The Clinical Value of HDL Function Measurements Ebtehaj, Sanam

University of Groningen

The Clinical Value of HDL Function Measurements Ebtehaj, Sanam

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The Clinical Value of HDL Function Measurements

Sanam Ebtehaj

This project was financially supported by:

University Medical Center Groningen, Graduate School of Medical Science

Cover and layout: Sanam Ebtehaj

Print by: Ridderprint BV, the Netherlands

Copyright ã 2019 Sanam Ebtehaj

All rights reserved. No part of this publication may be reproduced, stored or transmitted in any form or by any means, electronically or mechanically, including photocopy, recording or otherwise without the written permission of the author.

ISBN: 978-94-034-1740-0 (printed)

ISBN: 978-94-034-1739-4 (digital)

The Clinical Value of HDL Function Measurements

PhD thesis

to obtain the degree of PhD at the University of Groningen

on the authority of the Rector Magnificus Prof. E. Sterken

and in accordance with the decision by the College of Deans.

This thesis will be defended in public on Wednesday 3 July 2019 at 9.00 hours

by

Sanam Ebtehaj born on 19 September 1983

in Tabriz, Iran

Supervisors Prof. dr. F. Kuipers Prof. dr. U.J.F. Tietge

Assessment Committee Prof. dr. J.A. Kuivenhoven Prof. dr. S.J.L. Bakker Prof. dr. G. Theilmeier

Paranymphs

Shiva Shajari

Ali Saeed

CONTENTS

Chapter 1. General Introduction

Chapter 2. HDL cholesterol efflux does not predict cardiovascular risk in hemodialysis patients

Chapter 3. The predictive value of the anti-oxidative function of HDL for cardiovascular disease and graft failure in renal transplant recipients Chapter 4. The anti-inflammatory function of HDL is impaired in type 2 diabetes:

the role of hyperglycemia, paraoxonase-1, and low-grade inflammation Chapter 5. HDL function measurements provide clinical information independent of

established cardiovascular risk metrics - a cross-sectional study from the PROCAM-CT cohort

Chapter 6. General discussion and future perspective Chapter 7. Summary

Appendix. Acknowledments List of publications

9

39

61

85

103

123

141

149

154

1

CHAPTER 1

General Introduction

Chapter 1

Scope of the thesis

Cardiovascular disease (CVD) is the leading cause of morbidity and mortality globally

. Among other risk factors, it is well established that the concentration of cholesterol in high-density lipoproteins (HDL) is inversely correlated with the risk of CVD

.

Due to the variable functional capacity of HDL in several physiological and pathological processes, the link between HDL, HDL-cholesterol (HDL-C) level and CVD has been a subject of intense research, leading, however, to contrasting results. Traditionally, low levels of HDL-C and high levels of Low-Density Lipoprotein cholesterol (LDL-C) were first linked to increasing the risk of CVD

. Therefore, to prevent cardiovascular events pharmacological strategies lowering LDL-C were proposed to be complemented by approaches to increase levels of HDL- C. However, recent clinical trials have shown that HDL-C raising pharmacological therapies such as niacin

or CETP inhibitors do not reduce cardiovascular risk

. In addition, genetic studies indicated that lifelong reductions or increases of HDL-C levels due to variations at several loci with a direct impact on HDL metabolism do not translate into a respective increased or decreased risk for atherosclerotic CVD

. Combined, these data lead to a shift of focus in the cardiovascular field from HDL-C quantity measurements to efforts to determine the quality of HDL and its overall impact on diverse diseases. HDL possesses many features that conceivably contribute to the association between elevated HDL-C and protection from CVD

.

This thesis aims to assess the contribution of HDL functionalities to CVD risk and to specifically determine the predictive value of selected key anti-atherosclerotic functions of HDL for the risk of future CVD events.

10

General Introduction

Introduction

Lipoproteins are complex particles with a central core containing cholesterol esters (CE) and triglycerides (TG) surrounded by free cholesterol (FC), phospholipids, and apolipoproteins, which facilitate lipoprotein formation and function. These lipoprotein fractions are classified into the following groups based on density: very low-density lipoproteins (VLDL), <1.019 g/ml;

low-density lipoproteins (LDL), 1.019–1.063 g/ml; and high-density lipoproteins (HDL), >1.063 g/ml

. Each class varies in size, density, and lipid composition. Dyslipidemia is one of the important risk factors for development and progression of cardiovascular disease (CVD), mainly atherosclerotic coronary artery disease

