ApoA-I Protects Pancreatic ß-Cells From Cholesterol-Induced Mitochondrial Damage and Restores Their Ability to Secrete Insulin.

BACKGROUND: High cholesterol levels in pancreatic ß-cells cause oxidative stress and decrease insulin secretion. ß-cells can internalize apo (apolipoprotein) A-I, which increases insulin secretion. This study asks whether internalization of apoA-I improves ß-cell insulin secretion by reducing oxidative stress. METHODS: Ins-1E cells were cholesterol-loaded by incubation with cholesterol-methyl-ß-cyclodextrin. Insulin secretion in the presence of 2.8 or 25 mmol/L glucose was quantified by radioimmunoassay. Internalization of fluorescently labeled apoA-I by ß-cells was monitored by flow cytometry. The effects of apoA-I internalization on ß-cell gene expression were evaluated by RNA sequencing. ApoA-I-binding partners on the ß-cell surface were identified by mass spectrometry. Mitochondrial oxidative stress was quantified in ß-cells and isolated islets with MitoSOX and confocal microscopy. RESULTS: An F1-ATPase ß-subunit on the ß-cell surface was identified as the main apoA-I-binding partner. ß-cell internalization of apoA-I was time-, concentration-, temperature-, cholesterol-, and F1-ATPase ß-subunit-dependent. ß-cells with internalized apoA-I (apoA-I+ cells) had higher cholesterol and cell surface F1-ATPase ß-subunit levels than ß-cells without internalized apoA-I (apoA-I- cells). The internalized apoA-I colocalized with mitochondria and was associated with reduced oxidative stress and increased insulin secretion. The IF1 (ATPase inhibitory factor 1) attenuated apoA-I internalization and increased oxidative stress in Ins-1E ß-cells and isolated mouse islets. Differentially expressed genes in apoA-I+ and apoA-I- Ins-1E cells were related to protein synthesis, the unfolded protein response, insulin secretion, and mitochondrial function. CONCLUSIONS: These results establish that ß-cells are functionally heterogeneous, and apoA-I restores insulin secretion in ß-cells with elevated cholesterol levels by improving mitochondrial redox balance.

Identification of a Distinct Platelet Phenotype in the Elderly: ADP Hypersensitivity Coexists With Platelet PAR (Protease-Activated Receptor)-1 and PAR-4-Mediated Thrombin Resistance.

BACKGROUND: Thrombin (via PAR [protease-activated receptor]-1 and PAR-4) and ADP (via P2Y12 receptors) are potent endogenous platelet activators implicated in the development of cardiovascular disease. We aimed to assess whether platelet pathways alter with aging. METHODS: We characterized platelet activity in community-dwelling volunteers (n=174) in the following age groups: (1) 20 to 30 (young); (2) 40 to 55 (middle-aged); (3) ≥70 years (elderly). Platelet activity was assessed by aggregometry; flow cytometry (surface markers [P-selectin: alpha granule release, CD63: dense granule release, PAC-1: measure of conformationally active GPIIb/IIIa at the fibrinogen binding site]) measured under basal conditions and after agonist stimulation [ADP, thrombin, PAR-1 agonist or PAR-4 agonist]); receptor cleavage and quantification; fluorometry; calcium flux; ELISA. RESULTS: The elderly had higher basal platelet activation than the young, evidenced by increased expression of P-selectin, CD63, and PAC-1, which correlated with increasing inflammation (IL [interleukin]-1ß/IL-6). The elderly demonstrated higher P2Y12 receptor density, with greater ADP-induced platelet aggregation (P<0.05). However, elderly subjects were resistant to thrombin, achieving less activation in response to thrombin (higher EC50) and to selective stimulation of both PAR-1 and PAR-4, with higher basal PAR-1/PAR-4 cleavage and less inducible PAR-1/PAR-4 cleavage (all P<0.05). Thrombin resistance was attributable to a combination of reduced thrombin orienting receptor GPIbα (glycoprotein Ibα), reduced secondary ADP contribution to thrombin-mediated activation, and blunted calcium flux. D-Dimer, a marker of in situ thrombin generation, correlated with platelet activation in the circulation, ex vivo thrombin resistance, and circulating inflammatory mediators (TNF [tumor necrosis factor]-α/IL-6). CONCLUSIONS: Aging is associated with a distinctive platelet phenotype of increased basal activation, ADP hyperreactivity, and thrombin resistance. In situ thrombin generation associated with systemic inflammation may be novel target to prevent cardiovascular disease in the elderly.

