Dietary nitrate supplementation improves revascularization in chronic ischemia
Open accessAbstract
Background—: Revascularization is an adaptive repair mechanism that restores blood flow to undersupplied ischemic tissue. Nitric oxide plays an important role in this process. Whether dietary nitrate, serially reduced to nitrite by commensal bacteria in the oral cavity and subsequently to nitric oxide and other nitrogen oxides, enhances ischemia-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 levels were assessed. Enhanced green fluorescence protein transgenic mice were used for bone marrow transplantation. Dietary nitrate 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 apoptosis of potentially regenerative myoblasts in chronically ischemic tissue. The regenerative effects of nitrate treatment were abolished 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 enhance ischemia-induced revascularization.
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TL;DR
Dietary nitrate supplementation in mice with chronic hind-limb ischemia improved perfusion recovery and revascularization, increased circulating nitrite/S-nitrosothiols, mobilized endothelium-regenerating cells, and reduced myoblast apoptosis. These benefits were abolished by antiseptic mouthwash that eradicated oral nitrate-reducing bacteria.
Key findings
- Nitrate-treated mice showed greater perfusion recovery in the ischemic hind limb than controls (73±4% versus 59±4%; P<0.02), while mouthwash-treated nitrate mice did not (55±3%; P<0.02).
- Plasma S-nitrosothiols, nitrite, and nitrate increased with nitrate supplementation (S-nitrosothiol, 349±98 versus 9±1.9 nmol/L; nitrite, 5.8±1.7 versus 0.5±0.1 µmol/L; nitrate, 451±75 versus 40±17 µmol/L); mouthwash lowered nitrite and S-nitrosothiol levels.
- Capillary density was higher with nitrate than control (123±20 versus 59±15 cells per 1 mm²; P=0.045), and nitrate plus mouthwash showed no increase (57±7 cells per 1 mm²; P<0.02).
- Apoptotic myoblast nuclei were lower with nitrate than control (67±16 versus 197±19 cells per 1 mm²; P<0.001), with no nitrate benefit after mouthwash (169±26 cells per 1 mm²; P<0.02); nitrate also increased CD34⁺/Flk-1⁺ mobilization (P<0.001) and EGFP⁺/CD31⁺/CD45⁻ cell migration (P=0.029), both blocked by mouthwash.
Methods
- Male NMRI mice (14±3 weeks old, average body weight 30±6 g) received sodium nitrate in drinking water (1 g/L; ≈150 µmol/d) or sodium chloride control for 14 days; unilateral hind-limb ischemia was created by left femoral artery excision at day 7. Subgroups received antiseptic mouthwash (0.2% hexetidine) twice daily to suppress oral microflora.
- Outcomes included laser Doppler perfusion imaging, CD31/CD45 immunohistology for capillary density, TUNEL staining of isolated myoblasts for apoptosis, FACS for CD34⁺/Flk-1⁺ cells, EGFP bone marrow transplantation for migration of bone marrow-derived CD31⁺/CD45⁻ cells, and HPLC/chemiluminescence or immunoassay for plasma nitrate, nitrite, S-nitrosothiols, and tissue cGMP.
- Results are mean±SEM; comparisons used 1-way ANOVA with Bonferroni, Kruskal-Wallis with Mann-Whitney, or unpaired t tests, with P<0.05 considered significant.
Limitations
- The paper states that the exact identity of the final mediator(s) of the nitrate effects is still not settled and that multiple possible mechanisms exist; it also notes that unchanged tissue cGMP does not exclude cGMP-mediated effects because counterregulatory mechanisms may disguise them.
- Evident from the text: findings are from a mouse hind-limb ischemia model, not human patients, and some analyses used small groups (e.g., migration analysis n=3–4). The paper does not include a dedicated limitations section.
Model-written summary. Check it against the paper before relying on it.
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- Findingown data
Dietary nitrate increases migration of bone marrow-derived EGFP+/CD31+/CD45− cells to the site of active revascularization in ischemic hind-limb tissue of mice compared with low-nitrate diet.
“Blinded analysis of at least 5 visual fields per section revealed that animals treated with nitrate showed higher amounts of EGFP ϩ /CD31 ϩ /CD45 Ϫ cells at the site of active revascularization compared with animals on a low-nitrate diet (Pϭ0.029; Figure 6D and 6E).”
Quote in the paperSubstantial claim - Findingown data
Antibacterial mouthwash abolishes the nitrate-mediated increase in migration of bone marrow-derived EGFP+/CD31+/CD45− cells in mice.
“No effects on the migration of bone marrow-derived EGFP ϩ /CD31 ϩ /CD45 Ϫ cells were seen with nitrate when antiseptic mouthwash was used (Pϭ0.034; Figure 6D and 6E).”
Quote in the paperSubstantial claim - Findingown data
Dietary nitrate supplementation does not alter skeletal muscle tissue cGMP levels in mice.
