Designer high-density lipoprotein particles enhance endothelial barrier function and suppress inflammation.

High-density lipoprotein (HDL) nanoparticles promote endothelial cell (EC) function and suppress inflammation, but their utility in treating EC dysfunction has not been fully explored. Here, we describe a fusion protein named ApoA1-ApoM (A1M) consisting of apolipoprotein A1 (ApoA1), the principal structural protein of HDL that forms lipid nanoparticles, and ApoM, a chaperone for the bioactive lipid sphingosine 1-phosphate (S1P). A1M forms HDL-like particles, binds to S1P, and is signaling competent. Molecular dynamics simulations showed that the S1P-bound ApoM moiety in A1M efficiently activated EC surface receptors. Treatment of human umbilical vein ECs with A1M-S1P stimulated barrier function either alone or cooperatively with other barrier-enhancing molecules, including the stable prostacyclin analog iloprost, and suppressed cytokine-induced inflammation. A1M-S1P injection into mice during sterile inflammation suppressed neutrophil influx and inflammatory mediator secretion. Moreover, systemic A1M administration led to a sustained increase in circulating HDL-bound S1P and suppressed inflammation in a murine model of LPS-induced endotoxemia. We propose that A1M administration may enhance vascular endothelial barrier function, suppress cytokine storm, and promote resilience of the vascular endothelium.

Anti-Inflammatory Effect of High-Density Lipoprotein Blunted by Delivery of Altered MicroRNA Cargo in Patients With Rheumatoid Arthritis.

OBJECTIVE: High-density lipoprotein (HDL) has well-characterized anti-atherogenic cholesterol efflux and antioxidant functions. Another function of HDL uncharacterized in rheumatoid arthritis (RA) is its ability to transport microRNAs (miRNAs) between cells and thus alter cellular function. The study's purpose was to determine if HDL-miRNA cargo is altered and affects inflammation in RA. METHODS: HDL-microRNAs were characterized in 30 RA and 30 control participants by next generation sequencing and quantitative polymerase chain reaction. The most abundant differentially expressed miRNA was evaluated further. The function of miR-1246 was assessed by miRNA mimics, antagomiRs, small interfering RNA knockdown, and luciferase assays. Monocyte-derived macrophages were treated with miR-1246-loaded HDL and unmodified HDL from RA and control participants to measure delivery of miR-1246 and its effect on interleukin-6 (IL-6). RESULTS: The most abundant miRNA on HDL was miR-1246; it was significantly enriched two-fold on HDL from RA versus control participants. HDL-mediated miR-1246 delivery to macrophages significantly increased IL6 expression 43-fold. miR-1246 delivery significantly decreased DUSP3 1.5-fold and DUSP3 small interfering RNA knockdown increased macrophage IL6 expression. Luciferase assay indicated DUSP3 is a direct target of miR-1246. Unmodified HDL from RA delivered 1.6-fold more miR-1246 versus control participant HDL. Unmodified HDL from both RA and control participants attenuated activated macrophage IL6 expression, but this effect was significantly blunted in RA so that IL6 expression was 3.4-fold higher after RA versus control HDL treatment. CONCLUSION: HDL-miR-1246 was increased in RA versus control participants and delivery of miR-1246 to macrophages increased IL-6 expression by targeting DUSP3. The altered HDL-miRNA cargo in RA blunted HDL's anti-inflammatory effect.

Mechanistic insights into the anti-restenotic effects of HSP27 and HO1 modulated by reconstituted HDL on neointimal hyperplasia.

High-density lipoprotein (HDL) therapy has demonstrated beneficial effects in acute stroke and acute myocardial infarction models by reducing infarct size. In this study, we investigated the inhibitory effects of reconstituted HDL (rHDL) on neointimal hyperplasia and elucidated its underlying mechanism using a balloon injury rat model. Our finding revealed a significant 37% reduction in the intima to media ratio in the arteries treated with 80 mg/kg rHDL compared to those subjected to injury alone (p < 0.05), indicating a specific inhibition of neointimal hyperplasia. In vivo analysis further supported the positive effects of rHDL by demonstrating a reduction in smooth muscle cell (SMC) proliferation and an increase in endothelial cell (EC) proliferation. Additionally, rHDL treatment led to decreased infiltration of leukocytes and downregulated the expression of matrix metallopeptidase 9 (MMP9) in the neointimal area. Notably, rHDL administration resulted in decreased expression of VCAM1 and HIF1α, alongside increased expression of heme oxygenase 1 (HO1) and heat shock protein 27 (HSP27). Overexpression of HSP27 and HO1 effectively inhibited SMC proliferation. Moreover, rHDL-mediated suppression of injury-induced HIF1α coincided with upregulation of HSP27. Interestingly, HSP27 and HO1 had varying effects on the expression of chemokine receptors and rHDL did not exert significant effect on chemokine receptor expression in THP1 cells. These findings underscore the distinct roles of HSP27 and HO1 as potential regulatory factors in the progression of restenosis. Collectively, our study demonstrates that rHDL exerts a potent anti-neointimal hyperplasia effect by reducing leukocytes infiltration and SMC proliferation while promoting EC proliferation.

