Biomedical subjects

R T Dean

Publications and source records attributed to R T Dean.

At least 55 records · Page 3Linked to original sources

PubMed1997

Direct copper reduction by macrophages. Its role in low density lipoprotein oxidation.

Oxidation of low density lipoprotein (LDL) results in changes to the lipoprotein that are potentially atherogenic. Numerous studies have shown that macrophages cultured in vitro can promote LDL oxidation via a transition metal-dependent process, yet the exact mechanisms that are responsible for macrophage-mediated LDL oxidation are not understood. One contributing mechanism may be the ability of macrophages to reduce transition metals. Reduced metals (such as Fe(II) or Cu(I)) rapidly react with lipid hydroperoxides, leading to the formation of reactive lipid radicals and conversion of the reduced metal to its oxidized form. We demonstrate here the ability of macrophages to reduce extracellular iron and copper and identify a contributing mechanism. Evidence is provided that a proportion of cell-mediated metal reduction is due to direct trans-plasma membrane electron transport. Glucagon suppressed both macrophage-mediated metal reduction and LDL oxidation. Although metal reduction was augmented when cells were provided with a substrate for thiol production, thiol export was not a strict requirement for cell-mediated metal reduction. Similarly, while the metal-dependent acceleration of LDL oxidation by macrophages was augmented by thiol production, macrophages could still promote LDL oxidation when thiol export was minimized (by substrate limitation). This study identifies a novel mechanism that may contribute to macrophage-mediated LDL oxidation and may also reveal potential new strategies for the inhibition of this process.

Cells, Cultured↗

PubMed1997

Regulation of serum-induced lipid accumulation in human monocyte-derived macrophages by interferon-gamma. Correlations with apolipoprotein E production, lipoprotein lipase activity and LDL receptor-related protein expression.

The demonstration of lipid loaded macrophages in atherosclerotic tissue has led to the development of in vitro systems to elucidate the mechanisms involved in lipid accumulation. Here we have characterised the changes which occur in human monocyte-derived macrophage (MDM) lipids during culture in either human serum (HS) or foetal calf serum (FCS). MDM cultured in HS were rapidly converted to lipid filled foam cells, as assessed using HPLC analysis and oil red-O staining and compared with the same cells grown in FCS. However, the lipids which accumulated were predominantly triglycerides with smaller amounts of unesterified cholesterol (UC) and only traces of cholesteryl esters (CE). alpha-Tocopherol (alpha-TocH) was present at higher levels in MDM cultured in HS compared to the same cells grown in FCS. MDM lipid accumulation was dependent on the triglyceride-rich lipoprotein (TGRL) fraction of human serum; accordingly, supplementation of FCS with human TGRL also induced MDM lipid accumulation. The relationships between cellular lipid accumulation and secretion of apolipoprotein E (apo E) and lipoprotein lipase (LPL) as well as expression of the low density lipoprotein receptor-related protein (LRP) were also examined. MDM lipid accumulation was associated with increased apo E secretion but did not alter extracellular LPL activity. The lipid accumulation which was induced by HS was potently inhibited (but not reserved) by the inflammatory cytokine interferon-gamma (IFN gamma), and this was associated with decreased apo E production, LPL secretion and expression of LRP. These studies reveal striking differences in the lipid composition of MDM cultured in either HS or FCS, and indicate that oil red-O staining is not necessarily associated with cholesteryl ester accumulation in human macrophages. Furthermore, the effect that serum-induced lipid accumulation has on the specific MDM functions studied should be appreciated when developing in vitro macrophage models.

Apolipoproteins E↗

PubMed1997

Effects of CSF-1 on cholesterol accumulation and efflux by macrophages.

