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Background: In recent years, small animal arterial port-catheter systems have been implemented in rodents with reasonable success. The aim of the current study is to employ the small animal port-catheter system to evaluate the safety of multiple hepatic-artery infusions (HAI) of low-density lipoprotein-docosahexaenoic acid (LDL-DHA) nanoparticles to the rat liver., Methods: Wistar rats underwent surgical placement of indwelling HAI ports. Repeated administrations of PBS or LDL-DHA nanoparticles were performed through the port at baseline and days 3 and 6. Rats were sacrificed on day 9 at which point blood and various organs were collected for histopathology and biochemical analyses., Results: The port-catheter systems were implanted successfully and repeated infusions of PBS or LDL-DHA nanoparticles were tolerated well by all animals over the duration of the study. Measurements of serum liver/renal function tests, glucose and lipid levels did not differ between control and LDL-DHA treated rats. The liver histology was unremarkable in the LDL-DHA treated rats and the expression of hepatic inflammatory regulators (NF-κβ, IL-6 and CRP) were similar to control rats. Repeated infusions of LDL-DHA nanoparticles did not alter liver glutathione content or the lipid profile in the treated rats. The DHA extracted by the liver was preferentially metabolized to the anti-inflammatory DHA-derived mediator, protectin DX., Conclusion: Our findings indicate that repeated HAI of LDL-DHA nanoparticles is not only well tolerated and safe in the rat, but may also be protective to the liver., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Lipoproteins are very attractive natural-based transport systems suitable for applications in diagnostics and cancer therapy. Low- and high-density lipoproteins (LDL, HDL) were selected for hypericin (hyp) delivery in cancer cells. Hyp was used, as it is a well-known model for hydrophobic molecules, in order to estimate the LDL and HDL transport efficacy. We applied fluorescence techniques, absorption and Raman spectroscopy to characterize the state and alteration of LDL and HDL in the absence and presence of hyp. The fluorescence intensity of hyp loaded in lipoproteins was two times weaker in HDL than LDL. We demonstrated that there are faster redistribution kinetics of hyp from HDL than from LDL. As a consequence, hyp uptake by glioma and breast cancer cells was driven more via endocytosis when hyp was delivered by LDL than by HDL. Hyp induced photodynamic action was stronger when hyp was delivered by HDL than LDL. Ex ovo hyp fluorescence pharmacokinetics demonstrated differences in biodistributions of hyp in lipoproteins topical applications. However, hyp was successfully delivered to cancer cells grafted on quail's chorioallantoic membrane. The results presented in this paper could provide strategies to develop adequate and targeted anticancer therapy., (Copyright © 2018 Elsevier B.V. All rights reserved.)

A variety of diseases are caused by deficiencies in amounts or activity of key proteins. An approach that increases the amount of a specific protein might be of therapeutic benefit. We reasoned that translation could be specifically enhanced using trans-acting agents that counter the function of negative regulatory elements present in the 5' UTRs of some mRNAs. We recently showed that translation can be enhanced by antisense oligonucleotides (ASOs) that target upstream open reading frames. Here we report the amount of a protein can also be selectively increased using ASOs designed to hybridize to other translation inhibitory elements in 5' UTRs. Levels of human RNASEH1, LDLR, and ACP1 and of mouse ACP1 and ARF1 were increased up to 2.7-fold in different cell types and species upon treatment with chemically modified ASOs targeting 5' UTR inhibitory regions in the mRNAs encoding these proteins. The activities of ASOs in enhancing translation were sequence and position dependent and required helicase activity. The ASOs appear to improve the recruitment of translation initiation factors to the target mRNA. Importantly, ASOs targeting ACP1 mRNA significantly increased the level of ACP1 protein in mice, suggesting that this approach has therapeutic and research potentials., (© The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Scope: In animal studies, epigallocatechin gallate (EGCG), the dominant catechin in green tea, has been shown to improve cholesterol metabolism. However, the molecular mechanisms of EGCG underlying these functions have not been fully understood. In this study, we aimed to clarify the molecular mechanisms of the effect of EGCG on cholesterol metabolism mainly in HepG2 cells., Methods and Results: We found that EGCG induced a reduction of the extracellular proprotein convertase subtilisin/kexin 9 (PCSK9) level accompanied by an up-regulation of the LDL receptor (LDLR) in HepG2 cells. The EGCG-induced up-regulation of LDLR occurred via the extracellular signal-regulated kinase (ERK) signaling pathway. Moreover, we showed that EGCG induced a significant early reduction of the extracellular PCSK9 protein level. However, there were no significant changes in the PCSK9 mRNA and the intracellular PCSK9 protein levels induced by EGCG. Annexin A2 knockdown affected the basal LDLR expression and did not affect the EGCG-induced reduction of the extracellular PCSK9 protein level or the up-regulation of LDLR., Conclusion: Annexin A2 possesses an essential function for the basal LDLR expression in HepG2 cells. But, EGCG induces the suppression of PCSK9 accompanied by an up-regulation of LDLR in an annexin A2-independent manner. EGCG attenuates the statin-induced an increase in PCSK9 level., (© 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Increases in plasma LDL-cholesterol have unequivocally been established as a causal risk factor for atherosclerosis. Hence, strategies for lowering of LDL-cholesterol may have immediate therapeutic relevance. Here we study the role of human neutrophil peptide 1 (HNP1) in a mouse model of atherosclerosis and identify its potent atheroprotective effect both upon transgenic overexpression and therapeutic delivery. The effect was found to be due to a reduction of plasma LDL-cholesterol. Mechanistically, HNP1 binds to apolipoproteins enriched in LDL. This interaction facilitates clearance of LDL particles in the liver via LDL receptor. Thus, we here identify a non-redundant mechanism by which HNP1 allows for reduction of LDL-cholesterol, a process that may be therapeutically instructed to lower cardiovascular risk., (Copyright © 2017 3-V Biosciences. Published by Elsevier B.V. All rights reserved.)

Determination of LDL particle uptake into cells is a valuable technique in the field of cholesterol metabolism. This allows assessment of LDL uptake capacity in different adherent and non-adherent cells types, as well as the effect of cellular, genetic, or pharmacological perturbations on this process. Here, we detail a general procedure that describes the production of fluorescently-labeled LDL particles and quantitative and non-quantitative assays for determining cellular LDL uptake.

Foam cell formation, which is caused by imbalanced cholesterol influx and efflux by macrophages, plays a vital role in the occurrence and development of atherosclerosis. Humanin (HN), a mitochondria-derived peptide, can prevent the production of reactive oxygen species and death of human aortic endothelial cells exposed to oxidized low-density lipoprotein (ox-LDL) and has a protective effect on patients with in early atherosclerosis. However, the effects of HN on the regulation of cholesterol metabolism in RAW 264.7 macrophages are still unknown. This study was designed to investigate the role of [Gly14]-humanin (HNG) in lipid uptake and cholesterol efflux in RAW 264.7 macrophages. Flow cytometry and live cell imaging results showed that HNG reduced Dil-ox-LDL accumulation in the RAW 264.7 macrophages. A similar result was obtained for lipid accumulation by measuring cellular cholesterol content. Western blot analysis showed that ox-LDL treatment upregulated not only the protein expression of CD36 and LOX-1, which mediate ox-LDL endocytosis, but also ATP-binding cassette (ABC) transporter A1 and ABCG1, which mediate ox-LDL exflux. HNG pretreatment inhibited the upregulation of CD36 and LOX-1 levels, prompting the upregulation of ABCA1 and ABCG1 levels induced by ox-LDL. Therefore we concluded that HNG could inhibit ox-LDL-induced macrophage-derived foam cell formation, which occurs because of a decrease in lipid uptake and an increase in cholesterol efflux from macrophage cells., (Copyright © 2016 Elsevier Inc. All rights reserved.)

There has been increasing interest in fluorescence-based imaging techniques in clinical practice, with the aim to detect and visualize the tumour configuration and the border with healthy tissue. Strong photodynamic activity of hypericin (Hyp) can be improved by various molecular transport systems (e.g. LDL). Our aim was to examine pharmacokinetics of Hyp in the presence of LDL particles on ex ovo chorioallantoic membrane (CAM) of Japanese quail with implanted TE1 tumour spheroids (human squamocellular carcinoma). Spheroids were implanted on CAM surface on embryonal day 7 and after 24 hours formulations of free Hyp and Hyp:LDL 100:1 and 200:1 were topically applied. All experimental formulations in the fluorescent image very well visualized the tumour spheroid position, with gradual increase of fluorescence intensity in 6-h observation period. LDL transportation system exhibited clear superiority in fluorescence pharmacokinetics than free Hyp formulation by increasing tumour-normal difference. Our experimental results confirm that Hyp and Hyp:LDL complex is potent fluorophore for photodynamic diagnosis of squamocellular carcinoma.

