Biomarkers of dysfunctional changes in high-density lipoproteins in the assessment of lipid metabolism in hyperlipidemia modeling in vivo

Due to improper nutrition, high-density lipoproteins (HDLs) can be subjected to structural changes, acquiring a dysfunctional phenotype. Therefore, research efforts are currently focused on improving HDL functionality despite its blood level. The aim of this study was to evaluate the effect of phycocyanin concentrate (as part of a food matrix) on the functional properties of HDL. Male Wistar rats were fed a high-fat diet containing 2% cholesterol for 113 days. Experimental animals were treated with 30 and 300 mg/kg b.w. of phycocyanin concentrate mixed with soy protein isolate. Serum and hepatic cholesterol and triglyceride levels, and the content of protein, triglycerides, choline-containing phospholipids, malondialdehyde, sphingosine-1-phosphate, and paraoxonase-1 in HDL fractions were assessed. The decrease in protein in HDL particles is characteristic for dysfunctional phenotype of these particles. Phycocyanin concentrate diet prevented the depletion of protein in HDL particles, regardless of the dosage. The functionality of HDL is associated with paraoxonase-1 activity, which inhibits lipid peroxidation in lipoproteins. Our results have shown a significant increase in the level of paraoxonase-1 in HDL particles in groups treated with phycocyanin. HDL particles become more enriched with triglycerides with the development of hyperlipidemia. Triglycerides in HDL particles and in serum decreased by two times in animals receiving 30 mg/kg b.w. of phycocyanin. The MDA content in HDL particles decreased in all animals receiving a high-fat diet with the addition of 2% cholesterol. The introduction of 300 mg/kg of phycocyanin returned this indicator to the values of the Control group. Thus, biomarkers of dysfunctional changes in HDL in rodent hyperlipidemia models may be a useful tool for assessing lipid metabolism disorders. Also, the results confirm the potential ability to use phycocyanin concentrate as part of lipid-lowering products.

Cancer patients receiving doxorubicin (DOX) chemotherapy are at high risk of developing cardiac alterations, but the mechanisms remain elusive. Both breast cancer and DOX therapy are associated with dyslipidemia. We aimed to investigate whether changes in high-density lipoprotein (HDL) particles subclass distribution and functionalities are associated with DOX-induced cardiac alterations in breast cancer patients and tumor-bearing mice. HDL particles subclasses were assessed using the Lipoprint® system in breast cancer patients (n = 34) and tumor-bearing mice at baseline and after receiving DOX chemotherapy. HDL particles antioxidative properties were assessed by measuring paraoxonase-1 (PON1) activity. The ability of isolated HDL particles to protect against DOX-induced cytotoxicity was assessed in H9c2 cells. In breast cancer patients, DOX therapy reduced intermediate HDL particles subclasses, an effect that positively correlated with cardiac alterations. In mice, breast cancer shifted HDL particles subclasses distribution from intermediate to large HDL particles while DOX therapy increased small HDL particles. Both breast cancer and DOX therapy were associated with reduced PON1 activity. The decrease in intermediate HDL particles correlated with poorer cardiac function and lower PON1 activity. Interestingly, HDL particles isolated from tumor-bearing mice or from micereceiving DOX failed to protect H9c2 cells against DOX-induced cytotoxicity compared to HDL particles of healthy mice. In our study, a shift in HDL particles subclasses and functionalities correlated with cardiac alterations in cancer patients and mice treated with DOX. Consequently, our data warrant further research to explore whether targeting HDL particles may represent a therapeutic strategy to limit DOX-induced cardiotoxicity in breast cancer patients.

Disturbance in lipid metabolism has been suggested as a major pathogenic factor for age-related macular degeneration (AMD). Conventional lipid measures have been inconsistently associated with AMD. Other factors that can alter lipid metabolism include lipoprotein phenotype and genetic mutations. We performed a case-control study to examine the association between lipoprotein profile and neovascular AMD (nAMD) and whether the cholesterylester transfer protein (CETP) D442G mutation modulates these associations. Patients with nAMD had significantly higher concentrations of HDL and IDL compared with controls. The increase in HDL particles in nAMD patients was driven by an excess of medium-sized particles. Concurrently, patients with nAMD also had lower Apo A-1, lower VLDL and chylomicron lipoprotein. Many of these associations showed a dose-dependent association between controls, early AMD cases, and nAMD cases. Adjustment for the presence of the D442G mutation at the CETP locus did not significantly alter the increased AMD risk associated with HDL particle concentration. AMD is associated with variation in many lipoprotein subclasses, including increased HDL and IDL particles and decreased Apo A-1, VLDL, and chylomicron particles. These data suggest widespread systemic disturbance in lipid metabolism in the pathogenesis of AMD, including possible alterations in lipoprotein carrier capacity.

