Ann Thorac Surg 1997;63:656-662
© 1997 The Society of Thoracic Surgeons
Original Article: Cardiovascular
Effects of Warm Ischemia on Valve Endothelium
Giulio Pompilio, MD,
Gian Luca Polvani, MD,
Giuseppe Rossoni, PhD,
Massimo Porqueddu, MD,
Ferruccio Berti, PhD,
Isabella Barajon, MD,
Maria Grazia Petruccioli, PhD,
Anna Guarino, BSc,
Giovanni Aguggini, PhD,
Paolo Biglioli, MD,
Andrea Sala, MD
Department of Cardiac Surgery, University of Milan, and Italian Homograft Bank, Centro Cardiologico "I Monzino" Foundation IRCCS, and Departments of Pharmacology and Tossicology, Human Anatomy, and Animal Physiology, University of Milan, Milan, Italy
Accepted for publication September 27, 1996.
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Abstract
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Background. This study investigates the time-dependent resistance of the endothelium of porcine aortic and pulmonary valves to different periods of warm ischemia (WIT).
Methods. Twenty-five 9-month-old swine were divided after death into five groups of WIT (0, 6, 12, 24, and 36 hours). Aortic and pulmonary valves were removed and a total of 15 aortic and 15 pulmonary valve specimens were obtained for each WIT interval. Valves were then examined for (1) their viability rate by the trypan blue dye exclusion method at light microscopy (percent of viability compared with 0 hours of WIT); (2) ultrastructural signs of irreversible or reversible ischemic damage by transmission electron microscopy (cell disruption, dilation of endoplasmic reticulum, cytoplasmic edema, nuclear and mitochondrial changes); (3) endothelial function by pharmacologic evaluation of both the endothelial-releasing capacity of prostacyclin and the endothelial-dependent dynamic responses to relaxing (acetylcholine from 1 x 10-10 mol/L to 1 x 10-4 mol/L) in aortic and pulmonary valve segments precontracted with norepinephrine (1 x 10-6 mol/L) and contracting (NG-monomethyl-L-arginine, 1 x 10-4 mol/L) drugs.
Results. Our results showed an endothelial progressive time-dependent ischemic injury, which reached significance after 12 hours of exposure. Viability and functional data indicated that 6 hours of WIT only provoked slight endothelial damage (p > 0.05 respect to time 0 hours), with signs at transmission electron microscopy consistent with a reversible injury. At 12 hours of exposure, we observed a significant reduction (p < 0.05) with respect to time 0 of the viability rate of prostacyclin production and of the endothelium-dependent dynamic responses to acetylcholine and NG-monomethyl-L-arginine. These functional impairments, although significant, were not consistent, however, with a complete loss of viability. Transmission electron microscopic observations confirmed the appearance of signs of irreversible injury; nevertheless, some elements were found to be well preserved or presented reversible damage. After 24 hours of WIT, ultrastructural and functional data were consistent with a dramatic decrease compared with controls in endothelial viability and functions (p < 0.01). Finally, after 36 hours of WIT, there was a subtotal loss of viability, of functions (p < 0.001) and, at transmission electron microscopic observations, of the endothelial layer of the valves.
Conclusions. Our data show that the endothelial cells are resistant to short periods of WIT (up to 6 hours), and suggest that these cells can endure longer exposures, up to 12 hours of warm ischemia. Periods of 24 and 36 hours of WIT provoke progressive irreversible damage.
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Introduction
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At present there is general consensus that unstented homograft valves are an important device in valvular reconstructive procedures and have superior characteristics compared with other biological and mechanical valves. As is shown by clinical evidence and biomedical research, homograft valve durability has been related to cellular "viability" [1, 2]. The harvesting interval or "warm ischemic time" (WIT) has been recognized as the main determinant of cell survival [3].
