Transcriptional Upregulation of Mitochondrial Uncoupling Protein 2 Protects Against Oxidative Stress-Associated Neurogenic Hypertension
Rationale: Mitochondrial uncoupling proteins (UCPs) belong to a superfamily of mitochondrial anion transporters that uncouple ATP synthesis from oxidative phosphorylation and mitigates mitochondrial reactive oxygen species production.
Objective: We assessed the hypothesis that UCP2 participates in central cardiovascular regulation by maintaining reactive oxygen species homeostasis in the rostral ventrolateral medulla (RVLM), where sympathetic premotor neurons that maintain vasomotor tone located. We also elucidated the molecular mechanisms that underlie transcriptional upregulation of UCP2 in response to oxidative stress in RVLM.
Methods and Results: In Sprague–Dawley rats, transcriptional upregulation of UCP2 in RVLM by rosiglitazone, an activator of its transcription factor peroxisome proliferator-activated receptor (PPAR)γ, reduced mitochondrial hydrogen peroxide level in RVLM and systemic arterial pressure. Oxidative stress induced by microinjection of angiotensin II into RVLM augmented UCP2 mRNA or protein expression in RVLM, which was antagonized by comicroinjection of NADPH oxidase inhibitor (diphenyleneiodonium chloride), superoxide dismutase mimetic (tempol), or p38 mitogen-activated protein kinase inhibitor (SB203580) but not by extracellular signal-regulated kinase 1/2 inhibitor (U0126). Angiotensin II also induced phosphorylation of the PPARγ coactivator, PPARγ coactivator (PGC)-1α, and an increase in formation of PGC-1α/PPARγ complexes in a p38 mitogen-activated protein kinase–dependent manner. Intracerebroventricular infusion of angiotensin II promoted an increase in mitochondrial hydrogen peroxide production in RVLM and chronic pressor response, which was potentiated by gene knockdown of UCP2 but blunted by rosiglitazone.
Conclusions: These results suggest that transcriptional upregulation of mitochondrial UCP2 in response to an elevation in superoxide plays an active role in feedback regulation of reactive oxygen species production in RVLM and neurogenic hypertension associated with chronic oxidative stress.
- uncoupling proteins
- peroxisome proliferator-activated receptor
- oxidative stress
- blood pressure
Living organisms possess a variety of physiological protective mechanisms to counteract oxidative stress and to restore redox balance. Oxidative damage to cells that results from an imbalance of production over degradation of the reactive oxygen species (ROS), particularly superoxide anion (O2−·) and hydrogen peroxide (H2O2), is associated with a variety of cardiovascular diseases, including heart failure, atherosclerosis, and hypertension.1–3 Of note is that overproduction of O2−· and H2O2 in the central nervous system contributes to neural mechanisms of hypertension by increasing sympathetic outflow to the peripheral blood vessels.4,5
In addition to the degradative enzymes (eg, superoxide dismutase [SOD] and catalase) and low-molecular-weight antioxidants (eg, ascorbic acid and glutathione), the uncoupling proteins (UCPs) have emerged as important natural antioxidants in the maintenance of ROS homeostasis.6 UCPs belong to a superfamily of mitochondrial anion transporters that uncouple ATP synthesis from oxidative phosphorylation by causing proton leakage across the mitochondrial inner membrane, leading to energy dissipation and heat production.7 More importantly, the resultant decrease in proton electrochemical gradient across the inner mitochondrial membrane elicited by the UCPs mitigates mitochondrial ROS production.6,8 In mammals, 5 homologues, UCP1 to UCP5, have so far been cloned.9 Among them, dysfunction of UCP2 is suggested to be of considerable importance in cardiovascular pathophysiology associated with oxidative stress. Knockdown of ucp2 gene increases mitochondrial membrane potential and ROS production in murine endothelial cells.10 Oxidative stress is also greater in the thoracic aorta of mice that are subject to bone marrow transplant derived from UCP2−/− mice.11 Adenovirus-mediated overexpression of UCP2, on the other hand, decreases ROS generation in human aortic endothelial cells.12
In the rostral ventrolateral medulla (RVLM), where sympathetic premotor neurons for the maintenance of vasomotor tone are located,13 emerging evidence supports a pivotal role for oxidative stress in neural mechanism of hypertension.4,5,14 On the other hand, whereas UCP2 is expressed in a wide array of tissues, including the brain,15 the physiological significance of its antioxidant role in central regulation of cardiovascular phenotypes is yet to be identified. The present study was undertaken to assess the hypothesis that mitochondrial UCP2 participates in central cardiovascular regulation by maintaining ROS homeostasis in RVLM. We also elucidated the molecular mechanisms that underlie transcriptional regulation of UCP2 expression in response to oxidative stress in RVLM.
An expanded Methods section is available in the Online Data Supplement http://circres.ahajournals.org.
