HIGH-DENSITY LIPOPROTEIN - THE MYTH OF THE "GOOD CHOLESTEROL" Thimoteus Speer, Homburg/Saar, Germany

Chairs: Maurice Laville, Lyon, FranceErling B. Pedersen, Holstebro, Denmark

Prof. Thomoteus SpeerDepartment of Internal Medicine 4

Saarland University Hospital Homburg/Saar, Germany

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Dear Chairman, dear ladies and gentlemen good morning. As the title for this talk I havechosen a really provocative title 'High density lipoprotein and the myth of the goodcholesterol' and during this talk I'll try to explain to you why I've chosen this title.

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At first, we have to ask a question: What is HDL? This is the first mistake of this presentation,and hopefully, the last one. We have to ask: What are HDLs?

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HDL is not a single molecule or one unique particle, HDL is rather complex and I will show youhow.

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To answer this question I will at first dissect the word

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'high-density lipoprotein' in its parts.

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First, we have a lipid part, which consists of a monolayer of phospholipids and unesterifiedcholesterol on its outer surface

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and a core consisting of cholesterol esters and triglycerides inside the particle.

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Moreover, HDL contains a protein part, which is called in this case apolipoproteins.

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The most abundant Apolipoproteins in the HDL particles are Apolipoproteins A1 and A2 butthere are many, many more different proteins associated with the HDL particle.

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Analyses of the proteome of HDL by using proteomics made it possible to quantify and toidentify these proteins. You can see here some of the results of one of the first studiesexamining the HDL proteome. Some proteins are associated with a lipid metabolism, some areproteinase inhibitors, acute phase response proteins and moreover, complement regulatoryfactors.

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So, that we can say HDL is not equal to HDL because there is a strong remodelling influencedby many conditions such as kidney chronic disease, inflammation and coronary artery diseasewhich change the composition of HDL.

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Here are results from dialysis patients. They performed proteomics and compared the proteincomposition of the HDL particle to HDL from healthy controls. They found a significantupregulation, for example, of the surfactant protein B (SP-B) but also of the pro-inflammatoryprotein serum amyloid A.

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So, we are moving to the next part of the word HDL 'high density', this means a specificproperty of lipoproteins showing a density within a specific range. LDL, low density lipoproteinshave a density between 1.006 g/cm3 and 1.063 g/cm3 and HDL particles are characterized bya density between 1.063 g/cm3 and 1.210 g/cm3.

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This specific property makes it possible to use ultracentrifugation to isolate these HDL particlesfrom serum samples.

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It's a simple technique but time consuming. One can use potassium bromide added to theserum to adjust the density of 1.063 g/cm3 and then, we can achieve a separation betweenthe LDL and VLDL particles having a density below 1.063 g/cm3 and the HDL, also the albuminfraction with a density above 1.063 g/cm3. This isolated HDL can be used for further in vitroassays studying its function.

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So, how is HDL synthesized?

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As I've already told you, the main structural protein component of HDL is Apolipoprotein A1,which is synthesized by the liver and the intestine. Upon its secretion, this apoA1 acceptsphospholipids and free cholesterol from the intestine and from the liver leading to theformation of lipidated apoA1 also called pre-β-HDL, which further matures into the discoidalnascent HDL. This nascent HDL accepts further free cholesterols and phospholipids from

peripheral tissues, for example, macrophages but also from VLDL or chylomicrons. These freecholesterol molecules are esterified by the HDL-associated enzyme LCAT which forms thishydrophobic core of cholesterol esters in the HDL particle. Subsequently, these cholesterolesters are cleared in the liver either directly via interaction of the HDL particle with thescavenger receptor B1 or in an exchange between LDL via the LDL receptor.

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This process here of accepting cholesterol from peripheral tissues is called Reverse CholesterolTransport and this Reverse Cholesterol Transport can easily be measured.

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Here, cells, in this case macrophages, are loaded with radioactive labelled cholesterol and

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after a washing step to remove the excess of cholesterol which has not entered the cell, oneadds HDL

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as Cholesterol-acceptor into this system which induces the flux of cholesterol from the cell toHDL.

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Subsequently by scintillation counting,

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the radioactive activity can be measured intracellularly and bound to HDL in the supernatantas a measure for the Reverse Cholesterol Transport. But how relevant is this cholesteroltransport? Does it correlate with outcome?

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Here are the results of a study published in 2011. The cholesterol efflux or Reverse CholesterolTransport was determined in around 800 patients undergoing coronary angiography andhealthy subjects and divided in quartiles. Here you can see that the cholesterol efflux, theReverse Cholesterol Transport, led to a highly significant reduction in the odds ratio forcontrary artery disease.

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How does it look in patients with CKD? Here are two results of two recent trials examining thecholesterol efflux in dialysis patients and comparing it to healthy controls. You can see, bothstudies found that the cholesterol efflux was significantly reduced in dialysis patients ascompared to healthy subjects.

