Biochemical markers for CVD risk assessment

The present invention relates to assays for detection of biochemical markers valuable for diagnostic purposes in cardiovascular disease and prognosis of disease development, including biochemical markers indicative of the risk of cardiovascular events resulting from atherosclerotic development and plaque instability.

Worldwide, cardiovascular disease (CVD) is the leading cause of morbidity and mortality. At present, there are no effective and non-invasive diagnostic methods that allow for diagnosis and classification of patients into different risk-groups and for the diagnosis of low risk patients. Diagnostic and prognostic tools are composed mainly of multivariate analysis of simple markers, such as age, smoking and various lipid and lipoprotein concentrations. CVD covers several clinical syndromes, primarily, angina pectoris, myocardial infarction (coronary thrombosis) and stroke. All of these syndromes are usually the sequelae of complicated atherosclerosis . Atherosclerosis begins with intimal thickening in childhood and progresses to fatty streaks in the intima of arteries - these lesions are characterized as type I and II, respectively. Fatty streaks are the earliest macroscopically visible lesions in the development of atherosclerosis and occur among almost all human beings of all races and societies. In the non pathogenic state, endothelial cells (EC) resist adhesive interactions with leukocytes. However, the actions of proinflammatory cytokines and accumulated oxidized lipoprotein in the arterial wall during atherogenesis, initiate expression of adhesion molecules, such as intercellular adhesion molecules (ICAM)-I and vascular cell adhesion molecules (VCAM)-I, on the surface of aortic ECs. This allows for capturing and transmigration of leukocytes through the endothelial surface, into the intimal part of the vessel wall. The development of plaques involves an increasing number of smooth muscle cells (SMC) that undergo displacement and apoptosis, which

results in increased matrix turnover. The impaired collagen synthesis can result in a weakened fibrous cap and an atherosclerotic plaque that is more prone to rupture; however, most investigators believe that the actions of a proteolytic enzymes such as matrix metallo-proteases (MMPs) and other proteases importantly contribute to the risk of plaque rupture (Clarkson and Kaplan 509-28) .

Plaques are divisible into two different types: Vulnerable' and ^stabilized' plaques. However, for detailed histological analyses and molecular understanding, a more detailed classification is often used. There are three major stages in development of plaque: initiation, fatty streaks and the complex/advanced plaque (Stary H. C.) .

Atherosclerotic plaques develop within the intima of arteries, and may be classified depending on their composition and structure. This classification divides lesions into eight types (Stary H. C.) : I . Macrophages loaded with and enlarged by lipid droplets

(macrophage foam cells) are increased in the intima. II. Macrophage foam cells accumulate in the deep part of the proteoglycan layer along with lipid droplets within the intimal SMC. The layers of foam cells are visible as fatty streaks. In type II lesions monocytes penetrate the endothelial lining by monocyte chemo attractant proteins (mainly MCP-I) , which are over expressed in human atheroma. The early types of lesion (type I and II) can start in infancy and do not necessarily lead to plaque rupture. Furthermore, the development of atherosclerosis may end after the formation of type III lesion, and the formation of plaque is not predictable (Stary H. C.) . III. The type III lesion is determined as the intermediate lesion between the fatty streaks (type II) and the atheroma (type IV) . These lesions contain pools of extracellular lipid and thereby expand the spaces between the normally closely adjoining SMCs of the deep musculo-elastic layer of the intima. The pools of material may replace

proteoglycans and collagen fibres that normally reside here, but this occurs with little impact at this stage of atherogenesis .

IV. The atheroma is the first clinical sign of atherosclerosis. Displacement of SMCs in the intima of arteries by accumulating extracellular pools of lipids and disruption of the intimal architecture is a hallmark of a type IV lesion. The formation of the lipid cores is the end result of this SMC displacement. Formation of a lipid core accounts for the increased wall thickening. The lipid core is a large and well delineated region of the deep intima where the normal structural elements of this part of the arterial wall have been replaced by densely packed foam cell remnants, free lipids droplets, cholesterol crystals and calcium particles. SMCs normally resident in this area are decreased or completely absent at this stage of atherosclerosis progression. Any remnant SMCs become widely dispersed and have developed elongated cell bodies and very often unusually thick basement membranes. At this stage, the development of a layer overlying the lipid core begins. This layer consists of collagen and proteoglycan- rich intercellular matrix, SMCs with and without lipid droplets, macrophages, and foam cells. V. The response to type IV lesion is the formation of a reparative fibrous tissue matrix, forming a fibrous "cap".

Typically, these lesions will consist of layers of lipid cores and reparative tissue irregularly stacked on top of each other. Events such as hematoma and thrombus formation may additionally complicate these types of lesions. If not fatal, these lesion complications are integrated into the lesion and overgrown by a thin layer of reparative matrix tissue, consisting of collagens and proteoglycans. The content of extracellular matrix proteins collagen and proteoglycans increases in the atherosclerotic plaque during formation of the cap.

VI. The defects of the endothelium such as fissures, erosions, ulcerations, hematoma, thrombus, haemorrhage can if combined lead to more complicated lesion type designated type VI lesion. VII. The lesion is often referred to as calcified lesion, where more than 50% of the lesion consists of mineral. In addition to calcifications, these lesions contain abundance of reparative fibrous connective tissue. When the SMCs trapped in this undergo apoptosis and disintegrate; their mineralized organelles become a part of the calcification.

VIII. The fibrotic lesion follows the calcific lesion. The fibrotic lesion may consist entirely of collagen and no lipid. (Stary H. C. )

Cardiovascular events are often the result of plaque rupture, in which inflammation and the release of proteases weaken the shoulder regions of the fibrous cap and allow the fatty materials in the plaque to come into contact with the blood precipitating a mural thrombus (Clarkson and Kaplan) . Thinning of the fibrous cap by increased protease activity in the combination with decreased matrix production, is considered a hallmark of plaque instability increasing the risk of rupture. Vulnerability of plaques and their risk of rupture is an area of clinical interest. Definition of a vulnerable plaque (VP) is not standardized, but there is a general agreement stating existence of three histological hallmarks compared to stable plaque:

1) A larger lipid core (> 40 percent of total lesion) .

2) A thinner fibrous cap (65 - 150 micrometers) .

3) Large amount of acute inflammatory cells.

Major criteria for defining VP include: active inflammation (presence of monocytes, macrophages and T cells) , thin cap with large lipid core, endothelial denudation with superficial platelet aggregation, fissured plaque, and > 90% stenosis of the artery. Other minor criteria include: superficial calcified nodule, intraplaque haemorrhage, endothelial dysfunction, and outward remodelling (Shin, Edelberg, and Hong) .

Plaque complications, instability and rupture may be inhibited by medical treatment and/or lifestyle modification. In some cases, however, more invasive methods may be needed, i.e. angioplasty or bypass surgery. Presently, diagnostic tools are based on either static image analyses still under development or low-technology methods such as systolic and diastolic blood pressure levels related to the risk of CVD. The field has devoted much attention to the development of multivariate analysis that may better identify patients at high risk. One such model is the SCORE-model (Systematic Coronary Risk Evaluation model) . In 1994, with a revision in 2003, The European Atherosclerosis Society, The European Society of Cardiology and The European Society of Hypertension issued a set of recommendations regarding prevention of coronary heart diseases. This guideline is based on several assessment techniques, which have been developed to assess the risk of CVD in asymptomatic subjects, i.e. identification of asymptomatic high-risk patients. The SCORE-model integrates gender, age, smoking, systolic blood pressure and either total cholesterol or the cholesterol/HDL ratio as risk factors (Graham et al. ) .

In order to make a more detailed diagnosis, the SCORE model is not sufficient and imaging techniques are used. Imaging methods are therefore used mostly on patients in the high-risk group or during research.

IMAGING TECHNIQUES

Coronary angiography (CAG) is currently the gold standard imaging technique for defining the degree of stenosis. CAG images the lumen of the vessel in two dimensions, but is restricted only to the lumen and not the vessel wall thereby CAG can not distinguish between an artery with a stable plaque and an artery with a vulnerable plaque. CAG is often used to determine whether a patient needs surgery; angioplasty or bypass. In order to determine if a point of luminal narrowing is an advanced plaque,

other techniques are needed i.e. intravascular coronary ultrasound (IVUS) or angioscopy.

IVUS provides two-dimensional cross-sectional images of the plaque and vessel wall, and is considered as a method good for characterization of vessel wall and morphology and the degree of calcification, but poor for assessing the lipids in the lesion. However, IVUS is invasive and requires expertise and expense: therefore, its use is not wide spread. Angioscopy is another useful method in understanding and identifying atherosclerosis. Angioscopy is a direct visualization of plaque surface and has the capability of detecting colour of plaque and thrombosis. Angioscopy is, however, invasive and technically difficult, and so far it is has not been able to detect the degree of plaque extension. Another imaging technique that currently is receiving much attention is Magnetic Resonance imaging (MRI) . MRI is noninvasive and able to identify carotid plaque at high risk of stroke. On the other hand, MRI is not the best technique to image coronary arteries, because of small plaque sizes and location of the coronary arteries. Other imaging techniques are under development, i.e. elastography, thermography and optical coherence tomography (Schaar et al . ) .

The imaging techniques mentioned are all under development and alone, none can identify a vulnerable plaque, but they are useful tools in understanding both the molecular events and plaque turnover prior to rupture. Presently, the only opportunity to diagnose CVD at an early stage is to utilize a range of risk factors for established coronary heart disease, peripheral artery disease and cerebrovascular atherosclerotic disease of the patient in question, as well as close relatives of the patient.