“Quantification of cGMP levels by competitive enzyme immunoassay in excised skeletal muscle tissue revealed no impact of nitrate supplementation (4Ϯ5 fmol/mg tissue) or mouthwash procedure (9Ϯ7 fmol/mg tissue).”
Quote in the paperSubstantial claim - Findingown data
Antibacterial mouthwash lowers plasma nitrate levels in mice.
“We also observed lower amounts of plasma nitrate in mouthwash-treated animals.”
Quote in the paperNot a substantial claim - Findingown data
Antibacterial mouthwash abolishes the nitrate-mediated reduction in myoblast apoptosis in mice.
“Blocking of oral nitrate reduction by antibacterial mouthwash showed no beneficial effects of nitrate (169Ϯ26 versus 67Ϯ16 cells per 1 mm 2 ; PϽ0.02; Figure 5B and 5C).”
Quote in the paperSubstantial claim
- Findingown data
Dietary nitrate intake increases plasma nitrite levels in mice.
“Intake of nitrate by drinking water (Ϸ150 mol/d) over 7 days showed an increase in plasma S-nitrosothiol, nitrite, and nitrate levels compared with controls (S-nitrosothiol, 349Ϯ98 versus 9Ϯ1.9 nmol/L; nitrite, 5.8Ϯ1.7 versus 0.5Ϯ0.1 mol/L; nitrate, 451Ϯ75 versus 40Ϯ17 mol/L; Figure 2A and 2B).”
Quote in the paperSubstantial claim - Findingown data
Dietary nitrate intake increases plasma nitrate levels in mice.
“Intake of nitrate by drinking water (Ϸ150 mol/d) over 7 days showed an increase in plasma S-nitrosothiol, nitrite, and nitrate levels compared with controls (S-nitrosothiol, 349Ϯ98 versus 9Ϯ1.9 nmol/L; nitrite, 5.8Ϯ1.7 versus 0.5Ϯ0.1 mol/L; nitrate, 451Ϯ75 versus 40Ϯ17 mol/L; Figure 2A and 2B).”
Quote in the paperSubstantial claim - Findingown data
Antibacterial mouthwash lowers plasma nitrite and S-nitrosothiol concentrations in mice.
“The nitrite and S-nitrosothiol concentrations were markedly lower in mice that underwent an antibacterial mouthwash (Figure 2B).”
Quote in the paperSubstantial claim - Findingown data
Dietary nitrate increases mobilization of CD34+/Flk-1+ cells in peripheral blood of mice compared with controls.
“Quantitative FACS analysis of CD34 ϩ /Flk-1 ϩ cells in peripheral blood revealed that mobilization of this population was dramatically higher in mice receiving nitrate-enriched drinking water compared with the control group (PϽ0.001; Figure 6A-6C).”
Quote in the paperSubstantial claim - Findingown data
Antibacterial mouthwash abolishes the nitrate-mediated increase in mobilization of CD34+/Flk-1+ cells in mice.
“Again, no nitrate-mediated effects were seen in the group that received antibacterial mouthwash (PϽ0.01; Figure 6C).”
Quote in the paperSubstantial claim - Findingown data
Dietary nitrate intake increases plasma S-nitrosothiol levels in mice.
“Intake of nitrate by drinking water (Ϸ150 mol/d) over 7 days showed an increase in plasma S-nitrosothiol, nitrite, and nitrate levels compared with controls (S-nitrosothiol, 349Ϯ98 versus 9Ϯ1.9 nmol/L; nitrite, 5.8Ϯ1.7 versus 0.5Ϯ0.1 mol/L; nitrate, 451Ϯ75 versus 40Ϯ17 mol/L; Figure 2A and 2B).”
Quote in the paperSubstantial claim - Findingown data
Eradication of nitrate-reducing bacteria in the oral cavity by antiseptic mouthwash abolishes the nitrate-mediated improvement in perfusion recovery in mice.
“Eradication of the nitrate-reducing bacteria in the oral cavity through antiseptic mouthwash twice daily (Figure I in the online-only Data Supplement) did not show the nitrate-mediated positive effect on perfusion (55Ϯ3%; PϽ0.02; Figure 1B and 1C).”
Quote in the paperSubstantial claim - Findingown data
Dietary nitrate supplementation improves perfusion recovery in chronic hind-limb ischemia in mice.
“Perfusion recovery in the ischemic hind limb was significantly improved in mice treated with nitrate compared with controls (73Ϯ4% versus 59Ϯ4%; PϽ0.02; Figure 1B and 1C).”
Quote in the paperSubstantial claim - Findingown data
Antibacterial mouthwash and different nitrate content of drinking water do not influence water and food consumption or body weight in mice.
“However, antibacterial mouthwash procedures or the different nitrate content of drinking water had no influence on water and food consumption or body weight (Figure 3A-3D).”
Quote in the paperNot a substantial claim - Findingown data
Antiseptic mouthwash prevents the nitrate-mediated increase in capillary density.
