Ascorbic acid enhances low-density lipoprotein receptor expression by suppressing proprotein convertase subtilisin/kexin 9 expression.

Ascorbic acid, a water-soluble antioxidant, regulates various biological processes and is thought to influence cholesterol. However, little is known about the mechanisms underpinning ascorbic acid-mediated cholesterol metabolism. Here, we determined if ascorbic acid can regulate expression of proprotein convertase subtilisin/kexin 9 (PCSK9), which binds low-density lipoprotein receptor (LDLR) leading to its intracellular degradation, to influence low-density lipoprotein (LDL) metabolism. At cellular levels, ascorbic acid inhibited PCSK9 expression in HepG2 and Huh7 cell lines. Consequently, LDLR expression and cellular LDL uptake were enhanced. Similar effects of ascorbic acid on PCSK9 and LDLR expression were observed in mouse primary hepatocytes. Mechanistically, ascorbic acid suppressed PCSK9 expression in a forkhead box O3-dependent manner. In addition, ascorbic acid increased LDLR transcription by regulating sterol regulatory element-binding protein 2. In vivo, administration of ascorbic acid reduced serum PCSK9 levels and enhanced liver LDLR expression in C57BL/6J mice. Reciprocally, lack of ascorbic acid supplementation in L-gulono-γ-lactone oxidase deficient (Gulo-/-) mice increased circulating PCSK9 and LDL levels, and decreased liver LDLR expression, whereas ascorbic acid supplementation decreased PCSK9 and increased LDLR expression, ameliorating LDL levels in Gulo-/- mice fed a high fat diet. Moreover, ascorbic acid levels were negatively correlated to PCSK9, total and LDL levels in human serum samples. Taken together, these findings suggest that ascorbic acid reduces PCSK9 expression, leading to increased LDLR expression and cellular LDL uptake. Thus, supplementation of ascorbic acid may ameliorate lipid profiles in ascorbic acid-deficient species.

Function and Immunogenicity of Gene-corrected iPSC-derived Hepatocyte-Like Cells in Restoring Low Density Lipoprotein Uptake in Homozygous Familial Hypercholesterolemia.

Gene correction of induced pluripotent stem cells (iPSCs) has therapeutic potential for treating homozygous familial hypercholesterolemia (HoFH) associated with low-density lipoprotein (LDL) receptor (LDLR) dysfunction. However, few data exist regarding the functional recovery and immunogenicity of LDLR gene-corrected iPSC-derived hepatocyte-like cells (HLCs) obtained from an HoFH patient. Therefore, we generated iPSC-derived HLCs from an HoFH patient harbouring a point mutation (NM_000527.4:c.901 G > T) in exon 6 of LDLR, and examined their function and immunogenicity. From the patient's iPSCs, one homozygous gene-corrected HoFH-iPSC clone and two heterozygous clones were generated using the CRISPR/Cas9 method. Both types of iPSC-derived HLCs showed recovery of the function of LDL uptake in immunofluorescence staining analysis. Furthermore, these gene-corrected iPSC-derived HLCs showed little immunogenicity against the patient's peripheral blood mononuclear cells in a cell-mediated cytotoxicity assay. These results demonstrate that LDL uptake of iPSC-derived HLCs from HoFH can be restored by gene correction without the appearance of further immunogenicity, suggesting that gene-corrected iPSC-derived HLCs are applicable to the treatment of HoFH.

Paeoniflorin attenuates oxidized low-density lipoprotein-induced apoptosis and adhesion molecule expression by autophagy enhancement in human umbilical vein endothelial cells.