BackgroundMetabolic diseases such as obesity and diabetes are associated with changes in high-density lipoprotein (HDL) particles, including changes in particle size and protein composition, often resulting in abnormal function. Recent studies suggested that patients with non-alcoholic fatty liver disease (NAFLD), including individuals with non-alcoholic steatohepatitis (NASH), have smaller HDL particles when compared to individuals without liver pathologies. However, no studies have investigated potential changes in HDL particle protein composition in patients with NAFLD, in addition to changes related to obesity, to explore putative functional changes of HDL which may increase the risk of cardiovascular complications.MethodsFrom a cohort of morbidly obese females who were diagnosed with simple steatosis (SS), NASH, or normal liver histology, we selected five matched individuals from each condition for a preliminary pilot HDL proteome analysis. HDL particles were enriched using size-exclusion chromatography, and the proteome of the resulting fraction was analyzed by liquid chromatography tandem mass spectrometry. Differences in the proteomes between the three conditions (normal, SS, NASH) were assessed using label-free quantitative analysis. Gene ontology term analysis was performed to assess the potential impact of proteomic changes on specific functions of HDL particles.ResultsOf the 95 proteins identified, 12 proteins showed nominally significant differences between the three conditions. Gene ontology term analysis revealed that severity of the liver pathology may significantly impact the anti-thrombotic functions of HDL particles, as suggested by changes in the abundance of HDL-associated proteins such as antithrombin III and plasminogen.ConclusionsThe pilot data from this study suggest that changes in the HDL proteome may impact the functionality of HDL particles in NAFLD and NASH patients. These proteome changes may alter cardio-protective properties of HDL, potentially contributing to the increased cardiovascular disease risk in affected individuals. Further validation of these protein changes by orthogonal approaches is key to confirming the role of alterations in the HDL proteome in NAFLD and NASH. This will help elucidate the mechanistic effects of the altered HDL proteome on cardioprotective properties of HDL particles.

This study was designed to establish the mechanism responsible for the increased apolipoprotein (apo) A-II levels caused by the cholesteryl ester transfer protein inhibitor torcetrapib. Nineteen subjects with low HDL cholesterol (<40 mg/dl), nine of whom were also treated with 20 mg of atorvastatin daily, received placebo for 4 weeks, followed by 120 mg of torcetrapib daily for the next 4 weeks. Six subjects in the nonatorvastatin cohort participated in a third phase, in which they received 120 mg of torcetrapib twice daily for 4 weeks. At the end of each phase, subjects underwent a primed-constant infusion of [5,5,5-(2)H(3)]L-leucine to determine the kinetics of HDL apoA-II. Relative to placebo, torcetrapib significantly increased apoA-II concentrations by reducing HDL apoA-II catabolism in the atorvastatin (-9.4%, P < 0.003) and nonatorvastatin once- (-9.9%, P = 0.02) and twice- (-13.2%, P = 0.02) daily cohorts. Torcetrapib significantly increased the amount of apoA-II in the alpha-2-migrating subpopulation of HDL when given as monotherapy (27%, P < 0.02; 57%, P < 0.003) or on a background of atorvastatin (28%, P < 0.01). In contrast, torcetrapib reduced concentrations of apoA-II in alpha-3-migrating HDL, with mean reductions of -14% (P = 0.23), -18% (P < 0.02), and -18% (P < 0.01) noted during the atorvastatin and nonatorvastatin 120 mg once- and twice-daily phases, respectively. Our findings indicate that CETP inhibition increases plasma concentrations of apoA-II by delaying HDL apoA-II catabolism and significantly alters the remodeling of apoA-II-containing HDL subpopulations.

