Related Experiment Video
Updated: Aug 24, 2026
Isolation of Primary Patient-specific Aortic Smooth Muscle Cells and Semiquantitative Real-time Contraction Measurements In Vitro
Published on: February 15, 2022
CD14 glycoprotein expressed in vascular smooth muscle cells
Hyoung Chul Choi1, Kwang Youn Lee
1Department of Pharmacology, College of Medicine, Yeungnam University, Namgu, Daegu, Korea. hcchoi@med.yu.ac.kr
Abstract:
The inducible nitric oxide synthase (iNOS) expression in vascular smooth muscle cells is an important factor for pathogenesis of septic shock or multiple organ dysfunction syndrome. The mechanisms of iNOS expression in such conditions are partly known. This study tried to clarify the signal transduction of lipopolysaccharide (LPS) single stimulation that induces iNOS mRNA and protein in vascular smooth muscle cells (VSMC). VSMC were primarily cultured from rat aorta. The concentrations of nitrite in culture media were measured by the Griess reaction. Western blottings and immunoreaction for iNOS, nuclear factor kappaB (NFkappaB) p65, and CD14 protein were performed. mRNAs of iNOS and tumor necrosis factor (TNF) alpha were analyzed by RT-PCR. Genistein inhibited LPS induced early phase nitrite production, while pyrrolidine dithiocarbamate (PDTC) inhibited nitrite production at a late phase. PDTC significantly reduced NFkappaB p65 and iNOS protein expression by LPS. TNFalpha mRNA expression by LPS was not detected in VSMC. Membranous CD14 glycoprotein was detected in VSMC and soluble CD14 glycoprotein was not detected in fetal bovine serum added in culture media. These results suggest that CD14 glycoprotein is present on the cell membranes of VSMC, a non-myelomonocyte lineage, acting as an LPS receptor. Activations of tyrosine kinase and NFkappaB p65 are essential for iNOS expression by LPS single stimulation, while TNFalpha is not a concern to iNOS expression in VSMC.
Insights
Lipopolysaccharide (LPS) induces inducible nitric oxide synthase (iNOS) in vascular smooth muscle cells via CD14 and NF-kappaB activation. Tumor necrosis factor-alpha is not involved in this iNOS expression pathway.
Area of Science:
- Cell Biology
- Immunology
- Physiology
Background:
- Inducible nitric oxide synthase (iNOS) expression in vascular smooth muscle cells (VSMC) contributes to septic shock and multiple organ dysfunction syndrome.
- The precise signaling pathways for iNOS induction by lipopolysaccharide (LPS) in VSMC remain incompletely understood.
Purpose of the Study:
- To elucidate the signal transduction mechanisms by which single lipopolysaccharide (LPS) stimulation induces iNOS mRNA and protein expression in rat aortic VSMC.
- To investigate the role of CD14 and nuclear factor kappaB (NF-kappaB) in LPS-induced iNOS expression in VSMC.
Main Methods:
- Primary culture of rat aortic VSMC.
- Measurement of nitrite production using Griess reaction.
- Western blotting for iNOS, NF-kappaB p65, and CD14.
- RT-PCR analysis for iNOS and tumor necrosis factor-alpha (TNF-alpha) mRNA.
- Inhibition studies using genistein and pyrrolidine dithiocarbamate (PDTC).
Main Results:
- LPS induced nitrite production, NF-kappaB p65 activation, and iNOS protein expression in VSMC.
- Genistein inhibited early nitrite production, while PDTC inhibited late nitrite production and NF-kappaB/iNOS expression.
- CD14 glycoprotein was detected on VSMC membranes, suggesting its role as an LPS receptor.
- TNF-alpha mRNA expression was not detected in VSMC following LPS stimulation.
Conclusions:
- CD14 acts as an LPS receptor on VSMC, a non-myelomonocyte lineage.
- Tyrosine kinase and NF-kappaB p65 activation are critical for LPS-induced iNOS expression in VSMC.
- TNF-alpha is not a significant mediator of iNOS expression in VSMC under these conditions.
