[Evident placebo effect in refractory angina pectoris. Do refer to the placebo-controlled study at the Huddinge hospital!].
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Angina Pectoris↗
Biomedical subjects
A Rück
Publications and source records attributed to A Rück.
Effects of intramyocardial injection of phVEGF-A165 as sole therapy in patients with refractory coronary artery disease--12-month follow-up: angiogenic gene therapy.
OBJECTIVE: To test the safety and bioactivity of phVEGF-A165 after intramyocardial injection during 12-month follow-up. DESIGN: Open-labelled study. SUBJECTS: Inclusion criteria were angina pectoris, Canadian Cardiovascular Society (CCS) class III-IV, unamenable to further revascularization, ejection fraction (EF) >30%, perfusion defects extending over >10% of the anterolateral left ventricle wall detectable with adenosine single photon emission computerized tomography (SPECT) and at least one patent vessel visible by coronary angiography. Seven of 39 patients referred for gene therapy were included. INTERVENTION: Via a mini-thoracotomy under general anaesthesia. phVEGF-A165 was injected directly into the myocardium at four sites in the anterolateral region of the left ventricle. RESULTS: Operative procedures were uneventful. Perioperative release of myocardial markers and electrocardiogram (ECG) changes were detected in two patients. There were no perioperative deaths but one patient died 7 months postoperatively because of myocardial infarction. Plasma vascular endothelial growth factor (VEGF)-A levels increased two to threefold peaking 6 days postoperatively (P < 0.004) and returning to baseline by day 30. A significant reduction in angina pectoris was reported. The CCS class improved from 3.3+/-0.2 to 1.9+/-0.3 (P < 0.01) and nitroglycerine intake decreased from 39+/-15 to 12+/-5 tablets week(-1) (P < 0.001) 2 months after gene transfer. Improvements remained after 12 months when nitroglycerine consumption approached zero. Improved myocardial function in the phVEGF-A165 injection region was documented in all patients (P < 0.016) by tissue velocity imaging (TVI). Reduced reversible ischaemia was detected by adenosine SPECT in four patients. Improved collateralization was detected in four patients with coronary angiography. CONCLUSION: Intramyocardial injection of phVEGF-A165 is safe and may lead to improved myocardial perfusion and function with longstanding symptomatic relief in end-stage angina pectoris. Based on these results this therapeutic potential is being tested in a double-blind placebo controlled multicentre trial.
Aged↗
Myocardial Doppler tissue velocity improves following myocardial gene therapy with VEGF-A165 plasmid in patients with inoperable angina pectoris.
BACKGROUND: Myocardial tissue velocity and perfusion were studied in patients with severe angina pectoris following gene therapy by intramyocardial injection of phVEGF-A165 via thoracotomy. Plasma concentrations of VEGF-A increased postoperatively. Two months after treatment anginal status and myocardial tissue velocity improved and perfusion showed a tendency to improve. Tissue velocity imaging appears to be a sensitive, objective method for detecting changes in myocardial function following gene therapy. OBJECTIVE: To study effects on myocardial tissue velocity and perfusion in patients with angina pectoris following intramyocardial injection of phVEGF-A165 via thoracotomy. DESIGN: Open label, phase I/II. METHODS: Six patients with Canadian Cardiovascular Society (CCS) angina pectoris functional class III - IV and with major defects at adenosine stress single-photon emission computerized tomography (SPECT) were studied. In addition to SPECT, coronary angiography and dobutamine stress echocardiography with tissue Doppler velocity imaging were performed before and two months after gene transfer. RESULTS: Plasma concentrations of VEGF-A increased 2 to 3 times (P < 0.04) over baseline from 2 to 14 days after injection with normalization after 4 weeks. The CCS class improved about 40%, from 3.3 +/- 0.2 to 2.0 +/- 0.3 (P < 0.02) and nitroglycerine consumption decreased 30 - 40%, from 44 +/- 17 to 15 +/- 5 tablets per week (P < 0.05). The maximal systolic myocardial tissue velocity increased in all patients about 25% (P < 0.02) but did not reach the reference range. Myocardial perfusion at SPECT improved in four of the six patients. CONCLUSIONS: Anginal status, myocardial tissue velocity and perfusion can be improved by phVEGF-A165 intramyocardial injection. Tissue velocity imaging appears to be a sensitive, objective method for detecting changes in myocardial function following gene therapy.
