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. Author manuscript; available in PMC: 2013 Aug 12.
Published in final edited form as: Circ Res. 2012 Aug 20;111(9):1157–1165. doi: 10.1161/CIRCRESAHA.111.261750

Increased Superoxide and Endothelial NO Synthase Uncoupling in Blood Vessels of Bmal1-Knockout Mice

Ciprian B Anea 1,*, Bo Cheng 1,*, Shruti Sharma 1, Sanjiv Kumar 1, R William Caldwell 1, Lin Yao 1, M Irfan Ali 1, Ana M Merloiu 1, David W Stepp 1, Stephen M Black 1, David JR Fulton 1, R Daniel Rudic 1
PMCID: PMC3740771  NIHMSID: NIHMS491675  PMID: 22912383

Abstract

Rationale

Disruption of the circadian clock in mice produces vascular dysfunction as evidenced by impairments in endothelium-dependent signaling, vasomotion, and blood vessel remodeling. Although the altered function of endothelial NO synthase and the overproduction of reactive oxygen species are central to dysfunction of the endothelium, to date, the impact of the circadian clock on endothelial NO synthase coupling and vascular reactive oxygen species production is not known.

Objective

The goals of the present study were to determine whether deletion of a critical component of the circadian clock, Bmal1, can influence endothelial NO synthase coupling and reactive oxygen species levels in arteries from Bmal1-knockout (KO) mice.

Methods and Results

Endothelial function was reduced in aortae from Bmal1-KO mice and improved by scavenging reactive oxygen species with polyethylene glycol-superoxide dismutase and nonselectively inhibiting cyclooxygenase isoforms with indomethacin. Aortae from Bmal1-KO mice exhibited enhanced superoxide levels as determined by electron paramagnetic resonance spectroscopy and dihydroethidium fluorescence, an elevation that was abrogated by administration of nitro-L -arginine methyl ester. High-performance liquid chromatography analysis revealed a reduction in tetrahydrobiopterin and an increase in dihydrobiopterin levels in the lung and aorta of Bmal1-KO mice, whereas supplementation with tetrahydrobiopterin improved endothelial function in the circadian clock KO mice. Furthermore, levels of tetrahydrobiopterin, dihydrobiopterin, and the key enzymes that regulate biopterin bioavailability, GTP cyclohydrolase and dihydrofolate reductase exhibited a circadian expression pattern.

Conclusions

Having an established influence in the metabolic control of glucose and lipids, herein, we describe a novel role for the circadian clock in metabolism of biopterins, with a significant impact in the vasculature, to regulate coupling of endothelial NO synthase, production of superoxide, and maintenance of endothelial function. (Circ Res. 2012; 111:1157–1165.)

Keywords: Bmal1, circadian, endothelial NO synthase, superoxide, uncoupling

Endothelial NO synthase (eNOS) exerts a positive influence on the health of blood vessels, restraining cellular proliferation,1 platelet aggregation,2 pathological remodeling,3,4 and reducing blood pressure5,6 and vascular tone.7 However, changes within the endothelial cell microenvironment can cause eNOS to switch functionality to induce deleterious effects in the vasculature. Uncoupling of eNOS, which occurs when levels of the eNOS cofactor tetrahydrobiopterin (BH4) are suboptimal, triggers the production of superoxide in place of NO.8,9

A surprising connection has emerged between eNOS and the circadian clock, the collection of genes that regulate 24-hour rhythmic variations in gene expression and cellular function. Mice with mutations in the circadian clock have impairments in eNOS expression, phosphorylation, and the kinase Akt.10–13 Furthermore, mutation of discrete components of the circadian clock in mice (Bmal1-knockout[KO]/KO) and Period-2 (Per-2) isoform mutant mice causes endothelial dysfunction,10,14 pathological remodeling,10,15 and transplant arteriosclerosis.16 To date, little is known regarding the influence of the circadian clock on the mechanisms that govern endothelial function and, in particular, on a prominent cause of its dysfunction, oxidant stress.

Herein, we present data demonstrating increased superoxide production in the vasculature of Bmal1-KO mice. Furthermore, the balance of BH4 and dihydrobiopterin (BH2) is shifted to uncouple eNOS, which may be triggered through impairments in expression and circadian rhythm of 2 key enzymes in the control of biopterins, GTP cyclochydrolase-1 (GTPCH-1) and dihydrofolate reductase (DHFR). These results suggest that Bmal1 and the circadian clock regulate endothelial superoxide production by controlling coupling of eNOS to its cofactor BH4.

