Protective effect of defibrotide on perfusion induced endothelial damage.

In the present study, in vitro effects of Defibrotide (D) on perfusion-induced changes in the morphology of endothelium were investigated by scanning (SEM) and transmission (TEM) electron microscope. Human umbilical cord veins were incubated or perfused with platelet-rich plasma alone (PRP) or platelet-rich plasma with Defibrotide (PRP+D) at 3ml/min or 14ml/min and the changes observed were compared. SEM examination of luminal surfaces demonstrated that perfusion with high flow rates may damage endothelial cells and lead to morphological changes which may be prevented by the presence of Defibrotide in the perfusate. Also, the marked reduction in the number of adhered platelets on luminal surface of veins incubated or perfused with Defibrotide compared to veins treated with platelet-rich plasma only revealed that Defibrotide has anti-thrombotic effects. TEM examination of ruthenium red (RR) stained thin sections of veins demonstrated that perfusion disrupts the glycosaminoglcan (GAG) coat on endothelial cells. But the presence of D in the perfusate preserves the integrity of GAG, indicating further cytoprotective effects of the drug on endothelium.

Long-term effect of 17beta-estradiol and thrombin on tissue factor pathway inhibitor release from HUVEC.

In spite of the increasing evidence that estrogens have protective effects on the vascular system, the evidence that estrogens may contribute to the risk of thrombosis is still being debated. We investigated the effect of 17beta-estradiol (E2) on tissue factor pathway inhibitor (TFPI) release from of cultured human umbilical vein endothelial cells (HUVEC). In this study HUVEC were harvested by collegenase treatment and cultured in multiwelled plates with medium 199 supplemented with 10% fetal calf serum and antibiotics. The cells were incubated in the presence or absence of E2 (1 and 100 nM) with/without thrombin (4 U/mL) for 6 or 24 hours. After the incubations TFPI level of media were measured by IMUBIND Total Eliza kit. Our results demonstrates that E2 at physiological concentrations decreases the release of TFPI from HUVEC significantly. Thrombin also decreases TFPI antigen levels detected in culture media. When combined with thrombin the effect of estrogen is not visible due the much higher effectivity of thrombin in diminishing TFPI levels. These results show that E2 shifts the hemostatic balance towards the procoagulant phase through lowering the TFPI levels secreted by the endothelium.

Effects of single-shot and twin-shot shockwaves on urinary enzyme concentrations.

BACKGROUND AND OBJECTIVE: Extracorporeal shockwave lithotripsy (SWL) remains the first-line treatment of urinary calculi. However, a number of studies have shown that adverse effects on the kidneys and the surrounding tissues may be encountered in short- and long-term follow-up. The aim of this study was to compare the effects of single-shot and twin-shot SWL techniques to identify the safest modality in terms of urinary enzyme excretion. PATIENTS AND METHODS: In this prospective, investigator-blinded, randomized study, urinary enzymes, beta2-microglobulin, microalbumin, Na, K, Ca, and creatinine concentrations were analyzed in 59 consecutive patients. Measurements were performed in urine specimens collected immediately before and after the SWL procedure and also on the 3rd and 7th days after treatment, which was performed on a Dornier MFL-5000 lithotripter utilizing the twin-shot technique (Group 1; N = 30) or the single-shot technique (Group 2; N = 29) with 3000 shockwaves at 18 kV per treatment. RESULTS: Although there was no statistically significant difference in the results between the groups, urinary levels of microalbumin, alanine and aspartate aminotransferases, beta-2-microalbumin, gamma-glutamyltranspeptidase, Na, K, and Ca rose acutely after SWL, reaching maximum levels on the 3rd day, and returned to the baseline by the 7th day following the treatment in both groups. CONCLUSION: This study demonstrates that SWL performed by either a single-shot or twin-shot shockwave technique has a transient detrimental effect on renal function, as assessed by urine enzyme concentrations. It is recommended that the twin-shot shockwave technique be used in routine lithotripsy in consideration of the cost-effectiveness provided by the shorter treatment time.

The effect of losartan and captopril on glomerular basement membrane anionic charge in a diabetic rat model.

