[Show abstract][Hide abstract] ABSTRACT: In this study, we screened a library of 500 compounds for fungicidal activity via induction of endogenous reactive oxygen species (ROS) accumulation. Structure-activity relationship studies showed that piperazine-1-carboxamidine analogues with large atoms or large side chains substituted on the phenyl group at the R(3) and R(5) positions are characterized by a high ROS accumulation capacity in Candida albicans and a high fungicidal activity. Moreover, we could link the fungicidal mode of action of the piperazine-1-carboxamidine derivatives to the accumulation of endogenous ROS.
[Show abstract][Hide abstract] ABSTRACT: The mechanism of induction of cardiomyocyte (CM) dedifferentiation, as seen in chronic hibernating myocardium, is largely unknown. Recently, a cellular model was proposed consisting of long-term cocultures of adult rabbit CMs and cardiac fibroblasts in which typical structural characteristics of hibernation-like dedifferentiation could be induced. Only CMs in close contact with fibroblasts underwent these changes. In this study, we further investigated the characteristics of the fibroblast-CM interaction to seek for triggers and phenomena involved in CM dedifferentiation. Adult rabbit CMs were cocultured with cardiac or 3T3 fibroblasts. Heterocellular interactions and the structural adaptation of the CMs were quantified and studied with vital microscopy and electron microscopy. Immunocytochemical analysis of several adhesion molecules, i.e., N-cadherin, vinculin, beta1-integrin, and desmoplakin, were examined. Upon contact with CMs, fibroblasts attached firmly and pulled the former cells, resulting in anisotropic stretch. Quantification of the attachment sites revealed a predominant binding of the fibroblast to the distal ends of the CM in d 1 cocultures and a shift towards the lateral sides of the CMs on d 2 of coculture, suggesting a redistribution of CM membrane proteins. Immunocytochemical analysis of cell adhesion proteins showed that these were upregulated at the heterocellular contact sites. Addition of autologous and nonautologous fibroblasts to the CM culture similarly induced a progressive and accelerated structural adaptation of the CM. Dynamic passive stretch invoked by the fibroblasts and/or intercellular communication involving cell adhesion molecule expression at the interaction sites may play an important role in the induction of hibernation-like dedifferentiation of the cocultured adult rabbit CMs.
Cell Biochemistry and Biophysics 02/2006; 44(1):119-28. · 2.38 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The synthetic class of azole antimycotics constitutes the largest group of antifungal agents currently in clinical use. Widespread use of azoles has led to the rapid development of multiple drug resistance, which poses a major hurdle in antifungal therapy. The generally accepted mode of action of azoles is the inhibition of 14α-lanosterol demethylase, a key enzyme in ergosterol biosynthesis, resulting in depletion of ergosterol and accumulation of toxic 14α-methylated sterols in membranes of susceptible yeast species. For some azoles, their antifungal mode of action is not only characterized by inhibition of ergosterol biosynthesis. Recently, it was shown that generation of reactive oxygen species (ROS) is important for the antifungal activity of miconazole, pointing to an ancillary mode of action for this azole. We further analysed the effect of other azole antifungals on ROS generation in Candida albicans and could demonstrate that only miconazole induces ROS production in C. albicans. Furthermore, we show that the miconazole induced ROS production is probably caused by inhibition of the enzymes implicated in breakdown of peroxide radicals and hydrogen peroxide, i.e. peroxidase and catalase. Interestingly, only miconazole was found to exert its antifungal effect in a fungicidal way. In conclusion, further development of novel azole antimycotics, based on the chemical structure of miconazole and on its related ROS inducing capacity/fungicidal activity would be an interesting approach to address the problem of resistance occurrence.
[Show abstract][Hide abstract] ABSTRACT: Fibroblasts have been shown to couple to neonatal cardiomyocytes in heterocellular cultures through functional gap junctions. Our objective was to provide evidence for an additional type of heterocellular communication between fibroblasts and adult cardiomyocytes in vitro and in vivo.
