Ann Thorac Surg 1996;62:526-532
© 1996 The Society of Thoracic Surgeons
Original Articles: Cardiovascular
Assessment of Endothelial Preservation in Human Cell Cultures
Thomas Eberl, MD,
Rosmarie Steinlechner, PhD,
Paul Hengster, MD,
Manfred Herold, MD, PhD,
Hans Schröcksnadel, MD,
Willi Salvenmoser,
Martin Rhomberg, MD,
Erich Gnaiger, PhD,
Raimund Margreiter, MD
Departments of Transplant Surgery, Internal Medicine, and Gynecology and Obstetrics, University Hospital Innsbruck, and Department of Zoology, University of Innsbruck, Innsbruck, Austria
Accepted for publication April 6, 1996.
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Abstract
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Background. Impairment of microcirculation due to endothelial cell damage must be considered a limiting factor in organ preservation. The present study aims at a quantitative assessment of preservation-induced injury in cultured human endothelial cells.
Methods. Monolayer cultures of human umbilical vein endothelial cells were exposed to cold (4°C) hypoxic storage in University of Wisconsin solution, histidine-tryptophane-ketoglutarate solution, Euro-Collins solution, and saline solution. Cellular integrity was evaluated by viable cell count, ultrastructural analysis, and prostacyclin release after 24, 48, and 72 hours of storage and subsequent 6 hours of reincubation in culture medium at 37°C. Expression of intercellular adhesion molecule-1 was investigated after 6, 12, and 24 hours of cold preservation and after 6 hours of rewarming.
Results. Cellular viability was best maintained with University of Wisconsin and histidine-tryptophane-ketoglutarate solutions with no significant reduction of cell count up to 72 hours; Euro-Collins solution and saline solution caused a significant decline in cell numbers after 24 hours (p < 0.05). Morphology was best preserved by University of Wisconsin solution. Prostacyclin values were elevated after 24 hours in Euro-Collins solution and saline solution, after 48 hours in histidine-tryptophane-ketoglutarate, Euro-Collins, and saline solutions, and after 72 hours in Euro-Collins solution (p < 0.05, compared with University of Wisconsin solution). ICAM expression was weak after cold storage (24 hours) in University of Wisconsin solution, moderate after incubation in histidine-tryptophane-ketoglutarate and Euro-Collins solutions and intensive after storage in saline solution. In contrast, rewarming caused intensive expression of intercellular adhesion molecule-1 in all experimental groups as compared with controls, which showed baseline expression at any time.
Conclusions. From our results we conclude that in this model cellular integrity is best protected by University of Wisconsin solution, increased prostacyclin release is consistent with morphologic alterations and intercellular adhesion molecule-1 expression is clearly up-regulated in endothelial cells under reperfusion conditions after cold hypoxic storage.
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Introduction
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One of the basic requirements of organ transplantation is adequate preservation of grafts, usually by cold storage after initial flushing. The potential for recovery of organ function after ischemia depends on protection of both the parenchymal cells and the vascular endothelium. The latter represents the first target during cold flush-out and subsequent reperfusion. Endothelial damage causes changes in microcirculation after reperfusion by cellular swelling, platelet accumulation, impairment of procoagulant and anticoagulant properties, and leukocyte adherence.
The mechanisms responsible for the different ischemic tolerances of various transplantable organs are not fully understood [1]. There is increasing evidence that preservation of the liver is limited by the integrity of the sinusoidal lining [2]. In contrast, the tissue components responsible for ischemia tolerance of thoracic organs, in particular the lung, are still a matter of discussion. With respect to these organs there is some indication that the endothelium may play a crucial role [3].
In contrast to numerous animal models using whole organs or myocardial and parenchymal cells, only a few studies concerning the isolated endothelial compartment have been reported so far [4–7]. The aim of this study was to investigate the effects of various preservation solutions under hypoxic hypothermic conditions, including simulated reperfusion in the experimental protocol. All parameters were evaluated on monolayer cultures of human endothelial cells. Combined assessment of morphologic, biochemical, as well as immunologic findings was performed, which facilitates a more accurate evaluation of preservation injuries.