Aged↗
Methylene blue mediated photodynamic therapy in experimental colorectal tumors in mice.
Methylene blue (MB+) is a well-known dye in medicine and has been discussed as an easily applicable drug for the topical treatment during photodynamic therapy (PDT). The therapeutic response of MB+ was investigated in vivo by local injection of MB+ in a xenotransplanted subcutanous tumor (adeno-carcinoma, G-3) in female nude mice. MB+ in a concentration of 1% was applied both undiluted and diluted to 0.1 and 0.01% with isotonic sodium chloride. Treatment with 1% MB+ and subsequent irradiation at 662 nm with 100 J/cm2 led to complete tumor destruction in 79% of the treated animals. A decrease of the fluence rate from 100 to 50 mW/cm2 increased the phototoxic response as well as fractionated light application. Small sensitizer concentrations reduced the PDT effect significantly. It seems that the light induced reaction of MB+ could be correlated with the rapid production of reactive oxygen species. Below a threshold dose of MB+ oxidative damage of the tissue is prevented. However, above this dose, as a point of no return, MB+ acts as an extremely potent oxidant.
Adenocarcinoma↗
Fluorescence diagnosis of endometriosis on the chorioallantoic membrane using 5-aminolaevulinic acid.
The chorioallantoic membrane (CAM) is a useful model for the fluorescence diagnosis of experimentally induced endometriosis. In our experimental setup 75.7% of the histologically examined tissue preparations were viable and only 24.3% showed signs of necrosis on the CAM after various periods of incubation. Best results were obtained when grafting to the CAM was performed between days 7 and 9 and when implants were left on the CAM for 3-5 days (P < 0.05). We were able to demonstrate that 5-aminolaevulinic acid (ALA) is stored selectively in ectopic endometrium. The subsequent fluorescence of the endometrium shows a rapid increase that reaches a peak after 10-14 h which can be clearly differentiated from the weaker fluorescence of grafted normal peritoneum and fimbriae (P < 0.01).
Adult↗
Light-induced apoptosis involves a defined sequence of cytoplasmic and nuclear calcium release in AlPcS4-photosensitized rat bladder RR 1022 epithelial cells.
Oxidative stress induced by light activation of photosensitizers is regarded to have a role in triggering cell death pathways during photodynamic therapy (PDT). Reactive oxygen species have been proposed to act as signal transduction molecules activating downstream reactions that lead to apoptosis. Mainly debated is the cooperating role of other signaling systems like calcium or pH. The present work contributes to this discussion by studying PDT effects in cell cultures of rat bladder epithelial cells for the hydrophilic tetrasulfonated aluminum phthalocyanine (AlPcS4). Cells were coincubated with the photosensitizer and the calcium-sensitive probe Fluo-3. The light-induced reactions were analyzed with a confocal laser scanning microscope. The dynamics of the process during light activation was observed with subcellular resolution. A transient calcium elevation during the irradiation process was detected, especially in the cell's nuclei, followed by a more sustained increase. The evaluation of the energy-dose-dependent phototoxicity after an incubation time with the photosensitizer of 1 and 24 h, showed enhanced phototoxicity when the drug was present for 24 h. Surprisingly, stimulation of cell proliferation was observed at very low light doses (at 0.2 J/cm2) when the drug was incubated for 24 h (cell viability 160%). Induction of apoptosis could be observed after irradiation with fluences between 1 and 3 J/cm2. Apoptotic cells were identified with fluorescein isothiocyanate-labeled Annexin V, which binds to phosphatidylserine after its translocation to the outer plasma membrane. In the presence of the antioxidant pyrrolidinedithiocarbamate the transient calcium elevation was totally inhibited, as was the subsequent translocation of PS. In contrast, N-acetyl-L-cysteine did not suppress the transient calcium increase. Our data might be consistent with calcium regulated processes during AlPcS4-PDT and the involvement of oxygen radicals.
