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

Jerzy Krupinski123*, Manal Abudawood3, Sabine Matou-Nasri3, Raid Al-Baradie4, Eugen Bogdan Petcu5, Carlos Justicia6, Anna Planas6, Donghui Liu3, Norma Rovira1, Marta Grau-Slevin7, Julio Secades8 and Mark Slevin37

Author Affiliations

1 Cerebrovascular Diseases, Department of Neurology, Hospital Universitari Mútua Terrassa, Terrassa, Barcelona, Spain

2 Department of Medicine, University of Manchester, Manchester, UK

3 School of Healthcare Science, Manchester Metropolitan University, Manchester, UK

4 College of Applied Medical Science, Almajmaáh University, P.O. Box 1405, Almajmaah, 11952, Kingdom of Saudi Arabia

5 School of Medicine, Centre for Medicine and Oral Health campus, Gold Coast Campus, Griffith University, Queensland, QLD, 4222, Australia

6 Department of Brain Ischemia and Neurodegeneration, IIBB-CSIC, IDIBAPS, Rossello 161, Barcelona, E-08036, Spain

7 Cardiovascular Research Center, Hospital de Sant Pau and IIB-Sant Pau, CiberOBN-Instituto de Salud Carlos III, Barcelona, Spain

8 Ferrer Grupo, Barcelona, Spain

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Vascular Cell 2012, 4:20  doi:10.1186/2045-824X-4-20

Published: 10 December 2012



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.


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.


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.


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.

Citicoline; Angiogenesis; Apoptosis; Stroke; Ischaemia; IRS-1