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Eur J Cardiothorac Surg 2003;24:527-534
© 2003 Elsevier Science NL

Transcriptional profiling and growth kinetics of endothelium reveals differences between cells derived from porcine aorta versus aortic valve

R. Saeid Farivar*, Lawrence H. Cohn, Edward G. Soltesz, Tomislav Mihaljevic, James D. Rawn, John G. Byrne

Division of Cardiac Surgery, Brigham and Women's Hospital, Harvard Medical School, 75 Francis Street, Boston, MA 02115, USA

Received 18 September 2002; received in revised form 30 May 2003; accepted 16 June 2003.

* Corresponding author. Tel.: +1-617-285-5585; fax: +1-617-732-6559
e-mail: sfarivar{at}partners.org


   Abstract
Top
 Abstract
1. Introduction
 2. Materials and methods
 3. Results
 4. Discussion
 References
 
Objective: Valvular tissue and aorta calcify at different rates when placed as fresh homografts or cryopreserved allografts. Furthermore, differences between valvular endothelial cells and aortic endothelial cells are not well appreciated. We established primary cultures of valve and aortic endothelial cells derived from swine and tested transcriptional and proliferative differences on various extracellular matrices. Methods: Transcriptional profiling was performed on primary cultures of porcine valve and aortic endothelial cells. We extracted total RNA from both cell types and created cDNA libraries. We scored for 847 genes important in signal transduction pathways, and measured their expression on valve and aortic endothelial cells. To determine if there were functional differences between aortic and valvular cells, their growth rate was determined by cell counting on various extracellular matrices. Results: Of 847 genes investigated, 69 (8.1%) were transcriptionally active on aortic endothelial cells and 89 (10.5%) on valve endothelial cells. Common to both cell types were 55 genes, which represents 79.7% (55/69) of activated genes on aortic endothelial cells and 61.8% (55/89) of those in valve endothelial cells. Remarkable features of the analysis included Ephrin ligand and receptor specificity for cell type, a potential fibroblast growth factor autocrine loop in both cell types, as well as upregulation of the platelet-derived growth factor receptor in valvular cells. Aortic endothelial cells were noteworthy of upregulation of vascular endothelial cell growth factor-B and vascular cell adhesion molecule. Proliferation analysis revealed that valve endothelial cells grew more rapidly (12-fold over control) than aortic endothelial cells (3-fold over control). Furthermore, valve endothelial cells proliferated most rapidly on gelatin or collagen, whereas aortic endothelial cells were most proliferative on lysine or laminin. Conclusions: Valve and aortic endothelial cells have different transcriptional and proliferative profiles. The knowledge of these differences may be an exploitable strategy in the future rational design of artificially engineered valve surfaces and in the study of the valve antigenicity, immunogenicity and structural failure.

Key Words: Endothelial cell • Valve • Microarray • Cell culture • Pig


   1. Introduction
Top
 Abstract
 1. Introduction
2. Materials and methods
 3. Results
 4. Discussion
 References
 
Valve endothelium is antigenic [1–4], or able to display potentially immunogenic epitopes such as major histocompatibility complexes I and II [5] or the {alpha}Gal antigen [1]. Furthermore, it has been demonstrated in culture that valve endothelial cells may be immunogenic [2], or elicit a mixed lymphocyte reaction. In vivo, it has been shown that donor-specific humoral responses are elicited in homografts recipients [6], and it has been demonstrated that there is the expression of immunologically relevant molecules on human cardiac valve endothelium in situ [4]; yet the importance of this phenomenon is unclear since fresh homografts or cryopreserved allografts are routinely introduced in the United States without ABO typing or any immunosuppressives with a 70–85% freedom from failure at 8–10 years [7,8].

It is currently controversial whether a viable endothelial cell surface would improve or detract from the longevity of a valvular bioprosthesis [9,10]. While viable endothelium may provide appropriate coagulation factors, and nourish supporting fibroblasts (for matrix remodeling) via paracrine agents to provide a durable valve with excellent hemodynamic properties; the endothelium, if immunologically mismatched, may also sensitize the recipient to anti-valvular antibodies and cellular responses which could accelerate deterioration and structural failure.

It may be clinically relevant to determine if fresh homografts or cryopreserved allografts contain viable endothelial cells. Evidence suggests that the majority of cryopreservation techniques do not leave viable endothelial cells [11], although varying results have been reported since different antibiotics [12], temperature of storage [3], rate of cooling [13], and preservative [14] have been shown to affect endothelial cell viability. Furthermore, fresh homografts valves appear to have some viable endothelial adhesion molecules, whereas cryopreserved valves do not [3,15].

