scholarly journals Epigenetic Profiles of Primary Endothelial Cells from Patients with Low VWF Levels

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 983-983
Author(s):  
Christopher J. Ng ◽  
Alice Liu ◽  
Katrina J. Ashworth ◽  
Kenneth L. Jones ◽  
Jorge Di Paola
Keyword(s):  
Endothelial Cells ◽  
Single Cell ◽  
Rna Sequencing ◽  
Methylation Status ◽  
Transcriptional Analysis ◽  
Genome Wide ◽  
Single Cell Rna Sequencing ◽  
Age Range ◽  
Von Willebrand

Abstract Background Von Willebrand disease (VWD) type 1 is characterized by low von Willebrand factor (VWF) levels and mucocutaneous bleeding (MCB). Approximately 50% of patients with VWD type 1 exhibit mutations in VWF. However, a large number of patients with VWF levels between 30-50 IU/dL do not show mutations in VWF indicating that other mechanisms are involved. Blood outgrowth endothelial cells (BOECs) are a source of donor-specific endothelial cells and have demonstrated impairments in VWF release and packaging in patients with VWD. BOECs have not been evaluated in individuals with low VWF levels. Hypothesis/Objective We hypothesize that BOECs from individuals with low VWF levels will reveal unique VWF and genome wide epigenetic signatures that may explain the altered plasma VWF levels seen in these patients. Methods BOEC Derivation: Patients with low VWF levels and MCB (30-50 IU/dL) were enrolled in an IRB-approved study. The mononuclear layer from whole blood was isolated and plated onto collagen coated plates. After extended incubation, the presence of BOECs was confirmed by visual morphology and flow cytometry. VWF Transcriptional Analysis: 9 cells lines including: a) 2 BOEC cell lines from control individuals and a HUVEC cell line and c) BOECs from individuals with low VWF, were assayed via single cell RNA sequencing. Bioinformatic analysis included generalized transcriptional expression and single cell expression of VWF. RNA-sequencing expression data was filtered according to the following standardized algorithm. Cells that were defined as monocytes (TYROBP expression > 2 copies) were excluded. Following monocyte exclusions, cells were determined to be of endothelial origin if they demonstrated the presence of PECAM1, CDH5, ROBO4, ESAM, TIE1, or NOTCH4 transcripts, as previously reported by Butler et al. (Cell Reports, 2016). Epigenetic Profiling:Genomic DNA was extracted from BOECs and from peripheral leukocytes (paired to the BOEC draw sample) and analyzed for DNA methylation via an Illumina 850K methylation array. Results BOEC Derivation:A total of eight BOEC lines were generated, 6 from individuals with MCB and VWF levels between 30-50 IU/dL (5:1 female: male ratio, age range 11-54 years) and 2 from healthy controls (2 female, age range 22-39 years) with normal VWF levels and no symptoms of MCB. VWF Expression is decreased in Low VWF Samples: Overall transcript expression of VWF was significantly decreased in low VWF BOEC samples (5.341 transcripts/cell) vs. control endothelial cells (9.076 transcripts/cell), P <0.0001. Generalized Methylation Profiling:Via adjusted P-values, there were 129 methylation sites across multiple genes that were differentially methylated in Low VWF BOECs vs. control endothelial cells. A cluster plot demonstrates that the two control BOEC samples were generally clustered as compared to the other samples (Figure 1A). VWF Specific Methylation: The Illumina 850K array covers 70 prospective methylation sites in VWF, ranging from upstream of the transcriptional start site through the length of the gene. A previous report demonstrated that differences in 8 methylation sites in the VWF promoter correlated with VWF expression (Yuan et al. Nature Communications 2016). 7 of these sites are covered in our assay. Across all of those 7 sites, there was significant increased methylation of the CpG islands in the Low VWF BOECs when compared to the control endothelial cells (Figure 1B). Stability of VWF Methylation:To ensure that the isolation and culture of BOECS does not significantly affect the methylation status of VWF, we conducted a Pearson correlation analysis and demonstrated that peripheral leukocyte (at time of blood draw) and BOEC methylation is highly correlated at VWF specific methylation sites (R2 0.6, P = 0.0004) (Figure 1C). Conclusions Single cell RNA sequencing and genome wide methylation assays of BOECs from individuals with low VWF reveal significant differences in generalized methylation status when compared to BOECs from individuals with normal VWF levels and HUVECs. There is transcriptional downregulation of VWF in low VWF BOECs that is associated with hypermethylation of 7 specific VWF CpG sites in the VWF promoter. Additional sites are being evaluated. Finally, we validated the methylation status of BOECs by demonstrating high correlation with the methylation status of leukocytes from the same individuals. Figure 1 Figure 1. Disclosures Ng: Shire: Consultancy; CSL Behring: Consultancy.

