Comparison of arterial storage conditions for delayed arterial ring testing

Abstract

Objective: Arterial ring testing is the gold standard for measuring arterial function. Increased arterial tone through arterial contraction and impaired endothelial relaxation (endothelial dysfunction) are key metrics of impaired arterial health in peripheral arterial disease (PAD). To allow for comparative testing of arteries during standard laboratory hours, storage buffers and conditions have been used to extend the functional life of arteries. Various storage conditions have been compared, but there has not been a robust comparison or validation in human arteries. The objective of this work is to optimize storage of arterial segments for endothelial cell (EC) testing in a murine model and to test EC function in human PAD arteries. We hypothesized that certain storage conditions would be superior to others. Methods: Healthy murine aortas were harvested from 10- to 14-week-old C57/Bl6J male and female mice and compared under different storage protocols (24 hours) to immediate arterial testing. The storage conditions tested were: Opti-MEM (37°C or 4°C), Krebs-HEPES with 1.8 mmol/L or 2.5 mmol/L calcium (4°C), or Wisconsin (WI) solution at 4°C. Vascular function was evaluated by isometric force testing. Endothelium-dependent and -independent relaxation were measured after precontraction with addition of methacholine or sodium nitroprusside, respectively. Arterial contraction was stimulated with potassium chloride or phenylephrine. Analysis of variance was used to determine significance compared with immediate testing with P .95). Other storage conditions were inferior to freshly tested aortas (P .39). For human arterial testing, 28 tibial arteries were tested for relaxation and contraction with 16 arteries with peripheral artery occlusive disease (PAD with disturbed flow) and 12 without peripheral artery occlusive disease (PAD with stable flow), of which 14 were calcified and 14 were noncalcified. Endothelial-dependent relaxation data was measurable in 9 arteries and arterial contraction data was measurable in 14 arteries. When comparing flow conditions, arteries exposed to disturbed flow (n = 4) had significantly less relaxation (2% vs 59%; P = .03) compared with stable flow conditions (n = 5). In contrast, presence the (n = 6) or absence of calcification (n = 3) did not impact arterial relaxation. Arterial contraction was not different between groups in either comparison by flow (n = 9 disturbed; n = 5 stable) or calcification (n = 6 present; n = 8 absent). Conclusions: In healthy murine aortas, arterial storage for 24 hours in 4°C WI or 37°C Opti-MEM both preserved endothelium-dependent relaxation and maximum force of contraction. In human PAD arteries stored in 4° WI, flow conditions before arterial harvest, but not arterial calcification, led to differences in arterial relaxation in human PAD arteries. Arterial contractility was more robust (11/28 arteries) compared with arterial relaxation (7/28 arteries), but was not significantly different under flow or calcification parameters. This work defines ideal storage conditions for arterial ring testing and identifies that EC dysfunction from disturbed flow may persist in delayed ex vivo arterial testing. : Clinical Relevance: Delayed arterial ring testing is feasible and choice of preservation protocol matters. This strategy allows for physiological testing of human PAD arteries in a staged manner. Using this testing paradigm, flow-dependent changes to arterial function were found to be more powerful than mechanical stiffening owing to calcification for endothelial cell function in PAD arteries.

Keywords

• Peripheral arterial disease
• Arterial storage
• Endothelial function
• Arterial contraction