scholarly journals Long non-coding RNA TERRA influences DNA damage and survival of endothelial cells and cardiomyocytes

2020 ◽  
Vol 41 (Supplement_2) ◽  
Author(s):  
D Bink ◽  
T.P Pham ◽  
P Hofmann ◽  
S Dimmeler ◽  
R.A Boon
Keyword(s):  
Dna Damage ◽  
Endothelial Cells ◽  
Cell Function ◽  
Umbilical Vein ◽  
Heart Tissue ◽  
Funding Source ◽  
Caspase Activity ◽  
Endothelial Cell Function ◽  
Human Cardiomyocytes ◽  
Non Coding Rnas

Abstract   Ageing is the major risk factor for cardiovascular disease. Current therapies are mainly based on proteins, while targeting long non-coding RNAs (lncRNAs) is largely unexplored. Long non-coding RNAs are emerging as novel regulators of cellular functions and contributors to cardiovascular ageing. Although telomeres are heterochromatic regions, non-coding transcripts called Telomeric repeat-containing RNA (TERRA) are transcribed from the telomeres of most chromosomes. The transcription of TERRA starts at the subtelomere and ends in the telomere, leading to molecules of 0.2–10kb. This study aims to characterize the role of TERRA in the cardiovascular system. TERRA molecules from different chromosomes were upregulated in the hearts of old mice compared to young mice (p=0.002). An increased TERRA expression was also shown in heart tissue of patients with ischemic heart disease compared to donor heart tissue (p=0.001). In vitro an upregulation of the TERRA molecule transcribed from chromosome 20 (h20q-TERRA) was found in old passage human umbilical vein endothelial cells (HUVECs) (P15–17) compared to young HUVECs (P3) (p=0.014). IPSC-derived cardiomyocytes also increased the expression of h20q-TERRA with increasing passage (p=0.011). After knockdown of h20q-TERRA with LNA GapmeRs HUVECs show less sprout formation in a spheroid assay compared to negative control transfected HUVECs (p=0.002), without showing a change in migration (p=0.205) or proliferation (p=0.114). H20q-TERRA knockdown revealed that there was more apoptosis (p=0.015), more DNA damage as measured with the comet assay (p<0.001), increased γH2AX levels (p=0.029) and an increase in γH2AX colocalization with the telomere (p=0.011). The amount of phosphorylated P53 was also increased after knockdown (p=0.038), while it was decreased after TERRA overexpression (p=0.001). Inhibition of the ATM-yH2AX-P53 pathway did however not reduce the increased apoptosis after knockdown (KU60019 p=0.399; siP53 p=0.303). Silencing the m18-TERRA molecule in mouse endothelial H5V cells led to an increase in caspase activity (p=0.004) similar to what was shown in HUVECs. In addition, increased caspase activity (p=0.012), increased γH2AX levels (p<0.001) and increased γH2AX colocalization with the telomere (p=0.007) was also shown after silencing of h20q-TERRA in human cardiomyocytes. In summary, our data demonstrates that TERRA is upregulated with ageing and plays a role in endothelial and cardiomyocyte function and survival. These data show that TERRA transcripts are induced in cardiovascular ageing and are essential for endothelial cell function. Funding Acknowledgement Type of funding source: Public grant(s) – EU funding. Main funding source(s): Horizon 2020

Abstract 101: MiR-4260 Promotes the Proliferation, Migration, and Tube Formation of Human Umbilical Vein Endothelial Cells

2015 ◽  
Vol 117 (suppl_1) ◽  
Author(s):  
Qi Sun ◽  
Dongcao Lv ◽  
Qiulian Zhou ◽  
Yihua Bei ◽  
Junjie Xiao
Keyword(s):  
Endothelial Cells ◽  
Target Genes ◽  
Cell Function ◽  
Umbilical Vein ◽  
Tube Formation ◽  
Endothelial Cell Function ◽  
Human Umbilical Vein ◽  
And Migration ◽  
Umbilical Vein Endothelial Cells

