Abstract
Background and purposes - Citicoline is neuroprotective agent used in stroke patients. Citicoline induces angiogenesis i n vitro ; however, the molecular mechanisms induced by citicoline have not been fully investigated. The present study was designed to investigate the key signalling mechanisms through which citicoline modulates angiogenesis-associated with stroke recovery. Methods - In vitro angiogenesis assays: migration (Bodyen chamber, a chemotaxis assay and wound recovery), proliferation and differentiation into tube-like structures in Matrigel TM as well as spheroid development assays were carried out using human brain microvessel endothelial cells (HBMECs). Western blotting was performed on the protein extraction from HBMEC stimulated with citicoline. Citicoline signalling pathway was studied using a phospho-protein screening array performed by Kinexus. A Staurosporin/calcium ionophore-induced apoptosis assay was performed by seeding HBMEC on the glass coverslips. Cells were pre-incubated with citicoline and the apoptosis was induced by addition of the ionophore or staurosporin. Cells were examined by microscoy and/or stained using propidium iodide and Hoechst stain solution and then counted under fluorescence microscope. Transient MCAO was carried out on Sprague Dawley rats (n=4 per group) with and without citicoline treatment (1000mg/Kg) applied at the time of occlusion and subsequently every 3 days until euthanasia (21 days). Vascularity of the stroke-affected regions was examined with antibodies recognising the microvessels (CD34) and active cells (CD105). Results - Citicoline presented no mitogenic and chemotactic effects on HBMEC; however, it significantly increased wound recovery, the formation of tube-like structures in Matrigel TM -with a stronger effect than FGF-2, and enhanced spheroid development and sprouting. Citicoline induced the expression of ERK-1/2, known to be involved in last step of signalling pathways of angiogenesis. Kinexus results showed an over-expression of ASK-1, HER2, IRS-1 and Jun and inhibition of Hsp27, Integrin alpha4, MEK1 (MAP2K1) and Histone H2B proteins. Knock-down of IRS-1 with targeted siRNA inhibited the pro-angiogenic effects (sprouting and tube-formation) of citicoline in HBMECs. The percentage of surviving cells was higher in the presence of citicoline after inducing apoptosis. Histology of infarcted regions showed that citicoline significantly increased the numbers of new active (CD105-positive) microvessels. Conclusion - The findings demonstrate both a pro-angiogenic and protective effect of citicoline on HBMECs in vitro and following MCAO in vitro. Additionally, this molecule may play a key role in the modulation of angiogenesis thereby and ultimately, improving tissue function, revascularisation and neuronal survival after ischaemic stroke.
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