Myristicin regulates proliferation and apoptosis in oxidized low-density lipoprotein-stimulated human vascular smooth muscle cells and human umbilical vein endothelial cells by regulating the PI3K/Akt/NF-κB signalling pathway.

CONTEXT: Atherosclerosis (AS) is a chronic inflammatory disease. Human vascular smooth muscle cell (hVSMC) accumulation and human umbilical vein endothelial cell (HUVEC) dysfunction are associated with the pathogenesis of AS. This study explores whether myristicin plays a protective role in AS. MATERIALS AND METHODS: hVSMCs and HUVECs were stimulated with 100 µg/mL oxidized low-density lipoprotein (ox-LDL) to establish a cellular model of AS. Cell viability, lactate dehydrogenase (LDH) release and cell apoptosis were evaluated using MTT, LDH and flow cytometry assays, respectively. Cell migration and inflammatory cytokine release were assessed using Transwell assay and ELISA. RESULTS: Myristicin (5, 10, 25, and 50 µM) had no obvious effect on cell viability or the activity of LDH in hVSMCs, while 100 and 200 µM myristicin markedly suppressed hVSMCs viability and increased LDH release. Myristicin had no obvious effect on cell viability or the activity of LDH in HUVECs. Myristicin inhibited viability and increased apoptosis in ox-LDL-treated hVSMCs, but was associated with increased proliferation and inhibited apoptosis in HUVECs stimulated by ox-LDL. Additionally, myristicin markedly suppressed ox-LDL-induced hVSMCs migration and the release of inflammatory cytokines, including MCP-1, IL-6, VCAM-1 and ICAM-1, in HUVECs. Results also demonstrated that the promoting effects of ox-LDL on the PI3K/Akt and NF-κB signalling pathway in both hVSMCs and HUVECs were abolished by treatment with myristicin. DISCUSSION AND CONCLUSIONS: Myristicin regulated proliferation and apoptosis by regulating the PI3K/Akt/NF-κB signalling pathway in ox-LDL-stimulated hVSMCs and HUVECs. Thus, myristicin may be used as a new potential drug for AS treatment.

Kaempferol-induced GPER upregulation attenuates atherosclerosis via the PI3K/AKT/Nrf2 pathway.

CONTEXT: The effect of kaempferol, a regulator of oestrogen receptors, on atherosclerosis (AS) and the underlying mechanism is elusive. OBJECTIVE: To explore the effect and mechanism of kaempferol on AS. METHODS AND MATERIALS: In vivo, C57BL/6 and apolipoprotein E (APOE)-/- mice were randomly categorized into six groups (C57BL/6: control, ovariectomy (OVX), high-fat diet (HFD); APOE-/-: OVX-HFD, OVX-HFD + kaempferol (50 mg/kg) and OVX-HFD + kaempferol (100 mg/kg) and administered with kaempferol for 16 weeks, intragastrically. Oil-Red and haematoxylin-eosin (HE) staining were employed to examine the effect of kaempferol. In vitro, human aortic endothelial cells (HAECs) were pre-treated with or without kaempferol (5, 10 or 20 µM), followed by administration with kaempferol and oxidized low-density lipoprotein (ox-LDL) (200 µg/mL). The effect of kaempferol was evaluated using flow cytometry, and TdT-mediated dUTP Nick-End Labelling (TUNEL). RESULTS: In vivo, kaempferol (50 and 100 mg/kg) normalized the morphology of blood vessels and lipid levels and suppressed inflammation and apoptosis. It also activated the G protein-coupled oestrogen receptor (GPER) and PI3K/AKT/nuclear factor-erythroid 2-related factor 2 (Nrf2) pathways. In vitro, ox-LDL (200 µg/mL) reduced the cell viability to 50% (IC50). Kaempferol (5, 10 or 20 µM) induced-GPER activation increased cell viability to nearly 10%, 19.8%, 30%, and the decreased cellular reactive oxygen species (ROS) generation (16.7%, 25.6%, 31.1%), respectively, consequently attenuating postmenopausal AS. However, the protective effects of kaempferol were blocked through co-treatment with si-GPER. CONCLUSIONS: The beneficial effects of kaempferol against postmenopausal AS are associated with the PI3K/AKT/Nrf2 pathways, mediated by the activation of GPER.

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.

