Dariusz Sitkiewicz

Characterization of a novel, high-molecular weight, acidic, endothelin-1 inactivating metalloendopeptidase from the rat kidney.

Objective To characterize endothelin-1 inactivating peptidase (ET-1 peptidase) recently isolated from rat kidney. Methods ET-1 peptidase was purified from the membranes of whole Wistar-Kyoto (WKY) rat kidneys using differential centrifugation, detergent solubilization, ion-exchange chromatography, ultrafiltration and preparative electrophoresis. The enzyme activity in the presence of increasing concentrations of unlabelled peptides, inhibitors and other substances was determined at pH 5.5 and 37°C using fixed amounts of [125I]-ET-1 as the substrate. Results On non-denaturing gels, the purified enzyme migrated in the form of a compact, low-mobility (Rf 0.07), high relative molecular mass (approximately 250000) protein band. During denaturing polyacrylamide gel electrophoresis this protein separated into three fractions with apparent relative molecular masses 158 000,110 000 and 61 000. Using different buffers, the optimum pH for this enzyme was found to be 5.5. Zinc (3.7mmol/l), nickel (4.0 mmol/l), citrate (0.6mmol/l), phosphate (1.3 mmol/l) and barbital ions (2.5 mmol/l) inhibited ET-1 peptidase activity by 50%, whereas magnesium, calcium, cobalt, manganous, sodium and borate ions were without effect. The most powerful inhibitors of the enzyme included: phenanthroline [median inhibitory concentration (IC50) 28 μmol/l], phosphoramidon (IC50 8.0 μmol/l), thiorphan (IC50 32 μmol/l) and N-carboxymethyl-Phe-Leu (IC50 12 μmol/l). Also, bacitracin (25 μmol/l), cyclosporine A (20 μmol/l) and sodium dodecyl sulphate (0.5%) inhibited enzyme activity by 50%, whereas bestatin, puromycin, aprotinin, phenylmethylsulphonyl fluoride, amanitin (50–100 μmol/1) and cardiotoxin (25 μg/assay) had no effect. The Michaelis constant (Km) values of 70 and 66 nmol/l were found towards ET-1 and the ET(16–21) fragment, respectively, whereas the Km values in respect to big-ET-1, sarafotoxin S6b, sulphated cholecystokin octapeptide, gastrin, glucagon, insulin, gastric inhibitory peptide and growth hormone ranged from 1.5 to approximately 50fimol/l. The enzyme showed no apparent affinity for enkephalins, bradykinin, angiotensins, cholecystokinin tetrapeptides and kyotorphin. Conclusions The present data suggest that the ET-1 peptidase that we isolated from rat kidney displays inhibitory characteristics similar to that of other known metalloendopeptidases. However, this enzyme exhibits several unique properties such as high molecular mass, an apparent complex subunits structure, pH optimum at 5.5, and very high substrate specificity towards ET-1 and the ET(16–21) fragment compared with other peptides either related or unrelated to endothelin.

Purification of endothelin-1-inactivating peptidase from the rat kidney

Objective To identify and purify endothelin-1-inactivating peptidase from rat tissues Methods Subcellular fractions of rat kidney, aorta, heart, lung, liver and blood cells were prepared by differential centrifugation. Kidney membrane-bound peptidase was solubilized with Triton X-100, chromatographed on the diethyl-aminoethyl-ceilulose, ultrafiltered through a membrane of relative molecular mass 100 000 cutoff and subjected to electrophoresis on a non-denaturing polyacrylamide gel. The enzyme activity assay was performed at pH 5.5 using [125I]-endothelin-1 as the substrate. The trichloroacetic acid precipitation test, an endothelin-1 immunoreactivity assay, reverse-phase high-performance liquid chromatography and a receptor-binding assay were applied for the detection of degradation products Results High-activity endothelin-1-degrading peptidase coincided with the fraction from the kidney membranes of both Wistar-Kyoto and spontaneously hypertensive rats, but not with any other of the tissues that were studied. The membrane (0.5 μg protein/assay) degraded [125I]-endothelin-1 (5–100pmol/l) within a half-time of about 10min at 37°C. The enzyme was purified to an apparent homogeneity with non-denaturing gel electrophoresis, by which it was identified as a low-mobility (Rf 0.07) protein fraction of high relative molecular mass (>250 000). The optimum pH was 5.5, with a little activity found outside the range 5.0–7.0. The activity of the peptidase was inhibited by 0.5mmol/l 1,10 phenanthroline (half-maximal inhibitory concentration 0.03mmol/l), and by 1 mmol/l EDTA, implicating a metalloenzyme. Bestatin, puromycin, phenylmethylsulphonyl fluoride and thiorphan were without effect. Unlabelled endothelin-1 inhibited the degradation of [12Sl]-endothelin-1 (half-maximal inhibitory concentration 100nmol/l), whereas 100u.mol/l methionine enkephalin or angiotensin I did not. High-performance liquid chromatography analyses of the [125I]-endothelin-1 incubated with purified peptidase revealed a time-dependent accumulation of one major radioactive fraction that was soluble in trichloroacetic acid. This product (or products) was not further hydrolysed. It did not react with the endothelin antibodies or with the specific, myocardial membrane receptors Conclusion Our data suggest that the rat kidney contains an acidic metalloproteinase of high relative molecular mass that is able to hydrolyse endothelin-1 rapidly and efficiently in vitro. The enzyme may participate in the inactivation of circulating or tissue endothelins, or both

