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. 2017 Feb;76(2):450-457.
doi: 10.1136/annrheumdis-2016-209442. Epub 2016 Jul 18.

Endothelial progenitor dysfunction associates with a type I interferon signature in primary antiphospholipid syndrome

Affiliations

Endothelial progenitor dysfunction associates with a type I interferon signature in primary antiphospholipid syndrome

Robert C Grenn et al. Ann Rheum Dis. 2017 Feb.

Abstract

Objectives: Patients with antiphospholipid syndrome (APS) are at risk for subclinical endothelial injury, as well as accelerated atherosclerosis. In the related disease systemic lupus erythematosus, there is a well-established defect in circulating endothelial progenitors, which leads to an accrual of endothelial damage over time. This defect has been at least partially attributed to exaggerated expression of type I interferons (IFNs). We sought to determine whether these pathways are important in APS.

Methods: We studied 68 patients with primary APS. Endothelial progenitors were assessed by flow cytometry and functional assay. Type I IFN activity was determined by a well-accepted bioassay, while peripheral blood mononuclear cells were scored for expression of IFN-responsive genes.

Results: Endothelial progenitors from patients with APS demonstrated a marked defect in the ability to differentiate into endothelial cells, a phenotype which could be mimicked by treating control progenitors with APS sera. Elevated type I IFN activity was detected in the circulation of patients with APS (a finding that was then replicated in an independent cohort). While IgG depletion from APS sera did not rescue endothelial progenitor function, the dysfunction was successfully reversed by a type I IFN receptor-neutralising antibody.

Conclusions: We describe, for the first time to our knowledge, an IFN signature in primary APS and show that this promotes impaired endothelial progenitor function. This work opens the door to novel approaches that may mitigate vascular damage in APS, such as anti-IFN drugs.

Keywords: Antiphospholipid Syndrome; Atherosclerosis; Cytokines.

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Conflict of interest statement

The authors have no competing interests or conflicts to disclose. AUTHORSHIP AND CONFLICT OF INTEREST DISCLOSURES The authors have no competing interests or conflicts to disclose. R.C.G., S.Y., A.A.G., N.M.K., C.N.-A., and D.H.-R. conducted experiments and analyzed data. A.R.C., W.J.M., P.L.B., and J.S.K. analyzed data and designed the study. All authors participated in writing the manuscript, and gave approval before submission.

Figures

Figure 1
Figure 1
EPC/CACs show a diminished ability to differentiate into EC-like cells in primary APS. A, Classic EPCs (defined as CD34+CD133+) were quantified in peripheral blood of controls and primary APS patients by flow cytometry (units=EPCs/ml of blood). Each data point represents an individual control/patient. The mean for each group is represented by a horizontal line. B, PBMCs were isolated from either controls or primary APS patients, and then cultured under proangiogenic conditions for 12–14 days. EPC/CACs were scored for their ability to successfully differentiate into EC-like cells as determined by surface binding of both UEA-1 lectin and acetylated LDL. Again, means and individual data points are indicated. C, Representative photomicrographs from the experiments of panel B. UEA-1 lectin is stained green and acetylated LDL red. Scale bar=500 μm.
Figure 2
Figure 2
Primary APS sera interfere with the emergence of EC-like cells from control EPC/CACs. A, PBMCs were isolated from controls, and then cultured under proangiogenic conditions for 12–14 days. For the first 3 days of culture, media was supplemented with sera from either heterologous controls or primary APS patients. EPC/CACs were scored for their ability to successfully differentiate into EC-like cells as determined by surface binding of both UEA-1 lectin and acetylated LDL. Each data point represents an individual control/patient. The mean for each group is represented by a horizontal line. B, Representative photomicrographs from the experiments of panel A. UEA-1 lectin is stained green and acetylated LDL red. Scale bar=500 μm. C, Total IgG was depleted from 10 APS serum samples. These patients were all positive for anti-β2GPI IgG at baseline. For each group, the median, 25th/75th percentiles, and minimum/maximum are represented by box-and-whisker. Anti-β2GPI IgG was undetectable in the depleted samples. D, As in panel A, PBMCs were isolated from controls, and then cultured under proangiogenic conditions for 12–14 days. For the first 3 days of culture, media was supplemented with APS sera that were either depleted of IgG, or not. EPC/CACs were scored for their ability to successfully differentiate into EC-like cells as determined by surface binding of both UEA-1 lectin and acetylated LDL. Each pair of data points represents an individual patient.
Figure 3
Figure 3
Primary APS sera have elevated levels of type I IFNs based on an established bioassay. As described in Methods, HeLa cells were treated with sera from either controls or primary APS patients for 6 hours, and then harvested for RNA preparation. Expression of the type I IFN-regulated genes IFIT1 (panel A), IFI44 (panel B), and PRKR (panel C) was scored by quantitative PCR. Each data point represents an individual control/patient. The median for each group is represented by a horizontal line.
Figure 4
Figure 4
The PBMC compartment has evidence of a type I IFN signature in primary APS patients, similar to SLE patients. RNA was prepared from total PBMCs. Expression of the type I IFN-regulated genes IFIT1 (panel A), IFI44 (panel B), and PRKR (panel C) was scored by quantitative PCR. Each data point represents an individual control/patient. The median for each group is represented by a horizontal line. D, Data for primary APS patients are segregated based on whether the patients were negative or positive for anti-β2GPI IgG by the Sydney criteria. Again, medians and individual data points are indicated.
Figure 5
Figure 5
A neutralizing antibody against the type I IFN receptor restores normal EPC/CAC differentiation in primary APS patients. A, PBMCs were isolated from controls, and then cultured under proangiogenic conditions for 12–14 days. For the first 3 days of culture, media was supplemented with sera from either heterologous controls or primary APS patients. For these same days, media was additionally supplemented with the aforementioned IFN receptor-neutralizing antibody, or isotype control. EPC/CACs were scored for their ability to successfully differentiate into EC-like cells as determined by surface binding of both UEA-1 lectin and acetylated LDL. Each pair of data points represents an individual control/patient. B, Representative photomicrographs from the experiments of panel A. UEA-1 lectin is stained green and acetylated LDL red. Scale bar=500 μm.

Comment in

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