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R Pasqualini

Publications and source records attributed to R Pasqualini.

At least 55 records · Page 3Linked to original sources
PubMed1998

Cellular uptake mechanisms of 99mTcN-NOET in cardiomyocytes from newborn rats: calcium channel interaction.

BACKGROUND: Bis[N-ethoxy,N-ethyl(dithiocarbamato)]nitrido Tc (V) (TcN-NOET) is a new technetium complex proposed as a tracer of myocardial perfusion. However, its cellular uptake mechanisms are unknown, although membrane localization on rat heart preparations and preferential binding to polymorphonuclear neutrophils (PMNs) have been reported. Because of the central role of calcium in PMN actions, a relationship was hypothesized between this ion flux and TcN-NOET cellular uptake. METHODS AND RESULTS: The mechanisms of cellular uptake of TcN-NOET were investigated in newborn rat cardiomyocytes by study of the effect of calcium channel modulators on tracer binding. Nifedipine had no effect on tracer uptake at 1 minute. However, verapamil 0.1 micromol/L and diltiazem 0.5 micromol/L induced a 40% decrease in uptake. Conversely, Bay K 8644 0.25 micromol/L increased TcN-NOET uptake by 73%. Alterations in other membrane ion transports failed to modify tracer uptake, indicating the specificity of the relationship between TcN-NOET uptake and calcium channels. Kinetic studies indicated that cellular net accumulation of the tracer was slow (t1/2=28.5 minutes) and retention was prolonged (84% of initial activity retained after 120 minutes of washout). The energy dependence of TcN-NOET uptake was investigated after 60 minutes of metabolic inhibition by iodoacetic acid plus rotenone. The ATP decrease was not associated with reduction in tracer uptake at 1 minute (114.9+/-21.9% of control, P=NS). CONCLUSIONS: The decrease in uptake observed with verapamil and diltiazem, the increase with Bay K 8644, and the lack of effect with nifedipine suggest that TcN-NOET binds to L-type calcium channels in the open configuration, without entering cardiomyocytes. The kinetics of TcN-NOET accumulation and retention are slow, and the mechanism for cellular uptake is not energy-dependent. From a clinical point of view, the effect of concurrent treatment by calcium inhibitors on myocardial binding of TcN-NOET should be taken into account.

Animals↗
PubMed1998

Fibronectin matrix regulates activation of RHO and CDC42 GTPases and cell cycle progression.

Adherent cells assemble fibronectin into a fibrillar matrix on their apical surface. The fibril formation is initiated by fibronectin binding to the integrins alpha5 beta1 and alphav beta3, and is completed by a process that includes fibronectin self-assembly. We found that a 76- amino acid fragment of fibronectin (III1-C) that forms one of the self-assembly sites caused disassembly of preformed fibronectin matrix without affecting cell adhesion. Treating attached fibroblasts or endothelial cells with III1-C inhibited cell migration and proliferation. Rho-dependent stress fiber formation and Rho-dependent focal contact protein phosphorylation were also inhibited, whereas Cdc42 was activated, leading to actin polymerization into filopodia. ACK (activated Cdc42-binding kinase) and p38 MAPK (mitogen-activated protein kinase), two downstream effectors of Cdc42, were activated, whereas PAK (p21-activated kinase) and JNK/SAPK (c-Jun NH2-terminal kinase/ stress-activated protein kinase) were inhibited. III1-C treatment also modulated activation of JNK and ERK (extracellular signal-regulated kinases) in response to growth factors, and reduced the activity of the cyclin E-cdk2 complex. These results indicate that the absence of fibronectin matrix causes activation of Cdc42, and that fibronectin matrix is required for Rho activation and cell cycle progression.

Actins↗
PubMed1998

Molecular heterogeneity of the vascular endothelium revealed by in vivo phage display.