Low-Density Lipoprotein-Reactive T Cells Regulate Plasma Cholesterol Levels and Development of Atherosclerosis in Humanized Hypercholesterolemic Mice.

BACKGROUND: Atherosclerotic cardiovascular disease is a chronic inflammatory process initiated when cholesterol-carrying low-density lipoprotein (LDL) is retained in the arterial wall. CD4+ T cells, some of which recognize peptide components of LDL as antigen, are recruited to the forming lesion, resulting in T-cell activation. Although these T cells are thought to be proatherogenic, LDL immunization reduces disease in experimental animals. These seemingly contradictory findings have hampered the development of immune-based cardiovascular therapy. The present study was designed to clarify how activation of LDL-reactive T cells impacts on metabolism and vascular pathobiology. METHODS: We have developed a T-cell receptor-transgenic mouse model to characterize the effects of immune reactions against LDL. Through adoptive cell transfers and cross-breeding to hypercholesterolemic mice expressing the antigenic human LDL protein apolipoprotein B-100, we evaluate the effects on atherosclerosis. RESULTS: A subpopulation of LDL-reactive T cells survived clonal selection in the thymus, developed into T follicular helper cells in lymphoid tissues on antigen recognition, and promoted B-cell activation. This led to production of anti-LDL immunoglobulin G antibodies that enhanced LDL clearance through immune complex formation. Furthermore, the cellular immune response to LDL was associated with increased cholesterol excretion in feces and with reduced vascular inflammation. CONCLUSIONS: These data show that anti-LDL immunoreactivity evokes 3 atheroprotective mechanisms: antibody-dependent LDL clearance, increased cholesterol excretion, and reduced vascular inflammation.

Dietary advice to cardiovascular patients. A brief update for physicians.

It is important, in our opinion, to provide physicians with a brief update of scientifically-sound evidence in preventive nutrition, to be employed in their everyday practice, since the latest scientific and clinical advances in this area are generally not well known. Here, we review the most recent evidence in support of an optimal cardio-protective diet, and we identify the need to focus mainly on protective food which should be part of such diet, rather than on nutrients with negative effects to be limited (salt, saturated fats, simple sugars). We conclude that, to favor patient compliance, it is also necessary to underscore indications on the topics for which there is convincing and coherent literature, leaving other less-explored aspects to individual preferences.

MicroRNA-126 alleviates endothelial cells injury in atherosclerosis by restoring autophagic flux via inhibiting of PI3K/Akt/mTOR pathway.

MicroRNAs (miRNAs) have emerged as critical modulators of ECs function and play a vital role in the development of cardiovascular disease. Among them, miR-126 is a crucial regulator of atherosclerosis. Endothelial cells (ECs) death and autophagy have been described in cells to cope with the progression of atherosclerosis. Hence, the aim of this study is to investigate the effects of miR-126 on atherosclerosis in oxidized low-density lipoprotein (ox-LDL)-stimulated human umbilical vein endothelial cells (HUVECs) and the potential roles of autophagy flux in these processes. Our results showed that miR-126 level was significantly reduced in ox-LDL-treated HUVECs and miR-126 overexpression induced by miR-126 mimics remarkably blocked ox-LDL-induced HUVECs injury as evidenced by the reduced cell viability, and the increased LDH release, caspase-3 activity and apoptosis ratio. In addition, ox-LDL increased LC3-II, Beclin 1, and p62 expressions in HUVECs, while these changes were nullified in the presence of treatment with bafilomycin A1 (BafA1, an inhibit autophagic flux inhibitor). However, we found that ox-LDL-induced impaired autophagy flux was recused by miR-126 mimics. Subsequently, we found that Bafi A1 pretreatment reversed the protection of miR-126 mimics against ox-LDL-induced HUVECs injury. Finally, our results showed that miR-126 mimics rescued ox-LDL-induced impaired autophagy flux through inhibiting PI3K/Akt/mTOR signaling. Taken together, our findings suggested that miR-126 alleviates ox-LDL-induced HUVECs injury through restoring autophagy flux via repressing PI3K/Akt/mTOR pathway, and further implicate the potential therapeutic targets to reverse atherosclerosis.

FSD-C10 Shows Therapeutic Effects in Suppressing oxidized low-density lipoprotein (ox-LDL)-Induced Human Brain Microvascular Endothelial Cells Apoptosis via Rho-Associated Coiled-Coil Kinase (ROCK)/Mitogen-Activated Protein Kinase (MAPK) Signaling.