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Material and Methods
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Cultivation of Human Endothelial Cells
Human umbilical vein endothelial cell cultures were grown using a modification of the technique described by Jaffe and colleagues [8]. Cells were harvested from umbilical cords by incubation with collagenase type II (Sigma Chemicals Co, St. Louis, MO) for 15 minutes and plated into 25-cm2 culture flasks (Falcon, Becton Dickinson Co, Lincoln Park, NJ) precoated with 28 µg/mL of human fibronectin (HFN Inotech AG, Zürich, Switzerland). Subsequent cultivation was maintained at 37°C in a humidified atmosphere (5% CO2 in air) using endothelial cell growth medium (PromoCell, Heidelberg, Germany) supplemented by 2% fetal bovine serum, endothelial cell growth factor, and gentamycin plus amphotericin B. Cells were identified as endothelial by the typical cobblestonelike morphologic appearance and positive factor VIII rAG staining. Only cultures of high purity without fibroblast contamination were accepted for further experimentation. Trypsin–ethylendiamine tetraacetic acid solution (0.025/0.02%) was applied for subcultivation. Second and third passages were used for preservation experiments. Endothelial cells were seeded onto 24-well plates (2 cm2 per well) for cell counts and biochemical measurements, and glass coverslips (diameter, 12 mm) for morphologic and immunohistochemical screening. Surfaces were precoated with fibronectin. The initial seeding density was 25,000 ± 5,000 cells/cm2. Experiments were initiated after a cultivation period of 5 days, when a confluent monolayer was established.
Preservation Experiments
The following organ preservation solutions were tested: University of Wisconsin solution (UW) (Viaspan, DuPont Pharmaceuticals, Wilmington, DE), histidine-tryptophane-ketoglutarate solution (HTK) (Custodiol, Koehler Chemie GmbH, Alsbach, Germany), Euro-Collins solution (EC) (Fresenius AG, Bad Homburg, Germany), and 0.9% NaCl solution (saline solution). Cells grown in culture medium were used as controls. The medium for controls was changed at the beginning of each preservation experiment.
After removing the culture medium and washing the monolayer with phosphate-buffered saline solution (PBS), 1.5 mL of cold preservation solution (4°C) was added and the plates stored at 4°C. During the storage period, culture plates were gased with nitrogen to simulate hypoxia. Storage periods of 24, 48, and 72 hours were chosen for cell counts, morphologic and biochemical assessment, and 6-, 12-, and 24-hour periods for detection of intercellular adhesion molecule expression. After the particular cold storage period, reperfusion was simulated by removing the preservation fluid, washing with PBS and reincubation in culture medium at 37°C in an atmosphere of 5% CO2 in air for 6 hours (rewarming/reoxygenation). In controls the medium was changed at the corresponding times.
Cell Counts and Viability Assessment
Viability was assessed by trypan blue exclusion after removing the supernatant and washing with PBS. Cells were counted under an inverted microscope using an ocular grid (Zeiss, Oberkochen, Germany) [9]. Ten different areas were examined in each well and the results expressed as cells per centimeter squared. The total cell count and the number of viable cells were assessed in duplicate, with seven cell lines from different umbilical cords.
Morphologic Investigations
Cellular integrity was observed in vitro by means of a Zeiss Axiovert 135 inverse microscope (Zeiss) using an interference contrast technique described by Nomarski [10], which yields a three-dimensional image. After screening the area of each culture dish, photomicrographs of representative regions were selected. Morphologic changes of the monolayer (intercellular contacts) and cytoplasm (granulation, vacuole formation, disintegration, cellular swelling) were assessed blindly.
