Subpopulation Of Endothelial Cells Research Articles

Single-cell insights: pioneering an integrated atlas of chromatin accessibility and transcriptomic landscapes in diabetic cardiomyopathy

BackgroundDiabetic cardiomyopathy (DCM) poses a growing health threat, elevating heart failure risk in diabetic individuals. Understanding DCM is crucial, with fibroblasts and endothelial cells playing pivotal roles in driving myocardial fibrosis and contributing to cardiac dysfunction. Advances in Multimodal single-cell profiling, such as scRNA-seq and scATAC-seq, provide deeper insights into DCM’s unique cell states and molecular landscape for targeted therapeutic interventions.MethodsSingle-cell RNA and ATAC data from 10x Multiome libraries were processed using Cell Ranger ARC v2.0.1. Gene expression and ATAC data underwent Seurat and Signac filtration. Differential gene expression and accessible chromatin regions were identified. Transcription factor activity was estimated with chromVAR, and Cis-coaccessibility networks were calculated using Cicero. Coaccessibility connections were compared to the GeneHancer database. Gene Ontology analysis, biological process scoring, cell-cell communication analysis, and gene-motif correlation was performed to reveal intricate molecular changes. Immunofluorescent staining utilized various antibodies on paraffin-embedded tissues to verify the findings.ResultsThis study integrated scRNA-seq and scATAC-seq data obtained from hearts of WT and DCM mice, elucidating molecular changes at the single-cell level throughout the diabetic cardiomyopathy progression. Robust and accurate clustering analysis of the integrated data revealed altered cell proportions, showcasing decreased endothelial cells and macrophages, coupled with increased fibroblasts and myocardial cells in the DCM group, indicating enhanced fibrosis and endothelial damage. Chromatin accessibility analysis unveiled unique patterns in cell types, with heightened transcriptional activity in myocardial cells. Subpopulation analysis highlighted distinct changes in cardiomyocytes and fibroblasts, emphasizing pathways related to fatty acid metabolism and cardiac contraction. Fibroblast-centered communication analysis identified interactions with endothelial cells, implicating VEGF receptors. Endothelial cell subpopulations exhibited altered gene expressions, emphasizing contraction and growth-related pathways. Candidate regulators, including Tcf21, Arnt, Stat5a, and Stat5b, were identified, suggesting their pivotal roles in DCM development. Immunofluorescence staining validated marker genes of cell subpopulations, confirming PDK4, PPARγ and Tpm1 as markers for metabolic pattern-altered cardiomyocytes, activated fibroblasts and endothelial cells with compromised proliferation.ConclusionOur integrated scRNA-seq and scATAC-seq analysis unveils intricate cell states and molecular alterations in diabetic cardiomyopathy. Identified cell type-specific changes, transcription factors, and marker genes offer valuable insights. The study sheds light on potential therapeutic targets for DCM.

Single-cell ‘omic profiles of human aortic endothelial cells in vitro and human atherosclerotic lesions ex vivo reveal heterogeneity of endothelial subtype and response to activating perturbations

Heterogeneity in endothelial cell (EC) sub-phenotypes is becoming increasingly appreciated in atherosclerosis progression. Still, studies quantifying EC heterogeneity across whole transcriptomes and epigenomes in both in vitro and in vivo models are lacking. Multiomic profiling concurrently measuring transcriptomes and accessible chromatin in the same single cells was performed on six distinct primary cultures of human aortic ECs (HAECs) exposed to activating environments characteristic of the atherosclerotic microenvironment in vitro. Meta-analysis of single-cell transcriptomes across 17 human ex vivo arterial specimens was performed and two computational approaches quantitatively evaluated the similarity in molecular profiles between heterogeneous in vitro and ex vivo cell profiles. HAEC cultures were reproducibly populated by four major clusters with distinct pathway enrichment profiles and modest heterogeneous responses: EC1-angiogenic, EC2-proliferative, EC3-activated/mesenchymal-like, and EC4-mesenchymal. Quantitative comparisons between in vitro and ex vivo transcriptomes confirmed EC1 and EC2 as most canonically EC-like, and EC4 as most mesenchymal with minimal effects elicited by siERG and IL1B. Lastly, accessible chromatin regions unique to EC2 and EC4 were most enriched for coronary artery disease (CAD)-associated single-nucleotide polymorphisms from Genome Wide Association Studies (GWAS), suggesting that these cell phenotypes harbor CAD-modulating mechanisms. Primary EC cultures contain markedly heterogeneous cell subtypes defined by their molecular profiles. Surprisingly, the perturbations used here only modestly shifted cells between subpopulations, suggesting relatively stable molecular phenotypes in culture. Identifying consistently heterogeneous EC subpopulations between in vitro and ex vivo models should pave the way for improving in vitro systems while enabling the mechanisms governing heterogeneous cell state decisions.

