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Obesity is a universal epidemic and underlies comorbidities including type 2 diabetes and cardiovascular disease. Adipocytes store dietary metabolites within lipid droplets to avoid lipotoxicity and to provide energy to the body through metabolic processes such as lipid and glucose metabolism. Collaborative studies demonstrated that the extracellular matrix protein procollagen C-endopeptidase enhancer 2 (PCPE2) mediates the ability of scavenger receptor BI (SR-BI) to transport cholesterol into hepatocytes and adipocytes.It remains unknown if SR-BI and PCPE2 also play roles in other adipocyte metabolic processes and if their cooperative partnership is conserved. This knowledge gap prompted us to test the hypothesis that PCPE2 facilitates SR-BI’s ability to regulate metabolic processes in adipocytes. We validated a differentiated adipocyte model system using mesenchymal stem cells isolated from ears of wild-type (WT), SR-BI -/- , and PCPE2 - /- mice by demonstrating increased lipid droplet formation, mRNA expression of adipogenesis markers, and secretion of adiponectin over a 9-day post-differentiation period across genotypes. We used this model system to test the roles SR-BI and PCPE2in glucose and lipid metabolism. Compared to WT adipocytes, glucose uptake in SR-BI -/- and PCPE2 -/- adipocytes was decreased by 5.4% and 12.6%, respectively, despite increases in surface GLUT4 levels. We also observed that adipocytes lacking PCPE2 had an increased response to insulin through AKT signaling, which may be responsible for the increased surface expression of GLUT4. Loss of SR-BI or PCPE2 in adipocytes did not affect palmitic acid uptake, despite elevated mRNA expression of fatty acid transporters ( Cd36, Fabp4 ) compared to WT adipocytes. SR-BI -/- and PCPE2 -/- adipocytes showed an inverse response to insulin when measuring phosphorylation of fatty acid regulatory proteins (AMPK, ACC) compared to WT adipocytes, suggesting altered lipid metabolism in adipocytes lacking SR-BI or PCPE2. Together, our data suggest that, individually, SR-BI or PCPE2 is important for glucose uptake and fatty acid regulation, but not fatty acid uptake. Further investigations will determine whether a partnership is required for optimal metabolic function in adipocytes.

Objective: Small GTPase Rap1 (Ras-association proximate 1) is a novel, positive regulator of NO release and endothelial function with a potentially key role in mechanosensing of atheroprotective, laminar flow. Our objective was to delineate the role of Rap1 in the progression of atherosclerosis and its specific functions in the presence and absence of laminar flow, to better define its role in endothelial mechanisms contributing to plaque formation and atherogenesis. Approach and Results: In a mouse atherosclerosis model, endothelial Rap1B deletion exacerbates atherosclerotic plaque formation. In the thoracic aorta, where laminar shear stress–induced NO is otherwise atheroprotective, plaque area is increased in Athero-Rap1B iΔEC (atherogenic endothelial cell–specific, tamoxifen-inducible Rap1A+Rap1B knockout) mice. Endothelial Rap1 deficiency also leads to increased plaque size, leukocyte accumulation, and increased CAM (cell adhesion molecule) expression in atheroprone areas, whereas vascular permeability is unchanged. In endothelial cells, in the absence of protective laminar flow, Rap1 deficiency leads to an increased proinflammatory TNF-α (tumor necrosis factor alpha) signaling and increased NF-κB (nuclear factor kappa-light-chain-enhancer of activated B cells) activation and elevated inflammatory receptor expression. Interestingly, this increased signaling to NF-κB activation is corrected by AKTVIII—an inhibitor of Akt (protein kinase B) translocation to the membrane. Together, these data implicate Rap1 in restricting Akt-dependent signaling, preventing excessive cytokine receptor signaling and proinflammatory NF-κB activation. Conclusions: Via 2 distinct mechanisms, endothelial Rap1 protects from the atherosclerosis progression in the presence and absence of laminar flow; Rap1-stimulated NO release predominates in laminar flow, and restriction of proinflammatory signaling predominates in the absence of laminar flow. Our studies provide novel insights into the mechanisms underlying endothelial homeostasis and reveal the importance of Rap1 signaling in cardiovascular disease.

Cellular membranes are not homogenous mixtures of proteins; rather, they are segregated into microdomains on the basis of preferential association between specific lipids and proteins. These microdomains, called lipid rafts, are well known for their role in receptor signaling on the plasma membrane (PM) and are essential to such cellular functions as signal transduction and spatial organization of the PM. A number of disease states, including atherosclerosis and other cardiovascular disorders, may be caused by dysfunctional maintenance of lipid rafts. Lipid rafts do not occur only in the PM but also have been found in intracellular membranes and extracellular vesicles (EVs). Here, we focus on discussing newly discovered functions of lipid rafts and microdomains in intracellular membranes, including lipid and protein trafficking from the ER, Golgi bodies, and endosomes to the PM, and we examine lipid raft involvement in the production and composition of EVs. Because lipid rafts are small and transient, visualization remains challenging. Future work with advanced techniques will continue to expand our knowledge about the roles of lipid rafts in cellular functioning.

