A lot of nutritional studies, especially epidemiological, rely on certain biomarkers to assess disease risk. Many of these biomarkers are accepted with little challenge, and thus become very common to examine. Historically, jumping too quickly to conclusions from associations has confined our nation to a nutritional nut-house; people are confused because of mixed and ever changing messages. Has this happened with HDL as well?
We know a lot about various macronutrient combinations and many other dietary interventions affect HDL; it has been studied for decades. But after all of this time, is it a convincing theory: is HDL a role-player or an observer in the cardiovascular context?
A new paper by Vergeer et al. (1, free full text here) summarizes the evidence in 17 pages. Papers like these should be must reads for nutritionists just to get a sense of the complexity of these issues, as no abbreviated version will do them true justice. But I went through it and attempted a reduced summary below. I also color coded their quoted summaries in blue, so if you want a summary in a summary of a review, you can be ultra-lazy.
Epidemiology
The hypothesis, that low HDL-C contributes to cardiovascular disease (CVD), started in 1951 with a study showing low HDL-C in coronary artery disease patients, then took off after the Framingham results in 1977 corroborated. Since then, many epidemiological studies have shown a strong link between HDL-C and CVD. Many other variables also influence HDL-C and CVD risk (e.g. exercise, weight loss, cigarettes, stress, etc). However, even after adjustment for these variables in meta-analyses, low HDL-C is still strongly correlated with CVD. As with any meta-analysis, results such as these are as noted always subject to residual confounding. The authors note other related parameters that may have even stronger associations with CVD, such as apoA-I or HDL subclasses, but are still subject to the same limitations as analyses of HDL-C itself. In their words: “In conclusion, the epidemiological association between HDL and CVD is strong, and is largely responsible for the formulation of the HDL hypothesis, but in itself does not prove a causal relationship.”
Function
The Reverse Cholesterol Transport (RCT) pathway was established by showing that HDL can accept cholesterol. This led to the theory that HDL picks up cholesterol at peripheral cells and takes it to the liver for excretion. One of the mechanisms which I remember learning in biochem courses, is that cholesterol is transfered from HDL to VLDL which is then taken up through the LDL receptor pathway. Surprisingly, the theory is not holding up in mice: experiments when knocking out genes related to HDL metabolism, cholesterol excretions are unaltered. In humans, results are mixed, and it seems that HDL has little effect on cholesterol homeostasis.
An alternative role could be that HDL prevents CVD by removing cholesterol from macrophages (which can otherwise take up oxLDL and turn into foam cells promoting inflammation). This may be supported by data thus far, but interpretations seem difficult to translate to in vivo contexts. Studies on the cholesterol donating capacity of cells so far also do not support a link to CVD. In their words: “Altogether, there is no evidence for a role of HDL in the net removal of cholesterol from the body (or vascular wall) and subsequent excretion into feces in mice, while evidence in humans is very scarce. To date, there are no assays to measure cholesterol efflux that have proven value in predicting cardiovascular events in humans. This reflects the inherent difficulty of finding a measure for the complex dynamics of cellular cholesterol exchange in atherosclerotic lesions.”
In Vitro
HDL in vitro can inhibit endothelial adhesion molecule expression and monocyte transmigration, as well as leukocyte adhesion. HDL also has measured antioxidant/anti-inflammatory activity; HDL capacity for these factors distinguish CVD and healthy subjects in a couple studies, but it has not been studied further yet. There is some evidence that HDL inhibits apoptotic mechanisms which protect endothelial cells, and promotes nitric oxide production and coagulation thereby altering platelet function. Also of importance, HDL can vasodilate endothelial cells through nitric oxide; infusion studies show partial restoration of endothelial vasodilation in diseased contexts. In their words: “In conclusion, there is considerable evidence for a direct protective role of HDL in inflammatory, oxidative, apoptotic and thrombotic processes but these studies are primarily performed in in vitro settings. With respect to protection from endothelial dysfunction, some evidence suggests that in vitro findings may apply to in vivo situations as well, but studies are small. In each case, a direct link between ex vivo measurements (biomarkers) and cardiovascular risk is yet to be established. Since such evidence could strengthen the concept that HDL is an actor rather than a bystander in atherogenesis, an important challenge for the coming years will be the translation of HDL function assays from bench to bed-side. These assays are particularly needed in view of the ongoing testing of novel HDL drugs in clinical trials, where early indications of success or failure are absolutely vital.”
Animal Models
Various studies manipulating apoA-I, which is a large component of HDL, have shown that apoA-I may be protective against (or that a lack of it promotes) CVD. Human apoA-I gene transfers and overexpressions in animals consistently prevent CVD. In their words: “Taken together, the currently published animal data provide good evidence in favour of a direct atheroprotective effect of apoA-I.”