Low density lipoprotein (LDL), which is a principal carrier for the delivery of cholesterol, has been used as a great candidate for the delivery of drugs to tumor based on the great requirements for cholesterol of many cancer cells. Mimicking the structure and composition of LDL, we designed a synthetic low-density lipoprotein (sLDL) to encapsulate paclitaxel-alpha linolenic acid (PALA) for tumor therapy. The PALA loaded sLDL (PALA-sLDL) and PALA-loaded microemulsion (PALA-ME, without the binding domain for LDLR) displayed uniform sizes with high drug loading efficiency (> 90%). In vitro studies demonstrated PALA-sLDL exhibited enhanced cellular uptake capacity and better cytotoxicity to LDLR over-expressed U87 MG cells as compared to PALA-ME. The uptake mechanisms of PALA-sLDL were involved in a receptor mediated endocytosis and macropinocytosis. Furthermore, the in vivo biodistribution and tumor growth inhibition studies of PALA-sLDL were investigated in xenograft U87 MG tumor-bearing mice. The results showed that PALA-sLDL exhibited higher tumor accumulation than PALA-ME and superior tumor inhibition efficiency (72.1%) compared to Taxol® (51.2%) and PALA-ME (58.8%) but with lower toxicity. These studies suggested that sLDL is potential to be used as a valuable carrier for the selective delivery of anticancer drugs to tumor with low systemic toxicity., Competing Interests: All the authors declare no conflicts of interests.

Scope: Iron overload contributes to the pathogenesis of atherosclerosis and iron chelators are beneficial through their antioxidant properties. Hepatic iron loading increases cholesterol synthesis. Whether iron depletion could affect hepatic cholesterol metabolism is unknown., Methods and Results: We examined the effect of the iron chelator deferoxamine (DFO) on mRNA expression of genes involved in cholesterol metabolism and/or cholesterol uptake. Our results revealed that DFO increases LDL receptor (LDLR) mRNA levels in human hepatocyte-derived cell lines HepG2 and Huh7 cells, and in K562 cells. In HepG2 cells, we observed that DFO increases (i) LDLR-mRNA levels in a time- and dose-dependent manner, (ii) LDLR-protein levels; (iii) cell surface LDLR; and (iv) LDL uptake. In contrast, the mRNA levels of 3-hydroxy-3-methylglutaryl-coenzyme A reductase, sterol regulatory element-binding proteins, and the mRNA/protein levels of proprotein convertase subtilisin-kexin 9 were not modulated by DFO, suggesting that the LDLR regulation by DFO is not at the transcriptional or posttranslational levels. Since LDLR-mRNA was stabilized by DFO, a posttranscriptional mechanism is suggested for the DFO-mediated upregulation of LDLR., Conclusion: DFO induced an increase in LDLR expression by a posttranscriptional mechanism resulting in an enhancement of LDL uptake in HepG2 cells, suggesting increased LDLR activity as one of the underlying causes of the hypocholesterolemic effect of iron reduction., (© 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Background: Lectin-like oxidized low-density lipoprotein-1 (LOX-1) is the major receptor for oxidized low density lipoprotein (ox-LDL) uptake in human umbilical vein endothelial cells (HUVECs). Previously, we found that rapamycin inhibited ox-LDL accumulation in HUVECs, and this effect was related to its role in increasing the activity of autophagy-lysosome pathway. In this study, we determined whether rapamycin could also reduce ox-LDL uptake in HUVECs and investigated the underlying signaling mechanisms., Results: Flow cytometry and live cell imaging showed that rapamycin reduced Dil-ox-LDL accumulation in HUVECs. Furthermore, rapamycin reduced the ox-LDL-induced increase in LOX-1 mRNA and protein levels. Western blotting showed that rapamycin inhibited mechanistic target of rapamycin (mTOR), p70s6k and IκBα phosphorylation triggered by ox-LDL. Flow cytometry implied that mTOR, NF-κB knockdown and NF-κB inhibitors significantly reduced Dil-ox-LDL uptake. Moreover, immunofluorescent staining showed that rapamycin reduced the accumulation of p65 in the nucleus after ox-LDL treatment for 30 h. mTOR knockdown decreased LOX-1 protein production and IκBα phosphorylation induced by ox-LDL. NF-κB knockdown and NF-κB inhibitors reduced LOX-1 protein production, but did not inhibit mTOR phosphorylation stimulated by ox-LDL., Conclusions: These findings demonstrate that rapamycin reduce mTOR phosphorylation and subsequently inhibit NF-κB activation and suppresses LOX-1, resulting in a reduction in ox-LDL uptake in HUVECs.

Low-density lipoprotein (LDL) uptake is one of a number of tests used to demonstrate hepatocyte-like function after stem cell differentiation. Use of two compounds, LDL and acetylated LDL (AcLDL), has been described despite each having different mechanisms of uptake. Three primary hepatocyte cultures and three sets of mesenchymal stromal cell (MSC) cultures, derived from both adipose tissue and bone marrow, were harvested from dogs. Those cells were compared to commercially available human and mouse bone marrow-derived MSCs. LDL receptor expression was demonstrated by gene expression and immunofluorescence in all primary hepatocyte cultures, undifferentiated canine bone marrow MSCs and canine adipose MSCs. Undifferentiated human and mouse bone marrow MSCs also expressed the LDL receptor. In vitro, canine hepatocytes took up labeled LDL, but not AcLDL. All undifferentiated MSCs took up LDL, but not AcLDL. In conclusion, LDL and not AcLDL is a test of canine hepatocyte-like phenotype, but this is not tissue or species specific and, therefore, is not informative assay when testing proof of MSC to hepatocyte differentiation.

The glutathione (GSH)-dependent antioxidant system has been demonstrated to inhibit atherosclerosis. Macrophage CD36 uptakes oxidized low density lipoprotein (oxLDL) thereby facilitating foam cell formation and development of atherosclerosis. It remains unknown if GSH can influence macrophage CD36 expression and cellular oxLDL uptake directly. Herein we report that treatment of macrophages with l-buthionine-S,R-sulfoximine (BSO) decreased cellular GSH production and ratios of GSH to glutathione disulfide (GSH/GSSG) while increasing production of reactive oxygen species. Associated with decreased GSH levels, macrophage CD36 expression was increased, which resulted in enhanced cellular oxLDL uptake. In contrast, N-acetyl cysteine and antioxidant enzyme (catalase or superoxide dismutase) blocked BSO-induced CD36 expression as well as oxLDL uptake. In vivo, administration of mice with BSO increased CD36 expression in peritoneal macrophages and kidneys. BSO had no effect on CD36 mRNA expression and promoter activity but still induced CD36 protein expression in macrophages lacking peroxisome proliferator-activated receptor γ expression, suggesting it induced CD36 expression at the translational level. Indeed, we determined that BSO enhanced CD36 translational efficiency. Taken together, our study demonstrates that cellular GSH levels and GSH/GSSG status can regulate macrophage CD36 expression and cellular oxLDL uptake and demonstrate an important anti-atherogenic function of the GSH-dependent antioxidant system by providing a novel molecular mechanism., (© 2015 by The American Society for Biochemistry and Molecular Biology, Inc.)

TNF-α potently induces LOX-1 expression in THP-1 macrophages at concentrations between 1.25-50 ng/mL. The interplay between the two TNF receptors (TNFR1 and TNFR2) was apparent in the expression pattern of LOX-1 in response to TNF-α. Interestingly, R1 signal abrogation depleted both TNFR2 as well as LOX-1 transcript expression, suggesting that TNFR1 holds priority in the relative signaling mechanism between TNFR1 and TNFR2. TNF-α was also found to abrogate the oxidized-LDL (ox-LDL) mediated increase in intracellular pool of NO, a known downstream intermediate of LOX-1 pro-inflammatory signaling cascade. At the level of ox-LDL clearance, TNF-α inhibited the uptake (scavenging) of ox-LDL via LOX-1. Our study demonstrates the ability of TNF-α to enhance the signaling propensity of LOX-1 by increasing its expression and inhibiting its scavenging property.

