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Background: Heme oxygenase-1 (HO-1), en enzyme converting heme to carbon monoxide, iron and biliverdin has been recently implicated in regulation of angiogenesis. HO-1 expression can be achieved by stimulation with cobalt protoporphyrin (CoPPIX) or cobalt chloride (CoCl2). CoCl2 also mimics hypoxia, activating hypoxia inducible factor-1 (HIF-1). Here we determined the role of HO-1 in regulation of the expression of VEGF-A and PDGF-BB as well as IL-1β, IL-6 and IL-8 in human microvascular endothelial cells (HMEC-1). Results: CoPPIX induced HO-1 expression and strongly enhanced VEGF and IL-8 production, through the activation of VEGF and IL-8 promoters, as determined by ELISA and reporter gene assay, respectively. IL-6 protein synthesis was also highly up-regulated. Inhibition of HO activity by tin protoporhyrin (SnPPIX) decreased VEGF production, while, interestingly, it affected neither IL-6 nor IL-8 production. Regulation of IL-8 production after CoPPIX treatment may involve p38 MAPK, MEK1/2 and JNK kinase as IL-8 synthesis was diminished by specific kinase inhibitors SB202190, PD98059 and SP600125, respectively. Synthesis of VEGF was down-regulated by MEK1/2 inhibition. Notably, HO-1 induction by CoPPIX did not affect PDGF-BB and IL-1β production. CoCl2 induced HO-1 expression and enhanced VEGF synthesis, but it did not affect IL-8 production. However, in contrast to CoPPIX, the effect of CoCl2 on VEGF synthesis is not reversed by SnPPIX and seems to be mostly mediated by the activation of hypoxia-responsive element (HRE) of VEGF promoter. Stimulation of HRE, and concomitantly VEGF synthesis by CoCl2 is related to the enhancement of production of reactive oxygen species (ROS), determined by dichlorofluorescein oxidation. Treatment with N-acetylcysteine reversed CoCl2 effects. On the other hand, CoPPIX did not induce ROS generation neither it affected HRE of VEGF promoter. Conclusions: Our data indicate that production of VEGF is enhanced by induction of HO-1, while the enhancement of synthesis of IL-6 and IL-8 is not reliant on that pathway. In turn, PDGF-BB and IL-1β expression did not change after HO-1 induction. Interestingly, although both CoCl2 and CoPPIX induce HO-1, the effect of CoCl2 on VEGF does not involve HO-1 and is dependent on HIF-1 transcription factor, while the effect of CoPPIX does not involve HIF-1 but relies on HO-1. Supported by grant PBZ-KBN 107/P04/2004.
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J. Vasc. Biol. 42, Sup:2 (2005) p6