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    Role of citicoline in modulation of angiogenesis and apoptosis in vascular/human brain microvessel endothelial cells

    Alshammari, Dina (2014) Role of citicoline in modulation of angiogenesis and apoptosis in vascular/human brain microvessel endothelial cells. Doctoral thesis (PhD), Manchester Metropolitan University.


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    Background and purposes: Citicoline, also known as CDP-choline (cytidine-5-diphosphocholine), is a naturally-occurring endogenous nucleoside, which is one of the neuroprotective drugs that have been used as a therapy in stroke patients. However, the mechanisms through which it acts are not fully understood. In terms of analysis of the signalling mechanisms associated with citicoline-induced protection, it has been previously shown that citicoline may protect the ischemic neurons by suppressing caspase apoptotic pathway activation. Moreover, preliminary in vitro studies have shown that citicoline induces angiogenesis (the formation of new blood vessels from pre-existing capillaries). However, the possible beneficial effects of citicoline treatment on revascularization and angiogenesis after stroke have not been fully examined. The present study was designed to investigate the key signalling mechanisms through which citicoline modulates apoptosis and angiogenesis-associated with stroke recovery. Methods: An analysis of citicoline signalling pathway was studied from phospho-protein screening array done by Kinexus. In vitro angiogenesis assays: migration, proliferation and differentiation into tube-like structures in Matrigel™ ™ assays, have been used in human brain microvessel endothelial cells (hCMEC/D3). Western blotting was performed on protein extraction from hCMEC/D3 stimulated with citicoline. Analysis of apoptosis by flow-cytometry in hCMEC/D3. A hypoxia induced apoptosis assay was performed by seeding hCMEC/D3 on to glass coverslips in serum poor medium. Quantification of apoptotic cells were carried out under fluorescence microscopy using a combination of propidium iodide and DAPI stain solution. Apoptotic pathways in hCMEC/D3 stimulated with citicoline were examined by indirect immunofluorescence and real time PCR. Pharmacological inhibitor of Her2 (GW2974) was used to investigate the angiogenic signalling pathway by western blotting and Matrigel™ assay in hCMEC/D3 in the presence or absence of citicoline. Results: 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 Ser14 proteins. Citicoline induced EC migration and differentiation in poly-l-lysine and Matrigel™ . Using microarray screening, the Histone H2B (Ser14) appeared to be the main phosphor-protein expression blocked by citicoline in hCMEC/D3, and the expression of tyrosine-protein kinase erbB-2 receptor (Her2) appeared to be induced by citicoline in hCMEC/D3. Treatment with the Her2 inhibitor (GW2974) totally blocked citicoline induced endothelial tube formation in EC, whereas treatment with GW2974 in combination with FGF-2 did not affect FGF-2 induced endothelial tube formation. In cultured hCMEC/D3 treatment with GW2974 inhibited citicoline induced phosphor-Erk expression, whereas treatment with GW2974 in combination with FGF-2 did not affect FGF-2 induced phosphor-Erk expression. However, Citicoline had no mitogenic effects on hCMEC/D3. Phspho-Caspase-3 and phosphor-H2B (Ser14) expression were inhibited by citicoline in hCMEC/D3 whereas the expression of phosphor-Her2 and phosphor-Erk expression were increased. Moreover, citicoline treatment showed a decrease in number of apoptotic cells (positive PI staining) in hypoxia induced apoptosis compared to untreated cells. In cell migration assay, treatment with citicoline significantly increased cells migration in hCMEC/D3 compared to untreated cells on hypoxia conditions. Detection of apoptotic cells by flow cytometry showed inconclusive results in both treated and untreated hCMEC/D3. Results from indirect immunofluorescence showed a significant increase in active Caspase-3 and H2B (Ser14) expression in citicoline treated cells in comparison with untreated cells in hypoxia conditions. Results from PCR showed inhibition of pro-apoptotic genes including BNIP3, BNIP3L, caspase 4, caspase 8, caspase 9, CIDEB, DFFA, LTBR, TP53BP2, TRADD, and TRAF3 with citicoline treatment. They also show an over-expression of a number of anti-apoptotic genes including NAIP, NOD1, TNFRSF25, and TP53. Conclusions: A screening of phosphor-protein expression revealed that citicoline specifically over-expressed Her2 which demonstrated that citicoline plays a key role in Her2 induced angiogenesis. Blocking of Her2 pathway inhibited the formation of tube-like structures in citicoline treated cells and therefore citicoline induces angiogenesis through Her2 pathway, and that is important in terms of understanding the molecular pathway in which citicoline acts as a pro-angiogenic molecule in tissue remodelling after stroke. Citicoline decreased active caspase-3 and H2B (Ser14) expression, and positive PI staining which demonstrates a protective effect of citicoline against endothelial cells apoptosis. Citicoline also improved cell surivial by decreasing the expression ofBNIP3, BNIP3L, caspase 4, caspase 8, caspase 9, CIDEB, DFFA, LTBR, TP53BP2, TRADD, and TRAF3 genes and inducing the expression ofNAIP, NOD1, TNFRSF25, and TP53. Citicoline treatment significantly promotes wound healing in stroke mimicking conditions (hypoxia). Thus, the therapeutic properties of citicoline has the potential to promote vessel formation whilst reducing the risk cell death from hypoxic stress following ischemic stroke.

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