BACKGROUND ox-LDL-induced injury of brain microvascular endothelial cells (BMECs) is strongly associated with cerebral vascular diseases such as cerebral arterial atherosclerosis. ROCK inhibitor was proved to be anti-apoptotic and has been used in treating cerebral vascular diseases. Research on the neuroprotective effects of a novel ROCK inhibitor, FSD-C10, is still limited. The present study investigated the anti-apoptotic effect and underlying molecular mechanism of FSD-C10 in ox-LDL-mediated apoptosis of BMECs. MATERIAL AND METHODS ox-LDL and/or FSD-C10 were used to incubate immortalized human BMECs. MTT assay was used to assess cell viability. Cell apoptosis was evaluated by TUNEL assay. A colorimetric method was used to assess ROCK activity. Western blot analysis was used to examine the expression and phosphorylation levels of proteins. RESULTS ox-LDL incubation reduced the viability of BMECs by inducing cell apoptosis in a concentration-dependent manner. ROCK activity was also elevated by ox-LDL incubation in BMECs in a concentration-dependent manner. Expression level of Bcl2 was reduced while expression levels of Bax and active caspase3 were increased by ox-LDL treatment in a concentration-dependent manner. ox-LDL also increased the phosphorylation levels of p38, JNK, and ERK1/2 in a concentration-dependent manner. FSD-C10 treatment increased the cell viability by reducing apoptosis of BMECs exposed to ox-LDL. Moreover, FSD-C10 was found to suppress the phosphorylation levels of p38, JNK, and ERK1/2 and the expression levels of Bax and active caspase3 in ox-LDL treated BMECs. CONCLUSIONS FSD-C10 increases cell viability in ox-LDL-treated BMECs by reducing cell apoptosis. ROCK/MAPKs-mediated apoptosis appears to be the underlying molecular mechanism.

Ginkgolic acid exerts an anti-inflammatory effect in human umbilical vein endothelial cells induced by ox-LDL.

This present investigation examined the mitigating impact of Ginkgolic acid in the organization on oxidized low-density lipoproteinox-LDL (ox- LDL) animated in HUVECs, and to clear up its fundamental molecular components. The levels of nitric oxide (NO), prostaglandin E2 (PGE2), and pro-inflammatory cytokines were measured by Griess examine and catalyst connected immunosorbent test. The declarations of inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2), mitogen-initiated protein kinases (MAPKs), and Akt were measured utilizing Western smearing. ox-LDL-instigated was utilized as the HUVECs cell model of inflammation. Ginkgolic acid significantly inhibited the production of NO, PGE2, and pro-inflammatory cytokines in a dose-dependent manner and suppressed the expression of iNOS and COX-2 in ox-LDL-stimulated HUVECs cells. Ginkgolic acid strongly suppressed NF-κB by preventing degradation of inhibitor of κB-α as well as by inhibiting phosphorylation of Akt and MAPKs. Ginkgolic acid reduced LDL-stimulated inflammation in endothelial cells. These outcomes suggest that the anti-inflammatory properties of Ginkgolic acid are related to a down-control of iNOS, COX-2, and master provocative cytokines through the restraint of NF-κB pathway in ox- LDL-animated endothelial cells.

Hepatic Arterial Infusion of Low-Density Lipoprotein Docosahexaenoic Acid Nanoparticles Selectively Disrupts Redox Balance in Hepatoma Cells and Reduces Growth of Orthotopic Liver Tumors in Rats.

