Heterogeneity Of Endothelial Cells Research Articles

Single-cell transcriptomic analysis reveals the antiangiogenic role of Mgarp in diabetic retinopathy.

Diabetic retinopathy (DR) is a common vascular complication of diabetes mellitus and a leading cause of vision loss worldwide. Endothelial cell (EC) heterogeneity has been observed in the pathogenesis of DR. Elucidating the underlying mechanisms governing EC heterogeneity may provide novel insights into EC-specific therapies for DR. We used the single-cell data from the Gene Expression Omnibus database to explore EC heterogeneity between diabetic retinas and non-diabetic retinas and identify the potential genes involved in DR. CCK-8 assays, EdU assays, transwell assays, and tube formation assays were conducted to determine the role of the identified gene in angiogenic effects. Our analysis identified three distinct EC subpopulations in retinas and revealed that Mitochondria-localized glutamic acid-rich protein (Mgarp) gene is potentially involved in the pathogenesis of DR. Silencing of Mgarp significantly suppressed the proliferation, migration, and tube formation capacities in retinal endothelial cells. This study not only offers new insights into transcriptomic heterogeneity and pathological alteration of retinal ECs but also holds the promise to pave the way for antiangiogenic therapy by targeting EC-specific gene.

An 18-gene signature of recurrence-associated endothelial cells predicts tumor progression and castration resistance in prostate cancer.

The prognostic and therapeutic implications of endothelial cells (ECs) heterogeneity in prostate cancer (PCa) are poorly understood. We investigated associations of EC heterogeneity with PCa recurrence and castration resistance in 8 bulk transcriptomic and 4 single-cell RNA-seq cohorts. A recurrence-associated EC (RAEC) signature was constructed by comparing 11 machine learning algorithms through nested cross-validation. Functional relevances of RAEC-specific genes were also tested. A subset of ECs was significantly associated with recurrence in primary PCa and named RAECs. RAECs were characteristic of tip and immature cells and were enriched in migration, angiogenesis, and collagen-related pathways. We then developed an 18-gene RAEC signature (RAECsig) representative of RAECs. Higher RAECsig scores independently predicted tumor recurrence and performed better or comparably compared to clinicopathological factors and commercial gene signatures in multiple PCa cohorts. Of the 18 RAECsig genes, FSCN1 was upregulated in ECs from PCa with higher Gleason scores; and the silencing of FSCN1, TMEME255B, or GABRD in ECs either attenuated tube formation or inhibited PCa cell proliferation. Finally, higher RAECsig scores predicted castration resistance in both primary and castration-resistant PCa. This study establishes an endothelial signature that links a subset of ECs to prostate cancer recurrence and castration resistance.

Bone marrow cells contribute to seven different endothelial cell populations in the heart.

Understanding the mechanisms underlying vascular regeneration in the heart is crucial for developing novel therapeutic strategies for myocardial ischemia. This study investigates the contribution of bone marrow-derived cells to endothelial cell populations in the heart, and their role in cardiac function and coronary circulation following repetitive ischemia (RI). Chimeric rats were created by transplanting BM cells from GFP female rats into irradiated male recipients. After engraftment chimeras were subjected to RI for 17days. Vascular growth was assessed from recovery of cardiac function and increases in myocardial blood flow during LAD occlusion. After sorting GFP+ BM cells from heart and bone of Control and RI rats, single-cell RNA sequencing was implemented to determine the fate of BM cells. Our in vivo RI model demonstrated an improvement in cardiac function and myocardial blood flow after 17days of RI with increased capillary density in the rats subjected to RI compared to Controls. Single-cell RNA sequencing of bone marrow cells isolated from rats' hearts identified distinct endothelial cell (EC) subpopulations. These ECs exhibited heterogeneous gene expression profiles and were enriched for markers of capillary, artery, lymphatic, venous, and immune ECs. Furthermore, BM-derived ECs in the RI group showed an angiogenic profile, characterized by upregulated genes associated with blood vessel development and angiogenesis. This study elucidates the heterogeneity of bone marrow-derived endothelial cells in the heart and their response to repetitive ischemia, laying the groundwork for targeting specific subpopulations for therapeutic angiogenesis in myocardial ischemia.