Single-cell 'omic profiles of human aortic endothelial cells in vitro and human atherosclerotic lesions ex vivo reveal heterogeneity of endothelial subtype and response to activating perturbations.

Heterogeneity in endothelial cell (EC) sub-phenotypes is becoming increasingly appreciated in atherosclerosis progression. Still, studies quantifying EC heterogeneity across whole transcriptomes and epigenomes in both in vitro and in vivo models are lacking. Multiomic profiling concurrently measuring transcriptomes and accessible chromatin in the same single cells was performed on six distinct primary cultures of human aortic ECs (HAECs) exposed to activating environments characteristic of the atherosclerotic microenvironment in vitro. Meta-analysis of single-cell transcriptomes across 17 human ex vivo arterial specimens was performed and two computational approaches quantitatively evaluated the similarity in molecular profiles between heterogeneous in vitro and ex vivo cell profiles. HAEC cultures were reproducibly populated by four major clusters with distinct pathway enrichment profiles and modest heterogeneous responses: EC1-angiogenic, EC2-proliferative, EC3-activated/mesenchymal-like, and EC4-mesenchymal. Quantitative comparisons between in vitro and ex vivo transcriptomes confirmed EC1 and EC2 as most canonically EC-like, and EC4 as most mesenchymal with minimal effects elicited by siERG and IL1B. Lastly, accessible chromatin regions unique to EC2 and EC4 were most enriched for coronary artery disease (CAD)-associated single-nucleotide polymorphisms from Genome Wide Association Studies (GWAS), suggesting that these cell phenotypes harbor CAD-modulating mechanisms. Primary EC cultures contain markedly heterogeneous cell subtypes defined by their molecular profiles. Surprisingly, the perturbations used here only modestly shifted cells between subpopulations, suggesting relatively stable molecular phenotypes in culture. Identifying consistently heterogeneous EC subpopulations between in vitro and ex vivo models should pave the way for improving in vitro systems while enabling the mechanisms governing heterogeneous cell state decisions.

Characterization of Vascular Niche in Systemic Sclerosis by Spatial Proteomics.

Systemic sclerosis (SSc) is a connective tissue disease that can serve as a model to study vascular changes in response to inflammation, autoimmunity, and fibrotic remodeling. Although microvascular changes are the earliest histopathologic manifestation of SSc, the vascular pathophysiology remains poorly understood. We applied spatial proteomic approaches to deconvolute the heterogeneity of vascular cells at the single-cell level in situ and characterize cellular alterations of the vascular niches of patients with SSc. Skin biopsies of patients with SSc and control individuals were analyzed by imaging mass cytometry, yielding a total of 90 755 cells including 2987 endothelial cells and 4096 immune cells. We identified 7 different subpopulations of blood vascular endothelial cells (VECs), 2 subpopulations of lymphatic endothelial cells, and 3 subpopulations of pericytes. A novel population of CD34+;αSMA+ (α-smooth muscle actin);CD31+ VECs was more common in SSc, whereas endothelial precursor cells were decreased. Co-detection by indexing and tyramide signal amplification confirmed these findings. The microenvironment of CD34+;αSMA+;CD31+ VECs was enriched for immune cells and myofibroblasts, and CD34+;αSMA+;CD31+ VECs expressed markers of endothelial-to-mesenchymal transition. The density of CD34+;αSMA+;CD31+ VECs was associated with clinical progression of fibrosis in SSc. Using spatial proteomics, we unraveled the heterogeneity of vascular cells in control individuals and patients with SSc. We identified CD34+;αSMA+;CD31+ VECs as a novel endothelial cell population that is increased in patients with SSc, expresses markers for endothelial-to-mesenchymal transition, and is located in close proximity to immune cells and myofibroblasts. CD34+;αSMA+;CD31+ VEC counts were associated with clinical outcomes of progressive fibrotic remodeling, thus providing a novel cellular correlate for the crosstalk of vasculopathy and fibrosis.

