The Principal Eosinophil Peroxidase Oxidant, HOSCN, Potently Induces Endothelial Cell Tissue Factor Activity through Transcriptional Activation Dependent upon NF-kB and ERK1/2: Potential Mechanism for a Prothrombotic and Proinflammatory Endothelial Phenotype in the Hypereosinophilic Syndrome.

We have shown that SCN- is the principal substrate for EPO in vivo and its product, HOSCN, a weak sulfhydryl-reactive oxidant, is a uniquely potent (i.e. 10–1000-fold more than any other) phagocyte oxidant inducer of tissue factor (TF) activity in human umbilical vein endothelial cells (HUVEC). We here characterize the intracellular signaling pathways and transcription factors activated by HUVEC in response to this unusual oxidant. We exposed monolayers to increasing concentrations of various phagocyte-derived oxidants or 10 ng/ml LPS in M199 medium with 10% FCS and assayed TF in scraped, freeze-thawed extracts by one-stage clotting assay. We find that 50–150 μM HOSCN induces TF activity up to 100-fold at 4–6 hours, levels similar to those stimulated by LPS. In contrast, similar concentrations of HOBr, H2O2, and HOCl induced at most a < 2-fold increase in TF activity. Because the TF gene has upstream regulatory sequences that bind transcription factors AP-1, Egr-1 and NF-kB, we probed nuclear extracts of HOSCN- and LPS-treated HUVEC by EMSA using oligonucleotide probes specific for their respective factors. HOSCN induces potent (similar to 10 μg/ml LPS/ml) upregulation of NF-kB p50/p65 and the TF-specific c-rel/p65 heterodimer by 1–2 hours as confirmed by supershift assay. HOSCN induced modest activation of Egr-1 at 1 hour and strong activation of the c-Jun but not c-Fos AP-1 binding factors. Investigating intracellular kinase pathways upstream from these transcription factors, we probed Western blots of HOSCN-treated HUVEC extracts for the phosphorylated forms of ERK1/2 and SAPK/JNK and for I-kB. HOSCN stimulates a transient increase in pERK1/2 at 10–20 min followed by a pronounced secondary increase starting at 1h and peaking at 2–4 h as well as increasing pSAPK/JNK at 1-4h and promoting degradation of I-kB. The functional significance of ERK1/2 and NF-kB was confirmed using specific inhibitors of these respective pathways, U0126 and andrographolide, either of which nearly completely blocked HOSCN-induced TF expression. The critical role of NF-kB in this phenomenon was shown when wortmannin, which blocks the PI3K/Akt pathway that constitutively inhibits NF-kB, increased TF activity 10–30 fold but simultaneous addition of andrographolide blocked this increase. To extend our analysis of HOSCN on transcriptional activation, we screened 54 separate transcription factors using a Panomics TranSignal™Protein/DNA array and found that HOSCN induces >3-fold activation of Stat4, CDP, GRE, CBF, and Ets-1/PEA3, a pattern similar to but readily distinguishable from that of LPS. We conclude HOSCN strongly induces TF activity in endothelial cells through transcriptional activation dependent upon ERK 1/2 and NF-kB. Given the widespread genomic distribution of binding sites for these transcription factors and the types of genes they regulate, we hypothesize HOSCN generated by adherent and infiltrating eosinophils may provoke the development of a prothrombotic and proinflammatory endothelial/endocardial phenotype that promotes the pronounced thrombotic diathesis characteristic of the Hypereosinophilic Syndrome.