Human genetic studies have repeatedly associated ADAMTS7 with atherosclerotic cardiovascular disease. Subsequent investigations in mice demonstrated that ADAMTS7 is proatherogenic and induced in response to vascular injury. However, the cell-specific mechanisms governing ADAMTS7 proatherogenicity remain unclear. To determine which vascular cell types express ADAMTS7, we interrogated single-cell RNA-seq of human carotid atherosclerosis and found ADAMTS7 expression in smooth muscle cells (SMCs), endothelial cells (ECs), and fibroblasts. We subsequently created SMC- and EC-specific Adamts7 conditional KO and transgenic mice. Conditional KO of Adamts7 in either cell type did not reduce atherosclerosis, whereas transgenic induction in either cell type increased atherosclerosis. In SMC transgenic mice, this increase coincides with an expansion of lipid-laden SMC foam cells and a decrease in fibrous cap formation. RNA-seq of Adamts7-overexpressing SMCs revealed an upregulation of lipid genes typically assigned to macrophages. Mechanistically, ADAMTS7 increases SMC oxidized LDL uptake through CD36, whose expression is upregulated by PU.1. Assay for transposase-accessible chromatin using sequencing (ATAC-seq) and motif analysis revealed increased chromatin accessibility at AP-1-enriched regions, consistent with AP-1-dependent remodeling of PU.1-regulated lipid-handling loci. In summary, ADAMTS7 promotes atherosclerosis by driving SMC foam cell formation through an AP-1/PU.1/CD36 regulatory axis.

BACKGROUND:Atherosclerosis involves complex interactions between lipids, immune cells, vascular smooth muscle cells, and fibroblasts within the arterial wall. While significant advances in single-cell technologies have shed light on the roles of immune cells and vascular smooth muscle cells in plaque development, fibroblasts remain underexplored, leaving critical gaps in understanding their contributions to disease progression and plaque stability. Comprehensive characterization of fibroblast phenotypes in atherosclerosis is essential to unravel their diverse functions and to distinguish between subsets that may play protective versus pathogenic roles in the disease process.METHODS:Here, we utilized cellular indexing of transcriptomes and epitopes by sequencing to comprehensively profile fibroblast diversity in a mouse model of atherosclerosis. Mice were fed an atherogenic diet for 0, 8, 19, and 26 weeks, representing distinct stages of disease progression, enabling a detailed phenotypic characterization of fibroblasts throughout the course of atherosclerosis development.RESULTS:We identified 4 distinct fibroblast subpopulations, including a myofibroblast population closely resembling vascular smooth muscle cell-derived chondromyocytes. The proportions of these fibroblast subsets exhibited a modest decline as atherosclerosis progressed. Through multimodal analysis, we identified CD26 (cluster of differentiation) as a highly expressed and specific marker for one of these fibroblast subpopulations, distinguishing it from other subsets. Using a combination of flow cytometry and immunohistochemistry, we demonstrated that CD26+ fibroblasts predominantly reside in the adventitia of healthy arteries. During atherosclerosis progression, these cells expand into the intima and primarily localize within the fibrous cap of the lesion.CONCLUSIONS:Our multiomic analysis highlights the phenotypic diversity and dynamic changes of fibroblasts during atherosclerosis progression. Among these, CD26+ fibroblasts emerge as a distinct subpopulation that expands within atherosclerotic lesions and may play a critical role in promoting plaque stability through their migration into the fibrous cap.