Methods

Animals

All experiments were conducted in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals and approved and monitored by the Georgia Health Sciences University Institutional Animal Care and Use Committee. Congenic 12- to 16-week-old male, wild-type (WT) and Bmal1-KO littermate mice were generated from heterozygote breedings (Jackson Laboratories) and were used for all studies. Mice were housed under standard 12 hour light/dark conditions. Mice were anesthetized by intraperitoneal injection of ketamine and xylazine.

Measurement of Superoxide by EPR

Superoxide was quantified by electron paramagnetic resonance (EPR) spectroscopy. Thoracic aortae were dissected and immediately immersed in PBS and 25 μmol/L desferrioxamine. Samples were homogenized and protein concentrations quantified and normalized (2 mg/mL; bicinchoninic acid protein assay kit). Samples were then split into 2 volumes containing pegylated superoxide dismutase (SOD; 100 U/mL) or solvent (vehicle). These solutions were then incubated with methoxycarbonyl-2,2,5,5-tetramethylpyrrolidine (20 μmol/L) for 45 minutes. Samples were analyzed immediately with a MiniScope MS200 EPR (Magnettech, Berlin, Germany) at a microwave power of 40 mW, modulation amplitude of 1000 mG, and modulation frequency of 100 kHz. Separate studies were conducted to assess the impact of eNOS uncoupling by preincubating samples with 100 μmol/L nitro-L-arginine methyl ester (L-NAME) before measuring EPR amplitude. The amplitude of each EPR spectrum was analyzed using ANALYSIS software (version 2.02; Magnettech).

Dihydroethidium Staining

Aortae were excised and washed for 30 minutes in PBS. Vessels were bathed in dihydroethidium (DHE; 100 μmol/L, Invitrogen) for 30 minutes at 37°C. For control vessels, the O2 −scavenger 4,5-dihydroxy- 1,3-benzendisulfonate (Tiron) (100 μmol/L) was used. To assess the impact of eNOS uncoupling, separate sections were incubated with DHE and L-NAME (100 μmol/L). Vessels were removed, washed in ice-cold PBS for 1 hour, and immediately placed in optimal cutting temperature compound (Tissue-Tek, Redding, CA). The vessels were sectioned at a thickness of 10 μm and mounted on Fischer premium glass slides. Microscopy was performed with a Zeiss Axio Observer inverted microscope and digital camera at ×20 magnification. DHE fluorescence was detected using an excitation filter of 546/12 nm, emission wavelength of 590 nm, and a 580-nm beam splitter. Autofluorescence emitted by the elastic laminae was detected at an excitation wavelength of 425/40 nm, emission at 505/40 nm, and 460 nm beam splitter.

Functional Studies in Isolated Aortic Arteries

Twelve- to fifteen-week-old Bmal1-KO mice or age-matched littermate WT controls were anesthetized with ketamine/xylazine and subsequently exsanguinated. Mice were euthanized at 10:00 AM (zeitgeber time 3). Residual blood was removed by perfusing physiological saline by cardiac puncture. The thoracic aorta was carefully dissected free and excised from the aortic arch to the point of the diaphragm. Perivascular fat was carefully dissected, and the aorta was cut into rings (2 mm thickness) for placement into organ chambers containing Krebs buffer maintained under physiological conditions. The composition of Krebs-Henseleit solution (in mmol/L) was as follows: NaCl, 118.3; KCl, 4.7; CaCl2, 2.5; MgSO4 7H2O, 1.2; KH2PO4, 1.2; NaHCO3, 25; dextrose, 5.6; and equilibrated with 95%O2–5%CO2 to maintain pH of 7.4 at 37°C. The rings were suspended by 2 tungsten wires (25 μm diameter) and mounted in a vessel myograph system (6 mL chamber size; Multi Myograph, Danish Myo Technology). Isometric tension was measured using a force transducer coupled to data acquisition system. A resting tension of 1.0 g was used throughout the experiments. After an equilibration period of 60 minutes (during which time Krebs-Henseleit solution was changed every 10 minutes and the resting tension was readjusted), rings were precontracted with phenylephrine until a plateau was reached. Vessels were then washed with Krebs-Henseleit solution, and this was repeated at least 3× to stabilize the tissue. Aortae were then precontracted with phenylephrine and concentration-dependent responses to the endothelium-dependent dilator, acetylcholine (Ach; 1×10−9 to 5×10−4 mol/L). Separate studies were conducted to assess the impact of superoxide formation by preincubating aortic rings with 1000 U/mL SOD before measuring the relaxant response to Ach. To study the role of cyclooxygenase (COX), the vascular rings were incubated with indomethacin (1 μmol/L) for 30 minutes before measuring endothelium-dependent responses to Ach.