Crescenzo and colleagues [4], using a morphometric analysis with electronic microscopy, found a significant association between fibroblast cell injury and progressive WIT. Conversely, the role of endothelial cell viability for homograft valve durability remains unclear [5–7], although it has been suggested that endothelial cell retention could be a factor of homograft resistance to degenerative processes [7, 8]. Endothelial cell resistance to WIT is still debated, although vascular endothelium is thought to have a low resistance to ischemic injuries. Yankah and Hertzer [9] have reported an endothelial valve survival of 24% after exposure for 2 hours at room temperature, whereas other experiments describe the destruction of the endothelial layer within 24 to 48 hours postmortem [8, 10]. However, in rat models, the valve endothelium was shown to be viable for at least 40 hours postmortem [11]. More recently, functional studies on endothelial viability using assessment of prostacyclin (PGI2) production [12, 13] have been proposed to further evaluate the impact of valve disinfection and cryopreservation processes on endothelial cell viability, although functional evaluation of the endothelial cells estimates the endothelial viability as being lower than found by conventional techniques [11, 14].
In many homograft bank protocols, the harvesting time intervals are often different when donors with nonbeating hearts are available, ranging from 6 to more than 24 hours postmortem. The evidence of the role endothelium plays in homograft valve biology and the lack of detailed information on the ischemic response of valve endothelium to WIT have prompted us to seek a better understanding of endothelial viability at the moment of homograft explant. This study addresses the issue of characterizing the time-dependent resistance of the aortic and pulmonary valve endothelium to warm ischemic injury in a porcine model by determination of (1) the viability rate by the dye exclusion method; (2) ultrastructural changes by transmission electron microscopy; and (3) endothelial functions by the evaluation of both the releasing capacities of PGI2 and the dynamic responses to relaxing (acetylcholine [Ach]) and contracting (NG-monomethyl-L-arginine [L-NMMA]) drugs.
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Material and Methods
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Harvesting
Twenty-five 9-month-old Yorkshire and Poland China swine (mean weight, 27 kg) were killed in an animal laboratory by endovenous infusion of an overdose of penthotal. All animals were treated in accordance with the "Guide for the Care and Use of Laboratory Animals" published by the National Institutes of Health (NIH publication 85–23, revised 1985). Animals were then kept at room temperature (20°C), and WIT (time between death and specimen preparation) was measured accurately for each swine. Each animal was assigned to one of five time groups for a defined exposure to warm ischemia: 0, 6, 12, 24, and 36 hours. A total of 5 animals were studied for each WIT interval. The heart was quickly removed after death and placed in oxygenated and prewarmed (37°C) Krebs-Henseleit solution. The cusps of the aortic and pulmonary valves (AoVs and PuVs) were dissected. Each cusp was then divided into three segments with a surface area of approximately 20 to 30 mm2, for a total of 15 AoV and 15 PuV specimens for each WIT interval. Specimens were then examined for their viability (with dye exclusion method at light microscopy), for their ultrastructural morphology (by transmission electron microscopy), and for their endothelial function (with pharmacologic analysis).
Dye Exclusion Test
Aortic valve and PuV specimens were rinsed in Krebs-Henseleit solution for transport and immediately analyzed. Endothelial cells were harvested from porcine AoV and PuV leaflets by collagenase treatment as previously described [15]. The cells were detached from pieces of tissue by 20 minutes of incubation at 37°C in 0.1% collagenase type II in phosphate-buffered saline solution with calcium and magnesium and collected in one or two 10-mL centrifugal tubes, and the specimen was flushed with phosphate-buffered saline solution without collagenase [16]. The cells were centrifuged for 5 minutes at 200 g and resuspended in RMPI 1640 culture medium containing sodium bicarbonate, 200 mmol of L-glutamine, penicillin/streptomycin. The technique of dye exclusion was used for the evaluation of cell viability [17]. Trypan blue was tested by incubating cell suspension at room temperature with an equal volume of 0.5% trypan blue in phosphate-buffered saline solution (pH 7.4) for 1 minute and then by counting the number of unstained (viable) cells and of total cells (stained and unstained) at light microscopy (model Telaval 3; Zeiss, Germany). The degree of viability was expressed for 6, 12, 24, and 36 hours of WIT as a percentage of the viability rate of the group with 0 hours of WIT used as the control group. Cell growth obtained from each specimen was then monitored by light microscopy.