Site-specific genetic and functional signatures of aortic endothelial cells at aneurysm predilection sites in healthy and AngIIApoE-/- mice.

Aortic aneurysm is characterized by a pathological dilation at specific predilection sites of the vessel and potentially results in life-threatening vascular rupture. Herein, we established a modified "Häutchen method" for the local isolation of endothelial cells (ECs) from mouse aorta to analyze their spatial heterogeneity and potential role in site-specific disease development. When we compared ECs from aneurysm predilection sites of healthy mice with adjacent control segments we found regulation of genes related to extracellular matrix remodeling, angiogenesis and inflammation, all pathways playing a critical role in aneurysm development. We also detected enhanced cortical stiffness of the endothelium at these sites. Gene expression of ECs from aneurysms of the AngIIApoE-/- model when compared to sham animals mimicked expression patterns from predilection sites of healthy animals. Thus, this work highlights a striking genetic and functional regional heterogeneity in aortic ECs of healthy mice, which defines the location of aortic aneurysm formation in disease.

Single-Cell RNA Sequencing Reveals the Spatial Heterogeneity and Functional Alteration of Endothelial Cells in Chronic Hepatitis B Infection.

Chronic Hepatitis B virus (CHB) infection is a global health challenge, causing damage ranging from hepatitis to cirrhosis and hepatocellular carcinoma. In our study, single-cell RNA sequencing (scRNA-seq) analysis was performed in livers from mice models with chronic inflammation induced by CHB infection and we found that endothelial cells (ECs) exhibited the largest number of differentially expressed genes (DEGs) among all ten cell types. NF-κB signaling was activated in ECs to induce cell dysfunction and subsequent hepatic inflammation, which might be mediated by the interaction of macrophage-derived and cholangiocyte-derived VISFATIN/Nampt signaling. Moreover, we divided ECs into three subclusters, including periportal ECs (EC_Z1), midzonal ECs (EC_Z2), and pericentral ECs (EC_Z3) according to hepatic zonation. Functional analysis suggested that pericentral ECs and midzonal ECs, instead of periportal ECs, were more vulnerable to HBV infection, as the VISFATIN/Nampt- NF-κB axis was mainly altered in these two subpopulations. Interestingly, pericentral ECs showed increasing communication with macrophages and cholangiocytes via the Nampt-Insr and Nampt-Itga5/Itgb1 axis upon CHB infection, which contribute to angiogenesis and vascular capillarization. Additionally, ECs, especially pericentral ECs, showed a close connection with nature killer (NK) cells and T cells via the Cxcl6-Cxcr6 axis, which is involved in shaping the microenvironment in CHB mice livers. Thus, our study described the heterogeneity and functional alterations of three subclusters in ECs. We revealed the potential role of VISFATIN/Nampt signaling in modulating ECs characteristics and related hepatic inflammation, and EC-derived chemokine Cxcl16 in shaping NK and T cell recruitment, providing key insights into the multifunctionality of ECs in CHB-associated pathologies.

#1479 Single nucleus RNA-sequencing indicates functional heterogeneity of renal endothelial cells in chronic kidney disease