Endothelin-1 inactivating peptidase in the human kidney and urine.

Objective Recently, an apparently novel, specific endothelin-1 inactivating metalloendopeptidase (ET-1 peptidase) has been isolated from the rat kidney. In this study we attempted to determine whether the same or a similar peptidase is present in the human kidney, and whether the enzyme is excreted into the urine. The urinary ET-1 peptidase could serve as an indirect index of the renal endothelin system, both in physiology and pathophysiology. Methods Kidney specimens were obtained from part of nephrectomized kidneys unaffected by any neoplastic process from six adult patients. The enzyme was purified using differential centrifugation, detergent solubilization of the membrane proteins, ultrafiltration and nondenaturing gel electrophoresis. The enzyme activity assays were performed at pH 5.5 and 37°C in the presence of increasing concentrations of unlabelled peptides and inhibitors using a fixed amount of [125I]ET-1 as substrate. The degradation extent was quantified with trichloroacetic acid precipitation and high performance liquid chromatography. The degrading activity of ET-1 was determined in urine samples from adult patients with hypertension, children with chronic renal failure and those with stable renal allograft. Results ET-1 peptidase from the human kidney displays characteristics close to that of the rat ET-1 peptidase we have recently described (J. Hypertens 1994; 12:1155–1162). The enzyme, a membrane-bound metalloendopeptidase, exhibits low electro- phoretical mobility on nondenaturing gel (Rf 0.08); it is an apparently heterologous structure comprising three enzymatically inactive subunits, it has a pH optimum at 5.5, a nanomolar range affinity to the ET-1 (KM180 nmol/l) that is hydrolysed to two main degradation products, and a 10-100-fold lower affinity to big ET-1 (KM11.5 μmol/l), endothelin 11–21 fragment (KM15.3 μmol/l), endothelin antagonist Trp- Leu-Asp-Ile-Ile-Trp (KM3.1 μmol/l), gastrin (KM2.2 μmol/l) and cholecystokinin (KM4.0 μmol/l). Substance P, neuropeptide Y, atrial natriuretic peptide, bradykinin, angiotensin II and enkephalin were poor substrates for the enzyme. The most powerful inhibitors of the ET-1 peptidase included thiorphan (IC50 0.28 nmol/l), phosphoramidon (IC50 0.55 nmol/l), phenanthroline (IC50 11.5 μmol/l), cyclosporin (IC50 400 μmol/l), phosphate (IC50 1.2 mmol/l), citrate (IC50 0.6 mmol/l) and aniline naphthalene sulphonic acid (IC50 0.25 mmol/l). Our data suggest that three ET-1 degrading peptidases with optimal activity at pH 4.5, 5.5 and 7.0, respectively, are excreted into the urine. The enzyme with a pH optimum 4.5 is of lysosomal origin whereas the two other enzymes correspond by their pH optima to the renal ET-1 peptidase and neutral endopeptidase. We have found statistically significant increases (P < 0.001) in the activity of both lysosomal and ET-1 peptidase in the urine in patients with hypertension and in children with chronic renal failure compared with healthy subjects or children with stable renal allograft. Conclusions Human kidney contains an acidic, highly specific endothelin-1 inactivating metalloendopeptidase that may have a key role in the regulation of concentrations of renal and circulating endothelins. The enzyme is excreted into the urine where its activity seems to be increased in patients with hypertension and chronic renal failure; it may potentially serve as an indirect index of the renal endothelin system.