Transmission Electron Microscopy
The leaflets of the AoV and PuV were harvested from the animals for each warm ischemic interval. After a short fixation in toto of the leaflets pinned onto a plastic surface to avoid curling, performed with 3% glutalaldehyde in 0.12 mol/L phosphate buffer at pH 7.4, smaller samples were trimmed out and postfixed in the same fixative for 2 hours at 4°C. After a thorough washing in phosphate buffer, the samples were postfixed with 1% osmium tetroxide and processed for plastic embedding in epoxy resin. Semithin sections were collected on celloidin-coated slot grids to avoid curling of the outer margins of the section where the endothelium is located, and were stained with uranyl acetate and lead citrate.
Electron microscopy was performed with a Phillips CM10 and with a Jeol 100CX. To assess the extent of cellular damage, we followed the criteria outlined by Crescenzo and colleagues [4]; that is, cytoplasmic edema, dilation of the endoplasmic reticulum, mitochondrial swelling (as signs of reversible cellular injury), and mitochondrial flocculent densities, karyolysis, and disrupted plasma membrane (as signs of irreversible cell injury).
Organ Chamber Studies
The tissues were suspended by means of two 
-shaped stainless steel needles wires, one stationary and the other connected to a strain gauge force transducer (model 7004; Ugo Basile, Comerio-Va, Italy) coupled to a Basile pen-recorder (model 7070) for measurement of the isometric tension. In particular, cardiac valve specimens were placed in 10-mL organ baths containing Krebs-Henseleit solution with the following composition (in millimoles per liter): NaCl, 118; KCl, 4.7; MgSO4, 1.2; CaCl2, 2.5; Na2 HPO4, 1.2; NaHCO3, 25; glucose, 12; and EDTA 0.03, warmed at 37°C and gassed with a gas mixture of 95% O2 + 5% CO2 (pH 7.4). The vascular segments were left to equilibrate under a resting tension of 2 g for 1 hour, and the Krebs-Henseleit solution was changed every 20 minutes.
Protocol
After the equilibration period, tissues were subjected to two successive challenges with a maximum depolarizing 100 mmol/L potassium chloride (KCl) solution to establish the maximum contractile response of each preparation.
To study the endothelial function, we precontracted AoV and PuV segments with norepinephrine (NE; 1 x 10-6 mol/L) and, when the constrictor response reached a plateau, acetylcholine was added accumulatively (from 10-10 mol/L to 10-4 mol/L) to produce endothelium-dependent relaxation. The maximum relaxation produced by acetylcholine was expressed as a percentage of the NE contraction. After the washout of NE and acetylcholine, cardiac valve specimens were challenged with a single concentration of the nitric oxide synthase inhibitor L-NMMA (1 x 10-4 mol/L), and the tension developed was expressed as a percentage of the spasm induced by KCl.
Prostacyclin Assay
The release of PGI2 by the AoV and PuV segments was measured in the bath medium in basal tonus conditions of the preparation and at the end (approximately 20 minutes) of the maximal relaxing effect of acetylcholine, or at the end of the maximal contracting effect of L-NMMA. In particular, 2 mL of bath medium was collected every 20 minutes, frozen at -20°C, and stored until assayed for PGI2 content. The PGI2 content was measured quantitatively as 6-keto-prostaglandin F1 (the stable metabolite of PGI2), using a commercial kit (detection limit, 5 pg/mL), by a specific enzyme immunoassay described by Pradelles and co-workers [18]. The concentration of the autacoid found in the bath medium was expressed in picograms per milligram of wet tissue.
Drugs
Acetylcholine chloride, NG-monomethyl-L-arginine, norepinephrine, RMPI 1640-nutrient media solution, collagenase type II, L-glutamine, trypan blue, and penicillin/streptomycin were obtained from the Sigma Chemical Co, St. Louis, MO; 6-keto-PGF1a kit from Cayman Chemical Company, Ann Arbor, MI.
Statistical Analysis
All values in the figures and text are expressed as mean ± standard error of the mean. In all experiments, n is the number of pigs from which the cardiac valve segments and arterial wall rings were obtained. A two-way analysis of variance or two-tailed Student's t test was used to compare means between the different time groups and to analyze intragroup variations. A p value of less than 0.05 indicates a significant difference.
