HDL Protein Research Articles

Response to Comment on Mathew et al. Therapeutic Lifestyle Changes Improve HDL Function by Inhibiting Myeloperoxidase-Mediated Oxidation in Patients With Metabolic Syndrome. Diabetes Care 2018;41:2431–2437

We thank Holzer and Marsche (1) for their interest in our work on the impact of therapeutic lifestyle changes (TLC) on HDL function in patients with metabolic syndrome (2). They suggest that a modified isolation technique for HDL2 and HDL3 proposed by their group removes proteins such as albumin (most abundant plasma protein) and apolipoprotein (apo)B (the major constituent of LDL), thereby enhancing HDL purity. It is well known that HDL protein composition is highly heterogeneous, and different HDL isolation techniques—such as sequential ultracentrifugation (KBr or D2O/sucrose), size exclusion chromatography, immune affinity capture, ion exchange chromatography, or isoelectric focusing—yield variable combinations of ∼95 proteins, as reviewed (3) and as updated periodically on the website http://homepages.uc.edu/∼davidswm/HDLproteome.html. These proteins …

40 - Post-translational modification of ApoA-1 in metabolic syndrome

The increased cardiovascular morbidity and mortality associated with low cholesterol content of HDL is not improved by interventions that increase HDL cholesterol, suggesting that the vascular pathology results from dysfunction of the HDL apoproteins. In conditions associated with low HDL cholesterol, such as metabolic syndrome, increased oxidative stress leads to lipid peroxidation and formation of highly reactive dicarbonyl electrophiles, including malondialdehyde (MDA), isolevuglandins and 4-oxo-nonenal, that covalently react with the e-amine of lysines. Modification of ApoA-1 by MDA is known to disrupt reverse cholesterol transport. We measured the level of the lysine- MDA-lysine crosslinks in the HDL proteins of patients with metabolic syndrome with LC/tandem mass spectrometry utilizing a 13C-labelled internal standard and found an increased level of this lysine modification in comparison with healthy volunteers (metabolic syndrome, 5.5 ± 0.3 ng/mg protein; healthy subjects, 4.4 ± 0.2 ng/mg protein; P = 0.041). To address ApoA-1 more specifically, we characterized the relative reactivity of lysine sites on HDL- associated ApoA-1 and found that several lysines (aa 12, 23, 59 and 238/239) were preferentially modified by MDA. The probability that, in addition to MDA-K crosslinks, there are many other modifications of ApoA-1 lysines by carbonyl electrophiles was investigated by determining the loss of unmodified peptides that contained a lysine sensitive to carbonyl adduction. In metabolic syndrome, we found that the ratio of unmodified peptide 11-23 (K12) to an ApoA-1 peptide without a lysine was markedly reduced (0.44 ± 0.13 vs healthy controls 2.26 ± 0.32; P = 0.0005) indicating a global modification of this peptide by 80%. The proportion of unmodified 227-238 (K238) was reduced by 56% in ApoA-1 from metabolic syndrome patients (p = 0.0005). This represents a profound degree of post-translational modification of the molecule that regulates the function of HDL.

Dietary Supplementation with Trihexanoin Enhances Intestinal Function of Weaned Piglets.

Trihexanoin is a short-chain triglyceride (SCT). Many studies have reported that SCTs play important roles in the maintenance of intestinal epithelial structure and function. The present work was to investigate the effects of trihexanoin on growth performance, carbohydrate and fat metabolism, as well as intestinal morphology and function in weaned piglets. Twenty weaned piglets (21 ± 2 d) were randomly allocated to one of two treatment groups: The control group (basal diet supplemented with 0.5% soya oil); the TH group (basal diet supplemented with 0.5% trihexanoin). Dietary trihexanoin supplementation significantly reduced diarrhea rate; increased the concentrations of LDL, HDL and total protein in plasma; decreased cholesterol concentrations and glutamyl transpeptidase activity in plasma; improved intestinal morphologic structure; altered the mRNA levels and abundances of proteins related to glycogen and fat metabolism, mucosal barrier function, antioxidant capacity and water transport capacity; and altered the community of intestinal microflora. These results indicate that dietary trihexanoin supplementation could reduce diarrhea, regulate carbohydrate and fat metabolism, exert beneficial effects on the intestinal mucosal barrier, protect the intestinal mucosa from injuries, improve intestinal transport and absorption, and enhance antioxidant capacity. In conclusion, dietary supplementation with 0.5% trihexanoin improves the intestinal function and health of weaned piglets.

