JCL roundtable: Apolipoproteins as causative elements in vascular disease

Journal of Clinical Lipidology (2015) -, -–-

Clinical Lipidology Roundtable Discussion

JCL roundtable: Apolipoproteins as causative elements in vascular disease Q14

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W. Virgil Brown, MD*, Frank M. Sacks, MD, FNLA, Allan D. Sniderman, MD, FRCP (C), FRSC Emory University School of Medicine, Atlanta, GA, USA (Dr Brown); Department of Nutrition, Harvard T.H. Chan School of Public Health, Boston, MA, USA (Dr Sacks); and Division of Experimental Medicine, Department of Medicine, McGill University, Montreal, Quebec, Canada (Dr Sniderman) KEYWORDS: Apolipoproteins; Vascular disease; Cholesterol; ApoC3; ApoB

Abstract: In clinical lipidology, we have focused our major efforts in defining risk status and specifying the targets of therapy by using the cholesterol content of the lipoproteins. However, we now know that these measures are variable and that they may not reveal all the valuable information that can be used to treat our patients. The amount of cholesterol in each lipoprotein can be quite different in different patients. The number of particles containing apolipoprotein B (apoB) can be abnormally high with a value for low-density lipoprotein cholesterol, which is within our guidelines. Furthermore, the content of apoC3 in apoB-containing lipoproteins can predict risk without a close association with triglycerides or cholesterol. The genome-wide association studies and studies in special families with known genetic polymorphisms have been particularly revealing relationships between these 2 apoB and vascular risk. Ó 2015 National Lipid Association. All rights reserved.

Financial disclosures Dr Sacks received consulting honoraria from Pfizer and Omthera, and honorarium from Diichi-Sankyo. He received a research grant from AstraZeneca. Dr Sniderman received speaker honorarium from Cleveland HeartLab. Dr Brown: In clinical lipidology, we have focused our major efforts in defining risk status and specifying the targets of therapy by using the cholesterol content of the lipoproteins. However, we now know that these measures are variable and that they may not reveal all the valuable information that can be used to treat our patients. The amount of cholesterol in each lipoprotein * Corresponding author. Emory University School of Medicine, 3208 Habersham Rd, NW, Atlanta, GA 30305, USA. E-mail address: [email protected]

can be quite different in different patients. The number of particles containing apolipoprotein B (apoB) can be abnormally high with a value for low-density lipoprotein (LDL) cholesterol, which is within our guidelines. Furthermore, the content of apoC3 in apoB-containing lipoproteins can Dr Brown predict risk without a close association with triglycerides or cholesterol. The genome-wide association studies and studies in special families with known genetic polymorphisms have been particularly revealing relationships between these 2 apolipoproteins and vascular risk. For this Roundtable, we are pleased to have been joined by 2 experts in the study of these apolipoproteins

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2 and their potential relationship to vascular disease. We have recorded this conversation during the Spring Clinical Forum of the National Lipid Association. Dr Frank Sacks from Harvard Medical School and T.H. Chan School of Public Health at Harvard and Dr Allan Sniderman from McGill University have agreed to answer my questions. Dr Brown: The interest in elevated apolipoprotein B (apoB) as a major indicator of risk has been with us for many years. Dr Sniderman, do you think it is time for us to simplify this issue and give up on low-density lipoprotein–cholesterol (LDL-C) and simply measure apoB? Will that measure also cover the added information obtained from measuring non–high-density lipoprotein–cholesterol (HDL-C) in patients with elevated triglycerides (TG)? Dr Sniderman: You are so right. The debate has gone on for so long, but in my view, it is over. Or at least to the extent, it will be decided by science, it is over. There were many reasons the debate lasted so long and was so unproductive. The most obvious was that different studies produced different results, but the most important was that the conventional statistical methods were inappropriate to compare Dr Sniderman apoB, LDL-C, and non–HDLC as markers of cardiovascular risk. They were inappropriate because they were not designed to compare closely correlated variables such as LDL-C, non–HDL-C, and apoB. When apoB particles contain an average mass of cholesterol, which is the case most of the time, all 3 markers must predict cardiovascular risk equally well. Only when apoB particles contain more or less cholesterol than average can the predictions of LDL-C, non–HDL-C, and apoB differ. Discordance analysis is designed to compare markers when their predictions differ, not when they are the same.1 The advantage is that comparison is not diluted by all the instances when they are the same. Discordance analysis creates a head-to-head comparison of 2 markers: one predicts high risk; the other, low risk. If risk is high, the first one wins; if risk is low, the other does. There is no ambiguity as to the outcome. At least 6 discordance analyses have now been published, and all show apoB is superior to either LDL-C or non–HDL-C as a marker of cardiovascular risk. If evidence actually matters, that should settle it.1,2 But, we should not stop there because we can learn even more when the information from TG, cholesterol, and apoB is integrated because we can then confidently and easily identify each of the different apoB dyslipoproteinemias.3 In our obsession with risk, we have forgotten the importance of diagnosis, but accurate diagnosis is the key to effective care. Therefore, the right choice is not apoB and nothing else but apoB and everything else.3

