Dietary inorganic nitrate improves mitochondrial efficiency in humans

Nitrate, an inorganic anion abundant in vegetables, is converted in vivo to bioactive nitrogen oxides including NO. We recently demonstrated that dietary nitrate reduces oxygen cost during physical exercise, but the mechanism remains unknown. In a double-blind crossover trial we studied the effects of a dietary intervention with inorganic nitrate on basal mitochondrial function and whole-body oxygen consumption in healthy volunteers. Skeletal muscle mitochondria harvested after nitrate supplementation displayed an improvement in oxidative phosphorylation efficiency (P/O ratio) and a decrease in state 4 respiration with and without atractyloside and respiration without adenylates. The improved mitochondrial P/O ratio correlated to the reduction in oxygen cost during exercise. Mechanistically, nitrate reduced the expression of ATP/ADP translocase, a protein involved in proton conductance. We conclude that dietary nitrate has profound effects on basal mitochondrial function. These findings may have implications for exercise physiology- and lifestyle-related disorders that involve dysfunctional mitochondria.     

Dietary inorganic nitrate alleviates doxorubicin cardiotoxicity: mechanisms and implications

Doxorubicin (DOX) is one of the most powerful and widely prescribed chemotherapeutic agents to treat divergent human cancers. However, the clinical use of DOX is restricted due to its severe cardiotoxic side-effects. There has been ongoing search for cardioprotectants against DOX toxicity. Inorganic nitrate has emerged as a bioactive compound that can be reduced into nitrite and nitric oxide in vivo and in turn plays a therapeutic role in diseases associated with nitric oxide insufficiency or dysregulation. In this review, we describe a novel concept of using dietary supplementation of inorganic nitrate to reduce DOX-induced cardiac cellular damage and dysfunction, based on our recent promising studies in a mouse model of DOX cardiotoxicity. Our data show that chronic oral ingestion of sodium nitrate, at a dose equivalent to ~400% of the Acceptable Daily Intake of the World Health Organization, alleviated DOX-induced left ventricular dysfunction and mitochondrial respiratory chain damage. Such cardioprotective effects were associated with reduction of cardiomyocyte necrosis/apoptosis, tissue lipid peroxidation, and mitochondrial H(2)O(2) generation following DOX treatment. Furthermore, proteomic studies revealed enhanced cardiac expression of mitochondrial antioxidant enzyme - peroxiredoxin 5 in the nitrate-treated animals. These studies suggest that inorganic nitrate could be an inexpensive therapeutic agent for long-term oral administration in preventing DOX-induced cardiac toxicity and myopathy during the prolonged pathological process. Future clinical trials in the cancer patients undergoing DOX chemotherapy are warranted to translate these experimental findings into an effective new therapy in preventing the DOX-induced cardiomyopathy.     

Alterations in circulating angiogenic and anti‐angiogenic factors in type 2 diabetic patients with neuropathy

Diabetic peripheral neuropathy (DPN) is one of the most common diabetic chronic complications. There is an increased attention directed towards the role of angiogenic factors including vascular endothelial growth factor (VEGF) and anti‐angiogenic factors including soluble endoglin (sEng) as contributors to diabetic microvascular complications including neuropathy. The purposes of this study were to determine the role of these angiogenesis regulators in the prognosis of DPN. The study group included 60 patients with type 2 diabetes mellitus (T2DM) and 20 clinically healthy individuals. The patients were divided into two groups. Group I included 20 T2DM patients without peripheral neuropathy, and Group II consisted of 40 T2DM patients with DPN. In all groups, plasma VEGF, sEng and endothelin‐1 (ET‐1), nitric oxide and ET‐1 mRNA were estimated. Plasma levels of VEGF, sEng, ET‐1 and nitric oxide were significantly elevated in diabetic patients (Groups I and II) compared with healthy control subjects, with a higher increase in their levels in patients with DPN compared with diabetic patients without peripheral neuropathy. Measurement of plasma levels of angiogenesis‐related biomarkers in high‐risk diabetic patients might identify who later develop DPN, thus providing opportunities for early detection and targets for novel treatments. Copyright © 2013 John Wiley & Sons, Ltd.     

