A functional genomic framework to elucidate novel causal non-alcoholic fatty liver disease genes.

Background & Aims: Non-alcoholic fatty liver disease (NAFLD) is the most prevalent chronic liver pathology in western countries, with serious public health consequences. Efforts to identify causal genes for NAFLD have been hampered by the relative paucity of human data from gold-standard magnetic resonance quantification of hepatic fat. To overcome insufficient sample size, genome-wide association studies using NAFLD surrogate phenotypes have been used, but only a small number of loci have been identified to date. In this study, we combined GWAS of NAFLD composite surrogate phenotypes with genetic colocalization studies followed by functional in vitro screens to identify bona fide causal genes for NAFLD. Approach & Results: We used the UK Biobank to explore the associations of our novel NAFLD score, and genetic colocalization to prioritize putative causal genes for in vitro validation. We created a functional genomic framework to study NAFLD genes in vitro using CRISPRi. Our data identify VKORC1, TNKS, LYPLAL1 and GPAM as regulators of lipid accumulation in hepatocytes and suggest the involvement of VKORC1 in the lipid storage related to the development of NAFLD. Conclusions: Complementary genetic and genomic approaches are useful for the identification of NAFLD genes. Our data supports VKORC1 as a bona fide NAFLD gene. We have established a functional genomic framework to study at scale putative novel NAFLD genes from human genetic association studies.

Genome-Wide Genetic Associations Prioritize Evaluation of Causal Mechanisms of Atherosclerotic Disease Risk.

OBJECTIVE: The goal of this review is to discuss the implementation of genome-wide association studies to identify causal mechanisms of vascular disease risk. APPROACH AND RESULTS: The history of genome-wide association studies is described, the use of imputation and the creation of consortia to conduct meta-analyses with sufficient power to arrive at consistent associated loci for vascular disease. Genomic methods are described that allow the identification of causal variants and causal genes and how they impact the disease process. The power of single-cell analyses to promote genome-wide association studies of causal gene function is described. CONCLUSIONS: Genome-wide association studies represent a paradigm shift in the study of cardiovascular disease, providing identification of genes, cellular phenotypes, and disease pathways that empower the future of targeted drug development.

Comprehensive Integration of Multiple Single-Cell Transcriptomic Data Sets Defines Distinct Cell Populations and Their Phenotypic Changes in Murine Atherosclerosis.

BACKGROUND: The application of single-cell transcriptomic (single-cell RNA sequencing) analysis to the study of atherosclerosis has provided unique insights into the molecular and genetic mechanisms that mediate disease risk and pathophysiology. However, nonstandardized methodologies and relatively high costs associated with the technique have limited the size and replication of existing data sets and created disparate or contradictory findings that have fostered misunderstanding and controversy. METHODS: To address these uncertainties, we have performed a conservative integration of multiple published single-cell RNA sequencing data sets into a single meta-analysis, performed extended analysis of native resident vascular cells, and used in situ hybridization to map the disease anatomic location of the identified cluster cells. To investigate the transdifferentiation of smooth muscle cells to macrophage phenotype, we have developed a classifying algorithm based on the quantification of reporter transgene expression. RESULTS: The reporter gene expression tool indicates that within the experimental limits of the examined studies, transdifferentiation of smooth muscle cell to the macrophage lineage is extremely rare. Validated transition smooth muscle cell phenotypes were defined by clustering, and the location of these cells was mapped to lesion anatomy with in situ hybridization. We have also characterized 5 endothelial cell phenotypes and linked these cellular species to different vascular structures and functions. Finally, we have identified a transcriptomically unique cellular phenotype that constitutes the aortic valve. CONCLUSIONS: Taken together, these analyses resolve a number of outstanding issues related to differing results reported with vascular disease single-cell RNA sequencing studies, and significantly extend our understanding of the role of resident vascular cells in anatomy and disease.

Single-nuclei multiomic analyses identify human cardiac lymphatic endothelial cells associated with coronary arteries in the epicardium.