To assess whether human monocyte-specific colony-stimulating factor (CSF-1) might influence atherogenesis, CSF-1-induced macrophage responses that might contribute to enhanced clearance of low-density lipoprotein (LDL) or modified LDL were investigated. Careful account was made of cell preservation and increases in cell volume and protein (representing increased cell surface area, and thus endocytically active membrane) during culture with CSF-1. This permitted distinction between selective and nonspecific effects of CSF-1, the latter paralleling increases in cellular mass and volume. CSF-1 enhanced mouse peritoneal macrophage survival in vitro during exposure to lipoprotein-deficient serum with or without native LDL or acetylated LDL (Ac-LDL), as judged by maintenance of cellular DNA and cell numbers. In the presence of copper-oxidized LDL (Ox-LDL), such effects were very slight. In all conditions, CSF-1 increased cellular protein content. CSF-1 increased the uptake of both Ac-LDL and Ox-LDL calculated per culture, but this was entirely explicable by the increased cell protein, indicating that there was no selective enhancement of scavenger receptor or other routes for uptake of the modified LDLs. Similarly, CSF-1 also increased the accumulation of cholesterol and its esters nonspecifically. CSF-1 did have a marked and specific effect on the composition of cholesterol esters, decreasing the proportion of polyunsaturated esters relative to monounsaturated and saturated esters. Finally, cholesterol efflux induced by apolipoprotein A1 from Ac-LDL-loaded macrophages was not influenced by CSF-1. Thus, the enhanced macrophage catabolism of modified LDLs by CSF-1 is part of a nonspecific action on the cells but could contribute to a reduction in circulating cholesterol, observed in some situations of CSF-1 presentation in humans.

Animals↗

PubMed1997

Separation and characterization of cholesteryl oxo- and hydroxy-linoleate isolated from human atherosclerotic plaque.

In previous work we demonstrated that up to 30% of cholesteryl linoleate in homogenates of advanced human plaque samples is present in oxidized forms. Here we show that the material from plaque hexane extracts which co-elutes with cholesteryl hydroxy-linoleate on reversed phase HPLC (Anal Biochem 1993;213:79), is composed of several isomers of cholesteryl hydroxy- and cholesteryl oxo-octadecadienoate. Enzymatic hydrolysis and measurement of liberated cholesterol and disappearance of the esters revealed that almost all of the material consisted of unoxidized cholesterol esterified to oxidized derivatives of octadecadienoate. Semi-preparative reversed-phase HPLC was used to obtain sufficient quantities of this co-eluting material to undertake normal phase HPLC separation of these components. The nature of such separated and isolated compounds was identified, by co-chromatography with authentic standards, UV spectroscopy and chemical ionization and electron impact mass spectrometry, as cholesteryl hydroxy- and cholesteryl oxo-octadecadienoate. These oxidized fatty acids have been observed previously in plaque, in agreement with our new unambiguous demonstration of their presence as cholesteryl esters. The application of the methods described for the separation of the various forms of oxidized cholesteryl octadecadienoate may aid mechanistic studies of in vitro and in vivo lipoprotein lipid oxidation.

Arteriosclerosis↗

PubMed1997

Pre-clinical evaluation of technetium-99m platelet receptor-binding peptide.

UNLABELLED: P748 is a dimeric peptide which incorporates two high affinity GPIIb/IIIa receptor-binding domains and a novel 99mTc binding sequence, which provides the platelet imaging agent 99mTc-P748. The aim of this study was to evaluate 99mTc-P748 preclinically for use as a hot spot scintigraphic thrombus imaging agent. METHODS: Technetium-99m-P748 was prepared by either a ligand exchange or a one-vial kit. The oxorhenium congener, [ReO]P748, was prepared by ligand exchange from Bu4NReOBr4. The binding of P748 peptide and [ReO]P748 to GPIIb/IIIa receptors on activated platelets was assessed by their inhibition of ADP stimulated human platelet aggregation in platelet rich plasma (PRP). The localization of 99mTc-P748 in deep vein and pulmonary thrombi was assessed in a canine thrombosis model and the biodistribution of 99mTc-P748 was determined in rats. RESULTS: P748 peptide inhibited the aggregation of human platelets in PRP by 50% at a concentration (IC50) of 28 nM and [ReO]P748 had an IC50 of 36 nM showing the high in vitro receptor binding affinity of both the peptide and its rhenium complex (and by analogy its technetium complex). Technetium-99m-P748 was readily prepared at room temperature in 15 min in > or = 90% radiochemical yield and purity and provided definitive images of femoral vein thrombi within 20 min and pulmonary thrombi, within 1 hr in the canine model. Femoral vein thrombus-to-blood and thrombus-to-muscle ratios at 4 hr averaged 6.7 and 46, respectively. Pulmonary thrombus-to-blood and thrombus-to-normal lung ratios at 4 hr averaged 29 and 27, respectively. Dog and rat studies both showed rapid clearance of the radiotracer from the blood and with no significant hepatobiliary excretion but with notable early kidney retention. CONCLUSION: The combination of high in vitro receptor-binding affinity, high thrombus uptake and definitive in vivo images of both femoral vein and pulmonary thrombi show that 99mTc-P748 has considerable potential as a clinical imaging agent for the detection of venous thromboembolism.