Effect of smoking on circulating angiogenic factors in high risk pregnancies

Objective

Changes in maternal concentrations of the anti-angiogenic factors, soluble fms-like tyrosine kinase 1 (sFlt1) and soluble endoglin (sEng), and the pro-angiogenic placental growth factor (PlGF) precede the development of preeclampsia in healthy women. The risk of preeclampsia is reduced in women who smoke during pregnancy. The objective of this study was to investigate whether smoking affects concentrations of angiogenic factors (sFlt1, PlGF, and sEng) in women at high risk for developing preeclampsia.

Study Design

We performed a secondary analysis of serum samples from 993 high-risk women (chronic hypertension, diabetes, multifetal gestation, and previous preeclampsia) in a preeclampsia prevention trial. sFlt1, sEng and PlGF were measured in serum samples obtained at study entry, which was prior to initiation of aspirin (median 19.0 weeks'' [interquartile range of 16.0–22.6 weeks'']). Smoking status was determined by self-report.

Results

sFlt1 was not significantly different in smokers from any high-risk groups compared to their nonsmoking counterparts. PlGF was higher among smokers compared to nonsmokers among diabetic women (142.7 [77.4–337.3] vs 95.9 [48.5–180.7] pg/ml, p = 0.005) and women with a history of preeclampsia (252.2 [137.1–486.0] vs 152.2 [73.6–253.7] pg/ml, p = 0.001). sEng was lower in smokers with multifetal gestations (5.8 [4.6–6.5] vs 6.8 [5.5–8.7] ng/ml, p = 0.002) and trended lower among smokers with diabetes (4.9 [3.8–5.6] vs 5.3 [4.3–6.3] ng/ml, p = 0.05). Smoking was not associated with a lower incidence of preeclampsia in any of these groups.

Conclusions

In certain high-risk groups, smoking is associated with changes in the concentrations of these factors towards a pro-angiogenic direction during early pregnancy; however, there was no apparent association between smoking and the development of preeclampsia in our cohort.     

Distinct effects of unfractionated heparin versus bivalirudin on circulating angiogenic peptides

Background

Human studies of therapeutic angiogenesis, stem-cell, and progenitor-cell therapy have failed to demonstrate consistent clinical benefit. Recent studies have shown that heparin increases circulating levels of anti-angiogenic peptides. Given the widely prevalent use of heparin in percutaneous and surgical procedures including those performed as part of studies examining the benefit of therapeutic angiogenesis and cell-based therapy, we compared the effects of unfractionated heparin (UFH) on angiogenic peptides with those of bivalirudin, a relatively newer anticoagulant whose effects on angiogenic peptides have not been studied.

Methodology/Principal Findings

We measured soluble fms-like tyrosine kinase-1 (sFLT1), placental growth factor (PlGF), vascular endothelial growth factor (VEGF), and soluble Endoglin (sEng) serum levels by enzyme linked immunosorbent assays (ELISA) in 16 patients undergoing elective percutaneous coronary intervention. Compared to baseline values, sFLT1 and PlGF levels increased by 2629±313% and 253±54%, respectively, within 30 minutes of UFH therapy (p<0.01 for both; n = 8). VEGF levels decreased by 93.2±5% in patients treated with UFH (p<0.01 versus baseline). No change in sEng levels were observed after UFH therapy. No changes in sFLT1, PlGF, VEGF, or sEng levels were observed in any patients receiving bivalirudin (n = 8). To further explore the direct effect of anticoagulation on circulating angiogenic peptides, adult, male wild-type mice received venous injections of clinically dosed UFH or bivalirudin. Compared to saline controls, sFLT1 and PlGF levels increased by >500% (p<0.01, for both) and VEGF levels increased by 221±101% (p<0.05) 30 minutes after UFH treatment. Bivalirudin had no effect on peptide levels. To study the cellular origin of peptides after anticoagulant therapy, human coronary endothelial cells were treated with UFH and demonstrated increased sFLT1 and PlGF levels (ANOVA p<0.01 for both) with reduced VEGF levels (ANOVA p<0.05). Bivalirudin had no effect on peptide levels in vitro.

