The Major Phagocyte Peroxidase-Derived Oxidant HOSCN Stimulates Gene-Specific p38 MAPK- Dependent, NF-κb-Mediated Activation of Tissue Factor, VCAM-1 and ICAM-1 Expression In Endothelium.

Abstract

1481 Thiocyanate (SCN-) is, unexpectedly, the principal physiologic substrate for eosinophil peroxidase (EPO) and a major (i.e., accounting for 50% of H2O2 consumed) substrate for myeloperoxidase (MPO). The product of these reactions is HOSCN, a weak, exclusively sulfhydryl-reactive oxidant that we have previously shown to be a uniquely potent (up to 100-fold) oxidant transcriptional inducer of human umbilical vein endothelial cell (HUVEC) tissue factor (TF), ICAM-1 and VCAM-1 expression via a mechanism dependent upon NF-κB p65/p50 activation. We hypothesized that oxidative activation of p38 MAPK is a necessary and proximal step in the activation of NF-κB p65/p50 at the TF, VCAM-1 and ICAM-1 promoters. To test this we utilized the p38 MAPK-specific pyridinyl imidazole inhibitor SB 203580 (SB). In HUVEC monolayers exposed 4h to 150 μM HOSCN in M199 medium containing 10% FCS, SB strongly inhibited (>90%, ED50 300 nM) TF activity, decreased VCAM-1 expression assessed by western blot by > 50% but had no discernible effect upon ICAM-1 expression. qRT-PCR analysis confirmed that the effects of SB on these three molecules was transcriptionally mediated. Immunoprecipitation of HOSCN-treated HUVEC whole cell extracts with a polyclonal anti-phospho-p38 MAPK reagent showed rapid (within 1 min) phosphorylation of p38 MAPK that lasted >30 min with consequent kinase activity documented by detection of the downstream target product phospho HSP27. Confocal immunofluorescence confirmed the rapid and durable induction of phospho p38 MAPK from undetectable to abundant, primarily cytoplasmic, but also some nuclear localization. We further hypothesized that the disparate effects of SB on these three molecules reflects differences in the sequences of their NF-κB-binding sites that affect their ability to bind the p65 NF-kB subunit. Chromatin immunoprecipitation (ChIP) analysis of HUVEC treated for 3h with HOSCN using an anti-p65 antibody revealed that SB inhibited by 75% the 10-fold increase in binding of p65 to the authentic endogenous VCAM-1 and NF-κB binding loci but had no discernible effect on p65 binding to the ICAM-1 locus. An identical pattern was seen in HUVEC exposed 30 min to the prototypical inflammatory cytokine TNF. To test the physiologic significance of these findings we assessed the effect of SB upon PMN and eosinophil binding to HOSCN-exposed HUVEC monolayers treated as described above. SB pretreatment of HUVEC blocked by > 80% the 3–8-fold increase in binding of PMN and eosinophils that occurs in HOSCN-exposed monolayers. Blocking antibodies to ICAM-1 and VCAM-1 demonstrate that both of the adhesion molecules contribute to HOSCN-induced PMN and eosinophil adhesion. We conclude that HOSCN generated by PMN and eosinophils attached or subjacent to vascular endothelium has the capacity to rapidly activate endothelial p38 MAPK-dependent activation of p65 binding to the TF and VCAM-1, but not ICAM-1, NF-κB binding loci; and these differences manifest in physiologically relevant transcriptional regulation of protein expression. Therefore, p38 MAPK inhibitors may have anti-thrombotic and anti-inflammatory potential, the latter particularly in states, such as allergic inflammation, that are most dependent on VCAM-1. Disclosures: No relevant conflicts of interest to declare.

Hypothiocyanous Acid: Benign or Deadly?

“…The ability of HOSCN to influence cellular signaling pathways has also been reported in studies with HUVEC. HOSCN is a potent (up to 100-fold) transcriptional inducer of endothelial cell expression of tissue factor, and monocyte adhesion molecules (ICAM-1, VCAM-1, and E-selectin), via a process mediated by NF-κB activation. , Recent studies demonstrate that oxidative activation of p38 MAPK by HOSCN facilitates the activation of NF-κB in HUVEC …”

“…HOSCN alters the expression of the pro-inflammatory markers ICAM-1, VCAM-1, and E-selectin, to promote the recruitment and adhesion of leukocytes to the vascular endothelium and their subsequent migration into tissues. ,, In addition, the ability of HOSCN to induce macrophage apoptosis may contribute to plaque destabilization, as macrophage apoptotic cell death is reported to play a role in the conversion of subclinical atherosclerotic lesions to disrupted, vulnerable plaques. These plaques have the potential to rupture and trigger thrombosis, causing myocardial infarction and/or stroke. , Although HOSCN can induce changes in the cellular phenotype that may promote atherosclerosis, there is still a lack of direct evidence for a specific role of SCN – in disease development.…”