The effect of type 1 IFN on human aortic endothelial cell function in vitro: relevance to systemic lupus erythematosus

Abstract

Cardiovascular disease (CVD) is an important cause of morbidity and mortality in patients with systemic lupus erythematosus. The etiopathogenesis of premature CVD is not fully understood, but recently interferon-alpha (IFNα) has been implicated as a contributing factor. Since IFNα has been associated with both disease activity and endothelial dysfunction in lupus patients, we aimed to determine whether IFNα has direct effects on human aortic endothelial cell (HAoEC) function in vitro. We studied the function of IFNα2b-treated HAoECs in terms of cell proliferation, capillary-like network formation, and nitric oxide (NO) generation. Changes in gene expression were also analyzed using an exon gene array. IFNα2b regulated the expression of 198 genes, including recognized interferon-stimulated genes (ISGs). Gene ontology analysis showed over-representation of genes involved in antigen presentation and host response to virus but no significant changes in clusters of genes recognized as important in endothelial cell activation or dysfunction. HAoEC proliferation, tubule formation, and NO bioavailability were unchanged, suggesting that IFNα in isolation does not have a direct impact on aortic endothelial cell function.

FIG. 1.

Gene expression in human aortic endothelial cell (HAoEC) in response to IFNα2b. (A) IFNα2b (0.1–100 ng/mL) but not tumor necrosis factor-alpha (TNFα) (10 ng/mL) increases expression of the interferon-sensitive gene IFI44. Reverse transcription-PCR products shown for IFI44 and the reference gene GAPDH. (B) The fold change in IFI44 was similar across doses of IFN. (C) Gene ontology (GO) analysis of 198 differentially expressed transcripts by IFNα2b (fold ≥2, q ≤0.2) according to biological function reveals over-representation of genes with immunoregulatory function. (D) A more detailed GO analysis of the 63 genes initially assigned to immune function group shows marked expression of genes involved in viral replication and antigen presentation. (E) Tabulation of genes upregulated by IFNα2b by ≥15-fold includes the recognized ISGs IFIT1, IFI44L, and MX1.

FIG. 2.

HAoEC gene expression in response to IFNα2b and TNFα. (A) Venn diagram to show number of genes significantly upregulated by both IFNα2b and TNFα in the exon gene arrays. (B) The expression of 20 genes is significantly upregulated by both cytokines.

FIG. 3.

The effect of IFNα2b on endothelial cell proliferation and nitric oxide (NO) bioavailability. (A) IFNα2b had no effect on the number of HAoECs at 24, 48, or 72 h compared to vehicle control. Bars represent the mean and SE of the mean; combined results from n=3 independent experiments. (B) Cellular proliferation as measured by MTT assay shows a positive effect of vascular endothelial growth factor (VEGF) (20 ng/mL) and a negative effect of the NO scavenger PTIO (10 μM) on proliferation in both HAoEC and human venous endothelial cell (HUVEC) after 24 h. The absorbance values were normalized to vehicle-treated cells. (C) IFNα2b (10 ng/mL) had no effect on proliferation compared to vehicle control in HAoEC at 24, 48, or 72 h. (D) In contrast, IFNα2b significantly increased HUVEC proliferation at 24 h (P=0.047). This effect persisted to 48 h but was not statistically significant (P=0.134) at this time point. (E) The NO bioavailability (as measured by supernatant nitrite concentration) was not changed by IFNα2b (0.1–10 ng/mL) at either 6 or 24 h.

FIG. 4.

The effect of IFNα2b on endothelial cell tubule network formation. (A) HAoEC formed tubule networks in Matrigel with clear branches (arrowheads) and branch points/junctions (star). Network density was determined by the number of junctions in 3 random fields. Each experimental condition was conducted in triplicate and each graph represents n>3 independent experiments. Bars represent the mean (SE) number of junctions. (B) The density of the HAoEC network was significantly increased by 10 ng/mL VEGF-A (P=0.026) and decreased by 10 μM carboxy-PTIO (P=0.001). IFNα2b (10 ng/mL) had no effect on the HAoEC network density. (C) Similarly, the network density was not changed in HUVEC. Similar positive and negative effects of VEGF-A and PITO were seen in HUVEC (data not shown). (D-i) HAoEC tubule networks in three-dimensional (3D) type 1 collagen matrix were less complex than those on Matrigel. Representative image to show branch points/junctions (star) and branches (arrowheads). (D-ii, iii) Representative images of HAoEC networks in the presence of IFNα2b (10 ng/mL) or vehicle control show no difference in tubule network structure or density.