Over the past two decades, there has been an intense focus on the lowering of low-density lipoprotein (LDL) cholesterol for the treatment of atherosclerotic cardiovascular disease (CVD). Nonetheless, and despite the great progress that has been made in the development of LDL-lowering drugs, existing therapies reduce cardiovascular events by one third, there continues to be significant mortality and morbidity as a result of atherosclerosis.
- Protective Role of HDL particles via Reverse Lipid Transport
The major carriers for cholesterol in the blood are lipoproteins, including low-density lipoprotein (or LDL) particles, and high-density lipoprotein (or HDL) particles. In a healthy human body, there is a balance between the delivery and removal of cholesterol. The LDL particles deliver cholesterol to organs, where it can be used to produce hormones, maintain healthy cells, and be transformed into natural products that assist in the digestion of lipids. The HDL particles remove cholesterol from arteries and tissues to transport it back to the liver for storage, recycling, and elimination through a pathway called “Reverse Lipid Transport (RLT)”.
In the conventional model of atherosclerosis development, an imbalance develops in which there is too much cholesterol delivery by LDL particles (therefore often called “bad cholesterol") or too little removal by HDL particles (better known as “good cholesterol”). When people have a high level of LDL or a low level of HDL, the imbalance results in more cholesterol being deposited in the arteries than being removed. This imbalance in homeostasis can also be exacerbated by, among other factors, age, gender, high blood pressure, smoking, diabetes, obesity, genetic factors, physical inactivity, vascular disease of the extremities or the brain and the consumption of a high-fat diet. The excess cholesterol carried in the blood on LDL particles is deposited throughout the body but frequently ends up in the lining of arteries, especially those found in the heart. Repeated deposits of cholesterol can cause life-threatening complications such as vascular inflammation, plaque formation, and narrowed or blocked arteries. The blocking of these arteries can result in chest pain, heart attack and possibly death. Preventive strategies to limit the build-up of plaque by reducing circulating LDL cholesterol (e.g. statins, bile acid sequestering resins, etc.) have been shown to reduce cardiovascular events by one third and have therefore become the standard of care in cardiovascular risk management.
There is a tremendous unmet need for therapies which can move beyond the focus on LDL reduction alone.
A high LDL-to-HDL cholesterol ratio leads to the build up of lipid-rich plaques in the arterial walls. These plaques are highly vulnerable to rupture, leading to myocardial infarction or a stroke. Conversely, researchers believe that the regression of such plaques would have a major impact on reducing the risk of such acute cardiovascular events.
HDL therapies are designed to increase the number of HDL particles to promote the regression of atherosclerosis through the stimulation of the RLT pathway leading to cholesterol removal from vulnerable arterial plaques. HDL particle-elevating therapies can be combined with other lipid therapies, such as lipid-lowering drugs like statins, to further reduce the risk of cardiovascular events.
- HDL Structure
The population of HDL particles consists of particles of various sizes, depending on how much cholesterol each particle has mobilized and is carrying for transport to the liver for elimination. The newly formed smallest particles (also called pre-β HDL) are essentially “empty” and have the greatest ability to mobilize cholesterol. A natural pre-β HDL is a lipoprotein consisting of a protein called apolipoprotein A-I (apoA-I) and phospholipids interacting together to form a small discoidal particle. In a normal individual, in terms of cholesterol content, pre-β HDL particles represent only 1-10% of the total HDL-cholesterol. These small particles increase in size as they accumulate cholesterol, creating larger, “full,” and mature alpha-HDL particles capable of delivering that collected cholesterol to the liver for elimination.
HDL and Reverse Lipid Transport
As mentioned above, scientists believe the protective function of HDL particles can be explained by their role in the Reverse Lipid Transport (RLT) pathway. The RLT1 pathway is responsible for removal of cholesterol from arteries and its transport to the liver for elimination from the body in four basic steps.
The first step is the removal of cholesterol from arteries by the nascent HDL particle in a process termed “cholesterol removal.” Cholesterol is a membrane constituent that maintains structural domains that are important in the regulation of vesicular trafficking and signal transduction. In most cells, cholesterol is not catabolized. Thus, the regulation of cellular sterol efflux plays a crucial role in cellular sterol homeostasis. Cellular sterol can efflux to extracellular sterol acceptors by both nonregulated, passive diffusion mechanisms as well as by an active, regulated, energy-dependent process mediated by the ABCA1 transporter. In the second step, cholesterol is esterified to form a cholesteryl ester that is more tightly associated with the HDL particle as it is carried in the blood; this process is called “cholesterol conversion/esterification.” The third step is the transport and delivery of that esterified cholesterol to the liver in a process termed “cholesterol transport.” The final step is the transformation and disposal of cholesterol by the liver in a process termed “cholesterol elimination.” RLT is therefore the only natural mechanism capable of transporting cholesterol from vessel wall plaque back to the liver to be eliminated and consequently to reverse the build-up of plaque.