Circulating microribonucleic acids miR-1, miR-21 and miR-208a in patients with symptomatic heart failure: Preliminary results

BACKGROUND Cardiomyocytes produce a wide variety of bioactive molecules that regulate numerous physiological and pathophysiological processes. Recently, it has been recognized that changes in microribonucleic acid (miRNA) expression may lead to cardiac dysfunction. AIMS To assess the expression of circulating miRNAs (miR-1, miR-21 and miR-208a) in patients with symptomatic heart failure (HF), and to investigate the relationship between expression of these miRNAs and secretion of N-terminal pro-B-type natriuretic peptide (NT-proBNP) and galectin-3. METHODS Thirty-five patients in New York Heart Association (NYHA) class II/III (age: 68.8 ± 13.0 years) and 26 patients in NYHA class IV (age: 72.0 ± 10.4 years) hospitalized in the intensive coronary care unit participated in the study. Serum concentrations of miRNAs were measured by quantitative real-time polymerase chain reaction. Basic biochemical assays were carried out, and NT-proBNP and galectin-3 concentrations were measured in all serum samples. RESULTS miR-1 was downregulated in patients with symptomatic HF and its expression decreased with severity of NYHA class (P=0.007). In contrast, overexpression of miR-21 was seen in all patients, independent of HF severity. Results suggest no miR-208a leakage into the circulation in patients with symptomatic HF. There was an inverse relationship between miR-1 expression and NT-proBNP concentration (Spearmans rank correlation coefficient [r]=-0.389; P=0.023) in patients in NYHA class II/III. Overexpression of miR-21 correlated significantly with galectin-3 concentration (r=0.422; P=0.032). CONCLUSION Dysregulation of miR-1 and miR-21 expression may be essential for the development of HF; miR-1 might become a biomarker for predicting HF exacerbation.

Interindividual variability of atorvastatin treatment influence on the MPO gene expression in patients after acute myocardial infarction

Myeloperoxidase (MPO) and C-reactive protein (CRP) may play critical roles in generation of oxidative stress and the development of the systemic inflammatory response. The aim of the study was to determine the effect of atorvastatin therapy on the MPO gene expression and its plasma level in relation to lipids level lowering and an anti-inflammatory response in patients after acute myocardial infarction. The research material was represented by 112 samples. Thirty-eight patients with first AMI receiving atorvastatin therapy (40 mg/day) and followed up for one month were involved in the study. The relative MPO gene expression in peripheral blood mononuclear cells (PBMCs) was examined using RT-qPCR in 38 patients before-, 38 patients after-therapy and in 36 patients as the control group. The plasma concentrations of MPO and serum concentrations of biochemical parameters were determined using commercially available diagnostic tests. After one month of atorvastatin therapy, in 60.5% patients a decrease of MPO gene expression, whereas in 39.5% patients an increase, was observed. The plasma MPO levels behaved in the same way as the MPO gene expression. However, the serum lipids and CRP concentrations were significantly lower after one month of atorvastatin therapy in both groups of patients - with decreased and increased MPO gene expression. Atorvastatin exhibited a different effect on MPO gene expression and its plasma level. Short-term atorvastatin therapy resulted in lipid lowering and anti-inflammatory activity in patients after AMI, independently of its effect on MPO gene expression. The molecular mechanisms of this phenomenon are not yet defined and require further research.

Ischaemia modified albumin in patients with acute coronary syndrome and negative cardiac troponin I

Abstract Background. Approximately 40–60% of patients with acute coronary syndrome (ACS) have normal cardiac troponin I (cTnI) concentrations on admission. Ischaemia modified albumin (IMA) has been suggested as a new biomarker of myocardial ischaemia. Methods. A total of 43 patients presenting with symptoms suggestive of ACS but with normal (< 0.1 μg/L) cTnI concentrations and 45 healthy subjects were studied. The patients from the study group were divided into two groups: STEMI (n = 28) and NSTEMI (n = 15). All these patients were undergoing percutaneous coronary intervention (PCI) with stenting. The concentrations of cTnI, myoglobin and IMA were determined on admission and 4 h after PCI. Results. Mean (SD) IMA concentrations were higher in patients with ACS (114.39 ± 25.18 U/ml) as compared to the control group (96.24 ± 6.28 U/ml, p < 0.005). IMA concentrations ≥ 104.0 U/ml demonstrated 72.1% sensitivity and 75.6% specificity for the diagnosis of ACS. The area under the receiver operator characteristic curve was 0.766 (95% CI 0.664–0.868) for ACS patients (NSTEMI + STEMI). In both groups increased median (IQR) cTnI concentration after PCI was observed (STEMI patients to 65.4 (10.9–106.9) μg/L and NSTEMI to 17.6 (0.77–84.0) μg/L). In contrast, no increase in IMA concentration was observed. Conclusions. IMA may be a useful biomarker for the identification of ACS patients presenting with typical acute chest pain and/or abnormal electrocardiograms but negative cTnI.