Biochemical Evaluation
In the supernatant, basal release of prostaglandin I2 (prostacyclin) was quantified as a biochemical marker of endothelial cell activation. A radioimmune assay (Advanced Magnetics, Cambridge, MA) for 6-ketoprostaglandin F1 alpha, the stable end product of prostacyclin, was used. Blank values (solution without cells) were subtracted from the experimental values. Each measurement was performed in duplicate with seven independent cell lines. Results were expressed as pg/106 cells.
Determination of Intercellular Adhesion Molecule-1 Expression
The preservation solution or culture medium was removed, and the cells were washed twice with PBS, fixed in 70% methanol, and stored at -20°C. Immunohistochemical staining was performed within 2 days of fixation. The samples were washed twice with PBS and incubated with 50 µL supernatant of the 7F7 anti-intercellular adhesion molecule-1 (ICAM-1) monoclonal antibody [11] at room temperature for 45 minutes. After three further washing steps with PBS, conjugate fluorescein isothiocyanate-labeled goat antimouse IgG (Clonab Ig FITC; Biotest, Dreieich, Germany) was added in a dilution of 1:50 (45 minutes, room temperature). After three further washing steps, coverslips were examined under a Zeiss Axioplane microscope (Zeiss), with incident light fluorescence equipment and an excitation filter suitable for conjugated fluorescein isothiocyanate. Blank values representing background activity were obtained by incubation with PBS instead of the anti-ICAM-1 antibody and subsequent addition of Clonab conjugated fluorescein isothiocyanate-labeled IgG.
Results were expressed semiquantitatively as baseline, weak, moderate, or intensive staining. The ICAM expression was assessed in a blind test.
Statistical Analysis
Average values were obtained from duplicate measurements in each experiment and mean values calculated from the results of seven experiments (n = 7, for each solution). Results were expressed as mean values ± standard error of the mean. The experimental groups were compared by two-way analysis of variance, and differences were considered significant when p values were less than 0.05.
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Results
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Cell Counts
Counts of viable cells after 24, 48, and 72 hours of cold hypoxic storage and the respective rewarming/reoxygenation periods are shown in Figure 1
. Compared with controls, a significant decrease in cell number was observed after cold storage with EC and saline solution at all times (Fig 1A). After preservation plus 6 hours of rewarming/reoxygenation, a further reduction in cell number as compared with cold storage was apparent with EC and UW but not with saline solution and HTK (Fig 1B).
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Fig 1. . Viable cell counts (mean values ± standard error of the mean) after cold storage periods. (A) *p < 0.05 compared with control and University of Wisconsin solution [UW]; #p < 0.05 compared with control, University of Wisconsin solution, and histidine-tryptophane-ketoglutarate solution [HTK]) and after rewarming/reoxygenation. (B) *p < 0.05 compared with control; +p < 0.05 compared with control and HTK; #p < 0.05 compared with control, UW, and HTK; ^p < 0.05 compared with all other groups. (EC = Euro-Collins solution; SA = saline solution.)
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Morphology
The normal morphologic appearance of the control group is shown in Figure 2. The cells form a typical confluent monolayer owing to contact inhibition. Morphologic integrity was well preserved after storage in UW solution for 24 hours (Fig 3A) and was maintained for up to 48 hours. A slight retraction of the monolayer and granulation of the cytoplasm was seen after 72 hours. No signs of morphologic changes were observed after rewarming after 24 hours of cold storage in UW solution. However, a scattered retraction of the monolayer, vacuoles, and granulation of the cytoplasm were demonstrable after 48 and to a larger extent after 72 hours and rewarming/reoxygenation. Storage in HTK solution for 24 hours resulted in a clear retraction of the monolayer structure and granulation of the cytoplasm (Fig 3B). These alterations were further aggravated after 48 and 72 hours by cell swelling and the appearance of vacuoles. Morphologic recovery from 24 hours of cold storage was seen after rewarming/reoxygenation, whereas signs of metabolic stress (vacuoles, granules) persisted after 48 hours plus rewarming/reoxygenation. A severe retraction of the monolayer was visible after 72 hours of cold storage and rewarming/reoxygenation.
