Genes In Endothelial Cells Research Articles

Single-cell transcriptome profiling highlights the role of APP in blood vessels in assessing the risk of patients with proliferative diabetic retinopathy developing Alzheimer's disease.

Introduction: The incidence of diabetic retinopathy (DR) has been found to be associated with the risk of developing Alzheimer's disease (AD). In addition to the common properties of neurodegeneration, their progressions are involved with abnormal vascular functions. However, the interactions between them have not been fully understood. This study aimed to investigate the key factor for the underlying interactions and shared signaling pathways in the vasculature of DR and AD. Methods: We retrieved single-cell RNA sequencing (scRNA-seq) data regarding human fibrovascular membrane (FVM) of proliferative diabetic retinopathy (PDR) and human hippocampus vessels of AD from the NCBI-GEO database. GSEA analysis was performed to analyze AD-related genes in endothelial cells and pericytes of PDR. CellChat was used for predicting cell-cell communication and the signaling pathway. Results: The data suggested that amyloid-beta precursor protein (APP) signaling was found crucial in the vasculature of PDR and AD. Endothelial cells and pericytes could pose influences on other cells mainly via APP signaling in PDR. The endothelial cells were mainly coordinated with macrophages in the hippocampus vasculature of AD via APP signaling. The bulk RNA-seq in mice with PDR validated that the expression of APP gene had a significant correlation with that of the AD genome-wide association studies (GWAS) gene. Discussion: Our study demonstrates that the vasculopathy of PDR and AD is likely to share a common signaling pathway, of which the APP-related pathway is a potential target.

Single-cell RNA sequencing reveals hub genes of myocardial infarction-associated endothelial cells

BackgroundMyocardial infarction (MI) is a cardiovascular disease that seriously threatens human health. Dysangiogenesis of endothelial cells (ECs) primarily inhibits recovery from MI, but the specific mechanism remains to be further elucidated.MethodsIn this study, the single-cell RNA-sequencing data from both MI and Sham mice were analyzed by the Seurat Package (3.2.2). The number of ECs in MI and Sham groups were compared by PCA and tSNE algorithm. FindMarkers function of Seurat was used to analyze the DEGs between the MI and Sham groups. Then, the ECs was further clustered into 8 sub-clusters for trajectory analysis. The BEAM was used to analyze the branch point 3 and cluster the results. In addition, the DEGs in the microarray data set of MI and Sham mice were cross-linked, and the cross-linked genes were used to construct PPI networks. The key genes with the highest degree were identified and analyzed for functional enrichment. Finally, this study cultured human umbilical vein endothelial cells (HUVECs), established hypoxia models, and interfered with hub gene expression in cells. The impact of hub genes on the migration and tube formation of hypoxic-induced HUVECs were verified by Wound healing assays and tubule formation experiments.ResultsThe number and proportion of ECs in the MI group were significantly lower than those in the Sham group. Meantime, 225 DEGs were found in ECs between the MI and Sham groups. Through trajectory analysis, EC4 was found to play an important role in MI. Then, by using BEAM to analyze the branch point 3, and clustering the results, a total of 495 genes were found to be highly expressed in cell Fate2 (mainly EC4). In addition, a total of 194 DEGs were identified in Micro array dataset containing both MI and Sham mice. The hub genes (Timp1 and Fn1) with the highest degree were identified. Inhibiting Timp1 and Fn1 expression promoted the migration and tube formation of HUVECs.ConclusionsOur data highlighted the non-linear dynamics of ECs in MI, and provided a foothold for analyzing cardiac homeostasis and pro-angiogenesis in MI.

TNIK regulation of interferon signaling and endothelial cell response to virus infection.

Traf2 and Nck-interacting kinase (TNIK) is known for its regulatory role in various processes within cancer cells. However, its role within endothelial cells (ECs) has remained relatively unexplored. Leveraging RNA-seq data and Ingenuity Pathway Analysis (IPA), we probed the potential impact of TNIK depletion on ECs. Examination of RNA-seq data uncovered more than 450 Differentially Expressed Genes (DEGs) in TNIK-depleted ECs, displaying a fold change exceeding 2 with a false discovery rate (FDR) below 0.05. IPA analysis unveiled that TNIK depletion leads to the inhibition of the interferon (IFN) pathway [-log (p-value) >11], downregulation of IFN-related genes, and inhibition of Hypercytokinemia/Hyperchemokinemia [-log (p-value) >8]. The validation process encompassed qRT-PCR to evaluate mRNA expression of crucial IFN-related genes, immunoblotting to gauge STAT1 and STAT2 protein levels, and ELISA for the quantification of IFN and cytokine secretion in siTNIK-depleted ECs. These assessments consistently revealed substantial reductions upon TNIK depletion. When transducing HUVECs with replication incompetent E1-E4 deleted adenovirus expressing green fluorescent protein (Ad-GFP), it was demonstrated that TNIK depletion did not affect the uptake of Ad-GFP. Nonetheless, TNIK depletion induced cytopathic effects (CPE) in ECs transduced with wild-type human adenovirus serotype 5 (Ad-WT). Our findings suggest that TNIK plays a crucial role in regulating the EC response to virus infections through modulation of the IFN pathway.

