Abstract 15227: Egln1 Deficiency Induce Alveolar Macrophages Accumulation and Polarize Lung Interstitial Macrophages to a Pro-pah State Mediating Obliterative Pulmonary Vascular Remodeling and Severe PAH

Introduction: Previously we identified a mouse model (Tie2Cre-mediated disruption of Egln1 ) of Pulmonary arterial hypertension (PAH) with progressive obliterative vascular remodeling including vascular occlusion and plexiform-like lesions and right heart failure. Tie2Cre-meidated disruption of Egln1 in bone marrow contributes to the severity of PAH indicating the importance of the immune cells in the pathogenesis of PH. Hypothesis: Macrophage accumulation and polarization due to Egln1 deficiency contributes to obliterative pulmonary vascular remodeling and severe PAH in Egln1 Tie2Cre mice. Methods: Egln1 Tie2Cre mice with macrophage Fas-Induced apoptosis (MaFIA) transgene were generated (Egln1 Tie2Cre /MaFIA) and received AP20187 treatment to deplete the macrophages during the development of PAH afterwards the RVSP and RV/(LV+S) ratio were measured as the indicator of diseases severity. Flowcytometry and immunostaining were used to quantify different types of macrophages. Bulk RNA-seq and single cell RNA-seq were used to identify PAH-related pathway changes and population changes of lung macrophages in Egln1 Tie2Cre mice. BrdU staining was used to measure the smooth muscle cell (SMC) proliferation co-culturing with macrophages isolated from Egln1 Tie2Cre mice. Results: We observed that CD206+ Alternative Activated Macrophages (AAMs) are accumulated in the pulmonary vessel adventitia of IPAH patients as well as Egln1 Tie2Cre mice. These macrophages are Egln1 deficient and express elevated level of pro-PAH factors and thus promote SMC proliferation in vitro . Moreover, genetic depletion of total macrophages using the Egln1 Tie2Cre /MaFIA mouse model markedly inhibited obliterative pulmonary vascular remodeling and PAH. Single cell RNA-seq revealed that lung resident alveolar macrophages (Siglec-F+/Cd11c+) accumulated due to hyper-proliferation while recruited lung interstitial (Cx3cr1+/Aif1+) macrophages polarize to a pro-PAH state in Egln1 Tie2Cre mice. Conclusions: Egln1 deficiency in the HCs induced proliferation/accumulation of alveolar macrophages and polarized the recruited interstitial macrophages to AAM state which contributes to the vascular remodeling by secreting pro-PAH factors.

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Role of nitric oxide in heparin-induced attenuation of hypoxic pulmonary vascular remodeling

Journal of Applied Physiology ◽

10.1152/japplphysiol.00664.2001 ◽

2002 ◽

Vol 92 (5) ◽

pp. 2012-2018 ◽

Cited By ~ 6

Author(s):

Damian J. Horstman ◽

Lars G. Fischer ◽

Peter C. Kouretas ◽

Robert L. Hannan ◽

George F. Rich

Keyword(s):