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Fig 2. . Normal morphologic appearance of endothelial monolayer. (Control group; magnification x200 before 46% reduction.)
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In contrast to preservation in UW and HTK, cold storage in EC solution already caused severe cellular damage after 24 hours (Fig 3C
), with disruption of the monolayer, membrane damage with subsequent cellular swelling, and disintegration of the cytoplasm. Filamentous cytoplasmatic retraction fibers were visible in the intercellular space. The alterations were more pronounced after longer periods of storage. No recovery could be observed after reincubation in culture medium.
After storage in cold saline solution for 24 hours a severe retraction of the monolayer with only occasional cell contacts and cytoplasmatic swelling was detectable (Fig 3D). Longer storage periods led to complete destruction of cellular integrity. A partial recovery from 24 hours of storage was detectable after rewarming/reoxygenation but not after longer storage periods.
Prostacyclin Release
Basal prostacyclin release after experimental cold storage and reincubation periods is depicted in Figure 4. Significant differences were observed between UW and EC after 48 hours and between UW and control after 72 hours as well as between saline solution and controls after 48 hours or more and 6 hours of rewarming/reoxygenation.
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Fig 4. . Basal prostacyclin release (mean values ± standard error of the mean) after cold storage periods. (A) *p < 0.05 compared with University of Wisconsin solution [UW], and after rewarming/reoxygenation. (B) *p < 0.05 compared with controls. (EC = Euro-Collins solution; HTK = histidine-tryptophane-ketoglutarate solution.)
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Intercellular Adhesion Molecule-1 Expression
Baseline expression of ICAM-1 was observed in control cells at all times, as described for endothelial cells that are not activated [12]. University of Wisconsin and HTK-preserved cells showed baseline expression after 6 and 12 hours. After 24 hours, however, weak (Fig 5A) and moderate expression was visible in UW- and HTK-preserved cultures, respectively. Euro-Collins- and saline solution-treated cells showed weak ICAM-1 expression after 6 and 12 hours. Cold storage for 24 hours resulted in moderate expression in EC-treated cultures and intensive staining after preservation with saline solution.
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Fig 5. . Expression of intercellular adhesion molecule-1 on endothelial cells after preservation with University of Wisconsin solution. Weak expression (A) after cold storage for 24 hours and intensive staining after subsequent rewarming for 6 hours (B) was detected. (Immunohistochemical staining; original magnification, x400.)
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As a consequence of rewarming/reoxygenation after 6 and 12 hours of cold storage, moderate ICAM-1 expression was observed in the UW, HTK, and EC groups and weak expression in the saline group. Intensive expression of ICAM-1 was detectable in the UW and HTK groups after 24 hours and subsequent rewarming/reoxygenation (Fig 5B). Because of the severe cell membrane damage and subsequent unspecific staining, ICAM expression could not be analyzed in the EC and saline groups after 24 hours.
Expression of ICAM-1 was negative in the blank samples at all times. A summary of ICAM-1 expression is given in Table 1.
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Comment
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Long-distance procurement of thoracic organs is limited by short ischemia tolerance. In contrast to myocardial protection, which has been extensively investigated, little is known about the mechanisms involved in ischemic endothelial damage.
Several preservation effects on the endothelium can be studied with the cell culture technique. The relevance of our method, using human umbilical endothelial cells, is limited by its unspecificity concerning the heterogeneity of microvascular endothelium in different organs. Major properties and response-to-injury mechanisms of endothelial cells, however, are shared by the different subtypes [13, 14] and the human umbilical vein endothelial cell culture represents a well-established model [6–9, 13–17]. A significant advantage of this method should be the human origin of cells, which makes it possible to exclude species-related differences [7, 15].
