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Gian Luca Polvani
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Ann Thorac Surg 1996;61:667-673
© 1996 The Society of Thoracic Surgeons

Original Article: Cardiovascular

Retention of Endothelium-Dependent Properties in Human Mammary Arteries After Cryopreservation

Giulio Pompilio, MD, Gian Luca Polvani, MD, Carlo Antona, MD, Giuseppe Rossoni, PhD, Anna Guarino, Massimo Porqueddu, MD, Michel Buche, MD, Paolo Biglioli, MD, Andrea Sala, MD

Department of Cardiac Surgery, Italian Homograft Bank, and Department of Pharmacology, Chemotherapy and Medical Toxicology, University of Milan, Centro Cardiologico ``I Monzino'' Fundation IRCCS, Milan, Italy

Accepted for publication October 13, 1995.


   Abstract
Top
 Footnotes
 Abstract
Introduction
 Material and Methods
 Results
 Comment
 References
 
Background. We investigated the effects of cryopreservation and antibiotic treatment on endothelium-dependent vasomotor properties of human internal mammary arteries (IMAs).

Methods. Sixty IMA specimens from routine coronary artery bypass grafting procedures were randomly assigned to six groups. Group I (controls) were immediately tested after harvest. Remaining groups were prepared according to a stepwise design: group II, 6 hours of warm ischemia; group III, 6 hours of warm ischemia + 24 hours at 4°C (without antibiotics); group IV, 6 hours of warm ischemia + 24 hours of 4°C antibiotic disinfection; group V, 6 hours of warm ischemia + 24 hours at 4°C (without antibiotics) + cryopreservation; and group VI, 6 hours of warm ischemia + 24 hours of 4°C disinfection + cryopreservation. The IMA specimens were cut into rings and the tension of vascular smooth muscle was recorded. The IMA rings were contracted with norepinephrine (3 x 10-6 mol/L) and tested with cumulative concentrations of acetylcholine (from 1 x 10-9 to 1 x 10-5 mol/L), contracted with endothelin-1 (from 1 x 10-11 to 1 x 10-6 mol/L), and contracted with the nitric oxide-synthase inhibitor NG-monomethyl-L-arginine (1 x 10-4 mol/L). Rings were also tested for their capacity to generate 6-keto-prostaglandin F1 (the stable metabolite of prostacyclin), and endothelial cell viability rate was finally evaluated with the trypan blue dye exclusion method.

Results. Our results show that a complete cryopreservation protocol does not significantly modify (p > 0.05) the relaxant activity to acetylcholine in norepinephrine-precontracted IMA rings (controls; 90.2% ± 4.2% vs group VI, 77.1% ± 6.2%) or the vasoconstrictor response induced by endothelin-1 (controls, 62.6% ± 2.8% versus group VI, 73.7% ± 4.8%) and NG-monomethyl-L-arginine (controls, 22.4% ± 1.5% versus group VI, 18.9% ± 1.9%). Furthermore, IMA cryopreservation does not significantly modify (p > 0.05) the endothelial release of prostacyclin either in basal conditions (-20% versus controls) or during pharmacologic intervention with acetylcholine (-18% versus controls), endothelin-1 (-17% versus controls), and NG-monomethyl-L-arginine (-18% versus controls).

Conclusions. We conclude that the IMA endothelial function does not seem significantly injured by any of the current steps of disinfection and cryopreservation.


   Introduction
Top
 Footnotes
 Abstract
 Introduction
Material and Methods
 Results
 Comment
 References
 
Recent advances in surgical coronary artery revascularization procedures have emphasized the importance of arterial grafts because of the superior long-term patency rate of the internal mammary artery (IMA), compared with saphenous vein grafts [1]. Bilateral and sequential IMA [2], right gastroepiploic artery [3], and inferior epigastric artery [4] graftings were proposed to achieve the most complete arterial revascularization.