Abstract Background and Aims The burden of cardiovascular disease (CVD) in patients with chronic kidney disease (CKD) remains very high. Endothelial cell (EC) dysfunction is an important key player in the initiation and progression of both pathologies. More importantly, endothelial dysfunction occurs early in both disease courses and is potentially reversible, making it a promising target for effective preventive strategies. Recently, accumulating single-cell RNA sequencing-based evidence has highlighted EC heterogeneity in different pathophysiological contexts. However, while biological processes such as systemic inflammation, oxidative stress fibrosis are being defined as comm EC in CKD and CVD, a comprehensive understanding of at single-cell resolution is lacking. Here, we characterized renal EC subpopulations by single-nucleus RNA sequencing (snRNA-seq) to investigate alterations in CKD patients healthy controls and identify potential drivers of EC dysfunction in CKD. Method Droplet-based snRNA-seq (10X Genomics Chromium) was performed on cryopreserved kidney biopsy cores from 6 human patients with CKD (eGFR < 45 mL/min/1.73 m2) and 3 healthy controls. CKD patients included 2 patients with primary focal segmental glomerulosclerosis (FSGS) and 4 with secondary/maladaptive FSGS (2 patients with diabetes mellitus, 2 female patients in total); all patients had a Framingham 10-year cardiovascular risk score > 20, but no established CVD. Healthy control samples were pre-perfusion biopsies from patients undergoing living kidney donation (including 2 female patients). EC subclusters were annotated based on the expression of canonical EC marker genes. Differential gene expression analysis for patient samples vs. controls included all ECs and DEGs were filtered for log2FC > 0.25 or < −0.25 and adjusted P-value of < .05. Gene set enrichment analysis (GSEA) on the identified DEGs was performed using GO terms. Results A total of 4 081 ECs were present in the dataset, consisting of 5 EC subtypes in both the patient and control cohorts (Panel A). DEGs in CKD patients vs. controls included upregulated genes related to organogenesis and cell differentiation (MEF2C, MEIS2), vascular development (FLRT2), and angiogenesis regulators (VEGFC, PRCP, HDAC9) (Panel B). Notably, VEGFC and FLRT2, are involved in directing EC motility and vascular patterning. In accordance, GSEA showed enrichment for GO biological processes related to vascular endothelial growth factor signaling pathway, endothelial cell differentiation, endothelial development, regulation of angiogenesis, and endothelial cell migration (Panel C). This suggests a potential link between these genes and aberrant angiogenesis, analogous to previous findings in diabetic nephropathy. Some but not all of these changes were also reported for renal ECs of early diabetic nephropathy patients. Of note, the majority of patients studied in our cohort were non-diabetic, warranting further investigation into renal endothelial cell transcriptional alterations that are general to renal disease or specific to particular renal conditions such as diabetic nephropathy. Differential gene expression and GSEA analysis of the EC subclusters in CKD indicated involvement of the peritubular capillary and ascending vasa recta ECs in EC migration / angiogenesis-related responses, overlapping with the overall analysis described above. Glomerular capillary ECs showed upregulation of immune-related genes and processes, reflecting the inflammatory status of the kidney disease. Conclusion snRNA-seq analysis revealed heterogeneity and subtype-specific gene expression alterations of renal ECs from CKD patients compared to healthy controls. Differential functions include enhanced angiogenic (potentially compensatory) and immune-related responses. These findings suggest that specific biological functions may direct different roles of the renal EC subtypes in CKD and will be further validated for their cluster-specificity and for their therapeutic relevance.

O12 Macrophages enhance angiogenesis in an induced pluripotent stem cell-derived skin organoid model

Abstract Introduction and aims Hair-bearing skin organoids (SkOs) are a complex three-dimensional (3D) model that faithfully recapitulates human skin as a multicellular organ with appendage structures. The development of such models provides unique potential for investigating human skin development and translational dermatology. Although this model answers many demands of an investigative tool, one obvious limitation is the absence of immune cells in SkOs. Our aim is to generate coculture systems of SkOs with macrophages to investigate how macrophages influence skin development. Methods Our group, and others, generated single-cell (sc)RNA-Seq and spatial transcriptomic datasets to thoroughly characterize human skin cell populations across the human lifespan. We used these datasets to compare the dynamic tempo of induced pluripotent stem cell (iPSC)-derived SkO (n = 84) differentiation with human prenatal (n = 15) and adult skin. For functional assays, we generated 2D angiogenesis coculture assays with macrophages and endothelial cells (n = 3) and 3D coculture of SkOs with macrophages (n = 3). Macrophages, endothelial cells and SkOs were all derived from the same iPSC line following established protocols. SkO vasculature was analysed after immunowholemount staining of CD31+ endothelial cells. Results Analysis of scRNA-Seq data highlighted a paucity in endothelial cell heterogeneity in SkOs compared with prenatal skin. We hypothesized that this could be explained by the absence of immune cells, as spatial transcriptomics microenvironment analysis of prenatal skin demonstrated that endothelial cells collocate with macrophages. With this in mind, we generated functional assays to investigate interactions of macrophages and endothelial cells in skin development. The 2D and 3D assays revealed that presence of macrophages significantly increased endothelial cell sprouting and network formation. Conclusions Our findings suggest a vital role of macrophages in skin morphogenesis, specifically in relation to the guidance of tip cells during angiogenesis. In addition, we present the SkO as an excellent complex model for investigating skin development, cell–cell interactions, and disease modelling for translational research in regenerative medicine and beyond.