High density lipoprotein proteome is associated with cardiovascular risk factors and atherosclerosis burden as evaluated by coronary CT angiography

Background and aimsHigh density lipoprotein cholesterol (HDL-C) is associated with risk of cardiovascular disease (CVD); however, therapeutic manipulations of HDL-C have failed to reduce CVD events. This suggests that HDL-C and the atheroprotective capacity of HDL are not directly linked. The goal of this study was to evaluate the relationships between HDL-bound proteins and measures of atherosclerosis burden and HDL function. MethodsThe HDL proteome was analyzed using mass spectrometry in 126 human subjects, who had undergone coronary computed tomography angiography (CCTA) to quantify calcified (CB) and non-calcified (NCB) atherosclerosis burden. Partial least squares regression analysis was used to evaluate associations between HDL-bound proteins and CB, NCB, or cholesterol efflux capacity (CEC). ResultsSignificant overlap was found among proteins associated with NCB and CEC. Proteins that were associated with NCB displayed an inverse relationship with CEC, supporting a link between this protective function of HDL and clinical plaque burden. CB was associated with a set of proteins mostly distinct from NCB and CEC. When CVD risk factors were evaluated, BMI had a stronger influence on important HDL proteins than gender, age, or HDL-C. Most HDL proteins associated with function or atherosclerosis burden were not significantly correlated with HDL-C. ConclusionsThese findings indicate that the HDL proteome contains information not captured by HDL- C and, therefore, has potential for future development as a biomarker for CVD risk. Additionally, the proteome effects detected in this study may provide HDL compositional goals for evaluating new and existing HDL-modification therapies.

Apolipoprotein A-I deficiency alters the small intestine transcriptome and increases the expression of the antimicrobial enzyme DUOX2

Aim: The small intestine, together with the liver, is responsible for the synthesis of apoA-I, the most abundant protein of HDL. In addition, it has been recently shown that ilea of patients affected by Crohn’s disease have a genetic signature characterized by the down-regulation of the APOA1 gene and the up-regulation of DUOX2. DUOX2 is an antimicrobial enzyme that produces hydrogen peroxide in the intestine, and its expression is robustly up-regulated when a dysbiosis perturbs mucosal homeostasis.

Human ApoA-I overexpression ameliorates two main cardioprotective functions of HDL in db/db mice

Aim: The impact of ApoA-I, main protein of HDL, has been long examined. However, the impact of ApoA-I on atheroprotective action of dysfunctional HDL in diabetes is still limited. In this regard, we analyzed its consequences in important properties of HDL such as in vivo macrophage-specific reverse cholesterol transport (m-RCT) and ex vivo anti-oxidant protection in db/db mice

BMP1 5'UTR + 104T/C gene variation: can be a predictive marker for serum HDL and apoprotein A1 levels in male patients with coronary heart disease.

Apolipoprotein A1 (Apo A1), the major protein of HDL, is secreted as a proprotein and then is cleaved by C-terminal procollagen endoproteinase/bone morphogenetic protein-1 (BMP1). BMP1 stimulates the conversion of newly secreted proapo A1 to its phospholipid-binding form. Therefore, genetic variations of BMP1 gene may affect serum ApoA1 and HDL levels. We aimed to investigate the effects of the functional 5'UTR + 104 (T/C) variant of BMP1 on serum ApoA1 and HDL levels and risk of coronary heart disease (CHD) in this study. The BMP1 5'UTR + 104 (T/C) (rs143383) variation was determined in 131 male patients with CHD and 51 male controls by real-time polymerase chain reaction technique. ApoA1 levels were measured by immunoturbidimetry. The serum Apo-A1 levels were found higher in controls with the BMP1-CC genotype than those with the T-allele (p < 0.001). Our findings show the association of this variation with serum ApoA1 and HDL-C levels which increase in the order of CT < TT < CC in the controls. No effect was found on ApoA1 and HDL-C levels in CHD patients, as it was observed in the controls. However, the BMP1-TT genotype was associated with higher triglyceride (TG) levels as compared to C-allele (p = 0.009). These discrepancies could be due to statin therapy which has dominant effects on lowering cholesterol levels comparing to TG levels. Our results indicated that the BMP1 5'UTR + 104 (T/C) variation may affect the serum ApoA1 and lipoprotein levels depending on statin therapy so that contributes to the development of CHD.

