The Correlation of PPARα Activity and Cardiomyocyte Metabolism and Structure in Idiopathic Dilated Cardiomyopathy during Heart Failure Progression.

This study aimed to define relationship between PPARα expression and metabolic-structural characteristics during HF progression in hearts with DCM phenotype. Tissue endomyocardial biopsy samples divided into three groups according to LVEF ((I) 45-50%, n = 10; (II) 30-40%, n = 15; (III) <30%, n = 15; and control (donor hearts, >60%, n = 6)) were investigated. The PPARα mRNA expression in the failing hearts was low in Group (I), high in Group (II), and comparable to that of the control in Group (III). There were analogous changes in the expression of FAT/CD36 and CPT-1 mRNA in contrast to continuous overexpression of GLUT-4 mRNA and significant increase of PDK-4 mRNA in Group (II). In addition, significant structural changes of cardiomyocytes with glycogen accumulation were accompanied by increased expression of PPARα. For the entire study population with HF levels of FAT/CD36 mRNA showed a strong tendency of negative correlation with LVEF. In conclusion, PPARα elevated levels may be a direct cause of adverse remodeling, both metabolic and structural. Thus, there is limited time window for therapy modulating cardiac metabolism and protecting cardiomyocyte structure in failing heart.

Comparative and developmental anatomy of cardiac lymphatics.

The role of the cardiac lymphatic system has been recently appreciated since lymphatic disturbances take part in various heart pathologies. This review presents the current knowledge about normal anatomy and structure of lymphatics and their prenatal development for a better understanding of the proper functioning of this system in relation to coronary circulation. Lymphatics of the heart consist of terminal capillaries of various diameters, capillary plexuses that drain continuously subendocardial, myocardial, and subepicardial areas, and draining (collecting) vessels that lead the lymph out of the heart. There are interspecies differences in the distribution of lymphatic capillaries, especially near the valves, as well as differences in the routes and number of draining vessels. In some species, subendocardial areas contain fewer lymphatic capillaries as compared to subepicardial parts of the heart. In all species there is at least one collector vessel draining lymph from the subepicardial plexuses and running along the anterior interventricular septum under the left auricle and further along the pulmonary trunk outside the heart and terminating in the right venous angle. The second collector assumes a different route in various species. In most mammalian species the collectors run along major branches of coronary arteries, have valves and a discontinuous layer of smooth muscle cells.

Type of desmin expression in cardiomyocytes - a good marker of heart failure development in idiopathic dilated cardiomyopathy.

AIM: The aim of this study was to determine whether remodelling of the desmin (DES) cytoskeleton affects myocardial function and whether it could be a useful marker of disease progression in patients with idiopathic dilated cardiomyopathy (IDCM). MATERIAL AND METHODS: Endomyocardial biopsy was performed in 195 IDCM patients, and five to six specimens were collected from the left ventricle. DES expression was evaluated using tissue immunostaining and Western blotting. The study population was assigned to four groups according to DES expression type: I, normal DES staining at Z-lines giving a regular pattern of cross-striation (n = 57); IIA, increased DES staining with a regular pattern of cross-striation (n = 40); IIB, increased DES staining with an irregular pattern of cross-striation and/or the presence of aggregates (n = 56); and III, decreased/lack of DES staining (n = 42). Fibrosis, cardiomyocyte hypertrophy and ultrastructure were assessed for the four types of DES expression. RESULTS: The pathological types of DES expression (IIB or III) were associated with pathological changes in mitochondria and the contractile apparatus. Cardiomyocyte diameter and level of fibrosis were both significantly affected. DES expression type correlated with NYHA class, left ventricular end-diastolic diameter, left ventricular ejection fraction and the level of N-terminal pro-brain natriuretic protein. CONCLUSION: The type of immunohistochemical DES expression correlated with the level of myocardial injury at the cellular and organ levels. This correlation was similar to that observed between DES expression and the well-established biochemical, echocardiographic and clinical parameters of heart failure (HF). DES expression type could be used as an important diagnostic feature of HF development.

Microstructure and biocompatibility of titanium oxides produced on nitrided surface layer under glow discharge conditions.

