In this study, we have, for the first time, demonstrated both a vascular protective, and pro-angiogenic effect of citicoline using in vivo and in vitro models. Here we show the strong protective effect of citicoline on brain microvascular EC, demonstrated by the ability of citicoline to protect against either calcium ionophore or hypoxia-induced cell damage/apoptosis identified by nuclear uptake of propidium iodide. Previous studies have shown that citicoline was able to protect motor neurons of organotypic rat lumbar spinal cord cultures, from apoptosis following administration of the excitotoxic agent DL-threo-β-hydroxyaspartate (a glutamate analogue) . Similarly, citicoline with or without hypothermia, protected neurons against apoptosis in a rat model of transient focal MCAO through a mechanism including the modulation of Bcl-2, Bax and caspase-9 . In other cell types/sources, citicoline protected against cell damage from Kainic acid in retinal neurons  and indirectly, may protect lung adenocarcinoma cells (A549) against apoptosis [16, 17]. No studies until now have been carried out using EC. Our data suggests a strong protective effect against the damaging process of excitotoxicity and hypoxia, similar to that experienced after acute ischaemic stroke. In regard to the possible mechanism? Our kinexus phospho-protein screen identified a tenfold reduction in expression of Histone H2B (serine 14). Phosphorylation of histone H2B at serine 14 (S14), a posttranslational modification required for nuclear condensation, correlates with cells undergoing programmed cell death in vertebrates . The authors of this paper also identified a 34 kDa apoptosis-induced H2B kinase as caspase-cleaved Mst1 (mammalian sterile twenty) kinase. Mst1 can phosphorylate H2B at S14 in vitro and in vivo, and the onset of H2B S14 phosphorylation is dependent upon cleavage of Mst1 by caspase-3. These data reveal a histone modification that is uniquely associated with apoptotic chromatin and provide insights into a previously unrecognized physiological substrate for Mst1 kinase. Further experiments are needed to confirm that these findings represent a key novel mechanistic pathway for EC protection associated with citicoline treatment.
In addition, although citicoline had no effect on the chemotaxis of HBMEC/D3 determined by using the Boyden chamber method it significantly increased the number of migrating cells in the scratch wound healing assay. Citicoline also significantly increased the formation of tube-like structure in Matrigel producing a stronger effect than the known mitogenic factor FGF-2 (p <0.05). Although citicoline had no mitogenic and chemotactic effects on EC, it had a significant effect on cell differentiation and migration which are two of the key steps of the angiogenic process. This may be an extremely valuable novel finding in regard to understanding the potential mechanisms through which citicoline treatment results in patient recovery, since both protection of EC and induction and maintenance of angiogenesis is key to both short-term and chronic re-vascularization after stroke impacting indirectly but significantly also on neuronal survival and re-integration .
Western blotting demonstrated that citicoline induced pERK1/2 expression, a key mitogenic signalling protein known to be involved in angiogenesis and generally stimulated by growth factors through interaction with their receptors . This data demonstrated the potential of citicoline to activate intra-cellular signal transduction pathways and induce phosphorylation of down-stream angiogenic molecules; hence we investigated this ability in more detail by analysis of the Kinexus-phospho-protein Western screen following treatment of vascular EC with citicoline. Interestingly, treatment with citicoline modified the expression of only several of the >500 proteins on the array showing a degree of specificity. IRS-1 and Her2 were both phosphorylated in the presence of citicoline. These two proteins have not been implicated in stroke recovery pathways or stroke angiogenesis until now.
Here, we went on to demonstrate the importance of IRS-1 in mediating the angiogenic effects of citicoline in vitro, and further showed localization of p-IRS-1 in the vascular regions of peri-infarcted tissue of animals 21 days after MCAO but only following treatment with citicoline. The vessels were nearly always thin-walled neo-vessels with minimal or no pericyte coverage and CD105-positive suggesting dynamic activity and contribution to the re-vascularisation process.
Only recently, IRS-1 over-expression was attributed to increased angiogenesis in human EC in association with increased Akt and VEGF-A expression , whilst in vivo, antisense IRS-1 sequences delivered by sub-conjunctival injection inhibited rat corneal neovascularisation , and when delivered by means of eye-drops (GS-101) were found to be tolerable in a phase-1 clinical trial and may be sufficient to prevent neovascularisation in disease such as retinopathy and neovascular glaucoma . Therefore, IRS-1 represents a potent modulator of pro-angiogenic signalling cascades in vascular EC and as such, since we have shown both in vitro, and in the rat model of temporary MCAO that citicoline induces phosphorylation of IRS-1 and concomitant EC activation and increased vascularisation, this could be a key novel mechanism of action of citicoline.
A further interesting finding was the up-regulation of HER2 by citicoline in our vascular ECs in vitro. Although we have not investigated this in more detail within this piece of work or in our hCMEC, HER2 has previously been shown to be important in promotion of angiogenesis and concomitant tumour breast tumour growth , and is strongly implicated in activation of intracellular signalling pathways increasing the expression of VEGF and IL-8 . Therefore, further investigation is warranted to determine its importance in the induction of citicoline-associated angiogenesis and vascularisation after ischaemic stroke.