High-density lipoprotein protects vascular endothelial cells from indoxyl sulfate insults through its antioxidant ability.

Chronic kidney disease (CKD) patients have a high risk of cardiovascular disease. Indoxyl sulfate (IS) is a uremic toxin that has been shown to inhibit nitric oxide production and cause cell senescence by inducing oxidative stress. High-density lipoprotein (HDL) has a protective effect on the cardiovascular system; however, its impacts on IS-damaged endothelial cells are still unknown. This study aimed to explore the effects of exogenous supplement of HDL on vascular endothelial cells in a uremia-mimic environment. Tube formation, migration, adhesion, and senescence assays were used to evaluate the cell function of human aortic endothelial cells (HAECs). Reactive oxygen species generation was measured by using Amplex red assay. L-NAME and MCI186 were used as a nitric oxide synthase inhibitor and a free radical scavenger, respectively. HDL exerted anti-inflammatory and antioxidant effects via HIF-1α/HO-1 activation and IL-1ß/TNF-α/IL-6 inhibition in IS-stimulated HAECs. HDL improved angiogenesis ability through upregulating Akt/eNOS/VEGF/SDF-1 in IS-stimulated HAECs. HDL decreased endothelial adhesiveness via downregulating VCAM-1 and ICAM-1 in IS-stimulated HAECs. Furthermore, HDL reduced cellular senescence via upregulating SIRT1 and downregulating p53 in IS-stimulated HAECs. Importantly, the above beneficial effects of HDL were mainly due to its antioxidant ability. In conclusion, HDL exerted a comprehensive protective effect on vascular endothelial cells against damage from IS through its antioxidant ability. The results of this study might provide a theoretical basis for potential HDL supplementation in CKD patients with endothelial damage.

Clinical Pharmacokinetics and Pharmacodynamics of CSL112.

Cardiovascular diseases are the leading cause of death worldwide. Although there have been substantial advances over the last decades, recurrent adverse cardiovascular events after myocardial infarction are still frequent, particularly during the first year of the index event. For decades, high-density lipoprotein (HDL) has been among the therapeutic targets for long-term prevention after an ischemic event. However, early trials focusing on increasing HDL circulating levels showed no improvement in clinical outcomes. Recently, the paradigm has shifted to increasing the functionality of HDL rather than its circulating plasma levels. For this purpose, apolipoprotein-AI-based infusion therapies have been developed, including reconstituted HDL, such as CSL112. During the last decade, CSL112 has been extensively studied in Phase 1 and 2 trials and has shown promising results. In particular, CSL112 has been studied in the Phase 2b AEGIS trial exhibiting good safety and tolerability profiles, which has led to the ongoing large-scale Phase 3 AEGIS-II trial. This systematic overview will provide a comprehensive summary of the CSL112 drug development program focusing on its pharmacodynamic, pharmacokinetic, and safety profiles.

Beneficial Effect of Cuban Policosanol on Blood Pressure and Serum Lipoproteins Accompanied with Lowered Glycated Hemoglobin and Enhanced High-Density Lipoprotein Functionalities in a Randomized, Placebo-Controlled, and Double-Blinded Trial with Healthy Japanese.

This study evaluated the efficacy and safety of 20 mg of Cuban policosanol in blood pressure (BP) and lipid/lipoprotein parameters of healthy Japanese subjects via a placebo-controlled, randomized, and double-blinded human trial. After 12 weeks of consumption, the policosanol group showed significantly lower BP, glycated hemoglobin (HbA1c), and blood urea nitrogen (BUN) levels. The policosanol group also showed lower aspartate aminotransferase (AST), alanine aminotransferase (ALT), and γ-glutamyl transferase (γ-GTP) levels at week 12 than those at week 0: A decrease of up to 9% (p < 0.05), 17% (p < 0.05), and 15% (p < 0.05) was observed, respectively. The policosanol group showed significantly higher HDL-C level and HDL-C/TC (%), approximately 9.5% (p < 0.001) and 7.2% (p = 0.003), respectively, than the placebo group and a difference in the point of time and group interaction (p < 0.001). In lipoprotein analysis, the policosanol group showed a decrease in oxidation and glycation extent in VLDL and LDL with an improvement of particle shape and morphology after 12 weeks. HDL from the policosanol group showed in vitro stronger antioxidant and in vivo anti-inflammatory abilities. In conclusion, 12 weeks of Cuban policosanolconsumption in Japanese subjects showed significant improvement in blood pressure, lipid profiles, hepatic functions, and HbA1c with enhancement of HDL functionalities.