Vascular beds are known to differ in structure and metabolic function, but less is known about their molecular diversity. We have studied organ-specific molecular differences of the endothelium in various tissues by using in vivo screening of peptide libraries expressed on the surface of a bacteriophage. We report here that targeting of a large number of tissues with this method yielded, in each case, phage that homed selectively to the targeted organ. Different peptide motifs were recovered from each of these tissues. The enrichment in homing to the target organs relative to an unselected phage was 3-35-fold. Peptide sequences that conferred selective phage homing to the vasculature of lung, skin, and pancreas were characterized in detail. Immunohistochemistry showed that the phage localized in the blood vessels of their target organ. When tested, the phage homing was blocked in the presence of the cognate peptide. By targeting several tissues and by showing that specific homing could be achieved in each case, we provide evidence that organ- and tissue-specific molecular heterogeneity of the vasculature is a general, perhaps even universal, phenomenon. Our results also show that these molecular differences can serve as molecular addresses.

Animals↗
PubMed1998

Cancer treatment by targeted drug delivery to tumor vasculature in a mouse model.

In vivo selection of phage display libraries was used to isolate peptides that home specifically to tumor blood vessels. When coupled to the anticancer drug doxorubicin, two of these peptides-one containing an alphav integrin-binding Arg-Gly-Asp motif and the other an Asn-Gly-Arg motif-enhanced the efficacy of the drug against human breast cancer xenografts in nude mice and also reduced its toxicity. These results indicate that it may be possible to develop targeted chemotherapy strategies that are based on selective expression of receptors in tumor vasculature.

Animals↗
PubMed1998

Chemotherapy targeted to tumor vasculature.

Vasculature-targeted chemotherapy--the destruction of tumor blood vessels with cytotoxic agents--makes use of biochemical differences between angiogenic and resting blood vessels. This approach may minimize or eliminate some of the problems associated with conventional solid-tumor targeting, such as poor tissue penetration and drug resistance. Experiments with antiangiogenic and thrombotic factors have shown that eliminating tumor blood supply has dramatic antitumor effects in mice. Targeting chemotherapeutic agents to the tumor vasculature combines the blood vessel destruction with the usual antitumor activities of the drug, resulting in increased efficacy and reduced toxicity in experiments with tumor-bearing mice. Human clinical trials, which are soon to follow, will determine the final value of this approach.

Antineoplastic Agents↗
PubMed1997

Myocardial uptake and redistribution of 99mTc-N-NOET in dogs with either sustained coronary low flow or transient coronary occlusion: comparison with 201Tl and myocardial blood flow.

BACKGROUND: 99mTc-N-NOET (NOET) is a new myocardial perfusion imaging agent that redistributes over time. We sought to better define the redistribution kinetics of NOET using open-chest canine models of sustained low coronary flow (protocol 1) and transient coronary occlusion followed by reflow (protocol 2). METHODS AND RESULTS: In protocol 1 (n=10), NOET and 201Tl were injected during low flow in the left anterior descending coronary artery (LAD) that was sustained for 2 hours. Protocol 2 dogs (n=6) were injected with NOET during 20 minutes of LAD occlusion followed by 2 hours of reflow. In both protocols, serial NOET planar images were acquired, and myocardial flow and 2-hour tracer activities were determined by gamma-well counting. Defect resolution was observed on images in both protocols. Initial defect count ratios, reflecting flow disparity at injection (0.66+/-0.03 and 0.57+/-0.04, respectively), increased over 2 hours (0.73+/-0.02 and 0.75+/-0.04, respectively; P<.001 versus initial). Quantitative imaging showed that NOET redistribution resulted from greater clearance from normal areas versus low-flow or transiently occluded areas. In protocol 1, 2-hour NOET and 201Tl stenotic-to-normal tissue activity ratios were similar (0.76+/-0.06 versus 0.73+/-0.04, P=NS) and higher than injection flow ratios (0.52+/-0.06 and 0.56+/-0.07, respectively, P<.001), consistent with tracer redistribution. In protocol 2, NOET redistributed to an even greater extent (injection flow ratio, 0.27+/-0.04; 2-hour tissue activity ratio, 0.84+/-0.03, P<.001). CONCLUSIONS: NOET is the first 99mTc-labeled myocardial imaging agent with kinetics similar to 201Tl in experimental models, permitting redistribution imaging. NOET appears to be a promising agent for assessing patients with coronary artery disease.

