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.

Rates of obesity and its associated metabolic comorbidities continue to rise in the developed world. It is well established that in obesity, the distribution and not just amount of excess white adipose tissue (WAT) correlates with a person's risk for comorbidities such as coronary artery disease and type 2 diabetes. Thus, understanding the specific mechanisms that drive WAT development in specific adipose depots could elucidate novel mechanisms of metabolic disease. SNPs near the gene CEBPA have been associated with multiple cardiometabolic traits by human genome-wide association studies, including waist-to-hip ratio, suggesting that CEBPA regulates WAT distribution. CEBPA encodes a well characterized transcription factor (C/EBPα) that is long recognized as a master regulator of adipocyte differentiation, yet depot-specific roles for C/EBPα have not been previously described. To further investigate this genetic link, we generated mice with adipocyte-specific Cebpa knockout (Cebpa_ASKO) and found that these mice are almost entirely lacking gonadal WAT (gWAT) despite the inguinal WAT (iWAT) being present in near normal amounts. Despite developing, Cebpa_ASKO iWAT contains fewer and larger adipocytes, and fails to expand when challenged with high fat diet. RNA-seq and functional studies demonstrate evidence of altered lipid metabolism and adipocyte function in Cebpa_ASKO iWAT. Finally, Cebpa_ASKO mice have multiple other metabolic phenotypes, including lipid-laden BAT, increased hepatic triglycerides, and increased plasma cholesterol, all of which worsen with prolonged high fat diet feeding. Taken together, these data highlight depot-specific roles for C/EBPα in adipose tissue development, as well as the importance of adipocyte C/EBPα in maintaining metabolic homeostasis.

Background:Vascular smooth muscle cells (VSMCs) play a central role in atherosclerosis by undergoing phenotypic modulation from a quiescent, contractile state to a range of synthetic phenotypes, including fibroblast-like, macrophage-like, and lipid-laden foam cell-like states. However, a comprehensive multimodal characterization and understanding of the transcriptional programs driving these transitions remain incomplete. Methods:To comprehensively define the phenotypic diversity of VSMCs during atherosclerosis progression, we performed in-depth profiling using cellular indexing of transcriptomes and epitopes by sequencing (CITE-seq) and bulk RNA sequencing in a VSMC lineage-tracing atherosclerotic mouse model. Insights from these datasets guided the design of targeted in vitro experiments to investigate candidate regulatory mechanisms. Results:Single-cell multi-omics revealed extensive cellular heterogeneity within atherosclerotic plaques, including a rare population of VSMC-derived macrophage-like cells, whose presence was confirmed by histological analysis. These studies also identified a substantial population of VSMC-derived foam cells, comprising approximately 70% of all foam cells in the lesions. These cells exhibited activation of gene programs associated with lipid metabolism, proliferation, and tumor-like features. The transcription factor Bhlhe40 emerged as a key regulator of this phenotypic transition, with elevated expression in VSMC-derived foam cells during disease progression. Functional knockdown of Bhlhe40 suppressed VSMC phenotypic switching and foam cell characteristics, underscoring its potential role as a driver of VSMC modulation. Conclusions:These findings advance our understanding of VSMC phenotypic modulation in atherosclerosis and highlight Bhlhe40 as a key regulator of this process. Elucidating the mechanisms governing VSMC plasticity may offer new therapeutic opportunities to reduce cardiovascular risk by targeting disease-driving cellular transitions.

Background:Atherosclerotic cardiovascular disease (ASCVD) remains the leading cause of mortality in the western world despite the success of lipid lowering therapies, highlighting the need for novel lipid-independent therapeutic strategies. Genome-wide association studies (GWAS) have identified numerous genes associated with ASCVD that function in the vessel wall, suggesting that vascular cells mediate ASCVD, and that the genes and pathways essential for this vascular cell function may be novel therapeutic targets for the treatment of ASCVD. Furthermore, some of these implicated genes appear to function in the adventitial layer of the vasculature, suggesting these cells are able to potentiate ASCVD. Methods:To investigate the role of adventitial cells in atherosclerosis, we conducted single-cell RNA sequencing (scRNA-seq) of the aortic adventitia during atherogenesis in male Ldlr -/- mice via pools of three mice, two samples per condition. We cross-referenced the scRNA-seq data with human ASCVD GWAS to identify regulators of adventitial responses in ASCVD. These regulators were then validated in vitro in human adventitial fibroblasts. Results:We identified four adventitial fibroblast populations, all of which displayed shifts in population size and gene expression over the course of atherogenesis. SERPINH1, an ASCVD-linked GWAS gene, was differentially expressed in adventitial fibroblasts during atherogenesis. Knockdown of SERPINH1 in vitro reduced fibroblast migration and altered subcluster marker gene expression. Conclusions:These findings reveal dynamic changes in adventitial fibroblasts during atherosclerosis and suggest that reduced SERPINH1 expression disrupts adventitial fibroblast function, contributing to ASCVD progression.

