The X-linked epigenetic regulator UTX controls NK cell-intrinsic sex differences.

Viral infection outcomes are sex biased, with males generally more susceptible than females. Paradoxically, the numbers of antiviral natural killer (NK) cells are increased in males. We demonstrate that while numbers of NK cells are increased in male mice, they display decreased effector function compared to females in mice and humans. These differences were not solely dependent on gonadal hormones, because they persisted in gonadectomized mice. Kdm6a (which encodes the protein UTX), an epigenetic regulator that escapes X inactivation, was lower in male NK cells, while NK cell-intrinsic UTX deficiency in female mice increased NK cell numbers and reduced effector responses. Furthermore, mice with NK cell-intrinsic UTX deficiency showed increased lethality to mouse cytomegalovirus. Integrative multi-omics analysis revealed a critical role for UTX in regulating chromatin accessibility and gene expression critical for NK cell homeostasis and effector function. Collectively, these data implicate UTX as a critical molecular determinant of sex differences in NK cells.

Type V Collagen in Scar Tissue Regulates the Size of Scar after Heart Injury.

Scar tissue size following myocardial infarction is an independent predictor of cardiovascular outcomes, yet little is known about factors regulating scar size. We demonstrate that collagen V, a minor constituent of heart scars, regulates the size of heart scars after ischemic injury. Depletion of collagen V led to a paradoxical increase in post-infarction scar size with worsening of heart function. A systems genetics approach across 100 in-bred strains of mice demonstrated that collagen V is a critical driver of postinjury heart function. We show that collagen V deficiency alters the mechanical properties of scar tissue, and altered reciprocal feedback between matrix and cells induces expression of mechanosensitive integrins that drive fibroblast activation and increase scar size. Cilengitide, an inhibitor of specific integrins, rescues the phenotype of increased post-injury scarring in collagen-V-deficient mice. These observations demonstrate that collagen V regulates scar size in an integrin-dependent manner.

The transcription factor Fli1 restricts the formation of memory precursor NK cells during viral infection.

Natural killer (NK) cells are innate lymphocytes that possess traits of adaptive immunity, such as memory formation. However, the molecular mechanisms by which NK cells persist to form memory cells are not well understood. Using single-cell RNA sequencing, we identified two distinct effector NK cell (NKeff) populations following mouse cytomegalovirus infection. Ly6C- memory precursor (MP) NK cells showed enhanced survival during the contraction phase in a Bcl2-dependent manner, and differentiated into Ly6C+ memory NK cells. MP NK cells exhibited distinct transcriptional and epigenetic signatures compared with Ly6C+ NKeff cells, with a core epigenetic signature shared with MP CD8+ T cells enriched in ETS1 and Fli1 DNA-binding motifs. Fli1 was induced by STAT5 signaling ex vivo, and increased levels of the pro-apoptotic factor Bim in early effector NK cells following viral infection. These results suggest that a NK cell-intrinsic checkpoint controlled by the transcription factor Fli1 limits MP NK formation by regulating early effector NK cell fitness during viral infection.

Single-cell sequencing of human white adipose tissue identifies new cell states in health and obesity.

White adipose tissue (WAT) is an essential regulator of energy storage and systemic metabolic homeostasis. Regulatory networks consisting of immune and structural cells are necessary to maintain WAT metabolism, which can become impaired during obesity in mammals. Using single-cell transcriptomics and flow cytometry, we unveil a large-scale comprehensive cellular census of the stromal vascular fraction of healthy lean and obese human WAT. We report new subsets and developmental trajectories of adipose-resident innate lymphoid cells, dendritic cells and monocyte-derived macrophage populations that accumulate in obese WAT. Analysis of cell-cell ligand-receptor interactions and obesity-enriched signaling pathways revealed a switch from immunoregulatory mechanisms in lean WAT to inflammatory networks in obese WAT. These results provide a detailed and unbiased cellular landscape of homeostatic and inflammatory circuits in healthy human WAT.

The cellular architecture of the antimicrobial response network in human leprosy granulomas.

