Weitzberg, Malte Kelm and Tienush RassafPia Stock, Christos Rammos, Michael Niessen, Christian Heiss, Jon O. Lundberg, Eddie

Ulrike B. Hendgen-Cotta, Peter Luedike, Matthias Totzeck, Martina Kropp, Andreas Schicho,Dietary Nitrate Supplementation Improves Revascularization in Chronic Ischemia

Print ISSN: 0009-7322. Online ISSN: 1524-4539 Copyright © 2012 American Heart Association, Inc. All rights reserved.

is published by the American Heart Association, 7272 Greenville Avenue, Dallas, TX 75231Circulation doi: 10.1161/CIRCULATIONAHA.112.112912

2012;126:1983-1992; originally published online September 19, 2012;Circulation. 

http://circ.ahajournals.org/content/126/16/1983World Wide Web at:

The online version of this article, along with updated information and services, is located on the

http://circ.ahajournals.org/circulationaha/suppl/2012/09/19/CIRCULATIONAHA.112.112912.DC1.htmlData Supplement (unedited) at:

  http://circ.ahajournals.org//subscriptions/

is online at: Circulation Information about subscribing to Subscriptions: 

http://www.lww.com/reprints Information about reprints can be found online at: Reprints:

  document. Permissions and Rights Question and Answer this process is available in the

click Request Permissions in the middle column of the Web page under Services. Further information aboutOffice. Once the online version of the published article for which permission is being requested is located,

can be obtained via RightsLink, a service of the Copyright Clearance Center, not the EditorialCirculationin Requests for permissions to reproduce figures, tables, or portions of articles originally publishedPermissions:

at CONS SWISS CONSORTIA on July 21, 2014http://circ.ahajournals.org/Downloaded from at CONS SWISS CONSORTIA on July 21, 2014http://circ.ahajournals.org/Downloaded from at CONS SWISS CONSORTIA on July 21, 2014http://circ.ahajournals.org/Downloaded from at CONS SWISS CONSORTIA on July 21, 2014http://circ.ahajournals.org/Downloaded from

Vascular Medicine

Dietary Nitrate Supplementation ImprovesRevascularization in Chronic Ischemia

Ulrike B. Hendgen-Cotta, PhD*; Peter Luedike, MD*; Matthias Totzeck, MD; Martina Kropp, MSc;Andreas Schicho, MS; Pia Stock, MSc; Christos Rammos, MD; Michael Niessen, MS;

Christian Heiss, MD; Jon O. Lundberg, MD, PhD; Eddie Weitzberg, MD, PhD;Malte Kelm, MD; Tienush Rassaf, MD

Background—Revascularization is an adaptive repair mechanism that restores blood flow to undersupplied ischemictissue. Nitric oxide plays an important role in this process. Whether dietary nitrate, serially reduced to nitrite bycommensal bacteria in the oral cavity and subsequently to nitric oxide and other nitrogen oxides, enhancesischemia-induced remodeling of the vascular network is not known.

Methods and Results—Mice were treated with either nitrate (1 g/L sodium nitrate in drinking water) or sodium chloride(control) for 14 days. At day 7, unilateral hind-limb surgery with excision of the left femoral artery was conducted.Blood flow was determined by laser Doppler. Capillary density, myoblast apoptosis, mobilization of CD34�/Flk-1�,migration of bone marrow–derived CD31�/CD45�, plasma S-nitrosothiols, nitrite, and skeletal tissue cGMP levelswere assessed. Enhanced green fluorescence protein transgenic mice were used for bone marrow transplantation. Dietarynitrate increased plasma S-nitrosothiols and nitrite, enhanced revascularization, increased mobilization of CD34�/Flk-1� and migration of bone marrow–derived CD31�/CD45� cells to the site of ischemia, and attenuated apoptosisof potentially regenerative myoblasts in chronically ischemic tissue. The regenerative effects of nitrate treatment wereabolished by eradication of the nitrate-reducing bacteria in the oral cavity through the use of an antiseptic mouthwash.

Conclusions—Long-term dietary nitrate supplementation may represent a novel nutrition-based strategy to enhanceischemia-induced revascularization. (Circulation. 2012;126:1983-1992.)

Key Words: mouthwashes � revascularization � nitrates � nitric oxide � nitrites

Revascularization is a process aimed at maintaining andrestoring tissue viability during chronic ischemia. The

underlying signaling mechanisms are complex and includecytokines, chemokines, proteinases, and cell adhesion mole-cules.1 Members of the vascular endothelial growth factorfamily and the stromal cell–derived factor 1� have beendescribed to play a central role in initiating angiogenesis,vasculogenesis, and arteriogenesis.2–6

Editorial see p 1939Clinical Perspective on p 1992

Nitric oxide (NO) derived from endogenous enzymaticproduction via endothelial NO synthase is a well-knownproangiogenic molecule that regulates both vascular endothe-lial growth factor signaling and the recruitment of bonemarrow–derived endothelial progenitor cells.7–9 Disruptionof endothelial NO synthase impairs the release of endo-

thelial progenitor cells into the circulation and theirmigration to the site of injury,8 thus attenuating vascularregenerative processes.

Inorganic nitrate from dietary sources is converted in vivoto nitrite and then NO and other bioactive nitrogen ox-ides.10,11 Nitrate is found in considerable amounts in oureveryday diet, and leafy green vegetables such as spinach,lettuce, or beetroot have particularly high concentrations.12 Inthe oral cavity, commensal nitrate–reducing bacteria effec-tively reduce nitrate to nitrite, which is swallowed with �1 Lsaliva per day and thus continuously enters the circulation.13

The relevance of exogenous supplementation with dietarynitrate for cardiovascular functions has recently been shown.After a 3-day nitrate-enriched diet, diastolic blood pressurewas reduced significantly in healthy nonhypertensive volun-teers.14 A commercially available antibacterial mouthwashadministered daily for 1 week to rats supplemented with

Received April 17, 2012; accepted August 10, 2012.From the Division of Cardiology, Pulmonology and Vascular Medicine, Medical Faculty, University Hospital Dusseldorf, Dusseldorf, Germany

(U.B.H.-C., P.L., M.T., M.K., A.S., P.S., C.R., M.N., C.H., M.K., T.R.), and Karolinska Institute, Department of Physiology and Pharmacology,Stockholm, Sweden (J.O.L., E.W.).

