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Ann Thorac Surg 2000;70:1594-1600
© 2000 The Society of Thoracic Surgeons

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Original articles: cardiovascular

Cell composition of the human pulmonary valve: a comparative study with the aortic valve–the VESALIO* project^*

^a Departments of Experimental and Clinical Medicine and Biomedical Sciences, and Institute of Cardiovascular Surgery, University of Padua, and Fidia Research Laboratories, Padua, Italy

Address reprint requests to Dr Pauletto, Department of Experimental and Clinical Medicine, University of Padua, Via Giustiniani, 2-35128 Padua, Italy
e-mail: pauletto{at}ux1.unipd.it

	Abstract

Top Footnotes Abstract Introduction Material and methods Results Comment Acknowledgments References

 
Background. Cell populations present in human semilunar valves have not been investigated thoroughly. The aim of this study was to characterize the cell phenotypes in pulmonary valve leaflets (PVL) in comparison with aortic (AVL) valve leaflets.

Methods. AVL and PVL were dissected from hearts (n = 4) harvested from transplanted patients. Leaflets were processed for immunocytochemistry analysis and Western blotting procedures using a panel of monoclonal antibodies specific for cytoskeletal/contractile antigens.

Results. The fibrosa and the ventricularis layers of AVL had a higher cellularity than PVL. In PVL and AVL most cells were reactive for vimentin and nonmuscle (NM) myosin, though vimentin-positive cells were more abundant in AVL than in PVL. Sparse cells positive to antismooth muscle (SM) -actin, calponin, and anti-SM myosin antibodies were found only at the outer edge of fibrosa. In Western blotting, AVL and PVL extracts were shown to be equally reactive for vimentin, SM -actin, and NM myosin, whereas both valves were negative for SM myosin and SM22.

Conclusions. Three distinct cell phenotypes have been identified in both valves: fibroblasts, myofibroblasts, and fetal-type SM cells whose distribution is specifically related to the valve layers. Although PVL and AVL cell populations differ quantitatively, some minor qualitative differences exist for vimentin and NM myosin distribution. These data are essential for studies aimed at repopulating valve scaffolds by using tissue engineering technology.

	Introduction

Top Footnotes Abstract Introduction Material and methods Results Comment Acknowledgments References

 
In recent years, the pulmonary valve has generated great interest among cardiac surgeons owing to the promising results obtained with autotransplantation of the pulmonary valve into the aortic position for aortic valve replacement, according to the Ross procedure [1]. Moreover, pulmonary homografts have been proposed as alternative aortic valve substitutes [2]. Since 1985, the pulmonary homograft has also been the conduit of choice for right ventricular outflow tract reconstruction in congenital heart defects [3]. In children, aortic valve or root replacement by pulmonary autografts allows for the maintenance of viable cell populations within the valve cusps, making the autograft capable of growing harmonically together with the host [1]. In addition, the preservation of the physiologic cell environment may play a role in preventing calcific degeneration of valve cusps [4]. Cell seeding onto commercially available heart valve bioprostheses [5] or synthetic scaffolds [6] has been attempted using endothelial [5] or interstitial cells [6], or both [7], and arterial or dermal endothelial cells-myofibroblasts mixtures [8]. The selection of appropriate cells for in vitro cultures and subsequent seeding remains an unresolved aspect in this field. Whereas the mechanical properties of pulmonary valve leaflets (PVL) have been extensively studied and compared with those of their aortic valve leaflet (AVL) counterparts [2], the differentiation characteristics of the cells present in the AVL and PVL have not been investigated in detail.

We report on the cell typing analysis regarding the different layers of human PVL, in comparison with AVL, based on their differentiation features as established by the immunochemical and immunocytochemical patterns with a panel of specific monoclonal antibodies. This study should furnish some information about the basic cellular composition of the two valves that can be useful to evaluate the adaptive potential of PVL in the aortic position [9] and, hence, the long-term durability of the transplanted valve. This study should also be able to give a "cellular map" of cell phenotype distribution in the different layers of valves, which is relevant for studies aimed at repopulating bioprostheses from the commerce as well as for the seeding of cells on artificial scaffolds.