Granulomas are complex cellular structures composed predominantly of macrophages and lymphocytes that function to contain and kill invading pathogens. Here, we investigated the single-cell phenotypes associated with antimicrobial responses in human leprosy granulomas by applying single-cell and spatial sequencing to leprosy biopsy specimens. We focused on reversal reactions (RRs), a dynamic process whereby some patients with disseminated lepromatous leprosy (L-lep) transition toward self-limiting tuberculoid leprosy (T-lep), mounting effective antimicrobial responses. We identified a set of genes encoding proteins involved in antimicrobial responses that are differentially expressed in RR versus L-lep lesions and regulated by interferon-γ and interleukin-1ß. By integrating the spatial coordinates of the key cell types and antimicrobial gene expression in RR and T-lep lesions, we constructed a map revealing the organized architecture of granulomas depicting compositional and functional layers by which macrophages, T cells, keratinocytes and fibroblasts can each contribute to the antimicrobial response.

MYCT1 controls environmental sensing in human haematopoietic stem cells.

The processes that govern human haematopoietic stem cell (HSC) self-renewal and engraftment are poorly understood and challenging to recapitulate in culture to reliably expand functional HSCs1-3. Here we identify MYC target 1 (MYCT1; also known as MTLC) as a crucial human HSC regulator that moderates endocytosis and environmental sensing in HSCs. MYCT1 is selectively expressed in undifferentiated human haematopoietic stem and progenitor cells (HSPCs) and endothelial cells but becomes markedly downregulated during HSC culture. Lentivirus-mediated knockdown of MYCT1 prevented human fetal liver and cord blood (CB) HSPC expansion and engraftment. By contrast, restoring MYCT1 expression improved the expansion and engraftment of cultured CB HSPCs. Single-cell RNA sequencing of human CB HSPCs in which MYCT1 was knocked down or overexpressed revealed that MYCT1 governs important regulatory programmes and cellular properties essential for HSC stemness, such as ETS factor expression and low mitochondrial activity. MYCT1 is localized in the endosomal membrane in HSPCs and interacts with vesicle trafficking regulators and signalling machinery. MYCT1 loss in HSPCs led to excessive endocytosis and hyperactive signalling responses, whereas restoring MYCT1 expression balanced culture-induced endocytosis and dysregulated signalling. Moreover, sorting cultured CB HSPCs on the basis of lowest endocytosis rate identified HSPCs with preserved MYCT1 expression and MYCT1-regulated HSC stemness programmes. Our work identifies MYCT1-moderated endocytosis and environmental sensing as essential regulatory mechanisms required to preserve human HSC stemness. Our data also pinpoint silencing of MYCT1 as a cell-culture-induced vulnerability that compromises human HSC expansion.

Second-Strand Synthesis-Based Massively Parallel scRNA-Seq Reveals Cellular States and Molecular Features of Human Inflammatory Skin Pathologies.

High-throughput single-cell RNA-sequencing (scRNA-seq) methodologies enable characterization of complex biological samples by increasing the number of cells that can be profiled contemporaneously. Nevertheless, these approaches recover less information per cell than low-throughput strategies. To accurately report the expression of key phenotypic features of cells, scRNA-seq platforms are needed that are both high fidelity and high throughput. To address this need, we created Seq-Well S3 ("Second-Strand Synthesis"), a massively parallel scRNA-seq protocol that uses a randomly primed second-strand synthesis to recover complementary DNA (cDNA) molecules that were successfully reverse transcribed but to which a second oligonucleotide handle, necessary for subsequent whole transcriptome amplification, was not appended due to inefficient template switching. Seq-Well S3 increased the efficiency of transcript capture and gene detection compared with that of previous iterations by up to 10- and 5-fold, respectively. We used Seq-Well S3 to chart the transcriptional landscape of five human inflammatory skin diseases, thus providing a resource for the further study of human skin inflammation.

Mapping human haematopoietic stem cells from haemogenic endothelium to birth.