*Drs Hendgen-Cotta and Luedike contributed equally to this article.The online-only Data Supplement is available with this article at http://circ.ahajournals.org/lookup/suppl/doi:10.1161/CIRCULATIONAHA.

112.112912/-/DC1.Correspondence to Tienush Rassaf, MD, FESC, University Hospital Dusseldorf, Medical Faculty, Division of Cardiology, Pulmonology and Vascular

Medicine, Moorenstrasse 5, D-40225 Dusseldorf, Germany. E-mail Tienush.Rassaf@med.uni-duesseldorf.de© 2012 American Heart Association, Inc.

Circulation is available at http://circ.ahajournals.org DOI: 10.1161/CIRCULATIONAHA.112.112912

1983 at CONS SWISS CONSORTIA on July 21, 2014http://circ.ahajournals.org/Downloaded from

nitrate via drinking water has been shown to suppress the oralmicroflora to such an extent that the conversion of nitrate tonitrite in the oral cavity was strongly reduced. Consequently,the blood pressure–lowering and gastroprotective effects ofnitrate were abolished.15 In contrast to pharmacologicalapplication of the anion nitrite,16 dietary nitrate is nontoxiceven in higher doses, whereas nitrite can cause serious harmat considerably lower levels.

On the basis of the recently described NO-like effects ofdietary nitrate, we evaluated whether long-term dietary nitratesupplementation matched to the effect of a human diet rich invegetables would improve ischemia-induced revasculariza-tion and tissue regeneration using a mouse hind-limb ische-mia model.

MethodsChemicalsAll reagents were obtained from Sigma-Aldrich (Taufkirchen, Ger-many) unless indicated otherwise.

AnimalsMale Naval Medical Research Institute (NMRI) mice 14�3 weeksold with an average body weight of 30�6 g were obtained from thelocal animal house. C57BL/6-Tg(CAG-EGFP)C14-Y01-FM131Osbmice were kindly provided by Dr Masaru Okabe (Osaka University,

Osaka, Japan). All experiments were approved by the local ethicscommittee in compliance with the European Convention for theProtection of Vertebrate Animals Used for Experimental and otherScientific Purposes (Council of Europe Treaty Series No. 123).

Sodium Nitrate Supplementation andAntiseptic MouthwashSodium nitrate was added to the drinking water for 14 days at aconcentration of 1 g/L (�150 �mol according to the measured dailydrinking water consumption). This is equivalent to a rich vegetableintake in human.11 An equal concentration of sodium chloride servedas control. To suppress the resident microflora in the oral cavity ofthe animals, subgroups were treated twice daily for 14 days with acommercial antibacterial mouthwash solution (0.2% wt/vol hexeti-dine/water; Pfizer, Berlin, Germany). The control group received amouthwash with a water solution only. All mice were weighed aftersodium nitrate intake for 7 days. Water and food consumption wasrecorded daily. Animals underwent surgical excision of the leftfemoral artery at day 7. At day 14, mice were subjected to imaging,blood sampling, and histological analyses.

Hind-Limb Ischemia ModelUnder complete anesthesia after ketamine (45 mg/kg) and xylazine(10 mg/kg) injections, chronic ischemia was generated in compliancewith well-established unilateral hind-limb surgery protocols.17 Inbrief, after a 1-cm skin incision was made at the medial thigh, thefemoral artery was separated from the femoral vein and nerve. The partproximal to the outlet of the profunda femoris artery and the distalend (outlet of the saphenous artery) were ligated with 7–0 silk

Figure 1. Effect of dietary nitrate supplementation on perfusion recovery. A, Experimental protocol. B, Original laser Doppler perfusionimages (LDPIs) display hind-limb perfusion before and 7 days after excision of the femoral artery. Mice receiving nitrate (middle row)show increased perfusion compared with control animals (left row). Mice receiving antiseptic mouthwash (MW) did not exhibit thenitrate-induced gain of perfusion (right row). C, Nitrate-supplemented mice showed increased perfusion recovery in chronic hind-limbischemia (black bars; P�0.02); this effect was not observed in mice receiving antiseptic mouthwash (gray bars; P�0.02). Data areexpressed as mean�SEM (n�21–23).

1984 Circulation October 16, 2012

at CONS SWISS CONSORTIA on July 21, 2014http://circ.ahajournals.org/Downloaded from

sutures (Serag-Wiessner, Naila, Germany). The femoral artery wasthen excised between the ligations. Wound closures were conductedwith single-layer sutures with 4–0 Prolene threads. Immediatelyafter surgery, animals received a subcutaneous injection of buprenor-phine (0.05 mg/kg). Postoperative analgesia was maintained withbuprenorphine (0.05 mg/kg) every 12 hours for 2 days, and micewere closely monitored for any signs of distress.

Assessment of PerfusionPerfusion was assessed by laser Doppler perfusion imaging (Per-imed, Stockholm, Sweden) immediately after surgery and for thenext 7 days. Blood flow was measured as changes in laser frequency,represented by pixels of different colors. The mean hind-limbperfusion was calculated as the ratio of ischemic to nonischemic sideby a blinded observer.

Capillary Density MeasurementParaffin-embedded serial sections (6 �m) obtained from skeletalmuscles of the ischemic and nonischemic (control) hind limb wereused for immunostaining. To define capillaries on day 7 aftersurgery, a primary antibody against CD31 (platelet/endothelial celladhesion molecule-1; Santa Cruz Biotechnology, Santa Cruz, CA)was used to detect endothelial cells, followed by a rabbit anti-goatAlexa 647 secondary antibody. Double staining with CD45 antibod-ies was performed to unmask leukocytes. Tissues were mounted bythe use of Vectashield DAPI nuclear counterstain. Only CD31� andCD45� cells were calculated per 1-mm2 hind-limb section.

