Heparin

Human umbilical vein endothelial cells (HUVECs) were purchased from ATCC (Manassas, VA, USA) and cultured in F-12K Medium (Thermo Fisher Scientific, Waltham, MA, USA) supplemented with 10% FBS (GIBCO, Grand Island, NY, USA), 0.1 mg/mL heparin (Selleck Chemicals, Houston, TX, USA), and 30 μg/mL Endothelial Cell Growth Supplement (Thermo Fisher) at 37°C in a 5% CO₂ atmosphere.
To explore the effects of bleomycin (MedChem Express, NJ, USA) and Astragaloside IV (AS-IV, Solarbio, Beijing, China) on lipid metabonomics in HUVECs, the cells were treated with bleomycin (50 μM) to induce cell senescence; after which, they were coincubated with AS-IV (50 μM) and bleomycin to explore the effect of AS-IV. At the same time, the effect of different doses of bleomycin on LPC levels in HUVECs was detected. The cells were treated with different concentrations of LPC (0.1 μM, 0.25 μM, and 0.4 μM, Merck Millipore, Burlington, MA, USA) to examine the influence of LPC on cell senescence and damage. To further explore how AS-IV functions in the LPC-induced ferroptosis of endothelial cells, the cells were coincubated with LPC (0.4 μM) and AS-IV (50 μM) with or without FIN56 (Selleck, 5 μM).

Vemurafenib (vem, PLX-4032, #S1267) was purchased from Selleckchem, Houston, TX, USA.
Heparin (Heparin sodium salt from porcine intestinal mucosa, #4784), Geneticin/G418 (G418 disulfate salt, #A1720) and puromycin (puromycin dihydrochloride, #P8833) were purchased from Sigma-Aldrich, St. Louis, MO, USA.
All drugs were diluted according to the manufacturer’s instructions.

A total of 20 consecutive patients with CSF were recruited with the approval of the Ethics Committee of The Fifth Affiliated Hospital of Xinjiang Medical University (XYDWFYLS-2019-08). At the same time, 20 contemporary patients with angiographically normal coronary flow were recruited as controls. The exclusion criteria were adopted from those in a previous study [21 (link)]. Written informed consents were obtained from all patients enrolled.
We collected fasting 10 ml peripheral blood samples from all the study participants in the morning. The peripheral blood mononuclear cells were isolated by Ficoll gradient centrifugation and then inoculated on to a culture plate coated with human fibronectin (BD, U.S.A.). The M199 medium supplemented with 20% foetal bovine serum (Thermo Fisher Scientific, Waltham, MA, U.S.A.), 30 μg/ml endothelial cell growth supplements (Sigma–Aldrich, St. Louis, MO, U.S.A.), 90 μg/ml heparin (Selleck Chemicals, Houston, TX, U.S.A.), and 1% antibiotics solution was changed every 3 days. The adherent cells were screened for markers of peripheral blood EPCs, 7 days later. We identified Dil-AcLDL and FITC-UEA-I (FITC-lectin) (Sigma–Aldrich) double-stained positive cells as differentiated EPCs by IF staining. The purity of isolated EPCs was determined by flow cytometry using anti-CD34 and anti-VEGFR antibodies.

Bortezomib and endocytosis inhibitors, including heparin, chlorpromazine, amiloride, dynasore, wortmannin, omeprazole, and genistein were purchased from Selleck Chemicals (Houston, TX, USA). 5-(N-Ethyl-Nisopropyl) amiloride (EIPA) was obtained from Cayman Chemical (Ann Arbor, MI, USA). Antibodies against flotillin-1 (Flot1, D2V7J, 18634T, 1:1000), glyceraldehyde-3-phosphate dehydrogenase (GAPDH, D16H11, 5174S, 1:1000), calreticulin (D3E6, 12238T, 1:1000), caveolin-1 (CAV-1, D46G3, 3267T, 1:1000), and clathrin heavy chain (CLTC, D3C6, 4796S, 1:1000) were purchased from (Cell Signaling Technology, Danvers, MA, USA). Antibodies against dynamin-2 (DNM2, EPR9053, ab151555, 1:1000) and CD9 (EPR2949, ab92726, 1:2000) were bought from Abcam (Cambridge, United Kingdom). Anti-human CD63 antibody (Ts63, 10628D, 1:250) was obtained from Thermo Fisher Scientific (Waltham, MA, USA). IRDye 680RD or 800CW goat anti-mouse/rabbit IgG secondary antibodies (1:10000) were purchased from LI-COR Biosciences (Lincoln, NE, USA).