. It is well accepted that high plasma levels of LDL-C play a main role in initiating and propagating the process of atherosclerotic lesion development

22-24

. There is abundant evidence for the effectiveness of treatments that decrease LDL-C in the prevention of CVD. For instance, using HMG-CoA reductase inhibitors, known as statins, has become the mainstay of therapeutic strategies to reduce the risk of atherosclerotic CVD

. Despite the vastly proven anti-atherogenic benefits of statins, however, substantial residual CVD risk remains

It was supported by epidemiological and clinical studies, as well as meta- analyses, that individuals with low plasma levels of HDL-C are at increased risk of CVD events

27-30

. However, recent genetic studies as well as pharmacological intervention trials demonstrated that increased HDL-C levels do not translate into the expected reduction in CVD risk

Drugs that elevate HDL-C via different mechanisms such as fibrates, niacin

or cholesteryl ester transfer protein (CETP) inhibitors

have failed to consistently and significantly reduce the risk of major cardiovascular events in statin-treated patients with established CVD. For instance, in the context of niacin, two independent clinical trials with niacin added to statin therapy showed that among patients with atherosclerotic CVD there was no additive clinical benefit from the added niacin to statin therapy, despite significant increases in HDL-C level

. Thus far, clinical trials with inhibitors of CETP, a transfer protein promoting the exchange of CE for TG from HDL to apoB-containing lipoproteins in plasma, also have been disappointing. The first CETP inhibitor torcetrapib, was even prematurely stopped due to increased cardiovascular mortality

. Other CETP inhibitors such as dalcetrapib and evacetrapib, also failed to reduce cardiovascular events

. While these findings have casted doubt upon the importance of HDL-C modulation for CVD risk, it is becoming increasingly clear that HDL functionality measurements could represent viable targets for CVD risk reduction. Thus, efforts to determine defined anti-atherosclerotic functions of HDL represent an emerging concept for CVD risk prediction. An attractive novel intervention strategy might be to improve HDL functionality, rather than simply increasing HDL-C concentrations

. However, still more data are required before this concept can be clinically implemented. Therefore, the aim of this thesis is to provide more insight into the value of HDL functionality for cardiovascular risk.

1. High density lipoprotein structure and composition

HDL constitutes a heterogeneous group of particles that differ in size, shape, density, lipid and protein composition. The surface of these particles is composed of a monolayer of negatively charged phospholipids (phosphatidylcholine, phosphatidylserine, cardiolipin and phosphatidylethanolamine), FC and apolipoproteins, with a TG and CE-rich hydrophobic core

37

. HDL particles are the smallest lipoproteins with a mean size of 8–10 nm and a high density

Chapter 1

of 1.063–1.21 g/ml 37,38.

1.1 Subtypes of HDL

HDL can be, more or less arbitrarily, classified into two major subtypes by density using ultracentrifugation. These subtypes are large, lipid-rich HDL2 (1.063-1.125 g/ml) which can be further separated into HDL2b and HDL2a ; and small, protein rich HDL3 (1.125-1.21 g/ml) which can be further separated into HDL3a, HDL3b and HDL3c

.

1.2 Main proteins of HDL

HDL proteins can be categorized into four major subclasses namely, apolipoproteins, lipid transfer proteins, enzymes and minor proteins.

1.2.1 Apolipoproteins of HDL

Apolipoproteins interact with lipids to form lipoproteins

. Apolipoproteins play an important role in lipoprotein receptor recognition and the regulation of certain enzymes in lipoprotein metabolism

. The HDL particles consist of different apolipoproteins including apoA-I as well as apoA-II, apoA-IV, apoA-V, apoC-II, apoC-III, apoC-IV, apoD, apoE, apoF, apoH, apoJ, apoL and apoM on their surface

. The main and the most abundant apolipoprotein in HDL is apoA-I.

ApoA-I molecules account for approximately 70% of total HDL protein

. Almost all HDL particles are believed to contain apoA-I

. Both liver (about 70%) and the intestine (about 30%) synthesize apoA-I, which plays a key role in biogenesis and function of HDL 45. Major functions of apoA-I involve interaction with cellular receptors and activation of lecithin:cholesterol acyltransferase (LCAT), which transforms FC to CE

.