Determination of Myoblast ApoptosisSeven days after chronic hind-limb ischemia, the hind limbs wereremoved from mice, and the bones were dissected. The muscle was thenplaced in PBS to keep it moist. Subsequently, the muscle was mincedand the cells were digested with Pronase in PBS (10 mg/mL) with 1 mLHEPES Puffer (25 mmol/L) and Dulbecco modified Eagle medium(PromoCell, Heidelberg, Germany). Myoblasts were cultured in HAM’sF-10 medium (PromoCell) supplemented with 20% FCS, 10% equineserum, and 0.2% penicillin/streptomycin on collagen-coated dishes.After incubation and sufficient fibroblast adhesion, myoblasts werecarefully extracted. To verify the myoblast population, fluorescence-ac-tivated cell sorting (FACS) was performed on an FACS sorter (FACSCanto, BD, Franklin Lakes, NJ) with the integrin-�7 antibody(Antikoerper-Online GmbH, Aachen, Germany) for sorting. Cells ex-hibiting DNA fragmentation were identified by terminal transferasedUTP nick-end labeling (TUNEL) assay according to the manufactur-er’s recommendations (Roche Diagnostics, Mannheim, Germany). Cellswere visualized with a Nikon fluorescence microscope (Nikon Instru-ments Europe BV Germany, Dusseldorf, Germany), and TUNEL-positive (green) and total (blue) nuclei were counted in 3 separate fields.Apoptotic susceptibility was presented as the percentage of TUNEL-positive nuclei per total counted nuclei.

Determination of Peripheral BloodMononuclear CellsBlood samples were collected 7 days after hind-limb surgery.Peripheral blood mononuclear cells were isolated by Ficoll densitygradient centrifugation. To determine the isolated blood cells, weperformed FACS analyses using FITC rat anti-mouse CD 34 andAPC rat anti-mouse Flk-1 antibodies (BD, Heidelberg, Germany).

Bone Marrow TransplantationTo assess the migration of bone marrow–derived endothelium-regenerating cells into ischemic tissue, NMRI and enhanced greenfluorescence protein (EGFP) transgenic mice were used for bonemarrow transplantation. Bone marrow cells were obtained from thetibias and femurs of male EGFP� mice. A suspension of single cellswas created and prepared for transplantation. To generate chimeric

mice, 4 to 6�106 bone marrow cells were injected into the heart ofthe wild-type recipients in which bone marrow had been lethallyirradiated with 6.5 Gy (2�10 minutes). Four weeks later, wild-typemice were subjected to nitrate supplementation for 7 days andlong-term ligation of the femoral artery.

Figure 2. Effect of dietary nitrate supplementation on S-nitrosothiol(RSNO), nitrite (NO2

�), and nitrate (NO3�) levels in plasma. A,

Assessment of circulating RSNO, NO2�, and NO3

� levels. B, Cir-culating RSNO, NO2

�, and NO3� levels increase after nitrate sup-

plementation during a 7-day period. Mice receiving antisepticmouthwash (MW) displayed lower RSNO and NO2

� levels inplasma. Data are expressed as mean�SEM (n�6–8).

Hendgen-Cotta et al Dietary Nitrate Improves Revascularization 1985

at CONS SWISS CONSORTIA on July 21, 2014http://circ.ahajournals.org/Downloaded from

Determination of Bone Marrow–Derived CellsSeven days after long-term ligation of the femoral artery, paraffin-embedded serial sections (6 �m) obtained from skeletal muscleswere stained with rat anti-mouse CD31 antibody, followed by arabbit anti-goat Alexa 647 secondary antibody. Double staining withCD45 antibodies was performed to unmask inflammatory cells.Nuclei were counterstained with DAPI. EGFP�/CD31�/CD45�

cells were calculated per 1-mm2 hind-limb section.

Biochemical AnalysisPlasma nitrate, nitrite, and S-nitrosothiol levels were measured withhigh-performance liquid chromatography (ENO20, Eicom, Dublin,Ireland) and chemiluminescence18 and skeletal tissue cGMP levelwas determined with the BiotrakTM cGMP competitive enzymeimmunoassay system (GE Healthcare, Munich, Germany) followingthe manufacturer’s protocol. Tissue cGMP levels were expressed per1 mg wet tissue.19

Statistical AnalysisResults are presented as mean�SEM unless stated otherwise. Datawere analyzed by 1-way ANOVA and post hoc Bonferroni multiple-comparison tests and Kruskal-Wallis with Mann–Whitney test withGraphPad Prism 5 and IBM SPSS version 20 software to comparedifferences between multiple groups. The Student unpaired t test wasused to analyze 2 groups. A value of P�0.05 was consideredstatistically significant.

ResultsDietary Nitrate Improves Perfusion Recovery inChronic Hind-Limb IschemiaTo determine whether dietary nitrate supplementation augmentsthe recovery of tissue perfusion, we subjected mice to femoralartery ligation, and the effects on perfusion recovery wereassayed by laser Doppler perfusion imaging immediately aftersurgery and the next 7 days (Figure 1A and 1B). Perfusionrecovery in the ischemic hind limb was significantly improved inmice treated with nitrate compared with controls (73�4% versus59�4%; P�0.02; Figure 1B and 1C). Eradication of thenitrate-reducing bacteria in the oral cavity through antisepticmouthwash twice daily (Figure I in the online-only Data Sup-plement) did not show the nitrate-mediated positive effect onperfusion (55�3%; P�0.02; Figure 1B and 1C).

Dietary Nitrate Supplementation AugmentsS-Nitrosothiol, Nitrite, and Nitrate Levels inPlasma Without Affecting Feeding BehaviorIntake of nitrate by drinking water (�150 �mol/d) over 7 daysshowed an increase in plasma S-nitrosothiol, nitrite, and nitratelevels compared with controls (S-nitrosothiol, 349�98 versus9�1.9 nmol/L; nitrite, 5.8�1.7 versus 0.5�0.1 �mol/L; nitrate,

Figure 3. Effect of dietary nitrate supplementation on body weight and feeding behavior. A, Experimental protocol. Daily assessment ofwater, food consumption, and body weight. B through D, Dietary nitrate supplementation and daily mouthwash (MW) procedure had noeffect on water and food intake and body weight development. Data are expressed as mean�SEM (n�4–5).

1986 Circulation October 16, 2012

at CONS SWISS CONSORTIA on July 21, 2014http://circ.ahajournals.org/Downloaded from

451�75 versus 40�17 �mol/L; Figure 2A and 2B). The nitriteand S-nitrosothiol concentrations were markedly lower in micethat underwent an antibacterial mouthwash (Figure 2B). We alsoobserved lower amounts of plasma nitrate in mouthwash-treatedanimals. This could theoretically be due to a decrease in waterintake (the nitrate source) in mouthwash-treated animals. How-ever, antibacterial mouthwash procedures or the different nitratecontent of drinking water had no influence on water and foodconsumption or body weight (Figure 3A–3D).