The disadvantages of titanium implants are their low wear resistance and the release of titanium elements into surrounding tissue. These can be eliminated by modifying the surface by surface engineering methods, among them nitriding under glow discharge conditions which allow to produce diffusive surface layers. Their combining with an oxide layer might be valuable for biological events occurring at the bone implant interface. The aim of this study was to enhance the titanium biomaterial performance via combining nitriding and oxidizing treatments in one process under glow discharge conditions. The oxynitrided surface layers were produced at 680 degrees C. The obtained layer was TiO + TiN + Ti2N + alphaTi(N) type and about 4-microm thick and was of diffusive character. This layer significantly increased wear resistance and slightly corrosion resistance compared to that of the reference titanium alloy. The produced titanium oxide was about 400-nm thick and built from fine crystallites. This oxide exhibits bioactivity in SBF (simulated body fluid). Osteoblasts of Saos-2 line incubated on this surface exhibited good adhesion and proliferation and ALP release comparable with cells cultured on the reference titanium alloy and TiN + Ti2N + alphaTi(N) surface layers. A quantitative analysis of blood platelets adhering to this layer revealed their highest amount in comparison to that on both the nitrided surface layer and titanium alloy. The presented study provided a simple and reproducible method of combining oxidizing and nitriding under glow discharge in one process. Experimental data in vitro suggests that titanium alloy oxynitriding under low temperatures at glow discharge conditions improves titanium alloy properties and biocompatibility and tissue healing. Therefore, the layer of TiO + TiN +Ti2N + alphaTi(N) type could be valuable for long-term bone implants.

Combination of hydroxyapatite islets with Ti3P surface layer produced on titanium alloy for bone implants.

This study is concerned with the properties and bioactivity and biocompatibility of hydroxyapatite islets deposited on a new composite layer Ti3P+Ti2Ni type produced by a duplex method on Ti6Al4V titanium alloy. The microstructure and chemical composition of a produced surface layers and hydroxyapatite coating were investigated using scanning electron microscope equipped with EDS. Their bioactivity were examined in simulated body fluid and analyzed with XPS. Dissolution of hydroxyapatite was tested in culture medium during 12 days of incubation. Biocompatibility was investigated in osteoblast Saos2 line culture in contact with the tested material. Cell proliferation and activity were determined by the MTT test and measurement of alkaline phosphatase activity, respectively. Cell distribution was analyzed under a confocal microscope. The produced surface layers have a diffusion character with fine-grained structure and about 4 microm thick external zone of Ti3P. The experiments revealed higher bioactivity and biocompatibility of the Ti3P in comparison with reference titanium alloy. Hydroxyapatite islets were 0.8 mm in diameter and about 300 nm thick. They partially dissolved during the experiment what lead to formation on Ti3P between hydroxyapatite islets a precipitate containing Ca and P. Biocompatibility analyzed under confocal microscope in range of cell adhesion with osteoblast cells of Saos2 line revealed initial the highest osteoblast adhesion on Ti3P between hydroxyapatite islets and increasing on hydroxyapatite during following days. Cell were characterized by high proliferation and ALP activity. Therefore, the high bioactivity and biocompatibility of Ti3P and profitable hydroxyapatite properties make this composite layer promising for increasing implant fixation in vivo.

Expression of SDF-1-CXCR4 axis and an anti-remodelling effectiveness of foetal-liver stem cell transplantation in the infarcted rat heart.

SDF-1, a chemokine secreted by injured tissues, may be instrumental in chemoattracting CXCR4(+) stem cells (SCs) for repair of infarcted myocardium. We hypothesize that the myocardial SDF-1 expression determines also the engraftment and beneficial effects of SCs transplanted into the infarcted heart. Myocardial infarction (MI) was induced in rats by coronary artery ligation. The animals were either sacrificed at 2, 7, 16, 21 or 28 days after MI or were re-operated at 2, 7 or 14 days after MI to receive SCs transplantation, and were sacrificed 14 days later. SCs transplantation consisted of 3 x 15 microl injections of SCs isolated from foetal rat liver (FLSCs) into the myocardium bordering the infarction zone (5 x 10(6) cells/heart, labelled with PKH2 Green Fluorescent Cell Linker, approximately 20% CXCR4(+)). In the MI border zone, SDF-1 and CXCR4 immunostaining was transiently increased after MI, picking at 2 days and down regulating to the sham level by 21 days after MI. Simultaneously, an increased incorporation of CXCR4(+) and CD133(+) cells into capillaries was evident. AMD1300, a blocker of CXCR4, prevented the post-MI expression of CXCR4. In the MI border zone, the cardiomyocyte cross-sectional diameter increased and capillary/cardiomyocyte ratio decreased systematically during the 28 post-MI days, while an interstitial collagen accumulation demonstrated transient increase. FLSCs did not survive in the non-infarcted hearts. In infarcted hearts, FLSCs survived best when they were injected at 2 days after MI. The survival was negligible again when the injection was performed at 14 days after MI. FLSCs transplanted at 2 days after MI caused a further rise in SDF-1, CXCR4, and CD133 expression, compared with the untreated infarcted hearts. Only FLSCs transplanted at 2 days, but not later, attenuated cardiomyocyte hypertrophy and increased capillary/cardiomyocyte ratio in the MI border zone. These results suggest that myocardial signalling for homing of the endogenous and the exogenous SCs is transiently activated early after MI, that SDF-1 is instrumental in this process, and that there is only a narrow time-window after MI when SCs transplantation results in their efficient myocardial engraftment and beneficial anti-remodelling effect.