scholarly journals Single Cell RNA Sequencing of Blood Outgrowth Endothelial Cells from Patients with Low Von Willebrand Factor Reveal Multiple Novel Signaling Pathways

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 982-982
Author(s):  
Christopher J. Ng ◽  
Alice Liu ◽  
Katrina J. Ashworth ◽  
Kenneth L. Jones ◽  
Jorge Di Paola
Keyword(s):  
Endothelial Cells ◽  
Single Cell ◽  
Signaling Pathways ◽  
Rna Sequencing ◽  
Cell Lines ◽  
Mixed Model ◽  
Transcriptional Profiling ◽  
Bioinformatic Analysis ◽  
Single Cell Rna Sequencing ◽  
Age Range

Abstract Background The mechanisms that determine low VWF levels in patients with VWF levels between 30-50 IU/dL and no mutations in VWF are poorly understood. Hypothesis/Objective We hypothesize that the study of blood outgrowth endothelial cells (BOECs) from individuals with low VWF levels may reveal unique transcriptional profiles that contribute to the low VWF levels seen in these patients. Methods BOEC Derivation: Patients with low VWF levels and mucocutaneous bleeding (MCB) (30-50 IU/dL) were enrolled in an IRB-approved study. The mononuclear layer from whole blood was isolated and plated onto collagen coated plates. After extended incubation, BOECs were validated by visual inspection and flow cytometry. Endothelial Transcriptional Characterization: A total of 9 cells lines including those from individuals with low VWF and HUVEC and BOECs from individuals with normal VWF levels as control were assayed via single cell RNA sequencing. Bioinformatic analysis included generalized transcriptional expression, Ingenuity Pathway Analysis (IPA), and expression of VWF. RNA-sequencing expression data was filtered according to a standardized algorithm. Cells that were defined as monocytes (TYROBP expression > 2 copies) were excluded. Following monocyte exclusions, cells were determined to be of endothelial origin if they demonstrated the presence of transcripts of PECAM1, CDH5, ROBO4, ESAM, TIE1, or NOTCH4 as previously described by Butler et al. (Cell Reports 2016). Results BOEC Derivation: A total of eight BOEC lines were generated, 6 from individuals with MCB and VWF levels between 30-50 IU/dL (5:1 female: male ratio, age range 11-54 years) and 2 from healthy controls (2 female, age range 22-39 years) with normal VWF levels and no symptoms of MCB. Transcriptional Profiling of single endothelial cells from Low VWF Individuals: A 3D T-SNE plot that assesses unbiased differences in gene expression profiling was generated with each cell line represented by a different color (Figure 1A) demonstrating that individual cell lines have significant differences in their underlying transcriptional profiling. VWF Expression in Low VWF Samples: Overall expression of VWF was significantly decreased in low VWF BOEC samples (5.341 transcripts/cell) vs. control (9.076 transcripts/cell) ECs (figure 1B), P<0.0001. Further, histogram and mixed model (multiple gaussian) analysis of VWF expression revealed changes in generalized expression of VWF in Low VWF BOECs compatible with multiple populations of VWF-expressing BOECs, demonstrating cell mosaicism within each sample. IPA Analysis of Low VWF vs Control BOECs: IPA analysis demonstrated 64 pathways with a z-score difference >1 in Low VWF BOECs when compared to control BOECs (table 1), including multiple signaling pathways such as PI3kinase and AKT as well as several cytoskeleton pathways. Conclusions Single cell RNA sequencing of Low VWF BOECs reveal significant differences in transcriptional profiling when compared to control endothelial cell lines (control BOECs + HUVEC). BOECs from individuals with Low VWF levels demonstrate significantly lower VWF transcript expression than the control endothelial cells. Interestingly, BOECs from low VWF patients show significant differences in VWF transcript number within cells from the same individual demonstrating a degree of mosaicism previously described in murine endothelial cells. Finally, there are multiple cellular pathways that are differentially regulated in Low VWF BOECs as compared to control endothelial cells. Disclosures Ng: CSL Behring: Consultancy; Shire: Consultancy.

