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Ann Thorac Surg 1998;65:986-992
© 1998 The Society of Thoracic Surgeons

Endothelial-Dependent Dynamic and Antithrombotic Properties of Porcine Aortic and Pulmonary Valves

Giulio Pompilio, MDaa, Giuseppe Rossoni, PhDbb, Andrea Sala, MDaa, Gian Luca Polvani, MDaa, Ferruccio Berti, PhDbb, Luca Dainese, MDaa, Massimo Porqueddu, MDaa, Paolo Biglioli, MDaa

a Department of Cardiac Surgery, University of Milan, Centro Cardiologico "I Monzino" Foundation IRCCS, Milan, Italy
b Department of Pharmacology, Chemotherapy and Medical Toxicology, University of Milan, Centro Cardiologico "I Monzino" Foundation IRCCS, Milan, Italy

Accepted for publication October 16, 1997.

Address reprint requests to Dr Pompilio, Department of Cardiac Surgery, "I Monzino" Foundation, IRCCS Via Parea, 4, 20138 Milan, Italy


   Abstract
Top
 Abstract
Introduction
 Material and methods
 Results
 Comment
 Acknowledgments
 References
 
Background. In the present study, the endothelium-dependent antithrombotic and dynamic properties of porcine aortic (AoV) and pulmonary valves (PuV) were investigated.

Methods. Fifteen fresh AoV and 15 fresh PuV were obtained from 25 9-month-old swines. The valves were examined for endothelial function by pharmacologic evaluation (with and without endothelium) of both the endothelial-releasing capacity of prostacyclin and the endothelial-dependent dynamic response to relaxing (acetylcholine from 10-10 mol/L to 10-4 mol/L in AoV and PuV segments precontracted with norepinephrine [3 x 10-6 mol/L]) and contracting (endothelin-1, from 10-11 mol/L to 10-5 mol/L; and NG-monomethyl-L-arginine, 10-4 mol/L) drugs. The ultrastructural integrity of the endothelial valve layer was also examined with transmission electron microscopy.

Results. Acetylcholine caused potent relaxation in both AoV and PuV specimens with, but not in those without, endothelium. Endothelin-1 produced a concentration-dependent tension increase in AoV and PuV with and without endothelium. However, the intrinsic activity of the peptide significantly increased in tissues without endothelium. NG-monomethyl-L-arginine evoked a progressive increase in resting tension of the preparations, but the AoV and PuV without endothelium were less sensitive to the inhibition of the nitric oxide generation. Aortic and pulmonary valves with an intact endothelium showed a spontaneous ability to release prostacyclin. The basal release of this lipidic autacoid significantly decreased in cardiac valves without endothelium. This phenomenon was observed in both basal conditions, and under stimulation with the aforementioned drugs. Transmission electron microscopy showed the perfect preservation of endothelial cells in all the preparations examined.

Conclusions. Valvular endothelium of AoV and PuV seems to have similar antithrombotic and dynamic functions of vascular endothelium, actively participating in valvular homeostasis.


   Introduction
Top
 Abstract
 Introduction
Material and methods
 Results
 Comment
 Acknowledgments
 References
 
There is a growing interest in certain functional properties of cardiac valves as a result of a better understanding of the endothelial cell layer covering the surface of the valves and endocardium. Although morphologically distinct [1], valvular endothelium seems similar to vascular endothelium. Ku and colleagues [2] have demonstrated that canine aortic valves, which release endothelium-derived relaxing factors or nitric oxide, have the capability to inhibit platelet adhesion and aggregation in a manner similar to vascular endothelium [3–5]. This suggests that the antithrombotic properties of valvular endothelium are important to avoid the formation of fibrin platelet-thrombi on cardiac valves during the cardiac cycle.

Another interesting point is the potential capability of valvular endothelium to relax and contract, producing and releasing potent vasodilating (prostacyclin and nitric oxide) and vasoconstricting (endothelin) factors, similar to vascular endothelium [6]. Nevertheless, to date, in view of the supposed general lack of vascular smooth muscle cells in cardiac valve leaflets [2], these functions have not been extensively investigated. Messier and colleagues [7] recently demonstrated that the interstitial cells of porcine aortic valve leaflets are myofibroblasts, capable of contracting in response to vasoactive stimuli, and suggested that this cell population may be central to lifelong aortic leaflet durability. Moreover, in vivo dynamic measurements of the aortic valve cycle have shown that aortic valve leaflets have an intrinsic potential to contract, before the onset of aortic blood flow [8], suggesting the active role of valve leaflets in opening and closing the aortic valve.