Vasculogenesis of the embryonic heart: contribution of nucleated red blood cells to early vascular structures.

During embryogenesis, coronary vessels develop via vasculogenesis and angiogenesis. Vasculogenesis is formation in situ of primary vessels from angioblasts - endothelial cell progenitors, and angiogenesis is formation of vessels from the existing ones. In the embryonic heart vasculogenesis precedes and overlaps angiogenesis and lasts till the end of the fetal life. What is unique about heart vasculogenesis is the fact that nucleated blood cells accompany early angioblasts in a spatiotemporal way. Morphologically these structures resemble yolk sac blood islands, thus, they have been called blood-island-like structures. In addition, these early vascular structures (blood-island-like) are found in the heart before coronary vessel system connects with the systemic circulation. We present the recent data regarding endothelial cell properties and derivation during coronary vessel formation and hypotheses concerning a source of blood cells in early vascular structures of the heart; the latter has received little attention in the literature. This review summarizes current knowledge on the endothelial cell origination from epicardial mesothelium or liver primordium. This review also focuses on blood cell contribution to coronary vessel vasculogenesis. The role of proepicardium in the epicardial cover formation and the epicardium as a source of cellular components of coronary vasculature and interstitial fibroblasts is presented. It seems that blood cells and angioblasts, which form the early vascular structures do not derive from the same hemangioblastic precursor.

Ischemic preconditioning prevents endothelial dysfunction, P-selectin expression, and neutrophil adhesion by preventing endothelin and O2- generation in the post-ischemic guinea-pig heart.

Evidence indicates that ischemia/reperfusion (IR) results in endothelial dysfunction and neutrophil adhesion in the post-ischemic myocardium and that ischemic preconditioning (IPC), superoxide dismutase (SOD), and anti-endothelin-1 (ET-1) interventions prevent these effects. We tested the hypothesis that ET-1-induced superoxide (O(2)(-)) generation mediates endothelial injury and neutrophil accumulation in the IR heart, that IPC protects the endothelium and prevents the adhesion by attenuating post-ischemic ET-1, and thus O(2)(-), generation, and that the mitochondrial ATP-dependent potassium channel (mK(ATP)) triggers the IPC-induced protection. Langendorff-perfused guinea-pig hearts were subjected either to 30 min ischemia/35 min reperfusion (IR) or were preconditioned prior to IR with three cycles of either 5 min ischemia/5 min reperfusion or 5 min infusion/5 min wash-out of mK(ATP) opener diazoxide (0.5 microM). Neutrophils were infused to the hearts at 15-25 min of the reperfusion. Coronary flow responses to acetylcholine (ACh) and nitroprusside (SNP) served as measures of endothelium-dependent and -independent vascular function, respectively. Myocardial outflow of ET-1 and O(2)(-), P-selectin expression, neutrophil adhesion and functional recoveries were followed during reperfusion. IR augmented ET-1 and O(2)(-) outflow, P-selectin expression, and neutrophil adhesion, and impaired ACh response. These effects were attenuated or prevented by IPC and diazoxide, and 5-hydroxydecanoate (a selective mK(ATP) blocker) abolished the effects of IPC and diazoxide. SOD (150 U/ml) and tezosentan (5 nM, a mixed ET-1-receptor antagonist) mimicked the effects of IPC, although they had no effect on the ET-1 generation. The preventive effect of IPC, SOD and tezosentan on P-selectin expression preceded their effect on neutrophil adhesion. These data suggest that in guinea-pig heart: (i) ET-1-induced O(2)(-) generation mediates the post-ischemic endothelial dysfunction, P-selectin expression and neutrophil adhesion; (ii) IPC and diazoxide afford protection by attenuating the ET-1, and thus O(2)(-) generation; (iii) the mK(ATP) opening triggers the IPC protection; (iv) endothelial injury promotes post-ischemic neutrophil adhesion, but not vice versa.

