Senescence-associated (beta)-galactosidase reflects an increase in lysosomal mass during replicative ageing of human endothelial cells

2000 ◽  
Vol 113 (20) ◽  
pp. 3613-3622 ◽  
Author(s):  
D.J. Kurz ◽  
S. Decary ◽  
Y. Hong ◽  
J.D. Erusalimsky
Keyword(s):  
Endothelial Cells ◽  
Replicative Senescence ◽  
Umbilical Vein ◽  
Galactosidase Activity ◽  
Acridine Orange Staining ◽  
Lysosomal Ph ◽  
Protein Levels ◽  
Flow Cytometric ◽  
Umbilical Vein Endothelial Cells ◽  
Beta Galactosidase

Senescence-associated (beta)-galactosidase is widely used as a biomarker of replicative senescence. However, it remains unknown whether this is a distinct enzyme active at pH 6, and differentially expressed in senescence, or a manifestation of an increase in the classic acid lysosomal (beta)-galactosidase. Here we have investigated the origin of senescence-associated-(beta)-galactosidase activity by modifying the intracellular and lysosomal pH of young and senescent human umbilical vein endothelial cells and examining the effect of these manipulations on the levels of activity, using a flow cytometric assay. Lysosomal alkalinisation with chloroquine or bafilomycin A(1), as well as equilibration of the intracellular milieu to pH 6 with nigericin, caused a profound (92-99%) inhibition of the total intracellular (beta)-galactosidase activity. However, independent of pH alterations, senescent cells showed levels of (beta)-galactosidase activity three- to sixfold higher than young cells. This increase in activity occurred in parallel to an increase in (beta)-galactosidase protein levels. Acridine Orange staining revealed an increase in lysosomal content with replicative age, which correlated with the increase in (beta)-galactosidase. These findings demonstrate that senescence-associated (beta)-galactosidase is a manifestation of residual lysosomal activity at a suboptimal pH, which becomes detectable due to the increased lysosomal content in senescent cells.

scholarly journals Anti-Inflammatory and Anti-Apoptotic Effects of Stybenpropol A on Human Umbilical Vein Endothelial Cells

2019 ◽  
Vol 20 (21) ◽  
pp. 5383 ◽  
Author(s):  
Li Zhang ◽  
Feifei Wang ◽  
Qing Zhang ◽  
Qiuming Liang ◽  
Shumei Wang ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Cell Adhesion ◽  
Adhesion Molecule ◽  
Cell Adhesion Molecule ◽  
Umbilical Vein ◽  
Caspase 9 ◽  
Protein Levels ◽  
Tnf Α ◽  
Umbilical Vein Endothelial Cells ◽  
Cell Adhesion Molecule 1

Inflammation is a key mediator in the progression of atherosclerosis (AS). Benzoinum, a resin secreted from the bark of Styrax tonkinensis, has been widely used as a form of traditional Chinese medicine in clinical settings to enhance cardiovascular function, but the active components of the resin responsible for those pharmaceutical effects remain unclear. To better clarify these components, a new phenylpropane derivative termed stybenpropol A was isolated from benzoinum and characterized via comprehensive spectra a nalysis. We further assessed how this phenylpropane derivative affected treatment of human umbilical vein endothelial cells (HUVECs) with tumor necrosis factor-α (TNF-α). Our results revealed that stybenpropol A reduced soluble intercellular cell adhesion molecule-1 (sICAM-1), soluble vascular cell adhesion molecule-1 (sVCAM-1), interleukin-8 (IL-8), and interleukin-1β (IL-1β) expression by ELISA, inhibited apoptosis, and accelerated nitric oxide (NO) release in TNF-α-treated HUVECs. We further found that stybenpropol A decreased VCAM-1, ICAM-1, Bax, and caspase-9 protein levels, and increased the protein levels of Bcl-2, IKK-β, and IκB-α. This study identified a new, natural phenylpropane derivative of benzoinum, and is the first to reveal its cytoprotective effects in the context of TNF-α-treated HUVECs via regulation of the NF-κB and caspase-9 signaling pathways.

scholarly journals Evodiamine Attenuates P2X7-Mediated Inflammatory Injury of Human Umbilical Vein Endothelial Cells Exposed to High Free Fatty Acids

2018 ◽  
Vol 2018 ◽  
pp. 1-10 ◽  
Author(s):  
Yun Xue ◽  
Ting Guo ◽  
Lifang Zou ◽  
Yingxin Gong ◽  
Bing Wu ◽  
...  
Keyword(s):  
Insulin Resistance ◽  
Fatty Acids ◽  
Endothelial Cells ◽  
Free Fatty Acids ◽  
Inflammatory Responses ◽  
Umbilical Vein ◽  
Human Umbilical Vein ◽  
Beneficial Effects ◽  
Protein Levels ◽  
Umbilical Vein Endothelial Cells