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Results
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Dye Exclusion Method
A dye exclusion test with trypan blue was used to measure the degree of viability of endothelial cells after the different times of warm ischemia. As shown in Figure 1
, the control group (0 hours of WIT) averaged a cell viability of 81 ± 5.4 for AoV and 79 ± 6.1 for PuV, which was taken as 100% cell viability for the remaining groups [19]. Statistical analysis did not show a significant loss of endothelial viability after 6 hours of WIT (viability rate, 88.7% for AoV and 89.4% for PuV, p > 0.05 compared with controls). Conversely, after 12 hours of WIT a significant difference was observed with a viability rate of 47.9% for AoV and of 50.3% for PuV, respectively (p < 0.05 compared with 0 hours of WIT). This loss of cellular viability was increased after 24 hours (viability rate for AoV, 36.3%; for PuV, 35.3%, p < 0.05 compared with controls) and after 36 hours (viability rate for AoV, 15.5%; for PuV, 14.1%, p < 0.01 compared with controls). A significant difference in viability rate (p < 0.05) was also found between 6 and 12 hours, and from 24 to 36 hours of WIT in the two groups.
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Figure 1. . Percent of viability as determined by the dye exclusion method for porcine aortic ( AoV) and pulmonary (PuV) valves, harvested at different warm ischemic times. The control group (0 hours of warm ischemia) was taken as 100% of viability for the remaining groups. Each column represents the mean percent of viability ± standard error of the mean. (ap < 0.05; bp < 0.01 versus control group (time 0); 12 hours versus 6 hours, p < 0.05; 36 hours versus 24 hours, p < 0.05.)
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Transmission Electron Microscopy
Our ultrastructural findings indicate that there is an association between WITs and the extent of endothelial injury. The method of harvesting and processing the valve leaflets appeared to be very conservative, as at 0 hours of WIT the endothelium of the valves and the underlying tissue appeared to be very well preserved with no artifacts that could be imputed to the technical procedure. Endothelial cell organelles and cytoplasm did not show any apparent damage and the lining of the valve wall was continuous (Fig 2).
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Figure 2. . Ultrastructural features of endothelial cells at 0 hours of warm ischemia. The nucleus and the cytoplasm are well preserved and the endothelial lining of the valve wall is continuous. (x10,000 before 52% reduction.)
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After 6 hours of WIT, the cytoplasm of endothelial cells was still quite intact. Some edema and initial dilation of the endoplasmic reticulum could be seen, together with some retraction of the cytoplasmic appendages and increase of the luminal villous extroflexions. At 12 hours, endothelial cells were still visible on the surface of the valve leaflets. Many of them showed signs of clear-cut damage, consistent with irreversible cellular injury. Mitochondrial flocculent densities, chromatin condensation, delamination of the basal membrane, initial detachment, and some disruption of the plasma membrane were present. Nevertheless, some elements were in a comparatively less advanced stage of deterioration whereas others showed signs of damage consistent with reversible cellular injury (Fig 3A). At this stage, an intact lining of endothelial cytoplasmic profiles with visible membrane junctions was observed (Fig 3B).
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Figure 3. . (A) Endothelial cells after 12 hours of warm ischemia. Mild deterioration of the cytoplasmic structures is present. Initial disarrangements of the luminal cell membrane, cytoplasmic edema, dilation of the endoplasmic reticulum ( arrowhead), and mitochondria at different stages of damage (asterisks) can be seen. (x 26,700 before 57% reduction.) (B) Endothelial cytoplasmic appendages still cover long stretches of the valve wall and membrane junctions are preserved (arrowhead). (x 19,800 before 55% reduction.)
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After 24 hours, only irreversible signs of cellular injury were found, with disruption of the plasma membrane and karyolysis, although many cells appeared to maintain the integrity of the cytoplasmic profile and preserved membrane junctions (Fig 4). At 36 hours, a disruption of the endothelial layer with only some isolated endothelial cells with signs of irreversible damage, or endothelial fragments, was observed.
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Figure 4. . At 24 hours of warm ischemia severe cell damage is visible. Most of the cytoplasmic structures have degenerated and the cytoplasm appears "cleared." (x 14,400 before 50% reduction.)