A green-inspired method to prepare non-split high-density lipoprotein (HDL) carrier with anti-dysfunctional activities superior to reconstituted HDL.

Numerous studies have demonstrated that dysfunctional high-density lipoprotein (HDL), especially oxidized HDL (OxHDL), could generate multifaceted in vivo proatherogenic effects that run counter to the antiatherogenic activities of HDL. It thereby reminded us that the in vitro reconstituted HDL (rHDL) might encountered with oxidation-induced dysfunction. Accordingly, a green-inspired method was employed to recycle non-split HDL from human plasma fraction IV. Then it was compared with rHDL formulated by an ethanol-injection method in terms of physicochemical properties and anti-dysfunctional activities. Results exhibited that rHDL oxidation extent exceeded that of non-split HDL evidenced by higher malondialdehy content, weaker inhibition on low-density lipoprotein (LDL) oxidation and more superoxide anion. The reserved paraoxonase-1 activity on non-split HDL could partially explain for above experimental results. In the targeted transport mechanism experiment, upon SR-BI receptor inhibition and/or CD36 receptor blockage, the almost unchanged non-split HDL uptake in lipid-laden macrophage indicated its negligible oxidation modification profile with regard to rHDL again. Furthermore, compared to rHDL, better macrophage biofunctions were observed for non-split HDL as illustrated by accelerated cholesterol efflux, inhibited oxidized LDL uptake and lessened cellular lipid accumulation. Along with decreased ROS secretion, obviously weakened oxidative stress damage was also detected under treatment with non-split HDL. More importantly, foam cells with non-split HDL-intervention inspired an enhanced inflammation repression and apoptosis inhibition effect. Collectively, the anti-dysfunctional activities of non-split HDL make it suitable as a potential nanocarrier platform for cardiovascular drug payload and delivery.

Swertia mussotii prevents high-fat diet-induced non-alcoholic fatty liver disease in rats by inhibiting expression the TLR4/MyD88 and the phosphorylation of NF-κB.

CONTEXT: Swertia mussotii Franch. (Gentianaceae) is a source of the traditional Tibetan medicine, ZangYinChen, and is used to treat chronic hepatitis and many types of jaundice. OBJECTIVE: This study explored the therapeutic effects and mechanism of S. mussotii on non-alcoholic fatty liver disease in diet-induced hypercholesterolaemia. MATERIALS AND METHODS: After a week of adaptive feeding, 32 Sprague-Dawley rats were divided into four groups: (1) Control, (2) Control-S, (3) Model, and (4) Model-S. During the 12 experimental weeks, we established the Model using a high-fat diet. Control-S and Model-S were given 1.0 g/kg S. mussotii water extract via gavage starting in the fifth week until the end of experiment. RESULTS: When compared with Model rats, the S. mussotii water extract led to a reduction in high-density lipoproteins (43.9%) and albumin (13.9%) and a decrease in total cholesterol (54.0%), triglyceride (45.6%), low-density lipoproteins (8.6%), aspartate aminotransferase (11.0%), alanine aminotransferase (15.5%), alkaline phosphatase (19.1%), total protein (6.4%), and glucose (20.8%) in serum. A reduction in three cytokines (IL-1ß, IL-6, and TNFα) was detected. Histopathological examination showed that liver steatosis was significantly relieved in S. mussotii-treated high-fat diet rats. S. mussotii also caused a downregulation in the expression of TLR4 (43.2%), MyD88 (33.3%), and a decrease in phosphorylation of NF-κB. DISCUSSION AND CONCLUSIONS: Our findings indicate that S. mussotii may act as a potential anti-inflammation drug via inhibition of the TLR4/MyD88/NF-κB pathway. Further in vivo and in vitro studies are needed to validate its potential in clinical medicine.

Impact of Sacubitril/Valsartan on Lipid Parameters in Patients with Heart Failure with Reduced Ejection Fraction.

BACKGROUND AND OBJECTIVE: Sacubitril/valsartan, an angiotensin receptor-neprilysin inhibitor, has been shown to significantly reduce cardiovascular mortality, heart failure hospitalizations, and all-cause mortality in patients with heart failure with reduced ejection fraction. This study aims to investigate the long-term impact of the sacubitril/valsartan combination on lipid parameters in patients with heart failure with reduced ejection fraction. METHODS: For this single-center retrospective cross-sectional study, data of patients using sacubitril/valsartan because of heart failure with reduced ejection fraction were collected. In addition to routine controls, the patients' lipid levels were measured at 3-month intervals. The parameters that were obtained over 3 years included total cholesterol, high-density lipoprotein cholesterol, triglyceride, and N-terminal pro-B-type natriuretic peptide levels. RESULTS: A total of 192 patients with a functional capacity New York Heart Association II-V, and who were using sacubitril/valsartan because of heart failure with reduced ejection fraction, were included in this study. Independent of statin use, there was a decrease in total cholesterol levels (196.1 ± 44.8 mg/dL vs 161.5 ± 41.7 mg/dL, p < 0.001) and triglyceride levels (159.1 ± 10.4 mg/dL vs 121.4 ± 6.9 mg/dL, p < 0.001), and there was an improvement in high-density lipoprotein cholesterol levels (44.9 ± 1.9 mg/dL vs 48.2 ± 2.4 mg/dL, p < 0.001) when comparing baseline levels with third-year levels. CONCLUSIONS: Sacubitril/valsartan in patients with heart failure with reduced ejection fraction, independent of statin use, may cause a decrease in total cholesterol and triglyceride levels and an improvement in high-density lipoprotein cholesterol levels.

