Functional and Morphological Differences of Muscle Mitochondria in Chronic Fatigue Syndrome and Post-COVID Syndrome.

Patients suffering from chronic fatigue syndrome (CFS) or post-COVID syndrome (PCS) exhibit a reduced physiological performance capability. Impaired mitochondrial function and morphology may play a pivotal role. Thus, we aimed to measure the muscle mitochondrial oxidative phosphorylation (OXPHOS) capacity and assess mitochondrial morphology in CFS and PCS patients in comparison to healthy controls (HCs). Mitochondrial OXPHOS capacity was measured in permeabilized muscle fibers using high-resolution respirometry. Mitochondrial morphology (subsarcolemmal/intermyofibrillar mitochondrial form/cristae/diameter/circumference/area) and content (number and proportion/cell) were assessed via electron microscopy. Analyses included differences in OXPHOS between HC, CFS, and PCS, whereas comparisons in morphology/content were made for CFS vs. PCS. OXPHOS capacity of complex I, which was reduced in PCS compared to HC. While the subsarcolemmal area, volume/cell, diameter, and perimeter were higher in PCS vs. CFS, no difference was observed for these variables in intermyofibrillar mitochondria. Both the intermyofibrillar and subsarcolemmal cristae integrity was higher in PCS compared to CFS. Both CFS and PCS exhibit increased fatigue and impaired mitochondrial function, but the progressed pathological morphological changes in CFS suggest structural changes due to prolonged inactivity or unknown molecular causes. Instead, the significantly lower complex I activity in PCS suggests probably direct virus-induced alterations.

COVID-19 in Female and Male Athletes: Symptoms, Clinical Findings, Outcome, and Prolonged Exercise Intolerance-A Prospective, Observational, Multicenter Cohort Study (CoSmo-S).

BACKGROUND: An infection with SARS-CoV-2 can lead to a variety of symptoms and complications, which can impair athletic activity. OBJECTIVE: We aimed to assess the clinical symptom patterns, diagnostic findings, and the extent of impairment in sport practice in a large cohort of athletes infected with SARS-CoV-2, both initially after infection and at follow-up. Additionally, we investigated whether baseline factors that may contribute to reduced exercise tolerance at follow-up can be identified. METHODS: In this prospective, observational, multicenter study, we recruited German COVID elite-athletes (cEAs, n = 444) and COVID non-elite athletes (cNEAs, n = 481) who tested positive for SARS-CoV-2 by PCR (polymerase chain reaction test). Athletes from the federal squad with no evidence of SARS-CoV-2 infection served as healthy controls (EAcon, n = 501). Questionnaires were used to assess load and duration of infectious symptoms, other complaints, exercise tolerance, and duration of training interruption at baseline and at follow-up 6 months after baseline. Diagnostic tests conducted at baseline included resting and exercise electrocardiogram (ECG), echocardiography, spirometry, and blood analyses. RESULTS: Most acute and infection-related symptoms and other complaints were more prevalent in cNEA than in cEAs. Compared to cEAs, EAcon had a low symptom load. In cNEAs, female athletes had a higher prevalence of complaints such as palpitations, dizziness, chest pain, myalgia, sleeping disturbances, mood swings, and concentration problems compared to male athletes (p < 0.05). Until follow-up, leading symptoms were drop in performance, concentration problems, and dyspnea on exertion. Female athletes had significantly higher prevalence for symptoms until follow-up compared to male. Pathological findings in ECG, echocardiography, and spirometry, attributed to SARS-CoV-2 infection, were rare in infected athletes. Most athletes reported a training interruption between 2 and 4 weeks (cNEAs: 52.9%, cEAs: 52.4%), while more cNEAs (27.1%) compared to cEAs (5.1%) had a training interruption lasting more than 4 weeks (p < 0.001). At follow-up, 13.8% of cNEAs and 9.9% of cEAs (p = 0.24) reported their current exercise tolerance to be under 70% compared to pre-infection state. A persistent loss of exercise tolerance at follow-up was associated with persistent complaints at baseline, female sex, a longer break in training, and age > 38 years. Periodical dichotomization of the data set showed a higher prevalence of infectious symptoms such as cough, sore throat, and coryza in the second phase of the pandemic, while a number of neuropsychiatric symptoms as well as dyspnea on exertion were less frequent in this period. CONCLUSIONS: Compared to recreational athletes, elite athletes seem to be at lower risk of being or remaining symptomatic after SARS-CoV-2 infection. It remains to be determined whether persistent complaints after SARS-CoV-2 infection without evidence of accompanying organ damage may have a negative impact on further health and career in athletes. Identifying risk factors for an extended recovery period such as female sex and ongoing neuropsychological symptoms could help to identify athletes, who may require a more cautious approach to rebuilding their training regimen. TRIAL REGISTRATION NUMBER: DRKS00023717; 06.15.2021-retrospectively registered.