BACKGROUND:Monocytes are a critical innate immune system cell type that serves homeostatic and immunoregulatory functions. They have been identified historically by the cell surface expression of CD14 and CD16. However, recent single-cell studies have revealed that they are much more heterogeneous than previously realized. METHODS:We utilized cellular indexing of transcriptomes and epitopes by sequencing (CITE-seq) and single-cell RNA sequencing to describe the comprehensive transcriptional and phenotypic landscape of 437 126 monocytes. RESULTS:This high-dimensional multimodal approach identified vast phenotypic diversity and functionally distinct subsets, including IFN-responsive, MHCIIhi (major histocompatibility complex class II), monocyte-platelet aggregates, as well as nonclassical, and several subpopulations of classical monocytes. Using flow cytometry, we validated the existence of MHCII+CD275+ MHCIIhi, CD42b+ monocyte-platelet aggregates, CD16+CD99- nonclassical monocytes, and CD99+ classical monocytes. Each subpopulation exhibited unique characteristics, developmental trajectories, transcriptional regulation, and tissue distribution. In addition, alterations associated with cardiovascular disease risk factors, including race, smoking, and hyperlipidemia were identified. Moreover, the effect of hyperlipidemia was recapitulated in mouse models of elevated cholesterol. CONCLUSIONS:This integrative and cross-species comparative analysis provides a new perspective on the comparison of alterations in monocytes in pathological conditions and offers insights into monocyte-driven mechanisms in cardiovascular disease and the potential for monocyte subpopulation targeted therapies.

Vascular smooth muscle cells (VSMCs) play a central role in the development of atherosclerosis due in part to their capability to phenotypically transition into either a protective or harmful state. However, the ability to identify and trace VSMCs and their progeny in vivo is limited due to the lack of well-defined VSMC cell surface markers. Therefore, investigations into VSMC fate must utilize lineage-tracing mouse models, which are time-consuming and challenging to generate and not feasible in humans. Here, we employed CITE-seq to characterize the phenotypic expression of 119 cell surface proteins in mouse atherosclerosis. We found that CD200 is a highly expressed and specific marker of VSMCs, which persists even with phenotypic modulation. We validated our findings using a combination of flow cytometry, qPCR, and immunohistochemistry, all confirming that CD200 can identify and mark VSMCs and their derived cells in early to advanced mouse atherosclerotic lesions. Additionally, we describe a similar expression pattern of CD200 in human coronary and carotid atherosclerosis. Thus, our data support the use of CD200 as a lineage marker for VSMCs and VSMC-derived cells in mouse and human atherosclerosis.

Atherosclerotic lesions are cellularly heterogeneous tissue comprised of diverse cell types including smooth muscle cells (SMCs), endothelial cells, and immune cells. The development of single cell technologies has provided the granular resolution required to identify new cell types present in plaques, aiding in our understanding of the pathophysiology of the disease. Transdifferentiation of SMCs during atherosclerosis progression to a synthetic phenotype has a major influence on disease severity and development. Therefore, to deeply characterize and immunophenotype SMCs in atherosclerotic plaques we performed cellular indexing of transcriptomes and epitopes by sequencing (CITE-seq) on six patients and scRNA-sequencing on fifteen patients undergoing carotid endarterectomy. Our initial analysis identified 25 unique cell populations, which consisted of 4 SMC populations which comprised about 14% of all cells in the dataset. Three of the SMC populations appeared to have a contractile phenotype, with high expression of SMC-specific genes like ACTA2, MYH11, and CNN1. The fourth SMC cluster had a modulated phenotype with downregulation of SMC genes and an up regulation of fibroblast genes such as COL1A2 and LUM. We also identified novel and uncharacterized SMC-specific cell surface proteins such as CD29, CD142, and EGR. The modulated SMC cluster specifically expressed CD90, giving us the unique ability to differentiate these similar yet distinct cell types. We then performed a subclustering analysis on SMC and fibroblast populations, identifying 8 SMC subpopulations. This was followed by Canonical Pathway analysis with Ingenuity Pathway Analysis (IPA) using the most highly expressed genes in each cluster to identify enriched pathways. This revealed a diverse array of functional differences between SMC populations. Our data support the idea that SMCs have a vast array of phenotypic states and functions. Future work will uncover the relevance of these newly identified cell types in the development of atherosclerosis and clinical events.

