Oxidative stress-driven expression of myeloperoxidase in endothelial cells results in accumulation of markers of oxidative and nitrosative stress in vitro and in vivo

Endothelial cells (ECs), are able to manage with higher concentrations of ROS as well as reactive nitrogen species, respect to the other cell types. The unbalance between oxidizing species and antioxidant cellular defences has been implicated in the pathogenesis of cardiovascular diseases. While the role of MPO in the oxidative burst is well established, the effects of this enzyme on endothelial biology have only recently come to light. MPO can interact with endothelial –derived NO causing its depletion and favouring its conversion to nitrogen dioxide radical, a compound responsible of protein nitration generating 3-nitrotyrosine. Moreover, MPO is the only enzyme capable to form hypoclorous acid (HClO) at physiological concentrations of halides. For the present study we used primary cultures of human umbilical vein endothelial cells (HUVEC) and human endocardial endothelial cells (EEC), which were isolated respectively from umbilical cord vein and from the endocardial layer of hearts from patients affected by chronic heart failure (CHF). HUVEC and EEC were subjected to oxidative stress by incubation with H2O2 (60 μM) for 3 and 6 h. Treated and control cells were tested for MPO and 3 chlorotyrosine immunopositivity by immunocytochemistry (ICC) and MPO expression at the mRNA level by RT-PCR. HUVEC and EEC were isolated as homogeneous endothelial populations. LDH assays confirmed that the dose of hydrogen peroxide used was non-lethal. MPO was expressed only in treated ECs and absent in control ones. Moreover, 6h treatment showed a significant MPO increase compared to 3h treatment for HUVEC alone. ICC for 3-chlorotyrosine showed that only treated HUVEC and EEC were positively stained after 3h and 6h H2O2 exposure. Moreover after 6h we observed a significant increase in positivity ICC results were confirmed by detection of MPO mRNA only in treated cells. Moreover, we confirmed our in vitro results by in vivo detection of MPO, 3-chlorotyrosine and 3-nitrotyrosine by immunohistochemistry in the endocardial layer of hearts from CHF affected patients. Moreover, since prostacyclin synthase is one of the most prevalent nitrated proteins, we confirmed, by co-staining in immunofluorescence experiments, that this proteins is one of the potential targets of nitration in post-infarct CHF hearts This study demonstrated for the first time the expression of MPO by ECs after oxidative stress, both in vitro and in vivo. We can only speculate on the mechanism underlying MPO induction in endothelial cells following hydrogen peroxide exposure. Further experiments are being performed to establish if direct or indirect mechanisms take place in ECs exposed to oxidative stress.