Coupled single-cell and bulk RNA-seq analysis reveals the engulfment role of endothelial cells in atherosclerosis

The clearance of apoptotic cell debris, containing professional phagocytosis and non-professional phagocytosis, is essential for maintaining the homeostasis of healthy tissues. Here, we discovered that endothelial cells could engulf apoptotic cell debris in atherosclerotic plaque. Single-cell RNA sequencing (RNA-seq) has revealed a unique endothelial cell subpopulation in atherosclerosis, which was strongly associated with vascular injury-related pathways. Moreover, integrated analysis of three vascular injury-related RNA-seq datasets showed that the expression of scavenger receptor class B type 1 (SR-B1) was up-regulated and specifically enriched in the phagocytosis pathway under vascular injury circumstances. Single-cell RNA-seq and bulk RNA-seq indicate that SR-B1 was highly expressed in a unique endothelial cell subpopulation of mouse aorta and strongly associated with the reorganization of cellular adherent junctions and cytoskeleton which were necessary for phagocytosis. Furthermore, SR-B1 was strongly required for endothelial cells to engulf apoptotic cell debris in atherosclerotic plaque of both mouse and human aorta. Overall, this study demonstrated that apoptotic cell debris could be engulfed by endothelial cells through SR-B1 and associated with the reorganization of cellular adherent junctions and cytoskeleton.

Single-Cell RNA Sequencing Identifies Response of Renal Lymphatic Endothelial Cells to Acute Kidney Injury.

The renal lymphatic vasculature and the lymphatic endothelial cells that make up this network play important immunomodulatory roles during inflammation. How lymphatics respond to AKI may affect AKI outcomes. The authors used single-cell RNA sequencing to characterize mouse renal lymphatic endothelial cells in quiescent and cisplatin-injured kidneys. lymphatic endothelial cell gene expression changes were confirmed in ischemia-reperfusion injury and in cultured lymphatic endothelial cells, validating renal lymphatic endothelial cells single-cell RNA sequencing data. This study is the first to describe renal lymphatic endothelial cell heterogeneity and uncovers molecular pathways demonstrating lymphatic endothelial cells regulate the local immune response to AKI. These findings provide insights into previously unidentified molecular pathways for lymphatic endothelial cells and roles that may serve as potential therapeutic targets in limiting the progression of AKI. The inflammatory response to AKI likely dictates future kidney health. Lymphatic vessels are responsible for maintaining tissue homeostasis through transport and immunomodulatory roles. Owing to the relative sparsity of lymphatic endothelial cells in the kidney, past sequencing efforts have not characterized these cells and their response to AKI. Here, we characterized murine renal lymphatic endothelial cell subpopulations by single-cell RNA sequencing and investigated their changes in cisplatin AKI 72 hours postinjury. Data were processed using the Seurat package. We validated our findings by quantitative PCR in lymphatic endothelial cells isolated from both cisplatin-injured and ischemia-reperfusion injury, by immunofluorescence, and confirmation in in vitro human lymphatic endothelial cells. We have identified renal lymphatic endothelial cells and their lymphatic vascular roles that have yet to be characterized in previous studies. We report unique gene changes mapped across control and cisplatin-injured conditions. After AKI, renal lymphatic endothelial cells alter genes involved in endothelial cell apoptosis and vasculogenic processes as well as immunoregulatory signaling and metabolism. Differences between injury models were also identified with renal lymphatic endothelial cells further demonstrating changed gene expression between cisplatin and ischemia-reperfusion injury models, indicating the renal lymphatic endothelial cell response is both specific to where they lie in the lymphatic vasculature and the kidney injury type. In this study, we uncover lymphatic vessel structural features of captured populations and injury-induced genetic changes. We further determine that lymphatic endothelial cell gene expression is altered between injury models. How lymphatic endothelial cells respond to AKI may therefore be key in regulating future kidney disease progression.

Single-cell RNA sequencing analysis identifies one subpopulation of endothelial cells that proliferates and another that undergoes the endothelial-mesenchymal transition in regenerating pig hearts.

Background: In our previous work, we demonstrated that when newborn pigs undergo apical resection (AR) on postnatal day 1 (P1), the animals' hearts were completely recover from a myocardial infarction (MI) that occurs on postnatal day 28 (P28); single-nucleus RNA sequencing (snRNAseq) data suggested that this recovery was achieved by regeneration of pig cardiomyocyte subpopulations in response to MI. However, coronary vasculature also has a key role in promoting cardiac repair. Method: Thus, in this report, we used autoencoder algorithms to analyze snRNAseq data from endothelial cells (ECs) in the hearts of the same animals. Main results: Our results identified five EC clusters, three composed of vascular ECs (VEC1-3) and two containing lymphatic ECs (LEC1-2). Cells from VEC1 expressed elevated levels of each of five cell-cyclespecific markers (Aurora Kinase B [AURKB], Marker of Proliferation Ki-67 [MKI67], Inner Centromere Protein [INCENP], Survivin [BIRC5], and Borealin [CDCA8]), as well as a number of transcription factors that promote EC proliferation, while (VEC3 was enriched for genes that regulate intercellular junctions, participate in transforming growth factor β (TGFβ), bone morphogenic protein (BMP) signaling, and promote the endothelial mesenchymal transition (EndMT). The remaining VEC2 did not appear to participate directly in the angiogenic response to MI, but trajectory analyses indicated that it may serve as a reservoir for the generation of VEC1 and VEC3 ECs in response to MI. Notably, only the VEC3 cluster was more populous in regenerating (i.e., ARP1MIP28) than non-regenerating (i.e., MIP28) hearts during the 1-week period after MI induction, which suggests that further investigation of the VEC3 cluster could identify new targets for improving myocardial recovery after MI. Histological analysis of KI67 and EndMT marker PDGFRA demonstrated that while the expression of proliferation of endothelial cells was not significantly different, expression of EndMT markers was significantly higher among endothelial cells of ARP1MIP28 hearts compared to MIP28 hearts, which were consistent with snRNAseq analysis of clusters VEC1 and VEC3. Furthermore, upregulated secrete genes by VEC3 may promote cardiomyocyte proliferation via the Pi3k-Akt and ERBB signaling pathways, which directly contribute to cardiac muscle regeneration. Conclusion: In regenerative heart, endothelial cells may express EndMT markers, and this process could contribute to regeneration via a endothelial-cardiomyocyte crosstalk that supports cardiomyocyte proliferation.