Oxidized low-density lipoprotein (ox-LDL)-induced endothelial dysfunction is recognized as a driving force in the development of atherosclerosis (AS). Paeoniflorin (Pae), a typical traditional herbal medicine, possesses anti-inflammatory, antioxidative, antihyperglycaemic, and antiapoptotic properties. Our study aimed to investigate the effects of Pae on ox-LDL-induced injury of the human umbilical vein endothelial cells (HUVECs) and to explore its molecular mechanism. We found that ox-LDL stimulation inhibited cell viability, activated autophagy, and induced apoptosis and adhesion molecule expression in HUVECs. Pae rescued ox-LDL-induced viability reduction and enhanced the ox-LDL-induced autophagy activation in HUVECs. Pae inhibited ox-LDL-induced apoptosis and adhesion molecule expression by autophagy enhancement in HUVECs. In addition, inhibition of SIRT1 by EX-527 abolished the promoting effect of Pae on autophagy and restored the inhibitory effect of Pae on apoptosis and adhesion molecule expression in the presence of ox-LDL. In conclusion, Pae attenuated ox-LDL-induced apoptosis and adhesion molecule expression by autophagy enhancement via upregulation of SIRT1 in HUVECs, shedding light on the mechanism underlying the protective effect of Pae on ox-LDL-induced injury of HUVECs.

Lactoferrin-tagged quantum dots-based theranostic nanocapsules for combined COX-2 inhibitor/herbal therapy of breast cancer.

AIM: Herein, tumor-targeted quantum dots (QDs)-based theranostic nanocapsules (NCs) coloaded with celecoxib and honokiol were developed. Materials & methodology: The anionic CD44-targeting chondroitin sulfate and cationic low density lipoprotein (LDL)-targeting lactoferrin (LF) were sequentially assembled onto the surface of the positively charged oily core. As an imaging probe, highly fluorescent mercaptopropionic acid-capped cadmium telluride QDs were coupled to LF. RESULTS: In vitro, fluorescence of QDs was quenched (OFF state) due to combined electron/energy transfer-mediated processes involving LF. After intracellular uptake of NCs, fluorescence was restored (ON state), thus enabled tracing their internalization. The NCs demonstrated enhanced cytotoxicity against breast cancer cells as well as superior in vivo antitumor efficacy. CONCLUSION: We propose these multifunctional nanotheranostics for imaging and targeted therapy of breast cancer.

Folic acid delays development of atherosclerosis in low-density lipoprotein receptor-deficient mice.

Many studies support the cardioprotective effects of folic acid (FA). We aimed to evaluate the utility of FA supplementation in preventing the development of atherosclerotic in low-density lipoprotein receptor-deficient (LDLR-/-) mice and to elucidate the molecular processes underlying this effect. LDLR-/- mice were randomly distributed into four groups: control group, HF group, HF + FA group and the HF + RAPA group. vascular smooth muscle cells (VSMCs) were divided into the following four groups: control group, PDGF group, PDGF + FA group and PDGF + FA + RAPA group. Blood lipid levels, oxidative stress and inflammatory cytokines were measured. Atherosclerosis severity was evaluated with oil red O staining. Haematoxylin and eosin (H&E) staining was used to assess atherosclerosis progression. Immunohistochemical staining was performed with antismooth muscle α-actin (α-SMA) antibodies and anti-osteopontin (OPN) antibodies that demonstrate VSMC dedifferentiation. The protein expression of α-SMA, OPN and mechanistic target of rapamycin (mTOR)/p70S6K signalling was detected by Western blot analysis. FA and rapamycin reduced serum levels of total cholesterol, triacylglycerol, LDL, inhibiting oxidative stress and the inflammatory response. Oil red O and H&E staining demonstrated that FA and rapamycin inhibited atherosclerosis. FA and rapamycin treatment inhibited VSMC dedifferentiation in vitro and in vivo, and FA and rapamycin attenuated the mTOR/p70S6K signalling pathway. Our findings suggest that FA attenuates atherosclerosis development and inhibits VSMC dedifferentiation in high-fat-fed LDLR-/- mice by reduced lipid levels and inhibiting oxidative stress and the inflammatory response through mTOR/p70S6K signalling pathway.

Naoxintong Retards Atherosclerosis by Inhibiting Foam Cell Formation Through Activating Pparα Pathway.