Animals↗
PubMed1997

New bis(dithiocarboxylato) nitridotechnetium-99m radiopharmaceuticals for leucocyte labelling: in vitro and in vivo studies.

Dithiocarboxylate ligands were synthesized and characterised. New nitrido 99m-technetium complexes were obtained with these ligands and identified by thin layer chromatography. The nitrido complexes were tested in vitro in whole blood for leucocyte labelling and the design of the ligand was optimized. Best results were obtained with aliphatic linear ligands, containing 9 to 11 atoms of carbon. The in vivo experiment failed because an inflammated area could not be visualized by gamma imaging, the cell labelling mechanism being probably different.

Animals↗
PubMed1997

Alpha v integrins as receptors for tumor targeting by circulating ligands.

Phage displaying an Arg-Gly-Asp (RGD)-containing peptide with a high affinity for alpha v integrins homed to tumors when injected intravenously into tumor-bearing mice. A substantially higher amount of alpha v-directed RGD phage than control phage was recovered from malignant melanomas and breast carcinoma. Antibodies detected the alpha v-directed RGD phage in tumor blood vessels, but not in several normal tissues. These results show that the alpha v integrins present in tumor blood vessels can bind circulating ligands and that RGD peptides selective for these integrins may be suitable tools in tumor targeting for diagnostic and therapeutic purposes.

Amino Acid Sequence↗
PubMed1997

Increased membrane ratios of metabolite to precursor fatty acid in essential hypertension.

Desaturase enzymes are responsible for the conversion of essential fatty acids to the longer-chain eicosanoid precursors. These enzymes require zinc as an essential cofactor, and the following ratios-C20:4/C18:2, C20:5/C18:3, and C22:6/C20:5-are considered indexes of their activity. We analyzed these parameters in plasma and erythrocyte membranes of 105 essential hypertensive patients, 20 white coat hypertensive patients, and 100 age-matched normotensive control subjects. Dietary analysis excluded significant quantitative and qualitative differences in fatty acid dietary intake between essential hypertensive patients and normotensive control subjects. Zinc levels and C20:4/C18:2, C20:5/C18:3, and C22:6/ C20:5 ratios were significantly higher in essential hypertensive patients than control subjects, whereas white coat hypertensive patients showed intermediate values for all these parameters. These data provide evidence for an alteration in fatty acid metabolism of essential hypertensive patients, consistent with increased activity of desaturase enzymes. The consequent greater bioavailability of eicosanoid precursors, and in particular of arachidonic acid, could affect several vascular functions and have a bearing on the pathogenesis or complications of hypertension.

Adult↗
PubMed1997

Interaction of technetium-99m-N-NOET with blood elements: potential mechanism of myocardial redistribution.