Animals↗
Free radical-induced inactivation of creatine kinase: influence on the octameric and dimeric states of the mitochondrial enzyme (Mib-CK).
Free radicals of X-ray-induced water radiolysis, either directly or indirectly via their reaction products, reduce the activity of both dimeric cytoplasmic muscle-type creatine kinase (MM-CK) and octameric mitochondrial creatine kinase (Mi-CK) to virtually zero. Similarly values of the characteristic D(37)-dose of enzyme inactivation (dose required to reduce enzyme activity to 37%) were found for the two isoenzymes of CK under identical conditions. Octamer stability was not significantly affected within the dose range considered. However, both the dissociation of octamers into dimers by a transition-state analogue complex (TSAC), and the reassociation of the dimers into octamers, showed dose-dependent reduction. Binding of the TSAC to the active centre was found to protect the enzyme against inactivation by free radicals. No protection was observed for the radiation-induced decrease of the endogenous tryptophan fluorescence. The experimental results are in line with the following interpretation: (i) the reduction of Mi(b)-CK dimer association is due to free radical-induced modification of Trp-264, situated at the dimer/dimer interface; (ii) the active-site Trp-223 is not a prime target for free radicals and is not involved in the inactivation of the enzyme; (iii) the inhibition of TSAC-induced dissociation of Mi(b)-CK, like enzyme inactivation, is primarily due to a modification of the active-site Cys-278.
Animals↗
[Photodynamic ablation and argon-plasma coagulation of premalignant and early-stage malignant lesions of the oesophagus--an alternative to surgery?].
Eleven patients with high-grade mucosal dysplasia in a columnar-lined oesophagus and 2 patients with a squamous carcinoma (uT1) underwent photodynamic therapy (PDT) and argon-plasma coagulation (APC). For PDT, 5-aminolevulinic acid (5-ALA) was given orally (60 mg/kg) and treated endoscopically with a light dose of 150 J/cm2 (100 mW/cm2) at 635 nm 4-6 h later. A second PDT was performed under the same conditions 2-3 weeks later. Patients who received APC were treated 4 times on average to reach radical Barrett mucosa eradication. All patients were consistently given medication to suppress acid production. No complications occurred in either group. Follow-up endoscopies and multiple biopsies for 3-42 months showed squamous regeneration in the dysplastic columnar-lined oesophagus in all 11 patients. Two patients with squamous carcinoma were found to have a recurrence 9 and 12 months later. PDT with 5-ALA-induced endogenous prophyrins, as well as APC combined with omeprazole protection, can eradicate superficial high-grade dysplastic mucosa in Barrett's oesophagus. However, we did not succeed in destroying a manifest carcinoma.
Aged↗
Characterization of the chick chorioallantoic membrane model as a short-term in vivo system for human skin.
We report on the cultivation and characterization of human skin on the chorioallantoic membrane of chicken eggs with the aim of replacing animals in short-term investigations in dermatology. Adult human split-thickness skin was grafted onto the chorioallantoic membrane of 5-day chick embryos. Grafts and surrounding host tissue were examined daily by in vivo stereomicroscopy and in histological sections and were characterized using a panel of monoclonal antibodies. The skin grafts were completely incorporated into the chorioallantoic membrane 2 days after transplantation. A remarkable angiogenesis occurred towards the grafts. Skin tissues revascularized within 2 or 3 days by reperfusion of the existing graft vasculature. Anastomosis of host and graft blood vessels occurred and the transplanted skin was nourished by the host blood supply as indicated by nucleated chick erythrocytes in the skin vessels. The skin grafts on the chorioallantoic membrane preserved an almost entire human phenotype. Besides a fully differentiated human epidermis and dermis containing all the cellular and extracellular constituents such as skin immune cells, capillary vessels composed of human endothelial cells were enclosed by a basement membrane of human origin. The integrin expression pattern formed in human skin transplants 5 days after grafting was identical to that of human skin controls before grafting.