Blood Profiling of Athletes after COVID-19: Differences in Blood Profiles of Post-COVID-19 Athletes Compared to Uninfected Athletic Individuals-An Exploratory Analysis.

Blood profiling data in athletic populations and their respective responses to SARS-CoV-2 infection are lacking. Thus, this exploratory pilot study aimed to analyze and compare clinical blood markers in previously infected trained athletes (ATH; 30 m/29 f) and a not previously infected healthy athletic control group (HC; 12 m/19 f). The ATH group undertook a sports medical examination which included extended blood analyses. Blood profiles with a total of 74 variables were assessed (blood counts, pro-/inflammatory and immunological markers, and micronutrients), and the ATH group was compared to the age-matched, vaccinated HC group with comparable athletic back grounds, though without previous SARS-CoV-2-infections. The ATH group showed lower IgG, Troponin-T levels, and they had a lower complement/acute-phase protein activation. Furthermore, Vitamin D levels were lower and electrolyte/micronutrient concentrations were higher in ATH. Soluble transferrin receptor as a marker of erythrocyte turnover was decreased whereas PTT as a coagulation marker was increased. Subgroup analyses according to sex revealed more differences between the women of the ATH and HC groups (for 25 different variables) than between the men (for 5 different variables), especially for immunological and metabolic variables. In particular, the immune system and electrolyte/micronutrient status should be observed frequently and sex-specifically in this athletic cohort.

Monitoring of strength, inflammation and muscle function in allogenic stem-cell transplantation patients - a pilot study for novel biomarker and risk stratification determination.

Background: Low aerobic capacity is associated with an increased mortality risk in allogenic stem-cell transplantation (alloSCT) patients, but currently used risk scores in the pre-transplantation workup are still underestimating physical activity as a prognostic factor. Aim: To examine the physical condition, muscle function, blood inflammation and training adherence of alloSCT patients during inpatient time to identify potential biomarkers associated with development of myopathy and sarcopenia. Methods: Patients undergoing alloSCT were examined at four time points (T0: before alloSCT; Tha: hospital admission; T1: engraftment; T2: inpatient discharge). T0 included cardiopulmonary performance, body composition, grip and knee strength, motor skill tests (One-leg stand/Tinetti/Chair-rising), blood sampling (blood cell profiling and inflammation targets (Kynurenin/high sensitivity C-reactive Protein (hsCRP)/Tumor necrosis factor alpha (TNF-alpha)/Musclin/Galectin-3) and quality of life, state of health, fatigue, muscle weakness and physical activity by questionnaires (IPAQ/BSA/SARC-F/Fatigue). At T1 and T2, blood samples, grip strength and motor skill tests were repeated. Glucocorticoid dose and daily physical activity were documented during inpatient stay. Results: 26 of 35 included patients (4 females; age 55.58 ± 12.32 years; BMI 24.70 ± 3.27 kg/m2; VO2peak 16.55 ± 4.06 ml/min/kg) could proceed to alloSCT. Grip strength and Tinetti decreased from T0 until T2, no difference in Chair-rising test, One-leg and Tandem stand. All patients engrafted after 24.9 days ± 3.9 days. HsCRP and Kynurenine increased from T0 to T1, decreased at T2. TNF-alpha (T0vsT2/T1vsT2) and Musclin (T0vsT1) decreased. At T2, Galectin-3 was higher compared to T0/T1. Correlation analysis of grip strength and inflammatory markers revealed a positive correlation with TNF-alpha at T2. 50% of patients documented physical activity and questionnaire and reported a 50%-reduction of daily endurance and strength training between T1 to T2. Conclusion: Allogeneic stem-cell transplantation is associated with immune system vulnerability due to conditioning, increased inflammation and fatigue, and loss of muscle strength and function. In addition to hsCRP, Kynurenine seems to be a reliable biomarker to monitor acute and regenerative inflammation status of alloSCT patients, while Musclin and Galectin-3 may be added to physiological assessment regarding myopathy and sarcopenia. Grip strength and daily activity level should be documented by professionals to identify risk patients early and support them with optimal (exercise) therapy.

