Treating VSMCs with Ang II leads to altered HIF-1α proline hydroxylation, decreased pVHL binding, reduced HIF-1α ubiquitination, and proteasomal degradation leading to increased HIF-1α stabilization (Page et al., 2008
). ROS generation plays a pivotal role in these Ang II–mediated effects on the HIF-1 system. Our current work now identifies mitochondrial-derived ROS as essential intermediates leading to the stabilization of HIF-1α, the activation of HIF-1, increased in HIF-1 target gene expression, and associated cellular effects, such as VSMC migration.
It is well accepted that the NADPH oxidase plays a primary role in VSMC biology. noxROS have been implicated in various VSMC signaling mechanisms originating from AT1
receptor activation after Ang II stimulation (Garrido and Griendling, 2009
). noxROS have been implicated in increasing HIF-1α translation (Page et al., 2002
; Lauzier et al., 2007
). Additionally, a recent study showed the involvement of the NADPH oxidase in HIF-2 stabilization (Diebold et al., 2010
). Because Ang II–induced HIF-1α stability was ROS dependent, our first studies pushed us to investigate the role of the NADPH oxidase and noxROS in HIF-1α stabilization. Under our conditions, the NADPH oxidase does not play a major role in HIF-1α accumulation. This is shown here by targeting p22phox
by RNAi and using the specific NADPH inhibitor, VAS2870. However, siRNA against other NADPH oxidase subunits, including Nox1, Nox4 and p47phox
, were equally ineffective to block HIF-1α accumulation after Ang II treatment. (D. A. Patten, E. L. Pagé, and D. E. Richard, unpublished observations). These unexpected results led us to demonstrate here that mitochondrial/complex III–generated mtROS are indispensable for HIF-1 activation by Ang II. It is important to note that the NADPH oxidase and the mitochondria are not the only ROS-generating systems in VSMCs (San Martin and Griendling, 2010
). It is possible that the activity of other oxidases and ROS generators may affect HIF-1 activation at different levels. Continued studies in this area will clarify the role of other oxidases and ROS generators on HIF-1 activation in nonhypoxic conditions.
Our previous work described that DPI, which blocks ROS generation through its inhibitory effects on flavoprotein-containing enzymes, blocked HIF-1α accumulation during Ang II treatment. We suggested this was mediated through the inhibition of the NADPH oxidase. Not surprisingly, it has been reported that DPI is not a specific NADPH oxidase inhibitor (Hutchinson et al., 2007
; Aldieri et al., 2008
). Moreover, DPI inhibits all flavin-containing enzymes, including those found in the mitochondria (Li and Trush, 1998
). Here, our work shows that DPI does indeed block mtROS generation in VSMCs during Ang II treatment and indicates that DPI blocks HIF-1α stabilization by Ang II.
Our results show that mtROS are essential for blocking HIF-1α hydroxylation and pVHL binding during Ang II treatment. It is well accepted that hydroxylation of proline residues 564 and 402 (Pro402
) of human HIF-1α regulates pVHL-mediated proteasomal degradation (Ivan et al., 2001
; Masson et al., 2001
). Further studies indicated that the modification of only one of these proline residues is sufficient to stabilize HIF-1α (Chan et al., 2005
). Our previous work demonstrated that treating VSMCs with Ang II primarily suppressed the hydroxylation of Pro402
(Page et al., 2008
). In agreement with these observations, here we show that mtROS are responsible for decreasing Pro402
hydroxylation while Pro564
hydroxylation is mostly unaffected. Taken together, our results show that increased mtROS results in an inactivation of PHD.
is known to be an important mediator of the effects of Ang II on VSMC (Zafari et al., 1998
). Our previous studies demonstrated that H2
led to the inhibition of PHD activity, as measured by reduced pVHL/HIF-1α binding (Page et al., 2008
). This indicates that superoxide, produced in the mitochondria, is transformed into H2
, which effectively inhibits PHD activity. This inhibition most probably occurs due to the Fenton reaction and the oxidation of Fe2+
, which decreases available ascorbate, leading to decreased hydroxylation of HIF-1α on Pro402
, impaired binding of pVHL to HIF-1α, and HIF-1α stabilization under normal oxygen conditions. These results also suggest a crucial role of superoxide dismutase (SOD) in HIF-1 activation by Ang II. Preliminary evidence suggest that SOD activity is indeed involved because diethylthiocarbamate (DETC), a SOD inhibitor, led to a significant inhibition of HIF-1α accumulation under Ang II treatment in VSMCs (G. A. Robitaille and D. E. Richard, unpublished observations). Further studies are needed to clearly identify the role of different SOD enzymes in HIF-1 activation. Given the importance of mtROS in HIF-1 induction, mitochondrial SOD2 may be of particular interest.
Because Ang II can activate both noxROS and mtROS in VSMCs after Ang II treatment, our results indicate a specificity of mtROS in regulating HIF-1α stabilization. The reason for this specificity for mtROS remains to be elucidated. Theoretically, mtROS differ from noxROS by only the area in which it is produced and the local expression of SOD. We hypothesize that local mtROS/H2O2 gradients exist around the mitochondria which is responsible for inhibition of PHD activity and HIF-1α stabilization signaling.