Single-cell Transcriptomic Analysis Reveals an Immunosuppressive Network Between POSTN CAFs and ACKR1 ECs in TKI-resistant Lung Cancer.

Tyrosine kinase inhibitor (TKI) therapy, a principal treatment for advanced non-small cell lung cancer (NSCLC), frequently encounters the development of drug resistance. The tumor microenvironment (TME) plays a critical role in the progression of NSCLC, yet the relationship between endothelial cells (ECs) and cancer-associated fibroblasts (CAFs) subpopulations in TKI treatment resistance remains largely unexplored. The BioProject database PRJNA591860 project was used to analyze scRNA-seq data including 49 advanced-stage NSCLC samples across three different time points: pre-targeted therapy (naïve), post-partial response (PR) to targeted therapy, and post-progressive disease (PD) stage. The data involved clustering stromal cells into multiple CAFs and ECs subpopulations. The abundance changes and functions of each cluster during TKI treatment were investigated by KEGG and GO analysis. Additionally, we identified specific transcription factors and metabolic pathways via DoRothEA and scMetabolism. Moreover, cell-cell communications between PD and PR stages were compared by CellChat. ECs and CAFs were clustered and annotated using 49 scRNA-seq samples. We identified seven ECs subpopulations, with OIT3 ECs showing enrichment in the PR phase with a drug-resistance phenotype, and ACKR1 ECs being prevalent in the PD phase with enhanced cell adhesion. Similarly, CAFs were clustered into 7 subpopulations. PLA2G2A CAFs were predominant in PR, whereas POSTN CAFs were prevalent in PD, characterized by an immunomodulatory phenotype and increased collagen secretion. CellChat analysis showed that ACKR1 ECs strongly interacted with macrophage through the CD39 pathway and POSTN CAFs secreted Tenascin-C (TNC) to promote the progression of epithelial cells, primarily malignant ones, in PD. This study reveals that POSTN CAFs and ACKR1 ECs are associated with resistance to TKI treatment, based on single-cell sequencing.

Endothelial cell clonality, heterogeneity and dysfunction in pulmonary arterial hypertension.

Our understanding of the pathophysiology of pulmonary arterial hypertension (PAH) has evolved over recent years, with the recognition that endothelial cell (EC) dysfunction and inflammation play an integral role in the development of this disease. ECs within the pulmonary vasculature play a unique role in maintaining vascular integrity and barrier function, regulating gas exchange, and contributing to vascular tone. Using single-cell transcriptomics, research has shown that there are multiple, unique EC subpopulations with different phenotypes. In response to injury or certain stressors such as hypoxia, there can be a dysregulated response with aberrant endothelial injury repair involving other pulmonary vascular cells and even immune cells. This aberrant signaling cascade is potentially a primary driver of pulmonary arterial remodeling in PAH. Recent studies have examined the role of EC clonal expansion, immune dysregulation, and genetic mutations in the pathogenesis of PAH. This review summarizes the existing literature on EC subpopulations and the intricate mechanisms through which ECs develop aberrant physiologic phenotypes and contribute to PAH. Our goal is to provide a framework for understanding the unique pulmonary EC biology and pathophysiology that is involved in the development of PAH.

Single-cell profiling reveals transcriptomic signatures of vascular endothelial cells in non-healing diabetic foot ulcers.

