High resolution monitoring of valvular interstitial cell driven pathomechanisms in procalcific environment using label-free impedance spectroscopy

Figures

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  Figure 1. Electrochemical impedance spectroscopy (EIS) real-time monitoring of human valvular interstitial cells (VICs) in vitro in profibrotic or procalcifying environment. (A) Averaged time traces of VICs cultivated in control medium (CM), profibrotic medium (FM) or procalcifying medium (PM) for 20 days. n = 10. Mean ± SEM (SEM shown as continuous lines). (B) The relative collagen type 1 secretion and (C) Alizarin Red absorption of VICs in CM, PM or FM. n = 10. Association plots and Pearson correlation coefficients (r) for EIS and (D) collagen secretion or (E) Alizarin red absorption for the exact time traces of PM-treated VICs. Pearson correlation coefficient r was calculated for phase 1 (day 0 to 8) and phase 2 (day12 to 21) separately. Visualization of (F) near infrared molecular imaging for hydroxyapatite (HA) and (G) quantification of the HA-positive area during the 21 day culture period. The dashed lines indicate the inflection point between phase 1 and phase 2. CI, Cellular impedance.

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  Figure 2. Viability and cell death of VICs in PM. (A) Visualization of viable, Calcein-AM + and dead, propidiumiodide + VICs cultured in control or procalcifying medium over 21 days. (B) Calcein-AM quantification, (C) mitochondrial transmembrane potential Δψm and (F) relative macromolecular permeability of VICs in control or procalcifying medium normalized to t = 0. Mean ± SEM. n ≥ 4.

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  Figure 3. Myofibroblastogenesis of VICs in PM. (A) Visualization of viable, Calcein-AM+ and dead, propidiumiodide+ VICs cultured in control medium (CM) or procalcifying medium (PM) over 21 days. n = 5. (B) Gene expression of alpha smooth muscle actin (ACTA2) of VICs in CM and PM at day 7 and 21. ACTA2 gene expression was normalized to expression of hypoxanthine phosphoribosyltransferase 1 (HPRT1), n = 10. (C) Phalloidin staining of F-Actin cytoskeleton modulation of VICs in CM, PM, growth medium (GM) and profibrotic medium (FM). n = 3.

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  Figure 4. Electrochemical impedance spectroscopy (EIS) analysis of PM-induced sex- and donor-dependent effects in valvular interstitial cells (VICs). (A) Single and averaged time traces (bold lines) of either male (orange) or female (petrol) VICs in procalcifying medium PM for 20 days. (B) Minimal and (C) maximal EIS signals of male and female VICs in PM. Relative (D) collagen 1 secretion and (E) Alizarin Red absorption data from Figures 1B,C stratified by donor sex. Dashed lines indicate means without the impedance based extreme values (values >90 percentile were excluded). n = 5 vs. 5. CM, Control medium; FM, Profibrotic medium; CI, Cellular impedance.

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  Figure 5. Scheme of EIS-based ECM mineralization detection. Modified using (48).