Effect of 5-Azacitidine Treatment on Redox Status and Inflammatory Condition in MDS Patients

This study focused on the impact of the treatment with the hypomethylating agent 5-azacitidine on the redox status and inflammation in 24 MDS patients. Clinical and genetic features of MDS patients were recorded, and peripheral blood samples were used to determine the activity of the endogenous antioxidant defense system (superoxide dismutase, SOD; catalase, CAT; glutathion peroxidase, GPx; and reductase, GRd, activities), markers of oxidative damage (lipid peroxidation, LPO, and advanced oxidation protein products, AOPP). Moreover, pro-inflammatory cytokines and plasma nitrite plus nitrate levels as markers of inflammation, as well as CoQ10 plasma levels, were also measured. Globally, MDS patients showed less redox status in terms of a reduction in the GSSG/GSH ratio and in the LPO levels, as well as increased CAT activity compared with healthy subjects, with no changes in SOD, GPx, and GRd activities, or AOPP levels. When analyzing the evolution from early to advanced stages of the disease, we found that the GPx activity, GSSG/GSH ratio, LPO, and AOPP increased, with a reduction in CAT. GPx changes were related to the presence of risk factors such as high-risk IPSS-R or mutational score. Moreover, there was an increase in IL-2, IL-6, IL-8, and TNF-α plasma levels, with a further increase of IL-2 and IL-10 from early to advanced stages of the disease. However, we did not observe any association between inflammation and oxidative stress. Finally, 5-azacitidine treatment generated oxidative stress in MDS patients, without affecting inflammation levels, suggesting that oxidative status and inflammation are two independent processes.

Effect of 5-Azacitine Treatment on Redox Status and Inflammatory Condition in MDS Patients

This study focused on the impact of the treatment with the hypomethylating agent 5-Azacitidine on the redox status and inflammation in 24 MDS patients. Clinical and genetic features of MDS patients were recorded and peripheral blood samples were used to determine the activity of the endogenous antioxidant defense system (superoxide dismutase, SOD, catalase, CAT, glutathion peroxidase, GPx, and reductase, GRd, activities), markers of oxidative damage (lipid peroxidation, LPO, and advanced oxidation protein products, AOPP). Moreover, pro-inflammatory cytokines and plasma nitrite plus nitrate levels as markers of inflammation, and CoQ10 plasma levels, were also measured. Globally, MDS patients showed less redox status in terms of a reduction in the GSSG/GSH ratio and in the LPO levels, and increased CAT activity compared with healthy subjects, with not changes in SOD, GPx and GRd activities, and AOPP levels. When analyzed the evolution from early- to advanced stages of the disease, the GPx activity, GSSG/GSH ratio, LPO and AOPP increased, with a reduction in CAT. GPx changes were related with the presence of risk factors such as high-risk IPSS-R or mutational score. Besides, there was an increase in IL-2, IL-6, IL-8 and TNF-α plasma levels, with further increase of IL-2 and IL-10 from early to advanced stage of the disease. However, we did not observe any association between inflammation and oxidative stress. Finally, 5-azacitidine treatment generates oxidative stress in MDS patients, without affecting inflammation levels, suggesting that oxidative status and inflammation are two independent processes.

Comodulation of NO-Dependent Vasodilation by Erythroid Band 3 and Hemoglobin: A GP.Mur Athlete Study

GP.Mur, a red blood cell (RBC) hybrid protein encoded by glycophorin B-A-B, increases expression of erythroid band 3 (Anion Exchanger-1, SLC4A1). GP.Mur is extremely rare but has a prevalence of 1–10% in regions of Southeast Asia. We unexpectedly found slightly higher blood pressure (BP) among healthy Taiwanese adults with GP.Mur. Since band 3 has been suggested to interact with hemoglobin (Hb) to modulate nitric oxide (NO)-dependent hypoxic vasodilation during the respiratory cycle, we hypothesized that GP.Mur red cells could exert differentiable effects on vascular tone. Here we recruited GP.Mur-positive and GP.Mur-negative elite male college athletes, as well as age-matched, GP.Mur-negative non-athletes, for NO-dependent flow-mediated dilation (FMD) and NO-independent dilation (NID). The subjects were also tested for plasma nitrite and nitrate before and after arterial occlusion in FMD. GP.Mur+ and non-GP.Mur athletes exhibited similar heart rates and blood pressure, but GP.Mur+ athletes showed significantly lower FMD (4.8 ± 2.4%) than non-GP.Mur athletes (6.5 ± 2.1%). NO-independent vasodilation was not affected by GP.Mur. As Hb controls intravascular NO bioavailability, we examined the effect of Hb on limiting FMD and found it to be significantly stronger in GP.Mur+ subjects. Biochemically, plasma nitrite levels were directly proportional to individual band 3 expression on the red cell membrane. The increase of plasma nitrite triggered by arterial occlusion also showed small dependency on band 3 levels in non-GP.Mur subjects. By the GP.Mur comparative study, we unveiled comodulation of NO-dependent vasodilation by band 3 and Hb, and verified the long-pending role of erythroid band 3 in this process.