The cholesterol trafficking defect in Niemann-Pick type C (NPC) disease leads to impaired regulation of cholesterol esterification, cholesterol synthesis, and low density lipoprotein receptor activity. The ATP-binding cassette transporter A1 (ABCA1), which mediates the rate-limiting step in high density lipoprotein (HDL) particle formation, is also regulated by cell cholesterol content. To determine whether the Niemann-Pick C1 protein alters the expression and activity of ABCA1, we determined the ability of apolipoprotein A-I (apoA-I) to deplete pools of cellular cholesterol and phospholipids in human fibroblasts derived from NPC1+/+, NPC1+/-, and NPC1-/- subjects. Efflux of low density lipoprotein-derived, non-lipoprotein, plasma membrane, and newly synthesized pools of cell cholesterol by apoA-I was diminished in NPC1-/- cells, as was efflux of phosphatidylcholine and sphingomyelin. NPC1+/- cells showed intermediate levels of lipid efflux compared with NPC1+/+ and NPC1-/- cells. Binding of apoA-I to cholesterol-loaded and non-cholesterol-loaded cells was highest for NPC1+/- cells, with NPC1+/+ and NPC1-/- cells showing similar levels of binding. ABCA1 mRNA and protein levels increased in response to cholesterol loading in NPC1+/+ and NPC1+/- cells but showed low levels at base line and in response to cholesterol loading in NPC1-/- cells. Consistent with impaired ABCA1-dependent lipid mobilization to apoA-I for HDL particle formation, we demonstrate for the first time decreased plasma HDL-cholesterol levels in 17 of 21 (81%) NPC1-/- subjects studied. These results indicate that the cholesterol trafficking defect in NPC disease results in reduced activity of ABCA1, which we suggest is responsible for the low HDL-cholesterol in the majority of NPC subjects and partially responsible for the overaccumulation of cellular lipids in this disorder.

Plasma lipoprotein levels are predictors of risk for coronary artery disease. Lipoprotein structure-function relationships provide important clues that help identify the role of lipoproteins in cardiovascular disease. The compositional and conformational heterogeneity of lipoproteins are major barriers to the identification of their structures, as discovered using traditional approaches. Although electron microscopy (EM) is an alternative approach, conventional negative staining (NS) produces rouleau artifacts. In a previous study of apolipoprotein (apo)E4-containing reconstituted HDL (rHDL) particles, we optimized the NS method in a way that eliminated rouleaux. Here we report that phosphotungstic acid at high buffer salt concentrations plays a key role in rouleau formation. We also validate our protocol for analyzing the major plasma lipoprotein classes HDL, LDL, IDL, and VLDL, as well as homogeneously prepared apoA-I-containing rHDL. High-contrast EM images revealed morphology and detailed structures of lipoproteins, especially apoA-I-containing rHDL, that are amenable to three-dimensional reconstruction by single-particle analysis and electron tomography.

High-density lipoprotein (HDL) particles have multiple beneficial and cardioprotective roles, yet our understanding of their full structural and functional repertoire is limited due to challenges in separating HDL particles from contaminating plasma proteins and other lipid-carrying particles that overlap HDL in size and/or density. Here we describe a method for isolating HDL particles using a combination of sequential flotation density ultracentrifugation and fast protein liquid chromatography with a size exclusion column. Purity was visualized by polyacrylamide gel electrophoresis and verified by proteomics, while size and structural integrity were confirmed by transmission electron microscopy. This HDL isolation method can be used to isolate a high yield of purified HDL from a low starting plasma volume for functional analyses. This method also enables investigators to select their specific HDL fraction of interest: from the least inclusive but highest purity HDL fraction eluting in the middle of the HDL peak, to pooling all of the fractions to capture the breadth of HDL particles in the original plasma sample. We show that certain proteins such as lecithin cholesterol acyltransferase (LCAT), phospholipid transfer protein (PLTP), and clusterin (CLUS) are enriched in large HDL particles whereas proteins such as alpha-2HS-glycoprotein (A2HSG), alpha-1 antitrypsin (A1AT), and vitamin D binding protein (VDBP) are enriched or found exclusively in small HDL particles.