Purpose of review The primary cardioprotective function of high-density lipoprotein (HDL) is to remove excess cellular free cholesterol (FC) from peripheral tissues and deliver it to the liver. Here, we summarize recent research that examines apolipoprotein A-I (apoA-I) lipidation models by adenosine triphosphate binding cassette transporter A1 (ABCA1) and discuss its relevance in atherosclerotic cardiovascular disease (ASCVD). Recent findings The first step in HDL formation involves the interaction between apoA-I and ABCA1, where ABCA1 mediates the removal of FC and phospholipids from lipid-laden macrophages to form discoidal nascent HDL (nHDL). However, there are currently no clear-cut systematic models that characterize HDL formation. A number of recent studies have investigated the importance of apoA-I C- and N-terminal domains required for optimal cholesterol efflux and nHDL production. Furthermore, functional ABCA1 is required for direct or indirect binding to apoA-I where ABCA1 dimer-monomer interconversion facilitates apoA-I lipidation from plasma membrane microdomains. Microparticles are also another lipid source for apoA-I solubilization into nHDL. Summary ApoA-I and ABCA1 are key factors in macrophage-mediated cholesterol efflux and nHDL production. Understanding of the key steps in HDL formation may unlock the therapeutic potential of HDL and improve clinical management of ASCVD.

Obesity continues to be an epidemic in the United States, placing individuals at a higher risk of developing other comorbidities such as type 2 diabetes and cardiovascular disease. Adipocytes accumulate excess triglycerides and undergo hypertrophic expansion as a protective mechanism to avoid lipotoxicity. Adipocytes are also highly dynamic cells that perform many key metabolic processes including lipid and glucose metabolism. Recent collaborative studies demonstrated that SR-BI function may require the extracellular matrix protein procollagen C-endopeptidase enhancer 2 (PCPE2) in to regulate cholesterol transport in hepatocytes and adipocytes. However, the roles of SR-BI and PCPE2 in other adipocyte metabolic processes remain unclear. This knowledge gap prompted our novel hypothesis that PCPE2 facilitates SR-BI’s ability to regulate metabolic processes in adipocytes. To test our hypothesis, we have optimized an adipocyte model system where mesenchymal stem cells isolated from the outer ears of wild-type (WT), SR-BI knockout (SR-BI -/- ), and PCPE2 -/- mice were differentiated into adipocyte-like cells. We validated our adipocyte model system by demonstrating increased Oil Red O staining, mRNA expression of adipogenesis markers, and adiponectin secretion over a 9-day post-differentiation period for all three genotypes. This model system was then used to test the roles of SR-BI and PCPE2 on various metabolic functions. Preliminary data suggests that absence of SR-BI or PCPE2 slightly impairs fatty acid uptake into adipocytes despite elevated gene expression of fatty acid transporters (FABP4, CD36) compared to WT. These data further suggest that SR-BI may rely on PCPE2 to aid in transport of fatty acids. Adipocytes lacking SR-BI or PCPE2 also displayed higher levels of mitochondrial reactive oxygen species, indicating that SR-BI and PCPE2 may be important for maintaining healthy mitochondria in adipocytes. These changes in function do not appear to depend on SR-BI oligomerization, as BS 3 crosslinking studies demonstrated the presence of SR-BI oligomers in adipocytes despite the loss of PCPE2. Altogether, these data suggest that SR-BI and PCPE2 may be reliant on each other and are important for mediating metabolic processes in adipocytes.

Plaque psoriasis is a common inflammatory condition of the skin characterized by red, flaking lesions. Current therapies for plaque psoriasis target many facets of the autoimmune response, but there is an incomplete understanding of how oxidative damage produced by enzymes such as myeloperoxidase contributes to skin pathology. In this study, we used the Aldara (Imiquimod) cream model of plaque psoriasis in mice to assess myeloperoxidase inhibition for treating psoriatic skin lesions. To assess skin inflammation severity, an innovative mouse psoriasis scoring system was developed. We found that myeloperoxidase inhibition ameliorated psoriasis severity when administered either systemically or topically. The findings of this study support the role of oxidative damage in plaque psoriasis pathology and present potential new therapeutic avenues for further exploration.

RATIONALE: Single nucleotide polymorphisms (SNPs) near the ILRUN gene are genome-wide significantly associated with plasma lipid traits and coronary artery disease (CAD) but the biological basis of this association is unknown. OBJECTIVE: To investigate the role of ILRUN in plasma lipid and lipoprotein metabolism. METHODS AND RESULTS: ILRUN encodes a protein that contains a ubiquitin-associated (UBA)-like domain, suggesting that it may interact with ubiquitinylated proteins. We generated mice globally deficient for Ilrun (IlrunKO) and found they had significantly lower plasma cholesterol levels resulting from reduced liver lipoprotein production. Liver transcriptome analysis uncovered altered transcription of genes downstream of lipid-related transcription factors, particularly PPARα, and livers from IlrunKO mice had increased PPARα protein. Human ILRUN was shown to bind to ubiquitinylated proteins including PPARα, and the UBA-like domain of ILRUN was found to be required for its interaction with PPARα. CONCLUSIONS: These findings establish ILRUN as a novel regulator of lipid metabolism that promotes hepatic lipoprotein production. Our results also provide functional evidence that ILRUN may be the casual gene underlying the observed genetic associations with plasma lipids at 6p21 in human.