BACKGROUND & AIMS: Dietary intake of the natural omega-3 fatty acid docosahexaenoic acid (DHA) has been implicated in protecting patients with viral hepatitis B or C from developing hepatocellular carcinoma (HCC). Little is known about the effects of DHA on established solid tumors. Here we describe a low-density lipoprotein-based nanoparticle that acts as a transporter for unesterified DHA (LDL-DHA) and demonstrates selective cytotoxicity toward HCC cells. We investigated the ability of LDL-DHA to reduce growth of orthotopic hepatomas in rats. METHODS: AxC-Irish (ACI) rats were given intrahepatic injections of rat hepatoma cells (H4IIE); 24 tumor-bearing rats (mean tumor diameter, ∼1 cm) were subject to a single hepatic artery injection of LDL nanoparticles (2 mg/kg) loaded with DHA (LDL-DHA), triolein (LDL-TO), or sham surgery controls. Tumor growth was measured by magnetic resonance imaging and other methods; tumor, liver, and serum samples were collected and assessed by histochemical, immunofluorescence, biochemical, and immunoblot analyses. RESULTS: Three days after administration of LDL-TO or sham surgery, the control rats had large, highly vascularized tumors that contained proliferating cells. However, rats given LDL-DHA had smaller, pale tumors that were devoid of vascular supply and >80% of the tumor tissue was necrotic. Four to 6 days after injection of LDL-DHA, the tumors were 3-fold smaller than those of control rats. The liver tissue that surrounded the tumors showed no histologic or biochemical evidence of injury. Injection of LDL-DHA into the hepatic artery of rats selectively deregulated redox reactions in tumor tissues by increasing levels of reactive oxygen species and lipid peroxidation, depleting and oxidizing glutathione and nicotinamide adenine dinucleotide phosphate, and significantly down-regulating the antioxidant enzyme glutathione peroxidase-4. Remarkably, the redox balance in the surrounding liver was not disrupted. CONCLUSION: LDL-DHA nanoparticle selectively kills hepatoma cells and reduces growth of orthotopic liver tumors in rats. It induces tumor-specific necrosis by selectively disrupting redox balance within the cancer cell.

Synovial macrophages promote TGF-ß signaling and protect against influx of S100A8/S100A9-producing cells after intra-articular injections of oxidized low-density lipoproteins.

OBJECTIVE: Low-density lipoproteins (LDL) in inflamed synovium is oxidized and taken-up by synoviocytes. In this study, we investigate whether direct injection of oxidized LDL (oxLDL) into a normal murine knee joint induces joint pathology and whether synovial macrophages are involved in that process. DESIGN: Synovium was obtained from end-stage osteoarthritis (OA) patients in order to analyze LDL-uptake. Murine knee joints were injected five consecutive days with oxLDL, LDL, or vehicle (phosphate buffered saline (PBS)). This procedure was repeated in mice depleted of synovial macrophages by intra-articular injection of clodronate liposomes 7 days prior to the consecutive injections. Joint pathology was investigated by immunohistochemistry, flow cytometry (FCM) and synovial RNA expression and protein production. RESULTS: Synovial tissue of OA patients showed extensive accumulation of apolipoprotein B. Multiple injections of oxLDL in murine knee joints significantly increased TGF-ß activity in synovial wash-outs, but did not induce catabolic or inflammatory processes. In contrast, repeated injections of oxLDL in macrophage-depleted knee joints led to increased synovial thickening in combination with significantly upregulated protein and RNA levels of CCL2 and CCL3. FCM-analyses revealed increased presence of monocytes and neutrophils in the synovium, which was confirmed by immunohistochemistry. Also protein levels of S100A8/A9 were significantly increased in synovial wash-outs of oxLDL-injected joints, as was expression of aggrecanase-induced neo-epitopes. Interestingly, no raise in TGF-ß concentrations was measured in macrophage-depleted joints. CONCLUSIONS: OxLDL can affect joint pathology, since synovial macrophages promote anabolic processes after oxLDL injections. In absence of synovial macrophages, however, oxLDL induces production of pro-inflammatory mediators and aggrecanase activity combined with increased influx of monocytes and neutrophils.

Altered metabolism of LDL in the arterial wall precedes atherosclerosis regression.

RATIONALE: Plasma cholesterol lowering is beneficial in patients with atherosclerosis. However, it is unknown how it affects entry and degradation of low-density lipoprotein (LDL) particles in the lesioned arterial wall. OBJECTIVE: We studied the effect of lipid-lowering therapy on LDL permeability and degradation of LDL particles in atherosclerotic aortas of mice by measuring the accumulation of iodinated LDL particles in the arterial wall. METHODS AND RESULTS: Cholesterol-fed, LDL receptor-deficient mice were treated with either an anti-Apob antisense oligonucleotide or a mismatch control antisense oligonucleotide once a week for 1 or 4 weeks before injection with preparations of iodinated LDL particles. The anti-Apob antisense oligonucleotide reduced plasma cholesterol by ≈90%. The aortic LDL permeability and degradation rates of newly entered LDL particles were reduced by ≈50% and ≈85% already after 1 week of treatment despite an unchanged pool size of aortic iodinated LDL particles. In contrast, the size, foam cell content, and aortic pool size of iodinated LDL particles of aortic atherosclerotic plaques were not reduced until after 4 weeks of treatment with the anti-Apob antisense oligonucleotide. CONCLUSIONS: Improved endothelial barrier function toward the entry of plasma LDL particles and diminished aortic degradation of the newly entered LDL particles precede plaque regression.

