Aggregated low density lipoproteins (agLDL) contribute to massive intracellular cholesteryl ester (CE) accumulation in human vascular smooth muscle cells (VSMC). Our aim was to determine the conformational and physical structure of agLDL and elastic material produced either by control human VSMC or by agLDL-loaded human VSMC (agLDL-VSMC). At the conformational level scanned by FTIR spectroscopy, a new undefined, probably non-H-bonded, structure for tropoelastin produced by agLDL-VSMC is revealed. By differential scanning calorimetry, a decrease of water affinity and a drop of the glass transition associated with aggregated tropoelastin (from 200°C to 159°C) in the supernatant from agLDL VSMC are evidenced. This second phenomenon is due to an interaction between agLDL and tropoelastin as detected by the weak specific FTIR absorption band of agLDL in supernatant from agLDL-loaded VSMC. 1. Introduction VSMC in atherosclerotic lesion are unable to produce normal elastic fibers due to atherosclerotic risk factors such as diabetes and associated hyperglycemia, endothelial dysfunction, and inflammation [1, 2]. If experimental hypercholesterolemia decreases the wall elastin content in vivo [3] and in vitro systems [4], the role of hypercholesterolemia in the altered VSMC elastogenic capacity and the possible mechanisms involved are not yet elucidated. VSMC become foam cells through the uptake of diversely modified LDLs [5, 6], whereas the aggregation of LDLs (agLDL) seems to be a key condition for lipid accumulation in VSMCs [7, 8]. Intracellular cholesterol accumulation alters proteoglycan composition [9] and collagen assembly [10] in VSMC, but it is unknown whether intracellular lipid may change the physical characteristics of the tropoelastin synthesized by human VSMC. The aim of this work was to characterize agLDL as well as tropoelastin produced by agLDL-lipid-loaded human VSMC versus that produced by control VSMC using polymer characterization techniques that were previously shown to be efficient in checking the molecular architecture and chain dynamics of proteins [11, 12]. 2. Materials and Methods 2.1. Human VSMC Primary cultures of human VSMC were obtained from nonatherosclerotic areas of human coronary arteries from hearts explanted during heart transplantation at Hospital of Santa Creu i Sant Pau as previously described [5, 6]. Donors of the explanted hearts were men between 40 and 60 years old. Explants were incubated at 37°C in a humidified atmosphere of 5% CO2. Cells grown out of explants were suspended in a solution of trypsin/EDTA and
References
[1]
C. S. Fox, S. Coady, P. D. Sorlie et al., “Increasing cardiovascular disease burden due to diabetes mellitus: the Framingham Heart study,” Circulation, vol. 115, no. 12, pp. 1544–1550, 2007.
[2]
R. Cernes, R. Zimlichman, and M. Shargorodsky, “Arterial elasticity in cardiovascular disease: focus on hypertension, metabolic syndrome and diabetes,” Advances in Cardiology, vol. 45, pp. 65–81, 2008, Review.
[3]
T. Augier, P. Charpiot, C. Chareyre, M. Remusat, P. H. Rolland, and D. Gar？on, “Medial elastic structure alterations in atherosclerotic arteries in minipigs: plaque proximity and arterial site specificity,” Matrix Biology, vol. 15, no. 7, pp. 455–467, 1997.
[4]
E. Csonka, K. Szemenyei, M. Miskulin, and A. M. Robert, “Morphological examination of aortic endothelial and smooth muscle cells grown in vitro on collagen membranes,” Artery, vol. 8, no. 3, pp. 253–258, 1980.
[5]
V. Llorente-Cortés, J. Martínez-González, and L. Badimon, “LDL receptor-related protein mediates uptake of aggregated LDL in human vascular smooth muscle cells,” Arteriosclerosis, Thrombosis, and Vascular Biology, vol. 20, no. 6, pp. 1572–1579, 2000.
[6]
V. Llorente-Cortés, M. Otero-Vi？as, E. Hurt-Camejo, J. Martínez-González, and L. Badimon, “Human coronary smooth muscle cells internalize versican-modified LDL through LDL receptor-related protein and LDL receptors,” Arteriosclerosis, Thrombosis, and Vascular Biology, vol. 22, no. 3, pp. 387–393, 2002.
[7]
V. V. Tertov, A. N. Orekhov, I. A. Sobenin et al., “Three types of naturally occurring modified lipoproteins induce intracellular lipid accumulation due to lipoprotein aggregation,” Circulation Research, vol. 71, no. 1, pp. 218–228, 1992.