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.

Aberrant tumor vasculature. Facts and pitfalls.

Endothelial cells form a single cell layer lining the inner walls of blood vessels and play critical roles in organ homeostasis and disease progression. Specifically, tumor endothelial cells are heterogenous, and highly permeable, because of specific interactions with the tumor tissue environment and through soluble factors and cell-cell interactions. This review article aims to analyze different aspects of endothelial cell heterogeneity in tumor vasculature, with particular emphasis on vascular normalization, vascular permeability, metabolism, endothelial-to-mesenchymal transition, resistance to therapy, and the interplay between endothelial cells and the immune system.

Comprehensive transcriptional atlas of human adenomyosis deciphered by the integration of single-cell RNA-sequencing and spatial transcriptomics.

Adenomyosis is a poorly understood gynecological disorder lacking effective treatments. Controversy persists regarding "invagination" and "metaplasia" theories. The endometrial-myometrial junction (EMJ) connects the endometrium and myometrium and is important for diagnosing and classifying adenomyosis, but its in-depth study is just beginning. Using single-cell RNA sequencing and spatial profiling, we mapped transcriptional alterations across eutopic endometrium, lesions, and EMJ. Within lesions, we identified unique epithelial (LGR5+) and invasive stromal (PKIB+) subpopulations, along with WFDC1+ progenitor cells, supporting a complex interplay between "invagination" and "metaplasia" theories of pathogenesis. Further, we observed endothelial cell heterogeneity and abnormal angiogenic signaling involving VEGF and ANGPT pathways. Cell-cell communication differed markedly between ectopic and eutopic endometrium, with aberrant signaling in lesions involving PTN, TWEAK, and WNT cascades. This study reveals unique stem cell-like and invasive cell subpopulations within adenomyosis lesions identified, dysfunctional signaling, and EMJ abnormalities critical to developing precise diagnostic and therapeutic strategies.

Molecular and Spatial Signatures of Mouse Embryonic Endothelial Cells at Single-Cell Resolution.

Mature endothelial cells (ECs) are heterogeneous, with subtypes defined by tissue origin and position within the vascular bed (ie, artery, capillary, vein, and lymphatic). How this heterogeneity is established during the development of the vascular system, especially arteriovenous specification of ECs, remains incompletely characterized. We used droplet-based single-cell RNA sequencing and multiplexed error-robust fluorescence in situ hybridization to define EC and EC progenitor subtypes from E9.5, E12.5, and E15.5 mouse embryos. We used trajectory inference to analyze the specification of arterial ECs (aECs) and venous ECs (vECs) from EC progenitors. Network analysis identified candidate transcriptional regulators of arteriovenous differentiation, which we tested by CRISPR (clustered regularly interspaced short palindromic repeats) loss of function in human-induced pluripotent stem cells undergoing directed differentiation to aECs or vECs (human-induced pluripotent stem cell-aECs or human-induced pluripotent stem cell-vECs). From the single-cell transcriptomes of 7682 E9.5 to E15.5 ECs, we identified 19 EC subtypes, including Etv2+Bnip3+ EC progenitors. Spatial transcriptomic analysis of 15 448 ECs provided orthogonal validation of these EC subtypes and established their spatial distribution. Most embryonic ECs were grouped by their vascular-bed types, while ECs from the brain, heart, liver, and lung were grouped by their tissue origins. Arterial (Eln, Dkk2, Vegfc, and Egfl8), venous (Fam174b and Clec14a), and capillary (Kcne3) marker genes were identified. Compared with aECs, embryonic vECs and capillary ECs shared fewer markers than their adult counterparts. Early capillary ECs with venous characteristics functioned as a branch point for differentiation of aEC and vEC lineages. Our results provide a spatiotemporal map of embryonic EC heterogeneity at single-cell resolution and demonstrate that the diversity of ECs in the embryo arises from both tissue origin and vascular-bed position. Developing aECs and vECs share common venous-featured capillary precursors and are regulated by distinct transcriptional regulatory networks.

