Ann Thorac Surg 2002;73:1606-1614
© 2002 The Society of Thoracic Surgeons
Original article: general thoracic
Endothelial cell injury induced by preservation solutions: a confocal microscopy study
Francesco Alamanni, MDa,*,
Alessandro Parolari, MD, PhD*a,*,
Rossana Visigalli, BS, PhDb,
Ovidio Bussolati, MD, PhDb,
Patrizia Rubini, MD, PhDc,
Roberto Sala, MD, PhDb,
Luigi Bonati, MDc,
Gian Carlo Gazzola, MDb,
Paolo Biglioli, MDa,
Valeria Dall’Asta, BSb
a Department of Cardiac Surgery, Centro Cardiologico-Fondazione I Monzino IRCCS, University of Milan, Milan, Italy
b Department of Experimental Medicine, Unit of General and Clinical Pathology, University of Milan, Milan, Italy
c Institute of General Surgery and Organ Transplantation, University of Parma, Parma, Italy
Accepted for publication January 21, 2002.
* Address reprint requests to Dr Parolari, Department of Cardiac Surgery, University of Milan, Centro Cardiologico-Fondazione I Monzino IRCCS, Via Parea 4, 20138 Milan, Italy
e-mail: alessandro.parolari{at}cardiologicomonzino.it
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Abstract
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Background. We evaluated the effects of standard preservation solutions on cultured human greater saphenous vein endothelial cells.
Methods. Endothelial cells (eight strains) were preincubated for 6 or 24 hours at 4°C in Celsior, Euro-Collins, St. Thomas Hospital II, and University of Wisconsin solutions, reincubated in warm oxygenated culture medium 199, and observed up to 48 hours. Culture viability was assessed through cell counting and confocal microscopy of calcein loaded cells.
Results. Incubation in both Euro-Collins and St. Thomas, but not in Celsior or University of Wisconsin solutions, caused significant cells losses and diffuse morphological damages characterized by solution-specific distinctive alterations. Injury caused by 6-hour, but not by 24-hour treatment, was reversible.
Conclusions. The incubation with Celsior and University of Wisconsin solutions substantially preserved endothelial viability and proliferative capability. Conversely, a prolonged incubation in either Euro-Collins or St. Thomas solutions caused severe and potentially irreversible damage referable to the induction of, respectively, apoptotic or necrotic changes.
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Introduction
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One of the basic requirements for successful cardiac transplantation and myocardial preservation during cardiac surgery is adequate protection of the heart tissues, usually achieved by cold incubation with preservation or storage solutions. It is generally accepted that a satisfactory recovery of organ function critically depends on the achieved degree of successful protection of both myocytes and vascular endothelium.
Indeed, endothelial dysfunction can cause impaired vasomotor function after successful cardiac and heart transplant surgery [1]; in addition, several experimental evidences suggest that endothelial damage can be related to the preservation and cardioplegic protocols currently employed in the clinical practice [1–11].
The aim of this study is to quantify the degree of endothelial damage caused by the incubation in several standard solutions commonly employed for myocardial preservation and storage. The study has been performed in a model of human adult macrovascular endothelium in vitro, consisting of human endothelial cells derived from saphenous veins (HSVECs). The viability of endothelial cells, exposed to storage solutions under hypothermic conditions, was assessed either immediately after the treatment or after a 48-hour restoration of normal growth conditions, so as to simulate a long-term reperfusion. The effects of storage solutions were also evaluated through confocal laser scanning microscopy of cells preloaded with calcein acetoxy-methylester dye, a method developed by our laboratory [12–14] that allows the real-time assessment of cell membrane integrity as well as the evaluation of morphological changes associated with cell toxicity.
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Material and methods
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Cell culture
The endothelial cell strains have been obtained from remnants of greater saphenous vein harvested for coronary bypass procedures in 8 male patients, age range 50 to 65, who underwent elective coronary bypass graft procedures. The cultures were established according to the method of Jaffe and colleagues [15], with minor modifications [13]. HSVECs were routinely cultivated in M199 supplemented with 20% fetal bovine serum, heparin (90 U/ml), endothelial cell growth supplement (50 µg/ml) and glutamine (2 mM) on plasticware coated with 2 µg/cm2 collagen (Collagen S, Boehringer Mannheim Italia, Milan, Italy) under the following conditions: pH 7.4, atmosphere 5% CO2 in air, temperature 37°C. Culture medium was refreshed every 2 days. Cultures, periodically tested with confocal microscopy for endothelial markers, were homogeneously positive for von Willebrand factor and CD31 and internalized fluorescent acetylated low-density lipoprotein (LDL) [13]. The experiments were performed with subconfluent cultures between the third and the sixth passage in vitro.