BACKGROUND:Atherosclerotic plaques are complex tissues composed of a heterogeneous mixture of cells. However, our understanding of the comprehensive transcriptional and phenotypic landscape of the cells within these lesions is limited. METHODS:To characterize the landscape of human carotid atherosclerosis in greater detail, we combined cellular indexing of transcriptomes and epitopes by sequencing and single-cell RNA sequencing to classify all cell types within lesions (n=21; 13 symptomatic) to achieve a comprehensive multimodal understanding of the cellular identities of atherosclerosis and their association with clinical pathophysiology. RESULTS:We identified 25 cell populations, each with a unique multiomic signature, including macrophages, T cells, NK (natural killer) cells, mast cells, B cells, plasma cells, neutrophils, dendritic cells, endothelial cells, fibroblasts, and smooth muscle cells (SMCs). Among the macrophages, we identified 2 proinflammatory subsets enriched in IL-1B (interleukin-1B) or C1Q expression, 2 TREM2-positive foam cells (1 expressing inflammatory genes), and subpopulations with a proliferative gene signature and SMC-specific gene signature with fibrotic pathways upregulated. Further characterization revealed various subsets of SMCs and fibroblasts, including SMC-derived foam cells. These foamy SMCs were localized in the deep intima of coronary atherosclerotic lesions. Utilizing cellular indexing of transcriptomes and epitopes by sequencing data, we developed a flow cytometry panel, using cell surface proteins CD29, CD142, and CD90, to isolate SMC-derived cells from lesions. Lastly, we observed reduced proportions of efferocytotic macrophages, classically activated endothelial cells, and contractile and modulated SMC-derived cells, while inflammatory SMCs were enriched in plaques of clinically symptomatic versus asymptomatic patients. CONCLUSIONS:Our multimodal atlas of cell populations within atherosclerosis provides novel insights into the diversity, phenotype, location, isolation, and clinical relevance of the unique cellular composition of human carotid atherosclerosis. These findings facilitate both the mapping of cardiovascular disease susceptibility loci to specific cell types and the identification of novel molecular and cellular therapeutic targets for the treatment of the disease.

Background: One of the hallmarks of atherosclerosis progression is the uptake of oxidized lipids by cells to become Foam/Foamy cells - the most prominent being vascular smooth muscle cells (VSMC)-foam cells. Although studies have identified the ability of VSMC to become VSMC-foam cells, their transcriptomic profile and the phenotypic modulations which distinguish them from contractile VSMC remain poorly defined and understood. Hypothesis: We hypothesize that VSMC-foamy cells differ from contractile VSMC not only by modulation of genes shared by both cell types, but also by the expression of a unique set of genes which contribute to their formation and maintenance within atherosclerotic lesions. Method: To address this, we performed bulk RNA sequencing of atherosclerotic plaques from conditional VSMC-lineage tracing mice models on high fat diet. VSMC and VSMC-foam cells were isolated and sequenced at specific time points reflective of late and advanced atherosclerosis disease progression. Computational analysis enabled us identify differentially expressed genes, biological pathways and molecular functions which distinguished VSMC from VSMC-foam cells. Result and Conclusion: Our computational analysis identified differential genes and pathways unique to VSMC-foam cells which might be associated with disease progression. From our analysis, VSMC showed a lower expression of contractile VSMC marker genes and a higher expression of genes in synthetic/intermediary multipotent state, we term “SEM” state. Our study also showed that VSMC-foam cells also expressed unique genes which distinguished them from contractile VSMC. These genes not only serve as potential genetic markers, but might further explain the nuclear composition, as well as the inflammatory and metabolic profile of VSMC-foam cells. Defining and understanding the molecular mechanisms and drivers which promote VSMC-foam cell formation and maintenance in atherosclerosis, would aid in our understanding of the disease with potentials for more effective therapeutic solutions.

