Haecs

Manufactured by Merck Group

Sourced in United Kingdom

HAECs, or Human Aortic Endothelial Cells, are primary cells derived from the aortic tissue of human donors. These cells are used as an in vitro model to study the biology and function of endothelial cells, which line the interior of blood vessels. The core function of HAECs is to provide a physiologically relevant cell culture system for researchers to investigate endothelial cell processes, such as angiogenesis, vascular permeability, and response to stimuli.

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8 protocols using haecs

Cholesterol Modulation in Endothelial Cells

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HAECs were purchased from Lonza Group Ltd. (Material number: ml-2535) and were grown in M199 supplemented with 15% fetal bovine serum, 2 mM l-glutamine (Gibco), 50 µg/mL heparin, and 30 µg/mL EC growth factor (Becton Dickinson) in a 1% gelatin-coated tissue culture flask. The cells used in the experiments in this study were in the 7th and 10th passages. All the experiments were approved by the Ethics Committee of the University of Tokyo, Graduate School of Medicine.
Cholesterol reduction was induced by incubating HAECs with 10 mM methyl-β-cyclodextrin (MβCD, Sigma-Aldrich) dissolved in Hanks’ Balanced Salt solution (HBSS, Sigma-Aldrich) for 30 min at 37°C. Cholesterol enrichment was achieved by incubating HAECs with a complex of cholesterol (Sigma-Aldrich) and MβCD (1:7 molar ratio) dissolved in the complete culture medium for 6 h at 37°C. The final concentration of cholesterol was 100 µM. To inhibit the mitochondrial respiratory chain complex I, HAECs were treated with 5 µM rotenone (Sigma-Aldrich) dissolved in HBSS for 10 min at 37°C.

Yamamoto K., Shimogonya Y., Maeno R., Kawabe K, & Ando J. (2023). Endothelial cells differentially sense laminar and disturbed flows by altering the lipid order of their plasma and mitochondrial membranes. American Journal of Physiology - Cell Physiology, 325(6), C1532-C1544.

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Culturing Human Aortic Endothelial Cells

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HAECs were purchased (Invitrogen) and expanded in Medium 200 with low-serum growth supplement from the same supplier, 5% FBS, and 1% penicillin-streptomycin. Standard plastic tissue culture flasks were coated with 5 μg/ml fibronectin (Sigma-Aldrich, St. Louis, MO) for 2 h at 37°C, before seeding with HAECs. HAECs were detached from cultures at ~70% con-fluency with 0.05% trypsin/0.53 mM EDTA for subsequent plating.

Tran-Nguyen T.K., Chandra D., Yuan K., Patibandla P.K., Nguyen K.T., Sethu P., Zhang Y., Xue J., Mobley J.A., Kim Y.I., Shoushtari A., Leader J.K., Bon J., Sciurba F.C, & Duncan S.R. (2020). Glucose-Regulated Protein 78 Autoantibodies Are Associated with Carotid Atherosclerosis in Chronic Obstructive Pulmonary Disease Patients. ImmunoHorizons, 4(2), 108-118.

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Culturing Human Aortic Endothelial Cells

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Aortic Endothelial Cell Stimulation Assay

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Human aortic endothelial cells (HAECs) were purchased from Lonza (Basel, Switzerland) and cultured according to the manufacturer’s instructions. All experiments were performed using HAECs between the 2^th and the 5^th passage. HAECs were treated with: LPS (Sigma Aldrich, St. Louis, MO) at a concentration of 100 ng/mL for 6 hours; ox-PAPC (oxidation products of 1-palmitoyl-2-arachidonoyl-sn-glycerol-3-phosphatidylcholine, Hycult Biotech, Uden, The Netherlands), an antigenic epitope of oxidized LDL, at a concentration of 100 µg/mL for 6 hours; or long chain free fatty acids (FFA, oleic acid 500 µM+palmitic acid 500 µM) for 24 hours. Cells were incubated with 10 nM Eritoran for 30 minutes prior to treatments where indicated. Eritoran (Eisai Inc., Woodcliff Lake, NJ) is a synthetic analog of lipid A and a potent and specific antagonist of LPS action, which inhibits lipid A binding to MD2 and terminates MD2/TLR4-mediated signaling [17] (link). At the end of treatments, HAECs where collected and used for molecular analysis.

Menghini R., Campia U., Tesauro M., Marino A., Rovella V., Rodia G., Schinzari F., Tolusso B., di Daniele N., Federici M., Zoli A., Ferraccioli G, & Cardillo C. (2014). Toll-Like Receptor 4 Mediates Endothelial Cell Activation Through NF-κB but Is Not Associated with Endothelial Dysfunction in Patients with Rheumatoid Arthritis. PLoS ONE, 9(6), e99053.

