PAC1 deficiency reduces chondrogenesis in atherosclerotic lesions of hypercholesterolemic ApoE-deficient mice.

BACKGROUND: Induction of chondrogenesis is associated with progressive atherosclerosis. Deficiency of the ADCYAP1 gene encoding pituitary adenylate cyclase-activating peptide (PACAP) aggravates atherosclerosis in ApoE deficient (ApoE-/-) mice. PACAP signaling regulates chondrogenesis and osteogenesis during cartilage and bone development. Therefore, this study aimed to decipher whether PACAP signaling is related to atherogenesis-related chondrogenesis in the ApoE-/- mouse model of atherosclerosis and under the influence of a high-fat diet. METHODS: For this purpose, PACAP-/-/ApoE-/-, PAC1-/-/ApoE-/-, and ApoE-/- mice, as well as wildtype (WT) mice, were studied under standard chow (SC) or cholesterol-enriched diet (CED) for 20 weeks. The amount of cartilage matrix in atherosclerotic lesions of the brachiocephalic trunk (BT) with maximal lumen stenosis was monitored by alcian blue and collagen II staining on deparaffinized cross sections. The chondrogenic RUNX family transcription factor 2 (RUNX2), macrophages [(MΦ), Iba1+], and smooth muscle cells (SMC, sm-α-actin) were immunohistochemically analyzed and quantified. RESULTS: ApoE-/- mice fed either SC or CED revealed an increase of alcian blue-positive areas within the media compared to WT mice. PAC1-/-/ApoE-/- mice under CED showed a reduction in the alcian blue-positive plaque area in the BT compared to ApoE-/- mice. In contrast, PACAP deficiency in ApoE-/- mice did not affect the chondrogenic signature under either diet. CONCLUSIONS: Our data show that PAC1 deficiency reduces chondrogenesis in atherosclerotic plaques exclusively under conditions of CED-induced hypercholesterolemia. We conclude that CED-related chondrogenesis occurs in atherosclerotic plaques via transdifferentiation of SMCs and MΦ, partly depending on PACAP signaling through PAC1. Thus, PAC1 antagonists or PACAP agonists may offer therapeutic potential against pathological chondrogenesis in atherosclerotic lesions generated under hypercholesterolemic conditions, especially in familial hypercholesterolemia. This discovery opens therapeutic perspectives to be used in the treatment against the progression of atherosclerosis.

Effect of chronic intermittent hypoxia (CIH) on neuromuscular junctions and mitochondria in slow- and fast-twitch skeletal muscles of mice-the role of iNOS.

BACKGROUND: Obstructive sleep apnea (OSA) imposes vascular and metabolic risks through chronic intermittent hypoxia (CIH) and impairs skeletal muscle performance. As studies addressing limb muscles are rare, the reasons for the lower exercise capacity are unknown. We hypothesize that CIH-related morphological alterations in neuromuscular junctions (NMJ) and mitochondrial integrity might be the cause of functional disorders in skeletal muscles. METHODS: Mice were kept under 6 weeks of CIH (alternating 7% and 21% O2 fractions every 30 s, 8 h/day, 5 days/week) compared to normoxia (NOX). Analyses included neuromuscular junctions (NMJ) postsynaptic morphology and integrity, fiber cross-sectional area (CSA) and composition (ATPase), mitochondrial ultrastructure (transmission-electron-microscopy), and relevant transcripts (RT-qPCR). Besides wildtype (WT), we included inducible nitric oxide synthase knockout mice (iNOS-/-) to evaluate whether iNOS is protective or risk-mediating. RESULTS: In WT soleus muscle, CIH vs. NOX reduced NMJ size (- 37.0%, p < 0.001) and length (- 25.0%, p < 0.05) together with fiber CSA of type IIa fibers (- 14%, p < 0.05) and increased centronucleated fiber fraction (p < 0.001). Moreover, CIH vs. NOX increased the fraction of damaged mitochondria (1.8-fold, p < 0.001). Compared to WT, iNOS-/- similarly decreased NMJ area and length with NOX (- 55%, p < 0.001 and - 33%, p < 0.05, respectively) or with CIH (- 37%, p < 0.05 and - 29%, p < 0.05), however, prompted no fiber atrophy. Moreover, increased fractions of damaged (2.1-fold, p < 0.001) or swollen (> 6-fold, p < 0.001) mitochondria were observed with iNOS-/- vs. WT under NOX and similarly under CIH. Both, CIH- and iNOS-/- massively upregulated suppressor-of-cytokine-signaling-3 (SOCS3) > 10-fold without changes in IL6 mRNA expression. Furthermore, inflammatory markers like CD68 (macrophages) and IL1ß were significantly lower in CIH vs. NOX. None of these morphological alterations with CIH- or iNOS-/- were detected in the gastrocnemius muscle. Notably, iNOS expression was undetectable in WT muscle, unlike the liver, where it was massively decreased with CIH. CONCLUSION: CIH leads to NMJ and mitochondrial damage associated with fiber atrophy/centronucleation selectively in slow-twitch muscle of WT. This effect is largely mimicked by iNOS-/- at NOX (except for atrophy). Both conditions involve massive SOCS3 upregulation likely through denervation without Il6 upregulation but accompanied by a decrease of macrophage density especially next to denervated endplates. In the absence of muscular iNOS expression in WT, this damage may arise from extramuscular, e.g., motoneuronal iNOS deficiency (through CIH or knockout) awaiting functional evaluation.

