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Targeted drug delivery is an ongoing aspect of scientific research that is expanding through the design of micro- and nanoparticles. In this paper, we focus on spray dried microparticles as carriers for a repurposed lipophilic antioxidant (probucol). We characterise the microparticles and quantify probucol prior to assessing cytotoxicity on both control and cisplatin treated hair cells (known as House Ear Institute-Organ of Corti 1; HEI-OC1). The addition of water-soluble polymers to 2% β-cyclodextrin resulted in a stable probucol formulation. Ursodeoxycholic acid (UDCA) used as formulation excipient increases probucol miscibility and microparticle drug content. Formulation characterisations reveals spray drying results in spherical UDCA-drug microparticles with a mean size distribution of ∼5-12 μm. Probucol microparticles show stable short-term storage conditions accounting for only ∼10% loss over seven days. By mimicking cell culture conditions, both UDCA-probucol (67%) and probucol only (82%) microparticles show drug release in the initial two hours. Furthermore, probucol formulations with or without UDCA preserve cell viability and reduce cisplatin-induced oxidative stress. Mitochondrial bioenergetics results in lower basal respiration and non-mitochondrial respiration, with higher maximal respiration, spare capacity, ATP production and proton leak within cisplatin challenged UDCA-probucol groups. Overall, we present a facile method for incorporating lipophilic antioxidant carriers in polymer-based particles that are tolerated by HEI-OC1 cells and show stable drug release, sufficient in reducing cisplatin-induced reactive oxygen species accumulation., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Al-Salami H has been and is currently receiving funding from Beijing Nat-Med Biotechnology Co. Ltd. and Glanis PTY Ltd. All other authors have no completing interest to declare., (Copyright © 2024 American Pharmacists Association. Published by Elsevier Inc. All rights reserved.)

Hepatic fibrosis is a common pathological process in chronic liver diseases, characterized by excessive reactive oxygen species (ROS), activated hepatic stellate cells (HSCs), and massive synthesis of extracellular matrix (ECM), which are important factors in the development of liver cirrhosis, liver failure, and liver cancer. During the development of hepatic fibrosis, ECM collagen produced by activated HSCs significantly hinders medication delivery to targeted cells and reduces the efficiency of pharmacological therapy. In this study, we designed a multifunctional hyaluronic acid polymeric nanoparticle (HA@PRB/COL NPs) based on autophagy inhibitor probucol (PRB) and collagenase type I (COL) modification, which could enhance ECM degradation and accurately target HSCs through specificity binding CD44 receptor in hepatic fibrosis therapy. Upon encountering excessive collagen I-deposition formed barrier, HA@PRB/COL NPs performed the nanodrill-like function to effectively degrade pericellular collagen I, leading to greater ECM penetration and prominent HSCs internalization capacity of delivered PRB. In mouse hepatic fibrosis model, HA@PRB/COL NPs were efficiently delivered to HSCs through binding CD44 receptor to achieve efficient accumulation in fibrotic liver. Further, we showed that HA@PRB/COL NPs executed the optimal anti-fibrotic activity by inhibiting autophagy and activation of HSCs. In conclusion, our novel dual-functional co-delivery system with degrading fibrotic ECM collagen and targeting activated HSCs exhibits great potentials in the treatment of hepatic fibrosis in clinic. STATEMENT OF SIGNIFICANCE: The excess release of extracellular matrix (ECM) such as collagen in hepatic fibrosis hinders medication delivery and decreases the efficiency of pharmacological drugs. We aimed to develop a nano-delivery carrier system with protein hydrolyzed surfaces and further encapsulated an autophagy inhibitor (PRB) to enhance fibrosis-related ECM degradation-penetration and hepatic stellate cells (HSCs) targeting in hepatic fibrosis niche (HA@PRB/COL NPs). The COL of HA@PRB/COL NPs successfully worked as a scavenger to promote the digestion of the ECM collagen I barrier for deeper penetration into fibroid liver tissue. It also accurately targeted HSCs through specifically binding to the CD44 receptor and subsequently released PRB to inhibit autolysosome and ROS generation, thus preventing HSCs activation. Our HA@PRB/COL NPs system provided a promising therapeutic strategy for hepatic fibrosis in a clinic setting., Competing Interests: Declaration of Competing Interest The authors declare that they have no competing interest., (Copyright © 2023 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.)

The ATP binding cassette (ABC) transporter molecule ABCA1 participates in the cholesterol transport within and through cell membranes. We recently demonstrated that in dog spermatozoa, capacitation could be decreased with probucol (PRO), an ABCA1 specific antagonist. In this study, a dose-effect relationship of PRO on dog sperm capacitation, tyrosine phosphorylation and cholesterol efflux from the sperm plasma membrane was investigated. A total of 16 ejaculates from dogs of different breeds, aged 2-4 years were used. Sperm motility and membrane integrity in the main fraction was determined by CASA. Samples were stained with a boron dipyrromethene difluoride (BODIPY) fluorophore (P9672, Sigma- Aldrich, A) diluted in DMSO at a final concentration of 0.4 μM. All samples were divided into 5 aliquots, with 0, 100, 250, 500 and 1000 μM of PRO. After incubation at 37 °C for 2 h, PI was added and flow cytometry performed. All aliquots were examined for capacitation and acrosome reaction by using the CTC assay and tyrosine phosphorylation (TP). Membrane integrity was measured in all aliquots to investigate the effect of PRO on cell membranes. Membrane integrity did not differ between controls (0 μM), and 100, 250 and 500 μM PRO, but decreased with 1000 μM PRO (p < 0.05). Increasing PRO concentration decreased the percentage alive cells with cholesterol efflux per PRO group (0 μM: 77.8 ± 10.6%, 100 μM: 63.7 ± 11.7%, 250 μM: 52.1 ± 12.9%, 500 μM: 37.7 ± 11.6%, 1000 μM: 33.1 ± 14.4%; p < 0.05), decreased head and entire tail phosphorylated cells (0 μM: 34.6%, 1000 μM: 5.1% p < 0.05); and decreased the percentage capacitated cells (maximum with PRO 500 μM: capacitated vs. control: 54.2 ± 17% vs 25 ± 7.7%, p < 0.05). Conclusion: PRO decreased the cholesterol efflux, and decreased tyrosine phosphorylation and capacitation in a dose-dependent manner. This suggests a strong involvement of the ABCA1 transporter in different functional aspects of sperm capacitation in dogs., Competing Interests: Declaration of competing interest The authors declare no conflict of interest., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

The development of high-end targeted drugs and vaccines against modern pandemic infections, such as COVID-19, can take a too long time that lets the epidemic spin up and harms society. However, the countermeasures must be applied against the infection in this period until the targeted drugs became available. In this regard, the non-specific, broad-spectrum anti-viral means could be considered as a compromise allowing overcoming the period of trial. One way to enhance the ability to resist the infection is to activate the nonspecific immunity using a suitable driving-up agent, such as plant polysaccharides, particularly our drug Panavir isolated from the potato shoots. Earlier, we have shown the noticeable anti-viral and anti-bacterial activity of Panavir. Here we demonstrate the pro-inflammation activity of Panavir, which four-to-eight times intensified the ATP and MIF secretion by HL-60 cells. This effect was mediated by the active phagocytosis of the Panavir particles by the cells. We hypothesized the physiological basis of the Panavir proinflammatory activity is mediated by the indol-containing compounds (auxins) present in Panavir and acting as a plant analog of serotonin., (Copyright © 2021 Elsevier B.V. All rights reserved.)