Dr Brown: When you evaluate risk, what lipoprotein measurements do you discuss with a new patient? How do you fit ApoB into an effective scheme for clinical care? Dr Sniderman: I start by explaining that cholesterol is an oil, and oils cannot mix with water. Therefore, cholesterol is transported through plasma, which is water, in particles, which are coated with phospholipids and a single molecule of a protein named apoB, which can mix with water. I tell them that the cause of the atherosclerosis is retention of apoB particles within the arterial wall, and this initiates and sustains the development of the atherosclerotic process.4 I also tell them that cholesterol is not the only poison in the particle. The apoB itself and the phospholipids can be biologically modified and provoke injurious processes within the arterial wall. I tell them that the likelihood of a particle getting into the wall depends on their number within the lumen of the artery. The higher the number of the atherogenic particles, Q4 the more they will enter the arterial wall and the more they will be retained within the arterial wall. I have found that these patients understand these ideas without any difficulty. Dr Brown: Do you talk to them about cholesterol at all? Dr Sniderman: Absolutely. Dr Brown: So, what do you say? Dr Sniderman: I explain to my patients that the amount of cholesterol in these particles can vary substantially and that there are a large number of people with markedly increased numbers of particles but whose cholesterol levels are quite unimpressive because their individual particles contain less cholesterol than the average person in the population. Because the number of particles is increased, they are at higher cardiovascular risk. Their cholesterol level is misleadingly reassuring. The counterexamples are those who have high levels of cholesterol—LDL-C or non– HDL-C—but a normal number of apoB particles. They have very high content of cholesterol per particle. There are now strong and consistent data from a number of studies including Framingham that these individuals with low particle numbers but relatively high cholesterol are not at high risk. We should not be concerned about their cholesterol level.1,2 Both clinical insights are eminently worthwhile. Dr Brown: When a patient is referred because their Q5 family doctor told them their cholesterol was too high, was that an inappropriate measure? Why was the cholesterol even measured in the first place? Dr Sniderman: No, the doctor was just following the directions of the guidelines. There is nothing wrong with measuring cholesterol. What is wrong is stopping there. In my view, the guidelines have not kept up with physiology, technology, and epidemiology. Elevated cholesterol is a good, but imperfect, sign of an elevated number of low-density lipoprotein (LDL) particles. Elevated cholesterol, if apoB is increased, does substantially increase cardiovascular risk, whereas elevated cholesterol, if atherogenic particle number is normal, does not increase.1,2 Therefore, your doctor was following an outdated

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paradigm, which ignores the advances in the physiology and technology to measure the atherogenic potential of LDL. This debate is similar to the old argument of whether LDL-C should supplant total cholesterol. Total cholesterol is a good but imperfect surrogate for LDL-C, and LDL-C is a good, but imperfect, surrogate for LDL particle number. Dr Brown: So, what are you going to tell the patient when you treat the high cholesterol? What numbers are you going to give this patient to inform them of the magnitude of lipoprotein risk? What are the values you are trying to achieve to reach an acceptable lowering of risk? Dr Sniderman: The first thing I do is measure apoB to determine whether treatment is necessary or not. If apoB is elevated, then all things being equal, treatment needs to be seriously considered. Treatment is a collaborative process between the patient and the physician and involves diet, exercise, and lifestyle as well as pharmacologic agents if indicated. For the majority, my target for LDL-lowering therapy is an apoB , 75 mg/dL. For those at very high risk, my target is an apoB , 65 mg/dL. These are the equivalent population levels to the LDL-C and non–HDLC targets chosen by many recent guideline groups. The apoB targets chosen by many of the guideline groups are much too high. It seems that once 1 group selected values, the others just repeated them. Dr Brown: Are you not going to focus on the cholesterol at all but use a value of apoB to define the goals of therapy? Dr Sniderman: Except for remnant lipoprotein disorder (type III), apoB is my target. ApoB can be measured on non-fasting samples using equipment available in virtually any clinical chemistry laboratory. The assays are automated and standardized, and the procedure to measure apoB is not expensive.5 Dr Brown: Could you ever use other techniques, which measure particle number by physical means? Dr Sniderman: The technique that has been used the most is NMR. I have not used it personally, but it has been incorporated into a substantial number of studies. To the best of my knowledge, NMR gives the equivalent information as apoB. So, I regard NMR as an equivalent acceptable technique to measure LDL. Dr Brown: Is it always important for the physician to know the apoB before he or she starts therapy under all circumstances? Dr Sniderman: Absolutely. LDL-C is not adequate. Non–HDL-C is not adequate. As I mentioned previously, a substantial minority of those with elevated cholesterol have a normal apoB, and they are not at high cardiovascular risk. Non–HDL-C does not avoid this error. Moreover, without apoB, we cannot diagnose remnant lipoprotein disorder, one of the most important dyslipoproteinemias, (Fredrickson type III hyperlipoproteinemia).3 These people have high TG and high cholesterol but a normal apoB. This is a disorder, which demands treatment on diagnosis, but which cannot be diagnosed presently by almost any clinical