Platelet reactivity on aspirin, clopidogrel and abciximab in patients with acute coronary syndromes and reduced estimated glomerular filtration rate

However, deminished platelet function in renal impairment may be related to lower platelet reactivity in patients with ACS and renal dysfunction in comparison to patients with ACS and normal renal function [5–7]. Confirmation of the above hypothesis would improve our understanding of the potential mechanism behind increased tendency for bleeding in patients with ACS and renal dysfunction. Therefore, the aim of the study was to analyze platelet reactivity in relation to renal function in patients with ACS treated with dual (aspirin and clopidogrel) or triple (aspirin, clopidogrel and abciximab) antiplatelet therapy.

Significance of aspirin and clopidogrel resistance in patients undergoing percutaneous coronary interventions.

Dual antiplatelet therapy (aspirin plus clopidogrel) is mandatory in patients treated with coronary stent implantation. This strategy is highly effective in prevention of stent thrombosis until its struts are covered with endothelium. However, a substantial number of patients still suffer from recurrent ischemic coronary events despite adequate antiplatelet therapy. These events fall into three categories: stent thrombosis, in stent restenosis and events related to other non-stented coronary lesions. Some data suggest that beside other local and systemic factors resistance to aspirin and clopidogrel may be a possible cause of stent thrombosis and ischemic events in patients after coronary interventions. Several mechanisms of antiplatelet drug resistance have been reported including poor compliance, interactions with other drugs, genetic polymorphism or increased platelet turnover. More research is needed to adequately assess the clinical significance and prognostic value of antiplatelet drug resistance detected by laboratory tests in patients undergoing percutaneous intervention. We review published data on mechanisms and the clinical significance of aspirin and clopidogrel resistance in patients after coronary interventions.

Vitamin D receptor (VDR) polymorphism and the risk of cardiovascular events

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Expression of Platelet Surface Receptors and Early Changes in Platelet Function in Patients with STEMI Treated with Abciximab and Clopidogrel versus Clopidogrel Alone

BackgroundPlatelets play a crucial role in the pathogenesis of acute coronary syndromes (ACS). The efficacy of antiplatelet treatment is pivotal in the success of percutaneous coronary intervention (PCI) performed in patients with ACS.ObjectiveThe aim of the study was to investigate the effects of clopidogrel with or without abciximab on the expression of platelet surface receptors and platelet function in patients with ST-segment elevation myocardial infarction (STEMI) undergoing PCI.Materials and methodsThirty patients with STEMI were included in the study. During acute primary coronary intervention, patients received aspirin (acetylsalicylic acid) and clopidogrel in a loading dose of 300mg. Clopidogrel was the only antiplatelet therapy used by nine patients (group B). Twenty-one patients (group A) received additional abciximab. Blood samples were collected and analyzed twice: before and up to 22 hours after administration of antiplatelet therapy. The platelet aggregation was established as primary platelet-related hemostasis (closure time [CT] assessed using the PFA100 system). The absolute number of platelet surface antigens as CD41a, CD42a, CD42b, CD61, and CD62P were determined by flow cytometry analysis.ResultsThe study revealed a statistically significant increase in CT induced by adenosine diphosphate and adrenaline (epinephrine) +130 seconds (p < 0.0001) and +94 seconds (p < 0.0001), respectively, in group A patients post-therapy. While in group B the parameters of CT did not change after treatment. In addition, the absolute number of CD41a antigens (glycoprotein [GP] IIb/IIIa) increased significantly after treatment in group A. No significant changes were observed after treatment in the expression of CD62P (P-selectin) antigens in either treatment group. There was a significant reduction in the percentage of CD62P-positive platelets in group B after antiplatelet therapy.ConclusionsThe absolute number of GP IIb/IIIa receptors increases and platelets are not activated up to 12 hours after cessation of abciximab therapy. Treatment of STEMI patients undergoing PCI with a loading dose of clopidogrel reduces the percentage of active platelets but does not influence the CT.