Original articleConversion of biliverdin to bilirubin by biliverdin reductase contributes to endothelial cell protection by heme oxygenase-1—evidence for direct and indirect antioxidant actions of bilirubin
Introduction
“Endothelial dysfunction” has been identified as a hallmark of most cardiovascular complications [1], and it was demonstrated at a molecular level that oxidative stress is not only a by-product of cardiovascular diseases but in most cases is a cause and contributes to pathogenesis [2], [3]. These observations have important clinical implications, since patients with an impaired endothelial function have a higher risk of cardiovascular events [4].
Heme oxygenases are regarded as protective enzymes and are expressed as a constitutive isoform (HO-2) as well as an inducible isoform (HO-1) [5]. Antioxidant actions of HO-2 gained less attention in the past but especially neuronal protection seems to be a feature of this constitutive isoform [6]. For the inducible HO-1 (heat shock protein 32) it was shown that this enzyme is an essential constituent of the cellular antioxidant defense system, especially for prevention of cardiovascular and neurodegenerative disease, which also makes it an attractive target for therapeutic interventions [5], [7]. HO-1 null mice showed increased pulmonary hypertension in response to chronic hypoxia [8], pharmaceutical HO-1 induction improved diabetic complications [9], as well as nitroglycerin-induced vascular dysfunction (“nitrate tolerance”) [10]. Finally HO-1 gene transfer protected from ischemic heart damage [11]. In essence, HO-1 catalyzes the break-down of heme yielding free iron, carbon monoxide (CO) and biliverdin [5]. Free iron will induce ferritin, finally resulting in beneficial effects on the endothelium by decreased levels of free iron [12]. CO is a weak activator of sGC, a potent stimulator of BKCa channels, inhibits platelet aggregation and displays antiatherosclerotic, antiapoptotic as well as proangiogenic effects depending on the P38MAP-kinase pathway [5], [13]. Finally, biliverdin (BV) is converted to the potent antioxidant bilirubin (BR) by biliverdin reductase (BVR) [14], [15], a soluble enzyme which is expressed in all tissues in two forms: BVRA (dominant in adults) and BVRB (dominant in fetal state) [14], [16]. Although it was shown in numerous studies that CO has highly beneficial antioxidant effects (e.g. by using CO-containing air in metabolic cages or CO-releasing compounds [CORMs]) [17], [18], it cannot be excluded that, besides direct effects of CO on sGC, BK-channels and gene expression, a part of these beneficial effects is related to protection observed by hypoxia (“hypoxic preconditioning”) [19]. In summary, it is not exactly known to what extent the bilirubin and CO pathways contribute to the overall protection by HO-1, although few reports favor an important role of BVR in mediating the protective antioxidant effects of HO-1 [20], [21], [22]. However, this hypothesis is currently subject to ongoing discussion and two recent studies by Young as well as Maghzal provided pros [23], and cons for this concept [24].
With the present study, we aimed to test the role of BVR-dependent conversion of BV to BR for the overall protective antioxidant properties of the HO-1 pathway. For this purpose we used in vitro and biological models for oxidative stress. Since no specific inhibitors for BVR are known, siRNA was used to silence the BVR gene in primary endothelial cells (HUVECs) and accordingly suppress its activity. Special focus was put on the antioxidant properties of BV and BR since previous reports postulated an antioxidant catalytic cycle converting BV to BR via BVR and the reverse reaction would be mediated by reaction of BR with oxidants yielding BV. To distinguish between direct and indirect effects of BR and BV we also analyzed the effects of both compounds on endothelial expression of the BH4-synthesizing enzyme GTP-cyclohydrolase-1 (GCH-1).
Section snippets
Materials and methods
Detection of protein tyrosine nitration was performed by dot blot analysis as previously reported [25]. Superoxide-induced 2-hydroxyethidium formation was measured as described [26], [27]. The conversion (break-down) of bilirubin and biliverdin (50 µM) by various oxidants was tested by HPLC analysis. The HPLC system, flow, mobile phases and column are described in the online supplement. Human umbilical vein endothelial cells (HUVECs) were obtained from Academic Teaching Hospital in Frankfurt am
Bilirubin is a more potent inhibitor of peroxynitrite-mediated protein tyrosine nitration as compared to biliverdin
To assess the antioxidative properties of BR and BV two nitration systems for BSA were established. Tyrosine residues in BSA were either nitrated by authentic peroxynitrite or in situ generated ONOO− from the thermal decomposition of Sin-1, a more physiological model to assess peroxynitrite-mediated oxidations. Peroxynitrite-mediated tyrosine nitration involves tyrosyl-radical-intermediates as a consequence of homolytic bond cleavage in ONO-OH yielding hydroxyl (HO•) and nitrogen dioxide (•NO2)
Discussion
In the present study we show that BR at high concentrations is a superior antioxidant as compared to BV. Silencing of BVR caused increased oxidative stress and damage in HUVECs challenged by LPS indicating that chronic indirect antioxidant effects of BR may be as important as direct RONS scavenging capacity. In key experiments we could demonstrate that the protective effect of hemin-dependent induction of HO-1 is partially lost in BVR-deficient cells and completely lost in HO-1-silenced cells.
Acknowledgments
We thank Angelica Karpi, Nicole Papanaiou and Nicole Schramm for expert technical assistance and Margot Neuser as well as Thilo Weckmüller for graphical assistance. The support by the midwives of the Academic Teaching Hospital in Frankfurt am Main/Höchst and St. Vincenz Hospital in Mainz is gratefully acknowledged. The present work was supported by generous financial support by the Johannes Gutenberg University and School of Medicine Mainz (MAIFOR and Forschungsfonds grants to A.D.), by the
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T.J. and M.H. contributed equally to this study and should therefore both be considered as first author.