Pharmacological functions of salidroside in renal diseases: facts and perspectives.

Rhodiola rosea is a valuable functional medicinal plant widely utilized in China and other Asian countries for its anti-fatigue, anti-aging, and altitude sickness prevention properties. Salidroside, a most active constituent derived from Rhodiola rosea, exhibits potent antioxidative, hypoxia-resistant, anti-inflammatory, anticancer, and anti-aging effects that have garnered significant attention. The appreciation of the pharmacological role of salidroside has burgeoned over the last decade, making it a beneficial option for the prevention and treatment of multiple diseases, including atherosclerosis, Alzheimer's disease, Parkinson's disease, cardiovascular disease, and more. With its anti-aging and renoprotective effects, in parallel with the inhibition of oxidative stress and inflammation, salidroside holds promise as a potential therapeutic agent for kidney damage. This article provides an overview of the microinflammatory state in kidney disease and discuss the current therapeutic strategies, with a particular focus on highlighting the recent advancements in utilizing salidroside for renal disease. The potential mechanisms of action of salidroside are primarily associated with the regulation of gene and protein expression in glomerular endothelial cells, podocytes, renal tubule cells, renal mesangial cells and renal cell carcinoma cell, including TNF-α, TGF-β, IL-1β, IL-17A, IL-6, MCP-1, Bcl-2, VEGF, ECM protein, caspase-3, HIF-1α, BIM, as well as the modulation of AMPK/SIRT1, Nrf2/HO-1, Sirt1/PGC-1α, ROS/Src/Cav-1, Akt/GSK-3β, TXNIP-NLRP3, ERK1/2, TGF-β1/Smad2/3, PI3K/Akt, Wnt1/Wnt3a β-catenin, TLR4/NF-κB, MAPK, JAK2/STAT3, SIRT1/Nrf2 pathways. To the best of our knowledge, this review is the first to comprehensively cover the protective effects of salidroside on diverse renal diseases, and suggests that salidroside has great potential to be developed as a drug for the prevention and treatment of metabolic syndrome, cardiovascular and cerebrovascular diseases and renal complications.

Diagnostic and prognostic potential of long non-coding RNA NORAD in patients with acute deep vein thrombosis and its role in endothelial cell function

BackgroundDeep venous thrombosis (DVT) is the common clinical cardiovascular disease, and easily develops into post-thrombotic syndrome (PTS). The study aimed to examine the clinical value of long non-coding RNA NORAD gene in the development of DVT and PTS. In vitro, the underlying mechanism was explored.MethodsSerum levels of lncRNA NORAD gene in 85 DVT cases and 85 healthy individuals were tested. The role of lncRNA NORAD gene in human umbilical vein endothelial cells (HUVECs) proliferation, migration and inflammation was examined. The candidate downstream target gene was predicted via bioinformatic analysis. Gene ontology (GO) analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis were done for the function annotation and pathway enrichment.ResultsLncRNA NORAD gene was at high expression in the serum of DVT patients, it can distinguish DVT patients from healthy controls with the area under the curve of 0.919. Elevated expression of lncRNA NORAD gene in PTS patients was detected, DVT cases with high expression of lncRNA NORAD gene were more susceptible to PTS. LncRNA NORAD gene knockdown promoted HUVECs’ proliferation, migration while suppressing cell apoptosis and inflammation. MiR-93-5p served as a target of lncRNA NORAD gene, and its overexpression reversed the role of lncRNA NORAD gene in the biological function of HUVECs. The target genes of miR-93-5p were enriched in HIF-1 signaling, TGF-beta signaling and PI3K-Akt signaling, protein-protein interaction (PPI) network indicated STAT3, MAPK1 to be the key targets.ConclusionsUpregulation of expression of lncRNA NORAD gene was a potential diagnostic biomarker for DVT and related to the development of PTS. LncRNA NORAD/miR-93-5p axis was involved in the progress of DVT through regulating endothelial cell function.

