Shear Stress-Induced Microstructure Remodeling of Human Bicuspid Aortopathy Revealed by Nonlinear Optical Microscopy

Congenital defects and abnormal hemodynamics are widely accepted theories in bicuspid aortic valve (BAV)-associated aortopathy. However, current clinical criteria for surgical intervention in BAV patients fall short of direct correlation with neither factor. Here, we applied nonlinear optical (NLO) microscopy to investigate the microstructural changes of the aortic wall associated with bicuspid aortopathy at different stages. Fresh aortic specimens were collected from 24 study patients and assessed by NLO scanning layer-by-layer to probe the distribution of the extracellular matrix and smooth muscle cells (SMCs). NLO images demonstrated near-perfect concordance with traditional histology (Masson's and Weigert's staining), affirming its reliability for histologic assessment. In BAV-associated aortic dilatation (BAV-AD), collagen fibers exhibited better alignment with a helix-like pattern compared to tricuspid aortic valve (TAV) patients, while their content was markedly reduced relative to nondilated BAV (BAV-NA) cases. Notably, both collagen and elastin fibers in BAV patients displayed more ordered alignment in regions subjected to high wall shear stress than in low wall shear stress regions. Quantitative analysis of fiber anisotropy and relative abundance revealed distinct interpatient and spatial variations in collagen organization, underscoring the complex biomechanical landscape associated with aortic remodeling. Our results demonstrate that rapid and precise tomography of aortic architecture could be realized by NLO microscopy, serving as a potential histological marker to guide surgical intervention in bicuspid aortopathy.