Korean J Physiol Pharmacol.  2016 Mar;20(2):169-175. 10.4196/kjpp.2016.20.2.169.

High glucose and palmitate increases bone morphogenic protein 4 expression in human endothelial cells

• Hong OK
• Yoo SJ
• Son JW
• Kim MK
• Baek KH
• Song KH
• Cha BY
• Jo H
• Kwon HS

• KMID: 2388673
• DOI: http://doi.org/10.4196/kjpp.2016.20.2.169

Abstract

Here, we investigated whether hyperglycemia and/or free fatty acids (palmitate, PAL) aff ect the expression level of bone morphogenic protein 4 (BMP4), a proatherogenic marker, in endothelial cells and the potential role of BMP4 in diabetic vascular complications. To measure BMP4 expression, human umbilical vein endothelial cells (HUVECs) were exposed to high glucose concentrations and/or PAL for 24 or 72 h, and the effects of these treatments on the expression levels of adhesion molecules and reactive oxygen species (ROS) were examined. BMP4 loss-of-function status was achieved via transfection of a BMP4-specific siRNA. High glucose levels increased BMP4 expression in HUVECs in a dose-dependent manner. PAL potentiated such expression. The levels of adhesion molecules and ROS production increased upon treatment with high glucose and/or PAL, but this eff ect was negated when BMP4 was knocked down via siRNA. Signaling of BMP4, a proinflammatory and pro-atherogenic cytokine marker, was increased by hyperglycemia and PAL. BMP4 induced the expression of infl ammatory adhesion molecules and ROS production. Our work suggests that BMP4 plays a role in atherogenesis induced by high glucose levels and/or PAL.

Keyword

Bone morphogenic protein 4; Diabetes mellitus; Free fatty acid; High glucose

MeSH Terms

Atherosclerosis
Diabetes Mellitus
Diabetic Angiopathies
Endothelial Cells*
Fatty Acids, Nonesterified
Glucose*
Human Umbilical Vein Endothelial Cells
Humans*
Hyperglycemia
Reactive Oxygen Species
RNA, Small Interfering
Transfection
Fatty Acids, Nonesterified
Glucose
RNA, Small Interfering
Reactive Oxygen Species

Figure

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  Fig. 1 Glucose increased expression of BMP4 in dose-dependent manners.After 16~24 h plating, the cells were washed with DMEM media (containing 2% FBS, w/o growth factor), and then incubated with glucose (D-glucose 100, 500, 1000 mg/dL), and mannitol (D-glucose 100 mg/dL+Mannitol 25 mmol ) for 24 h. High glucose increase BMP4 expression in dose-dependent manner in HUVECs. Data are expressed as the mean±SEM of four independent observations in separate cell culture wells. *p<0.05 vs. 100 mg/dL glucose.

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  Fig. 2 High glucose and FFA increased expression of BMP4 in time-dependent manners.HUVECs incubated with HG, PAL, and HG/PAL combination with DMEM media (containing 2% FBS, w/o growth factor) for 24 h or 72 h. BMP4 increased by high glucose, and this increase in PAL accelerates more in time-dependent manner. Data are expressed as the mean±SEM of four independent observations in separate cell culture wells. *p<0.05 vs. NG, NG; 100 mg/dL glucose, HG; 500 mg/dL glucose, PAL; 500 µM palmitate.

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  Fig. 3 Activation of BMP4 induced by high glucose and FFA ef fects on various adhesion molecues.We used siRNA techniques to selectively deplete BMP4-dependent pathways and examine the effect of HG, PAL, HG/PAL with DMEM media (containing 2% FBS, w/o growth factor) for 72 h. Expression of adhesion molecules were increased by treatments with HG and/or PAL. When BMP4 was knockdowned by siRNA, this expression was blunted. Data are expressed as the mean±SEM of four independent observations in separate cell culture wells. *p<0.05, NG; 100mg/dL glucose, HG; 500 mg/dL glucose, PAL; 500 µM palmitate.

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  Fig. 4 High glucose and FFA induced BMP4 activation increase monocyte adhesion.We used siRNA techniques to selectively deplete BMP4-dependent pathways and examine the effect of HG, PAL, HG/PAL with DMEM media (containing 2% FBS, w/o growth factor) for 72 h. In monocyte adhesion assay, HG with PAL induced monocyte adhesion in HUVECs and it was prevented by BMP4 siRNA. Data are expressed as the mean±SEM of four independent observations in separate cell culture wells. *p<0.05 vs. NG, NG; 100 mg/dL glucose, HG; 500 mg/dL glucose, PAL; 500 µM palmitate.

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  Fig. 5 High glucose and FFA induced BMP4 activation increases ROS production.We used siRNA techniques to selectively deplete BMP4-dependent pathways and examine the effect of HG, PAL, HG/PAL with DMEM media (containing 2% FBS, w/o growth factor) for 72 h. HG and/or PAL increases ROS production in HUVECs, BMP4 knockdown ameliorated this response. Data are expressed as the mean±SEM of four independent observations in separate cell culture wells. *p<0.05, NG; 100 mg/dL glucose, HG; 500 mg/dL glucose, PAL; 500 µM FFA.

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