Conclusions/Significance

Circulating levels of sFLT1, PlGF, and VEGF are significantly altered by UFH, while bivalirudin therapy has no effect. These findings may have significant implications for clinical studies of therapeutic angiogenesis, stem-cell and progenitor-cell therapy.     

Platelet-activating factor mobilizes intracellular calcium in vascular smooth muscle cells

The effect of platelet-activating factor (PAF) on polyphosphoinositide metabolism and 45Ca2+ efflux was examined in a vascular smooth muscle cell line (A7r5). PAF stimulated a rapid but transient production of inositol trisphosphate and inositol bisphosphate which, in the presence of lithium, resulted in an accumulation of inositol monophosphate. In addition, PAF induced a rapid efflux of 45Ca2+ from preloaded cells, an effect which was concentration-dependent. These data suggest that PAF mobilizes intracellular Ca2+ via the production of inositol trisphosphate.     

Proton-sensing G protein-coupled receptor mobilizes calcium in human synovial cells

Lowered extracellular pH in a variety of tissues is associated with increased tissue destruction and initiation of inflammatory processes. Although the acid-sensing receptors described previously are ion channels, we describe a G protein-coupled proton-sensitive receptor that stimulates Ca(2+) release from intracellular stores in a tumor-derived synoviocyte cell line (SW982) and in primary cultures of human synovial cells from patients with inflammatory arthropathies. We established a link between proton-dependent receptor activation and intracellular Ca(2+) mobilization by demonstrating 1) dependence on the integrity of the intracellular Ca(2+) store, 2) independence from extracellular Ca(2+), and 3) proton-induced production of inositol phosphate and 4) by abolishing the effect with GTPase inhibitors. We propose that this G protein-coupled acid-sensing receptor linked to intracellular Ca(2+) mobilization in synoviocytes can contribute to downstream inflammatory and cellular proliferative processes in synovial fibroblasts. The acid-sensing receptor has distinct characteristics as a metabotropic G protein-coupled receptor on human synoviocytes in this emerging new class of receptors.     

5,6-Epoxyeicosatrienoic acid mobilizes Ca2+ in anterior pituitary cells

Luteinizing hormone releasing hormone stimulates the concomitant release of luteinizing hormone and 45Ca2+ from prelabeled anterior pituitary cells. Indomethacin (10 microM) and nordihydroguaiaretic acid (10 microM) had no effect on the luteinizing hormone releasing hormone-stimulated release of either luteinizing hormone or 45Ca2+. Eicosatetraynoic acid (10 microM) blocked both luteinizing hormone releasing hormone-stimulated luteinizing hormone secretion and luteinizing hormone releasing hormone-stimulated 45Ca2+ efflux. 5,6-Epoxyeicosatrienoic acid stimulated both luteinizing hormone secretion and 45Ca2+ efflux from anterior pituitary cells. Additionally, 5,6-epoxyeicosatrienoic acid closely mimics the ability of luteinizing hormone releasing hormone to increase intracellular free calcium. These results are consistent with the hypothesis that 5,6-EET alters calcium homeostasis in a manner similar to that observed during luteinizing hormone releasing hormone stimulation of luteinizing hormone release.     

Levels and values of circulating endothelial progenitor cells,soluble angiogenic factors,and mononuclear cell apoptosis in liver cirrhosis patients

Background

The roles of circulating endothelial progenitor cell (EPC) and mononuclear cell apoptosis (MCA) in liver cirrhosis (LC) patients are unknown. Moreover, vascular endothelial growth factor (VEGF) and stromal cell-derived factor (SDF)-1α are powerful endogenous substances enhancing EPC migration into circulation. We assessed the level and function of EPCs [CD31/CD34 (E₁), KDR/CD34 (E₂), CXCR4/CD34 (E₃)], levels of MCA, VEGF and SDF-1α in circulation of LC patients.