Inflammation and severity of depressive symptoms in physically active individuals after COVID-19 - An exploratory immunopsychological study investigating the effect of inflammation on depressive symptom severity.

Background: SARS-CoV-2 infection is a risk factor for the development of depressive symptoms such as lack of energy, loss of interest, and depressed mood. Inflammatory processes might underline this association. The aim of this study was to investigate the association between inflammatory markers and the severity of depression after SARS-CoV-2 infection and the predictive effect of inflammatory markers on the severity of depressive symptoms. Lifestyle factors and lifestyle-related diseases can influence inflammation and depressive symptoms. As these lifestyle factors and lifestyle-related diseases are less common in physically active individuals, they are a suitable population for investigating this research question. Methods: We investigated 61 at least moderate physically active individuals on average ∼6 months (SD = 4.22, range = 0.5-19 months) after SARS-CoV-2 infection (t0) and performed a follow-up after 3 months (t1). Depressive symptoms and biomarkers of inflammation (interleukin [IL]-1ß, IL-8, IL-10, Ferritin, Lipopolysaccharide-binding-protein [LBP], neutrophil-to-lymphocyte ratio [NLR], platelet-to-lymphocyte ratio [PLR], lymphocyte-to-monocyte ratio [LMR]) and kynurenine [KYN] were measured at both time points. Concentrations of inflammatory markers at t0 were used to predict the severity of depressive symptoms at t0 and t1. Results: Concentrations of KYN were negatively related to the severity of depressive symptoms at t0. Concentrations of LMR predicted higher depressive symptoms at t0 as well as at t1. Furthermore, individuals with lower concentrations of LBP at t0 showed a higher severity of depressive symptoms at t1. No correlation was found between severity of depressive symptoms and IL-1ß, IL-8, IL-10, ferritin, NLR, and PLR at both time points. Conclusions: KYN, LBP and LMR might be useful as a predictive factor of depressive symptoms in physically active individuals after SARS-CoV-2 infection. While the results for KYN confirm the current scientific evidence, our results highlight the importance of the innovative inflammatory markers LMR and LBP. LMR and LBP might be interesting targets for predicting the development of depressive symptoms in SARS-CoV-2 infected populations and should be further investigated in future studies.

Recovery of performance and persistent symptoms in athletes after COVID-19.

INTRODUCTION: After the acute Sars-CoV-2-infection, some athletes suffer from persistent, performance-impairing symptoms, although the course of the disease is often mild to moderate. The relation between cardiopulmonary performance and persistent symptoms after the acute period is still unclear. In addition, information about the development of this relationship is lacking. OBJECTIVE: To assess the prevalence of persistent symptoms over time and their association with the performance capability of athletes. METHODS: We conducted two cardiopulmonary exercise tests (CPET) in a three months interval with 60 athletes (age: 35.2±12.1 years, 56.7% male) after infection with Sars-CoV-2 (t0: study inclusion; t1: three months post t0). At each examination, athletes were asked about their persistent symptoms. To evaluate the change of Peak VO2/BM (Body Mass) between the time before infection and the first examination, the VO2/BM (predVO2) before infection was predicted based on anthropometric data and exercise history of the athletes. For data analysis, athletes were grouped according to their symptom status (symptom-free, SF; persistent symptoms, PS) and its progression from the first to the second examination 1) SF-SF, 2) PS-SF and 3) PS-PS. RESULTS: Comparing the SF and PS groups at t0, significant differences for Max Power/BM, Max Power/lbm (lean body mass), Peak VO2, Peak VO2/BM, Peak VO2/lbm, Peak VO2/HR, Peak VE, Peak Vt and VE/VCO2-Slope were observed. Regarding the progression over three months, an increase in Max Power/BM was shown in SF-SF and PS-SF (tendency). Max Power/lbm increased in SF-SF and PS-PS (tendency). A decrease of VE/VCO2-Slope in PS-PS was found. CONCLUSION: COVID-19 led to a decline in performance that was greater in PS than in SF. Additionally, PS had decreased ventilatory parameters compared to SF. Furthermore, an improvement over time was observed in some CPET parameters and a partial recovery was observed judging by the decrease in various symptoms.