Atherosclerosis is the common pathological basis of cardiovascular and cerebrovascular disease. This study aimed to investigate the effects of vascular endothelial growth factor (VEGF) and salvianolic acid B (SalB) on the permeability of the rabbit aortary endothelial cells (RAECs) and to figure out the possible underlying molecular mechanisms. The extravasation of (125)I-low density lipoprotein ((125)I-LDL) through the RAECs was significantly increased by VEGF and decreased by SalB. Meanwhile, the tight junction-associated proteins occludin and claudin-5 were found downregulated by VEGF and the caveolae structure proteins caveolin-1 and caveolin-2 upregulated, which were abolished by the infusion of SalB. In addition, a marked increase in levels of cGMP and protein kinase G-1 (PKG-1) as well as activation of nuclear factor-κB (NF-κB) p65 were found after VEGF infusion, which were attenuated by SalB. This study demonstrates that VEGF and SalB can alter the LDL permeability of the RAECs by a paracellular pathway (downregulation of occludin and claudin-5) and a transcellular pathway (upregulation of caveolin-1 and caveolin-2), in which the cGMP/PKG/NF-κB signal pathway is possibly involved. The experimental results provide a new method and basic knowledge of prevention and treatment for cardiovascular and cerebrovascular disease.

Familial hypercholesterolemia (FH) is a common autosomal codominant disease with a frequency of 1:500 individuals in its heterozygous form. The genetic basis of FH is most commonly mutations within the LDLR gene. Assessing the pathogenicity of LDLR variants is particularly important to give a patient a definitive diagnosis of FH. Current studies of LDLR activity ex vivo are based on the analysis of 125I-labeled lipoproteins (reference method) or fluorescent-labelled LDL. The main purpose of this study was to compare the effectiveness of these two methods to assess LDLR functionality in order to validate a functional assay to analyse LDLR mutations. LDLR activity of different variants has been studied by flow cytometry using FITC-labelled LDL and compared with studies performed previously with 125I-labeled lipoproteins. Flow cytometry results are in full agreement with the data obtained by the 125I methodology. Additionally confocal microscopy allowed the assignment of different class mutation to the variants assayed. Use of fluorescence yielded similar results than 125I-labeled lipoproteins concerning LDLR activity determination, and also allows class mutation classification. The use of FITC-labelled LDL is easier in handling and disposal, cheaper than radioactivity and can be routinely performed by any group doing LDLR functional validations.

Background/aims: Integrin activation and lymphocyte migration to the vascular intima is a key event in early atherosclerosis. α4β7 integrin (LPAM-1) and its ligand, mucosal addressin cell adhesion molecule (MAdCAM-1) are known to play an important role in homing of activated lymphocytes to gut-associated lymphoid tissues. However, it is unclear whether α4β7 integrin is involved in the pathogenesis of atherosclerosis., Methods: The expressions of α4β7 integrin and its ligands in atherosclerosis plaques from 12 week high fat diet (HFD) fed ApoE(-/-) and C57BL/6 mice were examined using immunofluorescent and immunohistochemical assays, respectively. We also generated ApoE/β7 double deficient mice and compared atherosclerotic lesion development in β7(+/+)ApoE(-/-) and β7(-/-)ApoE(-/-) mice that were fed with HFD for 12 weeks., Results: We found an upregulation of α4β7 integrin and its ligands VCAM-1 and MAdCAM-1 at atherosclerosis plaques in Apolipoprotein E deficient (ApoE(-/-)) mice fed with HFD for 12 weeks. Over the 12 week HFD period, peripheral blood lymphocyte (PBL) expression of α4β7 integrin increased in parallel with aortic lesion size. A removal of α4β7 integrin by genetic deletion of the β7 chain in the ApoE(-/-) mouse resulted in a markedly decreased 12 week-HFD atherosclerotic plaque area. β7(-/-) ApoE(-/-) macrophages showed reduced acetylated and native LDL uptake and phagocytic activity, revealing possible roles for α4β7 at two distinct stages of macrophage dysfunction during atherogenesis. Finally, a reduced activity of integrin downstream signalling components focal adhesion kinase (FAK) and MAPK/ERK1/2 in macrophage indicates their possible engagement during α4β7 integrin signalling in atherosclerosis., Conclusions: Together our results reveal a critical role of α4β7 in diet-induced atherosclerosis in mouse., (© 2014 S. Karger AG, Basel.)

Although chronic arsenic exposure is a well-known risk factor for cardiovascular diseases, including atherosclerosis, the molecular mechanism underlying arsenic-induced atherosclerosis remains obscure. Therefore, this study aimed to elucidate this molecular mechanism. We examined changes in the mRNA level of the lectin-like oxidized LDL (oxLDL) receptor (LOX-1) in a mouse aortic endothelial cell line, END-D, after sodium arsenite (SA) treatment. SA treatment significantly upregulated LOX-1 mRNA expression; this finding was also verified at the protein expression level. Flow cytometry and fluorescence microscopy analyses showed that the cellular uptake of fluorescence (Dil)-labeled oxLDL was significantly augmented with SA treatment. In addition, an anti-LOX-1 antibody completely abrogated the augmented uptake of Dil-oxLDL. We observed that SA increased the levels of the phosphorylated forms of nuclear factor of kappa light polypeptide gene enhancer in B cells (NF-κB)/p65. SA-induced upregulation of LOX-1 protein expression was clearly prevented by treatment with an antioxidant, N-acetylcysteine (NAC), or an NF-κB inhibitor, caffeic acid phenethylester (CAPE). Furthermore, SA-augmented uptake of Dil-oxLDL was also prevented by treatment with NAC or CAPE. Taken together, our results indicate that arsenic upregulates LOX-1 expression through the reactive oxygen species-mediated NF-κB signaling pathway, followed by augmented cellular oxLDL uptake, thus highlighting a critical role of the aberrant LOX-1 signaling pathway in the pathogenesis of arsenic-induced atherosclerosis., (© 2013.)

Aim: Hematopoietic stem cells, especially CD117(pos) cells, have been found to possess a regenerative potential in various tissues, in particular cardiac muscle. However, the characterization of the relevant ion currents of stem cells prior to implantation lacks documentation. Activation of angiotensin II type 2 receptor (AT2 R) can lead to further cell differentiation and receptor auto-expression and might thus influence electrophysiological properties of CD117(pos) stem cells. This study was designed to functionally characterize membrane currents of CD117(pos) cells under normal and AT2 R-stimulated conditions., Methods: CD117(pos) murine bone marrow stem cells were isolated with MACS technique and stimulated for the AT2 R with angiotensin II and losartan for 3-5 days prior to patch-clamp measurements. RT-PCR was used to determine channel expression. Endothelial properties were analysed with immunocytochemistry and acLDL uptake assay., Results: A well-expressed inward rectifying current (IKir ) was identified in cultured CD117(pos) cells. Furthermore, a ZD 7288 (HCN channel blocker)-sensitive current component was isolated. Voltage-dependent potassium currents and chloride currents were less expressed. A small fraction of cells demonstrated voltage- and time-dependent inward currents. In AT2 R-stimulated cells inward rectifying the hyperpolarization-induced inward currents were slightly attenuated on the translational level but showed increased mRNA expression. Cultured CD117(pos) cells express CD31 and VEGFR-2 and significantly increased the uptake of acLDL., Conclusions: CD117(pos) cells do not have properties of action potential-generating cells and moderately change their excitability during AT2 R stimulation. Electrophysiological and molecular properties of control and AT2 R-stimulated cells point to a differentiation to vascular endothelial cells. This could increase beneficial vascularization in injured tissues., (© 2013 Scandinavian Physiological Society. Published by John Wiley & Sons Ltd.)

To allow visualization of macrophage-rich and miniature-sized atheromas by magnetic resonance (MR) imaging, we have converted low-density lipoprotein (LDL) into MR-active nanoparticles via the intercalation of a 1,4,7,10-tetraazacyclodecane-1,4,7-triacetic acid (DO3A) derivative and the subsequent coordination reaction with Gd(3+). After careful removal of nonchelated Gd(3+), an MR-active LDL (Gd(3+)-LDL) with a remarkably high payload of Gd(3+) (in excess of 200 Gd(3+) atoms per particle) and a high relaxivity (r(1) = 20.1 s(-1) mM(-1) per Gd(3+) or 4040 s(-1) mM(-1) per LDL) was obtained. Dynamic light-scattering photon correlation spectroscopy (DLS) and cryo transmission electron microscope (cryoTEM) images showed that Gd(3+)-LDL particles did not aggregate and remained of a similar size (25-30 nm) to native LDL. Intravenous injection of Gd(3+)-LDL into an atherosclerotic mouse model (ApoE(-/-)) resulted in an extremely high enhancement of the atheroma-bearing aortic walls at 48 h after injection. Free Gd(3+) dissociation from Gd(3+)-LDL was not detected over the imaging time window (96 h). Because autologous LDL can be isolated, modified, and returned to the same patient, our results suggest that MR-active LDL can potentially be used as a noninfectious and nonimmunogenic imaging probe for the enhancement of atheroplaques presumably via the uptake into macrophages inside the plaque.

