Mercedes Camacho¹, Jose Martinez-Gonzalez², Cristina Rodriguez², Laura Siguero¹, Cristina Seriola¹, Jose-Maria Romero^1,3, Luis Vila¹

1Laboratory of Angiology, Vascular Biology and Inflammation, Institute of Biomedical Research, II-B Sant Pau, c/ Antoni Ma Claret 167, 08025 Barcelona, Spain, ²Centro de Investigacion Cardiovascular (CSIC-ICCC), Institute of Biomedical Research , II-B Sant Pau, c/ Antoni Ma Claret 167, 08025 Barcelona, Spain, ³Vascular Surgery Department, Institute of Biomedical Research, II-B Sant Pau, c/ Antoni Ma Claret 167, 08025 Barcelona, Spain

TABLE OF CONTENTS

1. Abstract

2. Introduction

3. Materials and methods

3.1. Synthesis of cPTIO and cPTI

3.2. Cell culture and treatment

3.3. Determination of arachidonic acid metabolism from endogenous substrate mobilized with thrombin

3.4. COX-1 and COX-2 protein levels

3.5. COX-2 mRNA levels

3.6. NO-dependence of the induction of COX-2 expression by cPTIO

3.7. Effect of radical scavengers on the induction of COX-2 expression by cPTIO

3.8. Signalling pathways involved in cPTIO-induced expression of COX-2

3.9. Statistics

4. Results and discussion

4.1. Prostanoid release by HUVEC exposed to cPTIO

4.2. Effect of cPTIO on the expression of COX-isoenzymes

4.3. Role of NO in the up-regulation of COX-2 expression induced by cPTIO

4.4. Redox states of cPTIO in HUVEC

4.5. Role of reactive oxygen species in the up-regulation of COX-2 by cPTIO

4.6. PGE2 released in response to cPTIO is synthesized by COX

4.7. Generation of nitrosylating species by cPTIO

4.8. Signalling pathways involved in the cPTIO-induced expression of COX-2

5. Acknowledgments

6. References

1. ABSTRACT

cPTIO (2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide) exerts beneficial actions on systemic inflammatory response. Besides its nitric oxide (NO) scavenging properties cPTIO could exert beneficial effects through modulation of arachidonic acid metabolism. We studied the effect of cPTIO on the biosynthesis of vasoactive prostaglandins (PG) by endothelial cells. Human cord umbilical vein endothelial cells (HUVEC) were treated with cPTIO, and expression of cycloxygenase (COX) isoenzymes in terms of mRNA and protein was determined by real-time-PCR and immunoblotting. Release of PGE₂ (as index of untransformed PGH₂ release) and 6-oxo-PGF_1alpha (PGI₂ stable metabolite) was determined by enzyme-immunoassay. cPTIO significantly increases the release of untransformed PGH₂ associated to the induction of COX-2 expression. Experiments with NO-synthase inhibitors and radical scavengers showed that induction of COX-2 by cPTIO was mediated by free radical species, likely caused by the mobilization of NO from cellular stores. Finally, using specific signal-transduction inhibitors we show the involvement of Src/PI3-K/PKC pathway. Additional effects other than a direct NO scavenging activity may confer therapeutic advantages to cPTIO as compared with NO-synthase inhibitors for the treatment of systemic inflammation-associated vascular hyporeactivity.