Alteration of HDL Protein Composition with Hemodialysis Initiation.

HDL particles obtained from patients on chronic hemodialysis exhibit lower cholesterol efflux capacity and are enriched in inflammatory proteins compared with those in healthy individuals. Observed alterations in HDL proteins could be due to effects of CKD, but also may be influenced by the hemodialysis procedure, which stimulates proinflammatory and prothrombotic pathways. We compared HDL-associated proteins in 143 participants who initiated hemodialysis within the previous year with those of 110 participants with advanced CKD from the Hemodialysis Fistula Maturation Study. We quantified concentrations of 38 HDL-associated proteins relative to total HDL protein using targeted mass spectrometry assays that included a stable isotope-labeled internal standard. We used linear regression to compare the relative abundances of HDL-associated proteins after adjustment and required a false discovery rate q value ≤10% to control for multiple testing. We further assessed the association between hemodialysis initiation and cholesterol efflux capacity in a subset of 80 participants. After adjustment for demographics, comorbidities, and other clinical characteristics, eight HDL-associated proteins met the prespecified false discovery threshold for association. Recent hemodialysis initiation was associated with higher HDL-associated concentrations of serum amyloid A1, A2, and A4; hemoglobin-β; haptoglobin-related protein; cholesterylester transfer protein; phospholipid transfer protein; and apo E. The trend for participants recently initiating hemodialysis for lower cholesterol efflux capacity compared with individuals with advanced CKD did not reach statistical significance. Compared with advanced CKD, hemodialysis initiation within the previous year is associated with higher concentrations of eight HDL proteins related to inflammation and lipid metabolism. Identified associations differ from those recently observed for nondialysis-requiring CKD. Hemodialysis initiation may further impair cholesterol efflux capacity. Further work is needed to clarify the clinical significance of the identified proteins with respect to cardiovascular risk. This article contains a podcast at https://www.asn-online.org/media/podcast/CJASN/2018_07_25_CJASNPodcast_18_8_W.mp3.

Glycation and Deamidation Result in HDL Dysfunction in Patients with Type 2 Diabetes

Diabetes is associated with HDL dysfunction and perturbed iron metabolism. ApoAI, transferrin (Trf) and ceruloplasmin (Cp) are the key HDL proteins involved in cholesterol efflux and iron metabolism. We tested the hypothesis that hyperglycemia-induced glycation contributes to HDL dysfunction and altered iron metabolism in diet-controlled patients with T2D. HDL from patients with T2D and matched healthy controls (n=9/group) was isolated and it’s anti-oxidant and cholesterol efflux properties were quantified. HDL proteome composition and post-translational modification of proteins were quantified by proteomics aproach. Metabolic 2H2O-labeling was applied to quantify HDL proteome dynamics. Patients with T2D and controls had similar lipid (triglycerides, total cholesterol, and HDL cholesterol) profile. HDL from T2D patients had reduced anti-oxidant (PON1 activity) and macrophage-cholesterol efflux capacity (P&amp;lt;0.05) and was enriched with glycated apoAI and transferrin (Tf), and deamidated ceruloplasmin (Cp), the key HDL proteins involved in cholesterol and iron transport. Both ApoAI and Trf glycation were directly correlated with HbA1c and the extent of glycations were associated with their increased degradation. ApoAI glycation was inversely correlated with cholesterol efflux activity of HDL. Trf glycations at Lys-2and Lys-534 sites involved in iron coordination, were 2-3 fold higher in T2D than in the controls. The PTM search on Cp revealed five asparagine deamidation sites in T2D patients with N-943 being located on one of the oxidation-prone motifs and may result in the loss of Cp’s ferroxidase activity. In vivo HDL flux study demonstrated that glycated apoAI and Tf, and deamidated Cp species were degraded 3, 10, and 2 fold faster than the respective non-modified native proteins. HDL dysfunction and oxidative stress in T2D is related to glycation- and deamidation-induced instability of HDL proteins, including ApoAI, Tf and Cp. Disclosure T. Kasumov: None. M. Golizeh: None. S. Kashyap: None.

