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.     

The expression and proangiogenic effect of nucleolin during the recovery of heat-denatured HUVECs

Background

The present study aims to examine the expression patterns and roles of nucleolin during the recovery of heat-denatured human umbilical vein endothelial cells (HUVECs).

Methods

Deep partial thickness burn model in Sprague–Dawley rats and the heat denatured cell model (52 °C, 35 s) were used. The expression of nucleolin was measured using Western blot analysis and real-time PCR. Angiogenesis was assessed using in vitro parameters including endothelial cell proliferation, transwell migration assay, and scratched wound healing. Gene transfection and RNA interference approaches were employed to investigate the roles of nucleolin.

Results

Nucleolin mRNA and protein expression showed a time-dependent increase during the recovery of heat-denatured dermis and HUVECs. Heat-denaturation time-dependently promoted cell growth, adhesion, migration, scratched wound healing and formation of tube-like structures in HUVECs. These effects of heat denaturation on endothelial wound healing and formation of tube-like structures were prevented by knockdown of nucleolin, whereas over-expression of nucleolin increased cell growth, migration, and formation of tube-like structures in cultured HUVEC endothelial cells. In addition, we found that the expression of vascular endothelial growth factor (VEGF) increased during the recovery of heat-denatured dermis and HUVECs, and nucleolin up-regulated VEGF in HUVECs.

Conclusions

The present study reveals that the expression of nucleolin is up-regulated, and plays a pro-angiogenic role during the recovery of heat-denatured dermis and its mechanism is probably dependent on production of VEGF.

General significance

We find a novel and important pro-angiogenic role of nucleolin during the recovery of heat-denatured dermis.     

Prevention of deep vein thrombosis after hip replacement: randomised comparison between unfractionated heparin and low molecular weight heparin.

OBJECTIVE--To evaluate the efficacy and safety of two subcutaneous prophylactic regimens for postoperative deep vein thrombosis after total hip replacement. DESIGN--Prospective open randomised multicentre trial. SETTING--28 European departments of orthopaedic surgery. INTERVENTION--All patients had bilateral phlebography 10 days after surgery. 31 patients receiving low molecular weight heparin and 29 receiving unfractionated heparin were excluded from the efficacy analysis for various reasons. PATIENTS--349 patients undergoing total hip replacement between September 1988 and May 1989. 174 patients received subcutaneously a low molecular weight heparin (Fraxiparine) with anti-factor Xa activity of 41 IU/kg/day for three days, then 62 IU/kg/day from day 4 to day 10. 175 patients received subcutaneous unfractionated heparin at intervals of eight hours; doses were adjusted to maintain the activated thromboplastin time at two to five seconds above control values. MAIN OUTCOME MEASURE--Total incidence of deep vein thrombosis and incidence of proximal deep vein thrombosis on bilateral phlebography. RESULTS--The total incidence of deep vein thrombosis was 16% in patients receiving unfractionated heparin and 12.6% in patients receiving low molecular weight heparin (p = 0.45), and the incidence of thrombosis of the proximal veins was 13.1% and 2.9% respectively (p less than 0.001). Four patients receiving unfractionated heparin and one receiving low molecular weight heparin developed pulmonary embolism. The incidence of bleeding complications was low and comparable in the two groups. CONCLUSION--Low molecular weight heparin is at least as effective as unfractionated heparin in preventing deep vein thrombosis and is more effective at preventing thrombosis of the proximal veins in patients undergoing hip replacement. Low molecular weight heparin is not more likely to cause bleeding complications and is simpler to give than unfractionated heparin.     

Analysis of sulfates on low molecular weight heparin using mass spectrometry: structural characterization of enoxaparin

Introduction: Structural characterization of low molecular weight heparin (LMWH) is critical to meet biosimilarity standards. In this context, the review focuses on structural analysis of labile sulfates attached to the side-groups of LMWH using mass spectrometry. A comprehensive review of this topic will help readers to identify key strategies for tackling the problem related to sulfate loss. At the same time, various mass spectrometry techniques are presented to facilitate compositional analysis of LMWH, mainly enoxaparin.

Areas covered: This review summarizes findings on mass spectrometry application for LMWH, including modulation of sulfates, using enzymology and sample preparation approaches. Furthermore, popular open-source software packages for automated spectral data interpretation are also discussed. Successful use of LC/MS can decipher structural composition for LMWH and help evaluate their sameness or biosimilarity with the innovator molecule. Overall, the literature has been searched using PubMed by typing various search queries such as ‘enoxaparin’, ‘mass spectrometry’, ‘low molecular weight heparin’, ‘structural characterization’, etc.