Adult↗
Direct demonstration of a specific interaction between cyclophilin-D and the adenine nucleotide translocase confirms their role in the mitochondrial permeability transition.
A fusion protein between cyclophilin-D (CyP-D) and glutathione S-transferase (GST) was shown to bind to purified liver inner mitochondrial membranes (IMMs) in a cyclosporin A (CsA)-sensitive manner. Binding was enhanced by diamide treatment of the IMMs. Immobilized GST-CyP-D avidly bound a single 30 kDa protein present in Triton X-100-solubilized IMMs; immunoblotting showed this to be the adenine nucleotide translocase (ANT). Binding was prevented by pretreatment of the CyP-D with CsA, but not with cyclosporin H. Purified ANT also bound specifically to GST-CyP-D, but porin did not, even in the presence of ANT.
Animals↗
Reconstituted adenine nucleotide translocase forms a channel for small molecules comparable to the mitochondrial permeability transition pore.
Highly purified adenylate translocase (ANT) from rat heart mitochondria was functionally reconstituted as ATP/ADP exchange carrier in asolectin/cardiolipin vesicles. The ANT preparations used were free of porin, cyclophilin D, and Bax as analysed immunologically and by activity measurements. After pre-loading the ANT-containing proteoliposomes with ATP, malate or AMP, a gradual release of the trapped compounds by increasing the external Ca2+ concentrations could be demonstrated. N-Methyl-Val-4-cyclosporin did not inhibit the Ca2+ dependent release of internal substances from ANT liposomes. This inhibitor was found to be specific for the mitochondrial permeability transition pore (MTP) in intact mitochondria or reconstituted MTP-like protein complexes (e.g. hexokinase, porin, ANT complex). However, ADP in concentrations > 20 microM inhibited the liberation of internal compounds, while in contrast, atractyloside (30 microM) and HgCl2 (5 microM) both induced permeability of the ANT-containing liposomes resulting in a release of trapped substances. These results strongly suggest that ANT itself is capable to adopt a pore-like structure under conditions known to induce the permeability transition in mitochondria.
Animals↗
Complexes between porin, hexokinase, mitochondrial creatine kinase and adenylate translocator display properties of the permeability transition pore. Implication for regulation of permeability transition by the kinases.
Complexes between hexokinase, outer membrane porin, and the adenylate translocator (ANT) were recently found to establish properties of the mitochondrial permeability transition pore in a reconstituted system. The complex was extracted by 0.5% Triton X-100 from rat brain membranes and separated by anion exchanger chromatography. The molecular weight was approximately 400 kDa suggesting tetramers of hexokinase (monomer 100kDa). By the same method a porin, creatine kinase octamer, ANT complex was isolated and reconstituted in liposomes. Vesicles containing the reconstituted complexes both retained ATP that could be used by either kinase to phosphorylate external creatine or glucose. Atractyloside inhibited this activity indicating that the ANT was involved in this process and was functionally reconstituted. Exclusively from the hexokinase complex containing liposome internal malate or ATP was released by addition of Ca2+ in a N-methylVal-4-cyclosporin sensitive way, suggesting that the hexokinase porin ANT complex might include the permeability transition pore (PTP). The Ca2+ dependent opening of the PTP-like structure was inhibited by ADP (apparent I(50), 8 microM) and ATP (apparent I(50), 84 microM). Also glucose inhibited the PTP-like activity, while glucose-6-phosphate abolished this effect. Although porin and ANT were functionally active in vesicles containing the creatine kinase octamer complex, Ca2+ did not induce a release of internal substrates. However, after dissociation of the creatine kinase octamer, the complex exhibited PTP-like properties and the vesicles liberated internal metabolites upon addition of Ca2+. The latter process was also inhibited by N-methylVal-4-cyclosporin. The activity of peptidyl-prolyl-cis-trans-isomerase (representing cyclophilin) was followed during complex isolation. Cyp D was co-purified with the hexokinase complex, while it was absent in the creatine kinase complex. The inhibitory effect of N-methylVal-4-cyclosporin on the creatine kinase complex may be explained by direct interaction with the creatine kinase dimer that appeared to support octamer formation.