Cardiac lymphatic vessels play important roles in fluid homeostasis, inflammation, disease, and regeneration of the heart. The developing cardiac lymphatics in human fetal hearts are closely associated with coronary arteries, similar to those in zebrafish hearts. We identify a population of cardiac lymphatic endothelial cells (LECs) that reside in the epicardium. Single-nuclei multiomic analysis of the human fetal heart reveals the plasticity and heterogeneity of the cardiac endothelium. Furthermore, we find that VEGFC is highly expressed in arterial endothelial cells and epicardium-derived cells, providing a molecular basis for the arterial association of cardiac lymphatic development. Using a cell-type-specific integrative analysis, we identify a population of cardiac lymphatic endothelial cells marked by the PROX1 and the lymphangiocrine RELN and enriched in binding motifs of erythroblast transformation specific (ETS) variant (ETV) transcription factors. We report the in vivo molecular characterization of human cardiac lymphatics and provide a valuable resource to understand fetal heart development.

Early clinical outcomes and molecular smooth muscle cell phenotyping using a prophylactic aortic arch replacement strategy in Loeys-Dietz syndrome.

OBJECTIVES: Patients with Loeys-Dietz syndrome demonstrate a heightened risk of distal thoracic aortic events after valve-sparing aortic root replacement. This study assesses the clinical risks and hemodynamic consequences of a prophylactic aortic arch replacement strategy in Loeys-Dietz syndrome and characterizes smooth muscle cell phenotype in Loeys-Dietz syndrome aneurysmal and normal-sized downstream aorta. METHODS: Patients with genetically confirmed Loeys-Dietz syndrome (n = 8) underwent prophylactic aortic arch replacement during valve-sparing aortic root replacement. Four-dimensional flow magnetic resonance imaging studies were performed in 4 patients with Loeys-Dietz syndrome (valve-sparing aortic root replacement + arch) and compared with patients with contemporary Marfan syndrome (valve-sparing aortic root replacement only, n = 5) and control patients (without aortopathy, n = 5). Aortic tissues from 4 patients with Loeys-Dietz syndrome and 2 organ donors were processed for anatomically segmented single-cell RNA sequencing and histologic assessment. RESULTS: Patients with Loeys-Dietz syndrome valve-sparing aortic root replacement + arch had no deaths, major morbidity, or aortic events in a median of 2 years follow-up. Four-dimensional magnetic resonance imaging demonstrated altered flow parameters in patients with postoperative aortopathy relative to controls, but no clear deleterious changes due to arch replacement. Integrated analysis of aortic single-cell RNA sequencing data (>49,000 cells) identified a continuum of abnormal smooth muscle cell phenotypic modulation in Loeys-Dietz syndrome defined by reduced contractility and enriched extracellular matrix synthesis, adhesion receptors, and transforming growth factor-beta signaling. These modulated smooth muscle cells populated the Loeys-Dietz syndrome tunica media with gradually reduced density from the overtly aneurysmal root to the nondilated arch. CONCLUSIONS: Patients with Loeys-Dietz syndrome demonstrated excellent surgical outcomes without overt downstream flow or shear stress disturbances after concomitant valve-sparing aortic root replacement + arch operations. Abnormal smooth muscle cell-mediated aortic remodeling occurs within the normal diameter, clinically at-risk Loeys-Dietz syndrome arch segment. These initial clinical and pathophysiologic findings support concomitant arch replacement in Loeys-Dietz syndrome.

A single-cell CRISPRi platform for characterizing candidate genes relevant to metabolic disorders in human adipocytes.