Measurement of Biopterin Levels by High-Performance Liquid Chromatography

Measurements of BH4 were performed by high-performance liquid chromatography analysis, after iodine oxidation under acidic or alkaline conditions, as previously described.17,18 Aortas were homogenized in an extraction buffer (50 mmol/L Tris-HCl, 1 mmol/L EDTA, 1 mmol/L dithiothreitol; pH 7.4) and divided into equal volumes between 2 centrifuge tubes containing either 1 mol/L NaOH (alkaline) or 1 mol/L H3PO4 (acid). A solution of 1% iodine in 2% potassium iodide was added to each tube, and samples were then incubated in the dark at room temperature for 90 minutes. H3PO4 (1 mol/L) was then added to the tubes containing NaOH. Excess iodine was removed from all samples by adding 2% ascorbic acid, and then samples were centrifuged at 15 000g for 10 minutes to remove the precipitated protein. Biopterins were determined by high-performance liquid chromatography in 5% methanol/95% water using a Spherisorb OctaDecylSilyl-1 column (Waters, Franklin, MA) and fluorescence detection (350 nm excitation, 450 nm emission). A standard curve of freshly made BH4 (Sigma, St. Louis, MO) was included using the same extraction buffer. The amount of biopterin obtained by oxidation in acid condition represents total biopterins, which is the sum of BH4, BH2, and free biopterin (BH4+BH2+B). In contrast, alkaline oxidation measures the sum of BH2 and free biopterin (BH2+B). Therefore, BH4 levels were calculated as the difference between values obtained under acidic and alkaline conditions. BH4 levels were normalized for protein concentration by Bradford assay and expressed as femtomoles per microgram protein.

Endothelial Cell Isolation

Aortae were isolated from 12- to 15-week-old mice, ligated at 1 end, perfused/inflated with collagenase type II (Worthington), and subsequently incubated for 60 minutes. Endothelial cells were flushed out with media and plated, cultured, and used at passage 3.

Western Blotting

Aortas were dissected, pulverized under liquid nitrogen, and extracted with the use of ice-cold lysis buffer. Endothelial cells were also lyzed in denaturing sample buffer. Samples were loaded on 10% SDS-PAGE gel and transferred onto nitrocellulose membranes. DHFR (BD biosciences) and GTPCH-1 (Novus Biologicals) protein expression was detected with rabbit anti-mouse polyclonal antibodies, followed by enhanced chemiluminescence (enhanced chemiluminescence kit, Amersham). Signals on x-ray films were quantified by use of the Image J software.

Chronic Treatment With BH4

Osmotic pumps (Alzet 2001, DURECT, Cupertino, CA) were implanted subcutaneously in the intrascapular region under anesthesia with ketamine/xylazine for delivery of BH4 dihydrochloride (1 μL/h; Calbiochem, EMD4 Biosciences) or vehicle (0.9% NaCl or PBS, both containing 0.1% bovine serum albumin) for 7 days (n=5 in each group). The concentration of BH4 in the pumps was calculated so that each mouse received 5 mg BH4 per day. After 7 days, mice were euthanized, and the thoracic aorta was carefully dissected to be used for endothelial function studies.

Thromboxane Analysis

Thromboxane (Txb2) and creatinine were measured and analyzed with commercially available ELISA kits from Cayman Chemical in the plasma of mice.

Statistics

All data are expressed as means±SEM. Differences among genotypes were compared by 1- or 2-way ANOVA or by Student t test, with Bonferroni correction test used as the post hoc test. P<0.05 was considered statistically significant.

Results

SOD Improves Endothelial Function in Bmal1-KO Mice

Although we and others have recently demonstrated that mice with mutation of the circadian clock exhibit endothelial dysfunction,10,14 the mechanisms involved remain unclear. Elevated oxidant stress exerts a significant contribution in endothelial dysfunction,19 and, as such, we sought to directly assess its impact on the vasculature of Bmal1-KO mice that have a disrupted circadian rhythm and clock. In aortic ring tissue bath studies, Ach (5×10−4 mol/L) induced 73±1.9% relaxation in WT mice. The relaxant response to Ach was severely blunted to 26.9±1.7% in Bmal1-KO mice (Figure 1A). Polyethylene glycol-SOD, a scavenger of superoxide, was able to partially restore endothelial function (45.2±10.6% relaxation, 5×10−4 mol/L) in Bmal1-KO mice (Figure 1B), suggesting a role of superoxide in endothelial dysfunction. In addition, indomethacin partially improved endothelial function in Bmal1-KO mice (Figure 1C), whereas endothelium-independent relaxation was not different between WT and Bmal1-KO mice (Figure 1D), as has been previously shown.10

Figure 1. Contribution of oxidant stress to endothelial dysfunction in Bmal1-knockout (KO) mice.