1.2.2 Lipid transfer proteins of HDL

Phospholipid transfer protein (PLTP) and CETP are both lipid transfer proteins found in HDL

. PLTP is synthesized in the placenta, pancreas, lung, kidney, heart, liver, skeletal muscle and brain. It transfers phospholipids from apoB-containing triglyceride-rich lipoproteins into HDL and also exchanges phospholipids between lipoproteins

. The particle size distribution and composition of HDL are regulated by PLTP in the circulation, which also plays an important role in controlling plasma HDL size distribution by converting it into larger and smaller particles

49,52,53

. The CETP is mainly secreted by the liver (and here apparently to a large proportion by Kupffer cells), adipose tissue and spleen

, and circulates in plasma mainly associated with HDL. CETP catalyzes the exchange of CE for TG between HDL and apoB-containing lipoproteins, such as VLDL, LDL, thus resulting in CE depletion and TG enrichment of HDL. Therefore, CETP activity results in a pro-atherogenic lipid profile with decreased plasma HDL-C levels and increased levels of cholesterol within apoB-containing lipoproteins

.

1.2.3 Enzymes of HDL

A recent analysis of HDL identified a large number of enzymes that are -transported on these lipoprotein particles and potentially influence functional properties of HDL such as LCAT, Paraoxonase-1 (PON-1), Lipoprotein-associated phospholipase A2 (Lp-PLA2) and Glutathione

12

General Introduction Selenoperoxidase 1 (GSP-x)

. LCAT is an enzyme that catalyzes the conversion of cholesterol to CE, which then causes the formation of mature spherical HDL particles. LCAT is essential for the development of mature HDL and it is critical for the maintenance of normal HDL metabolism. Recent research findings from animal and human studies have revealed a potential beneficial role of LCAT in reducing atherosclerosis, however, individuals with familial LCAT deficiency (complete loss of function) and fish eye disease (partial loss of function) are not uniformly at increased risk for premature atherosclerosis despite decreased levels of HDL- C and rapid catabolism of apoA-I and apoA-II

. PON-1 protects LDL and HDL from oxidation induced by either copper ions or free radicals. It hydrolyzes oxidized LDL (ox-LDL)-associated lipids, which stimulate the production of cytokines, IL-8 and monocyte colony-stimulating factors, inducing adhesion of monocytes to the endothelial surface. Furthermore, low serum PON-1 activity has been reported as a predictive factor for future cardiovascular events

. Lp- PLA2 is a calcium-independent hydrolytic enzyme that can hydrolyze oxidized phospholipids, which was initially thought to be atheroprotective because it degraded platelet activating factor. It is also named platelet activating factor acetylhydrolase (PAF-AH)

. Another enzyme on HDL is GSPx-1, which catalyzes the reduction of organic hydroperoxides and hydrogen peroxide (H2O2) by glutathione, and thereby protects cells against oxidative damage. Many biological metrics of HDL, such as vasodilation and the induction of endothelial NO production might be at least partially mediated by this enzyme

.

1.2.4 Minor proteins of HDL

HDL carries other groups of proteins such as acute-phase response proteins which are elevated in CVD

and T2DM

Serum Amyloid A (SAA) proteins are a family of apolipoproteins associated with HDL in plasma; some members are constitutively expressed, while others are important acute-phase reactants

. During the acute phase of the inflammatory response, the rise in SAA levels constitutes one of the most rapid and intense increases of all acute phase proteins. High levels of SAA can be seen in patients with acute and chronic inflammation

. Group IIA secretory phospholipase A2 (sPLA2-IIA) is an acute phase protein that is increased in the setting of acute and chronic inflammation

. In plasma sPLA2-IIA associates with HDL particles and hydrolysis of HDL phospholipids by sPLA2-IIA plays a role in modulating HDL metabolism and function

. Increased sPLA2-IIA expression by inflammatory stimuli results in lower circulating HDL-C levels during inflammatory responses in vivo.

. The plasma concentration of sPLA2-IIA, as well as its activity, is markedly increased in patients with persisting infections and chronic inflammatory diseases

.

2. Multiple biological activities of HDL

Cholesterol is exported from the liver on apoB-containing VLDL. These are remodeled in the

blood compartment by TG hydrolysis to LDL particles that are taken up into cells mainly via the

LDL receptor (LDL-R). To prevent accumulation of cholesterol in peripheral tissues, HDL has the

capacity to remove excess cholesterol in the arterial wall. This cholesterol is then transported

on HDL to the liver for final excretion into the bile and feces, either as FC or after metabolic

conversion into bile acids

. This pathway is known as Reverse Cholesterol Transport (RCT) and

the role of HDL to efflux the cholesterol is critical in the prevention of CVD. Nowadays, a second

Chapter 1

cholesterol elimination pathway is known to contribute to fecal excretion of cholesterol, the direct transintestinal cholesterol excretion (TICE) of plasma-derived cholesterol by enterocytes into the lumen of the small intestine

. However, the role of HDL herein seems to be minor

81,82

.Moreover, HDL particles possess other potent biological activities such as anti- inflammatory, anti-oxidative, anti-thrombotic, anti-apoptotic and, vasodilatory properties (figure 1). Such heterogeneity of biological roles led to the emergence of attempts to experimentally assay HDL functions with the aim to refine cardiovascular risk assessment associated with HDL. Via these properties, HDL can be a protective factor against various diseases such as preventing bacterial infections, diabetes or CVD among others

.