Lectin-like oxidized LDL (oxLDL) receptor-1 (LOX-1), a novel scavenger receptor highly expressed in human and experimental atherosclerotic lesions, is responsible for the uptake of oxLDL in vascular cells. We demonstrated previously that Tanshinone II-A (Tan), a pharmacologically active compound extracted from the rhizome of the Chinese herb Salvia miltiorrhiza Bunge, inhibits atherogenesis in hypercholesterolemic rats, rabbits, and apolipoprotein-E deficient (ApoE⁻/⁻) mice. However, the precise mechanism by which Tan protects against atherogenesis remains to be elucidated. Therefore, we hypothesized that Tan can suppress the uptake of oxLDL by diminishing the expression of LOX-1 via suppression of NF-κB signaling pathway, thereby contributing to reduced macrophage foam cell formation. In cultured murine macrophages, oxLDL induced LOX-1 expression at the mRNA and protein levels, was abrogated by addition of Tan or pyrrolidinedithiocarbamic acid ammonium salt (PDTC), a widely used inhibitor of NF-κB, suggesting the involvement of NF-κB. Tan also reduced LOX-1 expression in atherosclerotic lesions of ApoE⁻/⁻ mice fed a high cholesterol diet. Mechanistically, Tan suppressed the nuclear translocation of NF-κB P65 subunit and phosphorylation of IκB-α induced by oxLDL. Electrophoretic mobility shift assay (EMSA) demonstrated that Tan inhibited the nuclear protein binding to NF-κB consensus sequence. Functionally, we observed that Tan inhibited DiI-oxLDL uptake by macrophages in a fashion similar to that produced by LOX-1 neutralizing antibody. Our current findings reveal a novel mechanism by which Tan protects against atherogenesis and shed new light on the potential therapeutic application of Tan to the treatment and prevention of atherosclerotic cardiovascular diseases., (Copyright © 2012 Mosby, Inc. All rights reserved.)

To clarify the effect of concentration polarization of oxidative modification of low-density lipoproteins (ox-LDLs) on human smooth muscle cells (SMCs), the proliferation, ox-LDL uptake and apoptosis with SMCs cultured on permeable (the permeable group) or non-permeable membranes (the non-permeable group) were analysed by 3-(4, 5-dimethylthiazolyl-2)-2, 5-diphenyltetrazolium bromide (MTT) assay, spectrofluorometry and flow cytometry using a parallel-plate flow chamber technique. The concentration polarization of ox-LDLs at the surface of the cultured cell monolayer was assessed by confocal laser microscopy. The results showed that concentration polarization of ox-LDLs could indeed occur at the cultured cell monolayer surface of the permeable group, leading to an enhanced wall concentration of ox-LDLs that was over 15 per cent higher than the bulk concentration of the perfusion solution at a pressure of 100 mmHg. When concentration of ox-LDLs in the perfusion solution was less than or equal to 100 µg ml(-1), SMCs' proliferation was induced, while cell apoptosis was induced when its concentration was above 150 µg ml(-1). The uptake of ox-LDLs by the cultured cells was significantly higher for the permeable group than for the non-permeable group. In addition, the ox-LDL-induced cell death and apoptosis were much more severe in the permeable group than that in the non-permeable group. Therefore, the experimental study suggests that concentration polarization of ox-LDLs plays an adverse role in the vascular system owing to its toxicity to vascular cells, in turn enhance ox-LDL infiltration into the arterial wall and accelerate SMC apoptosis.

Tumor cells were targeted with Gd-loaded/LDL (low density lipoproteins) adducts consisting of ca 300 Gd(III) amphiphilic complexes incorporated in the lipophilic LDL particles. The long reorientational time of the Gd(III) complex in the supramolecular adduct yielded a relaxivity peak at ca 1 T, whereas its relaxivity at 7 T was 5 times less. The field-dependent relaxivity markedly affected the signal enhancement attainable at the two magnetic fields. As tumor cells showed up-regulation of LDL transporters, B16 melanoma cells were labeled with the Gd-loaded/LDL adduct. Each cell contained ca 2 × 10(9) Gd atoms. Upon dispersion of 5000 labeled cells in 1 μl of agar, signal intensity (SI) enhancements of about 30 and 7% were observed at 1 and 7 T, respectively. The results obtained on cellular systems were confirmed in vivo upon the administration of Gd-loaded/LDL particles to C57 mice bearing a transplanted melanoma (B16) tumor. From the herein reported results, one may conclude that, for slowly moving Gd complexes, it is possible to obtain in vivo sensitivity enhancements at 1 T several times higher than that attained at high fields., (Copyright © 2011 John Wiley & Sons, Ltd.)

The LDL receptor (LDLR) is an endocytic receptor that plays a major role in the clearance of atherogenic lipoproteins from the circulation. During the endocytic process, the LDLR first binds lipoprotein at the cell surface and then traffics to endosomes, where the receptor releases bound lipoprotein. Release is acid-dependent and correlates with the formation of an intramolecular contact within the receptor. Human mutations at residues that form the contact are associated with familial hypercholesterolemia (FH) and the goal of the present study was to determine the role of contact residues on LDLR function. We show that mutations at nine contact residues reduce the ability of the LDLR to support lipoprotein uptake. Unexpectedly, only four of the mutations (W515A, W541A, H562Y and H586Y) impaired acid-dependent lipoprotein release. The remaining mutations decreased the lipoprotein-binding capacity of the LDLR through either reduction in the number of surface receptors (H190Y, K560W, H562Y and K582W) or reduction in the fraction of surface receptors that were competent to bind lipoprotein (W144A and W193A). We also examined three residues, distal to the contact, which were predicted to be necessary for the LDLR to adopt the acidic conformation. Of the three mutations we tested (G293S, F362A and G375S), one mutation (F362A) reduced lipoprotein uptake. Together, these data suggest that the intramolecular interface plays multiple roles in LDLR function., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Background: Low-density lipoprotein (LDL) particles accumulate in the arterial wall and become oxidized during atherogenesis, leading to the formation of atherosclerotic plaques. The major protein of the LDL particle, apolipoprotein B-100 (apoB-100), becomes fragmented during oxidation and a target for the immune system., Methods: In this study we used in situ ellipsometry to monitor the adsorption of LDL to solid silica surfaces and the effects of oxidation on the structure of the adsorbed LDL layer. We additionally investigated the binding kinetics of two recombinant human antibodies with different specificities recognizing epitopes of apoB-100 in surface-bound native and CuCl₂-oxidized LDL (oxLDL). The latter process was studied by adsorbing LDL and then adding the antibody and CuCl₂ while continuously monitoring adsorbed amount and the thickness of the film. The molar ratios between the antibodies and surface-bound LDL and oxLDL were calculated from these data., Results: Our results indicate that oxidation of surface-bound LDL induces swelling of the layer, accompanied by a slight desorption. We further found that both antibodies were able to recognize LDL and oxLDL in its adsorbed orientation. Quantitative information was obtained on the number of available binding sites on surface-bound LDL and oxLDL for these two antibodies., General Significance: Using ellipsometry for real-time monitoring of adsorption, in situ oxidation of LDL and binding of specific recombinant antibodies to surface-bound LDL, will open up possibilities to map different conformations and orientations of LDL in the adsorbed state., (Copyright © 2010 Elsevier B.V. All rights reserved.)

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/⁻)), 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/⁻) 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., (Copyright © 2010 Elsevier B.V. All rights reserved.)

