Nanoparticle-based "Two-pronged" approach to regress atherosclerosis by simultaneous modulation of cholesterol influx and efflux.

Reduction of lipoprotein uptake by macrophages and stimulation of cholesterol efflux are two essential steps required for atherosclerotic plaque regression. We used the optimized mannose-functionalized dendrimeric nanoparticle (mDNP)-based platform for macrophage-specific delivery of therapeutics to simultaneously deliver SR-A siRNA (to reduce LDL uptake) and LXR ligand (LXR-L, to stimulate cholesterol efflux) - a novel "Two-pronged" approach to facilitate plaque regression. mDNP-mediated delivery of SR-A siRNA led to a significant reduction in SR-A expression with a corresponding decrease in uptake of oxLDL. Delivery of LXR-L increased expression of ABCA1/G1 and cholesterol efflux. Combined delivery of siRNA and LXR-L led to a significantly greater decrease in macrophage cholesterol content compared to either treatment alone. Administration of this in vitro optimized formulation of mDNP complexed with SR-A-siRNA and LXR-L (Two-pronged complex) to atherosclerotic LDLR-/- mice fed western diet (TD88137) led to significant regression of atherosclerotic plaques with a corresponding decrease in aortic cholesterol content.

Selective oxidative stress and cholesterol metabolism in lipid-metabolizing cell classes: Distinct regulatory roles for pro-oxidants and antioxidants.

Atherogenesis is associated with macrophage cholesterol and oxidized lipids accumulation and foam cell formation. However, two other major lipid-metabolizing cell classes, namely intestinal and liver cells, are also associated with atherogenesis. This study demonstrates that manipulations of cellular oxidative stress (by fatty acids, glucose, low-density lipoprotein, angiotensin II, polyphenolic antioxidants, or the glutathione/paraoxonase 1 systems) have some similar, but also some different effects on cholesterol metabolism in macrophages (J774A.1) versus intestinal cells (HT-29) versus liver cells (HuH7). Cellular oxidative stress was ≈3.5-folds higher in both intestinal and liver cells versus macrophages. In intestinal cells or liver cells versus macrophages, the cholesterol biosynthesis rate was increased by 9- or 15-fold, respectively. In both macrophages and intestinal cells C-18:1 and C-18:2 but not C-18:0, fatty acids significantly increased oxidative stress, whereas in liver cells oxidative stress was significantly decreased by all three fatty acids. In liver cells, trans C-18:1 versus cis C-18:1, unlike intestinal cells or macrophages, significantly increased cellular oxidative stress and cellular cholesterol biosynthesis rate. Pomegranate juice (PJ), red wine, or their phenolics gallic acids or quercetin significantly reduced cellular oxidation mostly in macrophages. Recombinant PON1 significantly decreased macrophage (but not the other cells) oxidative stress by ≈30%. We conclude that cellular atherogenesis research should look at atherogenicity, not only in macrophages but also in intestinal and liver cells, to advance our understanding of the complicated mechanisms behind atherogenesis. © 2015 BioFactors, 41(4):273-288, 2015.

Chrysin inhibits foam cell formation through promoting cholesterol efflux from RAW264.7 macrophages.

CONTEXT: Chrysin, a natural flavonoid, has been shown to possess multiple pharmacological activities including anti-atherosclerosis. OBJECTIVE: The effects of chrysin on foam cell formation and cholesterol flow in RAW264.7 macrophages were investigated in this work to explore the potential mechanism underlying its anti-atherogenic activity. MATERIALS AND METHODS: The inhibitive effect of chrysin on foam cell formation and cholesterol accumulation induced by oxidized low-density lipoprotein cholesterol (ox-LDL) was assessed by oil red O staining and intracellular total cholesterol and triglyceride quantification in RAW264.7 macrophages. The action of chrysin on cholesterol efflux and influx was tested by fluorescent assays. Real-time quantitative PCR was used to quantify the relative expression of cholesterol flow-associated genes and luciferase assay was applied to test the transcription activity of peroxisome proliferator-activated receptor gamma (PPARγ). RESULTS: Chrysin dose dependently inhibited the formation of foam cells and prevented the enhanced cholesterol accumulation by ox-LDL. Treatment with chrysin (10 µM) significantly enhanced cholesterol efflux and substantially inhibited cholesterol influx. Simultaneously, chrysin significantly increased the mRNA levels of PPARγ, liver X receptor alpha (LXRα), ATP-binding cassette, sub-family A1 (ABCA1), and sub-family G1 (ABCG1), decreased scavenger receptor A1 (SR-A1) and SR-A2, and increased the transcriptional activity of PPARγ. DISCUSSION AND CONCLUSION: Chrysin is a new inhibitor of foam cell formation that may stimulate cholesterol flow. Up-regulation of the classical PPARγ-LXRα-ABCA1/ABCG1 pathway and down-regulation of SR-A1 and SR-A2 may participate in its suppressive effect on intracellular cholesterol accumulation.