PRESENT BIOCHEMICAL MARKERS

At present, several biochemical markers are known as risk factors for atherosclerosis. Recently much attention has been directed to the measurement of biochemical marker concentrations in serum; both lipids such as total cholesterol, low-density

lipoprotein cholesterol (LDL-C) and the high-density lipoprotein cholesterol (HDL-C) and inflammatory markers such as C-Reactive Protein (CRP), Interleukin-6 (IL- 6) , Interleukin-18 (IL-18), Tumor Necrosis Factor-alpha (TNFα) , CD40, CD40 ligand (CD40L) and others. Among lipoprotein markers, there have been at least two noteworthy advances. The size of LDL particles seems to predict the degree of atherosclerosis progression. Increased concentrations of small LDL particles are more related to CVD risk than increased concentrations of large particles (Gardner, Fortmann, and Krauss) .

The level of HDL-C is strongly related to triglyceride, and high triglyceride level is correlated to a higher risk of CHD. A cohort study by Jeppesen et al . (2003) found high TG/low HDL-C as the strongest risk factors of IHD (ischemic heart disease) (Jeppesen et al. ) .

Lipid profiles are important for evaluation of risk factors, but do not allow understanding and measurement of the molecular events associated with plaque turnover. A number of biochemical markers have been suggested as risk factors for CVD, although not specific product of the disease. These include CRP and Bone natriuretic peptide (BNP) (see Table 1) . Table 1 summarizes some of the known markers of CVD.

Table 1: A selection of present biochemical markers in CVD.

Thus, a range of different biochemical markers have been suggested as markers of cardiovascular events. Wang et al (2006) have measured 10 different biochemical markers in 3200 patients participating in the Framingham study, described in Tabel 1. The conclusion was that the measurement of 10 biochemical markers only contributes moderately to diagnosis over and above standard risk factors. Of the 10 biochemical markers, B-type natriuretic peptide level, C-reactive protein level and the urinary albumin-to- creatinine ratio showed the best correlation between marker and death/cardiovascular events (Wang et al.) .

C-REACTIVE PROTEIN C-reactive protein (CRP) is an acute phase serum protein produced by the liver in response to different clinical conditions such as, inflammation, infection, or trauma (Gabay & Kushner 1999) , The production of CRP is induced by cytokines such as IL-6,

released from the affected or damaged tissues. The physiological role of CRP is yet unknown and discussions on its pro-or antiinflammatory actions are ongoing.

There is accumulating evidence that the CRP is a risk factor for CVD in humans. In a study by Ridker et al . 2002, CRP was shown to be a better predictor of future cardiovascular events than LDL cholesterol, in a large population consisting of 28,000 healthy women followed for eight years for the occurrences of acute myocardial infarction, stroke, coronary revascularization, or death from CVD. Many other studies have also reported that baseline CRP levels constitute an independent risk factor for cardiovascular events (Thompson et al . 1995, Mendall et al. 1996, Kuller et al . 1996, Ridker et al . 1997, Tracy et al . 1997, Ridker et al . 2000) .

It has been speculated that circulating CRP only reflects the general inflammation occurring in the atherosclerotic process and is not an active component in the pathogenesis of the disease. However, several lines of evidence also support the view that CRP has a role in atherogenesis . First, chronic infections giving rise to CRP are also associated with increased risk for CVD (Leinonen & Saikku 2002) . Secondly, we and others have identified CRP is in different levels of atherosclerotic lesions (Reynolds & Vance 1987, Hatanaka et al . 1995) . Finally, CRP has been shown to have proatherogenic properties in vitro: CRP may activate endothelial cells to produce adhesion molecules (Pasceri et al. 2000) . It may also decrease the production of eNOS in endothelial cells (Venugopal et al . 2002) and enhance the uptake of LDL by macrophages (Zwaka et al . 2001) .

BRAIN NATRIURETIC PEPTIDE Brain (B-type) natriuretic peptide (BNP) is a peptide hormone secreted by the ventricles of the heart in response to excessive stretching of cardiac myocytes in the ventricles. T-proBNP (the inactive N-terminal fragment) is, along with the active hormone (BNP) , released to the blood stream upon cleavage of proBNP. Both

BNP and NT-proBNP have been suggested as potential biochemical markers of cardiovascular events (Wang et al . ) .

CHEMOKINES Chemokines are also potential markers of CVD; chemokines are low molecular weight cytokines produced in inflammation. One major chemokine in relation to CVD is monocyte chemo attractant protein 1 (MCP-I) . MCP-I appears to play an early and important role in the recruitment of monocytes to atherosclerotic lesions. In a study using a monkey model of atherosclerosis, plasma concentrations of MCP-I were highly associated with plaque size and plaque complications (Register et al.) .

LIPIDS INCLUDING CHOLESTEROL Recently much attention has been directed to the measurement of cholesterol concentrations in serum; both total cholesterol, as well as the concentrations of low-density lipoprotein cholesterol (LDL-C) and the high-density lipoprotein cholesterol (HDL-C) . Among lipoprotein markers, there have been at least two noteworthy advances. First, LDL particle size seems to predict the degree of atherosclerosis progression. Increased concentrations of small LDL particles are more related to CVD risk than increased concentrations of large particles (Gardner et al) . Secondly, the cholesteryl oleate content of LDL particles may become a particularly important marker of CVD risk. In monkeys, enrichment of lipoprotein particle cores with cholesteryl oleate was strongly and positively associated with more severe coronary artery atherosclerosis (Rudel et al) and was additive to the contributions of LDL and HDL cholesterol concentrations. These findings in experimental animals are further supported by earlier human studies (Lawrie et al) that showed plasma lipoproteins with lower proportions of cholesteryl linoleate (and conversely higher proportions of cholesteryl oleate) are typical of patients with complications of CHD (coronary heart disease) compared to normal controls.

The level of HDL-C is strongly related to triglyceride, and high triglyceride level is correlated to a higher risk of CHD. A cohort study by Jeppesen et al) found high TG/low HDL-C as the strongest risk factors of IHD (ischemic heart disease) . These lipid profiles are important for evaluation of risk factors, but do not allow understanding and measurement of the molecular events associated with plaque turnover. A number of biochemical markers have been suggested as risk factors for CVD, although these are not the specific products of the disease. These include CRP and ApoE .

LIPOPROTEINS

The biomarker most commonly used to predict CVD is cholesterol concentration (both total and the cholesterol/HDL ratio) . These are used along with other risk factors, such as blood pressure and level of LDL. Both factors are used in the previously mentioned SCORE-model. The level of LDL is important as LDL transports cholesterol in the blood and accumulation of oxidized LDL can promote atherosclerosis (Graham et al) . In addition, a significant association between CHD and triglyceride (TG) levels are found, in which an increased risk of CHD was associated with increasing TG levels, independent of both LDL-C and HDL-C levels, although the level of cholesterol is viewed as one of the major risk factors of CVD (Jeppeson et al) .

APO-E

Apolipoprotein E is found in chylomicrons, VLDL, and HDL. It is mainly synthesised in the liver, but also in many other organs such as brain, spleen, kidney (Siest et al. 1995) . ApoE plays an essential role in lipoprotein metabolism by acting as a ligand for two receptors: the LDL receptor and Apo E specific chylomicron remnant receptor. The interaction between ApoE with these receptors gives a basis for the metabolic regulation of cholesterol. Polymorphism at the apoE gene locus results in three alleles found in most populations: ε2, ε3 and ε4 that determine six

apoE phenotypes. Isoforms differ from each other by one aminoacid at positions 112 and 158. Apo E2 has cysteine on both residues and E4 has arginine at both positions. Apo E3 contains cysteine at position 112 and arginine at 158. Allele frequencies differ in different populations. Some studies have assessed the possible relationship between apoE polymorphism and atherosclerosis. A meta-analysis of 14 observation studies demonstrated ε4 allele as associated with coronary disease among both men and women (Wilson et al . 1996) . Furthermore, ε4 allele has been associated with carotid artery atherosclerosis (Terry et al . 1996, Cattin et al . 1997, Haraki et al . 2002) .

ApoE is 299 amino acids long and transports lipoprotein, fat soluble vitamins, and cholesterol into the lymph system and further into the blood circulation. ApoE is primarily synthesized in the liver. Currently, there are seven mammalian receptors for ApoE which belong to conserved low density lipoprotein receptor gene family.

ADDITIONAL BIOCHEMICAL MARKERS Microalbuminurea (albumin to creatinine level) is also a potential and independent marker. The urinary albumin excretion rate is a marker of changes in the kidney and, when compared with a small creatinine elevation, it may indicate atherosclerosis (Wang et al. ) . Of the procollagen markers, the marker for type III collagen turnover rate (PIIINP) has been investigated as a prognostic marker for hypertension and has been associated with myocardial infarction. Satta et al . examined the correlation between abdominal aortic aneurysm (AAA) and the concentration of the procollagen (PIIINP) in blood. They showed that the turnover of collagen type III is increased in patients with AAA and may be due to enhanced synthesis, enhanced degradation or a combination of both. In the same experiment, the carboxyterminal propeptide of type I procollagen (PICP) was measured, and there was no accelerated synthesis of type I collagen in the aneurysm sac.