The contact areas in heterocellular co-cultures were evaluated by specific labeling and the intercellular communication was studied using preloading of fibroblasts with tracer molecules. Heterocellular fibroblast-cardiomyocyte contacts present in the in vitro setting and in the border zone of a rabbit myocardial infarction in vivo were further examined by electron microscopy.
Addition of fibroblasts preloaded with the fluorescent low molecular weight tracer calcein-AM to cultured myocytes indicated early dye transfer via connexin 43 functional gap junctions. At a later time-period after co-culturing, dye transfer of fibroblasts preloaded with the high molecular weight tracer dextran 10,000 suggested partial cell fusion. The membrane continuity giving rise to this partial cell fusion was confirmed by electron microscopy, clearly showing areas of intercytoplasmic contacts between fibroblasts and phenotypically adapted (dedifferentiated) cardiomyocytes. Fluorescein-labeled annexin V affinity studies revealed transient exposure of phosphatidylserine at the contact sites, suggesting that phosphatidylserine mediates the fusion process. Close contacts between cardiac fibroblasts and dedifferentiated cardiomyocytes accompanied by disruption of the basal lamina were observed in the border zone of a rabbit myocardial infarction in vivo.
Our results suggest that the partial cell fusion-type of heterocellular communication in our co-culture model and the contacts observed in vivo may lead to new insights in cardiovascular disease.
Cardiovascular Research 11/2005; 68(1):37-46. · 5.81 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Cardiomyocyte apoptosis is known to occur in infarct border zones, where cardiomyocyte dedifferentiation, as seen in hibernating myocardium, can also be observed. The aim of the study is to determine whether dedifferentiated cardiomyocytes represent a population of cells stably surviving or undergoing apoptosis.
Microinfarctions were induced in sheep (n=8) by intracoronary injection of polymer macrobeads. The sheep were killed when cardiac function was gradually decreased (ejection fraction 37+/-6%, mean+/-SEM), but not earlier than 6 weeks after embolization. Transmural biopsies were taken from embolized and remote areas, based on flow measurements with positron emission tomography. Cells were classified as dedifferentiated when sarcomere content was depleted by >10% and glycogen content increased. Apoptosis was detected using the Tdt-mediated nick-end labelling (TUNEL) method and activated caspase-3 immunolabelling. Dedifferentiated cardiomyocytes were identified by morphology and by immunohistochemical evaluation of dedifferentiation related expression patterns of desmin, titin, cardiotin and alpha-smooth muscle actin. Cardiomyocyte apoptosis was detected in both the infarction border zones and remote areas. Dedifferentiated cardiomyocytes accounted for up to 30% of the cells in embolized areas and were almost exclusively non-apoptotic.
In embolization induced microinfarcted tissue, dedifferentiated cardiomyocytes are preferentially spared to undergo apoptosis. It is hypothesized that dedifferentiated cardiomyocytes and apoptotic cardiomyocytes represent two different cell populations. The dedifferentiated cells can be considered as stable surviving cells.
European Heart Journal 06/2002; 23(11):849-57. · 14.72 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Our current knowledge of the pathophysiology of chronic hibernating myocardium is mainly based on results from clinical studies, because of the absence of appropriate and validated animal models. These clinical observations have given rise to two major controversies: the role of reduced blood flow and that of histological changes in the hibernating segments. In this review, these two subjects will be briefly discussed, and put into the perspective of findings emerging from recently developed animal models.
[Show abstract][Hide abstract] ABSTRACT: Little is known about the causal factors which induce the typical structural changes accompanying cardiomyocyte dedifferentiation in vivo such as in chronic hibernating myocardium. For identifying important factors involved in cardiomyocyte dedifferentiation, as seen in chronic hibernation, an in vitro model mimicking those morphological changes, would be extremely helpful.
Adult rabbit cardiomyocytes were co-cultured with cardiac fibroblasts. The typical changes induced by this culturing paradigm were investigated using morphometry, electron microscopy and immunocytochemical analysis of several structural proteins, which were used as dedifferentiation markers, i.e., titin, desmin, cardiotin and alpha-smooth muscle actin.