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But besides its role in the Reverse Cholesterol Transport, HDL directly interacts with theendothelium. In healthy endothelial cells the endothelial nitric oxide synthase produces nitricoxide, which diffuses to the vascular smooth muscle cells and induces VSMC relaxation. Incontrast, in patients with CKD several factors circulating in the serum of these patients such aslipoproteins, ADMA, angiotensin 2 or TNF-α promote the production of reactive oxygen speciesor other COX-derived factors. Moreover, also endothelin, which induces a contraction of thevascular smooth muscle cells initiating a process which is called endothelial dysfunction andwhich is known to be one first crucial step in the pathogenesis of atherosclerotic diseases.

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HDL from healthy subjects then interacts with several receptors on the surface of endothelialcells and increases the activity of eNOS to produce the vasodilatatory nitric oxide.

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Moreover, HDL inhibits a pro-coagulatory activation of the endothelium, inhibits pro-inflammatory endothelial activation and promotes the repair of endothelial lesions.

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Exactly these properties were studied by our group in a recently published project. Werecruited patients with different degrees of renal impairment and healthy controls and isolatedHDL from the serum

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using density gradient ultracentrifugation.

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Afterwards, we incubated human aortic endothelial cells with the isolated HDL and performedseveral in vitro and in vivo analyses.

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At first, we determined the endothelial nitric oxide production in response to HDL by usingelectron spin resonance (ESR) spectroscopy and we found, as it was previously known, thatHDL from healthy subjects increased endothelial nitric oxide production. Surprisingly, we founda strong inhibition of the basal nitric oxide production in response to HDL from patients withCKD. Interestingly, these changes were already present when we used HDL from patients withincipient CKD. To confirm that the CKD per se and not concomitant diseases, such as coronaryartery disease and diabetes, which are known to induce changes of the HDL functionality, wealso analysed samples from children with CKD. This is a collaboration with Rukshana Shrofffrom London. Also in these childre,n we found that HDL from children with CKD inhibited thenitric oxide production indicating that CKD and not only the concomitant diseases areresponsible for the adverse effects of HDL. Since the effect on endothelial nitric oxideproduction was that much pronounced, we decided to go next for an in vivo model.

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We used this HDL, injected it into mice, and measured the arterial blood pressure 90 minutesafter the injection of HDL. In this experiment, we found that healthy HDL significantly reducedthe arterial blood pressure within 90 minutes and HDL from CKD patients even increased thearterial blood pressure.

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Next, we also examined the effect of HDL on endothelial repair. Therefore, we used theCarotid Injury Model. In this model, the right common carotid artery is damaged by a bipolarforceps on a length of 4 mm and afterwards, HDL is injected into the mice. Three days later,the carotid arteries were harvested and the damaged area was stained by using Evans blue.

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We also found here that HDL from healthy subjects promoted the repair of endothelial lesionswhile this effect was totally abolished with HDL from CKD patients.

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Moreover, to examine the anti-inflammatory properties of HDL we performed a mononuclearcell endothelial adhesion assay. You can see here: Healthy HDL reduced TNF-α and inducedendothelial mononuclear cell adhesion while this effect was not present with HDL from CKDpatients.

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In more mechanistic studies we found that the small molecule symmetric dimethyl arginine,SDMA associates with the HDL particle from CKD patients only and that this abnormal HDL isthen recognised by TLR2 directly inducing endothelial superoxide production via NADPHoxidase and down-regulates the eNOS activity. So if we see the findings of our studies andstudies by other groups, these findings indicate that HDL seems to be dysfunctional.

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One question remains: Should we raise HDL serum levels? What should we do in the clinics?

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Currently, there are no studies in patients with CKD but at present there are several studiesexamining the effect on pharmaceutically racing HDL cholesterol serum levels. You can seehere some findings of the Dalhart programme using Dalcetrapib, a CETP inhibitor to raise HDL.This is the first slide of this study. Here you see the HDL cholesterol serum levels. CETPinhibition works within one month of treatment. Dalcetrapib significantly increased the HDLcholesterol serum levels. But when we look at the results,

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at the incidence of the primary outcome, there was virtually no difference between the placebotreatment or treatment with Dalcetrapib. This is somewhat surprising since you know higherHDL in the general population from data from epidemiological studies indicate that higherlevels are associated with an improved outcome. But what's the reason for these observationsin this trial?

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Then we go to a sub-study of the Dalheart programme performed in Zurich. Here theyexamined the flow-mediated vasodilation as a marker for endothelial function in patientsreceiving Dalcetrapib or in patients receiving placebo 12 and 36 weeks after initiation oftreatment. Also here there is no difference between the flow-mediated dilation betweenDalcetrapib and placebo treated patients. What is the reason for this? As I told you, higherconcentrations of HDL induce endothelial nitric oxide production and should therefore, alsoimprove the flow-mediated vasodilation. There's currently a frequent debate ongoing aboutthese CETP inhibitor studies. I don't want to contribute to this but there's just one small thing.

