The pathophysiology of atherosclerosis

Željko Reiner

Department of internal diseases, University hospital center Zagreb


It is well established that atherosclerosis is a process which begins at an early age and progresses at a variable rate, depending upon a wide variety of factors acting upon the arterial wall. It begins with over expression of endothelial adhesion molecules (VCAM-1, ICAM-1, selectins), chemotactic factors (MCP-1), growth factors (M-CSF) and cytokines (IL-2) that facilitate the recruitment and internalization of monocytes which are transformed into macrophages. They may later undergo apoptosis with the release of metalloproteinases and tissue factor. After formation of foam-cells from macrophages and smooth muscle cells migration from intima to media, a fibrous cap is formed which confers the stability of the atherosclerotic plaque and separates the lipid core from the vessel lumen. Today it is clear that the composition of the plaque, rather that the percent stenosis is a predictor of the patients’ destiny. Most fatal outcomes of coronary artery atherosclerosis result from the plaque fibrous cap rupture or superficial erosion. The propensity of plaque to disrupt depends upon inflammatory pathways which impinge on matrix metalloproteinases and oxidative stress. Inflammation has been implicated not only in the beginning of atherogenesis but in plaque neovascularity (vasa vasorum) and intraplaque hemorrhage as well, and inflammatory cell derived tissue factor is a key contributor to plaque thrombogenicity. Therefore, inflammation, atherogenesis and thrombogenesis are interdependent and all three contribute to clinical consequences.


The molecular basis of atherosclerosis

Elizabeta Topić

University Department of Chemistry, Sestre Milosrdnice University Hospital, Zagreb, Croatia


Numerous epidemiological studies have demonstrated the genetic component, along with many environmental risk factors, to play an important role in the development of cardiovascular and cerebrovascular diseases underlain by atherosclerosis. However, straight association between the genetic risk factors and the development of atherosclerosis cannot be determined due to the complex etiology of the latter.

The most relevant genes postulated to pose a risk for the development of atherosclerosis are the genes involved in the metabolism of lipids, blood pressure and homocysteine; those involved in thrombosis, inflammation and leukocyte adhesion; and the genes of the matrix metalloproteinase family responsible for vascular remodeling. Along with the candidate genes involved in impairments of the lipoprotein metabolism, blood pressure regulation or hemostasis, which were in the past two decades associated with atherosclerosis and endothelial dysfunction, research has currently been focused on the genes of matrix metalloproteinases, of renin-angiotensin system involved in hypertension, adhesion molecules and of interleukins that are directly or indirectly related to atherosclerotic lesions.

To date, more than 850 different gene variants have been tested in the studies of gene to atherosclerosis association; however, quite frequently yielding contradictory results. These discrepancies can be readily explained by the complex etiology of atherosclerosis, genetic factors involved in its development, population specific heredity as well as by the methodological approach in selection of risk groups and in particular study design. This implicates that studying genetic associations in such polygenic disorders is extremely difficult, because the genetic effects are small, hardly measurable and only manifesting in the interaction of multiple genes occurring in the course of the disease. To improve the strength of the studies investigating gene associations with the genesis of atherosclerosis and to upgrade clinical precision of the studies, nowadays, meta-analyses that process pooled results of a great number of published studies have been increasingly used In a meta-analysis, results of such independent studies are pooled and analyzed together as if yielded by a single large study including a population large enough to recognize the gene impact. Such an approach in genetic association studies with atherosclerosis as s polygenic disease will provide a reliable overview and a realistic picture of genetic profile of candidate genes that presents the true significance in recognition the molecular basis of atherosclerosis.


Inflammation and atherosclerosis

Ines Vukasović

University Department of Chemistry, Sestre Milosrdnice University Hospital, Zagreb, Croatia