SARS-CoV-2 Altered Hemorheological and Hematological Parameters during One-Month Observation Period in Critically Ill COVID-19 Patients.

Hematological and hemorheological parameters are known to be altered in COVID-19; however, the value of combined monitoring in order to deduce disease severity is only scarcely examined. A total of 44 acute SARS-CoV-2-infected patients (aCOV) and 44 age-matched healthy controls (Con) were included. Blood of aCOV was sampled at admission (T0), and at day 2 (T2), day 5 (T5), day 10 (T10), and day 30 (T30) while blood of Con was only sampled once. Inter- and intra-group differences were calculated for hematological and hemorheological parameters. Except for mean cellular volume and mean cellular hemoglobin, all blood cell parameters were significantly different between aCOV and Con. During the acute disease state (T0-T5), hematological and hemorheological parameters were highly altered in aCOV; in particular, anemic conditions and increased immune cell response/inflammation, oxidative/nitrosative stress, decreased deformability, as well as increased aggregation, were observed. During treatment and convalescence until T30, almost all abnormal values of aCOV improved towards Con values. During the acute state of the COVID-19 disease, the hematological, as well as the hemorheological system, show fast and potentially pathological changes that might contribute to the progression of the disease, but changes appear to be largely reversible after four weeks. Measuring RBC deformability and aggregation, as well as oxidative stress induction, may be helpful in monitoring critically ill COVID-19 patients.

Kynurenine serves as useful biomarker in acute, Long- and Post-COVID-19 diagnostics.

Introduction: In patients with SARS-CoV-2, innate immunity is playing a central role, depicted by hyperinflammation and longer lasting inflammatory response. Reliable inflammatory markers that cover both acute and long-lasting COVID-19 monitoring are still lacking. Thus, we investigated one specific inflammatory marker involved as one key player of the immune system, kynurenine (Kyn), and its use for diagnosis/detection of the Long-/Post-COVID syndrome in comparison to currently used markers in both serum and saliva samples. Material and methods: The study compromised in total 151 inpatients with a SARS-CoV-2 infection hospitalized between 03/2020 and 09/2021. The group NC (normal controls) included blood bank donors (n=302, 144f/158m, mean age 47.1 ± 18.3 years (range 18-75)). Two further groups were generated based on Group A (n=85, 27f/58m, mean age 63.1 ± 18.3 years (range 19-90), acute admission to the hospital) and Group B (n=66, 22f/44m, mean age 66.6 ± 17.6 years (range 17-90), admitted either for weaning or for rehabilitation period due to Long-COVID symptoms/syndrome). Plasma concentrations of Kyn, C-Reactive Protein (CRP) and interleukin-6 (IL-6) were measured on admission. In Group B we determined Kyn 4 weeks after the negative PCR-test. In a subset of patients (n=11) concentrations of Kyn and CRP were measured in sera and saliva two, three and four months after dismission. We identified 12 patients with Post-COVID symptoms >20 weeks with still significant elevated Kyn-levels. Results: Mean values for NC used as reference were 2.79 ± 0.61 µM, range 1.2-4.1 µM. On admission, patients showed significantly higher concentrations of Kyn compared to NC (p-values < 0.001). Kyn significantly correlated with IL-6 peak-values (r=0.411; p-values <0.001) and CRP (r=0.488, p-values<0.001). Kyn values in Group B (Long-/Post-COVID) showed still significant higher values (8.77 ± 1.72 µM, range 5.5-16.6 µM), whereas CRP values in Group B were in the normal range. Conclusion: Serum and saliva Kyn are reflecting the acute and long-term pathophysiology of the SARS-CoV-2 disease concerning the innate immune response and thus may serve a useful biomarker for diagnosis and monitoring both Long- and Post-COVID syndrome and its therapy.