Experiments were carried out in adult male Sprague–Dawley rats (280 to 305 g, n=345) purchased from the Experimental Animal Center of the National Applied Research Laboratories, Taiwan. All experimental procedures were carried out in compliance with the guidelines of our institutional animal care committee.
PPARγ-Dependent Transcriptional Upregulation of Mitochondrial UCP2 in RVLM
Upregulation of mitochondrial UCP2 expression in RVLM was elicited by microinjection bilaterally of an activator of its transcription factor PPARγ, rosiglitazone into this medullary site. Ligand specificity was ascertained by comicroinjection of a selective PPARγ inhibitor, GW9662. The temporal changes in UCP2 mRNA or protein expression in RVLM after rosiglitazone treatment were determined by real-time RT-PCR and Western blot analysis.
Reduction in Oxidative Stress and Arterial Pressure After Transcriptional Upregulation of Mitochondrial UCP2 in RVLM
The effect of transcriptional upregulation of UCP2 on mitochondrial level of H2O2 in RVLM and basal systemic arterial pressure (SAP) was examined at various time intervals after microinjection bilaterally into RVLM of rosiglitazone. H2O2 in the mitochondrial fraction was measured by an Amplex Red Hydrogen Peroxide/Peroxidase Assay Kit (Molecular Probe, Eugene, Ore). SAP was determined under conscious conditions by radiotelemetry.16 The specificity of PPARγ-induced transcriptional upregulation of UCP2 on H2O2 production in RVLM or SAP was confirmed by coadministration of GW9662.
Transcriptional Upregulation of Mitochondrial UCP2 Expression in RVLM by O2−·
We reported previously that angiotensin II (Ang II) induces O2−· production in RVLM via activation of NADPH oxidase.17 To evaluate the role of NADPH oxidase–derived O2−· in the expression of mitochondrial UCP2, Ang II was microinjected bilaterally into RVLM, alone or together with a NADPH oxidase inhibitor, diphenyleneiodonium chloride (DPI) (1 nmol); a SOD mimetic, tempol (50 nmol); or by gene knockdown of p22phox or p47phox subunit of the NADPH oxidase with their respective antisense oligonucleotide (ASON) (100 pmol).17 The involvement of cytosolic or mitochondrial O2−· in Ang II–induced mitochondrial UCP2 expression was further confirmed in animals that received gene transfer into RVLM of adenovirus encoding the cytosolic copper/zinc SOD (AdSOD1), mitochondrial manganese SOD (AdSOD2), or catalase (AdCAT).5 The specificity and efficacy of p22phox and p47phox ASON17 and AdSOD1, AdCAT, or AdSOD25 have been characterized in our previous studies.
Involvement of Mitogen-Activated Protein Kinases in Transcriptional Upregulation of UCP2 in RVLM by O2−·
We reported previously16,17 that activation of p38 mitogen-activated protein kinase (p38 MAPK) and extracellular signal-regulated protein kinase (ERK)1/2 by NADPH oxidase–derived O2−· mediates the cellular responses to Ang II in RVLM. To examine the engagement of these 2 signaling molecules in transcriptional regulation of UCP2 by O2−·, the temporal expression of UCP2 mRNA or protein in RVLM was determined on coadministration of p38 MAPK or ERK1/2 inhibitor SB203580 or U0126 bilaterally into RVLM with Ang II.
Phosphorylation of PGC-1α by MAPKs and UCP2 Upregulation in RVLM
We determined whether PGC-1α, a PPAR-interacting protein for the induction of UCP2,18 is a target of the activated MAPKs that leads to UCP2 upregulation in RVLM. Expression of phosphorylated PGC-1α or the formation of PGC-1α/PPARγ complex in nuclear protein extracted from RVLM was determined by Western blot analysis or immunoprecipitation followed by immunoblot, after microinjection bilaterally in RVLM of Ang II, alone or together with SB230580 or U0126.
Protective Role for UCP2 Against Ang II–Induced Pressor Response and Oxidative Stress in RVLM
We established a protective role for O2−·-dependent transcriptional activation of mitochondrial UCP2 in central cardiovascular regulation and ROS homeostasis in RVLM by determining the effect of microinjection into the bilateral RVLM or intracisternal infusion (1 or 5 μg · μL−1 · h−1) by osmotic minipump of rosiglitazone or an ASON against UCP2 on pressor responses to chronic intracerebroventricular (ICV) infusion of Ang II for 7 days and the elicited elevation in H2O2 in RVLM.
ROS Production and SAP After Transcriptional Downregulation of Mitochondrial UCP2 in RVLM
We investigated whether mitochondrial UCP2 in RVLM participates in tonic regulation of tissue level of ROS or SAP by determining the effect of microinjection bilaterally into RVLM of UCP2 ASON on mitochondrial level of H2O2 or SAP. The effectiveness of gene knockdown of UCP2 was confirmed by real-time RT-PCR analysis or Western blot analysis.