Methods

Blood sample was prospectively collected once for assessing EPC level and function, MCA, and plasma levels of VEGF and SDF-1α using flow cytometry and enzyme-linked immunosorbent assay (ELISA), respectively, in 78 LC patients and 25 age- and gender-matched healthy controls.

Results

Number of EPCs (E₁, E₂, E₃) was lower (all p < 0.0001), whereas SDF-1α level and MCA were higher (p < 0.001) in study patients compared with healthy controls. Number of EPCs (E₂, E₃) was higher but MCA was lower (all p < 0.05) in Child''s class A compared with Child''s class B and C patients, although no difference in VEGF and SDF-1α levels were noted among these patients. Chronic hepatitis B and esophageal varices bleeding were independently, whereas chronic hepatitis C, elevated aspartate aminotransferase (AST), and decompensated LC were inversely and independently correlated with circulating EPC level (all p < 0.03). Additionally, angiogenesis and transwell migratory ability of EPCs were reduced in LC patients than in controls (all p < 0.001).

Conclusion

The results of this study demonstrated that level, angiogenic capacity, and function of circulating EPCs were significantly reduced, whereas plasma levels of SDF-1α and circulating MCA were substantially enhanced in cirrhotic patients.     

Dietary nitrate supplementation enhances exercise performance in peripheral arterial disease

Peripheral arterial disease (PAD) results in a failure to adequately supply blood and oxygen (O(2)) to working tissues and presents as claudication pain during walking. Nitric oxide (NO) bioavailability is essential for vascular health and function. Plasma nitrite (NO(2)(-)) is a marker of vascular NO production but may also be a protected circulating "source" that can be converted to NO during hypoxic conditions, possibly aiding perfusion. We hypothesized that dietary supplementation of inorganic nitrate in the form of beetroot (BR) juice would increase plasma NO(2)(-) concentration, increase exercise tolerance, and decrease gastrocnemius fractional O(2) extraction, compared with placebo (PL). This was a randomized, open-label, crossover study. At each visit, subjects (n = 8) underwent resting blood draws, followed by consumption of 500 ml BR or PL and subsequent blood draws prior to, during, and following a maximal cardiopulmonary exercise (CPX) test. Gastrocnemius oxygenation during the CPX was measured by near-infrared spectroscopy. There were no changes from rest for [NO(2)(-)] (152 ± 72 nM) following PL. BR increased plasma [NO(2)(-)] after 3 h (943 ± 826 nM; P ≤ 0.01). Subjects walked 18% longer before the onset of claudication pain (183 ± 84 s vs. 215 ± 99 s; P ≤ 0.01) and had a 17% longer peak walking time (467 ± 223 s vs. 533 ± 233 s; P ≤ 0.05) following BR vs. PL. Gastrocnemius tissue fractional O(2) extraction was lower during exercise following BR (7.3 ± 6.2 vs. 10.4 ± 6.1 arbitrary units; P ≤ 0.01). Diastolic blood pressure was lower in the BR group at rest and during CPX testing (P ≤ 0.05). These findings support the hypothesis that NO(2)(-)-related NO signaling increases peripheral tissue oxygenation in areas of hypoxia and increases exercise tolerance in PAD.     

Nestin expression in pancreatic stellate cells and angiogenic endothelial cells

Nestin is an intermediate filament protein expressed by neuroepithelial stem cells and which has been proposed to represent also a marker for putative islet stem cells. The aim of this study was to characterize the cell type(s) expressing nestin in the rat pancreas. By immunohistochemistry, nestin positivity was localized exclusively in mesenchymal cells of normal and regenerating adult pancreas. In the latter condition, the number of nestin-positive cells and the intensity of nestin immunoreactivity were greatly increased. Most nestin-positive cells had the morphology of stellate cells, a type of pericyte associated with blood vessels which has been previously reported to occur in liver and pancreas. In addition, nestin positivity was present in endothelial cells from neocapillaries during pancreas regeneration, and in all blood vessels during morphogenesis in fetal pancreas. Nestin expression was not found in the ductal epithelial cells from which islet cells originate in fetal and regenerating pancreas. In primary pancreatic tissue explants, nestin-positive mesenchymal cells rapidly attached to plastic and proliferated. These cells also expressed desmin, vimentin, and glial fibrillary acidic protein which are known to represent stellate cell markers. In summary, nestin in the pancreas is primarily a marker for reactive stellate cells, or pericytes, and endothelial cells during active angiogenesis.     

