Evaluation of the Chemical Stability, Membrane Permeability and Antiproliferative Activity of Cyclic Diarylheptanoids from European Hornbeam (Carpinus betulus L.).

Four cyclic diarylheptanoids-carpinontriols A (1) and B (2), giffonin X (3) and 3,12,17-trihydroxytricyclo [12.3.1.12,6]nonadeca-1(18),2(19),3,5,14,16-hexaene-8,11-dione (4)-were isolated from Carpinus betulus (Betulaceae). Chemical stability of the isolated diarylheptanoids was evaluated as a function of storage temperature (-15, 5, 22 °C) and time (12 and 23 weeks). The effect of the solvent and the pH (1.2, 6.8, 7.4) on the stability of these diarylheptanoids was also investigated. Compounds 2 and 4 showed good stability both in aqueous and methanolic solutions at all investigated temperatures. Only 2 was stable at all three studied biorelevant pH values. Degradation products of 1 and 3 were formed by the elimination of a water molecule from the parent compounds, as confirmed by ultrahigh-performance liquid chromatography-high-resolution tandem mass spectrometry (UHPLC-HR-MS). The permeability of the compounds across biological membranes was evaluated by the parallel artificial membrane permeability assay (PAMPA). Compound 3 possesses a logPe value of -5.92 ± 0.04 in the blood-brain barrier-specific PAMPA-BBB study, indicating that it may be able to cross the blood-brain barrier via passive diffusion. The in vitro antiproliferative activity of the compounds was investigated against five human cancer cell lines, confirming that 1 inhibits cell proliferation in A2058 human metastatic melanoma cells.

Hyperthermia-induced doxorubicin delivery from thermosensitive liposomes via MR-HIFU in a pig model.

PURPOSE: Encapsulation of cytotoxic drugs for a localized release is an effective way to increase the therapeutic window of such agents. In this article we present the localized release of doxorubicin (DOX) from phosphatidyldiglycerol (DPPG2) based thermosensitive liposomes using MR-HIFU mediated hyperthermia in a swine model. MATERIALS AND METHODS: German landrace pigs of weights between 37.5 and 53.5 kg received a 30-min infusion of DOX containing thermosensitive liposomes (50 mg DOX/m2). The pigs' biceps femoris was treated locally in two separate target areas with mild hyperthermia using magnetic resonance guided high intensity focused ultrasound, starting 10 min and 60 min after initiation of the infusion, respectively. The pharmacokinetics and biodistribution of DOX were determined and an analysis of the treatment parameters' influence was performed. RESULTS: Compared to untreated tissue, we found a 15-fold and a 7-fold increase in DOX concentration in the muscle volumes that had undergone hyperthermia starting 10 min and 60 min after the beginning of the infusion, respectively. The pharmacokinetic analysis showed a prolonged circulation time of DOX and a correlation between the AUC of extra-liposomal DOX in the bloodstream and the amount of DOX accumulated in the target tissue. CONCLUSIONS: We have demonstrated a workflow for MR-HIFU hyperthermia drug delivery that can be adapted to a clinical setting, showing that HIFU-hyperthermia is a suitable method for local drug release of DOX using DPPG2 based thermosensitive liposomes in stationary targets. Using the developed pharmacokinetic model, an optimization of the drug quantity deposited in the target via the timing of infusion and hyperthermia should be possible.

Decoding the substrate supply to human neuronal nitric oxide synthase.