Membrane integrity was assessed by trypan blue exclusion. This represents an accurate and well-established method, although it is not an absolute indicator of cellular viability. A correlation between trypan blue staining and lactate dehydrogenase release was described by Rauen and colleagues [4].
The present results show that preservation of human endothelial cells under cold hypoxic storage is best provided by UW and HTK solutions. In contrast, EC solution and saline solution cause a significant reduction in viable cell counts and severe alteration of morphology. In terms of viable cell count, there was no significant difference between the UW and HTK groups, but morphologic investigations indicated considerable alterations of the monolayer structure in the HTK group. Loss of intercellular contacts in vivo may lead to exposure of the underlying extracellular matrix to blood circulation with subsequent platelet adherence, formation of microthrombi, and complement activation [13, 14]. In contrast, preservation with UW solution resulted in a morphologically characterized protection of cellular integrity.
Morphologic observations were supported by measurement of prostacyclin release. Prostaglandin I2 levels were elevated after cold storage under conditions known to cause cellular distress. Synthesis of prostaglandins, in particular of prostaglandin I2, represents one of the main biochemical pathways in endothelial cells [13]. Increased prostaglandin I2 levels resulting from exposure of endothelial cells to hypoxia and energy depletion have been described by several researchers [16, 17]. Stimulation of prostaglandin synthesis under hypoxia is a consequence of activation of phospholipase A by elevated cytosolic calcium [16] or oxygen radicals [17] with subsequent release of arachidonic acid from cell membranes. Prostacyclin was described as a new parameter for endothelial injury under hypothermic preservation conditions in the bovine model by our group and this assay was applied here with human umbilical vein endothelial cells [7]. The large variation of results is not surprising in view of the variability of prostaglandin production in different cell lines [16].
From our results obtained by morphologic and biochemical assessment of cellular viability it can be concluded that human umbilical vein endothelial cells are best preserved by UW solution. These findings are supported by experimental and clinical studies, which suggest a superiority of UW solution in preservation of liver, pancreas, and small bowel as well as cardiac and lung allografts [18, 19].
However, impairment of microcirculation by leukocyte adherence during reperfusion even after short cold storage in UW solution was reported by Gonzales and colleagues [20] using in vivo fluorescence microscopy. From this and other studies [21] it becomes clear that leukocyte–endothelial cell interaction is a critical factor in the early reperfusion period and can cause the capillary "no-reflow" phenomenon. There is evidence that leukocyte rolling and sticking to the endothelium is mediated by activation of adhesion molecules, which can be stimulated by hypoxia and oxygen radicals [12, 22, 23]. Among others, ICAM-1 is one of the most relevant molecules involved in endothelial–leukocyte interaction. Therefore, we investigated the expression of ICAM-1, which interacts with leukocyte function antigen on the cell surface of lymphocytes. After cold storage only weak-to-moderate expression was observed and correlated with deterioration of cellular integrity. In contrast, rewarming resulted in a considerable up-regulation of ICAM-1 on the cell membrane, but no correlation with the degree of cell damage was detectable in the absence of leukocytes.
There is increasing evidence that ischemic endothelial injury not only impairs microcirculation but can lead to increased graft immunogenicity by up-regulating adhesion molecules and HLA antigens [24, 25]. Recent experimental and clinical data suggest that ischemic endothelial damage may contribute to a higher incidence of acute rejection episodes as well as chronic obliterative transplant vasculopathy [24, 25].
The endothelial cell culture model could be used for further studies investigating the underlying mechanisms and as a screening method for new preservation solutions.
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Acknowledgments
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We gratefully thank Marie-Luise Kunc and Christine Schwitzer for technical assistance and Johannes Möss, MD, for providing the monoclonal anti-ICAM antibody.
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Footnotes
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Address reprint requests to Dr Margreiter, Department of Transplant Surgery, University Hospital Innsbruck, Anichstrasse 35, A-6020 Innsbruck, Austria.
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Abstract
Introduction