Obesity, defined as excess white adipose tissue (WAT), associates with a higher risk of metabolic disease and comorbidities, and continues to be on the rise in the developed world. However, different WAT depots associate with differing levels of cardiometabolic risk. Expansion of visceral WAT (vWAT) correlates with increased risk for cardiovascular disease and type 2 diabetes, while expansion of subcutaneous WAT (sWAT) does not. These observations suggest mechanistic differences between the two depots, and elucidating these mechanisms may provide insights into promoting cardiometabolic health in humans. Human GWAS have shown that SNPs near the gene CEBPA associate with waist-to-hip ratio, a metric of vWAT mass in humans, suggesting CEBPA plays a specific role in vWAT. CEBPA encodes the transcription factor C/EBPα, a known regulator of adipogenesis. We established adipocyte-specific Cebpa knockout (Cebpa_ASKO) mice and found they lack gonadal WAT (gWAT), the largest vWAT depot in mice. Conversely, sWAT is still present in Cebpa_ASKO mice, although with reduced mass (-23%, p<0.05) and larger adipocytes. Additionally, Cebpa_ASKO mice have reduced lipolysis (-26% NEFAs, p<0.05), plasma adiponectin (-89%, p<0.0001) and leptin (-82%, p<0.05), suggesting the remaining sWAT is dysfunctional. Together, these data suggest that C/EBPα has WAT depot-specific roles, being required for vWAT development, but only for function in mature sWAT. Given the myriad roles for vWAT in metabolism and the absence of vWAT in Cebpa_ASKO mice, we investigated downstream metabolic consequences of this phenotype. Cebpa_ASKO mice have increased brown adipose tissue (BAT) mass (+118%, p<0.001) due to increased ectopic lipid deposition. Cebpa_ASKO mice also have increases in plasma cholesterol (+20%, p<0.05), liver mass (+24%, p<0.01), and hepatic fat content (+329%, p<0.01). Overall, these results demonstrate that adipocyte C/EBPα significantly influences whole body metabolism. Ongoing studies center on delineating the different mechanisms through which C/EBPα functions in vWAT and sWAT. Understanding the depot-specific mechanisms of C/EBPα could have practical implications for new clinical targets of obesity and metabolic disease.

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.

Adipose tissue remodeling and dysfunction, characterized by elevated inflammation and insulin resistance, play a central role in obesity -related development of type 2 diabetes (T2D) and cardiovascular diseases. Long intergenic non -coding RNAs (lincRNAs) are important regulators of cellular functions. Here, we describe the functions of linc-ADAIN (adipose anti-inflammatory), an adipose lincRNA that is downregulated in white adipose tissue of obese humans. We demonstrate that linc-ADAIN knockdown (KD) increases KLF5 and interleukin-8 (IL -8) mRNA stability and translation by interacting with IGF2BP2. Upregulation of KLF5 and IL -8, via linc-ADAIN KD, leads to an enhanced adipogenic program and adipose tissue inflammation, mirroring the obese state, in vitro and in vivo . KD of linc-ADAIN in human adipose stromal cell (ASC) hTERT adipocytes implanted into mice increases adipocyte size and macrophage infiltration compared to implanted control adipocytes, mimicking hallmark features of obesity -induced adipose tissue remodeling. linc-ADAIN is an anti-inflammatory lincRNA that limits adipose tissue expansion and lipid storage.