BACKGROUNDS: We recently reported that Naoxintong (NXT), a China Food and Drug Administration (FDA)-approved cardiac medicine, could reduce the plaque size, but the underlying mechanism remains elusive now. OBJECTIVE: In this study, we investigated the effects of NXT on foam cell accumulation both in vivo and in vitro and explored related mechanisms. METHOD: THP-1 cells and bone marrow-derived macrophages were incubated with oxidized low-density lipoprotein (ox-LDL) with/without Naoxintong. ApoE-/- mice fed an atherogenic diet were administered to receive NXT for eight weeks. Macrophage-derived foam cell formation in plaques was measured by immunohistochemical staining. Expression of proteins was evaluated by Western blot. Lentivirus was used to knockdown PPARα in THP-1 cells. RESULTS: After NXT treatment, foam cell accumulation was significantly reduced in atherosclerotic plaques. Further investigation revealed that oxidized low-density lipoprotein (ox-LDL) uptake was significantly decreased and expression of scavenger receptor class A (SR-A) and class B (SR-B and CD36) was significantly downregulated post-NXT treatment. On the other hand, NXT increased cholesterol efflux and upregulated ATP-binding cassette (ABC) transporters (ABCA-1 and ABCG-1) in macrophages. Above beneficial effects of NXT were partly abolished after lentiviral knockdown of PPARα. CONCLUSION: Our findings suggest that NXT could retard atherosclerosis by inhibiting foam cell formation through reducing ox-LDL uptake and enhancing cholesterol efflux and above beneficial effects are partly mediated through PPARα pathway.

ox-LDL increases microRNA-29a transcription through upregulating YY1 and STAT1 in macrophages.

Macrophages and oxidized low-density lipoprotein (ox-LDL) have been verified playing vital roles in the pathogenesis of atherosclerosis (AS). Previous studies demonstrated that microRNA-29a (miR-29a) was upregulated in many atherogenic process and cells, thus acting as an important participant in AS. But the detailed regulation mechanism of miR-29a in AS has not been fully understood. In our study, we demonstrated a positive feedback loop of ox-LDL-SRA-miR-29a. Furthermore, we found that YY1 and STAT1 were upregulated in ox-LDL-stimulating macrophages followed by translocation in the nucleus and binding to the transcriptional promoter region of miR-29a, thus leading to the increase of miR-29a expression. In addition, we demonstrated that JAK1/2 signaling was involved in miR-29a upregulation. Finally, we found that miR-29a played important roles in the secretion of proinflammation factors and lipid uptake in macrophages. We uncovered the molecular mechanism and provide novel insights into the function and regulatory network of miR-29a expression regulated by ox-LDL in macrophages.

The effects of omega-3 fatty acids and vitamin E co-supplementation on gene expression of lipoprotein(a) and oxidized low-density lipoprotein, lipid profiles and biomarkers of oxidative stress in patients with polycystic ovary syndrome.

This study was conducted to determine the effects of omega-3 fatty acids and vitamin E co-supplementation on gene expression of lipoprotein(a) (Lp[a]) and oxidized low-density lipoprotein (Ox-LDL), lipid profiles and biomarkers of oxidative stress in women with polycystic ovary syndrome (PCOS). This randomized double-blind, placebo-controlled trial was done on 68 women diagnosed with PCOS according to the Rotterdam criteria aged 18-40 years old. Participants were randomly assigned into two groups to receive either 1000 mg omega-3 fatty acids from flaxseed oil containing 400 mg α-Linolenic acid plus 400 IU vitamin E supplements (n = 34) or placebo (n = 34) for 12 weeks. Lp(a) and Ox-LDL mRNA levels were quantified in peripheral blood mononuclear cells of PCOS women with RT-PCR method. Lipid profiles and biomarkers of oxidative stress were quantified at the beginning of the study and after 12-week intervention. Quantitative results of RT-PCR demonstrated that compared with the placebo, omega-3 fatty acids and vitamin E co-supplementation downregulated expressed levels of Lp(a) mRNA (P < 0.001) and Ox-LDL mRNA (P < 0.001) in peripheral blood mononuclear cells of women with PCOS. In addition, compared to the placebo group, omega-3 fatty acids and vitamin E co-supplementation resulted in a significant decrease in serum triglycerides (-22.1 ± 22.3 vs. +7.7 ± 23.6 mg/dL, P < 0.001), VLDL- (-4.4 ± 4.5 vs. +1.5 ± 4.7 mg/dL, P < 0.001), total- (-20.3 ± 16.6 vs. +12.2 ± 26.1 mg/dL, P < 0.001), LDL- (-16.7 ± 15.3 vs. +11.9 ± 26.1 mg/dL, P < 0.001) and total-/HDL-cholesterol (-0.5 ± 0.6 vs. +0.4 ± 0.8, P < 0.001). There were a significant increase in plasma total antioxidant capacity (+89.4 ± 108.9 vs. +5.9 ± 116.2 mmol/L, P = 0.003) and a significant decrease in malondialdehyde levels (-0.3 ± 0.4 vs. -0.008 ± 0.6 µmol/L, P = 0.01) by combined omega-3 fatty acids and vitamin E intake compared with the placebo group. Overall, omega-3 fatty acids and vitamin E co-supplementation for 12 weeks in PCOS women significantly improved gene expression of Lp(a) and Ox-LDL, lipid profiles and biomarkers of oxidative stress.

