The observation that ANP upregulates the expression of HO-1, reported to play a protective role in inflammation and oxidative stress, led us to focus on potential protective effects of this cardiovascular hormone on cytotoxicity induced by TNF-α, one of the key mediators in inflammatory disease. The results of the present work demonstrate that ANP does not significantly attenuate TNF-α-mediated apoptosis in HUVEC.
HO-1 induction has previously been reported to confer protection of endothelial cells against TNF-α-induced apoptosis (Brouard et al. 2000). However, most of these investigations were performed by expressing large amounts of HO-1 in the respective cells, induced by heavy metals, porphyrins or by viral transfer of the HO-1 gene. By means of such approaches it was indeed possible to demonstrate a cytoprotective effect against oxidant-induced injury (Yang et al. 1999) as well as against oxyhemoglobin-induced endothelial dysfunction (Eguchi et al. 2001). This observation is supported by studies performed in various other cell models. For instance, HO-1 overexpression by gene transfer has been reported to protect against TNF-α- induced apoptosis in murine L929 fibroblasts (Petrache et al. 2000) and to confer protection against heme/hemoglobin-induced toxicity in rabbit coronary microvessel endothelial cells (Abraham et al. 1995). Moreover, Motterlini et al. achieved protection against oxidative stress by curcumin-mediated HO-1 induction in bovine aortic endothelial cells (Motterlini et al. 2000).
On the other hand, there are groups that report that HO-1 expression does not necessarily have cytoprotective effects. This observation was recently described by Redaelli et al. who reported no protection from ischemia/reperfusion induced apoptosis despite the expression of high levels of HO-1 in rat liver grafts (Redaelli et al. 2002). Adenovirus-mediated HO-1 gene expression has even been described to stimulate apoptosis in rat vascular smooth muscle cells (Liu et al. 2002).
These contradictory data, reporting either protection by HO-1 or non-protective effects, might be ascribed to the interplay of other pathways in the signaling events determining cellular survival. In this regard, two signaling pathways were shown to play a crucial role in HO-1- mediated protection against TNF-α-induced cytotoxicity: activation of p38 MAPK (Brouard et al. 2000, Brouard et al. 2002) and activation of NF-κB (Brouard et al. 2002). Brouard et al. found the gaseous molecule CO to mediate the antiapoptotic effect of HO-1 and to act via the activation of a transduction pathway involving the activation of p38 MAPK (Brouard et al. 2000). NF-κB mediates the expression of a variety of early responsive anti-apoptotic genes after binding of TNF-α to the TNFR-1. Inhibition of NF-κB therefore promotes apoptosis induced by TNF-α, whereas NF-κB activation protects cells from cell death (Beg and Baltimore 1996, van Antwerp et al. 1996, Wang et al. 1996). Brouard et al. demonstrated that
4 DISCUSSION
HO-1/CO cooperates with NF-κB-dependent anti-apoptotic genes to protect endothelial cells from TNF-α-mediated apoptosis and that the ability of HO-1/CO to activate the p38 MAPK pathway was necessary for this effect (Brouard et al. 2002). This observation is further supported by the findings of Madrid et al. who reported that p38 MAPK together with IKK participates in the stimulation of the transactivation potential of the p65 subunit of NF-κB by the serine/threonine kinase Akt (Madrid et al. 2001).
However, both the p38 MAPK (Tsukagoshi et al. 2001, Kiemer et al. 2002) as well as the NF-
κB (Kiemer and Vollmar 1998, Kiemer et al. 2000b, Kiemer et al. 2002e) pathway have previously been shown to be inhibited by ANP. Due to the important role in cell survival attributed to these pathways, the HO-1 protein upregulated by ANP might not be sufficient to confer protection against TNF-α.
On this basis, controversial reports in the literature of either cytoprotective or cell damaging effects of ANP might also be explained. Observations exist about ANP to be either an inducer of apoptosis or to protect from cytotoxic cell damage. For instance, ANP was found to protect endothelial cells from lysophosphatidylcholine-induced cytotoxicity (Murohara et al. 1999), to attenuate kidney damage induced by different stimuli (Polte et al. 2002, Murakami et al. 1999), to reduce hepatic ischemia reperfusion injury (Gerbes et al. 1998), to prevent Kupffer cell-induced oxidant injury in the rat liver (Bilzer et al. 1999), and to reduce apoptosis in serum-deprived PC12 cells (Fiscus et al. 2001). On the other hand, there are reports that describe the induction of apoptosis in rat aortic endothelial cells (Suenobu et al. 1999) and in cardiac myocytes (Wu et al. 1997). These contradictory observations might be ascribed to the interplay of protective as well as deleterious signaling pathways modulated by ANP.
Since TNF-α alone already induces NF-κB activity and therefore the expression of antiapoptotic proteins, we tested another well-known apoptosis-inducing drug, etoposide, in the same experimental setting for its influence on NF-κB and cell viability. In contrast to TNF-α, etoposide does not activate NF-κB in HUVEC, as shown by our experiments. Besides, to our knowledge, no reports exists reporting the activation of NF-κB after etoposide in endothelial cells. Therefore, the NF-κB-regulated antiapoptotic mechanisms, described in response to TNF-α, do not influence apoptosis elicited by etoposide. Nevertheless, the results of the present work indicate that ANP does not confer significant protection against etoposide-mediated apoptosis in HUVEC, either.
Although ANP was not able to protect HUVEC against TNF-α- and etoposide-induced apoptosis, it had a strong effect on morphological changes occuring after treatment with TNF-α. The pronounced changes of cell morphology, including elongation of the cells and formation of gaps in the endothelial monolayer, were almost completely abrogated by ANP, indicating that ANP has a regulatory effect on TNF-α-exposed HUVEC. The mechanisms
4 DISCUSSION
underlying this effect were investigated in another project by Nina Weber and published by Kiemer et al. (Kiemer et al. 2002c).