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Culturing Endothelial Cells for Glycocalyx Research

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Human umbilical vein endothelial cells (HUVECs) and human aortic endothelial cells (HAECs) were purchased from Lonza (Slough, UK) and grown in medium 199 (Invitrogen, Paisley, UK) supplemented with foetal bovine serum (10%),

β

-endothelial cell growth factor (1 ng/ml), bovine neural extract (

3 μ g / ml

), thymidine (

1.25 μ g / ml

), heparin (10 U/ml), penicillin (100 U/ml) and streptomycin (

100 μ g / ml

). All supplements were purchased form Sigma. Before experiment, endothelial cells were collected by incubating with trypsin-EDTA solution (0.5%, Sigma, Dorset, UK) for 5 min and then plated on a glass, non-coating coverslip at a density of

2500 cells / {cm}^{2}

(HUVECs) and

6000 cells / {cm}^{2}

(HAECs). They were further cultured for a week, allowing full recovery of the glycocalyx on the cell surface (here defined as an intensive, continuous FITC-WGA binding layer) (Bai and Wang 2012 (link)).

Li W, & Wang W. (2017). Structural alteration of the endothelial glycocalyx: contribution of the actin cytoskeleton. Biomechanics and Modeling in Mechanobiology, 17(1), 147-158.

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Aortic Endothelial Cell Cytokine Interactions

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Human aortic endothelial cells (HAECs) were purchased from Lonza (Chicago, IL) and cultured in EGM-2 medium supplemented with 2% fetal bovine serum but without antibiotics. On the day before the study, the fetal bovine serum concentration was reduced to 1%. Potential interplay between Ang II, IL17A and TNFα was tested in HAECs treated for 24 hours with Ang II (Sigma, A9525), TNFα (Thermo Scientific, 1857619) and/or IL17A (Ebioscience, 14-8171).

Itani H., Dikalova A., McMaster W., Nazarewicz R., Bikineyeva A., Harrison D, & Dikalov S. (2016). Mitochondrial Cyclophilin D in Vascular Oxidative Stress and Hypertension. Hypertension, 67(6), 1218-1227.

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Cholesterol and TMEM16A Expression in HAECs

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HAECs were obtained from ScienCell, USA. HAECs were cultured in DMEM (Dulbecco’s Modified Eagle Medium, HyClone) supplemented with 10% FBS (fetal bovine serum, Biological Industries, Israel) and 1% penicillin–streptomycin in a humid incubator (37℃, 5% CO₂). To examine the effect of cholesterol or MβCD on TMEM16A expression, HAECs were treated with water-soluble cholesterol (0–25 μM; Sigma-Aldrich) or MβCD (10 mM; Sigma-Aldrich) for 0–48 h.

Ma K., Liu S., Liang H., Wang G., Wang T., Luo S., Gao K., Wang H., Liu M., Bai L, & Xiao Q. (2020). Ca2+-activated Cl− channel TMEM16A inhibition by cholesterol promotes angiogenesis in endothelial cells. Journal of Advanced Research, 29, 23-32.

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Oxalate Exposure in Aortic Cells

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Primary human aortic endothelial cells (HAECs) and human aortic smooth muscle cells (HASMCs) were purchased from Science Cell Research Laboratories (Carlsbad, CA, USA) and cultured as previously described [23] . In brief, HAECs were grown in endothelial culture medium (No. 1001, Science Cell) containing 5% fetal bovine serum (No. 0025), 1% endothelial cell growth supplement (No. 1052), and 1% penicillin/streptomycin solution (No. 0503) at 37°C with 5% CO 2 . All experiments were performed on HAECs between passages 2 and 5. HASMCs were cultured in smooth muscle cell medium (Cat. #1101) supplemented with 2% fetal bovine serum, 1% SMCGS, and 1% penicillin and streptomycin. Experiments were performed using cells from passages 3-6. HAECs and HASMCs were treated with normal medium, 100 μmol/L oxalate, 200 μmol/L oxalate, or and 500 μmol/L oxalate for 6 h or 24 h. All other reagents were obtained from Sigma.

Sun K., Tang X., Song S., Gao Y., Yu H., Sun N., Wen B, & Mei C. (2021). Hyperoxalemia Leads to Oxidative Stress in Endothelial Cells and Mice with Chronic Kidney Disease. Kidney & blood pressure research, 46(3).

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