ABSTRACT
To construct an artificial low-density lipoprotein (aLDL) that highly mimics low-density lipoprotein (LDL) in vivo, and deliver vincristine (VCR) - doxorubicin (DOX) simultaneously, the 100 nm and 35 nm DOX-VCR-aLDLs (DV-aLDLs) were constructed, then the physicochemical characteristics were evaluated. Through in vitro inverse gravity diffusion experiment, the tumour cake and sphere model experiment, draw a conclusion that the diffusion of 35 nm DV-aLDLs was stronger than 100 nm DV-aLDLs, and the tumour retention of 35 nm DV-aLDLs was better than the DV-solution. In addition, the three-dimension (3D) in vivo distribution imaging of aLDLs was performed on HepG-2 tumour-bearing nude mice, followed by the biodistribution and therapeutic efficacy on these xenograft models. Taking advantage of better diffusion capacity in tumour tissue, as well as the synergistic effect of VCR and DOX, the 35 nm DV-aLDL had the strongest efficacy and the lowest toxicity. High entrapment efficiency and stability, both active and passive targeting, making aLDL a potential carrier for tumour-targeted therapy at the same time.
Subject(s)
Antineoplastic Agents/pharmacology , Doxorubicin/pharmacology , Lipoproteins, LDL/chemistry , Vincristine/pharmacology , Animals , Antineoplastic Agents/chemistry , Cell Proliferation/drug effects , Cell Survival/drug effects , Dose-Response Relationship, Drug , Doxorubicin/chemistry , Drug Screening Assays, Antitumor , Female , Hep G2 Cells , Humans , Lipoproteins, LDL/chemical synthesis , Liver Neoplasms, Experimental/drug therapy , Liver Neoplasms, Experimental/metabolism , Liver Neoplasms, Experimental/pathology , MCF-7 Cells , Mice , Mice, Nude , Molecular Structure , Structure-Activity Relationship , Vincristine/chemistryABSTRACT
Although peptide chemistry has made great progress, the frequent occurrence of aspartimide formation during peptide synthesis remains a formidable challenge. Aspartimide formation leads to low yields in addition to costly purification or even inaccessible peptide sequences. Here, we report an alternative approach to address this longstanding challenge of peptide synthesis by utilizing cyanosulfurylides to mask carboxylic acids by a stable C-C bond. These functional groups-formally zwitterionic species-are exceptionally stable to all common manipulations and impart improved solubility during synthesis. Deprotection is readily and rapidly achieved under aqueous conditions with electrophilic halogenating agents via a highly selective C-C bond cleavage reaction. This protecting group is employed for the synthesis of a range of peptides and proteins including teduglutide, ubiquitin, and the low-density lipoprotein class A. This protecting group strategy has the potential to overcome one of the most difficult aspects of modern peptide chemistry.
Subject(s)
Peptides/chemistry , Peptides/chemical synthesis , Solid-Phase Synthesis Techniques/methods , Aspartic Acid/analogs & derivatives , Aspartic Acid/chemical synthesis , Aspartic Acid/chemistry , Carboxylic Acids/chemistry , Cyanides/chemistry , Lipoproteins, LDL/chemical synthesis , Lipoproteins, LDL/chemistry , Protein Folding , Ubiquitin/chemistryABSTRACT
In view of the interrelations between NO, Fe, and LDL in the cardiovascular system it appears interesting to find out, if the lipoprotein particles undergo the process of iron-nitrosylation, commonly observed for other proteins and what is the biological fate of iron-nitrosylated LDL particles. Iron-nitrosylated LDL preparation containing Fe(NO)2 motif (DNICLDL) was obtained and characterized for the first time. In order to test its interactions with potential target cells, DNICLDL was administered to the hepatoma HepG2 cells. The effects were referred to those induced by native LDL (nLDL) and oxidized LDL (oxLDL) particles. DNICLDL administration considerably increased total iron content in the studied cell line, but did not influence the level of calcein-chelatable ions. DNICLDL was found to be low toxic to cells. The study suggests that DNICLDL might be a potential transducer of iron. © 2017 BioFactors, 44(2):192-201, 2018.
