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

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.

Cell culture

Human brain micro-vessel endothelial cell (EC) line named hCMEC/D3 was grown in endothelial basal medium-2 (EBM-2) medium supplemented with growth factors and hydrocortisone as described previously [9]. Cells were seeded into T25 flasks pre-coated with 0.1% collagen and maintained in a humidified 5% CO₂ atmosphere at 37°C. Every three days reaching the confluence, the cells were detached under the enzymatic activity of the trypsin then the cells in suspension were centrifuged for 5 min at 1300 rpm then seeded into new pre-coated T25 flasks. Throughout the study, the cells used were between passages 28 and 38. Cells were routinely cultured as described in our previously published work [10].

Staurosporin/ionophore-induced apoptosis assay

In this assay, glass coverslips were sterilized in a bath of 100% ethanol for 10–20 min then left to air dry. The coverslips were put in a 24-well plate and pre-coated with 500 μl of 0.1% collagen in acetic acid and then incubated for 1 h_. hCMEC/D3 were cultured in complete medium at a concentration of 5 × 10⁴ cells/ml on collagen pre-coated coverslips for 4 h-incubation. Then, the medium was replaced with serum-free medium and the cells were incubated. After 24 h incubation, the cells were pre-incubated with 10 μM citicoline for 4 h prior to apoptosis induction. After the pre-incubation with citicoline, apoptosis was induced using calcium ionophore (10 μM/24 h); or staurosporin: 10 μM/4 h (concentrations determined from pilot studies as optimal) or by exposure to oxygen-deprivation (12 h, 1% O₂; hypoxia confirmed by up-regulation of HIF-1α as determined in our pilot studies). These concentrations have previously been shown to induce apoptosis in about 40-90% of the cell population. For staining, one hour before the termination of the experiment, propidium iodide (PI; 10 μg/ml) was added in each well as an indicator of DNA damage. After 1 h, the medium was discarded and the cells were washed with PBS then fixed with 4% paraformaldehyde for 20 min at room temperature. Subsequently, cells were washed three times with PBS and exposed to 1 μg/ml Hoechst 6024 stain solution diluted in PBS at room temperature for 30 min. Finally, the cells were washed three times with PBS and one drop of FluorSave™ reagent was added on frosted glass slides and the coverslips were put upside down on the drops. An average of six fields at x200 of magnification was photographed per coverslip using an Axivoert fluorescence microscope. The apoptotic index is expressed as the number of apoptotic cells relative to the total number of cells (% apoptotic cells). In this experiment, triplicate wells were run for each condition with controls consisting of untreated cells.