The pathogenesis of Alzheimer's disease (AD) is characterized by overproduction, impaired clearance, and deposition of amyloid-β peptides (Aβ) and connected to cholesterol homeostasis. Since the blood-brain barrier (BBB) is involved in these processes, we investigated effects of the retinoid X receptor agonist, bexarotene (Bex), and the peroxisome proliferator-activated receptor α agonist and antioxidant, astaxanthin (Asx), on pathways of cellular cholesterol metabolism, amyloid precursor protein processing/Aβ production and transfer at the BBB in vitro using primary porcine brain capillary endothelial cells (pBCEC), and in 3xTg AD mice. Asx/Bex downregulated transcription/activity of amyloidogenic BACE1 and reduced Aβ oligomers and ~80 kDa intracellular 6E10-reactive APP/Aβ species, while upregulating non-amyloidogenic ADAM10 and soluble (s)APPα production in pBCEC. Asx/Bex enhanced Aβ clearance to the apical/plasma compartment of the in vitro BBB model. Asx/Bex increased expression levels of ABCA1, LRP1, and/or APOA-I. Asx/Bex promoted cholesterol efflux, partly via PPARα/RXR activation, while cholesterol biosynthesis/esterification was suppressed. Silencing of LRP-1 or inhibition of ABCA1 by probucol reversed Asx/Bex-mediated effects on levels of APP/Aβ species in pBCEC. Murine (m)BCEC isolated from 3xTg AD mice treated with Bex revealed elevated expression of APOE and ABCA1. Asx/Bex reduced BACE1 and increased LRP-1 expression in mBCEC from 3xTg AD mice when compared to vehicle-treated or non-Tg treated mice. In parallel, Asx/Bex reduced levels of Aβ oligomers in mBCEC and Aβ species in brain soluble and insoluble fractions of 3xTg AD mice. Our results suggest that both agonists exert beneficial effects at the BBB by balancing cholesterol homeostasis and enhancing clearance of Aβ from cerebrovascular endothelial cells., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Background: Diabetic erectile dysfunction (DMED) is mainly attributed to oxidative stress, and Nrf2 plays an important role in cellular antioxidation and regulates NO production in the vascular endothelium. Probucol maintains endothelial function through its antioxidant activity. This study investigated the efficacy and mechanism of probucol in improving erectile function in streptozotocin-induced diabetic rats., Methods: In our study, thirty 12-week-old Sprague-Dawley male rats were fasted for 12 h. All rats received a 1-time injection of intraperitoneal streptozotocin(60 mg/kg) or vehicle. After 72 h, STZ-treated rats (with random blood glucose concentrations consistently greater than 16.7 mmol/L) were considered diabetic. The diabetic rats were randomly assigned into 2 groups and treated with daily gavage feedings of probucol at doses of 0 and 500 mg/kg for 12 weeks. A positive control group underwent intraperitoneal injection of normal saline followed by daily gavage of saline solution. Erectile function was assessed by electrical stimulation of the cavernous nerves with real-time intracavernous pressure measurement. After euthanasia, penile tissue was investigated using immunohistochemistry, Western blot, and ELISA to assess the proteins of Nrf2/HO-1/DDAH/PPAR-γ/eNOS pathways., Results: After treatment, the rats in the probucol group presented significantly improved erectile function (P < 0.05) than that of the diabetic group without probucol treatment (DM). Also, protein expression of Nrf2, DDAH, PPAR-γ, HO-1 and eNOS was significantly higher than that of the DM group (P < 0.05). CGMP concentrations and SOD concentrations of probucol-treated rats were higher than those of DM group (P < 0.05). The MDA levels and ADMA levels were significantly lower than those of DM group rats (P < 0.05)., Conclusion: Probucol can improve erectile function via activation of Nrf2, which coordinates the HO-1/DDAH/PPAR-γ/eNOS pathways in streptozotocin-induced diabetic rats., (Copyright © 2018 Elsevier Inc. All rights reserved.)

The cholesteryl ester transfer protein (CETP) system moves cholesteryl esters (CE) from high density lipoproteins (HDL) to lower density lipoproteins, i.e. very low-density lipoprotein (VLDL) and low-density lipoprotein (LDL) in exchange for triglycerides (TGs). This shuttle process will ultimately form complexes facilitating a bidirectional exchange of CE and TGs, the end process being CE delivery to catabolic sites. The CETP system is generally characteristic of higher animal species; lower species, not provided with this system, have higher and enlarged HDL enriched with apo E, suitable for tissue receptor interaction. Discovery of the CETP system has led to the development of agents interfering with CETP, thus elevating HDL-C and potentially preventing cardiovascular (CV) disease. Activation of CETP leads instead to reduced HDL-C levels, but also to an enhanced removal of CE from tissues. CETP antagonists are mainly small molecules (torcetrapib, anacetrapib, evacetrapib, dalcetrapib) and have provided convincing evidence of a HDL-C raising activity, but disappointing results in trials of CV prevention. In contrast, the CETP agonist probucol leads to HDL-C lowering followed by an increment of tissue cholesterol removal (reduction of xanthomas, xanthelasmas) and positive findings in secondary prevention trials. The drug has an impressive anti-inflammatory profile (markedly reduced interleukin-1β expression). Newer agents, some of natural origin, have additional valuable pharmacodynamic properties. The pharmacological approach to the CETP system remains enigmatic, although the failure of CETP antagonists has dampened enthusiasm. Studies on the system, a crossroad for any investigation on cholesterol metabolism, have however provided crucial contributions and will still be confronting any scientist working on CV prevention., (Copyright © 2018. Published by Elsevier B.V.)

Scavenger receptor class B type 1(SR-B1) and low density lipoprotein receptor (LDLR) play vital roles in cholesterol homeostasis. Previous studies indicated a strong link between cholesterol and adipose tissue (AT). In this study, adult male SR-B1 and LDLR double knockout (DKO) mice were fed with high fat diet (HFD) for 3 months. Interestingly, we found severe loss of AT in DKO mice fed with HFD. To reverse the AT loss in DKO mice, 1% probucol was added in HFD. In DKO mice on HFD, plasma total cholesterol (TC) and free cholesterol (FC) levels were increased 6 and 15 folds respectively compared with wild type (WT) mice. We found severe loss of AT in whole body of DKO mice compared with WT or single KO mice. In AT of DKO mice, histology showed the very small size of adipocytes and infiltration of inflammatory cells; Genes expressions related to fatty acid uptake, lipogenesis and adipogenesis were decreased; TUNEL analysis and related genes expressions of endoplasmic reticulum (ER) stress and inflammation were significantly higher than those of WT or single KO mice. Probucol could reduce increased TC and FC levels, and reverse the loss of fat and apoptosis of AT in DKO mice. AT loss in DKO mice with HFD was probably due to high levels of FC which led to apoptosis induced by ER stress and inflammation of AT. This study provided a novel utility of probucol in rescue of fat loss in DKO mice., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Background and Aims: Micro-particles (MP) are secreted by various cells. Their biological roles in health and in disease remain unknown. Here we describe formation of MP in the process of ABCA1-dependent cholesterol efflux in different cell types., Methods: The ATP-binding cassette transporter, subfamily A, member 1 (ABCA1) is the rate-limiting step in the biogenesis of high-density lipoproteins (HDL). We have found that ABCA1 and apoA-I contribute to the formation of MP. Using cell-based systems with overexpression and selective inactivation of ABCA1, pharmacological blockade and modulation of membrane cholesterol content, we characterized MP release from various cell lines. We studied MP release in BHK cells stably expressing ABCA1 under mifepristone control, human THP-1 macrophages and HepG2 cells without, or with incubation with human apoA-I., Results: ABCA1 mediates the production of MPs containing cholesterol. This was also confirmed in primary human monocyte-derived macrophages (MDMs). Adding apoA-I markedly increases MP release from cells. Inhibition of ABCA1 with probucol or decreasing plasma membrane cholesterol with methyl-β cyclodextrin (CDX) markedly reduced MP release and nascent HDL formation. MPs do not contain apoA-I, but contain flotilin-2, a marker of plasma membrane, and CD63, an exosome marker. MPs exhibit considerable size heterogeneity (50-250 nm)., Conclusions: We show that MPs are lipoprotein-sized structures created by the ABCA1 transporter, and contribute approximately 30% of ABCA1-and apoA-I mediated cholesterol efflux. In addition, we found that MPs release from cells consists, in part, of exosomes and depends on the same pathway used for HDL biogenesis., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Background: High density lipoprotein receptor SR-BI plays a vital role in cholesterol homeostasis. Depletion of SR-BI causes plasma free cholesterol (FC) accumulation, which disrupts erythrocytes membrane and might induce hemolytic anemia. Here we explored the effects of hypercholesteremia, induced by depletion of low density lipoprotein receptor (LDL-R) and high fat diet (HFD) feeding, on plasma FC and possible hemolysis in SR-BI knockout (KO) mice, and the therapeutic effects of a lipid-lowering drug probucol., Methods and Results: To determine the effects of LDL-R depletion, SR-BI KO mice were cross-bred with LDL-R KO mice to generate SR-BI/LDL-R double KO (dKO) mice. Compared to control wild type (WT), SR-BI KO and LDL-R KO mice fed normal chow diet (NCD), dKO mice fed NCD had increased plasma FC and developed macrocytic anemia, splenomegaly, jaundice and renal tubular hemosiderin deposition, indicating spontaneous hemolysis. To determine the effects of HFD feeding and probucol therapy, dKO and LDL-R KO mice were fed HFD containing 0.5% cholesterol and 20% fat with or without 1% probucol. HFD further increased plasma FC and aggravated hemolysis while probucol almost normalized plasma FC and corrected hemolysis in dKO mice., Conclusion: We demonstrated that in SR-BI KO mice, hypercholesteremia due to LDL-R deficiency significantly increased plasma FC and induced spontaneous hemolysis, which could be further exacerbated by HFD feeding. Probucol almost normalized plasma FC and corrected diet-aggravated hemolysis in SR-BI KO mice with LDL-R deficiency., (Copyright © 2015 Elsevier Inc. All rights reserved.)

