ANIMAL AND IN VITRO MODELS IN HUMAN DISEASESApplication of stereological methods for the quantification of VCAM-1 and ICAM-1 expression in early stages of rabbit atherogenesis
Introduction
Atherosclerosis is a progressive vascular disease characterized by endothelial dysfunction, upregulation of cell adhesion molecules, accumulation of lipids, macrophages, smooth muscle cells (SMCs), and fibrous tissue in the arterial intima. The process of atherogenesis is still not completely understood, but it is generally believed that inflammation plays an important role in all stages of atherosclerosis [21]. Chronic inflammatory reaction in the arterial wall is usually accompanied by the recruitment of macrophages and T lymphocytes in the arterial intima. This accumulation is strongly regulated by the expression of cell adhesion molecules, including VCAM-1 and ICAM-1. Cytokines such as interleukin-1 and tumor necrosis factor α, produced in many acute and chronic conditions, are important mediators of induced adhesion molecule expression [14]. Furthermore, hypercholesterolemia, hyperhomocysteinemia, diabetes mellitus, oxLDL, and shear stress are other inductors of overexpression of VCAM and ICAM [27], [28]. VCAM-1 and ICAM-1 have structural and functional similarities. Both are transmembrane glycoproteins and members of the immunoglobulin (Ig) gene superfamily. VCAM-1 has seven extracellular Ig-like domains, whereas ICAM-1 has five. Both bind integrin ligands, VCAM-1 binds α4 integrins, and ICAM-1 interacts with αLβ2 integrins (LFA-1) or with αMβ2 integrins (Mac-1) [24]. Both VCAM-1 and ICAM-1 can stabilize leukocyte adhesion to the endothelium and participate in diapedesis. Endothelial cells, phenotypically modulated (synthetic phenotype) smooth muscle cells, and macrophages are in general sources of VCAM-1 and ICAM-1 expression in atherosclerotic lesions [2], [9]. VCAM-1 and ICAM-1 are expressed by activated endothelial cells and play an important role in very early stages of atherogenesis [22] as well as in more advanced atherosclerotic lesions, where intimal cells and endothelial cells of intimal neovessels in atherosclerotic plaques express them [20]. Thus, VCAM-1 and ICAM-1 are crucial to early leukocytes recruitment and subsequent progression of atherogenesis in all stages of atherosclerosis.
Stereological methods are precise tools for the quantitative evaluation of the structure of three-dimensional (3D) objects, based mainly on observations made on two-dimensional (2D) sections or projections. These are practical tools based on sound mathematical and statistical principles. Stereology may also be regarded as a sampling theory for populations with a geometric structure. They can be used for the estimation of volume, volume density number, surface area, length, and many more [5]. The morphometric study is one of the most important considerations in the uniform characterization of the atherosclerotic lesion, and it is usually necessary for an objective evaluation of the atherogenic process.
There were two aims in the present study. Firstly, we wanted to introduce stereological methods that may be used for the quantification of immunohistochemical staining. Secondly, we wanted to quantify the expression of VCAM-1 and ICAM-1 in particular parts of the vessel wall during a time-dependent period of feeding with a high-cholesterol diet.
Section snippets
Animals and diet
Twenty-four adult male New Zealand White rabbits (2.0–3.0 kg; BIOTEST Konarovice) were randomly subdivided into three groups. In each group, six rabbits consumed an atherogenic diet (0.4% cholesterol, 3% fat, 19% proteins), and two rabbits consumed the standard diet (control animals). Rabbits obtained 150 g of the atherogenic or standard diet per day, and food intake was monitored every morning. The first group of animals was fed for 1 month (1-month cholesterol group), the second group was fed
Biochemical verification of hypercholesterolemia
The mean total serum cholesterol levels in experimental animals were increased almost 30-fold in comparison with the control group. There was a statistically significant increase between the experimental and control groups of animals (P<0.05; Fig. 2).
Aortic VCAM-1 and ICAM-1 expression during the period of feeding with an atherogenic diet
After 1 month of cholesterol consumption, VCAM-1 and ICAM-1 expression was predominantly localized in the endothelial cells of early lesions (Fig. 3A and B); however, it was observed even in endothelium where no lesions were found (Fig. 3C and D).
Discussion
The objective characterization of the atherogenic process is usually performed by using a wide spectrum of morphometric methods. Several methods have been developed, including noninvasive ultrasound methods [30], magnetic resonance imaging [26], and especially in computer-assisted image analysis [25]. Stereological principles and methods are widely used in various branches of biological research, including cardiology [19] and especially neuroscience [1], [16]. However, they are not used very
Uncited References
[24].
Acknowledgements
The authors wish to thank Dr. Myron Cybulsky for providing monoclonal antibodies against rabbit VCAM-1, ICAM-1, Mrs. B. Šlechtová, Mrs. Ing. Z. Müllerová for her skilful technical assistance, Mr. Bohuslav Manek for the revision of the English text and Mrs. Dr. L. Kubı́nová for her splendid assistance regarding stereological methods. This work was supported by the Grant Agency of the Czech Republic Grant no. 304/03/P049, the Charles University Grant Agency Grant no. 92/200C, and by the Ministry
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