Dietary Nitrate Increases Revascularization AfterLong-Term Ligation of the Femoral ArteryTo investigate whether the improvement in tissue perfusionwas mediated in part by increased revascularization, capillarydensity was determined 7 days after hind-limb surgery(Figure 4A). The capillary density was higher in the nitrate-treated group compared with controls (123�20 versus 59�15cells per 1 mm2; P�0.045; Figure 4A and 4B) and micereceiving nitrate and antibacterial mouthwash (57�7 cells per1 mm2; P�0.02; Figure 4A and 4B), as evident by CD31�

staining in skeletal muscle tissue.

Dietary Nitrate Attenuates Apoptosis in Myoblastsin Chronic Hind-Limb IschemiaTo evaluate the remaining regenerative capacity of tissue inchronic ischemia, hind limbs were removed from mice on day7 after surgical ligation of the femoral artery, and myoblastswere isolated and cultured and underwent TUNEL stainingfor the detection of apoptotic myoblasts (Figure 5A and 5B).The amount of apoptotic nuclei was lower in the nitrate-treated group compared with controls (67�16 versus 197�19cells per 1 mm2; P�0.001; Figure 5B and 5C). Blocking oforal nitrate reduction by antibacterial mouthwash showed nobeneficial effects of nitrate (169�26 versus 67�16 cells per1 mm2; P�0.02; Figure 5B and 5C).

Dietary Nitrate Increases the Mobilization andMigration of Endothelium-Regenerating CellsQuantitative FACS analysis of CD34�/Flk-1� cells in periph-eral blood revealed that mobilization of this population wasdramatically higher in mice receiving nitrate-enriched drink-ing water compared with the control group (P�0.001; Figure

Figure 4. Effect of dietary nitrate on revascularization. A, Experimental protocol. B, Representative original micrographs of hind-limbsections show amounts of endothelial cells per 1 mm2. Dietary nitrate–treated mice show higher numbers of CD31�/CD45� cells 7days after hind-limb surgery, whereas the mice receiving mouthwash (MW) did not exhibit this effect. Top row micrographs showCD31�/CD45�-stained cells (red); middle row, DAPI-stained nuclei (blue); bottom row, double-stained (merged) cells indicating endo-thelial cells. C, Quantitative analysis of capillary density displayed as CD31�/CD45�-stained cells per 1 mm2 hind limb. Bars showhigher capillary density in dietary nitrate–supplemented mice at day 7 (black bars; P�0.045). Treatment with an antiseptic mouthwashdid not exhibit the nitrate-mediated increase in capillary density (gray bars; P�0.02). Data are expressed as mean�SEM (n�6).

Hendgen-Cotta et al Dietary Nitrate Improves Revascularization 1987

at CONS SWISS CONSORTIA on July 21, 2014http://circ.ahajournals.org/Downloaded from

6A–6C). Again, no nitrate-mediated effects were seen in thegroup that received antibacterial mouthwash (P�0.01; Figure6C). After demonstrating the mobilizing effect on CD34�/Flk-1� cells with dietary nitrate supplementation, we inves-tigated the migration of this mobilized population into ische-mic tissue using NMRI and EGFP transgenic mice for bonemarrow transplantation. The NMRI recipients underwentlethal irradiation and subsequent EGFP� bone marrow celltransplantation to generate marked hematopoietic chimeras.After 4 weeks of recovery, these animals received nitratesupplementation and control treatment before long-term liga-tion of the femoral artery was performed (Figure 6A). Toassess migration of bone marrow–derived cells into theischemic tissue, we investigated the localization of EGFP�/CD31�/CD45� cells in paraffin sections of the hind limbs.Blinded analysis of at least 5 visual fields per section revealedthat animals treated with nitrate showed higher amounts ofEGFP�/CD31�/CD45� cells at the site of active revascular-ization compared with animals on a low-nitrate diet(P�0.029; Figure 6D and 6E). No effects on the migration ofbone marrow–derived EGFP�/CD31�/CD45� cells wereseen with nitrate when antiseptic mouthwash was used(P�0.034; Figure 6D and 6E).

Dietary Nitrate Does Not Alter TissuecGMP LevelsQuantification of cGMP levels by competitive enzyme im-munoassay in excised skeletal muscle tissue revealed noimpact of nitrate supplementation (4�5 fmol/mg tissue) ormouthwash procedure (9�7 fmol/mg tissue). This result isconsistent with recent findings in studies investigating therole of nitrite injections in ischemia,16 indicating that othernitrite-signaling pathways for regulating angiogenic activitymay exist.20 However, it does not exclude the existence ofcGMP-mediated effects because counterregulatory mecha-nisms may disguise NO-mediated changes in this secondmessenger.

DiscussionChronically ischemic tissue, a feature of peripheral andcoronary artery disease, requires a remodeling of the vascularnetwork to reconstitute and sustain its viability. The physio-logical repair response, however, is often not sufficient, andtherapeutic angiogenesis remains an unmet medical need. Inthe present study, we demonstrate that dietary nitrate supple-mentation improves revascularization through mechanisms

Figure 5. Effect of dietary nitrate on apoptosis in myoblasts. A, Experimental protocol. B, Terminal deoxynucleotidyl transferase dUTPnick-end labeling (TUNEL) staining of isolated myoblasts demonstrating lower amounts of apoptotic cells in mice receiving dietarynitrate supplementation (middle) compared with those receiving control treatment (left) or antiseptic mouthwash (MW; right). Top rowmicrographs show TUNEL-positive nuclei indicating apoptotic cells (green); middle row, DAPI-positive cells indicating nuclei (blue); bot-tom, double-stained cells (merged) indicating apoptotic cells. C, Quantitative analysis of TUNEL-stained myoblasts per hind limb dis-played as the ratio between ischemic and nonischemic hind limb and expressed as percent. Bars show significantly decreased apopto-sis in mice supplemented with dietary nitrate (black bar). Mice without dietary nitrate supplementation (white bar; P�0.001) or withantiseptic mouthwash (gray bar; P�0.02) did not show nitrate-mediated regenerative effects. Data are expressed as mean�SEM(n�9–11).

1988 Circulation October 16, 2012

at CONS SWISS CONSORTIA on July 21, 2014http://circ.ahajournals.org/Downloaded from

involving the mobilization and migration of endothelium-regenerating cells. This is accompanied by an attenuation ofmyoblast apoptosis.