Figure 1. Key anti-atherogenic functionalities of High-Density Lipoprotein. HDL can inhibit the process of atherosclerosis by (A) promoting cholesterol efflux by taking up cholesterol from foam cells, (B) anti- inflammatory activity by inhibiting the endothelial expression of adhesion molecules, (C) anti-oxidant activity by inhibiting LDL oxidation and (D) vasorelaxation by increasing the endothelial production of NO via induction of eNOS.

14

General Introduction

2.1 Cholesterol efflux capacity

Cholesterol efflux is the first step of RCT. It was originally described as a mechanism by which HDL transports excess FC from cells in the periphery, such as macrophages in the artery wall to the liver for excretion into the bile and feces. Macrophage foam cells are the primary cell type that is rich in cholesterol and are major cellular components of the early and advanced atherosclerotic lesion. It has been shown that HDL via RCT plays a major role in the reduction of atherosclerotic plaque formation and risk of CVD

. As mentioned previously, HDL-C plasma levels cannot always predict the risk of CVD events. However, the cholesterol efflux ability of HDL from foam cells has been proposed to be an important and better predictor of CVD risk

.Lowering the lipid content in macrophage foam cells is mechanistically expected to attenuate atherosclerosis and reduce inflammation

. Impaired HDL cholesterol efflux function might be a critical HDL function for CVD, therefore enhancing cholesterol efflux capacity might serve as a new therapeutic target. With respect to this concept, Rohatgi et al.

showed that cholesterol efflux capacity was inversely associated with incident cardiovascular events in a large, multiethnic population cohort

. In a cross-sectional study Khera et al. also showed that this metric of HDL has a strong inverse association with both carotid intima–media thickness and the likelihood of having angiographically confirmed coronary artery disease, even independent of HDL-C levels

. Therefore, cholesterol efflux capacity may serve as a predictive biomarker for CVD events

.In humans the efflux capacity is provided by several mechanisms.

Adenosine triphosphate-binding cassette, subfamily A, member 1 (ABCA1) transporter mediates efflux towards apoA-I or lipid-poor nascent HDL as acceptors. Larger HDL particles can serve as an acceptor of cell cholesterol provided by the ABCG1 transporter (Favari et al.

2009). In comparison with mature HDL, nascent HDL carries little CE, but more FC. The scavenger receptor class B type 1 (SR-B1) is a multifunctional protein, which facilitates a bidirectional transport of cholesterol in macrophages with medium or large HDL as the major involved subclasses

. In addition to these mechanisms also passive or aqueous diffusion was proposed to contribute to the net movement of cholesterol out of macrophage foam cells.

After integration of FC into nascent HDL, FC is esterified by the enzymatic activity of LCAT within HDL, potentially helping to keep the gradient of FC from the cells to the acceptors. Afterward, the excess cholesterol from peripheral cells is transported to the liver, where it can be taken up by three different ways namely; 1. SR-B1, which mediates selective uptake of HDL CE

, but not the protein component of HDL

, 2. Holoparticle uptake, HDL (lipids and proteins) can undergo holoparticles endocytosis involving the mitochondrial F1 ATPase as well as P2Y13 receptors

and 3. Through the action of CETP, CE of HDL can also be selectively transferred to apoB-containing lipoproteins in exchange for TG and returned to the liver via the LDL-R.

Consequently, the apoB-containing lipoproteins may either transport the cholesterol to the liver, reducing the risk of atherosclerosis or return it to the peripheral tissues, which will increase the risk of atherosclerosis

.