scholarly journals Single cell sequencing reveals endothelial plasticity with transient mesenchymal activation after myocardial infarction

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Lukas S. Tombor ◽  
David John ◽  
Simone F. Glaser ◽  
Guillermo Luxán ◽  
Elvira Forte ◽  
...  
Keyword(s):  
Myocardial Infarction ◽  
Endothelial Cells ◽  
Endothelial Cell ◽  
Single Cell ◽  
Rna Sequencing ◽  
Endothelial Cell Migration ◽  
Metabolic Adaptation ◽  
Endothelial Cell Proliferation ◽  
Mesenchymal Transition ◽  
Single Cell Rna Sequencing

AbstractEndothelial cells play a critical role in the adaptation of tissues to injury. Tissue ischemia induced by infarction leads to profound changes in endothelial cell functions and can induce transition to a mesenchymal state. Here we explore the kinetics and individual cellular responses of endothelial cells after myocardial infarction by using single cell RNA sequencing. This study demonstrates a time dependent switch in endothelial cell proliferation and inflammation associated with transient changes in metabolic gene signatures. Trajectory analysis reveals that the majority of endothelial cells 3 to 7 days after myocardial infarction acquire a transient state, characterized by mesenchymal gene expression, which returns to baseline 14 days after injury. Lineage tracing, using the Cdh5-CreERT2;mT/mG mice followed by single cell RNA sequencing, confirms the transient mesenchymal transition and reveals additional hypoxic and inflammatory signatures of endothelial cells during early and late states after injury. These data suggest that endothelial cells undergo a transient mes-enchymal activation concomitant with a metabolic adaptation within the first days after myocardial infarction but do not acquire a long-term mesenchymal fate. This mesenchymal activation may facilitate endothelial cell migration and clonal expansion to regenerate the vascular network.

scholarly journals Modeling cellular crosstalk and organotypic vasculature development with human iPSC-derived endothelial cells and cardiomyocytes

2020 ◽  
Author(s):  
Emmi Helle ◽  
Minna Ampuja ◽  
Alexandra Dainis ◽  
Laura Antola ◽  
Elina Temmes ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Shear Stress ◽  
Endothelial Cell ◽  
Single Cell ◽  
Rna Sequencing ◽  
Tissue Growth ◽  
Cell Phenotype ◽  
Cell Populations ◽  
Pump System ◽  
Single Cell Rna Sequencing

AbstractRationaleCell-cell interactions are crucial for the development and function of the organs. Endothelial cells act as essential regulators of tissue growth and regeneration. In the heart, endothelial cells engage in delicate bidirectional communication with cardiomyocytes. The mechanisms and mediators of this crosstalk are still poorly known. Furthermore, endothelial cells in vivo are exposed to blood flow and their phenotype is greatly affected by shear stress.ObjectiveWe aimed to elucidate how cardiomyocytes regulate the development of organotypic phenotype in endothelial cells. In addition, the effects of flow-induced shear stress on endothelial cell phenotype were studied.Methods and resultsHuman induced pluripotent stem cell (hiPSC) -derived cardiomyocytes and endothelial cells were grown either as a monoculture or as a coculture. hiPS-endothelial cells were exposed to flow using the Ibidi-pump system. Single-cell RNA sequencing was performed to define cell populations and to uncover the effects on their transcriptomic phenotypes. The hiPS-cardiomyocyte differentiation resulted in two distinct populations; atrial and ventricular. Coculture had a more pronounced effect on hiPS-endothelial cells compared to hiPS-cardiomyocytes. Coculture increased hiPS-endothelial cell expression of transcripts related to vascular development and maturation, cardiac development, and the expression of cardiac endothelial cell -specific genes. Exposure to flow significantly reprogrammed the hiPS-endothelial cell transcriptome, and surprisingly, promoted the appearance of both venous and arterial clusters.ConclusionsSingle-cell RNA sequencing revealed distinct atrial and ventricular cell populations in hiPS-cardiomyocytes, and arterial and venous-like cell populations in flow exposed hiPS-endothelial cells. hiPS-endothelial cells acquired cardiac endothelial cell identity in coculture. Our study demonstrated that hiPS-cardiomoycytes and hiPS-endothelial cells readily adapt to coculture and flow in a consistent and relevant manner, indicating that the methods used represent improved physiological cell culturing conditions that potentially are more relevant in disease modelling. In addition, novel cardiomyocyte-endothelial cell crosstalk mediators were revealed.