Exactly how Ang II increases mtROS remains to be elucidated. By using two ETC complex III inhibitors and a siRNA against the Rieske Fe-S, our results indicate an essential role of complex III. However, the exact sequence of events leading to complex III activation and mtROS generation after AT1
receptor activation remains unclear. In contrast to our findings, Kimura et al. (2005b)
have proposed that in cardiac tissue, mtROS are produced during Ang II treatment through NADPH oxidase-derived ROS-induced ROS release (RIRR). In other words, the initial burst of noxROS leads to increased mtROS. Our results indicate that in VSMCs, RIRR does not occur because inhibition of the NADPH oxidase does not block Ang II–induced mtROS generation as measured by MitoSOX nor does it impede HIF-1 induction. It is important to note that results from Kimura et al.
were based on data obtained using the classical NADPH oxidase inhibitor, apocynin. However, recent results now indicate that apocynin is not a simple NADPH oxidase inhibitor in vascular cells and can also act as an antioxidant (Heumuller et al., 2008
). Additionally, it was shown that the expression of a dominant negative form of Rac1 can suppress mtROS generation by Ang II (Nozoe et al., 2008
) Because Rac1 is required for proper NADPH oxidase assembly, this suggested that the NADPH oxidase is required for increasing mtROS (Griendling et al., 2000
). Although the role of Rac1 on HIF-1 induction and activation has been described (Hirota and Semenza, 2001
), Rac1 also regulates other cellular processes in VSMCs including cytoskeletal organization, gene transcription, cell proliferation and membrane trafficking through interactions with PI3K, p21-activated kinase (PAK), Ras, and p70 S6 kinase (Chou and Blenis, 1996
; Kaibuchi et al., 1999
; Liliental et al., 2000
; Sun et al., 2000
). Additionally, Rac1 has been shown to regulate HIF-1α mRNA expression through activation of the NF-κB pathway in different cell systems including VSMCs (Gorlach et al., 2003
; Diebold et al., 2008
; Kim et al., 2008
). Finally, previous studies have shown that Rac1 does not affect mtROS generation in TNF-treated endothelial cells (Deshpande et al., 2000
). Therefore, the effect of Rac1 on HIF-1 induction could potentially be attributed to any number of cellular processes not directly linked to mtROS generation. Finally, a recent study shows that inversely, mtROS can lead to NADPH oxidase activation (Lee et al., 2006
). Given these divergent studies and our results, further investigation is needed to clearly delineate the mechanisms by which mtROS generation is activated in VSMCs after Ang II treatment.
HIF-1 regulation by Ang II occurs through three independent mechanisms. Until now, we have been unable to determine which mechanism played a primordial role in HIF-1 induction and activation. Our work here partly defines the relative contribution of the increased translation, transcription, and stability of HIF-1α by Ang II. We can now speculate that increased HIF-1α stability is of primary importance for HIF-1 regulation by Ang II. Also, because the inhibition of the NADPH oxidase had little or no effect on HIF-1α accumulation, and we have previously demonstrated that noxROS are important for increased HIF-1α translation by Ang II; our results indicate that increased HIF-1α protein translation is not essential for the prolonged accumulation of HIF-1α levels under Ang II treatment (Page et al., 2002
). However, increased HIF-1α translation may be important for the rapid accumulation of HIF-1α after Ang II receptor activation.
Because mitochondrial oxidative damage is thought to contribute in a number of human degenerative diseases, the development of antioxidants that are targeted to the mitochondria has gained significant interest. Generally, mitochondrial antioxidants include an antioxidant moiety (ubiquinone, tocopherol, nitroxide) and a covalently attached lipophilic triphenylphosphonium cation that serves for specific uptake by the mitochondria (Smith et al., 2008
). MitoQ and SkQ1 are two compounds that have been developed to specifically suppress mtROS generation. These two compounds have an added advantage over other mitochondrial-targeted antioxidants because they can be regenerated by accepting electrons from the respiratory chain. MitoQ has been shown to inhibit HIF-1α accumulation during hypoxia (Bell et al., 2007
). However, recent evidence suggests that the concentration window for MitoQ's antioxidant properties is very small before displaying prooxidant properties (Antonenko et al., 2008
). Using a different antioxidant moiety (plastoquinone), SkQ1 demonstrated more potent antioxidant properties and a larger functional concentration window than MitoQ (Skulachev et al., 2009
). Here, we successfully used SkQ1 to decrease mtROS generation during Ang II treatment, which effectively inhibited HIF-1α accumulation. Our studies therefore confirm the effectiveness of mitochondrial antioxidants as inhibitors of HIF-1 activation under nonhypoxic conditions and point to interesting therapeutic leads. Our recent in vivo studies have demonstrated a potential role for nonhypoxic HIF-1 induction in vascular remodeling diseases (Lambert et al. 2010
). Mitochondrial antioxidants, such as SkQ1, will prove interesting tools for further investigation in Ang II–mediated pathophysiological effects which involve VSMC migration, such as vascular remodeling.
Mitochondrial-derived ROS have gained substantial interest in the regulation of HIF-1 induction and activation. The hypoxic activation of HIF-1α has been shown by two independent groups to be regulated by mtROS (Chandel et al., 2000
; Guzy et al., 2005
). Additionally, a nonhypoxic activator of HIF-1, thrombopoietin (TPO), has been shown to control HIF-1α levels through the generation of mtROS (Yoshida et al., 2008
). Although the NADPH oxidase has classically been shown as the primary ROS generator in VSMCs and noxROS to be involved in different signaling pathways, our study now identifies mtROS as an essential intermediate for PHD inactivation, HIF-1α stabilization, and HIF-1 activation when VSMCs are stimulated with Ang II. Taken together, these studies suggest that mitochondrial ROS are a common intermediate signal transducer between hypoxic and nonhypoxic stimuli leading to the activation of HIF-1.