Apelin-VEGF-C mRNA delivery as therapeutic for the treatment of secondary lymphedema

Secondary lymphedema (LD) corresponds to a severe lymphatic dysfunction leading to the accumulation of fluid and fibrotic adipose tissue in a limb. Here, we identified apelin (APLN) as a powerful molecule for regenerating lymphatic function in LD. We identified the loss of APLN expression in the lymphedematous arm compared to the normal arm in patients. The role of APLN in LD was confirmed in APLN knockout mice, in which LD is increased and associated with fibrosis and dermal backflow. This was reversed by intradermal injection of APLN-lentivectors. Mechanistically, APLN stimulates lymphatic endothelial cell gene expression and induces the binding of E2F8 transcription factor to the promoter of CCBE1 that controls VEGF-C processing. In addition, APLN induces Akt and eNOS pathways to stimulate lymphatic collector pumping. Our results show that APLN represents a novel partner for VEGF-C to restore lymphatic function in both initial and collecting vessels. As LD appears after cancer treatment, we validated the APLN-VEGF-C combination using a novel class of nonintegrative RNA delivery LentiFlash® vector that will be evaluated for phase I/IIa clinical trial.

Transcriptome analysis of cultured human vascular endothelial cells after γ-ray irradiation and correlation analysis with ATP, ADP, and adenosine as secondary soluble factors.

Vascular endothelial cells serve as barriers between blood components and subendothelial tissue and regulate the blood coagulation-fibrinolytic system. Ionizing radiation is a common physical stimulant that induces a bystander effect whereby irradiated cells influence neighboring cells through signalings, including purinergic receptor signaling, activated by adenosine 5'-triphosphate (ATP), adenosine 5'-diphosphate (ADP), and adenosine as secondary soluble factors. Human vascular endothelial EA.hy926 cells were cultured and irradiated with γ-rays or treated with ATP, ADP, or adenosine under non-toxic conditions. RNA-seq, gene ontology, and hierarchical clustering analyses were performed. The transcriptome analysis of differentially expressed genes in vascular endothelial cells after γ-ray irradiations suggests that the change of gene expression by γ-irradiation is mediated by ATP and ADP. In addition, the expression and activity of the proteins related to blood coagulation and fibrinolysis systems appear to be secondarily regulated by ATP and ADP in vascular endothelial cells after exposure to γ-irradiation. Although it is unclear whether the changes of the gene expression related to blood coagulation and fibrinolysis systems by γ-irradiation affected the increased hemorrhagic tendency through the exposure to γ-irradiation or the negative feedback to the activated blood coagulation system, the present data indicate that toxicity associated with γ-irradiation involves the dysfunction of vascular endothelial cells related to the blood coagulation-fibrinolytic system, which is mediated by the signalings, including purinergic receptor signaling, activated by ATP and ADP.

Identification and validation of ferroptosis-related genes for diabetic retinopathy.

Diabetic retinopathy (DR) is a leading cause of blindness, and ferroptosis may be an essential component of the pathological process of DR. In this study, we aimed to screen five hub genes (TLR4, CAV1, HMOX1, TP53, and IL-1B) using bioinformatics analysis and experimentally verify their expression and effects on ferroptosis and cell function. The online Gene Expression Omnibus microarray expression profiling datasets GSE60436 and GSE1025485 were selected for investigation. Ferroptosis-related genes that might be differentially expressed in DR were identified. Then, Gene Ontology (GO) enrichment, Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment, and protein-protein interaction (PPI) network analyses were conducted to characterize the differentially-expressed ferroptosis-related genes. After tissue-specific analyses and external dataset validation of hub genes, the mRNA and protein levels of hub genes in retinal microvascular endothelial cells (HRMECs) symbiotic with high glucose were verified using real-time quantitative PCR (qRT-PCR) and immunocytochemistry (ICC). Finally, hub genes were knocked down using siRNA, and changes in ferroptosis and cell function were observed. Based on the differential expression analysis, 19 ferroptosis-related genes were identified. GO and KEGG enrichment analyses showed that ferroptosis-related genes were significantly enriched in reactive oxygen species metabolic processes, necrotic cell death, hypoxia responses, iron ion responses, positive regulation of cell migration involved in sprouting angiogenesis, NF-kappa B signaling pathway, ferroptosis, fluid shear stress, and atherosclerosis. Subsequently, PPI network analysis and critical module construction were used to identify five hub genes. Based on bioinformatics analysis of mRNA microarrays, qRT-PCR confirmed higher mRNA expression of five genes in the DR model, and immunocytochemistry confirmed their higher protein expression. Finally, siRNA interference was used to verify the effects of five genes on ferroptosis and cell function. Based on bioinformatics analysis, five potential genes related to ferroptosis were identified, and their upregulation may affect the onset or progression of DR. This study sheds new light on the pathogenesis of DR.