“Treatment with an antiseptic mouthwash did not exhibit the nitrate-mediated increase in capillary density (gray bars; PϽ0.02).”
Quote in the paperSubstantial claim - Findingown data
Antiseptic mouthwash prevents the nitrate-induced accumulation of CD34+/Flk-1+ cells.
“Antiseptic mouthwash (MW) did not show this effect and distinctly prevented accumulation of CD34 ϩ /Flk-1 ϩ cells despite nitrate supplementation (PϽ0.01;nϭ7-10).”
Quote in the paperSubstantial claim - Findingown data
Dietary nitrate supplementation decreases apoptosis at the site of ischemia.
“This study identified that dietary nitrate supplementation increases mobilization and migration of regenerative cells, improves the regenerative capacities of chronically ischemic tissue, and decreases apoptosis at the site of ischemia.”
- Also stated in: § CLINICAL PERSPECTIVE · plain
“Bars show significantly decreased apoptosis in mice supplemented with dietary nitrate (black bar).”
Quote in the paperSubstantial claim - Also stated in: § CLINICAL PERSPECTIVE · plain
- Findingown data
Dietary nitrate supplementation increases circulating nitrate (NO3−) levels in mice over 7 days.
“Circulating RSNO, NO 2 Ϫ , and NO 3 Ϫ levels increase after nitrate supplementation during a 7-day period.”
Quote in the paperSubstantial claim - Findingown data
Dietary nitrate supplementation increases the amount of CD34+/Flk-1+ cells in peripheral blood compared with standard drinking water.
“The amount of CD34 ϩ /Flk-1 ϩ cells was higher in the nitrate-treated group compared with those receiving standard drinking water (PϽ0.001).”
Quote in the paperSubstantial claim - Findingown data
Dietary nitrate supplementation increases mobilization of regenerative cells.
“This study identified that dietary nitrate supplementation increases mobilization and migration of regenerative cells, improves the regenerative capacities of chronically ischemic tissue, and decreases apoptosis at the site of ischemia.”
Quote in the paperSubstantial claim - Findingown data
Dietary nitrate supplementation increases migration of regenerative cells.
“This study identified that dietary nitrate supplementation increases mobilization and migration of regenerative cells, improves the regenerative capacities of chronically ischemic tissue, and decreases apoptosis at the site of ischemia.”
Quote in the paperSubstantial claim - Findingown data
Dietary nitrate supplementation increases migration of EGFP+/CD31+/CD45− cells 7 days after hind-limb surgery.
“Mice receiving dietary nitrate showed an increased migration of EGFP ϩ /CD31 ϩ /CD45 Ϫ cells 7 days after hind-limb surgery (middle) compared with mice receiving the mouthwash procedure (right).”
Quote in the paperSubstantial claim - Findingown data
Dietary nitrate supplementation increases circulating nitrite (NO2−) levels in mice over 7 days.
“Circulating RSNO, NO 2 Ϫ , and NO 3 Ϫ levels increase after nitrate supplementation during a 7-day period.”
Quote in the paperSubstantial claim - Findingown data
Eradicating commensal bacteria in the oral cavity decreases circulating levels of bioactive nitrogen oxides.
“Eradicating the commensal bacteria in the oral cavity and thus interrupting the bioactivation of the ingested nitrate decreased circulating levels of bioactive nitrogen oxides and reversed all of these beneficial effects.”
Quote in the paperSubstantial claim- Rests on an assumption: Bioactivation of nitrate from dietary or endogenous sources is carried out mainly by commensal bacteria that express effective nitrate reductase enzymes and are located in the gastrointestinal tract and on body surfaces.
- Findingown data
Antibacterial mouthwash procedure eradicates oral bacterial flora in mice.
“MW procedure led to an eradication of oral bacteria (p < 0.02).”
Quote in the paperSubstantial claim - Findingown data
Antiseptic mouthwash reduces circulating RSNO and nitrite levels in mice.
“Mice receiving antiseptic mouthwash (MW) displayed lower RSNO and NO 2 Ϫ levels in plasma.”
Quote in the paperSubstantial claim - Findingown data
Nitrate-supplemented mice show increased perfusion recovery in chronic hind-limb ischemia.
“Nitrate-supplemented mice showed increased perfusion recovery in chronic hind-limb ischemia (black bars; PϽ0.02); this effect was not observed in mice receiving antiseptic mouthwash (gray bars; PϽ0.02).”
Quote in the paperSubstantial claim - Findingown data
Antiseptic mouthwash attenuates the nitrate-mediated increase in EGFP+/CD31+/CD45− cell migration in chronic hind-limb ischemia.
“Bars show significantly increased EGFP ϩ /CD31 ϩ /CD45 Ϫ cell migration in mice supplemented with dietary nitrate at day 7 (black bars; Pϭ0.029), whereas antiseptic mouthwash-treated mice displayed a markedly attenuated nitrate-mediated increase in EGFP ϩ /CD31 ϩ /CD45 Ϫ cell migration in chronic hind-limb ischemia (gray bars; Pϭ0.034; nϭ3-4).”