UNLABELLED: Technetium-99m-N-NOET is a new 99mTc-labeled, neutral cardiac perfusion imaging agent which has been shown to undergo apparent redistribution in animal models and in humans. The purpose of this study was to investigate the interaction of 99mTcN-NOET with red blood cells (RBCs) and to determine the effects of these interactions on myocardial uptake and clearance of 99mTcN-NOET. METHODS: After bolus administration, myocardial 99mTcN-NOET clearance was monitored for 1 hr using a sodium iodide detector in 22 isolated, buffer-perfused rat hearts. Hearts were perfused as follows: seven controls with Krebs-Henseleit (KH) buffer (Group 1), five hearts with KH containing RBCs (Group 2), five hearts with KH containing RBCs and albumin (Group 3), five hearts with KH containing RBCs and dextran (Group 4). In a separate protocol, RBCs were incubated in 99mTcN-NOET and then perfused through five hearts (Group 5). RESULTS: Technetium-99m-N-NOET myocardial uptake (%ID) was significantly lower in all RBC groups (RBCs = 5.0% +/- 1.7%; RBCs + albumin = 8.2% +/- 2.1%; RBCs + dextran = 4.0% +/- 0.8%; incubated RBCs = 8.8% +/- 1.5%) compared with controls (72.2% +/- 2.8%; p < 0.05). Retention (99.4% +/- 0.6%) was near linear in the KH control group with virtually no fractional clearance at 60 min. Retention in groups whose perfusates contained RBCs (RBCs = 62.2 +/- 4.2%; RBCs + albumin = 29.9 +/- 4.3%; RBCs + dextran = 69.3 +/- 3.6%) were all significantly lower than control. Addition of albumin to RBC perfusate resulted in significantly lower retention (29.9% +/- 4.3%; p < 0.01) than was observed in RBC perfusate alone (62.2% +/- 4.2%). Substitution of dextran for albumin produced retention similar to RBCs alone (69.3% +/- 3.6%; p = ns). In a separate protocol, RBC binding of 99mTcN-NOET was high (64.4% +/- 8.6%) in triple-washed RBCs. Technetium-99m-N-NOET bound to RBCs was subsequently extracted from red cells by the myocardium when those cells were infused into hearts. CONCLUSION: Technetium-99m-N-NOET has high binding affinity to blood elements and transfers bidirectionally between myocardium and blood. The interaction of 99mTcN-NOET with blood elements represents a potential mechanism of redistribution.

Animals↗
PubMed1996

Organ targeting in vivo using phage display peptide libraries.

Preferential homing of tumour cells and leukocytes to specific organs indicates that tissues carry unique marker molecules accessible to circulating cells. Organ-selective address molecules on endothelial surfaces have been identified for lymphocyte homing to various lymphoid organs and to tissues undergoing inflammation, and an endothelial marker responsible for tumour homing to the lungs has also been identified. Here we report a new approach to studying organ-selective targeting based on in vivo screening of random peptide sequences. Peptides capable of mediating selective localization of phage to brain and kidney blood vessels were identified, and showed up to 13-fold selectivity for these organs. One of the peptides displayed by the brain-localizing phage was synthesized and shown to specifically inhibit the localization of the homologous phage into the brain. When coated onto glutaraldehyde-fixed red blood cells, the peptide caused selective localization of intravenously injected cells into the brain. These peptide sequences represent the first step towards identifying selective endothelial markers, which may be useful in targeting cells, drugs and genes into selected tissues.

Amino Acid Sequence↗
PubMed1996

Comparison of reversed phase and reversed phase ion pair high performance liquid chromatography for analysis of TcO and TcN complexes of L,L-ethylene dicysteine di-ethylester and its acid analogues.

99mTc(V)-oxo complexes of L,L-ethylene dicysteine (L,L-EC) and its di-ester derivative L,L-ethyl cysteinate dimer (L,L-ECD) are useful tracer agents for evaluation of renal function and cerebral blood flow respectively. Labelling of these molecules with a 99mTc(V)-nitrido core instead of the 99mTc(V)-oxo core alters their biological and physiochemical behavior. In the reversed phase high performance liquid chromatography (HPLC) method presently used to analyse 99mTc(V)-oxo preparations of L,L-EC and L,L-ECD, the 99mTc(V)-nitrido-L,L-EC complex and the possible impurities of a 99mTc(V)-nitrido-L,L-ECD preparation were found to elute with the void volume. In this study, a reversed phase ion pair HPLC method has been developed that is useful for the analysis of both 99mTc(V)-oxo and 99mTc(V)-nitrido preparations of L,L-EC and L,L-ECD. Tetrabutylammonium hydroxide is used as a cationic ion pairing agent.

Chromatography, High Pressure Liquid↗
PubMed1996

Influence of a 99mTcN core on the biological and physicochemical behavior of 99mTc complexes of L,L-EC and L,L-ECD.