	Material and methods

Top Footnotes Abstract Introduction Material and methods Results Comment Acknowledgments References

 
Collection of samples
PVL and AVL were obtained from the explanted hearts of 4 men (age range 49 to 69 years) with a history of idiopathic dilative cardiomyopathy undergoing cardiac transplantation. In these patients, the anamnestic data were carefully reviewed to rule out cardiac valve disease or any other relevant systemic disease. Valves obtained from transplanted hearts were inspected for the presence of calcifications and myxomatous degeneration by conventional histologic examination. The different leaflets from AVL and PVL were then separated, immediately rinsed in sterile phosphate buffered saline (PBS), frozen in liquid nitrogen, and stored at -80°C until use.

Frozen specimens from each AVL and PVL were cut by a Leica Jung CM 3000 cryostat. To ascertain the integrity of valve tissue in each experiment one hematoxylin-eosin stained section, taken from a group of sections obtained from each leaflet, was observed under microscope before the other cryosections were submitted to the immunocytochemical or immunochemical procedures.

Immunocytochemical analysis
The following monoclonal antibodies were used (see Table 1) in indirect immunoperoxidase experiments: SM-E7, specific for both SM1- and SM2-myosin heavy chain (MyHC) isoforms; NM-G2 antiplatelet-type NM-MyHC (MyHC-Apla2); NM-F6 antiplatelet-type NM-MyHC (MyHC-Apla1); and E-11 anti-SM22, specific for the SM cell lineage specific 22-kDa protein [10, 11]. The antivimentin was purchased from Boehringer Mannheim (Mannheim, Germany). The anti-SM -actin and anticalponin antibodies were obtained from Sigma (St. Louis, MO). The combined use of SM-E7 and NM-F6 allows for the identification of adult-type (staining with SM-E7 alone) or fetal-type (staining with SM-E7 plus NM-F6) SM cells [12]. By contrast, NM-G2 antibody staining on SM cells is invariant throughout development [12].

Slides were then rinsed twice with PBS and incubated with the secondary antibody (rabbit IgG, antimouse IgG) conjugated with horseradish peroxidase (Dako; Dakopatts, Glostrup, Denmark) at 1:40 dilution under the conditions described above. After being washed in PBS, sections were incubated in 20 mg 3-amino-9-ethylcarbazole (Sigma) in 2.5 mL dimethylformamide solubilized in 50 mL sodium acetate buffer at pH 5.0, containing 25 µL H₂O₂. Bound antibodies were detected by indirect immunoperoxydase until distinct staining was microscopically detected with minimal background (about 10 minutes of incubation). After washing, slides were mounted in Elvanol and observed under an Olympus FX540 microscope.

The following control experiments were performed: (1) nonimmune IgG followed by the secondary antibody, and (2) the secondary antibody alone.

Image analysis
Countings of immunostained cells and of the totaling of cell numbers on cryosections were carried out using a 25x objective by the AT-IBAS Image Analysis System (Kontron, Eching, Germany). Positive immunoreactions resulted in a staining of well-defined groups of cells or isolated cells. The threshold level used in the image analysis procedure to differentiate positive cells from the background staining was established by comparison with the negative control mentioned above (nonimmune IgG and the secondary antibody alone). The surface of the scanned area was 26,000 µm².

The number of positive cells and their relative percentage were assessed in the three layers (fibrosa, spongiosa, and ventricularis) of valvular leaflets. From each layer of the different leaflets, three different fields were analyzed. Two different images were taken from each field. To define in detail positive cells from the background, each image was set up at two monochromatic background lights of 450 nm and 630 nm, respectively.

In the first step, the area occupied by negative or positive cells was determined. The total mean area covered by positive cells (stained in red) and the total mean area of hematoxylin-positive, blue-counterstained cells was then measured. The mean number of cells per area unit was estimated by the ratio between the total mean area covered by positive or negative cells and the corresponding mean cell surface. In each layer of the various leaflets, the mean area of 15 negative cells and of 15 positive cells randomly chosen was measured for each type of immunostaining using a computer-driven planimetry and a 40x objective (VIDS V, Cambridge, UK). Positive/negative cell ratios in the various layers of PVL and AVL were determined.