laboratory. However, any doctor can now make the diagnosis by measuring cholesterol, TG, and apoB.3 Without apoB, the diagnosis is almost always missed, and this is sad because this is not necessary any longer. Dr Brown: So if the ApoB is not elevated in dysbetalipoproteinemia, should I not be concerned about the cholesterol? Dr Sniderman: No, we certainly should be concerned. The apoB is normal because the number of LDL particles is much lower than normal because very low–density lipoprotein (VLDL) particles are not converted into LDL particles. This should not be surprising given that removal and conversion of VLDL particles is the key pathophysiological fault in the disorder. The high cholesterol in these patients reflects a 20- to 40-fold increase in the number of remnant particles.4 These cholesterol-enriched remnant particles are highly atherogenic. However, it is not generally appreciated just how massively elevated remnant particle number is in these patients and conversely, just how little remnant particle number is increased in most other situations. Dr Brown: Are there other measures that are needed to complement ApoB? How do measures of HDL-C and TG fit into the ApoB paradigm? Dr Sniderman: For me, an adequate assessment of lipoprotein status requires measurement of TG, the usual cholesterol markers—total cholesterol, LDL-C and non– HDL-C, and HDL-C plus apoB. That should be the standard minimum. I think we should be looking forward to see what else we can incorporate to refine our ability to identify the individual who is on a trajectory that has a high probability of leading to vascular disease. Dr Brown: What about lipoprotein(a)? Would you measure that also at the beginning, and how does that fit into the ApoB assay paradigm? Dr Sniderman: Lp(a) is an LDL particle, which is attached to a molecule of apo(a). There is considerable evidence linking Lp(a) to an increased risk of vascular disease but none to date demonstrating benefit with treatment. I do measure Lp(a) although I believe there is still work to do to improve the available assays. That said, Lp(a) matters. Dr Brown: As new methods of directly measuring LDLC have been developed, the standardization programs used by laboratories have revealed significant deviation from the standard calculation known as the Friedewald equation. (total cholesterol minus HDL-C and minus the TG divided by 5). The Center for Disease Control standardization laboratory has made comments about this noting that this old method is often more accurate in persons with TG below 400 mg/dL. They also have endorsed the accuracy of some of the apoB assays, at least those used in large high volume laboratories. We published a Roundtable in this Journal in which those comments were made by one of the directors at the Lipid Standardization Laboratory at the Center for Disease Control. Let us turn to this issue of TG and apoC3. We have learned, of course, over the years that many other proteins other than ApoB are involved in lipoprotein metabolism in