Intestinal HDL is hepatoprotective High-density lipoprotein (HDL) is important for cholesterol metabolism and may have anti-inflammatory and antimicrobial properties. Although HDL is mainly produced by the liver, the intestine is also a source. Han et al. show in mice that intestinal HDL is not routed to the systemic circulation. Rather, in the form of HDL3, it is directly transported to the liver through the hepatic portal vein. There, it sequesters bacterial lipopolysaccharide from the gut that can trigger inflammation and liver damage. In various models of liver injury, loss of enteric HDL exacerbated pathology. By contrast, drugs elevating intestinal HDL improved disease outcomes. HDL3 is enriched in human portal venous blood, suggesting that enteric HDL may be targetable for the treatment of liver disease. Science , abe6729, this issue p. eabe6729

Adiponectin exerts its atheroprotection by stimulating adenosine triphosphate binding cassette transporter A1 (ABCA1)-mediated cholesterol efflux to apolipoprotein A-I (apoA-I). However, involvement of the apoA-I residues in this process have not been studied. In Tamm-Horsfall 1 (THP-1) macrophages and baby hamster kidney (BHK) cells we assessed adiponectin's potential to restore cholesterol efflux in the presence of apoA-I and ABCA1 mutants, respectively. Adiponectin was unable to restore efflux from THP-1 macrophages in the presence of apoA-I carboxy-terminal domain (CTD) successive mutants from residues 187-243 versus apoA-I mutants alone. Furthermore, adiponectin did not significantly influence cholesterol efflux to apoA-I from BHK-ABCA1 mutant cells. Adiponectin appears to require functional apoA-I CTD residues 187-243 and wild-type ABCA1 to mediate efficient cholesterol efflux from THP-1 macrophages and BHK cells, respectively. Therefore, adiponectin cannot rescue defective cholesterol efflux in apoA-I- or ABCA1-mutant conditions, but rather increases cholesterol efflux in wild-type apoA-I conditions compared to apoA-I exposure alone.

The HDL (high-density lipoprotein) Workshop was established in 2009 as a forum for candid discussions among academic basic scientists, clinical investigators, and industry researchers about the role of HDL in cardiovascular disease. This ninth HDL Workshop was held on May 16 to 17, 2019 in Boston, MA, and included outstanding oral presentations from established and emerging investigators. The Workshop featured 5 sessions with topics that tackled the role of HDL in the vasculature, its structural complexity, its role in health and disease states, and its interaction with the intestinal microbiome. The highlight of the program was awarding the Jack Oram Award to the distinguished professor emeritus G.S. Getz from the University of Chicago. The tenth HDL Workshop will be held on May 2020 in Chicago and will continue the focus on intellectually stimulating presentations by established and emerging investigators on novel roles of HDL in cardiovascular and noncardiovascular health and disease states.

Apolipoprotein (apo)A-I is an organizing scaffold protein that is critical to high-density lipoprotein (HDL) structure and metabolism, probably mediating many of its cardioprotective properties. However, HDL biogenesis is poorly understood, as lipid-free apoA-I has been notoriously resistant to high-resolution structural study. Published models from low-resolution techniques share certain features but vary considerably in shape and secondary structure. To tackle this central issue in lipoprotein biology, we assembled a team of structural biologists specializing in apolipoproteins and set out to build a consensus model of monomeric lipid-free human apoA-I. Combining novel and published cross-link constraints, small-angle X-ray scattering (SAXS), hydrogen-deuterium exchange (HDX) and crystallography data, we propose a time-averaged model consistent with much of the experimental data published over the last 40 years. The model provides a long-sought platform for understanding and testing details of HDL biogenesis, structure and function.

Purpose of review Scavenger receptor BI (SR-BI) is classically known for its role in antiatherogenic reverse cholesterol transport as it selectively takes up cholesterol esters from HDL. Here, we have highlighted recent literature that describes novel functions for SR-BI in physiology and disease. Recent findings A large population-based study has revealed that patients heterozygous for the P376L mutant form of SR-BI showed significantly increased levels of plasma HDL-cholesterol and had increased risk of cardiovascular disease, demonstrating that SR-BI in humans is a significant determinant of cardiovascular disease. Furthermore, SR-BI has been shown to modulate the susceptibility to LPS-induced tissue injury and the ability of sphingosine 1 phosphate to interact with its receptor, linking SR-BI to the regulation of inflammation. In addition, important domains within the molecule (Trp-415) as well as novel regulators (procollagen C-endopeptidase enhancer protein 2) of SR-BI's selective uptake function have recently been identified. Moreover, relatively high expression levels of the SR-BI protein have been observed in a variety of cancer tissues, which is associated with a reduced overall survival rate. Summary The HDL receptor SR-BI is a potential therapeutic target not only in the cardiovascular disease setting, but also in inflammatory conditions as well as in cancer.