Characterization of atherosclerotic plaques in blood vessels with low oxygenated blood and blood pressure (Pulmonary trunk): role of growth differentiation factor-15 (GDF-15).

BACKGROUND: Growth differentiation factor (GDF)-15 is linked to inflammation, cancer, and atherosclerosis. GDF-15 is expressed in most tissues but is extremely induced under pathological conditions. Elevated serum levels are suggested as a risk factor and a marker for cardiovascular diseases. However, the cellular sources and the effects of GDF-15 on the cardiovascular system have not been completely elucidated including progression, and morphology of atherosclerotic plaques. Thus, this work aimed to characterize the influence of GDF-15 deficiency on the morphology of atherosclerotic plaques in blood vessels with low-oxygen blood and low blood pressure as the pulmonary trunk (PT), in hypercholesterolemic ApoE-/- mice. METHODS: GDF-15-/- ApoE-/- mice were generated by crossbreeding of ApoE-/-- and GDF-15-/- mice. After feeding a cholesterol-enriched diet (CED) for 20 weeks, samples of the brachiocephalic trunk (BT) and PT were dissected and lumen stenosis (LS) was measured. Furthermore, changes in the cellularity of the PT, amounts of apoptosis-, autophagy-, inflammation- and proliferation-relevant proteins were immunohisto-morphometrically analyzed. Additionally, we examined an atherosclerotic plaque in a human post mortem sample of the pulmonary artery. RESULTS: After CED the body weight of GDF-15-/-ApoE-/- was 22.9% higher than ApoE-/-. Double knockout mice showed also an 35.3% increase of plasma triglyceride levels, whereas plasma cholesterol was similar in both genotypes. LS in the BT and PT of GDF-15-/-ApoE-/- mice was significantly reduced by 19.0% and by 6.7% compared to ApoE-/-. Comparing LS in PT and BT of the same genotype revealed a significant 38.8% (ApoE-/-) or 26.4% (GDF-15-/-ApoE-/-) lower LS in the PT. Immunohistomorphometry of atherosclerotic lesions in PT of GDF-15-/-ApoE-/- revealed significantly increased levels (39.8% and 7.3%) of CD68 + macrophages (MΦ) and α-actin + smooth muscle cells than in ApoE-/-. The density of TUNEL + , apoptotic cells was significantly (32.9%) higher in plaques of PT of GDF-15-/-ApoE-/- than in ApoE-/-. Analysis of atherosclerotic lesion of a human pulmonary artery showed sm-α-actin, CD68+, TUNEL+, Ki67+, and APG5L/ATG+ cells as observed in PT. COX-2+ and IL-6+ immunoreactivities were predominantly located in endothelial cells and subendothelial space. In BT and PT of GDF15-/-ApoE-/- mice the necrotic area was 10% and 6.5% lower than in ApoE-/-. In BT and PT of GDF15-/-ApoE-/- we found 40% and 57% less unstable plaques than ApoE-/- mice. CONCLUSIONS: Atherosclerotic lesions occur in both, BT and PT, however, the size is smaller in PT, possibly due to the effect of the low-oxygen blood and/or lower blood pressure. GDF-15 is involved in atherosclerotic processes in BT and PT, although different mechanisms (e.g. apoptosis) in these two vessels seem to exist.

Effect of cholesterol re-supplementation and atorvastatin on plaque composition in the thoracic aorta of New Zealand white rabbits.