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.

Predicting mitophagy-related genes and unveiling liver endothelial cell heterogeneity in hepatic ischemia-reperfusion injury.

Hepatic Ischemia-Reperfusion Injury (HIRI) is a major complication in liver transplants and surgeries, significantly affecting postoperative outcomes. The role of mitophagy, essential for removing dysfunctional mitochondria and maintaining cellular balance, remains unclear in HIRI. To unravel the role of mitophagy-related genes (MRGs) in HIRI, we assembled a comprehensive dataset comprising 44 HIRI samples alongside 44 normal control samples from the Gene Expression Omnibus (GEO) database for this analysis. Using Random Forests and Support Vector Machines - Recursive Feature Elimination (SVM-RFE), we pinpointed eight pivotal genes and developed a logistic regression model based on these findings. Further, we employed consensus cluster analysis for classifying HIRI patients according to their MRG expression profiles and conducted weighted gene co-expression network analysis (WGCNA) to identify clusters of genes that exhibit high correlation within different modules. Additionally, we conducted single-cell RNA sequencing data analysis to explore insights into the behavior of MRGs within the HIRI. We identified eight key genes (FUNDC1, VDAC1, MFN2, PINK1, CSNK2A2, ULK1, UBC, MAP1LC3B) with distinct expressions between HIRI and controls, confirmed by PCR validation. Our diagnostic model, based on these genes, accurately predicted HIRI outcomes. Analysis revealed a strong positive correlation of these genes with monocytic lineage and a negative correlation with B and T cells. HIRI patients were divided into three subclusters based on MRG profiles, with WGCNA uncovering highly correlated gene modules. Single-cell analysis identified two types of endothelial cells with different MRG scores, indicating their varied roles in HIRI. Our study highlights the critical role of MRGs in HIRI and the heterogeneity of endothelial cells. We identified the macrophage migration inhibitory factor (MIF) and cGAS-STING (GAS) pathways as regulators of mitophagy's impact on HIRI. These findings advance our understanding of mitophagy in HIRI and set the stage for future research and therapeutic developments.

Transcriptional profiles of pulmonary artery endothelial cells in pulmonary hypertension

Pulmonary arterial hypertension (PAH) is characterized by endothelial cell (EC) dysfunction. There are no data from living patients to inform whether differential gene expression of pulmonary artery ECs (PAECs) can discern disease subtypes, progression and pathogenesis. We aimed to further validate our previously described method to propagate ECs from right heart catheter (RHC) balloon tips and to perform additional PAEC phenotyping. We performed bulk RNA sequencing of PAECs from RHC balloons. Using unsupervised dimensionality reduction and clustering we compared transcriptional signatures from PAH to controls and other forms of pulmonary hypertension. Select PAEC samples underwent single cell and population growth characterization and anoikis quantification. Fifty-four specimens were analyzed from 49 subjects. The transcriptome appeared stable over limited passages. Six genes involved in sex steroid signaling, metabolism, and oncogenesis were significantly upregulated in PAH subjects as compared to controls. Genes regulating BMP and Wnt signaling, oxidative stress and cellular metabolism were differentially expressed in PAH subjects. Changes in gene expression tracked with clinical events in PAH subjects with serial samples over time. Functional assays demonstrated enhanced replication competency and anoikis resistance. Our findings recapitulate fundamental biological processes of PAH and provide new evidence of a cancer-like phenotype in ECs from the central vasculature of PAH patients. This “cell biopsy” method may provide insight into patient and lung EC heterogeneity to advance precision medicine approaches in PAH.