The discovery that the apolipoprotein E (apoE) ε4 allele is genetically linked to both sporadic and familial late-onset Alzheimer's disease (AD) raises the possibility that a dysfunction of the lipid transport system could seriously affect lipid homeostasis in the brain of AD subjects. The presence of the ε4 allele has been associated with lower levels of apoE in both serum and brain tissues of normal and AD subjects. In an attempt to reverse the apoE deficit in AD, we identified and characterized several apoE inducer agents using a low-throughput in vitro screening assay. The most promising of these compounds is called probucol. Administration of probucol, an old cholesterol-lowering drug, in a pilot trial in mild-to-moderate sporadic AD led to a significant increase in cerebrospinal fluid (CSF) apoE levels and a decrease in CSF in both phosphorylated tau 181 and beta-amyloid 1-42 concentrations without significant modifications of lipid hydroperoxide levels., Competing Interests: statement All authors declare no conflicts. Written informed consent was obtained from all participants involved in the different studies discussed in this review. Approvals for the different studies were obtained from the Douglas Mental Health Institute Human Research Ethic Committee., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Previous studies have demonstrated that the ATP-binding cassette transporters (ABC)A1 and ABCG1 function in many aspects of cholesterol efflux from macrophages. In this current study, we continued our investigation of extracellular cholesterol microdomains that form during enrichment of macrophages with cholesterol. Human monocyte-derived macrophages and mouse bone marrow-derived macrophages, differentiated with macrophage colony-stimulating factor (M-CSF) or granulocyte macrophage colony-stimulation factor (GM-CSF), were incubated with acetylated LDL (AcLDL) to allow for cholesterol enrichment and processing. We utilized an anti-cholesterol microdomain monoclonal antibody to reveal pools of unesterified cholesterol, which were found both in the extracellular matrix and associated with the cell surface, that we show function in reverse cholesterol transport. Coincubation of AcLDL with 50 μg/ml apoA-I eliminated all extracellular and cell surface-associated cholesterol microdomains, while coincubation with the same concentration of HDL only removed extracellular matrix-associated cholesterol microdomains. Only at an HDL concentration of 200 µg/ml did HDL eliminate the cholesterol microdomains that were cell-surface associated. The deposition of cholesterol microdomains was inhibited by probucol, but it was increased by the liver X receptor (LXR) agonist TO901317, which upregulates ABCA1 and ABCG1. Extracellular cholesterol microdomains did not develop when ABCG1-deficient mouse bone marrow-derived macrophages were enriched with cholesterol. Our findings show that generation of extracellular cholesterol microdomains is mediated by ABCG1 and that reverse cholesterol transport occurs not only at the cell surface but also within the extracellular space.

ApoE plays an important role in lipoprotein metabolism. This study investigated the effects of adenovirus-mediated human apoE overexpression (AdhApoE3) on sterol metabolism and in vivo reverse cholesterol transport (RCT). In wild-type mice, AdhApoE3 resulted in decreased HDL cholesterol levels and a shift toward larger HDL in plasma, whereas hepatic cholesterol content increased (P < 0.05). These effects were dependent on scavenger receptor class B type I (SR-BI) as confirmed using SR-BI-deficient mice. Kinetic studies demonstrated increased plasma HDL cholesteryl ester catabolic rates (P < 0.05) and higher hepatic selective uptake of HDL cholesteryl esters in AdhApoE3-injected wild-type mice (P < 0.01). However, biliary and fecal sterol output as well as in vivo macrophage-to-feces RCT studied with (3)H-cholesterol-loaded mouse macrophage foam cells remained unchanged upon human apoE overexpression. Similar results were obtained using hApoE3 overexpression in human CETP transgenic mice. However, blocking ABCA1-mediated cholesterol efflux from hepatocytes in AdhApoE3-injected mice using probucol increased biliary cholesterol secretion (P < 0.05), fecal neutral sterol excretion (P < 0.05), and in vivo RCT (P < 0.01), specifically within neutral sterols. These combined data demonstrate that systemic apoE overexpression increases i) SR-BI-mediated selective uptake into the liver and ii) ABCA1-mediated efflux of RCT-relevant cholesterol from hepatocytes back to the plasma compartment, thereby resulting in unchanged fecal mass sterol excretion and overall in vivo RCT.

Objective: Cigarette smoking is associated with impaired neovascularization in response to ischemia. Potential mechanisms include increased generation of reactive oxygen species (ROS) and a reduction in the function of endothelial progenitor cells (EPCs). Here we tested the hypothesis that antioxidant therapies could stimulate EPC function and improve ischemia-induced neovascularization following cigarette smoke exposure., Methods and Results: C57Bl/6 mice exposed to cigarette smoke (MES) were fed a normal diet (controls) or a diet supplemented with probucol (0.5%) or a combination of vitamin C (25 g/l in drinking water) and vitamin E (0.1% in normal chow). After two weeks of treatment, hindlimb ischemia was surgically induced by femoral artery removal. Exposure to cigarette smoke was associated with a significant reduction of blood flow recuperation and vessel density in ischemic muscles. However, a complete rescue of neovascularization was demonstrated in MES treated with probucol or antioxidant vitamins. We found that antioxidant therapy in MES is associated with a significant reduction of oxidative stress levels both in the plasma and in ischemic muscles. Moreover, EPCs exposed to cigarette smoke extracts in vitro showed a significant impairment of their angiogenic activities (migration, adhesion, homing into ischemic tissues) that was completely rescued by probucol and antioxidant vitamins., Conclusions: Probucol and antioxidant vitamins rescue cigarette smoke-dependent impairment of ischemia-induced neovascularization. The mechanisms involve beneficial effects on oxidative stress levels in ischemic tissues together with an improvement of EPC functional activities. Antioxidant therapy could constitute a novel therapeutic strategy to promote vessel growth and reduce tissue ischemia in atherosclerotic diseases., (Copyright 2009 Elsevier Ireland Ltd. All rights reserved.)

Expression of ABCA1 is regulated by transcription of the gene and calpain-mediated proteolytic degradation, and inhibition ABCA1 degradation results in increased ABCA1 and HDL biogenesis in vitro. We examined whether this approach could be a potential antiatherogenic treatment. Although probucol inhibits both the activity and degradation of ABCA1, its oxidized products, spiroquinone and diphenoquinone, reduce degradation of ABCA1 without inhibiting its activity or altering transcription of the ABCA1 gene. Accordingly, both compounds enhanced apolipoprotein A-I/ABCA1-dependent generation of HDL in vitro, and increased hepatic ABCA1 and plasma HDL without increasing antioxidant activity in plasma when given to rabbits. Both compounds also decreased vascular lipid deposition in cholesterol-fed rabbits. We therefore conclude that stabilization of ABCA1 against calpain-mediated degradation is a novel and potentially important strategy to increase HDL formation and prevent atherosclerosis. Spiroquinone and diphenoquinone are potential seeds for development of such drugs.

Objective: To establish underlying molecular mechanisms of pro-atherogenic effects of probucol in apo E-KO mice., Methods: Affymetrix Gene Chip System, GenMAPP/MAPPFinder software and real-time PCR techniques were used to identify alterations in gene expression and biological pathways in the liver and aorta of both male apo E-KO and male wild-type mice treated with or without probucol (1%, w/w) for 18 weeks. Plasma levels of lipids, cytokines, liver function test, and the extent of atherosclerosis and liver histology were examined., Results and Conclusions: Probucol treatment paradoxically reduced plasma cholesterol levels, increased plasma cytokine levels and atherogenesis in apo E-KO mice. Three hundred and sixty genes/transcripts and 110 biological processes were significantly differentially expressed in the liver of probucol-treated apo E-KO mice. The response to biotic stimulus, immune response and inflammatory response were the most prominent processes expressed in the liver. The expression of 60 of these genes involved in immune response including inflammatory responses, antigen presentation, humoral immune response, immune cell activation, innate immune response, and regulation of immune response was over-expressed. Many of these genes were also over-expressed in the aorta of probucol-treated apo E-KO mice. Such effects of probucol were not observed in the liver and aorta of wild-type mice. A significant interaction between apo E deficiency and probucol treatment was observed. Histological examinations showed a significant infiltration of inflammatory cells in the liver of probucol-treated apo E-KO mice, but not in C57BL/6 mice. These findings suggest that probucol-induced atherogenesis may be mediated through a pro-inflammatory state.

Probucol is a unique hypolipidemic agent that increases cholesteryl ester transfer protein (CETP) activity. Enhanced CETP-mediated conversion of high-density lipoprotein (HDL) partly explains the probucol-induced decrease in HDL cholesterol and increase in plasma prebeta1-HDL (native lipid-poor HDL) concentrations. However, HDL cholesterol is reduced in patients that are completely deficient in CETP. Angiopoietin-like protein 3 (ANGPTL3) is an endogenous suppressor of endothelial lipase that promotes the hydrolysis of HDL phospholipids and may generate prebeta1-HDL. To determine whether probucol decreases ANGPTL3 and HDL phospholipids while increasing prebeta1-HDL, we measured these parameters before and after a 4-week probucol treatment in 39 hypercholesterolemic patients and age- and sex-matched controls. The median ANGPTL3 had decreased from 143 to 113 microg/L by week 4 (p<0.05). High-performance liquid chromatography revealed that probucol decreased the phospholipid content of very large (13.5-15 nm) and large (12.1 nm) HDL particles predominantly by 65% (p<0.01) and 53% (p<0.001), respectively. The change in ANGPTL3, but not CETP mass, was positively correlated with that in large HDL phospholipids (r=0.455, p<0.05). The absolute and relative concentrations of prebeta1-HDL increased by 14% (p<0.01) and 60% (p<0.001), respectively. The conversion rate of prebeta1-HDL into alpha-migrating HDL by lecithin-cholesterol acyltransferase did not change significantly. In conclusion, probucol decreases plasma ANGPTL3 and HDL phospholipids while increasing prebeta1-HDL. We speculate that probucol induces HDL remodeling via an endothelial lipase-mediated pathway.