Close apposition of fibroblasts with adult rabbit cardiomyocytes induced hibernation-like dedifferentiation, similar to the typical changes seen in chronic hibernation in vivo. Both changes in ultrastructure and in the protein expression pattern of dedifferentiation markers as seen in chronic hibernating myocardium were seen in the co-cultured cardiomyocytes.
Hibernation-like changes can be induced by co-culturing adult rabbit cardiomyocytes with fibroblasts. This cellular model can be a valuable tool in identifying and characterizing the pathways involved in the dedifferentiation phenotype in vivo, and already suggests that many of the structural changes accompanying dedifferentiation are not per se dependent on a decreased oxygen availability.
Cardiovascular Research 09/2001; 51(2):230-40. · 5.81 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The aim of this study was to evaluate the relationship between flow/metabolism, histology and functional follow-up in a sheep model of subacute myocardial infarction. In eight juvenile sheep, a myocardial infarction was induced by intracoronary injection of macrobeads. Left ventricular function was evaluated using echocardiography. 2-[18F]fluoro-2-deoxy-D-glucose (18F-FDG)/nitrogen-13-labelled ammonia (13NH3) positron emission tomography (PET) was performed at 6 weeks and 16 weeks after embolization. In five sheep, a dynamic carbon-11 acetate study was performed. In each animal, two regions of interest were defined on the polar map, corresponding to the embolized and the non-embolized region. After the final measurements, the hearts were processed for histological evaluation. PET revealed a moderately decreased flow and oxidative metabolism in the embolized region at 6 weeks, without significant changes at follow-up. At 6 weeks, 18F-FDG uptake in the embolized area was more severely decreased as compared to the flow index in the embolized area (P < 0.05). At 16 weeks, 18F-FDG metabolism had significantly recovered (P < 0.05). Serial echocardiography showed a persistent decrease in global and regional left ventricular function. Histology revealed a mix of micro-infarcted and viable tissue in the embolized region. In this model of subacute myocardial infarction, a PET "reversed mismatch" pattern was observed, with partial recovery of 18F-FDG uptake at follow-up. The histological counterpart of this PET pattern appears to be patchy necrosis.
European Journal of Nuclear Medicine 05/2001; 28(4):457-65.
[Show abstract][Hide abstract] ABSTRACT: The aim of this study was to evaluate the relationship between flow/metabolism, histology and functional follow-up in a sheep model of subacute myocardial infarction. In eight juvenile sheep, a myocardial infarction was induced by intracoronary injection of macrobeads. Left ventricular function was evaluated using echocardiography. 2-[18F]fluoro-2-deoxy-D-glucose (18F-FDG)/nitrogen-13-labelled ammonia (13NH3) positron emission tomography (PET) was performed at 6 weeks and 16 weeks after embolization. In five sheep, a dynamic carbon-11 acetate study was performed. In each animal, two regions of interest were defined on the polar map, corresponding to the embolized and the non-embolized region. After the final measurements, the hearts were processed for histological evaluation. PET revealed a moderately decreased flow and oxidative metabolism in the embolized region at 6 weeks, without significant changes at follow-up. At 6 weeks, 18F-FDG uptake in the embolized area was more severely decreased as compared to the flow index in the embolized area (P<0.05). At 16 weeks, 18F-FDG metabolism had significantly recovered (P<0.05). Serial echocardiography showed a persistent decrease in global and regional left ventricular function. Histology revealed a mix of micro-infarcted and viable tissue in the embolized region. In this model of subacute myocardial infarction, a PET "reversed mismatch" pattern was observed, with partial recovery of 18F-FDG uptake at follow-up. The histological counterpart of this PET pattern appears to be patchy necrosis.
European journal of nuclear medicine and molecular imaging 03/2001; 28(4):457-465. · 5.22 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Substantial evidence has accumulated that apoptosis, sometimes called "programmed cell death," is important in several cardiac diseases. Although most researchers focus on apoptosis in the hope that by understanding its mechanisms one can block this form of cell death, little attention has been given to programmed cell survival.
