Oxidized low-density lipoprotein and β-glycerophosphate synergistically induce endothelial progenitor cell ossification.
OBJECTIVE
To investigate the ability of ox-LDL to induce ossification of endothelial progenitor cells (EPCs) in vitro and explored whether oxidative stress, especially hypoxia inducible factor-1α (HIF-1α) and reactive oxygen species (ROS), participate in the ossific process.


METHODS
Rat bone marrow-derived endothelial progenitor cells (BMEPCs) were cultured in endothelial growth medium supplemented with VEGF (40 ng/mL) and bFGF (10 ng/mL). The cells were treated with oxidized low-density lipoprotein (ox-LDL, 5 μg/mL) and/or β-glycerophosphate (β-GP, 10 mmol/L). Calcium content and Von Kossa staining were used as the measures of calcium deposition. Ossific gene expression was determined using RT-PCR. The expression of osteocalcin (OCN) was detected with immunofluorescence. Alkaline phosphatase (ALP) activity was analyzed using colorimetric assay. Intercellular reactive oxygen species (ROS) were measured with flow cytometry.


RESULTS
BMEPCs exhibited a spindle-like shape. The percentage of cells that expressed the cell markers of EPCs CD34, CD133 and kinase insert domain-containing receptor (KDR) were 46.2%±5.8%, 23.5%±4.0% and 74.3%±8.8%, respectively. Among the total cells, 78.3%±4.2% were stained with endothelial-specific fluorescence. Treatment of BMEPCs with ox-LDL significantly promoted calcium deposition, which was further significantly enhanced by co-treatment with β-GP. The same treatments significantly increased the gene expression of core-binding factor a-1 (cbfa-1) and OCN, while decreased the gene expression of osteoprotegerin (OPG). The treatments also significantly enhanced the activity of ALP, but did not affect the number of OCN(+) cells. Furthermore, the treatments significantly increased ROS and activated the hypoxia inducible factor-1α (HIF-1α). In all these effects, ox-LDL acted synergistically with β-GP.


CONCLUSIONS
Ox-LDL and β-GP synergistically induce ossification of BMEPCs, in which an oxidizing mechanism is involved.
Center for Laboratory Diagnosis, Beijing Tiantan Hospital Affiliated to Capital Medical University, China.
Li
L
Liu
Zhi-zhong
ZZ
Liu
Hui
H
Chen
Guo-jun
GJ
Zhang
Yu-hua
YH
Kong
Xi-xiong
XX
Kang
D000818:Animals; D001483:Base Sequence; D017931:DNA Primers; D005994:Glycerophosphates / Q000378:metabolism*; D008077:Lipoproteins, LDL / Q000378:metabolism*; D010012:Osteogenesis; D018384:Oxidative Stress; D016133:Polymerase Chain Reaction; D051381:Rats; D017382:Reactive Oxygen Species / Q000378:metabolism; D013234:Stem Cells / Q000166:cytology*
D017931:DNA Primers; D005994:Glycerophosphates; D008077:Lipoproteins, LDL; D017382:Reactive Oxygen Species; C099252:oxidized low density lipoprotein; C031463:beta-glycerophosphoric acid