Animals↗

PubMed1997

Pharmacokinetic considerations in the development of peptide-based imaging agents.

Pharmacokinetic factors to be considered in the development of peptide-based imaging agents are reviewed. These include size, plasma protein binding, lipophilicity, resistance or susceptibility to proteolysis and radiolabel integrity. These factors are discussed in the context of several examples of thrombus and tumor imaging agents either commercially available or in development.

Animals↗

PubMed1997

7-Hydroperoxycholesterol and its products in oxidized low density lipoprotein and human atherosclerotic plaque.

7-Hydroperoxycholesterols (7OOHs) are intermediates in cholesterol oxidation and potential cytotoxins. A normal-phase HPLC method with UV (205 nm) detection was developed that could resolve 7 alpha OOH, 7 beta OOH, 7-ketocholesterol (7K), and the epimeric 7-hydroxycholesterols (7OHs). 7OOH formation was investigated when LDL was exposed to four different oxidizing systems: Cu2+; Ham's F-10; mouse peritoneal macrophages in Ham's F-10; and a metal-independent peroxyl-radical generating system (AAPH). With all four oxidizing systems, 7OOH (both free and esterified, mostly as the beta-isomer) was the major oxysterol formed at early times, with 7K dominating at later stages (> or = 24 h) in Cu-oxLDL. When LDL was oxidized in the presence of cells there was transfer of free oxysterols from LDL to the cells. Negligible 7OOH, but significant amounts of 7OH, accumulated in the cells suggesting efficient cellular reduction of 7OOH. Lipid extracts from eight plaque samples obtained from patients undergoing carotid endarterectomy were analyzed. Only trace amounts of 7OOH (< 0.02% of total cholesterol) could be detected using this normal-phase HPLC method with UV detection or with a more sensitive reverse-phase method utilizing chemiluminescence detection. 7K was the major 7-oxygenated sterol detected, at least 20-fold in excess of that calculated for 7OOH, followed by 7 beta OH and 7 alpha OH. The trace concentrations of 7OOH in plaque indicate its lability in biological/cellular systems and may signify the ability of cells in the artery wall to metabolize it further.

Animals↗

PubMed1996

Hydroxypropyl-beta-cyclodextrin-mediated efflux of 7-ketocholesterol from macrophage foam cells.

Atherosclerosis involves the arterial accumulation of lipid-laden "foam cells" containing oxidized and unoxidized sterols and their esters (Mattsson-Hulten, L., Lindmark, H., Diczfalusy, U., Bjorkhem, I., Ottosson, M., Liu, Y., Bondjers, G., and Wiklund, O. (1996) J. Clin. Invest. 97, 461-8). Oxidized sterols are probably critical to atherogenesis because they inhibit cholesterol removal from cells and are cytotoxic. We recently reported that there is deficient induction of cellular cholesterol efflux by apolipoprotein A-I, the main initial acceptor of cellular cholesterol from macrophages loaded in vitro with oxidized low density lipoprotein (Kritharides, L., Jessup, W., Mander, E., and Dean, R. T. (1995) Arterioscler. Thromb. 15, 276-289). There was an even more marked impairment of the release of 7-ketocholesterol which is a major oxysterol in these cells and in human atherosclerotic lesions. Here we show that hydroxypropyl-beta-cyclodextrin can induce selective efflux of 7-ketocholesterol. Efflux of 7-ketocholesterol was time and concentration dependent, and the rate of its removal was 50-fold greater for hydroxypropyl-beta-cyclodextrin than for apolipoprotein A-I. Over a defined range of concentrations (0-5 mg/ml), efflux of 7-ketocholesterol was preferred over that of cholesterol and occurred without cell toxicity. Efflux of free 7-ketocholesterol was associated with decreased intracellular free and esterified 7-ketocholesterol. Hydroxypropyl-beta-cyclodextrin also enhanced efflux of other oxysterols. The physical solubilization of 7-ketocholesterol by the cyclodextrin was much greater than that of cholesterol, in accordance with its differential effects on efflux. These data highlight the importance of extracellular sterol solubilization in the efflux of cellular oxysterols and the mobilization of intracellular free and esterified oxysterol pools in macrophages loaded with oxidized low density lipoprotein. Synthetic sterol-solubilizing agents such as hydroxypropyl-beta-cyclodextrin are thus potential prototypes for the further development of oxysterol-removing agents.