Rapid Release of Interleukin-1ß from Human Platelets Is Independent of NLRP3 and Caspase.

OBJECTIVE: Platelets are critical in mediating both rapid responses to injury and the development and progression of coronary disease. Several studies have shown that, after prolonged exposure to agonists, they produce and release inflammatory mediators including interleukin-1ß (IL-1ß), via the classical pathway (NLRP3 inflammasome and caspase-1 cleavage to release active IL-1ß) as described for leukocytes. This study aimed to determine whether there is rapid release of IL-1ß in response to soluble platelet agonists and whether such rapid release is NLRP3- and caspase-1-dependent. METHODS AND RESULTS: Using flow cytometry to detect platelet activation (and release of α and dense granule contents) and the combination of Western blotting, enzyme-linked-immunosorbent assay, and immunogold labeling transmission electron and immunofluorescence microscopy, we identified that resting human platelets contain mature IL-1ß. Platelets release IL-1ß within minutes in response to adenosine diphosphate (ADP), collagen, and thrombin receptor agonists, but not in response to conventional NLRP3 inflammasome agonists-lipopolysaccharide and adenosine triphosphate. The rapid release of IL-1ß in response to ADP and thrombin receptor agonists was independent of caspases (including caspase-1) and NLRP3. Immature and mature IL-1ß were identified as low-abundance proteins on transmission electron microscopy of human platelets, and were localized to the platelet cytosol, open canalicular system, and the periphery of α granules. CONCLUSION: Unlike monocytes and neutrophils, human platelets are capable of rapid agonist- and time-dependent release of IL-1ß by a mechanism which is independent of caspase-1 and NLRP3.

Effects of pre-eclampsia on HDL-mediated cholesterol efflux capacity after pregnancy.

Background and aims: Preeclampsia (PE) is associated with life-long increased risk of cardiovascular disease. One of the main protective functions of high-density lipoprotein (HDL) is its role in reverse cholesterol transport. HDL-mediated cholesterol efflux capacity (CEC) is decreased during pregnancy in women with PE. Whether this persists postpartum is unknown. Methods: Basal and transporter-specific CEC were determined 6 months postpartum in women who had a normotensive (n = 44) or a PE (n = 42) pregnancy. CEC was also measured in 23 normotensive and 20 PE women for whom samples were collected 24 months postpartum. Basal, ATP-binding cassette transporter-A1 (ABCA1)- and -G1 (ABCG1)-specific CEC were primarily determined using Chinese hamster ovary cells stably expressing human ABCA1 or ABCG1, and were also assessed using a J774 mouse macrophage cell line. Results: ABCA1-specific CEC was significantly lower in women who had PE 6 months postpartum (0.57 ± 0.1 vs 0.53 ± 0.08; p < 0.05), whilst basal and ABCG1-specific efflux were not significantly different. cAMP-specific CEC in J774 cells was also lower 6 months after PE (0.85 ± 0.21 vs 0.75 ± 0.25, p < 0.05). Although apoA-I, apoE, plasminogen and PON-1 levels were not significantly different in women who had PE compared with controls, ABCA1 efflux did correlate with apoA-l, HDL-C and apoE levels after a normal, and with apoA-l and HDL-C levels after a PE pregnancy. ABCA1-specific efflux decreased in all women between 6 and 24 months postpartum, by 11 ± 1.6% in women who had a normotensive pregnancy and 9 ± 1.3% in women who had PE. After adjustment for apoA-I levels, there was no significant difference in ABCA1-specific efflux between the groups at 6 months postpartum and in normotensive women over time, but remained significantly different between 6 and 24 months in women who had PE. Conclusions: ABCA1-mediated CEC is impaired 6 months postpartum after a PE pregnancy and decreases thereafter in both normotensive and PE pregnancies. ABCA1-mediated efflux is dynamic after pregnancy but is unlikely to explain the long-term increased CVD risk in women with PE.