Quote in the paperSubstantial claim - Findingown data
Dietary nitrate-treated mice show higher numbers of CD31+/CD45− endothelial cells 7 days after hind-limb surgery.
“Dietary nitrate-treated mice show higher numbers of CD31 ϩ /CD45 Ϫ cells 7 days after hind-limb surgery, whereas the mice receiving mouthwash (MW) did not exhibit this effect.”
Quote in the paperSubstantial claim - Findingown data
Dietary nitrate supplementation increases circulating S-nitrosothiol (RSNO) levels in mice over 7 days.
“This is supported by the marked increase in circulating S-nitrosothiols observed here after nitrate supplementation.”
- Also stated in: § CLINICAL PERSPECTIVE · plain
“Circulating RSNO, NO 2 Ϫ , and NO 3 Ϫ levels increase after nitrate supplementation during a 7-day period.”
Quote in the paperSubstantial claim - Also stated in: § CLINICAL PERSPECTIVE · plain
- Findingown data
Mice receiving mouthwash do not exhibit the nitrate-induced increase in CD31+/CD45− cells.
“Dietary nitrate-treated mice show higher numbers of CD31 ϩ /CD45 Ϫ cells 7 days after hind-limb surgery, whereas the mice receiving mouthwash (MW) did not exhibit this effect.”
Quote in the paperSubstantial claim - Findingcitation
The daily mouthwash intervention effectively suppressed the increase in circulating nitrite and S-nitrosothiol levels observed after intake of nitrate-rich food or dietary nitrate supplementation in drinking water.
“This daily intervention effectively suppressed the increase in circulating nitrite and S-nitrosothiol levels that is observed after intake of nitrate-rich food or dietary nitrate supplementation in drinking water.15,38,42”
Quote in the paperSubstantial claim0 of 3 sources checked - Findingown data
Dietary nitrate supplementation increases capillary density in mice at day 7.
“Bars show higher capillary density in dietary nitrate-supplemented mice at day 7 (black bars; Pϭ0.045).”
Quote in the paperSubstantial claim - Findingown data
The nitrate-induced increase in perfusion recovery is not observed in mice receiving antiseptic mouthwash.
“Nitrate-supplemented mice showed increased perfusion recovery in chronic hind-limb ischemia (black bars; PϽ0.02); this effect was not observed in mice receiving antiseptic mouthwash (gray bars; PϽ0.02).”
Quote in the paperSubstantial claim - Interpretationreasoning
The null effect of nitrate on cGMP does not exclude cGMP-mediated effects because counterregulatory mechanisms may disguise NO-mediated changes in cGMP.
“However, it does not exclude the existence of cGMP-mediated effects because counterregulatory mechanisms may disguise NO-mediated changes in this second messenger.”
Quote in the paperNot a substantial claim - Interpretationown data
Disruption of the nitrate-NO pathway by long-term eradication of oral microflora completely abolishes the beneficial effects of dietary nitrate supplementation.
“Intriguingly, disruption of the nitrate-NO pathway by long-term eradication of the oral microflora completely abolished the beneficial effects of the dietary nitrate supplementation.”
Quote in the paperSubstantial claim - Premisecitation
Recent research in animals and humans has confirmed beneficial effects of dietary nitrate.
“Recent research in animals and humans has confirmed the beneficial effects of dietary nitrate.14,[33][34][35][36][37][38]”
Quote in the paperSubstantial claim0 of 1 sources checked- cites 14,[33][34][35][36][37][38]
Not checked in the free run.
- Interpretationreasoning
Dietary nitrate ameliorates the capacity of adult skeletal muscles to regenerate myofibers after damage.
“Dietary nitrate ameliorates the remarkable capacity of adult skeletal muscles to regenerate myofibers after damage.”
Quote in the paperSubstantial claim - Premisecitation
The associative relation between increased plasma nitrite levels and increased mobilization of CD34+/Flk-1+ cells from bone marrow has also been observed in patients with coronary artery disease receiving nutrients that increased NO bioavailability.
“The associative relation, increased plasma nitrite levels and an increased mobilization of CD34 ϩ /Flk-1 ϩ cells from the bone marrow, has also been observed in patients with coronary artery disease receiving nutrients that increased NO bioavailability.40”
Quote in the paperNot a substantial claim0 of 1 sources checked- cites [40] source
Not checked in the free run.
- Interpretationreasoning
Pharmacological treatment with nitrite may offer an alternative therapeutic opportunity.
“Pharmacological treatment with nitrite, an oxidation product of NO, may offer an alternative therapeutic opportunity.”
Quote in the paperNot a substantial claim - Interpretationown data
Long-term dietary nitrate supplementation may represent a novel nutrition-based strategy to enhance ischemia-induced revascularization.
“Long-term dietary nitrate supplementation may represent a novel nutrition-based strategy to enhance ischemia-induced revascularization.”
