Faculty Collaboration Database

Visualization and quantification of mitochondrial structure in the endothelium of intact arteries. Cardiovasc Res 2019 Aug 01;115(10):1546-1556

Date

11/27/2018

Pubmed ID

30476208

Pubmed Central ID

PMC6648341

DOI

10.1093/cvr/cvy294

Scopus ID

2-s2.0-85072658081 (requires institutional sign-in at Scopus site)   19 Citations

Abstract

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

METHODS AND RESULTS: 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).

CONCLUSIONS: 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.

Author List

Durand MJ, Ait-Aissa K, Levchenko V, Staruschenko A, Gutterman DD, Beyer AM

Authors

Andreas M. Beyer PhD Associate Professor in the Medicine department at Medical College of Wisconsin
Matt Durand PhD Associate Professor in the Physical Medicine and Rehabilitation department at Medical College of Wisconsin

MESH terms used to index this publication - Major topics in bold

Animals
Cells, Cultured
Diabetes Mellitus, Experimental
Diabetic Angiopathies
Endothelial Cells
Glucose
Human Umbilical Vein Endothelial Cells
Humans
Luminescent Proteins
Mesenteric Arteries
Mice, Transgenic
Microscopy, Confocal
Microscopy, Electron
Mitochondria
Mitochondrial Dynamics
Single-Cell Analysis
Time Factors