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4 the plasma. ApoC3 has become of interest more recently because of a variety of studies in families and in large populations. Dr Sacks, could you give us a synopsis of this information linking ApoC3 to cardiovascular disease (CVD)? Dr Sacks: Sure. ApoC3 is a small protein 8.8 kilodaltons, compared with ApoB, which is huge, 516 kilodaltons. ApoC3 is present on a portion of VLDL and LDL, not on all particles, and many molecules of apoC3 can be present on each apoC3-containing particle. Unlike apoB, apoC3 is not required for the synthesis of VLDL or LDL by the liver. ApoC3 may play some role to enhance the liver’s assembly or secretion of VLDL or even of LDL, although data on this Dr Sacks potential effect are not in agreement. ApoC3, although, is associated with hypertriglyceridemia. ApoC3 itself is a good predictor of CVD even after adjustment for TG. In the August 2015 issue of JCL, a metaanalysis showed that the concentration of apoC3 in apoB lipoproteins, VLDL, and LDL, predicts CVD in several populations. What is more, we find that the concentration of LDL specifically that contains apoC3 is the strongest predictor of CVD among apoB lipoproteins. The apoB concentration of LDL that has apoC3 is more predictive than the apoC3 concentration itself, supporting a particle-based viewpoint on lipoproteins and CVD. These findings also support a species concept of lipoproteins based on the proteins that they carry. All VLDL and LDL particles do not have ApoC3. ApoC3 for unknown reasons localizes on a subset of VLDL and LDL. In people who have hypertriglyceridemia, VLDL and LDL that have apoC3 are the major species. In people who have, normal TG, apoC3-containing particles are a minority of the total particles. Dr Brown: Could you tell us more about these large population-based genetic studies, the genome-wide association studies (so-called GWAS) and the linkage of apoC3 to vascular outcomes. Are those convincing? Dr Sacks: I think these associations have really moved the ApoC3 field forward. It is one thing to say that ApoC3 predicts incident CVD or specific kinds of particles, but we know that ApoC3 is part of a whole metabolic complex that is there to move TG and cholesterol from liver to peripheral tissues. So, the mechanism of the association with lipid concentrations is not necessarily the mechanism of causation of vascular disease. The so-called Mendelian randomization studies are important in helping to understand these issues. A real breakthrough came with Pauline and Shuldiner discovering in the Old Order Amish population that those that were heterozygous for defective ApoC3 had 50% lower ApoC3 levels than those that had wild type. They had a more favorable lipoprotein pattern including lower LDL levels. They also had reduced coronary calcification and other indicators of disease. The

individuals with the genetic mutation that cause dysfunctional apoC3 had lower apoC3 levels throughout their life and appeared to benefit from this different exposure of the resulting plasma lipoproteins. There have been 2 recent population studies involving Q7 some 75, 000 individuals in the United States and over 100,000 Danes in Copenhagen, which found 3 or 4 specific mutations that reduced apoC3 concentrations and TG by about 40%. Concomitantly, in both studies, the prevalence of vascular disease was reduced by 40% as well. These were published in the New England Journal of Medicine (July 2014). Again, life-long reductions in apoC3 seem to be very beneficial. Dr Brown: I believe there have also been some Dutch family studies that have reproduced, in effect, Shuldiner’s study showing defective or nonfunctional ApoC3 being associated with less disease as well. Dr Sacks: Yeah. Dr Brown: What is your opinion as to the beneficial effect of less functional and/or lower the concentrations apoC3 in blood? Dr Sacks: There are several potential mechanisms. As you originally reported in 1972, apoC3 is an inhibitor of lipoprotein lipase activity. ApoC3 is also an inhibitor of hepatic lipase. Hepatic lipase is needed to convert VLDL to LDL, so, a high apoC3 level may increase VLDL levels partly through inhibiting hepatic lipase. ApoC3 blocks the interaction between ApoE and B100 on VLDL and LDL with hepatic receptors that clear these lipoproteins. ApoC3-containing VLDL and LDL are mainly metabolized to smaller particles before they are cleared from the circulation. However, if apoE is also present on an apoC3-containing particle, metabolism is divided between clearance and conversion to smaller particles. Detailed studies of human lipoprotein metabolism show that the interplay of apoC3 and apoE governs the metabolic fate of VLDL and LDL as they circulate. One interesting question is why do we have apoC3? Whether by inhibiting lipases or clearance, apoC3 prolongs the time that lipoproteins circulate, spreading out over time the distribution of free fatty acids to the heart muscle and fat where they are needed for energy. Dr Brown: My early research in the laboratory involved isolating and characterizing hepatic lipase. This is a very active enzyme on the surface of liver cells and liver endothelium. It is also inhibited by surface coat proteins including apoC1, apoCII, and apoC3. From work of Enholm, apoC3 seems to be the most effective of the 3 at hepatic lipase inhibition. I agree that we have not followed up sufficiently on some of those studies to fully understand the importance of these proteins in the interaction of TG-rich lipoproteins as they circulate through the liver. Dr Sniderman, what are your thoughts about ApoC3? Dr Sniderman: Well, obviously apoC3 is totally fascinating and I think is on the verge of being clinically applicable. I had a question for Frank along the lines that you were discussing. Do you think that apoC3 affects the