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Acetylcholine Activity
The results obtained in these series of experiments are reported in Figure 5. They indicated that in the group with 0 hours of WIT (control group), the NE-precontracted AoV and PuV specimens were very sensitive to acetylcholine activity. In fact, when the preparations were exposed to cumulative concentrations of acetylcholine (from 1 x 10-10 mol/L to 1 x 10-4 mol/L) a 92% ± 6.1% (AoV) and 84% ± 6% (PuV) reduction of the spasm induced by NE (1 x 10-6 mol/L) was observed (p < 0.001). No significant differences with respect to the controls were found in the maximal response to the muscarinic agonist in NE-precontracted AoV and PuV segments after 6 hours of WIT (81% ± 5.8%, p > 0.05 and 73% ± 5%, p > 0.05, with respect to time 0, respectively). Conversely, at 12 hours of ischemia, we observed a significant decrease of the relaxing response induced by acetylcholine both for AoV and PuV (65% ± 8%, p < 0.05 and 58% ± 6%, p < 0.05, compared with 0 hours of WIT, respectively). This reduction was greater after 24 hours of ischemia, with an average of 40% ± 3.2% (p < 0.01 compared with 0 hours of WIT) in the AoV and 35% ± 4% in the PuV specimens (p < 0.01 compared with controls), and dramatically increased after 36 hours to 13% ± 1.8% (p < 0.001, compared with controls) in the AoV and 10% ± 1.4% (p < 0.001, compared with controls) in the PuV segments, respectively. A significant difference was also found from 12 to 24 hours (p < 0.05) and from 24 to 36 hours (p < 0.01). No differences (p > 0.05) were found between AoV and PuV during the study.
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Figure 5. . Effect of maximal endothelium-dependent relaxation to acetylcholine (cumulative concentrations from 1 x 10-10 mol/L to 1 x 10-4 mol/L) on porcine aortic ( AoV) and pulmonary (PuV) valves harvested at different warm ischemic times. Each column represents the mean values ± standard error of the mean of five replications. (ap < 0.05; bp < 0.01; cp < 0.001 versus control group [0 hours of warm ischemia]; b versus a, p < 0.05; c versus b, p < 0.01.)
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L-NMMA Activity
When the AoV and PuV specimens were challenged with L-NMMA (1 x 10-4 mol/L), an inhibitor of nitric oxide synthase activity, a progressive and long-lasting increase in resting tension of the preparation occurred (Fig 6). In all the segments examined this phenomenon reached steady state after about 15 minutes. The maximum tension in the group with 0 hours of warm ischemia, both for the AoV and PuV, was 24% ± 2.1% (p < 0.001) of the contraction induced by 100 mmol/L of KCl (Fig 6). No significant difference in tension–contraction was found for either AoV or PuV specimens after 6 hours (22% ± 1.6% and 21% ± 1.8%, respectively, p > 0.05 compared with 0 hours of WIT) of WIT. At 12 hours of ischemia, a significant reduction of the tension–contraction was observed for both AoV and PuV specimens: 19% ± 1.5% and 17% ± 1.2%, respectively, p < 0.05 compared with 0 hours of WIT. After 24 and 36 hours of ischemia, the loss of viable endothelium provoked a more pronounced reduction in response compared with 0 hours of WIT in both the AoV and PuV segments: 11% ± 1.2% (p < 0.01) and 2% ± 0.3% (p < 0.001), and 10% ± 0.8% (p < 0.01) and 0.5% ± 2% (p < 0.001), respectively. A significant difference was also found from 12 to 24 hours (p < 0.05) and from 24 to 36 hours (p < 0.01). No differences were found between aortic and pulmonary specimens during the protocol (p > 0.05).
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Figure 6. . Effect of maximal endothelium-dependent vasocontriction to NG-monomethyl-L-arginine (1 x 10-4 mol/L) on porcine aortic (AoV) and pulmonary (PuV) valves harvested at different warm ischemic times. Each column represents the mean values ± standard error of the mean of five replications. (ap < 0.05; bp < 0.01; cp < 0.001 versus control group [0 hours of WIT]; b versus a, p < 0.05; c versus b, p < 0.01.)