Apolipoprotein A-I inhibits transformation of high density lipoprotein subpopulations during incubation of human plasma

Backgroundβeta-2 glycoprotein I (β2GPI, Apolipoprotein H) is a plasma glycoprotein best known as a major autoantigen in autoimmune disorders such as antiphospholipid syndrome (APS) and systemic lupus erythematosus (SLE), both of which confer elevated cardiovascular risk. Despite its prominence in autoimmunity, its role in lipid metabolism and potential sex-specific effects remain poorly understood.MethodsWe investigated β2GPI’s influence on lipoprotein profiles using β2GPI knockout (KO) and wild-type (WT) mice subjected to normal chow (NC) and high-fat (HF) diets, as well as plasma from β2GPI-deficient patients and aged and sex matched controls. Lipoprotein fractions were analyzed for cholesterol and apolipoprotein content, and protein interactions were assessed by co-immunoprecipitation.ResultsIn animal studies, female β2GPI KO mice—but not males—displayed significantly increased total plasma cholesterol on a HF diet and greater cholesterol content within HDL fractions. Apo E was enriched in HDL fractions from female KO mice under both NC and HF diets, and plasma Apo E was elevated in HF-fed female KOs. In WT females on HF diet, β2GPI was enriched in HDL fractions, and β2GPI co-immunoprecipitated with Apo E. In human studies, the β2GPI-deficient female patient exhibited increased HDL cholesterol, a shift toward larger HDL particles, and enriched Apo E in HDL fractions relative to controls. Co-immunoprecipitation confirmed β2GPI–Apo E interaction in human plasma, with binding requiring Domain V of β2GPI.ConclusionsOur findings identify β2GPI as a sex-specific regulator of HDL metabolism. In females, β2GPI modulates Apo E-containing HDL particles, influencing cholesterol distribution and lipoprotein composition. These results reveal a novel mechanism linking β2GPI to lipid homeostasis, with potential implications for cardiovascular risk in women with autoimmune disease. Targeting β2GPI–Apo E interactions may represent a therapeutic avenue for correcting dysregulated HDL metabolism in female-specific cardiometabolic and autoimmune contexts.

The docosahexaenoic acid derivatives, diHEP-DPA and TH-DPA, synthesized via recombinant lipoxygenase, ameliorate disturbances in lipid metabolism and liver inflammation in high fat diet-fed mice

The isolation of high density lipoprotein (HDL) particles by density‐based methods using a density range of 1.063–1.21 g/mL is considered to be the gold standard. However, it is known that several HDL subclasses, including discoidal/nascent HDL particles, as well as ApoE‐containing, and intestinally derived ApoA‐IV‐containing HDL particles, are found at a higher density of up to 1.25 g/mL. In addition, using density‐based approaches alone it is not possible to purify HDL particles from a range of other particles such as extracellular vesicles, which overlap in density but not size. In this study, a novel method was optimized to isolate a broader array of HDL particles in a purified fraction that minimizes contamination by extracellular vesicles. The method utilizes density‐based sequential flotation ultracentrifugation followed by size exclusion chromatography with an FPLC system to isolate HDL fractions of different sizes, which were then analyzed for their cholesterol efflux capacity and protein content. The results demonstrate a higher yield of HDL and an increased level of HDL from earlier fractions using the 1.25 density cut‐off compared to the 1.21 density cut‐off. The cholesterol efflux capacity of early and late eluting fractions from the 1.25 density cut‐off method is increased, suggesting that both smaller and larger higher‐density particles, both with increased efflux capacity, are being isolated with the new method. Gel electrophoresis and Western Blot results from these fractions indicate enrichment of ApoE as well as ApoA‐IV in HDL fractions collected using the 1.25 density cut‐off method, particularly from plasma samples collected in the postprandial vs. the fasted state. In conclusion, we demonstrate that the use of the optimized HDL isolation method yields a broader array of HDL particles, which may be lost when the 1.21 density cut‐off is used.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Reduction of plasma LCAT activity has been observed in several conditions in which the size of HDL particles is increased; however, the mechanism of this reduction remains elusive. We investigated the plasma activity, mass, and in vivo catabolism of LCAT and its association with HDL particles in human apolipoprotein A-I transgenic, scavenger receptor class B type I knockout (hA-ITg SR-BI-/-) mice. Compared with hA-ITg mice, hA-ITg SR-BI-/- mice had a 4-fold higher total plasma cholesterol concentration, which occurred predominantly in 13-18 nm diameter HDL particles, a significant reduction in plasma esterified cholesterol-total cholesterol (EC/TC) ratio, and significantly lower plasma LCAT activity, suggesting a decrease in LCAT protein. However, LCAT protein in plasma, hepatic mRNA for LCAT, and in vivo turnover of 35S-radiolabeled LCAT were similar in both genotypes of mice. HDL from hA-ITg SR-BI-/- mice was enriched in sphingomyelin (SM), relative to phosphatidylcholine, and had less associated [35S]LCAT radiolabel and endogenous LCAT activity compared with HDL from hA-ITg mice. We conclude that the decreased EC/TC ratio in the plasma of hA-ITg SR-BI-/- mice is attributed to a reduction in LCAT reactivity with SM-enriched HDL particles.