CITE-seq, a single-cell multi-omics technology that measures RNA and protein expression simultaneously in single cells, has been widely applied in biomedical research, especially in immune related disorders and other diseases such as influenza and COVID-19. Despite the proliferation of CITE-seq, it is still costly to generate such data. Although data integration can increase information content, this raises computational challenges. First, combining multiple datasets is prone to batch effects that need to be addressed. Secondly, it is difficult to combine multiple CITE-seq datasets because the protein panels in different datasets may only partially overlap. Integrating multiple CITE-seq and single-cell RNA-seq (scRNA-seq) datasets is important because this allows the utilization of as many data as possible to uncover cell population heterogeneity. To overcome these challenges, we present sciPENN, a multi-use deep learning approach that supports CITE-seq and scRNA-seq data integration, protein expression prediction for scRNA-seq, protein expression imputation for CITE-seq, quantification of prediction and imputation uncertainty, and cell type label transfer from CITE-seq to scRNA-seq. Comprehensive evaluations spanning multiple datasets demonstrate that sciPENN outperforms other current state-of-the-art methods.

OBJECTIVE:Time-restricted feeding (TRF), whereby caloric intake is limited to a <12-hour window, is a potential regimen to ameliorate metabolic syndrome and cardiovascular disease (CVD) risk co-occurring with aging and with obesity. Early TRF (eTRF; early morning feeding followed by overnight fasting) times calorie consumption with hepatic circadian gene expression rhythms. Brief TRF trials demonstrate that high-density lipoprotein (HDL) cholesterol increases similar to diet/exercise interventions, which may impart beneficial CVD effects. Using a nonhuman primate (NHP) model, the efficacy of eTRF to raise HDL and increase plasma cholesterol efflux capacity (CEC) (primarily mediated by cholesterol efflux to HDL particles, a process that is inversely associated with CVD risk) was examined. METHODS:Adult (8-16 years old, n = 25) and geriatric (≥17 years old) NHPs were randomized to ad libitum feeding or eTRF for 12 months, and relevant body composition, glycemic control, and plasma HDL cholesterol levels and CEC were measured. RESULTS:Impaired CEC was found in geriatric NHPs. eTRF induced larger-sized HDL particles, increased HDL apolipoprotein A-1 content, lowered triglyceride concentrations, and increased plasma CEC (primarily to HDL particles) in both adult and geriatric NHPs without changes in glycemic control or body composition. CONCLUSIONS:A beneficial effect of eTRF on increasing HDL CEC in NHPs was demonstrated.

Preemptive identification of unstable, ‘vulnerable’ atherosclerotic plaques is important for predicting the risk of thrombotic events. A new study shows that unbiased classification of human atherosclerotic plaques on the basis of transcriptomic features identifies subgroups with distinct biology and clinical presentation.

Background: Long noncoding RNAs (lncRNAs) have emerged as novel regulators of macrophage biology and inflammatory cardiovascular diseases. However, studies focused on lncRNAs in human macrophage subtypes, particularly human lncRNAs that are not conserved in rodents, are limited. Methods: Through RNA-sequencing of human monocyte–derived macrophages, we identified suppressor of inflammatory macrophage apoptosis lncRNA ( SIMALR ). Lipopolysaccharide/IFNγ (interferon γ) stimulated human macrophages were treated with SIMALR antisense oligonucleotides and subjected to RNA-sequencing to investigate the function of SIMALR . Western blots, luciferase assay, and RNA immunoprecipitation were performed to validate function and potential mechanism of SIMALR. RNAscope was performed to identify SIMALR expression in human carotid atherosclerotic plaques. Results: RNA-sequencing of human monocyte–derived macrophages identified SIMALR , a human macrophage-specific long intergenic noncoding RNA that is highly induced in lipopolysaccharide/IFNγ–stimulated macrophages. SIMALR knockdown in lipopolysaccharide/IFNγ stimulated THP1 human macrophages induced apoptosis of inflammatory macrophages, as shown by increased protein expression of cleaved PARP (poly[ADP-ribose] polymerase), caspase 9, caspase 3, and Annexin V+. RNA-sequencing of control versus SIMALR knockdown in lipopolysaccharide/IFNγ–stimulated macrophages showed Netrin-1 ( NTN1 ) to be significantly decreased upon SIMALR knockdown. We confirmed that NTN1 knockdown in lipopolysaccharide/IFNγ–stimulated macrophages induced apoptosis. The SIMALR knockdown-induced apoptotic phenotype was rescued by adding recombinant NTN1. NTN1 promoter-luciferase reporter activity was increased in HEK293T (human embryonic kidney 293) cells treated with lentiviral overexpression of SIMALR . NTN1 promoter activity is known to require HIF1α (hypoxia-inducible factor 1 subunit alpha), and our studies suggest that SIMALR may interact with HIF1α to regulate NTN1 transcription, thereby regulating macrophages apoptosis. SIMALR was found to be expressed in macrophages in human carotid atherosclerotic plaques of symptomatic patients. Conclusions: SIMALR is a nonconserved, human macrophage lncRNA expressed in atherosclerosis that suppresses macrophage apoptosis. SIMALR partners with HIF1α (hypoxia-inducible factor 1 subunit alpha) to regulate NTN1, which is a known macrophage survival factor. This work illustrates the importance of interrogating the functions of human lncRNAs and exploring their translational and therapeutic potential in human atherosclerosis.