Randomized phase 2 study of nivolumab (nivo) plus either standard or reduced dose bevacizumab (bev) in recurrent glioblastoma (rGBM).

2021 ◽  
Vol 39 (15_suppl) ◽  
pp. 2015-2015
Author(s):  
Manmeet Singh Ahluwalia ◽  
Yasmeen Rauf ◽  
Hong Li ◽  
Patrick Y. Wen ◽  
David M. Peereboom ◽  
...  
Keyword(s):  
Single Cell ◽  
Rna Sequencing ◽  
Low Dose ◽  
Methylation Status ◽  
Post Treatment ◽  
Dose Effect ◽  
Blood Samples ◽  
Treatment Standard ◽  
Anti Vegf ◽  
Single Cell Rna Sequencing

2015 Background: Trials with anti-PD1 in rGBM have shown limited efficacy. VEGF is highly up regulated proangiogenic growth factor in GBM contributing to tumor-associated immunosuppression. Preclinical data suggests a potential dose effect of anti-VEGF therapy on immunomodulation. Hence, a combination of anti-PD1 and anti-VEGF may be a promising approach in rGBM. Methods: 90 patients with first-recurrent GBM were randomized (1:1) to nivolumab (240 mg IV Q2 weeks) with bevacizumab at standard (10 mg/kg; Arm A) or at low dose (3 mg/kg; Arm B) IV Q2 weeks. Stratification included extent of resection, age, performance status and MGMT methylation status. Single cell RNA sequencing with CITE-seq was used to analyze blood samples from pre- and 8 weeks post-treatment among 8 responders and 8 non-responders. Progression-free survival (PFS) and overall survival (OS) were compared between two arms. Results: 90 patients (Median age 60.6 years ranged 27.4-86.4, 67.8% male, median KPS 80) were enrolled between May 2018 and Jan 2020. Patients were followed in median 7.7 months (Range 0.7, 28.2). 35 of 88 patients were MGMT methylated (2 indeterminate). Overall OS was not significantly different between arm A and arm B (1 year: 41.1 vs 37.7%, p = 0.14), while OS was better for arm A in age > 60 (At 1-year: 46.2% vs 23.8%; Median: 10.6 vs 5.9 months; P = 0.046). OS was no different in the two arms for age ≤ 60 years (At 1-year: 35.6% vs 56.4; Median 8.0 vs 12.4 months; P = 0.90). Single cell RNA sequencing with CITE-seq was used to analyze blood samples from 16 patients, baseline and 8 weeks post treatment. Standard dose bevacizumab treated patients had decreased myeloid derived suppressor cells and an inflammatory response gene signature at 8 weeks. Most frequent toxicities ( > 20%) included fatigue (45.6%), proteinuria (34.4 %), diarrhea (28.9%), hypertension (23.3%) and lipase increase (21.1%). Toxicities in grade 3-4 were hypertension (7.8%), fatigue (5.6) and other non-neurological toxicities including DVT, PE, infection, and abnormal liver function. Conclusions: Overall PFS and OS rates appear similar for nivolumab with either standard or low-dose bevacizumab compared to historical benchmarks of bevacizumab monotherapy. Nivolumab with standard bevacizumab may benefit older but not younger patients. Ongoing response evaluation and immunocorrelative data will be presented. Clinical trial information: NCT03452579.