Differences in Immune Response During Competition and Preparation Phase in Elite Rowers.

Background: Metabolic stress is high during training and competition of Olympic rowers, but there is a lack of biomedical markers allowing to quantify training load on the molecular level. We aimed to identify such markers applying a complex approach involving inflammatory and immunologic variables. Methods: Eleven international elite male rowers (age 22.7 ± 2.4 yrs.; VO2max 71 ± 5 ml·min-1·kg-1) of the German National Rowing team were monitored at competition phase (COMP) vs. preparation phase (PREP), representing high vs. low load. Perceived stress and recovery were assessed by a Recovery Stress Questionnaire for Athletes (RESTQ-76 Sport). Immune cell activation (dendritic cell (DC)/macrophage/monocytes/T-cells) was evaluated via fluorescent activated cell sorting. Cytokines, High-Mobility Group Protein B1 (HMGB1), cell-free DNA (cfDNA), creatine kinase (CK), uric acid (UA), and kynurenine (KYN) were measured in venous blood. Results: Rowers experienced more general stress and less recovery during COMP, but sports-related stress and recovery did not differ from PREP. During COMP, DC/macrophage/monocyte and T-regulatory cells (Treg-cell) increased (p = 0.001 and 0.010). HMGB1 and cfDNA increased in most athletes during COMP (p = 0.001 and 0.048), while CK, UA, and KYN remained unaltered (p = 0.053, 0.304, and 0.211). Pro-inflammatory cytokines IL-1ß (p = 0.002), TNF-α (p < 0.001), and the chemokine IL-8 (p = 0.001) were elevated during COMP, while anti-inflammatory Il-10 was lower (p = 0.002). Conclusion: COMP resulted in an increase in biomarkers reflecting tissue damage, with plausible evidence of immune cell activation that appeared to be compensated by anti-inflammatory mechanisms, such as Treg-cell proliferation. We suggest an anti-inflammatory and immunological matrix approach to optimize training load quantification in elite athletes.

Impact of A Six Week Training Program on Ventilatory Efficiency, Red Blood Cell Rheological Parameters and Red Blood Cell Nitric Oxide Signaling in Young Sickle Cell Anemia Patients: A Pilot Study.

Patients with sickle cell anemia (SCA) show impaired ventilatory efficiency, altered blood rheology, high levels of oxidative/nitrosative stress and enhanced hemolysis with large amounts of circulating free hemoglobin, which reduces nitric oxide (NO) bioavailability. The aim of the study was to investigate whether physical exercise could improve these physiological and biological markers described to contribute to SCA pathophysiology. Twelve SCA patients participated in a controlled six weeks training program with moderate volume (two sessions per week with 15-30 min duration per session) and intensity (70% of the first ventilatory threshold). Parameters were compared before (T0) and after (T1) training. Daily activities were examined by a questionnaire at T0 and one year after the end of T1. Results revealed improved ventilatory efficiency, reduced nitrosative stress, reduced plasma free hemoglobin concentration, increased plasma nitrite levels and altered rheology at T1 while no effect was observed for exercise performance parameters or hematological profile. Red blood cell (RBC) NO parameters indicate increased NO bioavailability which did not affect RBC deformability. Participants increased their daily life activity level. The data from this pilot study concludes that even low intensity activities are feasible and could be beneficial for the health of SCA patients.

Altered Hemorheology in Fontan Patients in Normoxia and After Acute Hypoxic Exercise.