The Modern Western Diet has been associated with the rise in metabolic and inflammatory diseases, including obesity, diabetes, and cardiovascular disease. This has been attributed, in part, to the increase in dietary omega-6 polyunsaturated fatty acid (PUFA) consumption, specifically linoleic acid (LA), arachidonic acid (ARA), and their subsequent metabolism to pro-inflammatory metabolites which may be driving human disease. Conversion of dietary LA to ARA is regulated by genetic variants near and within the fatty acid desaturase (FADS) haplotype block, most notably single nucleotide polymorphism rs174537 is strongly associated with FADS1 activity and expression. This variant and others within high linkage disequilibrium may potentially explain the diversity in both diet and inflammatory mediators that drive chronic inflammatory disease in human populations. Mechanistic exploration into this phenomenon using human hepatocytes is limited by current two-dimensional culture models that poorly replicate in vivo functionality. Therefore, we aimed to develop and characterize a three-dimensional hepatic construct for the study of human PUFA metabolism. Primary human hepatocytes cultured in 3D hydrogels were characterized for their capacity to represent basic lipid processing functions, including lipid esterification, de novo lipogenesis, and cholesterol efflux. They were then exposed to control and LA-enriched media and reproducibly displayed allele-specific metabolic activity of FADS1, based on genotype at rs174537. Hepatocytes derived from individuals homozygous with the minor allele at rs174537 (i.e., TT) displayed the slowest metabolic conversion of LA to ARA and significantly reduced FADS1 and FADS2 expression. These results support the feasibility of using 3D human hepatic cultures for the study of human PUFA and lipid metabolism and relevant gene-diet interactions, thereby enabling future nutrition targets in humans.

Background: Smooth muscle cells (SMCs) play significant roles in atherosclerosis via phenotypic switching, a pathological process in which SMC dedifferentiation, migration, and transdifferentiation into other cell types. Yet how SMCs contribute to the pathophysiology of atherosclerosis remains elusive. Methods: To reveal the trajectories of SMC transdifferentiation during atherosclerosis and to identify molecular targets for disease therapy, we combined SMC fate mapping and single-cell RNA sequencing of both mouse and human atherosclerotic plaques. We also performed cell biology experiments on isolated SMC-derived cells, conducted integrative human genomics, and used pharmacological studies targeting SMC-derived cells both in vivo and in vitro. Results: We found that SMCs transitioned to an intermediate cell state during atherosclerosis, which was also found in human atherosclerotic plaques of carotid and coronary arteries. SMC-derived intermediate cells, termed "SEM" cells (stem cell, endothelial cell, monocyte), were multipotent and could differentiate into macrophage-like and fibrochondrocyte-like cells, as well as return toward the SMC phenotype. Retinoic acid (RA) signaling was identified as a regulator of SMC to SEM cell transition, and RA signaling was dysregulated in symptomatic human atherosclerosis. Human genomics revealed enrichment of genome-wide association study signals for coronary artery disease in RA signaling target gene loci and correlation between coronary artery disease risk alleles and repressed expression of these genes. Activation of RA signaling by all-trans RA, an anticancer drug for acute promyelocytic leukemia, blocked SMC transition to SEM cells, reduced atherosclerotic burden, and promoted fibrous cap stability. Conclusions: Integration of cell-specific fate mapping, single-cell genomics, and human genetics adds novel insights into the complexity of SMC biology and reveals regulatory pathways for therapeutic targeting of SMC transitions in atherosclerotic cardiovascular disease.