Human genetic studies have linked the gene ADAMTS7 with coronary artery disease. In vivo studies in mice demonstrate that ADAMTS7 is proatherogenic, active in smooth muscle cells (SMCs), and induced in response to vascular injury. However, the mechanisms governing its proatherogenicity remain unclear. We generated an SMC specific Adamts7 transgenic mouse (TG_SMC) to mimic the induction of vascular ADAMTS7. This mouse was crossed onto the LDL KO background, and after 16 weeks of western diet feeding, TG_SMC mice had a 3.5-fold increase in en-face plaque (p < 0.01) compared to controls. TG_SMC aortas showed a 3-fold increase in foam cells (p = 0.013) as measured by flow cytometry, and 80% of these cells were SMCs. Subsequent ex vivo studies show increased oxLDL uptake in TG_SMC primary SMCs (1.15x, p < 0.01).To determine the underlying mechanism, we performed RNA-seq on primary SMCs and found that TG_SMCs have increased expression of lipid handling genes (Cd36, Fabp5, Trem2). Upstream regulator analysis identified the transcription factor PU.1 as a potential mediator of these changes, and siRNA knockdown of PU.1 attenuated the gene expression increases. Additionally, siRNA knockdown of Cd36, the most upregulated gene, and a known oxLDL receptor, ameliorated the Adamts7-mediated rise in SMC lipid uptake.We next generated an Adamts7 SMC-specific knockout model to test if SMC ADAMTS7 is solely responsible for its proatherogenic effect. Surprisingly, SMC-specific Adamts7 knockout did not affect atherosclerosis in mice. We performed RNAscope on atherosclerotic LDLR KO mice and found that Adamts7 is also expressed in endothelial cells (ECs) during atherogenesis. We bred our hyperlipidemic TG model to the EC-specific Cdh5-CreERT2 and found that EC overexpression of Adamts7 also increases en face plaque burden and foam cell formation (1.5x, p<0.05), demonstrating that vascular Adamts7, regardless of the source, increases atherosclerosis. In summary, our results indicate that Adamts7 promotes lipid uptake in vascular SMCs, thus revealing a novel mechanism through which ADAMTS7 mediates atherogenesis. Our findings support the growing body of literature demonstrating that therapeutic targeting of ADAMTS7 is a potential approach to reducing atherosclerosis.

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.

PURPOSE OF REVIEW:Genome-wide association studies have repeatedly linked the metalloproteinase ADAMTS7 to coronary artery disease. Here we aim to highlight recent findings surrounding the human genetics of ADAMTS7, novel mouse models that investigate ADAMTS7 function, and potential substrates of ADAMTS7 cleavage. RECENT FINDINGS:Recent genome-wide association studies in coronary artery disease have replicated the GWAS signal for ADAMTS7 and shown that the signal holds true even across different ethnic groups. However, the direction of effect in humans remains unclear. A recent novel mouse model revealed that the proatherogenicity of ADAMTS7 is derived from its catalytic functions, while at the translational level, vaccinating mice against ADAMTS7 reduced atherosclerosis. Finally, in vitro proteomics approaches have identified extracellular matrix proteins as candidate substrates that may be causal for the proatherogenicity of ADAMTS7. ADAMTS7 represents an enticing target for therapeutic intervention. The recent studies highlighted here have replicated prior findings, confirming the genetic link between ADAMTS7 and atherosclerosis, while providing further evidence in mice that ADAMTS7 is a targetable proatherogenic enzyme.