These recent observations on the antithrombotic and dynamic capacity of cardiac valves suggest that endothelium-dependent properties are central in determining valve functionality and durability. For a better comprehension of the functions of cardiac valve endothelium, we analyzed and compared porcine aortic and pulmonary valves (with or without endothelium), using a pharmacologic approach, by testing the endothelium-dependent relaxing response to acetylcholine and the endothelium-dependent constricting response to NG-monomethyl-L-arginine (a nitric oxide synthase inhibitor) and endothelin-1. In addition, we investigated the release of prostacyclin from porcine aortic and pulmonary valves (under basal and stimulated conditions) and the ultrastructural integrity of the endothelial valve layer (at electronic microscopy).


   Material and methods
Top
 Abstract
 Introduction
 Material and methods
Results
 Comment
 Acknowledgments
 References
 
Harvesting
Twenty-five 9-month-old Yorkshire and Poland China swine (weight, 25 ± 1.8 kg; BMG-Allevamento, Cividate Al Piano, BG, Italy) were killed by intravenous infusion with a penthotal sodium overdose. All the animals were cared for and 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). The heart was quickly removed after death (maximum time, 20 minutes) and placed in a cold Krebs-Henseleit solution. The cusps of the aortic and pulmonary valves (AoV and PuV) were dissected free from the connective tissue and divided into segments with a surface area of approximately 20 to 30 mm2. The endothelium of cardiac valves was either left intact or removed by gently rubbing against the teeth of a pair of forceps. A total of 15 AoV and 15 PuV specimens was then examined.

Organ chamber studies
Intact and endothelialized cardiac valves were suspended by means of two L-shaped stainless steel wires, one stationary and the other connected to strain gauge force transducer (model 7004, U. Basile, Comerio-VA, Italy) coupled to a U. Basile pen-recorder (model 7070) to measure the isometric tension.

In particular, cardiac valve specimens were placed in 10-mL organ baths containing the Krebs-Henseleit solution (composition in millimoles per liter: NaCl, 118; KCl, 4.7; MgSO4, 1.2; CaCl2, 2.5; KH2PO4, 1.2; NaHCO3, 25; glucose, 5.5, and EDTA, 0.03), warmed at 37°C and bubbled continuously with 95% O2 and 5% CO2 (pH 7.4). The valvular 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 depolarization of 0.1 mol/L potassium chloride (KCl) solution to establish the maximum contractile responses of each preparation. The AoV and PuV specimens were then submitted to the following pharmacologic study. (1) Acetylcholine activity in precontracted tissues: after the constrictor response of the valves specimens to norepinephrine (3 x 10-6 mol/L) had reached the plateau, the preparations were exposed to cumulative concentrations of acetylcholine (from 10-10 mol/L to 10-4 mol/L). The relaxant activity of the agonist was expressed as percentage reduction of the tension developed by norepinephrine; (2) endothelin-1 activity: the valve specimens were exposed to cumulative concentrations of endothelin-1 (from 10-11 mol/L to 10-5 mol/L) and the tension developed was recorded and expressed as a percentage of the contraction induced by KCl (0.1 mol/L); and (3) NG-monomethyl-L-arginine activity: the AoV and PuV specimens were challenged with NG-monomethyl-L-arginine (10-4 mol/L) and the tension developed was recorded and expressed as a percentage of the spasm induced by KCl (0.1 mol/L).

Prostacyclin assay
The release of prostacyclin from the valve specimens was evaluated directly in the incubation medium in basal tonus conditions of the preparation and at the end (approximately 20 minutes) of the pharmacologic interventions. Two milliliters of bath medium was collected every 20 minutes, frozen at -20°C, and stored until assayed for prostacyclin content. Prostacyclin was measured quantitatively as 6-keto-prostaglandin F1{alpha}(PGF1{alpha}) (a stable metabolite of prostacyclin) by a specific enzyme immunoassay (detection limit, 0.05 ng/mL) described by Pradelles and co-workers [9]. The assay is based on the competition between unlabeled 6-keto-PGF1{alpha} and a fixed quantity of peroxidase-labeled 6-keto-PGF1{alpha}, for a limited number of binding sites on a 6-keto-PGF1{alpha}-specific antibody. The concentration of the autacoid found in the bath medium was expressed in picograms per milligrams of wet tissue.