Abstract 166: Effects of Dietary Unsaturated Fat and Carbohydrate on the HDL Proteome and Metabolism of 9 HDL Proteins Across 6 HDL Size Fractions in Humans

Introduction: When dietary carbohydrate replaces fat, HDL-C and apoA1 decrease. The proportion in plasma of large HDL2 decreases while small HDL3 increases. These findings suggest that diet affects metabolically important attributes of HDL that produce the size changes. We studied in humans the effect of dietary carbohydrate and unsaturated fat on the HDL proteome and metabolism of 9 HDL proteins across 6 HDL sizes. Methods and Results: Twelve participants were placed on a controlled high unsaturated fat (HF) or high carbohydrate (HC) diet in a randomized crossover design. At the end of each 4-week diet period, subjects were infused with D3-Leu tracer, and blood was collected for 70 hrs. ApoA1-HDL was prepared by immunoaffinity purification, separated into 6 sizes α0, α1, α2, α3, preβ, and &lt;preβ by ND-PAGE, and in-gel trypsinized for mass spectrometry. We used label free quantification to characterize the HDL proteome. The proteome composition and distribution across the 6 HDL sizes were remarkably conserved in all subjects on both diets. Diet altered the abundance of several proteins on the major HDL sizes α2 and α3. The HC diet increased proteins involved in lipid metabolism (apoC3, apoC1, apoC2-C4) and acute phase response (SAA1, 4), and decreased antioxidant (PON1, 3) and protease inhibitor (SERPINA3, G1) proteins. We also monitored the metabolism of 9 proteins that likely affect HDL metabolism – apoA1, apoA2, apoA4, apoC3, apoE, apoM, apoJ, apoL1, and LCAT. We used high resolution parallel reaction monitoring and XPI software to measure tracer enrichment in these 9 proteins across the 6 HDL sizes. We found that the HC diet increased apoA2 and decreased apoA1 and apoA4 pool sizes on large HDL. The HC diet increased apoA1 and apoE turnover on large α1 and α2 and 3, respectively. Conclusions: Dietary carbohydrate when it replaces unsaturated fat alters the HDL proteome and metabolism of several HDL proteins on specific HDL sizes. Carbohydrate increases the abundance of proteins involved in lipid metabolism and the acute phase response, decreases antioxidant and protease inhibitor proteins, and enhances turnover of apoE and apoA1. This study suggests that diet modulates HDL function by affecting HDL composition and the metabolism of major HDL proteins.

Abstract 180: Missense Mutations in ABCA1 and CETP Associate with Changes in the HDL Proteome in Primate Half-Sibs Discordant for HDL Cholesterol Levels

HDL protein composition and corresponding function may impact cardiovascular disease risk. Identifying genetic variation that influences the HDL proteome may reveal modifiable HDL functions that impact this risk. We studied genetic determinants of the HDL proteome in a cohort of rhesus macaques enriched for extreme HDL cholesterol levels (HDL-c). We selected macaques from 2 distinct paternal half-sibships, each comprising 8 half-sib pairs matched for age-class and sex, but with large differences in HDL-c (N=22 genomes). HDL was isolated by ultracentrifugation and the protein cargo analyzed by mass spectrometry. Identified peptide sequences were compared to a Swiss-Prot canonical human protein database using BLAST to determine ortholog matches. Among the proteins identified, 64 are among the 229 reported in similar human studies, and 45 of these 64 are among the 95 highest-confidence HDL proteins tracked by the HDL Proteome Watch. We performed deep exome sequencing, and assessed predicted function for all genetic variants in macaques, among 23 genes associated with HDL disorders or variation in HDL-c in humans. We focused on the higher-impact variants most likely to have conserved effects between macaques and humans by proximity (i.e., &lt;10 bp) to known human mutations. This produced a set of 3 missense variants in ABCA1 associated with Tangier disease and HDL deficiency, and 4 missense variants in CETP associated with CETP deficiency, reduced CETP activity, and hyper- and hypoalphalipoproteinemias. Using a measured genotype approach, we tested for association of all 7 variants with adjusted spectral counts, while accounting for age and sex, and applied a false discovery rate (FDR) of 20% to all nominally significant results. After controlling for FDR, 3 missense variants in ABCA1 were significantly associated with spectral counts for VCAM1 , ITGA2 , and ITGB1 (nominal P-values 0.0005-0.0026), and 4 missense variants in CETP were significantly associated with spectral counts for APOA2 , APOC3 , C4BPA , and PLTP (nominal P-values 0.0002-0.0038). While our results require replication, these proteins suggest that changes in HDL-c in macaques are associated with changes in HDL that modulate vascular inflammation, immune response, and particle remodeling.