2.2 Anti-inflammatory activity

Inflammation refers to an elementary process of homeostasis and repair that, however, is also

a pathological mechanism that causes a variety of diseases

. It is well accepted that

atherosclerosis is a chronic inflammatory disorder characterized by the accumulation of

Chapter 1

cholesterol and immune cells (e.g. neutrophils, lymphocytes, and monocytes/macrophages) within the arterial wall

. This is associated with increased plasma concentrations of various inflammatory markers

and leads to plaque formation inside the arteries. The main consequence of plaque formation is a limited flow of oxygen-rich blood to the organs

. However, the main risk of plaque formation in the coronary arteries is plaque rupture

. More than two-thirds of heart attacks and strokes are caused by the rupture of atherosclerotic plaques

. Several studies have shown that HDL display multiple anti-inflammatory effects such as inhibition of cytokine-induced adhesion molecule expression, inhibition of monocyte adhesion to activated endothelial cells in vitro, reduction of neutrophil activation and infiltration in the arterial wall

. An early step in the inflammatory process is monocyte adhesion to activated endothelial cells due to damage or vascular inflammation

. Adhesion of monocytes is normally facilitated by the expression of several adhesion molecules, including vascular cell adhesion molecule-1 (VCAM-1) and intercellular adhesion molecule-1 (ICAM-1), which were demonstrated to be important in atherosclerotic lesion development

. Following attachment to endothelial cells, monocytes migrate into the intima, where they differentiate into macrophages. Then they produce a number of pro-inflammatory cytokines and reactive oxygen species that aggravate oxidation of LDL

. The mechanism underlying the anti- inflammatory function of HDL is known to be, at least partly, via inhibition of nuclear factor kappaB (NF-kB) signaling. In a similar way, SR-BI-dependent process S1P contained within HDL activates endothelial nitric oxide (eNOS) synthase and subsequently the production of NO, which decreases vascular inflammation

. The identification of apoM as the major carrier protein for HDL-bound S1P has resulted in an increased interest in the role of apoM as potential HDL-based CVD biomarker

. The anti-inflammatory activity of HDL may also involve hydrolysis of pro-inflammatory oxidized lipids from endothelial cells or from ox-LDL by HDL through the activity of Lp-PLA2, PON1 and LCAT

. However, different HDL subfractions have been suggested to have different effects, for example, small, dense, protein-rich HDL3 has been reported to have a higher capacity to inhibit VCAM-1 expression in endothelial cells in comparison with large, lipid-rich HDL2. Further it has been suggested that HDL can be dysfunctional to the point that it even increases inflammation, and thus can become a pro- inflammatory particle

. This will conceivably contribute to increase the risk of various diseases including CVD

.

2.3 Anti-oxidative activity

The anti-oxidant activity of HDL is achieved by its apolipoproteins, mainly apoA-I, but also apoA- II, apoA-IV, apoE, apoJ, and its associated enzymes. Oxidation of the accumulated LDL whitin the arterial wall is usually considered to contribute to the initiation and progression of atherosclerosis

. HDL exhibits potent capacity to protect LDL from free radical-induced oxidative damage and to inhibit ox-LDL-induced apoptosis of endothelial cells via transfer of oxidation products from LDL to HDL. The removal of oxidized phospholipids by HDL might be followed by subsequent degradation of oxidation products by HDL-associated enzymes or by uptake into the liver for degradation. PON1 inhibits formation of lipid peroxides in LDL

and may reduce active oxidized phospholipids in ox-LDL

. ApoA-I can also remove oxidized phospholipids from ox-LDL as well as from cells. PON1 hydrolyzes ox-LDL-associated

16

General Introduction compounds, and Lp-PLA2 hydrolyzes PAF, a potent lipid mediator with pro-inflammatory effects and thus participates in the degradation of oxidized phospholipids

.

2.4 Anti-thrombotic activity

Another potential beneficial function of HDL is its antithrombotic ability. Any damage to endothelial cells, together with an abnormal function of blood components can cause arterial thrombosis formation

. It is linked to dyslipidemia, activation of platelets and altered blood flow. Also, Lp(a) can result in a hypercoagulable state

and an increase in thrombotic disorders at higher levels

. HDL prevents thrombosis partly by promoting synthesis of NO which enhances the integrity of the endothelium and by reducing apoptosis of endothelial cells

132,133

. In addition, HDL inhibits the platelet activator thromboxane A2 formation and platelet activation factor synthase, which can reduce platelet aggregation

. HDL sphingosine, may inhibit the production of thrombin by promoting the integrity of the vessels and reducing apoptosis

. Furthermore, HDL stimulates the production of endothelial cell thrombomodulin which is an anticoagulant factor

. It has been reported that the administration of reconstituted HDL (rHDL) to humans and the infusion of apoA-I Milano into rats can inhibit platelet aggregation

. Antithrombotic activity of HDL may apply via different mechanisms such as inhibition of endothelial cell activation, thrombin generation and platelet activation

.