OBJECTIVE: Although angiotensin-converting enzyme inhibitors are known to reduce albuminuria by preserving glomerular basement membrane anionic content, the effects of angiotensin II receptor blockage are currently not known. The aim of this study was to evaluate the effects of captopril and losartan on glomerular basement membrane anionic charges in a diabetic rat model. DESIGN: After diabetes induction with streptozotocin, female Wistar rats were divided into three groups: group A, losartan 10 mg/kg by gavage (n = 8); group B, captopril 50 mg/l in drinking water (n = 6); group C, diabetic control rats (n = 8) given only tap water. Group D (eight rats) served as non-diabetic controls. At the end of 8 weeks, erythrocyte membrane charge, serum sialic acid, urinary glycosaminoglycan and albumin were measured and kidney specimens stained with Alcian blue in order to assess basement membrane glycosaminoglycans. RESULTS: Red blood cell anionic charges (ng Alcian blue/ 10(6) red blood cells) were 371.5+/-9.9 for group A, 443.5+/-7.1 for group B, 400.1+/-14.7 for group C, 468.7+/-4 for group D (AD, P<0.01; A>B P<0.01). Albuminuria (microg/day) was 778+/-221 for group A, 719+/-314 for group B, 1724+/-945 for group C, 393+/-263 for group D (A, B

The role of free prostate-specific antigen in the diagnosis of prostate cancer.

OBJECTIVE: To determine whether the free/total prostate-specific antigen (PSA) ratio can discriminate between patients with prostate cancer or benign prostatic hyperplasia (BPH). PATIENTS AND METHODS: A prospective study was conducted using free and total PSA assays in patients who underwent transrectal-ultrasound guided biopsies indicated by a total serum PSA level of > 4 ng/mL and/or a positive digital rectal examination. Sixty-nine men (median age 68 years, range 57-86) who presented to our out-patient department with symptoms of prostatism were included in the study. Blood samples were drawn from all patients before biopsy. RESULTS: Histopathological examination detected prostate cancer in 17 of 69 (25%) patients and 13 of these 17 patients had a free/total PSA ratio of < 0.15; only 12 of 52 (23%) patients with BPH had a ratio of < 0.15. Receiver operating characteristic analysis indicated a threshold free/total PSA ratio of < or = 0.15 was the optimum discriminatory level. In the whole study group, this threshold had sensitivity, specificity, positive- and negative-predictive values of 76%, 77%, 52% and 91%, respectively. There were 40 patients with serum PSA levels of 4-10 ng/mL and 17.5% (7/40) of these were diagnosed with cancer. Using a free/total PSA ratio of 0.15 would have failed to diagnose two patients of seven with prostate cancer but 30 patients would have avoided a biopsy. In this subgroup, the threshold ratio of 0.15 had sensitivity, specificity, positive- and negative-predictive values of 71%, 85%, 50% and 93%, respectively. The rates for a PSA density (PSAD) at a threshold of > or = 0.15 were 71%, 76%, 38%, 93%, respectively. CONCLUSION: These results indicate that using the free/total PSA ratio gives a significant improvement over PSAD and total PSA values alone in the diagnosis of prostate cancer: its use may also enhance the diagnostic accuracy in patients with intermediate PSA levels.

Defibrotide augments the anticandidial activity of NK cells.

1. The mechanism of action of Defibrotide, a fibrinolytic agent on Natural Killer (NK) cell cytotoxicity, was investigated through verapamil, TMB-8 cells and pertussis toxin. 2. Defibrotide increased the activity against Candida albicans cells (anticandidial activity), and it is determined that the calcium channels have a role in this effect. 3. Blockage of calcium channels reduced the anticandidial effect by 39.2%. Pertussis toxin led to a 10.7% inhibition, whereas the application of TMB-8 resulted in the stimulation of anticandidial activity. 4. It is concluded that defibrotide is a potent activator of NK cells.

17 beta-Estradiol increases intracellular free calcium concentrations of human vascular endothelial cells and modulates its responses to acetylcholine.

In this study, we have investigated the effect of 17 beta-estradiol (E2) on intracellular free calcium concentrations ([Ca2+]i) in human umbilical vein endothelial cells (HUVEC) using fura-2 fluorescence. E2 at concentrations of 1nM -1 microM was added subsequently to HUVEC cultures which were either deprived of estrogens or preincubated with E2 (100 nM) for 24 hours. In both groups of cultures, E2 stimulated significant increases in [Ca2+]i in a dose-dependent manner. The effects were more prominent in E2-deprived cells. Preincubation of cells with tamoxifen or the presence of it in the buffer during the experiments did not inhibit the response of the cells to E2. Experiments performed in Ca2+ free/EGTA buffer yielded transient increases in [Ca2+]i suggesting release of Ca2+ from intracellular stores was responsible for the initial peak, while sustained elevations were supported by Ca2+ influx from the extracellular space. Addition of La3+ abolished the sustained [Ca2+]i elevations. Carbachol (CCh) (1nM, 100 nM) did not induce changes in [Ca2+]i of estrogen-deprived cells but produced significant increases in [Ca2+]i of the same cells after incubation with E2 for 30 minutes. The cultures which were preincubated with E2 for 24 hours responded to carbachol directly. The results of our study indicate that E2 may modulate the functions of endothelial cells after only a brief exposure and also may be necessary for the response to acetylcholine especially at low concentrations.

