Gadolinium-Based Nanoparticles Can Overcome the Radioresistance of Head and Neck Squamous Cell Carcinoma Through the Induction of Autophagy.

Radiation therapy is a mainstay in the therapeutic management of Head and Neck Squamous Cell Carcinoma (HNSCC). Despite significant progress in this field, radioresistance still accounts for most treatment failures. Gadolinium-based nanoparticles (GBNs) have shown great promises as radiosensitizers but the underlying sensitizing mechanism is still largely unknown with regards to the disparities obtained in in vitro studies. In this study, we show that a new formulation of GBNs, AGuIX®, can radiosensitize HNSCC after cell uptake and further accumulation in lysosomes. Although radiation alone triggered late apoptosis and mitochondrial impairment, the pre-treatment with GBNs led to complex DNA damage and a specific increase of autophagic cell death. In addition, a significant radio-enhancement effect was obtained after the pre-conditioning of cells with a glutathione inhibitor before GBNs treatment and radiation exposure. Overall, our results provide additional information on the radio-enhancing properties of GBNs in the management of radioresistant HNSCC.

Comparison of the contractile responses of human coronary bypass grafts and monkey arteries to human urotensin-II.

Human urotensin-II (hU-II) is a cyclic peptide recently cloned in humans and present in human cardiac tissue and human arteries. The effects of hU-II were studied on human coronary bypass grafts in vitro. In three out of eight human mammary arteries, and two out of three human radial arteries, hU-II caused contraction; human saphenous veins did not respond to hU-II. When it exists, the contraction slowly develops and has a low-to-moderate intensity. All radial arteries obtained from young healthy non-human primates contracted strongly to hU-II.

Temperature-dependent basal tone in isolated human saphenous veins: implication of TP-receptors.

Cutaneous blood vessels are very sensitive to changes in environmental temperature. The influence of variations in local temperature on the mechanisms involved in the basal tone, present in isolated human saphenous veins has not yet been studied. In the present study, segments with and without endothelium of human saphenous veins obtained from coronary bypass surgery patients were mounted for isometric tension recording in oxygenated physiological salt solution (PSS). After stabilisation of the basal tone, the local temperature was rapidly either decreased from 37 degrees C to 24 degrees C (cooling) or increased from 37 degrees C to 42 degrees C (warming). When antagonists or inhibitors were used the preparations were incubated for 30 min with the drugs. During basal conditions, cooling caused relaxations of the saphenous vein segments with endothelium and warming caused contractions; the absence of the endothelium did not modify these responses. In veins without endothelium, the warming-induced contractions were significantly inhibited by verapamil (10 microM) and by the antagonist of TP-receptors (receptors for thromboxane A2) Bay u 3405 (1 microM). The warming induced contractions were not affected by cyclooxygenase or lipoxygenase inhibition. At 37 degrees C, the isoprostanes (8-iso-PGE2 and 8-iso-PGF2alpha) induced potent contractions that were significantly inhibited by Bay u 3405 (1 microM). The data show that a basal tone is present in isolated resting human saphenous vein segments at 37 degrees C. This basal tone is decreased by local cooling and enhanced by local warming and is not dependent on the presence of the endothelium. The warming-induced contraction of the veins is mediated by a non-cyclooxygenase, non-lipoxygenase metabolite (isoprostane?) that interacts with TP-receptors and via an extracellular calcium-dependent pathway.

Effect of chronic treatment with the inducible nitric oxide synthase inhibitor N-iminoethyl-L-lysine or with L-arginine on progression of coronary and aortic atherosclerosis in hypercholesterolemic rabbits.