Quote in the paperSubstantial claim - Interpretationown data
The effect of dietary nitrate supplementation on regenerative capacity depends critically on bacteria-dependent bioactivation of nitrate in the oral cavity.
“We show here that dietary nitrate supplementation increases the regenerative capacity of ischemic tissue and that this effect depends critically on bacteria-dependent bioactivation of nitrate in the oral cavity.”
Quote in the paperSubstantial claim- Rests on an assumption: Bioactivation of nitrate from dietary or endogenous sources is carried out mainly by commensal bacteria that express effective nitrate reductase enzymes and are located in the gastrointestinal tract and on body surfaces.
- Interpretationown data
Dietary nitrate supplementation improves revascularization in chronic ischemia.
“The present study provides the first evidence that dietary nitrate supplementation improves revascularization in chronic ischemia.”
- Also stated in: § Conclusions · hedged
“These results suggest that dietary nitrate supplementation may offer an attractive nutrition-based strategy to improve ischemiainduced revascularization.”
Quote in the paperSubstantial claim- Rests on an assumption: Under conditions of low oxygen tensions, nitrate and nitrite are physiologically recycled in blood and tissues to form nitric oxide and other bioactive nitrogen oxides that mediate cytoprotective signaling in the setting of pathological ischemia.
- Also stated in: § Conclusions · hedged
- Interpretationown data
Dietary nitrate supplementation increases the regenerative capacity of ischemic tissue.
“We show here that dietary nitrate supplementation increases the regenerative capacity of ischemic tissue and that this effect depends critically on bacteria-dependent bioactivation of nitrate in the oral cavity.”
- Also stated in: § CLINICAL PERSPECTIVE · plain
“This study identified that dietary nitrate supplementation increases mobilization and migration of regenerative cells, improves the regenerative capacities of chronically ischemic tissue, and decreases apoptosis at the site of ischemia.”
Quote in the paperSubstantial claim- Rests on an assumption: Under conditions of low oxygen tensions, nitrate and nitrite are physiologically recycled in blood and tissues to form nitric oxide and other bioactive nitrogen oxides that mediate cytoprotective signaling in the setting of pathological ischemia.
- Also stated in: § CLINICAL PERSPECTIVE · plain
- Interpretationown data
Eradicating commensal bacteria in the oral cavity reverses the beneficial effects of dietary nitrate supplementation.
“Eradicating the commensal bacteria in the oral cavity and thus interrupting the bioactivation of the ingested nitrate decreased circulating levels of bioactive nitrogen oxides and reversed all of these beneficial effects.”
Quote in the paperSubstantial claim- Rests on an assumption: Bioactivation of nitrate from dietary or endogenous sources is carried out mainly by commensal bacteria that express effective nitrate reductase enzymes and are located in the gastrointestinal tract and on body surfaces.
- The quote only partly states this claim. 1
- May combine two claims. 14
- Classified as probably not a substantial claim. 9
The quote only partly states this claim.
Findingown dataAntiseptic mouthwash prevents the nitrate-mediated decrease in apoptosis.
“Mice without dietary nitrate supplementation (white bar; PϽ0.001) or with antiseptic mouthwash (gray bar; PϽ0.02) did not show nitrate-mediated regenerative effects.”
Quote in the paperSubstantial claimMay combine two claims.
Interpretationown dataDietary nitrate supplementation improves revascularization through mechanisms involving mobilization and migration of endothelium-regenerating cells, accompanied by attenuation of myoblast apoptosis.
“In the present study, we demonstrate that dietary nitrate supplementation improves revascularization through mechanisms involving the mobilization and migration of endotheliumregenerating cells.This is accompanied by an attenuation of myoblast apoptosis.”
Quote in the paperSubstantial claimMay combine two claims.
PremisecitationNitrite as a reservoir can be further reduced to NO via numerous pathways in blood and tissue, and this process is accelerated under hypoxic conditions.
“Nitrite as a reservoir can be further reduced to NO via numerous pathways in blood and tissue, and this process is indeed accelerated under hypoxic conditions.44 -47”
Quote in the paperSubstantial claim0 of 4 sources checkedProbably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.
InterpretationcitationOral bacteria could directly catalyze nitrosation reactions from nitrate without intermediate nitrite formation, as protein S-nitros(yl)ation is an obligate concomitant process of anaerobic respiration on nitrate in E. coli.
“However, one cannot entirely rule out the possibility that oral bacteria could directly catalyze nitrosation reactions from nitrate without intermediate nitrite formation because recent data show that protein S-nitros(yl)ation is an obligate concomitant process of anaerobic respiration on nitrate in Escherichia coli.43”
Quote in the paperNot a substantial claim0 of 1 sources checked- cites [43] source
Not checked in the free run.
May combine two claims.
InterpretationcitationThe null effect of nitrate on cGMP is consistent with findings from nitrite injection studies in ischemia, indicating that other nitrite-signaling pathways for regulating angiogenic activity may exist.