Objective: This study was designed to investigate the efficacy of lipid-lowering agents in preventing steroid-induced osteonecrosis and the mechanism by which they do so in a rabbit model., Methods: Female Japanese white rabbits were randomly allocated to receive probucol (group P), pravastatin (group PS), simvastatin (group SS), or saline (group C) for 6 weeks (n = 15 in groups P, PS, and SS; n = 30 in group C). Methylprednisolone (20 mg/kg) was injected at 3 weeks after starting treatment, and the femurs were histologically examined bilaterally 3 weeks after methylprednisolone injection. Midazolam clearance was measured before treatment and before methylprednisolone injection to determine hepatic cytochrome P4503A (CYP3A) levels., Results: The incidence of osteonecrosis in the proximal metaphysis of the femurs in groups PS and SS was significantly lower than in group C (P < 0.05 and P < 0.0001, respectively), whereas it did not differ between groups P and C. It was significantly lower in group SS than in group PS (P < 0.05). Plasma concentrations of lipids (low-density lipoprotein, triglyceride, free fatty acid, and total cholesterol) in groups P, PS, and SS were significantly lower than in group C; and hepatic CYP3A levels were significantly higher in group SS than in groups P or PS after treatment (P < 0.005 for both)., Conclusions: Simvastatin and pravastatin significantly reduced the incidence of steroid-induced osteonecrosis in rabbits. Simvastatin was more effective in reducing the incidence of the disease, and increased CYP3A activity is a possible mechanism for this effect.

To investigate the effect of levofloxacin on carotid atherosclerosis, patients with hypercholesterolemia whose carotid atherosclerosis was not improved by probucol therapy (500 mg/day) for 24 months were enrolled. All patients were seropositive for anti C. pneumoniae IgA and/or IgG. Carotid atherosclerosis was evaluated by ultrasonic measurement of the maximum intima-media thickness (Max-IMT). All subjects were prescribed three courses of levofloxacin (each course, 400 mg/day for 2 weeks, followed by 14 days off drug treatment). At 12 months after combined therapy with probucol and levofloxacin, Max-IMT was significantly decreased compared with the value before treatment (P < 0.01). These results suggest that the combination therapy was effective for improving carotid atherosclerosis in C. pneumoniae-seropositive patients.

Background: Scavenger receptor class B type I (SR-BI) is a major receptor for high-density lipoproteins (HDL) in the liver. Overexpression of SR-BI attenuated experimental atherosclerosis in murine models, concomitant with a reduction in plasma HDL-cholesterol levels. Probucol is known to be a potent hypolipidemic drug to regress xanthoma formation and carotid atherosclerosis in conjunction with a marked reduction in HDL-cholesterol levels. However, the mechanism by which probucol affects atherosclerosis is not completely understood, and the effect of probucol on the expression of SR-BI was controversial. The aim of this study was to know the effect of probucol on HDL metabolism and SR-BI expression in the liver., Methods: Sixteen rabbits fed with high cholesterol diet for 8 weeks were randomly divided into two groups: (1) high cholesterol group (n = 8): maintained high cholesterol diet for 6 weeks; (2) probucol group (n = 8): the same cholesterol diet plus 1% probucol for 6 weeks. Control group (n = 8) was fed with normal diet for 14 weeks. The classical in situ two steps perfusion of the liver with collagenase IV was used to isolate the parenchymal hepatocytes. The selective uptake of HDL by hepatocytes was performed using the double radiolabelled HDL. Immunohistochemistry and reverse transcription polymerase chain reaction (RT-PCR) were used to evaluate SR-BI expression in the liver., Results: Compared with control group, rabbits fed with high cholesterol diet showed higher levels of serum total cholesterol (TC), low-density lipoprotein (LDL) cholesterol and HDL-C, all of which were significantly reduced by probucol treatment. The selective uptake of HDL CEs in probucol group (249.68 +/- 60.13 ng/mg cell protein) was about two folds higher as compared with the control group (122.47 +/- 54.06 ng/mg cell protein, P < 0.01) and high cholesterol group (104.92 +/- 47.91 ng/mg cell protein, P < 0.01), but it could not be reproduced in vitro. The expression of SR-BI were significantly decreased in the high cholesterol group (0.48 +/- 0.06) as compared with control group (0.65 +/- 0.06, P < 0.01). Probucol increased SR-BI expression (0.68 +/- 0.06, P < 0.01) as compared with high cholesterol group. The expression of SR-BI was positively associated with the selective CEs uptake (r = 0.47, P = 0.032)., Conclusions: Probucol up-regulates SR-BI expression and enhance the uptake of HDL CEs by hepatocytes, which may help us to understand the anti-atherogenic properties and the HDL-C-lowering effect of probucol.

In the present study, we examined whether the powerful antioxidant probucol (a clinically used lipid-lowering drug) would attenuate the oxidative stress and energy starvation in experimental model of heart failure (HF) using isoproterenol. Rats were injected subcutaneously with isoproterenol (2.4 mgkg-1) daily for 1 week, and then treated with probucol (61 mg/kg) daily for 2 weeks. Oxidative stress was assessed by measuring myocardial lipid peroxides level and antioxidant enzymes activities, glutathione peroxidase (GPx) and superoxide dismutase. In addition, cardiac metabolic damage was estimated by measuring myocardial ATP, ADP and AMP levels as well as ATP/ADP ratio. It was found that isoproterenol induced a significant increase in heart rate by approximately 30% as compared with the pre-value. These changes were significantly attenuated by post-treatment of rats with probucol. Also, isoproterenol induced several pathological changes including lymphocyte infiltration, myofibrillar hemorrhage and degeneration, and these changes were attenuated by probucol. In addition, animals treated with isoproterenol showed a significant increase in myocardial lipid peroxides level up to 163% and a significant decrease in myocardial GPx activity by 35% as compared with the control group. Probucol not only counteracted significantly the pronounced oxidative stress effect of isoproterenol but also it induced a significant increase in myocardial GPx as compared with the control group. The major new finding of the present study is that treatment with probucol induced a significant increase in myocardial ATP level (the source of energy) and ATP/ADP ratio. Moreover, there is a significant correlation between ATP/ADP ratio and myocardial probucol level. In conclusion, the cardioprotective effect of probucol in treatment of HF is a result of not only its antioxidant properties and an enhancement of endogenous antioxidant reserve (mainly GPx) but also an enhancement of myocardial energy state.

Excess intracellular free cholesterol (FC) is cytotoxic. This study examines prevention of FC-induced cytotoxicity in J774 macrophage foam cells by incubation with apolipoprotein AI (apoA-I). J774 were cholesterol enriched using acetylated low-density lipoprotein and FC/phospholipid (PL) dispersions. Treatment with an acyl coenzyme-A:cholesterol acyltransferase (ACAT) inhibitor, in the absence of extracellular acceptors, produced hydrolysis of stored esterified cholesterol (EC) and FC-induced cytotoxicity. Incubation of cells with ACAT inhibitor plus apoA-I resulted in FC efflux (0.39 +/- 0.02%/h) along with a reduction in cytotoxicity (26.30 +/- 5.80%), measured by adenine release. Small unilamellar vesicles (SUV) caused greater FC efflux (0.53 +/- 0.02%/h, P = 0.001), but a modest reduction in cytotoxicity (8.40 +/- 2.70%, P = 0.008). Co-incubation of ACAT inhibitor plus the cholesterol transport inhibitor U18666A or the antioxidant Probucol reduced efflux to apoA-I, but not to SUV. Pre-treatment of J774 foam cells with CTP-cAMP upregulates hormone sensitive lipase (HSL) and further upregulates ATP binding cassette A1 (ABCA1). Using mouse serum as a cholesterol acceptor, CTP-cAMP caused greater protection against FC-induced cytotoxicity compared to cells without pre-treatment, suggesting a role of ABCA1 in removal of cytotoxic FC. We conclude that a cytotoxic pool of FC is located in the plasma membrane, is readily available for efflux to apoA-I, and removal of cytotoxic cholesterol may involve ABCA1., (Copyright 2003 Elsevier Ireland Ltd.)

A novel vitamin E derivative that is freely soluble in water, 2-(alpha-D-glucopyranosyl)methyl-2,5,7,8-tetramethylchroman-6-ol (TMG), was evaluated for ability to inhibit development of atherosclerosis in Watanabe heritable hyperlipidemic (WHHL) rabbits or cholesterol-loaded New Zealand White rabbits. Although TMG rapidly entered the circulation blood after oral administration, the blood TMG concentration remained low, while neither TMG nor its metabolites appeared in the low-density lipoprotein (LDL) fraction. TMG did not decrease serum total cholesterol and the various lipoprotein-associated cholesterol fractions (very LDL-, or high-density lipoprotein- (HDL) cholesterol). TMG reduced the serum concentration of thiobarbituric acid-reactive substances (TBARS; an index of lipid peroxidation) in cholesterol-loaded rabbits but not WHHL rabbits. Nonetheless, TMG inhibited aortic atherosclerosis as effectively as probucol in both models. Our results indicate that TMG opposes progression of atherosclerosis not only by preventing oxidation of LDL, but also by presently unknown mechanisms. Even an antioxidant with no uptake by LDL apparently can inhibit development of atherosclerosis despite a very low serum concentration.

Loss of retinal pericytes is the initial deficit in the early stage of diabetic retinopathy. Glycated albumin (GA) forms under hyperglycemic conditions and exists in the retinal blood vessels of diabetic patients with retinopathy. In this study, using a 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide (MTT) reduction test, we investigated whether GA induces cytotoxicity in cultured bovine retinal pericytes, and whether the antioxidants, l-ascorbic acid, Trolox, and probucol, provide any protection from GA-mediated cytotoxicity. GA induced pericyte death in a dose-dependent manner. With increasing time, GA-induced cytotoxicity also increased despite no strict time dependence. Furthermore, this cell death was found to be mediated both by apoptosis, which was confirmed by apoptosis-specific fluorescent staining of nuclei and cell membranes, and mitochondrial damage, as elucidated by electron microscopy. All three antioxidants used in this study partially protected against GA-induced pericyte death, suggesting that oxidative stress plays a role in GA-induced pericyte death. The results indicate that GA induces cell death in cultured bovine retinal pericytes, and that certain antioxidants may reduce this cytotoxicity., ((c)2002 Elsevier Science (USA).)