2.5 Anti-apoptotic effects of HDL

One of the ongoing processes in atherosclerosis is cell death in response to endothelial injury

138

. The main stimulants are thought to be ox-LDL, pro-inflammatory cytokines, as well as growth factor deprivation. Ox-LDL stimulates apoptosis in endothelial cells by oxidative stress 139,140 contributing to atherosclerosis

. The production of oxidative stress causes dose- dependent levels of apoptosis

. A damaged endothelium is vulnerable to adhesion of inflammatory cells and platelets which can further contribute to atherosclerotic plaque formation

. It has been widely established that HDL prevents endothelial cell death by reducing apoptosis. The anti-apoptotic actions of HDL preserve the endothelial lining and prevent atherosclerosis

. HDL prevents activation of caspases 9 and 3 and apoptotic alterations of the plasma membrane such as increase of permeability and translocation of phosphatidylserine

. Furthermore, the anti-apoptotic capacity of HDL in endothelial cells has been attributed to apoA-1. In bovine aortic endothelial cells, HDL, especially ApoA-I, is able to prevent ox-LDL-induced apoptosis by blocking intracellular signaling involved in apoptosis. HDL also protects endothelial cells against growth factor deprivation-induced apoptosis

.

2.6 Vasorelaxation activity of HDL, (NO) production

The vascular endothelium is a large dynamic surface containing a multitude of diverse

receptors, which regulate and integrate a number of (patho)physiologically relevant signals

.

An important consequence of such signaling is the regulation of NO production

.

Hemodynamic shear stress on the endothelial cells also leads to NO generation

. NO is a key

molecule which maintains normal endothelial function and prevents CVD. HDL facilitates NO

production supposedly by using SR-B1 as a scaffold

. HDL can increase the abundance

of eNOS protein, the enzyme which catalyzes NO production resulting in vasorelaxation

.

Chapter 1

Moreover, NO inhibits platelet activation and adhesion of monocytes

.

3. Association of HDL with disease states 3.1 HDL and cardiovascular diseases

CVD is the leading cause of mortality and morbidity in the developed world

. Diabetes, obesity, hypertension, dyslipidemia and kidney diseases are the major risk factors for developing CVD

. This current and coming burden of disease underlines the need for further insight into the mechanisms that contribute to the pathogenesis of CVD. Further, such pathophysiological insights will expand the panels of mediators and pathways that can be used for prediction, prevention, and treatment. Numerous mechanisms by which HDL protects against the development of CVD have been recognized. As outlined above, cholesterol efflux capacity is one of the well-known anti-atherogenic functions of HDL

. Besides promoting cholesterol efflux, other functionalities of HDL, including anti-inflammatory and anti-oxidant, are potentially of major importance for CVD protection

. Endothelial cell dysfunction and foam cell formation are prominent features of atherosclerotic lesions and are widely recognized as early hallmarks of atherosclerotic plaque formation during initiation and progression of coronary artery disease (CAD)

. Arterial endothelial cells normally are resistant to adhesion of white blood cells such as leukocytes and monocytes

. Upon pathological triggers, (e.g. dyslipidemia, hypertension, diabetes and other inflammatory conditions) damaged endothelial cells will express adhesion molecules

. Then monocytes will adhere to the damaged endothelium via these adhesion molecules, which probably is one of the initial stages of plaque formation

. Consequently, monocytes will migrate into the vessel wall and differentiate into macrophages. Those monocyte-derived macrophages will take up the modified LDL, mainly ox-LDL in the arterial wall, and will form foam cells. These cells are known to contribute to plaque formation, progression and rupture

. Furthermore, damaged endothelium and chemoattractant mediators from activated macrophages stimulate leukocyte adhesion and subsequent migration into the intima (innermost layer of the artery)

. The main consequence of plaque formation is stenosis/plaque rupture in the artery, which leads to tissue ischemia or provokes thrombi, which interrupt local blood flow or embolize distal arteries.