Nitric Oxide ◽

Vascular Remodeling ◽

Pulmonary Vasculature ◽

Pulmonary Vascular Remodeling ◽

Antiproliferative Effects ◽

Coculture Model ◽

Pulmonary Arterial ◽

Nos Inhibitor

Heparin and nitric oxide (NO) attenuate changes to the pulmonary vasculature caused by prolonged hypoxia. Heparin may increase NO; therefore, we hypothesized that heparin may attenuate hypoxia-induced pulmonary vascular remodeling via a NO-mediated mechanism. In vivo, rats were exposed to normoxia (N) or hypoxia (H; 10% O2) with or without heparin (1,200 U · kg−1 · day−1) and/or the NO synthase (NOS) inhibitor N ω-nitro-l-arginine methyl ester (l-NAME; 20 mg · kg−1 · day−1) for 3 days or 3 wk. Heparin attenuated increases in pulmonary arterial pressure, the percentage of muscular pulmonary vessels, and their medial thickness induced by 3 wk of H. Importantly, althoughl-NAME alone had no effect, it prevented these effects of heparin on vascular remodeling. In H lungs, heparin increased NOS activity and cGMP levels at 3 days and 3 wk and endothelial NOS protein expression at 3 days but not at 3 wk. In vitro, heparin (10 and 100 U · kg−1 · ml−1) increased cGMP levels after 10 min and 24 h in N and anoxic (0% O2) endothelial cell-smooth muscle cell (SMC) coculture. SMC proliferation, assessed by 5-bromo-2′-deoxyuridine incorporation during a 3-h incubation period, was decreased by heparin under N, but not anoxic, conditions. The antiproliferative effects of heparin were not altered byl-NAME. In conclusion, the in vivo results suggest that attenuation of hypoxia-induced pulmonary vascular remodeling by heparin is NO mediated. Heparin increases cGMP in vitro; however, the heparin-induced decrease in SMC proliferation in the coculture model appears to be NO independent.

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Single cell analysis reveals the impact of age and maturation stage on the human oocyte transcriptome

10.1101/2020.09.25.309658 ◽

2020 ◽

Author(s):

Silvia Llonch ◽

Montserrat Barragán ◽

Paula Nieto ◽

Anna Mallol ◽

Marc Elosua-Bayes ◽

...

Keyword(s):

Single Cell ◽

Chromosome Segregation ◽

Maternal Age ◽

Maturation Stage ◽

P Value ◽

Rna Seq ◽

Transcript Levels ◽

Human Oocytes ◽

The Impact

AbstractStudy questionTo which degree does maternal age affect the transcriptome of human oocytes at the germinal vesicle (GV) stage or at metaphase II after maturation in vitro (IVM-MII)?Summary answerWhile the oocytes’ transcriptome is predominantly determined by maturation stage, transcript levels of genes related to chromosome segregation, mitochondria and RNA processing are affected by age after in vitro maturation of denuded oocytes.What is known alreadyFemale fertility is inversely correlated with maternal age due to both a depletion of the oocyte pool and a reduction in oocyte developmental competence. Few studies have addressed the effect of maternal age on the human mature oocyte (MII) transcriptome, which is established during oocyte growth and maturation, and the pathways involved remain unclear. Here, we characterize and compare the transcriptomes of a large cohort of fully grown GV and IVM-MII oocytes from women of varying reproductive age.Study design, size, durationIn this prospective molecular study, 37 women were recruited from May 2018 to June 2019. The mean age was 28.8 years (SD=7.7, range 18-43). A total of 72 oocytes were included in the study at GV stage after ovarian stimulation, and analyzed as GV (n=40) and in vitro matured oocytes (IVM-MII; n=32).Participants/materials, setting, methodsDenuded oocytes were included either as GV at the time of ovum pick-up or as IVM-MII after in vitro maturation for 30 hours in G2™ medium, and processed for transcriptomic analysis by single-cell RNA-seq using the Smart-seq2 technology. Cluster and maturation stage marker analysis were performed using the Seurat R package. Genes with an average fold change greater than 2 and a p-value < 0.01 were considered maturation stage markers. A Pearson correlation test was used to identify genes whose expression levels changed progressively with age. Those genes presenting a correlation value (R) >= |0.3| and a p-value < 0.05 were considered significant.Main results and the role of chanceFirst, by exploration of the RNA-seq data using tSNE dimensionality reduction, we identified two clusters of cells reflecting the oocyte maturation stage (GV and IVM-MII) with 4,445 and 324 putative marker genes, respectively. Next we identified genes, for which RNA levels either progressively increased or decreased with age. This analysis was performed independently for GV and IVM-MII oocytes. Our results indicate that the transcriptome is more affected by age in IVM-MII oocytes (1,219 genes) than in GV oocytes (596 genes). In particular, we found that genes involved in chromosome segregation and RNA splicing significantly increase in transcript levels with age, while genes related to mitochondrial activity present lower transcript levels with age. Gene regulatory network analysis revealed potential upstream master regulator functions for genes whose transcript levels present positive (GPBP1, RLF, SON, TTF1) or negative (BNC1, THRB) correlation with age.Limitations, reasons for cautionIVM-MII oocytes used in this study were obtained after in vitro maturation of denuded GV oocytes, therefore, their transcriptome might not be fully representative of in vivo matured MII oocytes.The Smart-seq2 methodology used in this study detects polyadenylated transcripts only and we could therefore not assess non-polyadenylated transcripts.Wider implications of the findingsOur analysis suggests that advanced maternal age does not globally affect the oocyte transcriptome at GV or IVM-MII stages. Nonetheless, hundreds of genes displayed altered transcript levels with age, particularly in IVM-MII oocytes. Especially affected by age were genes related to chromosome segregation and mitochondrial function, pathways known to be involved in oocyte ageing. Our study thereby suggests that misregulation of chromosome segregation and mitochondrial pathways also at the RNA-level might contribute to the age-related quality decline in human oocytes.Study funding/competing interest(s)This study was funded by the AXA research fund, the European commission, intramural funding of Clinica EUGIN, the Spanish Ministry of Science, Innovation and Universities, the Catalan Agència de Gestió d’Ajuts Universitaris i de Recerca (AGAUR) and by contributions of the Spanish Ministry of Economy, Industry and Competitiveness (MEIC) to the EMBL partnership and to the “Centro de Excelencia Severo Ochoa”.The authors have no conflict of interest to declare.