High resistance of IMA grafting to atherosclerotic degenerative processes has been attributed to its peculiar histologic structure [5] and to the biological properties of the endothelium [6]. Two characteristics of vascular endothelium of crucial importance have been identified: the antithrombotic and the vasodilator properties [7]. These functions may be achieved via secretion of vasoactive substances by the vascular endothelium, such as endothelium-derived relaxing factor or nitric oxide and prostacyclin (PGI2), which are well documented in both humans and animals to be the most important short-lived endothelial autocoids involved in the local control of vascular tone and prevention of platelet activation and thrombus formation [8]. It has been demonstrated that IMA endothelium produces more PGI2 than the saphenous vein and releases more nitric oxide than the saphenous vein [9]. In light of these findings, the other available arterial grafts employed in coronary operations (right gastroepiploic and inferior epigastric arteries) have also been investigated for their biological properties [10] in the hope of a similar long-term patency. However, the increasing number of multiple arterial graft procedures and coronary reoperations causes the search for available well-performing arterial coronary bypass conduits to continue.

Cryopreservation has been demonstrated with homograft valves to be a feasible and sure method of storing tissues while maintaining their structural integrity [11]. Cryopreserved allograft saphenous veins has been proposed as adjunctive grafts for myocardial revascularization, with acceptable results achieved in dogs [12]. This study investigates the retention of biological properties in IMAs after a complete process of disinfection and cryopreservation, by determination of both the endothelial releasing capacity of PGI2, and of the endothelial vasodynamic response to relaxing (acetylcholine [ACh]) and contracting (endothelin-1 [ET-1] and NG-monomethyl-L-arginine [L-NMMA]) drugs. Endothelial cell viability was also tested by the dye exclusion method.


   Material and Methods
Top
 Footnotes
 Abstract
 Introduction
 Material and Methods
Results
 Comment
 References
 
Study Design
A total of 60 left distal IMAs were obtained intraoperatively from patients undergoing elective coronary bypass grafting who had not received aspirin or other related compounds for 1 week. The study was designed to investigate IMA endothelial viability and function (in terms of releasing capacity and endothelium-dependent vasomotricity) in subsequent steps of a complete disinfection and cryopreservation treatment. These IMA segments were randomly divided into 6 groups (10 specimens for each group) according to the following processing steps: group I (control group), tested immediately after harvest (maximum, 30 minutes of warm ischemic time [WIT]); group II, 6 hours of WIT in saline solution (analogous to the maximum WIT accepted in non–heart-beating organ donors); in group III, as in group II + 24 hours at 4°C in Krebs-Henseleit solution (cold ischemic time; analogous to cold transport of donor tissue to processing centers, but in the absence of antibiotics); group IV: as in group II + 24 hours of low-dose antibiotic disinfection (cold disinfection time; virtually analogous to clinical protocols for heart valves), consisting of a serum-free RPMI 1640 nutrient media solution containing cefoxitin, 240 µg/mL, lyncomicin, 120 µg/mL, polymixin B sulfate, 100 µg/mL, and vancomycin, 50 µg/mL; group V, 6 hours of WIT and cold ischemia (identical to group III) but then cryopreserved as follows: 10% each of dimethysulfoxide and fetal calf serum in RMPI 1640, frozen at a rate of -1°C/min to -190°C (Kryo 10-16 series III; Planer Biomed, Sunbury Middlesex, UK), and stored in vapor-phase liquid nitrogen (-190°C) (17 K, Taylor-Wharton Cryogenics; Harsco Corp, Theodore, AL); and group VI, 6 hours of WIT and 24 hours of antibiotic disinfection at 4°C (identical to group IV) and then cryopreserved (as in group V). The cryopreservation technique was performed according to the modern ``slow freezing'' procedures (-1°C/min cryoprotected freezing and storage at -190°C). After 3 weeks of cryopreservation, IMAs of groups V and VI were thawed in 40°C saline solution for 15 minutes and cryoprotective agents were removed according to the current thawing protocols for heart valves [13]. The IMA segments of each group were then examined for their endothelial function (via pharmacologic analysis) and viability (via dye exclusion method).