Antioxidants have been proposed as a promising treatment for restenosis after percutaneous transluminal coronary angioplasty (PTCA), but their mechanism of action remains unclear. Here, we investigated the effect of antioxidants on gene expression in the artery after balloon denudation. We developed a sensitive ribonuclease (RNase) protection assay for the messenger RNA (mRNA) levels of immediate early (IE) genes (c-jun, c-fos and c-myc), as well as platelet-derived growth factor-A (PDGF-A), platelet-derived growth factor-beta receptor, transforming growth factor-beta 1, and vascular endothelial growth factor. New Zealand White rabbits were fed a 0.17% cholesterol diet containing vehicle, BO-653 or probucol, and balloon denudation for iliac arteries was performed. The iliac arteries were then removed at 4 h after the denudation, for IE genes, and 10 days after for growth factors and receptors. Both BO-653 and probucol significantly reduced neointimal thickening, compared with the control. In terms of gene expression, BO-653, but not probucol, significantly inhibited c-myc induction. On the other hand, probucol, but not BO-653, significantly inhibited PDGF-A expression. Neither treatment had any effect on the expression of other genes. These results suggest that antioxidants affect the gene expression of the neointimal response and that both BO-653 and probucol inhibit gene expression in specific manners.

Background: Due to the strong evidence on the involvement of active oxygen species in a variety of disorders, the role of antioxidants against oxidative stress has recently received increased attention., Methods: Twenty male rabbits were served a high-cholesterol (HC, 5 g/kg diet) diet or high-cholesterol diet supplemented with naringin (0.5 g/kg diet) or probucol (0.5 g/kg diet) for 8 weeks to compare the antioxidative effects of the citrus bioflavonoid (naringin) and antioxidative cholesterol-lowering drug (probucol)., Results: The plasma thiobarbituric acid-reactive substances (TBARS) concentration was not significantly different between the groups, whereas the hepatic TBARS concentration was significantly lower in the probucol group than in both normal and HC control or naringin group. Probucol and naringin supplementation led to an increase in the hepatic superoxide dismutase (SOD) and catalase (CAT) activities, and a decrease in the hepatic mitochondrial hydrogen peroxide (H(2)O(2)) content compared to the HC-control group. However, there was no difference in the cytosolic H(2)O(2) content or cytosolic glutathion peroxidase (GSH-Px) activity in the liver between the groups. Both naringin and probucol supplements significantly increased the plasma vitamin E concentration compared to the HC-control group. As regards the antioxidant enzyme gene expressions, naringin significantly increased the expression of three antioxidant enzyme mRNAs compared to the HC-control group, whereas probucol significantly increased the only SOD mRNA expression., Conclusions: The probucol supplement was very potent in the antioxidative defense system, whereas naringin exhibited a comparable antioxidant capacity based on increasing the gene expressions in the antioxidant enzymes, while also increasing the hepatic SOD and CAT activities, sparing plasma vitamin E, and decreasing the hepatic mitochondrial H(2)O(2) content.

Hematein is a compound isolated from Caesalpinia sappan that has been used in oriental medicine as both an analgesic and an anti-inflammatory agent. In this study, we examined the anti-atherogenic potential of hematein using cholesterol-fed New Zealand White (NZW) rabbits. NZW rabbits were divided into a hematein-supplemented (0.05% in diet) group (n=6), a probucol-supplemented (0.25% in diet) group (n=6), and a control group (n=6). After 8 weeks of treatments, the extent of the atherosclerotic lesions was significantly reduced in the hematein-supplemented group and the probucol-supplemented group without changing plasma lipoprotein levels. Hematein and probucol prevented the up-regulation of the vascular cell adhesion molecule-1 (VCAM-1) expression on the descending aorta induced by cholesterol diet. In culture, hematein also significantly inhibited the secretion of soluble VCAM-1 and of monocyte chemotactic protein-1 (MCP-1) respectively induced by tumor necrotic factor alpha (TNF-alpha) and mildly oxidized low density lipoprotein in human umbilical vein endothelial cell (HUVEC) culture. Also, hematein inhibited monocyte adhesion to endothelial cell and the activation of NF-kappaB in HUVECs stimulated with TNF-alpha. The results of the present study suggest that the anti-atherogenic effect of hematein is not related to control of the plasma lipid profile but probably related to the inhibition of VCAM-1 and MCP-1 expression resulting in an amelioration of lesion development in the rabbit.

We investigated the inhibitory effects of fluvastain (FV) and its metabolites (M-2, M-3, M-4, M-5, and M-7) on the formation of several reactive oxygen species (ROS), such as singlet oxygen (1O2), superoxide anion (O2-), hydroxy radical (*OH), hypochlorite ion (OCL-), and linoleic acid peroxide (LOO*). Inhibitory effects of pravastatin (PV), simvastatin (SV), probucol (PR) and alpha-tocopherol (TOC) were also tested. The inhibitory effects of 5-hydroxy FV (M-2) and 6-hydroxy FV (M-3) on the formation of 1O2, O2-, *OH, and OCL- were strongest. Scavenging of 1O2 by M-4, M-5, (+)-FV, and (-)-FV was also noted. The inhibitory effects of (+)-FV on the formation of 1O2 were comparable to those of (-)-FV, PV, SV, PR and M-7 had little or no inhibitory effect on the formation of several ROS. In conclusion, FV and its metabolites, particulary M-2 and M-3, have the potential to protect against oxidative stress mediated by several ROS.

An early event in atherogenesis is the adhesion of monocytes to endothelium via adhesion molecules, such as VCAM-1 and intracellular adhesion molecule-1 (ICAM-1). It has been suggested that VCAM-1 plays a very important role in the recruitment of monocytes in atherosclerosis. Probucol is a potent inhibitor of atherosclerosis in animal models. However, the mechanism of its antiatherogenic effect is poorly understood. The aim of our study was to evaluate whether probucol can influence the expression of endothelial cell adhesion molecules and endothelial adhesiveness. The study was performed on cultured human umbilical vein endothelial cells (HUVEC). HUVEC were pretreated with probucol (50 microM) at different time periods before stimulation with TNFalpha (100 U ml(-1)) or IL-1beta (100 U ml(-1)). The protein expression of VCAM-1 and ICAM-1 was measured by flow cytometry. VCAM-1 mRNA expression was measured by reverse transcription polymerase chain reaction (RT PCR). Probucol time dependently reduced agonist-induced VCAM-1 ( approximately 45%, 48 h) surface protein and mRNA expression ( approximately 40%, 48 h) in HUVEC, but not ICAM-1 surface protein expression. Decreased VCAM-1 expression was associated with reduction ( approximately 40%) of adherence between cytokine-stimulated HUVEC and peripheral blood mononuclear leukocytes (PBMC). Our results suggest that the antiatherogenic effect of probucol may, in part, be due to a downregulation of VCAM-1 expression.

Macrophage-derived foam cells are known to play an essential role in the development and progression of atherosclerotic lesions. Probucol prevents oxidative modification of low-density lipoprotein (LDL) and lowers plasma contents of LDL and high-density lipoprotein (HDL). A recent report using apoE -/- mice demonstrated that probucol treatment enhanced atherosclerosis in apoE -/- mice more rapidly than that in untreated apoE -/- mice, and a reduction in plasma cholesterol by probucol was not the cause of enhancement of atherosclerotic lesions in probucol-treated apoE -/- mice. Moreover, probucol was reported to inhibit apoA-I mediated cholesterol efflux from mouse macrophages. These reports suggested that probucol might directly affect cholesterol metabolism in mouse macrophages. Thus, we investigated the effects of probucol on cholesterol metabolism in mouse resident peritoneal macrophages. Probucol did not affect degradation of acetylated LDL (Ac-LDL), degradation of LDL and endogenous cholesterol synthesis in mouse macrophages. However, it significantly inhibited HDL-mediated cholesterol efflux. Moreover, probucol partially (30%) inhibited the binding of HDL to mouse macrophages, and significantly activated acyl-coenzyme A:cholesterol acyltransferase (ACAT). Our results suggested that probucol inhibited HDL-mediated cholesterol efflux by inhibiting the binding of HDL to mouse macrophages and reducing HDL-accessible free cholesterol content by ACAT activation, thereby worsening atherosclerotic lesions in apoE -/- mice. However, it remains unclear whether probucol inhibits HDL-mediated cholesterol efflux from human macrophages.

It has been reported that probucol is a lipid-lowering agent having a strong antioxidative effect and inhibitory action on vascular smooth muscle cell proliferation. In this work, we studied the effect of treatment with a 1% probucol diet on pulmonary hypertension induced by monocrotaline (MCT) in rats. Rats were fed a control or 1% probucol-supplemented diet for 7 days, then given a single subcutaneous injection of 60 mg/kg MCT or saline, and continuously fed the same diet for 20 days, respectively. MCT caused an increase in right ventricular systolic pressure (RVSP), an indicator of pulmonary hypertension, and central venous pressure (CVP) on day 20. In rats receiving a diet containing 1% probucol, RVSP was significantly lower than that in rats treated with control diet, and CVP remained essentially at the basal level. On day 20, MCT also caused an increase in the ratio of right ventricular (RV) to body weight (BW), compared to the control value, indicating the development of RV hypertrophy in MCT rats. RV hypertrophy was significantly inhibited in 1% probucol-treated rats. These findings suggest that chronic treatment with probucol effectively inhibits the progression of pulmonary hypertension in rats.