Single Cell RNA Sequencing (scRNA-seq) has proven to be a powerful technique to uncover heterogeneity from otherwise homogeneous tissues. Multiple studies have utilized scRNA-seq to examine the composition of atherosclerotic plaques in both humans and mice. However, most of these studies occurred in advanced atherosclerosis. We sought to expand this knowledge base by performing single-cell RNA sequencing at early atherosclerosis in mice. We crossed the Ldlr-/- hyperlipidemic mouse model to a smooth muscle cell (SMC) lineage tracing mouse (ROSA26:LSL-ZsGreen; MyH11-CreERT2). When treated with tamoxifen, SMCs expressing Myh11 are labeled green. At seven weeks of age, we treated these mice with five IP injections of 40mg/kg of tamoxifen. After one week of rest, we initiated the mice on a western diet. We dissected out the whole aorta at the time of harvest and digested the aorta for one hour with a cocktail of DNase, Hyaluronidase, and Liberase. Subsequently, we FACS sorted out both ZsGreen negative and ZsGreen positive cells and performed single-cell RNA seq using the 10x Genomics pipeline. We performed scRNA-seq with pools of three mice at four time points: after 1, 2, 3, and 4 weeks of western diet feeding. Clustering with CarDEC revealed three SMC populations. At four weeks of western diet, we observed an emergence of a SMC cluster characterized by increased expression of Vcam1, Dcn, Mgp, and Egr1. As expected, Gene Ontology analysis of the top 20 differentially expressed genes revealed this population to contain reduced markers of SMC differentiation. In addition, this population had reduced expression of Pura, a protein known to influence serum response factor and regulate smooth muscle actin expression. In summary, our studies indicate changes within SMCs, and the emergence of a dedifferentiated smooth muscle cell population after four weeks of western diet feeding. This study highlights that remodeling of the vasculature occurs even with short durations of hyperlipidemia. Further ongoing studies include expanding time points to longer durations of western diet and elucidating master regulators of this population with Virtual Inference of Protein activity by Enriched Regulon (VIPER).

The respiratory epithelium forms the first line of defense against inhaled pathogens and acts as an important source of innate cytokine responses to environmental insults. One critical mediator of these responses is the IL-1 family cytokine IL-33, which is rapidly secreted upon acute epithelial injury as an alarmin and induces type 2 immune responses. Our recent work highlighted the importance of the NADPH oxidase dual oxidase 1 (DUOX1) in acute airway epithelial IL-33 secretion by various airborne allergens associated with H2O2 production and reduction-oxidation-dependent activation of Src kinases and epidermal growth factor receptor (EGFR) signaling. In this study, we show that IL-33 secretion in response to acute airway challenge with house dust mite (HDM) allergen critically depends on the activation of Src by a DUOX1-dependent oxidative mechanism. Intriguingly, HDM-induced epithelial IL-33 secretion was dramatically attenuated by small interfering RNA- or Ab-based approaches to block IL-33 signaling through its receptor IL1RL1 (ST2), indicating that HDM-induced IL-33 secretion includes a positive feed-forward mechanism involving ST2-dependent IL-33 signaling. Moreover, activation of type 2 cytokine responses by direct airway IL-33 administration was associated with ST2-dependent activation of DUOX1-mediated H2O2 production and reduction-oxidation-based activation of Src and EGFR and was attenuated in Duox1 -/- and Src +/- mice, indicating that IL-33-induced epithelial signaling and subsequent airway responses involve DUOX1/Src-dependent pathways. Collectively, our findings suggest an intricate relationship between DUOX1, Src, and IL-33 signaling in the activation of innate type 2 immune responses to allergens, involving DUOX1-dependent epithelial Src/EGFR activation in initial IL-33 secretion and in subsequent IL-33 signaling through ST2 activation.

The arterial adventitia is a layer of cells outside the external lamina of blood vessels that contribute to the progression of coronary artery disease (CAD) like atherosclerosis. Adventitial fibroblasts are known regulators of vascular remodeling through their deposition of collagen fibrils around vessels during atherosclerotic lesion development. However, the molecular identity and function of the participating fibroblasts have not been well studied. We used single cell RNA sequencing (scRNA-seq) to characterize the transcriptome of adventitial fibroblasts and other cell types at multiple timepoints during atherogenesis in hyperlipidemic Ldlr knockout mice. Specifically, scRNA-seq was applied to Ldlr KO aortic adventitia after 0, 9, and 16 weeks of western diet (WD) feeding. Unbiased clustering analysis uncovered 9 different cell clusters, including 5 different fibroblast populations (Fibro1-5), and a single cluster each of macrophages, T cells, SMCs, and ECs. We observed that, as atherosclerosis progressed from 9 to 16 weeks of feedings, the proportion of total cells that were Fibro1, 3, and 4 decreased (33, 40, and 13%), while populations of Fibro2 and 5 slightly increased (13 and 18%). All of non-fibroblast populations (Macrophage, T cells, SMCs and ECs) increased from 9-16wks of diet. Next, we asked whether genes associated with coronary artery disease by human genome-wide association studies (GWAS) are expressed in different adventitial fibroblast clusters. We determined the expression of 266 genes implicated in CAD by GWAS and found 201 genes (76%) were expressed in adventitial cells at either/both 9 or 16wks. 34 genes displayed large changes in expression from 9 to 16wks. We identified five genes that were highly expressed in the adventitial Fibro5 cluster specifically, and additional other CAD GWAS genes specifically expressed in adventitial macrophages, SMCs, and ECs. In summary, we present here a cell atlas defined by scRNA-seq that reveals the heterogeneity amongst the fibroblasts in adventitial cells during atherogenesis. Additionally, we find that many CAD GWAS genes are expressed in adventitial cell populations, raising the intriguing possibility that CAD GWAS are identifying adventitial mechanisms that contribute to CAD.