MicroRNAs (miRNAs, miRs), endogenous small non-coding RNA, have been shown to act as essential regulators in angiogenesis which plays important roles in improving blood flow and cardiac function following myocardial infarction. The current study investigated the potential of miR-4260 in endothelial cell function and angiogenesis using human umbilical vein endothelial cells (HUVEC). Our data demonstrated that overexpression of miR-4260 was associated with increased proliferation and migration of HUVEC using EdU incorporation assay (17.25%±1.31 vs 25.78%±1.24 in nc-mimics vs miR-4260 mimics, respectively) and wound healing assay, respectively. While downregulation of miR-4260 inhibited the proliferation (17.90%±1.37 vs 10.66%±1.41 in nc-inhibitor vs miR-4260 inhibitor, respectively) and migration of HUVEC. Furthermore, we found that miR-4260 mimics increased (129.75±3.68 vs 147±3.13 in nc-mimics vs miR-4260 mimics, respectively), while miR-4260 inhibitor decreased the tube formation of HUVECs in vitro (123.25±2.17 vs 92±4.45 in nc-inhibitor vs miR-4260 inhibitor expression, respectively). Our data indicate that miR-4260 contributes to the proliferation, migration and tube formation of endothelial cells, and might be essential regulators for angiogenesis. Further study is needed to investigate the underlying mechanism that mediates the role of miR-4260 in angiogenesis by identifying its putative downstream target genes.

scholarly journals Up-regulation of the Notch ligand Delta-like 4 inhibits VEGF-induced endothelial cell function

Blood ◽  
2006 ◽  
Vol 107 (3) ◽  
pp. 931-939 ◽  
Author(s):  
Cassin Kimmel Williams ◽  
Ji-Liang Li ◽  
Matilde Murga ◽  
Adrian L. Harris ◽  
Giovanna Tosato
Keyword(s):  
Endothelial Cells ◽  
Endothelial Cell ◽  
Cell Function ◽  
Umbilical Vein ◽  
Endothelial Cell Proliferation ◽  
Vegf Receptor ◽  
Endothelial Cell Function ◽  
Notch Ligand ◽  
Neuropilin 1 ◽  
Umbilical Vein Endothelial Cells

AbstractDelta-like 4 (Dll4), a membrane-bound ligand for Notch1 and Notch4, is selectively expressed in the developing endothelium and in some tumor endothelium, and it is induced by vascular endothelial growth factor (VEGF)-A and hypoxia. Gene targeting studies have shown that Dll4 is required for normal embryonic vascular remodeling, but the mechanisms underlying Dll4 regulatory functions are currently not defined. In this study, we generated primary human endothelial cells that overexpress Dll4 protein to study Dll4 function and mechanism of action. Human umbilical vein endothelial cells retrovirally transduced with Dll4 displayed reduced proliferative and migratory responses selectively to VEGF-A. Expression of VEGF receptor-2, the principal signaling receptor for VEGF-A in endothelial cells, and coreceptor neuropilin-1 was significantly decreased in Dll4-transduced endothelial cells. Consistent with Dll4 signaling through Notch, expression of HEY2, one of the transcription factors that mediates Notch function, was significantly induced in Dll4-overexpressing endothelial cells. The γ-secretase inhibitor L-685458 significantly reconstituted endothelial cell proliferation inhibited by immobilized extracellular Dll4 and reconstituted VEGFR2 expression in Dll4-overerexpressing endothelial cells. These results identify the Notch ligand Dll4 as a selective inhibitor of VEGF-A biologic activities down-regulating 2 VEGF receptors expressed on endothelial cells and raise the possibility that Dll4 may be exploited therapeutically to modulate angiogenesis.

Heterodimerization of FGF-receptor 1 and PDGF-receptor-α: a novel mechanism underlying the inhibitory effect of PDGF-BB on FGF-2 in human cells

Blood ◽  
2006 ◽  
Vol 107 (5) ◽  
pp. 1896-1902 ◽  
Author(s):  
Debora Faraone ◽  
Maria S. Aguzzi ◽  
Gianluca Ragone ◽  
Katia Russo ◽  
Maurizio C. Capogrossi ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Growth Factor ◽  
Intracellular Signaling ◽  
Cell Function ◽  
Umbilical Vein ◽  
Pdgf Receptor ◽  
Inhibitory Effects ◽  
Fgf Receptor ◽  
Endothelial Cell Function