BACKGROUND: We examined the implications of iNOS in atherosclerosis progression using the selective inducible NO synthase (iNOS) inhibitor N:-iminoethyl-L-lysine (L-NIL) in hypercholesterolemic rabbits. METHODS AND RESULTS: Nine rabbits were fed a 0.3% cholesterol diet for 24 weeks (Baseline group); 25 animals were maintained on the diet and treated for 12 extra weeks with L-NIL (5 mg x kg(-1) x d(-1), L-NIL group, n=8), vehicle (Saline group, n=9), or L-arginine (2.25%, L-Arg group, n=8). In abdominal aortas of Saline rabbits, the lesions (53.7+/-5.7%, Baseline) increased to 75.0+/-5.0% (P:<0.05) but remained unaltered in the L-NIL group (63. 4+/-6.6%). Similar results were obtained for the intima/media ratio in thoracic aortas. In coronary arteries, the intima/media ratio was comparable in Baseline (0.68+/-0.18) and Saline (0.96+/-0.19) rabbits but decreased to 0.34+/-0.19 (P:<0.05) in L-NIL rabbits. L-Arginine had beneficial effects only in abdominal aortas. An increased thoracic aorta collagen content was found in Saline and L-Arg but not in L-NIL rabbits. In thoracic aortas of the Saline group, acetylcholine caused modest relaxations that slightly increased by L-arginine but not by L-NIL. Relaxations to nitroglycerin were ameliorated by L-NIL. CONCLUSIONS: This is the first study showing that chronic treatment with an iNOS inhibitor, L-NIL, limits progression of preexisting atherosclerosis in hypercholesterolemic rabbits. Increased intimal collagen accumulation may participate in iNOS-induced atherosclerosis progression.

Platelets and prostacyclin in arterial bypasses: implications for coronary artery surgery.

BACKGROUND: We investigated effects of platelets and prostacyclin formation in human internal mammary (IMA) and radial (RA) arteries. METHODS: IMA and RA segments were suspended in organ bath with increasing concentrations of platelets. Experiments were applied with and without ketanserin, a 5HT2 receptor antagonist, or U3405, a TXA2 receptor antagonist. The release of prostacyclin (PGI2) was assessed by enzyme immunoassay in vessels without endothelium, before and after contraction with angiotensin (AT) I-II. RESULTS: In IMA and RA with endothelium, platelets caused contractions, significantly enhanced in arteries without endothelium. Contractions to platelets were higher in RA than in IMA. U3405 reduced the platelet induced contractions in RA but not in IMA. Ketanserin inhibited the platelet induced contractions in IMA and RA. The basal release of PGI2 was more important in IMA than in RA. Addition of AT/I-II significantly reduced the release of PGI2 in IMA but not in RA. CONCLUSIONS: The RA responds more powerfully to platelets than IMA. Protective system with PGI2 seems to be more powerless in RA than in IMA. This accentuates the importance of antispastic and antiplatelet drugs when arteries are used for coronary artery bypass surgery.

Urinary nitrate excretion in cholesterol-fed rabbits: effect of a chronic treatment by N-iminoethyl-L-lysine, a selective inhibitor of inducible nitric oxide synthase.

To evaluate the influence of atherosclerosis on the global production of NO, we studied the effect of a 0.3% cholesterol-enriched diet on urinary nitrate excretion in rabbits during 69 weeks. To examine whether the inducible nitric oxide synthase (iNOS) present in atherosclerotic lesions could participate in NO excretion, hypercholesterolemic rabbits were treated chronically with the selective iNOS inhibitor, N-iminoethyl-L-lysine (L-NIL; 5 mg/kg/day). Urinary nitrate excretion was higher in hypercholesterolemic than in control rabbits throughout the study period and decreased progressively with time in both groups; L-NIL had no significant effect on urinary nitrate excretion. These data illustrate that systemic NO production is enhanced in hypercholesterolemia and that iNOS, present within the plaque, might not participate in this enhanced NO production.

Synthesis and biological evaluation of new tetrahydronaphthalene derivatives as thromboxane receptor antagonists.

New polysubstituted tetrahydronaphthalene derivatives were prepared as thromboxane receptor (TP-receptor) antagonists. Within this series of compounds S 18886 has been identified as an orally active, highly potent antagonist with a very long duration of action in different species.

New tetrahydronaphthalene derivatives as combined thromboxane receptor antagonists and thromboxane synthase inhibitors.