Nitric oxide, produced by the neuronal nitric oxide synthase (nNOS) from L-arginine is an important second messenger molecule in the central nervous system: It influences the synthesis and release of neurotransmitters and plays an important role in long-term potentiation, long-term depression and neuroendocrine secretion. However, under certain pathological conditions such as Alzheimer's or Parkinson's disease, stroke and multiple sclerosis, excessive NO production can lead to tissue damage. It is thus desirable to control NO production in these situations. So far, little is known about the substrate supply to human nNOS as a determinant of its activity. Measuring bioactive NO via cGMP formation in reporter cells, we demonstrate here that nNOS in both, human A673 neuroepithelioma and TGW-nu-I neuroblastoma cells can be fast and efficiently nourished by extracellular arginine that enters the cells via membrane transporters (pool I that is freely exchangeable with the extracellular space). When this pool was depleted, NO synthesis was partially sustained by intracellular arginine sources not freely exchangeable with the extracellular space (pool II). Protein breakdown made up by far the largest part of pool II in both cell types. In contrast, citrulline to arginine conversion maintained NO synthesis only in TGW-nu-I neuroblastoma, but not A673 neuroepithelioma cells. Histidine mimicked the effect of protease inhibitors causing an almost complete nNOS inhibition in cells incubated additionally in lysine that depletes the exchangeable arginine pool. Our results identify new ways to modulate nNOS activity by modifying its substrate supply.

Early urinary and plasma biomarkers for experimental diabetic nephropathy.

BACKGROUND: As the prevalence of diabetes rises, its complications such as diabetic nephropathy affect an increaseing number of patients. Consequently, the need for biomarkers in rodent models which reflect the stage and course of diabetic nephropathy is high. This article focuses on Heart-type fatty acid binding protein (H-FABP), osteopontin (OPN), nephrin, and Neutrophil gelatinase-associated lipocalin (NGAL) in urine, and kidney injury molecule (KIM)-1, clusterin, and tissue inhibitior of metalloproteinases (TIMP) 1 in plasma in uni-nephrectomized rats with streptocotozin-induced type 1 diabetes mellitus, a common animal model to explore renal impairment in the setting of diabetes mellitus. METHODS: 23 male Wistar rats were uni-nephrectomized and subsequently divided into two study groups. The diabetic group received streptozotocin (STZ) via tail-vein injection, the non-diabetic group received citrate buffer without STZ. Subsequently, blood glucose, body weight, and blood pressure were checked regularly. After 18 weeks, animals were placed in metabolic cages, blood and urine obtained and subsequently organs were harvested after sacrifice. RESULTS: Blood glucose levels were highly increased in diabetic animals throughout the experiment, whereas systolic blood pressure did not differ between the study groups. At study end, classical biomarkers such as urinary albumin and protein and plasma cystatin c were only slightly but not significantly different between groups indicating a very early disease state. In contrast, urinary excretion of H-FABP, OPN, nephrin, and NGAL were highly increased in diabetic animals with a highly significant p-value (p < 0.01 each) compared to non-diabetic animals. In plasma, differences were found for calbindin, KIM-1, clusterin, TIMP-1, and OPN. These findings were confirmed by means of the area under the receiver operating characteristic curve (ROC-AUC) analysis. CONCLUSIONS: In summary, our study revealed elevated levels of new plasma and urinary biomarkers (urinary osteopontin, urinary nephrin, urinary NGAL, urinary H-FABP, plasma KIM-1, plasma TIMP-1) in uni-nephrectomized diabetic rats, an established rat model of diabetic nephropathy. These biomarkers appeared even before the classical biomarkers of diabetic nephropathy such as albuminuria and urinary protein excretion. The new biomarkers might offer an advantage to urinary albumin and plasma cystatin c with respect to early detection.

Arginine transport in human erythroid cells: discrimination of CAT1 and 4F2hc/y+LAT2 roles.

