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Physiopathology
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User name	Brice Targat
Email	targat@ihes.fr
Phone / Fax number	+33(0)160926665 / +33(0)160926609
Location	IHES (Systems Epigenomics Group) - 35 route de Chartres - 91440 Bures sur Yvette, France

Scientific description

In sickle cell disease (SCD) hemoglobin S (HbS) polymerization renders red blood cells (RBC) both fragile and rigid and accounts for anemia and vasoocclusive crises (VOC). Abnormal RBC adhesion to vascular endothelial cells (VEC), in a context of chronic inflammation, cell activation and vascular tone abnormalities, is a major event in triggering VOC. Hydroxyurea (HU) is the only drug with a proven efficiency at decreasing VOC occurrence. HU decreases HbS polymerization and RBC adhesion. We studied HU effect on the other cellular partner of adhesion, i.e.VEC. HU-induced TrHBMEC transcriptome variations were analyzed by micro-arrays both in basal and pro-inflammatory conditions after 24h and 48h of treatment. Among the endothelial HU target genes we focused on those related to adhesion and inflammation phenomena. HU had no impact on adhesion genes as a whole, still expression of VCAM-1, a key adhesion receptor, was decreased. In contrast, HU had a significant effect on the inflammation gene cluster. It stimulates pro-inflammatory genes such as IL-1A, IL-1B, IL-6, IL-8, CCL2, CCL5 and CCL8 both at the mRNA and protein levels and also in HPMEC and HUVEC primary cells. This may suggest that HU increases inflammation in SCD patients to a threshold engaging an anti-inflammatory response.
Keywords: Treated TrHBME cell line

Technical description

Cell culture
For microarray experiments, we used the TrHBMEC line (Transformed Human Bone Marrow Endothelial Cells), a kind gift by B. Weskler [22]. Cells were cultured as previously described [18]. The use of TrHBMEC was restricted to cells between passage numbers 19 and 23.
For real-time quantitative PCR (RQ-PCR) experiments, we used primary cells between passage 3 and 8: HUVEC (Human Umbilical Vein Endothelial Cells), isolated from umbilical cords as previously described [23], and cultured in Endothelial Cell Growth Medium (PromoCell, Heidelberg, Germany); and HPMEC (Human Pulmonary Microcirculation Endothelial Cells) (PromoCell) cultured in Endothelial Cell Growth Medium MV2 (PromoCell).
All cells were grown to 90% of confluence and then exposed to a HC treatment combined or not with a treatment by pro-inflammatory cytokines (TNFÎ± and IFNÎ³) for 24h or 48h periods for the microarray assays and for 5h, 10h, 16h, 24h, and 48h for the RQ-PCR validation. HC (Sigma-Aldrich, St Louis, USA) was used at 250Î¼M (final concentration) and TNFÎ± and IFNÎ³ (R&D systems, Abingdon, UK) at 100U/ml.

RNA isolation for microarray experiments
RNA was isolated by the acid guanidinium thiocyanate (GNTC)-phenol-chloroforme extraction protocol. The protocol was slightly modified from the initial method proposed by Chomczynski[24]. All the centrifugation steps were realized at 4Â°C at 14,000 rpm. Cells were lyzed with 650Î¼l of GNTC (4M) with 7Î¼l/ml of Î² mercapto-ethanol. Then, one volume of water-saturated phenol with sodium citrate (0.1M, pH=4) and 0.3 volume of chloroform/isoamyl-alcohol (49:1, v:v) were added, successively. After homogenization by vortexing, the mixture was incubated at 4Â°C for 15 min and centrifuged for 20-30 min. The upper phase was collected, mixed with one volume of isopropanol, and incubated overnight at -20Â°C. The sample was then centrifuged for 20 min. The pellet was kept, washed with 70% ethanol, and centrifuged for 5-10 min. It was then solubilized with 30Î¼l of RNase-free H2O and a DNase I digestion step was realized (Qiagen, Hilden, Germany). The following day, the same protocol was carried out once more with an additional step: 620Î¼l of GNTC were added, and the aqueous phase (upper) was treated with chloroform/isoamyl-alcohol alone to avoid any phenol contamination. After vortexing and centrifugation, total RNA was precipitated by one volume of isopropanol overnight at -20Â°C. Then, the sample was centrifuged for 20 min at 14,000 rpm at 4Â°C. The pellet was washed with 70% ethanol, and centrifuged for 5-10 min. Finally, the RNA pellet was solubilized in 30Î¼l RNase-free water.
Quality and quantity of the isolated total RNA was determined using an Experion Analyzer (Biorad, Hercules, CA) as well as standard spectrophotometry.