The ontogeny of human haematopoietic stem cells (HSCs) is poorly defined owing to the inability to identify HSCs as they emerge and mature at different haematopoietic sites1. Here we created a single-cell transcriptome map of human haematopoietic tissues from the first trimester to birth and found that the HSC signature RUNX1+HOXA9+MLLT3+MECOM+HLF+SPINK2+ distinguishes HSCs from progenitors throughout gestation. In addition to the aorta-gonad-mesonephros region, nascent HSCs populated the placenta and yolk sac before colonizing the liver at 6 weeks. A comparison of HSCs at different maturation stages revealed the establishment of HSC transcription factor machinery after the emergence of HSCs, whereas their surface phenotype evolved throughout development. The HSC transition to the liver marked a molecular shift evidenced by suppression of surface antigens reflecting nascent HSC identity, and acquisition of the HSC maturity markers CD133 (encoded by PROM1) and HLA-DR. HSC origin was tracked to ALDH1A1+KCNK17+ haemogenic endothelial cells, which arose from an IL33+ALDH1A1+ arterial endothelial subset termed pre-haemogenic endothelial cells. Using spatial transcriptomics and immunofluorescence, we visualized this process in ventrally located intra-aortic haematopoietic clusters. The in vivo map of human HSC ontogeny validated the generation of aorta-gonad-mesonephros-like definitive haematopoietic stem and progenitor cells from human pluripotent stem cells, and serves as a guide to improve their maturation to functional HSCs.

Heterogeneous pdgfrb+ cells regulate coronary vessel development and revascularization during heart regeneration.

Endothelial cells emerge from the atrioventricular canal to form coronary blood vessels in juvenile zebrafish hearts. We find that pdgfrb is first expressed in the epicardium around the atrioventricular canal and later becomes localized mainly in the mural cells. pdgfrb mutant fish show severe defects in mural cell recruitment and coronary vessel development. Single-cell RNA sequencing analyses identified pdgfrb+ cells as epicardium-derived cells (EPDCs) and mural cells. Mural cells associated with coronary arteries also express cxcl12b and smooth muscle cell markers. Interestingly, these mural cells remain associated with coronary arteries even in the absence of Pdgfrß, although smooth muscle gene expression is downregulated. We find that pdgfrb expression dynamically changes in EPDCs of regenerating hearts. Differential gene expression analyses of pdgfrb+ EPDCs and mural cells suggest that they express genes that are important for regeneration after heart injuries. mdka was identified as a highly upregulated gene in pdgfrb+ cells during heart regeneration. However, pdgfrb but not mdka mutants show defects in heart regeneration after amputation. Our results demonstrate that heterogeneous pdgfrb+ cells are essential for coronary development and heart regeneration.

Single-cell profiling of prurigo nodularis demonstrates immune-stromal crosstalk driving profibrotic responses and reversal with nemolizumab.

BACKGROUND: Prurigo nodularis (PN) is a chronic neuroimmune skin disease characterized by bilaterally distributed pruritic hyperkeratotic nodules on extremities and trunk. Neuroimmune dysregulation and chronic scratching are believed to both induce and maintain the characteristic lesions. OBJECTIVES: This study sought to provide a comprehensive view of the molecular pathogenesis of PN at the single-cell level to identify and outline key pathologic processes and the cell types involved. Features that distinguish PN skin from the skin of patients with atopic dermatitis were of particular interest. We further aimed to determine the impact of the IL31RA antagonist, nemolizumab, and its specificity at the single-cell level. METHODS: Single-cell RNA-sequencing of skin from 15 healthy donors and nonlesional and lesional skin from 6 patients each with PN and atopic dermatitis, combined with spatial-sequencing using the 10x Visium platform. Integration with bulk RNA-sequencing data from patients treated with nemolizumab. RESULTS: This study demonstrates that PN is an inflammatory skin disease characterized by both keratinocyte proliferation and activation of profibrotic responses. This study also demonstrates that the COL11A1+ fibroblast subset is a major contributor to fibrosis and is predominantly found in the papillary dermis of PN skin. Activation of fibrotic responses is the main distinguishing feature between PN and atopic dermatitis skin. This study further shows the broad effect of nemolizumab on PN cell types, with a prominent effect driving COL11A1+ fibroblast and keratinocyte responses toward normal. CONCLUSIONS: This study provides a high-resolution characterization of the cell types and cellular processes activated in PN skin, establishing PN as a chronic fibrotic inflammatory skin disease. It further demonstrates the broad effect of nemolizumab on pathological processes in PN skin.