Figure 1

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Isometric tension development was assessed in aortic rings prepared from Bmal1-KO mice (black symbol) and littermate controls (white symbol). After preconstriction with phenylephrine, concentration response to acetylcholine (Ach) was measured. A, Representative traces and summary data of the Ach response from wild-type and Bmal1-KO mice (12–15 weeks old) are shown. B, Ach responses were assessed with and without 1000 U/mL superoxide dismutase (SOD), which improved the response to Ach in Bmal1-KO mice (n=5–7/group; *P<0.05). C, Indomethacin incubation (30 minutes) also improved relaxation response in rings from Bmal1-KO mice (n=4/group; *P<0.05). D, Relaxant responses to the endothelium-independent vasodilator sodium nitroprusside (SNP) were not different between groups (n=4/group). E, Bmal1-KO aortic rings were also assessed for their response to Ach from animals after chronic treatment with tetrahydrobiopterin (BH4). Chronic treatment with BH4 (5 mg/day, see Methods section) improved endothelial function comparable with the improvement seen when incubated with SOD in overlay representation of data (n=7/group; *P<0.05).

Increased Superoxide in Bmal1-KO Mice

As SOD improved endothelial function, we next undertook studies to directly measure superoxide, using EPR spectroscopy. Superoxide was significantly increased in the aorta from Bmal1-KO mice versus WT mice as determined by relative polyethylene glycol-SOD-inhibitable signal (Figure 2). Fluorescent detection of superoxide by DHE staining of the aorta in WT (Figure 3A) and Bmal1-KO mice (Figure 3B) revealed increased superoxide radical formation in Bmal1-KO mice, which was evident across the vascular wall. Recent evidence demonstrated that the antioxidant Tiron and the eNOS inhibitor L-NAME reduce superoxide production in pulmonary arteries of lambs undergoing experimental pulmonary hypertension.20 Similarly, in our studies, Tiron reduced DHE fluorescence in the Bmal1-KO mice (Figure 3C and 3D) as did L-NAME (Figure 3E and 3F), the latter providing evidence that uncoupling of eNOS might be a source of superoxide production.

Figure 2. Increased superoxide in Bmal1-knockout (KO) mice.

Figure 2

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A, Electron paramagnetic resonance (EPR) spectroscopy revealed a significant increase in superoxide signal from aorta of Bmal1-KO mice, shown as representative spectroscopy trace and quantification of EPR spectroscopy (B). n=10/group; *P<0.05 by 1-way ANOVA.

Figure 3. Increased dihydroethidium (DHE) fluorescence in Bmal1-knockout (KO) arteries.

Figure 3

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Histological, frozen, cross-sections of aorta were isolated and processed from wild-type (WT) and Bmal1-KO at Zeitgeber time (ZT) 3, and then incubated with DHE for 30 minutes at 37°C and subsequently visualized by fluorescence microscopy using fluorescein isothiocyanate filter to visualize the endogenous elastic laminar fluorescence of blood vessels and rhodamine red to visualize DHE-positive fluorophores. Although background fluorescence was present in WT mice (A) Bmal1-KO aorta exhibited increased DHE fluorescence (B) relative to WT mice. Incubation with the antioxidant Tiron had no notable effect on DHE fluorescence in WT aorta (C) but reduced DHE fluorescence in Bmal1-KO aorta (D). Nitro-L-arginine methyl ester (L-NAME) seemed to reduce baseline DHE fluorescence in WT mice (E) and even more robustly reduced the high DHE fluorescence in Bmal1-KO mice (F).

eNOS Uncoupling in Bmal1-KO Mice

The uncoupling of eNOS is an important source of superoxide production that occurs when levels of the eNOS cofactor, BH4, are reduced, causing production of superoxide in place of NO.8,9,21 These NOS-dependent increases in superoxide can be abolished when the NOS inhibitor L-NAME is administered.22,23 Indeed, administration of L-NAME (Figure 4A) and BH4 supplementation (Figure 4B) abolished the increase in superoxide signal in the aorta of Bmal1-KO mice, suggesting that eNOS uncoupling was the source of excess superoxide production in Bmal1-KO mice.