Since arginine metabolites, such as nitric oxide and polyamines, influence the expression of genes involved in erythroid differentiation, the transport of the cationic amino acid may play an important role in erythroid cells. However, available data only concern the presence in these cells of CAT1 transporter (system y(+)), while no information exists on the role of the heterodimeric transporters of system y(+)L (4F2hc/y(+)LAT1 and 4F2hc/y(+)LAT2) which operates transmembrane arginine fluxes cis-inhibited by neutral amino acids in the presence of sodium. Using erythroleukemia K562 cells and normal erythroid precursors, we demonstrate here that arginine transport in human erythroid cells is due to the additive contributions of a leucine-sensitive and leucine-insensitive component. In both cell types, leucine inhibition of arginine influx is much less evident in the absence of sodium, a hallmark of system y(+)L. In K562 cells, N-ethylmaleimide, a known inhibitor of CAT transporters (system y(+)), suppresses only a fraction of arginine influx corresponding to leucine-insensitive uptake. Moreover, in Xenopus oocytes coexpressing 4F2hc and y(+)LAT2, leucine exerts a marked inhibition of arginine transport, partially dependent on sodium, while no inhibition is seen in oocytes expressing CAT1. Lastly, silencing of SLC7A6, the gene for y(+)LAT2, lowers arginine transport and doubles the intracellular content of the cationic amino acid in K562 cells. We conclude that arginine transport in human erythroid cells is due to both system y(+) (CAT1 transporter) and system y(+)L (4F2hc/y(+)LAT2 isoform), which mainly contribute, respectively, to the influx and to the efflux of the cationic amino acid.

In human endothelial cells rapamycin causes mTORC2 inhibition and impairs cell viability and function.

AIM: Drug-eluting stents are widely used to prevent restenosis but are associated with late endothelial damage. To understand the basis for this effect, we have studied the consequences of a prolonged incubation with rapamycin on the viability and functions of endothelial cells. METHODS AND RESULTS: Human umbilical vein or aorta endothelial cells were exposed to rapamycin in the absence or in the presence of tumour necrosis factor alpha (TNFalpha). After a 24 h-incubation, rapamycin (100 nM) caused a significant cell loss associated with the increase of both apoptosis and necrosis, as quantified by propidium iodide staining, caspase 3 activity, and lactate dehydrogenase release. Rapamycin also impaired cell mobility, as assessed by a wound test, and promoted the formation of actin stress fibres, as determined with confocal microscopy. Moreover, the inhibitor prolonged TNFalpha-dependent E-selectin induction, inhibited endothelial nitric oxide synthase expression at both mRNA (quantitative real-time polymerase chain reaction) and protein level (enzyme-linked immunosorbent assay and western blot), and lowered bioactive nitric oxide output (RFL-6 reporter cell assay). Under the conditions adopted, rapamycin inhibited both mammalian target-of-rapamycin complexes (mTORC1 and mTORC2), as indicated by the reduced amount of raptor and rictor bound to mTOR in immunoprecipitates and by the marked hypophosphorylation of protein S6 kinase I (p70S6K) and Akt, determined by western blotting. The selective inhibition of mTORC1 by AICAR did not affect endothelial viability. CONCLUSION: A prolonged treatment with rapamycin impairs endothelial function and hinders cell viability. Endothelial damage seems dependent on mTORC2 inhibition.

Rapamycin stimulates arginine influx through CAT2 transporters in human endothelial cells.

In endothelial cells Tumor Necrosis Factor-alpha (TNFalpha) stimulates arginine transport through the increased expression of SLC7A2/CAT2 transcripts. Here we show that also rapamycin, an inhibitor of mTOR kinase, stimulates system y(+)-mediated arginine uptake in human endothelial cells derived from either saphenous (HSVECs) or umbilical veins (HUVECs). When used together with TNFalpha, rapamycin produces an additive stimulation of arginine transport in both cell models. These effects are observed also upon incubation with AICAR, a stimulator of Adenosine-Monophosphate-dependent-Protein Kinase (AMPK) that produces a rapamycin-independent inhibition of the mTOR pathway. Rapamycin increases the V(max) of high affinity arginine transport and causes the appearance of a low affinity component that is particularly evident if the treatment is carried out in the presence of TNFalpha. RT-qPCR studies have demonstrated that these kinetic changes correspond to the induction of both the high affinity transporter CAT2B and the low affinity isoform CAT2A. Western blot and immunocytochemical analyses indicate that, consistently, the expression of CAT2 proteins is also stimulated under the same conditions. These changes are associated with an increase of the intracellular arginine concentration but with a decrease of NO production. Thus, our data suggest that mTOR activity is associated with the repression of CAT2 expression at mRNA and protein level.