PROTEIN PROFILE OF PLAQUE

Human arteries can be divided into larger or elastic arteries, medium or muscular arteries, and small arteries. The walls of arteries are composed of intima, media and adventitia, separated by the internal elastic lamina and external elastic lamina. The intima consists of connective tissue, smooth muscle cells and a few isolated macrophages. The boundaries of the intima can be defined as a layer between the luminal surface of the endothelium and the internal elastic lamina.

The arterial intima can further be divided into two layers . The inner layer, called a proteoglycan layer, composed of abundant proteoglycans, smooth muscle cells and macrophages. The lower layer, musculoelastic layer, is composed of abundant smooth muscle cells and elastic fibers. In the normal conditions, the two layers of the intima are barely visible by light microscopy, but are distinct and prominent when intimal thickening occurs. The media is the muscular part of the arterial wall, composed of smooth muscle cells, elastin, collagen fibrils. The adventitia, outer layer, is highly microvascular and contains collagens, elastic fibrils, smooth muscle cells, and lymphatic channels.

Human atherosclerotic plaques are characterized by a lipid- rich core covered by a fibrous cap composed of fibrillar collagens, elastin, proteoglycans and SMC. Proteoglycans hyaluronan are major nonfibrillar components of the extracellular matrix that have the potential to affect lesion development by regulating events such as lipid accumulation, thrombosis, and cell proliferation and migration and by affecting the material properties of the tissue (Wight 1995) . Infiltrating ApoE and CRP are also present and we have demonstrated localisation of both in atherosclerotic plaques of coronary arteries at different stages of the atherosclerotic disease. ?? Reference

ApoE and CRP DISTRIBUTION IN HUMAN BEINGS

Table 2 below shows the distribution of ApoE and CRP in the human body.

Table 2:

Protein Sites of expression

APOE Blood, Serum, Plasma, Liver, Saliva, Monocyte, Cerebellum, Cerebrospinal Fluid, Frontal Cortex, Hippocampus, Temporal Cortex CRP Blood, Kidney, Liver, Peritoneal Fluid, Plasma, Serum

Table 3 below illustrates known interactions of ApoE and CRP with proteins demonstrated in vivo and/or in vitro.

Table 3

Protein Interactions with proteins ApoE Albumin, Amyloid beta A4 protein, Macroglobulin,

Microtubule associated protein tau, LDL receptor,

Cathepsin B, Neurofilament 3, Phospholipid transfer protein, Prion protein, VLDL receptors, Scavenger receptors class B, CRP Serum amyloid P, Complement factor H, Fibronectin 1,

Histone 1, FC gamma RI, FC gamma RIIb, CD32, Platelet glycoprotein VI, Leptin.

Non protein interaction : Calcium, Cholesterol

COLLAGEN DISTRIBUTION IN HUMAN BEINGS Collagen is widely distributed in the human body, i.e. ~ 30% of the protein mass in the human body is composed of collagen. In Table 4, the major collagen types are listed with their major tissue distribution.

Table 4

Collagen Tissue distribution type

I Skin, bone, tendon, ligament, cornea

II Cartilage, vitreous

III Skin, vessel, intestine, uterus

IV Basement membranes

V Bone, skin, cornea, placenta

VI Bone, cartilage, cornea, skin, vessel

VII Skin, bladder, oral mucosa, umbilical cord, amnion

VIII Descemet's membrane, vessel, bone, brain, heart, kidney, skin, cartilage

XIII Endothelial cells, skin, eye, heart, skeletal muscle

XIV Vessel, bone, skin, cartilage, eye, nerve, tendon, uterus

XXI Vessel, heart, stomach, kidney, skeletal muscle, placenta

Type I collagen is the most abundant collagen and is found in most connective tissue. It is especially important for the structure of bone and skin. The major content of collagen in the human body is distributed in the skin and bone, where the major collagenous components are type I and III collagens. Collagen type III is a major component of large arteries, and is also found in smaller amounts in tissues rich type I collagen. In addition, collagen type IV is found in the basement membrane and around blood vessels and nerves. The most common localization of type V collagen is within the characteristic collagen fibrils, in association with the collagen type I and III (Garrone et al) . Some collagens have a restricted tissue distribution: for example, type II, which are found almost exclusively in cartilage (Mayne R. ) .

Collagen fibrils often consist of more than one collagen type. For example, the type I collagen fibrils often contain small

amounts of types III, V and XII, whereas the type II collagen fibrils of cartilage also contain types IX and XI.

COLLAGENS IN ARTERIES In arteries, six types of collagen are found (types I, III, IV, V, VI and VIII) , where type I and III are the most abundant , 80 - 90 % of collagen content. Type I and III are also predominant in the vessel wall. They appear to be co-distributed in different amounts within all three layers of the artery wall, synthesis of collagen type I and III tends to be located in the intima (Mayne R) •

COLLAGENS AND OTHER STRUCTURAL PROTEINS IN PLAQUE - TURNOVER

During development of atherosclerotic plaques collagen is accumulated in the fibrous cap (Stary H. C.) . In a study by Katsuda et al (1992) collagen types I, III and IV were found in the thickening intima at all stages of the lesion in aortic human tissues. Collagen type VI was distributed in the basement membrane in the region of intimal cells and in advanced lesions also detected around the elongated SMC. Earlier studies of type I and III have provided evidence of an equal distribution in atherosclerotic arterial wall (Shekhonin et al) . According to McCullagh et al (1980) type III is the predominant collagen in normal human aortic media (appr. 70 % of the extractable collagen) . A recent study by Eriksen et al (2006) found a decrease of total collagen content in human aortic valve depending on the degree of stenosis. The molecular mechanism of stenosis is thought to be similar to atherosclerosis. In healthy aortic valves, the collagen content is mainly type I and III. During stenosis, the total content of collagen decreases, which is presumably caused by an increased turnover of collagen type I. Type I collagen accounted for approximately 60-70% of total collagen; whereas the proportion of type III collagen was 30-40% both in healthy valves and in calcified valves.

Type V collagen also increases in advanced atherosclerotic lesions and is distributed throughout the extracellular matrix in both aortic media and in the subendothelial region of the plaques (McCullagh et al) . There seems to be a consensus that the main collagen types to be found in atherosclerotic plaque are type I and III, whether they are equally distributed in healthy and atherosclerotic vessel remains to be further investigated.

In the study by Katsuda et al (1992) no collagen was detected in the center of the atheroma of more advanced lesions.

ELASTIN

Elastin is one of the most stable proteins in the body and is found in most connective tissue caused by its elasticity and resilience. Elastin dominates the protein content of the arterial wall, where it is the major extracellular matrix protein.

Elastin is the main component in elastic fibers and is related to calcification. Vascular calcification occurs at two distinct sites within the vessel wall: the intima and the media. Intimal calcification is related to atherosclerosis, mainly within the necrotic core. Calcified elastic fiber constitutes the plaque shoulder where the plaques are most prone to rupture; suggesting that calcification of elastic fiber may affect plaque stability (Bobryshev Y. V.) . In atherosclerosis, the content of elastic fibers decreases along with lipid deposition, this generates an enhanced susceptibility to elastin degrading enzymes. Thereby the content of elastin in contrast to collagen decreases as the lesion develops .

DISTRIBUTION OF ELASTIN IN HUMAN BEINGS

Table 5 shows the distribution of Elastin in the human body.

Table 5:

Protein Sites of expression Elastin Aorta and other Blood vessels, Lung, Skin Fibroblasts

Table 6 illustrates known interactions of Elastin with proteins demonstrated in vivo and/or in vitro.

Table 6:

Protein Interactions with proteins

Elastin Decorin, Elastase, Fibrillin, Fibulin, Lysozyme, Lysyl Oxidase, Galectin, Biglycan, Nidogen, Ficolin, Proteinase3.

PROTEOGLYCANS AS MATRIX COMPONENTS

Proteoglycans (PG) are polysaccharide-protein macromolecules localized predominately in the intercellular matrix of vessel wall (Salisbury and Wagner 1981) . PGs are macromolecules characterized by the presence of one, or more, long un-branched and highly polyanionic sugar side chains called GAGs, covalently attached to a core protein through a link region. The repeating unit of the GAG consists of an amino sugar, either N-acetyl-glucosamine (GIcNAc) or N-acetyl-galactosamine (GaINAc) , and a hexuronic acid, either glucouronic acid (GIcA) or iduronic acid (IdoA) . One or both of the sugars in the repeating unit contain one or more sulfate groups (Rodriguez-Lee 2007) . In addition to the GAG chains, most core proteins carry N- and/or O-linked oligosaccharides.

CLASSIFICATION AND NOMENCLATURE OF PGs

PGs are a very heterogeneous group of macromolecules. A single type of core protein can vary in the number and type of attached GAG chains. The length of the chains and the arrangement of the sulfated residues along the chains vary also. Four main classes of GAGs are distinguished according to the structure of the repeating disaccharide unit: chondroitin sulfate

(CS) and dermatan sulfate (DS) , heparin sulfate (HS) and heparin, hyaluronan, and keratin sulfate (KS) .

Hyaluronan is the simplest of GAGs. In contrast to all of the others, it does not contain any sulfated sugars. All of its disaccharide units are identical, its chain length is enormous and it is not linked to any core protein.

KS is a sulfated polylactosamine chain. KS-I has originally been described in cornea, and is N-linked to aspargine residues in the core protein, whereas KS-II or cartilage KS, is 0-linked to serine or threonine residues (Funderburgh 2000) .

