Tetrahydrobiopterin, L-Arginine and Vitamin C Act Synergistically to Decrease Oxidative Stress, Increase Nitric Oxide and Improve Blood Flow after Induction of Hindlimb Ischemia in the Rat Jinglian Yan, Guodong Tie, and Louis M Messina Department of Surgery, University of Massachusetts Medical School, Worcester, Massachusetts, United States of America Nitric oxide (NO) derived from endothelial nitric oxide synthase (eNOS) is a potent vasodilator and signaling molecule that plays an essential role in vascular remodeling of collateral arteries and perfusion recovery in response to hindlimb ischemia. In ischemic conditions, decreased NO bioavailability was observed because of increased oxidative stress, decreased L-arginine and tetrahy- drobiopterin. This study tested the hypothesis that dietary cosupplementation with tetrahydrobiopterin (BH4), L-arginine, and vita- min C acts synergistically to decrease oxidative stress, increase nitric oxide and improve blood flow in response to acute hindlimb ischemia. Rats were fed normal chow, chow supplemented with BH4 or L-arginine (alone or in combination) or chow supple- mented with BH4 + L-arginine + vitamin C for 1 wk before induction of unilateral hindlimb ischemia. Cosupplementation with BH4 + 2+ L-arginine resulted in greater eNOS expression, Ca -dependent NOS activity and NO concentration in gastrocnemius from the is- chemic hindlimb, as well as greater recovery of foot perfusion and more collateral artery enlargement than did rats receiving ei- ther agent separately. The addition of vitamin C to the BH4 + L-arginine regimen did further increase these dependent variables, although only the increase in eNOS expression reached statistical significances. In addition, rats given all three supplements demonstrated significantly less Ca2+-independent activity, less nitrotyrosine accumulation, greater glutathione:glutathione disul- fide (GSH:GSSG) ratio and less gastrocnemius muscle necrosis, on both macroscopic and microscopic levels. In conclusion, co- supplementation with BH4 + L-arginine + vitamin C significantly increased vascular perfusion after hindlimb ischemia by increas- ing eNOS activity and reducing oxidative stress and tissue necrosis. Oral cosupplementation of L-arginine, BH4 and vitamin C holds promise as a biological therapy to induce collateral artery enlargement. Online address: http://www.molmed.org doi: 10.2119/molmed.2011.00103 INTRODUCTION loss of eNOS-derived NO production At least two characteristics of eNOS Endothelial dysfunction is a hallmark (that is, functional inactivation of eNOS) render it susceptible to oxidative stress. of peripheral artery disease (PAD) (1). and inactivation of NO by a superoxide First, eNOS transcription, posttransla- – Central to the development of endothelial anion (O2 ) to form peroxynitrite tional modification and trafficking to the dysfunction, regardless of its cause, is a (OONO–) (2,3). It is likely that all three caveolae are attenuated by the accumula- reduction in the bioavailability of nitric mechanisms contribute to the endothelial tion of reactive oxygen species (ROS) oxide (NO) derived from endothelial ni- dysfunction characteristic of PAD because within the endothelial cell (4). Second, the tric oxide synthase (eNOS). Three funda- increased oxidative stress is a common eNOS cofactor tetrahydrobiopterin (BH4) mental mechanisms can compromise NO antecedent in the pathogenesis of this dis- is highly susceptible to oxidation (5). BH4 bioavailability: loss of eNOS expression, ease and can reduce NO bioavailability. maintains eNOS in its functional dimeric form; in the absence of BH4, eNOS be- comes uncoupled so that the electron flux is diverted away from the L-arginine bind- Address correspondence to Louis M Messina, Department of Surgery, University of Massa- ing site and instead reduces molecular chusetts Medical School, 55 Lake Avenue North, Worcester, MA 01655. Phone: 508-856- – oxygen, generating O2 (6). This circum- 5599; Fax: 508-856-8329; E-mail: [email protected]. stance initiates a vicious cycle, wherein Submitted March 22, 2011; Accepted for publication February 21, 2012; Epub – eNOS catalytic activity produces O2 , not www.molmed.org) ahead of print February 24, 2012. NO, worsening existent oxidative stress. These molecular characteristics of eNOS predict that several therapeutic options might prove effective for the 676 | YAN ET AL. | MOL MED 18:676-684, 2012 RESEARCH ARTICLE treatment of PAD, namely dietary sup- respectively. This action would enhance Preparation plementation with an antioxidant, or eNOS-derived NO production and, by Severe ischemia was induced in the – with the eNOS substrate L-arginine, or quenching existent O2 , reduce NO inac- left hindlimb. The femoral artery was lig- – with