BACKGROUND:Traditional and novel risk factors cannot sufficiently explain the differential susceptibility to cardiovascular disease (CVD). Epigenetics may serve to partially explain this residual disparity, with life course stressors shown to modify methylation of genes implicated in various diseases. Subclinical CVD is often comorbid with cognitive impairment (CI), which warrants research into the identification of common genes for both conditions.METHODS:We conducted a systematic review of the existing literature to identify studies depicting the relationship between life course stressors, DNA methylation, subclinical CVD, and cognition.RESULTS:A total of 16 articles (8 human and 8 animal) were identified, with the earliest published in 2008. Four genes (COMT, NOS3, Igfl1, and Sod2) were analyzed by more than one study, but not in association with both CVD and CI. One gene (NR3C1) was associated with both outcomes, albeit not within the same study. There was some consistency among studies with markers used for subclinical CVD and cognition, but considerable variability in stress exposure (especially in human studies), cell type/tissue of interest, method for detection of DNA methylation, and risk factors. Racial and ethnic differences were not considered, but analysis of sex in one human study found statistically significant differentially methylated X-linked loci associated with attention and intelligence.CONCLUSIONS:This review suggests the need for additional studies to implement more comprehensive and methodologically rigorous study designs that can better identify epigenetic biomarkers to differentiate individuals vulnerable to both subclinical CVD and associated CI.

Objective: The role of hepatocyte Abca1 (ATP binding cassette transporter A1) in trafficking hepatic free cholesterol (FC) into plasma versus bile for reverse cholesterol transport (RCT) is poorly understood. We hypothesized that hepatocyte Abca1 recycles plasma HDL-C (high-density lipoprotein cholesterol) taken up by the liver back into plasma, maintaining the plasma HDL-C pool, and decreasing HDL-mediated RCT into feces. Approach and Results: Chow-fed hepatocyte-specific Abca1 knockout (HSKO) and control mice were injected with human HDL radiolabeled with 125 I-tyramine cellobiose ( 125 I-TC; protein) and 3 H-cholesteryl oleate ( 3 H-CO). 125 I-TC and 3 H-CO plasma decay, plasma HDL 3 H-CO selective clearance (ie, 3 H- 125 I fractional catabolic rate), liver radiolabel uptake, and fecal 3 H-sterol were significantly greater in HSKO versus control mice, supporting increased plasma HDL RCT. Twenty-four hours after 3 H-CO-HDL injection, HSKO mice had reduced total hepatic 3 H-FC (ie, 3 H-CO hydrolyzed to 3 H-FC in liver) resecretion into plasma, demonstrating Abca1 recycled HDL-derived hepatic 3 H-FC back into plasma. Despite similar liver LDLr (low-density lipoprotein receptor) expression between genotypes, HSKO mice treated with LDLr-targeting versus control antisense oligonucleotide had slower plasma 3 H-CO-HDL decay, reduced selective 3 H-CO clearance, and decreased fecal 3 H-sterol excretion that was indistinguishable from control mice. Increased RCT in HSKO mice was selective for 3 H-CO-HDL, since macrophage RCT was similar between genotypes. Conclusions: Hepatocyte Abca1 deletion unmasks a novel and selective FC trafficking pathway that requires LDLr expression, accelerating plasma HDL-selective CE uptake by the liver and promoting HDL RCT into feces, consequently reducing HDL-derived hepatic FC recycling into plasma.

