Statistically verified methods for determining predictors of development of arterial hypertension depending on endothelial nitric oxide synthase T786C gene promoter polymorphism using lipid profile indicators.

Objective. Polymorphism investigation of T786C gene promoter of endothelial nitric oxide synthase (eNOS/NOS3) in the arterial hypertension is a promising field for determining the relationship between heredity, hypertension, and dyslipidemia, which still remains controversial. The purpose of the study was to investigate the lipid profile, which depends on the NOS3 T786C gene promotor region polymorphism in patients with arterial hypertension. Methods. The study involved 86 patients with arterial hypertension. The control group consisted of 30 basically healthy individuals. The lipid profile in the blood serum of the studied patients was measured by commercially available kits using Biochem FC-200 analyzer (HTI, USA). The allelic polymorphism of NOS3 T786C gene promoter was studied using a polymerase chain reaction technique with electrophoretic detection of the results. Results. An increase at the level of all atherogenic fractions in the blood was found in the group of patients carrying the CC genotype compared with carriers of the TT genotype of the NOS3 gene. The total cholesterol serum level in the group of carriers of the CC genotype of NOS3 T786C gene promoter increased by 33.3% compared with carriers of the TT genotype and it was almost twice as high as the control values. In the group of carriers in the CC genotype of the NOS3 gene, the serum level of triglycerides was statistically significantly higher (2.9 times) than in the group of carriers of the TT genotype. The low-density lipoprotein (LDL) and very low-density lipoprotein (VLDL) serum levels significantly increased in patients with arterial hypertension with the CC genotype by 1.6 and 4.6 times, respectively, compared with the TT genotype carriers. The high-density lipoprotein (HDL) serum level, as an antiatherogenic factor, was statistically significantly lower (by 45.8%) in the group of the CC genotype carriers of the NOS3 gene than in the group with carriers of the TT genotype (0.58±0.06 vs. 1.07±0.03 mmol/l.) Conclusions. The increase in all atherogenic and decrease in antiatherogenic lipid parameters of the lipidogram of patients with arterial hypertension and the deepening of dyslipidemia in carriers of the CC genotype compared with carriers of the TT genotype of the NOS3 T786C gene promoter is crucial in the development of dyslipidemia.

Serum inducible and endothelial nitric oxide synthase in coronary artery disease patients with Type 2 Diabetes mellitus.

OBJECTIVE: Coronary artery disease (CAD) is a well-known cause of morbidity and mortality in type 2 diabetes mellitus (T2DM). The role of endothelial nitric oxide synthase (eNOS) and inducible nitric oxide synthase (iNOS) in T2DM patients in relation to CAD is not well understood. We examined serum inducible and endothelial nitric oxide synthase activities in patients with T2DM in relation to the presence of coronary artery disease. PATIENTS AND METHODS: The present study was conducted in the Department of Physiology, King Saud University, Riyadh, Saudi Arabia. Subjects were grouped into control (Group A, n=87), T2DM without CAD (Group B, n=70), and T2DM patients with CAD (Group C, n=49). The selection of T2DM subjects was according to the American Diabetes Association (ADA). Serum iNOS, eNOS, hsCRP, nitrates and nitrites along with lipid profile were compared between different groups. Spearman's correlation and ROC analysis were also performed. RESULTS: Serum eNOS levels were significantly high in the control group (112.38±47.16 U/ml) than in DM without CAD (81.43±49.91 U/ml) and DM with CAD (84.80±43.32 U/ml, p<.001). Serum iNOS levels were significantly higher in DM with CAD (42.87±28.83 U/ml) compared to both control (22.08±11.77 U/ml) and DM without CAD (16.24±12.30 U/ml, p<.001). Additionally, the differences in nitrite and NO were not significant between the three groups (34.06±24.75, 33.02±21.50, 38.83±24.34 uM, p = .384), and (56.51±36.78, 49.89±28.83 vs. 55.77±30.34 uM, p=.416) respectively. ROC curve analysis revealed a sensitivity and specificity of 73.5% and 68.6% of iNOS level at a cutoff point of 21.1 U/ml to predict CAD in T2DM patients. The ROC analysis for iNOS, eNOS, and hs-CRP were .782 (p<.001), .574 (p=.170), and .726 (p<.001), respectively. CONCLUSIONS: Patients with T2DM have significantly higher levels of serum iNOS and lower levels of eNOS. However, iNOS levels were significantly higher in T2DM patients with concomitant CAD. Moreover, iNOS activity positively correlated with glycemic control and hsCRP. Therefore, iNOS could be an emerging future marker for CAD in T2DM patients and its antagonists could be useful in the management of these patients.

The serum levels of testosterone in coronary artery disease patients; relation to NO, eNOS, endothelin-1, and disease severity.