Abstract 555: Investigation of 4-oxo-2-nonenal Mediated HDL Dysfunction and the Use of Lipid Dicarbonyl Scavengers in Preserving HDL Function

Background: Lipid peroxidation products such as hydroxy-2-nonenal (HNE) are elevated in atherosclerosis and cause HDL dysfunction. Generated in parallel to HNE is 4-oxo-2-nonenal (ONE), a less studied lipid dicarbonyl possibly due to its far greater reactivity. The consequences of ONE modification of HDL have not been studied. We have recently found that scavengers of lipid dicarbonyls such as salicylamine (SAM) and pentylpyridoxamine (PPM) prevent HDL dysfunction induced by malondialdehyde and isolevuglandin (IsoLG). In this study, we examine the impact of ONE adduct formation on HDL structure-function, and the scavenging abilities of various small molecules in preventing ONE modification. Methods and Results: By Western blot analysis, ONE crosslinked apoA-I on HDL at a concentration of 3 ONE molecules for every 10 apoA-I proteins (0.3 molar equivalence, eq.), which is 100 fold lower than HNE but comparable to IsoLG. ONE-mediated crosslinking of HDL proteins preferentially produced a 39 kDa band on SDS-PAGE, likely an apoA-I/apoA-II heterodimer. ONE-modified HDL partially inhibited the ability of HDL to protect against the inflammatory response of macrophages (as shown in TNFα and IL-1β mRNA expression), but did not render HDL pro-inflammatory. At 3 eq., ONE dramatically decreased the ability of apoA-I to exchange among HDLs, from ~46.5% to only ~18.4% (P&lt;0.001). HDL-mediated macrophage cholesterol efflux was decreased by ~70.6% (P&lt;0.005) and 56.1% (P&lt;0.001) by HDL modified by 0.3 and 3 eq. ONE, respectively. Investigation of various scavenger analogues in protecting HDL from ONE modification showed that while SAM and its fluoro- and chloro- analogues partially prevented ONE-mediated HDL protein crosslinking, PPM nearly completely blocked crosslinking. PPM pretreatment of HDL prior to 3 eq. ONE modification was also able to restore HDL-mediated macrophage cholesterol efflux, from 56.1~ to ~83.0% (P&lt;0.01) while the inactive analogue pentylpyridoxine had no effect. Conclusions: Our study is the first to show that ONE causes HDL dysfunction, and demonstrates that not all modified HDLs result in the same “dysfunction”. We also demonstrate the use of PPM in preferentially scavenging ONE in biological systems.

Abstract 020: Regression of Atherosclerosis Through Manipulation of Vascular Macrophages; a Novel Gene-therapy Approach

Aggressive lipid lowering halts atherosclerotic plaque progression but does not lead to bulk plaque regression in humans. We have previously reported that gene transfection of the HDL protein apoAI into macrophages (MΦ-apoAI) decrease the rate of plaque development in atherosclerosis-prone mice. In this study, we hypothesized that MΦ-apoAI can promote regression of atherosclerosis synergistically with lipid-lowering. Atherogenic mice (LDLR -/- and LDLR -/- /MΦ-apoAI) were fed a high-fat diet to promote stage II/III atherosclerotic lesions (as baseline comparator), and then switched to an extreme lipid-lowering intervention (chow with MTTP inhibitor). After 3 weeks on the lipid-lowering diet, both groups showed reduced systemic and lesion inflammation, and reduced macrophage content on histology when compared to baseline. Upon lipid-lowering, vascular ultrasound showed that LDLR -/- /MΦ-apoAI mice has 21% improvement in aortic pulse transit time, and a 37.1% improvement in instantaneous aortic compliance (a surrogate marker for arterial elastic modulus), compared to LDLR -/- mice. Histologic evidence showed that LDLR -/- /MΦ-apoAI mice on lipid-lowering diet also had reduced lesion size (-24%), reduced macrophage content (-27.5%), increased M2/M1 ratio (+51%) and reduced necrotic core area (-28.6%), compared to LDLR -/- mice. Using transplantation of bone marrow cells from MΦ-apoAI mice into recipient mice with established lesions, we show that newly recruited cells are not major contributors to the regression afforded by MΦ-apoAI. Thus, to study the kinetics of pre-existing cells in the lesion during regression, we developed a contrast ultrasound-mediated gene delivery system to tag and trace lesion cells in LDLR -/- mice, which revealed that lesion macrophages expressing apoAI specifically migrate to mediastinal LN in a CCR7-dependent manner during regression. Expression of apoAI in pre-existing lesion macrophages, promotes regression of atherosclerosis beyond lipid-lowering alone, and increases CCR7-dependent egress of macrophages to adjacent lymph nodes. Ultrasound-mediated gene delivery can be a useful device to study the kinetics of cell in the artery wall and also represent a novel approach with application to human therapy.