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Single cell RNA sequencing of calvarial and long bone endocortical cells

10.1101/849224 ◽

2019 ◽

Cited By ~ 1

Author(s):

Ugur M. Ayturk ◽

Joseph P. Scollan ◽

Alexander Vesprey ◽

Christina M. Jacobsen ◽

Paola Divieti Pajevic ◽

...

Keyword(s):

Gene Expression ◽

Single Cell ◽

Cultured Cells ◽

Neutralizing Antibody ◽

Long Bone ◽

Appendicular Skeleton ◽

Rna Seq ◽

Isolated Cells

ABSTRACTSingle cell RNA-seq (scRNA-seq) is emerging as a powerful technology to examine transcriptomes of individual cells. We determined whether scRNA-seq could be used to detect the effect of environmental and pharmacologic perturbations on osteoblasts. We began with a commonly used in vitro system in which freshly isolated neonatal mouse calvarial cells are expanded and induced to produce a mineralized matrix. We used scRNA-seq to compare the relative cell type abundances and the transcriptomes of freshly isolated cells to those that had been cultured for 12 days in vitro. We observed that the percentage of macrophage-like cells increased from 6% in freshly isolated calvarial cells to 34% in cultured cells. We also found that Bglap transcripts were abundant in freshly isolated osteoblasts but nearly undetectable in the cultured calvarial cells. Thus, scRNA-seq revealed significant differences between heterogeneity of cells in vivo and in vitro. We next performed scRNA-seq on freshly recovered long bone endocortical cells from mice that received either vehicle or Sclerostin-neutralizing antibody for 1 week. Bone anabolism-associated transcripts were also not significantly increased in immature and mature osteoblasts recovered from Sclerostin-neutralizing antibody treated mice; this is likely a consequence of being underpowered to detect modest changes in gene expression, since only 7% of the sequenced endocortical cells were osteoblasts, and a limited portion of their transcriptomes were sampled. We conclude that scRNA-seq can detect changes in cell abundance, identity, and gene expression in skeletally derived cells. In order to detect modest changes in osteoblast gene expression at the single cell level in the appendicular skeleton, larger numbers of osteoblasts from endocortical bone are required.

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Enhancing droplet-based single-nucleus RNA-seq resolution using the semi-supervised machine learning classifier DIEM

10.1101/786285 ◽

2019 ◽

Cited By ~ 4

Author(s):

Marcus Alvarez ◽

Elior Rahmani ◽

Brandon Jew ◽

Kristina M. Garske ◽

Zong Miao ◽

...