Mechanism analysis of Buyang Huanwu decoction in treating atherosclerosis based on network pharmacology and invitro experiments.

Atherosclerosis (AS) is one of the main risk factors of ischemic cardiovascular and cerebrovascular diseases. Buyang Huanwu decoction (BYHWT) is a classic Chinese medicine prescription that is used for treating AS. However, the underlying pharmacological mechanism remains unclear. This study aims to clarify the molecular mechanism of BYHWT in treatment of AS through network pharmacology and invitro experiments. Molecular structure information and targets of core components of BYHWT were obtained from PubChem and UniProtKB databases. Genes involved in AS were obtained from DisGeNet, GeneCards and OMIM databases. The core targets of BYHWT in AS treatment were identified by protein-protein interaction (PPI) network analysis with STRING platform, and analyzed by gene ontology (GO) analysis and the Kyoto Encyclopedia of Genes and Genomics (KEGG) pathway enrichment analysis. Molecular docking was used to verify the binding affinity between the core targets and the bioactive ingredients. HUVEC viability, inflammatory response and mRNA expression levels of core target genes were evaluated by cell counting kit 8 assay, enzyme-linked immunosorbent assay (ELISA) and qRT-PCR. A total of 60 candidate compounds and 325 predicted target genes were screened. PPI network analysis suggested that TP53, SRC, STAT3, and AKT1 may be the core targets. BYHWT in AS treatment was associated with 46 signaling pathways. GA120, baicalein, and 3,9-di-o-methylnissolin had good binding affinity with core target proteins. Baicalein treatment could significantly promoted the viability and repress the inflammatory response of HUVEC cells stimulated by ox-LDL. In addition, Baicalein can regulate the expression of core targets including AKT1, MAPK1, PIK3CA, JUN, TP53, SRC, EGFR, and ESR1. In conclusion, BYHWT and its main bioactive component baicalein, inhibit inflammatory response and modulate multiple downstream genes of endothelial cells, and show good potential to block the progression of AS and cardiovascular/cerebrovascular diseases.

Integration of scRNA and bulk RNA-sequence to construct the 5-gene molecular prognostic model based on the heterogeneity of thyroid carcinoma endothelial cell.

Thyroid cancer (TC) is a kind of cancer with high heterogeneity, which leads to significant difference in prognosis. The prognostic molecular processes are not well understood. Cancer cells and tumor microenvironment (TME) cells jointly determine the heterogeneity. However, quite a little attention was paid to cells in the TME in the past years. In this study, we not only reveal that endothelial cells (ECs) are strongly associated with the progress of papillary thyroid cancer (PTC) using single-cell RNA-seq (scRNA-seq) data downloaded from Gene Expression Omnibus (GEO) and WGCNA, but also screen 5 crucial genes of ECs: CLDN5, ABCG2, NOTCH4, PLAT, and TMEM47. Furthermore, the 5-gene molecular prognostic model is constructed, which can predict how well a patient will do on PD-L1 blockade immunotherapy for TC and evaluate prognosis. Quantitative real-time polymerase chain reaction (qRT-PCR) analysis demonstrates that PLAT is decreased in TC and the increase of PLAT can restrain the migratory capacity of TC cells. Meanwhile, in TC cells, PLAT suppresses VEGFa/VEGFR2-mediated human umbilical vascular endothelial cell (HUVEC) proliferation and tube formation. Totally, we construct the 5-gene molecular prognostic model from the perspective of EC and provide a new idea for immunotherapy of TC.

Deletion of RAMP1 Signaling Enhances Diet-induced Obesity and Fat Absorption via Intestinal Lacteals in Mice.

Intestinal lymphatic vessels (lacteals) play a critical role in the absorption and transport of dietary lipids into the circulation. Calcitonin gene-related peptide and receptor activity-modifying protein 1 (RAMP1) are involved in lymphatic vessel growth. This study aimed to examine the role of RAMP1 signaling in lacteal morphology and function in response to a high-fat diet (HFD). RAMP1 deficient (RAMP1-/-) or wild-type (WT) mice were fed a normal diet (ND) or HFD for 8 weeks. RAMP1-/- mice fed a HFD had increased body weights compared to WT mice fed a HFD, which was associated with high levels of total cholesterol, triglycerides, and glucose. HFD-fed RAMP1-/- mice had shorter and wider lacteals than HFD-fed WT mice. HFD-fed RAMP1-/- mice had lower levels of lymphatic endothelial cell gene markers including vascular endothelial growth factor receptor 3 (VEGFR3) and lymphatic vascular growth factor VEGF-C than HFD-fed WT mice. The concentration of an absorbed lipid tracer in HFD-fed RAMP1-/- mice was higher than that in HFD-fed WT mice. The zipper-like continuous junctions were predominant in HFD-fed WT mice, while the button-like discontinuous junctions were predominant in HFD-fed RAMP1-/- mice. Deletion of RAMP1 signaling suppressed lacteal growth and VEGF-C/VEGFR3 expression but accelerated the uptake and transport of dietary fats through discontinuous junctions of lacteals, leading to excessive obesity. Specific activation of RAMP1 signaling may represent a target for the therapeutic management of diet-induced obesity.

