Biochemical and Biophysical Research Communications
Oxidized low density lipoprotein impairs endothelial progenitor cell function by downregulation of E-selectin and integrin αvβ5
Section snippets
Materials and methods
Cell culture. Peripheral blood mononuclear cells were isolated from buffy coats of human donors by density gradient centrifugation with Histopaque®-1077 (Sigma) as previously described [11]. Briefly, cells were plated on culture dishes coated with human fibronectin (Sigma) and maintained in EC basal medium-2 (EBM-2) (Clonetics) supplemented with endothelial growth medium SingleQuots and 5% fetal bovine serum (FBS; Sigma). After 4 days in culture, adherent cells were passaged and maintained in
Prevention of vascular structure formation by oxLDL
Applying a co-culture system on growth factor-reduced Matrigel®, VEGF acted as a strong promoter of capillary-like tubuli formation where EPC worked in concert with differentiated EC (HMEC) to form vessel-like structures (Fig. 1A). While the addition of nLDL had no effect on the organization of the co-culture system (data not shown), oxLDL in sub-apoptotic concentrations impaired network formation at 50 μg/ml (Fig. 1B) and totally inhibited formation of capillary-like network-like structures at
Discussion
In the present study, we investigated mechanisms responsible for the impaired angiogenic potential of EPC induced by oxLDL. Sub-apoptotic concentrations of oxLDL inhibited tube formation and impaired the integration of EPC into the vascular network of differentiated EC, an effect that was not seen when treated with nLDL. A possible mechanism for the observed impaired functional properties is the reduced expression of E-selectin and integrin αvβ5 on EPC by oxLDL as both molecules are relevant
Acknowledgments
Supported in part by the Swiss National Foundation (No. 3200B0-114100 to C.K.), the Swiss Heart Foundation to C.K. and the University of Bern, Switzerland (Grant-in-aid to C.K.).
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2010, AtherosclerosisCitation Excerpt :Adherent cells were trypsinized, passaged and maintained in culture till day 7. EPC were characterized by uptake of 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine-labeled acetylated low density lipoprotein (Dil-Ac-LDL, Harbor Bio-products) and BS-1 lectin (Sigma) staining, as well as flow cytometry analysis of the following surface markers: CD34, CD133, CD45, CD14, KDR, CD31, VE-cadherin (CD144) and MCAM (CD146) as published previously [8,9]. To produce human EPC and HUVEC conditioned medium (EPC-CM and HUVEC-CM, respectively), EPC and HUVEC were cultured for 72 h under hypoxic conditions (1.5% O2, 5% CO2, 93.5% N2) using a humidified gas-sorted anoxic incubator-gloved box (InVivo2 400, Ruskin, UK).