Previous evidence has shown that platelet-derived growth factor-BB (PDGF-BB) and fibroblast growth factor-2 (FGF-2) directly interact with high affinity, leading to potent reciprocal inhibitory effects on bovine endothelial cells and rat vascular smooth muscle cells. In this study, we report that PDGF-BB inhibits a series of FGF-2–induced events, such as proliferation of human umbilical vein endothelial cells (HUVECs), FGF-2 cellular internalization, phosphorylation of intracellular signaling factors including p38, rac1/cdc42, MKK4, and MKK3/6, and phosphorylation of FGF-receptor 1 (FGF-R1). PDGF-receptor-α (PDGF-Rα) was found to mediate PDGF-BB inhibitory effects because its neutralization fully restored FGF-2 mitogenic activity and internalization. Additional biochemical analyses, coimmunoprecipitation experiments, and FRET analysis showed that FGF-R1 and PDGF-Rα directly interact in vitro and in vivo and that this interaction is somehow increased in the presence of the corresponding ligands FGF-2 and PDGF-BB. These results suggest that FGF-R1/PDGF-Rα heterodimerization may represent a novel endogenous mechanism to modulate the action of these receptors and their ligands and to control endothelial cell function.

Bone morphogenetic protein 4 regulates microRNAs miR-494 and miR-126–5p in control of endothelial cell function in angiogenesis

2017 ◽  
Vol 117 (04) ◽  
pp. 734-749 ◽  
Author(s):  
Erika Saretzki ◽  
Franziska Pankratz ◽  
Bianca Engert ◽  
Sebastian Grundmann ◽  
Christoph Bode ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Endothelial Cell ◽  
Western Blot ◽  
Bone Morphogenetic Protein ◽  
Cell Function ◽  
Umbilical Vein ◽  
Endothelial Cell Proliferation ◽  
Bone Morphogenetic Protein 4 ◽  
Endothelial Cell Function ◽  
Morphogenetic Protein

SummaryMicroRNAs are small non-coding RNAs that negatively regulate posttranscriptional gene expression. Several microRNAs have been described to regulate the process of angiogenesis. Previously, we have shown that bone morphogenetic protein 4 (BMP4) increased the proangiogenic activity of endothelial cells. In this project, we now investigated how the pro-angiogenic BMP4 effect is mediated by microRNAs. First, we performed a microRNA array with BMP4-stimulated human umbilical vein endothelial cells (HUVECs). Among the topregulated microRNAs, we detected a decreased expression of miR-494 and increased expression of miR-126–5p. Next, we analysed the canonical Smad and alternative signalling pathways, through which BMP4 would regulate miR-126–5p and miR-494 expression. Furthermore, the functional effect of miR-494 and miR-126–5p on endothelial cells was investigated. MicroRNA-494 overexpression decreased endothelial cell proliferation, migration and sprout formation. Consistently, miR-494 inhibition increased endothelial cell function. As potential miR-494 targets, bFGF and BMP endothelial cell precursorderived regulator (BMPER) were identified and confirmed by western blot. Luciferase assays showed direct miR-494 binding in BMPER 3’UTR. In contrast, miR-126–5p overexpression increased pro-angiogenic endothelial cell behaviour and, accordingly, miR-126–5p inhibition decreased endothelial cell function. As a direct miR-126–5p target we identified the anti-angiogenic thrombospondin-1 which was confirmed by western blot analysis and luciferase assays. In the Matrigel plug assay application of antagomiR-494 increased endothelial cell ingrowth, whereas antagomiR-126–5p treatment decreased cell ingrowth in vivo. Taken together, through differential regulation of the anti-angiomiR-494 and the angiomiR-126–5p by BMP4 both microRNAs contribute to the pro-angiogenic BMP4 effect on endothelial cells.Supplementary Material to this article is available online at www.thrombosis-online.com.

scholarly journals Chronic increases in sphingosine kinase-1 activity induce a pro-inflammatory, pro-angiogenic phenotype in endothelial cells

2009 ◽  
Vol 14 (3) ◽  
Author(s):  
Vidya Limaye ◽  
Pu Xia ◽  
Chris Hahn ◽  
Malcolm Smith ◽  
Mathew Vadas ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Synovial Fluid ◽  
Signaling Pathway ◽  
Cell Function ◽  
Capillary Tube ◽  
Umbilical Vein ◽  
Sphingosine Kinase ◽  
Dominant Negative ◽  
Sphingosine Kinase 1 ◽  
Endothelial Cell Function