The treatment of diabetic foot ulcers (DFUs) poses a challenging medical problem that has long plagued individuals with diabetes. Clinically, wounds that fail to heal for more than 12 weeks after the formation of DFUs are referred to as non-healing/chronic wounds. Among various factors contributing to the non-healing of DFUs, the impairment of skin microvascular endothelial cell function caused by high glucose plays a crucial role. Our study aimed to reveal the transcriptomic signatures of non-healing DFUs endothelial cells, providing novel intervention targets for treatment strategies. Based on the GEO dataset (GSE165816), we selected DFU-Healer, DFU-Non-healer, and healthy non-diabetic controls as research subjects. Single-cell RNA transcriptomic sequencing technology was employed to analyze the heterogeneity of endothelial cells in different skin tissue samples and identify healing-related endothelial cell subpopulations. Immunofluorescence was applied to validate the sequencing results on clinical specimens. The number of endothelial cells and vascular density showed no significant differences among the three groups of skin specimens. However, endothelial cells from non-healing DFUs exhibited apparent inhibition of angiogenesis, inflammation, and immune-related signaling pathways. The expression of CCND1, ENO1, HIF1α, and SERPINE1 was significantly downregulated at the transcriptomic and histological levels. Further analysis demonstrated that healing-related endothelial cell subpopulations in non-healing DFUs has limited connection with other cell types and weaker differentiation ability. At the single-cell level, we uncovered the molecular and functional specificity of endothelial cells in non-healing DFUs and highlighted the importance of endothelial cell immune-mediated capability in angiogenesis and wound healing. This provides new insights for the treatment of DFUs.

Abstract 18360: Single-Cell Analysis Reveals Insights Into Visceral Adiposity in Mice With Diabetes-Associated Atherosclerosis

Introduction: - Visceral white adipose tissue (vWAT), once considered passive and uniform, is a dynamic and heterogeneous tissue involved in glucose and lipid metabolism and immune response. Changes in adipose tissue morphology, physiology, and cellular composition occur in response to dietary fat and carbohydrates, associated with insulin resistance, dyslipidemia, and type 2 diabetes. vWAT also has implications for vascular diseases such as atherosclerosis. However, the connection between vWAT metabolism and atherosclerosis in mouse models remains to be fully elucidated. Methods: - vWAT from male Ldlr -/- mice fed either a chow diet or high fat, high sucrose containing (HFSC) diet for 24 weeks were harvested. Single cell RNA sequencing, histology and immunofluorescence were used to analyze the cellular and transcriptomic profiles in the chow and HFSC groups. Results: - We identified distinct subpopulations of vascular endothelial cells (ECs), immune cells, and adipose stem and progenitor cells (ASPCs), revealing differences between healthy and diseased groups. Notably, we observed a significant increase in immune cell composition, accompanied by a decrease in ECs and progenitor cells in HFSC-fed mice. Within the macrophage population, we noted increased activated macrophages, B-cell derived macrophages, and collagen-expressing macrophage subclusters. In ECs, we observed increased lymphatic and vascular EC subgroups. Furthermore, inflammation and altered metabolism pathways were found in both macrophage and ECs. We identified novel markers, such as Lyz2 and Trem2, that were highly associated with diabetes associated atherosclerosis in male Ldlr -/- mice. Through cell-cell interaction analysis (CellChat), we discovered distinct interactions patterns associated with enriched pathways such as chemokine, Wnt, and BMP signaling in multiple cell types, including macrophages, mesothelial cells, ASPCs, pericytes, SMCs, and ECs. Conclusion: Our scRNAseq atlas reveals interactions amongst adipose cell types in lean and obese states, with the chemokine pathway emerging as a prominent player. This dataset serves as a valuable resource for studying genes and cell types involved in vWAT function in diabetes-associated atherosclerosis.

Abstract 15041: Single-Cell RNA Sequencing Reveals Four Distinctive Endothelial Subpopulations Through Mouse Endothelial Aging