Single-cell RNA sequencing of batch Chlamydomonas cultures reveals heterogeneity in their diurnal cycle phase.

The photosynthetic unicellular alga Chlamydomonas (Chlamydomonas reinhardtii) is a versatile reference for algal biology because of its ease of culture in the laboratory. Genomic and systems biology approaches have previously described transcriptome responses to environmental changes using bulk data, thus representing the average behavior from pools of cells. Here, we apply single-cell RNA sequencing (scRNA-seq) to probe the heterogeneity of Chlamydomonas cell populations under three environments and in two genotypes differing by the presence of a cell wall. First, we determined that RNA can be extracted from single algal cells with or without a cell wall, offering the possibility to sample natural algal communities. Second, scRNA-seq successfully separated single cells into nonoverlapping cell clusters according to their growth conditions. Cells exposed to iron or nitrogen deficiency were easily distinguished despite a shared tendency to arrest photosynthesis and cell division to economize resources. Notably, these groups of cells not only recapitulated known patterns observed with bulk RNA-seq but also revealed their inherent heterogeneity. A substantial source of variation between cells originated from their endogenous diurnal phase, although cultures were grown in constant light. We exploited this result to show that circadian iron responses may be conserved from algae to land plants. We document experimentally that bulk RNA-seq data represent an average of typically hidden heterogeneity in the population.

Placenta accreta spectrum disorder at single-cell resolution: a loss of boundary limits in the decidua and endothelium.

BACKGROUND: Placenta accreta spectrum disorders are associated with severe maternal morbidity and mortality. Placenta accreta spectrum disorders involve excessive adherence of the placenta preventing separation at birth. Traditionally, this condition has been attributed to excessive trophoblast invasion; however, an alternative view is a fundamental defect in decidual biology. OBJECTIVE: This study aimed to gain insights into the understanding of placenta accreta spectrum disorder by using single-cell and spatially resolved transcriptomics to characterize cellular heterogeneity at the maternal-fetal interface in placenta accreta spectrum disorders. STUDY DESIGN: To assess cellular heterogeneity and the function of cell types, single-cell RNA sequencing and spatially resolved transcriptomics were used. A total of 12 placentas were included, 6 placentas with placenta accreta spectrum disorder and 6 controls. For each placenta with placenta accreta spectrum disorder, multiple biopsies were taken at the following sites: placenta accreta spectrum adherent and nonadherent sites in the same placenta. Of note, 2 platforms were used to generate libraries: the 10× Chromium and NanoString GeoMX Digital Spatial Profiler for single-cell and spatially resolved transcriptomes, respectively. Differential gene expression analysis was performed using a suite of bioinformatic tools (Seurat and GeoMxTools R packages). Correction for multiple testing was performed using Clipper. In situ hybridization was performed with RNAscope, and immunohistochemistry was used to assess protein expression. RESULTS: In creating a placenta accreta cell atlas, there were dramatic difference in the transcriptional profile by site of biopsy between placenta accreta spectrum and controls. Most of the differences were noted at the site of adherence; however, differences existed within the placenta between the adherent and nonadherent site of the same placenta in placenta accreta. Among all cell types, the endothelial-stromal populations exhibited the greatest difference in gene expression, driven by changes in collagen genes, namely collagen type III alpha 1 chain (COL3A1), growth factors, epidermal growth factor-like protein 6 (EGFL6), and hepatocyte growth factor (HGF), and angiogenesis-related genes, namely delta-like noncanonical Notch ligand 1 (DLK1) and platelet endothelial cell adhesion molecule-1 (PECAM1). Intraplacental tropism (adherent versus non-adherent sites in the same placenta) was driven by differences in endothelial-stromal cells with notable differences in bone morphogenic protein 5 (BMP5) and osteopontin (SPP1) in the adherent vs nonadherent site of placenta accreta spectrum. CONCLUSION: Placenta accreta spectrum disorders were characterized at single-cell resolution to gain insight into the pathophysiology of the disease. An atlas of the placenta at single cell resolution in accreta allows for understanding in the biology of the intimate maternal and fetal interaction. The contributions of stromal and endothelial cells were demonstrated through alterations in the extracellular matrix, growth factors, and angiogenesis. Transcriptional and protein changes in the stroma of placenta accreta spectrum shift the etiologic explanation away from "invasive trophoblast" to "loss of boundary limits" in the decidua. Gene targets identified in this study may be used to refine diagnostic assays in early pregnancy, track disease progression over time, and inform therapeutic discoveries.

