iNOS expression requires NADPH oxidase‐dependent redox signaling in microvascular endothelial cells

Abstract

Redox regulation of inducible nitric oxide synthase (iNOS) expression was investigated in lipopolysaccharide and interferon-γ (LPS+IFNγ)-stimulated microvascular endothelial cells from mouse skeletal muscles. Unstimulated endothelial cells produced reactive oxygen species (ROS) sensitive to inhibition of NADPH oxidase (apocynin and DPI), mitochondrial respiration (rotenone) and NOS (L-NAME). LPS+IFNγ caused a marked increase in ROS production; this increase was abolished by inhibition of NADPH oxidase (apocynin, DPI and p47phox deficiency). LPS+IFNγ induced substantial expression of iNOS protein. iNOS expression was prevented by the antioxidant ascorbate, NADPH oxidase inhibition (apocynin, DPI and p47phox deficiency), but not by inhibition of mitochondrial respiration (rotenone) and xanthine oxidase (allopurinol). iNOS expression also was prevented by selective antagonists of ERK, JNK, Jak2, and NFκB activation. LPS+IFNγ stimulated activation/phosphorylation of ERK, JNK, and Jak2 and activation/degradation of IκB, but only the activation of JNK and Jak2 was sensitive to ascorbate, apocynin and p47phox deficiency. Ascorbate, apocynin and p47phox deficiency also inhibited the LPS+IFNγ-induced DNA binding activity of transcription factors IRF1 and AP1 but not NFκB. In conclusion, LPS+IFNγ-induced NFκB activation is necessary for iNOS induction but is not dependent on ROS signaling. LPS+IFNγ-stimulated NADPH oxidase activity produces ROS that activate the JNK-AP1 and Jak2-IRF1 signaling pathways required for iNOS induction. Since blocking either NFκB activation or NADPH oxidase activity is sufficient to prevent iNOS expression, they are separate targets for therapeutic interventions that aim to modulate iNOS expression in sepsis.

Lipopolysaccharide (LPS)-mediated Angiopoietin-2-dependent Autocrine Angiogenesis Is Regulated by NADPH Oxidase 2 (Nox2) in Human Pulmonary Microvascular Endothelial Cells

“…Peng et al (43) showed that LPSmediated TNF-␣ expression in cardiomyocytes was dependent on gp91phox and p38 kinase activation. Similarly, Wu et al (46) and Patel et al (45) showed Nox2 and Nox4 mediated LPS-dependent MAPK activation in aortic smooth muscle cells and skeletal muscle microvascular endothelial cells, respectively. Our data regarding HPMECs are consistent with the studies mentioned above and support the regulation of proinflammatory MAPK activation by Nox isoforms.…”

Evaluation of Vitamin C for Adjuvant Sepsis Therapy

“…Part of the evidence for this mechanism is that injection of ascorbate (200 mg/kg) prevents CLP from elevating the concentration of NO metabolites in plasma (64). Further, ascorbate injection blocks the increase by CLP of iNOS mRNA expression in microvascular endothelial cells at 3 h after CLP, apparently due to inhibition by ascorbate of NADPH oxidase activity (63,65). Parenteral ascorbate also blocks the increases in total NOS, iNOS, and neuronal NOS (nNOS) activities in skeletal muscle of CLP mice (68) (Fig.…”

“…Tyrosine residues in the catalytic subunit of PP2A (PP2Ac) become nitrated in microvascular endothelial cells exposed to LPS + IFNc, because this insult increases NADPH oxidase synthesis of superoxide, iNOS synthesis of NO, and consequently the production of peroxynitrite from superoxide and NO (62,65). The tyrosine nitration of PP2Ac increases the phosphatase activity of PP2A, which dephosphorylates serine and threonine residues in occludin (24,41).…”

“…For the ascorbate and DHAA treatments in cell cultures, the protection of the endothelial barrier is associated with increased intracellular ascorbate concentration and it depends on inhibition of both peroxynitrite formation and PP2A nitration/ activation (24). Administration of ascorbate prevents the induction by septic insult of NAPDH oxidase, and consequently blocks induction of iNOS, in microvascular endothelial cells (65). Thus, ascorbate slows peroxynitrite formation and nitration of PP2Ac.…”

“…of ascorbate (51,59). Cell culture experiments show that high concentrations of ascorbate prevent increases in the activity of NADPH oxidase and the production of superoxide and related oxidants in microvascular endothelial cells exposed to septic insult (24,65). By lowering reactive oxygen species levels, either NADPH oxidase deficiency or ascorbate administration may increase the effective concentration of NO within capillaries and thus prevent microthrombus formation during sepsis (59).…”