Subject(s)
Iron/metabolism , Lipoproteins, LDL/pharmacology , Nitrogen Oxides/pharmacology , Cations, Divalent , Cell Survival/drug effects , Electron Spin Resonance Spectroscopy , Fluoresceins/chemistry , Fluoresceins/pharmacology , Hep G2 Cells , Humans , Ion Transport , Iron/pharmacology , Iron Chelating Agents/chemistry , Iron Chelating Agents/pharmacology , Lipoproteins, LDL/chemical synthesis , Nitrogen Oxides/chemical synthesis , TransferrinABSTRACT
Increased level of low-density lipoprotein (LDL) strongly correlates with incidence of coronary heart disease. We synthesized novel molecularly imprinted polymers (MIP) as biomimetic specific receptors to establish rapid analysis of LDL levels. For that purpose the ratios of monomers acrylic acid (AA), methacrylic acid (MAA), and N-vinylpyrrolidone (VP), respectively, were screened on 10 MHz dual-electrode quartz crystal microbalances (QCM). Mixing MAA and VP in the ratio 3:2 (m/m) revealed linear sensor characteristic to LDL cholesterol (LDL-C) from 4 to 400 mg/dL or 0.10-10.34 mmol/L in 100 mM phosphate-buffered saline (PBS) without significant interference: high-density lipoprotein (HDL) yields 4-6% of the LDL signal, very-low-density-lipoprotein (VLDL) yields 1-3%, and human serum albumin (HSA) yields 0-2%. The LDL-MIP sensor reveals analytical accuracy of 95-96% at the 95% confidence interval with precision at 6-15%, respectively. Human serum diluted 1:2 with PBS buffer was analyzed by LDL-MIP sensors to demonstrate applicability to real-life samples. The sensor responses are excellently correlated to the results of the standard technique, namely, a homogeneous enzymatic assay (R(2) = 0.97). This demonstrates that the system can be successfully applied to human serum samples for determining LDL concentrations.
Subject(s)
Dimethylpolysiloxanes/chemistry , Lipoproteins, LDL/blood , Molecular Imprinting , Electrodes , Humans , Lipoproteins, LDL/chemical synthesis , Lipoproteins, LDL/chemistry , Serum Albumin/chemistryABSTRACT
Although angiotensin II potently affects blood pressure and fluid balance, it is also involved in deterioration in atherosclerotic cardiovascular disease. Recently, angiotensin AT(1) receptor blockers have been demonstrated to be effective in patients with atherosclerotic disease, but the exact mechanisms of these blockers are still controversial. Atherosclerotic plaques are characterized by cholesterol ester accumulation and acyl-CoA:cholesterol acyltransferase-1 (ACAT-1) expression, which are both parameters of degeneration of macrophage-derived foam cells. We examined the effects of angiotensin AT(1) receptor blockers on the formation of foam cells from macrophages. When macrophages from a human cell line were stimulated with oxidized low-density lipoprotein (oxLDL), the angiotensin AT(1) receptor blockers candesartan and losartan attenuated the intracellular accumulation of cholesterol ester and the increases in mRNA and protein levels of ACAT-1. Moreover, the increase in oxLDL-induced ACAT-1 was reduced by AG1478, an inhibitor of the epidermal growth factor (EGF) receptor. Additionally, oxLDL up-regulated the protein level of heparin-binding EGF-like growth factor (HB-EGF), a ligand of the EGF receptor. Inhibitors of angiotensin-converting enzyme affected neither cholesterol ester accumulation nor the expression of ACAT-1. Although oxLDL itself increased the secretion of angiotensin II, the amount of secreted angiotensin II was insufficient to induce expression of ACAT-1 protein. Thus, we first demonstrated that angiotensin AT(1) receptor blockers suppress ACAT-1 expression and cholesterol ester accumulation through an oxLDL-activated EGF receptor, but it is unclear how oxLDL activates angiotensin AT1 receptor in an angiotensin II-independent manner. The therapeutic mechanism of angiotensin AT(1) receptor blockers for atherosclerosis may be at least partially explained by our present results.
