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

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. Calcific aortic stenosis or calcific aortic valve disease (CAVD) is a progressive disease involving multiple signaling pathways, endothelial dysfunction, cytokine infiltration, collagen remodeling, as well as lipid and calcium deposition 1-4. The symptoms and markers for CAVD manifest via both degenerative (apoptotic) and active (osteogenic) mechanisms 5,6. Aortic valve endothelial and interstitial cells differentiate into an osteoblast-like phenotype, the extracellular matrix becomes thicker and stiffer and calcium mineralization occurs throughout the tissue 7,8. Aortic stenosis and sclerosis have an increased prevalence in the elderly and contribute to a 50% elevated risk of infarction and other potentially fatal cardiovascular pathologies 2,9,10. Currently, valve replacement is the preferred treatment method, as other strategies, such as the retardation of calcific progression, prevention and early diagnosis, are non-existent 11. Diagnostic techniques like echocardiography, cardiac MRI and cardiac CT are widely used, but are only sensitive during later stages of the disease once there is tissue mineralization, and hemodynamic and geometric impairment 12,13. Aortic valve interstitial cells (VICs) are the primary cells in the heart valves and are involved in tissue maintenance, repair and remodeling 14. VICs exist in a quiescent state under healthy conditions and are activated due to injury or disease 15 , potentially differentiating into an osteogenic-like phenotype to potentiate calcification 2. Current in vitro biochemical techniques to assess CAVD are typically destructive as they involve cell lysis or fixation and do not facilitate the longitu...

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

“…Our current study is the first instance when a TPEF 755–860 ratio has been shown to demonstrate tissue level changes occurring during early CAVD progression in a mouse model that was more sensitive than the TPEF Col-Cal ratio. Like we had previously demonstrated in our in vitro studies [ 13 , 28 , 52 ], in this study also we observed TPEF autofluorescence ratios decreased at 16 weeks in the pro-calcific mice commissures and correlated with increased calcification, osteogenic differentiation and proliferation. This is also similar to the findings of Quinn et al .…”

“…We have also shown previously that VICs under pathological stretch have reduced optical redox ratios compared to unstretched cells [ 13 ]. We have also demonstrated that when VICs are subjected to osteogenic conditions in vitro, their optical redox ratio decreased and correlated with gene expression of osteogenic markers like RUNX2, osteopontin and osteocalcin [ 28 ]. In the current study, we measured the TPEF 755–860 ratio (A 860/525 /(A 755/460 + A 860/525 )) from ex vivo tissue sections by obtaining the autofluorescence intensities at 755 nm and 860 nm excitation, which correspond to the NADH and FAD excitation wavelengths.…”

“…We have also demonstrated that this TPEF-based optical redox ratios in VICs was reduced as cellular stretching increased from normal to pathologic magnitudes [ 13 ]. Additionally, optical redox ratio altered and correlated with temporal changes in VIC phenotype during osteogenic de-differentiation, in vitro [ 28 ]. In addition to NADH and FAD, molecules such as collagen, calcium and lipids within tissues will contribute to the autofluorescence emission in the visible range [ 27 , 29 – 32 ].…”

Label-Free Multiphoton Microscopy for the Detection and Monitoring of Calcific Aortic Valve Disease

“…Our lab has previously reported that when VICs underwent a pathogenic phenotype shift, they experienced a decrease in ORR, suggesting a possible link between VIC pathology and its metabolic state (21,42,43,67). TPEF imaging of VICs under quiescent and osteogenic conditions revealed that the ORR decreased during early osteogenic differentiation (Figure 1F) and correlated with gene expression of osteogenic markers.…”

“…MPM-based approaches combining TPEF, SHG and CARS, for label-free imaging of an aortic valve have been previously demonstrated (41). TPEF has been used to assess aortic valve interstitial cell (VIC) proliferation (21,42), osteogenesis (43), and valve calcification in vitro and ex vivo (39). TPEF autofluorescence ratios have shown potential in assessing CAVD progression ex vivo (44).…”