Lamp1 mediates lipid transport, but is dispensable for autophagy in Drosophila.

The endolysosomal system not only is an integral part of the cellular catabolic machinery that processes and recycles nutrients for synthesis of biomaterials, but also acts as signaling hub to sense and coordinate the energy state of cells with growth and differentiation. Lysosomal dysfunction adversely influences vesicular transport-dependent macromolecular degradation and thus causes serious problems for human health. In mammalian cells, loss of the lysosome associated membrane proteins LAMP1 and LAMP2 strongly affects autophagy and cholesterol trafficking. Here we show that the previously uncharacterized Drosophila Lamp1 is a bona fide ortholog of vertebrate LAMP1 and LAMP2. Surprisingly and in contrast to lamp1 lamp2 double-mutant mice, Drosophila Lamp1 is not required for viability or autophagy, suggesting that fly and vertebrate LAMP proteins acquired distinct functions, or that autophagy defects in lamp1 lamp2 mutants may have indirect causes. However, Lamp1 deficiency results in an increase in the number of acidic organelles in flies. Furthermore, we find that Lamp1 mutant larvae have defects in lipid metabolism as they show elevated levels of sterols and diacylglycerols (DAGs). Because DAGs are the main lipid species used for transport through the hemolymph (blood) in insects, our results indicate broader functions of Lamp1 in lipid transport. Our findings make Drosophila an ideal model to study the role of LAMP proteins in lipid assimilation without the confounding effects of their storage and without interfering with autophagic processes.Abbreviations: aa: amino acid; AL: autolysosome; AP: autophagosome; APGL: autophagolysosome; AV: autophagic vacuole (i.e. AP and APGL/AL); AVi: early/initial autophagic vacuoles; AVd: late/degradative autophagic vacuoles; Atg: autophagy-related; CMA: chaperone-mediated autophagy; Cnx99A: Calnexin 99A; DAG: diacylglycerol; eMI: endosomal microautophagy; ESCRT: endosomal sorting complexes required for transport; FB: fat body; HDL: high-density lipoprotein; Hrs: Hepatocyte growth factor regulated tyrosine kinase substrate; LAMP: lysosomal associated membrane protein; LD: lipid droplet; LDL: low-density lipoprotein; Lpp: lipophorin; LTP: Lipid transfer particle; LTR: LysoTracker Red; MA: macroautophagy; MCC: Manders colocalization coefficient; MEF: mouse embryonic fibroblast MTORC: mechanistic target of rapamycin kinase complex; PV: parasitophorous vacuole; SNARE: soluble N-ethylmaleimide sensitive factor attachment protein receptor; Snap: Synaptosomal-associated protein; st: starved; TAG: triacylglycerol; TEM: transmission electron microscopy; TFEB/Mitf: transcription factor EB; TM: transmembrane domain; tub: tubulin; UTR: untranslated region.

Naringenin Protects against Hypertension by Regulating Lipid Disorder and Oxidative Stress in a Rat Model.

BACKGROUND: Naringenin, a natural resource-derived flavanone, exhibits a plethora of pharmacological properties. The present study aimed to investigate the effects of naringenin on obesity-associated hypertension and its underlying mechanism. METHODS: Obesity-associated hypertension rat model was established with a high-fat diet (HFD) and was administrated with naringenin (25, 50, 100 mg/kg). Body and fat weights were recorded and blood pressure was measured. Serum lipid parameters (cholesterol, low-density lipoprotein [LDL], high-density lipoprotein [HDL], and triglycerides), oxidative stress biomarkers (malondialdehyde [MDA], superoxide dismutase [SOD], nitrite oxide [NO], and glutathione [GSH]), and adipocytokines (leptin and adiponectin) were determined. The expressions of signal transducer and activator of transcription (STAT) 3 were determined by using Western blotting. RESULTS: Treatment with naringenin (100 mg/kg) reduced body and fat weight in HFD-induced rats. Besides, treatment with naringenin (50 and 100 mg/kg) reduced blood pressure and regulated lipid parameters by decreasing cholesterol, triglycerides, and LDL and increasing HDL. Treatment with naringenin (50 and 100 mg/kg) reduced serum MDA and NO, whereas it increased serum SOD and GSH. Furthermore, treatment with naringenin (50 and 100 mg/kg) regulated adipocytokines and decreased the phosphorylation of STAT3. CONCLUSION: Naringenin ameliorates obesity-associated hypertension by regulating lipid disorder and oxidative stress.