Insulin resistance and type 2 diabetes mellitus (T2DM) are highly prevalent around the world. Elevated concentrations of free fatty acids (FFAs) are closely related to insulin resistance and T2DM. P2X7 receptor is an ion channel gated by ATP, which is implicated in various scenarios including immune response, pain, and inflammation. In this study, we have explored whether P2X7 receptor is involved in pathological changes in human umbilical vein endothelial cells (HUVECs) induced by high FFA treatment, and the potential beneficial effects of evodiamine. Evodiamine could effectively suppress the enhanced expression of P2X7 receptor caused by high FFAs at both mRNA and protein levels. In addition, high FFA-induced cytotoxicity, the upregulated release of ATP, and production of reactive oxygen species (ROS) could be ameliorated by evodiamine in HUVECs. Evodiamine could also reverse the decreased NO formation and the increased adhesive events of immune cells at high FFAs. Moreover, evodiamine inhibited P2X7-dependent TNF-α expression and ERK 1/2 phosphorylation due to high FFAs. All these results indicated that evodiamine could correct the upregulated expression of P2X7 receptor induced under high FFA condition in HUVECs, and consequently suppressed oxidative stress and inflammatory responses.

scholarly journals YB-1 transferred by gastric cancer exosomes promotes angiogenesis via enhancing the expression of angiogenic factors in vascular endothelial cells

BMC Cancer ◽  
2020 ◽  
Vol 20 (1) ◽  
Author(s):  
Xiaoxia Xue ◽  
Jin Huang ◽  
Kai Yu ◽  
Xinyue Chen ◽  
Yini He ◽  
...  
Keyword(s):  
Gastric Cancer ◽  
Endothelial Cells ◽  
Vascular Endothelial Cells ◽  
Umbilical Vein ◽  
Angiogenic Factors ◽  
Mrna Levels ◽  
Protein Levels ◽  
Significant Difference ◽  
Umbilical Vein Endothelial Cells ◽  
Intercellular Communications

Abstract Background Angiogenesis is important for the progression of gastric cancer (GC). Y-box binding protein 1 (YB-1) predicts advanced disease and indicates neovasculature formation in GC tissues, while the related mechanisms remain elusive. Exosomes mediate intercellular communications via transferring various molecules including proteins, lipids, mRNAs, and microRNAs, while the cargos of GC exosomes and the related mechanisms in GC angiogenesis were rarely reported except for several microRNAs. Methods In this study, human umbilical vein endothelial cells (HUVECs) were, respectively, treated by the exosomes isolated from the YB-1 transfected and the control SGC-7901 cells (SGC-7901-OE-Exo and SGC-7901-NC-Exo), and their apoptosis, proliferation, migration, invasion, and angiogenesis were, sequentially, compared. The levels of angiogenic factors including VEGF, Ang-1, MMP-9 and IL-8 in the exosome-treated HUVECs and the GC-derived exosomes were, separately, detected using PCR and Western blotting as well as RNA sequencing assays. Results We observed the consistent level of YB-1 in the exosomes and their originated GC cells, and the internalization of exosomes into HUVECs. Comparing with SGC-7901-NC-Exo, SGC-7901-OE-Exo significantly inhibited the apoptosis but promoted the proliferation, migration, invasion, and angiogenesis of HUVECs, within which the increased mRNA and protein levels of VEGF, Ang-1, MMP-9 and IL-8 were demonstrated. Meanwhile, mRNA levels of VEGF, Ang-1, MMP-9 and IL-8 showed no significant difference between SGC-7901-NC-Exo and SGC-7901-OE-Exo, although statistically higher mRNA of YB-1 was detected in the SGC-7901-OE-Exo. Conclusions Our findings illustrate YB-1 as the key component of exosome to promote GC angiogenesis by upregulating specific angiogenic factors in the exosome-treated endothelial cells but not in the exosomes themselves.