Despite endeavors to promote blood vessel formation byadministration of proangiogenic factors, gene therapy, ortargeting of microRNAs, clinically applicable strategies havenot yet been developed or are still in a preclinical phase.21–24

Although proangiogenic properties of NO have been demon-strated, direct application of NO or classic NO donors in vivobears a high risk of failure owing to lack of targeted delivery,development of tolerance, cellular toxicity, and risk of hypo-tension.25–27 Pharmacological treatment with nitrite, an oxi-dation product of NO, may offer an alternative therapeuticopportunity. We have recently shown that the administrationof nitrite under hypoxia regulates cardiac energetics andfunctions resembling the characteristics described for acutehibernation.28 A cytoprotective role of nitrite in the setting of

myocardial, liver, kidney, and brain ischemia/reperfusioninjury has previously been demonstrated.19,29,30 In ischemicangiogenesis, a continuous pharmacological intervention withnitrite injections resulted in an increased vascular density inthe hind limb and stimulated endothelial cell proliferation.16

Nitrite has several properties that make it an attractive drugcandidate. Its conversion into bioactive nitrogen oxides isslow,31 and the effect is not subjective to tolerance.32 There-fore, one can deliver it even in fairly high doses without therisk of classic NO-related side effects such as hypotension. Inaddition, first-passage metabolism in the liver is minimalcompared with that of classic organic nitrates. Moreover, itsbioactivation is augmented in ischemic areas, thereby makingthe drug selective to ischemic areas.

Dietary nitrate is an effective way of delivering nitritesystemically, and with a half-life of 6 hours, it continuouslygenerates nitrite and bioactive nitrogen oxides like

Figure 6. Impact of dietary nitrate on mobilization and migration of endothelium-regenerating cells. A, Experimental protocol. B, Exem-plary flow cytometric analysis shows the total mononuclear cells in peripheral blood analyzed at day 7 after the onset of hind-limb is-chemia. The gated population displays isolated blood cells, stained with FITC rat anti-mouse CD34 and APC rat anti-mouse Flk-1. Band C, The amount of CD34�/Flk-1� cells was higher in the nitrate-treated group compared with those receiving standard drinkingwater (P�0.001). Antiseptic mouthwash (MW) did not show this effect and distinctly prevented accumulation of CD34�/Flk-1� cellsdespite nitrate supplementation (P�0.01; n�7–10). D, Representative original micrographs of hind-limb sections show amounts ofenhanced green fluorescence protein–positive (EGFP�)/CD31�/CD45� cells per 1 mm2. Mice receiving dietary nitrate showed anincreased migration of EGFP�/CD31�/CD45� cells 7 days after hind-limb surgery (middle) compared with mice receiving the mouth-wash procedure (right). E, Bars show significantly increased EGFP�/CD31�/CD45� cell migration in mice supplemented with dietarynitrate at day 7 (black bars; P�0.029), whereas antiseptic mouthwash–treated mice displayed a markedly attenuated nitrate-mediatedincrease in EGFP�/CD31�/CD45� cell migration in chronic hind-limb ischemia (gray bars; P�0.034; n�3–4). Data are expressed asmean�SEM. FACS indicates fluorescence-activated cell sorting.

Hendgen-Cotta et al Dietary Nitrate Improves Revascularization 1989

at CONS SWISS CONSORTIA on July 21, 2014http://circ.ahajournals.org/Downloaded from

S-nitrosothiols. Recent research in animals and humans hasconfirmed the beneficial effects of dietary nitrate.14,33–38

Intake of nitrate-rich foods such as leafy vegetables andfruits, abundant in the Mediterranean diet, may representan opportunity for disease prevention and health modula-tion of human physiological functions. The NO-like effectsof dietary nitrate range from lowering of blood pres-sure14,38 to a reduction in experimental myocardial infarctsize,33 inhibition of platelet aggregation,34 increase inexercise tolerance in peripheral arterial disease,35 andimprovement in intrinsic mitochondrial efficiency.36 Inaddition, nitrate supplementation offered cardioprotectionagainst doxorubicin-induced cardiomyopathy37 and pre-vented oxidative stress and cardiovascular and renal injuryin salt-induced hypertension.39

These findings from experimental animal studies andhuman clinical studies support the hypothesis that nitrate offood origin promotes cardiovascular health and thus presentsa novel mechanistic explanation for some of the well-knownhealth benefits of a diet rich in vegetables.

We demonstrate here that dietary nitrate supplementationstrongly augments perfusion recovery in chronic hind-limbischemia in vivo via a significant increase in capillarydensity. This improvement was associated with an increase incirculating nitrite and S-nitrosothiol concentrations, an ele-vated mobilization of CD34�/Flk-1� cells, and migration ofbone marrow–derived CD31�/CD45� cells into ischemictissue. The associative relation, increased plasma nitritelevels and an increased mobilization of CD34�/Flk-1� cellsfrom the bone marrow, has also been observed in patientswith coronary artery disease receiving nutrients that increasedNO bioavailability.40

In line with these findings, the results of the present studyfurther point to a distinct contribution of dietary nitratesupplementation on tissue viability. Dietary nitrate amelio-rates the remarkable capacity of adult skeletal muscles toregenerate myofibers after damage. This rapid repair processis carried out mainly by satellite cells with a contribution ofNO.41 Quiescent satellite cells become active and proliferateon injury and display the regenerative capacity of the muscle.Committed daughter cells, the myoblasts, continue to prolif-erate, followed by definite differentiation as initialized by acoordinated cellular signaling.