Mitochondrial Respiratory Enzyme Activity and Tissue ATP Content After Transcriptional Up- or Downregulation of Mitochondrial UCP2 in RVLM
The effect of microinjection bilaterally into RVLM of rosiglitazone or UCP2 ASON on enzyme activity of mitochondrial respiratory complex I to V, electron-coupling capacity between respiratory complexes, or tissue level of ATP in RVLM was assessed. Enzyme activity of mitochondrial respiratory complex I to V, NADH cytochrome c reductase (marker for electron coupling capacity between complexes I and III) or succinate cytochrome c reductase (marker for electron coupling capacity between complexes II and III) was determined by spectrophotometry.19 Tissue ATP concentration was measured by an ATP bioluminescence assay.19
Data are expressed as means±SEM. The statistical software SigmaStat (SPSS, Chicago, Ill) was used for data analysis. One-way or 2-way ANOVA with repeated measures was used to assess group means, as appropriate, to be followed by the Scheffé multiple-range test for post hoc assessment of individual means. P<0.05 was considered statistically significant.
Transcriptional Upregulation of Mitochondrial UCP2 in RVLM by PPARγ Activator
The fundamental premise for UCP2 to play a role in ROS homeostasis in RVLM and cardiovascular regulation is its presence in this neural substrate. Results from our first series of experiments established this premise by showing a moderate basal expression of UCP2 mRNA or protein in the mitochondrial fraction of tissues from RVLM (Figure 1). We further showed that determined 6 or 12 hours postinjection, microinjection bilaterally into RVLM of rosiglitazone (1 or 5 nmol), an activator of the transcription factor PPARγ, significantly upregulated UCP2 mRNA (Figure 1A) or protein (Figure 1B) expression. Comicroinjection bilaterally into RVLM of a PPARγ antagonist, GW9662 (100 or 500 pmol), discernibly attenuated the induced upregulation of UCP2 protein expression by rosiglitazone (5 nmol) (Figure 1C). GW9662 alone, however, did not affect UCP2 protein level in RVLM.
Transcriptional Activation of Mitochondrial UCP2 Decreases H2O2 Level in RVLM and Reduces SAP
Our second series of experiments investigated whether transcriptional upregulation of UCP2 regulates tissue level of ROS in RVLM. Given the abundance of manganese SOD in the mitochondria that rapidly dismutates O2−· to H2O2,20 the latter was used as an index of ROS production. Activation of UCP2 by rosiglitazone (1 or 5 nmol) significantly decreased H2O2 level in the mitochondrial fraction of RVLM (Figure 2A). The reduction in mitochondrial H2O2 detected 12 hours after administration of the PPARγ activator (5 nmol) was significantly reversed by comicroinjection into RVLM of GW9662 (100 or 500 pmol) (Figure 2B). Moreover, our third series of experiments showed that microinjection of rosiglitazone (1 or 5 nmol) bilaterally into RVLM resulted in a dose-related decrease in mean (M)SAP (Figure 3A), as measured by radiotelemetry under conscious conditions. This rosiglitazone-induced vasodepressor response, which was significant for at least 27 hours and returned to baseline 48 hours postinjection, was reversed by coadministration of GW9662 (500 pmol) into RVLM (Figure 3B). Transcriptional activation of UCP2, on the other hand, exerted minimal effect on HR (data not shown). Microinjection of rosiglitazone into areas adjacent to the confines of RVLM (eg, lateral reticular nucleus or spinal trigeminal nucleus) did not affect basal MSAP (+3.6±2.4 versus −4.5±3.1 mm Hg, n=5) or HR (−5±3 versus −7±4 bpm, n=5). Furthermore, we found in a separate experiment that pressor response induced by l-glutamate (2 nmol) in RVLM of anesthetized animals was comparable before and after microinjection of rosiglitazone (+16.3±2.5 versus +18.7±3.0 mm Hg, n=4) into the RVLM.
NADPH Oxidase–Derived O2−· Induces Transcriptional Upregulation of Mitochondrial UCP2 in RVLM
In our search for cellular signals that upregulate UCP2, we noted that an elevated production of O2−· induces mitochondrial UCP expression.21 Our previous work17 further indicated that activation of NADPH oxidase by Ang II is an important source of O2−· in RVLM. The fourth series of experiments therefore investigated whether NADPH oxidase–derived O2−· upregulates mitochondrial UCP2 transcription in RVLM. Microinjection bilaterally into RVLM of Ang II (100 pmol) resulted in upregulation of UCP2 mRNA (Figure 4A) or protein (Figure 4B) expression in RVLM that endured at least 24 hours. The Ang II–induced increase in UCP2 mRNA or protein level, detected 12 or 24 hours postinjection, was significantly antagonized by coadministration into RVLM of a NADPH oxidase inhibitor, DPI (1 nmol), or a SOD mimetic, tempol (50 nmol). In addition, the Ang II–induced mitochondrial UCP2 protein upregulation was also blunted in animals that received p22phox or p47phox ASON (100 pmol; Figure 4C), microinjected bilaterally into the RVLM 24 hours before Ang II administration, or in animals that received AdSOD1 or AdSOD2 (Figure 4D), but not AdCAT, 7 or 14 days after gene transfer. The specificity of our finding was further ascertained by the absence of the membranous p22phox or cytosolic p47phox in the mitochondrial fraction, and selective inhibition of protein expression by their respective ASON in the membranous or cytosolic fraction from RVLM (Online Figure I).
