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 apoptotis 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.