Intriguingly, disruption of the nitrate-NO pathway bylong-term eradication of the oral microflora completely abol-ished the beneficial effects of the dietary nitrate supplemen-tation. This daily intervention effectively suppressed theincrease in circulating nitrite and S-nitrosothiol levels that isobserved after intake of nitrate-rich food or dietary nitratesupplementation in drinking water.15,38,42 The fact that mouth-wash prevented increases in plasma nitrite and all observedbeneficial effects of nitrate provides significant mechanisticinsight because it suggests an important role of intermediatenitrite formation in the bioactivation of nitrate. However, onecannot entirely rule out the possibility that oral bacteria coulddirectly catalyze nitrosation reactions from nitrate withoutintermediate nitrite formation because recent data show thatprotein S-nitros(yl)ation is an obligate concomitant process ofanaerobic respiration on nitrate in Escherichia coli.43 It is

important to note that the exact identity of the final media-tor(s) of the nitrate effects is still not settled and that multiplepossible mechanisms exist. Nitrite as a reservoir can befurther reduced to NO via numerous pathways in blood andtissue, and this process is indeed accelerated under hypoxicconditions.44 – 47 In addition, nitrite forms other reactivenitrogen oxides, including S-nitrosothiols and nitrogenoxide, the latter promoting the formation of bioactivenitration products such as nitro fatty acids.11,48,49 Thisoxidative chemistry is emerging as an alternative nitrite-mediated signaling mechanism33,50 that could explain someof the NO-like effects of nitrate and nitrite seen undernormoxic conditions. The current data suggest thatS-nitros(yl)ation might be a crucial signal transductionpathway and that the formation of S-nitrosothiols mightaccount for the regenerative response to the dietary nitratesupplementation in chronic ischemia. This is supported bythe marked increase in circulating S-nitrosothiols observedhere after nitrate supplementation.

ConclusionsWe show here that dietary nitrate supplementation in-creases the regenerative capacity of ischemic tissue andthat this effect depends critically on bacteria-dependentbioactivation of nitrate in the oral cavity. These resultssuggest that dietary nitrate supplementation may offer anattractive nutrition-based strategy to improve ischemia-induced revascularization.

Sources of FundingThis study was supported in part by grants from the DeutscheForschungsgemeinschaft (DFG; Ra969/6-1 to Dr Rassaf and Ke405/5-1 to Dr Kelm). Dr Rassaf is a Heisenberg professor funded bythe DFG (Ra969/7-1). Dr Totzeck received a scholarship from theDeutsche Herzstiftung. Dr Luedike receives a stipend from theGerman Cardiac Society.

DisclosuresNone.

References1. Carmeliet P, Jain RK. Molecular mechanisms and clinical applications of

angiogenesis. Nature. 2011;473:298–307.2. Christman KL, Fang Q, Kim AJ, Sievers RE, Fok HH, Candia AF, Colley

KJ, Herradon G, Ezquerra L, Deuel TF, Lee RJ. Pleiotrophin inducesformation of functional neovasculature in vivo. Biochem Biophys ResComm. 2005;332:1146–1152.

3. Dimmeler S, Zeiher AM, Schneider MD. Unchain my heart: the scientificfoundations of cardiac repair. J Clin Invest. 2005;115:572–583.

4. Roy H, Bhardwaj S, Yla-Herttuala S. Biology of vascular endothelialgrowth factors. FEBS Lett. 2006;580:2879–2887.

5. Ruiz de Almodovar C, Luttun A, Carmeliet P. An SDF-1 trap for myeloidcells stimulates angiogenesis. Cell. 2006;124:18–21.

6. Springer ML, Ozawa CR, Banfi A, Kraft PE, Ip TK, Brazelton TR, BlauHM. Localized arteriole formation directly adjacent to the site of VEGF-induced angiogenesis in muscle. Mol Ther. 2003;7:441–449.

7. Ziche M, Morbidelli L, Choudhuri R, Zhang HT, Donnini S, Granger HJ,Bicknell R. Nitric oxide synthase lies downstream from vascular endo-thelial growth factor-induced but not basic fibroblast growth factor-induced angiogenesis. J Clin Invest. 1997;99:2625–2634.

8. Aicher A, Heeschen C, Mildner-Rihm C, Urbich C, Ihling C, Technau-Ihling K, Zeiher AM, Dimmeler S. Essential role of endothelial nitric

1990 Circulation October 16, 2012

at CONS SWISS CONSORTIA on July 21, 2014http://circ.ahajournals.org/Downloaded from

oxide synthase for mobilization of stem and progenitor cells. Nat Med.2003;9:1370–1376.

9. Morbidelli L, Chang CH, Douglas JG, Granger HJ, Ledda F, Ziche M.Nitric oxide mediates mitogenic effect of VEGF on coronary venularendothelium. Am J Physiol. 1996;270:H411–H415.

10. Lundberg JO, Gladwin MT, Ahluwalia A, Benjamin N, Bryan NS, ButlerA, Cabrales P, Fago A, Feelisch M, Ford PC, Freeman BA, Frenneaux M,Friedman J, Kelm M, Kevil CG, Kim-Shapiro DB, Kozlov AV, LancasterJR Jr, Lefer DJ, McColl K, McCurry K, Patel RP, Petersson J, Rassaf T,Reutov VP, Richter-Addo GB, Schechter A, Shiva S, Tsuchiya K, vanFaassen EE, Webb AJ, Zuckerbraun BS, Zweier JL, Weitzberg E. Nitrateand nitrite in biology, nutrition and therapeutics. Nat Chem Biol. 2009;5:865–869.

11. Lundberg JO, Weitzberg E, Gladwin MT. The nitrate-nitrite-nitric oxidepathway in physiology and therapeutics. Nat Rev Drug Discov. 2008;7:156–167.

12. Lundberg JO, Weitzberg E, Cole JA, Benjamin N. Nitrate, bacteria andhuman health. Nat Rev Microbiol. 2004;2:593–602.

13. Lundberg JO, Govoni M. Inorganic nitrate is a possible source forsystemic generation of nitric oxide. Free Radic Biol Med. 2004;37:395–400.

14. Larsen FJ, Ekblom B, Sahlin K, Lundberg JO, Weitzberg E. Effects ofdietary nitrate on blood pressure in healthy volunteers. N Engl J Med.2006;355:2792–2793.

15. Petersson J, Carlstrom M, Schreiber O, Phillipson M, Christoffersson G,Jagare A, Roos S, Jansson EA, Persson AE, Lundberg JO, Holm L.Gastroprotective and blood pressure lowering effects of dietary nitrate areabolished by an antiseptic mouthwash. Free Radic Biol Med. 2009;46:1068–1075.

16. Kumar D, Branch BG, Pattillo CB, Hood J, Thoma S, Simpson S, IllumS, Arora N, Chidlow JH Jr, Langston W, Teng X, Lefer DJ, Patel RP,Kevil CG. Chronic sodium nitrite therapy augments ischemia-inducedangiogenesis and arteriogenesis. Proc Natl Acad Sci U S A. 2008;105:7540–7545.

17. Couffinhal T, Silver M, Zheng LP, Kearney M, Witzenbichler B, IsnerJM. Mouse model of angiogenesis. Am J Pathol. 1998;152:1667–1679.