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proportion of VLDL particles that are converted to LDL particles? This is an area of great importance but great ignorance in the lipid area because the VLDL particle can be removed by the liver before it becomes an LDL particle or it can become an LDL particle. One event is benign, the other malignant. Have you not suggested that ApoC3 in some way is inhibiting the uptake process of VLDL and encouraging the conversion to LDL? The existence of the apoC3-LDL particle seems to point in that direction. Dr Sacks: I really agree with you that formation of the dense LDL phenotype in hypertriglyceridemia has a lot to do with the action of ApoC3. ApoC3 inhibits the removal of VLDL and large LDL from the circulation, directing the metabolism of these particles to dense LDL, which has a slow clearance rate. Dr Brown: We puzzled for a long time over why we needed 2 enzymes that are very close cousins. They have the same molecular weight and are highly conserved in terms of their amino acid sequence. The newly born human infant and some animals have lipoprotein lipase and hepatic lipase in the liver. The liver in humans and most mammals cease their lipoprotein lipase production very early in ex utero life. Perhaps, this physiological change evolved because the secretion of VLDL becomes important when exogenous calories begin to supply excess energy to the liver. If lipoprotein lipase persisted in the liver, this TG-rich particle with the activator of lipoprotein lipase (ApoC2), on its surface would be hydrolyzed in the space of Disse. This would create a futile cycle of energy due to the lipids returning immediately to the liver instead of their transport to peripheral tissues. The ApoC3 may also allow the particle to escape the hepatic lipase that also lines the space of Disse. It could have an even broader role in that it could inhibit uptake of VLDL by receptors for ApoB and ApoE as well. We have much evidence that it inhibits the uptake of VLDL and chylomicron remnants, but it may be more important as an inhibitor of nascent VLDL uptake by the liver. The uptake of remnants may depend greatly on first reducing their TG content through hepatic lipase action. Years ago, we studied 2 young women who were totally deficient in apoC3. Not only did they clear their TG 5 times the normal rate, but they also produced LDL at a normal rate. That suggests that the major issue is actually the hepatic lipase functionality, not the uptake and degradation of the entire remnant particle by liver. If the major issue was preventing uptake of remnant VLDL, why would LDL production rates in the plasma be normal? Furthermore, the administration of the antisense oligonucleotide (APOC3Rx from Isis Pharmaceuticals) results in marked reductions in apoC3 and TG but no significant reduction in apoB in human plasma. I am postulating that the evolutionary advantage of having ApoC3 is to protect newly formed VLDL. Lipoprotein lipase activity in the peripheral tissues is possible because of the very high–affinity interaction between ApoC2 and the lipase. This hydrolysis removes apoC3

and results in transfer to high-density lipoprotein (HDL). Our early experiments indicated that ApoC3 inhibition of the lipase was blocked by a very small amount of a degradation product of TG hydrolysis. In the early 1970s, we published that monoglycerides appeared to be that product preventing ApoC3 inhibition. We proposed that the monoolein used in these experiments became a component of the surface of the TG emulsion and blocked ApoC3 at that location. However, it could be acting as an allosteric effector by binding directly to apoC3, preventing its inhibitory action and allowing it to transfer to HDL. The experiments to test this hypothesis need to be done. Dr Sacks: Well, I think it needs to be looked into again. Now, in human kinetic studies, my laboratory has found that ApoC3-containing VLDL is converted quite readily to LDL. There is a normal rate of conversion. Now, VLDL that does not have apoC3 on it was also readily converted to LDL. VLDL that has both apoC3 and apoE has both conversion to LDL and clearance from the circulation. Finally, VLDL that has apoE but not apoC3 is very quickly removed from the circulation and does not become LDL. I agree then that a physiological role of apoC3 is to protect nascent VLDL from clearance. But too much apoC3, which many of us have, overplays this function causing abnormal VLDL and LDL metabolism. ApoE is an important part of this whole story. Dr Brown: I would agree with that. Dr Sacks: ApoE is a very high–affinity ligand for the LDL receptor, and it also has its own receptors on the liver. So, when a VLDL or a large LDL has ApoE on it, it gets removed quickly from the circulation. This was what we found in humans, and it agrees with animal models. Dr Brown: That is correct if it is normal ApoE, ApoE2 being one exception. Dr Sacks: That is true. Now, back to lipoprotein lipase. A really interesting study was published last year by a Swedish group in Journal of Biological Chemistry. They made lipid droplets that contained varying amounts of ApoC3, ApoC1, and lipoprotein lipase. What they found is that ApoC3 displaced lipoprotein lipase from the lipid droplet, and then, the displaced lipase gets inactivated by ANGPTL3. ApoC1 did the same thing. I found especially interesting that the concentration of ApoC3 or apoC1 has to be high to do that to lipoprotein lipase. Expressed as the ratio of apoC3 to apoC2, the ratio needed to be above Q8 5 to cause this LPL inhibition. The ratio of apoC2to apoC3 in circulating VLDL is about 1 to 2. May be the combined concentration of C3 and C1 relative to apoC2 is important because apoC3-containing VLDL also has apoC1 and apoC2. There is a lot of interplay between different molecular players. Dr Brown: The Isis, APOCIIIRx drug, has produced profound reductions of plasma TG in patients who are moderately to severely hypertriglyceridemic. Is there really a problem with too much ApoC3 in these people? Is excess ApoCIII the etiologic issue for such for hypertriglyceridemia in these patients with TG over 200 mg/dL? The

