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Inducible NO Synthase (iNOS) in human neutrophils, but not pulmonary microvascular endothelial cells (PMVEC) mediates septic protein leak in vitro.
Author(s) -
Shelton Jennifer L,
Wang LF,
Cepinskas G,
Sandig M,
McCormack D G,
Mehta S
Publication year - 2006
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.20.5.a1164-a
Subject(s) - peroxynitrite , albumin , chemistry , leak , nitric oxide synthase , pharmacology , nitric oxide , biochemistry , microbiology and biotechnology , biology , superoxide , enzyme , organic chemistry , environmental engineering , engineering
Sepsis is associated with injury of the PMVEC leading to protein leak and acute lung injury. The role of iNOS in human PMVEC injury has not been addressed. We isolated HPMVEC from lung tissue using magnetic bead‐bound anti‐PECAM antibody, and assessed septic Evans‐blue albumin leak across HPMVEC monolayers in the presence/absence of human neutrophils. HPMVEC were used at passage 3, seeded on 3 μm Transwell inserts and grown to confluence. We confirmed by RT‐PCR that iNOS is induced in human neutrophils and HPMVEC following stimulation with cytomix. Cytomix stimulated trans‐HPMVEC albumin leak was not attenuated by pre‐treatment with 1400W, a selective iNOS inhibitor, or L‐NAME, a non‐selective NOS inhibitor. In neutrophil‐HPMVEC co‐culture, cytomix stimulation increased albumin leak across HPMVEC by 5 fold. Pre‐treatment of neutrophil‐HPMVEC co‐cultures with 1400W or L‐NAME attenuated cytomix‐induced albumin leak by more than 75% (p <0.05). Treatment with Spermine Nonoate, an NO donor did not increase albumin leak across HPMVEC, However SIN‐1, a peroxynitrite donor increased albumin leak across HPMVEC monolayers by 15 fold. Scavenging of superoxide and peroxynitrite with PGSOD and FeTTPS respectively, significantly attenuated SIN‐1 induced trans‐HPMVEC albumin leak and cytomix stimulated leak in neutrophil‐HPMVEC co‐cultures. In conclusion, albumin leak across HPMVEC is dependent on iNOS activity in neutrophils and not in EC. SIN‐1 and cytomix‐stimulated neutrophil‐dependent trans‐HPMVEC albumin leak appear to be mediated by peroxynitrite.