scholarly journals Single-Cell RNA Sequencing Unveils Unique Transcriptomic Signatures of Organ-Specific Endothelial Cells

Circulation ◽  
2020 ◽  
Vol 142 (19) ◽  
pp. 1848-1862 ◽  
Author(s):  
David T. Paik ◽  
Lei Tian ◽  
Ian M. Williams ◽  
Siyeon Rhee ◽  
Hao Zhang ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Single Cell ◽  
Rna Sequencing ◽  
Expression Patterns ◽  
Receptor Expression ◽  
Transcriptional Networks ◽  
Sequencing Data ◽  
Tissue Specific ◽  
Functional Relationships ◽  
Single Cell Rna Sequencing

Background: Endothelial cells (ECs) display considerable functional heterogeneity depending on the vessel and tissue in which they are located. Whereas these functional differences are presumably imprinted in the transcriptome, the pathways and networks that sustain EC heterogeneity have not been fully delineated. Methods: To investigate the transcriptomic basis of EC specificity, we analyzed single-cell RNA sequencing data from tissue-specific mouse ECs generated by the Tabula Muris consortium. We used a number of bioinformatics tools to uncover markers and sources of EC heterogeneity from single-cell RNA sequencing data. Results: We found a strong correlation between tissue-specific EC transcriptomic measurements generated by either single-cell RNA sequencing or bulk RNA sequencing, thus validating the approach. Using a graph-based clustering algorithm, we found that certain tissue-specific ECs cluster strongly by tissue (eg, liver, brain), whereas others (ie, adipose, heart) have considerable transcriptomic overlap with ECs from other tissues. We identified novel markers of tissue-specific ECs and signaling pathways that may be involved in maintaining their identity. Sex was a considerable source of heterogeneity in the endothelial transcriptome and we discovered Lars2 to be a gene that is highly enriched in ECs from male mice. We found that markers of heart and lung ECs in mice were conserved in human fetal heart and lung ECs. We identified potential angiocrine interactions between tissue-specific ECs and other cell types by analyzing ligand and receptor expression patterns. Conclusions: We used single-cell RNA sequencing data generated by the Tabula Muris consortium to uncover transcriptional networks that maintain tissue-specific EC identity and to identify novel angiocrine and functional relationships between tissue-specific ECs.

scholarly journals Single-Cell Transcriptome Analysis Reveals Dynamic Cell Populations and Differential Gene Expression Patterns in Control and Aneurysmal Human Aortic Tissue

Circulation ◽  
2020 ◽  
Vol 142 (14) ◽  
pp. 1374-1388
Author(s):  
Yanming Li ◽  
Pingping Ren ◽  
Ashley Dawson ◽  
Hernan G. Vasquez ◽  
Waleed Ageedi ◽  
...  
Keyword(s):  
Gene Expression ◽  
Single Cell ◽  
Rna Sequencing ◽  
Aortic Wall ◽  
Genome Wide Association ◽  
Aortic Tissue ◽  
Sequencing Data ◽  
Genome Wide ◽  
Single Cell Rna Sequencing ◽  
Differential Gene

Background: Ascending thoracic aortic aneurysm (ATAA) is caused by the progressive weakening and dilatation of the aortic wall and can lead to aortic dissection, rupture, and other life-threatening complications. To improve our understanding of ATAA pathogenesis, we aimed to comprehensively characterize the cellular composition of the ascending aortic wall and to identify molecular alterations in each cell population of human ATAA tissues. Methods: We performed single-cell RNA sequencing analysis of ascending aortic tissues from 11 study participants, including 8 patients with ATAA (4 women and 4 men) and 3 control subjects (2 women and 1 man). Cells extracted from aortic tissue were analyzed and categorized with single-cell RNA sequencing data to perform cluster identification. ATAA-related changes were then examined by comparing the proportions of each cell type and the gene expression profiles between ATAA and control tissues. We also examined which genes may be critical for ATAA by performing the integrative analysis of our single-cell RNA sequencing data with publicly available data from genome-wide association studies. Results: We identified 11 major cell types in human ascending aortic tissue; the high-resolution reclustering of these cells further divided them into 40 subtypes. Multiple subtypes were observed for smooth muscle cells, macrophages, and T lymphocytes, suggesting that these cells have multiple functional populations in the aortic wall. In general, ATAA tissues had fewer nonimmune cells and more immune cells, especially T lymphocytes, than control tissues did. Differential gene expression data suggested the presence of extensive mitochondrial dysfunction in ATAA tissues. In addition, integrative analysis of our single-cell RNA sequencing data with public genome-wide association study data and promoter capture Hi-C data suggested that the erythroblast transformation-specific related gene( ERG ) exerts an important role in maintaining normal aortic wall function. Conclusions: Our study provides a comprehensive evaluation of the cellular composition of the ascending aortic wall and reveals how the gene expression landscape is altered in human ATAA tissue. The information from this study makes important contributions to our understanding of ATAA formation and progression.