CROP-Seq combines gene silencing using CRISPR interference with single-cell RNA sequencing. Here, we applied CROP-Seq to study adipogenesis and adipocyte biology. Human preadipocyte SGBS cell line expressing KRAB-dCas9 was transduced with a sgRNA library. Following selection, individual cells were captured using microfluidics at different timepoints during adipogenesis. Bioinformatic analysis of transcriptomic data was used to determine the knockdown effects, the dysregulated pathways, and to predict cellular phenotypes. Single-cell transcriptomes recapitulated adipogenesis states. For all targets, over 400 differentially expressed genes were identified at least at one timepoint. As a validation of our approach, the knockdown of PPARG and CEBPB (which encode key proadipogenic transcription factors) resulted in the inhibition of adipogenesis. Gene set enrichment analysis generated hypotheses regarding the molecular function of novel genes. MAFF knockdown led to downregulation of transcriptional response to proinflammatory cytokine TNF-α in preadipocytes and to decreased CXCL-16 and IL-6 secretion. TIPARP knockdown resulted in increased expression of adipogenesis markers. In summary, this powerful, hypothesis-free tool can identify novel regulators of adipogenesis, preadipocyte, and adipocyte function associated with metabolic disease.NEW & NOTEWORTHY Genomics efforts led to the identification of many genomic loci that are associated with metabolic traits, many of which are tied to adipose tissue function. However, determination of the causal genes, and their mechanism of action in metabolism, is a time-consuming process. Here, we use an approach to determine the transcriptional outcome of candidate gene knockdown for multiple genes at the same time in a human cell model of adipogenesis.

Single-cell transcriptome dataset of human and mouse in vitro adipogenesis models.

Adipogenesis is a process in which fat-specific progenitor cells (preadipocytes) differentiate into adipocytes that carry out the key metabolic functions of the adipose tissue, including glucose uptake, energy storage, and adipokine secretion. Several cell lines are routinely used to study the molecular regulation of adipogenesis, in particular the immortalized mouse 3T3-L1 line and the primary human Simpson-Golabi-Behmel syndrome (SGBS) line. However, the cell-to-cell variability of transcriptional changes prior to and during adipogenesis in these models is not well understood. Here, we present a single-cell RNA-Sequencing (scRNA-Seq) dataset collected before and during adipogenic differentiation of 3T3-L1 and SGBS cells. To minimize the effects of experimental variation, we mixed 3T3-L1 and SGBS cells and used computational analysis to demultiplex transcriptomes of mouse and human cells. In both models, adipogenesis results in the appearance of three cell clusters, corresponding to preadipocytes, early and mature adipocytes. These data provide a groundwork for comparative studies on these widely used in vitro models of human and mouse adipogenesis, and on cell-to-cell variability during this process.

Single-cell transcriptome dataset of human and mouse in vitro adipogenesis models.

Adipogenesis is a process in which fat-specific progenitor cells (preadipocytes) differentiate into adipocytes that carry out the key metabolic functions of the adipose tissue, including glucose uptake, energy storage, and adipokine secretion. Several cell lines are routinely used to study the molecular regulation of adipogenesis, in particular the immortalized mouse 3T3-L1 line and the primary human Simpson-Golabi-Behmel syndrome (SGBS) line. However, the cell-to-cell variability of transcriptional changes prior to and during adipogenesis in these models is not well understood. Here, we present a single-cell RNA-Sequencing (scRNA-Seq) dataset collected before and during adipogenic differentiation of 3T3-L1 and SGBS cells. To minimize the effects of experimental variation, we mixed 3T3-L1 and SGBS cells and used computational analysis to demultiplex transcriptomes of mouse and human cells. In both models, adipogenesis results in the appearance of three cell clusters, corresponding to preadipocytes, early and mature adipocytes. These data provide a groundwork for comparative studies on human and mouse adipogenesis, as well as on cell-to-cell variability in gene expression during this process.

Molecular mechanisms of coronary artery disease risk at the PDGFD locus.

Genome wide association studies for coronary artery disease (CAD) have identified a risk locus at 11q22.3. Here, we verify with mechanistic studies that rs2019090 and PDGFD represent the functional variant and gene at this locus. Further, FOXC1/C2 transcription factor binding at rs2019090 is shown to promote PDGFD transcription through the CAD promoting allele. With single cell transcriptomic and histology studies with Pdgfd knockdown in an SMC lineage tracing male atherosclerosis mouse model we find that Pdgfd promotes expansion, migration, and transition of SMC lineage cells to the chondromyocyte phenotype. Pdgfd also increases adventitial fibroblast and pericyte expression of chemokines and leukocyte adhesion molecules, which is linked to plaque macrophage recruitment. Despite these changes there is no effect of Pdgfd deletion on overall plaque burden. These findings suggest that PDGFD mediates CAD risk by promoting deleterious phenotypic changes in SMC, along with an inflammatory response that is primarily focused in the adventitia.