the eNOS cofactor BH4. Vitamin C, tivation by its reaction with O2 . ated between the inguinal ligament and or L-ascorbic acid, is a potent antioxidant In our previous work (19), we observed popliteal fossa, and the ligated section and has been shown to preserve BH4 decreased eNOS expression, decreased and its branches were excised. This pro- levels and enhance endothelial NO pro- bioavailable NO and increased oxidative cedure was carried out under anesthesia duction in vitro (7). ONOO– reacts with stress in hindlimb ischemic rats, and oral with 2% isofluorane. The untreated right BH4 6–10 times faster when in the pres- supplementation of BH4 increased the hindlimb served as an internal control ence of ascorbate. The intermediate prod- beneficial effect of eNOS gene transfer. for each rat. An additional group of uct of the reaction between ONOO– and The goal of this study was to test the hy- sham-operated rats were also used for BH4 is the trihydrobiopterin radical pothesis that combined dietary supple- selected assays. These rats underwent • (BH3 ), which is also reduced back to mentation with BH4, L-arginine and vita- isolation of the femoral artery in the left BH4 by ascorbate. Thus, ascorbate does min C will act synergistically to improve hindlimb under 2% isofluorane anesthe- not protect BH4 from oxidation but hindlimb perfusion recovery and preser- sia, but the artery was left intact. rather recycles the BH3 radical back to vation of tissue integrity in response to BH4 (8). Vitamin C levels are low in PAD severe hindlimb ischemia. To this end, we Study Design patients (9), and acute (10) or short-term generated severe hindlimb ischemia in All rats were fed standard chow in (11) vitamin C supplementation reduces the rat by means of femoral artery exci- powder form and water ad libitum PAD symptoms; however, cross-sectional sion. Measured dependent variables in- (Deans Feeds, Redwood City, CA, USA). epidemiological surveys have failed to cluded gastrocnemius muscle eNOS ex- Animals were randomly selected to re- find a clear link between long-term vita- pression and activity, hindlimb laser ceive normal chow (control), chow with min C intake and PAD symptoms or dis- Doppler perfusion and collateral artery a single added supplement (BH4 or ease progression (12,13). L-arginine sup- enlargement, and gastrocnemius nitroty- L-arginine), chow supplemented with plementation showed exciting promise in rosine accumulation and tissue necrosis. BH4 + L-arginine or chow supplemented short-term studies of PAD (14,15), but with BH4 + L-arginine + L-ascorbic acid. this effect was not observed in a subse- MATERIALS AND METHODS Dietary supplementation was com- quent long-term study by the same group menced 7 d before the induction of (16). BH4 improves eNOS- dependent va- Materials hindlimb ischemia and was continued sodilation in long-term smokers and pa- Dietary supplements included the fol- until sacrifice of the animal. Chow was tients with type 2 diabetes, conditions as- lowing: BH4 (10 mg/kg/d; Schircks Lab- replaced every 2 d. The time of sacrifice sociated with increased oxidative stress oratories, Jonas, Switzerland); L-arginine, varied with the measured endpoint (17,18). To our knowledge, BH4 has not provided as L-arginine α-ketoglutarate under consideration, as described below. been specifically evaluated as a therapeu- (hereafter L-arginine; 88.5 mg/kg/d; tic modality in PAD. Body Tech, North Bergen, NJ, USA); and Western Blotting An important gap in our understand- L-ascorbic acid (that is, vitamin C; 88.5 Rats were sacrificed 14 d after induc- ing of BH4, L-arginine and L-ascorbic mg/kg/d, Sigma-Aldrich, St. Louis, MO, tion of ischemia for measurement of acid in the prevention and treatment of USA). The dose of tetrahydrobiopterin gastrocnemius muscle eNOS, phosphory- PAD is the potential synergistic effect of was selected on the basis of a published lated eNOS (p-eNOS) and nitrotyrosine combined therapy, and there is convinc- report of its use in rats (35). And the dose expression. The timing of death was se- ing evidence to suggest that such an ap- of L-arginine and vitamin C was on the lected on the basis of previous work that proach would prove successful. For ex- basis of their clinical dose in patients. demonstrated maximal postischemic ample, supplementation with L-arginine change in these variables at this time (19). alone might prove deleterious in the face Animals Samples were homogenized in liquid ni- of endothelial oxidative stress inasmuch All protocols were approved by the trogen and transferred to NP-40 lysis as the resultant increase in eNOS cat- Institutional Animal Care and Use Com- buffer, comprised of 50 mmol/L HEPES – alytic activity might generate O2 , not mittee at the University