The liver X receptors (LXRs) sense oxysterols and regulate genes involved in cholesterol metabolism. Synthetic agonists of LXRs are potent stimulators of fatty acid synthesis, which is mediated largely by sterol regulatory element-binding protein-1c (SREBP-1c). Paradoxically, an improved hepatic lipid profile by LXR was observed in mice fed a Western high fat (HF) diet. To explore the underlying mechanism, we administered mice normal chow or an HF diet and overexpressed LXRalpha in the liver. The HF diet with tail-vein injection of adenovirus of LXRalpha increased the expression of LXR-targeted genes involved in cholesterol reverse transport but not those involved in fatty acid synthesis. A similar effect was also observed with the use of 22R-hydroxycholesterol, an LXR ligand, in cultured hepatocytes. Consequently, SREBP-1c maturation was inhibited by the HF diet, which resulted from the induction of Insig-2a. Importantly, increased cholesterol level suppressed the expression of 2,3-oxidosqualene cyclase (OSC), which led to an increase in endogenous LXR ligand(s). Furthermore, siRNA-mediated knockdown of OSC expression enhanced LXR activity and selectively up-regulated LXR-targeted genes involved in cholesterol reverse transport. Thus, down-regulation of OSC may account for a novel mechanism underlying the LXR-mediated lipid metabolism in the liver of mice fed an HF diet.

Hypertriglyceridaemia, low high density lipoprotein (HDL) cholesterol level and reduced LDL particle size are the major features of uraemic dyslipidaemia. They are also found in the Insulin Resistance Syndrome. To examine alterations in HDL composition in patients on chronic dialysis and their relationship with insulin resistance. HDL particle size was determined in 33 patients on chronic haemodialysis (HD), 27 on chronic ambulatory peritoneal dialysis (CAPD) and 32 control non-diabetic subjects (C) without renal disease by non-denaturing 3-30% polyacrylamide gradient gel electrophoresis. A weighted HDL particle size score was calculated taking into account both HDL particle size and percentage total HDL protein concentration of each HDL band of the individual. Lipid and apolipoliprotein concentrations were determined in HDL2 and HDL3 particles obtained by sequential ultracentrifugation. In a subset of 24 control subjects and 22 subjects on HD, insulin sensitivity was also determined by an intravenous glucose tolerance test (IVGTT). HDL particles were found to be more triglyceride enriched and apoAI depleted in subjects on HD even though plasma triglyceride level was highest in patients on CAPD. Five subpopulations of HDL particles were identified by gradient gel electrophoresis in all subjects combined. In the subgroup of subjects who underwent IVGTT, the weighted HDL particle size score correlated positively with HDL cholesterol level (r = 0.6, p < 0.0005), LDL particle size (r = 0.47, p < 0.001), and insulin sensitivity (r = 0.48, p < 0.001), and negatively with plasma triglyceride level (r = 0.37, p < 0.01). We conclude that even though HDL cholesterol is reduced to a similar level in subjects on both forms of dialysis for end stage renal failure, abnormalities of HDL composition are more marked in subjects on HD. Reduction in HDL particle size is linked with insulin resistance and accompanies reduction in LDL particle size and hypertriglyceridaemia.