Apolipoprotein E isoform-dependent effects on the processing of Alzheimer's amyloid-ß.

Since the identification of the apolipoprotein E (apoE) *ε4 allele as a major genetic risk factor for late-onset Alzheimer's disease, significant efforts have been aimed at elucidating how apoE4 expression confers greater brain amyloid-ß (Aß) burden, earlier disease onset and worse clinical outcomes compared to apoE2 and apoE3. ApoE primarily functions as a lipid carrier to regulate cholesterol metabolism in circulation as well as in the brain. However, it has also been suggested to interact with hydrophobic Aß peptides to influence their processing in an isoform-dependent manner. Here, we review evidence from in vitro and in vivo studies extricating the effects of the three apoE isoforms, on different stages of the Aß processing pathway including synthesis, aggregation, deposition, clearance and degradation. ApoE4 consistently correlates with impaired Aß clearance, however data regarding Aß synthesis and aggregation are conflicting and likely reflect inconsistencies in experimental approaches across studies. We further discuss the physical and chemical properties of apoE that may explain the inherent differences in activity between the isoforms. The lipidation status and lipid transport function of apoE are intrinsically linked with its ability to interact with Aß. Traditionally, apoE-oriented therapeutic strategies for Alzheimer's disease have been proposed to non-specifically enhance or inhibit apoE activity. However, given the wide-ranging physiological functions of apoE in the brain and periphery, a more viable approach may be to specifically target and neutralise the pathological apoE4 isoform.

Free Thiol ß2-GPI (ß-2-Glycoprotein-I) Provides a Link Between Inflammation and Oxidative Stress in Atherosclerotic Coronary Artery Disease.

OBJECTIVE: Atherosclerotic coronary artery disease is well recognised as an inflammatory disorder that is also influenced by oxidative stress. ß2-GPI (ß-2-glycoprotein-I) is a circulating plasma protein that undergoes post-translational modification and exists in free thiol as well as oxidized forms. The aim of this study was to assess the association between these 2 post-translational redox forms of ß2-GPI and atherosclerotic coronary artery disease. Approach and Results: Stable patients presenting for elective coronary angiography or CT coronary angiography were prospectively recruited. A separate group of patients after reperfused ST-segment-elevation myocardial infarction formed an acute coronary syndrome subgroup. All patients had collection of fasting serum and plasma for quantification of total and free thiol ß2-GPI. Coronary artery disease extent was quantified by the Syntax and Gensini scores. A total of 552 patients with stable disease and 44 with acute coronary syndrome were recruited. While total ß2-GPI was not associated with stable coronary artery disease, a higher free thiol ß2-GPI was associated with its presence and extent. This finding remained significant after correcting for confounding variables, and free thiol ß2-GPI was a better predictor of stable coronary artery disease than hs-CRP (high-sensitivity C-reactive protein). Paradoxically, there were lower levels of free thiol ß2-GPI after ST-segment-elevation myocardial infarction. CONCLUSIONS: Free thiol ß2-GPI is a predictor of coronary artery disease presence and extent in stable patients. Free thiol ß2-GPI was a better predictor than high-sensitivity C-reactive protein.

Increased ABCA1 (ATP-Binding Cassette Transporter A1)-Specific Cholesterol Efflux Capacity in Schizophrenia.