High-density lipoprotein reduces microglia activation and protects against experimental autoimmune encephalomyelitis in mice.

Studies have shown that high-density lipoprotein (HDL) is a powerful anti-atherosclerosis factor in vivo and in vitro, with anti-inflammatory effects, and it also plays an important role in the immune system and central nervous system (CNS). In this study, the BV2 microglia inflammation model and experimental autoimmune encephalomyelitis animal model were used to investigate the potential mechanism of HDL in multiple sclerosis. Our results show that HDL inhibits the activation of BV2 microglia in a model of BV2 microglia inflammation and were validated with primary microglia. HDL can down-regulate the expression of TNF-α, IL-6, iNOS and NO. Western blot results showed that HDL could reduce the expression levels of TLR4, CD14, MyD88 and NF-κB p65 LPS-induced microglia. In a mouse model of experimental autoimmune encephalomyelitis, hematoxylin-eosin (HE) staining showed decreased infiltration of inflammatory cells in brain and spinal cord tissues, and Luxol Fast Blue (LFB) staining showed significant improvement in spinal cord demyelination. We found that HDL reduced spinal cord and brain inflammation after EAE induction, inhibited the infiltration of CD68 and Iba-1 positive inflammatory cells, and reduced the production of multiple pro-inflammatory cytokines, including pro-inflammatory cytokines TNF-α, IL-6, and IL-1ß. Western blot showed that EAE mice HDL inhibited the activation of ERK1/2 and JNK in MAPK pathway and p-IκBα and P65 in NF-κB pathway. Taken together, our study suggests that HDL may influence microglia activation and inflammatory response in mice by regulating inflammatory signaling pathways, improving induction of experimental autoimmune encephalomyelitis, an animal model of multiple sclerosis, and provides further insights into HDL therapy for multiple sclerosis.

Mimic Lipoproteins Responsive to Intratumoral pH and Allosteric Enzyme for Efficient Tumor Therapy.

Discoid-reconstituted high-density lipoprotein (d-rHDL) is advantageous for tumor-targeted drug delivery due to its small size, long circulation, and efficient internalization into cancer cells. Nevertheless, an allosteric reaction catalyzed by serum lecithin-cholesterol acyltransferase (LCAT) may cause drug leakage from d-rHDL and reduce its targeting efficiency. Conversely, similar "structural weakening" catalyzed by acyl-coenzyme A-cholesterol acyltransferase (ACAT) inside tumor cells can stimulate precise intracellular drug release. Therefore, we synthesized and characterized a pH-sensitive n-butyraldehyde bi-cholesterol (BCC) to substitute for cholesterol in the d-rHDL particle, and bovine serum albumin (BSA) was used as the targeting agent. This dual pH- and ACAT-sensitive d-rHDL (d-d-rHDL) was small with a disk-like appearance. Morphological transformation observation, in vitro release assays, and differences in internalization upon LCAT treatment confirmed that BCC effectively inhibited the remodeling behavior and enhanced the tumor-targeting efficiency. The accumulation of d-d-rHDL in HepG2 cells was significantly higher than that in LO2 cells, and accumulation was inhibited by free BSA. The pH sensitivity was verified, and d-d-rHDL achieved efficient drug release in vitro and inside tumor cells after exposure to acidic conditions and ACAT. Confocal laser scanning microscopy demonstrated that d-d-rHDL escaped from lysosomes and became distributed evenly throughout cells. Moreover, in vivo imaging assays in a tumor-bearing mouse model demonstrated tumor-targeting properties of d-d-rHDL, and paclitaxel-loaded d-d-rHDL showed strong anticancer activity in these mice. This dual-sensitive d-d-rHDL thus combines structural stability in plasma and an intracellular pH/ACAT-triggered drug release to facilitate inhibition of tumor growth.

Reconstituted HDL-apoE3 promotes endothelial cell migration through ID1 and its downstream kinases ERK1/2, AKT and p38 MAPK.

