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Hydroxyurea stimulates production of pro-inflammatory cytokines in endothelial cells: Relevance to sickle cell disease.

ABSTRACT: 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

ORGANISM(S): Homo sapiens

PROVIDER: GSE11372 | GEO | 2009/04/09

SECONDARY ACCESSION(S): PRJNA106597

REPOSITORIES: GEO

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Project description:Effects of the matricellular protein CCN1 on macrophage inflammatory gene expression was tested in a macrophage cell line I-13.35. CCN1 (CYR61) is a matricellular protein that is highly expressed at sites of inflammation and wound repair. In these contexts, CCN1 can modify the activities of specific cytokines, enabling TNF-alpha to be cytotoxic without blocking NFkB activity and enhancing the apoptotic activity of FasL and TRAIL. Here we show that CCN1 supports the adhesion of macrophages through integrin alphaMbeta2 and syndecan-4, activates NFkB-mediated transcription, and induces a pro-inflammatory genetic program characteristic of classically activated M1 macrophages that participates in Th1 responses. The effects of CCN1 include upregulation of cytokines (TNFa, IL-1a, IL-1b, IL-6, IL-12b), chemokines (MIP-1a, MCP-3, Gro1, Gro2, IP-10), regulators of oxidative stress and complement (iNOS, C3), and downregulation of specific receptors (TLR4, IL-10rb) and anti-inflammatory factors (TGF-b1). CCN1 regulates this genetic program through at least two distinct mechanisms: an immediate-early response resulting from direct activation of NFkB by CCN1, leading to the synthesis of cytokines including TNFa and IP-10; and a delayed response resulting from CCN1-induced TNFa, which acts as an autocrine/paracrine mediator to activate the expression of other cytokines including IL-1b and IL-6. These results identify CCN1 as a novel component of the extracellular matrix that activates pro-inflammatory genes in macrophages, implicating its role in regulating macrophage function during inflammation. Macrophage cell line I-13.35 (from Tlr4-defective C3H/HeJ) was treated with purified recombinant CCN1 protein for 6 hr. Total RNA was isolated, converted to cRNA probes, and hybridized to a oligonucleotide array representing 113 murine inflammation-related genes (from SA Biosciences at www.sabiosciences.com, Cat. Number OMM-011.) The array is a nylon membrane based DNA microarray on which 60-mer oligonucleotides probes were printed. Gene expression profile of resting cells incubated in serum-free medium (containing albumin, BSA) was used as a control for basal levels of gene expression.

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Hydroxyurea stimulates production of pro-inflammatory cytokines in endothelial cells: Relevance to sickle cell disease.

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