Figure 4. Uncoupling of endothelial NO synthase (eNOS) and biopterin imbalance in Bmal1-knockout (KO) mice.

Figure 4

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Aorta from wild-type (WT) and Bmal1-KO mice were incubated with vehicle, 100 μmol/L nitro-L-arginine methyl ester (L-NAME), or 100 μmol/L tetrahydrobiopterin (BH4) and subsequently prepared and analyzed by electron paramagnetic resonance (EPR) spectroscopy. Bmal1-KO samples treated with vehicle had a significant increase in superoxide signal relative to WT samples (A), but this increase in polyethylene glycol (PEG)-superoxide dismutase (SOD) inhibitable signal was abolished in the presence of both L-NAME (A) and BH4 (B). Quantification of biopterin levels by high-performance liquid chromatography (HPLC) in lungs (C) and aorta (D) demonstrated that Bmal1-KO animals exhibited decreased BH4 levels and increased dihydrobiopterin (BH2) levels, with a resultant dampening of BH4:BH2 ratio. Chronic treatment of aorta with tempol in drinking water did not restore BH4 levels in Bmal1-KO animals (D) (n=7/group; *P<0.05 vs respective WT by 1-way ANOVA).

Because both absolute levels of BH424–26 and the ratio of BH4 and BH227 are associated with eNOS uncoupling and superoxide production, we sought to determine whether biopterin dynamics might underlie uncoupling. Using high- performance liquid chromatography to quantify biopterin in lung and aorta, we found that Bmal1-KO mice exhibited reduced levels of BH4, whereas BH2 levels were elevated, yielding a blunting of the BH4:BH2 ratio in lung (Figure 4C). Despite the alteration in BH4 and BH2 profile, total biopterins remained largely unchanged in the lung tissue (Figure 4C), which is characteristic of either oxidation of BH4 or a defect in recycling via the enzyme DHFR. In aorta, uncoupling was also evident as reductions in BH4 and elevations in BH2, while chronic treatment with tempol in drinking water failed to rescue BH4 levels in Bmal1-KO mice (Figure 4D). Again, total biopterins in Bmal1-KO mice (9.7±2.7 fmol/μg protein) were not significantly different from WT mice (11.8±3.3 fmol/μg protein) as observed in lung tissue. These data demonstrate that there is uncoupling of eNOS in the aortic and lung tissues of Bmal1-KO mice.

Reduced Expression of GTPCH-1 and DHFR in Bmal1-KO Mice

To determine whether enzymes involved in biopterin metabolism might underlie the defect in BH4 balance, we assessed GTPCH-1 and DHFR expression in cultured endothelial cells of Bmal1-KO mice. Expression of GTPCH-1, the rate-limiting enzyme in de novo BH4 biosynthesis, was reduced, albeit not significantly in Bmal1-KO mice versus WT mice (Figure 5A), and DHFR, the enzyme important in recycling of BH2 to BH4, did exhibit a significant reduction in expression in the endothelial cells of Bmal1-KO mice relative to WT mice (Figure 5B). To determine whether these differences were also found in vivo, we assessed expression of the enzymes in aorta over 24 hours to additionally assess whether there was a circadian rhythm in expression. GTPCH-1 protein expression did oscillate in aorta of WT mice (Figure 6A), a rhythm that was attenuated in Bmal1-KO mice (Figure 6B). DHFR also exhibited an oscillating rhythm in mouse aorta (Figure 6B) and also synchronized28 human aortic endothelial cells (Figure 6E). In WT mice, levels of BH4 (Figure 7) exhibited an oscillation that manifested as 2 peaks in a 48-hour time course. Again, the rhythm in DHFR was absent in Bmal1-KO animals (Figure 6D). Finally, supplementation of BH4 improved endothelial function in the aorta of Bmal1-KO mice (Figure 1E) to 48.1±4.2% relaxation, providing further evidence for eNOS uncoupling in the endothelial dysfunction in Bmal1-KO mice.

Figure 5. Decreased dihydrofolate reductase (DHFR) protein expression in Bmal1-knockout (KO) endothelial cells.

