Although HDL cholesterol concentrations are strongly and inversely associated with risk of coronary heart disease, interventions that raise HDL cholesterol do not reduce risk of coronary heart disease. HDL cholesterol efflux capacity—a prototypical measure of HDL function—has been associated with coronary heart disease after adjusting for HDL cholesterol, but its effect on incident coronary heart disease risk is uncertain.
Methods
We measured cholesterol efflux capacity and assessed its relation with vascular risk factors and incident coronary heart disease events in a nested case-control sample from the prospective EPIC-Norfolk study of 25 639 individuals aged 40–79 years, assessed in 1993–97 and followed up to 2009. We quantified cholesterol efflux capacity in 1745 patients with incident coronary heart disease and 1749 control participants free of any cardiovascular disorders by use of a validated ex-vivo radiotracer assay that involved incubation of cholesterol-labelled J774 macrophages with apoB-depleted serum from study participants.
Findings
Cholesterol efflux capacity was positively correlated with HDL cholesterol concentration (r=0·40; p<0·0001) and apoA-I concentration (r=0·22; p<0·0001). It was also inversely correlated with type 2 diabetes (r=–0·18; p<0·0001) and positively correlated with alcohol consumption (r=0·12; p<0·0001). In analyses comparing the top and bottom tertiles, cholesterol efflux capacity was significantly and inversely associated with incident coronary heart disease events, independent of age, sex, diabetes, hypertension, smoking and alcohol use, waist:hip ratio, BMI, LDL cholesterol concentration, log-triglycerides, and HDL cholesterol or apoA-I concentrations (odds ratio 0·64, 95% CI 0·51–0·80). After a similar multivariable adjustment the risk of incident coronary heart disease was 0·80 (95% CI 0·70–0·90) for a per-SD change in cholesterol efflux capacity.
Interpretation
HDL cholesterol efflux capacity might provide an alternative mechanism for therapeutic modulation of the HDL pathway beyond HDL cholesterol concentration to help reduce risk of coronary heart disease.
Funding
US National Institutes of Health, UK Medical Research Council, Cancer Research UK.
The anterior cingulate cortex plays a pivotal role in the cognitive and affective aspects of pain perception. Both endogenous and exogenous opioid signaling within the cingulate mitigate cortical nociception, reducing pain unpleasantness. However, the specific functional and molecular identities of cells mediating opioid analgesia in the cingulate remain elusive. Given the complexity of pain as a sensory and emotional experience, and the richness of ethological pain-related behaviors, we developed a standardized, deep-learning platform for deconstructing the behavior dynamics associated with the affective component of pain in mice-LUPE (Light aUtomated Pain Evaluator). LUPE removes human bias in behavior quantification and accelerated analysis from weeks to hours, which we leveraged to discover that morphine altered attentional and motivational pain behaviors akin to affective analgesia in humans. Through activity-dependent genetics and single-nuclei RNA sequencing, we identified specific ensembles of nociceptive cingulate neuron-types expressing mu-opioid receptors. Tuning receptor expression in these cells bidirectionally modulated morphine analgesia. Moreover, we employed a synthetic opioid receptor promoter-driven approach for cell-type specific optical and chemical genetic viral therapies to mimic morphine's pain-relieving effects in the cingulate, without reinforcement. This approach offers a novel strategy for precision pain management by targeting a key nociceptive cortical circuit with on-demand, non-addictive, and effective analgesia.
Tangier disease (TD) is an autosomal recessive disorder caused by mutations in the ATP-binding cassette transporter A1 (ABCA1). These result in a greatly reduced ability to transport cholesterol out of cells, leading to the accumulation of cholesterol in macrophages and many other body tissues expressing ABCA1. We recruited two TD individuals, TD1, compound heterozygote at S2046R/K531N, and TD2, homozygous for the E1005X/E1005X truncation mutation, as well as heterozygote/non-affected family members, and healthy controls. We reprogrammed PBMC of TD patients and controls to pluripotency by Sendai viral transduction with Oct3/4, Sox2, Klf4 and c-Myc. TD-iPSC expressed pluripotency markers including SSEA-3, SSEA-4, TRA-1.60, and TRA-1.81, and maintained a normal karyotype. TD iPSC differentiated efficiently to macrophages. iPSC-derived macrophages (IPSDM) characteristics paralleled those of primary PBMC-derived macrophages (HMDM) at morphological, phenotypic and transcriptomic levels. TD-IPSDM and HMDM showed no cholesterol efflux to apolipoprotein A-I (apoA-I) and impaired efflux to HDL 3 . Treatment of TD cells with LXR agonists, which upregulate ABCA1 expression, failed to enhance cholesterol efflux to apoA-I in TD-IPSDM and HMDM consistent with the absence of functional ABCA1. In both IPSDM and HMDM, the heterozygote ABCA1 mutation carrier had an intermediate defect in cholesterol efflux and a partial response to the LXR agonist consistent with the presence of one functional allele. Relative to control-IPSDM, TD-IPSDM also showed a higher cholesterol ester/total cholesterol (CE/TC) ratio upon acetylated-LDL loading. Compared with control-IPSDM, TD-IPSDM showed enhanced phagocytosis of zymosan particles and greater inflammatory response to ATP treatment in lipopolysaccharide (LPS)-primed IPSDM, evidenced by markedly elevated gene expression of IL-1beta, IL-6, IL-8 and CCL5, but not TNF-alpha. These observations provide further support for the utility of IPSDM in defining more subtle macrophage phenotypes that are less obvious manifestations of Mendelian disorders. We conclude that macrophages derived from TD IPS can effectively recapitulate pathologic hallmarks of the disease.