OBJECTIVE: Patients with schizophrenia have increased long-term mortality attributable to cardiovascular disease and commonly demonstrate features of mixed dyslipidemia with low HDL-C (high-density lipoprotein cholesterol). The removal of cholesterol from cells by HDL via specific ATP-binding cholesterol transporters is a major functional property of HDL, and its measurement as cholesterol efflux capacity (CEC) can predict cardiovascular risk. Whether HDL function is impaired in patients with schizophrenia is unknown. Approach and Results: We measured basal and ABCA1 (ATP-binding cassette transporter A1)- and ABCG1 (ATP-binding cassette transporter G1)-dependent CEC, comparing patients with schizophrenia with age- and sex-matched healthy controls, and related our findings to nuclear magnetic resonance analysis of lipoprotein subclasses. Total plasma cholesterol and LDL-C (low-density lipoprotein cholesterol) were comparable between healthy controls (n=51) and patients (n=120), but patients with schizophrenia had increased total plasma triglyceride, low HDL-C and apo (apolipoprotein) A-I concentrations. Nuclear magnetic resonance analysis indicated a marked (15-fold) increase in large triglyceride-rich lipoprotein particle concentration, increased small dense LDL particles, and fewer large HDL particles. Despite lower HDL-C concentration, basal CEC was 13.7±1.6% higher, ABCA1-specific efflux was 35.9±1.6% higher, and ABCG1 efflux not different, in patients versus controls. In patients with schizophrenia, ABCA1-specific efflux correlated with the abundance of small 7.8 nm HDL particles but not with serum plasminogen or triglyceride levels. CONCLUSIONS: Patients with schizophrenia have increased concentrations of atherogenic apoB-containing lipoproteins, decreased concentrations of large HDL particles, but enhanced ABCA1-mediated CEC. In this population, preventative strategies should focus on reducing atherogenic lipoproteins rather than increasing CEC.

Rate and Extent of Recovery from Reproductive and Cardiac Dysfunction Due to Androgen Abuse in Men.

CONTEXT: Androgen abuse impairs male reproductive and cardiac function, but the rate, extent, and determinants of recovery are not understood. OBJECTIVE: To investigate recovery of male reproductive and cardiac function after ceasing androgen intake in current and past androgen abusers compared with healthy non-users. METHODS: Cross-sectional, observational study recruited via social media 41 current and 31 past users (≥3 months since last use, median 300 days since last use) with 21 healthy, eugonadal non-users. Each provided a history, examination, and serum and semen sample and underwent testicular ultrasound, body composition analysis, and cardiac function evaluation. RESULTS: Current abusers had suppressed reproductive function and impaired cardiac systolic function and lipoprotein parameters compared with non- or past users. Past users did not differ from non-users, suggesting full recovery of suppressed reproductive and cardiac functions after ceasing androgen abuse, other than residual reduced testicular volume. Mean time to recovery was faster for reproductive hormones (anti-Mullerian hormone [AMH], 7.3 months; luteinizing hormone [LH], 10.7 months) than for sperm variables (output, 14.1 months) whereas spermatogenesis (serum follicle-stimulating hormone [FSH], inhibin B, inhibin) took longer. The duration of androgen abuse was the only other variable associated with slower recovery of sperm output (but not hormones). CONCLUSION: Suppressed testicular and cardiac function due to androgen abuse is effectively fully reversible (apart from testis volume and serum sex hormone binding globulin) with recovery taking between 6 to 18 months after ceasing androgen intake with possible cumulative effects on spermatogenesis. Suppressed serum AMH, LH, and FSH represent convenient, useful, and underutilized markers of recovery from androgen abuse.

TRAIL-Expressing Monocyte/Macrophages Are Critical for Reducing Inflammation and Atherosclerosis.