AbstractSphingosine kinase-1 (SK1) promotes the formation of sphingosine-1-phosphate (S1P), which has potent pro-inflammatory and pro-angiogenic effects. We investigated the effects of raised SK1 levels on endothelial cell function and the possibility that this signaling pathway is activated in rheumatoid arthritis. Human umbilical vein endothelial cells with 3- to 5-fold SK1 (ECSK) overexpression were generated by adenoviral and retroviralmediated gene delivery. The activation state of these cells and their ability to undergo angiogenesis was determined. S1P was measured in synovial fluid from patients with RA and OA. ECSK showed an enhanced migratory capacity and a stimulated rate of capillary tube formation. The cells showed constitutive activation as evidenced by the induction of basal VCAM-1 expression, and further showed a more augmented VCAM-1 and E selectin response to TNF compared with empty vector control cells (ECEV). These changes had functional consequences in terms of enhanced neutrophil binding in the basal and TNFstimulated states in ECSK. By contrast, over-expression of a dominant-negative SK inhibited the TNF-induced VCAM-1 and E selectin and inhibited PMN adhesion, confirming that the observed effects were specifically mediated by SK. The synovial fluid levels of S1P were significantly higher in patients with RA than in those with OA. Small chronic increases in SK1 activity in the endothelial cells enhance the ability of the cells to support inflammation and undergo angiogenesis, and sensitize the cells to inflammatory cytokines. The SK1 signaling pathway is activated in RA, suggesting that manipulation of SK1 activity in diseases of aberrant inflammation and angiogenesis may be beneficial.

scholarly journals Thymosin β4 regulates endothelial cell function via activating the AKT pathway

2021 ◽  
Vol 62 (4) ◽  
pp. 295-306
Author(s):  
Yong Tang ◽  
Hao Dong ◽  
Wenbin Lu ◽  
Xiaofeng Zhang ◽  
Xiao Shen ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Endothelial Cell ◽  
Cell Function ◽  
Umbilical Vein ◽  
Thymosin Β4 ◽  
Akt Inhibitor ◽  
Proliferation Markers ◽  
Endothelial Cell Function ◽  
Real Time Quantitative Pcr ◽  
And Migration

The vascular eendothelial cells are highly heterogeneous and associated with numerous diseases. Thymosin β4 (Tβ4) plays pleiotropic roles in endothelial cell differentiation, migration and angiogenesis. However, the underlying mechanisms played by Tβ4 in the regulation of endothelial cells have not yet been well investigated. In the present study, Tβ4 -GFP adenovirus, transfected into human umbilical vein endothelial cells (HUVECs), and cell morphology were analyzed by fluorescence microscopy. ELISA was used to determine the concentration of Tβ4 expression. Furthermore, the effects of Tβ4 overexpression on HUVECs proliferation, apoptosis and migration were investigated. Real-time quantitative PCR and western blot were conducted to examine mRNA and protein expression in HUVECs with Tβ4 overexpression. Moreover, the underlying molecular mechanism of Tβ4 in HUVECs function was tested through treatment with LY294002, a PI3K/AKT inhibitor. Overexpression of Tβ4 increased the cell ability of HUVECs, and up-regulated the expression of the proliferation markers PCNA and Cyclin D1. In addition, overexpression of Tβ4 reduced HUVECs apoptosis, both under normoxic and hypoxic conditions. Moreover, overexpression of Tβ4 increased the ability of HUVECs to migrate through the membrane and up-regulated levels of MMP-2 and MMP-9. The use of LY294002 decreased the p-AKT (Ser473) level, which was induced by Tβ4 overexpression. Importantly, LY294002 reduced Tβ4-induced HUVECs proliferation and migration. In conclusion, our results suggest that Tβ4 is a major regulator of HUVECs function by activating the AKT signaling pathway.

scholarly journals The CUL3-SPOP-DAXX axis is a novel regulator of VEGFR2 expression in vascular endothelial cells

2017 ◽  
Vol 7 (1) ◽  
Author(s):  
Tomohisa Sakaue ◽  
Iori Sakakibara ◽  
Takahiro Uesugi ◽  
Ayako Fujisaki ◽  
Koh-ichi Nakashiro ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Endothelial Cell ◽  
Cell Function ◽  
Vascular Endothelial Cells ◽  
Umbilical Vein ◽  
Vascular Endothelial Cell ◽  
Growth Factor Receptor ◽  
Vascular Endothelial ◽  
Death Domain ◽  
Endothelial Cell Function

