(6) Immunohistological Evaluation. Tissue staining was performed on transversal sections of human aortic valve leaflets and analyzed as described (Matilla L et al Sex-Related Signaling of Aldosterone/Mineralocorticoid Receptor Pathway in Calcific Aortic Stenosis. Hypertension 79: 1724-1737, 2022). All grossly calcified valves were decalcified in 10% formic acid solution (Sigma) for 24 h. Samples were dehydrated, embedded in paraffin, and cut in 5-μm-thick sections. Immunohistochemistry was performed following the protocol of Leica BOND-Polymer Re-fine Detection automatic immunostainer (Leica). All solutions were filled into the bottle-Bond Open Container (Leica) and registered on computer using the Leica Biosystem program. The immunostaining program protocol includes Fixative solution, Bond wash solution, Blocking with common immunohistochemistry blocker, and incubation with the primary antibody for ICAM-1 or VCAM-1 (Santa Cruz Biotechnology). Secondary antibodies poly-HRP-anti-mouse or poly-HRP-anti-rabbit IgG were used as appropriate. Positive immunoreactive signal was developed using an enhanced 3,3’-diaminobenzidine (DAB) system (Leica). Slides were counterstained with hematoxylin and mounted using DPX mounting media (Merck/Sigma-Aldrich). Imaging was performed using a Leica microscope at 50X and 400X magnification.

Ministerio de Ciencia e Innovacion (MICIN)/Agencia Estatal de Investigación (AEI)/10.13039/501100011033 [PID2020-113751RB-I00, PID2024-160809OB-I00]; Junta de Castilla y León [VA175P20, Gerencia Regional de Salud GRS2205/A/2020, and Programa Estratégico Instituto de Biomedicina y Genética Molecular-Escalera de Excelencia Ref. CLU-2019-0]; Fundación Eugenio Rodríguez Pascual FERP-2024-60. Instituto de Salud Carlos III [CIBER de Enfermedades Cardiovasculares (CIBERCV) CB16/11/00260, CB16/00483]. Ministerio de Ciencia, innovación y Universidades and Fondo Europeo de Desarrollo Regional (FEDER) [EQC2019-006686-P]; Junta de Castilla y Léon, Valladolid University, and FEDER [IR2020-1-UVA05]. European Union's Recovery and Resilience Facility-Next Generation, in the framework of the General Invitation of the Spanish Government’s public business entity Red.es to participate in talent attraction and retention programs within Investment 4 of Component 19 of the Recovery, Transformation and Resilience Plan. We acknowledge support from CSIC Network on Metabolic Diseases (COMETA) funded by the Consejo Superior de Investigaciones Científicas (CSIC), Spain. PhD fellowships from Valladolid University-Banco de Santander.

(1) Real-time metabolic analysis of human valve cells- It was performed in an Agilent Seahorse XFe24 metabolic analyzer (Agilent Technologies, Santa Clara, CA) using Cell Mito Stress and ATP assay kits. In total, 40,000 cells of endothelial valve cells or 30,000 cells of interstitial valve cells were seeded in a Seahorse XF24 Cell Culture Microplate and allowed to attach overnight. Then, cells were activated as indicated for 24 h. Before assay, media were replaced by Seahorse XF RPMI Medium, pH 7.4, supplemented with 2 mmol/L L-glutamine, 25 mmol/L D-glucose, and 1 mmol/L pyruvate. Cells were incubated for at least 45 minutes at 37°C in a non-CO2 incubator before the analysis. Data were analyzed with Seahorse Analytics software and normalized to the number of cells, evaluated by Hoechst staining in a Cytation 5 multimode reader (BioTek, Winooski, VT) at the end of the experiment. Data describe the indicated biological replicates performed in triplicate.

(5) Monocyte-valve endothelial cell (VEC) adhesion assays.VECs monolayers cultured in a 6-well plate were pre-incubated with metabolic modulators and then activated in activation media for 24 h under static conditions. In parallel, a monocytic cell line, THP-1 (ATCC®; Middlesex, UK Ref. TIB-202TM), was cultured in RPMI-1640 medium supplemented with 1% L-glutamine and 10% inactivated fetal bovine serum. For the adhesion assay, 1 x 106 cells/well of THP-1 in HBSS containing Ca2+ and Mg2+, were added to VECs monolayers and incubated for 7 min at 37oC. After two washes with HBSS, microphotographs of 16 sections were taken using a phase-contrast microscope (Nikon Eclipse TS100) connected to a digital camera. The number of adhered THP-1 cells was quantified using Image J software. Data were expressed as total number of THP-1cells adhered to the area of each section (mm2).

(3) Western blot assays for the immunodetection of proteins. Whole cell lysates were analyzed by separated by SDS-PAGE, 10% gels. Blots were incubated with primary antibodies recognizing the human form of intercellular adhesion molecule (ICAM)-1, vascular cell adhesion molecule (VCAM)-1. Data was normalized using antibodies recognizing the housekeeper gene β-tubulin. Densitometer FX and Quantity one software was used to acquire densitometry data for each blot. Images were exported to TIFF and the analysis of the band of interest was performed using Fiji software. Densitometry data were presented as arbitrary units normalized to β-tubulin. Data refer to the indicated biological replicates.

(2) Reversed transcriptase quantitative PCR (RT-PCR) for the gene expression analysis of several genes involved in metabolism and inflammation. Total RNA was extracted with Tri-Reagent and analyzed by RT–PCR. in a LightCycler480 (Roche Diagnostics) using KAPA SYBR FAST master mix kit (Merck Millipore). Transcript levels were expressed as relative to the housekeeping gene value, GAPDH (2−ΔCt, Ct=cycle threshold value). Data describe the number of biological replicates performed in duplicate.

(4) Quantitation of proinflammatory proteins secretion by ELISA It was detected in the supernatants of cells activated for 24 h by immunoassay kits following the manufacturer´s procedures: IL-6 (Diaclone, Besançon, France, # 950.030.048) and PGE2 (Arbor assay Inc, Arbor Assay, MI). The absorbance at 450 nm was measured using a microplate reader (Versamax, Molecular Devices), and data were normalized to total protein content measured by the bicinchoninic acid/BCA method.

Datasets of Figures 1-7 corresponding to a manuscript Glycolysis and hexosamine biosynthesis pathways in valve cells Sánchez-Bayuela T et al 2025

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