PGs can be classified according to several parameters:

Attached GAG chain (CS/DS- or HS containing PGs)

Topographic distribution in relation to the cell

(extracellular and basement membrane PGs, cell-associated PGs or intracellular PGs)

Core protein homology (hyalectans, small leucine-rich PGs

(SLRPs)

Chondroitin/dermatan sulfate PGs (Versican, aggrecan, neurocan, and brevican) belong to the family of hyaluronan-binding proteoglycans. This gene family is collectively termed hyalectans. Each family member has a characteristic distribution, with aggrecan prominent in cartilage, neurocan and brevican prominent in the central nervous system, and versican present in a variety of soft tissues, including arterial walls. The gene and protein structure of versican follows a domain template. The amino-terminal globular end (Gl) binds to GAG hyaluronan, and the carboxy-terminal globular domain (G3) resembles the selectin family of proteins, consisting of a C-type lectin adjacent to two epidermal growth factor (EGF) domains and a complement regulatory region. The middle region of versican core protein is encoded by two large exons that specify the CS attachment regions of versican. The region encoded by exon 7 is called αGAG, whereas the region encoded by exon 8 is called βGAG. Four mRNA transcripts arise from alternative splicing of versican, giving rise to VO, Vl, V2 , and V3 which differ in the

length of the core protein and the number of attached GAGs (Dours- Zimmermann and Zimmermann) . The number of potential GAG attachment sites in human versican is: 17-23 for VO, 12-15 for Vl, 5-8 for V2, and none for V3 (Wight 617-23) . Decorin and biglycan are members of SLRP-family that comprises at least nine members grouped into three classes (I, II, and III) and different subfamilies. They are all characterized by the presence of a central domain containing leucine-rich repeats to achieve strong presence of a central domain containing leucine-rich repeats to achieve strong protein-protein interactions. Decorin and biglycan are members of class I, and show highest amino-acid homology of the family (~57%) and are the only SLRPs with a propeptide. The propeptide is highly conserved across species and may function as a recognition sequence for xylosyltransferase, the first enzyme involved in synthesis of the GAG chain.

Versican, decorin, and biglycan are the major CS/DS PGs in the matrix of the mammalian arterial wall (Wight et al . 1986) . The size of Versican VO core protein is 370 kDa, which makes it roughly 10 times larger than decorin 36 kDa and biglycan 38 kDa. Side- chains show a wide range of sizes, but generally average around 40- 90 kDa each.

Heparan sulfate proteoglycans: HSPGs are divided into five distinct classes of cell-associated and pericellular PGs and they account for at least 95% of the HS of mammalian cell surfaces, basement membranes and ECMs. The cell-associated HSPGs include integral membrane syndecans and anchored glypicans. Pericellular HSPGs include mainly perlecan, agrin. These PGs are termed pericellular because of their close association with the plasmamembrane via integrins (Whitelock and Iozzo) . Perlecan is a modular HSPG that is expressed in nearly all basement membranes as well as mesenchymal organs and connective tissues and is one of the largest single-chain polypeptides found in vertebrate and invertebrate animals. The five modules of perlecan and its HS side-chains take part in a large number of molecular interactions such as with fibroblast growth factor - 2,

vascular endothelial growth factor (VEGF) , platelet-derived growth factor (PDGF), and other matrix proteins. The core protein of human perlecan is -470 kDa and, together with numerous 0-linked oligosaccharides and four HS side chains, it can reach a molecular weight of over 800 kDa (Knox and Whitelock) .

PROTEOGLYCAN DISTRIBUTION

Proteoglycans (PGs) are macromolecules distributed almost everywhere in the human body. The structure and size of PGs vary extremely. The basic structure of all PGs includes a core protein and at least one, but often many carbohydrate chains glycosaminoglycans (GAGs) . PGs can be found intracellularly, on the surface of cells, and in the extracellular matrix. The structural diversity of PGs suggests numerous biological functions, see Table 7.

Table 7:

Table 7 above gives an overview of PG distribution and function.

PROTEOGLYCANS IN ARTERIES

At least five types of PGs are present in the extracellular matrix of the artery wall; versican - which interacts with hyaluronan to form large aggregates; small-leucine rich decorin and biglycan, which interact with fibrillar matrix components like collagen and elastin; heparan sulphate - perlecan, which is a component of basal lamina and keratin sulphate - lumican (Talusan et al. ) .

Versican is one of several ECM molecules that accumulate in lesions of atherosclerosis. Although a number of studies indicate

that versican is clearly capable of binding to LDL, versican is generally not detected in the lipid-rich center of the necrotic core (Evanko et al . ) .

Lumican has been shown to directly bind to macrophages and to enhance macrophage migration. Lumican may therefore directly influence macrophage behavior in the vascular intima as well as stimulate the formation of the necrotic core, characteristic of advanced atherosclerotic lesions (Funderburgh et al . 1997) .

Biglycan is found in the fibrous cap. Versican and biglycan have affinity for LDL and form insoluble complexes, which accelerates oxidation of LDL. Biglycan may contribute to the pathogenesis of atherosclerosis by trapping lipoproteins in the artery wall. Changes in the proteoglycan metabolism of the intima of arteries constitute the initial lesions of atherosclerosis and the accumulation of proteoglycans play a fundamental role in the progression of atherosclerosis (Kunz J.) .

Perlecan was reported in human intimal hyperplasia as one of the central components of intimal extracellular matrix, by mass spectrometry-based analysis and by immunohistochemistry .

Table 8 illustrates distribution of some PGs in immunohistochemical stainings of PGs in normal and atherosclerotic arteries (Evanko et al) .

Table 8:

PROTEOGLYCAN INVOLVEMENT IN MATRIX REMODELLING

A study of atherosclerosis progression in nonhuman primates has demonstrated that accumulation of specific PGs varies with lesion severity and with the distribution of cells and growth factors, suggesting that different PGs play distinct roles during progression of atherosclerosis. Different levels of specific PGs may directly affect material properties of the tissue via their contribution to altering structural arrangements of fibrous matrix components such as elastin and collagen. Versican and hyaluronan show similar localization in the matrix, suggesting aggregate formation between the two in the atherogenesis . The marked increase in versican and hyaluronan in early lesions could suggest that they play a role in early atherosclerotic lesions, such as proliferation and migration of SMCs and leukocytes. Furthermore, versican and hyaluronan are principal matrix components of human restenotic lesions and have been shown to contribute to neo-intimal thickening after vascular injury in vitro. An abundance of versican early in atherogenesis could also predispose the extracellular matrix to increase lipid entrapment due to the binding of lipoproteins to chondroitin sulphate chains of versican. This idea is supported by co- localization of versican with apoprotein (a) and apolipoprotein E in transplant arteriopathy (Evanko et al) . Loss of versican from the plaque may result in matrix instability. This is further evidenced by upregulation of versican gene observed after vascular injury. Versican was also here identified in all stages of atherogenesis; in the intima of early developing plaques, but also throughout advanced lesions and at the borders of lipid-filled necrotic cores as well as at the plaque-thrombus interface (Wight and Merrilees 2005) . These observations implicate versican in lipid accumulation, inflammation, and thrombosis. Furthermore, versican plays an important role in the assembly of ECM and in control of elastic fiber fibrillogenesis, which is of fundamental importance in ECM remodelling during vascular disease (Wight and Merrilees 2005) .

The role of biglycan in arterial cell biology is unclear. Some immunohistochemical studies have showen biglycan association with collagen I and III staining in human restenotic lesions (Evanko et al.) . The importance of biglycan as matrix protein was further stated by the generation of BALB/cA mice homozygous for a null mutation of the biglycan gene, where 50% of biglycan-deficient male mice died suddenly within the first 3 months of life as a result of from aortic rupture. This observation suggests biglycan to be essential for the structural and functional integrity of the aortic wall, as well as a potential role of biglycan gene defects in the pathogenesis of aortic dissection and rupture in humans. (Heegaard et al. 2007)

Other studies indicate that biglycan is a major PG associated with elastin in primate arteries; these observations are similar to those of in human coronary arteriopathy (Evanko et al) .

Decorin has been shown to bind to collagen and regulate collagen fibril formation (Brown and Vogel) (Danielson et al . ) .

PROTEASE PROFILES

Proteases hydrolyse peptide bonds and are responsible for the degradation of extracellular matrix proteins such as collagen, proteoglycans and elastin in atheroma, see Table 9. In atherosclerotic plaques three main types are found: metallo- proteinases (i.e. MMPs), serine proteases and cysteine proteases (i.e. cathepsins) . Cathepsins and MMPs are responsible for degradation of all extracellular matrix proteins. As matrix is essential for plaque stability, its removal from the fibrous cap by proteases may invoke plaque rupture (Stary H. C) . In Table 9 a variety of proteases found in atherosclerotic plaque are listed.

Protease Degradation substrates

Cathepsin K Proteoglycans, elastin, collagen Cathepsin S Proteoglycans, elastin, collagen Cathepsin L Proteoglycans, Collagen type I Cathepsin B Proteoglycans MMP-I Collagen type I, II and III MMP- 2 Proteoglycans, elastin MMP- 3 Proteoglycans, collagen type III, elastin

MMP- 8 Proteoglycans, collagen type I, II and III

MMP- 9 Elastin, collagen type I and III MMP- 13 Proteoglycans, collagen type I, II and III

MMP- 18 Collagen type I

Table 9 Proteases detected in atherosclerotic plaques.