Subject(s)
Angiotensin II Type 1 Receptor Blockers/pharmacology , Benzimidazoles/pharmacology , Foam Cells/drug effects , Lipoproteins, LDL/pharmacology , Losartan/pharmacology , Tetrazoles/pharmacology , Acetyl-CoA C-Acetyltransferase/metabolism , Angiotensin II/metabolism , Angiotensin-Converting Enzyme Inhibitors/pharmacology , Biphenyl Compounds , Cells, Cultured , Cholesterol Esters/metabolism , Enzyme Inhibitors/pharmacology , ErbB Receptors/antagonists & inhibitors , Foam Cells/metabolism , Heparin-binding EGF-like Growth Factor , Humans , Intercellular Signaling Peptides and Proteins/metabolism , Lipoproteins, LDL/antagonists & inhibitors , Lipoproteins, LDL/chemical synthesis , Macrophages/drug effects , Macrophages/metabolism , Quinazolines/pharmacology , Triglycerides/metabolism , Tyrphostins/pharmacologyABSTRACT
Chronic Myeloid Leukemia (CML) stem/progenitor cells, which over-express Bcr-Abl, respond to imatinib by a reversible block in proliferation without significant apoptosis. As a result, patients are unlikely to be cured owing to the persistence of leukemic quiescent stem cells (QSC) capable of initiating relapse. Previously, we have reported that intracellular levels of imatinib in primary primitive CML cells (CD34+38(lo/â»)), are significantly lower than in CML progenitor cells (total CD34+) and leukemic cell lines. The aim of this study was to determine if potentially sub-therapeutic intracellular drug concentrations in persistent leukemic QSC may be overcome by targeted drug delivery using synthetic Low Density Lipoprotein (sLDL) particles. As a first step towards this goal, however, the extent of uptake of sLDL by leukemic cell lines and CML patient stem/progenitor cells was investigated. Results with non-drug loaded particles have shown an increased and preferential uptake of sLDL by Bcr-Abl positive cell lines in comparison to Bcr-Abl negative. Furthermore, CML CD34+ and primitive CD34+38(lo/â») cells accumulated significantly higher levels of sLDL when compared with non-CML CD34+ cells. Thus, drug-loading the sLDL nanoparticles could potentially enhance intracellular drug concentrations in primitive CML cells and thus aid their eradication.
Subject(s)
Drug Delivery Systems , Lipoproteins, LDL/chemistry , Lipoproteins, LDL/pharmacokinetics , Neoplastic Stem Cells/metabolism , Antigens, CD34/immunology , Antineoplastic Agents/administration & dosage , Cell Line, Tumor , Cell Membrane Permeability , Cells, Cultured , Drug Delivery Systems/methods , Hematopoietic Stem Cells/metabolism , Humans , Leukemia, Myelogenous, Chronic, BCR-ABL Positive/drug therapy , Leukocytes, Mononuclear/metabolism , Lipoproteins, LDL/chemical synthesis , Neoplastic Stem Cells/immunologyABSTRACT
Las enfermedades cardiovasculares (ECV) suponen la primera causa de muerteen los países desarrollados y se ha estimado que en el año 2010 también lideraránlas causas de muerte en los países en vías de desarrollo. Numerosos estudiosepidemiológicos han confirmado la relación entre colesterolemia y ECV, postulándoseque el descenso de los niveles séricos de colesterol produce una disminución de la incidencia y la prevalencia de muerte por cardiopatía isquémica yECV. Además se ha demostrado que la concentración elevada de lipoproteínas debaja densidad (LDL) es un factor de riesgo, mientras que la de lipoproteínas de altadensidad (HDL) es un factor protector frente a la ECV.Muchos autores han sugerido que esta acción beneficiosa de las HDL se debea que, unida a su molécula, existe una enzima denominada paraoxonasa (PON1).La PON1 es una enzima que presenta varias actividades in vitro: paraoxonasa, arilesterasay lactonasa. Parece inhibir la oxidación tanto de las LDL como de lasHDL y facilitar el transporte reverso del colesterol, disminuyendo así el riesgo deaterosclerosis.Su mecanismo de acción, propiedades catalíticas y sustratos naturales aun sedesconocen. Se han desarrollado algunos métodos espectrofotométricos y calorimétricospara determinar su actividad arilesterasa que aunque muy utilizados noson muy sensibles y los resultados obtenidos no son muy reproducibles. En estetrabajo se revisan muchos aspectos centrales referentes a esta enzima: mecanismosde acción, regulación por diferentes sustratos y mecanismos genéticos y dieta.Además se presenta un método que, utilizando como tampón un mimético desuero, permite obtener resultados más fiables y reproducibles de actividad arilesterasaen humanos y ratones. Por otra parte, se detectan posibles efectos de lospolimorfismos sobre los valores basales de actividad arilesterasa en individuos conriesgo cardiovascular incrementado
