Four weeks old heterozygous Fbn1C1039G/+ male animals were treated with TGFβ neutralizing antibody (anti-TGFβ, clone 1D11, Bio X Cell) or isotype (IgG, clone MOPC21, Bio X Cell) as previously shown [25 (link)]. One mg anti-TGFβ or isotype reagent was injected intraperitoneally on the first day, and then 200 μg was injected intraperitoneally every other day for 13 times. The ultrasound imaging of aortic root and the abdominal aorta were performed every week as described below. Aortic superoxide production and eNOS uncoupling activity were determined after 4 weeks injection as described below.
For in vitro experiments, male origin human aortic endothelial cells (HAECs) were isolated from male donor after obtaining permission for research applications by informed consent (Lonza, Walkersville, MD, USA). HAECs were cultured in EGM-2 media (EBM-2 basal medium with supplement pack, all reagents from Lonza (Walkersville, MD, USA). Cells of passages 4 to 6 were starved in EBM-2 basal medium overnight and then treated with 20 μg/mL anti-TGFβ (clone 1D11, Bio X Cell, West Lebanon, NH, USA) or IgG (clone MOPC21, Bio X Cell) for 24 h, prior to determination of protein expression levels of GTPCHI using Western blotting.
For in vitro experiments, male origin human aortic endothelial cells (HAECs) were isolated from male donor after obtaining permission for research applications by informed consent (Lonza, Walkersville, MD, USA). HAECs were cultured in EGM-2 media (EBM-2 basal medium with supplement pack, all reagents from Lonza (Walkersville, MD, USA). Cells of passages 4 to 6 were starved in EBM-2 basal medium overnight and then treated with 20 μg/mL anti-TGFβ (clone 1D11, Bio X Cell, West Lebanon, NH, USA) or IgG (clone MOPC21, Bio X Cell) for 24 h, prior to determination of protein expression levels of GTPCHI using Western blotting.