Distinct Expression of Nonmuscle Myosin IIB in Pulmonary Arteries of Patients With Aortic Stenosis vs Insufficiency Undergoing a Ross Procedure

The ascending aorta of male hypertensive bicuspid aortic valve patients preferentially associated with a cellular aneurysmal phenotype

“…Transthoracic and transesophageal echocardiograms were used to determine the phenotype and pathology of the aortic valve, the absolute diameter of the aortic annulus and ascending aorta. (Devereux et al, 1997 ; Emmott et al, 2021 ) All echocardiograms were performed and analyzed by American board‐certified anesthesiologists. In the male BAV cohort, 12 normotensive and hypertensive male BAV patients were associated with right‐left coronary cusp fusion whereas four normotensive and three hypertensive patients had a right‐non coronary artery cusp fusion.…”

“…Vascular tissue sections (6–8 μm thick) of the ascending aorta were stained using Masson's trichrome and Movat's pentachrome protocols to assess collagen and elastin levels, respectively (Emmott et al, 2021 ; Tardif et al, 2015 ). Within each tissue, five arbitrary regions (area; ~0,31 mm 2 ) were examined at 20× magnification to avoid biased sampling of the immunohistochemical analysis The surface area of blue‐stained collagen and black‐stained elastin fibers were measured using Image Pro Software to assess the average % of collagen and elastin levels.…”

“…Protein lysates were prepared from the ascending aorta of BAV patients and subjected to Western blot analysis (Emmott et al, 2021 ; Tardif et al, 2015 ). Antibodies used were mouse monoclonal anti‐calponin (1:5000; Santa Cruz Biotechnology); mouse monoclonal anti‐smooth muscle 22α (1:5000; Santa Cruz Biotechnology), rabbit polyclonal anti‐non‐muscle myosin IIB (1:5000; Cell Signaling Technologies), rabbit polyclonal anti‐matrix metalloproteinase‐2 (MMP‐2; recognizes proenzyme/cleaved enzyme, 1:5000; Cell Signaling Technologies), mouse monoclonal anti‐matrix metalloproteinase‐9 (MMP‐9, recognizes proenzyme, 1:1000; Thermo Fisher Scientific), tissue inhibitor of metalloproteinase‐1 (1:100; Santa Cruz Biotechnology), chicken polyclonal anti‐nestin (1:1000; Abcam), rabbit polyclonal anti‐p27 kip1 (1:2000; Santa Cruz Biotechnology), mouse monoclonal anti‐p21 cip1 (1:500; Santa Cruz Biotechnology), mouse monoclonal anti‐p53 (1:500; Santa Cruz Biotechnology), and a mouse monoclonal anti‐glyceraldehyde phosphate dehydrogenase (GAPDH, 1:50000; Ambion).…”

“…Thereafter, the appropriate secondary antibody‐conjugated to horseradish peroxidase (1:20,000; Jackson Immunoresearch) was added and bands visualized utilizing the ECL detection kit (Perkin Elmer). Films were scanned with Image J software ® and the target protein signal was depicted as arbitrary light units normalized to GAPDH (Emmott et al, 2021 ; Tardif et al, 2015 ).…”

“…Ascending aortic tissue sections (6–8 μm thick) were fixed and subjected to immunofluorescence (Emmott et al, 2021 ; Tardif et al, 2015 ). Tissues were incubated with chicken polyclonal anti‐nestin (1:100; Abcam) and rabbit monoclonal anti‐non‐muscle myosin IIB (1:100; Abcam).…”