Cardiovascular diseases (CVD) are the first cause of death in developed countriesand it is estimated that by 2010 they will also be the leading cause of death indeveloping countries. Epidemiologic studies have demonstrated that reduction oftotal serum cholesterol decreases prevalence and death rates associated withischemic cardiopathy and CVD. Furthermore, a high concentration of LDL isconsidered a risk factor, while high levels of HDL are thought to be a protectivefactor.Many authors have suggested that HDL-bound PON1 enzyme may conferthe protective effects to HDL. PON1 is an enzyme with several in vitro activities:paraoxonase, arylesterase, and lactonase. It has been reported that PON1 inhibitsLDL and HDL peroxidation, as well as it facilitates the cholesterol reverse transport,helping to inhibit the developing of atherosclerosis.Its native substrates, its in vivo mechanism of action and its moleculartargets in the human body are still unknown. Nevertheless, calorimetric andspectrophotometric methods, very often employed but reaching to low very precise and sensible results, to determine its arylesterase activity have been developed. Inthis paper several aspects of this enzyme such as the mechanism of action, theregulation by substrates, genes and diet, are reviewed. Moreover, we present amethod that uses a serum mimetic buffer that permits to obtain more precise andrepeatable results of the arylesterase activity in humans and mice. Furthermore therelationship between PON1 polymorphisms and arylesterase activity is also testedin subjects at increased CVD-risk
Subject(s)
Humans , Cardiovascular Diseases/enzymology , Enzymes/analysis , Enzymes/physiology , Cholesterol/analysis , Cholesterol/pharmacology , Myocardial Ischemia/drug therapy , Myocardial Ischemia/enzymology , Arteriosclerosis/drug therapy , Arteriosclerosis/enzymology , Polymorphism, Genetic , Lipoproteins, LDL/chemistry , Cholesterol/pharmacokinetics , Risk Factors , Lipoproteins, LDL/chemical synthesis , Cholesterol, LDL/pharmacologyABSTRACT
LDL oxidation may be important in atherosclerosis. Extensive oxidation of LDL by copper induces increased uptake by macrophages, but results in decomposition of hydroperoxides, making it more difficult to investigate the effects of hydroperoxides in oxidised LDL on cell function. We describe here a simple method of oxidising LDL by dialysis against copper ions at 4 degrees C, which inhibits the decomposition of hydroperoxides, and allows the production of LDL rich in hydroperoxides (626+/-98 nmol/mg LDL protein) but low in oxysterols (3+/-1 nmol 7-ketocholesterol/mg LDL protein), whilst allowing sufficient modification (2.6+/-0.5 relative electrophoretic mobility) for rapid uptake by macrophages (5.49+/-0.75 microg (125)I-labelled hydroperoxide-rich LDL vs. 0.46+/-0.04 microg protein/mg cell protein in 18 h for native LDL). By dialysing under the same conditions, but at 37 degrees C, the hydroperoxides are decomposed extensively and the LDL becomes rich in oxysterols. This novel method of oxidising LDL with high yield to either a hydroperoxide- or oxysterol-rich form by simply altering the temperature of dialysis may provide a useful tool for determining the effects of these different oxidation products on cell function.
Subject(s)
Dialysis/methods , Ketocholesterols/chemical synthesis , Lipid Peroxides/chemical synthesis , Lipoproteins, LDL/chemical synthesis , Animals , Cells, Cultured , Cold Temperature , Copper/chemistry , Hot Temperature , Humans , Lipoproteins, LDL/chemistry , Macrophages , MiceABSTRACT
This review evaluates drug delivery systems that involve intact plasma lipoproteins or some of their components. These complex macromolecules transport highly water-insoluble compounds (cholesteryl esters and triacylglycerols) in their natural environment - a property that renders them ideal carriers of hydrophobic drugs. Particular emphasis is placed on the application of lipoproteins as drug delivery agents in cancer chemotherapy. The history and present activity regarding lipoprotein-based formulations are reviewed, with the primary focus on the smaller sized (low and high density) lipoprotein-based formulations and their potential clinical and commercial value. The use of both native and synthetic lipoproteins as drug delivery agents are discussed from the standpoint of therapeutic efficacy, as well as commercial feasibility. The advantages of lipoprotein-based drug delivery formulations are compared with other drug delivery models, with the primary focus on liposomal preparations. Finally, an expert opinion is provided, regarding the potential use of lipoprotein-based formulations in cancer treatment, taking into consideration the major advantages (biocompatibility, safety, drug solubility) and the barriers (manufacturing protein components, financial interest, investments) to their commercial development.
