Ann Thorac Surg 2006;82:1742-1746
© 2006 The Society of Thoracic Surgeons
Original Articles: Cardiovascular
Effect of Thawing on Nitric Oxide Synthase III and Apoptotic Markers in Cryopreserved Human Allografts
Hans J. Geissler, MDa,*,
Uwe M. Fischer, MDa,
Saskia Foerster, MDa,
Andreas Krahwinkel, MSb,
Albert Antonyan, MDa,
Axel Kroener, MDa,
Klaus Addicks, MDb,
Uwe Mehlhorn, MDa,
Wilhelm Bloch, MDc
a Department of Cardiothoracic Surgery, University of Cologne, Cologne, Germany
b Department for Anatomy I, University of Cologne, Cologne, Germany
c Institute for Molecular and Cellular Sports Medicine, German Sports University, Cologne, Germany
Accepted for publication May 25, 2006.
* Address correspondence to Dr Geissler, Klinik für Herz- und Thoraxchirurgie, im Klinikum der Universität zu Köln, Joseph-Stelzmann-Str. 9, 50924 Köln (Email: hans.geissler{at}uk-koeln.de).
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Abstract
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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.
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Introduction
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Valved conduits for congenital heart surgery, valve replacement in acute endocarditis, and the Ross procedures are well-established indications for the use of cyropreserved allografts. Despite multiple reports on acceptable or excellent long-term graft performance, early degeneration of cryopreserved allograft heart valves has been observed in a variable and unpredictable extent [1–3]. A number of possible causes for cellular damage resulting in early allograft failure have been investigated; among others, immunologic host response, hypoxia, and chemical injury during preservation [4–7]. Although myocardial nitric oxide (NO) release has been predominantly investigated in ischemia-reperfusion injury, NO release from myocardium may be triggered by a number of unspeficic cellular injuries, as those associated with allograft processing. The role of NO release in myocardial injury is still controversial, with conflicting data on protective and deleterious effects [8]. However, one interesting effect of NO release is the induction of the apoptotic pathway [9, 10]. The occurrence of apoptosis in cryopreserved allografts has been shown experimentally and it has been identified as a plausible mechanism through which various forms of unspecific cell damage may result in major allograft cell loss [11, 12]. Therefore, the purpose of this study was to investigate the effect of thawing on myocardial constitutive nitric oxide synthase (NOS-III) and markers of the apoptotic pathway in human cyropreserved allografts.
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Material and Methods
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Allograft Heart Valves
All institutional protocols and regulations for organ and tissue donation were followed. The Ethics Committee of the Medical Faculty of the University of Cologne approved this study and waived the requirement for obtaining consent from next of kin for these studies.
Myocardial tissue from the ventricular outflow tract of ten human allograft heart valves (3 aortic and 7 pulmonary), unsuitable for human implantation due to structural valvular defects, was used for analysis. Structural valvular defects included inadvertent injury during preparation, beginning calcification or fibrotic thickening of semilunar leaflets. The allografts investigated had been treated in an identical fashion to those intended for implantation, including sterile harvest, 24 hour storage in a quadruple antibiotics solution at 4°C, controlled freezing, and storage in liquid nitrogen at –190°C (Table1). Mean donor age was 52.6 ± 8 years (7 male). Cause of brain death was traumatic in 5 cases, subarachnoidal bleeding in 3, and intracranial bleeding in 2.
Thawing Conditions and Experimental Groups
Immediately after removal from liquid nitrogen, the sterile wrapping of the allograft was opened and four evenly sized sections of myocardial tissue were excised. One section of myocardium was immediately fixated in 4% paraformaldehyde (PFA) at 4°C (group Control). Embedding for cryo-thin sections of Control group myocardium was started after 6 hours in PFA. The remaining three sections of myocardium were thawed according to the standard clinical protocol for allograft thawing used in the operating room (Table 2). During thawing after the standard clinical protocol no additive to Ringer's solution or Roswell Park Memorial Institute (RPMI) solution was used in the Standard group; in the Standard-LNA group the NOS-inhibitor L-NA was added to Ringer's solution and RPMI at a 10–4 M concentration and in the Standard-AT-II group angiotensin II was added at a 10–8 M concentration. After thawing, myocardium from the Standard, Standard-LNA, and the Standard-AT-II groups was fixated in 4% PFA at 4°C for 6 hours. Thereafter, myocardium was washed repeatedly in 0.1 M phosphate-buffered saline (PBS) and embedded over night in a 18% sucrose solution in 0.1M PBS. Sucrose-embedded myocardium was stored in tissue freezing medium at –80°C. For analysis, 8 µm thin sections were used.