MicroRNA 299-3p modulates replicative senescence in endothelial cells

2013 ◽  
Vol 45 (7) ◽  
pp. 256-267 ◽  
Author(s):  
Hui-Lan Jong ◽  
Mohd Rais Mustafa ◽  
Paul M. Vanhoutte ◽  
Sazaly AbuBakar ◽  
Pooi-Fong Wong
Keyword(s):  
Endothelial Cells ◽  
Target Genes ◽  
Replicative Senescence ◽  
Umbilical Vein ◽  
Gene Profiling ◽  
Cellular Processes ◽  
Migration Capacity ◽  
Umbilical Vein Endothelial Cells ◽  
Insight Into

MicroRNAs (miRNAs) regulate various cellular processes. While several genes associated with replicative senescence have been described in endothelial cells, miRNAs that regulate these genes remain largely unknown. The present study was designed to identify miRNAs associated with replicative senescence and their target genes in human umbilical vein endothelial cells (HUVECs). An integrated miRNA and gene profiling approach revealed that hsa-miR-299-3p is upregulated in senescent HUVECs compared with the young cells, and one of its target genes could be IGF1. IGF1 was upregulated in senescent compared with young HUVECs, and knockdown of hsa-miR-299-3p dose-dependently increased the mRNA expression of IGF1, more significantly observed in the presenescent cells ( passage 19) compared with the senescent cells ( passage 25). Knockdown of hsa-miR-299-3p also resulted in significant reduction in the percentage of cells positively stained for senescence-associated β-galactosidase and increases in cell viability measured by MTT assay but marginal increases in cell proliferation and cell migration capacity measured by real-time growth kinetics analysis. Moreover, knockdown of hsa-miR-299-3p also increased proliferation of cells treated with H2O2 to induce senescence. These findings suggest that hsa-miR-299-3p may delay or protect against replicative senescence by improving the metabolic activity of the senesced cells but does not stimulate growth of the remaining cells in senescent cultures. Hence, these findings provide an early insight into the role of hsa-miR-299-3p in the modulation of replicative senescence in HUVECs.

scholarly journals Oxidative Stress Induces HSP90 Upregulation on the Surface of Primary Human Endothelial Cells: Role of the Antioxidant 7,8-Dihydroxy-4-methylcoumarin in Preventing HSP90 Exposure to the Immune System

2018 ◽  
Vol 2018 ◽  
pp. 1-9 ◽  
Author(s):  
Elisabetta Profumo ◽  
Brigitta Buttari ◽  
Lavinia Tinaburri ◽  
Daniela D’Arcangelo ◽  
Maurizio Sorice ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Endothelial Cells ◽  
Umbilical Vein ◽  
Protein A ◽  
Heat Shock Protein 90 ◽  
Surface Expression ◽  
Enzyme Linked Immunosorbent Assay ◽  
Protein Levels ◽  
Umbilical Vein Endothelial Cells

We have previously demonstrated that human heat shock protein 90 (HSP90), an intracellular self protein, is the target of cellular and humoral autoimmune responses in patients with carotid atherosclerosis. In this study, we evaluated in vitro whether oxidative stress, a feature of atherosclerotic plaque, alters HSP90 expression in endothelial cells, thus inducing surface localization of this molecule and whether the antioxidant compound 7,8-dihydroxy-4-methylcoumarin (7,8-DHMC) is able to prevent oxidative stress-induced alterations of HSP90 localization. By the use of flow cytometry, immunofluorescence, enzyme-linked immunosorbent assay, and semiquantitative reverse-transcription polymerase chain reaction, we demonstrated that exposure of human umbilical vein endothelial cells (HUVEC) to the prooxidant compound H2O2 upregulated HSP90 surface expression and reduced its secretion without altering HSP90 gene expression and intracytoplasmic protein levels. Pretreatment of HUVEC with 7,8-DHMC prevented H2O2-induced alterations of HSP90 cellular distribution and secretion. Our results suggest that the strong oxidative conditions of atherosclerotic plaques promote the upregulation of HSP90 surface expression on endothelial cells, thus rendering the protein a possible target of autoimmune reactions. The antioxidant 7,8-DHMC, by preventing oxidative-stress-triggered HSP90 surface upregulation, may be useful to counteract possible autoreactive reactions to HSP90.

scholarly journals Apoptotic Vascular Endothelial Cells Become Procoagulant

Blood ◽  
1997 ◽  
Vol 89 (7) ◽  
pp. 2429-2442 ◽  
Author(s):  
Thomas Bombeli ◽  
Aly Karsan ◽  
Jonathan F. Tait ◽  
John M. Harlan
Keyword(s):  
Endothelial Cells ◽  
Kinase Inhibitor ◽  
Vascular Endothelial Cells ◽  
Umbilical Vein ◽  
Annexin V ◽  
Vascular Endothelial ◽  
Flow Cytometric ◽  
Heparan Sulfates ◽  
Umbilical Vein Endothelial Cells ◽  
Membrane Components