Long-term Cu exposure alters CYP450s activity and induces jejunum injury and apoptosis in broilers.

Copper (Cu) is an essential trace element that plays a crucial role in numerous physiopathological processes related to human and animal health. In the poultry industry, Cu is used to promote growth as a feed supplement, but excessive use can lead to toxicity on animals. Cytochrome P450 enzymes (CYP450s) are a superfamily of proteins that require heme as a cofactor and are essential for the metabolism of xenobiotic compounds. The purpose of this study was to explore the influence of exposure to Cu on CYP450s activity and apoptosis in the jejunum of broilers. Hence, we first simulated the Cu exposure model by feeding chickens diets containing different amounts of Cu. In the present study, histopathological observations have revealed morphological damage to the jejunum. The expression levels of genes and proteins of intestinal barrier markers were prominently downregulated. While the mRNA expression level of the gene associated with CYP450s was significantly increased. Additionally, apoptosis-related genes and proteins (Bak1, Bax, Caspase-9, Caspase-3, and CytC) were also significantly augmented by excessive Cu, while simultaneously decreasing the expression of Bcl-2. It can be concluded that long-term Cu exposure affects CYP450s activity, disrupts intestinal barrier function, and causes apoptosis in broilers that ultimately leads to jejunum damage.

Optimizing growth and antioxidant function in heat-stressed broilers with vitamin C and betaine supplementation.

This study investigates the potential of vitamin C (VC) and/or betaine (Bet) to enhance growth performance, regulate serum metabolism, and bolster antioxidant function aiming to mitigate the impact of heat stress (HS) on broilers. Two hundred Ross 308 broilers at 28 days of age were randomly assigned to five groups. The control group, housed at 24 ± 1℃, was fed a basal diet. High-temperature treatment groups, housed at 32 ± 1℃, received a basal diet with 0 (HS group), 250 mg/kg VC (HSVC group), 1000 mg/kg Bet (HSBe group), and 250 mg/kg VC + 1000 mg/kg Bet (HSVCBe group). On day 42, assessments were made on growth performance, muscle quality, serum biochemistry, and antioxidant function. Results revealed that HS significantly lowered (P < 0.05) average daily feed intake (ADFI), the degree of redness (a*) in muscles, and serum total superoxide dismutase (T-SOD) level. It also reduced (P < 0.01) average daily gain (ADG), and serum total antioxidant capacity (T-AOC) level, while increasing (P < 0.05) shear force, serum direct bilirubin (D-BIL), uric acid (UA), and malondialdehyde (MDA) levels compared with the control group. Dietary supplementation of VC and Bet, either alone or in combination, significantly decreased shear force and serum UA level, while increasing ADG and serum T-AOC, T-SOD level compared with the HS group (P < 0.05). In conclusion, the addition of VC and/or Bet to the diet proves effective in enhancing the growth performance of HS-exposed broilers through the positive regulation of serum chemical metabolism and the alleviation of oxidative damage.

Systematic evaluation of transcriptomics-based deconvolution methods and references using thousands of clinical samples.