[Show abstract][Hide abstract] ABSTRACT: Taxol-induced peripheral neuropathy is a commonly-occurring side-effect in the treatment of cancer patients with taxoteres or taxanes. Taxol is known to induce apoptosis in a number of tumor cells. This report documents that, similar to proliferating cells, taxol induces apoptosis in NGF-differentiated PC12 cells, as assessed by exogenous FITC-annexin-V binding and nuclear fragmentation. It is shown that PC12 cells that stably overexpress Bcl-2 are protected against the toxic effect of taxol, as evidenced by the XTT assay and by a decreased fraction of propididum iodide positive cells in a dye exclusion test. Also the number of annexin-V-positive cells and the number of fragmented nuclei are lower in the Bcl-2 transfected cells. The effect is similar to the protective effect of Bcl-2 against NGF deprivation in differentiated PC12 cells. Although taxol forced both wild-type and Bcl-2-overexpressing cells into a mitotic state, only in Bcl-2-overexpressing cells did this lead to the appearance of metabolically active, multi-nucleated cells. This suggests that Bcl-2 is able to induce an alternative escape pathway, downstream of the G2/M block, in taxol-treated differentiated PC12 cells.
[Show abstract][Hide abstract] ABSTRACT: BACKGROUND: Although several short-term animal models of stunning and hibernation have been studied extensively, it has been difficult to produce a consistent animal model of chronic hibernation. The aim of the present study was to develop a nonsurgical porcine stent model of coronary stenosis in order to investigate the relationship between chronic dysfunctional myocardium and viability using 2D-echo, dobutamine stress echo (DSE) and positron emission tomography (PET). METHODS AND RESULTS: Focal progressive coronary stenosis was induced by implantation of an oversized stent in the left anterior descending (LAD) and/or circumflex (LCX) coronary artery in a total of 115 pigs, according to various experimental protocols: copper stent in the LAD (group I, n = 5); noncoated stainless steel stent in the LAD combined with balloon overstretch (group II, n = 7); poly(organo)phosphazene-coated stent in the LAD (group III, n = 77); and poly(organo)phosphazene-coated stent in both the LAD and the LCX (group IV, n = 26). Occurrence of left ventricular dysfunction was evaluated weekly by 2D-echo. At the time of left ventricular dysfunction the presence of viable myocardium within the dysfunctional region was investigated with DSE and PET, and confirmed by histology. The degree of coronary artery stenosis was measured by quantitative coronary angiography and morphometry. Severe coronary artery stenosis in the presence of dysfunctional, but viable, myocardium was induced in groups III and IV (47% and 11% of the animals, respectively). CONCLUSIONS: The authors developed a nonsurgical porcine stent model of progressive coronary stenosis using an oversized polymer-coated stent resulting in chronically decreased myocardial function, with residual inotropic reserve and viable myocardium. This condition may arise from repetitive periods of ischemia, or from sustained hypoperfusion, or a combination of these processes eventually leading to myocardial hibernation.
International Journal of Cardiovascular Interventions 07/2000; 3(2):111-120.