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Prostacyclin Determination
Segments of AoV and PuV placed in a suitable organ bath released spontaneously assayable amounts of arachidonic acid metabolites first recognized as PGI2 in the incubation medium. The trend of this phenomenon was fairly constant throughout the various preparations and was quantified for the 0 hours of WIT group as 120.7 ± 13.3 pg for the AoV and 102.7 ± 13.5 pg for the PuV of 6-keto-PGF1 (a stable metabolite of PGI2) per milligram of wet tissue released every 20 minutes for a period of 2 hours. As shown in Table 1, the basal release of PGI2 was not significantly different after 6 hours of WIT: 111.2 ± 10.8 pg/mg for AoV and 95.5 ± 7.2 pg/mg for PuV (p > 0.05). Conversely, both for AoV and PuV, endothelial functional impairment provoked a significant reduction versus the control 0 hours of WIT group in PGI2 release after 12 hours (78.5 ± 9.7 pg/mg and 73.9 ± 8.6 pg/mg, respectively, p < 0.05), 24 hours (59.2 ± 4.7 pg/mg and 55.5 ± 6.2 pg/mg, respectively, p < 0.01), and 36 hours of WIT (22.9 ± 3.4 pg/mg and 22.6 ± 2.4 pg/mg, respectively, p < 0.001). A significant (p < 0.05) progressive loss in PGI2 production was also observed between 12 and 24 hours (p < 0.05) and 24 and 36 hours (p < 0.01) of WIT. No significant difference was found between AoV and PuV during the study.
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Table 1. . Generation of 6-Keto-Prostaglandin F1a in Porcine Isolated Pulmonary and Aortic Valves Harvested at Different Warm Ischemic Timesa
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Comment
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Most of the current harvesting and cryopreservation protocols of homograft valves are based on the premise that the matrix cell (fibroblast and smooth muscle cells) viability enhances valve durability [1, 2]. Therefore, efforts to increase homograft viability would seem necessary [3, 8]. Cellular injury associated with WIT was identified as the first determinant of cellular viability [4]. A more precise definition of fibroblast ischemic injury was recently achieved [4], with the demonstration of an ischemic time-dependent progression in fibroblast damage; during the first 12 hours of WIT, fibroblast reversible injury predominated with a positive correlation between increasing WIT and reversible cellular damage (with 62.2% of normal cells, 35.1% of reversible injury, and only 2.7% of irreversible injury at 12 hours); after 12 hours a marked increase (31.4% and 40% at WIT of 16 and 20 hours) in irreversible injury is reported. Functional studies on fibroblast response to WIT correlates well with morphometric findings [6] and with mechanisms of injury related to the depletion of high-energy phosphate intermediates [14].
More recently, experimental research on homograft valves has focused on the endothelial cells, and there is increasing interest in the properties of vascular endothelium [20]. The role that endothelium plays in the success of allograft valves is still not clear, because of the possible involvement of viable endothelial cells in immunologic reactions [21]. However, it has been suggested that the presence of viable endothelial cells at the time of implantation may delay the process of calcification and be an active factor in long-term survival of the cryopreserved human heart allograft valve [7]. The effects on endothelial cell viability of storage of porcine cardiac valves in 4°C nutrition medium or in liquid nitrogen at different temperatures have been extensively evaluated [22]. Conversely, there is little investigation correlating the time-dependent injury of valvular endothelial cells after periods of warm ischemia.
In 1969, a study of postmortem changes in endothelial cells of aortic homograft valve was performed by Innes and colleagues [5], using the surface staining technique. They found a high percentage (88%) of well-defined endothelium of fresh aortic valve specimens. If the cusps were stained later than 48 hours postmortem, destruction of the endothelium was observed [5]. Yankah and Hertzer [9] have studied the endothelial and fibroblast allograft viability at both room and 4°C storage temperatures, using the Alcian blue dye exclusion test. The control valve specimens obtained within 5 minutes after cardiac explantation showed 100% viable endothelial and fibroblast cells. Only 24% of the endothelium and 45% of the fibroblast were found alive after 2 hours of exposure at room temperature. However, another study by Yankah and colleagues [11], using the same approach, reported that the rat valve endothelium appeared to be viable for at least 40 hours postmortem. Other studies [8], more generally, showed that when heart valves obtained from experimental animals are properly handled, they retain viable endothelial cells. The endothelium is generally destroyed within 24 to 48 hours postmortem [8, 10]. In this study we determined the valvular endothelium-dependent response to WIT up to 36 hours of exposure with morphologic and functional analysis. Generally speaking, despite previous reports [5, 11], we found an unexpected resistance of valve endothelium to warm ischemia.