Rationale and Objectives: Targeted molecular probes offer the potential for greater specificity in cancer imaging with contrast-enhanced computed tomography (CT). We investigate a low-density lipoprotein (LDL) nanoparticle loaded with poly-iodinated triglyceride (ITG) in a proof of concept study of targeted x-ray imaging. LDLs are targeted to the LDL cell surface receptor (LDLR), which is overexpressed in several tumor types. The LDL-LDLR pathway presents a high-capacity and self-renewing transport system for molecular imaging in CT., Materials and Methods: ITG was synthesized and loaded into the core of LDL particles to form a reconstituted nanoparticle, hereafter referred to as (rITG)LDL. Particle size was measured by dynamic light scattering. The x-ray attenuation of the (rITG)LDL solution was measured with CT imaging and signal enhancement was calibrated for equivalent iodine concentration. Cultured human hepatoblastoma G2 (HepG2) cells, which overexpress LDLR, were incubated with (rITG)LDL with or without native LDL. The cells were imaged with CT to characterize particle sequestration., Results: Reconstitution of LDL with ITG was successful and did not compromise the targeting function of the particle. Measurement of the x-ray attenuation properties of the (rITG)LDL solution revealed an effective iodine concentration of 0.78 mg/mL. In vitro studies of HepG2 cells demonstrated a significant increase in CT image intensity over control cells when incubated with (rITG)LDL., Conclusion: The in vitro results of this study suggest that (rITG)LDL can provide adequate image enhancement for CT molecular imaging. Potential applications include breast imaging and small animal imaging at low x-ray energies. In vivo experiments will be required to verify that tumor uptake of (rITG)LDL is sufficient for enhanced detection. Use at higher x-ray energies, as used in conventional CT, will require a further increase in iodine loading., (Copyright © 2010 AUR. Published by Elsevier Inc. All rights reserved.)

This paper illustrates how principles of colloid science are useful in studying atherosclerosis. Accumulation of foam cells in the arterial intima is a key step in atherogenesis. The extent of foam cell formation is enhanced by low density lipoprotein (LDL) aggregates, and we have previously shown that the size of sphingomyelinase (Smase)-hydrolysis-induced aggregates depends directly on the concentration of ceramide generated in the LDL phospholipid monolayer, mediated by the hydrophobic effect. Here, we focus on the effect of LDL aggregate particle sizes on their subsequent uptake by macrophages. Our data show the first direct measurement of uptake as a function of aggregate size and the first direct comparison of uptake after Smase-catalyzed and vortex-mixing-mediated aggregation. Vortex-mixed aggregates with radii 20-77 nm showed maximal uptake approximately 118 microg sterol/mg protein at a 53 nm intermediate size, consistent with a mathematical model describing competition between aggregate surface area and volume. Smase-treated aggregates with radii 25-211 nm also showed maximal uptake at an intermediate size, approximately 58 microg sterol/mg protein for 132 nm particles, and fit a modified model that incorporated ceramide concentration expressed as aggregate size. This study shows that particle size is significant and composition may also be a factor in LDL uptake., (Copyright 2010 Elsevier Inc. All rights reserved.)

The development of atherosclerosis, a chronic inflammatory disease characterized by the formation of arterial fibrotic plaques, has been shown to be reduced by IL-33 in vivo. However, whether IL-33 can directly affect macrophage foam cell formation, a key feature of atherosclerotic plaques, has not been determined. In this study, we investigated whether IL-33 reduces macrophage foam cell accumulation in vivo and if IL-33 reduces their formation in vitro using THP-1 and primary human monocyte-derived macrophages. In Apolipoprotein E(-/-) mice fed on a high fat diet, IL-33 treatment significantly reduced the accumulation of macrophage-derived foam cells in atherosclerotic plaques. IL-33 also reduced macrophage foam cell formation in vitro by decreasing acetylated and oxidized low-density lipoprotein uptake, reducing intracellular total and esterified cholesterol content and enhancing cholesterol efflux. These changes were associated with IL-33-mediated reduction in the expression of genes involved in modified low-density lipoprotein uptake, such as CD36, and simultaneous increase in genes involved in cholesterol efflux, including Apolipoprotein E, thereby providing a mechanism for such an action for this cytokine. IL-33 also decreased the expression of key genes implicated in cholesterol esterification and triglyceride storage, including Acyl-CoA:cholesterol acyltransferase 1 and Adipocyte differentiation-related protein. Furthermore, using bone marrow-derived macrophages from ST2(-/-) mice, we demonstrate that the IL-33 receptor, ST2, is integral to the action of IL-33 on macrophage foam cell formation. In conclusion, IL-33 has a protective role in atherosclerosis by reducing macrophage foam cell formation suggesting that IL-33 maybe a potential therapeutic agent against atherosclerosis.

Background: The bone marrow contains a variety of blood vessels that have different functions in bone marrow maintenance and hematopoiesis. Arterioles control the flow of blood into bone marrow compartments, and sinusoids serve as a conduit to the bloodstream and as niches for megakaryocyte development. Most studies of bone marrow vasculature, including studies quantifying changes in the marrow vascular by microvascular density, do not differentiate between different types of marrow vessels. Recognizing changes in different types of blood vessels after chemotherapy exposure or during leukemia development has important physiological implications. We hypothesized that the functional heterogeneity of marrow vasculature could be recognized through the use of functional markers such as tyrosine kinase with immunoglobulin and epidermal growth factor homology domains-2 (Tie2) expression or 1,1-dioctadecyl -3,3,3,3-tetramethyl-indocarbocyanine perchlorate with acetylated low-density lipoprotein (DiI-Ac-LDL) uptake., Methods and Results: When transgenic mice with green fluorescent protein (GFP) expressed downstream of the Tie2 promoter were injected with Ac-LDL, Ac-LDL was specifically endocytosed by sinusoids, and Tie2 expression was more pronounced in the arteries, arterioles, and transitional capillaries. Combining these 2 functional endothelial markers and using confocal microscopy to obtain 3-dimensional images, we identified transitional zones where arterioles emptied into the sinusoids. Alternatively, coinjection of lectin with DiI-Ac-LDL has a similar result in normal mice, as seen in Tie2/GFP mice, and can be used to differentiate vessel types in nontransgenic mice., Conclusions: These results demonstrate that bone marrow vasculature is functionally heterogeneous. Methods to study changes in the marrow vasculature using microvascular density or quantifying changes in the vascular niche need to take this heterogeneity into account.

Etofibrate, a combination of fibric and nicotinic acid, is successfully used for the treatment of type IIb and IV hyperlipidemia. While an up-regulation of specific low density lipoproteins (LDL) binding sites in human platelets has been demonstrated, action on LDL-binding to the liver in patients and kinetic studies rare. This study aimed to investigate the influence of twice 500mg etofibrate daily given for 6 weeks on the in vivo binding of autologous LDL to the liver in 11 patients, 6 males, 5 females; aged 37-57 years, suffering from mixed hyperlipidemia. Etofibrate enhanced in vivo liver uptake of (123)I-LDL by 16.1% at mean, shortened plasma decay of LDL and improved lipid profile: serum total cholesterol was lowered by 14.9%, LDL-cholesterol was lowered by 22.2% and high-density lipoprotein (HDL)- cholesterol was increased by 10.9%. These findings are documenting a beneficial effect of the drug at the LDL liver receptor level in vivo.

Dietary flavonoid intake has been reported to be inversely associated with the incidence of coronary artery disease. To clarify the possible role of flavonoids in the prevention of atherosclerosis, we investigated the effects of some of these compounds, including fisetin, morin and myricetin, on the susceptibility of low-density lipoprotein (LDL) to oxidative modification and on oxLDL uptake in macrophages. The results demonstrated that fisetin had stronger inhibitory activity than the other two on inhibiting Cu(2+)-mediated LDL oxidation measured by thiobarbituric acid-reactive substances assay (TBARS), conjugated diene formation and electrophoretic mobility. The class B scavenger receptor, CD36, to which oxLDL binds, is present in atherosclerotic lesions. Treatment of U937-derived macrophages with myricetin (20 microM) significantly inhibited CD36 cell surface protein and mRNA expression (p<0.01). Fisetin, morin and myricetin (20 microM) also reduced the feed-forward induction of CD36 mRNA and surface protein expression by PPARgamma. The inhibition of CD36 by flavonols was mediated by interference with PPARgamma activation thus counteracting the deleterious autoamplification loop of CD36 expression stimulated by PPARgamma ligand. All three flavonols (10 and 20 microM) markedly decreased the uptake of 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanide perchlorate (DiI)-labeled oxLDL uptake in U937-derived macrophages dose-dependently. Current evidences indicate that fisetin, morin and myricetin not only prevent LDL from oxidation but also block oxLDL uptake by macrophages at least in part through reducing CD36 gene expression on macrophages. In conclusion, flavonols may play a role in ameliorating atherosclerosis.