Effects of dietary supplementation of different levels of gamma-aminobutyric acid on reproductive performance, glucose intolerance, and placental development of gilts.

This study aimed to investigate the effects of dietary gamma-aminobutyric acid (GABA) supplementation on reproductive performance, glucose intolerance, and placental development of gilts during mid-late gestation. Based on the principle of backfat thickness consistency, 124 gilts at 65 d of gestation were assigned to three dietary groups: CON (basic diet, n = 41), LGABA (basic diet supplemented with 0.03% GABA, n = 42), and HGABA (basic diet supplemented with 0.06% GABA, n = 41). The litter performance, glucose tolerance, placental angiogenesis, and nutrients transporters were assessed. The LGABA group improved piglet vitality and placental efficiency and decreased area under the curve of glucose tolerance test compared to the CON group (P < 0.05). Meanwhile, the LGABA group enhanced placental vessel density, platelet endothelial cell adhesion molecule-1 levels and gene expression of fibroblast growth factor 18 (P < 0.05). Furthermore, LGABA showed an uptrend in glucose transporter type 1 mRNA level (P = 0.09). Taken together, this study revealed that the dietary supplementation of 0.03% GABA can improve piglet vitality, glucose intolerance, and placental development of gilts.

Single-nucleus transcriptome sequencing reveals hepatic cell atlas in pigs

BackgroundAs the largest substantive organ of animals, the liver plays an essential role in the physiological processes of digestive metabolism and immune defense. However, the cellular composition of the pig liver remains poorly understood. This investigation used single-nucleus RNA sequencing technology to identify cell types from liver tissues of pigs, providing a theoretical basis for further investigating liver cell types in pigs.ResultsThe analysis revealed 13 cells clusters which were further identified 7 cell types including endothelial cells, T cells, hepatocytes, Kupffer cells, stellate cells, B cells, and cholangiocytes. The dominant cell types were endothelial cells, T cells and hepatocytes in the liver tissue of Dahe pigs and Dahe black pigs, which accounts for about 85.76% and 82.74%, respectively. The number of endothelial cells was higher in the liver tissue of Dahe pigs compared to Dahe black pigs, while the opposite tendency was observed for T cells. Moreover, functional enrichment analysis demonstrated that the differentially expressed genes in pig hepatic endothelial cells were significantly enriched in the protein processing in endoplasmic reticulum, MAPK signaling pathway, and FoxO signaling pathway. Functional enrichment analysis demonstrated that the differentially expressed genes in pig hepatic T cells were significantly enriched in the thyroid hormone signaling pathway, B cell receptor signaling pathway, and focal adhesion. Functional enrichment analysis demonstrated that the differentially expressed genes in pig hepatic hepatocytes were significantly enriched in the metabolic pathways.ConclusionsIn summary, this study provides a comprehensive cell atlas of porcine hepatic tissue. The number, gene expression level and functional characteristics of each cell type in pig liver tissue varied between breeds.

Abstract B046: Analyzing heterogeneity of tumor microenvironment of TNBC patients by meta-analysis of 7 breast cancer scRNAseq studies