Sex steroids mediate discrete effects on HDL cholesterol efflux capacity and particle concentration in healthy men

Exogenous testosterone decreases serum concentrations of high-density lipoprotein cholesterol (HDL-C) in men, but whether this alters cardiovascular risk is uncertain. To investigate the effects of testosterone and estradiol on HDL particle concentration (HDL-Pima) and metrics of HDL function. We enrolled 53 healthy men, 19 to 55years of age, in a double-blinded, placebo-controlled, randomized trial. Subjects were rendered medically castrate using the GnRH receptor antagonist acyline and administered either (1) placebo gel, (2) low-dose transdermal testosterone gel (1.62%, 1.25g), (3) full replacement dose testosterone gel (1.62%, 5g) or (4) full replacement dose testosterone gel together with an aromatase inhibitor for 4weeks. At baseline and end of treatment, serum HDL total macrophage and ABCA1-specific cholesterol efflux capacity (CEC), HDL-Pima and size, and HDL protein composition were determined. Significant differences in serum HDL-C were observed with treatment across groups (P=.01 in overall repeated measures ANOVA), with increases in HDL-C seen after both complete and partial testosterone deprivation. Medical castration increased total HDL-Pima (median [interquartile range] 19.1 [1.8] nmol/L at baseline vs 21.3 [3.1] nmol/L at week 4, P=.006). However, corresponding changes in total macrophage CEC and ABCA1-specific CEC were not observed. Change in serum 17β-estradiol concentration correlated with change in total macrophage CEC (β=0.33 per 10 pg/mL change in serum 17β-estradiol, P=.03). Testosterone deprivation in healthy men leads to a dissociation between changes in serum HDL-C and HDL CEC. Changes in serum HDL-C specifically due to testosterone exposure may not reflect changes in HDL function.

Type 2 diabetes is associated with loss of HDL endothelium protective functions.

Aims/HypothesisOne of the hallmarks of diabetes is impaired endothelial function. Previous studies showed that HDL can exert protective effects on endothelium stimulating NO production and protecting from inflammation and suggested that HDL in obese people with diabetes and dyslipidemia may have lower endothelial protective function. We aimed to investigate whether type 2 diabetes impairs HDL endothelium protective functions in people with otherwise normal lipid profile.MethodsIn a case-control study (n = 41 per group) nested in the Cooper Center Longitudinal Study we tested the ability of HDL to protect endothelium by stimulating endothelial nitric oxide synthase activity and suppressing NFκB-mediated inflammatory response in endothelial cells. In parallel we measured HDL protein composition, sphinogosine-1-phosphate and P-selectin.ResultsDespite similar levels of plasma HDL-C the HDL in individuals with type 2 diabetes lost almost 40% of its ability to stimulate eNOS activity (P<0.001) and 20% of its ability to suppress TNFα-dependent NFκB-mediated inflammatory response in endothelial cells (P<0.001) compared to non-T2D controls despite similar BMI and lipid profile (HDL-C, LDL-C, TC, TG). Significantly, the ability of HDL to stimulate eNOS activity was negatively associated with plasma levels of P-selectin, an established marker of endothelial dysfunction (r = −0.32, P<0.001). Furthermore, sphingosine-1-phosphate (S1P) levels were decreased in diabetic plasma (P = 0.017) and correlated with HDL-mediated eNOS activation.Conclusions/InterpretationsCollectively, our data suggest that HDL in individuals with type 2 diabetes loses its ability to maintain proper endothelial function independent of HDL-C, perhaps due to loss of S1P, and may contribute to development of diabetic complications.

