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 < .05. Under institutional review board approval, 28 PAD arteries were collected at amputation and underwent vascular function testing as described. 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.

Results: We found that 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 potassium chloride (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.

Keywords: Arterial contraction; Arterial storage; Endothelial function; Peripheral arterial disease.

Fig 1

Endothelial-dependent relaxation under different storage conditions. Relaxation of murine aortas under various storage conditions was compared with freshly tested aortas. (A) Endothelium-dependent relaxation curves of Fresh, Wisconsin (WI) at 4°C, and Opti-MEM at 37°C had preserved endothelial relaxation that was not seen in the other storage conditions. (B) When comparing maximum relaxation, Fresh, WI at 4°C, and Opti-MEM at 37°C, all exceeded the 30% threshold for intact endothelium-dependent relaxation. Aortas preserved in Krebs-HEPES with 1.8 mmol/L calcium and 2.5 mmol/L calcium as well as Opti-MEM at 4°C exhibited significantly depressed endothelium-dependent relaxation function. (C) To test the ability of vascular smooth muscle cells to relax independently of endothelial cells (EC), endothelium-independent relaxation curves demonstrate similar responses to increasing concentrations of sodium nitroprusside with all groups similar. (D) Maximum percentage endothelium-independent relaxation was similarly not affected by storage conditions. Total of six descending thoracic aortas (3 males and 3 females). Statistical testing with one-way analysis of variance. Lines represent mean and bars represent standard deviation. ∗P < .05. SNP, sodium nitroprusside.

Fig 2

Arterial force contraction under different storage conditions. Contraction forces of murine aortas were compared across storage conditions. (A) Force contraction curves in response to increasing concentrations of KCl. (B) There were no significant differences across groups, but maximum force contraction was more similar between Fresh, Opti-MEM at 37°C, and Wisconsin (WI) at 4°C than other conditions. (C) Force contraction curves in response to increasing concentrations of phenylephrine (PE). (D) Again, there were no significant differences across groups, but maximum force of contraction was more similar to Fresh in Krebs-HEPES with 2.5 mmol/L calcium, Opti-MEM at 37°C, and WI at 4°C than other conditions. A total of six descending thoracic aortas (3 males and 3 females). Statistical testing with one-way analysis of variance. Lines represent mean and bars represent standard deviation.

Fig 3

Arteries with peripheral artery obstructive disease (PAD) have impaired contractility. Contraction of PAD arteries compared with no-PAD arteries. Absolute force contraction curves in response to increasing concentrations of potassium chloride (KCl). Unlike healthy murine aortas, PAD arteries had decreased force of contraction compared with age and no-PAD controls. There were a total of six arteries (3 PAD and 3 non-PAD). PAD patients with mean age of 73.5 years old, 100% male, 100% with diabetes mellitus, 100% with hypertension, 33% with coronary artery disease, 33% with heart failure, 67% with hyperlipidemia, and 33% with end stage renal disease. Patients without PAD had mean age of 63.5 years old, were 33% male, 67% had diabetes mellitus, 33% had hypertension, 0% had coronary artery disease, 0% had heart failure, 33% had hyperlipidemia, and 0% had end-stage renal disease. Statistical testing was done with one-way analysis of variance. Dots represent mean with brackets denoting standard deviation. ∗P < .05.