patients with CAD
> 50% stenosis on angiography MI
PCI or CABG
Patients with CAD equivalent
Diabetes requiring medications
Peripheral vascular disease
Stroke
Lipid levels were not entry criteria

47. Exclusion Criteria
Therapy with a statin more potent than atorvastatin 20 mg/day (atorvastatin 40 or above, rosuvastatin any dose)
Therapy with ezetimibe, any other cholesterol absorption inhibitor, niacine, fibrate within the last 3 months
MI, CABG, PCI within the last 3 months
Age > 80 years
EF < 35% or CHF with NYHA class > 2
Creatinin clearance < 30 mL/min
CPK or SGPT > 2 times upper normal

48. Study Protocol
The statin taken by the patient was stopped and replaced by atorvastatin 40 mg/day
Patients were then randomized to ezetimibe 10 mg/day vs. placebo
Duration of treatment 8 weeks

49. MEASURMENTS
Standard lipid profile (Total cholesterol, VLDL, LDL, HDL)
LDL subfractions: small dense LDL and large buoyant LDL
Mean LDL particle size
Oxidized LDL
CPK, SGPT

50. End-Points Primary end-point Secondary end-points Safety end-points
Change in ox-LDL
Secondary end-points
Change in small dense LDL
Change in LDL particle size
Safety end-points
Elevation of CPK or SGPT more than twice upper normal

51. Inclusion Criteria n = 50 n = 50 Stenosis > 50% 19 (38%) 23 (46%)
Ezetimibe Placebo
n = 50 n = 50
Stenosis > 50% 19 (38%) 23 (46%)
Prior MI 18 (36%) 12 (24%)
PCI 23 (46%) 15 (30%)
CABG 26 (52%) 21 (42%)
Diabetes 18 (36%) 21 (42%)
Stroke 1 (2%) 1 (2%)
PVD 6 (12%) 5 (10%)
The number of inclusion criteria is superior to 100% because each patient may have more than 1 criterion that defines CAD

52. Baseline Characteristics
Ezetimibe Placebo
n = 50 n = 50
Age (year)
Male 44 (88%) 41 (82%)
Smoking 14 (28%) 12 (24%)
Hyperlipidemia 47 (94%) 43 (86%)
Hypertension 35 (70%) 38 (76%)
Fam. Hist. CAD 21 (42%) 14 (28%)
BMI (kg/m2)

53. Concomitant Medications
Ezetemibe Placebo
n = 50 n = 50
Aspirin 45 (90%) 43 (86%)
Clopidogrel 11 (22%) 11 (22%)
ACE inhb. or ARB 41 (82%) 34 (68%)
Beta-blockers 37 (74%) 35 (70%)
CCB 8 (16%) 18 (36%)*
Nitrates 10 (20%) 11 (22%)
Diuretics 6 (12%) 6 (12%)
OAD 15 (30%) 17 (34%)
Insulin 6 (12%) 6 (12%)
* P = 0.02

54. Statin Use at Baseline
90% of patients were using a statin prior to randomization
Simvastatin 53%
Atorvastatin 30%
Fluva or pravastatin 7%
None 10%

55. Change in LDL in the Placebo/Atorva group
Additional 10% reduction in LDL when shifting to atorva 40 mg/day

56. Change in LDL 20% additional reduction 10% additional reduction
P < 0.001
P < 0.001
20% additional reduction
10% additional reduction
Final LDL levels were lower in ezetimibe vs. placebo; p < 0.001

57. Change in Large, Buoyant LDL in the Placebo/Atorva Group
Additional 10% reduction in LDL when shifting to atorva 40 mg/day

58. Change in Large, Buoyant LDL
P < 0.001
P < 0.001
10% additional reduction
24% additional reduction
Final levels ezetimibe vs. placebo: p = 0.007

59. Change in Small Dense LDL in the Placebo/Atorva Group
Additional 36% reduction in LDL when shifting to atorva 40 mg/day

60. Change in Small Dense LDL
P < 0.001
P < 0.001
36% additional reduction
32% additional reduction
No difference between ezetimibe and placebo in lowering small dense LDL

61. Qualitative Lipid Analysis: Change in Particle Size
Particle Size in Angstrom
Placebo/atorva: p = 0.002
Ezetimibe/atorva p = 0.006
Prevalence of type A phenotype increased from 62% to 70% in the placebo group and from 58% to 74% in the ezetimibe group

62. Change in VLDL in the Placebo/Atorva Group

63. Change in VLDL
P = 0.07
P < 0.001

64. P = ns
P = ns

65. Change in Ox-LDL
p = NS p = 0.02
EZE
Placebo
EZE

67. Correlation Between the Changes in ox-LDL and Total LDL

68. Safety End-Point There was no elevation of CPK or SGPT in any patient of the 2 groups

69. Summary of Results: Change in Different Lipid Parameters
p = p = p = ns p = p = ns p = 0.02
Ezetimibe + Atorva
Placebo + Atorva
% reduction

70. Summary of Results: Effects of Atorvastatin and Ezetimibe on Various Lipid Parameters
LDL
Large LDL
Small dense LDL
Particle size
HDL
VLDL
Ox-LDL
More potent statin
Ezetimbe
The changes induced by statin are quantitative and qualitative
The Changes induced by ezetimibe are only quantitative

71. Conclusions
Aggressive reduction of LDL is currently recommended for high risk patients
Potent statins should be used as first line therapy
In our study, increasing the potency of statin therapy by switching to atorvastatin 40mg:
Did not affect ox-LDL
Resulted in quantitative and qualitative improvement in lipid profile
Was extremely safe and well tolerated
Ezetimibe in combination with atorvastatin:
Significantly decreased ox-LDL
Resulted in quantitative improvement in lipid profile