Subject(s)
Antineoplastic Agents/administration & dosage , Drug Delivery Systems/methods , Lipoproteins/chemistry , Animals , Antineoplastic Agents/chemistry , Antineoplastic Agents/therapeutic use , Humans , Lipoproteins, HDL/chemical synthesis , Lipoproteins, HDL/chemistry , Lipoproteins, LDL/chemical synthesis , Lipoproteins, LDL/chemistry , Neoplasms/drug therapyABSTRACT
Low density lipoprotein (LDL) is a normal plasma component, which is of interest in a number of research areas such as hypercholesterolaemia, drug targeting in cancer chemotherapy and as a lipid supplement in tissue culture systems. Currently, however, it can only be obtained by extraction from fresh plasma samples, which yields only small quantities. Synthetic LDL (sLDL) has been prepared using readily available lipid components coupled with a synthetic amphiphatic peptide molecule containing the apoprotein B receptor sequence. sLDL was capable of supporting the growth of Chinese Hamster Ovary (CHO) and fibroblast cells in serum-free culture media in a cholesterol-dependent manner that was related to the presence of the receptor peptide molecule. sLDL could be fluorescently labelled with 3,3'-dioctadecyloxalocarbocyanine perchlorate (DiO), and once labelled was assimilated by CHO and fibroblast cells in a time- and temperature-dependent manner that was dependent upon the presence of the receptor peptide. In addition, assimilation was reduced by an excess of unlabelled native LDL. The results indicated that the interaction of sLDL with CHO and fibroblast cells occurred via a receptor dependent system, most likely the LDL cellular receptor. sLDL is therefore a useful, easily obtained substitute for native LDL with potential applications in the areas of drug targeting to cells and serum-free tissue culture systems.
Subject(s)
Fibroblasts/metabolism , Lipoproteins, LDL/chemical synthesis , Lipoproteins, LDL/metabolism , Receptors, Lipoprotein/metabolism , Animals , CHO Cells , Carbocyanines/metabolism , Cell Culture Techniques , Cell Line , Cell Proliferation/drug effects , Cholesterol/analysis , Cricetinae , Culture Media, Serum-Free , Fibroblasts/cytology , Fibroblasts/physiology , Fluorescent Dyes/metabolism , Lipoproteins, LDL/chemistry , Microscopy, Confocal , Molecular Weight , Phospholipids/analysis , Temperature , Time Factors , Triglycerides/analysisABSTRACT
Lipid supplementation in serum-free tissue culture employs solubilization techniques to permit the addition of lipids, but these systems are potentially cytotoxic and do not present lipid in a natural form. In this research a simplified preparation method for synthetic low-density lipoprotein (sLDL) has been developed that involves microfluidization of a solvent lipid solution in a simple aqueous solution. This produces material with size and zeta potential characteristics similar to those of native LDL. sLDL supplementation in tissue culture media provides cholesterol concentrations higher than those achieved by 10% serum supplementation and existing chemically defined lipid supplements. sLDL stimulates NS0 and U937 cellular proliferation in completely serum-free media, the former in a lipid concentration dependent manner that is also related to both the receptor peptide structure employed and its concentration on the particle. The greatest NS0 cellular proliferation was obtained at the highest cholesterol concentration tested (0.5 mg/mL), which was 10 times higher than the cholesterol concentration achieved by standard 10% serum supplementation. U937 cellular proliferation was influenced by variation of sLDL's fatty acid constituents with a natural mixture producing maximal effect. Cell uptake studies in NS0 with fluorescently labeled sLDL indicated that assimilation is reduced by competition from native LDL. The planktonic nature of NS0 cell growth meant that cell binding and uptake experiments were difficult to conduct because of cellular aggregation. However, sLDL-induced U937 proliferation is ablated by the presence of an anti-LDL receptor antibody. The results indicate that sLDL uptake is via the LDL receptor and that sLDL can function as a lipid supplement for serum-free media capable of supplementation to cholesterol concentrations up to 0.5 mg/mL. Cellular uptake studies also suggest that sLDL will be useful for the targeting and delivery of materials to cells. sLDL therefore represents a new and promising synthetic biomimetic alternative to native LDL with multiple applications.