The development of atherosclerosis is a multifactorial process in which both elevated plasma cholesterol levels and proliferation of smooth muscle cells play a central role. Numerous studies have suggested the involvement of oxidative processes in the pathogenesis of atherosclerosis and especially of oxidised low density lipoproteins. Some epidemiological studies have shown an association between high dietary intake or high serum concentrations of vitamin E and lower rates of ischemic heart disease. Recently, the Cambridge Heart Antioxidant Study (CHAOS) reported strong protection by high vitamin E doses against the risk of fatal and non fatal myocardial infarction. Here we have shown that incubation of vascular smooth muscle cells in the presence of alpha-tocopherol resulted in inhibition of cell proliferation and protein kinase C activity. Since beta-tocopherol and probucol are not inhibitory, the effect of alpha-tocopherol is considered due to a non-oxidant mechanism. In order to understand the protective role of alpha-tocopherol against atherosclerosis in vivo the following rabbit studies were carried out. Atherosclerosis was induced by a vitamin E poor diet containing 2% cholesterol in a group of rabbit. The other groups had 2% cholesterol in the diet plus 50 mg/kg vitamin E i.m. or 1% probucol or 50 mg/kg vitamin E plus 1% probucol. After 4 weeks, aortas were removed and analysed by microscopy for atherosclerotic lesions. Samples of the media were analysed for protein kinase C activity. The aortas of cholesterol-fed rabbits showed typical atherosclerotic lesions, detected by microscopic examination, their media smooth muscle cells exhibited an increase in protein kinase C activity. Vitamin E fully prevented cholesterol induced atherosclerotic lesions and the induction of protein kinase C activity while probucol was not effective. These results show that the protective effect of vitamin E against hypercholesterolemic atherosclerosis is not produced by an other antioxidant such as probucol and, therefore, may not be linked to the antioxidant properties of this vitamin. The effects observed at the level of smooth muscle cells in vitro and ex-vivo suggests an involvement of signal transduction events in the protective effect of vitamin E against atherosclerosis.

Diabetes mellitus is associated with an increased risk of premature atherosclerosis, which may be due in part to an increased rate of low density lipoprotein (LDL) oxidation. Previous studies have shown that vitamin E, probucol, and lovastatin can reduce the oxidative susceptibility of LDL in normoglycemic animal models; however, few studies have investigated this in conjunction with aortic fatty streak lesion formation in diabetic hyperlipidemic models. Forty-eight Syrian hamsters were made diabetic by intraperitoneal injection of low dose streptozotocin. Diabetic animals (12 animals/groups) received a high saturated fat and cholesterol diet for 12.5 weeks. At 2.5 week of dietary treatments, the diet was supplemented with either: (1) 500 IU/day vitamin E (D+E); (2) 1% probucol w/w of the diet (D+P); (3) 25 mg/kg lovastatin (D+L); or (4) diabetic control (D). An age-matched group of hamsters (n=6) receiving the same diet but not made diabetic (ND) was used as control. At the end of the study, aortic arch foam cell-rich fatty streak lesion, plasma glucose, total cholesterol (TC), high density lipoprotein cholesterol (HDL-C), non-HDL-C, triglycerides (TG), phospholipids, alpha-tocopherol, plasma lipid peroxide and the susceptibility of LDL to copper-catalyzed oxidation were determined. Diabetes increased plasma glucose, and when combined with an atherogenic diet resulted in a further increase of plasma lipids. Vitamin E, probucol, and lovastatin significantly reduced plasma TG in the diabetic hamsters fed the atherogenic diet. Vitamin E treatment increased TC, probucol reduced HDL-C without affecting TC; whereas lovastatin reduced TC and selectively decreased non-HDL-C, and significantly reduced fatty streak lesion formation in the aortic arch. While vitamin E and probucol were effective in reducing several indices of oxidative stress including plasma lipid peroxides, cholesterol oxidation products and in vitro LDL oxidation, they had no effect on fatty streak lesion formation. Our results indicate that the LDL in diabetic animals is more susceptible to oxidation than in non-diabetic hamsters and that not only vitamin E and probucol but also lovastatin provide antioxidant protection. It appears that in this combined model of diabetes and hypercholesterolemia, progression of fatty streak lesion formation is mainly associated with changes in TC and non-HDL-C as affected by lovastatin, and is less dependent on the extent of LDL oxidation, changes in plasma TG level and oxidative stress status.

Unlabelled: Role of organic anion transporter 1 (OAT1) in cephaloridine (CER)-induced nephrotoxicity., Background: Cephaloridine (CER) has been used to elucidate the mechanisms of cephalosporin antibiotic-induced nephrotoxicity. Organic anion transporters have been thought to mediate CER uptake by the proximal tubule. The purpose of this study was to elucidate the possible involvement of organic anion transporter 1 (OAT1) in CER-induced nephrotoxicity., Methods: A mouse terminal proximal straight tubule (S3) cell line stably expressing rat OAT1 (S3 rOAT1) was established and used in this study. The cellular uptake of [14C]-para-aminohippuric acid (PAH), a prototype organic anion, and that of [14C]-CER were measured. The effects of CER on the viability of the cells and the amount of lipid peroxidation were estimated., Results: S3 rOAT1 expressed a functional organic anion transporter in the cytoplasmic membrane, and exhibited CER uptake activity. CER treatment resulted in a more significant decrease in the viability and a more significant increase in the amount of lipid peroxidation in S3 rOAT1 than in S3 cells transfected with an expression vector lacking the rOAT1 insert. Probenecid, an inhibitor of organic anion transport, and probucol, an antioxidant, significantly suppressed the decrease in viability and increase in the amount of lipid peroxidation in S3 rOAT1 treated with CER. The effects of various cephalosporin antibiotics on the uptake of [14C]PAH were correlated significantly with the effects of these drugs on cell viability., Conclusions: These results suggest that rOAT1 is, at least in part, responsible for the cellular uptake of CER and therefore CER-induced nephrotoxicity.

Background: Nitric oxide (NO) directly inhibits fluid and solute reabsorption in the proximal tubule. In the present study, we investigated the effect of NO on the Na+, K+-ATPase of opossum kidney (OK) cells, a proximal tubule cell line, and its mechanisms., Methods: Na+,K+-ATPase activity in the membrane fraction of OK cells was measured as the ouabain-sensitive ATP hydrolytic activity. The enzyme unit number on intact cells was measured by ouabain-binding assay., Results: Incubation with 0.5 mM sodium nitroprusside (SNP), a NO donor, for two hours inhibited the catalytic activity of the membrane-associated Na+,K+-ATPase in OK cells to 65.5 +/- 9.7% of control (N = 6, P < 0.05 vs. control). This effect of SNP was concentration- and time-dependent. The NO scavenger hemoglobin blunted, while another NO donor spermine NONOate (5 microM) mimicked this effect of SNP. At all concentrations and time points tested, SNP did not alter the molecular number of Na+,K+-ATPase on intact OK cells, indicating that NO inhibited the molecular activity of Na+,K+-ATPase. The soluble guanylate cyclase inhibitor, 1H-[1,2,4]oxadiazolo-[4, 3-a]quinoxalin-1-one (ODQ), blunted the inhibitory effect of SNP on the Na+,K+-ATPase activity. An exogenous cGMP analog similarly inhibited the Na+,K+-ATPase activity. Neither lipid soluble antioxidants vitamin E/probucol or thiol group compound DL-dithiothreitol (DTT) altered the inhibitory effect of SNP on the Na+,K+-ATPase activity., Conclusions: NO inhibited the molecular activity of the Na+,K+-ATPase of the OK proximal tubule cell line probably via cGMP-dependent mechanisms.

Gentamicin (GM) is widely used as a bactericidal agent for the treatment of severe gram negative infections, however, its clinical use is partially limited due to its nephrotoxicity. Recent evidence suggests a role of reactive oxygen metabolites in GM nephrotoxicity. The present study was designed to investigate a possible potential protective role of vitamin E and/or probucol against GM nephrotoxicity. GM was administered to rats in a single dose of (150 mg kg(-1)i.p.), while vitamin E (250 mg kg(-1)i.m.) and/or probucol (60 mg kg(-1)i.m.) were given once daily for 3 consecutive days prior to GM administration. GM-induced nephrotoxicity was evidenced by marked elevations in serum urea and creatinine levels, urinary activity of N-acetyl-beta- d -glucosaminidase (NAG) and gamma-glutamyl-transferase (gamma-GT). Also, GM caused significant increases in kidney content of malondialdehyde (MDA), and significant decreases in kidney content of reduced non-protein sulphydryls (NPSH) and superoxide dismutase (SOD) activity. Vitamin E pretreatment significantly lowered the elevated serum urea and creatinine levels, and urinary activity of NAG and gamma-GT. In addition, vitamin E ameliorated the rise in renal content of MDA and enhanced the renal NPSH content as well as SOD activity. Similarly, probucol significantly inhibited the elevations in urea and creatinine levels and enhanced renal NPSH content and SOD activity. Simultaneous use of vitamin E and probucol was more effective in mitigating disturbances in the assessed parameters. The present work indicates that, due to their antioxidant activity, vitamin E and probucol have potential protective effects against GM nephrotoxicity., (Copyright 1999 Academic Press.)