The gene tribbles pseudokinase 1 ( TRIB1 ) has been repeatedly linked to multiple human cardiometabolic traits through genome-wide association studies, including coronary artery disease, plasma cholesterol and triglycerides, and circulating liver transaminases, signaling that TRIB1 is a key regulator of liver metabolism and health. Studies in liver specific Trib1 KO mice have shown that hepatic TRIB1 regulates de novo lipogenesis and steatosis through the regulation of protein levels of the transcription factor C/EBPα, yet the mechanism governing this relationship in hepatocytes has not been investigated. We demonstrate here that human TRIB1 promotes the degradation of C/EBPα in both a COP1- and proteasome-dependent manner in human hepatoma cells. We also observe rapid degradation of TRIB1 protein in hepatoma cells, and find that this is also COP1- and proteasome-dependent. To identify hepatocyte-specific interacting partners of TRIB1 that regulate these processes, we performed tandem-affinity purification of TRIB1 in Huh7 cells and subsequent mass-spec analysis, and identified multiple novel TRIB1 binding partners including the pseudokinases serine/threonine kinase 38 (STK38) and 40 (STK40). We confirmed these interactions in vitro and found that STK40, but not STK38, is required for TRIB1-mediated proteasomal degradation of C/EBPα. Together, our results reveal that TRIB1 induces proteasomal degradation of CEBP/α in a COP1- and proteasome-dependent manner in human hepatocytes. Further, we identify the pseudokinase STK40 as a novel regulator of hepatic TRIB1 function, and ongoing work aims to investigate the role of STK40 in hepatic lipid metabolism. Finally, we show that TRIB1 is itself degraded by the proteosome in a COP1-dependent manner. As increased hepatic Trib1 confers a beneficial metabolic profile in mice, these findings could provide a novel avenue for therapeutic targeting of TRIB1 in the treatment of cardiometabolic disease. Overall, our findings add greater detail to the molecular mechanisms governing the regulation of metabolism by hepatic TRIB1, a gene which human genetics highlights as a crucial regulator of cardiometabolic traits in humans.

Purpose of review The pseudokinase Tribbles-1 (TRIB1) remains the focus of intense research since genome-wide association studies (GWAS) associated it with multiple cardiometabolic traits in humans, including plasma lipids and atherosclerosis. This review highlights recent advances in understanding the function of TRIB1 and what outstanding questions remain. Recent findings Studies performed in a myeloid-specific Trib1 mouse model show that Trib1 contributes to foam cell formation, underscoring the importance of continued research into tissue-specific functions of TRIB1. Investigations of TRIB1 function in a 3D hepatic organoid model demonstrate that hepatic TRIB1 functions elucidated in mouse models are recapitulated in these organoid systems. Lastly, a recent study showed berberine, an existing lipid-lowering drug, to be acting via a TRIB1-dependent mechanism, highlighting both a novel regulator of TRIB1 expression and the potential of studying TRIB1 through existing therapeutics. Summary TRIB1 remains one of the more fascinating loci to arise from cardiometabolic GWAS, given the constellation of traits it associates with. As genetic studies continue to link TRIB1 to metabolic phenotypes, more functional research on tissue-specific TRIB1, regulation of TRIB1 and its function in current therapies, as well as the reproduction of results from mice in human contexts are all necessary to increase our understanding of TRIB1 and its relevance.