OBJECTIVES: The changes in testosterone level and its correlation with the endothelial nitric oxide systems balance in patients with coronary artery disease (CAD) remains uncertain. Therefore, in our study, we aimed to evaluate the levels of testosterone, endothelin-1 (ET-1), nitric oxide (NO), and endothelial NOS (eNOS) in CAD patients, and control group to find the relationship between these parameters and disease severity. METHODS: Forty-four patients as CAD group with significant (≥50%) stenosis confirmed by angiography was included in the study, and 40 healthy men were included as the control group. According to the number of vessels obstruction, CAD severity was determined. The serum indicated parameters were assessed to discriminate between patients and controls. RESULTS: It was found that testosterone levels in the CDA group were significantly lower than those of the control group (p<0.05). In addition, the level of ET-1 in the CAD group was higher than that in the control group, but levels of NO and eNOS in observation were significantly lower than those in the control group (p<0.05). The correlation analysis revealed that testosterone was passivity correlated with serum NO levels (r=0.550, p=0.001). CONCLUSIONS: The current study reports that serum levels of testosterone are closely related to endothelial NO levels and might be of relevance to the pathogenesis of endothelial dysfunction and disease severity in CAD patients.

Fetal oxygen supply can be improved by an effective cross-talk between fetal erythrocytes and vascular endothelium.

In twin/multiple pregnancy, siblings experience an adverse intrauterine environment which forms the major etiological factor leading to pathological conditions. The status of the developing fetus is highly determined by the nitric oxide (NO) level, that facilitates vasodilation which in turn modulates the oxygen and nutrition supply. As the umbilical cord (UC) lacks innervation, activation of the endothelial nitric oxide synthase (NOS3) is fundamental to maintain adequate NO production. Recent ground breaking fact showed that under stress conditions, circulating red blood cells (RBCs) can actively produces NO as a "rescue mechanism". Therefore, this study majorly focused on the molecular mechanisms that affected the redox environment by altering NOS3 activation - both in the UC arteries and vein endothelium and RBCs - that have impacts on developmental parameters, like birth weight. In connection to that, we pursued the communication efficiency between the vessels' endothelium and the circulating RBCs in demand of bioavailable NO. Our results indicated that twinning itself at stage 33-35 weeks, does not reduce the NOS3 level and its phosphorylation status in the cord vessels. However, RBC-NOS3 activation is highly upregulated during this period - providing additional evidence for the active regulatory role of fetal RBCs in the rate of blood flow - and this functional activity highly correlates with the birth weight of the fetuses. Detailed analysis on NOS3 signalling at different time points of gestation could establish a benchmark in understanding of the pathophysiological mechanisms involved in the process of developing neonatal vascular diseases.

Priapism in sickle cell disease: Associations between NOS3 and EDN1 genetic polymorphisms and laboratory biomarkers.

Priapism is a urologic emergency characterized by an uncontrolled, persistent and painful erection in the absence of sexual stimulation, which can lead to penile fibrosis and impotence. It is highly frequent in sickle cell disease (SCD) associated with hemolytic episodes. Our aim was to investigate molecules that may participate in the regulation of vascular tone. Eighty eight individuals with SCD were included, of whom thirty-seven reported a history of priapism. Priapism was found to be associated with alterations in laboratory biomarkers, as well as lower levels of HbF. Patients with sickle cell anemia using hydroxyurea and those who received blood products seemed to be less affected by priapism. Multivariate analysis suggested that low HbF and NOm were independently associated with priapism. The frequency of polymorphisms in genes NOS3 and EDN1 was not statistically significant between the studied groups, and the presence of the variant allele was not associated with alterations in NOm and ET-1 levels in patients with SCD. The presence of the variant allele in the polymorphisms investigated did not reveal any influence on the occurrence priapism. Future studies involving larger samples, as well as investigations including patients in priapism crisis, could contribute to an enhanced understanding of the development of priapism in SCD.

Both Traditional and Stair Climbing-based HIIT Cardiac Rehabilitation Induce Beneficial Muscle Adaptations.

PURPOSE: There is a lack of knowledge as to how different exercise-based cardiac rehabilitation programming affects skeletal muscle adaptations in coronary artery disease (CAD) patients. We first characterized the skeletal muscle from adults with CAD compared with a group of age- and sex-matched healthy adults. We then determined the effects of a traditional moderate-intensity continuous exercise program (TRAD) or a stair climbing-based high-intensity interval training program (STAIR) on skeletal muscle metabolism in CAD. METHODS: Sixteen adults (n = 16, 61 ± 7 yr), who had undergone recent treatment for CAD, were randomized to perform (3 d·wk-1) either TRAD (n = 7, 30 min at 60%-80% of peak heart rate) or STAIR (n = 9, 3 × 6 flights) for 12 wk. Muscle biopsies were collected at baseline in both CAD and healthy controls (n = 9), and at 4 and 12 wk after exercise training in CAD patients undertaking TRAD or STAIR. RESULTS: We found that CAD had a lower capillary-to-fiber ratio (C/Fi, 35% ± 25%, P = 0.06) and capillary-to-fiber perimeter exchange (CFPE) index (23% ± 29%, P = 0.034) in Type II fibers compared with healthy controls. However, 12 wk of cardiac rehabilitation with either TRAD or STAIR increased C/Fi (Type II, 23% ± 14%, P < 0.001) and CFPE (Type I, 10% ± 23%, P < 0.01; Type II, 18% ± 22%, P = 0.002). CONCLUSION: Cardiac rehabilitation via TRAD or STAIR exercise training improved the compromised skeletal muscle microvascular phenotype observed in CAD patients.