Abstract Breast cancer can be classified into several types. Among them, triple-negative breast cancer (TNBC) is breast cancer that does not express estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor 2 (HER2). Since targeted therapy for breast cancer relies on the three receptors, treatment outcomes have been worst in TNBCs. However, the clinical benefit of immune-checkpoint inhibitors (ICIs) with chemotherapy over chemotherapy alone was demonstrated in several trials, opening a new avenue for patients with metastatic PD-L1+ TNBC. After several studies with ICIs, researchers found that the tumor microenvironment affects the response rate of immunotherapies. Therefore, to unravel the heterogeneity of the tumor microenvironment of TNBC patients, we collected 7 public single-cell RNA sequencing breast cancer studies. From 141,068 cells populating tumors and tumor-microenvironment (TMEs), we first identified epithelial cells separately. Using whole cells including epithelial and immune-stromal cells, we identified the Baylor-proposed TNBC molecular subtype of each patient. This approach with epithelial cells revealed that 19 basal-like immune-activated (BLIA), 5 basal-like immune-suppressed (BLIS), 2 luminal-androgen receptor (LAR), 3 mesenchymal (MES), and 3 unclassified subtypes. By comparing cell number and cell-cell interactions, the heterogeneity of TME-consisting cell populations between the TNBC subtypes was analyzed. Analysis of tumor-infiltrating lymphocytes revealed their difference in activation, expansion, and exhaustion programs across patients. Among the subtypes, the BLIA subtype had a greater amount of exhausted CD8+ T cells and regulatory T cells. This finding was consistent with cell-cell interaction analysis. In the case of the BLIA subtype, the interaction between regulatory T cells and CD8 T cells was more active than in other subtypes. Also, the interaction between cytotoxic cells (T cells and NK cells) and regulatory T cells were more active in the BLIA subtype. Additionally, tumor cells in the BLIA subtype seem to inhibit cytotoxic cells to a greater extent. Among the interactions between epithelial cells and exhausted CD8+ T cells, MDK interaction was higher in the BLIA subtype. Additionally, the expression of the MDK gene in endothelial cells and macrophages leads to upregulation in immune infiltration which causes more T cells in TME. Also, we found that the progression-free survival of patients differs due to the expression of the MDK gene. In conclusion, the MDK gene causes a T cell-enriched environment in TME, which leads to different progression-free survival. Citation Format: Kyungsoo Kim, Jee Hung Kim, Soong June Bae, Sung Gwe Ahn, Joon Jeong. Analyzing heterogeneity of tumor microenvironment of TNBC patients by meta-analysis of 7 breast cancer scRNAseq studies [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Tumor Immunology and Immunotherapy; 2023 Oct 1-4; Toronto, Ontario, Canada. Philadelphia (PA): AACR; Cancer Immunol Res 2023;11(12 Suppl):Abstract nr B046.

Activated CTHRC1 promotes glycolysis in endothelial cells: Implications for metabolism and angiogenesis

CTHRC1 is transiently expressed by activated fibroblasts during tissue repair and in certain cancers, and CTHRC1 derived from osteocytes is detectable in circulation. Because its biological activity is poorly understood, we investigated whether the N terminus of CTHRC1 encodes a propeptide requiring cleavage to become activated. The effects of full-length versus cleaved recombinant CTHRC1 on endothelial cell metabolism and gene expression were examined in vitro. Respirometry was performed on Cthrc1 null and wildtype mice to obtain evidence for biological activity of CTHRC1 in vivo. Cleavage of the propeptide observed in vitro was attenuated in the presence of protease inhibitors, and cleaved CTHRC1 significantly promoted glycolysis whereas full-length CTHRC1 was less effective. The respiratory exchange ratio was significantly higher in wildtype mice compared to Cthrc1 null mice, supporting the findings of CTHRC1 promoting glycolysis in vivo. Key enzymes involved in glycolysis were significantly upregulated in endothelial cells in response to treatment with CTHRC1. In healthy human subjects, 58% of the cohort had detectable levels of circulating full-length CTHRC1, whereas all subjects with undetectable levels of full-length CTHRC1 (with one exception) had measurable levels of truncated CTHRC1 (88 pg/ml to >400 ng/ml). Our findings support a concept where CTHRC1 induction in activated fibroblasts at sites of ischemia such as tissue injury or cancer functions to increase glycolysis for ATP production under hypoxic conditions, thereby promoting cell survival and tissue repair. By promoting glycolysis under normoxic conditions, CTHRC1 may also be a contributor to the Warburg effect characteristically observed in many cancers.

FAM222A, Part of the BET-Regulated Basal Endothelial Transcriptome, Is a Novel Determinant of Endothelial Biology and Angiogenesis-Brief Report.

BETs (bromodomain and extraterminal domain-containing epigenetic reader proteins), including BRD4 (bromodomain-containing protein 4), orchestrate transcriptional programs induced by pathogenic stimuli, as intensively studied in cardiovascular disease and elsewhere. In endothelial cells (ECs), BRD4 directs induced proinflammatory, proatherosclerotic transcriptional responses; BET inhibitors, like JQ1, repress these effects and decrease atherosclerosis. While BET effects in pathogenic conditions have prompted therapeutic BET inhibitor development, BET action under basal conditions, including ECs, has remained understudied. To understand BET action in basal endothelial transcriptional programs, we first analyzed EC RNA-Seq data in the absence versus presence of JQ1 before using BET regulation to identify novel determinants of EC biology and function. RNA-Seq datasets of human umbilical vein ECs without and with JQ1 treatment were analyzed. After identifying C12orf34, also known as FAM222A (family with sequence similarity 222 member A), as a previously unreported, basally expressed, potently JQ1-induced EC gene, FAM222A was studied in endothelial and angiogenic responses in vitro using small-interference RNA silencing and lentiviral overexpression, in vitro, ex vivo and in vivo, including aortic sprouting, matrigel plug assays, and murine neonatal oxygen-induced retinopathy. Resting EC RNA-Seq data indicate BETs direct transcriptional programs underlying core endothelial properties including migration, proliferation, and angiogenesis. BET inhibition in resting ECs also significantly induced a subset of mRNAs, including FAM222A-a unique BRD4-regulated gene with no reported EC role. Silencing endothelial FAM222A significantly decreased cellular proliferation, migration, network formation, aorta sprouting, and Matrigel plug vascularization through coordinated modulation of VEGF (vascular endothelial growth factor) and NOTCH mediator expression in vitro, ex vivo, in vivo; lentiviral FAM222A overexpression had opposite effects. In vivo, siFAM222A significantly repressed retinal revascularization in neonatal murine oxygen-induced retinopathy through similar angiogenic signaling modulation. BET control over the basal endothelial transcriptome includes FAM222A, a novel, BRD4-regulated, key determinant of endothelial biology and angiogenesis.