Recent evidences suggested atherosclerosis as a dynamic progressive disease arising from combination of endothelial dysfunction and inflammation. Inflammation represents the underlying mechanism leading to the formation of human atheroma and favouring both the destabilization of vulnerable plaques and the formation of occlusive thrombi. Inflammatory cytokines involved in vascular inflammation stimulate the generation of endothelial adhesion molecules, proteases, and other mediators, which may enter the circulation in soluble form. These primary cytokines also induce production of the messenger cytokine interleukin-6, which stimulates the liver to increase production of acute-phase reactants such as C-reactive protein. In addition, platelets and adipose tissue can generate inflammatory mediators relevant to atherothrombosis. Numerous studies indicated that modest changes in circulating CRP levels, as detected by highly sensitive methods (hsCRP), can be extremely useful in predicting cardiovascular and perhaps cerebrovascular diseases in apparently healthy individuals as well as in patients affected by atherosclerosis. Subjects manifesting with identical low density cholesterol and/or blood pressure levels have different rates of cardiovascular accidents on the basis of different circulating CRP concentrations. In high-risk secondary prevention settings such as acute coronary syndrome patients being treated with statin therapy, achieving low levels of plasma hsCRP concentration appears to be of similar importance as achieving low levels of LDL cholesterol.

In addition, cellular adhesive molecules, cytokines and chemokines, myeloperoxidase, soluble CD40 ligand, adiponectin, and matrix metalloproteinase 9 may provide additional information for cardiovascular risk stratification and prediction but requires confirmation in additional studies.


New biomarkers of acute myocardial ischemia

Nada Vrkić

University Department of Chemistry, Sestre Milosrdnice University Hospital, Zagreb, Croatia


Ischemia reflects both a vascular inflammatory response and myocardial injury.

About 40% of patients with ACS may not develop necrosis despite severe coronary artery disease and myocardial ischemia. Serial troponins and ECGs are very sensitive for the detection of myocardial infarction but they are insensitive for the detection of ischemia. Therefore, markers that could reliably detect ischemia even in the absence of necrosis (i.e., unstable angina) and/or before troponin level increase are desirable.

Several biomarkers are under investigation. All of them tend to be short-lived, increase promptly or within 3 h, and return to reference value within 3-24 h.

Ischemia-modified albumin (IMA) is a marker of impending myocyte necrosis. During ischemia, free-radical damage alters the N-terminus of albumin and affinity to bind cobalt is reduced. Studies have shown that the negative predictive value of IMA was over 95%, the sensitivity twice higher than an ECG and four times than troponin to detect patients with ACS at time of presentation, which is difficult to diagnose with other diagnostic methods.

Monitoring increased plasma unbound free fatty acid (UFFA) and their intracellular binding protein, heart-type fatty acid-binding protein (H-FABP) concentrations has been proposed as a biomarker for myocardial ischemia.

Ischemia can cause the release of mitochondrial proteins such nourin-1 (polypeptide 3 KD released rapidly by stressed myocytes; within 5 minutes by heart tissues in response to myocardial ischemia) and increased concentration of choline (released after stimulation by phospholipase D) Hypoxia also trigger release of BNP (naturally hormone B- natriuretic peptide) in absence of necrosis.

Current technologies, especially HPLC-MS analysis, can be used to identify clinically relevant perturbations in circulating metabolites. Application of metabolomics identifies changes in levels of metabolites belonging to the citric acid pathway. (central role in oxidative phosphorylation in the myocardium) and the urea cycle.

New markers of ischemia are yet to be evaluated. For more of them there is no standardized assay and no reference interval studies or consistent assay validation. None of them is specific to the myocardium. Therefore, their use is limited since other organs in ischemia may release these biomarkers, too. At present, none of these analytes are yet appropriate for routine clinical use.


Lipids in stroke

Ana-Maria Šimundić

University Department of Chemistry, Sestre Milosrdnice University Hospital, Zagreb, Croatia


As already shown in several major prospective clinical trials, cholesterol-lowering therapy is an efficient primary and secondary preventive measure in reducing the risk of stroke. The risk of recurrent stroke is significantly reduced in stroke patients who began regular treatment with the cholesterol-lowering statin drugs soon after the stroke. However, lipid testing and lipid lowering therapy in patients hospitalized for ischemic stroke is widely underused and the exact role and significance of lipids in stroke patients is still not well understood. Some investigators suggest a different role of HDL and LDL cholesterol in the aetiology of different stroke subtypes. Furthermore, it has been reported that serum lipid levels measured in early period after stroke admission are predictive of infarct volume and 6-month mortality rate, brain infarction subtype and early death from stroke. Nevertheless, there is not a widely accepted recommendation on the optimal time for lipid measurement. Studies on the lipid concentrations obtained during the early acute period of brain ischemia have so far produced conflicting results. Lipid concentrations have mostly been found lower in the early period after the stroke but there are also some reports in the literature on the elevated lipids in early stroke period. Lipid level in acute stroke is therefore still the unreliable measure of lipid status. The major finding in one our recent study on stroke patients was that higher serum triglycerides were associated with more severe stroke and substantial neurological deficit. Further studies should be done to explore the time dependent changes in lipid profile in acute ischemic stroke as well as the best time for lipid measurement, with the purpose of initiating the lipid lowering therapy.


