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Role of ecNOS-derived NO in mediating TNF-induced endothelial barrier dysfunction
K Bove1, P Neumann, N Gertzberg
1Research Service, Stratton Veterans Affairs Medical Center, Albany, New York 12208, USA.
Summary
Endothelial cell nitric oxide synthase (ecNOS) mediates acute, but not prolonged, increases in pulmonary microvessel permeability induced by tumor necrosis factor-alpha. This suggests ecNOS-derived nitric oxide (NO) is key for early barrier dysfunction.
Area of Science:
- Molecular Biology
- Physiology
- Endothelial Cell Biology
Background:
- Tumor necrosis factor-alpha (TNF-alpha) is known to increase endothelial permeability.
- The role of endothelial cell nitric oxide synthase (ecNOS) in TNF-alpha-induced changes in pulmonary microvessel endothelial cells (PEM) remains unclear.
- Understanding mediators of endothelial barrier function is crucial for treating inflammatory conditions.
Purpose of the Study:
- To investigate whether ecNOS mediates TNF-alpha-induced increases in nitric oxide (NO) and albumin permeability in PEM.
- To determine the time course of ecNOS expression and its relationship with endothelial barrier function following TNF-alpha stimulation.
- To elucidate the role of ecNOS-derived NO in acute versus prolonged TNF-alpha-induced endothelial dysfunction.
Main Methods:
- Pulmonary microvessel endothelial monolayers (PEM) were stimulated with TNF-alpha.
- Analysis of ecNOS mRNA and protein levels using RT-PCR and Western immunoblotting.
- Measurement of NO production and albumin clearance rate to assess NO levels and barrier function.
- Use of ecNOS antisense oligonucleotide and a NO agonist (Spermine-NONOATE) to probe mechanisms.
Main Results:
- TNF-alpha stimulation led to decreased ecNOS mRNA and biphasic changes in ecNOS protein levels (acute increase followed by decrease).
- Inhibition of ecNOS with antisense oligonucleotide prevented TNF-alpha-induced NO increase and albumin permeability at 4 hours.
- The protective effect of ecNOS inhibition on albumin permeability was lost at 24 hours, despite NO production being prevented.
- Inducible and brain NOS isotypes were not detected in PEM.
Conclusions:
- The ecNOS isotype mediates the acute generation of NO and subsequent endothelial barrier dysfunction in response to TNF-alpha.
- Prolonged increases in TNF-alpha-induced endothelial permeability are independent of NO derived from ecNOS.
- These findings highlight distinct mechanisms underlying acute and sustained endothelial responses to inflammatory stimuli.