An implanted port-catheter system for repeated hepatic arterial infusion of low-density lipoprotein-docosahexaenoic acid nanoparticles in normal rats: A safety study.

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-κß, 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.

Benefits of hypericin transport and delivery by low- and high-density lipoproteins to cancer cells: From in vitro to ex ovo.

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.

Antisense oligonucleotides targeting translation inhibitory elements in 5' UTRs can selectively increase protein levels.

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.

Assaying Low-Density-Lipoprotein (LDL) Uptake into Cells.

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.

Epigallocatechin gallate induces an up-regulation of LDL receptor accompanied by a reduction of PCSK9 via the annexin A2-independent pathway in HepG2 cells.

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.

Human Neutrophil Peptide 1 Limits Hypercholesterolemia-induced Atherosclerosis by Increasing Hepatic LDL Clearance.

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.

Gly[14]-humanin inhibits ox-LDL uptake and stimulates cholesterol efflux in macrophage-derived foam cells.

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.

Synthetic low-density lipoprotein (sLDL) selectively delivers paclitaxel to tumor with low systemic toxicity.

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.

Hypericin fluorescence kinetics in the presence of low density lipoproteins: study on quail CAM assay for topical delivery.

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.

Rapamycin Inhibits Oxidized Low Density Lipoprotein Uptake in Human Umbilical Vein Endothelial Cells via mTOR/NF-κB/LOX-1 Pathway.

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.