The main source of MMP expression in the plaque is suspected to be related to macrophage and SMC activity. Macrophages in plaques contain abundant MMP-I, -8, -9, and -13 and co-localize with sites of collagen and proteoglycan degradation in situ (Kunz J.) . Furthermore, own data suggest localization of MMP-8 and Cathepsin K in atherosclerotic plaques.

MATRIX METALLOPROTEINASES (MMP)

MMP is a large group of endopeptidases, capable of degrading most components of the ECM. Presently, more than 25 MMPs have been identified. Metallo-proteinases are characterized by an active site containing a metal atom, typically zinc, and are secreted as zymogens. Specific tissue inhibitors, TIMPs, regulate the activity of MMPs. A great variety of MMPs are found in the atherosclerotic plaques. They are most often located in macrophages bordering the

fibrous cap, within plaque shoulders in SMC and macrophages and are rarely identified within the fibrous cap (Kunz J. ) . ]

MMPs are classified in different groups according to their substrate specificity: Collagenases, which degrade fibrillar collagen, like collagen type I, II, III and V but also proteoglycans; Gelatinases, which degrade proteoglycans, collagen type IV, V, VII and elastin; Stromelysin that is active against proteoglycans and elastin (Rouis M) . These three subgroups are of particular interest with regards to matrix remodelling in atherosclerotic plaques.

GELATINASES

Insoluble elastin is digested by MMP-2 and -9, both belonging to the gelatinase-family of MMPs. MMP-9 has an important role affecting the size and composition of atherosclerotic plaque. In unstable human atherosclerotic plaques and in vulnerable regions of plaques, greater expression and concentration of MMP-9 have been observed. Moreover, MMP-9 is found intracellularly (indicating active synthesis) in coronary plaques more often in patients with unstable angina compared with those with stable angina. Blood MMP- 9 level increases in association with coronary atherosclerosis and predicts adverse cardiovascular events (Sundstrom and Vasan) . A recent study by Kuzuya et al (2006) indicates that MMP-2 is responsible for accumulation of SMC in the fibrous cap and thereby inducing plaque instability.

STROMELYSIN

MMP-3 belongs to the stromelysin proteases and is capable of degrading both elastin and proteoglycans. A study by Yamada et al (2002) indicates that MMP-3 may prove to be a reliable mean of predicting the genetic risk of myocardial infarction in women.

COLLAGENASES

MMP-I, -8 and -13 have all been identified in atherosclerotic plaques where they degrade proteoglycans and collagen types I and III. MMP-I, -8 and -13 are collagenases, which cleave collagen into two fragments that are further degraded by MMP-2, -3 or -9.

MMP-8 is expressed by neutrophils, not commonly found in human atheroma but has been identified in atherosclerotic plaques. MMP-8 may be partly responsible for degradation of the fibrous cap as MMP-8 has a preference for collagen type I (Herman et al) , having a three fold greater activity in degradation of collagen I than MMP-I and 13. This is supported by Turu et al (2006), in this study the content of MMP-8 in the plasma are significantly higher for patients with vulnerable plaques, than patients with stable plaques.

MMP-13 has been reported to cleave SLRPS, with high specificity for biglycan. Degradation of biglycan by MMP-13 at a specific cleavage site (...Gi ₇₇ ZVi ₇₈ ) has previously been demonstrated by Monfort et al . (2005) and proposed to play a important role in early detection of cartilage degradation in osteoarthritis.)

CATHEPSINS

Human cysteine cathepsins consist of 11 members, including cathepsins B, K, L, and S, and are predominantly expressed within the endosomalZlysosomal compartments of cells. Cathepsins are capable of catalysing the hydrolytic breakdown of proteoglycans, collagen and elastin.

In abdominal aortic aneurysm (AAA) high levels of cathepsins

S, K, and L were found compared to normal aorta. Normal human vascular SMC contain no detectable cathepsin K by immunostaining, but cells within atherosclerotic plaques are clearly positive.

Cathepsin K is localized in rupture-prone areas such as the fibrous cap, plaque shoulders and at the actual site of plaque ruptures

(Chapman et al) . Cathepsin S is found to co-localize with regions of increased elastin breakdown in atherosclerotic plaques, and

reduced atherosclerosis is observed in cathepsin S- and K-deficient mice (Liu et al) .

Both cathepsin L and K degrade several proteoglycans and collagen type I and II, cathepsin K degrades within covalently cross-linked triple helices, while cathepsin L cleaves only in the nonhelical telopeptide regions. Cathepsin K is localized in the fibrous cap and plaque shoulder. Cathepsin K expression in normal arteries is very low. Early human atherosclerotic lesions showed cathepsin K expression in the intimal and medial SMCs. In advanced atherosclerotic plaques, cathepsin K was localized mainly in macrophages and SMCs of the fibrous cap (Lutgens et al) . Cathepsin K protein levels were increased in atherosclerotic lesions when compared with normal arteries, whereas cathepsin K mRNA levels were similar in both atherosclerotic and normal arteries. Furthermore, it was shown that cathepsin K mRNA and protein levels were highest in advanced but stable human atherosclerotic plaques compared with early atherosclerotic lesions and lesions containing thrombus (Chapman et al) .

Cathepsin S is only sparsely expressed in intimal and medial SMCs in early human atherosclerotic lesion and fatty streaks. In advanced human atherosclerotic plaques cathepsin S was localized in macrophages and SMCs of the fibrous cap. EC lining the lumen of the vessel itself and the plaque microvessels also expressed cathepsin S. Furthermore, cathepsin S mRNA and protein levels were increased in human atheroma compared with normal arteries (Lutgens et al) . Cathepsin S can degrade proteoglycans, elastin and collagen (Liu et al) .

Presently, the determination of CVD risk is occurring at a late stage in atherosclerosis progression; a point in which there is a significant risk of fibrous plaque rupture. There is a need for diagnostic or prognostic assays that will provide information regarding atherosclerosis or CVD risk at both earlier stage and late stages. The findings of Katsuda et al (1992) suggest that there are enzymatic mechanisms for removal of collagens from advanced lesions, suggesting indeed a major role of neo-epitopes in

arteriosclerosis .

The present invention provides a method of bioassay for the quantification of peptide fragments comprising a neo-epitope formed by cleavage of a protein of an atherosclerotic plaque by a proteinase, said method comprising contacting a sample comprising said peptide fragments with an immunological binding partner having specific binding affinity for a said neo-epitope and determining the level of binding of said immunological binding partner to peptide fragments in said sample.

The result of said assay may produce an index indicative of the degree of risk in a particular patient of rupture of an atherosclerotic plaque or of the vulnerable status of the atherosclerotic plaques of a patient. Patients having a value for said index above a threshold level may be recommended for further investigation by plaque imaging methods (including those discussed above) or for the prescribing of medication for treatment of atherosclerosis or for surgical treatment of atherosclerosis, and such follow up investigations or treatment may form part of the method of the invention.

Proteins of the atherosclerotic plaque include lumican, versican, perlecan, decorin, biglycan, collagen type III, CRP, ApoE and elastin. Collagen type I is not considered to be proteins of the atherosclerotic plaque. Proteins present in the atherosclerotic plaque which are exposed there to proteases to a higher degree than elsewhere in the body are of particular interest .

Said immunological binding partner may have specific binding affinity for peptide fragments comprising a C-terminal neo-epitope or an N-terminal neo-epitope.

PROTEOGLYCAN ASSAYS

Said peptide fragments may be fragments of proteoglycans versican (SEQ ID NO 1) , lumican (SEQ ID NO 2) , perlecan (SEQ ID NO 3) , biglycan (SEQ ID NO 4) and decorin (SEQ ID NO 5), which are all

identified in normal and atherosclerotic arteries. Proteoglycans are some of the main proteins constituting atherosclerotic plaques and plaque cap together with elastin and collagens. The content of proteoglycans varies during the progression of atherosclerosis, which makes the potential neo-epitopes of proteoglycans a good marker of disease staging and disease progression. Since especially versican and lumican are not abundant in many other organs, this makes them more specific biochemical marker candidates.

Several candidate proteases may be responsible for the digestion of proteoglycans in the plaque, as the literature reports many different proteases in the atherosclerotic plaques. Most likely, this is the result of a large range of complicated processes eventually leading to plaque rupture. However, in our assessment, early phases may consist of a range of MMPs, whereas later stages may rely more on cathepsin degradation of the matrix, resulting in different neo-epitope profiles dependent on the stages of the disease. We have determined that the enzymes listed in table 4 generate lumican, versican, biglycan, perlecan and decorin resulting in at least following cleavage products:

Table 4. Protease generated peptides - cleavage products of proteoglycans. * indicates a site of cleavage.

Accordingly, in a method of the invention, said peptide fragments preferably comprise a neo-epitope formed by cleavage of versican, lumican, perlecan, decorin or biglycan by a protease at a site marked by the sign * in any one of the above partial sequences thereof.

Also, in a method of the invention, said peptide fragments preferably comprise a neo-epitope formed by cleavage of following proteoglycans: versican, lumican, perlecan, decorin and biglycan; by a (or more) protease (s) at a site in any one of the following partial sequences of versican, lumican, decorin, perlecan, and biglycan, or the immunological binding partner is specifically reactive with one of the following sequences:

Table 10 - Peptide fragments of proteoglycan cleavage.

Preferably, said immunological binding partner is not reactive with intact versican, lumican, decorin, perlecan, and biglycan. Preferably, said immunological binding partner is not reactive with a said sequence listed above if prolonged past the respective c- terminal and N-terminal ends of generated fragments.