Activation of classical protein kinase C decreases transport via systems y+ and y+L.

Activation of protein kinase C (PKC) downregulates the human cationic amino acid transporters hCAT-1 (SLC7A1) and hCAT-3 (SLC7A3) (Rotmann A, Strand D, Martiné U, Closs EI. J Biol Chem 279: 54185-54192, 2004; Rotmann A, Vekony N, Gassner D, Niegisch G, Strand D, Martine U, Closs EI. Biochem J 395: 117-123, 2006). However, others found that PKC increased arginine transport in various mammalian cell types, suggesting that the expression of different arginine transporters might be responsible for the opposite PKC effects. We thus investigated the consequence of PKC activation by phorbol-12-myristate-13-acetate (PMA) in various human cell lines expressing leucine-insensitive system y(+) [hCAT-1, hCAT-2B (SLC7A2), or hCAT-3] as well as leucine-sensitive system y(+)L [y(+)LAT1 (SLC7A7) or y(+)LAT2 (SLC7A6)] arginine transporters. PMA reduced system y(+) activity in all cell lines tested, independent of the hCAT isoform expressed, while mRNAs encoding the individual hCAT isoforms were either unchanged or increased. System y(+)L activity was also inhibited by PMA. The extent and onset of inhibition varied between cell lines; however, a PMA-induced increase in arginine transport was never observed. In addition, when expressed in Xenopus laevis oocytes, y(+)LAT1 and y(+)LAT2 activity was reduced by PMA, and this inhibition could be prevented by the PKC inhibitor bisindolylmaleimide I. In ECV304 cells, PMA-induced inhibition of systems y(+) and y(+)L could be prevented by Gö6976, a specific inhibitor of conventional PKCs. Thymelea toxin, which activates preferentially classical PKC, had a similar inhibitory effect as PMA. In contrast, phosphatidylinositol-3,4,5-triphosphate-dipalmitoyl, an activator of atypical PKC, had no effect. These data demonstrate that systems y(+) and y(+)L are both downregulated by classical PKC.

Role of neutral amino acid transport and protein breakdown for substrate supply of nitric oxide synthase in human endothelial cells.

Endothelial dysfunction is often associated with a relative substrate deficiency of the endothelial nitric oxide synthase (eNOS) in spite of apparently high intracellular arginine concentrations. For a better understanding of the underlying pathophysiological mechanisms, we aimed to characterize the intracellular arginine sources of eNOS. Our previous studies in human endothelial EA.hy926 cells suggested the existence of two arginine pools: pool I can be depleted by extracellular lysine, whereas pool II is not freely exchangeable with the extracellular space, but accessible to eNOS. In this study, we demonstrate that the eNOS accessible pool II is also present in human umbilical vein endothelial cells (HUVECs), but not in ECV bladder carcinoma cells transfected with an expression plasmid for eNOS. In the endothelial cells, one part of pool II (referred to as pool IIA) consisted of recycling of citrulline to arginine. This part could be depleted by neutral amino acids that match the substrate profile of system N transporter 1 (SN1), presumably by the removal of intracellular citrulline. SN1 was expressed in EA.hy926 cells and HUVECs as shown by real-time RT-PCR. The second part of pool II (referred to as pool IIB) could not be depleted by any of the cationic or neutral amino acids tested. Our data demonstrate that pool IIB is nourished by protein breakdown and thus represents a substrate pool likely to accumulate protein-derived endogenous inhibitors of eNOS. Preferential use of the arginine pool IIB under pathophysiological conditions might therefore explain the arginine paradox.