Single-Cell Gene-Regulatory Networks of Advanced Symptomatic Atherosclerosis.

BACKGROUND: While our understanding of the single-cell gene expression patterns underlying the transformation of vascular cell types during the progression of atherosclerosis is rapidly improving, the clinical and pathophysiological relevance of these changes remains poorly understood. METHODS: Single-cell RNA sequencing data generated with SmartSeq2 (≈8000 genes/cell) in 16 588 single cells isolated during atherosclerosis progression in Ldlr-/-Apob100/100 mice with human-like plasma lipoproteins and from humans with asymptomatic and symptomatic carotid plaques was clustered into multiple subtypes. For clinical and pathophysiological context, the advanced-stage and symptomatic subtype clusters were integrated with 135 tissue-specific (atherosclerotic aortic wall, mammary artery, liver, skeletal muscle, and visceral and subcutaneous, fat) gene-regulatory networks (GRNs) inferred from 600 coronary artery disease patients in the STARNET (Stockholm-Tartu Atherosclerosis Reverse Network Engineering Task) study. RESULTS: Advanced stages of atherosclerosis progression and symptomatic carotid plaques were largely characterized by 3 smooth muscle cells (SMCs), and 3 macrophage subtype clusters with extracellular matrix organization/osteogenic (SMC), and M1-type proinflammatory/Trem2-high lipid-associated (macrophage) phenotypes. Integrative analysis of these 6 clusters with STARNET revealed significant enrichments of 3 arterial wall GRNs: GRN33 (macrophage), GRN39 (SMC), and GRN122 (macrophage) with major contributions to coronary artery disease heritability and strong associations with clinical scores of coronary atherosclerosis severity. The presence and pathophysiological relevance of GRN39 were verified in 5 independent RNAseq data sets obtained from the human coronary and aortic artery, and primary SMCs and by targeting its top-key drivers, FRZB and ALCAM in cultured human coronary artery SMCs. CONCLUSIONS: By identifying and integrating the most gene-rich single-cell subclusters of atherosclerosis to date with a coronary artery disease framework of GRNs, GRN39 was identified and independently validated as being critical for the transformation of contractile SMCs into an osteogenic phenotype promoting advanced, symptomatic atherosclerosis.

Oncogenic KEAP1 mutations activate TRAF2-NFκB signaling to prevent apoptosis in lung cancer cells.

The Kelch-like ECH-associated protein 1 (KEAP1) - Nuclear factor erythroid 2 -related factor 2 (NRF2) pathway is the major transcriptional stress response system in cells against oxidative and electrophilic stress. NRF2 is frequently constitutively active in many cancers, rendering the cells resistant to chemo- and radiotherapy. Loss-of-function (LOF) mutations in the repressor protein KEAP1 are common in non-small cell lung cancer, particularly adenocarcinoma. While the mutations can occur throughout the gene, they are enriched in certain areas, indicating that these may have unique functional importance. In this study, we show that in the GSEA analysis of TCGA lung adenocarcinoma RNA-seq data, the KEAP1 mutations in R320 and R470 were associated with enhanced Tumor Necrosis Factor alpha (TNFα) - Nuclear Factor kappa subunit B (NFκB) signaling as well as MYC and MTORC1 pathways. To address the functional role of these hotspot mutations, affinity purification and mass spectrometry (AP-MS) analysis of wild type (wt) KEAP1 and its mutation forms, R320Q and R470C were employed to interrogate differences in the protein interactome. We identified TNF receptor associated factor 2 (TRAF2) as a putative protein interaction partner. Both mutant KEAP1 forms showed increased interaction with TRAF2 and other anti-apoptotic proteins, suggesting that apoptosis signalling could be affected by the protein interactions. A549 lung adenocarcinoma cells overexpressing mutant KEAP1 showed high TRAF2-mediated NFκB activity and increased protection against apoptosis, XIAP being one of the key proteins involved in anti-apoptotic signalling. To conclude, KEAP1 R320Q and R470C and its interaction with TRAF2 leads to activation of NFκB pathway, thereby protecting against apoptosis.

Cross-tissue omics analysis discovers ten adipose genes encoding secreted proteins in obesity-related non-alcoholic fatty liver disease.