Superoxide- and nitric oxide-derived species mediate endothelial dysfunction, endothelial glycocalyx disruption, and enhanced neutrophil adhesion in the post-ischemic guinea-pig heart.

The study was aimed at testing the hypothesis that a toxic product of the reaction between superoxide (O(2)(-)) and nitric oxide (NO) mediates, not only endothelial dysfunction, but also endothelium-glycocalyx disruption, and increased neutrophil (PMN) accumulation in the heart subjected to ischemia/reperfusion (IR) injury. Accordingly, we studied if scavengers of either O(2)(-) or NO, or a compound that was reported to attenuate cardiac production of peroxynitrite, would prevent endothelial injury and subsequent PNM adhesion in IR heart. Langendorff-perfused guinea-pig hearts were subjected to 30 min ischemia/35 min reperfusion, and infusion of PMN between 15 and 25 min of the reperfusion. Coronary flow responses to acetylcholine (ACh) and sodium nitroprusside (SNP) were used as measures of endothelium-dependent and -independent vascular function, respectively. PMN adhesion and endothelium glycocalyx ultrastructure were assessed in histological preparations. IR impaired the ACh, but not SNP, response by approximately 60%, caused endothelium-glycocalyx disruption, and approximately nine-fold increase in PMN adhesion. These alterations were prevented by superoxide dismutase (150 U/ml), NO synthase inhibitor, L-NAME (10 microM), NO scavenger, oxyhemoglobin (25 microM), and NO donor, SNAP (1 microM), and were not affected by catalase (600 u/ml). The glycocalyx-protective effect of these interventions preceded their effect on PMN adhesion. The data imply that PMN adhesion in IR guinea-pig heart is a process secondary to functional and/or structural changes in coronary endothelium, and that a toxic product of the reaction between superoxide and NO mediates these endothelial changes.

The role of endogenous nitric oxide in inhibition of ischemia/reperfusion-induced cardiomyocyte apoptosis.

The effect of nitric oxide (NO) synthase inhibition on apoptosis of cardiomyocytes during ischemia/reperfusion was investigated. Isolated perfused guinea-pig hearts were subjected to 35 min ischemia (I) followed by 30 min reperfusion (IR) in the presence or absence of NO synthase inhibitors, L-NAME or L-NMMA or a superoxide scavenger, SOD. Apoptosis was assessed by immunohistochemistry (TUNEL assay, Bax protein staining), by spectrophotometric measurement of cytochrome oxidase activity (COX), and by ultrastructural analysis. Inhibition of NOS significantly increased apoptosis with activation of Bax protein and decrease of COX. SOD infusion had a protective effect on these apoptotic markers. The results suggest that endogenous NO synthesis during I/R protects the heart against apoptotic cell death.

Improvement of titanium alloy for biomedical applications by nitriding and carbonitriding processes under glow discharge conditions.

Although titanium alloys are used in medicine, they present low wear resistance. In this paper we present the results of studies on surface layers produced by nitriding at three different temperatures, and by carbonitriding under glow discharge conditions in order to improve wear resistance, hardness, and to modulate microstructure and chemical composition of surface layers. A cell culture model using human fibroblasts was chosen to study the effect of such treatments on the cytocompatibility of these materials. The results showed that nitrided and carbonitrided surface layers were cytocompatible. Modulation of surface microstructure by temperature in the nitriding process and chemical composition of surface layers by carbonitriding led to differences in cellular behaviour. Cell proliferation appeared to be slightly reduced from the 6th day of culture on nitrided surfaces produced at 730 degrees C and 1000 degrees C, however after 12 days of culture, the best growth was on surface layers produced at 850 degrees C. The best viability was observed on the carbonitrided layer. The orientation and shape of the cells corresponded to surface topography. Nitriding and carbonitriding under glow discharge conditions may constitute interesting techniques allowing the formation of surface layers on parts with sophisticated shapes. They may also permit modulating surface topography in a way improving the features of titanium alloys for various applications in medicine.