BACKGROUND: The Fontan circulation is a unique palliation procedure for several congenital heart defects. Impaired exercise capacity has previously been demonstrated in these patients and also a higher risk for cardiopulmonary mortality. Hemorheology was shown to affect cardiopulmonary capacity and in turn to be affected by regular exercise and hypoxia but none of these have been investigated in Fontan patients so far. The aim of this study was to detect general differences in hemorheology in normoxia as well as possible altered hemorheological responses to hypoxia exposure and hypoxic exercise between Fontan patients and healthy controls. METHODS AND FINDINGS: 26 Fontan patients and 20 healthy controls performed an acute exercise test (AET) on a bicycle ergometer under hypoxia with ambient 15.2% oxygen saturation (sO2). Blood samples were taken at rest in normoxia (T0), at rest in hypoxia (T1), after maximum exhaustion in hypoxia (T2), and after 50 min recovery in normoxia (T3). Hemorheological and blood parameters were investigated. Additionally, arterial stiffness was tested at T0. Red blood cell (RBC) deformability, NOx, erythropoietin (EPO) concentration, RBC count, hemoglobin (Hb) concentration and hematocrit (hct) were significantly increased in Fontan patients compared to controls. Same was observed for arterial stiffness. No changes were observed for RBC aggregation, fibrinogen concentration, free radical levels and vascular endothelial growth factor (VEGF). Hypoxia exposure did not change parameters, whereas exercise in hypoxia increased aggregation and hct significantly in both groups. Fontan patients showed significantly increased aggregation-disaggregation balance compared to controls. CONCLUSION: Acute hypoxia exposure and exercise under hypoxia might have similar impact on hemorheology in Fontan patients and controls and was clinically well tolerated. Nevertheless, exercise alters aggregation and possibly hemodynamics which requires special attention in Fontan patients.

Effect of acute exercise on RBC deformability and RBC nitric oxide synthase signalling pathway in young sickle cell anaemia patients.

Sickle cell anaemia (SCA) is characterized by reduced red blood cell (RBC) deformability and nitric oxide (NO) bioavailability. The aim of the study was to investigate whether exercise might affect these parameters in SCA. SCA patients and healthy controls (AA) performed an acute submaximal exercise test until subjects reached the first ventilatory threshold (VT 1). Blood was sampled at rest and at VT 1. At rest, free haemoglobin level was higher and RBC count, haemoglobin and haematocrit were lower in SCA compared to AA. RBC deformability was lower in SCA. Exercise had no effect on the tested parameters. RBC NO level was higher in SCA compared to AA at rest and significantly decreased after exercise in SCA. This might be related to a reduction in RBC-NO synthase (RBC-NOS) activation which was only observed in SCA after exercise. Free radical levels were higher in SCA at rest but concentration was not affected by exercise. Marker for lipid peroxidation and antioxidative capacity were similar in SCA and AA and not affected by exercise. In conclusion, a single acute submaximal bout of exercise has no deleterious effects on RBC deformability or oxidative stress markers in SCA, and seems to modulate RBC-NOS signalling pathway.

Cryopreservation of red blood cells: Effect on rheologic properties and associated metabolic and nitric oxide related parameters.

AIM: High glycerol cryopreservation of red blood cells (RBCs) reduces metabolic processes at ultralow temperatures but less is known regarding the effect of cryopreservation on RBC nitric oxide (NO) metabolism, haemorheological properties, structural behaviour and membrane fragility. METHODS: Blood from ten healthy participants was sampled, glycerolized and stored at -80 °C (SB). Aliquots were thawed and further processed after 4, 8 and 12 weeks, respectively. At these time points, fresh blood (FB) was additionally sampled from each participant. FB/SB mixtures were prepared corresponding to transfusion of 1-3 blood bags. Additionally, mixtures were exposed to shear stress similar to that found in the circulation and deformability was measured to estimate possible behaviour of cryopreserved RBC in vivo. RESULTS: Ageing of RBC was reduced during cryopreservation. Markers for RBC metabolism (ATP, 2,3-DPG) were not altered but RBC sodium levels increased and potassium and calcium decreased, respectively. Mean cellular volume was higher and accordingly, mean cellular haemoglobin concentration was lower in SB. Deformability was altered during storage with less shear stress necessary to deform RBCs. Changes were also detectable in blood mixtures. Deformability remained unaltered in shear stress settings in FB and SB. RBC viscosity was reduced in SB. RBC-NOS content and phosphorylation sites as well as nitrite and RxNO levels seem not to be affected by the intervention. CONCLUSION: Cryopreservation maintains RBC metabolic function in vitro, but structure and function of cryopreserved RBC seems to be altered. Impact of these alterations in vivo seems to be less but needs further investigation.