Molecular mechanisms of coronary artery disease risk at the PDGFD locus.

Platelet derived growth factor (PDGF) signaling has been extensively studied in the context of vascular disease, but the genetics of this pathway remain to be established. Genome wide association studies (GWAS) for coronary artery disease (CAD) have identified a risk locus at 11q22.3, and we have verified with fine mapping approaches that the regulatory variant rs2019090 and PDGFD represent the functional variant and putative functional gene. Further, FOXC1/C2 transcription factor (TF) binding at rs2019090 was found to promote PDGFD transcription through the CAD promoting allele. Employing a constitutive Pdgfd knockout allele along with SMC lineage tracing in a male atherosclerosis mouse model we mapped single cell transcriptomic, cell state, and lesion anatomical changes associated with gene loss. These studies revealed that Pdgfd promotes expansion, migration, and transition of SMC lineage cells to the chondromyocyte phenotype and vascular calcification. This is in contrast to protective CAD genes TCF21, ZEB2, and SMAD3 which we have shown to promote the fibroblast-like cell transition or perturb the pattern or extent of transition to the chondromyocyte phenotype. Further, Pdgfd expressing fibroblasts and pericytes exhibited greater expression of chemokines and leukocyte adhesion molecules, consistent with observed increased macrophage recruitment to the plaque. Despite these changes there was no effect of Pdgfd deletion on SMC contribution to the fibrous cap or overall lesion burden. These findings suggest that PDGFD mediates CAD risk through promoting SMC expansion and migration, in conjunction with deleterious phenotypic changes, and through promoting an inflammatory response that is primarily focused in the adventitia where it contributes to leukocyte trafficking to the diseased vessel wall.

Discovery of Transacting Long Noncoding RNAs That Regulate Smooth Muscle Cell Phenotype.

BACKGROUND: Smooth muscle cells (SMC), the major cell type in atherosclerotic plaques, are vital in coronary artery diseases (CADs). SMC phenotypic transition, which leads to the formation of various cell types in atherosclerotic plaques, is regulated by a network of genetic and epigenetic mechanisms and governs the risk of disease. The involvement of long noncoding RNAs (lncRNAs) has been increasingly identified in cardiovascular disease. However, SMC lncRNAs have not been comprehensively characterized, and their regulatory role in SMC state transition remains unknown. METHODS: A discovery pipeline was constructed and applied to deeply strand-specific RNA sequencing from perturbed human coronary artery SMC with different disease-related stimuli, to allow for the detection of novel lncRNAs. The functional relevance of a select few novel lncRNAs were verified in vitro. RESULTS: We identified 4579 known and 13 655 de novo lncRNAs in human coronary artery SMC. Consistent with previous long noncoding RNA studies, these lncRNAs overall have fewer exons, are shorter in length than protein-coding genes (pcGenes), and have relatively low expression level. Genomic location of these long noncoding RNA is disproportionately enriched near CAD-related TFs (transcription factors), genetic loci, and gene regulators of SMC identity, suggesting the importance of their function in disease. Two de novo lncRNAs, ZIPPOR (ZEB-interacting suppressor) and TNS1-AS2 (TNS1-antisense 2), were identified by our screen. Combining transcriptional data and in silico modeling along with in vitro validation, we identified CAD gene ZEB2 as a target through which these lncRNAs exert their function in SMC phenotypic transition. CONCLUSIONS: Expression of a large and diverse set of lncRNAs in human coronary artery SMC are highly dynamic in response to CAD-related stimuli. The dynamic changes in expression of these lncRNAs correspond to alterations in transcriptional programs that are relevant to CAD, suggesting a critical role for lncRNAs in SMC phenotypic transition and human atherosclerotic disease.

Integrative single-cell analysis of cardiogenesis identifies developmental trajectories and non-coding mutations in congenital heart disease.