Experimental Setup
At the time of the experiment, IMAs from the six study groups (5 specimens for each group) were dissected free, cleaned of adherent connective tissue under a dissection microscope, cut into rings of about 5 mm in length, and suspended in an organ bath (10 mL) filled with Krebs-Henseleit solution (37°C) of the following composition (in mmol/L): NaCl, 118; KCl, 4.7; MgSO4, 1.2; CaCl2, 2.5; KH2PO4, 1.2; NaHCO3, 25; and glucose, 5.5. The solution was bubbled with a mixture of 95% O2 + 5% CO2, and the final pH was adjusted to 7.4.

The resting tension (about 1 g) of the vascular smooth muscle was recorded using an isometric force transducer (model 7004; Ugo Basile, Comerio-VA, Italy) coupled to a Basile pen-recorder (model 7070), and changes in tonus were expressed as percentage increases or decreases of the tension developed by norepinephrine (NE) or by KCl. After the equilibration period of 45 minutes the IMA segments obtained from the above indicated groups were submitted to the following pharmacologic study:

ACETYLCHOLINE ACTIVITY IN PRECONTRACTED TISSUES.
After the constrictor response of the IMA rings to NE (3 x 10-6 mol/L) had reached a plateau, the preparations were exposed to cumulative concentrations of ACh (from 1 x 10-9 to 1 x 10-5 mol/L). The relaxant activity of the agonist was expressed as a percentage of reduction of the tension developed by NE.

ENDOTHELIN-1 ACTIVITY.
The IMA rings were exposed to cumulative concentrations of ET-1 (from 1 x 10-11 to 1 x 10-6 mol/L) and the tension developed was recorded and expressed as a percentage of the contraction induced by KCl (100 mmol/L).

NG-MONOMETHYL-L-ARGININE ACTIVITY.
The IMA rings were challenged with the NO-synthase inhibitor [14] L-NMMA (1 x 10-4 mol/L) and the tension developed was recorded and expressed as a percentage of the spasm induced by KCl (100 mmol/L).

Prostacyclin Assay
The release of prostacyclin (PGI2) by the IMA rings was evaluated directly in the incubation medium in basal tonus conditions of the preparation and at the end (approximately 15 minutes) of the maximal vasorelaxant effect of ACh or at the maximal vasospasm effect of ET-1 and L-NMMA. In particular 2 mL of both media was collected and stored frozen (-20°C) until assayed for PGI2 content. The PGI2 content was quantitatively measured as 6-keto-prostaglandin (PG) F1{alpha} (the stable metabolite of PGI2) by a specific enzyme immunoassay described by Pradelles and associates [15], and the concentration of the autacoid determined was expressed in picograms per milligram of wet tissue.

Endothelial Viability
Internal mammary arteries in all six study groups (5 specimens in each group) were rinsed with Krebs-Henseleit solution for transport and immediately analyzed. Endothelial cells were harvested by collagenase treatment as previously described [16]. Cells were detached from pieces of tissue (2 x 2 mm) by a 20-minute incubation at 37°C in 0.1% collagenase type II in phosphate-buffered saline solution (D 5780; Sigma Chemical Company, Sigma Aldrich, Gallarate, Milan, Italy) with calcium and magnesium and collected in one or two 10-mL centrifugal tubes, flushing the piece with phosphate-buffered saline solution without collagenase [17]. The cells were centrifuged for 5 minutes at 200 g and resuspended in a culture medium RMPI 1640 containing sodium bicarbonate, 200 mmol of L-glutamine, fetal calf serum, and penicillin/streptomycin. The technique of dye exclusion was used for the evaluation of cell viability [18, 19]. 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 comparing the number of unstained (viable) cells with that of the total cells (stained and unstained) with a light microscope (model Telaval-3; Zeiss, Germany). The degree of viability was expressed for groups II through VI as a percentage of the viability rate of the control group (group I). A culture of the cells harvested was obtained for each specimen, and endothelial cells were identified at light microscopy on the basis of their appearance in tissue culture (``cobble-stone'' cells). Confluence was reached approximately 10 days after harvesting.