Low oxygen microenvironment and cardiovascular remodeling: Role of dual L/N.type Ca2+ channel blocker.

OBJECTIVE: Patients exposed to chronic sustained hypoxia frequently develop cardiovascular disease risk factors to ultimately succumb to adverse cardiovascular events. In this context, the present study intends to assess the role of cilnidipine (Cil), a unique calcium channel blocker that blocks both L-type and N-type calcium channels, on cardiovascular pathophysiology in face of chronic sustained hypoxia exposure. MATERIALS AND METHODS: The study involved Wistar strain albino rats. The group-wise allocation of the experimental animals is as follows - Group 1, control (21% O2); Group 2, chronic hypoxia (CH) (10% O2, 90% N); Group 3, Cil + 21% O2; and Group 4, CH (10% O2, 90% N) + Cil (CH + Cil). Cardiovascular hemodynamics, heart rate variability, and endothelial functions (serum nitric oxide [NO], serum endothelial nitric oxide synthase [NOS3], and serum vascular endothelial growth factor [VEGF]) were assessed. Cardiovascular remodeling was studied by histopathological examination of the ventricular tissues, coronary artery (intramyocardial), and elastic and muscular arteries. Normalized wall index of the coronary artery was also calculated. RESULTS AND CONCLUSION: The results demonstrated altered cardiovascular hemodynamics, disturbed cardiovascular autonomic balance, increased levels of VEGF and NOS3, and decreased bioavailability of NO on exposure to chronic sustained hypoxia. The histopathological examination further pointed toward cardiovascular remodeling. Treatment with Cil ameliorated the cardiovascular remodeling and endothelial dysfunction induced by CH exposure, which may be due to its blocking actions on L/N-type of calcium channels, indicating the possible therapeutic role of Cil against CH-induced cardiovascular pathophysiology.

The effects of regular aerobic exercise training on blood nitric oxide levels and oxidized LDL and the role of eNOS intron 4a/b polymorphism.

BACKGROUND: Nitric oxide (NO), oxidized LDL (OxLDL) and endothelial nitric oxide synthase intron 4a/b polymorphism (eNOSP) are related to atherosclerosis (AS). The present study investigated the effects of regular aerobic exercise training on the mentioned risk factors as well as blood lipids and lipoproteins (BLLPs) and the role of eNOSP, which is unclear. METHODS: The study was participated by 46 well trained male soccer referees as the athletic group (AG, age; 23.26 ± 2.84 years) and 43 sedentary controls (CG, age; 23.16 ± 3.28 years). Yoyo intermittent endurance (Yoyo IE-2 test) was performed to measure aerobic endurance levels of the participants. Serum NO, eNOS and oxidized LDL (OxLDL) levels (by ELISA method) and total oxidant /antioxidant status ratio (/TOS/TAS) as oxidative stress (OS) index (OSI) and BLLPs levels were determined. eNOSP was identified from genomic DNA samples with VNTR analysis. RESULTS: There is no significant difference between AG and CG including the genotype groups for NO, eNOS and BLLPs and eNOSP has no role. However, AG's NO (29%, p > .05) and TAS levels were significantly higher (p = .001) than those of CG, whereas OSI (p = .001) and OxLDL (p = .011) values were significantly lower. On the other hand, NO value of the athletic bb group was 29% higher compared with the control and the a carrier (aC = aa + ab) group. CONCLUSIONS: These findings suggest that regular aerobic exercise improves blood NO levels and antioxidant capacity, while decreasing OS levels including OxLDL, but not eNOS and BLLPs in the athletes. Although the polymorphism does not have a modifying effect on these effects, bb genotype group may benefit more from training for NO than aC group due to genetic tendency.

Cardiovascular Protection Variables Based on Exercise Intensity in Stable Coronary Heart Disease Patients After Coronary Stenting: A Comparative Study.