99mTc-nitrido complexes of L,L-ethylene dicysteine (99mTcN-L,L-EC) and 99mTcN-L,L-ethylene dicysteine diethylester (99mTcN-L,L-ECD) were prepared and their characteristics compared to those of the respective 99mTc-oxo complexes. 99mTcN-L,L-EC and 99mTcO-L,L-EC migrate to similar extents during electrophoresis at pH 12, but, at pH6, 99mTcN-L,L-EC migrates further than 99mTcO-L,L-EC. Renal excretion of 99mTcN-L,L-EC is inferior to that of 99mTcO-L,L-EC, indicating that the TcN-glycine sequence has lower affinity for the renal tubular system. Both 99mTcO-L,L-ECD and 99mTcN-L,L-ECD are neutral, but 99mTcN-L,L-ECD is hydrophilic and shows minimal brain uptake in both mice and the baboon.

Animals↗
PubMed1996

A polymeric form of fibronectin has antimetastatic effects against multiple tumor types.

Metastasis accounts for most deaths in cancer patients. Tumor cell adhesion to the extracellular matrix through integrins is thought to be a critical step in metastasis and a potential target for therapeutic intervention. We show here that treatment of human osteosarcoma, melanoma and carcinoma cells with a polymeric form of fibronectin (sFN), before inoculation into nude mice, prevented tumor formation. Intraperitoneally administered sFN significantly reduced lung colonization from intravenously injected tumor cells (experimental metastasis) and from subcutaneous tumors in nude mice (spontaneous metastasis). Treatment with sFN blocked cell spreading and migration in vitro suggesting a possible mechanism for the antimetastatic effect.

Animals↗
PubMed1996

Peptides in cell adhesion: powerful tools for the study of integrin-ligand interactions.

Many of the cell adhesion receptors recognize simple sequences that can be reproduced as synthetic peptides. This circumstance has led to a widespread use of peptides as modulators of cell adhesion. Peptides capable of binding to cell adhesion receptors, such as the integrins, can be used as mediators of cell attachment to a surface by coating the surface with the peptide. Peptides bound to a solid phase can also be used to isolate adhesion receptors by affinity chromatography. Alternatively, the peptides can be used to inhibit cell attachment to the natural ligands of the adhesion receptors. In either mode, adhesion peptides have proven to be highly useful probes in cell adhesion research and they also show promise as a new class of therapeutics. The purpose of this article is to review some of the properties and uses of adhesion peptides.

Binding Sites↗
PubMed1995

Synthesis and biodistribution of nitrido technetium-99m radiopharmaceuticals with dithiophosphinate ligands: a class of brain imaging agents.

The symmetrical complexes [99mTc][TcN(R2PS2)2] [R = CH3, CH2CH3, CH2CH2CH3, CH2(CH3)2], and the unsymmetrical complex [99mTc][TcN(Me2PS2)(Et2PS2)] have been prepared, at tracer level, through a two-step procedure involving the preliminary formation of a prereduced technetium nitrido intermediate followed by substitution reaction onto this species by the appropriate dithiophosphinate ligand [R2PS2]Na. The chemical identity of the resulting complexes have been established by comparison with the corresponding 99Tc-analogs prepared, at macroscopic level, by reacting the complex [99TcNCl4] [n-Bu4N] (n-Bu = n-butyl) with an excess of ligand in methanol, and characterized by elemental analyses and spectroscopic techniques. The complexes are neutral and lipophilic, and possess a square pyramidal geometry, with an apical Tc identical to N group and two dithiophosphinate ligands spanning the four positions on the basal plane through the four sulfur atoms of the > PS2 group. In vitro studies showed that these radiopharmaceuticals are stable in solution and that their chemical identity was not altered after incubation with rat blood. Biodistribution studies have been carried out in rats and primates. The results demonstrate that these compounds are significantly retained into the brain of these animals for a prolonged time. Planar gamma camera images have been obtained in monkeys showing a good visualization of the cerebral region. However, the existence of persistent blood activity yields a brain/blood ratio lower than that observed with other 99mTc-based brain perfusion imaging agents.

Animals↗