Among other gene manipulations studied with do not yield clear results include: ABCA1 and ABCG1, which influence de novo HDL synthesis in liver and small intestine and delivery of cholesterol to larger HDL, respectively; LCAT, which regulates HDL-C through esterification of free cholesterol; CETP, which mediates cholesteryl ester and triglyceride transfers between apoA-I and apoB; SR-B1, which is an HDL receptor in the liver, actually shows paradoxical results when knocked out with increased HDL-C but increased CVD and protection with overexpression; HL, which hydrolyzes triglycerides and phospholipids; EL, which primarily hydrolyzes HDL lipids; and LXR, which transcriptionally regulates other genes involved in cholesterol metabolism, which seems to be protective since it promotes cholesterol efflux from vascular macrophages. Contextual and methodological differences make conclusions difficult. Most of these studies do not show a consistent relationship between HDL-C and CVD. In their words: “Reviewing the data of major animal studies that have focused on primary modulators of HDL metabolism, it is clear that the vast majority of animal studies have thus far not provided evidence that HDL protects from atherosclerosis. Although many studies may have indicated that modulation of HDL metabolism is associated with atheroprotection, a careful analysis shows that this is often seen in the context of a simultaneous modulation of apoB containing lipoproteins which obscures the reported effects of HDL on atherosclerosis. The most positive and convincing data have been generated by studies of overexpression of human apoA-I in mice and apoA-I infusion in rabbits. Paradoxically, high HDL-C levels have also been associated with accelerated atherosclerosis in SR-BI-/- or LCAT transgenic animals. The capacity to reverse atherosclerosis in these mice through CETP expression underlines the interdependency of various HDL modulators to achieve atheroprotection. Finally, bone marrow transplantation experiments and studies with LXR agonists have provided good evidence that promotion of cellular cholesterol efflux from the vascular macrophage confers atheroprotection by promoting the initial steps of the RCT pathway. However, it should be noted that these processes bear no apparent relationship to plasma lipid levels, including those of HDL-C.”
Genetic Studies
With the genomic age well underway, we now can look at gene variations, HDL-C levels, and CVD risk. In family studies, 30-60% reductions in HDL-C because of mutations of several previously discussed genes is associated with an increased cIMT and CVD risk in some but not all studies. APOA1milano, which results in low HDL-C yet normal cIMT may suggest that the apoA-I version is more protective, but could also suggest that HDL is not protective. Genetic variations that yield a higher HDL-C level seem equivocal as well. A recent meta-analysis of 46 studies on CETP variations and CVD risk point to a link, however other explanations are possible. Recent GWAS have identified new SNPs which may assist in better predictive models of risk using multiple genes. In their words: “In conclusion, the published cross-sectional family-based candidate gene studies suffer from small numbers, ascertainment bias and lack of prospective follow-up. This means that the results may be skewed because samples are taken from families who have come to a physician’s attention, rather than from the general population. Bypassing referral bias and cross-sectional designs, large prospective population studies have indicated that non-synonymous mutations in ABCA1 and HL have no effect on CVD risk, nothwithstanding profound effects on HDL metabolism. Recent evidence obtained from large epidemiological and GWAS analyses do not support the HDL hypothesis either. In fact, a direct head-to-head comparison between genetic variants affecting either plasma LDL-C or HDL-C in a very large study171 indicated that only the former predict CVD.”
Clinical Trials
Various drugs and supplements have been tested on CVD which affect HDL. The authors review: fibric acid (PPARalpha agonists), which do seem to have a protective effect, but may be related to their triglyceride-lowering effects, not necessarily their HDL-C increasing effect. Nicotinic acid (niacin) also tends to have protective effects in some but not all studies so far, but also effects other lipid values beside HDL-C. HDL-like particles have thus far largely failed, a rApoA-Imilano/phospholipid complex infusion study showed promise but suffered from design problems. The authors note a need for better and longer term infusion studies, these would better clear up HDL’s relative role in pathology. CETP inhibitors have entered human trials, but the largest was terminated because of (likely) off-target toxicity unrelated to CETP inhibition. More trials are in progress. As previously discussed, apoA-I manipulation is promising in animal models, so apoA-I mimetics and synthesis stimulators are being tested. Though human data is still limited, animal studies are promising. These generally do not alter HDL-C levels, rather alter HDL function. It would seem that perhaps HDL level is not as important as its characteristics- perhaps future studies will find that nutritional effects on HDL have been examined inappropriately so far, testing HDL attributes may be a better idea. In their words: “In summary, there is evidence for fibrates and niacin as anti-atherosclerotic agents, but since both drugs have major effects on other blood lipids, this does not prove that HDL is causal to the observed atheroprotection. A large ongoing outcome trial with niacin (HPS2-THRIVE) may provide the statistical power to indirectly assess the effect of an increase in HDL-C in 2013. The use of apoA-I mimetic peptides has yet to be proven to have clinical relevance, whereas infusions of HDL-like particles have shown promise in preclinical studies and small-scale clinical trials, but have thus far not improved cardiovascular outcome in larger studies. To date, the most powerful tools to raise plasma HDL-C concentrations are CETP inhibitors. Whether these drugs decrease CVD risk may be learned from two large outcome trials of which the results are expected in 2012. Drugs that specifically upregulate apoA-I may be the ultimate means to prove that HDL protects from CVD in humans, but these compounds are in early clinical development.”
Conclusions?
Is HDL per se atheroprotective? According to this review, it would seem unlikely, but conclusions are difficult with limited human trials. There seems to be an interdependence of many genes, or possibly various components of HDL that may have a greater impact on CVD development or prevention than HDL level itself. After decades or research, we have a ways to go to elucidate the big picture. The HDL-CVD hypothesis certainly isn’t dead, but it remains very much a hypothesis.
Reference
1. Vergeer M, Holleboom AG, Kastelein JJ, & Kuivenhoven JA (2010). The HDL hypothesis – Does high-density lipoprotein protect from atherosclerosis? Journal of lipid research PMID: 20371550