18. Rassaf T, Bryan NS, Kelm M, Feelisch M. Concomitant presence ofN-nitroso and S-nitroso proteins in human plasma. Free Radic Biol Med.2002;33:1590–1596.

19. Hendgen-Cotta UB, Merx MW, Shiva S, Schmitz J, Becher S, Klare JP,Steinhoff HJ, Goedecke A, Schrader J, Gladwin MT, Kelm M, Rassaf T.Nitrite reductase activity of myoglobin regulates respiration and cellularviability in myocardial ischemia-reperfusion injury. Proc Natl Acad SciU S A. 2008;105:10256–10261.

20. Bryan NS, Fernandez BO, Bauer SM, Garcia-Saura MF, Milsom AB,Rassaf T, Maloney RE, Bharti A, Rodriguez J, Feelisch M. Nitrite is asignaling molecule and regulator of gene expression in mammaliantissues. Nat Chem Biol. 2005;1:290–297.

21. Doebele C, Bonauer A, Fischer A, Scholz A, Reiss Y, Urbich C,Hofmann WK, Zeiher AM, Dimmeler S. Members of themicroRNA-17-92 cluster exhibit a cell-intrinsic antiangiogenic functionin endothelial cells. Blood. 2010;115:4944–4950.

22. Tirziu D, Simons M. Angiogenesis in the human heart: gene and celltherapy. Angiogenesis. 2005;8:241–251.

23. Webster KA. Therapeutic angiogenesis: a complex problem requiring asophisticated approach. Cardiovasc Toxicol. 2003;3:283–298.

24. Mughal NA, Russell DA, Ponnambalam S, Homer-Vanniasinkam S.Gene therapy in the treatment of peripheral arterial disease. Br J Surg.2012;99:6–15.

25. Loh E, Stamler JS, Hare JM, Loscalzo J, Colucci WS. Cardiovasculareffects of inhaled nitric oxide in patients with left ventricular dysfunction.Circulation. 1994;90:2780–2785.

26. Kiss J, Kaapa P, Savunen T. Antioxidants combined with no donorenhance systemic inflammation in acute lung injury in rats. ScandCardiovasc J. 2007;41:186 –191.

27. Nakajima A, Ueda K, Takaoka M, Kurata H, Takayama J, Ohkita M,Matsumura Y. Effects of pre- and post-ischemic treatments with FK409,a nitric oxide donor, on ischemia/reperfusion-induced renal injury andendothelin-1 production in rats. Biol Pharm Bull. 2006;29:577–579.

28. Rassaf T, Flogel U, Drexhage C, Hendgen-Cotta U, Kelm M, Schrader J.Nitrite reductase function of deoxymyoglobin: oxygen sensor and reg-ulator of cardiac energetics and function. Circ Res. 2007;100:1749–1754.

29. Pluta RM, Dejam A, Grimes G, Gladwin MT, Oldfield EH. Nitriteinfusions to prevent delayed cerebral vasospasm in a primate model ofsubarachnoid hemorrhage. JAMA. 2005;293:1477–1484.

30. Lu P, Liu F, Yao Z, Wang CY, Chen DD, Tian Y, Zhang JH, Wu YH.Nitrite-derived nitric oxide by xanthine oxidoreductase protects the liveragainst ischemia-reperfusion injury. Hepatobiliary Pancreat Dis Int.2005;4:350–355.

31. Jansson EA, Huang L, Malkey R, Govoni M, Nihlen C, Olsson A,Stensdotter M, Petersson J, Holm L, Weitzberg E, Lundberg JO. Amammalian functional nitrate reductase that regulates nitrite and nitricoxide homeostasis. Nat Chem Biol. 2008;4:411–417.

32. Dejam A, Hunter CJ, Tremonti C, Pluta RM, Hon YY, Grimes G, PartoviK, Pelletier MM, Oldfield EH, Cannon RO 3rd, Schechter AN, GladwinMT. Nitrite infusion in humans and nonhuman primates: endocrineeffects, pharmacokinetics, and tolerance formation. Circulation. 2007;116:1821–1831.

33. Bryan NS, Calvert JW, Elrod JW, Gundewar S, Ji SY, Lefer DJ. Dietarynitrite supplementation protects against myocardial ischemia-reperfusioninjury. Proc Natl Acad Sci U S A. 2007;104:19144–19149.

34. Webb AJ, Milsom AB, Rathod KS, Chu WL, Qureshi S, Lovell MJ,Lecomte FM, Perrett D, Raimondo C, Khoshbin E, Ahmed Z, Uppal R,Benjamin N, Hobbs AJ, Ahluwalia A. Mechanisms underlying eryth-rocyte and endothelial nitrite reduction to nitric oxide in hypoxia: role forxanthine oxidoreductase and endothelial nitric oxide synthase. Circ Res.2008;103:957–964.

35. Kenjale AA, Ham KL, Stabler T, Robbins JL, Johnson JL, VanbruggenM, Privette G, Yim E, Kraus WE, Allen JD. Dietary nitrate supplemen-tation enhances exercise performance in peripheral arterial disease. J ApplPhysiol. 2011;110:1582–1591.

36. Larsen FJ, Schiffer TA, Borniquel S, Sahlin K, Ekblom B, Lundberg JO,Weitzberg E. Dietary inorganic nitrate improves mitochondrial efficiencyin humans. Cell Metab. 2011;13:149–159.

37. Zhu SG, Kukreja RC, Das A, Chen Q, Lesnefsky EJ, Xi L. Dietary nitratesupplementation protects against doxorubicin-induced cardiomyopathyby improving mitochondrial function. J Am Coll Cardiol. 2011;57:2181–2189.

38. Kapil V, Milsom AB, Okorie M, Maleki-Toyserkani S, Akram F, RehmanF, Arghandawi S, Pearl V, Benjamin N, Loukogeorgakis S, Macallister R,Hobbs AJ, Webb AJ, Ahluwalia A. Inorganic nitrate supplementationlowers blood pressure in humans: role for nitrite-derived NO. Hyper-tension. 2010;56:274–281.

39. Carlstrom M, Persson AE, Larsson E, Hezel M, Scheffer PG, Teerlink T,Weitzberg E, Lundberg JO. Dietary nitrate attenuates oxidative stress,prevents cardiac and renal injuries, and reduces blood pressure in salt-induced hypertension. Cardiovasc Res. 2011;89:574–585.