Transmission electron microscopy
To assess the presence of intact endothelial cells in aortic and pulmonary valve leaflets, a transmission electron microscopic analysis was randomly performed on freshly isolated specimens. After a short fixation in toto of the leaflets pinned onto a plastic surface to avoid curling, performed with 3% glutaraldehyde 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 absence of cellular damage, we excluded the presence of reversible cellular injury signs (cytoplasmatic edema, dilation of the endoplasmatic reticulum, mitochondrial swelling), or the presence of irreversible injury signs (mitochondrial flocculent density, karyolysis, and disrupted plasma membrane) [10].

Drugs
Acetylcholine chloride, NG-monomethyl-L-arginine, endothelin-1, norepinephrine chloride, L-glutamine, (Sigma Chemical Co, St. Louis, MO), penthotal sodium (Abbott S.p.A., Campoverde, LT, Italy), kit for 6-keto-PGF1{alpha} determination (Cayman Chemical Company, Ann Arbor, MI) were used in this study.

Statistical analysis
All values in the figures and text are expressed as mean values ± standard error of the mean. In each experiment, n is the number of pigs from which the cardiac valve segments were obtained. The maximal relaxation (Emax), the negative log molar concentration of a given vasodilator (acetylcholine) exhibiting 50% relaxation (pD2 value), was used to analyze the relaxations. The effective concentration of an agonist (endothelin-1) causing 25% (EC25 value) of the contraction of KCl was calculated for each valvular segment separately and was expressed as the negative log molar concentration. Because segments with and without endothelium of the same cardiac valves were studied in parallel, Student’s t test for paired observations was used for statistical comparison. Analysis of variance was used to compare more than two means. When a significant F value was obtained, Scheffé’s test was used to identify the differences among means. A p value of less than 0.05 indicates a significant difference.


   Results
Top
 Abstract
 Introduction
 Material and methods
 Results
Comment
 Acknowledgments
 References
 
Acetylcholine activity in precontracted tissues
The results obtained in this series of experiments are reported in Figure 1. They clearly indicated that norephinephrine-precontracted cardiac valve specimens of swine are very sensitive to acetylcholine-induced endothelium-dependent relaxation. In fact, in AoV and PuV with intact endothelium, acetylcholine (from 10-10 mol/L to 10-4 mol/L) caused potent relaxation with pD2 values of 7.6 ± 0.1 and 7.2 ± 0.2, and maximal relaxation reaching 93% ± 5% and 90% ± 6% of the contraction to KCl, respectively. In contrast, the activity of this agonist is precontracted preparations of AoV and PuV without endothelium was significantly less (p < 0.001) than observed in the segments with intact endothelium. In fact, the maximal relaxations reach 11% ± 2% and 5% ± 2% for AoV and PuV without endothelium, respectively (Fig 1).



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  		Fig 1. Relaxing activity of cumulative concentration–response curves to acetylcholine (from 10-10 mol/L to 10-4 mol/L) in porcine aortic (AoV) and pulmonary (PuV) valves, with (+E) and without (-E) endothelium, precontracted with norepinephrine (NE; 3 x 10-6 mol/L). Data are mean values ± standard error of the mean of five replications.

 
Endothelin-1 activity
Endothelin-1 produced a concentration-dependent tension increase in AoV and PuV with and without endothelium (Fig 2). The addition of an increasing concentration of endothelin-1 (from 10-11 mol/L to 10-5 mol/L) into organ baths containing the segments of cardiac valves with endothelium caused a concentration-dependent constriction of these tissues (AoV: ED25 = 7.3 ± 0.3; PuV: ED25 = 7.5 ± 0.2), with a peak effect equivalent to 30% ± 3% and 30% ± 2.4% of the maximal response to KCl for AoV and PuV, respectively. The intrinsic activity of the peptide significantly increased in tissues without endothelium. In fact, the ED25 observed in AoV and PuV were 8.4 ± 0.3 and 8.7 ± 0.4, respectively (Fig 2).