To define the multi-cellular epigenomic and transcriptional landscape of cardiac cellular development, we generated single-cell chromatin accessibility maps of human fetal heart tissues. We identified eight major differentiation trajectories involving primary cardiac cell types, each associated with dynamic transcription factor (TF) activity signatures. We contrasted regulatory landscapes of iPSC-derived cardiac cell types and their in vivo counterparts, which enabled optimization of in vitro differentiation of epicardial cells. Further, we interpreted sequence based deep learning models of cell-type-resolved chromatin accessibility profiles to decipher underlying TF motif lexicons. De novo mutations predicted to affect chromatin accessibility in arterial endothelium were enriched in congenital heart disease (CHD) cases vs. controls. In vitro studies in iPSCs validated the functional impact of identified variation on the predicted developmental cell types. This work thus defines the cell-type-resolved cis-regulatory sequence determinants of heart development and identifies disruption of cell type-specific regulatory elements in CHD.

Smad3 regulates smooth muscle cell fate and mediates adverse remodeling and calcification of the atherosclerotic plaque.

Atherosclerotic plaques consist mostly of smooth muscle cells (SMC), and genes that influence SMC phenotype can modulate coronary artery disease (CAD) risk. Allelic variation at 15q22.33 has been identified by genome-wide association studies to modify the risk of CAD and is associated with the expression of SMAD3 in SMC. However, the mechanism by which this gene modifies CAD risk remains poorly understood. Here we show that SMC-specific deletion of Smad3 in a murine atherosclerosis model resulted in greater plaque burden, more outward remodelling and increased vascular calcification. Single-cell transcriptomic analyses revealed that loss of Smad3 altered SMC transition cell state toward two fates: a SMC phenotype that governs both vascular remodelling and recruitment of inflammatory cells, as well as a chondromyocyte fate. Together, the findings reveal that Smad3 expression in SMC inhibits the emergence of specific SMC phenotypic transition cells that mediate adverse plaque features, including outward remodelling, monocyte recruitment, and vascular calcification.

Embryologic Origin Influences Smooth Muscle Cell Phenotypic Modulation Signatures in Murine Marfan Syndrome Aortic Aneurysm.

BACKGROUND: Aortic root smooth muscle cells (SMC) develop from both the second heart field (SHF) and neural crest. Disparate responses to disease-causing Fbn1 variants by these lineages are proposed to promote focal aortic root aneurysm formation in Marfan syndrome (MFS), but lineage-stratified SMC analysis in vivo is lacking. METHODS: We generated SHF lineage-traced MFS mice and performed integrated multiomic (single-cell RNA and assay for transposase-accessible chromatin sequencing) analysis stratified by embryological origin. SMC subtypes were spatially identified via RNA in situ hybridization. Response to TWIST1 overexpression was determined via lentiviral transduction in human aortic SMCs. RESULTS: Lineage stratification enabled nuanced characterization of aortic root cells. We identified heightened SHF-derived SMC heterogeneity including a subset of Tnnt2 (cardiac troponin T)-expressing cells distinguished by altered proteoglycan expression. MFS aneurysm-associated SMC phenotypic modulation was identified in both SHF-traced and nontraced (neural crest-derived) SMCs; however, transcriptomic responses were distinct between lineages. SHF-derived modulated SMCs overexpressed collagen synthetic genes and small leucine-rich proteoglycans while nontraced SMCs activated chondrogenic genes. These modulated SMCs clustered focally in the aneurysmal aortic root at the region of SHF/neural crest lineage overlap. Integrated RNA-assay for transposase-accessible chromatin analysis identified enriched Twist1 and Smad2/3/4 complex binding motifs in SHF-derived modulated SMCs. TWIST1 overexpression promoted collagen and SLRP gene expression in vitro, suggesting TWIST1 may drive SHF-enriched collagen synthesis in MFS aneurysm. CONCLUSIONS: SMCs derived from both SHF and neural crest lineages undergo phenotypic modulation in MFS aneurysm but are defined by subtly distinct transcriptional responses. Enhanced TWIST1 transcription factor activity may contribute to enriched collagen synthetic pathways SHF-derived SMCs in MFS.

Single-nucleus chromatin accessibility profiling highlights regulatory mechanisms of coronary artery disease risk.