Abstract 12226: Aging Impairs the Neuro-Vascular Interface in the Heart

Aging is a major risk factor for impaired cardiovascular function. The aging heart is characterized by vascular dysfunction, increased hypertrophy, fibrosis and electrophysiological alterations. However, the interaction between the different age-associated changes in cardiac remodelling is poorly define. Since vessels are aligned with nerves, and this interplay is critical for tissue homeostasis, we investigated whether an impairment of the neuro-vascular interface may contribute to age-associated pathologies in the heart. We show that aging reduces sympathetic and sensory nerve fibers, which both align to arteries and arterioles. Aging induced a dysregulation of vascular-derived neuro-regulatory genes, especially the axon repellent factor Sema3A, which was augmented by 2.4±0.1-fold in old heart endothelial cells. Consistently, endothelial Sema3a overexpression reduced axon density in vivo, thus mimicking the observed aged heart phenotype. Since cellular senescence was induced at the same time point when nerve density declined (16 months of age) and senescence endothelial cells showed a higher expression of Sema3a, we hypothesized that endothelial senescence may contribute to cardiac denervation. Indeed, supernatants of senescent endothelial cells reduced neurite outgrowth in vitro in a Sema3A-dependent manner. In vivo studies further confirmed that endothelial-specific induction of senescence by overexpression of Progerin induced left ventricular denervation and induced Sema3a expression. By contrast, the pharmacological removal of senescent cells by the senolytic drugs dasatinib and quercetin rescued age-induced denervation and prevented the age-induced induction of endothelial Sema3a expression. Functional studies additionally demonstrated that endothelial senescence reduced heart rate variability, while senolytics preserved heart rate variability, restored age-associated declines in sympathetic and parasympathetic activity and reduced age-associated increase in arrhythmias. These data suggest that senescence-associated regulation of neuro-regulatory genes in endothelial cells is associated with reduced nerve density and, thereby, contributes to age-associated cardiac dysfunction.

Abstract 18901: Critical Role of Endothelial Injury in the Initiation of Aortic Dissection

Introduction: Endothelium forms a protective barrier and maintains vascular hemostasis. However, the role of endothelial injury in aortic aneurysms and dissections (AAD) remains poorly understood. Receptor-interacting protein kinase 3 (RIP3)-mediated necroptosis and gasdermin D (GSDMD)-mediated pyroptosis trigger necrotic cell death. We hypothesize endothelial cell (EC) death induced by necroptosis and pyroptosis contributes to AAD formation. Methods: Endothelial integrity and gene expression were examined in ascending aortic tissues from ascending thoracic aortic aneurysm (ATAA) patients (n=9) and organ donor controls (n=8) by single-cell transcriptome analysis. Effects of EC death on AAD formation were determined in EC-specific Rip3 knockout (EC-Rip3 -/- , n=26), EC-specific Gsdmd knockout (EC-Gsdmd -/- , n=21), and necroptosis/pyroptosis inhibitor necrosulfonamide (NSA) treated mice (n=15) in sporadic AAD models induced by angiotensin II (Ang II) infusion. Evans blue staining and nanoparticle-mediated contrast-enhanced CT (n-CECT) detected endothelial hyperpermeability Results: Single-cell transcriptome and immunostaining analyses revealed significant upregulation of pro-death genes (e.g., RIP3 and GSDMD), but downregulation of cell junction genes (e.g., TJP1 and GJA1) in ECs of ATAA patients compared with controls. In the mouse AAD model, endothelial injury and hyperpermeability were detected 2-5 days after Ang II infusion and were associated with intramural nanoparticle accumulation, elastic fiber fragmentation, and macrophage infiltration. Importantly, EC-Rip3 -/- mice and EC-Gsdmd -/- mice showed preserved EC barrier function, reduced nanoparticle accumulation and elastic fiber fragmentation, and reduced AAD incidence (including aneurysm, dissection, and rupture) compared to their littermate controls (EC-Rip3 -/- mice: 52% vs 23.1%, P=0.033; EC-Gsdmd -/- mice: 52.6% vs 14.3%, P=0.01). Blocking necroptosis/pyroptosis by NSA treatment reduced the incidence (65% vs 27%; P=0.04) and severity of AAD. Conclusions: Endothelial injury and subsequent barrier dysfunction and infiltration are key features of AAD formation. Prevention of EC necrotic cell death can be a potential AAD therapeutic target.