PURPOSE: Our study aimed at determining and comparing the mechanism of cardiovascular protection variables in moderate-intensity continuous training (MICT) and high-intensity interval training (HIIT) in patients with stable coronary heart disease (CHD) after coronary stenting. PARTICIPANTS AND METHODS: This experimental study used the same subject and cross-over design, involving eleven stable CHD patients after coronary stenting. These were randomly divided into two groups; MICT for 29 minutes at 50-60% heart rate reserve and HIIT with 4x4 minute intervals at 60-80% heart rate reserve, each followed by three minutes of active recovery at 40-50% heart rate reserve. These were conducted three times a week for two weeks. The participants' levels of adrenaline, noradrenaline, endothelial nitric oxide synthase (eNOS), extracellular superoxide dismutase (EC-SOD) activity assayed, and flow-mediated dilatation (FMD) were examined before and after treatments were completed. RESULTS: The HIIT significantly increased the levels of noradrenaline and eNOS compared with MICT (p<0.05). Also, HIIT was better in maintaining EC-SOD activity and FMD compared with MICT (p<0.05). Through the noradrenalin pathway, HIIT had a direct and significant effect on eNOS and FMD (p<0.05) but MICT, through the noradrenaline pathways, had a direct and significant effect on eNOS (p<0.05), and through the EC-SOD activity pathways had a direct and significant effect on FMD (p<0.05). MICT reduced EC-SOD activity and also decreased the FMD value. CONCLUSION: HIIT is superior to MICT in increasing cardiovascular protection by increasing the concentrations of noradrenalin and eNOS, maintaining EC-SOD activity, and FMD in stable CHD patients after coronary stenting.

Chemical renal denervation-induced upregulation of the ACE2/Ang (1-7)/Mas axis attenuates blood pressure elevation in spontaneously hypertensive rats.

OBJECTIVE: Evidence has shown that the ACE2/Ang (1-7)/Mas axis plays an important role in the control of hypertension. Thus, we hypothesized that chemical renal denervation (RDN) could reduce blood pressure by regulating the ACE2/Ang (1-7)/Mas axis in spontaneously hypertensive rats. METHODS: Twelve rats were randomly divided into sham group and chemical RDN group. All the rats were sacrificed 4 weeks later. Plasma samples were collected to measure the renin-angiotensin system (RAS) activities and reactive oxygen species levels by radioimmunoassay, chromatometry and ELISA. Paraventricular nucleus (PVN) tissues were collected to examine the expression of the components of the ACE2/Ang (1-7)/Mas axis by western bolt and immunofluorescence. RESULTS: The systolic blood pressure (169.33 ± 7.50 vs 182.67 ± 7.00 mmHg, p < .05) and the diastolic blood pressure (97.50 ± 4.68 vs 109.33 ± 4.41 mmHg, p < .05) in the RDN group were obviously lower than the baseline levels, whereas the opposite results were observed in the sham group. The RDN group exhibited a significant reduction in the plasma ROS (91.59 ± 13.12 vs 72.34 ± 11.76 U/ml, p < .05) and NADPH oxidase (171.86 ± 1.14 vs 175.75 ± 1.74 nmol/ml, p < .001) compared with the sham group, while the plasma eNOS (3.47 ± 0.42 vs 2.49 ± 0.51 U/ml, p < .05) and NO (55.92 ± 8.10 vs 43.53 ± 4.58 µmol/L, p < .05) were increased. The expression of the components of the ACE2/Ang (1-7)/Mas axis was upregulated while the expression of the components of the ACE/Ang II/AT1 R axis was downregulated in the plasma and PVN in the RDN group. CONCLUSION: Our findings suggested that the reduction in blood pressure was regulated by chemical RDN-induced upregulation of the components of the ACE2/Ang (1-7)/Mas axis.

The Antithrombotic Effects of Low Molecular Weight Fragment from Enzymatically Modified of Laminaria Japonica Polysaccharide.

BACKGROUND Laminaria japonica polysaccharide (LJP), a fucose enriched sulfated polysaccharide has been demonstrated to have excellent anticoagulant and antithrombotic activities. However, the antithrombotic effect of low molecular weight polysaccharide from enzymatically modified of LJP (LMWEP) remains unknown. MATERIAL AND METHODS LMWEP was prepared by fucoidanase enzymatic hydrolysis, and the antithrombotic and anticoagulant activities, and the underlying mechanism were investigated thoroughly. Rats were randomly divided into 6 groups (8 rats in each group): the blank control group, the blank control group treated with LMWEP (20 mg/kg), the model group, the model group treated with heparin (2 mg/kg), the model group treated with LJP (20 mg/kg), and the model group treated with LMWEP (20 mg/kg). After 7 days of intravenous administration, blood was collected for biochemical parameters examinations. RESULTS LMWEP increased the activated partial thromboplastin time (APTT), thrombin time (TT), prothrombin time (PT), 6-keto prostaglandin F1alpha (6-Keto-PGF1alpha), and endothelial nitric oxide synthase (eNOS). In addition, LMWEP decreased fibrinogen (FIB), endothelin-1 (ET-1), thromboxane B2 (TXB2), erythrocyte sedimentation rate (ESR), and hematocrit (HCT). CONCLUSIONS LMWEP, an enzymatically modified fragment with a molecular weight of 25.8 kDa, is a potential antithrombotic candidate for treatment of thrombosis related diseases.