Keyword(s):

Gene Expression ◽

Single Cell ◽

Cell Types ◽

Supervised Machine Learning ◽

Data Sets ◽

Rna Seq ◽

Novel Approach ◽

Single Nucleus ◽

Downstream Analysis

AbstractSingle-nucleus RNA sequencing (snRNA-seq) measures gene expression in individual nuclei instead of cells, allowing for unbiased cell type characterization in solid tissues. Contrary to single-cell RNA seq (scRNA-seq), we observe that snRNA-seq is commonly subject to contamination by high amounts of extranuclear background RNA, which can lead to identification of spurious cell types in downstream clustering analyses if overlooked. We present a novel approach to remove debris-contaminated droplets in snRNA-seq experiments, called Debris Identification using Expectation Maximization (DIEM). Our likelihood-based approach models the gene expression distribution of debris and cell types, which are estimated using EM. We evaluated DIEM using three snRNA-seq data sets: 1) human differentiating preadipocytes in vitro, 2) fresh mouse brain tissue, and 3) human frozen adipose tissue (AT) from six individuals. All three data sets showed various degrees of extranuclear RNA contamination. We observed that existing methods fail to account for contaminated droplets and led to spurious cell types. When compared to filtering using these state of the art methods, DIEM better removed droplets containing high levels of extranuclear RNA and led to higher quality clusters. Although DIEM was designed for snRNA-seq data, we also successfully applied DIEM to single-cell data. To conclude, our novel method DIEM removes debris-contaminated droplets from single-cell-based data fast and effectively, leading to cleaner downstream analysis. Our code is freely available for use at https://github.com/marcalva/diem.

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CellMap: Characterizing the types and composition of iPSC-derived cells from RNA-seq data

10.1101/2021.05.24.445360 ◽

2021 ◽

Author(s):

Zhengyu Ouyang ◽

Nathanael Bourgeois ◽

Eugenia Lyashenko ◽

Paige Cundiff ◽

Patrick F Cullen ◽

...

Keyword(s):

Single Cell ◽

Induced Pluripotent Stem Cell ◽

Cell Types ◽

Model Systems ◽

Rna Seq ◽

Cell Type ◽

Fine Grained ◽

Single Nucleus ◽

Induced Pluripotent

Induced pluripotent stem cell (iPSC) derived cell types are increasingly employed as in vitro model systems for drug discovery. For these studies to be meaningful, it is important to understand the reproducibility of the iPSC-derived cultures and their similarity to equivalent endogenous cell types. Single-cell and single-nucleus RNA sequencing (RNA-seq) are useful to gain such understanding, but they are expensive and time consuming, while bulk RNA-seq data can be generated quicker and at lower cost. In silico cell type decomposition is an efficient, inexpensive, and convenient alternative that can leverage bulk RNA-seq to derive more fine-grained information about these cultures. We developed CellMap, a computational tool that derives cell type profiles from publicly available single-cell and single-nucleus datasets to infer cell types in bulk RNA-seq data from iPSC-derived cell lines.

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Abstract 14880: Single-cell Transcriptomes Identify Unique Endothelial Subpopulation (FABP4 + TMEM100 - ) With Lipid Metabolism Dysfunction in Pulmonary Arterial Hypertension

Circulation ◽

10.1161/circ.142.suppl_3.14880 ◽

2020 ◽

Vol 142 (Suppl_3) ◽

Author(s):

BIN LIU ◽

Jingbo Dai ◽

Li Shuai ◽

Dan Yi ◽

Youyang Zhao ◽

...

Keyword(s):