INTRODUCTION: Atherosclerotic Coronary Artery Disease (ASCAD) is the leading cause of mortality worldwide. Novel therapeutic approaches aiming to improve the atheroprotective functions of High Density Lipoprotein (HDL) include the use of reconstituted HDL forms containing human apolipoprotein A-I (rHDL-apoA-I). Given the strong atheroprotective properties of apolipoprotein E3 (apoE3), rHDL-apoE3 may represent an attractive yet largely unexplored therapeutic agent. OBJECTIVE: To evaluate the atheroprotective potential of rHDL-apoE3 starting with the unbiased assessment of global transcriptome effects and focusing on endothelial cell (EC) migration as a critical process in re-endothelialization and atherosclerosis prevention. The cellular, molecular and functional effects of rHDL-apoE3 on EC migration-associated pathways were assessed, as well as the potential translatability of these findings in vivo. METHODS: Human Aortic ECs (HAEC) were treated with rHDL-apoE3 and total RNA was analyzed by whole genome microarrays. Expression and phosphorylation changes of key EC migration-associated molecules were validated by qRT-PCR and Western blot analysis in primary HAEC, Human Coronary Artery ECs (HCAEC) and the human EA.hy926 EC line. The capacity of rHDL-apoE3 to stimulate EC migration was assessed by wound healing and transwell migration assays. The contribution of MEK1/2, PI3K and the transcription factor ID1 in rHDL-apoE3-induced EC migration and activation of EC migration-related effectors was assessed using specific inhibitors (PD98059: MEK1/2, LY294002: PI3K) and siRNA-mediated gene silencing, respectively. The capacity of rHDL-apoE3 to improve vascular permeability and hypercholesterolemia in vivo was tested in a mouse model of hypercholesterolemia (apoE KO mice) using Evans Blue assays and lipid/lipoprotein analysis in the serum, respectively. RESULTS: rHDL-apoE3 induced significant expression changes in 198 genes of HAEC mainly involved in re-endothelialization and atherosclerosis-associated functions. The most pronounced effect was observed for EC migration, with 42/198 genes being involved in the following EC migration-related pathways: 1) MEK/ERK, 2) PI3K/AKT/eNOS-MMP2/9, 3) RHO-GTPases, 4) integrin. rHDL-apoE3 induced changes in 24 representative transcripts of these pathways in HAEC, increasing the expression of their key proteins PIK3CG, EFNB2, ID1 and FLT1 in HCAEC and EA.hy926 cells. In addition, rHDL-apoE3 stimulated migration of HCAEC and EA.hy926 cells, and the migration was markedly attenuated in the presence of PD98059 or LY294002. rHDL-apoE3 also increased the phosphorylation of ERK1/2, AKT, eNOS and p38 MAPK in these cells, while PD98059 and LY294002 inhibited rHDL-apoE3-induced phosphorylation of ERK1/2, AKT and p38 MAPK, respectively. LY had no effect on rHDL-apoE3-mediated eNOS phosphorylation. ID1 siRNA markedly decreased EA.hy926 cell migration by inhibiting rHDL-apoE3-triggered ERK1/2 and AKT phosphorylation. Finally, administration of a single dose of rHDL-apoE3 in apoE KO mice markedly improved vascular permeability as demonstrated by the reduced concentration of Evans Blue dye in tissues such as the stomach, the tongue and the urinary bladder and ameliorated hypercholesterolemia. CONCLUSIONS: rHDL-apoE3 significantly enhanced EC migration in vitro, predominantly via overexpression of ID1 and subsequent activation of MEK1/2 and PI3K, and their downstream targets ERK1/2, AKT and p38 MAPK, respectively, and improved vascular permeability in vivo. These novel insights into the rHDL-apoE3 functions suggest a potential clinical use to promote re-endothelialization and retard development of atherosclerosis.

Effect of dietary supplementation of wildCumin (Bunium persicum) seeds on performance, nutrient digestibility and circulating metabolites in broiler chicks during the finisher phase.

The objective of the present study was to evaluate the impact of inclusion of wild cumin seeds (WCS) on performance, nutrient digestibility and blood profile in broilers during the finisher phase. For this purpose, 360, 14 days old chicks were randomly divided into four groups designated as CONT (control), 0.5WCS, 1.0WCS and 1.5WCS with 5 replicates, which were supplemented with WCS at the rate of 0, 0.5, 1, and 1.5% respectively. On the overall, feed intake was significantly (p < 0.01) higher in the CONT compared to the 1.0WCS. At the end of the finisher phase, and overall basis, body weight and feed conversion ratio were significantly (p < 0.05) higher in 0.5WCS. Crude protein apparent digestibility in the ileum was significantly (p < 0.05) higher in 0.5WCS compared to the CONT, while crude fat digestibility was significantly (p < 0.01) higher in 0.5WCS and 1.5WCS compared to the control. Similarly, serum triglyceride was significantly (p < 0.05) lower in 0.5WC but high density lipoprotein was significantly (p < 0.05) higher in the same group. It was concluded that wild Cumin at the rate of 0.5% was superior compared to the other treatments in the diet to improve the performance, nutrient digestibility and blood metabolites in broiler during the finisher phase.

Phospholipid nanoparticles: Therapeutic potentials against atherosclerosis via reducing cholesterol crystals and inhibiting inflammation.

