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

Vascular Cell

Mark Slevin⁷  Julio Secades³  Marta Grau-Slevin⁷  Norma Rovira¹  Donghui Liu²  Anna Planas⁵  Carlos Justicia⁵  Eugen Bogdan Petcu⁴  Raid Al-Baradie⁶  Sabine Matou-Nasri²  Manal Abudawood²  Jerzy Krupinski² 

[1] Cerebrovascular Diseases, Department of Neurology, Hospital Universitari Mútua Terrassa, Terrassa, Barcelona, Spain;School of Healthcare Science, Manchester Metropolitan University, Manchester, UK;Ferrer Grupo, Barcelona, Spain;School of Medicine, Centre for Medicine and Oral Health campus, Gold Coast Campus, Griffith University, Queensland, QLD, 4222, Australia;Department of Brain Ischemia and Neurodegeneration, IIBB-CSIC, IDIBAPS, Rossello 161, Barcelona, E-08036, Spain;College of Applied Medical Science, Almajmaáh University, P.O. Box 1405, Almajmaah, 11952, Kingdom of Saudi Arabia;Cardiovascular Research Center, Hospital de Sant Pau and IIB-Sant Pau, CiberOBN-Instituto de Salud Carlos III, Barcelona, Spain

关键词: IRS-1;    Ischaemia;    Stroke;    Apoptosis;    Angiogenesis;    Citicoline;   

Others  :  801931
DOI  :  10.1186/2045-824X-4-20

 received in 2012-09-10, accepted in 2012-11-27,  发布年份 2012

【 摘 要 】

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.

【 授权许可】

   
2012 Krupinski et al.; licensee BioMed Central Ltd.

【 图 表 】

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