Estimating cell type composition of blood and tissue samples is a biological challenge relevant in both laboratory studies and clinical care. In recent years, a number of computational tools have been developed to estimate cell type abundance using gene expression data. Although these tools use a variety of approaches, they all leverage expression profiles from purified cell types to evaluate the cell type composition within samples. In this study, we compare 12 cell type quantification tools and evaluate their performance while using each of 10 separate reference profiles. Specifically, we have run each tool on over 4000 samples with known cell type proportions, spanning both immune and stromal cell types. A total of 12 of these represent in vitro synthetic mixtures and 300 represent in silico synthetic mixtures prepared using single-cell data. A final 3728 clinical samples have been collected from the Framingham cohort, for which cell populations have been quantified using electrical impedance cell counting. When tools are applied to the Framingham dataset, the tool Estimating the Proportions of Immune and Cancer cells (EPIC) produces the highest correlation, whereas Gene Expression Deconvolution Interactive Tool (GEDIT) produces the lowest error. The best tool for other datasets is varied, but CIBERSORT and GEDIT most consistently produce accurate results. We find that optimal reference depends on the tool used, and report suggested references to be used with each tool. Most tools return results within minutes, but on large datasets runtimes for CIBERSORT can exceed hours or even days. We conclude that deconvolution methods are capable of returning high-quality results, but that proper reference selection is critical.

Single-Cell RNA-Seq Identifies Dynamic Cardiac Transition Program from ADCs Induced by Leukemia Inhibitory Factor.

Adipose-derived cells (ADCs) from white adipose tissue are promising stem cell candidates because of their large regenerative reserves and the potential for cardiac regeneration. However, given the heterogeneity of ADC and its unsolved mechanisms of cardiac acquisition, ADC-cardiac transition efficiency remains low. In this study, we explored the heterogeneity of ADCs and the cellular kinetics of 39,432 single-cell transcriptomes along the leukemia inhibitory factor (LIF)-induced ADC-cardiac transition. We identified distinct ADC subpopulations that reacted differentially to LIF when entering the cardiomyogenic program, further demonstrating that ADC-myogenesis is time-dependent and initiates from transient changes in nuclear factor erythroid 2-related factor 2 (Nrf2) signaling. At later stages, pseudotime analysis of ADCs navigated a trajectory with 2 branches corresponding to activated myofibroblast or cardiomyocyte-like cells. Our findings offer a high-resolution dissection of ADC heterogeneity and cell fate during ADC-cardiac transition, thus providing new insights into potential cardiac stem cells.

Transcriptional Evaluation of the Ductus Arteriosus at the Single-Cell Level Uncovers a Requirement for Vim (Vimentin) for Complete Closure.

OBJECTIVE: Failure to close the ductus arteriosus, patent ductus arteriosus, accounts for 10% of all congenital heart defects. Despite significant advances in patent ductus arteriosus management, including pharmacological treatment targeting the prostaglandin pathway, a proportion of patients fail to respond and must undergo surgical intervention. Thus, further refinement of the cellular and molecular mechanisms that govern vascular remodeling of this vessel is required. METHODS: We performed single-cell RNA-sequencing of the ductus arteriosus in mouse embryos at E18.5 (embryonic day 18.5), and P0.5 (postnatal day 0.5), and P5 to identify transcriptional alterations that might be associated with remodeling. We further confirmed our findings using transgenic mouse models coupled with immunohistochemistry analysis. RESULTS: The intermediate filament vimentin emerged as a candidate that might contribute to closure of the ductus arteriosus. Indeed, mice with genetic deletion of vimentin fail to complete vascular remodeling of the ductus arteriosus. To seek mechanisms, we turned to the RNA-sequencing data that indicated changes in Jagged1 with similar profile to vimentin and pointed to potential links with Notch. In fact, Notch3 signaling was impaired in vimentin null mice and vimentin null mice phenocopies patent ductus arteriosus in Jagged1 endothelial and smooth muscle deleted mice. CONCLUSIONS: Through single-cell RNA-sequencing and by tracking closure of the ductus arteriosus in mice, we uncovered the unexpected contribution of vimentin in driving complete closure of the ductus arteriosus through a mechanism that includes deregulation of the Notch signaling pathway.

Exposure to copper activates mitophagy and endoplasmic reticulum stress-mediated apoptosis in chicken (Gallus gallus) cerebrum.