In this study, heparin was covalently coupled by glutaraldehyde to Poly(vinyl alcohol) [PVA] in solid-liquid two-phase reaction system by two-step synthesis method to prepare a LDL-selective adsorbent. The parameters (the material ratio, reaction time and dosage of catalyzer) were investigated to evaluate their effect upon the immobilized amount of heparin onto the surface of PVA, IR was used to verify the covalent immobilization result and the heparin-modified PVA was also undergone the evaluation of its adsorption capability for low-density lipoprotein from hyperlipemia plasma, and its hemocompatibility was preliminarily evaluated by platelet adhesion test. Results showed: (1) under optimized reaction conditions the highest immobilization amount of heparin onto PVA surface within the experiments of this study has been obtained; (2) the optimized reaction conditions were: (i) at the refluxing temperature 78 degrees C; (ii) the material ratio of "PVA(g): 50% glutaraldehyde (ml)" was about "1:3"; (iii) the reaction time was about 5 h; and (iv) the amount of catalyzer (concentrated HCL) was about 1% of the 50% glutaraldehyde; (3) within the experiments of this study the highest immobilization amount would be up to 25 microg heparin on the surface of per g PVA granules; (4) the heparin-modified PVA granules showed significant adsorption for LDL under faintly alkaline environment (pH=7.2-9.5) ; (5) The result of platelet adhesion test showed no platelet adhered to its surface. Therefore, immobilization of heparin onto the surface of a support is one approach to prepare a kind of LDL adsorbent for blood purification.

Hypertriglyceridemia being an independent cardiovascular risk factor, we have compared the potential of sera from asymptomatic hypertriglyceridemic (HTG) type IIb, type IV or normolipidemic (NLP) subjects to promote both fractional cholesterol efflux and cellular cholesterol mass changes using macrophage foam cells. The J774 cells loaded with cholesterol by incubation with acetylated LDL were incubated in the absence or presence of cAMP to upregulate ABCA1 (ATP binding cassette transporter A1) and then incubated for 24h with 1% serum. Compared with NLP, type IV sera exhibited a major increase in ABCA1-dependent efflux while type IIb sera exhibited a moderate but not significant increased ABCA1-mediated efflux. Moreover, positive correlations were established between ABCA1-dependent efflux and the serum prebeta-HDL or TG concentrations. The major finding was that the sera from type IV induced higher total cholesterol and cholesteryl ester mass depletions from ABCA1-expressing cells compared with other groups. Moreover, negative correlations were obtained between total cholesterol or cholesteryl ester mass changes and serum prebeta-HDL levels. In conclusion, we demonstrated for the first time that the serum prebeta-HDL present in high proportions in type IV HTG subjects are not only responsible for higher cholesterol efflux potential but also for increased abilities to promote net removal of cholesterol from macrophage foam cells.

Changes in electrical charge and clearance rate of LDL after the formation of their complexes with bacterial LPS were studied in experiments on Wistar rats. It was found that binding of S. minnesota R595 LPS with (125)I-LDL sharply accelerated clearance of the greater part of LDL complexes, but on the other hand induced the appearance of an LDL-LPS subfraction with slower elimination rate compared to free LDL. Electrophoresis showed that after binding of LPS, LDL acquired a negative charge. These data suggest that the formation of LDL-LPS complexes is accompanied by modification of LDL due to which they acquire atherogenic properties.

Gd-DO3A-diph and Gd-AAZTAC17 are lipophilic magnetic resonance imaging (MRI) agents that display high affinity for low-density lipoprotein (LDL) particles. However, on binding to LDL, Gd-DO3A-diph shows a decreased hydration that results in a lower enhancement of water proton relaxation rate. Conversely, Gd-AAZTAC17 displays a strong relaxation enhancement at the imaging fields. Each LDL particle can load up to 100 and 400 UNITS of Gd-DO3A-diph and Gd-AAZTAC17, respectively. Their LDL adducts are taken up by human hepatoblastoma G2 (HepG2) and melanoma B16 tumor cells when added to the incubation medium. T(1) measurements of the labeled cells indicate that Gd-AAZTAC17 is significantly more efficient than Gd-DO3A-diph. Furthermore, it has been found that HepG2 hepatoma cells can internalize higher amounts of Gd-AAZTAC17 than B16 cells and the involvement of LDL receptors (LDLRs) has been demonstrated in competition assays with free LDL. Gd-AAZTAC17/LDL adduct proved to be an efficient probe in the magnetic resonance (MR) visualization of subcutaneous tumors in animal models obtained by injecting B16 melanoma cells into the right flank of mice. Finally, confocal microscopy validation of the distribution of LDL-based probes in the tumor has been obtained by doping the Gd-AAZTAC17/LDL adduct with a fluorescent phospholipid moiety.

Oxidative modification of LDL is causally involved in the development of atherosclerosis and occurs in vivo in the blood as well as within the vascular wall. The present study attempted to explore whether polyphenolic flavonoids influence monocyte-endothelium interaction and lectin-like oxidised LDL receptor 1 (LOX-1) expression involved in the early development of atherosclerosis. The flavones luteolin and apigenin inhibited THP-1 cell adhesion onto oxidised LDL-activated human umbilical vein endothelial cells (HUVEC), while the flavanols of (-)epigallocatechin gallate and (+)catechin, the flavonols of quercetin and rutin, and the flavanones of naringin, naringenin, hesperidin and hesperetin did not have such effects. Consistently, Western blot analysis revealed that the flavones at 25 microM dramatically and significantly abolished HUVEC expression of vascular cell adhesion molecule-1 and E-selectin evidently enhanced by oxidised LDL; these inhibitory effects were exerted by drastically down regulating mRNA levels of these cell adhesion molecules. In addition, quercetin and luteolin significantly attenuated expression of LOX-1 protein up regulated in oxidised LDL-activated HUVEC with a fall in transcriptional mRNA levels of LOX-1. In addition, quercetin and luteolin clearly blunted oxidised LDL uptake by HUVEC treated with oxidised LDL. The results demonstrate that the flavones luteolin and apigenin as well as quercetin were effective in the different initial steps of atherosclerosis process by inhibiting oxidised LDL-induced endothelial monocyte adhesion and/or oxidised LDL uptake. Therefore, certain flavonoids qualify as anti-atherogenic agents in LDL systems, which may have implications for strategies attenuating endothelial dysfunction-related atherosclerosis.

Blood lipoproteins (very low density--VLDL, low density--LDL and high density--HDL) penetrate into the myocardium through capillary endothelial layer via receptor mediated endocytosis (all lipoproteins), nonreceptor uptake, or along interendothelial gaps (LDL). In the myocardium lipoproteins are captured by interstitial macrophages and are subjected to degradation in secondary liposomes. Their action on myocardium results in development of perivascular swelling and constriction of substantial portion of capillaries. However part of capillaries (about 20%) stay in a condition of vasodilation. Destructive changes revealed (myelin figures, mild lysis of myofibrils) are presumably caused by activation of lysosomal proteinases. Corticosterone lowered coronary flow velocity, while parameters of working capacity of the heart remained at control level. Combined use of corticosterone and VLDL suppressed myocardial functional activity, to a great extent because of diminishment of coronary flow velocity. Corticosterone and LDL exerted less pronounced negative effect. Corticosterone and HDL caused improvement of parameters of cardiac working capacity.

The novel thiol-group-selective bifunctional 18F-labeling agent N-[6-(4-[18F]fluoro-benzylidene)aminooxyhexyl]maleimide ([18F]FBAM) has been developed. The bifunctional labeling precursor N-(6-aminoxyhexyl)maleimide containing a thiol-reactive maleimide group and a carbonyl-group-reactive aminooxy group was prepared in only three steps in a total chemical yield of 59%. Subsequent radiolabeling with 4-[18F]fluorobenzaldehyde gave the bifunctional 18F-labeling agent [18F]FBAM in a radiochemical yield of 29%. In a typical experiment, 3.88 GBq of [18F]fluoride could be converted into 723 MBq of [18F]FBAM within 69 min. Conjugation of [18F]FBAM with thiol groups was exemplified with the cysteine-containing tripeptide glutathione and with various apolipoproteins of human low-density lipoprotein (LDL) subfractions. The latter was evaluated with respect to the uptake of [18F]FBAM-LDL subfractions in human hepatoma cells (HepG2) in vitro. In vivo biodistribution studies in rats revealed high stability for [18F]FBAM-LDL subfractions. Moreover, the metabolic fate of [18F]FBAM-LDL subfractions in vivo was delineated by dynamic positron emission tomography studies using a dedicated small animal tomograph. Data were compared to former studies that used the NH2-reactive 18F-labeling agent N-succinimidyl-4-[18F]fluorobenzoate. The compound [18F]FBAM can be considered as an excellent prosthetic group for the selective and mild 18F labeling of thiol-group-containing biomolecules suitable for subsequent investigations in vitro and in vivo.