Abstract 13577: Burden of Cardiometabolic Risk Factors and Vascular Health

Introduction: Cardiometabolic risk factors (diabetes, obesity, and hypertension) are highly prevalent and contribute to cardiovascular disease (CVD). Endothelial dysfunction often precedes CVD development. Direct brachial vein endothelial cell (EC) harvesting enables the examination of vascular health via assessment of EC gene expression. Hypothesis: We hypothesized that individuals with greater cardiometabolic risk would demonstrate a more pro-inflammatory EC transcriptome. Methods: Adult participants without CVD underwent EC harvesting followed by RNA sequencing. We categorized individuals by number of risk factors (hypertension, diabetes, obesity) as 0 through 3. To evaluate the association between risk factor burden and outcome transcripts, we performed multivariable linear regression, adjusting for age, sex, and race/ethnicity. Results: Among 18 individuals (mean age 47 ± 14, 44% female, and 61% white), 17%, 44%, 28%, and 11% had 0,1,2, and 3 prevalent risk factors, respectively. EC RNA sequencing revealed 588 differentially expressed transcripts (p-adj &lt;0.05) with 585 upregulated, 3 downregulated, and excellent clustering ( Fig. a, b ). Gene ontology enrichment analysis demonstrated enriched and upregulated pathways of T-cell activation (NES = 2.22, p&lt;0.001), leukocyte differentiation (NES= 2.16, p&lt;0.001), and platelet aggregation (NES= 1.66, p=0.03) and downregulated pathways of endothelial cell proliferation (NES= -1.68, p=0.03). ( Fig. c ) Select upregulated genes (log 2FC &gt;3, p-adj&lt;0.05) included VCAM1, CEACAM1, ADAM 17, and CD99L2, with a graded increase in mean normalized counts with increasing number of risk factors. ( Fig. d ) Conclusions: We demonstrate a proinflammatory, pro-adhesive, and pro-coagulant EC transcriptome associated with increased cardiometabolic risk factor burden offering insight into potential mechanisms linking cardiometabolic risk factors with the development of CVD.

MicroRNA-10a enrichment in factor VIIa–released endothelial extracellular vesicles: potential mechanisms

BackgroundFactor VIIa induces the release of extracellular vesicles (EVs) from endothelial cells (EEVs). Factor VIIa–released EEVs are enriched with microRNA-10a (miR10a) and elicit miR10a-dependent cytoprotective responses. ObjectivesTo investigate mechanisms by which FVIIa induces miR10a expression in endothelial cells and sorts miR10a into the EVs. MethodsActivation of Elk-1 and TWIST1 expression was analyzed by immunofluorescence microscopy and immunoblot analysis. Small interfering RNA silencing approach was used to knock down the expression of specific genes in endothelial cells. EVs secreted from endothelial cells or released into circulation in mice were isolated by centrifugation and quantified by nanoparticle tracking analysis. Factor VIIa or EVs were injected into mice; mice were challenged with lipopolysaccharides to assess the cytoprotective effects of FVIIa or EVs. ResultsFVIIa activation of ERK1/2 triggered the activation of Elk-1, which led to the induction of TWIST1, a key transcription factor involved in miR10a expression. Factor VIIa also induced the expression of La, a small RNA-binding protein. Factor VIIa–driven acid sphingomyelinase (ASM) activation and the subsequent activation of the S1P receptor pathway were responsible for the induction of La. Silencing of ASM or La significantly reduced miR10a levels in FVIIa-released EEVs without affecting the cellular expression of miR10a. Factor VIIa–EEVs from ASM knocked-down cells failed to provide cytoprotective responses in cell and murine model systems. Administration of FVIIa protected wild-type but not ASM−/− mice against lipopolysaccharide-induced inflammation and vascular leakage. ConclusionOur data suggest that enhanced cellular expression of miR10a coupled with La-dependent sorting of miR10a is responsible for enriching FVIIa-released EVs with miR10a.