Objective: Multiple genome-wide association studies (GWAS) have identified SNPs in the 8q24 locus near TRIB1 that are significantly associated with plasma lipids and other markers of cardiometabolic health, and prior studies have revealed the roles of hepatic and myeloid Trib1 in plasma lipid regulation and atherosclerosis. The same 8q24 SNPs are additionally associated with plasma adiponectin levels in humans, implicating TRIB1 in adipocyte biology. Here, we hypothesize that TRIB1 in adipose tissue regulates plasma adiponectin, lipids, and metabolic health. Methods: We investigate the metabolic phenotype of adipocyte-specific Trib1 knockout mice (Trib1_ASKO) fed on chow and high-fat diet (HFD). Through secretomics of adipose tissue explants and RNA-seq of adipocytes and livers from these mice, we further investigate the mechanism of TRIB1 in adipose tissue. Results: Trib1_ASKO mice have an improved metabolic phenotype with increased plasma adiponectin levels, improved glucose tolerance, and decreased plasma lipids. Trib1_ASKO adipocytes have increased adiponectin production and secretion independent of the known TRIB1 function of regulating proteasomal degradation. RNA-seq analysis of adipocytes and livers from Trib1_ASKO mice indicates that alterations in adipocyte function underlie the observed plasma lipid changes. Adipose tissue explant secretomics further reveals that Trib1_ASKO adipose tissue has decreased ANGPTL4 production, and we demonstrate an accompanying increase in the lipoprotein lipase (LPL) activity that likely underlies the triglyceride phenotype. Conclusions: This study shows that adipocyte Trib1 regulates multiple aspects of metabolic health, confirming previously observed genetic associations in humans and shedding light on the further mechanisms by which TRIB1 regulates plasma lipids and metabolic health.

CCAAT/enhancer-binding protein alpha (C/EBPa) is a transcription factor known to mediate glucose and lipid metabolism. Hepatic protein levels of C/EBPa are controlled by the pseudokinase Tribbles-1 ( TRIB1 ), a gene which has repeatedly been linked to plasma lipids and coronary artery disease by human genome-wide association studies. Previous work has shown that genetic perturbation of hepatic Trib1 in mice alters plasma lipids. However, it is unknown if C/EBPa governs the relationship between Trib1 and plasma lipids. To investigate this, we first reasoned that if C/EBPa does govern this relationship, then human CEBPA should also be a GWAS hit for plasma lipids in existing data. Indeed, there is a GWAS locus for HDL cholesterol and triglycerides (TGs) at Chr19q13.11, with the lead SNP located 80kb downstream of CEBPA in an intron of the annotated gene PEPD . To see if this GWAS locus is identifying C/EBPa, we performed CRISPR deletion and CRISPR interference (CRISPRi) of the SNP locus in human hepatocytes and found that doing so reduced CEBPA gene expression (CRISPR: -85.5%, p<0.01; CRISPRi: -29.7%, p<0.005), confirming that the 19q13 GWAS locus regulates CEBPA gene expression. We next sought to determine how hepatic C/EBPa regulates lipids by performing hepatic knockout of Cebpa via AAV-Cre treatment in adult mice. Hepatic knockout of C/EBPa significantly reduced plasma lipids (-21%, p<0.005). RNA-seq analysis of livers from these mice identified changes in expression of known regulators of plasma lipids, including reduced Pcsk9 expression (-60.5%, p<0.005). Subsequent ELISA analysis confirmed that mice lacking hepatic C/EBPa have reduced circulating PCSK9 (-54%, p<0.005). Crossing these mice to a transgenic mouse expressing human apoB confirmed that these mice have reduced non-HDL cholesterol (-49.7%, p<0.01). Ongoing studies seek to confirm the PCSK9 phenotype in human hepatoma cells, while also investigating how C/EBPa may regulate plasma TGs and HDL cholesterol. In summary, we demonstrate here that human CEBPA is likely a causal gene at the 19q13 plasma lipid GWAS locus, and that in mice, Cebpa is a novel regulator of plasma PCSK9 and non-HDL cholesterol. These data illustrate the translational relevance of targeting hepatic C/EBPa in adult animals.