This report describes the in vitro and ex vivo antioxidant properties of a new antioxidant, CGP 2881. This compound is structurally similar to probucol, in that both compounds contain bis-tertiary butyl phenyl groups. However, CGP 2881 consistently inhibited CuSO4 (Cu2+)- and macrophage (MO)-induced oxidation of human low density lipoproteins (LDL) more potently than equimolar concentrations of probucol. CGP 2881 (1 mumol/l) prolonged the lag phase of diene formation during Cu(2+)-induced LDL oxidation by 3.4 versus 1.5-fold prolongation with 1 mumol/l probucol (P < 0.05 vs CGP 2881). The IC50 for inhibiting the formation of Cu(2+)-induced thiobarbituric acid-reactive substances (TBARS) was 0.15 mumol/l for CGP 2881, versus approximately 10 mumol/l for probucol. The IC50 for MO-induced oxidation of LDL (TBARS) was 0.64 mumol/l. In contrast, 1 mumol/l probucol failed to inhibit MO-induced oxidation of LDL. Treatment of cholic acid/cholesterol-fed rats with CGP 2881 (50 mg/kg per day, orally for 5 days) inhibited ex vivo Cu(2+)-induced oxidation (TBARS) of the very low density lipoproteins (VLDL) + LDL lipoprotein fraction by 93% versus vehicle controls (P < 0.0001), and prolonged the lag phase for Cu(2+)-induced diene formation by 3.4-fold over vehicle-treated controls. Five days of orally administered CGP 2881 reduced plasma total cholesterol and LDL cholesterol levels to 55 and 54% of vehicle-treated controls, respectively (P < 0.05). In contrast, probucol had no appreciable effect on plasma total cholesterol or LDL cholesterol levels, unless administered for longer than 5 days. Treatment of hypercholesterolemic rabbits with 50 mg/kg per day orally for 5-12 days delayed the lag phase of diene formation during LDL oxidation by 4.3-fold over controls. However, the relative antioxidant potencies of CGP 2881 and probucol seen with oral administration to hypercholesterolemic rabbits were reversed when the compounds were given intravenously. In addition, the effects of these antioxidants were potentiated when given to normocholesterolemic rabbits compared to hypercholesterolemic animals. These data establish that CGP 2881 demonstrates hypolipidemic activity and is a substantially more potent antioxidant than probucol (in vitro and ex vivo). CGP 2881 may be useful as a new antioxidant tool in the effort to better understand the atherogenicity of oxidized LDL (oxLDL).

Background and Aim: Substantial evidence suggests that oxidative modifications of low density lipoproteins (LDL) critically contribute to the pathogenesis and progression of human atherosclerosis. Oxidized LDL (oxLDL) are present in atherosclerotic plaques and contain oxysterols that exhibit a variety of adverse biological activities. Antioxidants have also been shown to prevent LDL modification. We have therefore assessed the efficacy of virgin olive oil phenolic compounds in preventing oxidative modifications of human LDL oxidized by UV light., Methods and Results: Cholesterol oxides formed during LDL photo-oxidation were determined by UV-HPLC in the presence of different concentrations of phenolic compounds and their pure components (tyrosol and oleuropein), and probucol, a widely used synthetic antioxidant. Electrophoretic mobility was also assayed. The results demonstrate that phenolic compounds are much more potent in preventing cholesterol oxide formation and apoproteic moiety modification than their pure components and probucol., Conclusions: The beneficial effects of a Mediterranean diet may be ascribable not only to the high unsaturated/saturated fatty acid ratio characteristic of olive oil, but also to the unique antioxidant properties of its phenolic compounds.

We investigated the effect of probucol on the intracellular pH ([pH]i) and proliferation of human umbilical vein endothelial cells (HUVEC), as well as their production of prostacyclin (PGI2). The addition of probucol produced a biphasic shift in [pH]i, with a brief initial acidification followed by a rapid alkaline shift. After pretreatment with EGTA, the initial decrease in [pH]i was abolished, and the subsequent increase was inhibited. After pretreatment with amiloride, only the increase of [pH]i was abolished. These results suggest that the probucol-induced increase of [pH]i was mainly dependent on Na+/H+ exchange and partly on extracellular Ca2+. In contrast, the addition of LDL produced a decrease of [pH]i. Under Ca2+-free condition, [pH]i was further decreased by LDL. In cells pretreated with amiloride, however, [pH]i was not further decreased by LDL. It was found that probucol promoted cell proliferation, and LDL inhibited cell proliferation. Addition of probucol also enhanced prostacyclin generation by HUVEC. This enhancement of PGI2 generation resulted from increased release of Ca2+ from the storage sites, due not only to increased production of inositol 1,4,5-triphosphate (IP3) but also to the increase of [pH]i. These findings may help to explain the antiatherosclerotic action of probucol.

The anti-atherogenic and cholesterol-lowering drug probucol (0.5-1%) or quercetin (1%), a natural antioxidant, was given to cholesterol-fed (1.5%) mice for a period of 6 weeks and to Watanabe heritable hyperlipidemic (WHHL) rabbits for a period of 8 weeks to investigate the oxidative changes in plasma and lipoproteins. Oxidation was measured as the total amount of malondialdehyde (nmol MDA/g protein) by a very specific MDA-HPLC method. A large and significant increase in MDA was seen in LDL from probucol treated WHHL rabbits (1778.7+/-585.5 nmol/g vs. 394.4+/-144.5 nmol/g, P < 0.001) and cholesterol-fed mice (579.7 + 47.3 nmol/g vs. 408.1+/-85.8 nmol/g, P < 0.05) as compared to controls while LDL cholesterol was lowered (WHHL rabbits: P < 0.05; mice: P < 0.01). In WHHL rabbits VLDL oxidation was determined additionally, and also revealed a large increase in the probucol group (2102.7+/-1156.1 nmol/g vs. 455.0+/-207.8 nmol/g, P< 0.01). In contrast, the oxidation of plasma and HDL from probucol treated animals was not statistically significantly increased, implying that probucol mediates a selective oxidation of atherogenic cholesterol-transporting lipoproteins. Quercetin treated animals did not show increased oxidation of LDL (and VLDL in rabbits) and cholesterol levels were not decreased. Furthermore, no protective antioxidant effect of quercetin was seen. In conclusion, the results suggest that a prooxidant mechanism rather than antioxidative effects influences lipoprotein metabolism in these animals. It is hypothesized that the oxidation of lipoproteins might be a physiological mechanism performed by macrophages or other cells for uptake and degradation (by macrophages and liver) of excessive amounts of LDL or VLDL and that probucol oxidizes atherogenic lipoproteins and thereby leads to a decrease in cholesterol levels.

Although numerous transgenic mouse models for atherosclerosis have been developed recently, little is known about their response to hypolipidaemic or anti-atherosclerotic agents. We investigated the effect of the known hypocholesterolaemic and anti-atherosclerotic drug probucol on serum lipids, lipoproteins and atherosclerosis in fat-fed low density lipoprotein (LDL) receptor deficient mice. Probucol at doses of 0.2 and 1% in the diet which are similar to those used in the mouse by other investigators reduced serum cholesterol by 26 and 37%, respectively. Probucol also reduced serum triglyceride levels by 33 and 47% at doses of 0.2 and 1%, respectively. The decrease in serum cholesterol and triglycerides was mainly due to a decrease of these lipids in VLDL and or chylomicrons. Despite these potentially beneficial changes in serum lipids atherosclerotic lesion areas in the aortic root were unchanged in the probucol treated mice. After 12 weeks treatment most of the mice receiving probucol had swollen feet and tails due to oedema. Histological examination of the base of the hearts from the probucol treated mice revealed lipid droplets within the reticuloendothelial and other interstitial cells. There was also an interstitial subacute inflammatory cell infiltration associated with the lipid deposition. The oedema induced by probucol could be the result of cardiac insufficiency due to interstitial lipidosis and inflammation in the base of the heart together with the extensive atherosclerotic lesions in the aortic sinus.

The in vivo direct antiatherogenic activity of the antioxidant probucol (200 mg/kg per day) or the beta-blocker with antioxidant properties carvedilol (10 and 20 mg/kg per day) was tested in the same animal in two different types of atherosclerotic lesion (proliferative and fatty lesions) induced in cholesterol-fed rabbits (1%). Drugs were given daily mixed with standard diet for 8 weeks; body weight and plasma lipid profile were not different among groups throughout the study. Aortic fatty lesions were induced by cholesterol feeding (n = 25 in each group) and their extent expressed as % of aorta inner surface covered by plaques was significantly reduced by both drugs (28.2+/-9.6%, P <0.05, 19.9+/-6.2%, P <0.01 for low- and high-dose carvedilol, respectively; 22.3+/-7.6%, P <0.01 for probucol, versus 41.6+/-10.7% in control rabbits). Proliferative lesions were obtained by positioning a hollow silastic collar around one carotid artery 6 weeks after dietary and drug treatments started (n = 5 in each group). The neointimal formation, mostly composed by myocytes, was determined by measuring cross-sectional thickness ratio of intimal (I) and medial (M) tissue of fixed arteries. In untreated animals, collared arteries resulted in a significant neointimal cell accumulation compared to the sham (1.10+/-0.14 versus 0.02+/-0.01) without change in medial thickness. I/M ratio was reduced by about 50% in animals treated with probucol (0.51+/-0.1) and carvedilol (0.66+/-0.21 and 0.52+/-0.1 in the low- and high-dose group, respectively). Total plasma TBARS were more than 50% lower in both probucol- and high-dose carvedilol-treated rabbits. Results show that pharmacological pretreatment with antioxidants directly inhibits early atherogenic processes, representing a potentially useful approach in the prevention of atherosclerosis.