Accumulation of cholesterol and increased demand for zinc in serum-deprived RPE cells.

PURPOSE: Having observed that confluent ARPE-19 cells (derived from human RPE) survive well in high-glucose serum-free medium (SFM) without further feeding for several days, we investigated the expression profile of RPE cells under the same conditions. METHODS: Expression profiles were examined with microarray and quantitative PCR (qPCR) analyses, followed by western blot analysis of key regulated proteins. The effects of low-density lipoprotein (LDL) and zinc supplementation were examined with qPCR. Immunofluorescence was used to localize the LDL receptor and to examine LDL uptake. Cellular cholesterol levels were measured with filipin binding. Expression patterns in primary fetal RPE cells were compared using qPCR. RESULTS: Microarray analyses of gene expression in ARPE-19, confirmed with qPCR, showed upregulation of lipid and cholesterol biosynthesis pathways in SFM. At the protein level, the cholesterol synthesis control factor SRBEF2 was activated, and other key lipid synthesis proteins increased. Supplementation of SFM with LDL reversed the upregulation of lipid and cholesterol synthesis genes, but not of cholesterol transport genes. The LDL receptor relocated to the plasma membrane, and LDL uptake was activated by day 5-7 in SFM, suggesting increased demand for cholesterol. Confluent ARPE-19 cells in SFM accumulated intracellular cholesterol, compared with cells supplemented with serum, over 7 days. Over the same time course in SFM, the expression of metallothioneins decreased while the major zinc transporter was upregulated, consistent with a parallel increase in demand for zinc. Supplementation with zinc reversed expression changes for metallothionein genes, but not for other zinc-related genes. Similar patterns of regulation were also seen in primary fetal human RPE cells in SFM. CONCLUSIONS: ARPE-19 cells respond to serum deprivation and starvation with upregulation of the lipid and cholesterol pathways, accumulation of intracellular cholesterol, and increased demand for zinc. Similar trends are seen in primary fetal RPE cells. Cholesterol accumulation basal to RPE is a prominent feature of age-related macular degeneration (AMD), while dietary zinc is protective. It is conceivable that accumulating defects in Bruch's membrane and dysfunction of the choriocapillaris could impede transport between RPE and vasculature in AMD. Thus, this pattern of response to serum deprivation in RPE-derived cells may have relevance for some aspects of the progression of AMD.

[Effects of electroacupuncture on the expression of oxidized low-density lipoprotein and its receptors in rats with coronary atherosclerotic heart disease].