Atherosclerosis and risk stratification in acute myocardial infarction

Birgit Herzig

Abbott GmbH&Co.KG, Wiesbaden, Germany


Troponin, myeloperoxidase (MPO) and homocysteine are markers for the diagnosis of cardiac diseases. While cardiac troponin assays are established tool for exclusion of coronary disease, MPO is a brand new marker for identifying high risk patients with arterosclerotic plaque ruptures.

Abbott Diagnostics offers fully automated troponin, homocysteine and MPO assays on AxSYM (Trp I, Hcy) and Architect platforms (Trp I, Hcy and MPO).

Nowadays Troponin is regarded as myocardial infarction (MI) marker of choice. Cardiac troponins I (TnI) and (TnT) are myofibrillar proteins of heart muscle cells. According to the ESC/ACC (European Society of Cardiology/American College of Cardiology) Consensus Guidelines the 99th percentile is recommended as cut-off.

Inclusion of at least two monoclonal antibodies against epitopes at the N-terminal part of the heart specific region of troponin seemed critical for optimum performance. Significantly superior clinical sensitivity was obtained with the Architect TnI assay vs. Roche TnT and Immulite TnI assays (GUSTO IV study).

MPO plays a major role in the regulation and termination of inflammatory processes. It is a hemoproteine stored in the leukocytes and will be secreted during phagocyte activation. MPO has important role in the atherosclerotic process. It is both a disease marker in atherosclerosis with vulnerable plaques and an event marker for plaque rupture and thus helps in the early diagnosis of acute myocardial infarction.

Homocysteine (Hcy) is a sulphur containing amino acid and part of methionine metabolic pathways. Elevated Hcy levels have a pro-oxidative effect and increase the risk for atherothrombotic disease, dementia, Alzheimer’s disease, depression and osteoporosis.


Glycated Hemoglobin A1c

Lucija Božić

Abbott Laboratories


WHO (World Health Organization) recognizes three main forms of diabetes:

type 1–10% of total diabetes cases has type 1. Cause is in loss of the insulin-producing beta cells of the islets of Langerhans of the pancreas leading to a deficiency of the insulin

type 2 – combination of defective insulin secretion and insulin resistance

gestational diabetes (comprises 2–5% of all pregnant women). Diabetes symptoms usually improve or disappear after delivery.

Diabetes mellitus is characterised by recurrent or persistent hyperglycemia and is diagnosed by any one of the following:

fasting plasma glucose level at or above 7 mmol/L.

fasting plasma glucose level at or above 11,1 mmol/L two hours after 75g oral glucose load as in a glucose tolerance test.

random plasma glucose level at or above 11,1 mmol/L.

Depending on the assay used, HbA1c is approximately 4 to 6% in nondiabetics, 6 to 8% in controlled diabetics and can be as much as 20% in uncontrolled diabetics

The current recommended goal for HbA1c in patients with diabetes is < 7.0%, which as defined as “good glycemic control” (American Diabetes Association Guidelines).

In 2007 Abbott Diagnostics launched new, fully automated immunoassay AxSYM HbA1c for the quantitative determination of percent hemoglobin A1c (HbA1c)-without pretreatment.

The Sample is whole blood [sodium fluoride/potassium oxalate (fluoride oxalate) and sodium fluoride/sodium EDTA (fluoride EDTA)]. Test is performed on Abbott analyzer AxSYM. Percent HbA1c measurements are used in the clinical management of diabetes to assess the long-term efficacy of diabetic control.

Standard AxSYM HbA1c calibrators are traceable to the IFCC reference calibrators and are aligned with the NGSP/DCCT (National Glycohemoglobin Standardization Program/Diabetes Control and Complications Trial) which ensure good reliability and confidence in result.