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.

Comprehensive scRNA-seq Model Reveals Artery Endothelial Cell Heterogeneity and Metabolic Preference in Human Vascular Disease.

Vascular disease is one of the major causes of death worldwide. Endothelial cells are important components of the vascular structure. A better understanding of the endothelial cell changes in the development of vascular disease may provide new targets for clinical treatment strategies. Single-cell RNA sequencing can serve as a powerful tool to explore transcription patterns, as well as cell type identity. Our current study is based on comprehensive scRNA-seq data of several types of human vascular disease datasets with deep-learning-based algorithm. A gene set scoring system, created based on cell clustering, may help to identify the relative stage of the development of vascular disease. Metabolic preference patterns were estimated using a graphic neural network model. Overall, our study may provide potential treatment targets for retaining normal endothelial function under pathological situations.

Specialized Retinal Endothelial Cells Modulate Blood-Retina Barrier in Diabetic Retinopathy.

Endothelial cells (EC) play essential roles in retinal vascular homeostasis. This study aimed to characterize retinal EC heterogeneity and functional diversity using single-cell RNA sequencing. Systematic analysis of cellular compositions and cell-cell interaction networks identified a unique EC cluster with high inflammatory gene expression in diabetic retina; sphingolipid metabolism is a prominent aspect correlated with changes in retinal function. Among sphingolipid-related genes, alkaline ceramidase 2 (ACER2) showed the most significant increase. Plasma samples of patients with non-proliferative diabetic retinopathy (NPDR) with or without diabetic macular edema (DME) and active PDR were collected for mass spectrometry analysis. Metabolomic profiling revealed that the ceramide levels were significantly elevated in NPDR-NDME/DME and further increased in active PDR compared with control patients. In vitro analyses showed that ACER2 overexpression retarded endothelial barrier breakdown induced by ceramide, while silencing of ACER2 further disrupted the injury. Moreover, intravitreal injection of the recombinant ACER2 adeno-associated virus rescued diabetes-induced vessel leakiness, inflammatory response, and neurovascular disease in diabetic mouse models. Together, this study revealed a new diabetes-specific retinal EC population and a negative feedback regulation pathway that reduces ceramide content and endothelial dysfunction by upregulating ACER2 expression. These findings provide insights into cell-type targeted interventions for diabetic retinopathy.

Endothelial heterogeneity and their relevance in cardiac development and coronary artery disease

Micro- and macrovascular endothelial cells (ECs) are characterized by structural and functional heterogeneity, which is also reflected in their secretory activity. The root of this heterogeneity and related regulatory mechanisms are still poorly understood. During embryogenesis, microvascular ECs participate in organogenesis prior to the development of the fetal circulation, suggesting that ECs are capable of releasing paracrine trophogens, termed angiocrine factors (AFs). These are angiocrine growth factors, adhesion molecules, and chemokines, which are intended to promote morphogenesis and repair of the adjacent parenchyma/stroma where the vessels are located. There is a tissue and organ-specificity of AFs that traces the heterogeneity of ECs. This AF heterogeneity also traces how ECs respond to pathological conditions or exposure to cardiovascular risk factors. The study of the mechanisms that regulate endothelial and paracrine heterogeneity and that contribute to endotheliopathy represents a broad and as yet understudied area of research. A better understanding of the cellular and molecular mechanisms that regulate this heterogeneity, leading to endotheliopathy is an exciting challenge. In this brief review we will discuss experimental advances in the heterogeneity of ECs and their AF, with a focus on their involvement in the pathogenesis of coronary artery disease.