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  		Fig 2. Vasoconstrictor activity of cumulative concentration–response curves to endothelin-1 (from 10-11 mol/L to 10-5 mol/L) in porcine aortic (AoV) and pulmonary (PuV) valves, with (+E) and without (-E) endothelium. Data are shown as mean values ± standard error of the mean of five replications and expressed as percent of the maximal contraction to potassium cloride (KCl; 0.1 mol/L).

 
NG-monomethyl--arginine activity
In AoV and PuV with, but not in those without, endothelium, NG-monomethyl-L-arginine (10-4 mol/L) evoked a progressive increase in resting tension in all preparations. In all segments of the cardiac valves examined, this phenomenon reached the study state at about 18 to 20 minutes and the maximal contraction evoked was 24% ± 2.8% and 22% ± 3.2% of the maximal response to KCl for AoV and PuV, respectively (Fig 3). On the contrary, the AoV and PuV without endothelium were less sensitive to the inhibition of nitric oxide generation. In fact, the contraction of the segments without endothelium after NG-monomethyl-L-arginine challenge, was 92% (p < 0.001 versus AoV with endothelium) and 84% (p < 0.001 versus PuV with endothelium) lower, respectively (Fig 3).



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  		Fig 3. Vasoconstrictor activity of NG-monomethyl-L-arginine (L-NMMA; 1 x 10-4 mol/L) in porcine aortic (AoV) and pulmonary (PuV) valves, with (white columns) and without (black columns) endothelium. Each column represents the mean values ± standard error of five replications and the data are expressed as percent of the maximal contraction to potassium cloride (KCl; 0.1 mol/L).

 
Prostacyclin determination
Cardiac valves with an intact endothelium, placed in a suitable organ bath, showed a spontaneous ability to release in the incubation medium assayable amounts of arachidonic acid metabolites, first recognized as prostacyclin. The trend of this phenomenon was fairly constant throughout the various preparations and, in basal tonus condition, was quantified respectively for AoV and PuV as 120.7 ± 13.3 pg/mg and 102.7 ± 13.5 pg/mg of 6-keto-PGF1{alpha} (a stable metabolite of prostacyclin) released every 20 minutes for a period of 2 hours (Table 1). The basal release of this lipidic autacoid significantly decreased in cardiac valves without endothelium. In fact, the amount of 6-keto-PGF1{alpha}, determined in AoV and PuV segments without endothelium, was 53% and 51% lower (p < 0.01) than that obtained in cardiac valves with an intact endothelium (see Table 1).


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  		Table 1. Generation of 6-Keto-Prostaglandin F1{alpha} From Porcine-Isolated Aortic and Pulmonary Valves (with or without endothelium) at Baseline and During the Pharmacologic Intervention With Acetylcholine, Endothelin-1, and NG-Monomethyl-L-Arginine

 
When AoV and Puv segments with endothelium were exposed to a cumulative concentration of acetylcholine (from 1 x 10-10 to 1 x 10-4 mol/L), the total amount of 6-keto-PGF1{alpha} found in the incubation medium (see Table 1), at the end of the maximal vasorelaxant effect of acetylcholine, had almost doubled (from 120.7 ± 13.3 pg/mg to 192.1 ± 192.1 pg/mg for AoV and from 102.7 ± 13.5 pg/mg to 165.3 ± 22.7 pg/mg for PuV). The enhancement of 6-keto-PGF1{alpha} observed in cardiac valves with endothelium was 51% and 48% lower (p < 0.01) when the acetylcholine was added to AoV and PuV without endothelium (see Table 1).

Similar results were obtained when endothelin-1 (from 1 x 10-11 to 1 x 10-5 mol/L) was added to the organ bath containing AoV and PuV with endothelium. As shown in Table 1, the total amount of 6-keto-PGF1{alpha} measured in the incubation medium, at the end of the maximal vasoconstrictor effect of endothelin-1, increased about fourfold (from 120.7 ± 13.3 pg/mg to 418.5 ± 54.2 pg/mg for AoV and from 102.7 ± 13.5 pg/mg to 450.8 ± 20.5 pg/mg for PuV) and the enhancement of this arachidonic acid metabolite was significantly (p < 0.01) reduced by 80% and 83% when endothelin-1 was added to AoV and PuV without endothelium.