[Show abstract][Hide abstract] ABSTRACT: It has been suggested that Ca(2+)content of atrial cardiomyocytes is increased at the onset of atrial fibrillation (AF). Whether this phenomenon is transient is currently unknown. Therefore, in this study the time-related changes in Ca(2+)location in atrial myocytes from goats with chronic AF have been investigated. The distribution of calcium was assessed with the electron microscope using the cytochemical phosphate-pyroantimonate and oxalate-pyroantimonate methods in atrial biopsies from goats in sinus rhythm and goats with 1-16 weeks of burst-pacing-induced AF. In atrial myocytes from control goats in sinus rhythm, a normal Ca(2+)distribution was observed, with regular deposits along the sarcolemma (an average of 3.4 deposits per microm at a regular distance). The number of sarcolemma-bound Ca(2+)deposits substantially increased after 1 and 2 weeks of atrial fibrillation. After this period the amount of Ca(2+)precipitate decreased at 4 and 8 weeks, and became below control level at 16 weeks. A similar time-related redistribution of Ca(2+)occurred in mitochondria. Whereas mitochondria from control goats displayed very few Ca(2+)deposits (average 4.0 deposits per micro m(2)), their number markedly increased after 1 and 2 weeks of atrial fibrillation, which indicates cellular Ca(2+)overload. From 4 weeks, Ca(2+)deposits reached control levels and were below control level after 16 weeks of atrial fibrillation (2.5 deposits per microm(2)). Our findings are consistent with the previously observed Ca(2+)overload early after the onset of atrial fibrillation. The present study shows that this overload persists for at least 2 weeks, after which the cardiomyocytes apparently adapt to a new Ca(2+)homeostasis, thereby avoiding Ca(2+)overload. This protection against Ca(2+)overload co-occurs with dedifferentiation like cellular remodeling.
Journal of Molecular and Cellular Cardiology 04/2000; 32(3):355-64. · 5.22 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Is the 'smart heart' smart enough? Since the introduction of the term 'hibernating myocardium', this has been referred to as the 'smart heart', however more recently several publications have suggested that cell death accompanies the hibernation process, so that revascularisation of patients with hibernating myocardium should be performed without delay. Other data, however, point to cellular dedifferentiation instead of cellular degeneration, which means that cardiac hibernation is an adaptive mechanism capable of preserving the myocardial viability for a prolonged period. In an attempt to find an answer to the above-mentioned question, this review summarises and discusses the findings in this field, also giving attention to possible explanations for the discrepant findings.
Cardiovascular Research 03/2000; 45(3):696-703. · 5.81 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The purpose of this study was to examine the effect of antagonists of different subtypes of Ca(2+) channels (nimodipine and flunarizine) and two types of Ca(2+) chelating agents (the cell permeant Ca(2+) chelator 1,2-bis(2-aminophenoxy)ethane-N,N,N', N'-tetraacetic acid acetoxymethylester (BAPTA-AM) and the cell non-permeant Ca(2+) chelator EGTA) on neurite retraction and cell death of nerve growth factor (NGF)-differentiated PC12 cells after NGF deprivation. We demonstrated that flunarizine and nimodipine, but not BAPTA-AM and EGTA, provided protection against cell death due to NGF deprivation. Using time-lapse videomicroscopy and quantitative image analysis, we found that retraction of neurites was an early and fast phenomenon after removal of NGF. None of the compounds tested (flunarizine, nimodipine, BAPTA-AM, EGTA) could prevent the retraction of neurites.
European Journal of Pharmacology 11/1999; 384(1):61-70. · 2.68 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Similar structural changes in the myocardium can be observed in chronic hibernating myocardium and in myocardium taken from hearts suffering chronic atrial fibrillation. We investigated whether or not these changes are indicative of apoptosis.
Myocardial biopsies from 28 strictly selected patients with chronic hibernating myocardium and heart samples from 13 goats with pacing-induced chronic atrial fibrillation were used. Special attention was paid to processing the tissues immediately (fixation/freezing) in order to prevent artificial degenerative changes, thereby excluding false positive identification of apoptosis. Infarcted areas or infarcted border zones were excluded from our study. Apoptosis was detected with light and electron microscopy and terminal deoxynucleotidyl transferase nick end-labelling. Immunohistochemistry was used for detecting Bcl-2, P53 and PCNA-proteins associated with apoptosis/DNA damage.
The results obtained for chronic hibernating left ventricular myocardium were similar to those for chronic fibrillating atrial myocardium. No apoptotic nuclei, as characterised by extensive chromatin clumping, could be observed in normal or dedifferentiated cardiomyocytes under the electron microscope. The end-labelling assay did not reveal any cardiomyocytes with damaged DNA. Nor could we find any evidence of substantial expression of Bcl-2, P53 or PCNA, a result indicative of the absence of apoptotic threat or DNA damage.