HDL normally transports about 50–70% of plasma sphingosine 1-phosphate (S1P), and the S1P in HDL reportedly mediates several HDL-associated biological effects and signaling pathways. The HDL receptor, SR-BI, as well as the cell surface receptors for S1P (S1PRs) may be involved partially and/or completely in these HDL-induced processes. Here we investigate the nature of the HDL-stimulated interaction between the HDL receptor, SR-BI, and S1PR1 using a protein-fragment complementation assay and confocal microscopy. In both primary rat aortic vascular smooth muscle cells and HEK293 cells, the S1P content in HDL particles increased intracellular calcium concentration, which was mediated by S1PR1. Mechanistic studies performed in HEK293 cells showed that incubation of cells with HDL led to an increase in the physical interaction between the SR-BI and S1PR1 receptors that mainly occurred on the plasma membrane. Model recombinant HDL (rHDL) particles formed in vitro with S1P incorporated into the particle initiated the internalization of S1PR1, whereas rHDL without supplemented S1P did not, suggesting that S1P transported in HDL can selectively activate S1PR1. In conclusion, these data suggest that S1P in HDL stimulates the transient interaction between SR-BI and S1PRs that can activate S1PRs and induce an elevation in intracellular calcium concentration.

Cellular membranes are not homogenous mixtures of proteins; rather, they are segregated into microdomains on the basis of preferential association between specific lipids and proteins. These microdomains, called lipid rafts, are well known for their role in receptor signaling on the plasma membrane (PM) and are essential to such cellular functions as signal transduction and spatial organization of the PM. A number of disease states, including atherosclerosis and other cardiovascular disorders, may be caused by dysfunctional maintenance of lipid rafts. Lipid rafts do not occur only in the PM but also have been found in intracellular membranes and extracellular vesicles (EVs). Here, we focus on discussing newly discovered functions of lipid rafts and microdomains in intracellular membranes, including lipid and protein trafficking from the ER, Golgi bodies, and endosomes to the PM, and we examine lipid raft involvement in the production and composition of EVs. Because lipid rafts are small and transient, visualization remains challenging. Future work with advanced techniques will continue to expand our knowledge about the roles of lipid rafts in cellular functioning.

Apolipoprotein A1 (ApoA1), the major constituent of the high-density lipoprotein (HDL) molecule, exhibits anti-inflammatory properties. Our laboratory has previously shown that ApoA1 protects against switching of regulatory T (Treg) cells to atherogenic T follicular helper cells in Western diet-fed mice. However, the role of ApoA1 in modulating Treg cell homeostasis in the absence of atherosclerosis remains uncharacterized. Here, we show that ApoA1 is required for normal Treg cell homeostasis and functioning at steady state. Specifically, lack of ApoA1 decreased the numbers of both natural and induced Treg cells and also lowered Treg cell-based homeostatic proliferation and suppressive functions. Importantly, these changes occurred without affecting other T cell populations. Finally, we determined that the observed phenotypes were caused by changes to cholesterol content and reduced interleukin-2 (IL-2) receptor signaling in ApoA1-deficient Treg cells. Overall, our results show that ApoA1-HDL is necessary for Treg cell homeostasis and functioning.

Identifying the causal gene(s) that connect genetic variation to a phenotype is a challenging problem in genome-wide association studies (GWASs). Here, wse develop a systematic approach that integrates mouse liver co-expression networks with human lipid GWAS data to identify regulators of cholesterol and lipid metabolism. Through our approach, we identified 48 genes showing replication in mice and associated with plasma lipid traits in humans and six genes on the X chromosome. Among these 54 genes, 25 have no previously identified role in lipid metabolism. Based on functional studies and integration with additional human lipid GWAS datasets, we pinpoint Sestrin1 as a causal gene associated with plasma cholesterol levels in humans. Our validation studies demonstrate that Sestrin1 influences plasma cholesterol in multiple mouse models and regulates cholesterol biosynthesis. Our results highlight the power of combining mouse and human datasets for prioritization of human lipid GWAS loci and discovery of lipid genes.