Coronary artery disease (CAD) is a complex inflammatory disease involving genetic influences across cell types. Genome-wide association studies have identified over 200 loci associated with CAD, where the majority of risk variants reside in noncoding DNA sequences impacting cis-regulatory elements. Here, we applied single-nucleus assay for transposase-accessible chromatin with sequencing to profile 28,316 nuclei across coronary artery segments from 41 patients with varying stages of CAD, which revealed 14 distinct cellular clusters. We mapped ~320,000 accessible sites across all cells, identified cell-type-specific elements and transcription factors, and prioritized functional CAD risk variants. We identified elements in smooth muscle cell transition states (for example, fibromyocytes) and functional variants predicted to alter smooth muscle cell- and macrophage-specific regulation of MRAS (3q22) and LIPA (10q23), respectively. We further nominated key driver transcription factors such as PRDM16 and TBX2. Together, this single-nucleus atlas provides a critical step towards interpreting regulatory mechanisms across the continuum of CAD risk.

Human Coronary Plaque T Cells Are Clonal and Cross-React to Virus and Self.

BACKGROUND: Coronary artery disease is an incurable, life-threatening disease that was once considered primarily a disorder of lipid deposition. Coronary artery disease is now also characterized by chronic inflammation' notable for the buildup of atherosclerotic plaques containing immune cells in various states of activation and differentiation. Understanding how these immune cells contribute to disease progression may lead to the development of novel therapeutic strategies. METHODS: We used single-cell technology and in vitro assays to interrogate the immune microenvironment of human coronary atherosclerotic plaque at different stages of maturity. RESULTS: In addition to macrophages, we found a high proportion of αß T cells in the coronary plaques. Most of these T cells lack high expression of CCR7 and L-selectin, indicating that they are primarily antigen-experienced memory cells. Notably, nearly one-third of these cells express the HLA-DRA surface marker, signifying activation through their TCRs (T-cell receptors). Consistent with this, TCR repertoire analysis confirmed the presence of activated αß T cells (CD4

Integration of genetic colocalizations with physiological and pharmacological perturbations identifies cardiometabolic disease genes.

BACKGROUND: Identification of causal genes for polygenic human diseases has been extremely challenging, and our understanding of how physiological and pharmacological stimuli modulate genetic risk at disease-associated loci is limited. Specifically, insulin resistance (IR), a common feature of cardiometabolic disease, including type 2 diabetes, obesity, and dyslipidemia, lacks well-powered genome-wide association studies (GWAS), and therefore, few associated loci and causal genes have been identified. METHODS: Here, we perform and integrate linkage disequilibrium (LD)-adjusted colocalization analyses across nine cardiometabolic traits (fasting insulin, fasting glucose, insulin sensitivity, insulin sensitivity index, type 2 diabetes, triglycerides, high-density lipoprotein, body mass index, and waist-hip ratio) combined with expression and splicing quantitative trait loci (eQTLs and sQTLs) from five metabolically relevant human tissues (subcutaneous and visceral adipose, skeletal muscle, liver, and pancreas). To elucidate the upstream regulators and functional mechanisms for these genes, we integrate their transcriptional responses to 21 relevant physiological and pharmacological perturbations in human adipocytes, hepatocytes, and skeletal muscle cells and map their protein-protein interactions. RESULTS: We identify 470 colocalized loci and prioritize 207 loci with a single colocalized gene. Patterns of shared colocalizations across traits and tissues highlight different potential roles for colocalized genes in cardiometabolic disease and distinguish several genes involved in pancreatic ß-cell function from others with a more direct role in skeletal muscle, liver, and adipose tissues. At the loci with a single colocalized gene, 42 of these genes were regulated by insulin and 35 by glucose in perturbation experiments, including 17 regulated by both. Other metabolic perturbations regulated the expression of 30 more genes not regulated by glucose or insulin, pointing to other potential upstream regulators of candidate causal genes. CONCLUSIONS: Our use of transcriptional responses under metabolic perturbations to contextualize genetic associations from our custom colocalization approach provides a list of likely causal genes and their upstream regulators in the context of IR-associated cardiometabolic risk.