Angiogenesis assays: cell proliferation

hCMEC/D3 cells were seeded at a concentration of 8 × 10⁴ cells/ml in 500 μl of complete basal medium in each well of 24-well plate. After 4 h, the medium was changed to serum-poor medium (SPM) containing 1% FBS containing different concentrations of citicoline (1 μM, 10 μM and 100 μM; NOTE; pilot experiments were carried out using 1-100 μM citicoline and optimized for the use of 10 μM subsequently as this produced the most prominent responses). After 72 h incubation, cells were washed with PBS and detached with trypsin. Cells were counted in a Coulter counter at least three times for each well. In this experiment, cells were treated in triplicate for each experimental condition. Cell migration (Boyden Chamber: chemotaxis assay)- Inserts of Transwell Costar® porous membranes were coated with 0.1% collagen and left for air dry in a 24-well plate. (Wound recovery)-cells were seeded onto plastic coverslips and when confluent, were scraped with a razor to induce straight lesions. Cells were seeded in 100 μL of serum- poor medium containing 1% FBS at a concentration of 7.3 × 10⁴ cells/ml. The inserts containing hCMEC/D3 were placed in a 24-well plate containing 500 μl of serum poor medium containing 1% FBS and supplemented with either citicoline (10 μM/ml) or FGF-2 (25 ng/ml), used as a positive control. Cells were treated in triplicate for each experimental condition. (Boyden chamber)-After 24 h incubation, the medium was removed from the insert and the cells which had migrated through the pores to the bottom side of the insert were fixed with 4% paraformaldahyde. Cotton swabs were soaked with PBS and used in order to remove the cells that did not migrate. After fixation, the migrated cells were stained with Giemsa (3 minutes). An optical microscope was used to count randomly five microscopic fields from each insert. (Wound healing)-after 24 h the plastic coverslips were fixed in paraformaldehyde and cell migration examined under a microscope at low power. Tube-like structure formation in Matrigel- EC (2 × 10⁶ cells/ml) were mixed with equal volume of growth factor-reduced Matrigel™ containing citicoline (10 mM) or fibroblast growth factor-2 (FGF-2) (25 ng/ml). Any material in the contact with the gel was cold to avoid the gel polymerization. In a 48-well plate, a total amount of 35 μl of this mixture was placed in a tear-like drop in the middle of the well and left to polymerize for 1 h incubation. Then, 0,5 ml of complete medium was added into each well. After 24 h incubation, 4% paraformaldahyde was added to fix the endothelial tube-like structures embedded in the gel. Five areas from each well were counted under optical microscope. Spheroids were produced by inoculation of centrifuged endothelial cells into matrigel with or without addition of citicoline or FGF-2 as a positive control and their development including sprout formation monitored as described above over a period of 7 days. At least 10 sprouts were measured for each condition and the experiments repeated at least 3 times.

Protein extraction and western blotting

EC (3 × 10⁵ cells/ml; 2 ml) were seeded in complete medium in 6-well plate and incubated for 48 h. Then, the medium was replaced with SPM containing 1% FBS. After 24 h incubation, the cells were stimulated with citicoline at 1-10-50 μM for 10 min then the cells were immediately washed with 1 ml of PBS and gently lysed on ice in 50 μl of ice-cold homogenized lysis buffer (PH 7.2). The cells then were scraped, the cell lysate proteins collected and transferred into 0.5 ml micro-centrifuge tubes. Samples were sonicated four times for 10 seconds each time with 10–15 second intervals on ice to rapture the cells and to shear nuclear DNA. Cell lysates were centrifuged at 20,000 g for 20 min and the protein concentration was determined using the BioRad protein assay.

In our preliminary un-published studies, we showed that citicoline also strongly induced angiogenesis in our bovine aortic EC (BAEC) and for this reason we performed a phospho-site protein expression screen using Kinex™ antibodies microarray KAM1.3 and performed by Kinexus (Bioinformatics Corporation, Vancouver, Canada). The most relevant phospho-protein expression up- or down-regulated by citicoline were confirmed by Western blotting (IRS-1, HER2 and Histone H2B; see additionally supplied data). Since phosphorylation of IRS-1 was most apparent when translated to our hCMEC/D3 cells, we investigated its role in intracellular signalling in more detail here. Standard siRNA transfection (using lipofectamine) was employed to induce a transient down-regulation of IRS-1 RNA (approximately 85-90%-data not included; Figure 1B and C). After 24 h exposure to siRNA or scrambled sequences, cells were used for analysis of tube-like structure formation described previously. The experiment was repeated twice in triplicate wells and a representative example shown.

Figure 1   ( Jump back to reading ◀ )

Transient rat MCAO

Four male Wistar rats (Charles River) were used in each group (control and citicoline treated), with a weight of 280-320 g at the beginning of the study. Animals were kept on a 12/12 hours light/darkness cycle, with access to food and water ad libitum.