1 - Tall, AR. "An overview of reverse cholesterol transport". Eur. Heart J. 19 Suppl A: A31–5 (February 1998)
- Epidemiology Data – Protective Role of Reverse Lipid Transport in CV Disease
Epidemiological studies have historically demonstrated that the risk of developing cardiovascular disease appeared to be higher in patients with low HDL-cholesterol independent of the level of LDL-cholesterol, even when patients are treated with the best available care. This observation can be explained by the role the HDL particle plays in the RLT pathway, the only natural mechanism capable of removing cholesterol from peripheral tissues and delivering it back to the liver for elimination. HDL particles mediate the flux of cholesterol through the RLT and therefore act to counterbalance the delivery of cholesterol to the vessel wall by the LDL particles. A deficiency in this protective mechanism results in the development of cardiovascular disease.
Clinical trialists and cardiologists have long relied upon the data from several large epidemiological landmark studies as strong evidence of the protective role of the HDL system in attenuating the risk of cardiovascular events.
The Framingham study2, a widely recognized epidemiological study, demonstrated a strong correlation between levels of HDL-cholesterol and the risk of coronary heart disease. As shown in the figure below, the lower the HDL-C level the higher the incidence of cardiovascular events independent of the level of LDL-C. A consistent finding was identified when apoA-I was used as a biomarker instead of HDL-C to demonstrate this inverse relationship with cardiovascular risk.
Figure 4 - Studies Framingham (left) and PROCAM (right)
In the AFCAPS/TexCAPS3 trial authors concluded that HDL-cholesterol should be included in the assessment of risk even at normal LDL-cholesterol concentrations. Another 21-year epidemiological study4 has shown that low HDL-cholesterol, even in the absence of elevated LDL-cholesterol, is an independent risk factor for the development of new or recurrent coronary heart disease events.
The Veterans Affairs HDL Intervention Trial (VA-HIT)5 supported this inverse relationship of HDL-cholesterol with cardiovascular risk and additionally suggested that modulation of HDL-C levels might impact CV risk. This clinical study provided the first evidence that a higher on-treatment HDL-cholesterol was significantly associated with a lower incidence of cardiovascular related events.
According to the National Cholesterol Education Program (NCEP) guidelines6, a low HDL-cholesterol (<35 mg/dL) is considered to be an independent risk factor for cardiovascular disease and has an impact in the decision of clinicians to initiate and maintain LDL-cholesterol-lowering medication (i.e. statins) as part of a total program of minimizing long-term cardiovascular risk.
2 - Gordon, T., Castelli, W. P., Hjortland, M. C., Kannel, W. B. and Dawber, T. R. High-density lipoprotein as a protective factor against coronary heart disease. The Framingham Study. Am. J. Med., 62, 707-714 (1977)
3 - Downs, J. R., Clearfield, M., Weis, S., Whitney, E., Shapiro, D. R., Beere, P. A., Langendorfer, A., Stein, E. A., Kruyer, W. and Gotto, A. M. Jr for the AFCAPS/TexCAPS Research Group Primary prevention of acute coronary events with lovastatin in men and women with average cholesterol levels. Results of AFCAPS/TexCAPS. JAMA, 279, 1615-1622 (1998).
4 - Goldbourt U, Yaari S, Medalie JH. Isolated low HDL cholesterol as a risk factor for coronary heart disease mortality. A 21-year follow-up of 8,000 men. Arterioscler ThrombVasc Biol; 17: 107–13. (1997)
5 - Rubins, H. B., Robins, S. J., Collins, D., Andersen, J. W., Elam, M. B., Faas, F. H., Linares, E., Schaefer, E. J., Schectman, G., Wilt, T. J. and Wittes, J. T. Gemfibrozil for the secondary prevention of coronary heart disease in men with low levels of high-density lipoprotein cholesterol. N. Engl. J. Med., 341, 410-418 (1999)
6 - http://www.nhlbi.nih.gov/guidelines/cholesterol/atglance.pdf
- HDL-C and HDL particle number
However, it has recently become increasingly apparent to the scientific community that the HDL-C measurement does not fully quantify or capture the essence of the HDL-related risk. Initially, Katheresan7 found that Mendelian randomization studies could identify patients with genetically high HDL-C who did not necessarily have greater protection from CV events. Several widely-published recent clinical trials did not demonstrate clinical benefit from medicines that increase HDL-cholesterol (but not necessarily the HDL particle number) - specifically the CETP-inhibitors8 - 9 and Niacin10.
Perceived by many as deleterious for the HDL hypothesis, these findings, despite being initially counterintuitive, do in fact make sense given what we know about the mechanism of RLT. The explanation for this phenomenon lies in understanding that the clinically-used “HDL-cholesterol” test, which is part of the standard “lipid profile” and which has been used by cardiologists, lipidologists, and primary care physicians for decades for the management of cholesterol, does not represent an actual measure of the Reverse Lipid Transport capacity available to regress atherosclerosis or even the actual number of HDL particles; instead the HDL-cholesterol (HDL-C) clinical test quantifies only the amount of cholesterol carried in transit within the total population of HDL particles in circulation. Thus, HDL-C is at best only an indirect marker of the HDL particle number and as a test is prone to bias the interpretation of a patient’s true RLT capacity. By way of example, in evaluating a patient’s given HDL-C value, one cannot determine if the measured amount of cholesterol in the HDL fraction (HDL-C) is distributed among a small number of HDL particles which are full (and therefore containing minimum additional RLT capacity) or if the same amount of cholesterol is distributed among a larger number of HDL particles which are only partially full and thus still have the capacity to receive cholesterol (a large amount of available RLT capacity).