When the AoV and PuV with endothelium were challenged with NG-monomethyl-L-arginine (1 x 10-4 mol/L), the total amount of 6-keto-PGF1{alpha} found in the incubation medium (see Table 1), at the maximal vasospasm effect of NG-monomethyl-L-arginine, had more than doubled (from 120.7 ± 13.3 pg/mg to 308.4.1 ± 33.5 pg/mg for AoV and from 102.7 ± 13.5 pg/mg to 267.3 ± 20.5 pg/mg for PuV). The increase in 6-keto-PGF1a, measured in cardiac valves with an intact endothelium significantly decreased (p < 0.01) in tissues without endothelium (73% for AoV and 73% for PuV).

Transmission electron microscopy
We randomly examined, by means of transmission electron microscopy, the integrity of the endothelial layer of control aortic and pulmonary valve specimens immediately after harvesting. A total of six specimens for each valve was studied. Ultrastructural features of endothelial cells show a complete preservation of the nucleus, the cytoplasm, and the endothelial lining (Fig 4).



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  		Fig 4. Ultrastructural features of endothelial cells of an aortic valve leaflet. The nucleus and the cytoplasm are well preserved, and the endothelial lining of the valve is continuous. (x23,200 before 52% reduction.)

 

   Comment
Top
 Abstract
 Introduction
 Material and methods
 Results
 Comment
Acknowledgments
 References
 
The concept of a passive role of cardiac valve leaflets in regulating the forward flow of blood during the cardiac cycle has recently been reviewed. Cardiac valves are now evaluated for more complex functions [7]. Two aspects has been identified as central for valvular homeostasis: the antithrombotic capacity [2] and the intrinsic contractility [7, 8]. It has been shown that canine cardiac valves actively contribute to prevent local platelet and thrombus formation and adhesion by releasing antithrombogenic substances [2]. Moreover, an active role of the aortic valve in opening and closing during the cardiac cycle has been recently demonstrated [8], and this intrinsic potential to open correlates well with histologic findings of the presence of myofibroblast in cardiac valve leaflets [7]. To date, however, the exact pathophysiologic role of the interaction between the active substances released by valvular endothelium and valve functionality has not been fully investigated.

We studied the capacity of valvular endothelium to generate and maintain antiplatelet and dynamic properties in porcine aortic and pulmonary valve leaflets. It would seem that porcine valvular endothelium is very similar to vascular endothelium. Our results suggest that the endothelial cell layer covering both aortic and pulmonary valves possesses highly active metabolic functions, which modulate the degree of contraction of the underlying smooth muscle cells and the production of antithrombotic substances.

In endothelial cells, nitric oxide is cleaved from its precursor, the amino acid L-arginine, by specific enzymes [11]. Nitric oxide stimulates the formation of cyclic GMP in smooth muscle cells, causing vasodilatation, and in platelets, causing inhibition of aggregation [12, 13]. By means of the methylated amino acid NG-monomethyl-L-arginine, the pathway for the formation of nitric oxide can be inhibited [14]. Valvular endothelium of porcine aortic and pulmonary valves must continuously release nitric oxide, as the inhibitor of nitric oxide formation, NG-monomethyl-L-arginine [15, 16], evoked endothelium-dependent contractions in aortic and pulmonary valve specimens with, but not in those without, endothelium. This suggests that, as with vascular endothelium, the basal release of endothelium-derived nitric oxide modulates the relaxation of these valve preparations. Previous studies have shown the release of endothelium-derived relaxing factors from canine cardiac valves [2] and from porcine mitral valves [17]. Moreover, cultured porcine endocardial cells were previously found to possess the ability to synthesize nitric oxide [18]. The present results provide evidence that there is a similar activation of the endothelial L-arginine pathway in porcine aortic and pulmonary valves.

The addition of acetylcholine is known to enhance the release of endothelium-derived relaxing factors in endothelial cells from blood vessels [19]. To study further the involvement of nitric oxide in endothelial-mediated cardiac valve relaxation, acetylcholine was added to valve preparations. Acetylcholine produced concentration-dependent relaxation in AoV and PuV precontracted with norepinephrine. This marked relaxing response to acetylcholine was not observed in the control endothelium-denuded valves, confirming that this agonist activates the endothelial L-arginine pathway and that nitric oxide accounts for the relaxation caused by acetylcholine. The fact that acetylcholine produced a potent endothelium-dependent relaxation in porcine cardiac valves confirms the previous observations in dogs [2].