“This result is consistent with recent findings in studies investigating the role of nitrite injections in ischemia, 16 indicating that other nitrite-signaling pathways for regulating angiogenic activity may exist.20”
Quote in the paperSubstantial claim0 of 2 sources checkedMay combine two claims.
InterpretationreasoningMouthwash prevention of plasma nitrite increases and all observed beneficial effects of nitrate suggests an important role of intermediate nitrite formation in the bioactivation of nitrate.
“The fact that mouthwash prevented increases in plasma nitrite and all observed beneficial effects of nitrate provides significant mechanistic insight because it suggests an important role of intermediate nitrite formation in the bioactivation of nitrate.”
Quote in the paperSubstantial claimProbably not a substantial claim: may not pass S3 (load-bearing).
PremisecitationAdministration of nitrite under hypoxia regulates cardiac energetics and functions resembling acute hibernation.
“We have recently shown that the administration of nitrite under hypoxia regulates cardiac energetics and functions resembling the characteristics described for acute hibernation.28”
Quote in the paperNot a substantial claim0 of 1 sources checked- cites [28] source
Not checked in the free run.
May combine two claims.
PremisecitationProangiogenic properties of NO have been demonstrated, but direct application of NO or classic NO donors in vivo bears a high risk of failure owing to lack of targeted delivery, development of tolerance, cellular toxicity, and risk of hypotension.
“Although proangiogenic properties of NO have been demonstrated, direct application of NO or classic NO donors in vivo bears a high risk of failure owing to lack of targeted delivery, development of tolerance, cellular toxicity, and risk of hypotension.[25][26][27]”
Quote in the paperSubstantial claim0 of 1 sources checked- cites [25][26][27]
Not checked in the free run.
May combine two claims.
PremisecitationContinuous pharmacological intervention with nitrite injections increases vascular density in the hind limb and stimulates endothelial cell proliferation in ischemic angiogenesis.
“In ischemic angiogenesis, a continuous pharmacological intervention with nitrite injections resulted in an increased vascular density in the hind limb and stimulated endothelial cell proliferation.16”
Quote in the paperSubstantial claim0 of 1 sources checked- cites [16] source
Not checked in the free run.
May combine two claims.
PremisecitationNitrite's conversion into bioactive nitrogen oxides is slow and not subject to tolerance, allowing high doses without classic NO-related side effects such as hypotension.
“Its conversion into bioactive nitrogen oxides is slow, 31 and the effect is not subjective to tolerance.32 Therefore, one can deliver it even in fairly high doses without the risk of classic NO-related side effects such as hypotension.”
Quote in the paperSubstantial claim0 of 2 sources checkedProbably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.
PremisecitationThe NO-like effects of dietary nitrate include lowering blood pressure, reducing experimental myocardial infarct size, inhibiting platelet aggregation, increasing exercise tolerance in peripheral arterial disease, and improving intrinsic mitochondrial efficiency.
“The NO-like effects of dietary nitrate range from lowering of blood pressure 14,38 to a reduction in experimental myocardial infarct size, 33 inhibition of platelet aggregation, 34 increase in exercise tolerance in peripheral arterial disease, 35 and improvement in intrinsic mitochondrial efficiency.36”
Quote in the paperNot a substantial claim0 of 4 sources checkedClassified as probably not a substantial claim.
PremisecitationA cytoprotective role of nitrite in myocardial, liver, kidney, and brain ischemia/reperfusion injury has previously been demonstrated.
“A cytoprotective role of nitrite in the setting of myocardial, liver, kidney, and brain ischemia/reperfusion injury has previously been demonstrated.19,29,30”
Quote in the paperSubstantial claim0 of 3 sources checkedProbably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.
PremisecitationNitrate supplementation offers cardioprotection against doxorubicin-induced cardiomyopathy and prevents oxidative stress and cardiovascular and renal injury in salt-induced hypertension.
“In addition, nitrate supplementation offered cardioprotection against doxorubicin-induced cardiomyopathy 37 and prevented oxidative stress and cardiovascular and renal injury in salt-induced hypertension.39”
Quote in the paperNot a substantial claim0 of 2 sources checkedMay combine two claims.
InterpretationreasoningFindings from animal and human studies support the hypothesis that nitrate of food origin promotes cardiovascular health and provides a mechanistic explanation for health benefits of a vegetable-rich diet.
“These findings from experimental animal studies and human clinical studies support the hypothesis that nitrate of food origin promotes cardiovascular health and thus presents a novel mechanistic explanation for some of the well-known health benefits of a diet rich in vegetables.”
Quote in the paperSubstantial claimClassified as probably not a substantial claim. May combine two claims.
Study design and conductown dataMigration analysis was blinded and at least 5 visual fields per section were analyzed.
“Blinded analysis of at least 5 visual fields per section revealed that animals treated with nitrate showed higher amounts of EGFP ϩ /CD31 ϩ /CD45 Ϫ cells at the site of active revascularization compared with animals on a low-nitrate diet (Pϭ0.029; Figure 6D and 6E).”