A large amount of copper (Cu) used in production activities can lead to the enrichment of Cu in the environment, which can cause toxicity to animals. However, the toxicity mechanism of Cu on the cerebrum is still uncertain. Hence, a total of 240 chickens were separated into four groups in this study to reveal the potential connection between mitophagy and endoplasmic reticulum (ER) stress-mediated apoptosis in the chicken cerebrum in the case of excess Cu exposure. The cu exposure situation was simulated by diets containing various levels of copper (11 mg/kg, control group; 110 mg/kg, group I; 220 mg/kg, group II and 330 mg/kg, group III) for 49 days. The results of histology showed that vacuolar degeneration was observed in the treated groups, and the mitochondria swell and autophagosomes formation were found under excess Cu treatment. Additionally, the expression of mitophagy (PINK1, Parkin, LC3I, LC3II and p62) and ER stress (GRP78, PERK, ATF6, IRE1α, XBP1, CHOP, and JNK) indexes were significantly upregulated under excess Cu exposure. Furthermore, the mRNA and protein expression of Bcl-2 were decreased, while Bak1, Bax, Caspase12, and Caspase3 were increased compared to the control group. In summary, this study demonstrated that an overdose of Cu could induce mitophagy and ER stress-mediated apoptosis in the chicken cerebrum. These findings revealed an important potential connection between Cu toxicity and cerebrum damage, which provided a new insight into Cu neurotoxicity.

Single-cell Transcriptomics Reveals Dynamic Role of Smooth Muscle Cells and Enrichment of Immune Cell Subsets in Human Abdominal Aortic Aneurysms.

OBJECTIVE: To determine cell-specific gene expression profiles that contribute to development of abdominal aortic aneurysms (AAAs). BACKGROUND: AAAs represent the most common pathological aortic dilation leading to the fatal consequence of aortic rupture. Both immune and structural cells contribute to aortic degeneration, however, gene specific alterations in these cellular subsets are poorly understood. METHODS: We performed single-cell RNA sequencing (scRNA-seq) analysis of AAAs and control tissues. AAA-related changes were examined by comparing gene expression profiles as well as detailed receptor-ligand interactions. An integrative analysis of scRNA-seq data with large genome-wide association study data was conducted to identify genes critical for AAA development. RESULTS: Using scRNA-seq we provide the first comprehensive characterization of the cellular landscape in human AAA tissues. Unbiased clustering analysis of transcriptional profiles identified seventeen clusters representing 8 cell lineages. For immune cells, clustering analysis identified 4 T-cell and 5 monocyte/macrophage subpopulations, with distinct transcriptional profiles in AAAs compared to controls. Gene enrichment analysis on immune subsets identified multiple pathways only expressed in AAA tissue, including those involved in mitochondrial dysfunction, proliferation, and cytokine secretion. Moreover, receptor-ligand analysis defined robust interactions between vascular smooth muscle cells and myeloid populations in AAA tissues. Lastly, integrated analysis of scRNA-seq data with genome-wide association study studies determined that vascular smooth muscle cell expression of SORT1 is critical for maintaining normal aortic wall function. CONCLUSIONS: Here we provide the first comprehensive evaluation of single-cell composition of the abdominal aortic wall and reveal how the gene expression landscape is altered in human AAAs.

Single-cell RNA sequencing to study vascular diversity and function.

PURPOSE OF REVIEW: Single-cell RNA sequencing (scRNA-seq) can capture the transcriptional profile of thousands of individual cells concurrently from complex tissues and with remarkable resolution. Either with the goal of seeking information about distinct cell subtypes or responses to a stimulus, the approach has provided robust information and promoted impressive advances in cardiovascular research. The goal of this review is to highlight strategies and approaches to leverage this technology and bypass potential caveats related to evaluation of the vascular cells. RECENT FINDINGS: As the most recent technological development, details associated with experimental strategies, analysis, and interpretation of scRNA-seq data are still being discussed and scrutinized by investigators across the vascular field. Compilation of this information is valuable for those using the technology but particularly important to those about to start utilizing scRNA-seq to seek transcriptome information of vascular cells. SUMMARY: As our field progresses to catalog transcriptomes from distinct vascular beds, it is undeniable that scRNA-seq technology is here to stay. Sharing approaches to improve the quality of cell dissociation procedures, analysis, and a consensus of best practices is critical as information from this powerful experimental platform continues to emerge.