Experimental protocol
At the beginning of each experiment, cells were washed three times with warm oxygenated culture medium 199 (M199) and then incubated for 6 or 24 hours with one of the following solutions: normothermic (37°C) M199 (Sigma-Aldrich Italia, Milan, Italy); hypothermic (4°C) M199 (Sigma-Aldrich Italia); hypothermic (4°C) Celsior (Imtix Sangstat, Lyon, France); hypothermic (4°C) Euro-Collins (SALF, Bergamo, Italy); hypothermic (4°C) St. Thomas II (SALF); or hypothermic (4°C) Wisconsin (Dupont Pharma, Cologno Monzese, Italy).
The composition of these solutions is shown in Table 1.
At the end of the treatment, reperfusion was simulated by removing the preservation fluid, washing three times with warm M199, and incubating cells with complete culture medium at 37°C in an atmosphere of 5% CO2 in air up to 48 hours. In normothermic controls maintained in M199, the medium was changed at the corresponding times with the same procedures to eliminate possible differences in cell counts due to repetitive washing of the endothelial cell monolayer.
The assessment of culture conditions, performed both by direct cell count and confocal microscopy, was performed at the following times: 1) before incubation of the cell cultures with the different storage solutions (baseline); 2) after 6 or 24 hours of incubation with storage solutions at 4°C (6-hour or 24-hour incubation); 3) after 24 hours of reperfusion in complete culture medium at 37°C (24-hour reperfusion); 4) after 48 hours of reperfusion in complete culture medium at 37°C (48-hour reperfusion).
Direct cell count
Cell loss was assessed by determining cell number of the endothelial population, using a cell counter ZM (Coulter Electronics Ltd, Luton, UK) after trypsinization of the cultures. For these experiments, 50 x 103 HSVECs were seeded into 2-cm2 wells of disposable 24-well trays (Nunc AS, Rosklide, Denmark) and grown for 3 to 5 days in complete growth medium, renewed 24 hours before the experiment. Under these conditions HSVEC cultures were subconfluent at the time of the experiment. Cell number was expressed as cells per cm2 of culture surface and presented as mean ± 1 standard deviation. Repeated measure analysis of variance tests, with Bonferroni correction, were used for statistical evaluation. A commercial statistical software (SPSS Version 8.0, SPSS Inc, Chicago, IL) was used; in selected cases, the percentage changes from the baseline were calculated. A p value less than 0.05 was considered statistically significant.
Confocal microscopy
The viability of HSVECs was also assessed through confocal laser scanning microscopy of cells preloaded with calcein acetoxy-methylester (calcein-AM, Molecular Probes Inc, Eugene, OR). This poorly fluorescent fluorescein derivative passively crosses the cell membrane as an electrically neutral form to be converted by intracellular esterases into the negatively charged, fluorescent calcein that is retained in the intracellular compartment as long as plasma membrane is intact. However, the dye rapidly leaks from cells with compromised membranes, even in the presence of residual intracellular esterase activity [13]. Therefore, the maintenance of intracellular calcein fluorescence can be taken as evidence of the preservation of cell membrane integrity, and the method can be effectively employed to discriminate between apoptosis and necrosis [13, 14].
Confocal studies were carried out with a Molecular Dynamics Multiprobe 2001 system (Molecular Dynamics, Inc, Sunnyvale, CA) equipped with an argon laser and based on a Nikon inverted microscope (Nikon Phase Contrast 2, Nikon Instruments SPA, Florence, Italy). HSVECs (Corning Costar Italia, Milano, Italy), seeded into a 10-cm2 dish (COSTAR) at a density of 5 x 104 cells/cm2 3 to 5 days before the experiment, were placed, after the treatments detailed for each experiment, in a thermostatted chamber with complete growth medium supplemented with 2 µM calcein-AM. After the stabilization of the signal, usually reached within 30 minutes, cells underwent observation with laser intensity, photomultiplier setting, and gain kept constant. Cultures were observed through a 20x objective with a pinhole set at 100 µm (excitation wavelength of 514 nm employing a primary beamsplitter at 535 nm and a final barrier filter 570 long-pass).