Drugs
Acetylcholine chloride, L-NMMA, NE, RMPI 1640-nutrient media solution, collagenase type II, L-glutamine, trypan blue, and penicillin/streptomycin were obtained from Sigma Chemical Company, St. Louis, MO; endothelin-1 from Peninsula Laboratory, Belmont, CA; 6-keto-PGF1{alpha} kit from Cayman Chemical Company, Ann Arbor, MI; and fetal calf serum from International PBI, MI, Italy.

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 patients from whom the IMA segments were obtained. A two-way analysis of variance or two-tailed Student's t test was used to compare means between different groups and to analyze intragroup variation [20]. A p value of less than 0.05 indicated a significant difference.


   Results
Top
 Footnotes
 Abstract
 Introduction
 Material and Methods
 Results
Comment
 References
 
Acetylcholine Activity in Precontracted Tissues
The results obtained in these series of experiments are reported in Figures 1 and 2GoGo. They indicated that in the control group (group I), the NE-precontracted IMA rings were very sensitive to ACh activity. In fact, when the preparations were exposed to cumulative concentrations of ACh (from 1 x 10-9 to 1 x 10-5 mol/L) a 90.2% ± 4.2% reduction of the spasm induced by NE (3 x 10-6 mol/L) was observed (p < 0.001).



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  		Fig 1. . Representative illustrations showing the effect of endothelium-dependent relaxation induced by acetylcholine (ACH) in norepinephrine (NE; 3 x 10-6 mol/L)-constricted ring preparations of human internal mammary artery and the endothelium-dependent vasocontraction induced by endothelin-1 (ET-1) or by NG-monomethyl-L-arginine (L-NMMA). The relaxant activity of ACH was expressed a percent reduction of the tension developed by NE, whereas the responses to ET-1 and L-NMMA were expressed a percentage increase of the tension developed by potassium chloride (KCl; 100 mmol/L). (* Washing.)

 


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  		Fig 2. . Maximal endothelium-dependent relaxation induced by acetylcholine (cumulative concentrations from 1 x 10-9 to 1 x 10-5 mol/L) in norepinephrine (NE; 3 x 10-6 mol/L)-constricted ring preparations of human internal mammary artery obtained from six different study groups. Columns represent the mean values ± standard error of the mean of five internal mammary artery segments for each group. For explanation of the groups, see text. No significant differences (p > 0.05) among the groups were found.

 
No significant difference with respect to controls (group I) was found in the maximal response to the muscarinic agonist in NE-precontracted IMA rings after 6 hours of WIT (group II, 88.3% ± 3.8%; p > 0.05), after 24 hours of cold ischemia without or with antibiotic disinfection (group III, 86.3% ± 3.5%, and group IV, 85.7% ± 4.1%; p > 0.05), or after crypreservation without or with antibiotic disinfection protocol (group V, 78.8% ± 2.1%, and group VI, 77.1 ± 2.8%; p > 0.05) (see Fig 2Go).

Endothelin-1 Activity
The addition of an increasing concentration of ET-1 (from 1 x 10-11 to 1 x 10-6 mol/L) to the organ bath containing IMA rings from group I (controls) induced a dose-dependent constriction of smooth muscle cell with a peak effect equivalent to 62.6% ± 2.8% (p < 0.001) of the vasospasm induced by 100 mmol/L of KCl (Figs 1, 3GoGo). A nonsignificant increase of vasoconstriction (p > 0.05 versus controls) was found in the remaining five groups: 63.7% ± 3.2% for group II, 65.9% ± 3.5% for group III, 66.5% ± 3.4% for group IV, 70.9% ± 4.3% for group V, and 73.7% ± 4.8% for group VI (see Fig 3Go).



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  		Fig 3. . Maximal endothelium-dependent vasoconstriction induced by endothelin-1 (cumulative concentrations from 1 x 10-11 to 1 x 10-6 mol/L) in human internal mammary artery rings obtained from six different study groups. Columns represent the mean values ± standard error of the mean of five internal mammary artery segments for each group. For explanation of the groups, see text. No significant differences (p > 0.05) among the groups were found.

