Korean J Physiol Pharmacol.  2008 Oct;12(5):231-236. 10.4196/kjpp.2008.12.5.231.

Heparin Attenuates the Expression of TNF alpha-induced Cerebral Endothelial Cell Adhesion Molecule

• Lee JH
• Kim CH
• Seo GH
• Lee J
• Kim JH
• Kim DG
• Ahn YS

• KMID: 1486104
• DOI: http://doi.org/10.4196/kjpp.2008.12.5.231

Abstract

Heparin is a well-known anticoagulant widely used in various clinical settings. Interestingly, recent studies have indicated that heparin also has anti-inflammatory effects on neuroinflammation-related diseases, such as Alzheimer's disease and meningitis. However, the underlying mechanism of its actions remains unclear. In the present study, we examined the anti-inflammatory mechanism of heparin in cultured cerebral endothelial cells (CECs), and found that heparin inhibited the tumor necrosis factor alpha(TNF alpha)-induced and nuclear factor kappa B (NF-kappa B)-dependent expression of adhesion molecules, such as intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1), which are crucial for inflammatory responses. Heparin selectively interfered with NF-kappa B DNA-binding activity in the nucleus, which is stimulated by TNF alpha. In addition, non-anticoagulant 2,3-O desulfated heparin (ODS) prevented NF-kappa B activation by TNF alpha, suggesting that the anti-inflammatory mechanism of heparin action in CECs lies in heparin's ability to inhibit the expression of cell adhesion molecules, as opposed to its anticoagulant actions.

Keyword

Heparin; NF-kappa B; ICAM-1; VCAM-1; Cerebral endothelial cells; Anti-inflammation

MeSH Terms

Alzheimer Disease
Cell Adhesion Molecules
Endothelial Cells
Heparin
Intercellular Adhesion Molecule-1
Meningitis
NF-kappa B
Tumor Necrosis Factor-alpha
Vascular Cell Adhesion Molecule-1
Cell Adhesion Molecules
Heparin
Intercellular Adhesion Molecule-1
NF-kappa B
Tumor Necrosis Factor-alpha
Vascular Cell Adhesion Molecule-1

Figure

• 

  Fig. 1. Heparin inhibited TNFα -induced expression of adhesion molecules and NF-κ B activation in cerebral endothelial cells. (A) Mouse cerebral endothelial cells (bEnd.3) were pretreated with heparin (1 mg/ml for 24 h) and then exposed to TNFα (10 ng/ml) for 4 h. mRNA levels of ICAM-1 and VCAM-1 were determined using real-time RT-PCR. ∗∗p<0.01 (B) The NF-α B DNA-binding activity of nuclear extracts of TNF α-stimulated cells (10 ng/ml for 30 min) in the presence or absence of pretreatment with heparin was analyzed using electrophoretic mobility shift assay (EMSA). Super-shift assay showed bands containing p65 and p50, which are major components of NF-κ B. ∗∗p<0.01 (C) DNA-binding activity of Sp-1 was observed. Supershift assay indicated a Sp-1 specific band.

• 

  Fig. 2. Heparin directly inhibited NF-κ B DNA-binding activity in vitro without affecting nuclear translocation. (A) An aliquot of nuclear extracts from TNF α-induced bEnd.3 cells was directly mixed with heparin in vitro. DNA-binding activities of NF-κ B and Sp-1 were analyzed using EMSA. ∗∗p<0.01 versus no heparin treatment group. (B) Cells were pretreated with heparin (1 mg/ml for 24 h) and then exposed to TNF α (10 ng/ml) for 30 min. An aliquot of nuclear extracts was immunoblotted with anti p65 and p50 antibodies. Sp-1 protein in the nuclear extracts was used as a loading control. (C) TNF α-stimulated CEC lysates with or without pretreatment with heparin were separated by polyacrylamide gel electrophoresis and transferred to a PVDF membrane, which was successively probed with IκBα antibody. Actin protein in the whole cell lysates was used as a loading control.

• 

  Fig. 3. Non-anticoagulant 2,3-O desulfated heparin (ODS) inhibited TNF α-induced NF-κ B DNA-binding. Cells were pretreated with ODS (1 mg/ml for 24 h) or heparin (1 mg/ml for 24 h). NF-κ B DNA-binding activity of nuclear extracts of TNF α-induced cells (10 ng/ml for 30 min) were analyzed using EMSA. ∗∗p<0.01 versus TNF α treatment group.

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