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

Abstract

BackgroundCiticoline 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.MethodsWe 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.ResultsCiticoline 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.ConclusionsThe 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 and Vitamin B12 Eye Drops in Type 1 Diabetes: Results of a 3-year Pilot Study Evaluating Morpho-Functional Retinal Changes

“…Nevertheless, studies using in vivo and in vitro models support that citicoline has a potential vascular protective effect in the brain microvascular endothelium [33], and the mechanism of its action involves protection against cell damage/apoptosis induced by calcium ionophore or hypoxia. Citicoline was an effective treatment in other neurodegenerative conditions such as Alzheimer disease, amyotrophic lateral sclerosis, stroke, and Parkinson disease [34][35][36], while in the case of DR, it showed potential effects in reduction of impairment of the entire neurovascular unit [22].…”

Apelinergic system in endothelial cells and its role in angiogenesis in myocardial ischemia

“…A link between AMPK and apelin was reported also for energy metabolism in adipose tissue [84] and myocardium [85] while involving additional proteins, such as insulin receptor substrate-1 (IRS-1) [85]. IRS-1 suggests itself potentially to be involved in the apelin-AMPK pathway in ECs, as it had been described to be expressed in ECs [86] and has an ability to mediate angiogenic effects [87].…”

Citicoline affects serum angiostatin and neurospecific protein levels in patients with atrial fibrillation and ischemic stroke

“…Nevertheless, there are limited published reports describing the molecular mechanisms through which it acts, and reliable molecular markers of citicoline action remain undiscovered. The data of in vitro studies [36] have shown that citicoline protects human brain microvascular endothelial cells (hCMEC/D3) against apoptosis and excitotoxic damage, strongly induces angiogenesis and significantly increases vascularisation in stroke affected regions of rats following the model of middle cerebral artery occlusion (MCAO) through a signalling pathway involving activation of the insulinreceptorsubstrate1. Another study [25] indicates that CDPcholine improves functio nal recovery after permanent MCAO in association with reductions in lesion volume, cell death and low density lipoprotein receptorrelated protein (LRP) expression.…”