Image processing was performed on a Silicon Graphics Personal Iris workstation (Image Space Software, Molecular Dynamics, Cupertino, CA). Intensities were expressed on a scale from black (zero) to white (maximal intensity, 255) and images were rendered in a 256 red, green, blue pseudocolors scale from black (zero), through different hues of blue, yellow, and red, to white (255) or in gray tonalities.
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Results
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Cell count
When HSVECs were incubated for 6 hours in different myocardial storage and preservation solutions at 4°C (Fig 1),
a statistically significant cell loss was observed in cultures incubated in Euro-Collins (-64% compared to baseline) and St. Thomas II solutions (-39%); on the contrary, no significant cell loss was detected after hypothermic incubation in M199, Celsior, or Wisconsin solutions. After a 24-hour reperfusion, cell number was still significantly lower than baseline for HSVECs incubated in Euro-Collins (-40%), and St. Thomas II solutions (-26%), while HSVECs incubated in warm (37°C) or hypothermic (4°C) M199, as well as in Celsior and Wisconsin solutions, showed a significant growth (+93%, +36%, +42%, and +49%, respectively). After 48 hours of reperfusion, cultures preincubated in all media but Euro-Collins solution exhibited significant increases in cell number, ranging from +91%, for cells incubated in St. Thomas II solution, to +196% for cells maintained in M199 at 37°C. Intergroup comparisons of the cell counts at each time point are reported in Table 2.
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Fig 1. Two sets of cells were treated in parallel for each strain of human saphenous vein endothelial cells (HSVECs). At time 0, medium was substituted with normothermic culture medium (37°C M199), with hypothermic culture medium (4°C M199), or with the indicated hypothermic (4°C) storage solutions. After a 6-h treatment, cells were incubated in normothermic M199 up to 48 h. At the indicated times, cell number was determined as described in Material and Methods. Histograms are means of eight triplicate values each obtained in a distinct strain of HSVECs with SD.
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Data obtained after a preservation treatment prolonged for 24 hours are shown in Figure 2,
and the corresponding cell count comparisons at the different time points are displayed in Table 3. Marked cell losses were exhibited by endothelial cultures incubated in Euro-Collins (-64%) and in St. Thomas II solutions (-84%), while not statistically significant changes were observed in cultures incubated in hypothermic M199 (-11%), Celsior (-15%), and Wisconsin (-6%) solutions. Cultures incubated under control, normal culture conditions underwent a 45% increase of cell number. After 24 hours of reperfusion, cell number was still lower than baseline for cells incubated in Euro-Collins (-45%), and St. Thomas II solutions (-74%), while cultures treated with hypothermic M199, Celsior, or University of Wisconsin solutions had not only recovered baseline cell numbers, but underwent a significant proliferation, with counts ranging from +114% to +129% of the pretreatment value. After 48 hours of reperfusion, endothelial cultures incubated in hypothermic M199, Celsior, and Wisconsin solutions had cell numbers markedly higher than baseline (+235%, +224%, and +235%, respectively), while cells remained very sparse in cultures incubated in Euro-Collins (-85%) and St. Thomas II (-87%).
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Fig 2. Two sets of cells were treated in parallel for each strain of human saphenous vein endothelial cells (HSVECs). At time 0, medium was substituted with normothermic culture medium (37°C M199), with hypothermic culture medium (4°C M199), or with the indicated hypothermic (4°C) storage solutions. After a 24-h treatment, cells were incubated in normothermic M199 up to 48 h. At the indicated times, cell number was determined as described in Material and Methods. Histograms are means of eight triplicate values each obtained in a distinct strain of HSVECs with SD.
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Confocal microscopy
Figure 3
shows a representative confocal image of calcein-loaded HSVECs after 6 hours of incubation with the various preservation solutions before (A-F) or after (G-L) a 48-hour reperfusion period. Control cells (A and G) were clearly labeled by the loaded dye and exhibited a characteristic endothelial morphology. As expected from previous results [12, 13], fluorescence was homogeneously distributed throughout the cytoplasm, with a high signal zone roughly corresponding to the nucleus. In images obtained immediately after 6 hours of incubation in Euro-Collins solution (Fig 3D), gross alterations in cell number, shape, and dimensions were evident: a major fraction of the cell population was no longer detectable; very few cells exhibited a spared morphology, while the remaining had lost endothelial features, exhibiting more or less markedly altered shapes or appearing as small, rounded elements with a very high level of intracellular fluorescence. In St. Thomas II solution (Fig 3E) cells showed a lower than control, homogeneous level of fluorescence with a few high-signal elements assuming a rounded shape. However, cell loss appeared less marked than in Euro-Collins-treated cultures. HSVECs incubated with Celsior or Wisconsin solutions (Fig 3C and D) maintained their typical shape with no evident morphological alteration or cell loss. A 48-hour reperfusion under normal growth conditions (Fig 3G-L) caused a substantial recovery in all the endothelial subpopulations with a good restoration of cell shape. However, even at this time point, cell populations treated with Euro-Collins (Fig 3J) or St. Thomas (Fig 3K) exhibited a lower density than endothelial cultures incubated in Celsior or Wisconsin solutions (Fig 3I and L, respectively).