Crude extract preparation, electrophoresis, and Western blotting
This procedure was performed on a pool of three PVL and three AVL leaflets using the following antibodies: antivimentin, anti SM-22, anti-SM -actin, SM-E7, NM-F6, and NM-G2.

Tissues were collected in an Eppendorf tube and proteins in the tissue were extracted for 2 minutes at 100°C in Laemmli’s sample buffer solution using a ratio of 1 mg/2 mL extraction solution. The solubilized proteins were separated from the unextracted material by centrifugation at 14,000g for 10 minutes.

Comparable amounts of total proteins or total MyHC from the various extracts to be used in electrophoresis and Western blotting experiments were determined by the procedure of Sandri and coworkers [13]. Briefly, small amounts of each extract were electrophoresed in 12.5% (for Western analysis with antivimentin, anti-SM22, or anti-SM -actin) or 5% (for Western analysis with anti-SM and anti-NM myosin antibodies and MyHC identification) SDS gels in order to obtain a single band when the gels were stained with the Coomassie blue.

Identical amounts of each extract to be loaded in the final gels were calculated by a comparison with a calibration curve, obtained using bovine serum albumin as a standard. The amounts selected were in the linear range of this calibration curve. Three different extractions from each tissue and four electrophoresis runs per extraction were analyzed for both total MyHC and specific MyHC isoform quantification.

Densitometry of MyHC separated on SDS gels was carried out as described above. The Western blotting procedure was that detailed in Chiavegato and associates [14]. Bound antibodies to antigens, blotted on the nitrocellulose paper, were revealed by indirect immunoperoxidase staining using rabbit IgG to mouse IgG coupled to horseradish peroxidase (Dako; Dakopatts, Glostrup, Denmark). Bound primary-secondary antibody complex was revealed by chemiluminescence using the Amersham ECL kit (Amersham, Buckinghamshire, UK).

Statistical analysis
The statistical analysis was performed using the STSC-Statgraphics 4.0 Package (STSC Inc, Hill AFB, UT). In the anterior, posterior, and septal PVL the reactivity to monoclonal antibodies was evaluated by analysis of variance (ANOVA). Results were then confirmed by Kruskal-Wallis testing.

In the comparison of PVL versus AVL, Student’s t test was used to evaluate the differences occurring in the reactivity to the monoclonal antibodies to the three layers (fibrosa, spongiosa, and ventricularis) and in the total cell number between layers from the two valves. Differences in the total cell number between layers were confirmed by the nonparametric test of Mann and Whitney, whereas the total cell number among anterior, posterior, and septal PVL was established by ANOVA. Differences of p value of at least less than 0.05 have been considered significant.

	Results

Top Footnotes Abstract Introduction Material and methods Results Comment Acknowledgments References

 
Cell density in the different layers of the PVL leaflets
The assessment of the cells present in the three layers of anterior, posterior, and septal cusps showed a lower number of cells in the spongiosa compared with the other layers. Differences, however, were not statistically significant (not shown). Comparative studies carried out in AVL, showed that both the fibrosa and the ventricularis layers had a cellularity higher than PVL, but differences were significant (p < 0.012) for ventricularis only (not shown).

Distribution of cell differentiation markers in semilunar valves
As a general feature, both PVL and AVL cells express, although to a different extent, vimentin and NM myosin isoforms in all three layers (fibrosa, spongiosa, and ventricularis; see Figure 4 for a schematic representation of the distribution patterns). In addition, to this "default" cell distribution there is a "spotted" (PVL) or "continuous" (AVL) immunostaining at the outer edge of fibrosa characterized by small clusters or a thin layer of cells, respectively, reactive with anti-SM -actin, anti-SM22, anticalponin, and anti-SM myosin antibodies (Fig 1). Intermingled with this cell population in both valves and at the same level of fibrosa, there is another cell phenotype that lacks the SM myosin content but maintains the expression of the other SM cell-related markers (see Fig 1 and Fig 4).

