To quantify the mitochondrial structure of ECs in intact arteries vs. cultured cells.

Cre-stop mito-Dendra2 mice, expressing the fluorescent protein Dendra2 in the mitochondrial matrix only, were used to label EC mitochondria using Cre-recombinase under the control of the VE-cadherin promoter. Conduit arteries, resistance arterioles and veins were fixed, mounted on glass slides and fluorescent images were obtained using a laser scanning confocal microscope (ex 488 nm; em 550 nm). ImageJ was used to calculate form factor (FF) and aspect ratio (AR) of the mitochondrial segments. Mitochondrial fragmentation count (MFC) was calculated by counting non-contiguous mitochondrial particles and dividing by the number of pixels which comprise the mitochondrial network. Primary aortic EC cultures (48 h on culture plates) were generated to compare the mitochondrial structure of cultured ECs vs. intact arteries. Aortic segments were also exposed to high glucose overnight (33 mM) ex vivo, and separate groups of mice were either infused with a high-glucose saline solution (300 mM) via tail vein catheter for 1 h or injected with streptozotocin (STZ; 50 mg/kg) to cause hyperglycaemia. Compared with cultured ECs, the mitochondria of ECs from the intact aorta were more fragmented (MFC: 6.4 ± 2.5 vs. 18.6 ± 9.4, respectively; P < 0.05). The mitochondrial segments of ECs within the aorta were more circular in shape (FF: 3.5 ± 0.75 vs. 1.8 ± 0.30, respectively; P < 0.05) and had less branching (AR: 2.9 ± 0.60 vs. 2.0 ± 0.25, respectively; P < 0.05) compared with cultured ECs. Ex vivo exposure of the intact aorta to high glucose overnight caused mitochondrial fission compared with normal glucose conditions (5 mM; MFC: 25.5 ± 11.1 high glucose vs. 11.0 ± 3.6 normal glucose; P < 0.05). Both 1-h infusion of high glucose saline (MFC: 22.4 ± 4.3) and STZ treatment (MFC: 40.3 ± 14.2) caused mitochondrial fission compared with freshly fixed aortas from control mice (MFC: 18.6 ± 9.4; P < 0.05 vs. high-glucose infusion and STZ treatment).

Using a novel mouse model, we were able to, for the first time, obtain high resolution images of EC mitochondrial structure in intact arteries. We reveal the endothelial mitochondrial network is more fragmented in the intact aorta compared with cultured ECs, indicating that mitochondria assume a more elongated and branched phenotype in cell culture.