Sortilin is a multi-ligand sorting receptor involved in trafficking of proteins from the Golgi apparatus to the lysosome and has been widely shown to be associated with plasma lipid traits and coronary artery disease. While over expression of sortilin in the liver reduces VLDL production, the reported effects of the genetic loss of sortilin on apolipoprotein B100 (apoB) and VLDL secretion have been contradictory and perplexing; loss of sortilin has been shown in different studies to result in both increased and decreased apoB/VLDL secretion. These conflicting studies were carried out in a variety of different models and used different methods of knocking down sortilin expression. To attempt to further clarify the role of sortilin, we explored the role of sortilin deficiency on apoB secretion in the hepatocyte by utilizing 2 in vitro models of sortilin knockdown; primary hepatocytes from Sort1-/- mice and siRNA -treated McA-RH7777 (McA) cells. In both primary hepatocytes and McA cells, loss of sortilin alone was not associated with any change in apoB secretion. The previously reported increases in VLDL secretion occurred on either the background of apoB over expression or in livers of mice on a high fat diet, suggesting the requirement for a metabolic stress. We found that apoB secretion was increased with Sort1 knockdown as compared to control in isolated primary hepatocytes from Apobec1-/-; hAPOB Tg mice and McA cells stably over expressing apoB. We then sought to increase apoB secretion by lipid loading with oleic acid (OA). While OA increased apoB secretion in all cells, there was no effect of Sort1 knockdown in this context. However, when the cells were further treated with either palmitic acid, proteasomal inhibitors, or tunicamycin (an ER stress inducer), there was an observed increase in apoB secretion with Sort1 knockdown, suggesting that sortilin regulates apoB secretion only when both apoB secretion is increased and the cell is stressed. Based on this data, we propose that hepatic sortilin regulates the post-ER fate of apoB for degradation and export and acts to coordinate intracellular apoB metabolism in response to the number and quality of apoB particles that reach the Golgi and the level of post-ER pre-secretory proteolysis activity.
Congenital orbital teratoma associated with a PTCH1 mutation syndrome (NBCCS), is a rare autosomal dominant disorder caused by mutations in the PTCH1 gene and shows a high level of penetrance and variable expressivity.The syndrome is characterized by developmental abnormalities or neoplasms and is diagnosed with 2 major criteria, or with 1 major and 2 minor criteria.Here, we report a new clinical manifestation associated with this syndrome in a boy affected by NBCCS who had congenital orbital teratoma at birth.Later, at the age of 15 years, he presented with 4 major and 4 minor criteria of NBCCS, including multiple basal cell carcinoma and 2 odontogenic keratocysts of the jaw, both confirmed by histology, more than 5 palmar pits, calcification of the cerebral falx, extensive meningeal calcifications, macrocephaly, hypertelorism, frontal bosses, and kyphoscoliosis.PTCH1 mutation analysis revealed the heterozygous germline mutation c.290dupA.This mutation generated a frameshift within exon 2 and an early premature stop codon (p.Asn97LysfsX43), predicting a truncated protein with complete loss of function.Identification of this mutation is useful for genetic counseling.Although the clinical symptoms are well-known, our case contributes to the understanding of phenotypic variability in NBCCS, highlighting that PTCH1 mutations cannot be used for predicting disease burden and reinforces the need of a multidisciplinary team in the diagnosis, treatment, and follow-up of NBCCS patients.