Interestingly, after 6 hours of WIT almost all of the endothelial cells were still intact at transmission electron microscopy, with a low percentage of reversible injured cells; at this stage, viability rate as shown by trypan blue exclusion was 88.7% and 89.4% for AoV and PuV, respectively (p > 0.05 compared with 0 hours of WIT). The appearance of ultrastructural signs of irreversible endothelial injury was seen at transmission electron microscopy after 12 hours of WIT, although in some observations endothelial cells still showed ultrastructural integrity, or reversible ischemic damage. At this stage, the viability rate with trypan blue was 47.9% for AoV and 50.3% for PuV (p < 0.05 compared with 0 hours of WIT). The pharmacologic tests with acetylcholine and L-NMMA, confirming a preservation of endothelial functions after 6 hours, suggested a significant loss of viability after 12 hours of exposure to warm ischemia. In fact, after 12 hours of exposure, a significant reduction of endothelial-dependent dynamic response to acetylcholine was observed: 65% ± 8% for AoV (p < 0.05) and 58% ± 6% for PuV (p < 0.05). The level of L-NMMA activity corresponds to the response to acetylcholine. Furthermore, PGI2 release from valve endothelium decreases very little after 6 hours of ischemia, and significantly more after 12 hours (inhibition rate, 28% for AoV and 35% for PuV, p < 0.05 compared with 0 hours of WIT).
These findings better define a supposed generic high sensitivity of this type of cell to short periods of ischemia, previously reported in occasional transmission electron microscopic observations [4], and with a considerable well-known resistance and retention of vascular endothelial-dependent functions after short periods of global normothermic ischemia [19, 23]. A significant reduction in endothelial viability and functions after this critical threshold was expected. However, partial retention of valvular endothelium-dependent properties after 12 hours of WIT was observed, along with ultrastructural evidence of endothelial cells, which were potentially still viable. These findings suggest that the capacity of valvular endothelium to resist ischemic injuries is perhaps even greater.
After 24 hours of exposure to WIT, there was a dramatic decrease in endothelial viability and functions, with a severe decrease in viability rate measured by the trypan blue method (AoV, 36.3%; PuV, 35.3%, p < 0.01 compared with 0 hours of WIT and p < 0.05 compared with 12 hours of WIT) associated with ultrastructural evidence of a very high rate of irreversible cellular injury. These findings were confirmed by functional evaluation, with a significant reduction of the endothelial-dependent relaxing and contracting response to acetylcholine and L-NMMA (for both AoV and PuV, p < 0.01 compared with 0 hours of WIT and p < 0.05 compared with 12 hours of WIT). PGI2 production was also severely reduced after 24 hours of WIT (p < 0.01 compared with 0 hours of WIT), as well as during the interval from 12 to 24 hours of exposure (p < 0.05). After 36 hours of ischemia, the valvular endothelial layer was almost completely destroyed, and both ultrastructural and functional evaluations indicate the subtotal loss of viability (p < 0.001 compared with 0 hours of WIT). These results correspond well to with the previously documented findings of endothelial cell intolerance to long periods of warm ischemia [11].
In conclusion, functional and morphologic data seem to demonstrate great resistance and retention of functions of valvular endothelial cells to short periods of WIT (up to 6 hours), and suggest capacity to partially resist longer exposures, up to 12 hours of WIT. Periods of ischemia of 24 hours provoke irreversible cellular damage, and at 36 hours a subtotal loss of the endothelial layer of the AoV and PuV is observed. These findings could be a contribution to the reconsideration of the problem of cellular viability in the field of viable homograft valves. Resistance of endothelium to ischemic injuries suggests that intervals of WIT of up to 12 hours before homograft harvesting maintain an acceptable cellular integrity.
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Acknowledgments
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This study is dedicated to the memory of Juan Climaco Rodriguez, MD. Special acknowledgment goes to Massimo Marzani: without his kind assistance in the care of the animal laboratory, this study would have been impossible.
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Footnotes
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Address reprint requests to Dr Pompilio, Department of Cardiac Surgery, "I Monzino" Foundation, Via Parea 4, 20138 Milan, Italy.
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References
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Abstract
Introduction
