Figure 4.

Treatment with Cyclosporine A Results in Closure of the mPTP and Subsequent Maturation of the Mitochondria

(A) Schematic overview of mature and immature mitochondria. Cyclosporine A (CsA) binds to cyclophilin D (CYPD) and thereby closes the mitochondrial permeability transition pore (mPTP). This prevents leakage of ROS and intermembrane space (IMS) proteins due to mitochondrial outer membrane permeabilization during the opening of mPTP.

(B) To determine the state of the mPTP in hiPSC-ECs, the cobalt/calcein AM (green) quenching method was used. hiPSC-EC NCRM1 treated with CsA for 30 min prevented calcein leakage, indicating that CsA closed the mPTP.

(C) TEM image shows the ultrastructure of mitochondria of hiPSC-ECs treated with 500 nM CsA during differentiation.

(D) Representative cross-sectional confocal images stained for MitoTracker red (oxidative mitochondria) and MitoTracker green (mitochondria) of hiPSC-ECs NCRM1 treated with CsA. (E) Quantification of percentage of mature mitochondria on TEM stitches (21 cells/group). (F and G) Quantitative analysis of mitochondrial morphology by analyzing MitoTracker confocal images. Individual particles (mitochondria) were analyzed for circularity and lengths of major and minor axes. From these values, aspect ratio (AR; major/minor) (F) and form factor (FF; perimeter²/4π × area) (G) were calculated. Both FF and AR have a minimal value of 1 when a particle is a small perfect circle, and the values increase as the shape becomes elongated. AR is a measure of mitochondrial length, and an increase of FF represents increase in length and branching (10 cells/group).

(H) Quantitative analysis of the area stained by MitoTracker red (oxidative mitochondria) divided by the area stained for MitoTracker green (mitochondria) (10 cells/group).

Values are presented as mean ± SEM of n = 3–5 independent experiments. One-way ANOVA was performed; ^∗p < 0.05, ^∗∗p < 0.001, ^∗∗∗p < 0.0001.