Pulmonary Arterial Hypertension ◽

Arterial Hypertension ◽

Single Cell ◽

Vascular Remodeling ◽

Molecular Mechanisms ◽

Premature Death ◽

Right Heart Failure ◽

Fatty Acid Binding ◽

Pulmonary Arterial ◽

Endothelial Plasticity

Introduction: Pulmonary arterial hypertension (PAH) is a disaster disease characterized by obliterative vascular remodeling and persistent increase of vascular resistance, leading to right heart failure and premature death. Understanding the cellular and molecular mechanisms will help develop novel therapeutic approaches for PAH patients. Hypothesis: We hypothesis that endothelial plasticity or distinct cell populations are critical for obstructive vascular remodeling in the pathogenesis of PAH. Methods: Here we applied single-cell RNA sequencing (ScRNA-seq) to profile the pulmonary cells in a severe mouse model ( Egln1 Tie2Cre mice) of PAH. Human hPAEC from idiopathic PAH patients and healthy donors were used to measure FABP4 and FABP5 expression. siRNA mediated knockdown of FABP4 and FABP5 was performed to study cell proliferation and apoptosis. Mice with Fabp4 and Fabp5 deletion ( Fabp45 -/- ) and wild type (WT) mice were incubated with hypoxia (10% O 2 ) to induced PAH. Egln1 Tie2Cre mice were bred with Fabp45 -/- mice to generate Egln1 Tie2Cre / Fabp45 -/- mice. Results: We identified five distinct EC subpopulations in both WT and Egln1 Tie2Cre mice via scRNA-seq. Unexpectedly, the number of Cluster (EC2, 49.8%) was markedly increased in Egln1 Tie2Cre lung compared with WT lung (2.8%). EC2 cluster (mainly from Egln1 Tie2Cre lung) was characterized by little expression of Tmem100 , Cldn5 , Tspan7 , Calcrl and Foxf1 and high expression of Fabp4, Cdh13, Sparl1 and Fabp5 . Fatty acid-binding protein (FABP) 4 and FABP5 (FABP4-5) were highly induced in PAECs from IPAH patients. Knockdown of FABP4-5 reduced EC proliferation and starvation-induced Caspase 3/7 activity. Fabp45 -/- mice were protected from hypoxia-induced PAH compared to WT mice. Moreover, Egln1 Tie2Cre / Fabp45 -/- mice also exhibited a reduction of RVSP and RV hypertrophy compared to Egln1 Tie2Cre mice. Conclusions: ScRNA-seq analysis identifies a unique endothelial population (FABP4 + TMEM100 - ) highly enriched in the lung of severe PAH mice. Knockdown of FABP4-5 reduces EC proliferation starvation-induced injury. Genetic deletion of FABP4-5 protects from hypoxia and Egln1 deficiency-induced PAH in mice.

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Cellular Pathways Promoting Pulmonary Vascular Remodeling by Hypoxia

Physiology ◽

10.1152/physiol.00039.2019 ◽

2020 ◽

Vol 35 (4) ◽

pp. 222-233

Author(s):

Larissa A. Shimoda

Keyword(s):

Pulmonary Vascular Resistance ◽

Vascular Resistance ◽

Vascular Remodeling ◽

Pulmonary Arterial Pressure ◽

Right Heart Failure ◽

Right Heart ◽

Pulmonary Vascular Remodeling ◽

Vascular Stiffening ◽

Pulmonary Arterial ◽

Cellular Pathways

Exposure to hypoxia increases pulmonary vascular resistance, leading to elevated pulmonary arterial pressure and, potentially, right heart failure. Vascular remodeling is an important contributor to the increased pulmonary vascular resistance. Hyperproliferation of smooth muscle, endothelial cells, and fibroblasts, and deposition of extracellular matrix lead to increased wall thickness, extension of muscle into normally non-muscular arterioles, and vascular stiffening. This review highlights intrinsic and extrinsic modulators contributing to the remodeling process.

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Bone Marrow Transplant Induces Pulmonary Vascular Remodeling in Mice.

Blood ◽

10.1182/blood.v114.22.4480.4480 ◽

2009 ◽

Vol 114 (22) ◽

pp. 4480-4480

Author(s):

Jason M Aliotta ◽

Michael Del Tatto ◽

Mark Dooner ◽

Mandy Pereira ◽

Peter J. Quesenberry

Keyword(s):