Red Blood Cell and Endothelial eNOS Independently Regulate Circulating Nitric Oxide Metabolites and Blood Pressure

Background: Current paradigms suggest that nitric oxide (NO) produced by endothelial cells (ECs) via endothelial nitric oxide synthase (eNOS) in the vessel wall is the primary regulator of blood flow and blood pressure. However, red blood cells (RBCs) also carry a catalytically active eNOS, but its role is controversial and remains undefined. This study aimed to elucidate the functional significance of red cell eNOS compared to EC eNOS for vascular hemodynamics and NO metabolism. Methods: We generated tissue-specific "loss-" and "gain-of-function" models for eNOS by using cell-specific Cre-induced gene inactivation or reactivation. We created two founder lines carrying a floxed eNOS (eNOS flox/flox ) for Cre-inducible knock out (KO), as well as gene construct with an inactivated floxed/inverted exon (eNOS inv/inv ) for a Cre-inducible knock in (KI), which respectively allow targeted deletion or reactivation of eNOS in erythroid cells (RBC eNOS KO or RBC eNOS KI mice) or endothelial cells (EC eNOS KO or EC eNOS KI mice). Vascular function, hemodynamics, and NO metabolism were compared ex vivo and in vivo . Results: The EC eNOS KOs exhibited significantly impaired aortic dilatory responses to acetylcholine, loss of flow-mediated dilation (FMD), and increased systolic and diastolic blood pressure. RBC eNOS KO mice showed no alterations in acetylcholine-mediated dilation or FMD but were hypertensive. Treatment with the NOS inhibitor L-NAME further increased blood pressure in RBC eNOS KOs, demonstrating that eNOS in both ECs and RBCs contributes to blood pressure regulation. While both EC eNOS KOs and RBC eNOS KOs had lower plasma nitrite and nitrate concentrations, the levels of bound NO in RBCs were lower in RBC eNOS KOs as compared to EC eNOS KOs. Crucially, reactivation of eNOS in ECs or RBCs rescues the hypertensive phenotype of the eNOS inv/inv mice, while the levels of bound NO were restored only in RBC eNOS KI mice. Conclusions: These data reveal that eNOS in ECs and RBCs contribute independently to blood pressure homeostasis.

Hydroxyurea improves nitric oxide bioavailability in humanized sickle cell mice

Despite advancements in disease management, sickle cell nephropathy, a major contributor to mortality and morbidity in patients, has limited therapeutic options. Previous studies indicate hydroxyurea, a commonly prescribed therapy for sickle cell disease (SCD), can reduce renal injury in SCD but the mechanisms are uncertain. Because SCD is associated with reduced nitric oxide (NO) bioavailability, we hypothesized that hydroxyurea treatment would improve NO bioavailability in the humanized sickle cell mouse. Humanized male 12-wk-old sickle (HbSS) and genetic control (HbAA) mice were treated with hydroxyurea or regular tap water for 2 wk before renal and systemic NO bioavailability as well as renal injury were assessed. Untreated HbSS mice exhibited increased proteinuria, elevated plasma endothelin-1 (ET-1), and reduced urine concentrating ability compared with HbAA mice. Hydroxyurea reduced proteinuria and plasma ET-1 levels in HbSS mice. Untreated HbSS mice had reduced plasma nitrite and elevated plasma arginase concentrations compared with HbAA mice. Hydroxyurea treatment augmented plasma nitrite and attenuated plasma arginase in HbSS mice. Renal vessels isolated from HbSS mice also had elevated nitric oxide synthase 3 (NOS3) and arginase 2 expression compared with untreated HbAA mice. Hydroxyurea treatment did not alter renal vascular NOS3, however, renal vascular arginase 2 expression was significantly reduced. These data support the hypothesis that hydroxyurea treatment augments renal and systemic NO bioavailability by reducing arginase activity as a potential mechanism for the improvement on renal injury seen in SCD mice.