Involvement of p38 MAPK in UCP2 Upregulation in RVLM by NADPH Oxidase-Derived O2−·
We reported previously16,17 that phosphorylation of p38 MAPK and ERK1/2 via NADPH oxidase–derived O2−· mediates Ang II–induced cellular responses in RVLM. Thus, our fifth series of experiments examined the roles of p38 MAPK or ERK1/2 in transcriptional upregulation of UCP2 by NADPH oxidase–derived O2−· in RVLM. Coadministration bilaterally into RVLM of p38 MAPK inhibitor, SB203580 (500 nmol), but not ERK1/2 inhibitor, U0126 (500 nmol), significantly antagonized the Ang II–induced upregulation of mitochondrial UCP2 mRNA (Figure 5A) or protein (Figure 5B) expression. Neither inhibitor, however, affected the basal expression of UCP2 mRNA or protein in RVLM.
Phosphorylation of PGC-1α by p38 MAPK is Essential for UCP2 Upregulation in RVLM
By being phosphorylated on activation of p38 MAPK,22 the nuclear coactivator PGC-1α, a PPAR-interacting protein for the induction of UCP2,18 presents itself as a reasonable interposing signal in the cascade of events that lead to transcriptional upregulation of UCP2 by NADPH oxidase–derived O2−·. Microinjection bilaterally into RVLM of Ang II (100 pmol) induced significant phosphorylation of PGC-1α at its threonine residue (Figure 6A) and an increase in the expression of PGC-1α/PPARγ complexes (Figure 6B) in RVLM, examined 15, 30, or 60 minutes posttreatment. This Ang II–induced PGC-1α phosphorylation (Figure 6A) or the increase in association between PGC-1α/PPARγ (Figure 6B), detected 30 minutes posttreatment, was significantly blunted by tempol (50 nmol) or SB203580 (500 nmol) but not U0126 (500 nmol).
Superoxide-Dependent Upregulation of Mitochondrial UCP2 in RVLM During Chronic Oxidative Stress
We reported previously that chronic infusion of Ang II induces p38 MAPK phosphorylation and oxidative stress in RVLM.16 Our seventh series of experiments therefore examined whether expression of mitochondrial UCP2 is regulated by O2−· under this condition of chronic oxidative stress. Compared with animals that received artificial cerebrospinal fluid (aCSF) infusion, protein expression of UCP2 in RVLM was significantly upregulated 7 days after animals were subject to ICV infusion of Ang II (100 μg · μL−1 · h−1) (Online Figure II). This Ang II–induced UCP2 upregulation was blunted by microinjection bilaterally into RVLM of an Ang II type 1 receptor antagonist, losartan (2 nmol), or by gene transfer of AdSOD1 or AdSOD2 into RVLM (Online Figure II).
UCP2 Protects Against Chronic Oxidative Stress in RVLM and Hypertension
Our eighth series of experiments established a causal role for O2−·-dependent transcriptional activation of mitochondrial UCP2 in central cardiovascular regulation and ROS homeostasis in RVLM. Consistent to our previous findings,16 chronic ICV infusion of Ang II (100 μg · μL−1 · h−1) for 7 days elicited a gradual increase in MSAP that became significant between days 5 and 7 (Figure 7A). The Ang II–induced long-term pressor response was significantly potentiated in animals subject to microinjection bilaterally into RVLM of UCP2 ASON (100 pmol), but not UCP2 sense oligonucleotide (SON) (100 pmol), given on day 4 after infusion of the octapeptide. On the other hand, concomitant intracisternal infusion of rosiglitazone (1 or 5 μg · μL−1 · h−1) by osmotic minipump for 7 days, which increased UCP2 protein expression in the mitochondrial but not cytosolic fraction from RVLM (Online Figure III), significantly attenuated the long-term pressor response (Figure 7B) that was reversed by UCP2 ASON treatment (Figure 7C). In addition, whereas gene knockdown of UCP2 (100 pmol) in RVLM enhanced, chronic infusion of rosiglitazone blunted the elevated tissue level of mitochondrial H2O2 in RVLM detected on day 7 after Ang II infusion (Figure 7D).