Expert commentary: This section highlights clinically relevant areas that need improvement to achieve satisfactory commercialization of LMWHs. It also primarily emphasizes the advancements in instrumentation related to mass spectrometry, and discusses building automated software for data interpretation and analysis.     

A longitudinal study of leptin during development in the male rhesus monkey: the effect of body composition and season on circulating leptin levels

The objective of this study was to examine longitudinal changes in serum leptin concentrations during development and to correlate those changes with sexual development in male rhesus monkeys housed under natural environmental conditions. Blood samples were drawn from 8 control animals approximately every other month from 10 to 30 mo of age and thereafter monthly through 80 mo of age. Leptin levels declined through the juvenile period until the onset of puberty and were negatively correlated with body weight. Seven of the eight animals became sexually mature during the breeding season of their fourth year of life. Puberty was delayed in the other animal until the subsequent breeding season. There were no significant fluctuations in leptin levels prior to or in association with the pubertal rise in LH and testosterone (T) secretion. During the peripubertal period, levels of leptin varied between 2 and 3 ng/ml. The animal that exhibited delayed puberty had the lowest body weight and highest leptin levels during this period. With the achievement of sexual maturity, leptin levels varied seasonally, with peak levels in the late winter (Jan-Mar) and a nadir in the late summer (Aug-Sept). A late winter rise in leptin was also evident in most of the animals during Years 2 and 3, but not during Year 4. In the fall of Years 5 and 6, the seasonal rise in leptin concentrations lagged 3-4 mo behind the seasonal increase in LH and T. In the fall of Year 5, but not thereafter, leptin levels were positively related to percent body fat and negatively correlated with lean body mass. The data do not support the hypothesis that increasing leptin concentrations trigger the onset of puberty in the male rhesus monkey. During the juvenile period and after sexual maturation, but not during the peripubertal period, leptin secretion varied with season in the animals; but the environmental factors that cue or drive this rhythm remain to be determined.     

The effect of heparin on motility parameters and protein phosphorylation during bovine sperm capacitation

It is generally accepted that incubation with heparin is required to induce capacitation of ejaculated bovine spermatozoa in vitro. The capacitation process implicates many biochemical events, and is correlated with modified sperm motility and the phosphorylation of specific proteins on tyrosine residues. To better understand the molecular basis of heparin-induced capacitation, bovine spermatozoa were incorporated with a radioactive substrate of protein kinases [gamma32P]-ATP, to observe protein phosphorylation dynamics over time. Sperm motion parameters including the percentage of motile spermatozoa, amplitude of lateral head displacement (ALH) and flagellar beat cross frequency (BCF) were assessed to determine whether the protein phosphorylation patterns induced by heparin also promote changes in motility. Capacitation was confirmed using the chlortetracycline fluorescence assay and the appearance of 'pattern B' stained spermatozoa. Evaluation of the different motility parameters during capacitation reveal that heparin has a marked negative effect, over time, on the percentage of motile spermatozoa, consistent with hyperactivation. Indeed, the presence of heparin greatly increases the BCF of bull spermatozoa and induces a significant increase in the ALH compared to spermatozoa incubated without heparin. We detected several sperm proteins that are phosphorylated over time. A 45 kDa protein is the most intensely phosphorylated of the sperm proteins. However, it is visible regardless of the presence of heparin. Interestingly, a second phosphorylated protein of approximately 50 kDa undergoes more intense phosphorylation with heparin than without. In summary, the present study demonstrated that heparin induces physiological changes in several sperm motility parameters including ALH, BCF and the percentage of motile spermatozoa. Heparin also increases the intensity of phosphorylation of a 50 kDa sperm protein. These results suggest that capacitation of bovine spermatozoa and capacitation-associated motility changes may be regulated by a mechanism that includes protein phosphorylation, and that a presently unknown protein kinase is involved.     

Effect of air ionization on heart rate and perceived exertion during a bicycle exercise test. A double-blind cross-over study.