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If we look into the inclusion criteria for this Dalcetrapib study, you can see that they used acreatinine threshold of 2.2 mg/dL and that even 11% of the study participants had a GFRbelow 60 ml/min. If we consider the results of our in vitro studies and studies by other groups,it makes some sense that increasing dysfunctional HDL does not improve or should notimprove the outcome.

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But currently, there are no studies in kidney disease patients per se. Here is just one resultfrom our group together with the Luric Consortium around Professor März, we analysed datafrom 3300 patients undergoing coronary angiography which were all followed for about 10years. We examined the relationship between increasing concentrations of high densitylipoproteins in patients with different categories of their GFR. In patients with a normal GFRabove 90 ml/min, you see that higher concentrations of HDL significantly reduced the hazardsratio for cardiovascular mortality during follow-up. In patients with a GFR of between 60 and90 ml/min there was no significant effect on increasing HDL concentrations on cardiovascularmortality. In patients with a GFR below 60 ml/min, there was even a small trend towards ahigher cardiovascular mortality. These epidemiological data can prove our experimentalfindings but of course, interventional studies are necessary in CKD patients to examine theprognostic value of HDL cholesterol serum levels.

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So, I can summarise that HDL is a particle, which exhibits a complex structure. Its compositionis not static but it's permanently remodelled under different disease conditions such as CKD.CKD changes the composition of the HDL particle, which deteriorates its vasoprotectiveproperties. Higher levels of HDL serum cholesterol are not associated with improved outcomesin CKD and it is necessary to develop novel assays to measure the vascular function of HDLinstead of its static serum levels to be also able to examine the effect on different therapies onHDL composition. Thanks.

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Chairman: So thank you very much Doctor Speer for this very nice talk. It's now open todiscussion. Are there any questions or comments? David Wheeler?

Question: Timo, David Wheeler. Very nice. Is there anything we could do to make HDL behaveproperly in CKD? So, should our therapeutic strategy be to make HDL behave itself rather thanto behave as a particle that's going to damage the endothelium? And have you got anysuggestions as to how we could do that?

Prof. Speer: At the moment there is nothing known about any therapeutic interventions tochange the remodelling of the HDL particle. There are some studies in patients with coronarydisease which indicate for example, that niacin changes the abnormal vasofunction of HDL butin CKD there are no data available at the moment but it's really necessary, yes.

Question: Thank you.

Question: Shoji from Japan. A very interesting story but perhaps dysfunctional HDL is relatedto inflammation right? But, as I presented before in Japan dialysis patients higher HDLcholesterol was associated with lower risk of coronary disease. So perhaps your data and mydata are different in terms of the basal information. Japanese patients are known to have lessinflammation. Do you have any comments on this?

Prof. Speer: Here in the Luric study, only people from Germany are included and we have noinformation as to whether they really have CKD, most of them have but we have only theinformation of the renal functionAs I said, I cannot say CKD is associated with a worseoutcome I can only say that a reduced kidney function accurately determined by using the newCKD eGFR including cystatin C and creatinine has no effect on the outcome.

Question: Thank you.

Question: Could you comment on the utility on the current assays for HDL in accuratelyestimating the level of abnormal HDL and the normal HDL?

Prof. Speer: That's a really good question because this is quite difficult. At the moment to dothese functional analyses, it is necessary to isolate the HDL particles by ultracentrifugation.This takes around 5 days so it is quite time consuming. At the moment it is possible tomeasure the activity of some HDL associated enzymes for example, the paraoxanase-1 activityis suggested to be a marker of the functionality of HDL but for the assays I presented, nitricoxide and so on, it is quite difficult to perform these analyses in routine testing.

Question: HDL is so complex. Could you share regards your speculation as to whether what isthe crucial principal changing the function of HDL? What is it? Would it be serum amyloid A,would it be paraoxanase? What causes the mess? Is it SDMA?

Prof. Speer: The answer to this question is complex as the question itself or the HDL particleitself. Yes, there are many studies in patients with CKD showing that HDL is dysfunctional insome way, endothelial effects, effects on mononuclear cells and every manuscript, every paperdescribes one mechanism, they show serum amyloid A and I have shown SDMA but I think thatthere's a interaction of the effects. I don't think that in the earliest stages of CKD, for example,in stage 2 that we have complex changes of the proteome of HDL. I rather think we havechanges in small molecules associated with HDL not necessarily covalently bound to the HDLparticle. Maybe it also depends on the underlying disease. Maybe it's different in diabetic CKDpatients as compared to patients with glomerulonephritis, for example.

Chairman: Thank you. Are there any more questions or comments? Doctor Speer, you suggestthat novel assays are necessary to measure the endothelial dysfunction in these patients. Areyou aware of data regarding the release of endothelial particles in the blood of CKD patientsand the relationship with the modification of HDL?

Prof. Speer: This is interesting, one knows that HDL reduces endothelial apoptosis but themicroparticles themselves have not been examined in the setting of HDL or in cells treatedwith HDL but it's interesting, yes.

Chairman: If there are no more questions, we have to thank the speakers and my co-moderators and close the session. Thank you very much for your attention.