BACKGROUND: Effects of re-supplementation of a cholesterol-enriched diet (CEDrs) on size, cholesterol content and morphology of already existing plaques are not known to date. METHODS: A group of rabbits received standard chow (SC) for 6 weeks ("negative control"; for plasma lipid measurements only). Group I-IV received 2% CED (induction) for 6 weeks; thereafter, groups II-IV have been fed a SC (= cholesterol withdrawal) for 68 weeks. Afterwards, feeding of groups II-IV was continued as follows: Group II - 10 weeks SC, group III - 4 weeks 0.5% CED (~re-supplementation), afterwards 6 weeks SC (~withdrawal again); group IV - 4 weeks 0.5% CED (re-supplementation) + atorvastatin (2.5 mg/kg body weight/day), afterwards 6 weeks SC (~withdrawal again) + atorvastatin. Plasma lipids, but also plaque size, morphology and cholesterol contents of thoracic aortas were quantified. RESULTS: After CEDrs, plasma cholesterol levels were increased. However, after withdrawal of CEDrs, plasma cholesterol levels decreased, whereas the cholesterol content of the thoracic aorta was increased in comparison with the group without CEDrs. Plaque size remained unaffected. Atorvastatin application did not change plasma cholesterol level, cholesterol content of the thoracic aorta and plaque size in comparison with the group without drug treatment. However, atorvastatin treatment increased the density of macrophages (MΦ) compared with the group without treatment, with a significant correlation between densities of MΦ (Mac-1+) and apoptotic (TUNEL+; TP53+), antigen-presenting (HLA-DR+) or oxidatively stressed (SOD2+) cells. CONCLUSIONS: In rabbits with already existing plaques, CEDrs affects plaque morphology and cellular composition, but not plaque size. Despite missing effects on plasma cholesterol levels, cholesterol content of the thoracic aorta and size of already existing atherosclerotic plaques, atorvastatin treatment transforms the already existing lesions to a more active form, which may accelerate the remodelling to a more stable plaque.

[Chronic hypoxia and cardiovascular risk : Clinical significance of different forms of hypoxia]. / Chronische Hypoxie und kardiovaskuläres Risiko : Klinische Bedeutung verschiedener Hypoxieformen.

It is of fundamental importance to differentiate whether chronic hypoxia occurs intermittently or persistently. While chronic intermittent hypoxia (CIH) is found typically in patients with obstructive sleep apnea (OAS), chronic persistent hypoxia (CPH) is typically diagnosed in patients with chronic lung disease. Cardiovascular risk is markedly increased in patients with CIH compared to patients with CPH. The frequent change between oxygen desaturation and reoxygenation in patients with CIH is associated with increased hypoxic stress, increased systemic inflammation, and enhanced adrenergic activation followed by endothelial dysfunction and increased arteriosclerosis. The pathophysiologic consequences of CPH are less well understood. The relationship between CPH and the development of pulmonary hypertension, pulmonary heart disease as well as polycythemia has been established.

Mechanisms of the anti-proliferative and anti-inflammatory effects of the herbal fixed combination STW 5 (Iberogast®) on colon adenocarcinoma (HT29) cells in vitro.

INTRODUCTION: Several conventional pharmaceuticals like non-steroidal anti-inflammatory drugs (NSAIDS) or selective cyclooxygenase-2 (COX-2) inhibitors have been demonstrated to exert anti-proliferative effects and to induce apoptosis in a variety of cell lines, e.g. colon, stomach, or prostate cancer cells. STW 5 (Iberogast(®)), a combination of nine plant extracts, is widely used in the treatment of gastrointestinal disorders, including functional dyspepsia and irritable bowel syndrome for which the involvement of an inflammatory etiology is discussed. To investigate the possible anti-proliferative effects, STW 5 and its components have been tested by using the colon-carcinoma cell line HT-29. The analyses have been performed in comparison to acetylsalicylic acid (ASA) and diclofenac (Diclo), which are well-known to reduce colon carcinoma risk. RESULTS: STW 5 showed significant anti-proliferative and pro-apoptotic effects on HT-29 cancer cells, similar to NSAIDs under test. However, using the LDH assay, STW 5 revealed significantly lower cytotoxicity than Diclo at same concentrations. In contrast to NSAIDs, STW 5 induced COX-1/COX-2, caspase-3 and Bax mRNA expressions in HT-29 and blocked LPS mediated translocation of the NF-κB p65 from the cytoplasm into the nucleus in PMA-differentiated THP-1 macrophages. These effects might be relevant, e.g. for prevention of undesirable side effects like gastric erosions. CONCLUSION: Our data suggest that the pro-apoptotic effect of STW 5 on HT-29 cells is involving multiple targets and is possibly due to an activation of the caspase cascade via mitochondrial destabilization. Active concentrations of STW 5 are, in relation to therapeutic doses, comparable to those of ASA and Diclo, suggesting a similar favorable effect on colon carcinoma risk.