 
NG-Monomethyl-L-Arginine Activity
When the IMA rings harvested from group I (control group) 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 preparations occurred (see Fig 1Go). In all IMA rings examined, this phenomenon reached the steady state around 15 minutes, and the maximum tension developed by the smooth muscle of these vessels in group I was 22.4% ± 1.5% (p < 0.001) of the contraction induced by 100 mmol/L of KCl (Figs 1 and 4GoGo). The loss of viable endothelium in the later processing groups did not provoke a significant reduction (p > 0.05) in tension-contraction with respect to the controls: group II, 21.7% ± 1.2%; group III, 21.4% ± 1.3%; group IV, 21.2% ± 2.1%; group V, 19.3% ± 1.6%; and group VI, 18.9% ± 1.9% (see Fig 4Go).



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  		Fig 4. . Maximal endothelium-dependent vasoconstriction induced by NG-monomethyl-L-arginine (1 x 10-4mol/L), an inhibitor of nitric oxide-synthase activity, in human internal mammary artery rings obtained from six different study groups. Columns represent the mean values ± standard error of the mean of five internal mammary artery segments for each group. For explanation of the groups, see text. No significant differences (p > 0.05) among the groups were found.

 
Prostacyclin Determination
Rings of IMA, placed in a suitable organ bath, showed a spontaneous ability to release assayable amounts of arachidonic acid metabolites first recognized as prostacyclin (PGI2) in the incubation medium. The trend of this phenomenon was fairly constant throughout the various preparations and was quantified, for the control group (group I), as 89 ± 7 pg of 6-keto-PGF1{alpha} (a stable metabolite of PGI2) per milligram (n = 5) released every 15 minutes for a period of 2 hours. As shown in Table 1Go the phenomenon was not significantly different (p > 0.05) for groups II through VI with respect to the control group; in fact, the basal release of 6-keto-PGF1{alpha} (n = 5 for each group) was 86 ± 5 pg/mg for group II, 85 ± 6 pg/mg for group III, 82 ± 5 pg/mg for group IV, 77 ± 5 pg/mg for group V, and 71 ± 8 pg/mg for group VI. When IMA rings of the control group (group I) were exposed to a cumulative concentration of ACh (from 1 x 10-9 to 1 x 10-5 mol/L) the total amount of 6-keto-PGF1{alpha} found in the incubation medium, at the maximum vasorelaxant effect of ACh, was augmented about twofold (from 89 ± 7 to 150 ± 13 pg/mg; p < 0.001) (see Table 1Go). The enhancement of the 6-keto-PGF1{alpha} observed in the control group was not significantly different (p > 0.05) for groups II through VI; in fact, the rate of biosynthesis of this metabolite decreased by 2% in group II, 5% in group III, 10% in group IV, 14% in group V, and 18% in group VI (see Table 1Go).


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  		Table 1. . Generation of 6-Keto-Prostaglandin F1{alpha} by Segments of Internal Mammary Artery of Study Groups in Basal Tonus Condition and During Pharmacologic Intervention With Acetylcholine, Endothelin-1, and NG-monomethyl-L-Argininea
 
Similar results were obtained when ET-1 (from 1 x 10-11 to 1 x 10-6 mol/L) was added to the organ bath containing IMA rings of different groups. As shown in Table 1Go, the immunoreactive 6-keto-PGF1{alpha} in control group (group I), found in the incubation medium at the maximum vasospasm effect of ET-1, was increased fivefold (from 89 ± 7 to 450 ± 38 pg/mg; p < 0.001) and the enhancement of this arachidonic acid metabolite was not statistically different (p > 0.05) from that obtained in group II (446 ± 30 pg/mg), group III (431 ± 18 pg/mg), group IV (414 ± 27 pg/mg, group V (396 ± 36 pg/mg), and group VI (374 ± 35 pg/mg) (see Table 1Go).

Furthermore, when the IMA rings harvested from group I (control group) were challenged with L-NMMA (1 x 10-4 mol/L) the total amount of 6-keto-PGF1{alpha} found in the incubation medium, at the maximal vasospasm effect of L-NMMA, was augmented about threefold (from 89 ± 7 to 260 ± 22 pg/mg; p < 0.001) (see Table 1Go). The increase in 6-keto-PGF1{alpha} measured in the control group was not significantly different (p > 0.05) for group II (251 ± 17 pg/mg), group III (244 ± 14 pg/mg), group IV (231 ± 18 pg/mg), group V (221 ± 20 pg/mg), and group VI (214 ± 20 pg/mg) (see Table 1Go).