An optimal bioengineered valve graft would display an exact donor-recipient matched valve endothelium. To understand more fully the biology of valve endothelium, we have established primary cultures of porcine valve endothelial cells and compared these to aortic endothelial cells. We have performed transcriptional profiling and cell proliferation assays of these two cell types; and we find clear differences between these two cell types which may be useful in the future rational design of artificial valve surfaces.


   2. Materials and methods
Top
 Abstract
 1. Introduction
 2. Materials and methods
3. Results
 4. Discussion
 References
 
2.1. Materials
Type I- and type IV-collagen (rat tail), fibronectin (human), poly-D-lysine (synthetic peptide), and laminin (mouse) coated six-well plates were purchased from Becton Dickinson (Bedford, MA). Microarrays and gene specific primer kits were purchased from R&D Systems (Minneapolis, MN). [{alpha}-32P]dCTP was purchased from Perkin Elmer (Boston, MA). Pigs were adult swine from Parson's Farms (Royalton, MA).

2.2. Cell culture
Primary cultures of porcine valvular endothelial cells were prepared using modifications of previously published methods [16],[1]. All animals received humane card in accordance with the Harvard University Protocol on animals, the Guide for the Care and Use of Laboratory Animals (revised 1996), and in accordance with the European Convention on Animal Care. Our institutional animal committee approved all animal experiments. Hearts were removed under aseptic conditions from domestic adult swine (50 kg) that were euthanized by 87 mg/kg sodium pentobarbital (Abbott Laboratories). The valve leaflets were removed from the aortic and mitral valve, and placed into phosphate buffered saline supplemented with antibiotics (100 U/ml penicillin G and 20 µg/ml gentamicin). A margin of leaflet was left attached to the annulus. Valve leaflets were washed free of red blood cells by three washes with phosphate-buffered saline (PBS)/antibiotic solution. The valve leaflets are then placed in a 0.6 mg/ml collagenase solution (from Clostridium histolyticum, Sigma, St Louis, MO) in PBS supplemented with 100 U/ml penicillin G and 20 µg/ml gentamicin at 37 °C for 45 min. The valve leaflet was then gently shaken for 1 min in medium 199 (Gibco BRL Life Technologies, Gaithersburg, MD). Cells which dislodged were then aliquoted into a 96-well plate, onto which complete media was added. Media was changed every 48 h. Cells were checked for expansion every day. Wells which contained cells with fibroblastic or smooth muscle cell morphology and growth characteristics, as evinced by spindle morphology or lack of contact inhibition, were discarded. Cells with typical cobblestone morphology were considered potential endothelial cells and then expanded and confirmed to be of endothelial cell origin by immunohistochemistry with the Ricinus communis A (RCA) lectin (a mammalian pan endothelial marker) and confirmed not to be smooth muscle cells by staining with anti-smooth muscle cell {alpha}-actin (Dako, Carpinteria, CA) [1]. These were used for subsequent experiments. Complete media consisted of medium 199 with Earle's salts supplemented with 10% heat-inactivated fetal calf serum (Hyclone, UT), penicillin 100 U/ml, streptomycin 0.1 mg/ml and L-glutamine (2 mM), endothelial cell growth factor (100 µg/ml, Boehringer Mannheim), and heparin (50 µg/ml, Sigma). Cells were subcultured at near confluence using a gentle trypsin (500 BAEE)–EDTA (180 µg/ml) detachment procedure in the standard manner. Valve endothelial cells were routinely cultured on gelatin coated dishes, while aortic endothelial cells were grown on standard tissue culture dishes.

Gelatin preparation of tissue culture dishes was performed by adding 0.5 ml/cm2 of bovine gelatin (2% solution, type B, Sigma) to the surface of the culture dish. Gelatin was allowed to equilibrate for 10 min at room temperature, at which point the excess gelatin was discarded by vacuum suction removal. Dishes were then stable at 4 °C for at least 1 week.

For aortic endothelial cells, the cells were placed into collagenase solution for 30 min at 37 °C after the aorta was incised along the longitudinal axis. After incubation, the surface was lightly scraped with a scalpel. The collected endothelial cells were placed in complete medium. A similar procedure to valve endothelial cells was used to aliquot the cells and observe morphology for expansion. We have previously demonstrated that these cells stain with endothelial markers, and are free of contaminating smooth muscle cells [1].