A stable HL60 subline having the potential for monocytic differentiation was established by use of GM-CSF. HL60, a human promyelocytic cell line, has frequently been employed for research in the fields of monocytic differentiation and atherosclerosis because of its potential to differentiate into either granulocytes or monocytes. However, HL60 are frequently seen to change their phenotype during long-term culture. To date, many sublines or variants of HL60 cells have been established. However, most of them display diminished or complete loss of activities that characterize parental cells. The present study was conducted to establish a stable HL60 subline with the potential for monocytic differentiation. Firstly, a single HL60 cell was isolated by limiting dilution, and was successfully proliferated by incubation with GM-CSF. Secondly, from this population, cells were selected that had the ability to generate superoxide after VD-induced monocytic differentiation. Cells obtained in this manner (designated HL60/DU-1) exhibited expression of CD14 and CD11b and suppression of CD3 expression after monocytic differentiation. NBT positivity showed a consistent level of over 971% after a 6-day challenge with VD throughout the experimental period of 12 months. HL60/DU-1 cells, which were cryopreserved in liquid nitrogen for 6 months, thawed and re-cultured, exhibited over 97% NBT positivity. Carvedilol and probucol, which exhibit antioxidative activity, inhibited superoxide release from the differentiated HL60/DU-1 cells. HL60/DU-1 cell line is a promising model for the study of monocytic differentiation and the effects of oxygen radicals.

Probucol is a powerful inhibitor of atherosclerosis in a number of animal models. However, it is unknown whether this is due to the strong antioxidant protection of low density lipoprotein (LDL), to antioxidant effects in the artery wall, or to cellular effects not shared by other antioxidants. To investigate whether murine models are suitable to study the antiatherogenic mechanisms of probucol, three experiments following different protocols were carried out in 135 male and female LDL receptor-deficient (LDLR-/-) mice. Treatment groups received a high (0.5%) or low (0.025%) dose of probucol, or low-dose probucol plus a high dose (0.1%) of vitamin E for periods ranging from 6 to 26 weeks. In all experiments, probucol strongly protected LDL against ex vivo oxidation (lag times exceeding 1400 min in 0.5% probucol-treated mice). Treatment with 0.5% probucol significantly lowered both HDL-cholesterol and plasma apolipoprotein (apo)A-I concentrations. In all three experiments, treatment with 0.5% probucol consistently increased the size of lesions in the aortic origin, from 1.3-fold (n.s.) to 2.9-fold (P < 0.05) in female mice and from 3.6- to 3.7-fold in males (P < 0.001). Even treatment with 0.025% probucol increased atherosclerosis 1.6-fold in male mice (P < 0.01). Addition of the high dose of vitamin E did not attenuate the pro-atherogenic effect of 0.025% probucol. In conclusion, probucol not only failed to decrease but actively increased atherogenesis in LDLR-/- mice in a dose-dependent manner, even though it provided a very strong antioxidant protection of LDL. This suggests that the reduction of atherosclerosis observed in other animal models is due to intracellular effects of probucol not found in mice, to differences in the metabolism of probucol, and/or to an overriding atherogenic effect of the decrease in HDL in murine models.

Recent data indicate that hypercholesterolemia increases endothelial superoxide anion (.O2-) production, and that this diminishes the bioactivity of nitric oxide produced in the endothelium. Probucol, a drug commonly employed for treatment of hypercholesterolemia, has antioxidant properties and inhibits oxidation of low density lipoproteins in vitro. We tested the hypothesis that probucol would decrease vascular .O2- production and improve endothelium-dependent relaxations in cholesterol-fed rabbits. Rabbits were divided into four groups: 1) a control group fed a standard diet; 2) a probucol group fed a standard diet containing 0.3% probucol; 3) a hypercholesterolemic group fed a diet containing 0.5% cholesterol; 4) a hypercholesterolemia-probucol group fed a diet containing 0.5% cholesterol and 0.3% probucol. The cholesterol-rich diet markedly increased plasma total cholesterol level and lipid peroxidation in the plasma, as reflected by thiobarbituric acid-reactive substances (TBARS). This concentration of probucol did not lower plasma cholesterol, but markedly reduced TBARS in the plasma of cholesterol-fed rabbits. Aortic segments from cholesterol-fed rabbits produced 1.8-fold more .O2- (assessed by lucigenin-enhanced chemiluminescence) and decreased endothelium-dependent vascular relaxations to acetylcholine compared to vessels from normal rabbits. In cholesterol-fed rabbits, probucol treatment normalized both .O2- production and endothelium-dependent relaxations to acetylcholine. In control rabbits, probucol had no effect on either of these parameters. We conclude that probucol treatment may prevent .O2(-)-induced inactivation of endothelium-derived nitric oxide and reduce vascular oxidant stress via reducing the level of .O2-.

Tissue factor (TF) plays a central role in the initial activation of the extrinsic coagulation pathway and is thought to be involved in the development of atherosclerosis and thrombosis. The effect of advanced glycosylation end products (AGEs) on TF expression and its mechanism were assessed by flow cytometric analysis. Human macrophage-like U937 cells, which were shown to contain mRNA encoding the receptors of advanced glycosylation end products (RAGE), expressed TF in a dose-dependent manner on incubation with AGE-albumin. AGE-albumin-induced TF expression was completely inhibited by the anti-oxidant agents, catalase and probucol. TF expression in peripheral monocytes from normal volunteers was also increased by AGE-albumin. Finally, TF expression in monocytes from individuals with diabetes mellitus, in whom the concentration of circulating AGEs is reported to be increased, was higher than that in monocytes from normal controls. These results suggest that AGE-induced TF expression in macrophages/monocytes is mediated by oxidant stress. AGEs may promote thrombosis and the development of atherosclerosis by inducing TF expression in monocytes in patients with diabetes mellitus.

Adriamycin (ADR) is a potent, broad-spectrum chemotherapeutic agent whose clinical use is limited by its cardiotoxicity. Since the pathogenesis of ADR-induced cardiomyopathy may involve free radicals and lipid peroxidation, the antioxidant, melatonin (MEL) may protect against toxic effects of ADR. We therefore tested this hypothesis using a rat model of ADR-induced cardiomyopathy. Sprague-Dawley rats were given ADR (cumulative dose, 15 mg/kg), MEL (cumulative dose, 84 mg/kg), ADR+MEL, ADR plus probucol (PRB, cumulative dose, 90 mg/kg), or vehicle alone, according to known regimens. The rats were maintained for 3 weeks following treatment, after which their cardiac performance was measured. Following sacrifice, their myocardial ultrastructure was examined, and their myocardial lipid peroxidation was assessed. Mortality was observed only in rats treated with ADR alone. When compared to control rats, surviving rats in the ADR group showed significant decreases in ratio of heart to body weight, arterial pressure, and left ventricular fractional shortening as well as a significant accumulation of ascites. The amount of myocardial thiobarbituric acid reactive substances was significantly higher in ADR-treated than in control rats. Both antioxidants, MEL and PRB, significantly prevented these ADR-induced changes. Electron microscopic examination revealed myocardial lesions indicative of ADR-induced cardiomyopathy in the ADR-treated rats. In contrast, treatment of these rats with MEL or PRB preserved myocardial ultrastructure. By preventing lipid peroxidation, MEL may be highly effective in protecting against ADR-induced cardiomyopathy.

Oxidation of LDL plays all important role in atherogenesis. The lag-time in the formation of conjugated dienes provides a sensitive measure of the resistance of plasma LDL to oxidation and is widely assumed to be an indicator of atherogenic risk. To test this assumption, we investigated whether different antioxidants yielding similar lag-times result in similar reduction of atherosclerosis. A 6-months intervention study was carried out in three groups of 10 LDL receptor-deficient rabbits each. Because previous studies indicated that antioxidants that reduce atherosclerosis resulted in very long lag-times, the first group was treated with an antioxidant combination containing 1,000 IU vitamin E, 0.05% probucol analogue (BM15.0639), and 0.025% probucol. The lag-times achieved throughout the intervention period by this combination (952 +/- 39 min) were matched in a second group (829 +/- 46 min) treated with a variable dose of probucol (0.0575-0.11%, average 0.091%). A third, untreated group served as a control. Plasma cholesterol levels of all groups were matched. Even though both treatments yielded similar antioxidant protection of plasma LDL, 0.091% probucol reduced aortic atherosclerosis by 51.7% compared to the untreated group (P < 0.005), whereas the antioxidant combination failed to reduce lesion formation. Thus, the lag-time is clearly not correlated with the antiatherogenic efficacy of different antioxidants. However, a weak correlation was found within the group treated with probucol only. Our results suggest that the degree of antioxidant protection of plasma LDL may not be a good indicator of the atherogenic risk, in general, and that probucol reduces atherogenesis by mechanisms not shared by all antioxidants.