The immunological binding partner may be one specifically reactive with a C-terminal or N-terminal neo-epitope formed by cleavage of type versican, lumican, decorin, perlecan, and biglycan .

Suitable immunological binding partners may therefore be specifically reactive with any of the following sequences in Table 11 at the N terminal of a peptide:

Table 11 N-terminal sequences of protease generated peptide fragments of proteoglycans.

or with any of the following sequences in Table 12, at the C- terminal of a peptide:

Table 12. C-terminal fragments of protease generated peptide fragments of proteoglycans.

Further cleavage sites defining neo-epitopes that may be assayed in a similar manner can be identified by exposing proteoglycans or other atherosclerotic plaque proteins to any of the enzymes described herein and isolating and sequencing peptides thereby produced.

COLLAGEN ASSAYS

Said peptide fragments may be fragments of Type III collagen (SEQ ID NO 153) , preferably of mature Type III collagen, i.e. not of collagen type III propeptide. The main proteins in the atherosclerotic plaques are collagen type I and III as well as elastin, whereas proteoglycan contributes only to a minor extent to the matrix of the plaque. Of the three major proteins found in atherosclerotic plaques collagen type I and III are dominant, whereas elastin dominates the protein profile in arteries, but not the main protein component in the plaque. Collagen type I is abundant throughout the human body, whereas type III has a more restricted tissue location, and thereby in our view constitutes a more specific candidate as biochemical marker.

Several candidate proteases may be responsible for the digestion of collagen in the plaque as the literature reports many different proteases in the atherosclerotic plaque. Most likely, this is the result of the large range of complicated processes eventually leading to plaque rupture. However, in our assessment, early phases may consist of a range of MMPs, whereas later stages may rely more on cathepsin K degradation of the matrix, resulting in different neo-epitope profiles dependent on the levels of disease. We have determined that the enzymes listed in the following table cleave type III collagen at at least the following cleavage sites (marked *) :

Accordingly, in a method of the invention, said peptide fragments preferably comprise a neo-epitope formed by cleavage of Type III collagen by a protease at a site marked by the sign * in any one of the above partial sequences of Type III collagen.

Also, in a method of the invention, said peptide fragments preferably comprise a neo-epitope formed by cleavage of collagen type III by a (or more) protease (s) at a site in any one of the above partial sequences of collagen type III between the *s, or the immunological binding partner is specifically reactive with a sequence extending between the *s in any entry in the above table.

Preferably, said immunological binding partner is not reactive with intact type III collagen. Preferably, said immunological binding partner is not reactive with a said sequence listed above if prolonged past the respective cleavage site .

The immunological binding partner may be one specifically reactive with a C-terminal or N-terminal neoepitope formed by cleavage of type III collagen.

Suitable immunological binding partners may therefore be specifically reactive with any of the following sequences at the N terminal of a peptide: (Sequence ID Nos follow each sequence)

GIPGAP 343 GDPGPP 408 LAGPPG 470 ERGLPG 534 IAGITG 598

IKGHRG 344 RGLAGP 409 TGARGL 471 ITGARG 599

VKGESG 345 SRGAPG 410 KGDAGQ 472 ITGARG 535 LRGGAG 600

HAGAQG 346 GKSGDR 411 LQGLPG 473 ERGLPG 536 KDGTSG 763

DGTSGH 347 VGGLAG 412 IAGITG 474 PGAPGG 537 VGGLAG 601

IGSPGP 348 AIGSPG 413 AGQQGA 475 GPPGVA 538 AGPPGM 602

LSGERG 349 GLAGPP 414 LAGPPG 476 GLAGPP 539 PQGPPG 603

VKGERG 350 RGLAGP 415 GLSGER 477 VGGLAG 540 SPGGKG 604

GARGLA 351 KGESGK 416 VKGERG 478 SPGAQG 541 GAPGEK 605

GAPGEK 352 IAGITG 417 IKGPAG 479 QPGVMG 542 IKGPAG 606

IKGPAG 353 PGAPGL 418 ERGLPG 480 IPGAPG 543 FRGPAG 607

GIPGQP 354 ERGLPG 419 VGGLAG 481 VKGERG 544 DAGAPG 608

GPPGPT 355 INGSPG 420 GPPGEP 482 GPPGIN 545 GLPGPP 609

KNGETG 356 LPGIAG 421 INGSPG 483 GINGSP 546 PGENGK 610

LKGENG 357 NTGAPG 422 LMGARG 484 LRGGAG 547 QQGAIG 611

GDPGPP 358 LRGGAG 423 KNGETG 485 YQGPPG 548 VAGPPG 612

DKGEPG 359 GHAGAQ 424 GERGAP 486 GIPGFP 549 PGMKGH 613

FPGARG 360 GFPGAR 425 PGPPGP 487 FPGMKG 550 PGDKGE 614

GDKGET 361 GRNGEK 426 GQPGDK 488 GPPGEN 551

GGAGEP 362 GERGSP 427 PGVPGA 489 AAGFPG 552

GPPGPP 363 GKDGES 428 GARGND 490 PGVSGP 553 GSDGQP 615

PGAPGQ 491 GFPGAP 554 GARGND 616

GAAGEP 365 IAGITG 429 GPPGPP 492 GLSGER 555 GARGPP 617

GLSGER 366 IAGITG 493 GGAGPP 556 GPKGDA 618

IAGITG 367 GPKGDA 430 VGGLAG 557 PGPQGH 619

PGFPGM 368 GSPGGP 431 GPAGIP 494 GLAGPP 558

GIAGIT 369 GPPGPP 495 PGPPGI 559 GITGAR 620

GARGLA 370 GLPGPP 432 RPGLPG 496 GAPGPM 560 GSRGAP 621

GLAGPP 371 GPPGSN 497 GARGLA 561 GAPGEK 622

GPPGVA 372 SGDRGE 433 PGFPGM 498 GLAGPP 562 PQGLQG 623

GPAGIP 373 GLAGPP 434 GPPGEN 499 PGFRGP 563 KGSPGA 624

PGPPGI 374 GAPGFR 435 GLPGPP 500 GPVGPS 564 PGPQGP 625

GAPGPQ 375 GFPGNP 436 PGPPGI 501 AGQPGE 565 GPTGPI 626

GDAGQP 376 NGEKGE 437 PGFRGP 502 GAPGFR 566 GSPGER 627

HAGAQG 377 GLAGPP 438 GIAGIT 503 PGFPGM 567

GPPGAP 378 GITGAR 439 GIPGAP 504 GLAGPP 568 GSPGAQ 628

DGTSGH 440 GITGAR 505 GLAGPP 629

GLAGPP 379 GPPGSN 441 GAPGEK 506 SGDRGE 569 GAPGEK 630

AIGPSG 380

or with any of the following sequences at the C-terminal of a peptide :

GPPGPA 381 NGDPGI 442 SPGPQG 507 AGIPGF 570 GMPGPR 631

SPGPAG 382 PGPQGV 443 PPGPQG 508 ERGAAG 571 PGPLGI 632

AAGTPG 383 IPGAPG 444 ERGPPG 509 PGPPGT 572 GNRGER 633

TSGHPG 384 SPGPQG 445 APGLRG 510 HPGSPG 573 PSGPPG 634

PSGPPG 385 GSPGPA 446 GPAGPP 511 SPGPQG 574 GLPGLA 635

QGPPGP 447 SPGPQG 512 GLAGTA 575 PGPPGT 636

PGLMGA 386 LAGPPG 513 GPPGPQ 576 GPPGPQ 637

SPGPQG 387 IPGFPG 448 FPGPKG 514 GFPGMK 577 FPGMKG 638

GPAGIP 449 PPGPAG 515 EKGPAG 578 GPPGIC 640

PPGPPG 388 FPGARG 450 PGPQGL 579 GAPGLM 641

GAIGPS 389 GIPGAP 451 FPGAPG 580

PPGPAG 390 ERGPPG 452 LPGAAG 516 APGPLG 581 EPGPRG 642

SPGPKG 391 PSGPPG 453 IPGQPG 517

PPGPAG 392 GPPGIN 454 GAPGPA 518 GFPGMK 582 GHRGFD 643

LPGPPG 393 GTSGHP 455 GHRGFD 519 PGLPGI 583 GAAGIK 644

GAPGLK 394 GLPGIA 456 PGPKGD 520 GHRGFD 584 GEPGPR 645

GLPGPP 395 GANGLP 457 GPPGPS 521 PGPKGN 585 GIPGAP 646

GPPGIK 396 TAGFPG 458 PPGAPG 522 GEVGPA 586 GAPGLM 647

GPPGVA 397 EGGPPG 459 GEVGPA 523 GPPGPS 587 TGARGL 648

TPGLQG 398 PQGLPG 460 GAPGLR 524 GLPGLA 588 MPGPRG 649

GTPGLQ 399 APGPLG 461 GPQGVK 525 GSPGYQ 589 GMKGHR 650

GFPGAR 400 GSPGPQ 462 GMPGPR 526 EMGPAG 590 GPQGVK 651

QPGPPG 401 GAAGAR 463 GAAGIK 527 GKPGAN 591 PGANGL 652

TGAPGS 402 GVKGER 464 PGAAGF 528 GMPGPR 592 GTGGPP 653

SPGPQG 403 GDAGAP 465 GPAGER 529 GITGAR 593 GPPGPR 654

GPAGPR 404 RGFDGR 466 GIAGPR 530 AGPRGA 594 GGKGER 655

APGLMG 405 GLSGER 467 TGARGL 531 GPAGAN 595 PQGVKG 656

GNRGER 406 GSPGPQ 468 VKGESG 532 GKPGAN 596 PGANGL 657

TGAPGS 407 GVKGER 469 TGERGA 533 GFPGAR 597

Further cleavage sites defining neoepitopes that may be assayed in a similar manner can be identified by exposing collagen type III or another atherosclerotic plaque protein to any of the enzymes described herein and isolating and sequencing peptides thereby produced.

