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Title: The role of prenylation in vascular calcification
Author: Ponnusamy, Arvind
ISNI:       0000 0004 5352 7391
Awarding Body: University of Manchester
Current Institution: University of Manchester
Date of Award: 2014
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Vascular calcification is an active, regulated process in which vascular smooth muscle cells (VSMCs) differentiate into osteoblast-like cells and deposit a mineralised matrix. It is well established that patients with chronic kidney disease and diabetes have poor cardiovascular outcomes which is thought to be due to increased vascular calcification and atherosclerosis. Previous work from the host laboratory has shown that nitrogen-containing bisphosphonates attenuate vascular calcification by inhibiting farnesyl pyrophosphate synthase, depleting cells of farnesyl pyrophosphate and geranylgeranyl pyrophosphate which are essential for the prenylation and activation of small GTPases such as Ras and Rho. Therefore, the initial aim of this study was to determine the effects of farnesyl transferase inhibitors (FTI-277 and manumycin A) on vascular calcification using a well validated in vitro model in which VSMCs are induced to deposit a mineralised matrix in the presence of β-glycerophosphate. FTI-277 significantly inhibited β-glycerophosphate-induced calcification of VSMCs in vitro (p<0.001), and this inhibition occurred in a dose- and time-dependent manner. In contrast, manumycin A had no effect on this process. Active Ras pull-down assays and western blotting confirmed that both FTI-277 and manumycin A decreased Ras GTPase activation in VSMCs. Western blot analysis also showed that FTI-277, but not manumycin A, increased Akt phosphorylation in these cells. To determine whether FTI-277 inhibited mineralisation by promoting PI3K/Akt signalling, VSMCs were induced to mineralise in the presence of FTI-277 plus either wortmannin (a PI3K inhibitor), SH6 (an Akt inhibitor) or vehicle alone. These reagents decreased the inhibitory effect of FTI-277 on mineral deposition by VSMCs, demonstrating that the effects of FTI-277 can be negated, at least partially, by preventing downstream PI3K or Akt signalling. Incubation of VSMCs with wortmannin alone significantly promoted mineral deposition by VSMC (p<0.001). In addition, FTI-277 significantly inhibited phosphate-induced apoptosis of human VSMC (p<0.05), and prevented PDGF-induced VSMC migration. FTI-277 also inhibited β-glycerophosphate-induced Runx2, Msx 2 and alkaline phosphatase mRNA expression (p<0.05), promoted matrix Gla protein mRNA expression (p<0.05), and maintained α-smooth muscle actin expression. Using an ex vivo assay, it was also demonstrated that FTI-277 inhibits phosphate-induced mineralisation of aortic rings from rats with early and end stage renal disease and sham-operated controls. To determine the effects of more specific Ras inhibition on VSMC mineralisation, Farnesyl Thiosalicylic Acid (FTS) was used. FTS (40 µM) inhibited mineralisation (p<0.001), decreased Ras GTPase activation and increased Akt phosphorylation but inhibited Erk phosphorylation in vitro. However lower doses of FTS increased mineralisation (p<0.001), but had no effect on Ras activation, or on Akt or Erk phosphorylation. FTS also appeared to delay PDGF-induced VSMC migration and induce apoptosis of human VSMCs in the absence of serum. Ex vivo studies demonstrated that FTS (40 µM) inhibited phosphate induced mineralisation of aortic rings from rats with end stage renal disease and sham-operated controls. This study demonstrates that farnesylation, or Ras specifically, could be a potential novel therapeutic target for the inhibition of vascular calcification.
Supervisor: Canfield, Ann; Kalra, Philip Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available