Techniques to harvest diseased human peripheral arteries and measure endothelial function in an ex vivo model

ObjectiveEndothelial dysfunction has been studied in animal models. However, direct evidence of endothelial function from human vessels is limited. Our objectives were to optimize methods in harvesting human arteries from amputation specimens, determine endothelial function, and measure responsiveness to l-arginine, a nitric oxide precursor.MethodsFresh amputation specimens were transferred expeditiously from the operating room to the bench laboratory for dissection and arterial harvest in an Investigational Review Board-approved protocol. Popliteal and tibial vessels were examined in pilot experiments leading to the use of the anterior tibial artery in consecutive experiments. Human lower extremity anterior tibial artery segments were harvested from 14 amputation specimens. Specimens were rapidly collected and divided for endothelial-dependent relaxation (EDR) studies in a tissue bath apparatus, immunohistochemistry, and intravascular ultrasound-derived virtual histology. A total of 47 ring segments were studied. The data were compared with two-way analysis of variance.ResultsHuman lower extremity arteries exhibited low responsiveness to acetylcholine (EDR, 24.9%; acetylcholine, 10−4). L-arginine supplementation enhanced EDR by 38.5% (P < .0001). N-nitro-L-arginine methyl ester abrogated EDR (P < .0001) in vessels exposed to L-arginine. Arterial responsiveness was intact in all vessels (endothelial independent relaxation to sodium nitroprusside, 113.2% ± 28.1%). Histology and immunohistochemistry confirmed intact endothelium by morphometric analysis, cluster of differentiation 31, endothelial nitric oxide synthase, and arginase II staining. Intravascular ultrasound-derived virtual histology indicated atheroma burden was 11.9 ± 4.7 mm3/cm, and plaque stratification indicated fibrous morphology was predominant (59.9%; necrotic core, 16.9%; calcium, 11.2%). Variations in plaque morphology did not correlate with endothelial function or responsiveness to L-arginine.ConclusionsHuman lower extremity arteries demonstrate low baseline endothelial function in patients requiring amputation. Endothelial dysfunction is improved by L-arginine supplementation in an ex vivo model. These results support strategies to increase local levels of nitric oxide in human vessels.

Clinical RelevanceEndothelial dysfunction is thought to be the precursor to atherogenesis. Although endothelial function has been studied extensively in animal models and in human coronary vessels, direct evidence for endothelial function in the peripheral arteries of patients with peripheral arterial disease is limited. This study shows direct evidence of endothelial dysfunction in arteries harvested from amputation specimens. In addition, the dysfunction can be improved with local addition of L-arginine. The ex vivo model described offers a flexible assay to study pharmaceutical and biologic agents in human arteries before expensive and time-consuming clinical trials.