scholarly journals Single-Cell RNA Sequencing Reveals Renal Endothelium Heterogeneity and Metabolic Adaptation to Water Deprivation

2019 ◽  
Vol 31 (1) ◽  
pp. 118-138 ◽  
Author(s):  
Sébastien J. Dumas ◽  
Elda Meta ◽  
Mila Borri ◽  
Jermaine Goveia ◽  
Katerina Rohlenova ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Endothelial Cell ◽  
Oxidative Phosphorylation ◽  
Single Cell ◽  
Rna Sequencing ◽  
Water Deprivation ◽  
Metabolic Adaptation ◽  
Single Cell Rna Sequencing

BackgroundRenal endothelial cells from glomerular, cortical, and medullary kidney compartments are exposed to different microenvironmental conditions and support specific kidney processes. However, the heterogeneous phenotypes of these cells remain incompletely inventoried. Osmotic homeostasis is vitally important for regulating cell volume and function, and in mammals, osmotic equilibrium is regulated through the countercurrent system in the renal medulla, where water exchange through endothelium occurs against an osmotic pressure gradient. Dehydration exposes medullary renal endothelial cells to extreme hyperosmolarity, and how these cells adapt to and survive in this hypertonic milieu is unknown.MethodsWe inventoried renal endothelial cell heterogeneity by single-cell RNA sequencing >40,000 mouse renal endothelial cells, and studied transcriptome changes during osmotic adaptation upon water deprivation. We validated our findings by immunostaining and functionally by targeting oxidative phosphorylation in a hyperosmolarity model in vitro and in dehydrated mice in vivo.ResultsWe identified 24 renal endothelial cell phenotypes (of which eight were novel), highlighting extensive heterogeneity of these cells between and within the cortex, glomeruli, and medulla. In response to dehydration and hypertonicity, medullary renal endothelial cells upregulated the expression of genes involved in the hypoxia response, glycolysis, and—surprisingly—oxidative phosphorylation. Endothelial cells increased oxygen consumption when exposed to hyperosmolarity, whereas blocking oxidative phosphorylation compromised endothelial cell viability during hyperosmotic stress and impaired urine concentration during dehydration.ConclusionsThis study provides a high-resolution atlas of the renal endothelium and highlights extensive renal endothelial cell phenotypic heterogeneity, as well as a previously unrecognized role of oxidative phosphorylation in the metabolic adaptation of medullary renal endothelial cells to water deprivation.

scholarly journals Single-cell transcriptomics of the human retinal pigment epithelium and choroid in health and macular degeneration

2019 ◽  
Vol 116 (48) ◽  
pp. 24100-24107 ◽  
Author(s):  
Andrew P. Voigt ◽  
Kelly Mulfaul ◽  
Nathaniel K. Mullin ◽  
Miles J. Flamme-Wiese ◽  
Joseph C. Giacalone ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Retinal Pigment Epithelium ◽  
Macular Degeneration ◽  
Single Cell ◽  
Rna Sequencing ◽  
Pigment Epithelium ◽  
Retinal Pigment ◽  
Age Related Macular Degeneration ◽  
Age Related ◽  
Single Cell Rna Sequencing