CRP AND ApoE ASSAYS

Said peptide fragments may be fragments of CRP (SEQ ID NO 658; or ApoE (SEQ ID NO 659) . For ApoE, preferably the chosen fragments occur in all of the identified isotypes of ApoE, ε2, ε3 and ε4.

Even though both CRP and ApoE are abundant throughout the human body, their localization in the atherosclerotic tissue

exposes them to the action of local proteases. These molecules are thereby good and specific candidates as biochemical markers. Several candidate proteases may be responsible for the digestion of CRP and ApoE in the plaque as the literature reports many different proteases in the atherosclerotic plaque. Most likely, this is the result of the large range of complicated processes eventually leading to plaque rupture. However, early phases may consist of a range of MMPs, whereas later stages may rely more on cathepsin K degradation of the matrix, resulting in different neo-epitope profiles dependent on the levels of disease. We have through a range of in vitro cleavages of pure native proteins determined that the enzymes listed in the following table cleave CRP and ApoE at at least following cleavage sites (marked

* ⁾ :

Table 13. CRP and APOE fragments generated by specific proteases .

Accordingly, in a method of the invention, said peptide fragments preferably comprise a neo-epitope formed by cleavage of CRP and ApoE by a protease at a site marked by the sign * in any one of the following partial sequences of CRP and APOE, or the immunological binding partner is specifically reactive with a sequence defined between the *s in one of the following sequences:

Table 14. Cleavage fragments of CRP and APOE.

ApoE fragments

A*KVEQAVETEPEPELR* Q

A*KVEQAVETEPEPELR* Q

V*AEVRAKLEEQAQQI *R

A*KVEQAVETEPEPELR* Q

A*MLGQSTEELRV*R (M-oxidized)

E*QAVETEPEPELR*Q

R*QQTEWQSGQRWE*L

L*AVYQAGAREGAERGLS *A

R*AKLEEQAQQIR*L

A*KLEEQAQQIRLQ*A

A*KVEQAVETEPEPELR* Q

K*VEQAVETEPEPELR*Q

E*QAVETEPEPELR*Q

D*EVKEQVAEVRAKLE*E

CRP fragments

K*ESDTSYVSLKAPLT *K

G*GNFEGSQSLVGDIG *N

A*LKYEVQGEVFTKPQ *L

G*IVEFWVDGKPRV*R

R*KAFVFPKE*S

K*YEVQGEVFTKPQLWP*-

D*SFGGNFEGSQS*L

D*FVLSPDEINT*I

S*LKKGYTVGAEA* S

A*FGQTDMSRKA*F

S*LKKGYTVGAEAS* I

G*EVFTKPQLWP*-

S*IILGQEQDSFGGN* F

K*YEVQGEVFTKPQ*L

Suitable immunological binding partners may therefore be specifically reactive with any of the following sequences at the N terminal of a peptide:

or with any of the following sequences at the C-terminal of a peptide :

Further cleavage sites defining neo-epitopes that may be assayed in a similar manner can be identified by exposing CRP and AppoE or another atherosclerotic plaque protein to any of the enzymes described herein and isolating and sequencing peptides thereby produced.

ELASTIN ASSAYS

Said peptide fragments may be fragments of elastin (SEQ ID NO 735) . Even though elastin is abundant throughout the human body, its localization in the atherosclerotic tissue exposes it to the action of local proteases, which is why these molecules are good and specific candidates as biochemical markers of atherosclerotic plaque turnover.

Several candidate proteases may be responsible for the digestion of elastin in the plaque as the literature reports many different proteases in the atherosclerotic plaque. Most likely,

this is the result of the large range of complicated processes eventually leading to plaque rupture. However, early phases may consist of a range of MMPs, whereas later stages may rely more on cathepsin K degradation of the matrix, resulting in different neo- epitope profiles dependent on the levels of disease. We have through a range of in vitro cleavages of pure native proteins determined that the enzymes listed in the following table cleave elastin at at least following cleavage sites (marked *) :

Table 15. Elastin fragments generated by specific proteases.

Accordingly, in a method of the invention, said peptide fragments preferably comprise a neo-epitope formed by cleavage of elastin by a protease at a site marked by the sign * in any one of the following partial sequences of Elastin, or the immunological binding partner is specifically reactive with a sequence defined between the *s in one of the following sequences:

Table 16. Cleavage fragments of Elastin.

Elastin fragments

A*RPGVGVGGI PTYGVGAGG* F

G*LPYTTGKLPYGYGPG*G

G*VAPGVGVAPGVGVAPGIGPGGVA*A

G*GAGVPGVPGAI PGI GGIAGVG* T

G*GAGVPGVPGAI PGI GGIAGVG* T G*VGI SPEAQAAAAAK*A G*VGI SPEAQAAAAAK*A

A*RPGVGVGGI PTYGVGAGG* F

G*LPYTTGKLPYGYGPG*G

Suitable immunological binding partners may therefore be specifically reactive with any of the following sequences at the N terminal of a peptide:

or with any of the following sequences at the C-terminal of a peptide :

Further cleavage sites defining neo-epitopes that may be assayed in a similar manner can be identified by exposing elastin or another atherosclerotic plaque protein to any of the enzymes described herein and isolating and sequencing peptides thereby produced. Assays for more than one of the peptides described above may be conducted separately and their results combined or more than one of the peptides described above may be measured together.

The result of an assay according to the invention may be combined with one or more other measured biomarkers to form a composite index of diagnostic or prognostic value.

The term ^immunological binding partner' as used herein includes polyclonal and monoclonal antibodies and also specific binding fragments of antibodies such as Fab or F(ab') ₂ . Thus, said immunological binding partner may be a monoclonal antibody or a fragment of a monoclonal antibody having specific binding affinity.

The term ^λ protein' used herein includes lipoproteins and proteoglycans and other protein- (non-protein) naturally occurring conjugates .

Generally, all previously known immunoassay formats can be used in accordance with this invention including heterogeneous and homogeneous formats, sandwich assays, competition assays, enzyme linked assays, radio-immune assays and the like. Thus, optionally, said method is conducted as a competition immunoassay in which said immunological binding partner and a competition agent are incubated in the presence of said sample and the competition agent competes with the peptide fragments in the sample to bind to the immunological binding partner.

Said competition agent may be a synthetic peptide or a purified native peptide formed by cleavage of the protein to which the neo-epitope belongs to reveal said neo-epitope. Thus, the peptide may be derived from any of versican, lumican, perlecan, decorin, biglycan, collagen type III, ApoE, CRP or elastin.

One suitable method could be a competition immunoassay using monoclonal antibodies or antibody binding fragments binding to neo- epitopes of fragments of any of these proteins or neo-epitopes on peptide fragments from other proteins derived from atherosclerotic plaques. Appropriately selected synthetic peptides coated onto the solid surface of a microtitre plate could compete with the sample for binding to the monoclonal antibodies or binding fragments.

Alternatively, purified, native fragments from one or more of these proteins carrying the neo-epitope recognised by the monoclonal antibody or binding fragment could be used on the solid surface. Yet another alternative is to immobilise the monoclonal antibody or binding fragment on the solid surface and then co-incubate the sample with a synthetic peptide appropriately linked to a signal molecule, e.g. horseradish peroxidase or biotin. The sample may be a sample of urine, serum, blood, plasma or other, e.g. atherosclerotic plaque biopsy. In certain preferred methods, the sample is a patient derived sample, and the method further comprises comparing the determined level of said binding of said peptide fragments with values characteristic of (a) comparable healthy individuals and/or (b) a pathological atherosclerotic condition and optionally associating a higher level of the measured peptide (normally indicated by a higher level of binding) with a more severe degree of a said condition .

An aspect of the present invention relates to the development of monoclonal antibodies recognising neo-epitopes as described above. This can be achieved by immunising mice with synthetic peptides originating from the amino acid sequence of the protein molecule concerned (including the sequences listed above or sequences terminating therein) , fusing the spleen-cells from selected mice to myeloma cells, and testing the monoclonal antibodies for binding to neo-epitopes on relevant synthetic

peptides. Specificity for neo-epitopes can be ensured by requiring reactivity with a synthetic peptide and a lack of reactivity with either a C-prolongated form of the immunising peptide (for a C- terminal neo-epitope) or an N-terminal prolongated form of the immunising peptide (for an N-terminal neo-epitope) . Antibodies for neo-epitopes may also be evaluated to establish a lack of binding capacity to native protein. Alternatively, specificity for a neo- epitope can be ensured by requiring the reactivity of the antibody to be negatively dependent on the presence of biotin or other functional groups covalently linked to one of the terminal amino acids .

The invention includes an immunological binding partner which is specifically immunoreactive with a neo-epitope formed by cleavage of a said protein by a protease at an end-site in any one of the partial sequences set out above, and may be for instance a monoclonal antibody or a binding fragment thereof.

