Epigallocatechin gallate (egcg)

The purity of all standards was >98%. Gallic acid, hydroxycinnamic acids (chlorogenic, caffeic, p-coumaric, and ferulic acids), the flavones luteolin, apigenin, luteolin-7-O-glucoside, and apigenin-7-O-glucoside, the flavonols quercetin (hydrate) and quercetin-3-O-rutinoside (rutin hydrate), the flavanones naringenin and diosmetin, the flavanols (±)-catechin (hydrate), (−)-epigallocatechin, (−)-epicatechin, and their gallate derivatives [(−)-catechin gallate, (−)-epicatechin gallate and (−)-epigallocatechin gallate], and the isoflavone daidzein were from Cayman Chemical Co. (Ann Arbor, MI, USA). The flavone diosmetin was from Extrasynthese Co. (Lyon, France). Methanol Chromasolv for HPLC was from Sigma Chemical (St. Louis, MO, USA).

Unless otherwise stated, the reagents used in this study were from Sigma Aldrich (Poole, Dorset, UK). Sodium citrate-containing vacutainers were from Greiner Bio-One. Paraformaldehyde solution was from Alfa Aesar. Epigallocatechin gallate (EGCG), epicatechin gallate (ECG), catechin gallate (CG), epicatechin (EC), catechin (C), gallocatechin gallate (GCG), epigallocatechin (EGC) and gallocatechin (GC) were from Cayman Chemicals (Michigan, USA).

iPSC cardiomyocytes at day 6 post initial plating were transfected with 100 ng plasmid DNA using ViaFect transfection reagent (E4981, Promega) and the following human DYRK1A and SRSF6 expression constructs: DYRK1A human clone (NM_001396, SC314641, Origene), DYRK1A human tagged ORF Clone (NM_001396, RG212584, Origene), and SRSF6 cDNA ORF clone N-HA tag (HG19052-NY, Sinobiological). Control cultures were transfected with ~ 100 ng of empty PCMV6XL5 vector (Origene).
Drug treatments were initiated 24 h post-transfection with expression constructs. Daunorubicin (DAU, 14159, Cayman Chemical) and epigallocatechin gallate (EGCG, 709035, Cayman Chemical) were added to culture media for a total incubation time of 14 h. DMSO vehicle was added to controls. After treatments with DAU, iPSC cardiomyocytes were washed with PBS, and incubated in fresh culture medium, or medium supplemented with EGCG for 24 h. Cell viability was determined with the CellTiter-Glo luminescent viability kit (G7570, Promega).

Human umbilical vein endothelial cells (HUVECS), lineage EA.hy 926, obtained from the Rio de Janeiro Cell Bank (BCRJ, Duque de Caxias, RJ, Brazil), were used in this study. The cells were cultivated in culture flasks at 37 °C with 5% CO₂ using a medium supplemented with 10% (v/v) fetal bovine serum (FBS), 50 μg/mL of penicillin, 100 μg/mL of streptomycin and 0.5 μg/mL of amphotericin B until they reached 80–90% confluency. For the experiments, the EC were seeded in culture plates in the same medium until they reached the necessary confluency. Subsequently, the culture medium was removed, and the cells were washed with PBS and incubated in culture medium without FBS and with 10% (v/v) of HP and PE plasma for 24 h at 37 °C in 5% CO₂. In the treated cells, the same procedures were performed, but in the presence or absence of EGCG (Cayman Chemical^®, Ann Arbor, MI, USA) (100 μM) during the last 60 min of incubation and L-NAME (Cayman Chemical^®, Ann Arbor, MI, USA) (100 μM), NOS-inhibiting substance, and LY294002 (Cayman Chemical^®, Ann Arbor, MI, USA) (30 μM), a PI3K inhibitor, 30 min before incubation with plasma. These procedures were performed prior to all experiments described below.

To assess the impact of shear stress on endothelial cells, EC were cultured and seeded on modified 100 mm culture plates. These plates had a 60 mm plate attached to the center and had been previously sterilized. The culture plates were maintained at 37 °C with 5% CO₂ until the cells adhered and reached the desired confluence. Subsequently, laminar fluid flow was applied to the culture plates using the AO-330D shaking equipment (Gehaka, São Paulo, SP, Brazil) at a predetermined rotation frequency. This generated a constant shear stress ranging from 6–40 dynes/cm² (equivalent to rotations per second: 199 rpm), which represented physiological conditions. After 48 h in the shaker, the cells were incubated with the PE plasma and rotated again for 24 h, totaling 72 h of rotation. EGCG (Cayman Chemical^®, Ann Arbor, MI, USA) (100 μM) was added in the last 60 min. After this period, the NO production was assessed by detecting nitrite levels in the culture supernatant. Additionally, an iron reduction (as described above) was conducted to analyze the antioxidant capacity of the supernatant.

Phenylephrine, acetylcholine chloride, sodium nitrite, sodium nitroprusside, and Tween-20 were purchased from Sigma Chemicals (St Louis, MO, USA). Angiotensin II was purchased from Sigma Chemicals (USA). EGCG was purchased from Cayman. NaCl was purchased from Calbiochem® Merck (Darmstadt, Germany). MgSO₄, KCl, KH₂PO₂, glucose and CaCl₂ were purchased from BDH Laboratory Supplies (Poole, UK). Bovine serum albumin (BSA) was purchased from Santa Cruz (Dallas, Texas, USA). All compounds were dissolved in deionized water. The concentrations stated are expressed as final molar concentrations in the buffer.