Subject(s)
Cell Culture Techniques , Culture Media, Serum-Free/chemistry , Lipoproteins, LDL/chemical synthesis , Lipoproteins, LDL/pharmacology , Animals , Biomimetics , Cell Proliferation , Chemistry, Physical/methods , Culture Media, Serum-Free/pharmacology , Humans , Lipoproteins, LDL/pharmacokinetics , Mice , Microscopy, Confocal , Tumor Cells, Cultured , U937 Cells/drug effectsABSTRACT
AIMS/HYPOTHESIS: Previous studies have implicated the glycoxidative modification of low-density lipoprotein (LDL) by glucose and aldehydes (apparently comprising both glycation and oxidation), as a causative factor in the elevated levels of atherosclerosis observed in diabetic patients. Such LDL modification can result in unregulated cellular accumulation of lipids. In previous studies we have characterized the formation of glycated, but nonoxidized, LDL by glucose and aldehydes; in this study we examine whether glycation of LDL, in the absence of oxidation, gives rise to lipid accumulation in arterial wall cell types. METHODS: Glycated LDLs were incubated with macrophage, smooth muscle, or endothelial cells. Lipid loading was assessed by HPLC analysis of cholesterol and individual esters. Oxidation was assessed by cholesterol ester loss and 7-ketocholesterol formation. Cell viability was assessed by lactate dehydrogenase release and cell protein levels. RESULTS: Glycation of LDL by glycolaldehyde and methylglyoxal, but not glucose (in either the presence or absence of copper ions), resulted in cholesterol and cholesterol ester accumulation in macrophage cells, but not smooth muscle or endothelial cells. The extent of lipid accumulation depends on the degree of glycation, with increasing aldehyde concentration or incubation time, giving rise to greater extents of particle modification and lipid accumulation. Modification of lysine residues appears to be a key determinant of cellular uptake. CONCLUSIONS/INTERPRETATION: These results are consistent with LDL glycation, in the absence of oxidation, being sufficient for rapid lipid accumulation by macrophage cells. Aldehyde-mediated "carbonyl-stress" may therefore facilitate the formation of lipid-laden (foam) cells in the artery wall.
Subject(s)
Acetaldehyde/analogs & derivatives , Acetaldehyde/chemistry , Lipoproteins, LDL/chemical synthesis , Lipoproteins/blood , Pyruvaldehyde/chemistry , Animals , Glycation End Products, Advanced , Humans , Indicators and Reagents , RatsABSTRACT
BACKGROUND: Endothelial dysfunction is thought to play a crucial role in the pathogenesis of atherosclerosis induced by ox-LDL. Recently, a variety of evidence suggested that endothelial progenitor cells (EPCs) participated in neovascularization and reendothelialization. However, effects of ox-LDL on EPCs number and activity are ill understood. METHODS: Total mononuclear cells (MNCs) were isolated from peripheral blood by Ficoll density gradient centrifugation, and then the cells were plated on fibronectin-coated culture dishes. After 7 days culture, attached cells were stimulated with ox-LDL (to make a series of final concentrations: 25 microg/mL, 50 microg/mL, 100 microg/mL, 200 microg/mL), native LDL (100 microg/mL) or vehicle control for the respective time points (6 h, 12 h, 24 h and 48 h). EPCs were characterized as adherent cells double positive for DiLDL-uptake and lectin binding by direct fluorescent staining under a laser scanning confocal microscope. EPCs were further documented by demonstrating the expression of KDR, VEGFR-2 and AC133 with flow cytometry. Proliferation, migration and in vitro vasculogenesis activity of EPCs were assayed by MTT assay, modified Boyden chamber assay and in vitro vasculogenesis kit, respectively. EPCs adhesion assay was performed by replating those on fibronectin-coated dishes, and then counting adherent cells. RESULTS: Incubation of isolated human EPCs with ox-LDL decreased the number of EPCs in concentration-dependent manner, maximum at 200 microg/mL (approximately 70% reduction, P < 0.001). In time-course experiments performed with an ox-LDL concentration of 100 microg/mL, decrease of EPCs number became apparent at 12 hours and reached the maximum at 24 hours (approximately 50% reduction, P < 0.01). In addition, ox-LDL dose and time dependently impaired EPC proliferative, migratory, adhesive and in vitro vasculogenesis capacity. CONCLUSION: The results of the present study defined a novel mechanism of action of ox-LDL: the reduction of EPCs with decreased functional activity.
