Label-free metabolic biomarkers for assessing valve interstitial cell calcific progression

Ishita Tandon,Shelby Johns,Kyle P Quinn,Kartik Balachandran,Olivia I Kolenc,Delaney Cross,Isaac Vargas

doi:10.1038/s41598-020-66960-4

Ishita Tandon, Shelby Johns + Show 5 more

Open Access

https://doi.org/10.1038/s41598-020-66960-4

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Journal: Scientific Reports	Publication Date: Jun 25, 2020
Citations: 7	License type: open-access

Affiliation: University of Arkansas at Fayetteville

Abstract

Calcific aortic valve disease (CAVD) is the most common form of valve disease where the only available treatment strategy is surgical valve replacement. Technologies for the early detection of CAVD would benefit the development of prevention, mitigation and alternate therapeutic strategies. Two-photon excited fluorescence (TPEF) microscopy is a label-free, non-destructive imaging technique that has been shown to correlate with multiple markers for cellular differentiation and phenotypic changes in cancer and wound healing. Here we show how specific TPEF markers, namely, the optical redox ratio and mitochondrial fractal dimension, correlate with structural, functional and phenotypic changes occurring in the aortic valve interstitial cells (VICs) during osteogenic differentiation. The optical redox ratio, and fractal dimension of mitochondria were assessed and correlated with gene expression and nuclear morphology of VICs. The optical redox ratio decreased for VICs during early osteogenic differentiation and correlated with biological markers for CAVD progression. Fractal dimension correlated with structural and osteogenic markers as well as measures of nuclear morphology. Our study suggests that TPEF imaging markers, specifically the optical redox ratio and mitochondrial fractal dimension, can be potentially used as a tool for assessing early CAVD progression in vitro.

Highlights

Calcific aortic valve disease (CAVD) is the most common form of valve disease where the only available treatment strategy is surgical valve replacement
Our results showed that Two-photon excited fluorescence (TPEF) metrics correlated with the early markers of CAVD progression and suggest that TPEF microscopy can be utilized as a label-free non-destructive tool for assessing CAVD progression in vitro
Positive Alizarin Red S (ARS) staining on day 28 confirmed that the nodules present in the osteogenic cultures by day 28 were calcific in nature (Fig. 1b)

Summary

Introduction

Calcific aortic valve disease (CAVD) is the most common form of valve disease where the only available treatment strategy is surgical valve replacement. We show how specific TPEF markers, namely, the optical redox ratio and mitochondrial fractal dimension, correlate with structural, functional and phenotypic changes occurring in the aortic valve interstitial cells (VICs) during osteogenic differentiation. Our study suggests that TPEF imaging markers, the optical redox ratio and mitochondrial fractal dimension, can be potentially used as a tool for assessing early CAVD progression in vitro. We seeded porcine aortic VICs as monolayers in quiescent versus osteogenic media on two dimensional soft or stiff substrates We examined these samples using TPEF microscopy to quantify ORR and mitochondrial FD and simultaneously characterized the CAVD progression in our model using traditional end-point biomarkers, such as calcific nodule quantification, gene expression, cell proliferation and apoptosis. Our results showed that TPEF metrics correlated with the early markers of CAVD progression and suggest that TPEF microscopy can be utilized as a label-free non-destructive tool for assessing CAVD progression in vitro

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