Functional activity and transmembrane signaling mechanisms after cryopreservation of human internal mammary arteries.

PURPOSE

Cryopreserved human blood vessels are important tools in bypass surgery. However, several in vitro studies have demonstrated diminished postthaw functional activity. Therefore the aim of this study was to investigate the consequences of various freezing/thawing protocols and the role of protein kinase C in the postthaw functional activity of cryopreserved human arteries.

METHODS

In vitro responses of frozen/thawed human internal mammary arteries (IMA) were used to investigate the functional activity after thawing at 15 degrees, 30 degrees, and 100 degrees C/min and after different prefreezing equilibration times (10, 60, 120, 240 minutes) with the cryomedium (Krebs-Henseleit solution containing 1.8 mol/L dimethyl sulfoxide and 0.1 mol/L sucrose) at room temperature followed by cryostorage at -196 degrees C.

RESULTS

Prefreezing equilibration for 10 to 120 minutes diminished maximal alpha-adrenoceptor-mediated responses to noradrenaline to approximately 60%, and equilibration for 240 minutes attenuated noradrenaline effects to less than 25% of that produced by unfrozen controls. Contractile responses were slightly better when thawing was performed at 15 degrees C/min compared with 100 degrees C/min. The postthaw sensitivity to direct activation of protein kinase C by phorbol 12,13-dibutyrate was enhanced. Compared with unfrozen tissues (pD2 = 7.36 +/- 0.07, n = 32) maximal sensitization to phorbol 12,13-dibutyrate was observed in IMA that had been frozen after 60 minutes of equilibration with the cryomedium (pD2 = 8.31 +/- 0.09, n = 30). Responses to phorbol 12,13-dibutyrate of cryopreserved IMA were highly susceptible to blockade of calcium influx by nifedipine, whereas those of unfrozen IMA were resistant to nifedipine. Against noradrenaline nifedipine was equipotent in cryopreserved (pD'2 = 7.75 +/- 0.15, n = 8) and unfrozen IMA (pD'2 = 7.70 +/- 0.10, n = 6). Endothelium-dependent relaxant responses to acetylcholine were significantly attenuated after cryopreservation (Emax = 26% +/- 5%, n = 4) compared with unfrozen IMA (Emax = 71% +/- 4%, n = 4, p < 0.001); endothelium-independent relaxant responses to sodium nitroprusside were unchanged.

CONCLUSIONS

Cryopreservation of human IMA under the conditions applied in this study (1) attenuated endothelial cell function and (2) induced an activation of protein kinase C, thereby increasing calcium influx through dihydropyridine-sensitive calcium channels. These experimental data suggest that postoperative administration of calcium channel blockers alone or combined with long-acting nitrates should effectively prevent the development of spasms in arterial grafts.