OBJECTIVE: To explore action mechanism of electroacupuncture for coronary atherosclerotic heart disease (CHD) in order to provide experimental support for clinical acupoint selection. METHODS: Among sixty clean-grade healthy male Wistar rats, twenty-four cases were randomly selected as a normal control group and an electroacupuncture (EA) preconditioning group, 12 cases in each one. Then rats in the EA preconditioning group and the rest 36 rats were fed with high fat diet for 12 weeks to duplicate the CHD model. When the models were successfully established, the rats were randomly divided into a model control group, an EA group and a medication group, 12 cases in each one. EA was applied with Hwa-to SDZ-IV apparatus in the EA preconditioning group at "Neiguan" (PC 6) and "Xinshu" (BL 15), 1 mA in current intensity, 2 Hz in frequency, 30 min per times, once every other day for 14 weeks. When model was established, the same acupoint and method was used in the EA group for 2 weeks while intragastric administration of atorvastatin mixed suspension, 0.25 mg/kg, once a day, was applied in the medication group for 2 weeks. The content of oxidized low-density lipoprotein (oxLDL) in the serum was tested by double antibody enzyme-linked immunosorbent assay (ELISA) while content of lectin-like oxidized low density lipoprotein receptor-1 (LOX-1) in coronary arterial tissue was test by western blot method. Expression of LOX-1 mRNA was tested by fluorogenic quantitative polymerase chain reaction (PCR). RESULTS: After model was duplicated successfully, the content of oxLDL in the serum and the expression of LOX-1 and its mRNA in coronary arterial tissue in the model control group were increased significantly compared with those in the normal control group (all P < 0.01). Compared with the model control group, the content of oxLDL in the serum and the expression of LOX-1 and its mRNA in coronary arterial tissue in the EA preconditioning group, EA group and medication group were significantly reduced (all P < 0.01). CONCLUSION: The electroacupuncture at "Neiguan" (PC 6) and "Xinshu" (BL 15) could effectively reduce the content of oxLDL in the serum and expression of LOX-1 and its mRAN in coronary arterial tissue in CHD rats. The oxidative modificatory low-density lipoprotein and its specific receptor system could be one of the ways to prevention and treatment of acupuncture for CHD.

Milk-derived peptides, Val-Pro-Pro and Ile-Pro-Pro, attenuate atherosclerosis development in apolipoprotein e-deficient mice: a preliminary study.

Milk-derived peptides, Val-Pro-Pro (VPP) and Ile-Pro-Pro (IPP), have angiotensin I-converting enzyme inhibitory activities and blood pressure-lowering effects. We examined the effects of these peptides on the development of atherosclerosis in apolipoprotein E-deficient [apoE(-/-)] mice. For 31 weeks, six-week-old male apoE(-/-) mice received a diet that included one of the following: fermented milk containing both VPP and IPP; casein hydrolysate containing both of these peptides; synthesized VPP; synthesized IPP; enalapril; captopril; or control diet. At the end of feeding, blood biochemistry, aortic atherogenesis, and gene expression by DNA microarray analysis were evaluated. There were no significant changes in the plasma lipid levels and 8-isoprostane, a marker of oxidative stress. The area ratio of intima to media in the aortic arch was significantly lower in the fermented milk, casein hydrolysate, synthesized VPP, enalapril, and captopril groups than in the control group. As is common with diets containing VPP and/or IPP, we observed reductions in mRNA expression of inflammatory cytokines, such as interleukin (IL)-6 and IL-1ß, oxidized low-density lipoprotein receptor, and transcription regulators. These results suggest that a continuous intake of VPP and IPP might be beneficial for preventing atherosclerosis caused by hypercholesterolemia.

Nigella sativa thymoquinone-rich fraction greatly improves plasma antioxidant capacity and expression of antioxidant genes in hypercholesterolemic rats.

The antioxidant activities of the thymoquinone-rich fraction (TQRF) extracted from Nigella sativa and its bioactive compound, thymoquinone (TQ), in rats with induced hypercholesterolemia were investigated. Rats were fed a semipurified diet supplemented with 1% (w/w) cholesterol and were treated with TQRF and TQ at dosages ranging from 0.5 to 1.5 g/kg and 20 to 100 mg/kg body wt, respectively, for 8 weeks. The hydroxyl radical (OH(.))-scavenging activity of plasma samples collected from experimental rats was measured by electron spin resonance. The GenomeLab Genetic Analysis System was used to study the molecular mechanism that mediates the antioxidative properties of TQRF and TQ. Plasma total cholesterol and low-density-lipoprotein cholesterol levels were significantly decreased in the TQRF- and TQ-treated rats compared to untreated rats. Feeding rats a 1% cholesterol diet for 8 weeks resulted in a significant decrease in plasma antioxidant capacity, as measured by the capacity to scavenge hydroxyl radicals. However, rats treated with TQRF and TQ at various doses showed significant inhibitory activity toward the formation of OH(.) compared to untreated rats. Upon examination of liver RNA expression levels, treatment with TQRF and TQ caused the up-regulation of the superoxide dismutase 1 (SOD1), catalase, and glutathione peroxidase 2 (GPX) genes compared to untreated rats (P<0.05). In support of this, liver antioxidant enzyme levels, including SOD1 and GPX, were also apparently increased in the TQRF- and TQ-treated rats compared to untreated rats (P<0.05). In conclusion, TQRF and TQ effectively improved the plasma and liver antioxidant capacity and enhanced the expression of liver antioxidant genes of hypercholesterolemic rats.