BACKGROUND: Non-alcoholic fatty liver disease (NAFLD) is a fast-growing, underdiagnosed, epidemic. We hypothesise that obesity-related inflammation compromises adipose tissue functions, preventing efficient fat storage, and thus driving ectopic fat accumulation into the liver. METHODS: To identify adipose-based mechanisms and potential serum biomarker candidates (SBCs) for NAFLD, we utilise dual-tissue RNA-sequencing (RNA-seq) data in adipose tissue and liver, paired with histology-based NAFLD diagnosis, from the same individuals in a cohort of obese individuals. We first scan for genes that are differentially expressed (DE) for NAFLD in obese individuals' subcutaneous adipose tissue but not in their liver; encode proteins secreted to serum; and show preferential adipose expression. Then the identified genes are filtered to key adipose-origin NAFLD genes by best subset analysis, knockdown experiments during human preadipocyte differentiation, recombinant protein treatment experiments in human liver HepG2 cells, and genetic analysis. FINDINGS: We discover a set of genes, including 10 SBCs, that may modulate NAFLD pathogenesis by impacting adipose tissue function. Based on best subset analysis, we further follow-up on two SBCs CCDC80 and SOD3 by knockdown in human preadipocytes and subsequent differentiation experiments, which show that they modulate crucial adipogenesis genes, LPL, SREBPF1, and LEP. We also show that treatment of the liver HepG2 cells with the CCDC80 and SOD3 recombinant proteins impacts genes related to steatosis and lipid processing, including PPARA, NFE2L2, and RNF128. Finally, utilizing the adipose NAFLD DE gene cis-regulatory variants associated with serum triglycerides (TGs) in extensive genome-wide association studies (GWASs), we demonstrate a unidirectional effect of serum TGs on NAFLD with Mendelian Randomization (MR) analysis. We also demonstrate that a single SNP regulating one of the SBC genes, rs2845885, produces a significant MR result by itself. This supports the conclusion that genetically regulated adipose expression of the NAFLD DE genes may contribute to NAFLD through changes in serum TG levels. INTERPRETATION: Our results from the dual-tissue transcriptomics screening improve the understanding of obesity-related NAFLD by providing a targeted set of 10 adipose tissue-active genes as new serum biomarker candidates for the currently grossly underdiagnosed fatty liver disease. FUNDING: The work was supported by NIH grants R01HG010505 and R01DK132775. The Genotype-Tissue Expression (GTEx) Project was supported by the Common Fund of the Office of the Director of the National Institutes of Health, and by NCI, NHGRI, NHLBI, NIDA, NIMH, and NINDS. The KOBS study (J. P.) was supported by the Finnish Diabetes Research Foundation, Kuopio University Hospital Project grant (EVO/VTR grants 2005-2019), and the Academy of Finland grant (Contract no. 138006). This study was funded by the European Research Council under the European Union's Horizon 2020 research and innovation program (Grant No. 802825 to M. U. K.). K. H. P. was funded by the Academy of Finland (grant numbers 272376, 266286, 314383, and 335443), the Finnish Medical Foundation, Gyllenberg Foundation, Novo Nordisk Foundation (grant numbers NNF10OC1013354, NNF17OC0027232, and NNF20OC0060547), Finnish Diabetes Research Foundation, Finnish Foundation for Cardiovascular Research, University of Helsinki, and Helsinki University Hospital and Government Research Funds. I. S. was funded by the Instrumentarium Science Foundation. Personal grants to U. T. A. were received from the Matti and Vappu Maukonen Foundation, Ella och Georg Ehrnrooths Stiftelse and the Finnish Foundation for Cardiovascular Research.

Exploring the genetic basis of coronary artery disease using functional genomics.

Genome-wide Association Studies (GWAS) have identified more than 300 loci associated with coronary artery disease (CAD), defining the genetic risk map of the disease. However, the translation of the association signals into biological-pathophysiological mechanisms constitute a major challenge. Through a group of examples of studies focused on CAD, we discuss the rationale, basic principles and outcomes of the main methodologies implemented to prioritize and characterize causal variants and their target genes. Additionally, we highlight the strategies as well as the current methods that integrate association and functional genomics data to dissect the cellular specificity underlying the complexity of disease mechanisms. Despite the limitations of existing approaches, the increasing knowledge generated through functional studies helps interpret GWAS maps and opens novel avenues for the clinical usability of association data.

HAS3-induced extracellular vesicles from melanoma cells stimulate IHH mediated c-Myc upregulation via the hedgehog signaling pathway in target cells.