Endogenous UCP2 Does Not Tonically Affect Mitochondrial H2O2 Level in RVLM or SAP
We further used gene knockdown to decipher whether endogenous UCP2 in RVLM exerts a tonic regulatory effect on ROS level and cardiovascular functions. Microinjection bilaterally into RVLM of UCP2 ASON (100 pmol), similar to SON (100 pmol), elicited no significant alterations in mitochondrial H2O2 level in RVLM, examined 6, 12, or 24 hours posttreatment (Online Figure IV, A). Likewise, basal MSAP (Online Figure IV, B) or HR (data not shown) monitored by radiotelemetry in animals under conscious condition over 24 hours was not significantly affected. Real-time RT-PCR and Western blotting analysis confirmed that UCP2 ASON treatment, but not SON, effectively suppressed UCP2 mRNA (Online Figure IV, C) or protein (Online Figure IV, D) expression in RVLM during the same posttreatment intervals.
Transcriptional Up- or Downregulation of Mitochondrial UCP2 in RVLM Does Not Affect Mitochondrial Respiratory Enzyme Activity or ATP Production
Enzyme activity of mitochondrial respiratory complex I to V, NADH cytochrome c reductase, or succinate cytochrome c reductase, as well as tissue ATP content in RVLM, assessed 12 or 24 hours after microinjection bilaterally into RVLM of rosiglitazone (5 nmol) or UCP2 ASON (100 pmol), was comparable to those from their corresponding control groups (Online Figure V).
The present study provided novel evidence for an active role for mitochondrial UCP2 in RVLM in ROS homeostasis and central cardiovascular regulation under conditions of oxidative stress. Our results support the notion that mitochondrial UCP2 participates actively in a cellular adaptive program for feedback control of ROS production in RVLM and the associated neurogenic hypertension during oxidative stress. To our knowledge, this is the first report that unveils the functional significance of mitochondrial UCP2 in protection against brain oxidative stress-associated hypertension.
Several antioxidant systems are present in the cell to counteract oxidative effects and to restore redox balance. In addition to the documented ROS scavenging enzymes and low molecular weight antioxidants, whether mitochondrial UCP functions as a natural antioxidant defense against oxidative stress is still debatable.23 The present study provided novel in vivo evidence to support an antioxidant role for UCP2 and revealed its functional significance in neural control of cardiovascular phenotype. We found in RVLM, where mitochondrial oxidative stress plays a pivotal role in neural mechanism of hypertension,19 that the rosiglitazone-promoted decrease in H2O2 level was completely reversed by the PPARγ inhibitor GW9662 at a time point when MSAP was statistically insignificant from vehicle controls. Furthermore, observations of temporally correlated sequential upregulation of UCP2 mRNA and protein by rosiglitazone at low dose, together with the antagonism of the prolong duration of rosiglitazone-induced hypotension by gene knockdown of UCP2, strongly support the notion that transcriptional upregulation of mitochondrial UCP2 underpins the reduction in mitochondrial H2O2 and the induced hypotension. These observations also deemed unlikely the possibility that the effects demonstrated are the consequences to a potential pleiotropic action of rosiglitazone.24 A less than 20% decrease in mitochondrial membrane potential by UCP is able to inhibit H2O2 production by more than 50%.25 Mitochondrial ROS production in the brain is also significantly lower in transgenic mice that overexpress UCP226 or after adenovirus-mediated gene transfer of UCP2 in aortic endothelial cells.12
It is generally accepted that O2−· induces expression of UCPs, including UCP2,21 although the underlying cellular events remain largely unknown. In this regard, we reported previously17 that Ang II induces O2−· in RVLM via activation of NADPH oxidase. The present study took advantage of this cellular event to unveil the molecular mechanism that underlies the O2−·-dependent transcriptional upregulation of mitochondrial UCP2. We found that Ang II at a dose that induces O2−· production17 also increased UCP2 mRNA and protein expression in RVLM. The delayed antagonism of Ang II–induced UCP2 protein upregulation 24 hours after treatment with the NADPH oxidase inhibitor DPI or the SOD mimetic tempol, together with the blockade of Ang II–induced UCP2 mRNA expression 12 hours after the same treatment, again suggests that NADPH oxidase–derived O2−· regulates UCP2 protein expression at the transcription level. It is intriguing to note that the Ang II–induced UCP2 upregulation was significantly blunted by p22phox or p47phox ASON and gene transfer of AdSOD1, indicating that O2−· derived from extramitochondrial compartments may play an active role in transcriptional upregulation of mitochondrial UCP2 in RVLM. The extramitochondrial origin of the NADPH oxidase–derived O2−· was further confirmed by observations that p22phox and p47phox subunits are only found in the membranous and cytosolic fractions from RVLM. In myocardium of the failing