Circulating tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) levels are reduced in patients with cardiovascular disease, and TRAIL gene deletion in mice exacerbates atherosclerosis and inflammation. How TRAIL protects against atherosclerosis and why levels are reduced in disease is unknown. Here, multiple strategies were used to identify the protective source of TRAIL and its mechanism(s) of action. Samples from patients with coronary artery disease and bone-marrow transplantation experiments in mice lacking TRAIL revealed monocytes/macrophages as the main protective source. Accordingly, deletion of TRAIL caused a more inflammatory macrophage with reduced migration, displaying impaired reverse cholesterol efflux and efferocytosis. Furthermore, interleukin (IL)-18, commonly increased in plasma of patients with cardiovascular disease, negatively regulated TRAIL transcription and gene expression, revealing an IL-18-TRAIL axis. These findings demonstrate that TRAIL is protective of atherosclerosis by modulating monocyte/macrophage phenotype and function. Manipulating TRAIL levels in these cells highlights a different therapeutic avenue in the treatment of cardiovascular disease.

A critical appraisal of the measurement of serum 'cholesterol efflux capacity' and its use as surrogate marker of risk of cardiovascular disease.

The 'cholesterol efflux capacity (CEC)' assay is a simple in vitro measure of the capacities of individual sera to promote the first step of the reverse cholesterol transport pathway, the delivery of cellular cholesterol to plasma HDL. This review describes the cell biology of this model and critically assesses its application as a marker of cardiovascular risk. We describe the pathways for cell cholesterol export, current cell models used in the CEC assay with their limitations and consider the contribution that measurement of serum CEC provides to our understanding of HDL function in vivo.

Triglyceride-Rich Lipoproteins.

Plasma triglyceride levels are causally related to an increased risk of cardiovascular disease. Here, the authors summarize their understanding of triglyceride-rich lipoprotein metabolism, previous and newly identified regulators, and their relevance as candidate targets for therapeutic intervention.

Cell-specific production, secretion, and function of apolipoprotein E.

Apolipoprotein E (apoE) is a 34-kDa glycoprotein that is secreted from many cells throughout the body. ApoE is best known for its role in lipoprotein metabolism. Recent studies underline the association of circulating lipoprotein-associated apoE levels and the development for cardiovascular disease (CVD). Besides its well-established role in pathology of CVD, it is also implicated in neurodegenerative diseases and recent new data on adipose-produced apoE point to a novel metabolic role for apoE in obesity. The regulation of apoE production and secretion is remarkably cell and tissue specific. Here, we summarize recent insights into the differential regulation apoE production and secretion by hepatocytes, monocytes/macrophages, adipocytes, and the central nervous system and relevant variations in apoE biochemistry and function.

The E3 ubiquitin ligase, HECTD1, is involved in ABCA1-mediated cholesterol export from macrophages.

The ABC lipid transporters, ABCA1 and ABCG1, are essential for maintaining lipid homeostasis in cells such as macrophages by exporting excess cholesterol to extracellular acceptors. These transporters are highly regulated at the post-translational level, including protein ubiquitination. Our aim was to investigate the role of the E3 ubiquitin ligase HECTD1, recently identified as associated with ABCG1, on ABCG1 and ABCA1 protein levels and cholesterol export function. Here, we show that HECTD1 protein is widely expressed in a range of human and murine primary cells and cell lines, including macrophages, neuronal cells and insulin secreting ß-cells. siRNA knockdown of HECTD1 unexpectedly decreased overexpressed ABCG1 protein levels and cell growth, but increased native ABCA1 protein in CHO-K1 cells. Knockdown of HECTD1 in unloaded THP-1 macrophages did not affect ABCG1 but significantly increased ABCA1 protein levels, in wild-type as well as THP-1 cells that do not express ABCG1. Cholesterol export from macrophages to apoA-I over time was increased after knockdown of HECTD1, however these effects were not sustained in cholesterol-loaded cells. In conclusion, we have identified a new candidate, the E3 ubiquitin ligase HECTD1, that may be involved in the regulation of ABCA1-mediated cholesterol export from unloaded macrophages to apoA-I. The exact mechanism by which this ligase affects this pathway remains to be elucidated.

Short-term cooling increases serum triglycerides and small high-density lipoprotein levels in humans.

