Huvecs

The source and culture of RAW264.7 cells and fresh bone marrow cells (BMDMs) were the same as described in a previous paper [47 (link)]. HUVECs (Sciencell, Carlsbad, CA, USA) were cultured in endothelial cell medium (ECM, Sciencell, Carlsbad, CA, USA) containing 10% FBS, 1% endothelial cell growth supplement (ECGS), 1% penicillin and streptomycin, and maintained in a humidified atmosphere with 5% CO₂ at 37 °C. After the density of the HUVECs reached 80%, the cells were incubated in serum-free medium for 24 h prior to the cell experiments.
RAW264.7 cells, BMDMs, and HUVECs were divided into the CON group, ALD group (10⁻⁷ mol/L aldosterone was administered for 24 h), and ESA group (cells were pretreated with 10⁻⁶ mol/L esaxerenone 2 h prior to aldosterone treatment). In some experiments, HUVECs were induced with 50 ng/mL VEGFA (Proteintech, Wuhan, China, Cat#: HZ-1038) and treated with or without the VEGFA receptor blocker bevacizumab (4 μg/mL) (MCE, Shanghai, China, Cat#: HY-P9906), and HUVECs were treated with bevacizumab after aldosterone induction. HUVECs were induced with 10 ng/mL TGF-β1 (MCE, Shanghai, China, Cat#: HY-P70543) and treated with or without the TGF-β1 receptor blocker LY2109761 (2 × 10⁻⁶ mol/L) (MCE, Shanghai, China, Cat#: HY-12075), and HUVECs were treated with LY2109761 after aldosterone induction.

HEK was isolated from circumcised foreskins of adolescent volunteers as described previously [45 (link), 46 (link)] and cultured in Keratinocyte Growth Medium 2 supplemented SupplementPack (Promo Cell, Germany). The HaCaT cells and HUVECs were purchased from Procell (Wuhan, China) and cultured in Roswell Park Memorial Institute 1640 (RPMI-1640) and Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin solution. To model psoriasis, cells were stimulated with recombinant human IL17 (Sino Biological, China) and/or TNF-α (Proteintech, USA) in serum-free medium for 24 h. To prepare HaCaT culture supernatant for culturing HUVECs, half of the medium was replaced with fresh serum-free medium after 16 h of cytokine treatments. The supernatant was collected 8 h later and centrifuged at 3,000 rpm for 10 min.

Human umbilical vein endothelial cells (HUVECs) were purchased from Cyagen (#HUVEC-2001). The HUVECs were cultured in a 1% gelatin coated plate in complete endothelial cell medium (ECM, #1001, ScienCell) containing 5% FBS, and endothelial growth factor and were maintained in a humidified chamber with 5% CO₂ at 37°C. Human umbilical vein smooth muscle cells (HVSMCs) were obtained from Otwo Biotech (#HTX2305) and maintained in DMEM supplemented with 10% FBS and 1% P/S. To establish the EndMT model, HUVECs were stimulated with 10 ng/ml TGF-β2 (#100-35B, Proteintech) in conditioned medium supplemented with 2.5% FBS for four consecutive days. For DSY application, HUVECs pretreated with TGF-β2 for 2 days were incubated with different doses of DSY for a further 2 days.