ASSOCIATIONS BETWEEN EFFICACY OF THE THERAPY AND CIRCADIAN FLUCTUATIONS OF ENDOTHELIAL NITRIC OXIDE SYNTHASE WITH TOLL-LIKE RECEPTORS 2 EXPRESSION, AND NOS3 POLYMORPHISM IN FEMALES WITH RHEUMATOID ARTHRITIS.

Despite significant progress in treatment of rheumatoid arthritis (RA), a considerable part of patients remains resistant to the current therapy, apparently for the reasons of undefined mechanisms of its pathogenesis. Recently, the disturbances of circadian regulation of inflammatory processes in RA have been highlighted as important ones. Endothelial nitric oxide synthase (NOS3) and soluble toll-like receptors 2 (sTLR2) take part in the regulation of angiogenesis, osteoclastogenesis, immune responses but their circadian rhythms and predictive significance in RA patients are still unknown. Aim - to estimate the associations between efficacy of treatment and the circadian rhythms of NOS3 and sTLR2, and NOS3 polymorphism in females with rheumatoid arthritis, Ukraine. 97 RA patients (100% female) aged 46.3±8.89 years with disease duration 8.44±6.52 years were examined. All patients as a disease-modifying therapy received methotrexate (MTX) orally in a dose ≤15 mg/week, folic acid 5 mg/week, NSAIDs and corticosteroids (CS) ≤10 mg/day by prednisone. Doses of MTX, NSAIDs and CS were stable 4 weeks prior to the enrolment and during the whole period of study. The efficacy end points included DAS28, RAID and American College of Rheumatology response criteria (ACR20/50/70). Serum levels of NOS3 and sTLR2 were determined at 08:00 and 20:00 using Cloud-Clone Corp kits (USA). NOS3 T-786С polymorphism was determined by Real-Time PCR. The SPSS22 software package was used for statistical processing of the results. The study was performed in accordance to the bioethical standards. After 12-week treatment among RA patients were revealed 52.6% ACR 20 responders and 47.4% non-responders. Opposite diurnal variation of NOS3 and sTLR2 serum levels were found in RA patients. There were significant differences in NOS3/sTLR2 ratio at 08:00 accordingly to NOS3 T786C genotype. The disturbances in daily variability of NOS3 or sTLR2 serum levels were more significant in non-responders compare to responders. Decrease of NOS3/sTLR2 ratio was a predictor of non-response to treatment in RA patients (ß=0.366, р=0.000). In RA patients the disturbances of circadian rhythms of endothelial nitric oxide synthase or toll-like receptors 2 expression are associated with an increase of resistance to disease-modifying therapy with methotrexate.

Circadian fluctuations of endothelial nitric oxide synthase activity in females with rheumatoid arthritis: a pilot study.

Rheumatoid arthritis (RA) is a disease associated with circadian disorders of steroid hormones or cytokine secretion which induce inflammatory, destructive and proliferative processes in the synovial joints. Angiogenesis plays an important role in RA, but circadian rhythms of the angiogenic mediator production, especially endothelial nitric oxide synthase (NOS3), are still unclear. NOS3 takes part in regulation of endothelial functions, inflammation, and bone remodeling process. Studying circadian rhythms of NOS3 production in RA patients will make an improvement in understanding the angiogenic-inflammatory pathways relevant to rheumatic diseases. The aim of the study was to test the hypothesis of a diurnal variation in circulating levels of NOS3 in RA patients. A cross-sectional monocentric pilot study of circadian variability of endothelial nitric oxide synthase in a Ukrainian population was conducted between March and July 2017. We examined 36 RA patients (100% women) and 34 age-matched healthy women without joint diseases and autoimmune diseases (control). Blood samples were collected four times per day (at 08:00; 14:00; 20:00 and 02:00) for two consecutive days. Serum NOS3 concentration was measured by ELISA (Cloud-Clone Corp kit). The study was conducted in compliance with bioethical standards. The SPSS22 software package was used for statistical processing of the results. A diurnal variation in circulating levels of NOS3 in healthy women was established, with peak values appearing in the evening and acrophase at 20:00, and low values in the morning, with batiphase at 08:00. In patients with RA serum, NOS3 levels were substantially decreased throughout the day compared to the control. In RA patients, a diurnal variation in circulating levels of NOS3 was also established. However, the variability of NOS3 production was higher in RA patients than in the control group. For example, in RA patients the difference between morning/evening values of NOS3 was 1.3 times higher (p < 0.05) than in the control. Negative correlations were found between the morning NOS3 levels and RA activity markers such as DAS28 and the number of tender and swollen joints. The diurnal variation in circulating levels of NOS3 in women with RA as well as in healthy women was found. However, in RA patients, a decrease in NOS3 production was observed, especially in the morning, which was associated with an increase in the disease activity. Thus, the circadian rhythm of circulating NOS3 can be opposite to the circadian rhythm of secretion of main inflammatory regulators in RA.

Dynamic changes of angiopoietins and endothelial nitric oxide supply during fluid resuscitation for major gyn-oncological surgery: a prospective observation.