Adenosine Diphosphate↗
Mass spectrometric mapping of ion channel proteins (porins) and identification of their supramolecular membrane assembly.
Mass spectrometric peptide mapping, particularly by matrix-assisted laser desorption-ionization (MALDI-MS), has recently been shown to be an efficient tool for the primary structure characterization of proteins. In combination with in situ proteolytic digestion of proteins separated by one- and two-dimensional sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), mass spectrometric peptide mapping permits identification of proteins from complex mixtures such as cell lysates. In this study we have investigated several ion channel membrane proteins (porins) and their supramolecular assembly in mitochondrial membranes by peptide mapping in solution and upon digestion in the gel matrix. Porins are integral membrane proteins serving as nonspecific diffusion pores or as specific systems for the transport of substrates through bacterial and mitochondrial membranes. The well-characterized porin from Rhodobacter capsulatus (R.c.-porin) has been found to be a native trimeric complex by the crystal structure and was used as a model system in this study. R.c.-porin was characterized by MALDI-MS peptide mapping in solution, and by direct in situ-gel digestion of the trimer. Furthermore, in this study we demonstrate the direct identification of the noncovalent complex between a mitochondrial porin and the adenine nucleotide translocator from rat liver, by MALDI-MS determination of the specific peptides due to both protein sequences in the SDS-PAGE gel band. The combination of native gel electrophoresis and mass spectrometric peptide mapping of the specific gel bands should be developed as a powerful tool for the molecular identification of protein interactions.
Electrophoresis, Polyacrylamide Gel↗
Some new aspects of creatine kinase (CK): compartmentation, structure, function and regulation for cellular and mitochondrial bioenergetics and physiology.
Creatine kinase (CK) isoenzymes, specifically located at places of energy demand and energy production, are linked by a phosphocreatine/creatine (PCr/Cr) circuit, found in cells with intermittently high energy demands. Cytosolic CKs, in close conjunction with Ca(2+)-pumps, play a crucial role for the energetics of Ca(2+)-homeostasis. Mitochondrial Mi-CK, a cuboidal-shaped octamer with a central channel, binds and crosslinks mitochondrial membranes and forms a functionally coupled microcompartment with porin and adenine nucleotide translocase for vectorial export of PCr into the cytosol. The CK system is regulated by AMP-activated protein kinase via PCr/Cr and ATP/AMP ratios. Mi-CK stabilizes and cross-links cristae- or inner/outer membranes to form parallel membrane stacks and, if overexpressed due to creatine depletion or cellular energy stress, forms those crystalline intramitochondrial inclusions seen in some mitochondrial cytopathy patients. Mi-CK is a prime target for free radical damage by peroxynitrite. Mi-CK octamers, together with CK substrates have a marked stabilizing and protective effect against mitochondrial permeability transition pore opening, thus providing a rationale for creatine supplementation of patients with neuromuscular and neurodegenerative diseases.
Adenine Nucleotides↗
The molecular structure of mitochondrial contact sites. Their role in regulation of energy metabolism and permeability transition.
