title: Endothelium and vascular smooth muscle function in internal mammary artery after cryopreservation.
abstract: An optimal cryopreserved arterial conduit should have anatomic and physiologic characteristics similar to those of the fresh artery. We have cryopreserved canine internal mammary artery (IMA) with intact, prostacyclin (PGI2)-producing endothelial cells, but the underlying vascular smooth muscle appeared nonfunctional. Thus the aim of this study was to evaluate which steps of the cryopreservation method compromise vascular smooth muscle function in cryopreserved IMA. Isometric tension recording responses to vasoconstrictor agonists were used to evaluate the level of vascular muscle integrity, while endothelial function was assessed by relaxation responses to acetylcholine and by PGI2 production. These variables were measured in vessels rewarmed to 37 degrees C, following initial exposure to different stages of an eight-step computerized cryopreservation protocol (n = 10). In this protocol, tissue temperature was lowered to -10 degrees C (steps 1-2), rapidly reduced to -12 degrees C (steps 3-4), reduced further to -40 degrees C at a rate of -0.5 degrees C/min (steps 5-6), lowered to -70 degrees C (step 7), and finally equilibrated at - 196 degrees C by immersion in liquid nitrogen (step 8). Compared to fresh IMA, the amplitude of dose-dependent contractions to norepinephrine (NE 10(-9) to 10(-4) M) was step-wise depressed after cooling to < or = -12 degrees C (steps 3-4), showing only 75 and 19% of maximal contraction after steps 3-4 and step 7, respectively. Similarly, depolarization-induced contractions to KCl (10 to 60 mM) also were progressively depressed after step-wise cryopreservation, showing reduced contractile amplitudes even after steps 1-2 (-10 degrees C). Vascular muscle agonist sensitivity was unchanged (KCl) or only mildly influenced (NE) by these same temperature reductions. In contrast, endothelial-dependent relaxation to acetylcholine and PGI2 production were maintained after all steps in the cryopreservation process, and baseline PGI2 production was higher in cryopreserved IMA. These data indicate that IMA smooth muscle cell viability is poorly preserved after cooling to below -10 to -12 degrees C of the cryopreservation process, whereas endothelial cell function appears intact after profound cooling to -196 degrees C. The loss of vascular smooth muscle responsiveness coupled with the protection afforded by an intact endothelium may provide an arterial conduit less susceptible to vasospasm. Such a graft, however, would lack the dynamic properties of flow regulation in response to the metabolic needs of the myocardium.
authors.affiliation: Department of Cardiothoracic Surgery, Medical College of Wisconsin, Milwaukee 53226, USA.
authors.forename: G H
authors.initials: GH
authors.lastname: Almassi
authors.forename: B
authors.initials: B
authors.lastname: Farahbakhsh
authors.forename: T
authors.initials: T
authors.lastname: Wooldridge
authors.forename: N J
authors.initials: NJ
authors.lastname: Rusch
authors.forename: G N
authors.initials: GN
authors.lastname: Olinger
mesh_terms: D000109:Acetylcholine / Q000494:pharmacology; D000818:Animals; D015925:Cryopreservation; D004285:Dogs; D004730:Endothelium, Vascular / Q000502:physiology*; D011464:Epoprostenol / Q000096:biosynthesis; D066298:In Vitro Techniques; D008323:Mammary Arteries / Q000502:physiology; D009131:Muscle, Smooth, Vascular / Q000502:physiology*; D009638:Norepinephrine / Q000494:pharmacology; D014661:Vasoconstriction; D014664:Vasodilation / Q000187:drug effects
chemical_list: D011464:Epoprostenol; D000109:Acetylcholine; D009638:Norepinephrine