40. Heiss C, Jahn S, Taylor M, Real WM, Angeli FS, Wong ML, Amabile N,Prasad M, Rassaf T, Ottaviani JI, Mihardja S, Keen CL, Springer ML,Boyle A, Grossman W, Glantz SA, Schroeter H, Yeghiazarians Y.Improvement of endothelial function with dietary flavanols is associatedwith mobilization of circulating angiogenic cells in patients with coronaryartery disease. J Am Coll Cardiol. 2010;56:218–224.

41. Dhawan J, Rando TA. Stem cells in postnatal myogenesis: molecularmechanisms of satellite cell quiescence, activation and replenishment.Trends Cell Biol. 2005;15:666–673.

42. Larsen FJ, Weitzberg E, Lundberg JO, Ekblom B. Dietary nitrate reducesmaximal oxygen consumption while maintaining work performance inmaximal exercise. Free Radic Biol Med. 2010;48:342–347.

43. Seth D, Hausladen A, Wang YJ, Stamler JS. Endogenous proteinS-nitrosylation in E. coli: regulation by OxyR. Science. 2012;336:470–473.

44. Totzeck M, Hendgen-Cotta UB, Luedike P, Berenbrink M, Klare JP,Steinhoff HJ, Semmler D, Shiva S, Williams D, Kipar A, Gladwin MT,Schrader J, Kelm M, Cossins AR, Rassaf T. Nitrite regulates hypoxic vaso-dilation via myoglobin-dependent nitric oxide generation. Circulation. 2012;126:325–334.

45. Gladwin MT, Schechter AN, Kim-Shapiro DB, Patel RP, Hogg N, ShivaS, Cannon RO 3rd, Kelm M, Wink DA, Espey MG, Oldfield EH, PlutaRM, Freeman BA, Lancaster JR Jr, Feelisch M, Lundberg JO. Theemerging biology of the nitrite anion. Nat Chem Biol. 2005;1:308–314.

46. Lundberg JO, Weitzberg E. No generation from nitrite and its role invascular control. Arterioscler Thromb Vasc Biol. 2005;25:915–922.

47. van Faassen EE, Bahrami S, Feelisch M, Hogg N, Kelm M, Kim-ShapiroDB, Kozlov AV, Li H, Lundberg JO, Mason R, Nohl H, Rassaf T,Samouilov A, Slama-Schwok A, Shiva S, Vanin AF, Weitzberg E, ZweierJ, Gladwin MT. Nitrite as regulator of hypoxic signaling in mammalianphysiology. Med Res Rev. 2009;29:683–741.

48. Luedike P, Hendgen-Cotta UB, Sobierajski J, Totzeck M, Reeh M, DeworM, Lue H, Krisp C, Wolters D, Kelm M, Bernhagen J, Rassaf T. Car-

Hendgen-Cotta et al Dietary Nitrate Improves Revascularization 1991

at CONS SWISS CONSORTIA on July 21, 2014http://circ.ahajournals.org/Downloaded from

dioprotection through S-nitros(yl)ation of macrophage migration inhibi-tory factor. Circulation. 2012;125:1880–1889.

49. Khoo NK, Freeman BA. Electrophilic nitro-fatty acids: anti-inflammatorymediators in the vascular compartment. Curr Opin Pharmacol. 2010;10:179–184.

50. Jadert C, Petersson J, Massena S, Ahl D, Grapensparr L, Holm L,Lundberg JO, Phillipson M. Decreased leukocyte recruitment byinorganic nitrate and nitrite in microvascular inflammation andNSAID-induced intestinal injury. Free Radic Biol Med. 2012;52:683– 692.

CLINICAL PERSPECTIVEWith the worldwide increase in cardiovascular diseases in recent decades, the need for novel preventive and noninvasivetherapeutic strategies has grown tremendously. In this context, there is accumulating evidence that inorganic nitrate fromdietary sources is able to influence the hallmarks of cardiovascular functions, including blood pressure regulation. Thebioactivation of nitrate from dietary or endogenous sources is carried out mainly by commensal bacteria that expresseffective nitrate reductase enzymes and are located in the gastrointestinal tract and on body surfaces. Under conditions oflow oxygen tensions, nitrate and nitrite are physiologically recycled in blood and tissues to form nitric oxide and otherbioactive nitrogen oxides that mediate cytoprotective signaling in the setting of pathological ischemia. The present studyprovides the first evidence that dietary nitrate supplementation improves revascularization in chronic ischemia. This studyidentified that dietary nitrate supplementation increases mobilization and migration of regenerative cells, improves theregenerative capacities of chronically ischemic tissue, and decreases apoptosis at the site of ischemia. Eradicating thecommensal bacteria in the oral cavity and thus interrupting the bioactivation of the ingested nitrate decreased circulatinglevels of bioactive nitrogen oxides and reversed all of these beneficial effects. These data underscore the potentialtherapeutic value of inorganic nitrate and suggest the possible application of a nutritional approach in the prevention andtreatment of cardiovascular diseases.

1992 Circulation October 16, 2012

at CONS SWISS CONSORTIA on July 21, 2014http://circ.ahajournals.org/Downloaded from

1

SUPPLEMENTAL MATERIAL

Dietary Nitrate Supplementation Improves Revascularization in Chronic Ischemia

Ulrike B. Hendgen-Cotta, PhD1*, Peter Luedike, MD1*, Matthias Totzeck, MD1, Martina

Kropp, MSc1, Andreas Schicho, MS1, Pia Stock, MSc1, Christos Rammos, MD1, Michael

Niessen, MS1, Christian Heiss, MD1, Jon O. Lundberg, MD, PhD2, Eddie Weitzberg, MD,

PhD2, Malte Kelm, MD1, Tienush Rassaf, MD1†

Supplemental Figure 1

2

Figure legend

Supplemental Figure 1. Antibacterial mouthwash procedure eradicated oral bacterial

flora. Oral smear tests were performed before and after mouthwash (MW) procedure. Mice

received 3 s of oral MW via an hexitidine containing aerosol can. Aerobic (ae) an anaerobic

(an) bacteria were cultured on standard agar plates and colony forming units (CFU) were

counted. MW procedure led to an eradication of oral bacteria (p < 0.02). Data are expressed

as mean ± SEM (n = 3).