The influence of ionization of air on heart rate (HR) and ratings of perceived exertion (RPE) during bicycle exercise was studied in nine healthy medical students selected according to a randomized schedule from the class of 90 students. The exercise tests were performed both under negative and positive ionization. The study was made with a double-blind, cross-over design. The body surface exposed to ionic current was made large by reducing the clothing of the subject. A significant overall tendency to lower HR and RPE values under negative ionization was observed (p less than 0.01, sign tests). The RPE values were significantly lower (p less than 0.01, paired t-test and the Wilcoxon test) under negative than under positive ionization at the maximal work load level but not at other relative load levels. However, when separately tested at each relative load level HR values did not differ significantly in negative and positive ionization. The results of this pilot study indicate that ionic composition of the air can modify the RPE and possibly also HR during exercise; negative air ionization seems to be beneficial compared with positive ionization. The mechanisms involved are obscure, but we suggest that negative ionization of air may increase oxidative metabolism through generation of a superoxide radical (O2-) that is reduced to H2O2 by superoxide dismutases.     

Promigratory and proangiogenic effects of AdipoRon on bone marrow-derived mesenchymal stem cells: an in vitro study

Objectives

To investigate the effect of AdipoRon on major factors involved in survival, migration and neovascularization of rat bone marrow-derived mesenchymal stem cells.

Results

AdipoRon promoted the MSCs viability. Real-time PCR indicated that the expression of cyclooxygenase-2 (COX-2), hypoxia-inducible factor-1 (HIF-1) C-X-C chemokine receptor type 4 (CXCR4), C–C chemokine receptor type 2 (CCR2), vascular endothelial growth factor matrix metalloproteinase-2 (MMP-2) and MMP-9 were upregulated in AdipoRon-treated MSCs compared to control groups. Prostaglandin E2 (PGE2) level, as well as migration ability of MSCs (scratch assay) was enhanced by AdipoRon preconditioning.

Conclusion

Preconditioning of MSCs with AdipoRon prior to transplantation could enhance cell survival, angiogenesis and migration via activating the COX-2/PGE2/HIF-1 pathway and other contributing factors.

     

Quantitative study of various factors influencing the migration of lymphocytes in vitro: glucocortico-steroid, PHA, cyclosporin A and heparin

Various agents known to influence the lymphocyte recirculation in vivo were investigated for their effect on the migration of lymphocytes in 3-D collagen gel. Both glucocortico-steroid and cyclosporin A were shown to inhibit the migration in a dose dependent manner. In contrast heparin and PHA treated lymphocytes migrated at a significantly faster rate.     

Moderate antiproteinuric effect of add-on aldosterone blockade with eplerenone in non-diabetic chronic kidney disease. A randomized cross-over study

Background

Reduction of proteinuria and blood pressure (BP) with blockers of the renin-angiotensin system (RAS) impairs the progression of chronic kidney disease (CKD). The aldosterone antagonist spironolactone has an antiproteinuric effect, but its use is limited by side effects. The present study evaluated the short-term antiproteinuric effect and safety of the selective aldosterone antagonist eplerenone in non-diabetic CKD.

Study Design

Open randomized cross-over trial.

Setting and Participants

Forty patients with non-diabetic CKD and urinary albumin excretion greater than 300 mg/24 hours.

Intervention

Eight weeks of once-daily administration of add-on 25–50 mg eplerenone to stable standard antihypertensive treatment including RAS-blockade.

Outcomes & Measurements

24 hour urinary albumin excretion, BP, p-potassium, and creatinine clearance.

Results

The mean urinary albumin excretion was 22% [CI: 14,28], P<0.001, lower during treatment with eplerenone. Mean systolic BP was 4 mmHg [CI: 2,6], P = 0.002, diastolic BP was 2 mmHg [CI: 0,4], P = 0.02, creatinine clearance was 5% [CI: 2,8], P = 0.005, lower during eplerenone treatment. After correction for BP and creatinine clearance differences between the study periods, the mean urinary albumin excretion was 14% [CI: 4,24], P = 0.008 lower during treatment. Mean p-potassium was 0.1 mEq/L [CI: 0.1,0.2] higher during eplerenone treatment, P<0.001. Eplerenone was thus well tolerated and no patients were withdrawn due to hyperkalaemia.

Limitations

Open label, no wash-out period and a moderate sample size.

Conclusions

In non-diabetic CKD patients, the addition of eplerenone to standard antihypertensive treatment including RAS-blockade caused a moderate BP independent fall in albuminuria, a minor fall in creatinine clearance and a 0.1 mEq/L increase in p-potassium.