BACKGROUND: Cold exposure and ß3-adrenergic receptor agonism, which both activate brown adipose tissue, markedly influence lipoprotein metabolism by enhancing lipoprotein lipase-mediated catabolism of triglyceride-rich lipoproteins and increasing plasma high-density lipoprotein (HDL) levels and functionality in mice. However, the effect of short-term cooling on human lipid and lipoprotein metabolism remained largely elusive. OBJECTIVE: The objective was to assess the effect of short-term cooling on the serum lipoprotein profile and HDL functionality in men. METHODS: Body mass index-matched young, lean men were exposed to a personalized cooling protocol for 2 hours. Before and after cooling, serum samples were collected for analysis of lipids and lipoprotein composition by 1H-nuclear magnetic resonance. Adenosine triphosphate-binding cassette A1 (ABCA1)-mediated cholesterol efflux capacity of HDL was measured using [3H]cholesterol-loaded ABCA1-transfected Chinese hamster ovary cells. RESULTS: Short-term cooling increased serum levels of free fatty acids, triglycerides, and cholesterol. Cooling increased the concentration of large very low-density lipoprotein (VLDL) particles accompanied by increased mean size of VLDL particles. In addition, cooling enhanced the concentration of small LDL and small HDL particles as well as the cholesterol levels within these particles. The increase in small HDL was accompanied by increased ABCA1-dependent cholesterol efflux in vitro. CONCLUSIONS: Our data show that short-term cooling increases the concentration of large VLDL particles and increases the generation of small LDL and HDL particles. We interpret that cooling increases VLDL production and turnover, which results in formation of surface remnants that form small HDL particles that attract cellular cholesterol.

Measurement of Macrophage-Specific In Vivo Reverse Cholesterol Transport in Mice.

Reverse cholesterol transport (RCT) is one of the main processes that is thought to protect against cardiovascular disease. RCT constitutes the removal of cholesterol from peripheral sites, its transport through the plasma compartment for delivery to the liver for excretion. Here, we describe an in vivo RCT method that incorporates these steps, measuring movement of cholesterol from macrophages to the plasma, the liver, and finally to the feces in mice.

N-glycosylation of human sphingomyelin phosphodiesterase acid-like 3A (SMPDL3A) is essential for stability, secretion and activity.

Sphingomyelin phosphodiesterase acid-like 3A (SMPDL3A) is a recently identified phosphodiesterase, which is a secreted N-linked glycoprotein. SMPDL3A is highly homologous to acid sphingomyelinase (aSMase), but unlike aSMase cannot cleave sphingomyelin. Rather, SMPDL3A hydrolyzes nucleotide tri- and diphosphates and their derivatives. While recent structural studies have shed light on these unexpected substrate preferences, many other aspects of SMPDL3A biology, which may give insight into its function in vivo, remain obscure. Here, we investigate the roles of N-glycosylation in the expression, secretion and activity of human SMPDL3A, using inhibitors of N-glycosylation and site-directed mutagenesis, with either THP-1 macrophages or CHO cells expressing human SMPDL3A. Tunicamycin (TM) treatment resulted in expression of non-glycosylated SMPDL3A that was not secreted, and was largely degraded by the proteasome. Proteasomal inhibition restored levels of SMPDL3A in TM-treated cells, although this non-glycosylated protein lacked phosphodiesterase activity. Enzymatic deglycosylation of purified recombinant SMPDL3A also resulted in significant loss of phosphodiesterase activity. Site-directed mutagenesis of individual N-glycosylation sites in SMPDL3A identified glycosylation of Asn69 and Asn222 as affecting maturation of its N-glycans and secretion. Glycosylation of Asn356 in SMPDL3A, an N-linked site conserved throughout the aSMase-like family, was critical for protection against proteasomal degradation and preservation of enzymatic activity. We provide the first experimental evidence for a predicted 22 residue N-terminal signal peptide in SMPDL3A, which is essential for facilitating glycosylation and is removed from the mature protein secreted from CHO cells. In conclusion, site-specific N-glycosylation is essential for the intracellular stability, secretion and activity of human SMPDL3A.