Purpose: In this study we examined the uptake of circulating lipoproteins into the retina, using a naturally fluorescent cholesterol analog for imaging and deuterated cholesterol for quantification by mass spectroscopy. The purpose of this study was to better understand cholesterol uptake, transport and homeostasis in the retina., Methods: Human low density lipoprotein (LDL) and high density lipoprotein (HDL) were labeled with the fluorescent cholesterol analog cholesta-5,7,9(11)-trien-3beta-ol (CTL) and deuterated cholesterol (25,26,26,26,27,27,27-[2H]cholesterol, D7Ch). Rats were injected intravenously with CTL-LDL, CTL-HDL and D7Ch-LDL. Fluorescent confocal microscopy was used to image the uptake of CTL and mass spectroscopy was used to quantify D7Ch. Immunohistochemistry and fluorescent confocal microscopy were used to localize apoB (an LDL marker protein) and LDL receptor (LDLR) protein in rat and monkey retinas., Results: CTL-specific fluorescence was imaged by confocal microscopy in the retinal pigment epithelium (RPE), choriocapillaris and parts of the neural retina within 2 h post-injection and was visualized in the photoreceptor outer segments by 4 h. Replacing LDL with HDL as the CTL carrier gave a less robust and more delayed labeling of retinal layers. Human apolipoprotein B (apoB) was also localized in the rat choriocapillaris and RPE by 4 h post-injection. Human apoB was detected by immunoblot analysis in the rat retina primarily as a about 70 kDa protein, suggesting proteolytic degradation. LDL-mediated uptake of cholesterol was quantified by mass spectroscopy using deuterated cholesterol in place of CTL. In addition, apoB and LDLR were localized in monkey retina by immunohistochemistry., Conclusions: The retina is capable of rapid uptake of circulating LDL via an LDLR-mediated process primarily occurring in the RPE and also possibly Müller cells. Despite the dominance of HDL over LDL in rat serum, LDL appears to be the preferred carrier for cholesterol transport to and uptake by the retina. The results also suggest that blood-borne LDL represents a significant contributor to the steady-state levels of cholesterol and possibly other lipids in the retina.

Objective: Atherosclerosis is a chronic inflammatory disease with immune cell infiltration. Various cytokines and chemokines have been characterized as pro- or antiatherogenic factors. Interleukin-20 (IL-20) belongs to the IL-10 family and is a proinflammatory cytokine involved in the pathogenesis of psoriasis. However, the association between IL-20 and atherosclerosis is undetermined. Therefore, we sought to investigate whether IL-20 is associated with atherosclerosis., Methods and Results: We examined the expression of IL-20 and its receptor complex IL-20R1/IL-20R2 in atherosclerotic lesions of humans and mice using immunohistochemical staining. IL-20 was expressed in macrophage-rich areas. Both IL-20 and IL-20R1/IL-20R2 were expressed by endothelial cells lining the intimal microvessels, vasa vasorum, but rarely in nonatherosclerotic arteries. We used reverse-transcription polymerase chain reaction to analyze gene expression. IL-20 transcripts increased in hypoxic monocytes and monocytes treated with oxidized low-density lipoprotein. The expression of IL-20R1 and IL-20R2 was also upregulated by human umbilical vein endothelial cells in response to hypoxic treatment. Incubating IL-20 with human umbilical vein endothelial cells upregulated CXCL9 and CXCL11 transcripts. Furthermore, in vivo administration of IL-20 expression vector using intramuscular electroporation promoted atherosclerosis in apolipoprotein E-deficient mice., Conclusions: Our data suggest that IL-20 is a proatherogenic cytokine that contributes to the progression of atherosclerosis.

Diabetes is associated with increased oxidative stress and atherosclerosis development. In the present study, we investigated the effects of pomegranate juice (PJ; which contains sugars and potent anti-oxidants) consumption by diabetic patients on blood diabetic parameters, and on oxidative stress in their serum and macrophages. Ten healthy subjects (controls) and 10 non-insulin dependent diabetes mellitus (NIDDM) patients who consumed PJ (50ml per day for 3 months) participated in the study. In the patients versus controls serum levels of lipid peroxides and thiobarbituric acid reactive substances (TBARS) were both increased, by 350% and 51%, respectively, whereas serum SH groups content and paraoxonase 1 (PON1) activity, were both decreased (by 23%). PJ consumption did not affect serum glucose, cholesterol and triglyceride levels, but it resulted in a significant reduction in serum lipid peroxides and TBARS levels by 56% and 28%, whereas serum SH groups and PON1 activity significantly increased by 12% and 24%, respectively. In the patients versus controls monocytes-derived macrophages (HMDM), we observed increased level of cellular peroxides (by 36%), and decreased glutathione content (by 64%). PJ consumption significantly reduced cellular peroxides (by 71%), and increased glutathione levels (by 141%) in the patients' HMDM. The patients' versus control HMDM took up oxidized LDL (Ox-LDL) at enhanced rate (by 37%) and PJ consumption significantly decreased the extent of Ox-LDL cellular uptake (by 39%). We thus conclude that PJ consumption by diabetic patients did not worsen the diabetic parameters, but rather resulted in anti-oxidative effects on serum and macrophages, which could contribute to attenuation of atherosclerosis development in these patients.

We have demonstrated that vascular endothelial growth factor (VEGF) is expressed in capillary pericytes of the developing corpus luteum (CL) and others have shown that basic fibroblast growth factor (FGF2) and angiopoietins (ANGPT) are present in the CL. VEGF and FGF2 target endothelial cells to initiate angiogenesis and stimulate nitric oxide (NO) production. Conversely, NO may increase VEGF expression by vascular smooth muscle cells and pericytes. To investigate the relationship between these angiogenic factors and NO in the CL, microvascular pericytes and endothelial cells were isolated from CL collected from superovulated ewes (n = 5) on d 9 of the estrous cycle. Pericytes were identified by their morphology in culture and by immunofluorescent staining for smooth muscle cell actin. Pericytes were incubated with or without varying doses of the NO-donor DETA-NO for 8 h. Then, total cellular RNA was extracted from the cells and evaluated for expression of mRNA for VEGF, FGF2, ANGPT1, ANGPT2, and NO receptor, guanylate cyclase 1, soluble beta3 (GUCY1B3), using real-time quantitative RT-PCR. NO caused a dose-dependent increase in VEGF (p < 0.001), FGF2 (p < 0.001), ANGPT2 (p < 0.06), and GUCY1B3 (p < 0.03) mRNA expression. Expression of mRNA for ANGPT1 in luteal pericytes was not affected by the NO treatment. These data provide further evidence of the role of the luteal pericyte and NO in angiogenic factor expression, and of the potential interactions of pericytes with endothelial cells via NO production.

The toxic oil syndrome (TOS) caused by ingestion of rapeseed oil adulterated with aniline is characterized by symptoms of an allergic and/or autoimmune illness associated with vessel wall lesions similar to those of atherosclerosis. Fatty acid esters of 3-(N-phenylamino)-1,2-propanediol (PAP) have been incriminated as the etiologic agents of TOS. However, the pathogenesis of TOS is yet unknown. Here, we addressed whether PAP fatty acid esters are incorporated into lipoproteins, which after transport to vascular endothelial cells are taken up to initiate TOS vasculopathy. After loading (14)C-dioleyl-ester of PAP into LDL labeled with 1,1'-dioctadecyl-3,3,3',3'-tetramethylindolcarbocyanine (DiI) we assessed receptor mediated endocytosis and intracellular localization of these lipopoproteins in vascular endothelial cells. Our data suggest that these lipoprotein-derivatives are internalized into endothelial cells by LDL receptor mediated endocytosis. Confocal microscopy revealed that DiI-LDL loaded with dioleyl-ester of PAP and incubated for 60 min with endothelial cells colocalizes with the lysosomotropic compound LysoTracker Green, indicating that internalized PAP-loaded LDL are targetted to the endolysosomal compartment for further processing. Subcellular fractionation of endothelial-like ECV-304 cells after incubation with LDL loaded with the (14)C-dioleyl-ester of PAP for 6h showed that the radioactive label accumulated in fractions containing endosomes, the Golgi apparatus and the endoplasmic reticulum.

