Objective
To establish a better understanding of the factors contributing to the proliferation of arterial smooth muscle cells (ASMC in order to permit new approaches to the inhibition of cell growth and the development of new therapeutic approaches. A better understanding of the molecular mechanism behind glycosaminoglycans (GAGs)- induced inhibition of growth factors will create possibilities for tailored construction of molecules preventing the local accumulation of growth factors in arterial wall and subsequent proliferation of human ASMC.
BACKGROUND:
Cardiovascular disease due to artherosclerosis is the major cause of mortality and morbidity in the industrialised world. The atheroslerotic process leads to enlargement of arterial wall from the accumulation of lipids, cells and extracellular matrix. Up to 50% of the atherosclerotic wall enlargement consists of ASMC and intercellular tissue. Furthemore, after successful organ transplantations and reconstructive vascular interventions (arterial by-pass surgery and balloon dilatation), 20-50%develop restenosis within 3-12 months, mainly due to a very rapid ASMC proliferation. Presently, there is no effective the rapy to control this phenomenon. The increase in arterial SMCs mass has been associated with activity of the platelet-derived growth factor (PDGF). There are two isoforms of the PDGF-A chain, where the longer A-chain isoform contains a sequence with a high proportion of basic amino acids in its terminal extension. Similarly to PDGF-A, PDGF-B occurs in long and short forms due toproteolytic processing. Proliferation of hASMC is also under influence of several other cytokines such as fibroblast growth factors (FGFs), interleukins and alpha-thrombin. Glycosaminoglycans (GAGs) affect the rate of proliferation of a number of different cell types and among them the ASMC. Heparin-like GAGs bind PDGF and inhibit its mitogenic effect onhASMC in vitro.
RECENT RESULTS:
Studies with Surface Plasmon Resonance Analysis (SPRA) and purifiedre combinant PDGF-AA isomers showed the requirement of the basic carboxyterminal extension with three essential amino acids (two basic and one polar) for high affinity binding to heparin. This interaction involves an allosteric binding mechanism increasing the affinity for heparin significantly. Looking for the potential source of the long PDGF-A isomerin artherial lesion it was shown that the differentiation of monocyte derived macrophages up regulate its expression (RT-PCR). Structural elements similar to the heparin-binding motif in PDGF-A, can be identified in other heparin-binding molecules (low density lipoprotein (LDL)-bound apo-betaprotein, FGF, PDGF-B). In vitro, human ASMC produce proteoglycans (PGs) with different GAG side chains; heparan (HS), chondroitin (CS) anddermatan sulfates (DS). Among them, HS shows the highest binding affinityto PDGF. The smallest heparin and HS-fragment binding to the long PDGF-Achain (membrane binding assay, affinity chromatography) consists of 6-8monosaccharide units and the PDGF A-GAG interaction is mediated viaN-sulfated saccharide domains containing both 2-O- and 6-O-sulfate groups.
This fragment reminds of bFGF binding heparin structure and presumably of the low density lipoproteins (LDL)-binding fragment, since PDGF and LDL compete for similar sites on ASMC-derived GAGs. In spite of the deposition, the interaction of heparin-binding molecules with PGs induces association between the HSPGs and the cytoskeleton (electron microscopy, in situ hybridisation, immunohistochemistry) with possible effects on cell signalling mechanisms. Analysis of PDGF-A and -B knockout mice (transgenic technique) show that distinct subsets of SMC are critically dependent one ither of the two PDGFs during embryonic development.
CONTINUATION OF THE PROJECT:
Using the methods described above, the mechanism of the interaction between PDGF-AA and GAGs is going to be defined in detail with use of recombinant PDGF-A mutants. The binding of recombinant PDGF-B long and short homodimers to heparin will be evaluated and the mechanism defined. We are going to define the smallest PDGF-AA and PDGF-BB binding heparin and HS-fragments and the required sulfatation pattern for this interaction. Using the NMR technique we want to identify the essential details of high order structure of PDGFGAG complex in solubon.
Furthermore, we are going to complete the estimation of the expression of PDGF isomers and PDGF receptors in in vitro cultures of cells accumulating in arterial intima during developmentof atherosclerosis/restenosis (hASMC, macrophages, endothelial oells).We are going to quantify this expression in normal artenal wall and atherosclerotic/restenotic lesions and to investigate the effect of different macrophage-derived cytokines on the expression of the long splicing PDGF-A isomer. To investigate the relative importance of PDGF contra other growth factors/cytokines for hASMC growth, we are going to use the anti-sense technique to inhibit expression of PDGF-receptors and eliminate the effect of PDGF on cells. We plan to evaluate the effect of PDGF-GAG interaction on PDGF-receptors and signalling mechanism and investigate its role and potential importance for embryogenesis.
BIOLOGICAI IMPORTANCE:
Our results, received up to now, implicate that the long but not the shortPDGF-A homodimer would be liable to accumulate locally on cell-associated GAGs and proteoglycans (PGs). An ensuing continous release of the long PDGF isoform from PG due to displacement by for instance LDL, FGF orproteolytic enzymes, could stimulate hASMC to proliferation and lead to conclusion of the artery. Thus the natural GAGs may have an importantmodulatory role for the maintenance of a normal ASMC population. Failure in this role will lead to proliferative arterial disease seen in atherosclerosis, hypertension and in post-operative restenosis.
Keywords: Cardiovascular disease, Atherosclerosis, Occlusion, Smooth MuscleCell Proliferation, Growth Factors, Glycosaminoglycans, Proteoglycans, Heparin.
Fields of science (EuroSciVoc)
CORDIS classifies projects with EuroSciVoc, a multilingual taxonomy of fields of science, through a semi-automatic process based on NLP techniques. See: The European Science Vocabulary.
CORDIS classifies projects with EuroSciVoc, a multilingual taxonomy of fields of science, through a semi-automatic process based on NLP techniques. See: The European Science Vocabulary.
- medical and health sciences clinical medicine angiology vascular diseases
- medical and health sciences clinical medicine surgery
- medical and health sciences clinical medicine cardiology cardiovascular diseases arteriosclerosis
- natural sciences chemical sciences organic chemistry amines
- medical and health sciences clinical medicine transplantation
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Coordinator
413 45 Göteborg
Sweden
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