Trial Registration

Clinicaltrials.gov {"type":"clinical-trial","attrs":{"text":"NCT00430924","term_id":"NCT00430924"}}NCT00430924     

Oviduct fluid and heparin induce similar surface changes in bovine sperm during capacitation: A flow cytometric study using lectins

Eight different lectins conjugated to fluorescein isothiocyanate (FITC) were used to screen for sperm plasma membrane changes during in vitro capacitation of bovine sperm. Analysis of lectin binding to sperm was done using flow cytometry. Of the eight lectins, only Triticum vulgaris (wheat germ agglutinin, WGA) binding to sperm was altered with capacitation. Capacitation of bovine sperm by heparin was found to decrease WGA binding to sperm by 78% (P < 0.05). The effect of capacitation by oviduct fluid was next compared with capacitation by heparin for changes in WGA binding to sperm. The effect of inhibiting capacitation with glucose on WGA binding was also determined. WGA-bound sperm were detected by flow cytometry as being present in two fluorescence peaks defined as low fluorescence (A) or high fluorescence (B) intensity. The percentage of sperm in peak A was greater for heparin and oviduct fluid-treated sperm compared to sperm incubated under noncapacitating conditions in only culture medium (P < 0.001). Capacitation with either heparin or oviduct fluid was inhibited by glucose as assessed by the ability of lysophosphatidylcholine (100 μg/ml) to induce acrosome reactions. Glucose also reduced the percentage of sperm in peak A for both heparin- and oviduct fluid-treated sperm (P < 0.01). We conclude that heparin or oviduct fluid induced changes on the sperm plasma membrane during capacitation. Binding sites for WGA on sperm were either structurally altered or lost during capacitation. © 1996 Wiley-Liss, Inc.     

The effect of isolated valgus moments on ACL strain during single-leg landing: A simulation study

Valgus moments on the knee joint during single-leg landing have been suggested as a risk factor for anterior cruciate ligament (ACL) injury. The purpose of this study was to test the influence of isolated valgus moment on ACL strain during single-leg landing. Physiologic levels of valgus moments from an in vivo study of single-leg landing were applied to a three-dimensional dynamic knee model, previously developed and tested for ACL strain measurement during simulated landing. The ACL strain, knee valgus angle, tibial rotation, and medial collateral ligament (MCL) strain were calculated and analyzed. The study shows that the peak ACL strain increased nonlinearly with increasing peak valgus moment. Subjects with naturally high valgus moments showed greater sensitivity for increased ACL strain with increased valgus moment, but ACL strain plateaus below reported ACL failure levels when the applied isolated valgus moment rises above the maximum values observed during normal cutting activities. In addition, the tibia was observed to rotate externally as the peak valgus moment increased due to bony and soft-tissue constraints. In conclusion, knee valgus moment increases peak ACL strain during single-leg landing. However, valgus moment alone may not be sufficient to induce an isolated ACL tear without concomitant damage to the MCL, because coupled tibial external rotation and increasing strain in the MCL prevent proportional increases in ACL strain at higher levels of valgus moment. Training that reduces the external valgus moment, however, can reduce the ACL strain and thus may help athletes reduce their overall ACL injury risk.     

Differentiation characteristics of circulating and bone marrow hematopoietic stem cells and the effect of thymus factors

Circulating hemopoietic stem cells (HSC) considerably differ from bone marrow HSC in active erythroid differentiation. After thymectomy of adult animals the number and differentiation of blood HSC remain unchanged, whereas during the cloning of bone marrow cells, a decrease in the number of granulocytic colonies is revealed. In in-vitro experiments, thymalin does not influence the number or differentiation of circulating HSC. On the contrary, in experiments made in vivo, it dramatically lowers erythroid specialization of blood HSC in thymectomized and sham-operated mice, which is followed by the diminution of the total number of circulating HSC. Differentiation of thymectomized mice bone marrow stem cells is completely normalized after thymalin injection. Sham-operated and thymectomized animals' HSC stimulated by thymalin injection become similar to bone marrow cells of normal mice as regards the trend of differentiation. Thymalin injection is likely to change the bone marrow HSC differentiation profile, thereby preventing the release of the cells with erythroid-oriented differentiation from the bone marrow to blood. The influence of thymalin on HSC is mediated by the environmental component which is present in the bone marrow and absent from the peripheral blood.     