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publication in the patients with proven lipoprotein lipase deficiency certainly suggests that hepatic lipase is inhibited by ApoC3 in such patients as well. Dr Sacks: Yes, the anti-apoC3 had a spectacular effect published in New England Journal recently, a spectacular effect in reducing the TG in 3 patients with lipoprotein lipase absence, the familial chylomicronemia syndrome. It is a devastating disease and can cause early death from pancreatitis. So, we need a drug like that specifically for patients with disorders of the lipoprotein lipase system. These patients have a very low level of LDL because they cannot process nascent chylomicrons and VLDL to smaller particles. Reducing the synthesis of apoC3 may allow hepatic lipase to process small VLDL down to a size that the liver can get rid of. The anti-apoC3 drug also operates this way in moderate hypertriglyceridemic and even in normolipidemic people, greatly reducing TG levels, as we would expect from the metabolic studies of apoC3-containing VLDL in humans that I mentioned. So, I think that apoC3 affects lipoprotein metabolism in normal humans. What is really exciting about that is that ApoC3 treatment may be useful to reduce TG and CVD in higher risk dyslipidemic patients who have, moderately elevated TG. Dr Brown: I believe you would agree that we need a small molecule that would be effective at inhibiting C3 concentrations and that would be more acceptable in the clinic for those with moderate but risky elevations in TG. However, I think this is a wonderful discovery and provides a great tool to study these issues that you and I have been involved with and speculating about over the years. Dr Sacks: One point I would just like to add, which we have not discussed is HDL and ApoC3. So interestingly, HDL as VLDL exists within a subspecies that has C3 on it. So, what we find is that ApoC3 is associated with about 10% of the ApoA1. If we look at the C3 containing HDL, it does not have a protective association with coronary disease. In fact, it actually has an adverse effect, it predicts coronary disease like an atherogenic particle. So that sort of goes along with this interest in the study of HDL speciation that several laboratories are working on. With more in-depth knowledge, it could be that the function of HDL particles system can be understood in more detail, and we could have a better target for treatment or a better predictor of risk. Dr Brown: If ApoC3 is inappropriately elevated and produces moderate hypertriglyceridemia, in the 200 to 500 mg/dL range, where we know most of the coronary disease relationships exist, it seems we have 2 potential accelerating or consequential features. One is more small particles in the ApoB range and a change in HDL composition that you have just mentioned. Dr Sacks: Since we published 2 years ago that ApoC3containing HDL predicted increased CVD, opposite to the relation of the more prevalent HDL that does not have apoC3, we have several additional prospective studies soon to be submitted for publication confirming the