Figure 5

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Expression levels of GTP cyclochydrolase-1 (GTPCH-1) (first step limiting enzyme in de novo synthesis of tetrahydrobiopterin [BH4]) and DHFR (main enzyme used by endothelial cells in dihydrobiopterin [BH2] to tetrahydrobiopterin [BH4] recycling pathway) were analyzed by immunoblotting in mouse endothelial cell lysates. Western blotting revealed no significant change in GTPCH-1 levels (A) and a significant reduction in DHFR protein levels (B) in endothelial cells isolated from Bmal1-KO mouse aorta (each band shown is an individual endothelial cell culture plate from the representative n=5 per group; *P<0.05).

Figure 6. Dihydrofolate reductase (DHFR) and GTP cyclochydrolase-1 (GTPCH-1) exhibit a circadian rhythm that is abolished in vasculature of Bmal1-knockout (KO) mice.

Figure 6

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GTPCH-1 and DHFR protein level expression oscillates in mouse aorta samples in wild-type (WT) animals with GTPCH-1 (A) peaking at 11 AM (Zeitgeber time [ZT] 4), whereas DHFR (B) peaks at 7 PM (ZT12) (n=3/group). In Bmal1-KO aortic samples, GTPCH-1 (C) and DHFR (D) protein oscillation was abolished (equal amounts of protein were loaded for WT and Bmal1-KO samples). E, Cultured human aortic endothelial cells were synchronized by horse serum shock as described.28,59 At the end of serum shock (ZT0), cells were lyzed beginning at ZT2 and at 6-hour intervals thereafter for 24 hours. Lysates were then immunoblotted for DHFR. DHFR protein expression displays a 24-hour circadian rhythm peaking at ZT14 as quantified by densitometry (right, n=5 per time point; *P<0.05 vs WT). Changes were quantified by densitometry (*P<0.05 vs WT; ZT intimates that experiments were performed under conditions where mouse room illumination follows standard light/dark cycles, where ZT4, 12, and 20 correspond to 11 AM, 7 PM, and 3 AM).

Figure 7. Rhythmic oscillation in tetrahydrobiopterins.

Figure 7

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Aortae were harvested from wild-type (WT) mice over a 48-hour time span in 6-hour intervals. Aorta was then processed for biopterin measurements by high-performance liquid chromatography (HPLC). Tetrahydrobiopterin (BH4) levels exhibited 2 peaks over the 48-hour time course (n=7–10 per time point; *P<0.05 vs ZT5, 1 way ANOVA and t test).

Discussion

The circadian clock is a negative feedback loop, driven by transcription factors Bmal1 and clock (or Npas2) that induce the Per and Cryptochrome genes which subsequently restrain this oscillatory cycle. The clock genes are ubiquitously expressed and exhibit rhythms in blood vessels.29–32 Cumulative evidence has demonstrated that this 24-hour oscillator or timer exerts a significant influence in the control of the vasculature, whereby disruption of circadian clock components results in thrombosis,10,33,34 pathological remodeling,10 vascular stiffness,15 impaired angiogenesis, and altered endothelial progenitor cell function.13 In addition, Bmal1-KO, clock mutant mice, and Per2 mutant mice all exhibit endothelial dysfunction, which may be a precursor to the chronic phenotypes.10,14,15 In part, the mechanisms underlying these impairments may involve the significant influence that the circadian clock exerts on key pathways in endothelial cell signaling. Circadian clock mutant mice exhibit misregulation of Akt and eNOS,10–12,14 which have a key role in endothelial tone regulation. Herein, we also demonstrate impaired control of super-oxide production and uncoupling of eNOS in Bmal1-KO mice.

Our data in the vasculature of Bmal1-KO mice demonstrating increased superoxide levels by EPR and DHE are consistent with increased DHE fluorescence in kidney and spleen of Bmal1-KO mice.35 In those studies, there was an age-dependent increase in DHE fluorescence in Bmal1-KO mice, but the elevations were evident even in young Bmal1-KO mice, consistent with our observations. In flies, the connection between oxidant stress and the circadian clock has also been described. A Drosophila mutant of the core clock component Per exhibited increased H2O2-induced mortality, which coincided with a decrease in catalase expression,36 whereas exposure to paraquat that catalyzes superoxide production blunted circadian oscillation in peripheral tissues of Drosophila, suggesting a reciprocal relationship between the circadian clock and oxygen radicals.37 In addition, SOD-1 was shown to have circadian rhythm in the liver of mice, an oscillation that was abolished in Per-2 mutant mice.38 Thus, the interaction between the circadian clock and oxidant stress is evident from fly to mouse, suggesting the fundamental significance of this interaction.