Microarray technology
Transcriptome profiles were acquired in form of technical and biological replicates using an Applied Biosystems 1700 Genome Survey Microarray platform (Applied Biosystems, Forster City, USA). This platform has been demonstrated to cover an increased signal dynamic range, display higher sensitivity, and provide more robust gene expression estimates when compared to the leading competing technologies due to its chemiluminescence-based detection chemistry [25]. All the experimental data were generated using the Human Genome Survey (HGS) arrays (Version 1 and 2) which measure gene expression levels of 29,918 validated human genes.

RNA labeling, hybridization, and detection
RNA labeling, hybridization, and detection were done following the protocols supplied by Applied Biosystems together with the corresponding kits. 15-20Î¼g of total RNA sample were first subjected to chemiluminescence RT labeling. Labeled cDNAs were then hybridized and detected according to the supplied protocols. For each treatment condition, at least two RNA samples from two independent cell cultures were analyzed in at least two independent technical replicates.

Data preprocessing and primary analysis
The Applied Biosystems Expression Array System Software v1.1.1. was used to acquire the chemiluminescence and fluorescence images and primary data analysis. Additionally, we renormalized the resulting data according to the median, once more, after having removed probes for which the Applied Biosystems Software has set flags equal to or greater than 212, indicating compromised or failed measurements, as well as signals from control spots.

Standard transcriptome analysis
Subtraction profiles were calculated according to standard procedures with the following modifications: data for technical replicates were averaged with weights anti-proportional to their coefficient of variance estimates. Biological replicates from different biological conditions were compared in an "everyone-against-everyone" scheme and log2 fold-change estimates ("logQ", "L") where then determined as averages of weighted individual logQ values. The weights were anti-proportional to the variance over the individual logQ values. For these inter-assay comparisons, the NeONORM [26] method was used for normalization with k=0.2. P-values were determined based on a normal distribution hypothesis of signal intensities using standard ANOVA methods. Multiple probes for a single gene, cross-reactivity of a single probe to several genes, as well as the resolution of probe-ID annotations was done according to the standards defined previously [27].

Pathway analysis
In a first approach, we focused our pathway analysis on genes related to inflammation and cellular adhesion, the two phenomena in which VECs are clearly key actors in SCD. Thus, we tested whether or not genes associated with these two phenomena were statistically significantly enriched or deprived within the four groups of HC-sensitive genes identified above. To this end we distributed the genes represented on the HGS microarrays into three distinct, non-overlapping groups based upon the GO/Kegg/PANTHER gene annotations: (i) a group of 372 genes related to inflammation, (ii) a group of 1,232 genes related to cellular adhesion, and (iii) a random set of genes representative of the remainder genes represented on the array. This distribution was based on the GO terms for "molecular function" and "biological process" and the lists of the genes composing the two first groups are given as Supplementary Data Files 5&6. P-values for enriched and deprived-representation of genes of pathways were calculated using a binominal distribution.

Reference

Laurance S, Pellay FX, Dossou-Yovo OP, Verger E, Krishnamoorthy R, Lapoumeroulie C, Benecke A, Elion J (2010) Hydroxycarbamide stimulates the production of pro-inflammatory cytokines by endothelial cells: Relevance to sickle cell disease. Pharmacogenetics & Genomics 20(4):257-268.