During atherogenesis, other cell types are involved such as mast cells and smooth muscle cells

163,164

. Their activation occurs in response to stimuli that are secreted by other cell types, for instance macrophages. Atherosclerotic plaques consist of various cell types like accumulated macrophage-derived foam cells, smooth muscle cells, et cetera. Apoptosis in the core of the plaque, mostly of macrophages, further promotes the accumulation of cellular debris and extracellular lipids, forming a lipid-rich pool called the necrotic core of the plaque

. As mentioned monocyte adhesion and migration through damaged endothelial cells is the initial stage of atherosclerosis and a main feature of CVD

. HDL has been shown to inhibit the expression of adhesion molecules on vascular endothelial cells which prevents monocyte adhesion and migration. Accumulating information from in vitro and in vivo studies confirmed the inverse correlation between HDL and the expression of adhesion molecules by vascular endothelial cells. In one of these studies, Clay MA et al. 2001 showed that the expression of adhesion molecules (E-selectin and VCAM-1) on human vascular endothelial cells was

18

General Introduction upregulated upon TNF-α stimulation. The same study showed that the TNF-α-induced increase in the expression of adhesion molecules was robustly inhibited by exposure to rHDL

. Reduced expression of adhesion molecules followed by reduced binding of inflammatory cells has been shown to result in efficient inhibition of atherosclerotic plaque formation

. Modified LDL, mainly ox-LDL particles, and their endocytosis by macrophages are important in atherosclerotic plaque formation and progression

. It has been shown that HDL, via carrying enzymes such as paraoxonase, inhibits LDL particle oxidation which may result in reduced local inflammation (inhibition of pro-inflammatory cytokines production by arterial wall resident cells) and uptake of ox-LDL by macrophages, consequently decreasing inflammation-triggered foam cell formation

.

3.2 HDL and diabetes

Diabetes is a considerable contributor to the global burden of disease

. Vascular complications include retinopathy and nephropathy, peripheral vascular disease (PVD), stroke, and coronary artery disease (CAD)

. Patients with diabetes are dyslipidemic, which is accompanied by a low concentration of HDL-C. It has been reported that patients with diabetes have abnormalities in the level as well as structure of lipoprotein particles

. Patients with type 2 diabetes mellitus (T2DM) have a substantially increased risk of atherosclerotic CVD

. Recent cell-based studies suggest that HDL has the potential to modulate glucose metabolism in humans with T2DM

. Apart from HDL, several studies indicated an association between LDL size and CAD. LDL particles were proposed to be more or less atherogenic dependent on their size

3. Small LDL particles are more atherogenic compared to large particles. In diabetes, the small dense LDL predominate, which carry a higher risk for vascular disorders.

They are more susceptible to oxidation and able to get into the artery wall where plaques form, thus promoting atherosclerosis

. Ox-LDL induces the expression of adhesion molecules and consequently the release of endothelium-derived inflammatory mediators

. As mentioned, high TG and low HDL-C constitute the main pattern of lipid abnormality in diabetes.

Beside lower HDL-C level, HDL not only loses the ability to prevent LDL oxidation, but also it can potentially even be converted from an anti-inflammatory to a pro-inflammatory particle

. Production of oxidized lipids in diabetic lipoproteins has been ascribed to increased ROS production in hyperglycemic states

. Gathering all together, the cardioprotective ability of HDL is impaired in diabetes characterized by a reduction of cholesterol efflux and in turn increased intracellular cholesterol in tissues which is known as initial step in cardiovascular disorders. Thus, T2DM patients usually have been demonstrated to display reduced cholesterol efflux capacity

. In addition to the reduction of cholesterol efflux capacity, impaired anti- inflammatory and antioxidant properties were found in HDL from patients with T2DM

.

3.3 HDL and chronic kidney disease

Chronic kidney disease (CKD) is usually divided into five different stages based on kidney

function

. Patients with CKD are at high risk of premature CVD

, and it is more likely that

these patients will die of CVD than progress to end stage renal disease (ESRD)

. People

with advanced kidney failure, who have progressed to ESRD usually require hemodialysis,

peritoneal dialysis or transplantation

. The risk of CVD varies with the degree of renal

Chapter 1

function impairment. It has been shown that CVD in CKD patients has a positive correlation with the degree of renal function impairment

. Features that are usually associated with CKD are inflammation, oxidative stress, and dyslipidemia; the combination of these factors is highly associated with the acceleration of atherosclerosis formation

. Dyslipidemia in these patients is characterized by increased levels of plasma TG and VLDL-C, together with decreased concentration of HDL-C, while LDL-C and total cholesterol (TC) are within the normal range, or even reduced

. Lacquaniti et al. reported in 2010 that decreased levels of HDL-C or apoA-I are frequently observed in such patients possibly due to the down-regulation of hepatic apoA-I synthesis. In addition, patients with ESRD suffer from an age-adjusted 30-fold increase in CVD mortality

. However, in advanced CKD, circulating lipid and lipoprotein levels do not predict CVD outcomes as in the general population. Therefore, it is possible that functional properties of lipoproteins, particularly of HDL, may be altered providing clinical information beyond cholesterol content. Therefore, studying HDL function may unravel proatherogenic mechanisms by which CKD patients develop premature atherosclerosis and CVD

.