Characterization of immortalized human dermal microvascular endothelial cells (HMEC-1) for the study of HDL functionality

BackgroundPrimary cultures endothelial cells have been used as models of endothelial related diseases such atherosclerosis. Biological behavior of primary cultures is donor-dependent and data could not be easily reproducible; endothelial cell lines are emerging options, particularly, human dermal microvascular endothelial cells (HMEC-1), that should be validated to substitute primary cultures for the study of HDL functions.MethodsMorphology, size and granularity of cells were assessed by phase contrast microscopy and flow cytometry of HMEC-1. The adhesion molecules, ICAM-1and VCAM-1 after TNF-α stimulation, and endothelial markers CD105 endoglin, as well as HDL receptor SR-BI were determined by flow cytometry. Internalization of HDL protein was demonstrated by confocal microscopy using HDL labeled with Alexa Fluor 488. HUVECs were used as reference to compared the characteristics with HMEC-1.ResultsHMEC-1 and HUVEC had similar morphologies, size and granularity. HMEC-1 expressed endothelial markers as HUVECs, as well as functional SR-B1 receptor since the cell line was able to internalize HDL particles. HMEC-1 effectively increased ICAM-1 and VCAM-1 expression after TNF-α stimulation. HUVECs showed more sensibility to TNF-α stimulus but the range of ICAM-1 and VCAM-1 expression was less homogeneous than in HMEC-1, probably due to biological variation of the former. Finally, the expression of adhesion molecules in HMEC-1 was attenuated by co-incubation with HDL.ConclusionHMEC-1 possess characteristics of endothelial cells, similar to HUVECs, being a cell line suitable to evaluate the functionality of HDL vis-à-vis the endothelium.

High-Density Lipoprotein Function in Cardiovascular Disease and Diabetes Mellitus.

Cardiovascular disease (CVD) is the most common cause of death worldwide.1 Major CVD risk factors are hypertension, smoking, physical inactivity, abnormal glucose levels/diabetes mellitus, and dyslipidemia. Among these, dyslipidemia characterized by a low level of HDL-C (high-density lipoprotein cholesterol) is strongly and inversely correlated with CVD risk,2–4 and low HDL-C levels is part of the atherogenic dyslipidemia complex associated with diabetes mellitus.5 These observations triggered intense interest in increasing HDL-C levels for therapeutic intervention of CVD.6,7 However, several recent lines of evidence now suggest that the association between HDL-C levels and CVD status may be indirect or more complicated than previously recognized.7 Thus, human genetic studies demonstrate that genetically altered HDL-C levels do not necessarily translate to an altered risk of CVD.8–12 Phase III clinical trials of drugs that elevate HDL-C, such as niacin and CETP (cholesteryl ester transfer protein) inhibitors, also have largely failed to reduce CVD events in statin-treated subjects with established CVD.13–15 For several CETP inhibitors, improvement in CVD prevention was unsuccessful because of off-target effects (torcetrapib) or lack of efficacy (dalcetrapib and evacetrapib).14,16–18 The exception is the recent REVEAL trial (Randomized Evaluation of the Effects of Anacetrapib Through Lipid-Modification), which included more subjects and was performed for longer than previous CETP inhibitor trials. The REVEAL demonstrated that the CETP inhibitor anacetrapib exhibited beneficial effects on cardiovascular outcomes on top of those of statin therapy, although the risk reduction was moderate19,20 and might have been due, at least in part, to a reduction in non-HDL-C rather than elevated HDL-C.21 Considering these cumulative observations, it remains uncertain whether increased HDL-C directly impacts atherosclerosis and the …

Studies in genetically modified mice implicate maternal HDL as a mediator of fetal growth.