BACKGROUND: Atherosclerosis-related cardiovascular diseases (CVDs) are the leading cause of mortality worldwide. Cholesterol crystals (CCs) induce inflammation in atherosclerosis and are associated with unstable plaques and poor prognosis, but no drug can remove CCs in the clinic currently. METHODS: We generated a phospholipid-based and high-density lipoprotein (HDL)-like nanoparticle, miNano, and determined CC-dissolving capacity, cholesterol efflux property, and anti-inflammation effects of miNano in vitro. Both normal C57BL/6J and Apoe-deficient mice were used to explore the accumulation of miNano in atherosclerotic plaques. The efficacy and safety of miNano administration to treat atherosclerosis were evaluated in the Ldlr-deficient atherosclerosis model. The CC-dissolving capacity of miNano was also detected using human atherosclerotic plaques ex vivo. FINDINGS: We found that miNano bound to and dissolved CCs efficiently in vitro, and miNano accumulated in atherosclerotic plaques, co-localized with CCs and macrophages in vivo. Administration of miNano inhibited atherosclerosis and improved plaque stability by reducing CCs and macrophages in Ldlr-deficient mice with favorable safety profiles. In macrophages, miNano prevented foam cell formation by enhancing cholesterol efflux and suppressed inflammatory responses via inhibiting TLR4-NF-κB pathway. Finally, in an ex vivo experiment, miNano effectively dissolved CCs in human aortic atherosclerotic plaques. INTERPRETATION: Together, our work finds that phospholipid-based and HDL-like nanoparticle, miNano, has the potential to treat atherosclerosis by targeting CCs and stabilizing plaques. FUNDING: This work was supported by the National Institutes of Health HL134569, HL109916, HL136231, and HL137214 to Y.E.C, HL138139 to J.Z., R21NS111191 to A.S., by the American Heart Association 15SDG24470155, Grant Awards (U068144 from Bio-interfaces and G024404 from M-BRISC) at the University of Michigan to Y.G., by the American Heart Association 19PRE34400017 and Rackham Helen Wu award to M.Y., NIH T32 GM07767 to K. H., Barbour Fellowship to D.L.

Lack of Neuroprotective Effects of High-Density Lipoprotein Therapy in Stroke under Acute Hyperglycemic Conditions.

INTRODUCTION: The pleiotropic protective effects of high-density lipoproteins (HDLs) on cerebral ischemia have never been tested under acute hyperglycemic conditions. The aim of this study is to evaluate the potential neuroprotective effect of HDL intracarotid injection in a mouse model of middle cerebral artery occlusion (MCAO) under hyperglycemic conditions. METHODS: Forty-two mice were randomized to receive either an intracarotid injection of HDLs or saline. Acute hyperglycemia was induced by an intraperitoneal injection of glucose (2.2 g/kg) 20 min before MCAO. Infarct size (2,3,5-triphenyltetrazolium chloride (TTC)-staining), blood-brain barrier leakage (IgG infiltration), and hemorrhagic changes (hemoglobin assay by ELISA and hemorrhagic transformation score) were analyzed 24 h post-stroke. Brain tissue inflammation (IL-6 by ELISA, neutrophil infiltration and myeloperoxidase by immunohisto-fluorescence) and apoptosis (caspase 3 activation) were also assessed. RESULTS: Intraperitoneal D-glucose injection allowed HDL- and saline-treated groups to reach a blood glucose level of 300 mg/dl in the acute phase of cerebral ischemia. HDL injection did not significantly reduce mortality (19% versus 29% in the saline-injected group) or cerebral infarct size (p = 0.25). Hemorrhagic transformations and inflammation parameters were not different between the two groups. In addition, HDL did not inhibit apoptosis under acute hyperglycemic conditions. Conclusion: We observed a nonsignificant decrease in cerebral infarct size in the HDL group. The deleterious consequences of reperfusion such as hemorrhagic transformation or inflammation were not improved by HDL infusion. In acute hyperglycemia, HDLs are not potent enough to counteract the adverse effects of hyperglycemia. The addition of antioxidants to therapeutic HDLs could improve their neuroprotective capacity.

l-Glutamine supplementation enhances glutathione peroxidase and paraoxonase-1 activities in HDL of exercising older individuals.

