Functional assessment of cryopreserved pig aortas for pharmaceutical research

Several in vitro studies have demonstrated diminished post-thaw functional activity. Therefore, the aim of this study was to investigate the consequences of thawing and storage method used on the post-thaw functional activity of cryopreserved pig aortas with the aim of adjusting the freezing and thawing protocol so that the vascular segments are preserved in the best possible state, maintaining structure and functionality so that they can later be transplanted with success. In vitro responses of frozen, thawed pig aortas were used to investigate the functional activity after thawing at 15 degrees C and 100 degrees C/min and after storage in gas or liquid phase of liquid nitrogen. Cryopreservation was performed in RPMI 1640 medium + 10% dimethylsulfoxide and the rate of cooling was -1 degrees C/min, until -150 degrees C was reached.After thawing the maximal contractile responses to all the contracting agonists tested (KCl, noradrenaline) were in the ranges of 13-27% compared with the responses in unfrozen pig aortas. Contractile responses were slightly better when thawing was performed at 15 degrees C/min compared with 100 degrees C/min. The endothelium independent relaxant responses to sodium nitroprusside were reduced ( P < 0.05). Cryostorage of pig arteries also resulted in a loss of the endothelium-dependent relaxant response to acetylcholine. The cryopreservation method used provided a limited preservation of pig aorta contractibility, a reduction of the endothelium independent relaxant responses, and no apparent preservation of the endothelium-dependent relaxation. It is possible that further refinements of the cryopreservation protocol might allow better post-thaw functional recovery of pig aortas.     

Endothelium-independent vasorelaxation effects of 16-O-acetyldihydroisosteviol on isolated rat thoracic aorta

Aims

The aim of this study is to investigate the vasorelaxant effect of 16-O-acetyldihydroisosteviol (ADIS) and its underlying mechanisms in isolated rat aorta.

Main methods

Rat aortic rings were isolated, suspended in organ baths containing Kreb's solution, maintained at 37 °C, and mounted on tungsten wire and continuously bubbled with a mixture of 95% O₂ and 5% CO₂ under a resting tension of 1 g. The vasorelaxant effects of ADIS were investigated by means of isometric tension recording experiment.

Key findings

ADIS (0.1 μM–3 mM) induced relaxation of aortic rings pre-contracted by phenylephrine (PE, 10 μM) and KCl (80 mM) with intact-endothelium (E_max = 79.26 ± 3.74 and 79.88 ± 3.79, respectively) or denuded-endothelium (E_max = 88.05 ± 3.69 and 78.22 ± 6.86, respectively). In depolarization Ca²⁺-free solution, ADIS inhibits calcium chloride (CaCl₂)-induced contraction in endothelium-denuded rings in a concentration-dependent manner. In addition, ADIS attenuates transient contractions in Ca²⁺-free medium containing EGTA (1 mM) induced by PE (10 μM) and caffeine (20 mM). By contrast, relaxation was not affected by tetraethylammonium (TEA, 5 mM), 4-aminopyridine (4-AP, 1 mM), glibenclamide (10 μM), barium chloride (BaCl₂, 1 mM), and 1H-[1,2,3]oxadiazolo[4,3-α]quinoxalin-1-one (ODQ, 1 μM).

Significance

These findings reveal the vasorelaxant effect of ADIS, through endothelium-independent pathway. It acts as a Ca^2 + channel blocker through both intracellular and extracellular Ca^2 + release.     

Functional assessment of cryopreserved human femoral arteries for pharmaceutical research

An established method for cryopreservation that might preserve the vascular and endothelial responses of human femoral arteries (HFAs) to be transplanted as allografts was studied. HFAs were harvested from multiorgan donors and stored at 4 degrees C in saline solution before cryostorage. Thirty HFA rings were isolated and randomly assigned to one control group of unfrozen HFAs (eight rings) and one group of cryopreserved HFAs (22 rings).Cryopreservation was performed in RPMI solution containing dimethylsulfoxide (DMSO) and the rate of cooling was -1 degrees C/min until -40 degrees C and faster rates until -150 degrees C was reached. The contractile and relaxant responses of unfrozen and frozen/thawed arteries were assessed in organ bath by measurement of isometric force generated by the HFAs.After thawing, the maximal contractile responses to the contracting agonist tested (noradrenaline) were in the range of 43% of the responses in unfrozen HFAs. The endothelium-independent responses to sodium nitroprusside were not altered whereas the endothelium-dependent relaxant responses to acetylcholine were weakly altered.The cryopreservation method used provided a limited preservation of contractility of HFAs, a good preservation of the endothelium-independent relaxant responses, and a good preservation of endothelium-dependent relaxation. It is possible that further refinements of the cryopreservation protocol, such as a slower rate of cooling and a more controlled stepwise addition of DMSO, might allow better post-thaw functional recovery.     

Aqueous extract of Schizandra chinensis fruit causes endothelium-dependent and -independent relaxation of isolated rat thoracic aorta

An aqueous extract of Schizandra chinensis fruit (ScEx) has long been used to promote the vascular health of postmenopausal women in Korea. This study investigated the ability of ScEx to relax rat aorta constricted with norepinephrine (NE) and the mechanism(s) of such relaxation. ScEx induced partial, endothelium-dependent relaxation. In particular, the relaxation induced by lower concentrations of ScEx (0.1 and 0.3 mg/ml) was largely endothelium-dependent, and was essentially abolished by N^G-nitro-l-arginine, methylene blue, 1H-[1,2,3] oxadiazole [4,4-a] quinoxalin-1-one, indomethacin, or ICI 182,780. The results indicate that the response to ScEx involves enhancement of the nitric oxide (NO)-cGMP system, and that it occurs via estrogen receptors. The magnitude of the inhibition with these treatments decreased with increasing ScEx concentration, however, indicating that other vasorelaxation mechanisms are involved, which depend on the ScEx concentration. Calcium concentration-dependent contraction curves in high potassium depolarization medium were shifted significantly to the right and downward after incubation with ScEx (0.3 and 1.0 mg/ml), implying that ScEx is also involved in inhibition of the extracellular calcium influx to vascular smooth muscle. These data demonstrate that ScEx caused both endothelium-dependent and -independent vasorelaxation, which may contribute to understanding the cardiovascular protective effect of ScEx.