Anti-inflammatory effects of the willow bark extract STW 33-I (Proaktiv(®)) in LPS-activated human monocytes and differentiated macrophages.

INTRODUCTION: Willow bark extract is frequently used in the treatment of painful rheumatological diseases, such as arthritis and back pain. Its effect has been attributed to its main component salicin, but pharmacological studies have shown that the clinical efficacy of the willow bark extract cannot be explained by its salicin content alone. Therefore different modes of action have been suggested for the anti-inflammatory effect of willow bark extract. Here, we report in vitro data revelling the effect and mode of action of the aqueous willow bark extract STW 33-I as well as a water-soluble fraction (fraction E [Fr E]) in comparison with well-known non-steroidal anti-inflammatory drugs (NSAIDs) like aspirin (ASA) and diclofenac (Diclo) on pro-inflammatorily activated human monocytes and differentiated macrophages. RESULTS: STW 33-I and the water-soluble Fr E showed concentration-dependent and significant anti-inflammatory effects in lipopolysaccharide-activated monocytes. Both inhibited the intracellular protein expression of tumour necrosis factor-alpha (TNFα) as well as the mRNA expression of TNFα and cyclooxygenase 2 (COX-2), and the release of nitric oxide (NO). In addition, apoptosis of pro-inflammatorily activated monocytes was induced. Furthermore, treatment of activated macrophages with STW 33-I inhibited the nuclear translocation of the p65 subunit of the nuclear transcription factor-kappa B (NF-κB p65). CONCLUSIONS: The present in vitro investigations suggest a significant anti-inflammatory activity of willow bark water extract STW 33-1 and of its water-soluble fraction by inhibiting pro-inflammatory cytokines (TNFα), COX-2 and nuclear translocation of the transcription factor NF-κB in pro-inflammatorily activated monocytes. Our results provide further evidence for the therapeutic use of STW 33-I in inflammation-related disorders.

Novel systemic cardiovascular disease biomarkers.

Motivated by the challenge of risk assessment in a heterogeneous population and guided by advances in our knowledge of the pathobiology of cardiovascular diseases (CVD), basic and clinical scientists have maintained substantial interest in the development and application of novel biomarkers for risk stratification of CVD. In particular, strategies to identify and combine multiple biomarkers, which may reflect diverse pathobiological contributors to the onset and complications of CVD, have been arising as an approach to improve more effectively the risk assessment and target therapy. Moreover, comparative evaluations of novel markers are necessary to estimate these candidates for integration into present and future strategies. In this review we consider the recent in-depth knowledge and advances with the use of systemic biomarkers in the area of CVD with special attention on inflammatory markers and those that can predict an individual arteriosclerotic disease stage.

The early immunological response to acute ischemic stroke: differential gene expression in subpopulations of mononuclear cells.

UNLABELLED: Peripheral blood mononuclear cells (PBMCs), i.e. lymphocytes, monocytes and macrophages are key players in the development of innate and adaptive immune responses. However, little is known about their properties in patients with acute stroke. EXPERIMENTAL PROCEDURES: We presently characterized the early time course of PBMC subpopulations in 19 patients with acute ischemic stroke and symptom onset below 6 h compared to 19 age-matched healthy subjects. Immediately after acute ischemic stroke, as well as 1 and 3 days thereafter, PBMC subpopulations (cluster of differentiation [CD]3+, CD14+, CD19+, CD68+) were isolated by magnetic bead system and the expression of proinflammatory (CD40, tumor necrosis factor-alpha [TNFalpha]), proapoptotic (caspase-3 [CPP32], poly(ADP-ribose) polymerase [PARP]) and adhesion relevant (CD38) genes was measured by quantitative polymerase chain reaction (PCR). Furthermore, besides routine parameters, plasma levels of oxidized low-density lipoproteins (oxLDL) were studied. RESULTS: In comparison to healthy subjects, patients revealed (i) twofold elevated plasma oxLDL concentrations, (ii) decreased (15%) blood cholesterol levels, and (iii) a 40% decrease in total number of lymphocytes. Furthermore, the majority of PBMC subpopulations revealed an increased expression of proinflammatory, proapoptotic or adhesion-relevant genes. Significant positive correlations were observed between expression of most of these genes in PBMCs and individual plasma oxLDL concentrations. CONCLUSION: Elevated expression of proinflammatory, proapoptotic and adhesion genes in subsets of PBMCs after ischemic stroke may contribute to an immunodepressive syndrome, possibly due to increased plasma oxLDL levels.