[Apoptosis and hepatic cell proliferation in autoimmune hepatitis and chronic viral hepatitis C in children: analysis of APO-1/Fas (CD95), bcl-2 and Ki67 expression]. / Apoptoza i proliferacja komórek watrobowych w autoimmunologicznym zapaleniu watroby i przewleklym wirusowym zapaleniu watroby typu C u dzieci: analiza ekspresji APO-1/Fas (CD95), bcl-2 i Ki67.

Hepatocyte damage in autoimmune hepatitis (AIH) and chronic viral hepatitis C (CVH) is attributed to an immune response. We analysed liver biopsy specimens from 4 children with AIH type I, 3 children with AIH type II and 2 children with CVH, using ApopDetek in situ hybridisation method and Mabs anti CD95, Ki67, bcl-2 by means of APAAP technique. The histological appearance of apoptotic bodies in both conditions was similar. The proliferation activity of the hepatocytes was elevated in cases of CVH and less extensive in AIH. Immunohistochemical analysis suggested that the liver damage in AIH and CVH could be mediated by CD95 system as a mechanism of T-cell mediated cytotoxicity.

Effect of ischemic preconditioning on endothelial dysfunction and granulocyte adhesion in isolated guinea-pig hearts subjected to ischemia/reperfusion.

It has been demonstrated that ischemic preconditioning (IPC) affords protection against the post-ischemic endothelial dysfunction. Here, a hypothesis was tested that IPC, by protecting the endothelium, prevents also the adherence of granulocytes (PMNs) in the post-ischemic heart. Langendorff-perfused guinea-pig hearts were subjected to 30 min ischemia/30 min reperfusion (IR) and peritoneal PMNs were infused between 15 and 25 min of the reperfusion. Acetylcholine (ACh)-induced coronary vasodilatation and nitrite outflow were used to measure endothelial function and coronary flow response to sodium nitroprusside (SNP) served as a measure of endothelium-independent vascular function. The endothelial adherence of PMNs to the coronary microvessels was assessed in histological preparation of the myocardium. In the hearts subjected to IR, ACh-induced vasodilatation and nitrite outflow were reduced by 55% and 69%, respectively, SNP response remained unaltered, and 22% of microvessels were occupied by PMNs, as compared to 2% in the sheam perfused hearts. These alterations were attenuated by IPC (3 x 5 min ischemia). A selectin blocker, sulfatide, prevented IR-induced PMNs adherence and did not affect the responses to ACh and SNP. These data demonstrate that IR leads to the endothelial dysfunction and to the selectin-mediated PMNs adhesion in the isolated guinea-pig and that IPC attenuates both alterations. We speculate that the pro-adhesive effect of IR is secondary to the endothelial injury and that the anti-PMNs action represents a novel cardioprotective mechanism of IPC.

Ultrastructural study of calcium shift in ischemic/reperfused rat heart under treatment with dimethylthiourea, diltiazem and amiloride.

Among factors underlying reperfusion injury are oxygen free radicals and Ca2+ influx via gated calcium channel or via Na+/H(+)-Na+/Ca2+ exchange which lead to calcium overload. The aim of the study was to ultrastructurally visualize the distribution of Ca2+ and to compare binding of calcium by the sarcolemma and calcium accumulation in mitochondria under therapy with an OH scavenger, dimethylthiourea (DMTU), Na+/H+ exchange inhibitor, amiloride, and calcium channel blocker, diltiazem, given alone or in combination to ischemic/reperfused hearts. Isolated working hearts subjected to 40 min ischemia and 30 min reperfusion were perfused with drugs added to the perfusate 15 min before ischemia and administered for the rest of the perfusion period. The cytochemical phosphate pyroantimonate method for localization of Ca2+ was used, and calcium distribution was analyzed with a computer image analyzer. All drugs given alone improved sarcolemmal ability to bind calcium. The best results were obtained with amiloride. All of the combined therapies gave even better results, but calcium accumulation in mitochondria diminished only with diltiazem therapy given alone or in combination with DMTU. Since the presence of Ca2+ deposits on the sarcolemma is believed to represent its normal function, and calcium sequestration by mitochondria reflects an increase in cytosolic calcium load, the lack of correlation between sarcolemmal and mitochondrial Ca2+ distribution might suggest impaired mechanisms of lowering cytoplasmic calcium or the existence of some mechanism other than Na+/Ca2+ exchange, mediated by activated Na+/H+ exchange.