Endothelial Viability
A dye-exclusion test with trypan blue was used to estimate the viability of endothelial cells in all study groups. The control group (group I) averaged a cell viability of 87.6% ± 5.8%, which was taken as 100% cell viability for the remaining groups. The analysis of variance did not demonstrate a significant loss of cell viability in groups II through VI with respect to controls. Viability rates were 97.7% in group II, 96.5% in group III, 94.6% in group IV, 85.7% in group V, and 84.8% in group VI (p > 0.05).


   Comment
Top
 Footnotes
 Abstract
 Introduction
 Material and Methods
 Results
 Comment
References
 
This experimental study was designed to cryopreserve human IMA without altering the functional properties of its endothelium. We tried to mimic favorable harvest conditions in which the WIT ranged from 30 minutes to 6 hours, correlating with a brief, or relatively brief, period between cessation of donor heartbeat and procurement, such as might occur in a multiple organ harvesting context or in non–heart-beating organ donors. The IMA specimens exposed to short ischemia (40 minutes; group I) conveniently served as a control group with which to compare the endothelial function after later processing steps.

A minimal decrease in PGI2 production with respect to group I occurred in group II IMA segments after 6 hours of WIT in basal conditions and during the different pharmacologic interventions caused by ACh, ET-1, and L-NMMA. The viability test confirms that the viability of endothelial cells is still preserved after 6 hours of WIT. Other studies have demonstrated an unexpected tolerance of the endothelium to ischemia in spite of a much lower resistance to reperfusion injuries [21].

Twenty-four hours of storage at 4°C (group III) in the presence of low-dose antibiotic sterilization (group IV) did not significantly affect IMA endothelial viability and function. These observations agree with well-documented previous data for homograft valves, with regard both to the high resistance of endothelial cells to cold ischemia and to the maintenance of a high viability rate and metabolic capabilities [22], and with regard to relatively low toxicity of the endothelium described by the current protocols of valve sterilization with a low-dose combination of antibiotics [23]. On the other hand, the necessity of a complete sterilization procedure is today universally recognized in all protocols of homograft and tissue storage for implantation, and should not be disregarded although the tissue harvesting from heart-beating and cadaveric donors must always be performed in sterile conditions.

The effects of cryopreservation on the structural integrity of endothelium have been widely investigated. Researchers focusing on the ultrastructural issues of cryopreserved endothelial cells reported a relatively good maintenance of endothelial integrity after freezing [22]. In venous allografts, cryopreservation did not significantly affect the chemical and physical properties of the venous surface, although a percentage of damaged cells was observed after freezing [24]. The functional properties of the cryopreserved endothelium have been investigated more recently: Elmore and associates [25] reported that cryopreserved vein did not present an enhanced thrombogenicity when compared with fresh controls, whereas smooth muscle cells showed a diminished reactivity to vasodilators. Brockbank and colleagues [24] also described that endothelial cells of vein allograft were shown to be viable after 3 weeks' cryopreservation. Similar findings for homograft valves have been reported by Lang and co-workers [11], demonstrating that preservation of cardiovascular tissue by cryopreservation methods did not significantly alter the metabolic status of the endothelial layer. However, data on functional behavior of saphenous grafts and heart valves are not necessarily comparable with those on arterial grafts—and in particular with the IMA—because of the difference in wall thickness, cellular composition, and vasa vasorum present in the arterial wall.