View larger version (47K): [in this window] [in a new window]   Fig 4. Schematic representation of the various cell populations present in the fibrosa, spongiosa, and ventricularis of aortic valve leaflets (AVL) and pulmonary valve leaflets (PVL) as determined by immunocytochemical experiments. Fetal-type SM cells and myofibroblasts are segregated in the outermost layer of fibrosa, whereas different fibroblast subtypes are expressed in the ventricularis of AVL versus PVL and in the spongiosa of both valves as well. The slight difference existing in vimentin distribution between AVL and PVL at the level of fibrosa is not reported in the figure. The up arrows and down arrows indicate a high or low number of vimentin-positive cells, respectively.

View larger version (117K): [in this window] [in a new window]   Fig 1. Immunocytochemistry on cryosections from pulmonary valve leaflets (A, C, E, G) and aortic valve leaflets (B, D, F, H) reacted with anti-SM -actin (A, B), anti-SM22 (C, D), anticalponin (E, F), and anti-SM(-MyHC) myosin (G, H), monoclonal antibodies. Note the continuous subendocardial layer of positive cells in A (green arrows) and clusters of positive cells in C, E, G, and H (red arrows) in the outermost part of the fibrosa. In aortic valve leaflets, the layer of positive subendocardial cells is generally thicker than that of pulmonary valve leaflets (x 400).

A quantitative comparison between PVL and AVL indicated that vimentin-positive cells were less abundant in PVL than in AVL. This was particularly evident in the spongiosa as well as in the ventricularis (Fig 2A). Other differential features between the two valves included the lack of reactivity with NM-F6 in the ventricularis (Fig 2B) and the reduced reactivity with NM-G2 in the fibrosa (Fig 2C) of AVL.

View larger version (20K): [in this window] [in a new window]   Fig 2. Bar graph showing the distribution of positive cells (number of cells/µm2) with (A) antivimentin, (B) NM-F6 anti-MyHC-Apla1, and (C) NM-G2 anti-MyHC-Apla2 in fibrosa, spongiosa, and ventricularis from aortic valve leaflets (white bars) and pulmonary valve leaflets (black bars). Mean ± SD is reported.

View larger version (57K): [in this window] [in a new window]   Fig 3. Western blotting analysis of crude extracts from (1) aortic valve leaflets and (2) pulmonary valve leaflets electrophoresed in (A) 12.5% or (B) 5% SDS-gels, reacted with antivimentin, anti-SM22, and anti-SM ()-actin is shown in panel A. In panel B, the pattern obtained with anti-SM(-MyHC), anti-MyHC-Apla1, and anti-MyHC-Apla2 antibodies is shown. The band reactive with the anti-NM myosin antibodies is about 200-kDa apparent mass. Note that SM22 and SM-MyHC antigens are undetectable under the conditions used in this experiment; a, b, and c indicated the Western blots.

	Comment

Top Footnotes Abstract Introduction Material and methods Results Comment Acknowledgments References

This study, aimed precisely at revealing the composition and dislocation of the different cell phenotypes in semilunar valves, shows that three major cell phenotypes were identified in both semilunar valves, independent from the cusps analyzed: SM cells, myofibroblasts, and fibroblasts (Fig 4). Similarity between PVL and AVL is also confirmed by Western blotting but it is only with the immunocytochemical approach that the subtle differences in the cell population localization appear. In fact, SM cells and myofibroblasts are essentially localized to the fibrosa, whereas fibroblasts are uniquely segregated in the ventricularis. The spongiosa is in general the poorest cell layer and displays a slightly different fibroblast pattern. Based on our previous studies, SM cells identified here resemble the arterial SM cells found in the early stages of development [11], whereas the accompanying myofibroblasts seem to belong to the VA subclass established by Sappino and associates [15]. Interestingly, fibroblasts show a unique composition in the spongiosa and differ in the two types of valves at the level of the ventricularis layer where vimentin is high in AVL and low in PVL, and MyHC-Apla1 is present exclusively in PVL (Fig 4). It is worth noting that it is the ventricularis, but not the other two valve layers, that shows a statistically significant increase in cellularity in AVL compared with PVL. Thus, the well-known differences in the cusp thickness between the two valves might be solely from the increased ventricularis "size" in AVL, which, interestingly enough, is accompanied by a thicker layer of myofibroblasts/fetal-type SM cells in the fibrosa (Fig 1).