Introduction: Genome-wide association studies revealed a robust association between genetic variants at the LIPA (lysosomal acid lipase) locus and coronary artery diseases (CAD). eQTL studies support that the risk alleles of LIPA CAD variants are associated with higher LIPA mRNA and enzyme activity in human monocytes, but not other blood or vascular cells, suggesting that increased myeloid LIPA may confer CAD risk. Herein, we aim to establish the causality of the variant-to-function relationship for the LIPA locus and elucidate how increased myeloid LIPA impact atherosclerosis in vivo. Hypothesis: We hypothesized that causal variants in LIPA lead to increased LIPA expression and enzyme activity in macrophages and myeloid-specific overexpression of Lipa promotes atherosclerosis. Results: We first confirmed that in human monocyte-derived macrophages, LIPA mRNA, protein and enzyme activity are higher in the risk allele carriers of CAD variants. High-resolution HiC revealed an intronic enhancer region showing strong interaction with the LIPA promoter. Within the enhancer region, both rs1320496 and rs1412445 had independent association with CAD and their risk alleles led to increased enhancer activity measured by luciferase assay. Risk allele of rs1320496 also demonstrated increased binding for PU.1, a myeloid-specific transcription factor. To establish how increased myeloid LIPA impact atherosclerosis, we generated myeloid-specific Lipa overexpression mice ( Lipa Tg , Ldlr -/- ). Lipa Tg significantly increased atherosclerotic lesion size without affecting plasma cholesterol level. scRNA-seq analysis showed that Lipa Tg led to reduced lipid-enriched yet increased inflammatory macrophage subsets, and upregulation of genes in chemokine signaling. This is further confirmed by reduced neutral lipid accumulation in both plaque and peritoneal macrophages in Lipa Tg mice. Mechanistically, Lipa Tg led to reduced expression of modified-LDL receptors, increased expression of inflammatory chemokines, and increased circulating IL18 and CXCL2 that likely drive immune cells infiltration during atherosclerosis. Conclusions: We for the first time established that LIPA risks alleles drive increased myeloid LIPA and aggravate atherosclerosis.

Genome-wide association studies in humans link ADAMTS7 with coronary artery disease. Subsequent studies in mice showed ADAMTS7 to be proatherogenic, as whole-body knockout (KO) reduced atherosclerosis independent of lipid-lowering. Further studies show that Adamts7 expression is temporarily induced in response to vascular injury, and Adamts7 KO reduces primary smooth muscle cell (SMC) migration ex vivo. However, both the mechanism through which ADAMTS7 influences atherosclerosis progression and the responsible cell type remain unclear. As ADAMTS7 is secreted, we sought to determine its cleavage targets. We generated an immortalized human SMC line with doxycycline-inducible expression of GFP or ADAMTS7. We analyzed conditioned media from these cells via terminal amine isotopic labeling of substrates (TAILS) proteomics. This method labels new protein N termini, enabling the identification of cleavage products. Overexpression of Adamts7 enriched extracellular matrix (ECM) peptides such as Col1a1, Col1a2, Col4a2, and fibronectin. Furthermore, KEGG analysis identified ECM receptor interaction and focal adhesion formation as highly enriched pathways in the dataset. These results suggest ADAMTS7 alters SMCs by modulating signaling between cell and ECM. To examine the cell type that confers the proatherogenic effects of Adamts7, we generated a conditional transgenic ADAMTS7 mouse on the Ldlr knockout background. Given previously described roles for ADAMTS7 in SMC migration and vascular reendothelialization, we crossed these mice to either the Tie2-Cre or Myh11-CreERT2 to overexpress ADAMTS7 in endothelial and SMCs respectively. We found >3-fold increase (p = 0.0002) in plaque burden by en face staining in overexpression of Adamts7 in SMCs compared to controls. In contrast, transgenic overexpression of Adamts7 in endothelial cells is embryonic or perinatally lethal (Chi-squared = 0.0018). In summary, our transgenic study affirms the proatherogenic effect of increased Adamts7 expression in SMCs in vivo. Furthermore, our TAILS data suggests a dynamic role of ADAMTS7 in cleaving ECM. These studies fill critical knowledge gaps in our understanding of ADAMTS7 biology and help inform potential therapeutic avenues to targeting this enzyme.