2-Hydroxypropyl-beta-cyclodextrin↗

PubMed1996

Sterol efflux is impaired from macrophage foam cells selectively enriched with 7-ketocholesterol.

The aim of the present study was to investigate whether impairment of cholesterol efflux previously found from mouse peritoneal macrophages loaded with oxidized low density lipoprotein (OxLDL) could be ascribed to the presence of oxysterols in these cells. 7-Ketocholesterol (7KC), the major oxysterol present in OxLDL-loaded cells, was selectively incorporated into unoxidized LDL, which was subsequently acetylated to produce a high uptake form. Mouse macrophages incubated with 7KC-enriched acetylated LDL (7kAcLDL) did not reveal cytotoxicity judged by cell protein and trypan blue exclusion. A large proportion of cellular 7KC was esterified, indicating that it is a substrate for acyl CoA:cholesterol acyltransferase. Cholesterol efflux from mouse macrophages loaded with 7kAcLDL, using apoA-I as a sterol acceptor, was impaired in cells containing >50 nmol of 7KC/mg of cell protein compared with cells loaded with oxysterol-free acetylated LDL. Thus impairment of cholesterol efflux could be reproduced in cells loaded with 7kAcLDL containing similar proportions of 7KC as OxLDL. 7KC itself was exported very poorly, even when the levels of 7KC in the cells were low. These results suggest that oxysterols present in foam cells in vitro can affect reverse sterol transport and may be potentially important in foam cell formation in vivo.

Animals↗

PubMed1996

Somatostatin receptor-binding peptides labeled with technetium-99m: chemistry and initial biological studies.

The synthesis of peptides which possess a high affinity for the somatostatin receptor and contain a chelator for the radionuclide technetium-99m is described. The target compounds were designed such that they would form stable, oxotechnetium(V) chelate complexes in which the Oxorhenium(V) chelate complexes of these peptides were prepared as nonradioactive surrogates for the technetium complexes. Peptide oxorhenium complexes and Tc-99m complexes eluted closely upon HPLC analysis. The receptor-binding affinities of both the free and rhenium-coordinated species were measured in vitro. The binding affinities of the free peptides (Ki's in the 0.25 - 10 nM range) compared favorably with [DTPA]octreotide (Ki = 1.6 nM), which, as the indium-111 complex, is already approved for somatostatin receptor (SSTR)-expressing tumor imaging in the United States and Europe. Furthermore, the rhenium-coordinated peptides had binding affinities which, in many cases, were higher than those of the corresponding free peptides, with several complexes having a Ki's of 0.1 nM. Some of the more potent SSTR-binding peptides were labeled with technetium-99m and assessed in an in vivo study with tumor-bearing rats. The 99m Tc-labeled peptides prepared in this study should be useful as SSTR-expressing tumor-imaging agents due to their high SSTR-binding affinities, ease of preparation, and, because they are low molecular weight peptides, expected pharmacokinetics characterized by rapid tracer excretion from the body resulting in high-contrast images.

Amino Acid Sequence↗

PubMed1996

Thrombus imaging using technetium-99m-labeled high-potency GPIIb/IIIa receptor antagonists. Chemistry and initial biological studies.

Platelet-specific compounds which are radiolabeled with gamma-emitting radionuclides may be particularly useful for the noninvasive in vivo detection of thrombi. The synthesis of peptides which are potent inhibitors of platelet aggregation and which contain a chelator for the radionuclide technetium-99m are described. The target compounds were designed such that stable, oxotechnetium(V) species could be prepared where the site of metal coordination was well defined. A strategy was employed where the pharmacophore-Arg-Gly-Asp-(RGD), or RGD mimetic, was constrained in a ring which was formed by the S-alkylation of a cysteine residue with an N-terminal chloroacetyl group. Binding affinities were enhanced by the replacement of arginine with the arginine mimetics S-(3-aminopropyl)cysteine and 4-amidinophenylalanine. Further enhancements could be obtained by the synthesis of oligomers which contained two or more rings containing receptor binding regions. The increase in binding affinity seen was more than that expected from a simple stoichiometric increase of pharmacophore. The most potent compounds described had IC50s of approximately 0.03 microM for the inhibition of human platelet aggregation. Two of the more potent peptides (P280 and P748) were labeled with technetium-99m and assessed in a canine thrombosis model. The 99m Tc complexes of the peptides prepared in this work hold promise as thrombus imaging agents due to their high receptor binding affinity, ease of preparation, and expected rapid pharmacokinetics.