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Fig 3. Confocal images of human saphenous vein endothelial cells (HSVECs) after 6 h of hypothermic preservation and 48 h of additional simulated reperfusion. The incubation medium of HSVECs was substituted for 6 h with normothermic (37°C) culture medium (M199; A and G), hypothermic (4°C) culture medium (M199; B and H), Celsior (C and I), Euro-Collins (D and J), St. Thomas II (E and K), or Wisconsin (F and L) solutions. Cell loading with calcein-AM and confocal observation of representative fields were performed at the end of the treatment (A-F) or after a subsequent 48-h incubation in warm M199 (G-L). The experiment, repeated three times, yielded comparable results. Intensities were expressed on a scale from black (zero) to white (maximal intensity, 255); images were rendered in a 256 RGB pseudocolors scale (L, inset) from black (zero), through different hues of blue, yellow and red, to white (255). (Bar = 20 µm.)
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A more detailed confocal image was obtained after 5 hours of treatment with Euro-Collins and Wisconsin, so as to document the earlier phase of the endothelial damage observed under the two conditions (Fig 4).
The image confirms that the morphological features associated to the damage caused by the two treatments were markedly different. In cultures incubated with Euro-Collins solution (Fig 4B), several cells exhibited small membrane blebs and initial fragmentation. In contrast, St. Thomas-treated cells (Fig 4C) exhibited a widespread loss of endothelial morphology (compare with control, (Fig 4A) with rounding of some elements.
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Fig 4. Confocal images of human saphenous vein endothelial cells (HSVECs) after 5 h of hypothermic preservation. The incubation medium of cells was substituted for 5 h with normothermic culture medium (M199; A, control), hypothermic Euro-Collins (B), or hypothermic St. Thomas II (C) solutions. Cell loading with calcein-AM and confocal observation of representative fields were performed at the end of the treatment. (Bar = 10 µm.)
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Figure 5
reports the confocal images obtained after 24 hours of treatment with the various storage solutions, before (Fig 5A-F) or after (Fig 5G-L) a 48-hour reperfusion period. While these images confirm the relative preservation of endothelial cell morphology and viability after incubation in Celsior and Wisconsin solutions, they indicate that an irreversible endothelial damage was caused by a 24-hour treatment with Euro-Collins or St. Thomas II solutions. Also at this late time point, the morphological changes caused by the two treatments were definitely distinct. In HSVEC populations incubated in Euro-Collins solution (Fig 5D) grossly altered, calcein-positive elements were clearly seen which had lost any morphological features of endothelial cells. Many of these bodies appeared as small round-shaped elements endowed with extremely high levels of fluorescence. In contrast, after prolonged incubation with St. Thomas solution (Fig 5E), no viable, calcein-positive cell was detectable, as if the decrease in calcein level, detected at earlier times (Figs 3E, 4C), had proceeded to a complete loss of the dye from the cells or all cells had detached from the culture substratum. Even after a 48-hour reperfusion period (Fig 5G-L), no recovery of the endothelial populations was detected in cells treated with either Euro-Collins (Fig 5J) or St. Thomas (Fig 5K) for 24 hours.
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Fig 5. Confocal images of human saphenous vein endothelial cells (HSVECs) after 24 h of hypothermic preservation and an additional 48 h of simulated reperfusion. The incubation medium of cells was substituted for 24 h with normothermic culture medium (M199; A and G), hypothermic culture medium (M199, B and H), Celsior (C and I), Euro-Collins (D and J), St. Thomas II (E and K), or Wisconsin (F and L) solutions. Cell loading with calcein-AM and confocal observation of representative fields were performed at the end of the treatment (A-F) or after a subsequent 48-h incubation in warm M199 (G-L). For other details, see the legend to Figure 3. (Bar = 20 µm.)
