Characterisation of Ki11502 as a potent inhibitor of PDGF beta receptor-mediated proteoglycan synthesis in vascular smooth muscle cells.
ABSTRACT Platelet-derived growth factor (PDGF) receptor signalling is implicated in cardiovascular diseases such as atherosclerosis and restenosis. PDGF expression levels are elevated in atherosclerotic lesions and play a key role in migration and proliferation of vascular smooth muscle cells in the neointima. PDGF stimulates glycosaminoglycan elongation on vascular proteoglycans biglycan and decorin, a process implicated in the aetiology of atherosclerosis. We investigated the ability of the specific kinase inhibitor Ki11502 to inhibit PDGF beta receptor phosphorylation and proteoglycan synthesis in human vascular smooth muscle cells. Ki11502 inhibited PDGF-mediated tyrosine phosphorylation of the PDGF beta receptor autophosphorylation site and at least six other receptor-associated proteins. Ki11502 also caused a concentration-dependent inhibition of PDGF-stimulated [(3)H]-thymidine incorporation. Total proteoglycan synthesis was assessed as incorporation of [(35)S]-sulfate. PDGF-induced a two-fold increase in [(35)S]-sulfate incorporation into proteoglycans secreted over 24h and was inhibited in a concentration-dependent manner by Ki11502. PDGF treatment resulted in a statistically significant (P<0.01) increase in total proteoglycan core protein secretion. Treatment of cells with Ki11502 (300 nM) had no effect on basal core protein secretion and completely abolished the PDGF-stimulated component. Analysis of isolated cleaved glycosaminoglycan chains by size-exclusion chromatography demonstrated that PDGF stimulated the synthesis and secretion of proteoglycans with elongated glycosaminoglycan chains and this effect was inhibited by Ki11502. Inhibition was also seen in the length of xyloside-glycosaminoglycan chains. The results demonstrate that Ki11502 is a potent and selective inhibitor of PDGF beta receptor phosphorylation, proliferation and proteoglycan synthesis in human vascular smooth muscle cells.
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ABSTRACT: The modern concept of the development of atherosclerosis implies that the underlying pathogenesis of this disease is vascular remodeling as a response of the vessel wall to hypertension associated with hyperlipidemia and subsequent inflammation. However, even though this disease has been investigated for decades, both from a basic and clinical research aspect, there are still many doubts as to what the initial phase of the disease is. In contemporary literature there are an increasing number of papers that stress the importance of the extracellular matrix (ECM) of the blood vessels connective tissue, particularly proteoglycans, in the formation of early atherosclerotic lesions of human coronary arteries.Archives of Biological Sciences 03/2011; 63(2):333-343. · 0.61 Impact Factor
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ABSTRACT: Among the family of receptor tyrosine kinases (RTKs), platelet-derived growth factor receptor (PDGFR) has attracted increasing attention as a potential target of anti-tumor therapy in colorectal cancer (CRC). To study the function of PDGFRβ in CRC cell lines, SW480, DLD-1 and Caco-2 cells showing high PDGFRβ expression were used for receptor down-regulation by small interfering RNA (siRNA) and using the pharmacological inhibitor of PDGFRβ Ki11502. Blockade of PDGFRβ using both approaches led to moderate inhibition of proliferation and diminished activation of the downstream PI3K-signaling pathway in all three cell lines. Surprisingly, incubation with Ki11502 resulted in an arrest of SW480 cells in the G2 phase of the cell cycle, whereas the siRNA approach did not result in this effect. To address this difference, we analyzed the involvement of the PDGFRβ family member c-KIT in Ki11502 effectiveness, but siRNA and proliferation studies in SW480 and DLD-1 cells could not prove the involvement of c-KIT inactivation during Ki11502 treatment. Hence, an RTK activation antibody array on SW480 cells led us to the identification of the non-receptor tyrosine kinase SRC, which is inactivated after Ki11502 treatment but not after the siRNA approach. Further studies using the SRC-specific inhibitor PP2 showed that SRC inhibition upon treatment with the inhibitor Ki11502 is responsible for the observed effects of Ki11502 in SW480 and DLD-1 CRC cells. In summary, our results demonstrate that the inhibition of PDGFRβ alone using siRNA has only moderate cellular effects in CRC cell lines; however, the multi-target inhibition of PDGFRβ, c-KIT and SRC, e.g., using Ki11502, represents a promising therapeutic intervention for the treatment of CRC.Oncotarget 07/2013; 4(7):1037-49. · 6.63 Impact Factor
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ABSTRACT: Atherosclerosis is the major underlying process of cardiovascular disease. Atherosclerosis commences with an initial pre-inflammatory phase of the trapping and accumulation of lipids in the vessel wall and this is followed by an inflammatory response. The trapping of lipids occurs via binding to proteoglycans, specifically biglycan, with hyperelongated glycosaminoglycan (GAG) chains. Models have been developed to study the aetiology as well as the effectiveness of medical and experimental interventions in preventing atherosclerosis. ApoE is an apolipoprotein associated with removal of lipids from peripheral tissues. Disruption of the ApoE gene in C57BL/6 mice produces mice which are ApoE-/- (ApoE deficient) and show elevated plasma lipids and accelerated development of atherosclerosis which is exacerbated on a high fat diet. These mice are widely used in studies of atherosclerosis. We investigated the possibility that changes in the size of biglycan might participate in the development of atherosclerosis in ApoE-/- mice. We prepared Aortic Smooth Muscle Cell (ASMC) cultures by the digestion technique from ApoE-/- and ApoE+/+ mice and studied the size of biglycan secreted by both cell types. The biglycan secreted by ASMCs for ApoE-/- mice was larger than that from ApoE+/+ ASMCs. The difference was not eliminated by treatment of cells with a high concentration of Platelet Derived Growth Factor (PDGF) or by treatment with the PDGF antagonist, imatinib. The size difference was however observed in the small free GAG chains (xyloside GAGs) secreted in cells supplemented with exogenous xyloside as a cellular assay of GAG synthesizing capacity. This result suggests that there is a hyperactivity of the fundamental GAG synthesizing capacity in the ASMCs from ApoE-/- mice. These data suggest that hyperelongated biglycan might be contributing to lipid accumulation in ApoE-/- mice used in studies of atherosclerosis and that some of the medical interventions might have an action to reverse this hyperelongation response.Clinical and Experimental Pharmacology. 01/2013; 3(3):125 - 130.