Introduction: Endothelial cell (EC) dysfunction is a hallmark of vascular aging, contributing to age-related vascular diseases. Elucidating EC aging at the single cell level has significant scientific and therapeutic potential. Research Question: What is the landscape of EC aging at the single cell level? Methods: Mouse aortas were harvested from 1-, 10-, and 20 month-old mice (n=2 males and 2 females at each time point). Pooled single-cell suspensions were prepared, and PECAM1+ cells were enriched by magnetic-activated cell sorting. Results: Single cell RNA sequencing revealed 879 ECs, categorized into six subpopulations (EC 1-6). Of the three canonical EC markers ( Pecam1 , Cdh5, and Kdr ) EC1 and 2 showed higher expression of Pecam1 , whereas EC3 and 4 showed higher expression of Cdh5 and Kdr . ECs from young (1 month-old), middle-aged (10 month-old), and elderly mice (20 month-old) were enriched in EC1, EC3, and EC4, respectively. Gene regulatory network analysis identified specific regulons for each EC subcluster. EC1 displayed progenitor-like features ( Isl1 as one of the top regulons, Procr and Cd34 as highly expressed genes) with high angiogenic potential ( Twist1 as one of the top regulons, Timp2 , Mmp14 , and Mmp2 as highly expressed genes) and the longest G1 phase suggesting its quiescence. EC3 cells exhibited high proliferative status demonstrated by G2M or S phase, accompanied by upregulation of stress-responsive TF Atf3 and NF-κB family members. EC4 demonstrated pronounced Pparg regulon activity. Genes directly regulated by Pparg ( Scarb1 and Cd36 ), and genes involved in fatty acid metabolism and atherogenesis ( Tcf15 , Fabp4 , Gpihbp1 , Lpl , and Meox2) were highly expressed in EC4 and EC3. Conclusions: Our analysis identified four distinct EC subpopulations through the aging process of mouse aortic ECs: EC1, Pecam1 h progenitor-like EC; EC2, Pecam1 h common EC; EC3, Cdh5 h Kdr h inflammatory atherogenic EC; and EC4, Cdh5 h Kdr h non-inflammatory atherogenic EC.

Single-Cell RNA Sequencing Reveals That Adaptation of Human Aortic Endothelial Cells to Antiproliferative Therapies Is Modulated by Flow-Induced Shear Stress.

Endothelial cells (ECs) are capable of quickly responding in a coordinated manner to a wide array of stresses to maintain vascular homeostasis. Loss of EC cellular adaptation may be a potential marker for cardiovascular disease and a predictor of poor response to endovascular pharmacological interventions such as drug-eluting stents. Here, we report single-cell transcriptional profiling of ECs exposed to multiple stimulus classes to evaluate EC adaptation. Human aortic ECs were costimulated with both pathophysiological flows mimicking shear stress levels found in the human aorta (laminar and turbulent, ranging from 2.5 to 30 dynes/cm2) and clinically relevant antiproliferative drugs, namely paclitaxel and rapamycin. EC state in response to these stimuli was defined using single-cell RNA sequencing. We identified differentially expressed genes and inferred the TF (transcription factor) landscape modulated by flow shear stress using single-cell RNA sequencing. These flow-sensitive markers differentiated previously identified spatially distinct subpopulations of ECs in the murine aorta. Moreover, distinct transcriptional modules defined flow- and drug-responsive EC adaptation singly and in combination. Flow shear stress was the dominant driver of EC state, altering their response to pharmacological therapies. We showed that flow shear stress modulates the cellular capacity of ECs to respond to paclitaxel and rapamycin administration, suggesting that while responding to different flow patterns, ECs experience an impairment in their transcriptional adaptation to other stimuli.

Uncovering the heterogeneity and cell fate decisions of endothelial cells after myocardial infarction by single‐cell sequencing

AbstractBackgroundThe plasticity of endothelial cells (ECs) is crucial for tissue response to injury. Myocardial infarction can profoundly affect EC function, leading to a shift toward mesenchymal differentiation.MethodsWe utilized human single‐nucleus RNA sequencing data to investigate the dynamic changes and cellular interactions between normal and post‐infarction ECs.ResultsWe identified two distinct subpopulations of ECs: A transient subpopulation characterized by short‐term mesenchymal gene expression and a long‐term subpopulation characterized by myocardial gene expression. Trajectory analysis revealed the differentiation pathways and potential roles over time and space. Furthermore, we uncovered the proliferation, differentiation, hypoxic, and inflammatory responses of ECs to injury.ConclusionsOur study provides a comprehensive and detailed characterization of endothelial cell states, highlighting the role of activated endothelial cell subpopulations in promoting inflammation and tissue repair after infarction.

Endothelial CD34 expression and regulation of immune cell response in-vitro

Endothelial cells cover the lining of different blood vessels and lymph nodes, and have major functions including the transport of blood, vessel homeostasis, inflammatory responses, control of transendothelial migration of circulating cells into the tissues, and formation of new blood vessels. Therefore, understanding these cells is of major interest. The morphological features, phenotype and function of endothelial cells varies according to the vascular bed examined. The sialomucin, CD34, is widely used as an endothelial marker. However, CD34 is differentially expressed on endothelial cells in different organs and in pathological conditions. Little is known about regulation of endothelial CD34 expression or function. Expression of CD34 is also strongly regulated in-vitro in endothelial cell models, including human umbilical vein endothelial cells (HUVEC) and endothelial colony forming cells (ECFC). We have therefore analysed the expression and function of CD34 by comparing CD34high and CD34low endothelial cell subpopulations. Transcriptomic analysis showed that CD34 gene and protein expressions are highly correlated, that CD34high cells proliferate less but express higher levels of IL-33 and Angiopoietin 2, compared with CD34low cells. Higher secretion levels of IL-33 and Angiopoietin 2 by CD34high HUVECs was confirmed by ELISA. Finally, when endothelial cells were allowed to interact with peripheral blood mononuclear cells, CD34high endothelial cells activated stronger proliferation of regulatory T lymphocytes (Tregs) compared to CD34low cells whereas expansion of other CD4+-T cell subsets was equivalent. These results suggest that CD34 expression by endothelial cells in-vitro associates with their ability to proliferate and with an immunogenic ability that favours the tolerogenic response.