Objective— Aim of this study was to evaluate changes in LCAT (lecithin:cholesterol acyltransferase) concentration and activity in patients with an acute coronary syndrome, to investigate if these changes are related to the compromised capacity of HDL (high-density lipoprotein) to promote endothelial nitric oxide (NO) production, and to assess if rhLCAT (recombinant human LCAT) can rescue the defective vasoprotective HDL function. Approach and Results— Thirty ST-segment–elevation myocardial infarction (STEMI) patients were enrolled, and plasma was collected at hospital admission, 48 and 72 hours thereafter, at hospital discharge, and at 30-day follow-up. Plasma LCAT concentration and activity were measured and related to the capacity of HDL to promote NO production in cultured endothelial cells. In vitro studies were performed in which STEMI patients’ plasma was added with rhLCAT and HDL vasoprotective activity assessed by measuring NO production in endothelial cells. The plasma concentration of the LCAT enzyme significantly decreases during STEMI with a parallel significant reduction in LCAT activity. HDL isolated from STEMI patients progressively lose the capacity to promote NO production by endothelial cells, and the reduction is related to decreased LCAT concentration. In vitro incubation of STEMI patients’ plasma with rhLCAT restores HDL ability to promote endothelial NO production, possibly related to significant modification in HDL phospholipid classes. Conclusions— Impairment of cholesterol esterification may be a major factor in the HDL dysfunction observed during acute coronary syndrome. rhLCAT is able to restore HDL-mediated NO production in vitro, suggesting LCAT as potential therapeutic target for restoring HDL functionality in acute coronary syndrome.

Rap1, a ubiquitously expressed small GTPase, integrates signals from multiple receptors and promotes activation and signaling by cell adhesion receptors. In vivo , endothelial (EC) Rap1 is required for normal vessel formation, angiogenesis and dynamic regulation of EC barrier. Two Rap1 isoforms, Rap1A and Rap1B, share 95% identity, and EC deletion of both isoforms leads to embryonic lethality due to cardiovascular defects. We have recently demonstrated that Rap1 is a novel regulator of shear sensing in endothelium and that it is essential for nitric oxide release, and that Rap1 deficiency leads to EC dysfunction. These findings demonstrated that Rap1 plays an essential role in vasoprotective response of endothelium in response to shear stress. The objective of this study is to determine the specific role of Rap1 response to laminar, vasoprotective flow and pro-inflammatory, disturbed flow by examining the effect of EC deletion of Rap1B, the more prominent Rap1 isoform, on progression of atherosclerosis. To investigate the role of Rap1 in progression of atherosclerosis, we crossed apolipoprotein E-deficient (ApoE -/- ) mice with tamoxifen-inducible, EC specific Rap1B-knockout (Cadh5-CreERT +/0 ; Rap1B f/f ) to generate atherogenic EC-Rap1B KO (Athero-Rap1B KO) mice. We hypothesized that Rap1B deficiency will exacerbate progression of atherosclerosis. Athero-Rap1B KO and littermate controls (Cadh5-Cre-negative mice or mice injected with carrier oil only) were fed high fat, western diet (21.2% fat, 0.2% cholesterol) for 16 weeks. No difference in total cholesterol levels were observed between Athero- Rap1B KO and control mice. Atherosclerotic lesion formation was visualized with oil O red and plaque area was quantified in en-face preparations. (n=5-9). We observed increased lesion area in the descending aorta of Athero-Rap1B KO mice (0.7351 ± 0.1065 vs 0.4866 ± 0.09812; p =0.0584, one-tailed t-test, Welch corrected) and a strong trend towards larger lesion area in the total aorta (2.142 ± 0.5058 vs 1.267 ± 0.1718; p=0.0679), and the arch of the aorta (1.489 ± 0.4343 vs 0.7807 ± 0.1152; p=0.0747) compared with control mice. These findings demonstrate that Rap1B deficiency accelerates progression of atherosclerosis and show that Rap1 plays a vasoprotective role in both laminar flow and disturbed flow areas.

Adipose tissue undergoes distinct structural remodeling in response to increases in fat mass, as in obesity. During this process, the extracellular matrix (ECM) plays a pivotal role with respect to both adipocyte receptor signaling and structural remodeling. One ECM protein, procollagen endopeptidase enhancer 2 (PCPE2), has been shown by our lab to work in partnership with the scavenger receptor class B type I (SR-BI) to regulate adipocyte cholesterol homeostasis. To investigate the functional impact of the partnership between SR-BI and PCPE2, we studied mature adipocytes differentiated from murine embryonic fibroblasts (MEF) and compared to our adipose specific PCPE2 knockout mice. Our studies showed the presence of an SR-BI-PCPE2 protein complex based on the co-immunoprecipitation of PCPE2 with SR-BI in both differentiated MEFs and mouse adipose tissue. Furthermore, stimulated emission depletion (STED) microscopy showed SR-BI and PCPE2 localized on the lipid droplet surface separated by distances ranging from 24 – 90 nm, suggesting that the SR-BI-PCPE2 complex may contain other proteins. Most significantly, a loss of SR-BI function was shown in PCPE2-deficient adipocytes by the SR-BI mediated HDL binding and cholesteryl ester (CE) uptake assays. Compared to differentiated MEFs from Ldlr -/- mice, binding of HDL to differentiated MEFs from Ldlr -/- Pcpe2 -/- mice was reduced by 75%, and HDL cholesteryl oleyl ether uptake was reduced by 50%. Disruption of adipocyte SR-BI function in the absence of PCPE2 also reduced free cholesterol efflux indicating substantial disruption to intracellular cholesterol homeostasis. Moreover, analysis of RNAseq from several human clinical studies show that PCPE2 mRNA expression is positively correlated with fat mass in specific adipose depots, while negatively correlated with plasma insulin, glucose and triglyceride levels, with the latter two more robust in type 2 diabetic populations (FDR < 0.004). Overall, these results suggest that the partnership between SR-BI and PCPE2 in adipocytes plays an important role in cholesterol trafficking and homeostasis which influences fat mass expansion, glucose tolerance and insulin resistance.