Ischemic preconditioning and superoxide dismutase protect against endothelial dysfunction and endothelium glycocalyx disruption in the postischemic guinea-pig hearts.

UNLABELLED: The effect of ischemic preconditioning and superoxide dismutase (SOD) on endothelial glycocalyx and endothelium-dependent vasodilation in the postischemic isolated guinea-pig hearts was examined. Seven groups of hearts were used: group 1 underwent sham aerobic perfusion; group 2 was subjected to 40 min global ischemia without reperfusion; group 3, 40 min ischemia followed by 40 min reperfusion; group 4 was preconditioned with three cycles of 5 min global ischemia followed by 5 min of reperfusion (IPC), prior to 40 min ischemia; group 5 was subjected to IPC prior to standard ischemia/ reperfusion; group 6 underwent standard ischemia/reperfusion and SOD infusion (150 U/ml) was begun 5 min before 40 min ischemia and continued during the initial 5 min of the reperfusion period; group 7 was subjected to 80 min aerobic perfusion with NO-synthase inhibitor, L-NAME, to produce a model of endothelial dysfunction independent from the ischemia/reperfusion. Coronary flow responses to acetylcholine (ACh) and sodium nitroprusside (SNP) were used as measures of endothelium-dependent and endothelium-independent vascular function, respectively. Reduction in coronary flow caused by NO-synthase inhibitor, L-NAME, served as a measure of a basal endothelium-dependent vasodilator tone. After completion of each experimental protocol, the hearts were stained with ruthenium red or lanthanum chloride for electron microscopy evaluation of the endothelial glycocalyx. While ischemia led only to a slightly flocculent appearance of the glycocalyx, in ischemia/reperfused hearts the glycocalyx was disrupted, suggesting that it is the reperfusion injury which leads to the glycocalyx injury. Moreover, the coronary flow responses to ACh and L-NAME were impaired, while the responses to SNP were unchanged in the ischemia/reperfused hearts. The disruption of the glycocalyx and the deterioration of ACh and L-NAME responses was prevented by IPC. In addition, the alterations in the glycocalyx and the impairment of ACh responses were prevented by SOD. The glycocalyx appeared to be not changed in the hearts subjected to 80 min aerobic perfusion with L-NAME. IN CONCLUSION: (1) the impairment of the endothelium-dependent coronary vasodilation is paralleled by the endothelial glycocalyx disruption in the postischemic guinea-pig hearts; (2) both these changes are prevented by SOD, suggesting the role of free radicals in the mechanism of their development; (3) both changes are prevented by IPC. We hypothesize, therefore, that alterations in the glycocalyx contribute to the mechanism of the endothelial dysfunction in the postischemic hearts.

Clinical, biochemical and histological analysis of seven patients with cholesteryl ester storage disease.

Lysosomal acid lipase (LAL) deficiency leads to two phenotypically different diseases: cholesteryl ester storage disease (CESD) and Wolman's disease. Lysosomal acid lipase hydrolyzes cholesteryl esters and triglycerides. Deficiency of LAL results in intralysosomal storage of cholesteryl esters and triglycerides. CESD has a chronic and benign course and is characterized by hepatomegaly and mild hypercholesterolemia. It leads to fibrosis (cirrhosis) and early atherosclerosis. This report presents the clinical, biochemical and microscopic data of seven patients with CESD followed up over 10 years. The physical development of all the study children remained within the normal range; 7 patients had hepatomegaly and 6 also had splenomegaly. Three patients had normal cholesterol, triglycerides and transaminases values; the other four had slightly elevated levels for these parameters. The activity of LAL in all patients was reduced to below 30% of the lower normal value. Histologically, cholesteryl crystals and lipid storage vacuoles in Kupffer cells were present in all examined patients except one. Accumulation of cholesteryl esters was visible on thin-layer chromatography of lipid extracts obtained from liver biopsies.

Ultrastructural demonstration of endothelial glycocalyx disruption in the reperfused rat heart. Involvement of oxygen free radicals.