A pyridine group was linked to the tetrahydronaphthalene moiety of the derivatives described in the preceding paper, to afford new combined thromboxane receptor (TP-receptor) antagonists and synthase inhibitors. The most interesting compound 2f inhibits TXA2 synthase with an IC50 value of 0.64 microM and the aggregation of human platelets with an IC50 value of 0.063 microM and shows a long duration of action in different species after oral administration.

Identification of enteroviruses by indirect immunofluorescence using monoclonal antibodies.

BACKGROUND: Serum neutralization (Nt) is used most often to type enterovirus isolates, but it is labor-intensive, expensive, and supplies of reference antisera for Nt are limited. Alternative methods of enterovirus typing are needed. OBJECTIVES: To investigate the use of indirect immunofluorescence (IFA) with commercially available monoclonal antibodies (MAbs) as an alternative to Nt for the identification of enteroviruses. STUDY DESIGN: Two MAb blends (one for coxsackie B viruses and one for echoviruses 4, 6, 9, 11, 30, and 34) and a coxsackie A9 MAb were used to screen 465 clinical isolates over a period of two years. Virus isolates which tested positive with one of the blends were typed with the individual MAbs of the respective blend. Individual MAbs for polioviruses 1, 2, and 3 acquired late in the study were used to screen 45 viral isolates. RESULTS: The antibodies identified 251/465 (54%) of the total number of isolates tested. IFA results for 451 of 465 viral isolates were in agreement with conventional identification methods. The sensitivity of the IFA screen using the MAb blends and coxsackie A9 MAb was 93% and the specificity was 99%. Thirteen discrepant isolates were negative by IFA, with twelve positive by Nt for echovirus 30 and one isolate positive by Nt for coxsackie A9. The remaining discrepant isolate was positive by IFA for both coxsackie A9 and coxsackie B5, but positive by Nt for coxsackie A9 only. CONCLUSIONS: IFA is highly specific for the identification of enteroviruses, but may not be sensitive enough to identify all strains within an enterovirus type. Procedures which utilize an IFA screen and confirm final results by Nt decrease turnaround time and reduce the number of cell culture tubes required for the identification of each enterovirus isolate.

Cloning and characterization of the human neutrophil-activating peptide (ENA-78) gene.

The neutrophil-activating peptide (ENA-78) is an inflammatory chemokine which is produced concomitantly with interleukin-8 (IL-8) in response to stimulation with either interleukin-1 (IL-1 beta) or tumor necrosis factor-alpha (TNF-alpha). We have identified a full-length ENA-78 cDNA and isolated its genomic clone. The gene was found to consist of four exons and three introns and its structure resembles the IL-8 gene. The human ENA-78 gene was mapped to chromosome 4q13-q21, the same locus as several other inflammatory cytokine genes. The transcription initiation site was mapped to a position 96 base pairs (bp) upstream from the translation initiation site. A fusion gene containing 125 bp upstream of exon 1 linked to a luciferase reporter gene was expressed in the human embryonic 293 cell line. The expression of the reporter gene was induced by TNF-alpha, IL-1 beta, or phorbol 12-myristate 13-acetate. The 125-bp promoter region contained the cis-regulatory elements for enhancer binding protein-like factor (C/EBP) and the nuclear factor (NF-kappa B). Transfection of 293 cells with deletion mutants demonstrated that the NF-kappa B element, but not the C/EBP site, is sufficient for expression and induction by either TNF-alpha or IL-1 beta. In contrast, the IL-8 gene requires both elements. This report demonstrates that ENA-78 and IL-8 genes shared great similarity in genomic structure and chromosome location. However, these two genes may be regulated by distinct mechanisms.

New N alpha-guanidinobenzoyl derivatives of hirudin-54-65 containing stabilized carboxyl or phosphoryl groups on the side chain of phenylalanine-63.