Abstract Whereas unperturbed endothelial cells provide potent anticoagulant properties, exposure to inflammatory and atherogenic stimuli can rapidly lead to a procoagulant behavior. Because recent studies provide evidence that apoptosis of vascular cells may occur under conditions such as atherosclerosis and inflammation, we investigated whether apoptotic endothelial cells may contribute to the development of a prothrombotic state. In this report, it is shown that both adherent and detached apoptotic human umbilical vein endothelial cells (HUVECs) become procoagulant. Apoptosis was induced by staurosporine, a nonspecific protein kinase inhibitor, or by culture in suspension with serum deprivation. Both methods resulted in similar findings. As assessed by flow cytometric determination of annexin V binding, HUVECs undergoing cell death exhibited typically a more rapid exposure of membrane phosphatidylserine (PS) than DNA fragmentation. Depending on the stage of apoptosis, this redistribution of phospholipids was found to induce an increase of the activity of the intrinsic tenase complex by 25% to 60%. Although apoptotic cells did not show antigenic or functional tissue factor (TF ) activity, when preactivated with lipopolysaccharide, TF procoagulant activity increased by 50% to 70%. At 8 hours after apoptosis induction, antigenic thrombomodulin, heparan sulfates, and TF pathway inhibitor decreased by about 83%, 80%, and 59%, respectively. The functional activity of these components was reduced by about 36%, 52%, and 39%, respectively. Moreover, the presence of apoptotic HUVECs led to a significant increase of thrombin formation in recalcified citrated plasma. In conclusion, apoptotic HUVECs, either adherent or in suspension, become procoagulant by increased expression of PS and the loss of anticoagulant membrane components.

scholarly journals 17β-estradiol upregulates striatin protein levels via Akt pathway in human umbilical vein endothelial cells

PLoS ONE ◽  
2018 ◽  
Vol 13 (8) ◽  
pp. e0202500 ◽  
Author(s):  
Shuhui Zheng ◽  
Peng Sun ◽  
Haimei Liu ◽  
Runmei Li ◽  
Lingli Long ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Umbilical Vein ◽  
Akt Pathway ◽  
Human Umbilical Vein ◽  
Protein Levels ◽  
17Β Estradiol ◽  
Umbilical Vein Endothelial Cells

scholarly journals Role of thiosulfate in hydrogen sulfide-dependent redox signaling in endothelial cells

2017 ◽  
Vol 313 (2) ◽  
pp. H256-H264 ◽  
Author(s):  
Anna Leskova ◽  
Sibile Pardue ◽  
John D. Glawe ◽  
Christopher G. Kevil ◽  
Xinggui Shen
Keyword(s):  
Endothelial Cells ◽  
Hydrogen Sulfide ◽  
Umbilical Vein ◽  
Culture Conditions ◽  
Redox Signaling ◽  
Endothelial Cell Proliferation ◽  
Protein Levels ◽  
Hypoxic Conditions ◽  
High Doses ◽  
Umbilical Vein Endothelial Cells

Recent reports have revealed that hydrogen sulfide (H2S) exerts critical actions to promote cardiovascular homeostasis and health. Thiosulfate is one of the products formed during oxidative H2S metabolism, and thiosulfate has been used extensively and safely to treat calcific uremic arteriopathy in dialysis patients. Yet despite its significance, fundamental questions regarding how thiosulfate and H2S interact during redox signaling remain unanswered. In the present study, we examined the effect of exogenous thiosulfate on hypoxia-induced H2S metabolite bioavailability in human umbilical vein endothelial cells (HUVECs). Under hypoxic conditions, we observed a decrease of GSH and GSSG levels in HUVECs at 0.5 and 4 h as well as decreased free H2S and acid-labile sulfide and increased bound sulfide at all time points. Treatment with exogenous thiosulfate significantly decreased the ratio of GSH/GSSG to total sulfide of HUVECs under 0.5 h of hypoxia but significantly increased this ratio in HUVECs under 4 h of hypoxia. These responses reveal that thiosulfate has different effects at low and high doses and under different O2 tensions. In addition, treatment with thiosulfate also diminished VEGF-induced cystathionine-γ-lyase expression and reduced VEGF-induced HUVEC proliferation under both normoxic and hypoxic conditions. These results indicate that thiosulfate can modulate H2S metabolites and signaling under various culture conditions that impact angiogenic activity. Thus, thiosulfate may serve as a unique sulfide donor to modulate endothelial responses under pathophysiological conditions involving angiogenesis. NEW & NOTEWORTHY This report provides new evidence that different levels of exogenous thiosulfate dynamically change discrete sulfide biochemical metabolite bioavailability in endothelial cells under normoxia or hypoxia, acting in a slow manner to modulate sulfide metabolites. Moreover, our findings also reveal that thiosulfate surprisingly inhibits VEGF-dependent endothelial cell proliferation associated with a reduction in cystathionine-γ-lyase protein levels.

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