Quote in the paperSubstantial claimMay combine two claims.
Study design and conductown dataDietary nitrate supplementation and daily mouthwash have no effect on water intake, food intake, and body weight development in mice.
“Dietary nitrate supplementation and daily mouthwash (MW) procedure had no effect on water and food intake and body weight development.”
Quote in the paperNot a substantial claimMay combine two claims.
Interpretationown dataS-nitros(yl)ation and formation of S-nitrosothiols may account for the regenerative response to dietary nitrate supplementation in chronic ischemia.
“The current data suggest that S-nitros(yl)ation might be a crucial signal transduction pathway and that the formation of S-nitrosothiols might account for the regenerative response to the dietary nitrate supplementation in chronic ischemia.”
Quote in the paperSubstantial claimProbably not a substantial claim: may not pass S3 (load-bearing).
Study design and conductown dataMyoblast population was verified by FACS with integrin-α7 antibody.
“To verify the myoblast population, fluorescence-activated cell sorting (FACS) was performed on an FACS sorter (FACS Canto, BD, Franklin Lakes, NJ) with the integrin-␣7 antibody (Antikoerper-Online GmbH, Aachen, Germany) for sorting.”
Quote in the paperNot a substantial claimProbably not a substantial claim: may not pass S3 (load-bearing).
Study design and conductown dataPerfusion was calculated by a blinded observer.
“The mean hind-limb perfusion was calculated as the ratio of ischemic to nonischemic side by a blinded observer.”
Quote in the paperNot a substantial claimProbably not a substantial claim: may not pass S3 (load-bearing).
PremisecitationThe rapid repair process of skeletal muscle is carried out mainly by satellite cells with a contribution of NO.
“This rapid repair process is carried out mainly by satellite cells with a contribution of NO. 41”
Quote in the paperNot a substantial claim0 of 1 sources checked- cites [41] source
Not checked in the free run.
The authors state this as an assumption.
Whether dietary nitrate enhances ischemia-induced remodeling of the vascular network is not known.
“Whether dietary nitrate, serially reduced to nitrite by commensal bacteria in the oral cavity and subsequently to nitric oxide and other nitrogen oxides, enhances ischemia-induced remodeling of the vascular network is not known.”
The paper relies on this without support.
The physiological repair response is often not sufficient, and therapeutic angiogenesis remains an unmet medical need.
“The physiological repair response, however, is often not sufficient, and therapeutic angiogenesis remains an unmet medical need.”
The paper relies on this without support.
Chronically ischemic tissue requires remodeling of the vascular network to reconstitute and sustain its viability.
“Chronically ischemic tissue, a feature of peripheral and coronary artery disease, requires a remodeling of the vascular network to reconstitute and sustain its viability.”
The paper relies on this without support.
Nitrite's first-passage metabolism in the liver is minimal compared with classic organic nitrates, and its bioactivation is augmented in ischemic areas, making it selective to ischemic areas.
“In addition, first-passage metabolism in the liver is minimal compared with that of classic organic nitrates.Moreover, its bioactivation is augmented in ischemic areas, thereby making the drug selective to ischemic areas.”
The authors state this as an assumption.
The exact identity of the final mediator(s) of the nitrate effects is still not settled and multiple possible mechanisms exist.
“It is important to note that the exact identity of the final mediator(s) of the nitrate effects is still not settled and that multiple possible mechanisms exist.”
The paper relies on this without support.
Bioactivation of nitrate from dietary or endogenous sources is carried out mainly by commensal bacteria that express effective nitrate reductase enzymes and are located in the gastrointestinal tract and on body surfaces.
“The bioactivation of nitrate from dietary or endogenous sources is carried out mainly by commensal bacteria that express effective nitrate reductase enzymes and are located in the gastrointestinal tract and on body surfaces.”
The paper relies on this without support.
Under conditions of low oxygen tensions, nitrate and nitrite are physiologically recycled in blood and tissues to form nitric oxide and other bioactive nitrogen oxides that mediate cytoprotective signaling in the setting of pathological ischemia.
“Under conditions of low oxygen tensions, nitrate and nitrite are physiologically recycled in blood and tissues to form nitric oxide and other bioactive nitrogen oxides that mediate cytoprotective signaling in the setting of pathological ischemia.”
The paper relies on this without support.
Inorganic nitrate from dietary sources is able to influence the hallmarks of cardiovascular functions, including blood pressure regulation.
“In this context, there is accumulating evidence that inorganic nitrate from dietary sources is able to influence the hallmarks of cardiovascular functions, including blood pressure regulation.”
Claims other papers make about this one
Each is checked against this paper’s own text: does it say what the citing paper says it does?
These reactions can beneficially regulate cardio-metabolic function and will be attenuated or abolished by chemical or physical destruction of the oral microbiota.
“These reactions can beneficially regulate cardio-metabolic function and will be attenuated or abolished by chemical or physical destruction of the oral microbiota[60,75,76,97–99].”