We have found that in the vasculature, a source of super-oxide excess during circadian dysfunction is the uncoupling of eNOS. The first indication that eNOS was uncoupled was that the NOS inhibitor L-NAME reduced superoxide, suggesting that in Bmal1-KO mice, eNOS predominately generates superoxide in place of NO. More direct evidence for the uncoupling of eNOS was the observed imbalance in BH4 and BH2 levels in tissues of Bmal1-KO mice. Indeed, there is evidence that both reduction of BH424–26 and imbalance of the BH4:BH2 ratio27 are signatures of eNOS uncoupling. Bmal1-KO mice exhibited both; BH4 levels were reduced and BH2 levels were elevated, resulting in an altered ratio indicative of a defect in biopterin dynamics.

Recent data have revealed that the enzymes involved in biopterin metabolism may directly contribute to oxidant stress and eNOS uncoupling. Small interfering RNA knockdown of DHFR reduces BH4 and increases BH2, resulting in increased oxidant stress via eNOS uncoupling.39,40 Comparable with the exogenous knockdown of DHFR, our data demonstrate that endogenous circadian clock dysfunction also blunts biopterin synthesis and recycling to cause uncoupling of eNOS. Although the rate-limiting enzyme, GTPCH-1, exhibited only a modest reduction in unsynchronized endothelial cells of Bmal1-KO mice, extended 24-hour sampling in aorta revealed a circadian rhythm in GTPCH-1 in WT mice and blunted rhythm in Bmal1-KO mice. Similarly, DHFR, which recycles BH2 to BH4, exhibited a circadian rhythm that was Bmal1-dependent. Although BH4 levels did exhibit a rhythm, the peak-to-peak time interval was ≈30 hours, not 24 hours as would be expected of a circadian rhythm. There are numerous factors that could underlie this difference. One, variability can be incurred depending on the resolution of the time intervals, which may relate to differences in the time span at which peak intervals occur. Two, levels of BH4 were measured, as opposed to gene transcripts, whose regulation involves additional complexity. Three, there are experimental limitations. For example, numerous mice in these studies were euthanized at a single time point, with each mouse requiring additional time for processing, providing another potential source for the difference. Despite this rhythm in BH4, no significant oscillation in BH2 or superoxide was observed (Online Figure I). This may further underscore the complexity in control of superoxide and biopterins, which involve a cascade of enzymes including NADPH oxidases and antioxidant enzymes. It is also possible that a dysfunctional circadian rhythm may suppress the overall function of BH4 synthesis and regeneration pathway at all times, perhaps comparable with sleep apnea, which occurs only at night, but the metabolic perturbations that occur persist throughout the 24-hour cycle.41,42 As such, it may be that the circadian clock exerts a regulatory role that only becomes unmasked during clock dysfunction, in face of counterbalancing mechanisms.

Although both SOD and BH4 supplementation partially improved endothelial dysfunction in Bmal1-KO mice, complete restoration of function was not achieved. Additional tonic influences also underlie the impaired endothelial function in circadian clock mutant mice, including Akt-eNOS axis signaling10,13 and COX signaling,14,15 which we and others have shown to be aberrant in circadian clock mutant mice. Interestingly, indomethacin, a nonselective COX inhibitor, also partially improved endothelial function as measured by the response to Ach in Bmal1-KO mice. In contrast, in Per-2 mutant mice, indomethacin does not affect Ach-induced relaxation, whereas it does improve ionomycin-induced relaxation.14 This may reflect differences in the levels of superoxide production in the vasculature between Bmal1-KO mice and Per isoform mutant mice. Indeed, COX inhibitors have been shown to improve endothelial function under numerous pathological conditions,43,44 including states of elevated oxidative stress45 and in conditions where eNOS is uncoupled.46 This may be, in part, because of the inhibition of vasconstricting prostaglandins but may also involve secondary mechanisms that control oxidant stress. COX may increase superoxide via the production of thromboxane,47 and it may also alter reactive oxygen species production from NADPH oxidases,48 which may in turn oxidize BH4 to cause eNOS uncoupling49 and further exacerbate superoxide production. Indeed, we found that thromboxane levels were increased in the blood of Bmal1-KO mice (Figure 8), and, COX-1, which is critical in thromboxane formation,50 has been shown to be elevated in Per-2 mutant mice.14 However, COX-2, which we have shown is increased in cultured endothelial cells of Bmal1-KO mice,15 can also mediate thromboxane production, albeit modestly.50 Temporal regulation of COXs by the circadian clock may emerge as an additional variable, controlling the complex actions of prostaglandins that act to both protect50–52 and harm cardiovascular function.53–55 In addition, studies are needed to address the involvement of the circadian clock in regulating mechanisms that directly control oxygen radical and peroxide formation, including NADPH oxidases and antioxidant enzymes that may additionally impact superoxide production directly as has been demonstrated in the NADPH oxidase-1–dependent uncoupling of eNOS.56 Although the circadian clock is established to exert a significant influence in the metabolic control of glucose and lipids,32,57,58 herein, we describe a novel role for the circadian clock in metabolism of biopterins, with a significant impact in the vasculature, to regulate coupling of eNOS, production of superoxide, and maintenance of endothelial function.