Abstract Ageing conveys the greatest risk for cardiovascular disease (CVD) development, which is the dominant cause of mortality in developed nations. High density lipoprotein (HDL) particles mediate reverse cholesterol transport, are anti-inflammatory and their function predicts CVD. We observed lower plasma cholesterol efflux capacity in geriatric vervet monkeys (p=0.03) when consuming either healthy or Western diets. Adult (n=16) and geriatric (n=19) monkeys were stratified into groups fed Western diet on ad libitum (Ad Lib) or early time restricted feeding (eTFR) schedules. eTRF supplied excess food only between 6am to 2pm. Housing, seasonality and fasting conditions for data and sample collections were equivalent. After 6 months, cholesterol efflux to HDL was greater in eTRF monkeys (p=0.01), with no age by group interaction. Efflux media and plasma was chromatographically separated to confirm labelled cholesterol, and enzymatically measured cholesterol, respectively, was affiliated with HDL particles. eTRF monkeys had higher total plasma cholesterol levels (p=0.03) which was due to greater cholesterol amounts associated with only HDL, and resulted in HDL particles that were larger. eTRF resulted in robustly better HDL function such that measures from geriatric individuals were comparable to younger adults. Additionally, no differences in adiposity was observed in eTRF monkeys. Few interventions are known to raise HDL levels, and more importantly, are confirmed to improve HDL function. Our study is to date the largest, longest, controlled eTRF evaluation in primates and we show that positive biological effects are observable in HDL isolated from both adult and geriatric individuals independently of weight change.

OBJECTIVE:Adipose tissue cholesterol increases with adipocyte triglyceride content and size during development of obesity. However, how adipocyte cholesterol affects adipocyte function is poorly understood. The aim of this study was to evaluate the role of the cellular cholesterol exporter, Abca1 (ATP-binding cassette transporter A1), on adipose tissue function during diet-induced obesity.APPROACH AND RESULTS:Adiponectin Cre recombinase transgenic mice were crossed with Abca1flox/flox mice to generate ASKO (adipocyte-specific Abca1 knockout) mice. Control and ASKO mice were then fed a high-fat, high-cholesterol (45% calories as fat and 0.2% cholesterol) diet for 16 weeks. Compared with control mice, ASKO mice had a 2-fold increase in adipocyte plasma membrane cholesterol content and significantly lower body weight, epididymal fat pad weight, and adipocyte size. ASKO versus control adipose tissue had decreased PPARγ (peroxisome proliferator-activated receptor γ) and CCAAT/enhancer-binding protein expression, nuclear SREBP1 (sterol regulatory element-binding protein 1) protein, lipogenesis, and triglyceride accretion but similar Akt activation after acute insulin stimulation. Acute siRNA-mediated Abca1 silencing during 3T3L1 adipocyte differentiation reduced adipocyte Abca1 and PPARγ protein expression and triglyceride content. Systemic stimulated triglyceride lipolysis and glucose homeostasis were similar between control and ASKO mice.CONCLUSIONS:Adipocyte Abca1 is a key regulator of adipocyte lipogenesis and lipid accretion, likely because of increased adipose tissue membrane cholesterol, resulting in decreased activation of lipogenic transcription factors PPARγ and SREBP1.

The chemotherapy drug doxorubicin (DOX) is well known to induce cardiac and skeletal muscle dysfunction. Previous studies demonstrate that exercise can mitigate dysfunction, reduce myocardial DOX accumulation, and depress markers of oxidative stress, but a putative mechanism is unknown. The aim of this study was to determine whether multidrug resistance protein (MRP) expression contributes to the protective effects of exercise against DOX-induced muscular dysfunction. Lower left ventricle (LV) and soleus DOX concentrations were observed in exercised animals, and MRP- 1, MRP-2, and MRP-7 expression was significantly increased in the LV with exercise. No MRP variations were apparent in skeletal muscles following the exercise protocol. As a marker of oxidative stress, malondialdehyde+4 hydroxyalkenal levels were analyzed, and exercise reduced both cardiac and skeletal muscle levels from exercised trained animals treated with DOX had significantly lower levels than SED-DOX. This study suggests increased MRP expression with exercise may contribute to exercise-induced protection in cardiac muscle but not skeletal muscle.