We report on the synthesis and pharmacological properties of a new series of thrombin inhibitors derived from hirudin carboxyl-terminal fragments. Two (arylphosphono)phenylalanines, p-PO3H2-L-Phe1 and m-PO3H2-L-Tyr, and one (carboxymethyl)phenylalanine, p-CH2COOH-L-Phe, were prepared and incorporated into position 63 of the modified hirudin's C-terminal dodecapeptide using the Fmoc solid-phase synthesis strategy. Substitution by any one of the residues led to very active analogs which doubled the thrombin time at low micromolar concentration (Ctt2) in vitro (1 microM < Ctt2 < 3 microM) and potently increased the activated partial thromboplastin time (APTT) ex vivo. These compounds displayed a higher potency in vitro and a longer duration of action in vivo than both the corresponding sulfated or phosphorylated tyrosine counterparts.

Diet-induced atherosclerosis inhibits release of noradrenaline from sympathetic nerves in rabbit arteries.

Contractile responses to sympathetic nerve stimulation and exogenous noradrenaline were compared in aortas and pulmonary arteries of control rabbits and rabbits fed a cholesterol-rich diet (0.3%) for 16 or 30 weeks. The diet-induced atherosclerosis reduced the contractions to increasing concentrations of exogenous noradrenaline (0.1 nM to 10 microM) in both arteries, and the reduction was more pronounced after 30 weeks of the hypercholesterolemia. The contractions produced with increasing frequencies of electrical stimulation (1-32 Hz) were nearly abolished in the atherosclerotic arteries. Labeling of the aorta and the pulmonary arteries with [3H]noradrenaline resulted in accumulation of radioactivity in both control and atherosclerotic blood vessels. After mounting the labeled blood vessels for superfusion, a basal efflux of [3H]noradrenaline and of 3H-metabolites was detected. In the atherosclerotic arteries, a decreased efflux of the intraneuronal deaminated metabolites 3,4-dihydroxyphenyl glycol (DOPEG) and 3,4-dihydroxymandelic acid (DOMA) was detected. Electrical stimulation at 1 Hz (pulmonary artery) or 2 Hz (aorta) caused an augmented efflux of total 3H from the control arteries; this was mostly due to release of intact [3H]noradrenaline. The electrical impulses evoked significantly less (16 weeks) or no (30 weeks) release of [3H]noradrenaline in the atherosclerotic arteries. These data illustrate that diet-induced atherosclerosis exerts an inhibitory action on the sympathetic nerve terminals in the aorta and the pulmonary artery of the rabbit. This effect, together with an inhibitory effect at the postjunctional level results in a loss of the responsiveness to nerve stimulation. The atherosclerotic process also inhibits the intraneuronal deamination of the sympathetic transmitter.

Modulation of the activity and assessment of the receptor selectivity in a series of new RGD-containing peptides.

We have investigated the structure-activity relationship of a series of new synthetic RGD analogs and their potential use as specific platelet aggregation inhibitors. Twelve short linear peptides showed high potency to inhibit aggregation in ADP-stimulated dog platelets. In order to assess the selectivity of these analogs towards platelet integrin GPIIb-IIIa, a new cell adhesion inhibition system was devised which was able to discriminate between the two closely related beta 3-integrins of the vasculature, GPIIb-IIIa (alpha IIb beta 3), present in platelets, and the vitronectin receptor (alpha v beta 3), expressed in endothelial cells and platelets. As reported for other peptides by Scarborough et al. (1993, J. Biol. Chem. 268, 1066), the analogs containing lysine instead of arginine in position 1 showed increased selectivity towards GPIIb-IIIa. One of them, in which the piperidine carboxylic group was attached to the N-terminus of KGDW, not only strongly inhibited platelet aggregation, but also selectively abolished cell adhesion mediated by GPIIb-IIIa without effect on the vitronectin receptor.

Evidence for induction of nonendothelial NO synthase in aortas of cholesterol-fed rabbits.