Abstract Vascular endothelial cell growth factor receptor 2 (VEGFR2) is an essential receptor for the homeostasis of endothelial cells. In this study, we showed that NEDD8-conjugated Cullin3 (CUL3)-based ubiquitin E3 (UbE3) ligase plays a crucial role in VEGFR2 mRNA expression. Human umbilical vein endothelial cells treated with MLN4924, an inhibitor of NEDD8-activating enzyme, or with CUL3 siRNA drastically lost their response to VEGF due to the intense decrease in VEGFR2 expression. Moreover, speckle-type POZ protein (SPOP) and death-domain associated protein (DAXX) were involved in the CUL3 UbE3 ligase complex as a substrate adaptor and a substrate, respectively. Knockdown of SPOP and CUL3 led to the upregulation of DAXX protein and downregulation of VEGFR2 levels. These levels were inversely correlated with one another. In addition, simultaneous knockdown of SPOP and DAXX completely reversed the downregulation of VEGFR2 levels. Moreover, the CUL3-SPOP-DAXX axis had the same effects on NOTCH1, DLL4 and NRP1 expression. Taken together, these findings suggest that the CUL3-SPOP-DAXX axis plays a very important role in endothelial cell function by targeting key angiogenic regulators.

Abstract 16549: Trimethylamine N-Oxide-Derived Endothelial Microvesicles Induce a Dysfunctional Endothelial Cell Phenotype

Circulation ◽  
2020 ◽  
Vol 142 (Suppl_3) ◽  
Author(s):  
Vinicius Garcia ◽  
L. Madden Brewster ◽  
Kelly A Stockelman ◽  
Hannah K Fandl ◽  
Clay Rahaman ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Endothelial Cell ◽  
Cell Function ◽  
Umbilical Vein ◽  
Major Adverse Cardiovascular Events ◽  
Cell Phenotype ◽  
No Production ◽  
Endothelial Cell Function ◽  
Cell Expression ◽  
In Cells

Introduction: Gut microbiota-generated increases in trimethylamine N-oxide (TMAO) has been linked to atherosclerosis and major adverse cardiovascular events. Experimental and clinical studies suggest that TMAO impairs endothelial cell function resulting in a proatherogenic endothelial phenotype. Endothelial microvesicles (EMVs) are anucleate vesicles shed constitutively by the endothelium aiding in cell-to-cell communication, activate repair or defense mechanisms, and/or stimulate immune responses. Under pathologic conditions, however, EMVs are released in greater number and their functional phenotype is more likely to evoke dysfunctional cellular effects. The experimental aims of this study were to determine: 1) if TMAO stimulates EMV release from endothelial cells in vitro ; and 2) the effects of TMAO-generated EMVs on endothelial cell inflammation, apoptosis, autophagy and nitric oxide (NO) production. Methods: Human umbilical vein endothelial cells (HUVECs) were treated with TMAO (100 μmol) for 24 h. EMVs released into the supernatant from cells treated with TMAO or vehicle were isolated and quantified by flow cytometry. Fresh HUVECs were treated with either TMAO-derived or control EMVs for 24 h. Results: EMV release was significantly higher in cells treated with TMAO compared with control (55±3 vs 28±3 EMV/μL). TMAO-generated EMVs induced significantly higher release of interleukin (IL)-6 (33.9±2.6 vs 20.2±1.3 pg/mL) and IL-8 (45.4±2.6 vs 33.1±1.6 pg/mL) and active NF-κB p65 (Ser536) (17.4±1.3 vs 7.7±1.0 AU) expression than control EMVs. TMAO EMVs significantly increased cell expression of apoptotic proteins caspase-9 (198.5±19.4 vs 119.7±11.2 AU) and active caspase-3 (17.7±2.8 vs 11.3±0.7 AU) and markedly depressed eNOS activity (10.6±0.9 vs 6.9±0.6 AU) and NO production (7.7±0.4 vs 5.5±0.6 μmol/L). In addition, cell autophagy was dysregulated by TMAO EMVs; cell expression of Beclin-1 (53.8±6.4 vs 36.6±3.6 AU), p62 (34.0±0.8 vs 16.9±1.2 AU) and LC3BII/LC3BI (15.0±0.1 vs 7.5±0.8 AU) were significantly elevated in cells treated with TMAO vs control EMVs. Conclusions: TMAO-generated EMVs adversely affect major functional characteristics of endothelial cells potentially contributing to the proatherogenic profile of TMAO.

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