Effect of Terplex/VEGF-165 gene therapy on left ventricular function and structure following myocardial infarction. VEGF gene therapy for myocardial infarction.

BACKGROUND: We used a novel lipopolymeric gene delivery system, TeplexDNA, to transfect myocardium with plasmid vascular endothelial growth factor-165 (pVEGF) and evaluated the ability of pVEGF to preserve left ventricular function and structure after coronary ligation in a rabbit model. METHODS: New Zealand white rabbits underwent circumflex coronary ligation after direct intramyocardial injection of either Terplex alone or Terplex + 50 microg pVEGF-165. Serial echocardiography and histologic studies were performed (n = 12/group). Mortality did not differ between groups. The data is reported as the mean +/- standard deviation. RESULTS: Over the 21 days following coronary ligation, pVEGF-165-treated animals demonstrated significant improvement in fractional shortening (20-25%, p = 0.02), long axis two-dimensional ejection fraction (42-51%, p=0.02) and short axis m-mode ejection fraction (46-54%, p = 0.02). No significant improvements were noted in the control group. VEGF-treated animals had a 50% increase in peri-infarct vessel density and a trend towards a smaller infarct size (20% vs. 29%, p = 0.10). In animals receiving pVEGF-165, the diastolic ventricular area increased from 1.87 +/- 0.24 cm2 prior to ligation to 2.19 +/- 0.23 cm2 at 21 days following ligation, compared to an increase from 1.84 +/- 0.38 to 2.54 +/- 0.55 cm2 over the same period in control animals (p = 0.03). Similarly, the systolic ventricular area in VEGF-165 animals increased from 1.06 +/- 0.26 cm2 prior to ligation to 1.50 +/- 0.29 cm2 at 21 days following ligation, compared to an increase from 1.16 +/- 0.30 to 1.86 +/- 0.43 cm2 over the same period in the control animals (p = 0.04). CONCLUSION: TerplexDNA mediated delivery of plasmid VEGF administered at the time of coronary occlusion improves left ventricular function and reduces left ventricular dilation following myocardial infarction.

The effect of supplementation with an antioxidant preparation on LDL-oxidation is determined by haptoglobin polymorphism.

The genetic polymorphism of haptoglobin (Hp) is an independent risk factor in the pathogenesis of atherosclerosis, a condition in which decreased resistance to in vitro oxidation of LDL-cholesterol is observed. We hypothesised that the Hp polymorphism is one of the factors modulating the resistance to Cu(2+)-induced oxidation of LDL during antioxidant supplementation. In this study, 74 middle-aged subjects with increased oxidative stress were allocated to either matched placebo or oral antioxidative treatment (Quatral) once daily for 16 weeks. Study parameters were increase of lag phase (DeltaLAG) and the ratio of lag phase during treatment period versus baseline (relative oxidation resistance, ROR), measured by Cu(2+)-induced oxidation of isolated LDL. Hp phenotypes were determined by starch gel electrophoresis. A significant and persistent increase of DeltaLAG (P < 0.05) and ROR (P < 0.01) were observed after 16 weeks of active treatment versus placebo. Interindividual differences in both parameters were significantly associated with the Hp polymorphism: in the active treatment group, DeltaLAG and ROR were significantly higher in Hp 1-1 subjects (P < 0.01) compared to Hp 2-1 and 2-2. Our data demonstrate that Hp phenotype is one of the modulating factors determining the increased resistance to Cu(2+)-induced oxidation of LDL during antioxidative treatment.