TNF-alpha and the IFN-gamma-inducible protein 10 (IP-10/CXCL-10) delivered by parvoviral vectors act in synergy to induce antitumor effects in mouse glioblastoma.

Interferon-gamma-inducible protein 10 is a potent chemoattractant for natural killer cells and activated T lymphocytes. It also displays angiostatic properties and some antitumor activity. Tumor necrosis factor-alpha (TNF-alpha) is a powerful immunomodulating cytokine with demonstrated tumoricidal activity in various tumor models and the ability to induce strong immune responses. This prompted us to evaluate the antitumor effects of recombinant parvoviruses designed to deliver IP-10 or TNF-alpha into a glioblastoma. When Gl261 murine glioma cells were infected in vitro with an IP-10- or TNF-alpha-transducing parvoviral vector and were subcutaneously implanted in mice, tumor growth was significantly delayed. Complete tumor regression was observed when the glioma cells were coinfected with both the vectors, demonstrating synergistic antitumor activity. In an established in vivo glioma model, however, repeated simultaneous peritumoral injection of the IP-10- and TNF-alpha-delivering parvoviruses failed to improve the therapeutic effect as compared with the use of a single cytokine-delivering vector. In this tumor model, cytokine-mediated immunostimulation, rather than inhibition of vascularization, is likely responsible for the therapeutic efficacy.

Muscular adaptations to computer-guided strength training with eccentric overload.

AIMS: In order to investigate the muscular adaptations to a novel form of strength training, 18 male untrained subjects performed 4 weeks of low resistance-high repetition knee extension exercise. METHODS: Nine of them trained on a conventional weight resistance device (Leg curler, CON/ECC group), with loads equivalent to 30% of the concentric one-repetition maximum (1RM) for both the concentric and eccentric phase of movement. The other nine trained on a newly developed computer-driven device (CON/ECC-OVERLOAD group) with the concentric load equivalent to 30% of the concentric 1RM and the eccentric load equivalent to 30% of the eccentric 1RM. RESULTS: Training resulted in significantly (P < or = 0.05) increased peak torque and a tendency (P=0.092) to increased muscle cross-sectional area for the CON/ECC-OVERLOAD but not the CON/ECC group, while strength endurance capacity was significantly (P < or = 0.05) increased in the CON/ECC group only. RT-PCR revealed significantly increased myosin heavy chain (MHC) IIa and lactate dehydrogenase (LDH) A mRNAs, a tendency for increased MHC IIx mRNA (P = 0.056) and high correlations between the changes in MHC IIx and LDH A mRNAs (r=0.97, P=0.001) in the CON/ECC-OVERLOAD group. CONCLUSIONS: These results indicate a shift towards a more type II dominated gene expression pattern in the vasti laterales muscles of the CON/ECC-OVERLOAD group in response to training. We suggest that the increased eccentric load in the CON/ECC-OVERLOAD training leads to distinct adaptations towards a stronger, faster muscle.

Effects of low-resistance/high-repetition strength training in hypoxia on muscle structure and gene expression.

To test the hypothesis that severe hypoxia during low-resistance/high-repetition strength training promotes muscle hypertrophy, 19 untrained males were assigned randomly to 4 weeks of low-resistance/high-repetition knee extension exercise in either normoxia or in normobaric hypoxia ( FiO(2) 0.12) with recovery in normoxia. Before and after the training period, isokinetic strength tests were performed, muscle cross-sectional area (MCSA) measured (magnetic resonance imaging) and muscle biopsies taken. The significant increase in strength endurance capacity observed in both training groups was not matched by changes in MCSA, fibre type distribution or fibre cross-sectional area. RT-PCR revealed considerable inter-individual variations with no significant differences in the mRNA levels of hypoxia markers, glycolytic enzymes and myosin heavy chain isoforms. We found significant correlations, in the hypoxia group only, for those hypoxia marker and glycolytic enzyme mRNAs that have previously been linked to hypoxia-specific muscle adaptations. This is interpreted as a small, otherwise undetectable adaptation to the hypoxia training condition. In terms of strength parameters, there were, however, no indications that low-resistance/high-repetition training in severe hypoxia is superior to equivalent normoxic training.