Figure 8. Increased thromboxane in Bmal1-knockout (KO) mice.

Figure 8

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Plasma was isolated from wild-type (WT) and Bmal1-KO mice at 6 PM. Thromboxane quantified by ELISA was increased in Bmal1-KO mice vs WT mice, when normalized to creatinine (bar graphs) or without normalization (numerals over bar graph) (n=5 per time point; *P<0.05 vs WT).

Supplementary Material

Supplement. Supplemental Figure. Time course of BH2 and superoxide production in aorta of WT mice.

A 48-hour time course was conducted in WT mice undergoing aortic harvest at 6 hour intervals and BH2 levels were assessed, but no significant circadian variation was observed (n=7–10 per time point). Similarly, aorta were harvested, and assayed for superoxide but again, no significant oscillation was observed (n=5).

Novelty and Significance.

What Is Known?

  • A circadian clock that malfunctions can cause endothelial dysfunction.

  • Broken clocks can also cause structural impairments in blood vessels, indicative of vascular disease.

  • Superoxide production, one source of which is from uncoupled endothelial NO synthase (eNOS), can induce both endothelial dysfunction and vascular disease.

What New Information Does This Article Contribute?

  • Mice with genetic disruption of the circadian clock component have increased vascular superoxide production.

  • Circadian clock dysfunction results in eNOS uncoupling.

  • Levels of tetrahydrobiopterins are reduced in Bmal1-knockout mice, and the key enzymes that are important in the generation and recycling of tetrahydrobiopterins, GTP cyclohydrolase I and dihydrofolate reductase are controlled by the circadian rhythms.

Circadian rhythm dysfunction that can manifest through sleep apnea, shift work, and nondipping hypertension has a profound effect to cause cardiovascular disease. The circadian clock is the molecular mechanism that controls 24-hour rhythms, among which the most critical component is the transcription factor Bmal1. Although recent work has demonstrated that broken circadian clocks can cause dysfunction in the vasculature, little is known regarding the underlying mechanisms. The current work reveals that impairment in Bmal1 causes an increase in superoxide production in blood vessels. We find that a key cofactor, tetrahydrobiopterin, for the enzyme eNOS is reduced in Bmal1-knockout mice, causing uncoupling of eNOS and elevation in superoxide production. Furthermore, the key enzymes in control of tetrahydrobiopterins, GTP cyclohydrolase I and dihydrofolate reductase are misregulated by Bmal1 dysfunction. These data suggest a novel role for the circadian clock in metabolism of biopterins, with a significant impact in the vasculature, to regulate coupling of eNOS, production of superoxide, and maintenance of endothelial function, which may have an important influence in the mechanisms underlying vascular disease.

Acknowledgments

Sources of Funding

This work was supported by the National Institutes of Health (HL089576 to R.D. Rudic, HL070215 to R.W. Caldwell, and HL067841 to S.M. Black).

Non-standard Abbreviations and Acronyms

BH2

dihydrobiopterin

BH4

tetrahydrobiopterin

DHFR

dihydrofolate reductase

eNOS

endothelial NO synthase

GTPCH-1

GTP cyclohydrolase-1

ROS

reactive oxygen species

ZT

zeitgeber time

Footnotes

Disclosures

None.

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Supplementary Materials

Supplement. Supplemental Figure. Time course of BH2 and superoxide production in aorta of WT mice.

A 48-hour time course was conducted in WT mice undergoing aortic harvest at 6 hour intervals and BH2 levels were assessed, but no significant circadian variation was observed (n=7–10 per time point). Similarly, aorta were harvested, and assayed for superoxide but again, no significant oscillation was observed (n=5).

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