Assay of circulating hyaluronic acid in the rat: study of diurnal variation and effect of anesthesia

Serum hyaluronic acid (HA) may provide a good marker for the severity of joint disease in the rat since a positive correlation was observed in experimental models of arthritis. However, little is known about its physiological variation in rats. In the present work, we do not find any circadian rhythm of HA in healthy Sprague-Dawley rats in contrast to that observed in humans, whose serum levels vary during daytime. Furthermore, the influence of blood sampling conditions on HA concentrations was evaluated in conscious animals and by using different anesthetics. The greater reproducibility for the assay of HA is observed with the intracardiac puncture under ether inhalation. Blood sample collection in the absence of anesthesia leads to a significant increase in serum levels of HA, which could be attributed partly to enhanced joint movements generated by psychological stress.     

Comparison of efficacy and safety of low molecular weight heparins and unfractionated heparin in initial treatment of deep venous thrombosis: a meta-analysis.

OBJECTIVE--To compare the efficacy and safety of low molecular weight heparins and unfractionated heparin in the initial treatment of deep venous thrombosis for the reduction of recurrent thromboembolic events, death, extension of thrombus, and haemorrhages. DESIGN--Meta-analysis of results from 16 randomised controlled clinical studies. SUBJECTS--2045 patients with established deep venous thrombosis. INTERVENTION--Treatment with low molecular weight heparins or unfractionated heparin. MAIN OUTCOME MEASURES--Incidences of thromboembolic events (deep venous thrombosis or pulmonary embolism, or both); major haemorrhages; total mortality; and extension of thrombus. RESULTS--A significant reduction in the incidence of thrombus extension (common odds ratio 0.51, 95% confidence interval 0.32 to 0.83; P = 0.006) in favour of low molecular weight heparin was observed. Non-significant trends also in favour of the low molecular weight heparins were observed for the recurrence of thromboembolic events (0.66, 0.41 to 1.07; P = 0.09), major haemorrhages (0.65, 0.36 to 1.16; P = 0.15), and total mortality (0.72, 0.46 to 1.4; P = 0.16). CONCLUSIONS--Low molecular weight heparins seem to have a higher benefit to risk ratio than unfractionated heparin in the treatment of venous thrombosis. These results, however, remain to be confirmed by using clinical outcomes in suitably powered clinical trials.     

Hesperidin displays relevant role in the nutrigenomic effect of orange juice on blood leukocytes in human volunteers: a randomized controlled cross-over study

Background

We previously showed, in healthy, middle-aged, moderately overweight men, that orange juice decreases diastolic blood pressure and significantly improves postprandial microvascular endothelial reactivity and that hesperidin could be causally linked to the observed beneficial effect of orange juice. The objective was to determine the effect of chronic consumption of orange juice on the gene expression profile of leukocytes in healthy volunteers and to assess to what extent hesperidin is involved in the effect of orange juice.

Methodology/Principal Findings

Volunteers were included in a randomized, controlled, crossover study. Throughout three 4-week periods, volunteers consumed daily: 500 ml orange juice, 500 ml control drink plus hesperidin or 500 ml control drink and placebo. Blood samplings were performed on 10 overnight-fasted subjects after the 4-week treatment period. Global gene expression profiles were determined using human whole genome cDNA microarrays. Both orange juice and hesperidin consumption significantly affected leukocyte gene expression. Orange juice consumption induced changes in expression of, 3,422 genes, while hesperidin intake modulated the expression of 1,819 genes. Between the orange juice and hesperidin consumption groups, 1,582 regulated genes were in common. Many of these genes are implicated in chemotaxis, adhesion, infiltration and lipid transport, which is suggestive of lower recruitment and infiltration of circulating cells to vascular wall and lower lipid accumulation.

Conclusions

This study shows that regular consumption of orange juice for 4 weeks alters leukocyte gene expression to an anti-inflammatory and anti-atherogenic profile, and hesperidin displays a relevant role in the genomic effect of this beverage.

Trial Registration

ClinicalTrials.gov {"type":"clinical-trial","attrs":{"text":"NCT 00983086","term_id":"NCT00983086"}}NCT 00983086     

The cortical effect of chewing gum during hand movements: A functional MRI study

Abstract

Nine right-handed normal subjects were recruited for this study. We compared the cortical activation during execution of hand movements (right finger flexion–extension) with that during execution of hand movements while chewing gum (right side chewing). We found that execution of hand movements while chewing gum induced less activation in the contralateral SM1 than hand movements alone. Based on our findings, it appears chewing gum during execution of hand movements enhanced the efficiency of hand movements.