The aim of our study was to examine the effects of the inhibitor of nitric oxide (NO)-synthase, nitro-L-arginine (L-NNA), in atherosclerotic aortas obtained from cholesterol-fed rabbits. In the atherosclerotic aortas, L-NNA (100 microM) caused endothelium-independent contractions that were not observed in aortas from control rabbits. L-NNA (100 microM) significantly enhanced the contractile responses to norepinephrine and 5-hydroxytryptamine (5-HT) in atherosclerotic aortas with and without endothelium; in control aortas, L-NNA only augmented the response to 5-HT when the endothelium was present. The concentration-dependent increases in the norepinephrine-induced contractions caused by L-NNA (1 to 100 microM) could be reversed by L-arginine (1 mM) both in atherosclerotic aortas with and without endothelium. L-NMMA also evoked concentration-dependent augmentations of the norepinephrine-induced contraction; the effect of L-NMMA was equipotent to that of L-NNA. Finally, L-NNA (100 microM) prevented the paradoxical endothelium-independent contraction to hypoxia in atherosclerotic aortas. These data strongly suggest that nonendothelial NO synthase has been induced in the aortas of the hyperlipidemic rabbit.

Hypoxia causes an abnormal contractile response in the atherosclerotic rabbit aorta. Implication of reduced nitric oxide and cGMP production.

Both atherosclerotic lesions and hypoxia alter the contractile properties of the arterial wall and, in particular, may interfere with the relaxation mechanisms dependent or not on the endothelium. The present study was designed to test the effect of severe hypoxia on the contractile behavior of the atherosclerotic rabbit aorta. Segments of aortas obtained from control, cholesterol-fed, or Watanabe hereditary hyperlipidemic rabbits were mounted in organ chambers for isometric tension recording. A change of the bath PO2 from "normoxic" conditions (95% O2-5% CO2) to "hypoxic" conditions (95% N2-5% CO2) caused relaxation in the precontracted control aortas (by approximately 85%) but a transient contraction (approximately 20% of the maximal contraction obtained with 30 mM KCl) followed by a relaxation in the precontracted atherosclerotic aortas. Both types of responses were observed in aortas contracted with aggregating platelets, 5-hydroxytryptamine (5-HT), norepinephrine, endothelin, and prostaglandin F2 alpha. The hypoxic contractions in atherosclerosis were not dependent on the presence of an intact endothelium. They could not be antagonized by blockers of alpha-adrenoceptors, 5-HT2 receptors, histamine receptors, thromboxane receptors, and muscarinic cholinoreceptors. Inhibitors of cyclooxygenase, lipoxygenase, Na+, K(+)-ATPase, and free radical scavengers or an activator of endothelium-derived relaxing factor did not significantly affect the hypoxic contraction; the absence of effect of some inhibitors of protein synthesis seems to rule out the involvement of endothelin, angiotensin II, and bradykinin. The hypoxic contraction was not influenced by omission of Ca2+ from the medium or by inhibition of Ca2+ influx but was prevented by blockade of intracellular Ca2+. The inhibitor of nitric oxide synthase (nitro-L-arginine, 100 microM) and the guanylyl cyclase inhibitor (methylene blue, 10 microM) both enhanced the initial contractile responses to 5-HT to a similar extent as hypoxia and completely prevented the hypoxic contraction in the atherosclerotic tissues. The cyclic nucleotide analogues 8-bromo-cGMP and dibutyryl cAMP also inhibited the hypoxic contraction in the atherosclerotic aorta. The cGMP levels were markedly decreased and the cAMP levels were moderately decreased in the aortas of the cholesterol-fed rabbits as compared with the control aortas. Hypoxia further decreased cGMP but not the cAMP levels in atherosclerotic aortas with and without endothelium. Our data thus demonstrate the occurrence of an unusual vasoconstrictor response in atherosclerotic arteries; this constrictor response depends on the availability of intracellular Ca2+ and seems to be due to the further inhibition of an already impaired cGMP production.(ABSTRACT TRUNCATED AT 400 WORDS)

Phosphoramidon inhibits the conversion of big ET-1 into ET-1 in the pithed rat and in isolated perfused rat kidneys.