Intercellular communication is fundamental to the survival and maintenance of all multicellular systems, whereas dysregulation of communication pathways can drive cancer progression. Extracellular vesicles (EVs) are mediators of cell-to-cell communication that regulate a variety of cellular processes involved in tumor progression. Overexpression of a specific plasma membrane enzyme, hyaluronan synthase 3 (HAS3), is one of the factors that can induce EV shedding. HAS3, and particularly its product hyaluronan (HA), are carried by EVs and are known to be associated with the tumorigenic properties of cancer cells. To elucidate the specific effects of cancerous, HAS3-induced EVs on target cells, normal human keratinocytes and melanoma cells were treated with EVs derived from GFP-HAS3 expressing metastatic melanoma cells. We found that the HA receptor CD44 participated in the regulation of EV binding to target cells. Furthermore, GFP-HAS3-positive EVs induced HA secretion, proliferation and invasion of target cells. Our results suggest that HAS3-EVs contains increased quantities of IHH, which activates the target cell hedgehog signaling cascade and leads to the activation of c-Myc and regulation of claspin expression. This signaling of IHH in HAS3-EVs resulted in increased cell proliferation. Claspin immunostaining correlated with HA content in human cutaneous melanocytic lesions, supporting our in vitro findings and suggesting a reciprocal regulation between claspin expression and HA synthesis. This study shows for the first time that EVs originating from HAS3 overexpressing cells carry mitogenic signals that induce proliferation and epithelial-to-mesenchymal transition in target cells. The study also identifies a novel feedback regulation between the hedgehog signaling pathway and HA metabolism in melanoma, mediated by EVs carrying HA and IHH.

Correlative light and electron microscopy is a powerful tool to study interactions of extracellular vesicles with recipient cells.

Extracellular vesicles (EVs) and their interactions with recipient cells constitute a rapidly growing research area. However, due to the limitations in current methodologies, the mechanisms of these interactions are still unclear. Microscopic studies of EVs are challenging, because their typical diameter is near the resolution limit of light microscopy, and electron microscopy has restricted possibilities for protein labelling. The objective of this study was to combine these two techniques to demonstrate in detail the interactions of EVs by recipient cells. Hyaluronan synthase 3 (HAS3) is an integral transmembrane protein that is enriched in EVs. In this work, GFP-HAS3 was utilized to study the interactions of EVs with the recipient cells. Surprisingly, confocal analysis correlation with scanning electron microscopy (SEM) revealed that most of the EVs were indeed lying on the recipient cell's plasma membrane, while the level of EV-derived intracellular signal was low. Immunoelectron microscopy supported this finding. Furthermore, hyaluronan oligosaccharides decreased the numbers of bound EVs, suggesting that CD44 participates in the regulation of their binding. This study indicates that correlative light and electron microscopy is a reliable method to analyze EV interactions with recipient cells. Detailed 3D confocal imaging of EV carrying a GFP-label on their plasma membrane combined with high-resolution electron microscopy provides significantly more information than either of the techniques alone. In the future studies it is crucial to utilize these techniques and their combinations to solve in detail the ambiguous fate of EV in target cells. Furthermore, live cell imaging at high resolution will be required to obtain definite answers on the detailed mechanisms of binding, fusion and endocytosis of EVs.

Extracellular vesicles are integral and functional components of the extracellular matrix.

Extracellular vesicles (EV) are small plasma membrane-derived particles released into the extracellular space by virtually all cell types. Recently, EV have received increased interest because of their capability to carry nucleic acids, proteins, lipids and signaling molecules and to transfer their cargo into the target cells. Less attention has been paid to their role in modifying the composition of the extracellular matrix (ECM), either directly or indirectly via regulating the ability of target cells to synthesize or degrade matrix molecules. Based on recent results, EV can be considered one of the structural and functional components of the ECM that participate in matrix organization, regulation of cells within it, and in determining the physical properties of soft connective tissues, bone, cartilage and dentin. This review addresses the relevance of EV as specific modulators of the ECM, such as during the assembly and disassembly of the molecular network, signaling through the ECM and formation of niches suitable for tissue regeneration, inflammation and tumor progression. Finally, we assess the potential of these aspects of EV biology to translational medicine.

Human mesenchymal stem cells secrete hyaluronan-coated extracellular vesicles.