Amino Acid Sequence↗

PubMed1996

Biodistribution and autoradiographic localization of I-125--labeled synthetic Peptide in aortic atherosclerosis in cholesterol-fed rabbits.

I-125 labeled SP4 is a synthetic oligopeptide derived from apolipoprotein B of low-density lipoprotein that has been shown to localized in atherosclerotic plaques in experimental animals. However, its biodistribution and mechanism of localization need to be further elucidated. Twenty-four cholesterol-fed (CF) and 20 normal (NL) New Zealand White rabbits were injected with I-125-SP4 and killed 15 to 30 min (6 NL; 6 CF) or 2 h (14 NL; 18 CF) later. We obtained aortic autoradiograms and activity concentrations (% injected dose/gm) in aortic segments and other tissues. The uptake of I-125-SP4 was higher in CF than in NL rabbits in all aortic segments (p < 0.05). I-125-SP4 was cleared rapidly in both CF and NL rabbits with 60 to 70% of the injected dose cleared from the blood by 1 h. No statistically significant differences in radiotracer biodistribution were observed between NL and CF rabbits although activity tended to be higher in the liver, gallbladder, and intestine in NL rabbits and in the kidney and spleen in CF rabbits. Silver grains were distributed mainly on foam cells of the fatty streaks in aortic microautoradiograms from two additional rabbits that had been injected with I-125-SP4. There were 23,518 plus minus 15,878 (SD) grains/mm(2) in fatty plaques but only 14,669 plus minus 11,035 grains/mm(2) in media muscle (p < 0.0001 [9 sections, 17 areas evaluated] in an atherosclerotic animal) in injected animals and 13,439 plus minus 5,565 grains/mm(2) in media muscle (two sections, four areas) in the normal control animals (NS versus media of atherosclerotic animal). I-125-SP4 specifically localizes in aortic atherosclerotic plaques in CF rabbits. There is no significant difference in tissue distribution between normal and CF rabbits except in the aorta. Preliminarily, it appears that the site of tracer uptake is on foam cells and this suggests the possibility of relative specificity for fatty plaque.

Journal Article↗

PubMed1996

The iron-selective chelator desferal can reduce chelated copper.

It was shown that the iron-selective chelator desferal (desferrioxamine mesylate:DFO) can reduce Cu(II) as judged by measuring the formation of the complex between Cu(I) and a specific chelator for this species, neocuproine (NC), in phosphate buffer. It was found that under optimal conditions, 3 moles of Cu(II) could be reduced per mole of DFO. Studies of the kinetics of Cu(II) reduction by DFO revealed that the rate of Cu(II) reduction by DFO was considerably slower than that by ascorbate. In the case of both reductants, even in the absence of NC, Cu(I) complexes remained in aqueous solutions for at least 30 min. DFO could also reduce Cu complexed to histidine. The results presented highlight the interpretive dangers which can arise in studies involving multiple transition metals, especially in the presence of multiple chelators. Specifically, when desferal is used, it is important to be aware that any copper present may become reduced, and that any Cu(I) formed might participate in ongoing redox reactions.

Animals↗

PubMed1996

Preclinical evaluation of technetium-99m-labeled somatostatin receptor-binding peptides.

