Label-free optical biomarkers detect early calcific aortic valve disease in a wild-type mouse model
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RESEARCH ARTICLE
Open Access
Label‑free optical biomarkers detect early calcific aortic valve disease in a wild‑type mouse model Ishita Tandon1, Shelby Johns1, Alan Woessner1, Jessica Perez1, Delaney Cross1, Asya Ozkizilcik1, Timothy J. Muldoon1, Srikanth Vallurupalli2, Muralidhar Padala3, Kyle P. Quinn1 and Kartik Balachandran1*
Abstract Background: Calcific aortic valve disease (CAVD) pathophysiology is a complex, multistage process, usually diagnosed at advanced stages after significant anatomical and hemodynamic changes in the valve. Early detection of disease progression is thus pivotal in the development of prevention and mitigation strategies. In this study, we developed a diet-based, non-genetically modified mouse model for early CAVD progression, and explored the utility of two-photon excited fluorescence (TPEF) microscopy for early detection of CAVD progression. TPEF imaging provides label-free, non-invasive, quantitative metrics with the potential to correlate with multiple stages of CAVD pathophysiology including calcium deposition, collagen remodeling and osteogenic differentiation. Methods: Twenty-week old C57BL/6J mice were fed either a control or pro-calcific diet for 16 weeks and monitored via echocardiography, histology, immunohistochemistry, and quantitative polarized light imaging. Additionally, TPEF imaging was used to quantify tissue autofluorescence (A) at 755 nm, 810 nm and 860 nm excitation, to calculate TPEF 755–860 ratio (A860/525/(A755/460 + A860/525)) and TPEF Collagen-Calcium ratio (A810/525/(A810/460 + A810/525)) in the murine valves. In a separate experiment, animals were fed the above diets till 28 weeks to assess for later-stage calcification. Results: Pro-calcific mice showed evidence of lipid deposition at 4 weeks and calcification at 16 weeks at the valve commissures. The valves of pro-calcific mice also showed positive expression for markers of osteogenic differentiation, myofibroblast activation, proliferation, inflammatory cytokines and collagen remodeling. Pro-calcific mice exhibited lower TPEF autofluorescence ratios, at locations coincident with calcification, that correlated with increased collagen disorganization and positive expression of osteogenic markers. Additionally, locations with lower TPEF autofluorescence ratios at 4 and 16 weeks exhibited increased calcification at later 28-week timepoints. Conclusions: This study suggests the potential of TPEF autofluorescence metrics to serve as a label-free tool for early detection and monitoring of CAVD pathophysiology. Keywords: Calcific aortic valve disease, Valve calcification, Two-photon excited fluorescence microscopy, Wild-type mouse model Background Calcific aortic valve disease (CAVD) is the most common form of valvulopathy in the western world, with a *Correspondence: [email protected] 1 Department of Biomedical Engineering, University of Arkansas, 122 John A. White Jr. Engineering Hall, Fayetteville, AR 72701, USA Full list of author information is available at the end of the article
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