Dietary nitrate increases gastric mucosal blood flow and mucosal defense

Salivary nitrate from dietary or endogenous sources is reduced to nitrite by oral bacteria. In the acidic stomach, nitrite is further reduced to bioactive nitrogen oxides, including nitric oxide (NO). In this study, we investigated the gastroprotective role of nitrate intake and of luminally applied nitrite against provocation with diclofenac and taurocholate. Mucosal permeability ((51)Cr-EDTA clearance) and gastric mucosal blood flow (laser-Doppler flowmetry) were measured in anesthetized rats, either pretreated with nitrate in the drinking water or given acidified nitrite luminally. Diclofenac was given intravenously and taurocholate luminally to challenge the gastric mucosa. Luminal NO content and nitrite content in the gastric mucus were determined by chemiluminescence. The effect of luminal administration of acidified nitrite on the mucosal blood flow was also investigated in endothelial nitric oxide synthase-deficient mice. Rats pretreated with nitrate or given nitrite luminally had higher gastric mucosal blood flow than controls. Permeability increased more during the provocation in the controls than in the nitrate- and nitrite-treated animals. Dietary nitrate increased luminal NO levels 50 times compared with controls. Nitrate intake also resulted in nitrite accumulation in the loosely adherent mucous layer; after removal of this mucous layer, blood flow was reduced. Nitrite administrated luminally in endothelial nitric oxide synthase-deficient mice increased mucosal blood flow. We conclude that dietary nitrate and direct luminal application of acidified nitrite decrease diclofenac- and taurocholate-induced mucosal damage. The gastroprotective effect likely involves a higher mucosal blood flow caused by nonenzymatic NO production. These data suggest an important physiological role of nitrate in the diet.     

Dietary nitrate inhibits stress-induced gastric mucosal injury in the rat

Dietary nitrate is reduced to nitrite by some oral bacteria and the resulting nitrite is converted to nitric oxide (NO) in acidic gastric juice. The aim of this study is to elucidate the pathophysiological role of dietary nitrate in the stomach. Intragastric administration of nitrate rapidly increased nitrate and NO in plasma and the gastric headspace, respectively. Water-immersion-restraint stress (WIRS) increased myeloperoxidase (MPO) activity in gastric mucosa and induced hemorrhagic erosions by a nitrate-inhibitable mechanism. In animals that had received either cardiac ligation or oral treatment with povidone-iodine, a potent bactericidal agent, administration of nitrate failed to increase gastric levels of NO and to inhibit WIRS-induced mucosal injury. WIRS decreased gastric mucosal blood flow by a mechanism which was inhibited by administration of nitrate. These data suggested that the enterosalivary cycle of nitrate and related metabolites consisted of gastrointestinal absorption and salivary secretion of nitrate, its conversion to nitrite by oral bacteria and then to NO in the stomach might play important roles in the protection of gastric mucosa from hazardous stress.     

Serum rapidly mobilizes calcium from an intracellular pool in quiescent fibroblastic cells

Addition of dialysed fetal bovine serum to quiescent cultures of Swiss 3T3 cells loaded with 45Ca2+ causes a very rapid increase in the rate of 45Ca2+ efflux from an intracellular pool. Exposure to serum for 2 min leads to a fall of 0.59 nmol Ca2+/mg protein in the intracellular Ca2+ content of the cells. Inhibitors of mitochondrial function prevent the stimulation of 45Ca2+ efflux by serum. The stimulation of 45Ca2+ efflux by serum is also observed in quiescent cultures of Rat-1, Swiss 3T6 and BHK cells and in secondary cultures of whole mouse embryo fibroblasts.