We investigated the effects of EP2306 and EP2302, two novel 2-biphenylmorpholine derivatives, on squalene synthase activity in rabbit and human liver microsomes, lipid biosynthesis, low-density lipoprotein (LDL) receptor expression and LDL protein uptake as well as apoB secretion in HepG2 cells. Both EP2306 and EP2302 inhibited squalene synthase activity dose-dependently. In rabbit liver microsomes, the IC50 values were 33 microM for EP2306 and 0.6 microM for EP2302 whereas in human liver microsomes, they were 63 microM for EP2306 and 1 microM for EP2302. Both EP2300 compounds inhibited cholesterol production by HepG2 cells dose dependently with IC50 values of 13.3 microM for EP2306 and 3 microM for EP2302. Furthermore, both EP2300 compounds and simvastatin significantly reduced triglyceride synthesis and apoB secretion and increased LDL receptor expression and LDL uptake in HepG2 cells. In summary, we have shown that EP2300 compounds are potent inhibitors of squalene synthase activity in rabbit and human liver microsomes and also they are effective inhibitors of cholesterol and triglyceride biosynthesis in HepG2 cells. These results suggest that EP2306 and EP2302 might prove to be useful for lipid-lowering and treatment of atherosclerosis in vivo.

Nanoscale particles could be synthetically designed to potentially intervene in lipoprotein matrix retention and lipoprotein uptake in cells, processes central to atherosclerosis. We recently reported on lipoprotein interactions of nanoscale micelles self-assembled from amphiphilic scorpion-like macromolecules based on a lauryl chloride-mucic acid hydrophobic backbone and poly(ethylene glycol) shell. These micelles can be engineered to present varying levels of anionic chemistry, a key mechanism to induce differential retentivity of low-density lipoproteins (LDL) (Chnari, E.; Lari, H. B.; Tian, L.; Uhrich, K. E.; Moghe, P. V. Biomaterials 2005, 26, 3749). In this study, we examined the cellular interactions and the ability of carboxylate-terminated nanoparticles to modulate cellular uptake of differentially oxidized LDL. The nanoparticles were found to be highly biocompatible with cultured IC21 macrophages at all concentrations examined. When the nanoparticles as well as LDL were incubated with the cells over 24 h, a marked reduction in cellular uptake of LDL was observed in a nanoparticle concentration-dependent manner. Intermediate concentrations of nanoparticles (10(-6) M) elicited the most charge-specific reduction in uptake, as indicated by the difference in uptake due to anionic and uncharged nanoparticles. At these concentrations, anionic nanoparticles reduced LDL uptake for all degrees of oxidation (no oxidation, mild, high) of LDL, albeit with qualitative differences in the effects. The anionic nanoparticles were particularly effective at reducing the very high levels of uptake of the most oxidized level of LDL. Since complexation of LDL with anionic nanoparticles is reduced at higher degrees of LDL oxidation, our results suggest that anionic nanoparticles interfere in highly oxidized (hox) LDL uptake, likely by targeting cellular/receptor uptake mechanism, but control unoxidized LDL uptake by mechanisms related to direct LDL-nanoparticle complexation. Thus, anionically functionalized nanoparticles can modulate the otherwise unregulated internalization of differentially oxidized LDL.

The ability of human postprandial triacylglycerol-rich lipoproteins (TRLs), isolated after meals enriched in saturated fatty acids (SFAs), n-6 PUFAs, and MUFAs, to inhibit the uptake of 125I-labeled LDL by the LDL receptor was investigated in HepG2 cells. Addition of TRLs resulted in a dose-dependent inhibition of heparin-releasable binding, cell-associated radioactivity, and degradation products of 125I-labeled LDL (P < 0.001). SFA-rich Svedberg flotation rate (Sf) 60-400 resulted in significantly greater inhibition of cell-associated radioactivity than PUFA-rich particles (P = 0.016) and total uptake of 125I-labeled LDL compared with PUFA- and MUFA-rich particles (P < 0.02). Normalization of the apolipoprotein (apo)E but not apoC-III content of the TRLs removed the effect of meal fatty acid composition, and addition of an anti-apoE antibody reversed the inhibitory effect of TRLs on the total uptake of 125I-labeled LDL. Real time RT-PCR showed that the SFA-rich Sf 60-400 increased the expression of genes involved in hepatic lipid synthesis (P < 0.05) and decreased the expression of the LDL receptor-related protein 1 compared with MUFAs (P = 0.008). In conclusion, these findings suggest an alternative or additional mechanism whereby acute fat ingestion can influence LDL clearance via competitive apoE-dependent effects of TRL on the LDL receptor.

Functional role of CD44, a principal receptor of hyaluronan, and glycated albumin for differentiation of resting human monocytes isolated by counterflow centrifugal elutriation was investigated. Flow cytometric analysis revealed that amadori-modified glycated albumin induced expression of CD44 as well as macrophage scavenger receptors (MSRs) such as CD36 and CD68 on resting monocytes. Crosslinking of CD44 on monocytes also induced MSR expression. Furthermore, CD44 crosslinking and/or glycated albumin enhanced the uptake of oxidized-low density lipoprotein in monocytes and foam cell formation. Taken together, engagement of CD44 (e.g., hyaluronan) and glycated albumin induced the differentiation of resting monocytes into foam macrophages through the induction of MSRs, implying that CD44 could be involved in atherosclerotic lesions of those such as diabetic patients.

Background: Human mesenchymal stem cells (MSC) possess powerful ex vivo expansion and versatile differentiation potential, placing themselves at the forefront of the field of stem cell-based therapy and transplantation. Of high clinical relevance is the endothelial differentiation potential of MSC, which can be used to treat various forms of ischemic vascular disease., Methods: We investigated whether human umbilical cord blood (UCB)-derived MSC are able to differentiate in vitro along an endothelial lineage, by using flow cytometry, RT-PCR and immunofluorescence analyzes, as well as an Ab array method., Results: When the cells were incubated for up to 3 weeks in the presence of VEGF, EGF and hydrocortisone, they began to express a variety of endothelial lineage surface markers, such as Flk-1, Flt-1, VE-Cadherin, vWF, VCAM-1, Tie-1 and Tie-2, and to secrete a specific set of cytokines. Differentiated cells were also found to be able to uptake low-density lipoprotein and form a tubular network structure., Discussion: These observations have led us to conclude that UCB-derived MSC retain endothelial potential that is suitable for basic and clinical studies aimed at the development of vasculature-directed regenerative medicine.

In blood circulation, low density lipoproteins (LDL) can undergo modification, such as oxidation, and become key factors in the development of atherosclerosis. Although the liver is the major organ involved in the elimination of oxidized LDL (oxLDL), the identity of the receptor(s) involved remains to be defined. Our work aims to clarify the role of the scavenger receptor class B type I (SR-BI) in the hepatic metabolism of mildly and standardly oxLDL as well as the relative contribution of parenchymal (hepatocytes) and nonparenchymal liver cells with a special emphasis on CE-selective uptake. The association of native LDL and mildly or standardly oxLDL labeled either in proteins or in cholesteryl esters (CE) was measured on primary cultures of mouse hepatocytes from normal and SR-BI knock-out (KO) mice. These in vitro assays demonstrated that hepatocytes are able to mediate CE-selective uptake from both LDL and oxLDL and that SR-BI KO hepatocytes have a 60% reduced ability to selectively take CE from LDL but not towards mildly or standardly oxLDL. When lipoproteins were injected in the mouse inferior vena cava, parenchymal and nonparenchymal liver cells accumulated more CE than proteins from native, mildly and standardly oxLDL, indicating that selective uptake of CE from these lipoproteins occurs in vivo in these two cell types. The parenchymal cells contribute near 90% of the LDL-CE selective uptake and SR-BI for 60% of this pathway. Nonparenchymal cells capture mainly standardly oxLDL while parenchymal and nonparenchymal cells equally take up mildly oxLDL. An 82% reduction of standardly oxLDL-CE selective uptake by the nonparenchymal cells of SR-BI KO mice allowed emphasizing the contribution of SR-BI in hepatic metabolism of standardly oxLDL. However, SR-BI is not responsible for mildly oxLDL metabolism. Thus, SR-BI is involved in LDL- and standardly oxLDL-CE selective uptake in parenchymal and nonparenchymal cells, respectively.