Mst1 participates in the atherosclerosis progression through macrophage autophagy inhibition and macrophage apoptosis enhancement.

Emerging evidence favors the notion that macrophage autophagy plays a prominent role in the pathogenesis of vulnerable plaque, suggesting the therapeutic potential of targeting autophagy in atherosclerosis. Here ApoE(-/-) mice were crossed with Mst1 knockout or Mst1 Tg mice to generate ApoE(-/-):Mst1(-/-) and ApoE(-/-):Mst1Tg mice. All animals were fed high-fat-diet for 4months to induce arterial atherosclerosis. Murine macrophage RAW264.7 cells were subjected to ox-LDL (50µg/mL) in an effort to examine the cellular mechanisms. A significant increase in the levels of Mst1 and p-Mst1 was observed in the aorta of ApoE(-/-) mice. Mst1 knockout significantly reduced atherosclerotic area, decreased lipid core area and macrophage accumulation as compared with ApoE(-/-) mice. Along the same line, Mst1 overexpression increased plaque area, lipid core and macrophage accumulation as compared with ApoE(-/-) mice. Mst1 deficiency significantly increased levels of Beclin1 and LC3II, while decreased that of p62 in aortic atherosclerosis. Moreover, in vitro data indicated that Mst1 knockdown prompted more typical autophagosomes upon ox-LDL challenge. Mst1 knockdown also enhanced autophagic flux as evidenced by GFP-mRFP-LC3 staining, increased LC3-II expression and decreased p62 expression in the presence of bafilomycin A1. Mst1 knockdown decreased, while Mst1 overexpression increased macrophage apoptosis upon ox-LDL exposure. In conclusion, Mst1 deficiency diminishes atherosclerosis and stabilizes atherosclerotic plaques in ApoE(-/-) mice. Mst1 may participate in atherosclerosis progression through inhibition of macrophage autophagy and promotion of macrophage apoptosis.

The roles of p62/SQSTM1 on regulation of matrix metalloproteinase-9 gene expression in response to oxLDL in atherosclerosis.

OBJECTIVE: Matrix metalloproteinase-9 (MMP-9) plays an important role in the remodeling of the extracellular matrix in atherosclerosis plaques. Autophagy protects macrophages against the processes of vascular disease. Our research explores how autophagy plays roles in macrophages to secret MMP-9. METHODS AND RESULTS: In response to increased doses of oxLDL or CQ we monitored the autophagic flux. Our results revealed that oxLDL was dynamically associated with autophagy and 100 µg/ml oxLDL blocked autophagic flux in THP-1 cells. Moreover p62/SQSTM1 knocking down and CQ respectively inhibited and increased MMP-9 transcriptional expression. These effects were mediated by inhibition of NF-κB. CONCLUSION: Abundant oxLDL blocked autophagic flux resulting in the aggregation of p62/SQSTM1. Then p62/SQSTM1 was involved in gene expression of MMP-9 via NF-κB-dependent signaling, and thus featuring novel plaque vulnerability properties of the atherosclerotic plaque. Understanding the mechanism that selectively modulates p62/SQSTM1 will provide a novel strategy for anti-atherogenesis.

Lp-PLA2 silencing protects against ox-LDL-induced oxidative stress and cell apoptosis via Akt/mTOR signaling pathway in human THP1 macrophages.