To determine the effect of post-ischaemic reperfusion on the ultrastructure of the endothelial glycocalyx and the role of oxygen free radicals, isolated working rat hearts were subjected to 20 min ischaemia followed by 3 or 30 min of reperfusion. Ruthenium red and lanthanum chloride were used to delineate the endothelial glycocalyx, and histochemical manganese/diaminobenzidine (Mn+2/DAB) or iron/diaminobenzidine (Fe+2/DAB) techniques were applied to visualize superoxide and hydrogen peroxide in myocardial capillaries. We found that ischaemia alone led to only a slightly flocculent appearance of the glycocalyx and its disruption was not observed until the onset of reperfusion. Prolongation of reperfusion to 30 min had no further effect on the ultrastructure of the glycocalyx. The ultrastructure of endothelial cells was normal. The disruption of the glycocalyx correlated in time and place with the appearance of Mn+2/DAB and Fe+2/DAB reaction products on the luminal surface of endothelial cells. Treatment with 5 mM N-(2-mercaptopropionyl)-glycine (MPG), an .OH radical scavenger, starting before ischaemia prevented the disruption of the glycocalyx, while 100 mM 3-morpholinosydnonimine (SIN-1), capable of generating both NO and -O2 simultaneously when applied at the time of reperfusion, increased the mean density of capillaries positively stained with Mn+2/DAB and Fe+2/DAB, and caused substantial disruption of the glycocalyx and damage to endothelial cells, which was not prevented by MPG. Our results suggest that the onset of reperfusion is critical for injury to the endothelial glycocalyx. Most probably the hydroxyl radical derived from the Fenton reaction is responsible for this injury. Peroxynitrite and/or nitric dioxide, if present upon reperfusion, may also account for damage of the endothelial glycocalyx.

Combined therapy with dimethylthiourea, diltiazem and amiloride/dimethylamiloride in the ischemic/reperfused heart.

OBJECTIVES: The relative contribution of oxygen free radicals, disturbances in calcium homeostasis and Na+/H+ exchange in the development of injury in the ischemic/reperfused heart was investigated. The study was designed to assess whether these factors initiate independent mechanisms of injury or, alternatively, they share a common mechanism of toxicity. METHODS: Isolated working rat hearts were subjected to different periods (30-55 min) of global ischemia and then were reperfused for 30 min. We compared the effects of oxygen radical scavengers (10 mM dimethylthiourea, DMTU and 0.6 mM desferrioxamine), inhibitors of Na+/H+ exchange (0.15 mM amiloride and 15 microM dimethylamiloride, DMA) and of 0.1 microM diltiazem, which was used to limit calcium overload, given alone or in combination, on the rate of myocardial injury development (recovery of hemodynamic function, LDH release, incidence of severe arrhythmias and structural integrity of cardiomyocytes were estimated at reperfusion following different periods of ischemia). RESULTS: All interventions studied, when given alone, provided nearly equivalent cardioprotection. DMTU or desferrioxamine when applied in combination with diltiazem provided additive cardioprotection, relatively limited, however, as compared to the remarkable cardioprotection achieved by DMTU or desferrioxamine in combination with amiloride. CONCLUSIONS: All mechanisms studied may contribute in an equal manner to the rate of injury development in the ischemic/reperfused heart. The oxygen free radicals-induced myocardial injury may be partially attributed to some disturbance in intracellular calcium homeostasis, possibly calcium overload, whereas the damaging effect of the Na+/H+ exchange activated upon reperfusion is probably largely related to some other mechanism.

Vitamin B-6 deficiency alters rat enterocyte calcium homeostasis but not duodenal transport.

Isolated enterocytes were used as differential transporting cells to examine calcium homeostasis in control and vitamin B-6-deficient rats. Kinetic analysis of calcium fluxes, as well as biochemical determinations, indicated that enterocytes from control animals had high concentrations of cytosol ionized calcium (318.5 +/- 22.4 nmol/L) and a large pool of exchangeable calcium (2.72 nmol/mg protein, or 86% of total cell calcium). Vitamin B-6 deficiency resulted in a 44% reduction in total cellular calcium (1.71 +/- 0.24 vs. 3.07 +/- 0.29 nmol/mg protein), a 69% reduction in total exchangeable calcium (0.85 vs. 2.72 nmol/mg protein) and a 56% reduction in cytosol ionized calcium concentration (141.4 +/- 13.5 vs. 318.5 +/- 22.4 nmol/L). Calcium fluxes between all cellular compartments were markedly diminished as a result of vitamin B-6 deficiency. However, vitamin B-6 deficiency did not affect the basic morphological or functional features of the enterocytes, such as cell viability, cell volume, membrane permeability and protein content. Moreover, intestinal calcium transport in vivo was not affected during vitamin B-6 deficiency, perhaps due to the greater paracellular ion movement compensating for the lower transcellular transport.