divergent risk of HDL with apoC3 vs HDL without apoC3. About 10% of HDL particles have apoC3, more in people with obesity than normal body weight. There is Q9 much to learn about the complexity of HLD particle composition and function. Dr Brown: What do you believe to be the role of cholesterol ester transfer protein (CETP) in changing the cholesterol content of LDL-C and HDL? Does this increase of Q10 decrease the functionality of HDL in the presence of high TG? Dr Sniderman: CETP is involved in core lipid exchanges between VLDL, the TG-rich lipoproteins, and LDL, and HDL. But this transfer is driven more by TG than by the mass of CETP. When there is hypertriglyceridemia, even small amounts of CETP seem sufficient to deplete cholesterol ester from the LDL and HDL particles. Dr Brown: One of the things that was in the ISIS data Q11 was that LDL cholesterol actually went up significantly in those with moderate to severe hypertriglyceridemia when ApoC3 concentrations were markedly reduced. Whole particles were not being removed since apoB did not fall, but in some patients, the cholesterol content was dramatically increased. It doubled in 2 of the lipoprotein lipase–deficient patients. The cholesterol content of HDL also rose. Detailed analytical studies of the particle distribution and the apolipoproteins in HDL would be of interest with the ApoCIII suppression. Dr Sniderman: Chylomicron particles are a source of HDL proteins and phospholipids and TG. As they are metabolized, nascent HDL particles are generated. Actually, a great deal of what otherwise appear to be disconnected observations fit together. So, a lot of observations that seem disconnected actually are part of the complex processing of TG-rich lipoproteins in the plasma. They will probably fit together much better than it might appear. Dr Brown: I have been concerned that the recent move to simplify the clinical guidelines could prove to result in a reduced emphasis on study of the complexity of lipoprotein metabolism. I believe we have learned much from the myriad of genetic disorders we see in lipid clinic. To me, these experiments of nature should teach that much is yet to learn that could prove very helpful in treating our patients at risk of vascular disease. It is certainly true that the overwhelming problem for most physicians is simply to lower the apoB-containing lipoproteins. Statins are a highly effective start in that effort. However, the clinical trials with the highest doses of statins have been only partially successful. Patients with high TG and low HDL have remained with high event rates. A greater depth of understanding and more specific therapeutic tools in the disorders of VLDL and HDL metabolism are needed to offer more comprehensive treatment. Dr Sacks: I think that the new information on apolipoproteins is going to prove to be useful. They could add significant information on risk prediction, and they raise the possibility for better targets of therapy than simply lowering the cholesterol synthesis and increasing LDL

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receptors. I think the lipoprotein field is very exciting, and a lot of new things could emerge both on risk prediction and in therapy. Dr Sniderman: Unfortunately, we will not be able to incorporate many of the most important physiological advances into clinical practice until we update the statistical tools we use to assess their importance because many are not well suited for the purposes they have been put to. We doctors do not understand the intricacies of statistical methods just as most statisticians do not understand the physiological intricacies of the biomarkers they have been studying. Thus, demonstrating a significant increase in the area under the curve of a receiver operating curve has been the sine qua non for all the guideline groups to accept a new marker as clinically useful. It turns out, however, this approach is not valid for markers that are correlated such as LDL-C, non–HDL-C, and apoB. Thus, the guidelines continue to hang on to the old methods without providing a stimulus for more physiological thinking that could advance our field. Dr Brown: Would you not agree that we are often unable to explain to the patient the basic cause of their lipid problem and therefore defining the target of the treatment and appropriate goals of the therapy in individuals is still quite short of ideal for many. The new guidelines are focused on a single therapy with one size fits most if not all patients. Assess total vascular risk and write a prescription that is the maximum safe dose is the paradigm. With this paradigm, all you need is simple data acquisition and a computer. You prescribe the appropriate dose of statin and you have dealt appropriately with the problem—statin deficiency. The doctor’s role is then to convince the patient to take the statin. How are we going to be able to use new information if we do not think carefully about the abnormal biochemistry and physiology? What is the role of new drugs? Dr Sniderman: I agree we have a guideline problem. Guidelines do not guide so much as they order. Whether they wish to, guideline recommendations have become the standard of care. Their authority is based on the widespread assumption that the process by which guidelines produce their recommendations is objective, impersonal, and error-free and driven by rigorous gradation of all the available evidence, of which randomized clinical trials are the highest form. Evidence from other sources such as physiological experiments, epidemiology, genetics, and clinical experience are the lowest and irrelevant. If so, the recommendations of different guidelines should be very similar. That is, if the process is truly scientific, the results—the recommendations—should be replicable. We have tested this assumption by comparing the 6 most recent cholesterol guidelines. Notwithstanding that all deal with essentially the same evidence base, many major recommendations differ substantially. It appears, therefore, that guideline recommendations are not only shaped by the evidence but also by the views of those who