heart, upregulation of UCP2 is closely associated with an increase in NADPH oxidase–derived O2−·.27 Furthermore, we found that dismutation of O2−· to H2O2 by the SOD1 transgene reversed, whereas conversion of H2O2 to H2O after overexpression of catalase by gene transfer did not affect the Ang II–induced UCP2 upregulation. These observations are interpreted to suggest that it is Ang II–induced O2−· but not H2O2 that induces the expression of mitochondrial UCP2 in RVLM. It is noteworthy that Ang II–induced UCP2 upregulation was also appreciably blunted by overexpression of SOD2 transgene in the RVLM. These results indicate that in addition to extramitochondrial sources, UCP2 expression may be regulated by O2−· generated in the mitochondrial compartment. We recognize that O2−· derived from xanthine oxidase increases the expression of UCP3, a homolog of UCP2 in skeletal muscle cells.28 Whereas its role in O2−·-dependent transcriptional upregulation of mitochondrial UCP2 in RVLM remains to be identified, we noted that that xanthine oxidase plays a minor role in Ang II–induced O2−· production.17,29
The O2−·-induced UCP2 expression is not antagonized by inhibitors of mitochondrial ATP-sensitive potassium channel, adenine translocase, or mitochondrial permeability transition pores, suggesting that it is specific to the UCP.20 We reported previously16,17 that activation of p38 MAPK and ERK1/2 underlies the manifestation of Ang II–induced cellular responses in RVLM via NADPH oxidase–derived O2−·. Of those 2 MAPKs, the present study demonstrated that p38 MAPK, but not ERK1/2, acts as a key regulator of O2−·-dependent UCP2 transcription in RVLM under oxidative stress. We found that p38 MAPK inhibitor (SB203580), but not ERK1/2 inhibitor (U0126), significantly prevented the Ang II–induced upregulation of mitochondrial UCP2 mRNA or protein. Our results further indicated that the induction of UCP2 transcription by p38 MAPK is accomplished at 2 levels: phosphorylation of threonine residue of the PPARγ coactivator, PGC-1α, and enhancement of the ability of the latter to bind with PPARγ. Activated p38 MAPK directly phosphorylates PGC-1α at its threonine 262, serine 265, and threonine 298 residues.25 The resultant potentiation of PGC-1α docking to PPARγ25 leads to a conformational change that permits the induction of UCPs by its transcription factors.30 Our observations of an increase in formation of PGC-1α/PPARγ complex in nuclear extract from RVLM after Ang II in a p38 MAPK–dependent manner are in line with those observations.
Functional evaluations in the present study further demonstrated, for the first time, that O2−·-dependent transcriptional upregulation of mitochondrial UCP2 in RVLM plays an active role in feedback antagonism of oxidative stress and the associated hypertension. Production of O2−· and H2O2 via activation of NADPH oxidase in RVLM plays an important role in mediating chronic pressor response after ICV infusion of Ang II.16 Using a loss-of-function approach, we found that Ang II–induced production of mitochondrial H2O2 and chronic pressor response were further augmented after gene knock down of UCP2 in RVLM by its ASON. Conversely, gain-of-function experiments revealed that transcriptional upregulation of mitochondrial UCP2 in RVLM by acute and chronic administration of the PPARγ activator rosiglitazone attenuated the tissue level of H2O2 and ameliorated the pressor response induced by Ang II. In addition, UCP2 gene knockdown attenuated the rosiglitazone-promoted inhibition of Ang II–induced hypertension. These results, together with our demonstration of a minor role for endogenous UCP2 at RVLM in regulation of basal ROS production and SAP, strongly support the notion that O2−·-dependent transcriptional upregulation of mitochondrial UCP2 in RVLM participates actively in a feedback adaptive program to reduce ROS production in RVLM and hypertension associated with chronic oxidative stress. Rosiglitazone treatment was reported to reduce blood pressure in hypertensive patients31 via reduction in ROS production in the vascular smooth muscle cells32 or endothelial cells.33 Our observations that rosiglitazone-induced vasodepressor response returned to baseline values 48 hours postinjection and that application of the PPARγ activator into areas adjacent to the confine of RVLM did not affect basal MSAP and HR suggest that the elicited cardiovascular responses were not the results of nonspecific neuro-cardiovascular toxicity. The elicitation of similar degree of pressor response by l-glutamate in RVLM before and after rosiglitazone treatment further indicates that the neural circuitry is functionally intact following transcriptional activation of UCP2. Because ICV Ang II infusion resulted in an increase in MSAP under the condition of UCP2 upregulation in RVLM, oxidative stress in other areas of brain may also participate in central Ang II–induced hypertension. In this regard, oxidative stress in the subfornical34 and hypothalamic areas35 in the forebrain also contributes to neural mechanism of Ang II–induced hypertension.