Enhanced iodide transport after transfer of the human sodium iodide symporter gene is associated with lack of retention and low absorbed dose.

Transfer of the sodium iodide symporter (hNIS) has been proposed as a new principle of cancer gene therapy. Using clinically relevant doses of (131)I for the treatment of NIS-expressing prostate carcinoma cells, we investigated the kinetics and the absorbed doses obtained in these tumors. hNIS-expressing cell lines accumulated up to 200 times more iodide when compared to wild-type cells. However, a rapid efflux of the radioactivity (80%) occurred during the first 20 min after replacement of the medium. In rats, the hNIS-expressing tumors accumulated up to 20 times more iodide when compared to contralateral transplanted wild-type tumors. After 24 h and doses of 550, 1200 or 2400 MBq/m(2) hNIS-expressing tumors lost 89, 89 and 91% of the initial activity, respectively. Dosimetric calculations showed that 1200 MBq/m(2) resulted in 3+/-0.5 Gy (wild-type tumor 0.15+/-0.1 Gy) and 2400 MBq/m(2) resulted in 3.1+/-0.9 Gy (wild-type tumor 0.26+/-0.02 Gy). Although transduction of the hNIS gene induces iodide transport in rat prostate adenocarcinoma a rapid efflux occurs, which leads to a low absorbed dose in genetically modified tumors. With regard to a therapeutic application additional conditions need to be defined leading to iodide trapping.

Plasma cystine concentration and redox state in aging and physical exercise.

Because redox-regulated signalling pathways are often modulated by the thiol/disulfide redox state (REDST), changes in plasma REDST may possibly account for pathological processes. We, therefore, investigated the mechanisms that account for changes in the plasma REDST as derived in first approximation from the cystine and acid soluble thiol (mainly cysteine) concentrations. Elderly subjects (studies A) and younger subjects after intensive physical exercise (IPE) (study B) i.e. subjects in conditions typically associated with decreased insulin responsiveness, showed, on the average, an increase in the plasma total free amino acid (TAA) concentration to approximately 3000 microM, including an increase in cystine but no increase in the thiol concentration if compared with controls. The REDST was decreased accordingly. A study on the postabsorptive amino acid exchange rates across the lower extremities (study C) indicated that a TAA level > or =2800 microM supports a balanced net protein synthesis even under conditions of weak insulin stimulation, suggesting that high TAA levels may prevent the release of cysteine into the blood in the postabsorptive state. Collectively, these studies indicate that the age-related oxidative shift in plasma REDST may result from the decrease in amino acid clearance capacity and may be aggravated by excessive physical exercise.

Glucose transport and apoptosis after gene therapy with HSV thymidine kinase.

The relation between tumour metabolism and induction of apoptosis by gene therapy was investigated in a rat Morris hepatoma (MH3924A) model expressing the HSV thymidine kinase (HSVtk) gene. In vivo the amount of glucose transporter (GLUT1 and GLUT3 isoforms) expressing cells was determined in tumours of untreated and treated animals using immunohistochemistry. In vitro uptake studies with 2-fluoro-2-deoxy-D-glucose (FDG), 3-O-methylglucose and thymidine (TdR) and a TUNEL (TdT-mediated dUTP nick end labelling) assay for the assessment of apoptosis were done immediately and 24 h after treatment of the recombinant cells with different doses of ganciclovir (GCV). Immunohistochemistry revealed a significant increase in GLUT1 in treated tumours which showed enhanced transport activity for FDG. In vitro the FDG and 3-O-methylglucose uptake increased to 186% when compared with that of the non-treated cells immediately after incubation with GCV. However, 24 h later the FDG uptake had declined to its normal level, whereas the accumulation of 3-O-methylglucose remained elevated. The uptake of TdR, which was determined simultaneously, decreased in the acid-insoluble fraction of the cells to 27% and 11%, respectively, immediately and 24 h after therapy, while in the acid-soluble fraction it increased to 229% and to 167%, respectively. Employing the TUNEL technique, 25% of cells were found to be apoptotic 24 h after the termination of GCV treatment. Inhibition of glucose transport by cytochalasin B or competition with deoxyglucose resulted in a 78% (cytochalasin B) and 88% (deoxyglucose) decrease in FDG uptake in the recombinant hepatoma cells and in an increase in the apoptotic cell fraction. It is concluded that inhibition of enhanced glucose transport in GCV-treated cells increased apoptosis. Therefore, enhanced glucose transport seems to represent a stress reaction of tumour cells dedicated for the prevention of cell death.