Atherosclerosis is a disease of the large- and medium-size arteries that is characterized by the formation of atherosclerotic plaques, in which foam cells are the characteristic pathological cells. However, the key underlying pathomechanisms are still not fully elucidated. In this study, we investigated the role of lipoprotein-associated phospholipase A2 (Lp-PLA2) in ox-LDL-induced oxidative stress and cell apoptosis, and further, elucidated the potential machanisms in human THP1 macrophages. Flow cytometry and western blot analyses showed that both cell apoptosis and Lp-PLA2 expression were dose-dependently elevated after ox-LDL treatment for 24 h and also time-dependently increased after 50 mg/L ox-LDL incubation in THP1 macrophages. In addition, Lp-PLA2 silencing decreased ox-LDL-induced Lp-PLA2 and CD36 expression in THP1 macrophages. We also found that the levels of oil red O-staining, triglyceride (TG) and total cholesterol (TC) were significantly upregulated in ox-LDL-treated THP1 cells, but inhibited by Lp-PLA2 silencing. Furthermore, ox-LDL treatment resulted in significant increases of ROS and MDA but a marked decrease of SOD, effects that were reversed by Lp-PLA2 silencing in THP1 cells. Lp-PLA2 silencing reduced ox-LDL-induced cell apoptosis and caspase-3 expression in THP1 cells. Moreover, Lp-PLA2 siRNA transfection dramatically lowered the elevated levels of p-Akt and p-mTOR proteins in ox-LDL-treated THP1 cells. Both PI3K inhibitor LY294002 and mTOR inhibitor rapamycin decreased the augmented caspase-3 expression and TC content induced by ox-LDL, respectively. Taken together, these results revealed that Lp-PLA2 silencing protected against ox-LDL-induced oxidative stress and cell apoptosis via Akt/mTOR signaling pathway in human THP1 macrophages.

Cellular Cholesterol Accumulation Facilitates Ubiquitination and Lysosomal Degradation of Cell Surface-Resident ABCA1.

OBJECTIVE: By excreting cellular cholesterol to apolipoprotein A-I, ATP-binding cassette transporter A1 (ABCA1) mediates the biogenesis of high-density lipoprotein in hepatocytes and prevents foam cell formation from macrophages. We recently showed that cell surface-resident ABCA1 (csABCA1) undergoes ubiquitination and later lysosomal degradation through the endosomal sorting complex required for transport system. Herein, we investigated the relevance of this degradation pathway to the turnover of csABCA1 in hypercholesterolemia. APPROACH AND RESULTS: Immunoprecipitation and cell surface-biotinylation studies with HepG2 cells and mouse peritoneal macrophages showed that the ubiquitination level and degradation of csABCA1 were facilitated by treatment with a liver X receptor (LXR) agonist and acetylated low-density lipoprotein. The effects of an LXR agonist and acetylated low-density lipoprotein on the degradation of csABCA1 were repressed completely by treatment with bafilomycin, an inhibitor of lysosomal degradation, and by depletion of tumor susceptibility gene 101, a major component of endosomal sorting complex required for transport-I. RNAi analysis indicated that LXRß inhibited the accelerated lysosomal degradation of csABCA1 by the LXR agonist, regardless of its transcriptional activity. Cell surface coimmunoprecipitation with COS1 cells expressing extracellularly hemagglutinin-tagged ABCA1 showed that LXRß interacted with csABCA1 and inhibited the ubiquitination of csABCA1. Immunoprecipitates with anti-ABCA1 antibodies from the liver plasma membranes showed less LXRß and a higher ubiquitination level of ABCA1 in high-fat diet-fed mice than in normal chow-fed mice. CONCLUSIONS: Under conditions of high cellular cholesterol content, csABCA1 became susceptible to ubiquitination by dissociation of LXRß from csABCA1, which facilitated the lysosomal degradation of csABCA1 through the endosomal sorting complex required for transport system.

Overexpression of LOXIN Protects Endothelial Progenitor Cells From Apoptosis Induced by Oxidized Low Density Lipoprotein.

Human endothelial progenitor cells (hEPC) are adult stem cells located in the bone marrow and peripheral blood. Studies have indicated that hEPC play an important role in the recovery and repair of injured endothelium, however, their quantity and functional capacity is reduced in several diseases including hypercholesterolemia. Recently, it has been demonstrated that hEPC express lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1) and its activation by oxidized low-density lipoprotein (ox-LDL) induces cellular dysfunction and apoptosis. This study aimed to investigate whether overexpression of LOXIN, a truncated isoform of LOX-1 that acts as a dominant negative, plays a protective role against ox-LDL-induced apoptosis in hEPC. Human endothelial progenitor cells exposed to ox-LDL showed a significant increase in LOX-1 expression, and apoptosis began at ox-LDL concentrations above 50 µg/mL. All hEPC apoptosed at 200 µg/mL ox-LDL. High LOXIN expression was generated using adenoviral systems in hEPC and SiHa cells transduced with 100 colony-forming units per cell. Transduced LOXIN localized to the plasma membrane and blocked ox-LDL uptake mediated by LOX-1. Overexpression of LOXIN protected hEPC from ox-LDL-induced apoptosis, and therefore maybe a novel way of improving hEPC function and quantity. These results suggest that adenoviral vectors of LOXIN may provide a possible treatment for diseases related to ox-LDL and vascular endothelium dysfunction, including atherosclerosis.