Contact sites between the outer and peripheral inner membrane of mitochondria are involved in protein precursor uptake and energy transfer. Hexokinase and mitochondrial creatine kinase could be attributed by different techniques to the energy transfer contacts. Kinetic analyses suggested a functional interaction between the kinases, outer membrane pore protein, and inner membrane adenylate translocator (ANT). This suggestion was strongly supported by isolation of hexokinase and creatine kinase complexes that were constituted of kinase oligomers, porin and ANT. Phospholipid vesicles carrying reconstituted kinase-porin-ANT complexes enclosed internal ATP in contrast to vesicles containing free porin only. This indicated that unspecific transport through porin was regulated by its interaction with a specific antiporter, ANT. A direct interaction between porin and ANT in the hexokinase complex conferred the reconstituted system with permeability properties reminiscent of the mitochondrial permeability transition (PT) pore. In the creatine kinase complex this interaction between porin and ANT was replaced by contact of both with the kinase octamer. Thus PT-pore-like functions were not observed unless the creatine kinase octamer was dissociated, suggesting that the ANT was locked in the antiporter state by interaction with the octamer. Indeed, reconstituted pure ANT showed PT-pore-like properties concerning Ca2+ sensitivity. However, as cyclophilin was missing, sensitivity against cyclosporin was not observed.
Animals↗
Photochemotherapy of experimental colonic tumours with intra-tumorally applied methylene blue.
INTRODUCTION: Phototoxicity of intra-tumoral injected methylene blue (MB+) was studied in 48 experimental colonic tumours in comparison with photosan-3, Zn-phthalocyanine and tetrasulphanated ClAl-phthalocyanine. METHODS: In mice. xenotransplanted subcutaneous tumours about 1 cm in diameter were treated photodynamically twice, with different sensitisers. The irradiation was performed at the sensitiser-specific wavelength, and a density of 100 mW/cm2 and a dose of 100 J/cm2. RESULTS: Light alone without sensitiser did not induce any effect in mice tumours. Surprisingly, Al-phthalocyanine could only be used for intratumoral injections because of toxic effects after intravenous applications in nude mice. Using MB+ (1%), 75% of the tumours were destroyed by a single photodynamic treatment (PDT). In addition, toxicity of MB+ was most intense when compared with Zn-phthalocyanine and photosan-3. However, after the second PDT, there was no statistically significant difference among these sensitisers. Dark toxicity of MB+ (1%) could be well demonstrated by sufficient sensitiser incorporation without irradiation, which led to a stationary tumour volume up to 3 weeks after injection. CONCLUSION: Intra-tumoral MB+ PDT is a potential treatment for inducing necrosis in vivo. With regard to tumour tissue, the selectivity of MB+ is high and depends on a precise local injection of the dye.
Animals↗
Complexes between hexokinase, mitochondrial porin and adenylate translocator in brain: regulation of hexokinase, oxidative phosphorylation and permeability transition pore.
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Animals↗
Nonlinear dynamics of intracellular methylene blue during light activation of cell cultures.
Methylene blue (MB+) is a well-known dye in medicine and has been discussed as an easily applicable drug for topical treatment in photodynamic therapy (PDT). Methylene blue can potentially be used as a redox indicator to detect the important redox reactions that are induced during PDT. The kinetics of this process was analyzed on a subcellular level with confocal laser scanning microscopy. BKEz-7 endothelial cells were incubated 4 h with 1 microM MB+. The fluorescence dynamics of MB+ during irradiation with 633 nm light was observed with subcellular resolution. Images were acquired at 0.5 s intervals (frame rate 1 image/0.5 s). Fluorescence was observed in the red channel of the laser scanning microscope. Synchronously, the phase-contrast image was visualized with the green channel. Morphological changes could therefore be correlated with the dynamics of MB+. In addition, the light-dose-dependent phototoxicity at 633 nm irradiation was determined by viable cell counting. After an induction period (phase I), fast fluorescent spikes could be observed in the whole cytoplasm, which decayed with a time constant of about 20 s (phase II), followed by a period of nearly constant fluorescence intensity (phase III) and exponential photobleaching (phase IV). Phase II exhibits highly nonlinear kinetics, which is hypothesized to correlate probably with a nonlinear quantal production of reactive oxygen species (ROS). Morphological cell changes were not observed during phase II. During phase III, a pycnotic cell nucleus developed. From the determination of viable cells we can conclude that a light dose applied within phase II was only sublethal in correlation with morphological observations. Overproduction of ROS leading finally to cell killing during phases III and IV is discussed.
Animals↗