[8]
A. Nermine, E. Ismail, M. Z. Alavi, and S. Moore, “Lipoprotein-proteoglycan complexes from injured rabbit aortas accelerate lipoprotein uptake by aterial smooth muscle cells,” Atherosclerosis, vol. 105, no. 1, pp. 79–87, 1994.
[9]
P. Vijayagopal, J. E. Figueroa, Q. Guo, J. D. Fontenot, and Z. Tao, “Marked alteration of proteoglycan metabolism in cholesterol-enriched human arterial smooth muscle cells,” Biochemical Journal, vol. 315, no. 3, pp. 995–1000, 1996.
[10]
M. J. Frontini, C. O'neil, C. Sawyez, B. M. C. Chan, M. W. Huff, and J. G. Pickering, “Lipid incorporation inhibits src-dependent assembly of fibronectin and type i collagen by vascular smooth muscle cells,” Circulation Research, vol. 104, no. 7, pp. 832–841, 2009.
[11]
V. Samouillan, A. Lamure, E. Maurel, J. Dandurand, C. Lacabanne, and M. Spina, “Dielectric characterization of collagen, elastin, and aortic valves in the low temperature range,” Journal of Biomaterials Science, Polymer Edition, vol. 11, no. 6, pp. 583–598, 2000.
[12]
V. Samouillan, J. Dandurand-Lods, A. Lamure, E. Maurel, C. Lacabanne, and M. Spina, “Thermal analysis characterization of aortic tissues for cardiac valve bioprostheses,” Journal of Biomedical Materials Research, vol. 46, pp. 531–538, 1999.
[13]
D. Krilov, M. Balarin, M. Kosovi？, O. Gamulin, and J. Brnjas-Kraljevi？, “FT-IR spectroscopy of lipoproteins—a comparative study,” Spectrochimica Acta A: Molecular and Biomolecular Spectroscopy, vol. 73, no. 4, pp. 701–706, 2009.
[14]
L. Debelle, A. J. P. Alix, S. M. Wei et al., “The secondary structure and architecture of human elastin,” European Journal of Biochemistry, vol. 258, no. 2, pp. 533–539, 1998.
[15]
F. Bonnier, D. Bertrand, S. Rubin et al., “Detection of pathological aortic tissues by infrared multispectral imaging and chemometrics,” Analyst, vol. 133, no. 6, pp. 784–790, 2008.
[16]
X. Hu, X. Wang, J. Rnjak, A. S. Weiss, and D. L. Kaplan, “Biomaterials derived from silk-tropoelastin protein systems,” Biomaterials, vol. 31, no. 32, pp. 8121–8131, 2010.
[17]
L. B. Dyksterhuis, E. A. Carter, S. M. Mithieux, and A. S. Weiss, “Tropoelastin as a thermodynamically unfolded premolten globule protein: the effect of trimethylamine N-oxide on structure and coacervation,” Archives of Biochemistry and Biophysics, vol. 487, no. 2, pp. 79–84, 2009.
[18]
E. J. Podet, D. R. Shaffer, S. H. Gianturco, W. A. Bradley, C. Y. Yang, and J. R. Guyton, “Interaction of low density lipoproteins with human aortic elastin,” Arteriosclerosis and Thrombosis, vol. 11, no. 1, pp. 116–122, 1991.
[19]
A. M. Tamburro, A. Pepe, B. Bochicchio, D. Quaglino, and I. P. Ronchetti, “Supramolecular amyloid-like assembly of the polypeptide sequence coded by exon 30 of human tropoelastin,” The Journal of Biological Chemistry, vol. 280, no. 4, pp. 2682–2690, 2005.
[20]
L. Debelle, A. J. P. Alix, M.-P. Jacob et al., “Bovine elastin and κ-elastin secondary structure determination by optical spectroscopies,” The Journal of Biological Chemistry, vol. 270, no. 44, pp. 26099–26103, 1995.
[21]
R. Prassl, B. Schuster, P. M. Abuja, M. Zechner, G. M. Kostner, and P. Laggne, “A comparison of structure and thermal behavior in human plasma lipoprotein(a) and low-density lipoprotein. Calorimetry and small-angle X-ray scattering,” Biochemistry, vol. 34, no. 11, pp. 3795–3801, 1995.
