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NO reduces PMN adhesion to human vascular endothelial cells due to downregulation of ICAM-1 mRNA and surface
S Lindemann1, M Sharafi, M Spiecker
1Department of Medicine II, Johannes Gutenberg-University, Mainz, Germany.
Abstract:
Reperfusion damage is largely due to the adherence of polymorphonuclear leukocytes to the endothelium initiated by adhesion molecule upregulation. The reduced endothelial nitric oxide release during ischemia may be involved in the upregulation of intercellular adhesion molecule 1. In this study, we tested if nitric oxide donors suppress polymorphonuclear leukocyte adherence to activated endothelial cells by inhibition of the intercellular adhesion molecule 1 surface expression. Confluent human umbilical vein endothelial cells were stimulated with tumor necrosis factor alpha (300 U/mL) after preincubation with increasing concentrations of the nitric oxide donors CAS 1609 (0.005-5 mM/L) and 3-(4-morpholinyl)-sydnonimine (0.01-1 mM/L). Intercellular adhesion molecule 1 surface expression was measured in a cell surface enzyme-linked immunosorbent assay, intercellular adhesion molecule 1 mRNA by Northern analysis. Human saphenous vein endothelial cells were transfected with the inducible nitric oxide synthase gene and stimulated with tumor necrosis factor alpha (300 U/mL). Fluorescein green-labeled polymorphonuclear leukocytes adhering to activated human umbilical vein endothelial cells/human saphenous vein endothelial cells were quantified by epifluorescent microscopy. The intercellular adhesion molecule 1 surface expression of activated human umbilical vein endothelial cells/human saphenous vein endothelial cells was significantly diminished to 40 to 60% of the maximum after treatment with CAS 1609, 3-(4-morpholinyl)-sydnonimine, or transfection with the inducible nitric oxide synthase gene. Intercellular adhesion molecule 1 mRNA was diminished by CAS 1609 and 3-(4-morpholinyl)-sydnonimine in the same manner. The functional relevance of our data was shown by reduction of polymorphonuclear leukocyte adherence to activated human umbilical vein endothelial cells/human saphenous vein endothelial cells following treatment with CAS 1609 and 3-(4-morpholinyl)-sydnonimine or transfection with inducible nitric oxide synthase. Tumor necrosis factor-induced polymorphonuclear leukocyte adherence was abolished by blocking antibody against intercellular adhesion molecule 1. Thus, exogenous or endogenous substitution of nitric oxide diminishes the expression of endothelial intercellular adhesion molecule 1 and its mRNA following tumor necrosis factor alpha stimulation. This results in a reduced polymorphonuclear leukocyte adherence to activated endothelium.
Insights
Nitric oxide donors and gene therapy reduce intercellular adhesion molecule 1 (ICAM-1) expression and polymorphonuclear leukocyte adherence to activated endothelial cells. This finding offers a potential therapeutic strategy for reperfusion injury by mitigating inflammatory responses.
Area of Science:
- Cardiovascular Biology
- Immunology
- Molecular Medicine
Background:
- Reperfusion damage involves polymorphonuclear leukocyte (PMN) adherence to endothelium, mediated by adhesion molecule upregulation.
- Reduced nitric oxide (NO) during ischemia may increase intercellular adhesion molecule 1 (ICAM-1) expression.
Purpose of the Study:
- To investigate if NO donors inhibit PMN adherence to activated endothelial cells by reducing ICAM-1 surface expression.
- To explore the role of exogenous and endogenous NO in modulating ICAM-1 expression and PMN adherence.
Main Methods:
- Human umbilical vein endothelial cells (HUVECs) and saphenous vein endothelial cells (HSECs) were treated with NO donors (CAS 1609, 3-(4-morpholinyl)-sydnonimine) or transfected with inducible NO synthase (iNOS) gene.
- Tumor necrosis factor alpha (TNF-α) stimulated cells to induce inflammation.
- ICAM-1 surface expression, mRNA levels, and PMN adherence were quantified using ELISA, Northern analysis, and epifluorescent microscopy.
Main Results:
- NO donors and iNOS gene transfection significantly reduced ICAM-1 surface expression (40-60%) and mRNA levels in TNF-α-stimulated endothelial cells.
- PMN adherence to activated endothelial cells was significantly reduced by NO treatment or iNOS expression.
- Blocking ICAM-1 abolished TNF-α-induced PMN adherence, confirming ICAM-1's critical role.
Conclusions:
- Exogenous or endogenous NO effectively diminishes endothelial ICAM-1 expression and mRNA following TNF-α stimulation.
- Reduced ICAM-1 expression leads to decreased PMN adherence to activated endothelium.
- NO-based therapies hold promise for mitigating reperfusion injury by controlling inflammatory cell adhesion.