review the evidence. That is, they are evidence-based and observer-based. This is not to say that the guideline process is not of enormous value. But, it is a human process, conceived by humans and executed by humans and therefore subjected to all the limitations that follow. The remedies are the same remedies as in all scientific work: opening the process to challenge by diverse views and appreciating that the gathering and interpretation of evidence is a much more subtle and challenging process than many believe. Randomized controlled clinical trials are not a perfect source of knowledge, nor are they the only legitimate source of knowledge. Dr Brown: And it is also not scientific because to me, the basis of science is having theory that one can use until a thoughtful challenge refines the theory and redirects our thought. Experimentation is only truly valuable if it addresses the theory in a meaningful way. Repeating an apparently successful intervention sets the stage for mimicry but not for advancement. Much of the art in any profession is built on repeating what apparently works, but in medicine, we must also continue a scientific approach as well or we will remain only partially successful. Dr Sacks: I really agree with you. I felt that the new guidelines de-emphasized the biology and the pathobiology of hyperlipidemia and atherosclerosis of which we know so much. I was disappointed that the guidelines relegated pathophysiology and causation to such a minor role. I think that is why they departed from an LDL goal. The present study compared to previous guidelines did Q12 not address dyslipidemia, high TG, and low HDL. Fibrate treatment has consistently reduced CVD in people who have high TG, for example, above 200 mg/dL and low HDL-C. There is also plenty of known pathophysiology that links dyslipidemia and atherosclerosis and the effect of fibrates to move metabolism in a normal direction. ApoC3 is a key player in this regard. Dr Brown: Exactly. That is the case in point that I think makes what we have talked about today so relevant. The underlying pathophysiology, which goes to the disease process, is the gold mine. The information we already have—the higher TG, the low HDLs, small dense particles—is not in the guidelines. Yet, we have very adequate evidence that these coexisting phenomena confer increase in risk even after statin treatment. Trial after trial has shown that. There was a near doubling of risk in the Q13 ACCORD trial if your plasma lipoproteins have these particular properties while taking an adequate dose of statins to produce low LDL-C. Dr Brown: I want to thank both of you for sharing your knowledge and insights into the importance of understanding the clinical import of both ApoB and ApoC3. Your studies in humans have provided a strong base for further studies related to developing these measures as risk factors and targets of treatment in patients with higher TG and low HDL values.

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Uncited references 6–20.

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9. Jørgensen AB, Frikke-Schmidt R, Nordestgaard BG, Tybjærg-Hansen A. Loss-of-function mutations in APOC3 and risk of ischemic heart disese. N Engl J Med. 2014;371:32–41. 10. The TG and HDL Working Group of the Exome Sequencing Project, National Heart, Lung, and Blood Institute. Loss-of-function mutations in APOC3, triglycerides, and coronary disease. N Engl J Med. 2014; 371:22–31. 11. Esther M, Ooi M, Barrett PHR, Chan DC, Watts GF. Apolipoprotein C-III: understanding an emerging cardiovascular risk factor. Clin Sci. 2008;114:611–624. 12. Gaudet D, Brisson D, Tremblay K, et al. Targeting APOC3 in the familial chylomicronemia syndrome. N Engl J Med. 2014;374: 2200–2206. 13. Zheng C. Updates on apolipoprotein CIII: fulfilling promise as a therapeutic target for hypertriglyceridemia and cardiovascular disease. Curr Opin Lipidol. 2014;25:35–39. 14. Sniderman AD, Lamarche B, Contois JH, de Graaf J. Discordance analysis and the Gordian Knot of LDL and non-HDL cholesterol versus apoB. Curr Opin Lipidol. 2014;25:461–467. 15. Pencina MJ, D’Agostino RB, Zdrojewski T, et al. Apolipoprotein B improves risk assessment of future coronary heart disease in the Framingham Heart Study beyond LDL-C and non-HDL-C. Eur J Prev Cardiol. 2015;22:1321–1327. 16. Sniderman A, Couture P, de Graaf J. Diagnosis and treatment of apolipoprotein B dyslipoproteinemias. Nat Rev Endocrinol. 2010;6: 335–346. 17. Williams KJ, Tabas I. The response-to-retention hypothesis of early atherogenesis. Arterioscler Thromb Vasc Biol. 1995;15:551–561. 18. AACC Lipoproteins and Vascular Diseases Division Working Group on Best Practices, Cole TG, Contois JH, Csako G, et al. Clin Chem. 2013;59(5):752–770. 19. Sniderman AD, Furberg CD. Why guideline-making requires reform. Q1 JAMA. 2009;301:429–431. 20. Sacks FM. The crucial roles of apolipoproteins E and C-III in apoB lipoprotein metabolism in normolipidemia and hypertriglyceridemia. Curr Opin Lipidol. 2015;26(1):56–63.

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