Immunohistochemistry for NOS-III, Caspase-3, and Poly-ADP-ribose Polymerase(PARP)
Eight micrometer slices from biopsy samples were placed in a bathing solution of 3% hydrogen peroxide and methanol for 20 minutes, and then cells were lysed with 0.25% Triton X-100 (Rohm & Haas Co, Philadelphia, PA) in ammonium chloride (0.5 mol/L). Thereafter, specimens were treated with 5% bovine serum albumin solution in tris-buffered saline (TBS) 0.05 mol/L. Before each step, thin sections were rinsed three times in TBS buffer 0.05 mol/L. Incubation with primary rabbit anti-NOS-III antibody (1:1500; Biomol, Hamburg, Germany), detecting the activated and translocated NOS-III [8], rabbit anti- guanosine monophosphate (anti-cGMP) (1:700; Biogenesis, Poole, England), rabbit anti-active caspase-3 (1:500, Pharmingen, San Diego, CA), and rabbit anti-PARP (1:250, Promega, Madson, WI) were performed in a TBS-based solution of 0.8% bovine serum albumin for 12 hours at 4°C. After rinsing with TBS, the sections were incubated with the corresponding secondary biotinylated goat anti-rabbit antibody (1:400, DAKO, Glostrup, Denmark) for 1 hour at room temperature. A streptavidin-horseradish-peroxidase complex was then applied as a detection system (1:150) for 1 hour. Finally, staining was developed for 10 to 20 minutes with 3,3-diaminobenzidine tetrahydrochloride in PBS 0.1 mol/L .
Densitometry (NOS-III, Caspase-3, and PARP)
For quantitative density analysis, the gray values of 50 cardiomyocytes from 10 randomly selected areas were measured. The staining intensity was reported as the mean measured cardiac myocyte gray value minus the background gray value. The background was measured at two cell-free areas of the slice. For staining intensity detection a Zeiss Axiophot microscope (Jena, Germany) connected to a camera was used, and analysis was performed by using the Optimas 6.01 image-analysis program (Optimas 6.01, Media Cybernetics, Silver Spring, MD) installed on a personal computer.
Colorimetric Caspase-3 Assay
Whereas caspase-3 activation was analyzed by immunohistochemistry, caspase functionality was investigated by a colorimetric caspase-3 assay (BioVision, Mountain View, CA). The assay was performed for the groups Standard, Standard-LNA, and Standard-AT-II. No assay was conducted for the group Control, as exposure to PFA would have terminated any caspase-3 related reaction.
Three evenly sized sections of myocardial tissue were excised from the ventricular outflow tract. After thawing under the various conditions according to the experimental groups, myocardial tissue was resuspended in 50 µL of chilled cell lysis buffer and incubated on ice for 10 minutes. After microcentrifugation (10.000 g) for 1 minute, protein concentration of the supernatent was assayed. Protein (50 µg) were diluted with 50 µL of chilled cell lysis buffer and 50 µL of 2X reaction buffer (containing 10 mM dithiothretol [DTT]) was added. Thereafter, 200 µM DEVD-pNA substrate was added and incubated at 37°C for 90 minutes. Samples were read at 450 nm in a microtiter plate reader.
Statistical Analysis
Data were analyzed for statistical significance using the 2-tailed Student t test for paired samples with nine degrees of freedom. A p value less than 0.05 was considered significant. Statistical analyses were performed with the software package SPSS for Windows, release 10.0.7 (SPSS Inc, Chicago, IL).
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Results
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Staining intensity assessed by TV densitometry for activated NOS-III, cGMP, active caspase-3, and PARP are depicted in Figures 1 and 2,
respectively. Analyses of gray units for activated NOS-III immunostaining showed significant differences between groups. Direct fixation of myocardium in PFA resulted in a significantly lower number of gray units for NOS-III (group Control; 34.9 ± 11.1) than PFA fixation after thawing after the standard clinical protocol (group Standard; 43.6 ± 14.7; p < 0.001). Likewise, standard thawing with addition of the NOS-inhibitor L-NA (group Standard-LNA; 33.9 ± 12.4) showed a significantly lower gray scale for activated NOS-III than standard thawing with addition of the NOS-stimulator angiotensin II (group Standard-AT-II; 42.9 ± 11.9, p < 0.001). For activated NOS-III gray scales, no significant differences were seen for the comparison between the groups Standard versus Standard-AT-II and Control vs Standard-LNA, whereas comparison of Control vs Standard-AT-II and Standard-LNA vs Standard showed significant differences. Analyses of gray units after cGMP immunostaining showed similar significant differences between groups as for activated NOS-III (Fig 1).
Myocardium from all groups showed a substantial level of activated caspase 3 and PARP-cleavage. The differences, however, between groups were not significant (Fig 2).
Colorimetric assay of caspase-3 activity showed no significant differences between the Standard, Standard-LNA, and the Standard-AT-II group (Fig 3).