UNLABELLED: We report here the results of studies on the in vitro receptor binding affinity, in vivo tumor uptake and biodistribution of two 99m-Tc-labeled peptides. METHODS: Peptides P587 and P829 were synthesized by N-alpha-Fmoc peptide chemistry, purified by reversed-phase HPLC and characterized by fast-atom bombardment mass spectrometry. The peptides were labeled with 99mTc by ligand exchange from 99mTc-glucoheptonate. In vitro somatostatin receptors (SSTR)-binding affinities of P587, P829 and their oxorhenium complexes, [DTPA]octreotide and In-[DTPA]octreotide were determined in an inhibition assay using AR42J rat pancreatic tumor cell membranes and 125I-[Tyr3]somatostatin-14 as the probe. In vivo single- and dual-tracer studies of 99mTc peptides and 111In-[DTPA]octreotide were carried out using Lewis rats bearing CA20948 rat pancreatic tumor implants. RESULTS: Technetium-99m-P587 and 99mTc-P829 of high-specific activity (>60 Ci (2.2 TBq)/mmole) were prepared in >90% radiochemical yield. P587 and P829 had a Ki = 2.5 nM and 10 nM, respectively. [ReO]P587 and [ReO]P829, representing the 99mTc complexes, had Ki = 0.15 nM and 0.32 nM, respectively. In comparison, [DTPA]octreotide and In-[DTPA]octreotide had Ki = 1.6 and 1.2 nM, respectively. In vivo tumor uptake of 99mTc-P587 and 99mTc-P829 was high (4.1 and 4.9%ID/g at 90 min postinjection compared to 2.9% for 111In-[DTPA]octreotide). Tumor/blood and tumor/muscle ratios at 90 min postinjection were 6 and 33 for 99mTc-P587, 21 and 68 for 99mTcP829, and 22 and 64 for 111In-[DTPA]octreotide. CONCLUSION: The high SSTR-binding affinity and high receptor-specific and saturable in vivo tumor uptake indicate that 99mTc-P587 and 99mTc-P829 are promising radiotracers for the clinical detection of SSTR-expressing tumors and other tissues by 99mTc gamma scintigraphy.

Animals↗

PubMed1996

Technetium-99m-white blood cell-specific imaging agent developed from platelet factor 4 to detect infection.

UNLABELLED: We have developed a leukocyte-avid, 99mTc-labeled peptide (P483H) as a potential imaging agent for infection. P483H contains the heparin-binding region of platelet factor-4 (PF-4) and a lysine-rich sequence for rapid renal clearance. Technetium-99m-P483H was evaluated for its ability to selectively label white blood cells (WBCs) in vitro and to detect focal E. coli infections in rabbits. METHODS: Technetium-99m-P483H was incubated with citrated whole human blood, layered onto WBC isolation media and subjected to density gradient centrifugation to measure WBC-associated radioactivity. Indium-111-WBCs and 99mTc-gluceptate were used as controls. In the in vivo model, E. coli infected rabbits were imaged and necropsied 4 hr after administration of 99mTc-P483H. Infected and contralateral control muscles were evaluated for %ID, %ID/g, Imax (muscle sample showing the highest uptake, i.e., %ID/g) and Imax-to-blood and Imax-to-control muscle ratios. Indium-111-WBCs, 111In-DTPA, 131I-albumin (HSA), 99mTc-nanocolloid, 67Ga and 99mTc-gluceptate were evaluated as in vivo controls. RESULTS: Technetium-99m-P483H associated predominantly with WBCs in vitro, and 99m-Tc-P483H provided high contrast images of infection in vivo. In vitro, 73% of 99mTc-P483H radioactivity was associated with WBCs. Technetium-99m-P483H outperformed 111In-WBCs, 111In-DTPA, 131I-albumin, 99mTc-nanocolloid, 67Ga-citrate and 99mTc-gluceptate with an infection Imax average of 0.062 %ID/g (+/- 0.029; n = 48). Technetium-99m-P483H also outperformed all controls, including 111In-WBCs, 111In-DTPA, 131I-albumin, 99mTc-nanocolloid, 67Ga-citrate and 99mTc-gluceptate. The Imax-to-blood and Imax-to-control muscle ratios for 99mTc-P483H averaged 3.1 (+/- 2.4) and 26.8 (+/- 16.8), respectively, and again outperformed all controls. CONCLUSION: Technetium-99m-P483H associates predominantly with WBCs in vitro and identified focal infections in vivo within 4 hr versus conventional imaging agents. Additionally, the agent showed rapid blood clearance and exclusive renal excretion, which provides a clear abdominal field for imaging abdominal infections.

Animals↗

PubMed1996

Thrombus imaging with a technetium-99m-labeled activated platelet receptor-binding peptide.