Regulation effect of quercetin on cholesterol influx and efflux from RAW264.7 macrophage / 国际药学研究杂志

Objective: To investigate the effects of quercetin on cholesterol accumulation and cholesterol flow in RAW264.7 macrophages and explore the potential mechanism underlying its anti-atherogenic activity. Methods: The inhibitory effect of quercetin on cholesterol accumulation induced by oxidized low-density lipoprotein (ox-LDL) was assessed by oil red O staining and total cholesterol (TC) specific kits in RAW264.7 macrophages. And the action of cholesterol efflux and influx was tested by fluorescent assays. Cholesterol flow-associated genes expression was detected by real-time quantitative PCR (RT-PCR). Results: Quercetin significantly inhibited the cholesterol accumulation. Treatment with quercetin (10 μmol/L) significantly enhanced cholesterol efflux and substantially inhibited cholesterol influx. RT-PCR showed that quercetin significantly increased the mRNA levels of peroxisome proliferator-activated receptor γ (PPARγ), liver X receptor alpha (LXRα), ATP-binding cassette, subfamily A1 (ABCA1) and subfamily G1 (ABCG1), decreased scavenger receptor (SR)-A1 and SR-A2. Conclusion: Quercetin might be a new inhibitor on intracellular cholesterol accumulation. Upregulation of the classical PPARγ-LXRα-ABCA1/ ABCG1 pathway and down-regulation of SR-A1 and SR-A2 may participate in its suppressive effect on intracellular cholesterol accumulation.

Inhibition of epipinoresinol on macrophage foam and potential mechanisms / 中草药

Objective: To clarify the potential inhibitive effect of epipinoresinol on macrophage foam and potential mechanisms. Methods: The inhibition of epipinoresinol on foam cell formation after stained with oil red O was assessed by Image-Pro Plus and Microplate Reader, and the effect on cholesterol efflux and influx was tested by fluorescence detection to obtain cholesterol inflows-time curve and outflow rate. Additionally the cholesterol flow-associated genes expression was checked by real-time PCR (RT-PCR). Results: Epipinoresinol dose-dependently inhibited the enhanced cholesterol accumulation elicited by oxidized low-density lipoprotein cholesterol (ox-LDL) in RAW264.7 cells. Treatment with epipinoresinol significantly enhanced the cholesterol efflux mediated by high-density lipoprotein (HDL) and substantially inhibited the cholesterol influx. RT-PCR showed that epipinoresinol significantly increased the mRNA levels of PPARγ, LXRα, ABCA1, and ABCG1, decreased those of SR-A1 and SR-A2. Conclusion: Epipinoresinol is a new inhibitor on foam cell formation that may stimulate the cholesterol efflux through up-regulating the PPARγ-LXRα-ABCA1/ABCG1 pathway and prevent cholesterol influx through down-regulating SR-A1 and SR-A2, which may be useful on atherosclerosis treatment.

Effect of oxidized low density lipoprotein on CD36 expression in macrophage / 医学研究生学报

Objective: To investigate the effect of oxLDL on CD36 expression and cholesterol influx in THP-1 macrophage. Methods: THP1 macrophage were treated with 50mg/L oxLDL for 0,12,24,36,48 h respectively.Oil red O staining was used to observe the intracellular lipid droplets.\labeled Cholesterol influx was determined by FJ-2107P type liquid scintillator.CD36 mRNA and protein level were determined by reverse transcription-polymerase chain reaction(RT-PCR) and Western blotting respectively. Results:We found that oxLDL elevated CD36 in both protein and mRNA levels and increased cholesterol influx in a time-dependent manner.The levels of cholesterol influx were 23.5%、(27.8%、)39.7%、44.1% and 49.7% respectively. Conclusion: oxLDL may upregulate CD36 expression and increase cholesterol influx.