Abstract

Endothelial microparticles (EMPs) are complex structures with pleiotropic properties and are emerging as an index of endothelial damage; however, further work to determine the effect of EMPs on vascular smooth muscle cells (VSMC) is needed. We have shown that elevated EMPs are detected in Systemic Lupus Erythematosus (SLE) and carotid artery disease patients, who present accelerated vascular ageing and calcification. This study aims to investigate the molecular components of EMPs and whether they modulate vascular calcification and osteogenic differentiation of VSMCs in vitro.

EMPs were generated in vitro using human aortic endothelial cells (AoEMPs) by serum starvation (24 h) followed by TNF-alpha stimulation (10 ng/ml; 24 h), isolated by ultracentrifugation and quantified using flow cytometry. Human coronary artery smooth muscle cells (HCASMCs) were incubated with 10⁶ AoEMPs/ml in osteoinductive media (5 mM BGP and 2.6 mM CaCl_²) for 21 days. Calcification was assessed by alizarin red staining and calcium deposition assays. Conditioned media was collected at 7, 14 and 21 days to identify markers of bone metabolism using Bioplex array technology and ELISA. AoEMPs were also subjected to proteomic and miR screening to identify relevant molecules and pathways.

AoEMP-treated HCASMCs showed enhanced calcification after 21 days, by both alizarin red staining (p < 0.005) and calcium deposition assays (p < 0.05). In addition, secreted osteocalcin and osteoprotegerin levels were elevated in AoEMP-treated cells after 7 and 21 days respectively. ELISA determined that HGF, a key protein in vascular calcification, was elevated in media from the AoEMP-treated group compared to controls at 14 and 21 days. HGF levels were also higher in SLE patient plasma (p < 0.05) compared to healthy controls, suggesting that HGF may be involved in the vascular calcification observed in SLE patients. Furthermore, proteomic screening identified HGF in AoEMPs, whilst miR screening highlighted miR-3148 in both AoEMPs and SLE plasma and is predicted to target osteoprotegerin gene expression.

We conclude that AoEMPs enhance calcification and the reprogramming of HCASMCs to an osteogenic pathway in vitro, which may be in part, linked with HGF and miR-3148, supporting their role in vascular calcification in SLE and carotid artery disease. Further studies are required to determine how AoEMPs contribute to pathophysiological mechanisms in vivo and whether the circulating property of AoEMPs may represent a new biomarker in vascular calcification.