Investigation of a potential scintigraphic marker of apoptosis: radioiodinated Z-Val-Ala-DL-Asp(O-methyl)-fluoromethyl ketone.

The imaging of apoptosis represents an attractive diagnostic goal in the area of tumor therapy, degenerative diseases and organ transplantation. Since caspases play a key role during the early period of the intracellular signal cascade of cells undergoing apoptosis we considered benzyloxycarbonyl-Val-Ala-DL-Asp(O-methyl)-fluoromethyl ketone [Z-VAD-fmk], a pan-caspase inhibitor, as a potential apoptosis imaging agent. Applying the Tl(TFA)(3)/[131I]iodide method Z-VAD-fmk was successfully labeled at the benzyloxycarbonyl protecting group. The success of radioiodination, however, depended on the presence of carrier iodide resulting in specific radioactivities of 2.6 GBq/micromol and the formation of a mixture of the 2- and 4-iodophenyl derivative (61%) which could not be separated by HPLC. Uptake measurements were performed with Morris hepatoma cells (MH3924Atk8) which showed expression of the Herpes Simplex Virus thymidine kinase (HSVtk) gene. Apoptosis was induced by treatment of the cells with 25 microM ganciclovir. The TUNEL assay revealed 1.3 +/-0.3 and 23 +/-1.1% apoptotic cells immediately and 24 h after therapy, respectively. A two-fold increase of [131I]IZ-VAD-fmk uptake was found at the end of treatment with the HSVtk/suicide system which constantly remained elevated for the following 4 hours. The slow cellular influx and lack of uptake saturation of [131I]IZ-VAD-fmk are evidence for simple diffusion as transport mechanism. In addition, the absolute cellular uptake of [131I]IZ-VAD-fmk was found to be low. This quality was related to the rather high lipophilicity of [131I]IZ-VAD-fmk causing unspecific binding to macromolecules in the medium. Instead of using an inhibitor, synthetic caspase substrates are currently investigated which may accumulate in the apoptotic cell by metabolic trapping thereby enhancing the imaging signal.

Expression of growth differentiation factor-15/ macrophage inhibitory cytokine-1 (GDF-15/MIC-1) in the perinatal, adult, and injured rat brain.

We and others have recently cloned a new member of the transforming growth factor-beta superfamily, growth differentiation factor-15/ macrophage inhibitory cytokine-1 (GDF-15/MIC-1). Using in situ hybridization and immunohistochemistry, we determined the distribution of GDF-15/MIC-1 mRNA and protein in the perinatal and cryolesioned adult rat brain. The choroid plexus epithelium of all ventricles represents the site of strongest and almost exclusive mRNA expression in the normal perinatal and adult brain. The newborn rat brain reveals GDF-15/MIC-1 immunoreactivity (ir) in ependymal cells lining the ventricles, in the striatal subventricular zone, and in populations of nonneural cells of the thalamic/hippocampal lamina affixa, in addition to that in the choroid plexus. Unilateral cryogenic cortical lesioning induced a significant increase of GDF-15/MIC-1 mRNA expression and ir at the lesion site and expression in presumed neurons within the dorsal thalamic area. At the lesion site, GDF-15/MIC-1-producing cells showed immuncytochemical features of neurons, macrophages, and activated microglial cells. Fluorescent microscopy revealed both intra- and extracellular GDF-15/MIC-1 ir. Up-regulation of GDF-15/MIC-1 in activated macrophages (Mstraight phi) is also supported by RT-PCR, ICC, and Western blot experiments showing pronounced induction of GDF-15/MIC-1 expression (mRNA and protein) in retinoic acid/phorbol ester-stimulated human M phi. Our data suggest that 1) GDF-15/MIC-1 is secreted into the cerebrospinal fluid and 2) in the newborn brain may penetrate through the ependymal lining and act on developing neurons and/or glial cells. As a constituent of cells in the lamina affixa, the protein might be involved in the regulation of mesenchyme-epithelial interactions. Finally, GDF-15/MIC-1 may also act within the antiinflammatory cytokine network activated in CNS lesions.