Our observation that transcriptional up- or downregulation of UCP2 exerted minimal effects on mitochondrial electron transport chain activity and tissue ATP content in RVLM implies that the primary function of UCP2 is not to promote gross thermogenesis or energetic inefficiency in the mitochondria of RVLM. This notion is in concordance with the consensus that a “mild” uncoupling caused by activation of UCP2 leads only to limited increases in proton conductance in the inner membrane of mitochondria, resulting in slightly increased oxidative phosphorylation rate but maintained production of ATP.36,37 These observations also deemed unlikely the possibility that the regulatory actions of UCP2 on ROS level and hemodynamic functions are secondary to induced bioenergetic deficiency in RVLM. We reported recently19 that impairment of mitochondrial electron transport chain activity increases O2−·, leading to chronic oxidative stress in RVLM. Because UCP2 exerted minimal effects on electron transport chain activity, it is unlikely that this source of ROS is the target for its antioxidant effect. UCP2 is engaged in regulation of redox balance by decreasing proton electrochemical gradient across the inner mitochondrial membrane.6,8 It follows that UCP2 may exert its antioxidant actions via this mode of mitochondrial action.
We recognize that results obtained from the mitochondrial fractions in this study depend on the purity of our preparations. In this regard, isolation of brain mitochondria by discontinuous Percoll density gradient38 has been reported to yield approximately 90% recognizable mitochondria with little contamination of other organelles. We also confirmed the purity of the isolated mitochondria by showing a lack of cytosolic marker proteins in the mitochondrial fraction. We are also aware that acute injection of reagents such as tempol or DPI exerted effects on UCP2 expression 12 or 24 hours after administration. We interpret these seemingly long-lasting pharmacological effects by noting that the signaling cascade that interposes between stimulation of angiotensin type 1 receptor by Ang II, production of NADPH oxidase–derived O2−·, activation of p38 MAPK, and transcriptional upregulation of UCP2 requires 12 to 24 hours to materialize. It follows that the same amount of time is required to manifest the effects of interrupting individual steps in this signaling cascade by tempol (O2−·) or DPI (NADPH oxidase) on Ang II–induced UCP2 expression.
In conclusion, the present study provided the first in vivo evidence for a direct link between brain mitochondrial UCP2 and central regulation of arterial pressure. Specifically, we demonstrated that transcriptional upregulation of mitochondrial UCP2 activated by an elevation in mitochondrial and NADPH oxidase–derived extramitochondrial O2−· plays an active role in feedback regulation of ROS production in RVLM and hypertension associated with chronic oxidative stress (Figure 8). Epigenetic studies reported a positive association between UCP2 gene polymorphism and increased oxidative stress in patients with hypertension.39 Because of the important role of mitochondrial oxidative stress in RVLM in neural mechanism of hypertension,19 our observed antioxidant action of mitochondrial UCP2 is of considerable importance in protection against neurogenic hypertension associated with brain oxidative stress. Our results also suggest the possibility of UCP2 as a target molecule for investigations on the etiology and treatment of neurogenic hypertension.
Sources of Funding
This study was supported by research grants 98-2923-B-182A-001-MY3 (to S.H.H.C., A.Y.W.C., and J.Y.H.C) and NSC-97-2320-B-075B-002-MY3 (to J.Y.H.C.) from the National Science Council and VGHKS 98-98 (J.Y.H.C.) and VGHKS97-20 (Y.-H.H.) from Kaohsiung Veterans General Hospital, Taiwan, Republic of China.
Original received April 9, 2009; revision received September 2, 2009; accepted September 3, 2009.
Gao L, Wang W, Li YL, Schultz HD, Liu D, Cornish KG, Zucker IH. Superoxide mediates sympathoexcitation in heart failure: roles of Ang II and NAD(P)H oxidase. Circ Res. 2004; 95: 937–944.
Taniyama Y, Griendling KK. Reactive oxygen species in the vasculature: molecular and cellular mechanisms. Hypertension. 2003; 42: 1075–1081.
Paravicini TM, Touyz RM. Redox signaling in hypertension. Cardiovasc Res. 2006; 71: 247–258.
Kimura Y, Hirooka Y, Sagara Y, Ito K, Kishi T, Shimokawa H, Takeshita A, Sunagawa K. Overexpression of inducible nitric oxide synthase in rostral ventrolateral medulla causes hypertension and sympathoexcitation via an increase in oxidative stress. Circ Res. 2005; 96: 252–260.
Arsenijevic D, Onuma H, Pecqueur C, Raimbault S, Manning BS, Miroux B, Couplan E, Alves-Guerra MC, Goubern M, Surwit R, Bouillaud F, Richard D, Collins S, Ricquier D. Disruption of the uncoupling protein-2 gene in mice reveals a role in immunity and reactive oxygen species production. Nat Genet. 2000; 26: 435–439.
Blanc J, Alves-Guerra MC, Esposito B, Rousset S, Gourdy P, Ricquier D, Tedgui A, Miroux B, Mallat Z. Protective role of uncoupling protein 2 in atherosclerosis. Circulation. 2003; 107: 388–390.