Bone Marrow ◽

Smooth Muscle ◽

Bone Marrow Transplant ◽

Vascular Remodeling ◽

Conflicts Of Interest ◽

Marrow Cell ◽

Marrow Transplant ◽

Pulmonary Vessel ◽

Pulmonary Vascular Remodeling ◽

Cell Conversion

Abstract Abstract 4480 Objective Pulmonary complications are common in bone marrow transplant (BMT) recipients and often of an infectious etiology or attributed to intensive conditioning regimens. Cases of pulmonary arterial hypertension have been described in the setting of autologous BMT but are believed to be the result of acquired infections or previously-administered chemotherapy. Whether transplanted marrow cells themselves are toxic to the recipient's lungs is unknown. Methods To address this, non-irradiated female BALB/C mice were transplanted with 5-6×107 whole bone marrow (WBM) cells from male BALB/C mice daily for four days on week zero, then again on week eight (4.5×108 cells total/mouse), or an equal volume of diluent (control). On week 24, transplanted mice received 1000 cGy of chest-only radiation or no radiation. On week 32, histochemical and immunohistochemical analyses were performed on recipient's lungs. Results Bone marrow chimerism was not significantly different in the irradiated and non-irradiated cohorts at the time of sacrifice (average 45.11+6.25% Y chromosome+, all mice). Recipient lungs contained few non-hematopoietic donor marrow-derived cells. These cells were exclusively epithelial (Y+/cytokeratin+), primarily type II pneumocytes (Y+/prosurfactant C+), while no endothelial (Y+/von Willebrand+) or vascular smooth muscle (Y+/alpha-actin+) cells were identified. Irradiated and non-irradiated cohorts had similar quantities of these cells (0.80+0.22% vs. 0.51+0.08% Y+/cytokeratin+; 0.37+0.08% vs. 0.32+0.12% Y+/prosurfactant C+, p>0.2). Pulmonary vessel wall thickness-to-blood vessel diameter ratios (PVWT/D) were significantly increased in the non-irradiated cohort compared with controls and these ratios were further increased in the irradiated cohort (141+5.75%, 161+5.34% of control, p<0.05 comparing all groups). Conclusions These data indicated that marrow cell infusion alone results in pulmonary vascular remodeling and this effect is augmented by radiation injury. These changes are independent of transplanted marrow cell conversion to pulmonary vascular endothelial, smooth muscle cells. These studies suggest that transplanted cells may be lung toxic entities in the setting clinical BMT. Disclosures: No relevant conflicts of interest to declare.

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Mechanisms of right heart disease in pulmonary hypertension (2017 Grover Conference Series)

Pulmonary Circulation ◽

10.1177/2045893217753121 ◽

2017 ◽

Vol 8 (1) ◽

pp. 204589321775312 ◽

Cited By ~ 1

Author(s):

Kewal Asosingh ◽

Serpil Erzurum

Keyword(s):

Heart Disease ◽

Right Ventricle ◽

Vascular Remodeling ◽

Cardiac Tissue ◽

Direct Consequence ◽

Right Heart Failure ◽

Right Heart ◽

Pulmonary Vascular Remodeling ◽

Right Ventricle Dysfunction ◽

The Right

Current dogma is that pathological hypertrophy of the right ventricle is a direct consequence of pulmonary vascular remodeling. However, progression of right ventricle dysfunction is not always lung-dependent. Increased afterload caused by pulmonary vascular remodeling initiates the right ventricle hypertrophy, but determinants leading to adaptive or maladaptive hypertrophy and failure remain unknown. Ischemia in a hypertrophic right ventricle may directly contribute to right heart failure. Rapidly enlarging cardiomyocytes switch from aerobic to anaerobic energy generation resulting in cell growth under relatively hypoxic conditions. Cardiac muscle reacts to an increased afterload by over-activation of the sympathetic system and uncoupling and downregulation of β-adrenergic receptors. Recent studies suggest that β blocker therapy in PH is safe, well tolerated, and preserves right ventricle function and cardiac output by reducing right ventricular glycolysis. Fibrosis, an evolutionary conserved process in host defense and wound healing, is dysregulated in maladaptive cardiac tissue contributing directly to right ventricle failure. Despite several mechanisms having been suggested in right heart disease, the causes of maladaptive cardiac remodeling remain unknown and require further research.

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