The invention includes a cell line producing a monoclonal antibody against a C-terminal or N-terminal neo-epitope formed by cleavage of an atherosclerotic plaque protein at the end-sites of sequences in any one of the partial sequences o set out above.

The invention further provides a peptide comprising a C- terminal or N-terminal neo-epitope formed by cleavage of a said protein in any one of the partial sequences of these proteins set out above. Such a peptide may be conjugated as a hapten to a carrier for producing an immune response to said peptide, or immobilised to a solid surface or conjugated to a detectable marker for use in an immunoassay.

The invention further comprises an isolated nucleic acid molecule coding for a peptide comprising a C-terminal or N-terminal neo-epitope formed by cleavage of a said protein in any one of the partial sequences set out above.

The invention further comprises a vector comprising a nucleic acid sequence comprising an expression signal and a coding sequence which codes for the expression of a peptide comprising a C-terminal or N-terminal neo-epitope formed by cleavage of a said protein in

any one of the partial sequences set out above and further includes a host cell transformed with such a vector and expressing a said peptide .

Yet another aspect of the invention relates to kits, which can be used conveniently for carrying out the methods described above. Such kits may include (1) a microtitre plate coated with synthetic peptide; (2) a monoclonal antibody or antibody binding fragment of the invention reactive with said synthetic peptide; and (3) a labelled anti-mouse IgG immunoglobulin. Alternatively, such kits may include (1) a microtitre plate coated with purified native protein fragments; (2) a monoclonal antibody recognising a neo- epitope on fragments of any one of said proteins, and reactive with said purified fragments; and (3) a labelled anti-mouse IgG immunoglobulin. Alternatively, such kits may include (1) a microtitre plate coated with streptavidin; (2) a synthetic peptide linked to biotin; (3) a monoclonal antibody recognising a neo- epitope on said protein fragments and reactive with said synthetic peptide; and (4) a labelled anti-mouse IgG immunoglobulin. Yet another alternative could be kits including (1) a microtitre plate coated with streptavidin; (2) a synthetic peptide linked to biotin; (3) a monoclonal antibody recognising a neo-epitope on said protein fragments (and reactive with said synthetic peptide) and conjugated to horseradish peroxidase.

Thus, the invention includes an immunoassay kit comprising an immunological binding partner as described herein, and a competition agent which binds said immunological binding partner, and optionally one or more of a wash reagent, a buffer, a stopping reagent, an enzyme label, an enzyme label substrate, calibration standards, an anti-mouse antibody and instructions for conducting a said immunoassay.

The assays described herein are useful in the diagnosis of atherosclerotic disease in patients. In addition, the tests are useful for the assessment of disease progression, and the monitoring of response to therapy. The immunological binding partners of the invention may also be used in immunostaining to

show the presence or location of cleavage products of any atherosclerotic plaque protein described herein.

The invention will be further explained and illustrated with reference to the accompanying drawings, in which:

Figure 1 shows Biglycan staining (magnifications 2, 4, 4 and 10 x respectively) using a monoclonal mouse antibody on an aortic sample with type III lesion.

Figure 2 shows Cathepsin K staining (magnifications 2, 4, 10 and 10 x respectively) using a monoclonal mouse antibody on an aortic sample with type III lesion.

Figure 3 shows Biglycan staining (magnifications 2, 4, 10 and 10 x respectively) using a monoclonal mouse antibody on an aortic sample with type V lesion.

Figure 4 shows Cathepsin K staining (magnifications 2, 4, 10 and 10 x respectively) using a monoclonal mouse antibody on an aortic sample containing type V lesion.

Figure 5 shows cleavage products of biglycan generated by proteases: MMP2, MMP3, MMP8, cathepsin K, cathepsin S, cathepsin B, and cathepsin L. M=Rainbow marker, -enz = no enzyme digestion, run on a gel in Example 2.

Figures 6 to 8 show competition study results obtained in Example 4.

Figure 9 shows competition study results obtained in Example 6.

Figures 10 and 11 show competition study results obtained in Example 7.

Example 1 .

For analysis of localization of proteoglycans and proteases we performed immunohistochemical stainings of human arterial samples derived from left coronary descending arteries (LAD) .

In the following, co-localization of Cathepsin K protease and biglycan is demonstrated.

Immunohistochemical staining as seen in Figures 1 and 2 revealed a co-localization of biglycan and cathepsin K. This may suggest that biglycan is a preferred substrate of cathepsin K.

The same immunohistochemical staining was performed on the aortic samples, where the atherosclerotic plaque was formed and as a result of this normal aortic architecture was replaced by macrophage foam cell infiltrates and calcifications. The results of these immunostainings are collected in Figures 3 and 4.

Immunohistochemical staining of biglycan and cathepsin K were shown to co-localize in a progressed atherosclerotic lesion. These results together generate hypothesis of specific cathepsin K cleavage sites in biglycan, resulting in increased neo-epitope generation in atherosclerotic lesions. To test this hypothesis, we cleaved biglycan with different proteases.

Example 2.

Degradation of biglycan for assessment of degradation fragments. Biglycan from bovine articular cartilage (B8041 - Sigma-Aldrich) was cleaved by following proteases: MMP2, MMP3, MMP8, Cathepsin K, Cathepsin S, Cathepsin B, and Cathepsin L. Fragments of proteoglycans generated by enzymatic cleavage of above mentioned proteases were separated on 10% NuPage® Bis-Tris gels and afterwards silver-stained by "Silver Express" - silver staining kit (Invitrogen cat.nr. LC6100, lot . nr .341099) . Results of separation of proteolytically derived and biglycan and silver-stainings are represented by Figure 5.

Example 3

Mice were immunised with collagen type III derived peptides conjugated with ovalbumin. Sera were screened for reactivity with screening peptide sequences conjugated to biotin. Monoclonal antibody secreting clones were produced and screened using the screening sequences. Clones were checked for lack of reactivity with elongated versions of the target peptide which are continued with the adjacent sequences from collagen type III (deselection peptide) and for lack of reactivity with a nonsense peptide. None of the clones positive for the target sequences reacted with either the elongated or the nonsense sequences.

The target sequences, immunogens, screening sequences and deselection sequences were as follows:

Example 4

Reactivity of collagen type III neo-epitope monoclonal antibodies with human urine

The reactivity of selected monoclonal antibody clones from example 3 with human urine was determined in a competition assay format using the immunising peptides as competition agent. In a typical procedure, 96 well streptavidin coated plates were coated for 30 min with 10 ng/mL Biotin-peptide in PBS-BTE at 20° C with shaking and washed 5x in washing buffer. 20 μl of diluted sample was added (either urine or peptide solution) . 100 μL of unpurified antibody solution (supernatant from cell culture) diluted as detailed below was added. The plates were incubated for 1 hr at 2O ⁰ C with shaking at 300 rpm and were then washed 5x in washing buffer. 100 μL secondary antibody-POD (1:5000) was added and incubated for lhr at 2O ⁰ C with shaking at 300 rpm before washing 5x in washing buffer. 100 μL TMB was added and incubated for 15 min in darkness shaking at 300 rpm before adding 100 μL stopping solution. The plates were read at 450 nm on an ELISA reader with 650 nm as reference. Competition therefore occurred between the peptide on the plate and peptide in solution for the antibody and the amount of plate bound antibody was determined by the peroxidase colour forming reaction. Results are seen in Figure 6 for four different clones. It can be seen that the antibodies each detect relevant sequences in urine .

Further competition studies were performed on one selected clone to test competition for antibody binding between the immunising peptide and native collagen type III cleaved in vitro by MMP9. Results are shown in Figure 7 for the cleaved collagen, the peptide KNGETG and an elongated version of that sequence. It can be seen that the antibody binds the immunising peptide sequence and the enzyme cleaved collagen, but not the extended sequence. Further competition studies on the same clone are seen in Figure 8 where the competition agents were the peptide KNGETG, human serum, rat serum, FCS (fetal calf serum) , and atherosclerotic plaque extracts respectively. It is seen that the antibody is

reactive with the peptide, the plaque extract and human serum, but not the rat serum or FCS.

Example 5

Raising of anti-sera to decorin, biglycan and versican sequences

Anti-sera were raised and monoclonal antibodies were obtained as in Example 3, but using the following immunogens, screening sequences and deselection sequences:

Example 6 Reactivity of decorin neo-epitope monoclonal antibody with human urine

A competition ELISA was carried out generally as in Example 5 using one anti-decorin unpurified monoclonal antibody (NB62)

Results are shown in Figure 9. Reactivity is seen against the peptide sequence against which the antibody was raised and selected and against urine, but not against the irrelevant peptide sequence NBl 8.

Example 7

Reactivity of versican neo-epitope monoclonal antibody with human urine

A competition ELISA was carried out generally as in Example 5 using two anti-versican unpurified monoclonal antibody clones raised against sequence (NB64) .

Results are shown in Figures 10 and 11 for the respective clones. In each case reactivity is seen against the peptide sequence against which the antibody was raised and selected and against urine, but not against the irrelevant peptide sequence NBl 8.

In this specification, unless expressly otherwise indicated, the word 'or' is used in the sense of an operator that returns a true value when either or both of the stated conditions is met, as opposed to the operator 'exclusive or' which requires that only one of the conditions is met. The word 'comprising' is used in the sense of 'including' rather than in to mean 'consisting of . All prior teachings acknowledged above are hereby incorporated by reference .

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