To investigate further the endothelium-dependent dynamic capacities of aortic and pulmonary valves, the addition of endothelin-1 was studied. Endothelin-1 is a peptide produced and released by endothelial cells, which exhibits potent vasoconstrictor effects on smooth muscle cells of the vascular bed [20]. Indeed, vascular endothelium possesses a competent regulatory mechanism against the vasoconstrictive action of endothelin-1, thanks to the release of endothelium-derived nitric oxide [21] and prostacyclin [22]. The overall in vivo systemic response to endothelin-1 includes a short-lived vasodepressor phase, attributed to the release of nitric oxide and prostacyclin, followed by a long-lasting increase in blood pressure [23]. The addition of endothelin-1 to our preparation caused a similar dose-dependent constriction of aortic and pulmonary valves, which is, however, less marked in cases of intact endothelium. Therefore, valvular endothelium seems to possess the capacity to release the constrictor peptide endothelin-1 and to regulate constrictive and relaxing responses of valve leaflets with the synergic action of endothelin-1, nitric oxide, and prostacyclin. As for vascular endothelium, Ca2+ mobilization from intracellular binding sites caused by endothelin-1 seems to be the explanation of nitric oxide and prostacyclin synthesis in valvular tissue.

Finally, the results obtained from these experiments indicate that the specimens of porcine aortic and pulmonary valves release a substantial amount of immunoreactive 6-keto-PGF1{alpha}, the stable metabolite of prostacyclin. The basal release of this lipidic autacoid (120.7 ± 13.3 pg/mg and 102.7 ± 13.5 pg/mg for AoV and PuV, respectively), is comparable with that produced by saphenous veins (130 ± 5 pg/mg), and inferior to that produced by internal mammary arteries (251.7 ± 6.3 pg/mg) [22].

This biochemical event is probably important in valvular homeostasis. In fact, prostacyclin, the major member of the prostaglandin family formed by endothelial cells [24], exhibits antiplatelet and vasodilator activity and plays a role in the regulation of leukocyte accumulation in the vessel walls, as well as in the control of smooth muscle proliferation and cholesterol metabolism [25]. The present findings also indicate that aortic and pulmonary valves have a similar capacity to accumulate 6-keto-PGF1{alpha} in the medium, similar to the vascular endothelium [26]. These results suggest that in vivo conditions, chemical or mechanical perturbations (such as pulsatile pressure and endogenous mediators) of the endothelial cell membrane of these valves, may lead to a more advantageous activation of the eicosanoid system, with preferential generation of prostacyclin. Interestingly, endothelium denuded valves still maintain a limited capacity to release prostacyclin, suggesting that interstitial cells (fibroblasts, smooth muscle cells) also release antithrombotic molecules.

Another point of interest arising from the present results is that the spontaneous generation of 6-keto-PGF1{alpha} by aortic and pulmonary valves is greatly enhanced by endothelin-1. This observation is an indirect indication of a competent modulator mechanism in valvular endothelium against vasoconstrictive stimuli.

The present findings may have important consequences not only in terms of the thrombogenicity and functionality of cardiac valves, but also for the valve leaflet capacity to resist structural degeneration. Both nitric oxide and prostacyclin have the capacity to inhibit smooth muscle cell proliferation and to control cholesterol metabolism [25, 27]. Therefore, valvular endothelium seems to be a main factor in determining the resistance of native cardiac valves to atherosclerotic degeneration. This hypothesis correlates well with the preservation of allograft valve substitute viability in cardiac operations; the more viable the allograft valve cellular components at the moment of the implant, the longer its long-term durability [28].

In conclusion, our study contributes to the characterization of the endothelium-dependent properties of AoV and PuV. Valvular endothelium seems to have similar functions to vascular endothelium, and in our opinion, more importance will be attributed in the future to the role of valvular endothelium in cardiac valve homeostasis.


   Acknowledgments
Top
 Abstract
 Introduction
 Material and methods
 Results
 Comment
 Acknowledgments
References
 
We thank Anna Guarino and Alessandra Corti for their help in writing this article, and Isabella Barajon for her assistance with electron microscopy.


   References
Top
 Abstract
 Introduction
 Material and methods
 Results
 Comment
 Acknowledgments
 References
 

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