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Original Article Glycation of Matrix Proteins in the Artery Inhibits Migration of Smooth Muscle Cells from the Media to the Intima

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Original Article Glycation of Matrix Proteins in the Artery Inhibits Migration of Smooth Muscle Cells from the Media to the Intima Original Article Glycation of Matrix Proteins in the Artery Inhibits Migration of Smooth Muscle Cells from the Media to the Intima (glycation / advanced glycation end products / collagen / elastin / myocytes / atherosclerosis) A. KUZAN1, O. MICHEL1, A. GAMIAN1,2 1Department of Medical Biochemistry, Wroclaw Medical University, Wrocław, Poland 2Laboratory of Medical Microbiology, Ludwik Hirszfeld Institute of Immunology and Experimental Therapy, Polish Academy of Sciences, Wroclaw, Poland Abstract. Formation and growth of atherosclerotic Introduction plaques have serious clinical consequences. One mechanism that occurs during atherogenesis is mi- Smooth muscle cells (SMC) are predominantly lo- gration of smooth muscle cells from the middle layer cated in the middle layer of the artery, where they play a of the artery to the intima, where they proliferate crucial role: they determine the contractility of the tis- and are transformed into foam cells. This degenera- sue, hereby adjusting the blood flow through the artery. tive process is accompanied by glycation, by which In such a case, they are called contractile smooth muscle proteins are modified and change the biomechanical cells, which are characterized by the richness in actin and biochemical properties. The aim of the study filaments. They may, however, undergo phenotype was to determine whether glycation of collagen and switching and migration and become cells that reside in elastin building the walls of blood vessels alters the the intima. Then, they are called synthetic type SMC, adhesion and rate of myocyte migration. In vitro ex- are rich in rough endoplasmic reticulum and character- periments included migration assays and immuno- ized by active metabolism (Stary et al., 1992, 1994; cytochemical staining with anti α-actin, β-catenin Tukaj, 2010). Such a change in the phenotype is associ- anti-collagen type IV antibodies. It turns out that ated with a number of impacts: intensive proliferation of there is a tendency to decrease the number of cells these cells occurs in the intima, secretion of inflamma- that had migrated through the barrier consisting tory mediators and apoptosis rate are increased, matrix of glycated proteins as compared to the control. protein synthesis is disturbed (Tukaj, 2010); all this in- Adversely, the morphology of the cells cultured in creases atherogenesis. Smooth muscle cells, similarly to the presence of glycated substrates is changed. The macrophages, after the uptake of large amounts of lipids lower intensity of β-catenin staining indicates lower become foam cells. After their death, the lipid content is adhesiveness of such cells. It is proposed that glyca- poured into the intercellular space to form a fatty core tion inhibits migration of smooth muscle cells from (Stary et al., 1992, 1994). the media to the intima, which represents part of the Glycation is a non-enzymatic process, spontaneously anti-atherogenic mechanism. occurring in the body or outside of a living organism, which involves reaction of reducing sugars (glucose, fructose, glucose-6-phosphate (G6P), and other) with Received November 20, 2016. Accepted April 3, 2017. the amino groups of proteins. Due to the fact that only a The research was supported by the grant for young scientists small part of the sugars is present in the form of a chain funded by the Wroclaw Medical University No. Pbmn134. having a free aldehyde group under physiological con- ditions, the process occurs very slowly, over many Corresponding author: Aleksandra Kuzan, Department of Medi- weeks or months (Slatter et al., 2008). Therefore, the cal Biochemistry, Wroclaw Medical University, Chałubińskiego problem of malfunction of the glycated protein mainly 10, Wrocław 50-368, Poland. Phone: (+48 71) 784 13 87; e-mail: [email protected] concerns long half-life proteins, such as collagen and other extracellular matrix proteins (Xiao et al., 2007). Abbreviations: AGE – advanced glycation end products, ECM – It is noteworthy that among modifications undergone extracellular matrix, EGF – epidermal growth factor, FGFb – by Amadori products arising from glycation is also con- basic fibroblast growth factor, G6P – glucose-6-phosphate, MGO – methylglyoxal, MMP – matrix metalloproteinases, MTT – densation. The reaction of these compounds with amino 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, acids of other proteins leads to the formation of PBS – phosphate-buffered saline, PDGF – platelet-derived growth crosslinks between proteins and induces formation of factor, RGD – tripeptide Arg-Gly-Asp, SMC – smooth muscle large protein complexes. The result of this phenomenon cells. in the case of fibrous proteins is increased thickness of Folia Biologica (Praha) 63, 105-114 (2017) 106 A. Kuzan et al. Vol. 63 the fibrils, and consequently, increased stiffness of the (ATCC, Dziekanów Leśny, Poland). The cell culture fibre, reduced solubility and lower