Involvement of free radicals in the cardioprotective effect of defibrotide.

Ischemia followed by reperfusion has deleterious effects on myocardial tissue and a wide range of drugs have been investigated to modulate these changes. Defibrotide (polydeoxyribonucleotides from bovine lung), a drug with antithrombotic and fibrinolytic activities, has also proven to be cardioprotective against myocardial ischemia/reperfusion damage. However, the mechanism of this protective effect has not been clarified yet. The aim of this study was to determine whether this effect is due to protection against free radical induced changes. The experimental model in rabbits includes coronary artery ligation for 60 min followed by a reperfusion period of 45 min. In this model, free radical damage was estimated by different parameters of lipid peroxidation such as diene conjugation, carbonyl content, and thiobarbituric acid reactive substances, together with protein oxidation determinations. The results demonstrate that defibrotide prevents free radical induced changes after myocardial ischemia/reperfusion.

Urinary leucine aminopeptidase is a more sensitive indicator of early renal damage in non-insulin-dependent diabetics than microalbuminuria.

We measured urinary activity of leucine aminopeptidase (EC 3.4.11.2) and creatinine concentrations (Cr, in mmol) in samples of second morning urine from 25 healthy subjects and 59 non-insulin-dependent diabetic (NIDD) subjects. If NIDD subjects are grouped according to their Alb/Cr ratio into normoalbuminuria (group A, Alb/Cr < 2.8 mg/mmol), microalbuminuria (group B, Alb/Cr 2.8-26.8 mg/mmol), and macroalbuminuria (group C, Alb/Cr > 26.8 mg/mmol), LAP/Cr ratios in all three groups exceeded those for healthy age-matched controls. Moreover, this ratio was higher in group B than in group A. The value for LAP/Cr was clearly abnormal (i.e., exceeded the upper limit of normal, log normal + 2 SD, found in healthy subjects) in 44% of group A. In the first 10-year period Of NIDD, prevalance of abnormal LAP/Cr ratio was 61.3%, whereas that of microalbuminuria was 35.5%. We have also found a LAP/Cr ratio abnormality of 91% in group B. Evidently, LAP/Cr may be increased early in NIDD subjects and be a more sensitive predictor of incipient nephropathy than microalbuminuria.

Protective effects of cilazapril against free radical injury in myocardial ischaemia-reperfusion.

Cilazapril is a prodrug which is rapidly hydrolysed to the pharmacologically active cilazaprilat following absorption to the bloodstream. In clinical pharmacological studies, administration of cilazapril resulted in potent, reversible, selective and competitive angiotensin converting enzyme inhibition. In this study, we have examined the protective effect of cilazapril on a myocardial ischaemia-reperfusion model by using different parameters of lipid peroxidation and protein oxidation. We have observed increased levels of diene conjugates, carbonyls and malondialdehyde as well as protein carbonyls after ischaemia-reperfusion, whereas protein sulphydryl groups were decreased. Our results clearly demonstrate that cilazapril, a non-sulphydryl, long-acting angiotensin converting enzyme inhibitor, has free-radical-scavenging potential in a model comparable to the clinical situation observed in humans.

Apolipoprotein E Genotyping in Turkish Myocardial Infarction Survivors and Healthy Controls.

The apolipoprotein (Apo) E gene is known to be polymorphic. Three common alleles determine six phenotypes which can easily be detected by restriction fragment length polymorphism. We performed apo E genotyping in myocardial infarction survivors and healthy controls for the first time in the Turkish population. DNA was amplified by polymerase chain reaction (PCR) and the PCR product was digested with restriction enzymes HhaI to detect apo E2, E3, E4 and with TaqI to detect apo E1. Relative allele frequency for the patient group was found to be 0.91 for E3, 0.07 for E2, 0.02 for E4 and for the control group 0.875 for E3, 0.067 for E2, 0.058 for E4. Copyright 1995 S. Karger AG, Basel

Investigation of the fibrinolytic activity of defibrotide fractions.

1. Defibrotide is a drug which is a mixture of DNA fragments of various lengths. To characterize the active component which is responsible for the fibrinolytic effect, we fractionated defibrotide by column chromatography and investigated direct and endothelium dependent fibrinolytic activity of the major fractions by euglobulin lysis time (ELT) measurements. 2. Defibrotide and its fractions added to human plasma samples did not change ELT. However addition of conditioned media of human umbilical vein endothelial cell cultures which were incubated with defibrotide or its high molecular weight fractions shortened the ELT significantly (P < 0.001), whereas small molecular weight fractions did not effect the ELT. 3. Our results show that high molecular weight defibrotide fractions have fibrinolytic activity and the effect is endothelium dependent.

Morphological changes in carotid arteries of rabbits induced by defibrotide infusion.