Expert focus has thus begun to shift to find better biomarkers of the RLT system and the functionality of HDL particles, and, given that real-time measures of the cholesterol-carrying capacity of the HDL system at any one time are not yet technically feasible, measures of the HDL particle number and particle size have emerged as reasonable surrogates. It is now perfectly understandable that the reason clinical trialists had historically used the HDL-C test to explore the epidemiology around the protective role of the HDL system in atherosclerosis prevention was not because it was necessarily the best measure of RLT, but rather because it was the only test that had been widely available and had been collected in large populations and in clinical trials. The HDL-C test had in fact proven to be sufficient for the purposes of risk stratification and decisions regarding the initiation of statins for LDL-cholesterol and overall cardiovascular risk management, so it is only now that it has proven to be insufficient for guiding the experimental assessment and manipulation of the RLT system itself. Nicholls and Puri contend eloquently that we should not “throw the baby out with the bathwater” when it comes to elucidating which test to use when quantifying the role of HDL functionality and the HDL particle number on cardioprotection .
Given the growing mechanistic evidence that the HDL particle has an important function in removal of cholesterol from the vessel wall, attention has returned back to the epidemiology studies to identify the best biomarkers which will provide better measures of the HDL particle number and RLT function and which better correlate with cardiovascular risk. The actual HDL particle number would be perhaps the best test, but such an assay requires NMR spectroscopy under carefully-controlled conditions and has therefore been used only a benchtop research tool up until now.
Attention has therefore returned to apoA-I as a more easily-measured surrogate for the particle number, since there is usually a stoichiometric ratio of two molecules of apoA-I per pre-beta HDL particle and a maximum of four molecules of apoA-I per mature, spherical HDL particle.
The INTERHEART study, a 5-year worldwide case control study in more than 10,000 patients, confirmed the relevance of apoA-I levels for predicting the risk of cardiovascular events. Indeed, the apoA-I plasma concentration can be considered as a proxy for small HDL particles, and therefore it correlates a higher level of pre-β HDL particles with a reduction in cardiovascular risks.
This conclusion was further demonstrated in the AMORIS study, a Swedish prospective study in more than 175,000 patients followed for 6 years as well as in the PRIME study in more than 10,000 patients.
The evidence for the protective role of HDL particles was most recently reinforced by results of the MESA study. The Multi-Ethnic Study of Atherosclerosis , an observational clinical study in more than 5,500 men and women, demonstrated that in fact HDL-C did not correlate with the incidence of cardiovascular events, and instead that the best predictor of CV events is an inverse association with the HDL particle number. Specifically, MESA showed that higher HDL particle numbers are directly linked with lower levels of atherosclerosis by carotid intima-media thickness (cIMT) ultrasound measurements as well as with a lower incidence of coronary heart disease events (myocardial infarction, CHD death, and angina).
Mora et al. demonstrated the superior predictive value of the HDL particle number (HDL-P) for cardiovascular events in statin-treated patients in JUPITER, a placebo-controlled trial of rosuvastatin in 17,802 asymptomatic men and women. Among rosuvastatin-allocated individuals, no statistically significant association was seen with CVD in relation to baseline HDL-C (adjusted HR 0.96, 95% CI 0.72-1.29 per 1-SD of 15.3 mg/dL), apoA-I (0.84, 95% CI 0.65-1.10 per 30.2 mg/dL), or size (1.07, 95% CI 0.82-1.39 per 0.52 nm), while baseline HDL-P had a statistically significant association (0.78, 95% CI 0.61-0.99 per 6.32 umol/L). Among rosuvastatin-allocated individuals, on-treatment HDL-P had a statistically significant and somewhat stronger association with CVD (0.73, 0.57-0.93, p=0.01) than HDL-C (0.82, 0.63-1.08, p=0.16) or apoA-I (0.86, 0.67-1.10, p=0.22) .
Indeed, these two most recent studies have reinvigorated confidence among many experts and have refocused attention upon the HDL particle itself as the protective agent against cardiovascular disease by providing key evidence that the most important biomarker for tracking the protective capacity against plaque accumulation and cardiovascular events is the number of HDL particles, more so than the HDL-cholesterol. The pre-β HDL particle is the engine that drives this RLT pathway, leading to the removal of cholesterol from the body and regression of atherosclerotic plaque. It is for this reason that HDL mimetic therapy offers such great therapeutic potential: providing additional cholesterol elimination capacity in the form of synthesized pre-β HDL particles should enhance the flux of cholesterol through the RLT pathway and reconstitute this natural process of cholesterol elimination, particularly for individuals in whom this endogenous pathway is deficient or absent.
Therapies that increases the number of HDL particles or act as HDL mimetics, while maintaining all of the beneficial features of natural HDL particles, without side effects, have significant potential to treat CVD.
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