Subject(s)
Endothelial Cells/drug effects , Lipoproteins, LDL/pharmacology , Stem Cells/drug effects , Adult , Blood Cell Count , Cell Adhesion/drug effects , Cell Movement/drug effects , Cell Proliferation/drug effects , Coronary Artery Disease/pathology , Endothelial Cells/physiology , Endothelial Cells/ultrastructure , Flow Cytometry , Humans , Lipoproteins, LDL/chemical synthesis , Neovascularization, Physiologic/drug effects , Stem Cells/physiology , Stem Cells/ultrastructure , Tetrazolium Salts , ThiazolesABSTRACT
Free radicals damage both lipids and proteins and evidence has accumulated for the presence of both oxidised lipids and proteins in aged tissue samples as well as those from a variety of pathologies including atherosclerosis, diabetes, and Parkinson's disease. Oxidation of the protein and lipid moieties of low-density lipoprotein is of particular interest due to its potential role in the unregulated uptake of lipids and cholesterol by macrophages; this may contribute to the initial stage of foam cell formation in atherosclerosis. In the study reported here, we examined the comparative time-courses of lipid and protein oxidation during copper-ion-mediated oxidation of low-density lipoprotein. We show that there is an early, lipid-mediated loss of 40-50% of the Trp residues of the apoB100 protein. There is no comparable loss over an identical period during the copper-ion-mediated oxidation of lipid-free BSA. Concomitant with Trp loss, the antioxidant alpha-tocopherol is consumed with subsequent extensive lipid peroxidation. Further changes to the protein, including the copper-ion-dependent 3.5-fold increase in 3,4-dihydroxyphenylalanine and the copper-ion-independent 3-5-fold increase in o-tyrosine, oxidation products of Tyr and Phe, respectively, only occur after maximal lipid peroxidation. Long incubation periods result in depletion of 3,4-dihydroxyphenylalanine, presumably reflecting further oxidative changes. Overall, copper-ion-mediated oxidation of LDL appears to proceed initially by lipid radical-dependent processes, even though some of the earliest detectable changes occur on the apoB100 protein. This is followed by extensive lipid peroxidation and subsequent additional oxidation of aromatic residues on apoB100, though it is not yet clear whether this late protein oxidation is lipid-dependent or occurs as a result of direct radical attack.
Subject(s)
Copper/chemistry , Lipids/chemistry , Lipoproteins, LDL/chemistry , Antioxidants/chemistry , Apolipoprotein B-100 , Apolipoproteins B/chemistry , Dihydroxyphenylalanine/chemical synthesis , Dihydroxyphenylalanine/chemistry , Humans , Lipid Peroxidation/physiology , Lipids/analysis , Lipoproteins, LDL/chemical synthesis , Lipoproteins, LDL/isolation & purification , Oxidation-Reduction , Phenylalanine/chemistry , Spectrometry, Fluorescence , Time Factors , Tryptophan/chemistry , Tyrosine/chemistry , alpha-Tocopherol/chemistryABSTRACT
A synthetic LDL (sLDL) has been prepared by combining a lipid microemulsion with amphipathic peptides containing the apoprotein B receptor domain. The biological properties of sLDL have been investigated using the U937 in vitro cell proliferation assay. sLDL exhibits a concentration dependent and saturable stimulation of U937 proliferation. By utilizing different amphipathic peptides, variable proliferation is achieved, indicating a specific interaction between sLDL and the U937 LDL receptor are possible. U937 proliferation is reduced by the addition of an anti-LDL receptor antibody, indicating that sLDL is assimilated via the LDL receptor pathway. The behavior of sLDL mimics that of native LDL, and this approach represents a viable technique for the production of an sLDL particle on a large scale for research and general application.
Subject(s)
Lipids/chemistry , Lipoproteins, LDL/metabolism , Receptors, LDL/metabolism , Receptors, Lipoprotein/chemistry , Cell Division/physiology , Emulsions/chemistry , Humans , Lipoproteins, LDL/chemical synthesis , Lipoproteins, LDL/chemistry , Lymphoma , Particle Size , Peptides/chemical synthesis , Peptides/chemistry , Tumor Cells, Cultured , U937 CellsABSTRACT
PURPOSE: Atherosclerosis is the underlying factor leading to such cardiovascular diseases (CVD) as stroke, aneurysm, and myocardial infarction. The early detection of atherosclerotic plaques is considered to be crucial for successful prevention and/or therapeutic and dietary intervention of CVD. Current diagnostic practice, on the other hand, can only detect the problem at an advanced stage. The purpose of this study was to examine the potential of using a radiolabeled cholesterol ester analog/acetylated low density lipoprotein (AcLDL) conjugate as a diagnostic agent for the early and non-invasive detection of atherosclerosis and for the monitoring of the effects of drug therapy. METHODS: Cholesteryl iopanoate (CI), a cholesterylester analog, was synthesized, radiolabeled, and incorporated into AcLDL. Early atherosclerotic lesions were induced in New Zealand White rabbits. 125[-CI/AcLDL was injected intravenously at 2 microCi/kg. Blood samples were taken at different time intervals after injection and clearance of the injected drug from blood was studied. The rabbits were sacrificed after 72 hours and the distribution of radioactivity in various organs was investigated. Aortae of both atherosclerotic lesion and control rabbits were removed for Sudan IV staining and autoradiography in order to confirm the formation of the atherosclerotic lesion and localization of radioactivity. RESULTS: The injected drug was found to be cleared from blood following a two compartment model. Radioactivity in the atherosclerotic aorta was found to be about 8 times higher than that in normal aorta, suggesting that the proposed diagnostic probe was selectively taken up by the atherosclerotic lesion. The autoradiography and staining confirmed that the localization of the proposed probe was superimposed with the atherosclerotic lesion site. CONCLUSIONS: The results suggested that incorporation of CI into AcLDL resulted in the selective localization of CI at the atherosclerotic plaque areas. CI/AcLDL labeled with appropriate radioisotope has the potential to be used as a probe for visualization of early atherosclerotic lesion using scintigraphy technology.