The ischemic lesion was induced by transient (90 minutes) occlusion of the right middle cerebral artery (MCAO), using the intraluminal thread occlusion method, described elsewhere [11], under isofluorane anesthesia. The occlusion period and the successful reperfusion of the right MCA were controlled by continuous recordings of ipsilateral laser-Doppler flowmetry (LDF). Animals were intraperitoneally treated with either citicoline (1000 mg/Kg) or saline. Drugs were daily administered until day 7. The first dose was given 15 minutes before reperfusion. 21 days after occlusion animals were anesthetized and perfused through the heart with heparinized saline, followed by 4% paraformaldehyde. Brains were cryoprotected with 30% sucrose, frozen at −40°C and 8-μm thick coronal sections were cut in a cryostat. 1, 7 and 21 days after occlusion rats were anesthetized and MRI T2-weighted images were acquired at 7 T (Bruker BioSpin), in order to study evolution of lesion volume. There was no mortality in this experimental group. Procedures followed were in accordance with institutional guidelines.

Immunohistochemistry

Immunohistochemical staining was used to determine the relative numbers of microvessels (anti-CD31) and active vessels (anti-CD105) in control and citicoline treated stroked brain tissue after 21 days (n = 5). The mean number of vessels from five areas of stroke-affected tissue was counted microscopically (× 20) in eight sections per animal. Double immunofluorescence was used to assess the distribution of the phospho-protein IRS-1 in relation to active microvessels in the stroke region (CD105/endoglin mouse monoclonal antibody). After incubation with primary antibodies for 1 h at room temperature (1:100), sections were washed and then incubated with the appropriate secondary antibodies (1:50) − fluorescein isothiocyanate-conjugated sheep anti-mouse IgG (Jackson) or tetramethylrhodamineisothiocyanate-conjugated rabbit anti-goat (Jackson). Images were captured with Nikon 80i Digital Microscope using Nis Elements 3.21 software with multichannel capture option. Negative control slides were included where the primary antibody was replaced with PBS.

Statistical analysis

All in vitro experiments were performed at least three times and the results are expressed as the means ± S.D. Section analysis by immunohistochemistry involved counting the numbers of microvessels from 5 regions within the infarcted tissue and from 3 separate sections (1, 5, and 10 from serial sections) for each condition. Statistical significance was tested by Student’s t-test and data were considered significant when p ≤ 0.05.

Citicoline protects hCMEC/D3 against cell damage/apoptosis

EC were pre-incubated with 10 μM citicoline for 4 h. Then, the cells were incubated with 1-10 μM staurosporin in DMSO for 4 h and 24 h, respectively. These concentrations were chosen in order to identify appropriate levels of cell damage/apoptosis and were classified as preliminary studies. In these experiments, the numbers of apoptotic cells were approximately 40% and 60% at 10 μM (4 h and 24 h respectively) (data not shown). Therefore, 10 μM/4 h was the chosen treatment in order to evaluate whether citicoline could protect the cells against cell death pathways. Pre-treatment of hCMEC/D3 cells with citicoline significantly decreased the number of damaged/apoptotic cells, determined by counting the numbers of PI-positive nuclei at the end of the experiment, under these conditions (Figure 1A; P < 0.05). Since hypoxia is a key feature of acute ischaemic stroke and a major activator of cell apoptotic pathways, we examined its effect on our EC in the presence of citicoline (10 μM/4 h pre-incubation). Again, the presence of citicoline in the media significantly protected the cells against apoptosis/cell damage (Figure 1B; p < 0.05). Increased expression of and release of Ca²⁺ Kainic acid and glutamate also leads to increased cell death after stroke. Ionophore treatment of EC is known to induce Ca influx and rapid dephosphorylation of eNOS at Thr495 resulting in eNOS activation. For this reason, we exposed our EC to the Ca ionophore A23187 (10 μM/24 h), after pre-treatment with citicoline. Figure 1C shows that the Ca ionophore caused significant reduction in survival of cells after 24 h. However in the presence of citicoline, the effects were reversed and the cells remained attached and morphologically identical to control-untreated cells (p < 0.05). The possible mechanism through which citicoline provides this protection is discussed later in this results section.