Citicoline induces angiogenesis improving survival of vascular/human brain microvessel endothelial cells through pathways involving ERK1/2 and insulin receptor substrate-1

Krupinski, J ORCID: https://orcid.org/0000-0002-5136-8898, Abudawood, M, Matou-Nasri, S, Al-Baradie, R, Petcu, EB, Justicia, C, Planas, A, Liu, D, Rovira, N, Grau-Slevin, M, Secades, J and Slevin, M ORCID: https://orcid.org/0000-0003-3767-4861 (2012) Citicoline induces angiogenesis improving survival of vascular/human brain microvessel endothelial cells through pathways involving ERK1/2 and insulin receptor substrate-1. Vascular Cell, 4 (1). p. 20. ISSN 2045-824X

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Abstract

BACKGROUND: Citicoline is one of the neuroprotective agents that have been used as a therapy in stroke patients. There is limited published data describing the mechanisms through which it acts. METHODS: We used in vitro angiogenesis assays: migration, proliferation, differentiation into tube-like structures in Matrigel™ and spheroid development assays in human brain microvessel endothelial cells (hCMEC/D3). Western blotting was performed on protein extraction from hCMEC/D3 stimulated with citicoline. An analysis of citicoline signalling pathways was previously studied using a Kinexus phospho-protein screening array. A staurosporin/calcium ionophore-induced apoptosis assay was performed by seeding hCMEC/D3 on to glass coverslips in serum poor medium. In a pilot in vivo study, transient MCAO in rats was carried out with and without citicoline treatment (1000 mg/Kg) applied at the time of occlusion and subsequently every 3 days until euthanasia (21 days). Vascularity of the stroke-affected regions was examined by immunohistochemistry. RESULTS: Citicoline presented no mitogenic and chemotactic effects on hCMEC/D3; however, it significantly increased wound recovery, the formation of tube-like structures in Matrigel™ and enhanced spheroid development and sprouting. Citicoline induced the expression of phospho-extracellular-signal regulated kinase (ERK)-1/2. Kinexus assays showed an over-expression of insulin receptor substrate-1 (IRS-1). Knock-down of IRS-1 with targeted siRNA in our hCMEC/D3 inhibited the pro-angiogenic effects of citicoline. The percentage of surviving cells was higher in the presence of citicoline. Citicoline treatment significantly increased the numbers of new, active CD105-positive microvessels following MCAO. CONCLUSIONS: The findings demonstrate both a pro-angiogenic and protective effect of citicoline on hCMEC/D3 in vitro and following middle cerebral artery occlusion (MCAO) in vivo.