Subject(s)
Arteriosclerosis/diagnosis , Cholesterol Esters , Lipoproteins, LDL , Radiopharmaceuticals , Animals , Cholesterol Esters/chemical synthesis , Female , Humans , Lipoproteins, LDL/chemical synthesis , Rabbits , Radiopharmaceuticals/chemical synthesisABSTRACT
Flow cytometry can be used to quantify the accumulation of ceroid in macrophages, the result of cellular handling of certain lipoproteins. Using P388D1 cells, a murine-derived macrophage-like cell line, the effect of the lipophilic antioxidant, DL-alpha-tocopherol, upon the uptake and accumulation of ceroid by the cells was monitored on culture with artificial lipoproteins containing a single lipid species. Ceroid accumulation was greater for artificial lipoprotein composed of BSA complexed with cholesteryl arachidonate, than with cholesteryl linoleate. alpha-Tocopherol inhibited the ceroid accumulation, which was also dependent upon cell density. Thus, since these findings are similar to recent observations in primary cultures of murine peritoneal macrophages, it would appear that macrophage-like cell lines such as P388D1 cells are appropriate for the study of potential agonists and antagonists of lipid oxidation. Culture of P388D1 cells with oxidised human low-density lipoprotein (LDL) also resulted in ceroid formation, shown to be dependent upon the level of LDL oxidation as assessed by thiobarbituric acid-reactivity, the xylenol orange assay of peroxides and gas chromatographic analysis of cholesterol and fatty acid content. Ceroid accumulation reflected changes in the level of LDL oxidation better than did the cell association of oxidised radiolabelled LDL, monitored as that bound and retained by the cell.
Subject(s)
Ceroid/analysis , Lipoproteins, LDL/metabolism , Macrophages/metabolism , Animals , Cell Line , Cholesterol/analysis , Fatty Acids/analysis , Iodine Radioisotopes , Lipid Peroxidation , Lipoproteins, LDL/chemical synthesis , Mice , Peroxides/analysis , Thiobarbituric Acid Reactive Substances/analysisABSTRACT
The stereoselective and saturable binding between receptor and ligand molecules plays an important role in many biological processes. Therefore ligand-receptor interactions have been increasingly studied in recent years by means of radionuclide labelled ligand molecules of high radiochemical purity and high specific activity. This contribution describes radiochemical development work of such radioligands performed at the Austrian Research Centre Seibersdorf in recent years: Tc99m-Neo-Galacto-Albumin was prepared as ligand for the Hepatic Binding Protein receptor, I-123-Low Density Lipoprotein (LDL) for LDL-receptors in the liver. Since LDL is involved in the origin of atherosclerosis it was subsequently labelled with Tc99m and In-111 as well. A pyrrolidine-methyl-benzamide derivative was separated into its stereoisomers and the active form labelled with I-123. It exhibited specific uptake in Dopamine D-2 receptor containing brain tissue. A benzazepine derivative was likewise labelled with I-123 as potential ligand for D-1 receptors.
Subject(s)
Isotope Labeling , Radioligand Assay , Albumins/chemical synthesis , Benzamides/chemical synthesis , Carrier Proteins , Dopamine , Humans , Indium Radioisotopes , Iodine Radioisotopes , Lipoproteins, LDL/chemical synthesis , Organotechnetium Compounds/chemical synthesis , Pyrrolidines/chemical synthesis , Serum Albumin , Urea/analogs & derivativesABSTRACT
Low-density lipoprotein particles are potential drug carriers, but only lipophilic drug species partition into the core of the system. In this paper the polar drug methotrexate has been coupled to the exterior protein of low density lipoprotein (LDL) particles using the reagent 1-ethyl-3(3'-dimethylaminopropyl) carbodiimide HCl. The coupled system was sized by photon correlation spectroscopy and the in vitro activity of the complex determined against L1210 cells maintained in medium supplemented with fetal calf serum. The reaction between methotrexate and low density lipoprotein is variable but quantifiable, about ten drug molecules being attached to each LDL particle, resulting in an increase in the radius and polydispersity of the particles. The activity of the complex against L1210 murine leukaemia cells has been demonstrated in vitro, but it is 30 times less active than free drug.