BACKGROUND: Despite goal-directed hemodynamic therapy, vascular function may deteriorate during surgery for advanced abdominal tumor masses. Fluid administration has been shown to be associated with distinct changes in serum levels of functional proteins. We sought to determine how serum total protein and angiopoietin (ANG) levels change during major abdominal tumor surgery. In addition, ex vivo endothelial nitric oxide synthase (eNOS) activation as well as NO bioavailability in vivo were assessed. METHODS: 30 patients scheduled for laparotomy for late-stage ovarian or uterine cancer were prospectively included. Advanced hemodynamic monitoring as well as protocol-driven goal-directed fluid optimization were performed. Total serum protein, ANG-1, -2, and soluble TIE2 were determined pre-, intra-, and postoperatively. Phosphorylation of eNOS was assessed in microvascular endothelial cells after incubation with patient serum, and microvascular reactivity was determined in vivo by near-infrared spectroscopy and arterial vascular occlusion. RESULTS: Cardiac output as well as preload gradually decreased during surgery and were associated with a median total fluid intake of 12.8 (9.7-15.4) mL/kg*h and a postoperative fluid balance of 6710 (4113-9271) mL. Total serum protein decreased significantly from baseline (66.5 (56.4-73.3) mg/mL) by almost half intraoperatively (42.7 (36.8-51.5) mg/mL, p < 0.0001) and remained at low level. While ANG-1 showed no significant dilutional change (baseline: 12.7 (11.9-13.9) ng/mL, postop.: 11.6 (10.8 -13.5) ng/mL, p = 0.06), serum levels of ANG-2 were even increased postoperatively (baseline: 2.2 (1.6-2.6) ng/mL vs. postop.: 3.4 (2.3-3.8) ng/mL, p < 0.0001), resulting in a significant shift in ANG-2 to ANG-1 ratio. Ex vivo phosphorylation of eNOS was decreased depending on increased ANG-2 levels and ANG-2/1 ratio (Spearman r = - 0.37, p = 0.007). In vivo, increased ANG-2 levels were associated with impaired capillary recruitment and NO bioavailability (Spearman r = - 0.83, p = 0.01). CONCLUSIONS: Fluid resuscitation-associated changes in serum vascular mediator profile during abdominal tumor surgery were accompanied by impaired eNOS activity ex vivo as well as reduced NO bioavailability in vivo. Our results may explain disturbed microvascular function in major surgery despite goal-directed hemodynamic optimization.

Coagulant Effects and Mechanism of Schefflera heptaphylla (L.) Frodin.

Schefflera heptaphylla (L.) Frodin, are commonly used in anti-inflammatory, analgesic, traumatic bleeding and hemostasisas. In this paper, the coagulation effect of the ethanol extract (Set), ethyl acetate phase (Sea) and n-butanol phase (Sbu) was evaluated by prothrombin time (PT), activated partial thromboplastin time (APTT), thrombin time (TT) and fibrinogen content (FIB) assays in vitro. Then, Three main lupanine triterpenes (compounds A-C) were isolated and identified from Sea and Sbu by a bioassay-guided method and their structure were identified as 3α-Hydroxy-lup-20(29)-ene-23, 28-dioic acid, betulinic acid 3-O-sulfate and 3α-Hydroxy-lup-20(29)-ene-23, 28-dioic acid 28-O-(α-l-rhamnopyranosyl(1→4)-O-ß-d-glucopyranosyl(1→6))-ß-d-glucopyranoside) by spectroscopic data analysis. Among of them, compound B was confirmed to have significant coagulant effect in vitro. Furthermore, the pro-coagulation mechanism of S. heptaphylla extracts and compound B were investigated by measuring whole blood viscosity (WBV), plasma viscosity (PV), erythrocyte sedimentetion rate (ESR), pack cell volume (PCV), APTT, PT, TT, and FIB in vivo. Meanwhile, the levels of thromboxane B2 (TXB2), 6-keto prostaglandin F1α (6-keto-PGF1α), endothelial nitric oxide synthase (eNOS) and (endothelin-1) ET-1 were detected. The bleeding time (BT) was tested by tail bleeding method, which proved the traumatic bleeding and hemostasis activities of S. heptaphylla. The pharmacology experiments showed that the Set, Sea, Sbu and compound B has significant pro-coagulation effect. In addition, compound B might be the main constituent of pro-coagulation in S. heptaphylla These results could support the fact that S. heptaphylla could be used traditionally to cure traumatic bleeding, and the pro-coagulation effects were associated with the regulation of vascular endothelium active substance and hemorheology parameters.

MicroRNA-423 may regulate diabetic vasculopathy.