Bone angiocrine factors.

Angiogenesis in the bone is unique and involves distinctive signals. Whether they are created through intramembranous ossification or endochondral ossification, bones are highly vascularized tissues. Long bones undergo a sequence of processes known as endochondral osteogenesis. Angiogenesis occurs during the creation of endochondral bone and is mediated by a variety of cells and factors. An initially avascular cartilage template is invaded by blood vessels from the nearby subchondral bone thanks to the secreted angiogenic chemicals by hypertrophic chondrocytes. Vascular endothelial growth factor (VEGF), one of several angiogenic molecules, is a significant regulator of blood vessel invasion, cartilage remodeling, and ossification of freshly created bone matrix; chondrocyte proliferation and hypertrophy are facilitated by the production of VEGFA and VEGF receptor-2 (VEGFR-2), which is stimulated by fibroblast growth factors (FGFs). NOTCH signaling controls blood capillaries formation during bone maturation and regeneration, while hypoxia-inducible factor 1 alpha (HIF1-a) promotes chondrocyte development by switching to anaerobic metabolism. To control skeletal remodeling and repair, osteogenic cells release angiogenic factors, whereas endothelial cells secrete angiocrine factors. One of the better instances of functional blood vessels specialization for certain organs is the skeletal system. A subpopulation of capillary endothelial cells in the bone regulate the activity of osteoprogenitor cells, which in turn affects bone formation during development and adult homeostasis. Angiogenesis and osteogenesis are strictly connected, and their crosstalk is essential to guarantee bone formation and to maintain bone homeostasis. Additionally, pathological processes including inflammation, cancer, and aging include both bone endothelial cells and angiocrine factors. Therefore, the study and understanding of these mechanisms is fundamental, because molecules and factors involved may represent key targets for novel and advanced therapies.

Endothelial to mesenchymal transition in neonatal hyperoxic lung injury: role of sex as a biological variable.

Bronchopulmonary dysplasia (BPD) is characterized by an arrest in alveolarization, abnormal vascular development, and variable interstitial fibroproliferation in the premature lung. Endothelial to mesenchymal transition (EndoMT) may be a source of pathological fibrosis in many organ systems. Whether EndoMT contributes to the pathogenesis of BPD is not known. We tested the hypothesis that pulmonary endothelial cells will show increased expression of EndoMT markers upon exposure to hyperoxia and that sex as a biological variable will modulate differences in expression. Wild-type (WT) and Cdh5-PAC CreERT2 (endothelial reporter) neonatal male and female mice (C57BL6) were exposed to hyperoxia (0.95 [Formula: see text]) either during the saccular stage of lung development (95% [Formula: see text]; postnatal day 1-5 [PND1-5]) or through the saccular and early alveolar stages of lung development (75% [Formula: see text]; PND1-14). Expression of EndoMT markers was measured in whole lung and endothelial cell mRNA. Sorted lung endothelial cells (from room air- and hyperoxia-exposed lungs) were subjected to bulk RNA-Seq. We show that exposure of the neonatal lung to hyperoxia leads to upregulation of key markers of EndoMT. Furthermore, using lung sc-RNA-Seq data from neonatal lung we were able to show that all endothelial cell subpopulations including the lung capillary endothelial cells show upregulation of EndoMT-related genes. Markers related to EndoMT are upregulated in the neonatal lung upon exposure to hyperoxia and show sex-specific differences. Mechanisms mediating EndoMT in the injured neonatal lung can modulate the response of the neonatal lung to hyperoxic injury and need further investigation.NEW & NOTEWORTHY We show that neonatal hyperoxia exposure increased EndoMT markers in the lung endothelial cells and this biological process exhibits sex-specific differences.