Looking at the map of three major phenotypes found in the semilunar valves (Fig 4), one might suggest that the cell phenotype distribution is reminiscent of the vascular wall organization (particularly of the large veins) inasmuch as the myofibroblast/SM cell dislocation in the fibrosa seems to correspond to the medial layer and the fibroblast localization is that of adventitial layer. This hypothesis is also supported by the finding that in rabbits kept on a cholesterol-enriched diet, valve interstitial cells accumulate cholesterol-esters [16] as occurs for "phenotypically modulated, synthetic" SM cells of the arterial wall during atherogenesis [11]. Given this structural similarity between semilunar valves and vascular wall, it might be that, as occurs in the arterial wall of hypertensive animals [11], the establishment of fetal-type SM cell/myofibroblast phenotype in the valves is dependent on the pressure level. Thus, cell phenotypic differences in PVL versus AVL might be reasonably attributed to different pressure levels in the pulmonary versus systemic circulation.

Our study is in concordance with that of Filip and colleagues [16] who showed that interstitial cells from cardiac valves possess characteristics similar to SM cells, namely, abundance of actomyosin filaments, cGMP-dependent proteinkinase, contractile response to epinephrine and angiotensin II. Bairati and De Biasi [17] also showed bundles of SM cells within porcine leaflets using electron microscopy that were not found in other studies performed in cultures, possibly because of "phenotypic modulation" of cells grown in vitro [18]. In porcine AVL, studies by Messier and associates [19] showed the concomitant presence of dual structural and functional phenotypes of interstitial cells, possessing the characteristics of both fibroblasts (matrix secretion) and SM cells (contraction to various agents including endothelin-1), and hence defined as myofibroblasts. Moreover, the same authors found that the higher level of reactivity to the anti-SM -actin antibodies was present in the subendocardial layer.

Our study defines in detail the composition and distribution of cell populations in human semilunar valves but what remains to be elucidated is the significance of the specific distribution of fetal-type SM cells/myofibroblasts in the subendocardial layer of fibrosa and the fibroblasts in the ventricularis. Recent data obtained in our laboratory indicate that a phenotypic cell conversion can occur in the adventitia of injured arterial wall by which fibroblasts can become myofibroblasts and fetal-type SM cells [11]. Thus, it might be that, as occurs in the arterial wall, the different levels of shear stress levels acting on the ventricularis versus fibrosa layer dictate and maintain such cell phenotypic organization.

An obvious limitation to this study is representated by the number of valves examined and the use of valve leaflets harvested from hearts of transplanted patients. Unfortunately, valve samples collected at autopsy are unsuitable for immunocytochemical studies owing to the ischemic time elapsed from death to harvesting. Moreover, valves dissected from hearts explanted for primary dilated cardiomyopathy are routinely used as homografts (homovital) for left ventricular outflow tract or right ventricular outflow tract reconstruction. The excellent long-term results presented by the Yacoub group [20] with such homovital procedures prove that these valves should be considered as normal. Finally, for homograft banking purpose, we have extensively studied AVL and PVL harvested from the hearts of transplanted patients (domino valves) by means of light and electron microscopy. We were unable to detect any major differences in terms of morphology and structure among these valves when compared with valves harvested from nonbeating-heart donors.

	Acknowledgments

Top Footnotes Abstract Introduction Material and methods Results Comment Acknowledgments References

 
This study was supported by grant no. 9806193973, entitled "Cell repopulation of homograft and xenograft cardiac valves and prevention of dystrophic calcification by means of tissue engineering technology and anticalcific agents" from the MURST (Rome, Italy). We thank Mrs Sarah Hansen for her excellent editorial work.

	Footnotes

Top Footnotes Abstract Introduction Material and methods Results Comment Acknowledgments References

 
^* Vitalitate Exornatum Succedaneum Aorticum Labore Ingegnoso Obtinebitur

	References

Top Footnotes Abstract Introduction Material and methods Results Comment Acknowledgments References

 

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