Extracellular vesicles (EVs) secreted by stem cells are potential factors mediating tissue regeneration. They travel from bone marrow stem cells into damaged tissues, suggesting that they can repair tissue injuries without directly replacing parenchymal cells. We have discovered that hyaluronan (HA) synthesis is associated with the shedding of HA-coated EVs. The aim of this study was to test whether bone marrow-derived hMSCs secrete HA-coated EVs. The EVs secreted by MSCs were isolated by differential centrifugation and characterized by nanoparticle tracking analysis. Their morphology and budding mechanisms were inspected by confocal microscopy and correlative light and electron microscopy. Hyaluronan synthesis of hMSCs was induced by lipopolysaccharide and inhibited by RNA interference and 4-methylumbelliferone. It was found that the MSCs have extremely long apical and lateral HA-coated filopodia, typical for cells with an active HA secretion. Additionally, they secreted HA-coated EVs carrying mRNAs for CD44 and all HAS isoforms. The results show that stem cells have a strong intrinsic potential for HA synthesis and EV secretion, and the amount of HA carried on EVs reflects the HA content of the original cells. These results show that the secretion of HA-coated EVs by hMSCs is a general process, that may contribute to many of the mechanisms of HA-mediated tissue regeneration. Additionally, an HA coat on EVs may regulate their interactions with target cells and participate in extracellular matrix remodeling.

EMT induced by EGF and wounding activates hyaluronan synthesis machinery and EV shedding in rat primary mesothelial cells.

The mesothelium is a membrane that forms the lining of several body cavities. It is composed of simple squamous mesothelial cells that secrete a glycosaminoglycan-rich lubricating fluid between inner organs. One of the most abundant glycosaminoglycans of those fluids is hyaluronan, which is synthesized on a plasma membrane and especially on apical filopodia of cultured cells. Our recent study showed that similar hyaluronan-rich protrusions are found in mesothelial lining in vivo, which suggests that hyaluronan synthesis in plasma membrane protrusions is a general process. However, the mesothelial lining was negative for the hyaluronan receptor CD44 while in many previous studies cultured mesothelial cells have been shown to express CD44. To further explore these findings we induced epithelial to mesenchymal transition in primary rat mesothelial cells by EGF-treatment and scratch wounding. Surprisingly, the results showed that at a normal epithelial, confluent stage the mesothelial cells are negative for CD44, but EMT induced by EGF or wounding activates CD44 expression and the whole hyaluronan synthesis machinery. In addition to typical EMT-like morphological changes, the growth of apical filopodia and budding of extracellular vesicles (EVs) were induced. In summary, the results of this study show that the activation of hyaluronan synthesis machinery, especially the expression of CD44 is strongly associated with EMT induced by EGF and wounding in mesothelial cells. Moreover, EMT enhances the secretion of EVs that carry CD44 and hyaluronan, which may be important regulators in EV interactions with their targets and ECM remodeling. The results of the present study also suggest that CD44 is a potential marker for EVs, especially those secreted from cells during tissue repair and pathological processes.

UDP-sugar substrates of HAS3 regulate its O-GlcNAcylation, intracellular traffic, extracellular shedding and correlate with melanoma progression.

Hyaluronan content is a powerful prognostic factor in many cancer types, but the molecular basis of its synthesis in cancer still remains unclear. Hyaluronan synthesis requires the transport of hyaluronan synthases (HAS1-3) from Golgi to plasma membrane (PM), where the enzymes are activated. For the very first time, the present study demonstrated a rapid recycling of HAS3 between PM and endosomes, controlled by the cytosolic levels of the HAS substrates UDP-GlcUA and UDP-GlcNAc. Depletion of UDP-GlcNAc or UDP-GlcUA shifted the balance towards HAS3 endocytosis, and inhibition of hyaluronan synthesis. In contrast, UDP-GlcNAc surplus suppressed endocytosis and lysosomal decay of HAS3, favoring its retention in PM, stimulating hyaluronan synthesis, and HAS3 shedding in extracellular vesicles. The concentration of UDP-GlcNAc also controlled the level of O-GlcNAc modification of HAS3. Increasing O-GlcNAcylation reproduced the effects of UDP-GlcNAc surplus on HAS3 trafficking, while its suppression showed the opposite effects, indicating that O-GlcNAc signaling is associated to UDP-GlcNAc supply. Importantly, a similar correlation existed between the expression of GFAT1 (the rate limiting enzyme in UDP-GlcNAc synthesis) and hyaluronan content in early and deep human melanomas, suggesting the association of UDP-sugar metabolism in initiation of melanomagenesis. In general, changes in glucose metabolism, realized through UDP-sugar contents and O-GlcNAc signaling, are important in HAS3 trafficking, hyaluronan synthesis, and correlates with melanoma progression.