The red-vine-leaf extract AS195 increases nitric oxide synthase-dependent nitric oxide generation and decreases oxidative stress in endothelial and red blood cells.

The red-vine-leaf extract AS195 improves cutaneous oxygen supply and the microcirculation in patients suffering from chronic venous insufficiency. Regulation of blood flow was associated to nitric oxide synthase (NOS)-dependent NO (nitric oxide) production, and endothelial and red blood cells (RBC) have been shown to possess respective NOS isoforms. It was hypothesized that AS195 positively affects NOS activation in human umbilical vein endothelial cells (HUVECs) and RBC. Because patients with microvascular disorders show increased oxidative stress which limits NO bioavailability, it was further hypothesized that AS195 increases NO bioavailability by decreasing the content of reactive oxygen species (ROS) and increasing antioxidant capacity. Cultured HUVECs and RBCs from healthy volunteers were incubated with AS195 (100 µmol/L), tert-butylhydroperoxide (TBHP, 1 mmol/L) to induce oxidative stress and with both AS195 and TBHP. Endothelial and red blood cell-nitric oxide synthase (RBC-NOS) activation significantly increased after AS195 incubation. Nitrite concentration, a marker for NO production, increased in HUVEC but decreased in RBC after AS195 application possibly due to nitrite scavenging potential of flavonoids. S-nitrosylation of RBC cytoskeletal spectrins and RBC deformability were increased after AS195 incubation. TBHP-induced ROS were decreased by AS195, and antioxidative capacity was significantly increased in AS195-treated cells. TBHP also reduced RBC deformability, but reduction was attenuated by parallel incubation with AS195. Adhesion of HUVEC was also reduced after AS195 treatment. Red-vine-leaf extract AS195 increases NOS activation and decreases oxidative stress. Both mechanisms increase NO bioavailability, improve cell function, and may thus account for enhanced microcirculation in both health and disease.

Increase in Red Blood Cell-Nitric Oxide Synthase Dependent Nitric Oxide Production during Red Blood Cell Aging in Health and Disease: A Study on Age Dependent Changes of Rheologic and Enzymatic Properties in Red Blood Cells.

AIM: To investigate RBC-NOS dependent NO signaling during in vivo RBC aging in health and disease. METHOD: RBC from fifteen healthy volunteers (HC) and four patients with type 2 diabetes mellitus (DM) were separated in seven subpopulations by Percoll density gradient centrifugation. RESULTS: The proportion of old RBC was significantly higher in DM compared to HC. In both groups, in vivo aging was marked by changes in RBC shape and decreased cell volume. RBC nitrite, as marker for NO, was higher in DM and increased in both HC and DM during aging. RBC deformability was lower in DM and significantly decreased in old compared to young RBC in both HC and DM. RBC-NOS Serine1177 phosphorylation, indicating enzyme activation, increased during aging in both HC and DM. Arginase I activity remained unchanged during aging in HC. In DM, arginase I activity was significantly higher in young RBC compared to HC but decreased during aging. In HC, concentration of L-arginine, the substrate of RBC-NOS and arginase I, significantly dropped from young to old RBC. In DM, L-arginine concentration was significantly higher in young RBC compared to HC and significantly decreased during aging. In blood from healthy subjects, RBC-NOS activation was additionally inhibited by N5-(1-iminoethyl)-L-Ornithine dihydrochloride which decreased RBC nitrite, and impaired RBC deformability of all but the oldest RBC subpopulation. CONCLUSION: This study first-time showed highest RBC-NOS activation and NO production in old RBC, possibly to counteract the negative impact of cell shrinkage on RBC deformability. This was even more pronounced in DM. It is further suggested that highly produced NO only insufficiently affects cell function of old RBC maybe because of isolated RBC-NOS in old RBC thus decreasing NO bioavailability. Thus, increasing NO availability may improve RBC function and may extend cell life span in old RBC.