AimsVascular calcification is a major cause of morbidity and mortality. Fibroblast growth factor-2 (FGF-2) plays an instructive role in osteogenesis and bone development, but its role in vascular calcification was unknown. Therefore, we investigated the involvement of FGF-2 in vascular calcification and determined the mechanism by which it regulates this process.Methods and resultsWe demonstrate that FGF-2 expression is increased in vascular smooth muscle cells (VSMCs) induced to deposit a mineralized matrix by incubation with β-glycerophosphate. FGF-2 is also localized to sites of calcification within human atherosclerotic plaques. The expression of syndecan-4, a heparan sulfate proteoglycan which regulates FGF-2 signalling, is also increased in mineralizing VSMCs and co-localizes with FGF-2 in human calcified atherosclerotic plaques. Exogenous FGF-2 inhibits VSMC mineralization, and this inhibition is reduced when syndecan-4 expression is knocked-down using siRNA. Biochemical inhibition of FGFR signalling using a pan FGFR inhibitor (BGJ398) or knocking-down syndecan-4 expression in VSMCs using siRNA increases VSMC mineralization. These increases are prevented by inhibiting transforming growth factor-β (TGFβ) signalling with SB431542, suggesting cross-talk between FGF-2 and TGFβ signalling is crucial for the regulation of VSMC mineralization. Syndecan-4 can also regulate FGF-2 signalling directly via protein kinase Cα (PKCα) activation. Biochemical inhibition of PKCα activity using Gö6976, or siRNA-mediated suppression of PKCα expression increases VSMC mineralization; this increase is also prevented with SB431542. Finally, the ability of FGF-2 to inhibit VSMC mineralization is reduced when PKCα expression is knocked-down.ConclusionThis is the first demonstration that syndecan-4 promotes FGF-2 signalling, and in turn, suppresses VSMC mineralization by down-regulating TGFβ signalling. Our discoveries that FGF-2 and syndecan-4 expression is increased in mineralizing VSMCs and that PKCα regulates FGF-2 and TGFβ signalling in VSMCs suggests that the syndecan-4/FGF-2/TGFβ signalling axis could represent a new therapeutic target for vascular calcification.

The main effector of the renin angiotensin system (RAS), angiotensin II (AngII), is a key mediator of this remodelling. AngII has previously been shown to upregulate microRNA-132 (miR-132) expression in rat VSMC which may contrib-3 Konstantinos Stellos.

(ROS) are known to play a central role in the tissue damage caused by ischaemia-reperfusion injury (IRI), yet the use of antioxidant supplements in large scale clinical trials has been shown to have no beneficial effect and indeed some studies have suggested that the addition of exogenous ROS may decrease infarct size. This suggests that mitochondria may exhibit a biphasic response to ROS, termed mitohormesis, characterised by beneficial effects at low doses and detrimental effects at high doses. To investigate this phenomenon, a tool is required to precisely titrate mitochondrial ROS. MitoParaquat is a novel mitochondria-targeted molecule that redox cycles at complex I to produce superoxide, closely mimicking the production of superoxide as the proximal ROS species in vivo. Here it is used to investigate the role of ROS in protection against acute myocardial IRI. Methods Male C57BL6/J mice aged 8-10 weeks were administered MitoParaquat or vehicle only control by intravenous injection 15 min before the induction of 30 min myocardial ischaemia by ligation of the left anterior descending coronary artery. After 2 hours of reperfusion, infarct size was determined by tripheyltetrazolium chloride staining. Results MitoParaquat decreased infarct size relative to vehicle only control (42.3±4.3%) at doses of 1 nmol (30.0±4.0%), 100 pmol (22.7±2.3%), and 10 pmol (24.2±2.0%). At 1 pmol, no significant difference from vehicle only control was observed (41.5±8.6%), and at 5 nmol it was found to be lethal. There was no significant difference in the area at risk between any groups. Conclusions First and foremost, the generation of low doses of exogenous ROS by MitoParaquat is shown to be protective against acute myocardial IRI in vivo. MitoParaquat is shown to exhibit a hormetic dose response curve, with protection conferred only in an intermediate dose range with high doses found to be lethal and infarcts from low dose not significantly different from control. Further work is required to determine the mechanism by which this cardioprotection occurs.