To test the hypothesis that microRNAs may play a role in diabetic retinopathy, we measured the levels of different markers [microRNAs, vascular endothelial growth factor (VEGF), nitric oxide (NO), and total antioxidant capacity (TAO)] in patients with type 2 diabetes mellitus (T2DM) and microvascular complications. Sixty-nine patients were recruited: 22 healthy subjects, ten T2DM patients without retinopathy, 22 with nonproliferative diabetic retinopathy, and 15 with proliferative diabetic retinopathy (PDR). Serum levels of NO, VEGF, TAO and 16 candidate microRNAs were measured. Additionally, the mRNA levels of endothelial nitric oxide synthase (eNOS), induced NOS (iNOS), C reactive protein (CRP), VEGF, tumor necrosis factor α (TNFα), PON2, p22, and SOD2 were measured in human vascular endothelial cells cultured in the presence of pooled sera from the subject groups. Plasma miR-423 levels showed a significant ~ twofold decrease in patients with PDR compared to controls. P lasma NO levels were significantly higher in retinopathy, VEGF levels were significantly lower, and TAO was significantly decreased. eNOS mRNA levels were lower in the cells of T2DM patients without retinopathy, but higher in PDR. PON2, p22, and SOD2 mRNA levels were all significantly lower in PDR. CRP, TNFα, iNOS, and VEGF mRNA levels showed no significant association with disease status. Lowered miR-423 levels in diabetic patients showed a correlation with VEGF and an inverse correlation between NO and eNOS expression. Our findings suggest a cross talk between miR-423 and VEGF signaling, affecting eNOS function. miR-423 may be involved in the regulation of diabetic vascular retinal proliferation.

Dysregulation of the endothelial nitric oxide pathway is associated with airway inflammation in COPD.

BACKGROUND: Chronic obstructive pulmonary disease (COPD) is related to endothelial dysfunction and the impaired generation of nitric oxide (NO) from L-arginine by the endothelial NO synthase (eNOS). The relationship between eNOS dysfunctionality and airway inflammation is unknown. We assessed serum asymmetric and symmetric dimethylarginine (ADMA and SDMA) and nitrite/nitrate concentrations, indicators of eNOS function, in patients with COPD and correlated them with markers of inflammation. METHODS: We recruited 15 control smokers, 29 patients with stable and 32 patients with exacerbated COPD requiring hospitalization (20 of them were measured both at admission and discharge). Serum L-arginine, ADMA, SDMA, nitrite and nitrate were measured and correlated with airway inflammatory markers (fractional exhaled nitric oxide concentration - FENO, sputum nitrite and nitrate, sputum cellularity), serum C-reactive protein - CRP, white blood cell count, lung function and blood gases. ANOVA, t-tests and Pearson correlation were used (mean ± SD or geometric mean ± geometric SD for nitrite/nitrate). RESULTS: Serum L-arginine/ADMA, a marker of substrate availability for eNOS, was lower in stable (214 ± 58, p < 0.01) and exacerbated COPD (231 ± 68, p < 0.05) than in controls (287 ± 64). The serum concentration of SDMA, a competitor of L-arginine transport, was elevated during an exacerbation (0.78 ± 0.39 µM) compared to stable patients (0.53 ± 0.14 µM, p < 0.01) and controls (0.45 ± 0.14 µM, p < 0.001). ADMA correlated with blood neutrophil percentage (r = 0.36, p < 0.01), FENO (r = 0.42, p < 0.01) and a tendency for positive association was observed to sputum neutrophil count (r = 0.33, p = 0.07). SDMA correlated with total sputum inflammatory cell count (r = 0.61, p < 0.01) and sputum neutrophil count (r = 0.62, p < 0.01). Markers were not related to lung function, blood gases or CRP. L-arginine/ADMA was unchanged, but serum SDMA level decreased (0.57 ± 0.42 µM, p < 0.05) after systemic steroid treatment of the exacerbation. Serum nitrite level increased in stable and exacerbated disease (4.11 ± 2.12 and 4.03 ± 1.77 vs. control: 1.61 ± 1.84 µM, both p < 0.001). CONCLUSIONS: Our data suggest impaired eNOS function in stable COPD, which is transiently aggravated during an exacerbation and partly reversed by systemic steroid treatment. Serum ADMA and SDMA correlate with airway inflammatory markers implying a possible effect of anti-inflammatory therapy on endothelial dysfunction. Serum nitrite can serve as a compensatory pool for impaired endothelial NO generation.

Impaired L-arginine metabolism marks endothelial dysfunction in CD73-deficient mice.