Regulatory T (Treg) cells contribute to the anti-inflammatory response during atherogenesis. Here we show that during atherogenesis Treg cells lose Foxp3 expression and their immunosuppressive function, leading to the conversion of a fraction of these cells into T follicular helper (Tfh) cells. We show that Tfh cells are pro-atherogenic and that their depletion reduces atherosclerosis. Mechanistically, the conversion of Treg cells to Tfh cells correlates with reduced expression of IL-2Rα and pSTAT5 levels and increased expression of IL-6Rα. In vitro, incubation of naive T cells with oxLDL prevents their differentiation into Treg cells. Furthermore, injection of lipid-free Apolipoprotein AI (ApoAI) into ApoE−/− mice reduces intracellular cholesterol levels in Treg cells and prevents their conversion into Tfh cells. Together our results suggest that ApoAI, the main protein in high-density lipoprotein particles, modulates the cellular fate of Treg cells and thus influences the immune response during atherosclerosis., Regulatory T (Treg) cells contribute to the anti-inflammatory response during atherogenesis. Here Gaddis et al. show that Apolipoprotein AI prevents the conversion of Treg cells into pro-atherogenic T follicular helper cells, and thus regulates the immune response during atherogenesis.

The formation of the atherosclerotic plaque that is characterized by the accumulation of abnormal amounts of cholesterol-loaded macrophages in the artery wall is mediated by both inflammatory events and alterations of lipid/lipoprotein metabolism. Reverse transport of cholesterol opposes the formation and development of atherosclerotic plaque by promoting high density lipoprotein (HDL)-mediated removal of cholesterol from peripheral macrophages and its delivery back to the liver for excretion into the bile. Although an inverse association between HDL plasma levels and the risk of cardiovascular disease (CVD) has been demonstrated over the years, several studies have recently shown that the antiatherogenic functions of HDL seem to be mediated by their functionality, not always associated with their plasma concentrations. Therefore, assessment of HDL function, evaluated as the capacity to promote cell cholesterol efflux, may offer a better prediction of CVD than HDL levels alone. In agreement with this idea, it has recently been shown that the assessment of serum cholesterol efflux capacity (CEC), as a metric of HDL functionality, may represent a predictor of atherosclerosis extent in humans. The purpose of this narrative review is to summarize the current evidence concerning the role of cholesterol efflux capacity that is important for evaluating CVD risk, focusing on pharmacological evidences and its relationship with inflammation. We conclude that HDL therapeutics are a promising area of investigation but strategies for identifying efficacy must move beyond the idea of simply raising static HDL-cholesterol levels and toward methods of measuring the dynamics of HDL particle remodeling and the generation of lipid-free apolipoprotein A-I (apoA-I). In this way, apoA-I, unlike mature HDL, can promote the greatest extent of cholesterol efflux relieving cellular cholesterol toxicity and the inflammation it causes.

Background Subcutaneously injected lipid‐free apoA‐I (apolipoprotein A‐I) reduces accumulation of lipid and immune cells within the aortic root of hypercholesterolemic mice without increasing high‐density lipoprotein–cholesterol concentrations. Lymphatic vessels are now recognized as prerequisite players in the modulation of cholesterol removal from the artery wall in experimental conditions of plaque regression, and particular attention has been brought to the role of the collecting lymphatic vessels in early atherosclerosis‐related lymphatic dysfunction. In the present study, we address whether and how preservation of collecting lymphatic function contributes to the protective effect of apoA‐I. Methods and Results Atherosclerotic Ldlr −/− mice treated with low‐dose lipid‐free apoA‐I showed enhanced lymphatic transport and abrogated collecting lymphatic vessel permeability in atherosclerotic Ldlr −/− mice when compared with albumin‐control mice. Treatment of human lymphatic endothelial cells with apoA‐I increased the adhesion of human platelets on lymphatic endothelial cells, in a bridge‐like manner, a mechanism that could strengthen endothelial cell–cell junctions and limit atherosclerosis‐associated collecting lymphatic vessel dysfunction. Experiments performed with blood platelets isolated from apoA‐I‐treated Ldlr −/− mice revealed that apoA‐I decreased ex vivo platelet aggregation. This suggests that in vivo apoA‐I treatment limits platelet thrombotic potential in blood while maintaining the platelet activity needed to sustain adequate lymphatic function. Conclusions Altogether, we bring forward a new pleiotropic role for apoA‐I in lymphatic function and unveil new potential therapeutic targets for the prevention and treatment of atherosclerosis.