SORT1 is strongly associated with plasma lipid traits and coronary artery disease. The protein it encodes, sortilin is a multi-ligand receptor involved in trafficking of proteins from the Golgi to the lysosome. Hepatic sortilin overexpression reduces VLDL secretion, but the effects of loss of sortilin function on apolipoprotein B100 (apoB) and VLDL secretion have been contradictory and perplexing. Conflicting studies have variously reported increased and decreased apoB/VLDL secretion in response to loss of sortilin, but used a variety of different models and methods of ablating sortilin function. To resolve this, we first measured VLDL secretion in Sort1 knockout (KO) mice on a chow diet and observed no differences in either apoB or TG secretion. ApoB secretion was similarly unaffected in Sort1 KO primary hepatocytes or in McA-RH7777 cells treated with siRNA to knockdown sortilin. We concluded that in a basal secretory state, loss of sortilin alone does not impact apoB secretion, leading us to test if differences in secretory state might explain the discrepancies in published results. We found that when hAPOB was overexpressed, Sort1 knockdown further increased its secretion. The reports of increased VLDL secretion with Sort1 knockout occurred with apoB overexpression, or on high fat diet (HFD); consistent with our hypothesis that secretory overload or metabolic stress are required to uncover the deficiency phenotype. We placed Sort1 KO mice on a 45% HFD for 12 weeks and observed a significant increase in VLDL secretion compared to controls. In addition, when we lipid-loaded hepatocytes with oleic acid or palmitic acid, apoB secretion was increased, and Sort1 knockdown further increased this compared to control. We treated cells with Tunicamycin to induce ER stress, and found that apoB secretion was decreased in the control group was observed, but not in the absence of Sort1, suggesting that sortilin regulates apoB secretion the cell is stressed. Based on these data, we propose that hepatic sortilin regulates the post-ER fate of apoB for either degradation or export; thereby coordinating intracellular apoB metabolism in response to the number and quality of apoB particles that reach the Golgi, and the level of post-ER pre-secretory proteolysis activity.
Objective— Plasma levels of high-density lipoprotein cholesterol (HDL-C) are strongly inversely associated with coronary artery disease (CAD), and high HDL-C is generally associated with reduced risk of CAD. Extremely high HDL-C with CAD is an unusual phenotype, and we hypothesized that the HDL in such individuals may have an altered composition and reduced function when compared with controls with similarly high HDL-C and no CAD. Approach and Results— Fifty-five subjects with very high HDL-C (mean, 86 mg/dL) and onset of CAD at the age of ≈60 years with no known risk factors for CAD (cases) were identified through systematic recruitment. A total of 120 control subjects without CAD, matched for race, sex, and HDL-C level (controls), were identified. In all subjects, HDL composition was analyzed and HDL cholesterol efflux capacity was assessed. HDL phospholipid composition was significantly lower in cases (92±37 mg/dL) than in controls (109±43 mg/dL; P =0.0095). HDL cholesterol efflux capacity was significantly lower in cases (1.96±0.39) than in controls (2.11±0.43; P =0.04). Conclusions— In people with very high HDL-C, reduced HDL phospholipid content and cholesterol efflux capacity are associated with the paradoxical development of CAD.
Elevated plasma concentrations of HDL cholesterol (HDL-C) are associated with protection from atherosclerotic cardiovascular disease. Animal models indicate that decreased expression of endothelial lipase (LIPG) is inversely associated with HDL-C levels, and genome-wide association studies have identified LIPG variants as being associated with HDL-C levels in humans. We hypothesized that loss-of-function mutations in LIPG may result in elevated HDL-C and therefore performed deep resequencing of LIPG exons in cases with elevated HDL-C levels and controls with decreased HDL-C levels. We identified a significant excess of nonsynonymous LIPG variants unique to cases with elevated HDL-C. In vitro lipase activity assays demonstrated that these variants significantly decreased endothelial lipase activity. In addition, a meta-analysis across 5 cohorts demonstrated that the low-frequency Asn396Ser variant is significantly associated with increased HDL-C, while the common Thr111Ile variant is not. Functional analysis confirmed that the Asn396Ser variant has significantly decreased lipase activity both in vitro and in vivo, while the Thr111Ile variant has normal lipase activity. Our results establish that loss-of-function mutations in LIPG lead to increased HDL-C levels and support the idea that inhibition of endothelial lipase may be an effective mechanism to raise HDL-C.
Opioids initiate dynamic maladaptation in brain reward and affect circuits that occur throughout chronic exposure and withdrawal that persist beyond cessation. Protracted withdrawal is characterized by negative affective behaviors such as heightened anxiety, irritability, dysphoria, and anhedonia, which pose a significant risk factor for relapse. While the ventral tegmental area (VTA) and mu-opioid receptors (MORs) are critical for opioid reinforcement, the specific contributions of VTA
