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Heme oxygenase-1 (HO-1; encoded by Hmox1), a downstream target of the Nrf2 transcription factor, has been postulated to be a negative regulator of osteoclasts (OCLs) differentiation. Here, we further explored such a hypothesis by examining HO-1 effects in different stages of osteoclastogenesis. We confirmed the inhibition of the expression of OCLs markers by Nrf2. In contrast, both the lack of the active Hmox1 gene or HO-1 silencing in OCLs precursor cells, bone marrow macrophages (BMMs), decreased their differentiation towards OCLs, as indicated by the analysis of OCLs markers such as TRAP. However, no effect of HO-1 deficiency was observed when HO-1 expression was silenced in BMMs or RAW264.7 macrophage cell line pre-stimulated with RANKL (considered as early-stage OCLs). Moreover, cobalt protoporphyrin IX (CoPPIX) or hemin, the known HO-1 inducers, inhibited OCLs markers both in RANKL-stimulated RAW264.7 cells and BMMs. Strikingly, a similar effect occurred in HO-1−/− cells, indicating HO-1-independent activity of CoPPIX and hemin. Interestingly, plasma of HO-1−/− mice contained higher TRAP levels, which suggests an increased number of bone-resorbing OCLs in the absence of HO-1 in vivo. In conclusion, our data indicate that HO-1 is involved in the response of bone marrow macrophages to RANKL and the induction of OCLs markers, but it is dispensable in early-stage OCLs. However, in vivo HO-1 appears to inhibit OCLs formation.

Aims: Nuclear factor E2-related factor 2 (Nrf2), a key cytoprotective transcription factor, regulates also proangiogenic mediators, interleukin-8 and heme oxygenase-1 (HO-1). However, hitherto its role in blood vessel formation was modestly examined. Particularly, although Nrf2 was shown to affect hematopoietic stem cells, it was not tested in bone marrow-derived proangiogenic cells (PACs). Here we investigated angiogenic properties of Nrf2 in PACs, endothelial cells, and inflammation-related revascularization. Results: Treatment of endothelial cells with angiogenic cytokines increased nuclear localization of Nrf2 and induced expression of HO-1. Nrf2 activation stimulated a tube network formation, while its inhibition decreased angiogenic response of human endothelial cells, the latter effect reversed by overexpression of HO-1. Moreover, lack of Nrf2 attenuated survival, proliferation, migration, and angiogenic potential of murine PACs and affected angiogenic transcriptome in vitro. Additionally, angiogenic capacity of PAC Nrf2−/− in in vivo Matrigel assay and PAC mobilization in response to hind limb ischemia of Nrf2−/− mice were impaired. Despite that, restoration of blood flow in Nrf2-deficient ischemic muscles was better and accompanied by increased oxidative stress and inflammatory response. Accordingly, the anti-inflammatory agent etodolac tended to diminish blood flow in the Nrf2−/− mice. Innovation: Identification of a novel role of Nrf2 in angiogenic signaling of endothelial cells and PACs. Conclusion: Nrf2 contributes to angiogenic potential of both endothelial cells and PACs; however, its deficiency increases muscle blood flow under tissue ischemia. This might suggest a proangiogenic role of inflammation in the absence of Nrf2 in vivo, concomitantly undermining the role of PACs in such conditions. Antioxid. Redox Signal. 20, 1693–1708.