Atherosclerosis is a chronic inflammatory disease of large and medium-sized arteries and the underlying cause of cardiovascular disease, a major cause of mortality worldwide. The over-accumulation of modified cholesterol-containing low-density lipoproteins (e.g. oxLDL) in the artery wall and the subsequent recruitment and activation of macrophages contributes to the development of atherosclerosis. The excessive uptake of modified-LDL by macrophages leads to a lipid-laden "foamy" phenotype and pro-inflammatory cytokine production. Modified-LDLs promote foam cell formation in part by stimulating de novo lipid biosynthesis. However, it is unknown if lipid biosynthesis directly regulates foam cell pro-inflammatory mediator production. Lipin-1, a phosphatidate phosphohydrolase required for the generation of diacylglycerol during glycerolipid synthesis has recently been demonstrated to contribute to bacterial-induced pro-inflammatory responses by macrophages. In this study we present evidence demonstrating the presence of lipin-1 within macrophages in human atherosclerotic plaques. Additionally, reducing lipin-1 levels in macrophages significantly inhibits both modified-LDL-induced foam cell formation in vitro, as observed by smaller/fewer intracellular lipid inclusions, and ablates modified-LDL-elicited production of the pro-atherogenic mediators tumor necrosis factor-α, interleukin-6, and prostaglandin E2. These findings demonstrate a critical role for lipin-1 in the regulation of macrophage inflammatory responses to modified-LDL. These data begin to link the processes of foam cell formation and pro-inflammatory cytokine production within macrophages.

Purpose of review The purpose of this study is to provide an update on the role HDL apolipoprotein A-I plays in reducing the risk of cardiovascular disease (CVD) and how it relates to reverse cholesterol transport (RCT). Recent findings Despite numerous studies showing that plasma HDL cholesterol concentrations are correlated with a reduced risk of CVD, pharmacologic elevation of HDL has not shown any beneficial effects to date. In contrast, studies correlating the measure of an individual's plasma cholesterol efflux capacity show greater promise as a tool for assessing CVD risk. Although ATP-binding cassette transporter 1-mediated lipidation of apoA-I is considered the principal source of plasma HDL, it represents only one side of the RCT pathway. Equally important is the second half of the RCT pathway in which the liver scavenger receptor class B1 selectively removes HDL cholesteryl esters for excretion. The combined action of the two enzyme systems is reflected in the overall steady-state concentration of plasma HDL cholesterol. For example, reduced ATP-binding cassette transporter 1-mediated production of nascent HDL lowers plasma HDL concentration, just as an increase in cholesteryl ester uptake by scavenger receptor class B1 reduces HDL levels. Thus, the complexity of intravascular HDL metabolism suggests that steady-state plasma HDL concentrations do not provide adequate information regarding an individual's HDL quality or function. Herein, we describe a new player, procollagen C-endopeptidase enhancer 2, which shows atheroprotective function and influences both sides of RCT by enhancing production and catabolism of HDL cholesteryl esters. Summary The discovery of a new molecule, procollagen C-endopeptidase enhancer 2, implicated in the regulation of HDL cholesteryl ester concentrations suggests that the extracellular matrix and the proteins that regulate its function represent a new and as yet unexplored realm of HDL cholesterol metabolism.

Tissue cholesterol accumulation, macrophage infiltration, and inflammation are features of atherosclerosis and some forms of dermatitis. HDL and its main protein, apoAI, are acceptors of excess cholesterol from macrophages; this process inhibits tissue inflammation. Recent epidemiologic and clinical trial evidence questions the role of HDL and its manipulation in cardiovascular disease. We investigated the effect of ectopic macrophage apoAI expression on atherosclerosis and dermatitis induced by the combination of hypercholesterolemia and absence of HDL in mice. Hematopoietic progenitor cells were transduced to express human apoAI and transplanted into lethally irradiated LDL receptor(-/-)/apoAI(-/-) mice, which were then placed on a high-fat diet for 16 weeks. Macrophage apoAI expression reduced aortic CD4(+) T-cell levels (-39.8%), lesion size (-25%), and necrotic core area (-31.6%), without affecting serum HDL or aortic macrophage levels. Macrophage apoAI reduced skin cholesterol by 39.8%, restored skin morphology, and reduced skin CD4(+) T-cell levels. Macrophage apoAI also reduced CD4(+) T-cell levels (-32.9%) in skin-draining lymph nodes but had no effect on other T cells, B cells, dendritic cells, or macrophages compared with control transplanted mice. Thus, macrophage apoAI expression protects against atherosclerosis and dermatitis by reducing cholesterol accumulation and regulating CD4(+) T-cell levels, without affecting serum HDL or tissue macrophage levels.