3.4 HDL and rheumatoid arthritis (RA)

Rheumatoid arthritis (RA) is a chronic inflammatory autoimmune disease in which there is chronic inflammation with fibrosis and the eventual destruction of cartilage and bone. Patients with RA have accelerated atherosclerosis associated with increased morbidity and mortality from CVD

. Reduction of of HDL-C levels is the most common lipid abnormality in RA, which is probably due to active inflammation. Inflammation can reduce the anti-atherogenic properties of HDL and it has been shown that HDL can even become pro-inflammatory in conditions like RA

. An increased risk of CVD among RA patients is described in several studies, liklely as a result of increased general inflammation per se but also perhaps secondary to side effects of medication or decreased physical activity

. A recent study proposed that pro-inflammatory HDL is significantly more common in patients with RA than in healthy controls, and pro-inflammatory HDL are unable to prevent the oxidation of LDL and the recruitment of monocytes, and thus might enhance the inflammatory reaction

.

20

General Introduction

Outline of the thesis

The overall aim of this thesis is to provide a better understanding of the value of HDL functionality measurements for cardiovascular risk assessment.

Although HDL-C levels are firmly and positively associated with the risk of future CVD events in large population studies, recent disappointing results from population genetics as well as from pharmacological intervention studies aiming to raise HDL-C concentrations have resulted in a shift in focus of HDL research from quantity to quality

. Several previous studies demonstrated in support of such a concept that HDL can lose its ability to counteract the development of atherosclerosis in certain disease states and may even become pro- atherogenic

. In addition, recent studies illustrated the potential of laboratory assays of HDL function for the development of new biomarkers for CVD

. Further, such assays could be suitable to also measure the effectiveness of current and future treatment regimens.

This thesis addressed HDL functionality in patient studies with relevance for CVD.

Chapter 1 provides a summary of HDL structure and metabolism in humans and introduces the concept of HDL function.

Chapter 2 examined cholesterol efflux from macrophage foam cells towards HDL from patients with Type 2 diabetes on hemodialysis participating in the 4D (Die Deutsche Diabetes Dialyse) Study to test, if the HDL cholesterol efflux capacity is predictive for cardiovascular risk.

Cholesterol efflux capacity of HDL was shown to correlate inversely with atherosclerotic CVD in populations with normal kidney function. Patients with ESRD suffer an exceptionally high cardiovascular risk not fully explained by traditional risk factors. However, the results of this chapter indicate that HDL cholesterol efflux capacity is not a suitable prognostic cardiovascular risk marker in diabetic patients on hemodialysis.

Chapter 3 examined the predictive value of HDL anti-oxidant properties for cardiovascular mortality, all-cause mortality, and graft failure in a longitudinal cohort study of renal transplant recipients (RTR). This population was specifically chosen since RTRs have a 4-6-fold higher risk of CVD than the general population and traditional CVD risk factors including HDL-C also do not fully explain this increase in risk. Therefore, RTR represent a patient group with considerably accelerated atherosclerosis. The prospective study presented in this chapter demonstrates that in RTR the anti-oxidative capacity of HDL does not predict cardiovascular or all-cause mortality.

There is an association with graft failure, however, not independent of baseline kidney function and inflammatory load.

Chapter 4 determined the impact of T2DM on the anti-inflammatory function of HDL and aimed to delineate potential factors that impact on this metric of HDL functionality. The HDL anti-inflammatory capacity is substantially impaired in T2DM, at least partly attributable to the degree of hyperglycemia, decreased PON-1 activity and enhanced low-grade chronic inflammation. Decreased anti-inflammatory protection capacity of HDL conceivably contributes to the increased atherosclerosis risk associated with T2DM.

In a cross-sectional study of PROCAM-CT including subjects with a high estimated CVD risk,

three key functional properties of HDL were tested, namely cholesterol efflux, anti-

inflammatory and anti-oxidative properties of HDL (chapter 5). Individual and combined HDL

functionalities and their association with PROCAM-risk, intima media thickness measurement,

Chapter 1

and Agatston score as measures of CVD risk were studied. Notably, there was no association between these measures of HDL function and PROCAM risk in this specific patient population.

Finally, chapter 6 summarizes the findings described in this thesis in the context of current literature and discusses perspectives for future research.

22

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