Procedure for Human Saphenous Veins <em>Ex Vivo</em> Perfusion and External Reinforcement

Abstract

The mainstay of contemporary therapies for extensive occlusive arterial disease is venous bypass graft. However, its durability is threatened by intimal hyperplasia (IH) that eventually leads to vessel occlusion and graft failure. Mechanical forces, particularly low shear stress and high wall tension, are thought to initiate and to sustain these cellular and molecular changes, but their exact contribution remains to be unraveled. To selectively evaluate the role of pressure and shear stress on the biology of IH, an ex vivo perfusion system (EVPS) was created to perfuse segments of human saphenous veins under arterial regimen (high shear stress and high pressure). Further technical innovations allowed the simultaneous perfusion of two segments from the same vein, one reinforced with an external mesh. Veins were harvested using a no-touch technique and immediately transferred to the laboratory for assembly in the EVPS. One segment of the freshly isolated vein was not perfused (control, day 0). The two others segments were perfused for up to 7 days, one being completely sheltered with a 4 mm (diameter) external mesh. The pressure, flow velocity, and pulse rate were continuously monitored and adjusted to mimic the hemodynamic conditions prevailing in the femoral artery. Upon completion of the perfusion, veins were dismounted and used for histological and molecular analysis. Under ex vivo conditions, high pressure perfusion (arterial, mean = 100 mm Hg) is sufficient to generate IH and remodeling of human veins. These alterations are reduced in the presence of an external polyester mesh.

Evaluating intimal hyperplasia under clinical conditions

“…Specifically, the ratio of intima to media, a parameter which has been associated with an increased risk of atherosclerosis and carotid stenosis [18], was more increased in the venous patch than in the host artery due to increased numbers of SMCs, myofibroblasts, and secretory fibroblasts. These findings are in agreement with the data we reported in previous publications, aimed at defining the cellular mechanisms leading to IH, including in an ex vivo system in which segments of human saphenous veins were exposed to arterial conditions [16,17,19,20]. The new data document that, over a relatively short time period (28 days), the new pig model allows for the longitudinal monitoring of IH development under haemodynamic conditions which closely mimic those prevailing in the vessels submitted to angioplasty in the human clinic and which faithfully reproduce the cellular landmarks of the pathological alterations of human veins grafted on the arterial compartment.…”

Connexin43 Inhibition Prevents Human Vein Grafts Intimal Hyperplasia

“…Using our validated ex-vivo vein perfusion model [ 13 , 16 ], human saphenous veins grafts were exposed to high-pressure (mean = 100mmHg) and high flow (180-200ml/min), mimicking the femoral artery hemodynamic. As previously established [ 16 ], 3 and 7 days of arterial perfusion resulted in neointima formation ( Fig 2A upper panel and 2B) and reduced media thickness ( Fig 2A upper panel and 2C).…”

“…One part was immediately fixed in either formalin (one half) or rapidly frozen in liquid nitrogen (the other half). The two others parts from the same vein were perfused using an ex-vivo perfusion system (EVPS) [ 13 , 14 , 16 ], with or without an external mesh reinforcement (ProVena, B.Braun Medical SA) to model and control (mesh) the development of IH as previously described [ 13 ] (more details on the method can be found at: http://www.jove.com/video/52079/procedure-for-human-saphenous-veins-ex-vivo-perfusion-external ). A pulsatile, cardioid signal at 60 pulses per minute with constant amplitude was setup up via the computer software which independently pilots the gearing pump.…”

“…In this setting, we identified divergent patterns of vein graft remodeling under low versus high flow and shear stress [ 12 ]. To further study the impact of hemodynamic forces after human vein arterial engraftment, we developed an ex-vivo human vein perfusion system (EVPS) [ 13 , 14 ] and observed a 7-day high-pressure perfusion (mean = 100 mmHg) to be sufficient for IH formation. The VSMC forming the media layer of the vessels are highly specialized contractile cells featuring a very low proliferation rate under physiological conditions [ 15 ].…”

Clueless with fewer cues from endothelin. Commentary: Venous endothelin guides sympathetic innervation of the developing mouse heart

“…To go further, rapid changes in blood flow and the resultant shear stress during development of the cardiovascular system can be a factor of downregulation for endothelin 1 in arteries, and most likely in veins (Morawietz et al, 2000 ; Longchamp et al, 2014 ). An abnormal shear stress could hence contribute to the perturbation of cardiac innervation as described above.…”