M6A methylation reader IGF2BP2 activates endothelial cells to promote angiogenesis and metastasis of lung adenocarcinoma

BackgroundLung adenocarcinoma (LUAD) is a common type of lung cancer with a high risk of metastasis, but the exact molecular mechanisms of metastasis are not yet understood.MethodsThis study acquired single-cell transcriptomics profiling of 11 distal normal lung tissues, 11 primary LUAD tissues, and 4 metastatic LUAD tissues from the GSE131907 dataset. The lung multicellular ecosystems were characterized at a single-cell resolution, and the potential mechanisms underlying angiogenesis and metastasis of LUAD were explored.ResultsWe constructed a global single-cell landscape of 93,610 cells from primary and metastatic LUAD and found that IGF2BP2 was specifically expressed both in a LUAD cell subpopulation (termed as LUAD_IGF2BP2), and an endothelial cell subpopulation (termed as En_IGF2BP2). The LUAD_IGF2BP2 subpopulation progressively formed and dominated the ecology of metastatic LUAD during metastatic evolution. IGF2BP2 was preferentially secreted by exosomes in the LUAD_IGF2BP2 subpopulation, which was absorbed by the En_IGF2BP2 subpopulation in the tumor microenvironment. Subsequently, IGF2BP2 improved the RNA stability of FLT4 through m6A modification, thereby activating the PI3K-Akt signaling pathway, and eventually promoting angiogenesis and metastasis. Analysis of clinical data showed that IGF2BP2 was linked with poor overall survival and relapse-free survival for LUAD patients.ConclusionsOverall, these findings provide a novel insight into the multicellular ecosystems of primary and metastatic LUAD, and demonstrate that a specific LUAD_IGF2BP2 subpopulation is a key orchestrator promoting angiogenesis and metastasis, with implications for the gene regulatory mechanisms of LUAD metastatic evolution, representing themselves as potential antiangiogenic targets.

Multicellular communities are perturbed in the aging human brain and Alzheimer's disease.

The role of different cell types and their interactions in Alzheimer's disease (AD) is a complex and open question. Here, we pursued this question by assembling a high-resolution cellular map of the aging frontal cortex using single-nucleus RNA sequencing of 24 individuals with a range of clinicopathologic characteristics. We used this map to infer the neocortical cellular architecture of 638 individuals profiled by bulk RNA sequencing, providing the sample size necessary for identifying statistically robust associations. We uncovered diverse cell populations associated with AD, including a somatostatin inhibitory neuronal subtype and oligodendroglial states. We further identified a network of multicellular communities, each composed of coordinated subpopulations of neuronal, glial and endothelial cells, and we found that two of these communities are altered in AD. Finally, we used mediation analyses to prioritize cellular changes that might contribute to cognitive decline. Thus, our deconstruction of the aging neocortex provides a roadmap for evaluating the cellular microenvironments underlying AD and dementia.

Endothelial CCRL2 induced by disturbed flow promotes atherosclerosis via chemerin-dependent β2 integrin activation in monocytes.

Chemoattractants and their cognate receptors are essential for leukocyte recruitment during atherogenesis, and atherosclerotic plaques preferentially occur at predilection sites of the arterial wall with disturbed flow (d-flow). In profiling the endothelial expression of atypical chemoattractant receptors (ACKRs), we found that Ackr5 (CCRL2) was upregulated in an endothelial subpopulation by atherosclerotic stimulation. We therefore investigated the role of CCRL2 and its ligand chemerin in atherosclerosis and the underlying mechanism. By analyzing scRNA-seq data of the left carotid artery (LCA) under d-flow and scRNA-seq datasets GSE131776 of ApoE-/- mice from the Gene Expression Omnibus database, we found that CCRL2 was upregulated in one subpopulation of endothelial cells in response to d-flow stimulation and atherosclerosis. Using CCRL2-/-ApoE-/- mice, we showed that CCRL2 deficiency protected against plaque formation primarily in the d-flow areas of the aortic arch in ApoE-/- mice fed high-fat diet (HFD). Disturbed flow induced the expression of vascular endothelial CCRL2, recruiting chemerin, which caused leukocyte adhesion to the endothelium. Surprisingly, instead of binding to monocytic CMKLR1, chemerin was found to activate β2 integrin, enhancing ERK1/2 phosphorylation and monocyte adhesion. Moreover, chemerin was found to have protein disulfide isomerase (PDI)-like enzymatic activity, which was responsible for the interaction of chemerin with β2 integrin, as identified by a Di-E-GSSG assay and a proximity ligation assay (PLA). For clinical relevance, relatively high serum levels of chemerin were found in patients with acute atherothrombotic stroke (AAS) compared to healthy individuals. Our findings indicate that d-flow-induced CCRL2 promotes atherosclerotic plaque formation via a novel CCRL2-chemerin-β2 integrin axis, providing potential targets for the prevention or therapeutic intervention of atherosclerosis.