Previous studies [9, 10] have demonstrated that, compared with the saphenous vein, the IMA endothelial cells have a more responsive endogenous nitrate and eicosanoid system. Nitric oxide and PGI2 potentiate each other's action, and in this way the endothelium of the coronary bypass grafts may have a protective role in preventing vasospasm and thrombus formation [6] in IMA conduits and can explain the excellent long-term patency. We found a nonsignificant difference in activation of the endothelial L-arginine pathway in cryopreserved IMAs with respect to the fresh controls, as reflected in a nonsignificant diminished relaxation to ACh in cryopreserved IMA segments [9]; again, the maintenance of inhibition of the L-arginine pathway for the formation of nitric oxide by means of the methylated amino acid L-NMMA [14] and the maintenance of the ET-1–mediated modulation of vascular tone [26]—neither significantly affected by cryopreservation—are evidence of endothelial preservation.

The great enhancement provoked by ET-1 on the generation of 6-keto-PGF1{alpha} is further indirect proof of an acceptable retention of endothelial functionality in the cryopreserved IMA, which is likely to maintain a competent modulatory mechanism against vasoconstrictive stimuli. Therefore, if compared with the control group of fresh specimens (group I), PGI2 production was reduced only to 20% (p > 0.05) in basal tonus condition and only to 17% to 18% (p > 0.05) during the various pharmacologic interventions. Finally, the dye-exclusion test confirmed a nonsignificant loss of endothelial cell viability (-15.2%; p > 0.05) compared with controls.

Theoretically, the fact that cryopreserved IMA does not significantly lose its secretive capacity and vasodynamic reactivity appears of crucial importance in conduits used as coronary artery bypass graft, if it is true that the biological properties strongly affect its patency and function [6]. However, there is an obvious gap between the theoretic evidence and the applicability of the cryopreserved IMA in the clinical practice of myocardial revascularization. As for homograft veins employed in coronary artery bypass grafting, the immunologic issue will probably be advocated as the major determinant of patency [12]. The immunologic properties of human vascular endothelium are well known, due to the ABO blood group and HLA A,B antigens. Chronic rejection of the endothelium, resulting in intimal thickening with an adventitial fibroblastic reaction leading to eventual thrombosis of the graft, has been suggested as the mechanism that reduces the long-term patency of cryopreserved venous allografts [27]. In the study by Deaton and associates [12] on cryopreserved allograft venous conduits, an overall patency rate of 74.8% at 3 months of implantation was demonstrated in dogs that received cyclosporine. However, controversy still exists over the importance of endothelial viability in the cryopreserved venous graft performances, because it was hoped that, similar to cryopreserved heart valves, vascular tissues when thawed would have viable cells within them and these cells would continue to maintain an active role within their substructures [12]. The viability of endothelium of cryopreserved IMAs at the moment of implantation may then be crucial for patency and adaptability as a coronary artery bypass graft. Of course, only experimental studies can determine the impact of the immunologic issue on early and late patency of cryopreserved IMAs.

The research on suitability and extensive use of arterial grafts, ie, expanded use of IMA [2], right gastroepiploic artery [3], and inferior epigastric artery [4] grafts, is today one of the most important issues for cardiac surgeons, due to the sharp increase in complete arterial revascularization procedures. The growing number of coronary reoperations makes this issue even more crucial. Alternative experimental conduits have been proposed; we propose use of cryopreserved IMA arteries.

In conclusion, we think that the retention of the biological properties in the endothelium of cryopreserved IMAs may represent a theoretic background for their employment in myocardial revascularization procedures. Of course, further experimental studies are required to prove this, to investigate the immunologic issues, and finally, to examine the early and long-term patency and functional behavior.


   Footnotes
Top
 Footnotes
Abstract
 Introduction
 Material and Methods
 Results
 Comment
 References
 
Address reprint requests to Dr Pompilio, Department of Cardiac Surgery, University of Milan, Centro Cardiologico ``I Monzino'' Fundation IRCCS, Via Parea 4, 20138, Milano, Italy.


   References
Top
 Footnotes
 Abstract
 Introduction
 Material and Methods
 Results
 Comment
 References
 

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  7. Busse R, Fleming I. The endothelial organ. Curr Opin Cardiol 1993;8:719–27.
  8. Radovki MW, Palmer RMJ, Moncada S. The anti-aggregating properties of vascular endothelium: interactions between prostacyclin and nitric oxide. Br J Pharmacol 1987;92:693–6.
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