From Enterosalivary nitrate metabolism and the microbiome: Intersection of microbial metabolism, nitric oxide and diet in cardiac and pulmonary vascular health (2017) · cites this paper as [98]
Nitrate supplementation prevents or limits ischemia-reperfusion injury and endothelial dysfunction in both animal and human models of ischemia-reperfusion.
“while nitrate supplementation prevents or limits ischemia-reperfusion injury and endothelial dysfunction in both animal[98,120–124] and human[97,125] models of ischemia-reperfusion.”
From Enterosalivary nitrate metabolism and the microbiome: Intersection of microbial metabolism, nitric oxide and diet in cardiac and pulmonary vascular health (2017) · cites this paper as [98]
Oral administration of nitrate and nitrite provides protection from ischemia-reperfusion injury.
“protection from ischemia-reperfusion injury[97,98,272]”
From Enterosalivary nitrate metabolism and the microbiome: Intersection of microbial metabolism, nitric oxide and diet in cardiac and pulmonary vascular health (2017) · cites this paper as [98]
Self-check
Percentages, sums and reported differences re-computed from the paper’s own numbers.
Each contradiction between two passages listed here was confirmed by an independent check.
Myoblast apoptosis is reported in incompatible measures
MajorThe Results report the Figure 5C apoptosis values as cells per square millimeter, but the figure legend says Figure 5C expresses apoptosis as a percentage ratio between ischemic and nonischemic hind limbs. These are different measures for the same reported comparison.
“The amount of apoptotic nuclei was lower in the nitratetreated group compared with controls (67Ϯ16 versus 197Ϯ19 cells per 1 mm 2 ; PϽ0.001; Figure 5B and 5C).”
“C, Quantitative analysis of TUNEL-stained myoblasts per hind limb displayed as the ratio between ischemic and nonischemic hind limb and expressed as percent.”
The citation lists haven’t been fetched yet
References 50 · Cited by 110
- Authors
- Hendgen-Cotta, U. B., Luedike, P., Totzeck, M., Kropp, M., Schicho, A., Stock, P., Rammos, C., Niessen, M., Heiss, C., Lundberg, J. O., Weitzberg, E., Kelm, M., & Rassaf, T.
- Affiliations
- Karolinska Institutet (SE)
- ORCID
- Venue
- Circulation
- Published
- 2012-10-16
- Volume
- 126
- Issue
- 16
- Pages
- 1983-1992
- ISSN
- 0009-7322 (ISSN-L)
- 1524-4539
- OpenAlex
- W2065872869
- Semantic Scholar
- f37dcdf8ed7ee71e28e4e9546e3a59d6fa4fe566
- Cited by
- 110
- References
- 50
- Citations by year
- 2026: 1 · 2025: 5 · 2024: 4 · 2023: 4 · 2022: 2 · 2021: 5 · 2020: 10 · 2019: 6 · 2018: 8 · 2017: 13 · 2016: 9 · 2015: 15 · 2014: 12 · 2013: 14 · 2012: 1
- FWCI
- 8.97
- Type
- journal article
- Language
- en
- Open access
- bronze
- doi.org
- Locations
- Circulation (publishedVersion)
- PubMed (publishedVersion)
- Archive ouverte UNIGE (University of Geneva) (publishedVersion, other-oa)
- Archive ouverte UNIGE (University of Geneva) (submittedVersion, cc-by)
- Topics
- Health Sciences › Medicine › Physiology › Nitric Oxide and Endothelin Effects
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Angiogenesis and VEGF in Cancer
- Health Sciences › Medicine › Surgery › Peripheral Artery Disease Management
- Keywords
- dietary nitrate, sodium nitrate, revascularization, chronic limb ischemia, hindlimb ischemia, nitric oxide, S-nitrosothiols, nitrite, skeletal muscle regeneration, cyclic GMP, bone marrow transplantation, antiseptic mouthwash
- MeSH terms
- Animals, Animal Feed, Cell Movement, Chronic Disease, Disease Models, Animal, Femoral Artery, Cyclic GMP, Hindlimb, Ischemia, Ligation, Mice, Inbred C57BL, Mice, Transgenic, Nitrates, Nitric Oxide, Nitrites, Regeneration, Regional Blood Flow, Bone Marrow Transplantation, Laser-Doppler Flowmetry, Dietary Supplements, S-Nitrosothiols, Myoblasts, Green Fluorescent Proteins, Mice
About these checks
Registry, retraction, Cites and statistics results are computed, not judged, with one exception: a printed reference that no index lists is read once by a model before it counts as a citation error. Claims are extracted by a model and kept only when their quote appears verbatim in the text; each cited source is then checked, with a model’s help, for whether it says what the claim relies on. Those verdicts can be wrong — read the claim’s quote and the cited source before relying on one.
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Classified by jev-1.13-20260917 (gold-guideline-v0.2) on 2026-10-09.
Statistics and arithmetic are recomputed, not judged.
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