Endothelin-1 (ET-1) is a powerful renal vasoconstrictor peptide that could be implicated in acute renal failure. The aim of this study was to test the effects of the endothelin-converting enzyme (ECE) inhibitor phosphoramidon on pressor responses to ET-1 and its precursor, big ET-1, in isolated perfused rat kidneys and in pithed rats. In Tyrode-perfused rat kidneys, both big ET-1 (0.2-0.4 nmol) and ET-1 (0.01-0.03 nmol) evoked dose-dependent constrictions. Phosphoramidon (10 microM) selectively inhibited the pressor responses to big ET-1 without altering those to ET-1, norepinephrine, angiotensin I (AT-I), or angiotensin II (AT-II). The metalloprotease inhibitor thiorphan, but not the angiotensin-converting enzyme (ACE) inhibitor perindoprilate, also selectively inhibited the renal constrictions caused by big ET-1 but not those induced by ET-1. In vivo, both big ET-1 and ET-1 (0.5-2 nmol/kg) evoked pressor responses that were augmented by indomethacin (15 mg/kg) and L-NNA (1 mg/kg/min). Phosphoramidon selectively inhibited the pressor responses to big ET-1 (ID50: 78 micrograms/kg/min) without affecting those to ET-1, AT-I, or AT-II. These data illustrate that the pressor responses to big ET-1 in the rat, both in vivo and in vitro, are due to its conversion into ET-1 by a phosphoramidon-sensitive ECE. In the rat, phosphoramidon selectively inhibits ECE but not ACE both in vitro and in vivo.

Effect of vasodilators, including nitric oxide, on the release of cGMP and cAMP in the isolated perfused rat kidney.

In isolated Tyrode-perfused rat kidneys, the release of the cyclic nucleotides cAMP and cGMP was measured in response to several vasodilators, including nitric oxide (NO). During vasoconstrictions induced by methoxamine, a basal release of both cyclic nucleotides was detected in the renal effluent (357 +/- 32 fmol/min for cGMP and 3097 +/- 219 fmol/min for cAMP). Injection of acetylcholine (ACh; 11 nmol), sodium nitroprusside (SNP; 0.8 nmol) and atrial natriuretic factor (ANF; 80 pmol) caused a marked release of cGMP. The cGMP release induced by ACh was not altered by indomethacin (3 microM) but was markedly reduced by the NO synthase inhibitor nitro-L-arginine (L-NNA; 200 microM). Authentic NO (0.16-80 nmol) caused dose-dependent vasodilatations that were accompanied by increases in the overflow of cGMP. The vasodilatations caused by forskolin (6 nmol) and prostacyclin (PGI2; 3-52 nmol) were not accompanied by an overflow of cGMP. The vasodilator responses to 5-hydroxytryptamine (5-HT; 0.25-2 mumol), obtained in presence of the 5-HT2 receptor blocker ritanserin (10 nM) and the 5-HT3 blocker ICS 205930 (10 nM), were markedly reduced by L-NNA; however, they were not accompanied by the renal release of cGMP. Both forskolin and PGI2 induced the release of cAMP from perfused rat kidneys; ACh, 5-HT and 5-carboxamidotryptamine (5-CT) also evoked a significant release of cAMP into the renal effluent. The release of cAMP induced by ACh and 5-HT was reduced by indomethacin and L-NNA. Higher doses of NO released cAMP from the perfused rat kidneys. Our data illustrate that both cAMP and cGMP can be released by vasodilator substances into the venous effluent of isolated perfused rat kidneys. The dilator responses to 5-HT were sensitive to the NO synthase inhibitor L-NNA and were accompanied by the release of cAMP and not by the release of cGMP. Our data suggest that the dilator responses may be due to NO released from endothelial cells, which then activates adenylyl cyclase either directly or indirectly.

Venous and arterial endothelial cells respond differently to thrombin and its endogenous receptor agonist.

The effects of thrombin and a peptide mimicking the amino terminus of its receptor, Res (42-55), on vascular reactivity were compared in isolated canine blood vessels. In saphenous veins contracted with endothelin-1, both thrombin and Res (42-55) caused relaxation in rings with endothelium and contraction in rings without endothelium. In coronary arteries, thrombin caused similar responses while Res (42-55) only caused contraction. These data suggest that different thrombin receptors are present on venous and arterial endothelial cells.