Cardiomyocyte dedifferentiation, but not extensive degeneration through apoptosis, can be observed in chronic hibernating myocardium and chronic fibrillating atrium. Dedifferentiation may be the best way to survive prolonged exposure to the unfavourable conditions imposed by increased wall stress, a relative lowered oxygen environment, or both.
Cardiovascular Research 09/1999; 43(4):947-57. · 5.81 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: To create a model of chronic heart failure in a large animal.
Heart failure was induced in sheep by single intracoronary injection of polymer macrobeads, which were administered into left main coronary artery (n=3) or selectively into left anterior descending (n=4) or left circumflex (n=5) coronary artery. The animals were followed by echocardigraphy for 20 weeks. Measurements comprised fractional area change (FAC), and diastolic ventricular area (EDVA) and regional wall-thickening fraction (WT%).
EDVA increased from 14.2+/-2.1 cm2 prior to embolization to 16.9+/-3.1 cm2 on day 1 (p<0.05), and remained significantly increased until completion of the follow-up period. FAC dropped from 47.9+/-4.6% at baseline to 29.3+/-4.4% on day 1 (p<0.001) and remained significantly depressed until 20 weeks later. In 9 selectively embolized animals WT% of the embolized area decreased from 33.8+/-8.0% at baseline to 5.3+/-2.6% on day 1 and remained significantly decreased.
A simple model of chronic heart failure was developed. It shows relatively high stability over time and may prove beneficial in experimental work on ventricular assist devices.
The International journal of artificial organs 08/1999; 22(7):499-504. · 1.45 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Left ventricular biopsies from 21 patients with clinically diagnosed chronic hibernating myocardium (CHM) were examined by light- and electron microscopy. A mean of 27% of cardiomyocytes were structurally altered and were characterized as hibernating, because of reduced amount of myofibrils and increased glycogen content. Electron microscopy of these cells showed reduction of T-tubules and numerous small mitochondria, but few changes associated with degeneration, acute ischemia or apoptosis. The structural changes found in CHM are reminiscent of dedifferentiation rather than degeneration. The expression patterns of some structural proteins show resemblance with those in embryonic cardiomyocytes. Histochemically, mitochondrial NADH-oxidase and proton translocating ATPase activities were absent, whereas cytochrome c activity was present. Intracellular calcium distribution indicated normally bound sarcolemmal calcium and absence of excess mitochondrial calcium accumulation. Nuclear chromatin ranged from normal to dispersed with only a few nuclei that were clumped. These results suggest that cardiomyocytes from human CHM hearts are structurally altered, but viable, and lack morphologic and cytochemical characteristics suggestive of apoptosis or acute ischemia.
Molecular and Cellular Biochemistry 10/1998; 186(1-2):159-68. · 2.39 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: There is increasing evidence that apoptosis in postmitotic neurons is associated with a frustrated attempt to reenter the mitotic cycle. Okadaic acid, a specific protein phosphatase inhibitor, is currently used in models of Alzheimer's research to increase the degree of phosphorylation of various proteins, such as the microtubule-associated protein tau. Okadaic acid induces programmed cell death in the human neuroblastoma cell lines TR14 and NT2-N, as evidenced by fragmentation of DNA and attenuation of this process by protein synthesis inhibitors. In differentiated TR14 cells, okadaic acid increases the fraction of cells in the S phase, induces the appearance of cyclin B1 and cyclin D1 markers of the cell cycle, and triggers a time-dependent increase in DNA fragmentation after release of a thymidine block. Fully differentiated NT2-N cells are forced to enter the mitotic cycle as shown by DNA staining. Chromatin condensation and chromosome formation are initiated, but the cells fail to complete their mitotic cycle. These data suggest that okadaic acid forces differentiated neuronal cells into the mitotic cycle. This pattern of cyclin up-regulation and cell cycle shift is compared with apoptosis induced by neurotrophic factor deprivation in differentiated rat pheochromocytoma PC12 cells.
Journal of Neurochemistry 04/1998; 70(3):1124-33. · 4.24 Impact Factor