Label-free optical biomarkers detect early calcific aortic valve disease in a wild-type mouse model

Ishita Tandon,Alan Woessner,Kartik Balachandran,Jessica Perez,Srikanth Vallurupalli,Kyle P Quinn,Delaney Cross,Asya Ozkizilcik,Shelby Johns,Muralidhar Padala,Timothy J Muldoon

doi:10.1186/s12872-020-01776-8

Abstract

BackgroundCalcific 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.MethodsTwenty-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.ResultsPro-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.ConclusionsThis study suggests the potential of TPEF autofluorescence metrics to serve as a label-free tool for early detection and monitoring of CAVD pathophysiology.

Highlights

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
The early pathogenic processes in CAVD are defined by the phenotypic transformation of valve endothelial (VEC) and interstitial (VIC) cells via cellular activation, osteogenic differentiation [5,6,7], as well as maladaptive extracellular matrix (ECM) remodeling [8, 9]
We have demonstrated that this two-photon excited fluorescence (TPEF)-based optical redox ratios in VICs was reduced as cellular stretching increased from normal to pathologic magnitudes [13]

Summary

Introduction

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. CAVD pathophysiology is marked by infiltration of cytokines, lipid deposition, and calcific nodule formation [4, 10, 11]. These early pathogenic processes are simulated using two- and three-dimensional in vitro models and in vivo models that mimic the CAVD process and valve milieu to varying degrees of accuracy [7, 12,13,14,15]. Assmann and colleagues have shown that a diet regimen supplemented with vitamin D, cholesterol and dicalcium phosphate caused significant calcification of the aortic root in Wistar rats [18]. We exploited a similar dietary regimen to develop a wild-type mouse model for early CAVD

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Conclusion