Effect of thawing on nitric oxide synthase III and apoptotic markers in cryopreserved human allografts.

Background: Previous investigations suggested apoptosis as a contributing factor to early failure of allograft heart valves. As myocardial apoptosis may be induced by nitric oxide (NO) release, this study investigated NO synthase [NOS-III] activation and apoptosis induction in human cryopreserved allografts during the thawing process.

Methods: Frozen myocardial tissue from ten human allograft heart valves, unsuitable for implantation, was submitted to the following conditions: (1) thawing in paraformaldehyde (Control), thawing according to the standard clinical protocol (Standard), standard-thawing with addition of the NOS-inhibitor N-omega-nitro-l-arginine (L-NA; Standard-LNA), and standard thawing with the NOS-stimulator angiotensin II (Standard-AT-II). Cryo-thin sections were investigated by immunostaining for activated NOS-III, cyclic guanosine monophosphate (cGMP), activated caspase-3, and poly-ADP-ribose polymerase (PARP). Quantitative analyses was performed by television densitometry.

Results: For activated NOS-III, gray unit values were significantly higher in the Standard and Standard-AT-II group than in the Control and Standard-LNA groups (p < 0.001). Gray unit values for cGMP, a downstream NO-signal-pathway molecule, showed results grossly corresponding to NOS-III activation. Activated caspase-3 and PARP showed high levels of expression in all groups with no significant differences.

Conclusions: Significant activation of NOS-III and subsequent NO-cGMP signal pathway occurs in human cryopreserved allografts during the thawing process and can be significantly reduced by a NOS-III inhibitor administered during thawing. Activation of the apoptosis pathway is also present after thawing, which was not correlated to NOS-III activation. Further experimental investigation focused on the time course and mechanisms of apoptosis and NOS-III activation are required to evaluate NOS and(or) apoptosis inhibitors as therapeutic strategies for improved allograft preservation.