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Comment
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In this study, we have investigated the effects on endothelial cultures of hypothermic incubation for periods similar to those employed in current clinical practice. The experiments were performed with human saphenous vein endothelial cells (HSVECs), an endothelial model, previously employed to evaluate cell toxicity of storage solutions [16], that is closer to human "adult" endothelium than umbilical vein cells (HUVECs). Two independent methods were employed, cell counting and assessment of endothelial cell morphology and viability, performed with confocal microscopy of living cells preloaded with the vital dye calcein.
We show that a prolonged incubation in two solutions commonly employed for organ storage and preservation, Euro-Collins and St. Thomas, has toxic effects on endothelial cell cultures. Under the same experimental conditions in vitro, good endothelial preservation was instead observed with Celsior and Wisconsin solutions. The in vitro experimental conditions adopted clearly differ from the situation of explanted whole organs, in which previous comparisons among the storage solutions employed here yielded somewhat conflicting results [17]. Nevertheless, the data presented here provide evidence at the cell level for the conclusions reached by several recent reports that point to good preservation outcomes for organs incubated either in Wisconsin or in Celsior solutions [18–20].
At variance with most previous studies [8, 21], cultures were also evaluated after a prolonged restoration of normal growth conditions (up to 48 hours) so as to assess their posttreatment proliferative capability. Cells exposed to Celsior and Wisconsin solutions exhibit a prompt recovery of their proliferative capabilities. On the contrary, endothelial damage induced by a 24-hour preservation treatment in either Euro-Collins or St. Thomas II solution was not followed by any recovery, implying that these storage treatments severely impair cell proliferative capability and thus render the damage irreversible.
Confocal microscopy images have clearly documented that marked, thus far uncharacterized, differences exist between the morphological alterations induced by Euro-Collins and St. Thomas solutions. In Euro-Collins solution, cell shrink and break in small bodies intensively positive for calcein signal. These morphological features definitely recall the apoptotic changes detected in other cell models with the same method employed here [14, 22]. Therefore, these observations indicate that the well-known toxic effects of Euro-Collins solution on endothelium [23, 24] should be attributed to the induction of apoptosis. In contrast, the generalized and irreversible loss of calcein exhibited by cultures treated with St. Thomas II solution for 24 hours points to severe membrane damage and, therefore, to the occurrence of necrosis.
It is noteworthy that an excellent endothelial preservation was achieved with either an intracellular-type (Wisconsin) or an extracellular-type (Celsior) solution. On the other hand, poor results were also obtained with either type of solution, because St. Thomas II has an "extracellular" composition, while Euro-Collins shows "intracellular" features. Moreover, although hypertonicity could contribute to the toxic effects exerted by Euro-Collins solution, as expected by our previous results [13], isotonic St. Thomas II solution also exerted severe endothelial injury. Therefore, we may speculate that formula features other than solution type and tonicity are also relevant for an optimal endothelial preservation. Components such as antioxidants or organic compounds, present in both Celsior and Wisconsin solutions, may account for the protective effects observed. The protocols and methods adopted in the present study would permit a ready identification of such components.
The rational formulation of solutions specifically designed to ensure good endothelial preservation would be of great relevance. Endothelium is indeed the first tissue to be in contact with storage solutions, and the role of endothelial damage in determining postoperative heart or graft dysfunction has been well documented [25–27]. Moreover, ischemia-reperfusion endothelial injury has been repeatedly associated with surgical procedures such as cardiovascular surgery and heart transplantation, where current storage and preservation techniques only allow a limited time for procedure completion. On the other hand, cardioplegic and storage solutions are mainly aimed to protect the cardiomyocyte and, therefore, they are not necessarily suitable for the optimal preservation of vascular endothelium [8]. However, due to the differences between the in vitro conditions adopted in the present study and the situation of endothelium in explanted organs, the results presented here should be considered with some caution and cannot be directly extrapolated to clinical organ preservation. Therefore, how the toxic effects documented here are involved in the endothelial dysfunction encountered in transplanted organs deserves further investigation.
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Acknowledgments
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This work was supported in part by grants from the Italian Ministry of Health (ICS49.2/RF97-24 and ICS030.6/RF00-45) and by a grant from Fondazione Cassa di Risparmio di Parma.
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Footnotes
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* Drs Alamanni and Parolari contributed equally to this study. 
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References
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Abstract
Introduction
