Role of vitamin D in endothelial repair mechanisms in systemic lupus erythematosus

Abstract

BackgroundPatients with systemic lupus erythematosus (SLE) have an increased prevalence of cardiovascular disease (CVD). Vitamin D deficiency is common in SLE and is an independent risk factor for CVD in the general population. We have previously shown that low vitamin D is associated with arterial stiffness in patients with SLE. Myeloid angiogenic cells (MACs) have an important role in endothelial repair in animal models. Preliminary studies have shown impaired function of these cells in SLE. Myeloid cells respond to vitamin D in vitro and express functional vitamin D receptors. We aimed to assess whether vitamin D has beneficial effects on MAC function and thus endothelial repair. MethodsPeripheral blood mononuclear cells (PBMCs) from vitamin D deficient (<20 ng/mL) patients with SLE, or vitamin D replete (>20 ng/mL) healthy controls, were cultured for 7 days on fibronectin in endothelial growth media with or without 1,25(OH)2D3 (0·1–100 nM). For survival studies, the number of cells able to take up LDL was counted in random fields. Surface CD marker expression (CD14, CD68, CD86, CCR7, CD206) in MACs was determined with real-time quantitative PCR. Angiogenic factor secretion was measured with a Bio-Plex human angiogenesis suspension array (Bio-Rad Laboratories, CA, USA). Angiogenic function of MACs was determined with human aortic endothelial cells in a Matrigel angiogenesis model. Data were analysed using linear regression, paired t tests, and one-way ANOVA where appropriate. FindingsMACs expressed high levels of CD206, and low CCR7, compared with PBMCs, consistent with an M2 macrophage phenotype. Phagocytosis was demonstrated by MAC uptake of fluorescein isothiocyanate-labelled beads. MAC secreted interleukin 8 (mean 13·9 ug/mL, SD 20·1), vascular endothelial growth factor (37·2 pg/mL, 26·1), hepatocyte growth factor (1414 pg/mL, 568), leptin (209 pg/mL, 144), and platelet-derived growth factor (564 pg/mL, 365) but not granulocyte-colony stimulating factor or angiopoetin. 1,25(OH)2D3 (0·1–100 nM) increased the number of MACs from patients with SLE after 8 days in those with a low number (<150 per field) at baseline (p=0·037) in a dose-dependent manner. This effect was replicated in healthy MACs treated with 0·1 ng/mL interferon-alpha2b (p≤0·001). Expression of all macrophage markers was reduced by 1,25(OH)2D3 (10 nM) but the M2 marker CD206 was preferentially reduced compared with M1 markers CCR7 (p=0·008) and CD86 (p=0·043). Conditioned media from vitamin D (10 nM) treated MACs increased endothelial tubule network density in the angiogenesis model (mean relative density 1·43 [SD 0·12] vs 1·66 [0·15], p=0·012) whereas vitamin D alone did not (0·96 [0·10], p=0·582). Vitamin D did not, however, increase the concentration of any of the angiogenic factors measured in the Bio-Plex array. InterpretationResults from this study suggest that MACs are a population of M2 macrophages with angiogenic capacity in vitro, and that vitamin D both increases the number of MACs and changes the surface maker profile in SLE. The angiogenic capacity of MACs was increased by vitamin D ex vivo, but this was not due to changes in expression of common angiogenic factors. Further work will identify the mechanism by which vitamin D might augment endothelial repair in patients with SLE. FundingUK Medical Research Council.