Preventive effects of nitrate-rich beetroot juice supplementation on monocrotaline-induced pulmonary hypertension in rats

Beetroot (Beta vulgaris L.) has a high level of nitrate; therefore, its dietary intake could increase nitric oxide (NO) level in the body, possibly preventing the development of pulmonary hypertension (PH). In this study, we examined the effects of beetroot juice (BJ) supplementation on PH and the contribution of nitrate to such effects using a rat model of monocrotaline (MCT, 60 mg/kg s.c.)-induced PH. Rats were injected subcutaneously with saline or 60 mg/kg MCT and were sacrificed 28 days after the injection. In some rats injected with MCT, BJ was supplemented from the day of MCT injection to the day of sacrifice. First, MCT-induced right ventricular systolic pressure elevation, pulmonary arterial medial thickening and muscularization, and right ventricular hypertrophy were suppressed by supplementation with low-dose BJ (nitrate: 1.3 mmol/L) but not high-dose BJ (nitrate: 4.3 mmol/L). Of the plasma nitrite, nitrate, and their sum (NOx) levels, only the nitrate levels were found to be increased by the high-dose BJ supplementation. Second, in order to clarify the possible involvement of nitrate in the preventive effects of BJ on PH symptoms, the effects of nitrate-rich BJ (nitrate: 0.9 mmol/L) supplementation were compared with those of the nitrate-depleted BJ. While the former exerted preventive effects on PH symptoms, such effects were not observed in rats supplemented with nitrate-depleted BJ. Neither supplementation with nitrate-rich nor nitrate-depleted BJ affected plasma nitrite, nitrate, and NOx levels. These findings suggest that a suitable amount of BJ ingestion, which does not affect systemic NO levels, can prevent the development of PH in a nitrate-dependent manner. Therefore, BJ could be highly useful as a therapy in patients with PH.

IL-10 and TGF-β Induced Arginase Expression Contributes to Deficient Nitric Oxide Response in Human Visceral Leishmaniasis

Nitric oxide (NO) is an anti-microbial effector of the innate immune system which plays major role in non-specific killing of various pathogens including protozoan parasites. However, due to subversion of the host’s immune processes by pathogens, suboptimal production of NO is frequently found in many infection models. Previous studies have shown suppressed NO production during Leishmania donovani infection, the causative agent of visceral leishmaniasis (VL). Availability of L-Arginine, a semi-essential amino acid is required for inducible nitric oxide synthase (iNOS) mediated NO production. However, arginase is another enzyme, which if expressed concomitantly, may strongly compete for L-Arginine, and suppress NO production by iNOS. In the present study, plasma nitrite and arginase levels were measured in VL patients before and after successful drug treatment, endemic and non-endemic healthy donors. We observed significantly lower NO levels in the plasma of VL patients as compared to endemic controls, which improved significantly post-treatment. Significantly elevated arginase activity was also observed in the plasma of VL patients, which may be associated with NO deficiency. VL patients also showed significantly higher levels of IL-10 and TGF-β, which are known to regulate expression of arginase in various immune cells. In vitro studies with human peripheral blood mononuclear cells (PBMCs) further corroborated the role of IL-10 and TGF-β in arginase mediated suppression of NO production.

Photobiomodulation 30 min or 6 h Prior to Cycling Does Not Alter Resting Blood Flow Velocity, Exercise-Induced Physiological Responses or Time to Exhaustion in Healthy Men