susceptibility to en- was maintained in a dedicated medium augmented with zymatic digestion (Avery and Bailey, 2008). Further- special supplements contained in the Vascular Smooth more, modifications of the side chains of amino acids Muscle Growth Cell Kit (ATCC). building scleroproteins entail not only structural changes, but also induce alterations in the location of electrostatic Glycation of the protein-sugar mixtures charges in the molecules, which in turn can affect inter- Mixtures of proteins, collagen and elastin were sub- actions with other proteins (Jabłońska-Trypuć and jected to the glycation process with simultaneous micro- Czerpak, 2007). As a result, this contributes to the limi- wave stimulation. The protein-sugar compounds (glu- tation of binding with specific integrins, which consid- cose, glucose-6-phosphate (G6P) and proteins – collagen erably weakens the interaction of collagen with the and/or elastin in specified concentration) were exposed cells. This is particularly important, inter alia, in the de- to microwave irradiation at 640W, in cycles: heating velopment of vascular damage or in wound healing in (30 s microwave action) and cooling (1 min at –20 °C). diabetes (Avery and Bailey, 2006). Cumulative length of the microwave action was 10 and In collagen glycation, attention should also be paid to 30 min. the fact that different types of collagen may vary in sus- The protein mixture included collagen type I (Sircol ceptibility to this non-enzymatic process. As previously Bovine Collagen Reference Standard, acid soluble type I, mentioned, the process concerns only long half-life pro- Biocolor, Carrickfergus, UK), type II collagen (collagen teins. The half-life, however, is not equal for different from chicken sternal cartilage, type II, Sigma-Aldrich, types of collagen. The turnover of type II collagen pre- Poznan, Poland), type IV collagen (collagen from hu- sent in the cartilage is approx. 100 years, whereas for man placenta, Bornstein and Traub Type IV, Sigma) and type I collagen occurring in the skin reaches approx. elastin (elastin from bovine neck ligament, Sigma). An 10 years, for collagen building bone it amounts only to equal volume of glucose (Sigma) or G6P sodium salt 1–2 years, and the turnover of collagen from periodontal (Sigma) in concentration 400 mM was added to the ob- ligaments takes only one day (Avery and Bailey, 2006). tained mixture. Subsequently, the mixture was parti- It is undoubtable that smooth muscle cells play a sig- tioned into three parts, the first of which was irradiated nificant role in the pathogenesis of atherosclerosis for 10 min, the other for 30 min, and the third was left as (Tukaj, 2010). Numerous studies in the last decade fo- a control. cused on this aspect of atherogenesis (Butoi et al., 2014; The amount of advanced glycation products was Chiong et al., 2014; Wall and Bornfeldt, 2014; Che- measured using specific spectrophotometric and fluores- pelenko, 2015; Monk and George, 2015, and many oth- cent properties: absorbance at 440 nm and fluorescence ers). Nevertheless, only few studies are devoted to the at an excitation wavelength of 370 nm and emission issue of migration of smooth muscle cells in the context 400–480 nm. Exemplary fluorescence spectra can be of the influence of advanced glycation end products on found in Fig. 1. It may be concluded that glycation oc- this process. It is generally postulated that advanced curs with much higher efficiency with the involvement glycation end products (AGE) stimulate migration of of glucose-6-phosphate than in the presence of glucose. SMC within atherosclerotic plaques (Dan et al., 2004; Chung et al., 2013). MTT assay – cytotoxity test of the collagen/ Research in this stream focuses predominantly on the collagen-elastin mixtures investigation of the mechanism leading to such effect and relates mainly to low molecular weight of AGE or The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetra- small glycated proteins (mostly on glycated bovine se- zolium bromide (MTT) assay was performed in order to rum albumin). Meanwhile, the question of the influence exclude the influence of the mixture components on the of glycation of long, fibrous, long half-life proteins from viability of smooth muscle cells derived from an artery. extracellular matrix on the migration of SMC appears to Cells were grown in a 96-well plate (5000 cells/well in be still unexplored. From the studies concerning the gly- 200 µl). After 24 h post seeding, the medium was re- cation of these proteins it follows that these structures placed with a medium containing glucose, G6P, colla- are stiffer than the protein unmodified by glycation. gen, or their mixtures. After an additional 24 and 48 h, Presumptively, in this way they will provide a more in- the culture medium was removed and 100 µl of MTT accessible barrier for SMC, thereby slowing their migra- solution at a concentration of 0.5 mg/ml was added to tion. This would constitute an anti-atherogenic mecha- each well. After 2 h of incubation, 100 µl of isopropanol nism, in contrast to many other mechanisms stimulating acidified with HCl was added to each well in order to growth of the atherosclerotic plaque, which is attributed dissolve formazan crystals.
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