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Comment
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Our data show significant NOS-III activation and cGMP production during the thawing process in human cryopreserved allografts. We further demonstrate apoptosis signal-pathway induction as shown by increased caspase-3 activity and PARP cleavage.
NOS-III Activation in Cryopreserved Allograft Heart Valves
Nitric oxide release has been associated with various physiological and pathophysiological processes in cardiac function. Thus, we hypothesized also a potential role for NO production in early degeneration of cryopreserved allograft heart valves. As myocardial NO production mainly follows NOS-III (eNOS)-activation, which is expressed constitutively in both endothelial cells and cardiomyocytes [13], we focused on eNOS-activation during the thawing process in cryopreserved human allografts.
We observed substantial activation of NOS-III in all investigated groups and a correlated production of cGMP. The cGMP production gives strong evidence for the activation of soluble guanylate cyclase by NO [14]. Standard thawing protocol even resulted in significantly higher activation of NOS-III and its NO-cGMP signal pathway as compared with controls, which was effectively prevented by NOS-inhibition. The NOS stimulation with angiotensin II, however, did not further activate NOS-III and NO-cGMP signal pathways which could be explained in two ways: either NOS-III activation already reaches maximum during standard thawing procedure and cannot further be stimulated or the cryopreserved allografts do not respond to angiotensin II during the thawing process.
Thus, as we can clearly demonstrate, increased NOS-III activation and the subsequent activation of NO-cGMP signal pathway during the thawing process, hypotheses on a significant role of NO in the early phase of allograft function are warranted.
Intensive research into the effects of NO on myocardial ischemia-reperfusion has accumulated abundant data on both protective and harmful impacts. While protective effects mainly involve vascular tone regulation [15] and inhibition of thrombocyte accumulation [16] or inhibition of cytokine-release by neutrophils [17], deleterious influence directly targets cellular function by DNA-synthesis inhibition, alteration of mitochondrial function, or inhibition of the ribonucleotide reductase [17, 18].
Furthermore, NO release has been associated with apoptotic cell death [10], which also occurs in endothelial cells and cuspal connective tissue of implanted allografts and seems to be one cause of their loss of cellularity [11].
In order to investigate a potential connection between NO production and apoptosis induction we analyzed the allografts for caspase-3 activation and cleavage of PARP. As the thawing process according to protocol occurred within minutes we focused on one key enzyme (caspase-3) and substrate (PARP) of the apoptosis signal pathway rather than apoptosis termination (eg, terminal dUTP nick end-labeling).
We found substantial caspase-3 activity and PARP cleavage in each group without significant differences among groups. These findings are supported by the results of the colorimetric caspase-3 assay, which showed no significant difference in caspase functionality between groups. This indicates, at least for the present levels of NOS-III activation, that there is no correlation between NO generation and apoptosis induction. However, NO as potential apoptosis inducer cannot be excluded as apoptosis initiation might already have reached maximum at NOS-III levels below controls.
Furthermore, we cannot preclude mechanisms other than NO release as potential apoptosis inducers such as ischemia, hypothermia, reactive oxygen species, and others. Especially for ischemia and(or) ischemia-reperfusion we and others could demonstrate myocardial apoptosis induction in both endothelial cells and cardiomyocytes [10]. Hence, additional studies using apoptosis inhibitors are necessary to determine the exact time course of apoptosis induction.
The allografts investigated in this study have all beeen classified as unsuitable for implantation, due to structural deficiencies. In most cases this meant beginning calcification or fibrotic thickening of semilunar leaflets. Although it is theoretically possible that these deficient valves respond differently on the molecular level to the impact of organ harvest and cryopreservation than unaffected valves, there is no evidence to support this notion. However, allografts suitable for human implantation are generally not available for investigation.
In summary, thawing of cryopreserved human cardiac allografts increases NOS-III activation and could thus lead to graft injuries mediated by NO release. Apoptosis induction can also be found in these grafts and might contribute to graft degeneration in the early phase after implantation. However, future studies are required to further elucidate the following: (1) to what extent NOS-III activation is altered in cryopreserved allografts as compared with normal, nonfailing myocardium; and (2) at which time point apoptosis induction occurs and which mechanisms are involved. These data are necessary for the design of future studies using NOS inhibitors and(or) apoptosis inhibitors as potential new therapeutic strategies in allograft protection.
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Acknowledgments
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This study has been supported by a grant from the Dr.-Karl-Wilder-Foundation, Berlin, of Gesamtverband der Deutschen Versicherungswirtschaft e.V.
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References
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Abstract
Introduction
) and cyclic guanosine monophosphate (cGMP; 
) assessed by TV densitometry (gray units). (*p < 0.033 vs control;
p < 0.005 vs standard; 
p < 0.005 vs Standard-LNA.)