The human retinal pigment epithelium (RPE) and choroid are complex tissues that provide crucial support to the retina. Disease affecting either of these supportive tissues can lead to irreversible blindness in the setting of age-related macular degeneration. In this study, single-cell RNA sequencing was performed on macular and peripheral regions of RPE-choroid from 7 human donor eyes in 2 independent experiments. In the first experiment, total RPE/choroid preparations were evaluated and expression profiles specific to RPE and major choroidal cell populations were identified. As choroidal endothelial cells represent a minority of the total RPE/choroidal cell population but are strongly implicated in age-related macular degeneration (AMD) pathogenesis, a second single-cell RNA-sequencing experiment was performed using endothelial cells enriched by magnetic separation. In this second study, we identified gene expression signatures along the choroidal vascular tree, classifying the transcriptome of human choriocapillaris, arterial, and venous endothelial cells. We found that the choriocapillaris highly and specifically expresses the regulator of cell cycle gene (RGCC), a gene that responds to complement activation and induces apoptosis in endothelial cells. In addition, RGCC was the most up-regulated choriocapillaris gene in a donor diagnosed with AMD. These results provide a characterization of the human RPE and choriocapillaris transcriptome, offering potential insight into the mechanisms of choriocapillaris response to complement injury and choroidal vascular disease in age-related macular degeneration.

scholarly journals Single-Cell RNA Sequencing Reveals Expanded Clones of Islet Antigen-Reactive CD4+ T Cells in Peripheral Blood of Subjects with Type 1 Diabetes

2017 ◽  
Vol 199 (1) ◽  
pp. 323-335 ◽  
Author(s):  
Karen Cerosaletti ◽  
Fariba Barahmand-pour-Whitman ◽  
Junbao Yang ◽  
Hannah A. DeBerg ◽  
Matthew J. Dufort ◽  
...  
Keyword(s):  
T Cells ◽  
Type 1 Diabetes ◽  
Single Cell ◽  
Rna Sequencing ◽  
Peripheral Blood ◽  
Cd4 T Cells ◽  
Single Cell Rna Sequencing ◽  
Islet Antigen

scholarly journals Single-Cell RNA Sequencing Reveals Endothelial Cell Transcriptome Heterogeneity under Homeostatic Laminar Flow

2020 ◽  
Author(s):  
Ziqing Liu ◽  
Dana L Ruter ◽  
Kaitlyn Quigley ◽  
Yuchao Jiang ◽  
Victoria L Bautch
Keyword(s):  
Endothelial Cells ◽  
Endothelial Cell ◽  
Laminar Flow ◽  
Single Cell ◽  
Rna Sequencing ◽  
Protein Translation ◽  
Flow Conditions ◽  
Expression Levels ◽  
Cell Behaviors ◽  
Single Cell Rna Sequencing

ABSTRACTObjectiveEndothelial cells that form the innermost layer of all vessels exhibit heterogeneous cell behaviors and responses to pro-angiogenic signals that are critical for vascular sprouting and angiogenesis. Once vessels form, remodeling and blood flow lead to endothelial cell quiescence, and homogeneity in cell behaviors and signaling responses. These changes are important for the function of mature vessels, but whether and at what level endothelial cells regulate overall expression heterogeneity during this transition is poorly understood. Here we profiled endothelial cell transcriptomic heterogeneity, and expression heterogeneity of selected proteins, under homeostatic laminar flow.Approach and ResultsSingle-cell RNA sequencing and fluorescence microscopy were used to characterize heterogeneity in RNA and protein gene expression levels of human endothelial cells under homeostatic laminar flow compared to non-flow conditions. Analysis of transcriptome variance, Gini coefficient, and coefficient of variation showed that more genes increased RNA heterogeneity under laminar flow relative to genes whose expression became more homogeneous. Analysis of a subset of genes for relative protein expression revealed that most protein profiles showed decreased heterogeneity under flow. In contrast, the magnitude of expression level changes in RNA and protein was coordinated among endothelial cells in flow vs. non-flow conditions.ConclusionsEndothelial cells exposed to homeostatic laminar flow showed increased cohort heterogeneity in RNA expression levels, while cohort expression heterogeneity of selected cognate proteins decreased under laminar flow. These findings suggest that EC homeostasis is imposed at the level of protein translation and/or stability rather than transcriptionally.

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