Comparison of arterial storage conditions for delayed arterial ring testing

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 utilized 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. Healthy murine aortas were harvested from 10-14-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 1.8mM or 2.5mM 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 (MCh) or sodium nitroprusside (SNP), respectively. Arterial contraction was stimulated with potassium chloride (KCl) or phenylephrine (PE). ANOVA was used to determine significance compared to immediate testing with P<.05. Under IRB approval, 28 PAD arteries were collected at amputation and underwent vascular function testing as described above. Disturbed flow conditions were determined by indirect (upstream occlusion) flow to the harvested tibial arteries. Stable flow arteries had in-line flow. Arterial calcification was quantified manually as present or not present. 4°C WI and 37°C Opti-MEM best preserved endothelium-dependent relaxation and performed similarly to immediately testing aortas (termed fresh for freshly tested) (P>.95). Other storage conditions were inferior to freshly tested aortas (P<.05). Vascular smooth muscle function was tested by endothelial-independent relaxation and contractility. All storage conditions preserved endothelial-independent relaxation and contractility similar to freshly tested arteries. However, 4°C WI and 37°C Opti-MEM storage conditions most closely approximated the maximum force of contraction of freshly tested arteries in response to KCl (P>.39). For human arterial testing, 28 tibial arteries were tested for relaxation and contraction with 16 PAOD arteries (PAD with disturbed flow) and 12 non-PAOD arteries (PAD with stable flow) of which 14 were calcified and 14 were non-calcified. 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% versus 59%; P=.03) compared to stable flow conditions (N=5). In contrast, presence (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). 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 degrees WI, flow conditions prior to 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 to 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.