PurposeThe aim of the current study was to investigate the effects of photobiomodulation therapy (PBMT) applied 30 min or 6 h prior to cycling on blood flow velocity and plasma nitrite concentrations at rest, time to exhaustion, cardiorespiratory responses, blood acid-base balance, and K+ and lactate concentrations during exercise.MethodsIn a randomized, crossover design, 13 healthy untrained men randomly completed four cycling bouts until exhaustion at the severe-intensity domain (i.e., above respiratory compensation point). Thirty minutes or 6 h prior to the cycling trials, participants were treated with PBMT on the quadriceps, hamstrings, and gastrocnemius muscles of both limbs using a multi-diode array (11 cm × 30 cm with 264 diodes) at doses of 152 J or a sham irradiation (with device turned off, placebo). Blood samples were collected before and 30 min or 6 h after treatments to measure plasmatic nitrite concentrations. Doppler ultrasound exams of the femoral artery were also performed at the same time points. Cardiorespiratory responses, blood acid-base balance, and K+ and lactate concentrations were monitored during exercise sessions.ResultsPBMT did not improve the time to exhaustion (p = 0.30). At rest, no differences were found in the peak systolic velocity (p = 0.97) or pulsatility index (p = 0.83) in the femoral artery, and in plasma nitrite concentrations (p = 0.47). During exercise, there were no differences for any cardiorespiratory response monitored (heart rate, p = 0.15; oxygen uptake, p = 0.15; pulmonary ventilation, p = 0.67; carbon dioxide output, p = 0.93; and respiratory exchange ratio, p = 0.32), any blood acid-base balance indicator (pH, p = 0.74; base excess, p = 0.33; bicarbonate concentration, p = 0.54), or K+ (p = 0.22) and lactate (p = 0.55) concentrations.ConclusionsPBMT at 152 J applied 30 min or 6 h before cycling at severe-intensity did not alter resting plasma nitrite and blood flow velocity in the femoral artery, exercise-induced physiological responses, or time to exhaustion in healthy untrained men.

The effects of local versus systemic passive heating on the acute inflammatory, vascular and glycaemic response

The aim of this study was to compare the acute cardiometabolic and perceptual responses between local and whole-body passive heating. Using a water-perfused suit, ten recreationally active males underwent three 90 min conditions: heating of the legs with upper-body cooling (LBH), whole-body heating (WBH) and exposure to a thermoneutral temperature (CON). Blood samples were collected before and up to 3h post-session to assess inflammatory markers, while a 2h oral glucose tolerance test was initiated 1h post-session. Femoral artery shear rate and perceptual responses were recorded at regular intervals. The interleukin (IL)-6 incremental area under the curve (iAUC) was higher for LBH (1096±851 pg/mL*270min) and WBH (833±476 pg/mL*270min) compared with CON (565±325 pg/mL*270min; p<0.047). Glucose concentrations were higher after WBH compared with LBH and CON (p<0.046). Femoral artery shear rate was higher at the end of WBH (1713±409 L/min) compared with LBH (943±349 L/min; p<0.001), and higher in LBH than CON (661±222 L/min; p=0.002). Affect and thermal comfort were more negative during WBH compared with LBH and CON (p<0.010). In conclusion, local passive heating elevated shear rate and the IL-6 iAUC. However, while resulting in more positive perceptual responses, the majority of the included cardiometabolic markers were attenuated compared with WBH. The increase in the interleukin-6 incremental area under the curve in response to passive heating is not reduced by upper-body cooling. Novelty: • Upper-body cooling attenuates the plasma nitrite, interleukin-1ra and femoral artery shear rate response to passive heating. • Upper-body cooling leads to more positive perceptual responses to passive heating.

Comparison of pancreatic microcirculation profiles in spontaneously hypertensive rats and Wistar-kyoto rats by laser doppler and wavelet transform analysis

Pancreatic microcirculatory dysfunction emerged as a novel mechanism in the development of hypertension. However, the changes of pancreatic microcirculation profiles in hypertension remain unknown. Pancreatic microcirculatory blood distribution pattern and microvascular vasomotion of spontaneously hypertensive rats (SHRs) and Wistar Kyoto rats (WKYs) were determined by laser Doppler. Wavelet transform analysis was performed to convert micro-hemodynamic signals into time-frequency domains, based on which amplitude spectral scalograms were constructed. The amplitudes of characteristic oscillators were compared between SHRs and WKYs. The expression of eNOS was determined by immunohistochemistry, and plasma nitrite/nitrate levels were measured by Griess reaction. Additionally, endothelin-1, malondialdehyde, superoxide dismutase and interleukin-6 were determined by enzyme-linked immunosorbent assay. SHRs exhibited a lower scale blood distribution pattern with decreased average blood perfusion, frequency and amplitude. Wavelet transform spectral analysis revealed significantly reduced amplitudes of endothelial oscillators. Besides reduced expression of eNOS, the blood microcirculatory chemistry complements micro-hemodynamic profiles as demonstrated by an increase in plasma nitrite/nitrate, endothelin-1, malondialdehyde, interleukin-6 and a decrease of superoxide dismutase in SHRs. Here, we described abnormal pancreatic microcirculation profiles in SHRs, including disarranged blood distribution pattern, impaired microvascular vasomotion and reduced amplitudes of endothelial oscillators.