UNLABELLED: The objective of this work was the preclinical evaluation of 99mTc-P280, a 99mTc-labeled peptide having high affinity and specificity for the GPIIb/IIIa receptor expressed on activated platelets, for use as a thrombus imaging agent. METHODS: The affinity and specificity of P280 peptide for the GPIIb/IIIa receptor was assessed by the inhibition of ADP-stimulated human platelet aggregation, the inhibition of the binding of fibrinogen to the GPIIb/IIIa receptor and the inhibition of the binding of vitronectin to the vitronectin receptor. P280 peptide was radiolabeled with 99mTc by ligand exchange using 99mTc-glucoheptonate. The ability of 99mTc-P280to detect thrombi in vivo was assessed using a canine venous thrombosis model and the biodistribution of 99mTc-P280 was determined in rats and rabbits. RESULTS: P280 peptide had an IC50 of 79 nM for the inhibition of aggregation of human platelets in platelet rich plasma, an IC50 of 6.8 nM for the inhibition of fibrinogen binding to the GPIIb/IIIa receptor and an IC50 of 13 microM for the inhibition of vitronectin binding to the vitronectin receptor, showing the high in vitro receptor binding affinity and specificity of the peptide. 99mTc-P280 was readily prepared in > or = 90% radiochemical and yield purity and provided images of femoral vein thrombin in the canine model by 1 hr postinjection (thrombus-to-blood ratio of 4.4 and thrombus-to-muscle ratio of 11 at 4 hr). Dog, rat and rabbit studies all showed rapid clearance of the radiotracer from the blood and rapid renal excretion. CONCLUSION: The combination of high in vitro receptor-binding affinity and specificity, in vivo thrombus imaging and fast clearance support the evaluation of 99mTc-280 as a clinical imaging agent.

Animals↗

PubMed1996

Free and esterified oxysterol: formation during copper-oxidation of low density lipoprotein and uptake by macrophages.

We have defined the lipid composition of copper-oxidized LDL (Cu-oxLDL) and a macrophage-foam cell model generated by the uptake of this modified lipoprotein. An HPLC method previously developed by our group for the measurement of lipid oxidation products of LDL was extended to permit the analysis of an array of 7-ketocholesteryl esters. Gas chromatography was used for the quantitation of oxysterols (free and esterified) in Cu-oxLDL and their subsequent uptake by macrophages. LDL (1.0 mg protein/ml) was oxidized using Cu(II) (20 microM) for up to 48 h at 37 degrees C. Resident mouse peritoneal macrophages were incubated with 24 h Cu-oxLDL (50 micrograms/ml) for 24 h. In 24 h Cu-oxLDL, cholesterol comprised approximately 50% of total sterols, 7-ketocholesterol comprised approximately 30% with five other oxysterols comprising the remainder (7 alpha- and 7 beta-hydroxycholesterol, cholesterol alpha- and beta-epoxides, and 6 beta-hydroxycholesterol). Macrophages that were incubated with 24 h Cu-oxLDL displayed a profile of oxysterols remarkably similar to that of 24 h Cu-oxLDL itself. The majority of cholesteryl esters and 7-ketocholesteryl esters in Cu-oxLDL and in Cu-oxLDL-loaded macrophages contained fatty acyl chains which are presumed oxidized. This work represents a comprehensive survey of free and esterified oxysterols in Cu-oxLDL and Cu-oxLDL-loaded macrophages and provides a basis for exploring how oxysterols are metabolized by macrophages and authentic human foam cells, and how, in turn, these oxysterols influence cellular metabolism.

Animals↗

PubMed1995

Biological fate of amino acid, peptide and protein hydroperoxides.

In the course of searching for a suitable marker for studying protein oxidation, we have successfully elucidated the structures of three valine hydroperoxides, i.e. beta-hydroperoxyvaline, (2S,3S)-gamma-hydroperoxyvaline and (2S,3R)-gamma-hydroperoxyvaline, which are novel products of protein oxidation. The corresponding valine hydroxides were obtained by sodium borohydride reduction [Fu, Hick, Sheil and Dean (1995) Free Rad. Biol. Med. 19, 281-292]. We hypothesized that valine hydroxides might be the major biological degradation products of valine hydroperoxides and, as such, could be useful markers for the study of protein oxidation in vivo. The aim of this study was to investigate the fate of valine hydroperoxide in selected biological systems by the use of chemiluminescence detection of hydroperoxides and HPLC analysis of O-phthaldialdehyde derivatives of amino acid residues. The degradation of hydroperoxides present on gamma-radiolysed solutions of valine, Pro-Val-Gly, or BSA occurred in the presence of: (1) transition metals (Fe2+, Fe3+, or Cu2+), (2) the detoxifying enzyme GSH peroxidase, (3) human plasma, and (4) J774 mouse monocyte macrophage cells. The major degradation product of valine hydroperoxide recovered in each case was found to be a valine hydroxide. These results suggest that valine hydroxide (derived from the hydroperoxide) may well be a useful in vivo marker for studying protein damage under oxidative stress.

Amino Acid Sequence↗