Defibrotide is an antithrombotic and profibrinolytic drug which modulates endothelial function. The drug increases prostacyclin and tissue plasminogen activator while it decreases plasminogen activator inhibitor synthesis by endothelial cells. In this study, in vivo effects of defibrotide on the morphology of endothelial cells and vessel wall of the healthy rabbits were investigated by light and electron microscopy. The examination of the carotid arteries of healthy rabbits after infusion of saline or defibrotide (10 mg/kg/hr) in saline solution for three hours revealed that the drug had induced dramatic morphological changes in all the test animals while no change was observed in control group. The changes observed after defibrotide administration, such as the decrease in hill and valley-like appearance of endothelial surface, and thinning of the intimal layer provides evidence for the vasorelaxant effect of the drug, while the decrease in the number of blood cells adhering to the endothelial surface confirms the antithrombotic effect of defibrotide. Finally the decrease in the number of crater-like structures may be due to the cytoprotective effect of the drug.

Short-term bioeffects of extracorporeal shockwave lithotripsy.

Safety guidelines for shockwave delivery during extracorporeal shockwave lithotripsy (SWL) are not yet clear. Renal functions were assessed by using urinary N-acetyl-beta-D-glucosaminidase (NAG), lactate dehydrogenase (LDH), alanine aminotransferase (ALT; EC.2.6.1.2), aspartate aminotransferase (AST; EC. 2.6.1.1), and gamma-glutamyltransferase (GGT) as well as sodium, potassium, and calcium concentrations in respect to tubular functions after SWL with the Dornier MFL 5000 unit in 32 patients. In order to monitor glomerular function, we determined microalbuminuria. Transient glomerular and tubular damage occurs in SWL-treated kidneys. The minimum interval between two shockwave treatments should be at least 7 days.

Subcellular localization of radioactively labelled defibrotide in cultured endothelial cells.

Defibrotide is a new antithrombotic and fibrinolytic drug which is obtained by controlled depolymerization of mammalian DNA. In various models of arterial and venous thrombosis, it has been shown that it induces tissue plasminogen activator [tPA] and prostacyclin [PGI2] release from the vessel wall. We have previously shown the presence of specific binding sites with a Kd of 4.2 micrograms/ml for radioactively labelled defibrotide. The present study was undertaken to identify the location of the binding site. Confluent cultures of endothelial cells from human umbilical vein were incubated with media containing 3H-acetyl-defibrotide for various intervals of time. Cells were then washed and harvested nonenzymatically. Subcellular location of 3H-defibrotide was investigated by fractionating cells on discontinuous sucrose gradient and measuring the distribution of radioactivity. 5'-nucleotidase enzyme activity was also measured to ensure the location of membrane fraction. Our results suggest that the major location of 3H-defibrotide in endothelial cells is the plasma membrane. On the other hand, nuclei also contain a considerable amount of the drug which suggests a mechanism where binding to a membrane protein is followed by internalization.

Cumene hydroperoxide-induced chemiluminescence in human erythrocytes: effect of antioxidants and sulfhydryl compounds.

1. The time-course of cumene hydroperoxide-induced changes in lipid peroxidation, protein sulfhydryl groups and chemiluminescence intensity was determined in human erythrocytes. 2. Increase in lipid peroxidation was maximal within 60 min of incubation and was paralleled by a decrease in protein sulfhydryl groups and an increase in chemiluminescence formation. 3. A standard assay system was established to investigate the protective effects of antioxidants and scavenger compounds on cumene hydroperoxide-induced chemiluminescence formation. 4. Chain-breaking antioxidants (i.e. butylated hydroxytoluene) and sulfhydryl compounds (i.e. dithiothreitol) were able to suppress chemiluminescence formation. 5. Our results suggested that secondary free radicals, as well as sulfhydryl groups of proteins are involved in cumene hydroperoxide-induced chemiluminescence formation.

Effect of chronic halothane exposure on lipid peroxidation, osmotic fragility and morphology of rat erythrocytes.

Effect of chronic halothane exposure on hepatic and erythrocyte lipid peroxidation and erythrocyte osmotic fragility and morphology were determined in rats exposed to 0.4% halothane, 8 h per day for 40 days. Hepatic lipid peroxidation was increased in the halothane-treated group compared to controls. Lipid peroxidation was not increased by halothane exposure in erythrocytes without hydrogen peroxide, but after peroxide supplementation lipid peroxidation increased more in the erythrocytes of halothane-exposed rats than in control rats. We have observed significant morphological changes in erythrocytes from halothane-treated rats. In addition, erythrocytes of halothane-treated rats were more fragile in saline solutions compared to those of controls. Our results suggest that chronic halothane exposure is not only hepatotoxic but also affects erythrocyte membrane structure and stability.