Studies in humans have shown a direct association between maternal plasma cholesterol concentrations and infant birthweight. Similarly, previous studies in our laboratory have shown that chow-fed mice lacking apolipoprotein (apo) A-I, the major protein in HDL, have low HDL-cholesterol (HDL-C) concentrations and smaller fetuses in midgestation. In the current study, we measured fetal weights in mice with varying levels of apoA-I gene dose (knockout, wild-type, and transgenic) and examined metabolic pathways known to affect fetal growth. As expected, we found the differences in apoA-I expression led to changes in HDL particle size and protein cargo as well as plasma cholesterol concentrations. Fetal masses correlated directly with maternal plasma cholesterol and apoA-I concentrations, but placental masses and histology did not differ between groups of mice. There was no significant difference in glucose or amino acid transport to the fetus or in expression levels of the glucose (glucose transporter 1 and 2) or amino acid (sodium-coupled neutral amino acid transporter 1 and 2) transporters in whole placentas, although there was a trend for greater uptake of both nutrients in the whole fetal unit (fetus + placenta) of mice with greater apoA-I levels; significant differences in transport rates occurred when mice without apoA-I (knockout) vs. mice with apoA-I (wild-type and transgenic) were compared. Glucose tolerance tests were improved in the mice with the highest level of apoA-I, suggesting increased insulin-induced uptake of glucose by tissues of apoA-I transgenic mice. Thus, maternal HDL is associated with fetal growth, an effect that is likely mediated by plasma cholesterol or other HDL-cargo, including apolipoproteins or complement system proteins. A direct role of enhanced glucose and/or amino acid transport cannot be excluded.-Rebholz, S. L., Melchior, J. T., Davidson, W. S., Jones, H. N., Welge, J. A., Prentice, A. M., Moore, S. E., Woollett, L. A. Studies in genetically modified mice implicate maternal HDL as a mediator of fetal growth.

A thumbwheel mechanism for APOA1 activation of LCAT activity in HDL[S

APOA1 is the most abundant protein in HDL. It modulates interactions that affect HDL's cardioprotective functions, in part via its activation of the enzyme, LCAT. On nascent discoidal HDL, APOA1 comprises 10 α-helical repeats arranged in an anti-parallel stacked-ring structure that encapsulates a lipid bilayer. Previous chemical cross-linking studies suggested that these APOA1 rings can adopt at least two different orientations, or registries, with respect to each other; however, the functional impact of these structural changes is unknown. Here, we placed cysteine residues at locations predicted to form disulfide bonds in each orientation and then measured APOA1's ability to adopt the two registries during HDL particle formation. We found that most APOA1 oriented with the fifth helix of one molecule across from fifth helix of the other (5/5 helical registry), but a fraction adopted a 5/2 registry. Engineered HDLs that were locked in 5/5 or 5/2 registries by disulfide bonds equally promoted cholesterol efflux from macrophages, indicating functional particles. However, unlike the 5/5 registry or the WT, the 5/2 registry impaired LCAT cholesteryl esterification activity (P < 0.001), despite LCAT binding equally to all particles. Chemical cross-linking studies suggest that full LCAT activity requires a hybrid epitope composed of helices 5-7 on one APOA1 molecule and helices 3-4 on the other. Thus, APOA1 may use a reciprocating thumbwheel-like mechanism to activate HDL-remodeling proteins.

Cell-free Biochemical Fluorometric Enzymatic Assay for High-throughput Measurement of Lipid Peroxidation in High Density Lipoprotein.

Low high-density lipoprotein cholesterol (HDL-C) levels are one of the most powerful independent negative predictors of atherosclerotic cardiovascular disease (CVD). The structure and function of HDL rather than HDL-C may more accurately predict atherosclerosis. Several HDL protein and lipid compositional changes that impair HDL function occur in inflammatory states such as atherosclerosis. HDL function is usually determined by cell based assays such as cholesterol efflux assay but these assays have numerous drawbacks lack of standardization. Cell-free assays may give more robust measures of HDL function compared to cell-based assays. HDL oxidation impairs HDL function. HDL has a major role in lipid peroxide transport and high amount of lipid peroxides is related to abnormal HDL function. Lipid-probe interactions should be considered when interpreting the results of non-enzymatic fluorescence assays for measuring the lipid oxidative state. This motivated us to develop a cell-free biochemical enzymatic method to assess HDL lipid peroxide content (HDLox) that contributes to HDL dysfunction. This method is based on the enzyme horseradish peroxidase (HRP) and the fluorochrome Amplex Red that can quantify (without cholesterol oxidase) the lipid peroxide content per mg of HDL-C. Here a protocol is describedfor determination of HDL-lipid peroxidation using the fluorochrome reagent. Assay variability can be reduced by strict standardization of experimental conditions. Higher HDLox values are associated with reduced HDL antioxidant function. The readout of this assay is associated with readouts of validated cell-based assays, surrogate measures of cardiovascular disease, systemic inflammation, immune dysfunction, and associated cardiovascular and metabolic risk phenotypes. This technical approach is a robust method to assess HDL function in human disease where systemic inflammation, oxidative stress and oxidized lipids have a key role (such as atherosclerosis).