BACKGROUND: Oxidative stress is an important factor in the formation of atherosclerotic plaques. High-density lipoprotein (HDL) harbors paraoxonase-1 (PON-1) and glutathione peroxidase (GPx), key enzymes in the protection against the harmful effects of oxidative stress. Although exercise training can increase both HDL-c content and its antioxidant action, and glutamine (Gln) intake also promotes GPx-based defenses, the association between exercise training and Gln in the regulation of PON-1 activity was not explored. Therefore, the objective of this study was to investigate the effects of Gln supplementation on the redox balance and on the total HDL antioxidant capacity by evaluation of the activity of PON-1 and GPx enzymes in physically exercised elderly individuals compared to non-exercised ones. METHODS: Fifty-one practitioners of a combined exercise training program (CET, age: 71.9 ± 5.7 years) and 32 non-practitioners (NP, age: 73 ± 6.3 years) participated in the study. CET and NP groups were separated into 2 subgroups according to the supplementation: Gln, 0.3 g/kg/day + 10 g maltodextrin (CET-Gln, n = 26; and NP-Gln, n = 16) or placebo, 10 g maltodextrin (CET-PL, n = 25; and NP-PL, n = 16). Blood samples were drawn at baseline and after 30 days after commencement of the supplementation for biochemical and enzyme activity analyses. RESULTS: Increased HDL-c, total peroxidase (PRx), and GPx activities were found in both CET-Gln and NP-Gln after the supplementation period, compared to baseline, in opposition to CET-PL and NP-PL groups. PON-1 activity increased only in CET-Gln. In both CET-Gln and NP-Gln groups, there was a reduction of the total peroxides/PRx, iron/PRx, and total peroxides/GPX ratios after supplementation. In CET-Gln, thiobarbituric acid-reactive substances (TBARS)/PRx and TBARS/GPx ratios were also lower after supplementation. CET-Gln and CET-PL subgroups had lower glycemia than NP-Gln and NP-PL, either at baseline or after the supplementation periods. The other parameters were unchanged after supplementation [total cholesterol, LDL-c, triglycerides, non-HDL cholesterol, total peroxides, TBARS, iron serum, Trolox-equivalent antioxidant capacity (TEAC), and uric acid]. CONCLUSIONS: Gln supplementation can increase glutathione peroxidase activity regardless the individuals were physically active or sedentary, but the PON-1 activity only increased in physically active individuals. These results show the potential of Gln supplementation in the maintenance of the vascular redox balance, with potential implications for atherogenesis protection.

A Novel Relative High-Density Lipoprotein Index to Predict the Structural Changes in High-Density Lipoprotein and Its Ability to Inhibit Endothelial-Mesenchymal Transition.

Therapeutic elevation of high-density lipoprotein (HDL) is thought to minimize atherogenesis in subjects with dyslipidemia. However, this is not the case in clinical practice. The function of HDL is not determined by its concentration in the plasma but by its specific structural components. We previously identified an index for the prediction of HDL functionality, relative HDL (rHDL) index, and preliminarily explored that dysfunctional HDL (rHDL index value > 2) failed to rescue the damage to endothelial progenitor cells (EPCs). To confirm the effectiveness of the rHDL index for predicting HDL functions, here we evaluated the effects of HDL from patients with different rHDL index values on the endothelial-mesenchymal transition (EndoMT) of EPCs. We also analyzed the lipid species in HDL with different rHDL index values and investigated the structural differences that affect HDL functions. The results indicate that HDL from healthy adults and subjects with an rHDL index value < 2 protected transforming growth factor (TGF)-ß1-stimulated EndoMT by modulating Smad2/3 and Snail activation. HDL from subjects with an rHDL index value > 2 failed to restore the functionality of TGF-ß1-treated EPCs. Lipidomic analysis demonstrated that HDL with different rHDL index values may differ in the composition of triglycerides, phosphatidylcholine, and phosphatidylinositol. In conclusion, we confirmed the applicability of the rHDL index value to predict HDL function and found structural differences that may affect the function of HDL, which warrants further in-depth studies.

Effect of High-Density Lipoprotein from Healthy Subjects and Chronic Kidney Disease Patients on the CD14 Expression on Polymorphonuclear Leukocytes.

In uremic patients, high-density lipoprotein (HDL) loses its anti-inflammatory features and can even become pro-inflammatory due to an altered protein composition. In chronic kidney disease (CKD), impaired functions of polymorphonuclear leukocytes (PMNLs) contribute to inflammation and an increased risk of cardiovascular disease. This study investigated the effect of HDL from CKD and hemodialysis (HD) patients on the CD14 expression on PMNLs. HDL was isolated using a one-step density gradient centrifugation. Isolation of PMNLs was carried out by discontinuous Ficoll-Hypaque density gradient centrifugation. CD14 surface expression was quantified by flow cytometry. The activity of the small GTPase Rac1 was determined by means of an activation pull-down assay. HDL increased the CD14 surface expression on PMNLs. This effect was more pronounced for HDL isolated from uremic patients. The acute phase protein serum amyloid A (SAA) caused higher CD14 expression, while SAA as part of an HDL particle did not. Lipid raft disruption with methyl-ß-cyclodextrin led to a reduced CD14 expression in the absence and presence of HDL. HDL from healthy subjects but not from HD patients decreased the activity of Rac1. Considering the known anti-inflammatory effects of HDL, the finding that even HDL from healthy subjects increased the CD14 expression was unexpected. The pathophysiological relevance of this result needs further investigation.