Apoptosis and changes in glucose transport early after treatment of Morris hepatoma with gemcitabine.

Apoptosis has been described as an energy-consuming process. This combined in vivo/in vitro study investigated the effects of the antineoplastic agent gemcitabine on tumour metabolism and on the induction of apoptosis. Dynamic positron emission tomography (PET) measurements of fluorine-18 fluorodeoxyglucose (FDG) uptake were done in rats bearing Morris hepatoma prior to and after therapy with 90 mg gemcitabine/kg b.w. Furthermore, thymidine (TdR) incorporation into the DNA of these tumours was determined. In vitro measurements of FDG and TdR uptake were performed immediately and 24 h after the end of gemcitabine treatment, and the amount of apoptotic cells was determined using the TUNEL reaction. In vivo an increase in FDG transport and phosphorylation occurred early after gemcitabine treatment, although TdR incorporation into the DNA of the tumours declined. In vitro, an enhanced glucose transport, an increase in TdR uptake in the cytoplasm and a decrease in TdR incorporation in the nucleic acid fraction early after treatment occurred. Inhibition of glucose transport caused an increase in the amount of apoptotic cells. The increase in glucose uptake and TdR metabolism early after therapy is interpreted as a stress reaction of the tumour cells, protecting the cells from apoptosis during this early period after exposure to cytotoxic drugs like gemcitabine.

Intercaval block in normal canine hearts : role of the terminal crest.

BACKGROUND: The intriguing monotony in the occurrence of intercaval conduction block during typical atrial flutter suggests an anatomic or electrophysiological predisposition for conduction abnormalities. METHODS AND RESULTS: To determine the location of and potential electrophysiological basis for conduction block in the terminal crest region, a high-density patch electrode (10x10 bipoles) was placed on the terminal crest and on the adjacent pectinate muscle region in 10 healthy foxhounds. With a multiplexer mapping system, local activation patterns were reconstructed during constant pacing (S(1)S(1)=200 ms) and introduction of up to 2 extrastimuli (S(2), S(3)). Furthermore, effective refractory periods were determined across the patch. If evident through online analysis, the epicardial location of conduction block was marked for postmortem verification of its endocardial projection. Marked directional differences in activation were found in the terminal crest region, with fast conduction parallel to and slow conduction perpendicular to the intercaval axis (1.1+/-0.4 versus 0.5+/-0.2 m/s, P<0.01). In the pectinate muscle region, however, conduction velocities were similar in both directions (0.5+/-0.3 versus 0.6+/-0.2 m/s, P=NS). Refractory patterns were relatively homogeneous in both regions, with local refractory gradients not >30 ms. During S(3) stimulation, conduction block parallel to the terminal crest was inducible in 40% of the dogs compared with 0% in the pectinate muscle region. CONCLUSIONS: Even in normal hearts, inducible intercaval block is a relatively common finding. Anisotropic conduction properties would not explain conduction block parallel to the intercaval axis in the terminal crest region, and obviously, refractory gradients do not seem to play a role either. Thus, the change in fiber direction associated with the terminal crest/pectinate muscle junction might form the anatomic/electrophysiological basis for intercaval conduction block.

Ceramide induces aSMase expression: implications for oxLDL-induced apoptosis.

Sphingomyelinase (SMase) stimulation and subsequent ceramide generation are suggested to be involved in signal transduction of stress-induced apoptosis. We now show that apoptosis of human macrophages (MPhi) and fibroblasts initiated by oxidized low density lipoproteins (minimally modified LDL, mmLDL) is associated with an increase in acid SMase (aSMase, E.C. 3.1.4.12) expression and ceramide concentration. Application of a novel, potent, and specific inhibitor of aSMase expression (NB6) diminished the effects of mmLDL and C6-ceramide treatment by inhibiting transcription via Sp1 and AP-2. Moreover, apoptosis was abolished after mmLDL and C6-ceramide treatment of hereditary aSMase-deficient fibroblasts (from Niemann-Pick patients). We suggest that in mmLDL-initiated apoptosis 1) enhanced ceramide generation via aSMase appears to be required as well as 2) a positive feedback control of aSMase expression by the increase in intracellular ceramide concentration.