The mechanism of cryoinjury: In vitro studies on human internal mammary arteries

Abstract

1 The mechanism of cryoinjury was investigated in human internal mammary arteries (IMA) by monitoring contractile responses to ET-1 and KCl. 2 For cryopreservation segments of IMA were equilibrated for 20 min with the cryomedium (RPMI 1640 culture medium containing 1.8 M DMSO and 0.1 M sucrose), frozen at a mean cooling rate of 1.38C min 71 to 7708C and stored in liquid nitrogen. Before use, samples were thawed slowly and the cryomedium removed by dilution. 3 Compared to unfrozen controls, ET-1 stimulated frozen/thawed IMA with similar e cacy but at 3 fold lower concentrations (P50.001). Addition of ET-1 (100 nM) induced maximal contraction of unfrozen IMA within 10 min, declining thereafter to 25% after 90 min. In frozen/thawed IMA the ET-1-induced contraction was sustained but could be reversed if protein kinase C was blocked by staurosporine (100 nM). Responses to ET-1 of cryostored IMA were 5 fold more susceptible to blockade by nifedipine than those of controls. 4 After cryostorage the e cacy of KCl was diminished to 40% (P50.05) and the KCl curve was shifted to the left (2 fold, P50.001). In both unfrozen and cryostored IMA the KCl (60 mM) e ect was sustained and equally susceptible to nifedipine. 5 It is suggested that the smooth muscle cell of IMA is receptive to physical forces which occur during cryopreservation. These forces modify transmembrane signal transduction and intracellular pathways, that are common to pharmacological agonists thereby changing vascular responses to several contractile agonists after thawing. British Journal of Pharmacology (2000) 130, 636 ± 640 Keywords: Human internal mammary artery; cryopreservation; cryoinjury; endothelin-1; depolarization; protein kinase C activation; calcium in¯ux; staurosporine; nifedipine Abbreviations: ANOVA, one-way analysis of variance; DAG, diacylglycerol; DMSO, dimethylsulphoxide; EC 50 , concentration of an agonist producing 50% of E max ; EDTA, ethylenediaminetetraacetatic acid disodium salt; E max , maximal contractile e ect; ET-1, endothelin-1; IC 50 , drug concentration of an inhibitor producing 50% inhibition of E max of an agonist; IMA, internal mammary artery; IP 3 , phosphoinositide 4,5-trisphosphate; KH, Krebs-Henseleit; pD 2 , negative logarithm of the molar concentration of agonist producing 50% of E max ; PGF 2a , prostaglandin

Cryopreservation of vascular tissues

“…68 Experiments on human IMA demonstrated that the freezing/ thawing procedures besides inducing activation of PKC cause an increase in calcium-influx into the arterial smooth muscle. 35,85 Evidence for cryopreservation-induced enhanced Ca 2+ -influx comes from the observation that in cryopreserved IMA (1) activation of PKC through direct stimulation of DAG by phorbol dibutyrate (PDBu) occurs at significantly lower concentrations than in unfrozen controls and (2) that this phenomenon can be completely reversed by the dihydropyridine-sensitive Ca 2+ -channel blocker nifedipine. 35 Similar observations have been made with endothelin-1, the effect of which is resistant to nifedipine before but highly sensitive to blockade of dihydropyridine-sensitive Ca 2+channels after cryopreservation.…”

Intraindividual comparison of human radial and internal thoracic arteries in vitro and the effect of preoperative calcium blocker therapy

“…According to in vitro experiments marked organ differences with respect to sensitivity to Ca 2+ eB seem to exist. While nifedipine attenuates high potassium-induced contractions in RA with an IC 50 of about 250 nM [15] it is about 100 times more potent (IC 50 ¼ 2.6 nM) when used to block high potassium effects in ITA [16]. Therefore, the present results suggested that the differences in contractile responses to KCl observed in the intraindividual comparison study were due to a residual effect of preoperative Ca 2+ eB therapy in ITA.…”

The Structural and Cellular Viability in Cryopreserved Rabbit Carotid Arteries

“…In contrast to endothelial cells, VSMCs in the vessels appear to be more resistant to the procurement and storage process [56]. Not only VSMCs' functionalities, including sensitivity to the agonists [51,57,58], but viability for regeneration during in vitro culture process also has been preserved.…”