Effects of Ethyl Pyruvate in Preventing the Development of Diet-induced Atherosclerosis by Blocking the HMGB1 Expression in ApoE-Deficient Mice.

Ethyl pyruvate (EP) is a lipid derivative of pyruvate and known as an anti-inflammatory agent effective to inhibit many diseases in experimental models. To test the hypothesis that Ethyl pyruvate might prevent atherosclerosis development by blocking the high-mobility group box-1 (HMGB1) expression, 8-week-old ApoE-deficient (ApoE-/-) mice were fed a high-fat diet and treated with EP (50 mg/Kg) or Physiological saline for 12 weeks. THP-1 cells were then differentiated into macrophages by treated with Phorbol-12-myristate-13-acetate (PMA, 100 ng/mL) and incubated with oxidized low-density lipoprotein (oxLDL) only or added with EP (5 mM) for 24 hour. In ApoE-/- mice, EP markedly reduced aortic sinus atherosclerosis lesions with no influence to serum lipid levels. Inhibited HMGB1 expression, decreased macrophages content and reduced Toll-like-receptor2 (TLR2), TLR4, monocyte chemotactic protein 1 (MCP-1) and Tumor Necrosis Factor α (TNF-α) mRNA levels were also observed at the same time. In vitro, EP decreased oxLDL uptake in macrophages and inhibited HMGB1 expression induced by oxLDL. In conclusion, our study indicated that EP was an effective agent against the development of diet-induced atherosclerosis in ApoE-deficient mice by way of blocking the HMGB1 expression.

Cytoprotective Effects of Oleanolic Acid in Human Umbilical Vascular Endothelial Cells is Mediated Via UCP2/ROS/Cytochrome C/AIF Pathway.

The aim of this study is to assess the potential protective effect of oleanolic acid (OA) against ox-LDL induced damage in human umbilical vascular endothelial cells (HUVECs) and investigate potential mechanism of action including antioxidative effects and inhibition of mitochondria apoptosis pathway. Cell counting kit 8 was used to evaluate the viability of HUVECs. 2', 7'-DCFH-DA staining and flow cytometry was used to assess the levels of intracellular reactive oxygen species in HUVECs. The protein expression levels of uncoupling protein 2, cytochrome C, and apoptosis induction factors were measured by western blotting. The results indicated that OA treatment alleviated ox-LDL induced cytotoxicity in HUVECs and ameliorated the reactive oxygen species levels. Western blotting results demonstrated that OA treatment increased the expression level of uncoupling protein 2 and decreased the release of cytochrome C and apoptosis induction factors from mitochondria to cytoplasm, suggesting inhibition of mitochondria apoptosis pathway. In conclusion, OA could protect HUVECs from ox-LDL-induced cytotoxicity; its antioxidant property and inhibition of mitochondria apoptosis are likely crucial contributors.

Impact of selective LDL apheresis on serum chemerin levels in patients with hypercholesterolemia.

BACKGROUND: Selective low-density lipoprotein (LDL) apheresis is commonly used to treat patients with familial hypercholesterolemia (FH). Chemerin is an adipokine with putative roles in the regulation of lipid metabolism. METHODS: In our pilot study, we measured serum chemerin levels by enzyme-linked immunosorbent assay in six severe heterozygous FH patients before and after their first LDL apheresis treatments using the technique of direct adsorption of lipoproteins (DALI). RESULTS: The first treatment sessions decreased serum chemerin levels by an average of 27.26 %. While following one patient, 12 months of regular LDL apheresis resulted in a permanent reduction in his serum chemerin level. Changes in the lipoprotein subfractions measured by gel electrophoresis (Lipoprint) correlated with the reduction of chemerin levels. Furthermore, we eluted and then measured chemerin bound to the DALI column. CONCLUSION: We conclude that LDL apheresis decreases the circulating level of chemerin by binding the protein to the column and thus improves lipoprotein subfraction pattern.

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.