Changes in the ecto-5'-nucleotidase activity-an extracellular nucleotide catabolic enzyme may lead to the inflammation and endothelial dysfunction. We investigated the effect of CD73 deletion on the endothelial function and L-arginine metabolism in various age groups of mice. 1-,3-,6-, and 12-month-old, male C57BL/6 J wild type (WT) and C57BL/6 J CD73-/- (CD73-/-) mice were used. Blood samples were used for the analysis of adenine nucleotide concentrations. Serum samples were analyzed for the concentration of amino acids, Interleukin 6 (IL-6), Intercellular Adhesion Molecule 1 (ICAM-1), Vascular Cell Adhesion Molecule 1 (VCAM-1), and endothelial nitric oxide synthase (eNOS) level. Serum and aortic nitrate/nitrite, as well as aortic arginase and NOS activity in endothelial cells (EC) were evaluated. CD73 deletion led to age-dependent increase in IL-6, ICAM-1, and VCAM-1 concentration compared to WT. All CD73-/- mice age groups were characterized by reduced L-Arginine concentration and eNOS level. Significantly lower NOS activity was noticed in EC isolated from CD73-/- mice lungs in comparison to EC isolated from WT lungs. The L-Arginine/ADMA ratio in the CD73-/- decreased in age-dependent manner in comparison to WT. The nitrate/nitrite ratio was reduced in serum and in aortas of 6-month-old CD73-/- mice as compared to WT. The ornithine/arginine and ornithine/citrulline ratios were increased in CD73-/- compared to controls. Blood (erythrocyte) Adenosine-5'-triphosphate and Adenosine-5'-diphosphate levels were reduced in favor to higher blood Adenosine-5'-monophosphate concentration in CD73-/- mice in comparison to WT. The CD73 deletion leads to the development of age-dependent endothelial dysfunction in mice, associated with impaired L-arginine metabolism. CD73 activity seems to protect endothelium.

Associations of the NOS3 rs1799983 polymorphism with circulating nitric oxide and lipid levels: a systematic review and meta-analysis.

BACKGROUND: Circulating nitric oxide (NO) and lipid levels are closely associated with coronary artery disease (CAD). It is unclear whether the rs1799983 polymorphism in endothelial nitric oxide synthase (NOS3) gene is associated with plasma levels of NO and lipids. This systematic review and meta-analysis (Preferred Reporting Items for Systematic Reviews and Meta-Analyses) aimed to clarify the relationships between the rs1799983 polymorphism and plasma levels of NO and lipids. METHODS: Sixteen studies (2702 subjects) and 59 studies (14 148 subjects) were identified for the association analyses for NO and lipids, respectively. Mean difference (MD) and 95% CI were used to estimate the effects of the rs1799983 polymorphism on plasma NO and lipid levels. The primary outcome variable was NO, and the secondary outcomes included triglycerides, total cholesterol (TC), low-density lipoprotein cholesterol (LDL-C) and high-density lipoprotein cholesterol (HDL-C). RESULTS: Carriers of the T allele had lower levels of NO (MD -0.27 µmol/L, 95% CI -0.42 to -0.12 µmol/L, p<0.001) and HDL-C (MD -0.07 mmol/L, 95% CI -0.14 to -0.00 mmol/L, p=0.04), and higher levels of TC (MD 0.13 mmol/L, 95% CI 0.06 to 0.20 mmol/L, p<0.001) and LDL-C (MD 0.14 mmol/L, 95% CI 0.05 to 0.22 mmol/L, p=0.002) than the non-carriers. Triglyceride levels were comparable between the genotypes. CONCLUSION: The association between the NOS3 rs1799983 polymorphism and CAD may be partly mediated by abnormal NO and lipid levels caused by the T allele.

Evaluation of Some Cytokines and Gene Expressions in Pre-eclampsia.

BACKGROUND AND OBJECTIVE: Preeclampsia(PE) is adisordercharacterized byhypertensionandproteinuria. There is accumulating evidence that this is a disease of the endothelium. Angiogenic factors may be responsible for the regulation of placental vascular development. Clinicians cannot predict pre-eclampsia prior to the onset symptoms. An ideal bio-marker for pre-eclampsia prediction is during the first trimester. This study investigated the serum levels of tumor necrosis factor-α (TNF-α), C-reactive protein (CRP) and the gene expressions of vascular endothelial growth factor (VEGF), endothelial nitric oxide synthase (eNOS) and p53 in PE trying to find out potential bio-markers for prediction and diagnosis of PE. MATERIAL AND METHODS: A total of 100 female volunteers were involved in this study and their ages were ranged from 25-35 years. They were divided into three groups: Group (1) was 20 healthy non-pregnant women, group (2) was 20 pregnant women normal pregnancies and group (3) was 60 preeclamptic patients. The study participants were enrolled at the Department of Obstetrics and Gynaecology at Mansoura University Hospital, Mansoura, Egypt. The study was approved by the Research Ethics Committee (Faculty of Science, Al Azhar University, Egypt) approved on the March 15, 2014) all women gave written informed consent. Serum levels of CRP, IL-10 and TNF-α were evaluated, in addition to the gene expression of VEGF, eNOS and p53. RESULTS: Significant elevations in the serum levels of blood pressure, TNF-α and CRP were observed in PE patients. Additionally, the gene expression of VEGF, eNOS and P53 were down-regulated in preeclampsia. CONCLUSION: Elevated serum levels of TNFα and CRP, in addition to the down-regulation of eNOS may be used as good predictors for preeclampsia. The TNF-α and VEGF gene were recommended used as markers for PE to be added to routine testes of pregnant women.