HDLs appear to affect regulatory T cell (Treg) homeostasis, as suggested by the increased Treg counts in HDL-treated mice and by the positive correlation between Treg frequency and HDL-cholesterol levels in statin-treated healthy adults. However, the underlying mechanisms remain unclear. Herein, we show that HDLs, not LDLs, significantly decreased the apoptosis of human Tregs in vitro, whereas they did not alter naïve or memory CD4+ T cell survival. Similarly, oleic acid bound to serum albumin increased Treg survival. Tregs bound and internalized high amounts of HDL compared with other subsets, which might arise from the higher expression of the scavenger receptor class B type I by Tregs; accordingly, blocking this receptor hindered HDL-mediated Treg survival. Mechanistically, we showed that HDL increased Treg ATP concentration and mitochondrial activity, enhancing basal respiration, maximal respiration, and spare respiratory capacity. Blockade of FA oxidation by etoxomir abolished the HDL-mediated enhanced survival and mitochondrial activity. Our findings thus suggest that Tregs can specifically internalize HDLs from their microenvironment and use them as an energy source. Furthermore, a novel implication of our data is that enhanced Treg survival may contribute to HDLs’ anti-inflammatory properties.

The article by Mao et al1 shows that apolipoprotein A-I binding protein (AIBP) limits angiogenesis through regulation of Notch 1 signaling. Deletion of AIBP increased vascular density and improved perfusion in a murine ischemia model. AIBP expression was increased in patients with ischemic cardiomyopathy. In an independent study, this protein, renamed NAXE, has been shown to participate in recovering S-tetrahydro-nicotinamide adenine dinucleotide (S-NADHX) by epimerizing R-NADHX. So the question becomes how does an enzyme that participates in metabolite repair by recovering reducing intracellular equivalents participate in angiogenesis? Article, see p 1727 AIBP has been reported to avidly bind apolipoprotein A-1 (apoA-I), the principal protein component of high-density lipoprotein (HDL). Loss of AIBP was found to accelerate angiogenesis without affecting the integrity of vessel walls. Using Apoa1bp−/− mice, the present article has provided evidence that AIBP plays a role in angiogenesis that involves cholesterol metabolism and Notch signaling.1 These studies suggest that AIBP downregulates Notch 1 by a process that depends on γ-secretase activity. γ-Secretase is an …

Scavenger receptor class B member 1 (SR-BI) functions as a HDL receptor and stimulates reverse cholesterol transport (RCT) via selective uptake of HDL cholesterol esters (HDL-CE). Procollagen c-endopeptidase enhancer protein 2 (PCPE2) is an extracellular matrix glycoprotein encoded by the PCOLCE2 gene and most highly expressed in adipose tissue, heart, and aorta. PCPE2 has been suggested to be atheroprotective secondary to mechanisms involved in lipid metabolism. Our lab previously reported that SR-BI mediated cholesterol ester uptake is reduced in the absence of PCPE2 despite its increased expression levels. Subsequent HDL turnover studies suggested an overall reduction in catabolism, consistent with larger HDL particles observed, is responsible for the paradox. More recently, human data from the TwinsUK study showed strong correlations between PCOLCE2 and adipose tissue distribution with BMI included as a covariate. Furthermore, despite similar overall body weights, our lab observed a significant reduction in visceral fat pad development in PCPE2-deficient mice fed a Western diet for 25 weeks when compared to wild type mice. In addition, our lab has generated PCPE2 knockout cell lines from murine preadipocyte 3T3-L1 cells (PCPE2 -/- 3T3-L1) and Chinese hamster ovary (CHO) cells using CRISPR-Cas9 and siRNA technology, respectfully. In addition to both cell lines showing increased SR-BI expression, differentiated PCPE2 -/- 3T3-L1 cells have impaired lipid droplet formation. To elucidate the relationship between PCPE2 and SR-BI function and their effects on adipose tissue development, we performed RNA sequencing on visceral adipose tissue from PCPE2-deficient male mice fed a Western diet for 12 weeks. Genes exhibiting significant expression alterations (FDR

HDL is cardioprotective, but plasma HDL levels do not necessarily predict cardiovascular outcomes. The major HDL-associated protein apoA-I picks up its cholesterol from cells within extravascular compartments to return it to plasma and then bile. Yet, tools are lacking to quantify the important step of HDL transit through extravascular spaces. Here, we developed recombinant photoactivatable apoA-I to quantify endogenous HDL recirculation. Using the tool, we studied HDL passage through skin in healthy mice versus those with experimental psoriasis, wherein collagen density increased in the skin in a CD4 + T cell-dependent manner. In control mice, photoactivated HDL mobilized to plasma within 2 h but was retained in collagen-enriched skin of mice with psoriasis. These data suggest that cardiovascular comorbidity in psoriasis might be linked to T cell-mediated structural changes in skin that impedes systemic recirculation of HDL. This new tool is likely to find wide application in HDL research.