Lee KU, Lee IK, Han J, Song DK, Kim YM, Song HS, Kim HS, Lee WJ, Koh EH, Song KH, Han SM, Kim MS, Park IS, Park JY. Effects of recombinant adenovirus-mediated uncoupling protein 2 overexpression on endothelial function and apoptosis. Circ Res. 2005; 96: 1200–1207.
Ross CA, Ruggiero DA, Park DH, Joh TH, Sved AF, Fernandez-Pardal J, Saavedra JM, Reis DJ. Tonic vasomotor control by the rostral ventrolateral medulla: effect of electrical and chemical stimulation of the area containing C1 adrenaline neurons on arterial pressure, heart rate and plasma catecholamine and vasopressin. J Neurosci. 1984; 4: 474–494.
Chan SHH, Wang LL, Tseng HL, Chan JYH. Upregulation of AT1 receptor gene on activation of protein kinase Cβ/nicotinamide adenine dinucleotide diphosphate oxidase/ERK1/2/c-fos signaling cascade mediates long-term pressor effect of angiotensin II in rostral ventrolateral medulla. J Hypertens. 2007; 25: 1845–1861.
Chan SHH, Hsu KS, Huang CC, Wang LL, Ou CC, Chan JYH. NADPH oxidase-derived superoxide anion mediates angiotensin II-induced pressor effect via activation of p38 mitogen-activated protein kinase in the rostral ventrolateral medulla. Circ Res. 2005; 97: 772–780.
Chan SHH, Wu KLW, Chang AYW, Tai MH, Chan JYH. Oxidative impairment of mitochondrial electron transport chain complexes in RVLM contributes to neurogenic hypertension. Hypertension. 2009; 53: 271–227.
Doughan AK, Harrison DG, Dikalov SI. Molecular mechanisms of angiotensin II-mediated mitochondrial dysfunction. Linking mitochondrial oxidative damage and vascular endothelial dysfunction. Circ Res. 2008; 102: 488–496.
Johnson-Cadwell LI, Jekabsons MB, Wang A, Polster BM, Nicholls DG. ‘Mild Uncoupling’ does not decrease mitochondrial superoxide levels in cultured cerebellar granule neurons but decreases spare respiratory capacity and increases toxicity to glutamate and oxidative stress. J Neurochem. 2007; 101: 1619–1631.
Andrews ZB, Horvath B, Barnstable CJ, Elsworth J, Yang L, Beal MF, Roth RH, Matthews RT, Horvath TL. Uncoupling protein-2 is critical for nigral dopamine cell survival in a mouse model of Parkinson’s disease. J Neurosci. 2005; 25: 184–191.
Guo P, Nishiyama A, Rahman M, Nagai Y, Noma T, Namba T, Ishizawa M, Murakami K, Miyatake a, Kimura S, Mizushige K, Abe Y, Ohmori K, Kohno M. Contribution of reactive oxygen species to the pathogenesis of left ventricular failure in Dahl salt-sensitive hypertensive rats: effects of angiotensin II blockade. J Hypertens. 2006; 24: 1097–1104.
Silveira LR, Pilegaard H, Kusuhara K, Curi R, Hellsten Y. The concentration induced increase in gene expression of peroxisome proliferator-activated receptor (PPAR)-γ coactivator 1α (PGC-1α), mitochondrial uncoupling protein 3 (UCP3) and hexokinase II (HKII) in primary rat skeletal muscle cells is dependent on reactive oxygen species. Biochim Biophys Acta. 2006; 1763: 969–976.
Raji A, Seely EW, Bakins SA, Williams GH, Simonson DC. Rosiglitazone improves insulin sensitivity and lowers blood pressure in hypertensive patients. Diabetes Care. 2003; 26: 172–178.
Duan SZ, Usher MG, Bortensen RM. Peroxisome proliferator-activated receptor-γ-mediated effects in the vasculature. Circ Res. 2008; 102: 283–294.
Sorrentino SA, Bahlmann FH, Besler C, Müller M, Schulz S, Kirchhoff N, Doerries C, Horváth T, Limbourg A, Limbourg F, Fliser D, Haller H, Drexler H, Landmesser U. Oxidant stress impairs in vivo reendothelialization capacity of endothelial progenitor cells from patients with type 2 diabetes mellitus: restoration by the peroxisome proliferator-activated receptor-gamma agonist rosiglitazone. Circulation. 2007; 116: 163–173.
Puigserver P, Adelmant G, Wu Z, Fan M, Xu J, O'Malley B, Spiegelman BM. Activation of PPAR gamma coactivator-1 through transcriptional factor docking. Science. 1999; 286: 1368–1371.
Zimmerman MC, Lazartigues E, Sharma RV, Davisson RL. Hypertension caused by angiotensin II infusion involves increased superoxide production in the central nervous system. Circ Res. 2004; 95: 210–216.