2.3. Microarray analysis
Total RNA was extracted from aortic and valve endothelial cells as previously described [17]. In brief, endothelial cells were lysed with 4 ml Tri-Reagent per 10-cm petri dish of confluent cells. Chloroform was added at 1/5 final volume and the solution was vigorously vortexed. Nucleoprotein complexes were then allowed to dissolve by letting the homogenate sit for 15 min on ice. The solution was centrifuged for 30 min at 4 °C at 12 000xg. The aqueous layer was then removed and mixed with an equal volume of isopropanol. The solution was precipitated overnight at -20 °C. Subsequently, the isopropanol/RNA mixture was centrifuged at 12 000xg for 30 min at 4 °C. The resulting RNA pellet was washed with a total of 6 ml of 70% ethanol and recentrifuged at 7500xg at 4 °C for 15 min. The RNA pellet extracted from cell culture was dissolved in 40 µl of water. RNA was quantified by absorbance at 260 nm, with a 260 nm/280 nm ratio of 1.7–1.9. Cells were routinely used when 80% confluent, and in the third to fifth passage. Cells were routinely harvested 24 h after the media was changed.

Radioactive cDNA libraries were then constructed using gene-specific primers for 847 genes of interest (Cytokine Expression Array, R&D systems) by reverse transcription in the presence of [{alpha}-32P]dCTP. Two micrograms of total RNA was mixed with primers. The sample was denatured at 92 °C and then allowed to cool to 42 °C over 20 min. Once at 42 °C, reverse transcription buffer (50 mM Tris–HCl, pH 8.5 (at 22 °C), 8 mM MgCl2, 30 mM KCl, 1 mM dithiothreitol), nucleotides (10 mM each of dATP, dGTP, dTTP, and 100 uM dCTP), ribonuclease inhibitor (30 U), [{alpha}-32P]dCTP (20 µCi) and Superscript II reverse transcriptase (Gibco BRL, 1 U) were added to the buffer mixture. The reaction was allowed to proceed for 90 min at 42 °C. All probes were purified of unincorporated nucleotides by spin purification through Sephadex G25 spin columns. Incorporation of radioactive nucleotides was typically 25–50% efficient.

Blocking and prehybridization of arrays was performed for 1 h at 60 °C in 5x SSPE (0.9 M NaCl; 0.05 M sodium phosphate, pH 7.4; 0.005 M EDTA, pH 8), 2% sodium dodecyl sulfate, 5x Denhardt's solution (0.1% Ficoll 400, 0.1% polyvinylpyrollidine, 0.1% bovine serum albumin) and 100 µg/ml sonicated and denatured salmon testes DNA. Hybridization was performed at 60 °C, using a degenerate screening procedure, since gene specific primers were optimized for human cDNA. Complementary DNA was then hybridized to the microarray blot at 60 °C for 18 h. Three washes were performed with the first at 1x SSPE for 20 min at 60 °C, the 0.5x SSPE for 20 min at 60 °C, then 0.25x SSPE for 10 min at 60 °C. Finally, blots were exposed to a Molecular Dynamics Storage Phosphor Screen for 72 h. Images were then scanned and stored at 88 µm resolution, and analyzed using Total Lab software (Amersham Pharmacia). The linear range of phosphorimage analysis using a 633 nm HeNe laser was at least six orders of magnitude higher than autoradiography film plus densitometry (Molecular Dynamics Application Note #51), and was used for all analyses. Background was minimal with high-stringency washes, and was removed by the spot edge averaging technique. Housekeeping genes were used to normalize results between blots, and negative controls were pUC19 DNA, and bacterial DNA, which was completely negative in all cases. ß-Actin was used to normalize results, and its densitometric count was arbitrarily set at 6000 units. All experiments were performed three times, with different batches of cells, with the average counts being reported. Genes which were not activated in all three experiments were excluded. This represented less than 10% of activated genes (typically 6–8 genes were not activated in all three blots).

2.4. Cell counting
To determine the relative rate of cell growth, the kinetics of cells on various plates was measured for PAEC (porcine aortic endothelial cells) and PVEC (porcine valve endothelial cells). Coatings tested included human fibronectin, rat tail collagen (type I and type IV), poly-D-lysine, mouse laminin, bovine gelatin as well as tissue culture treated plastic dishes (as control). All specialty extracellular matrix coatings tested have been previously reported to promote the growth of various endothelial cell lineages.

Cells were inoculated from passage 2–4 at a density of 1x105 cells/ml into a six-well plate and allowed to adhere for 24 h. After 24 h, non-adherent cells were removed and the media was changed. Media was changed thereafter every 48 h with complete media, including endothelial cell growth factor, heparin and 20% serum. Cells were counted in ten random medium-powered fields (200x) using an Olympus inverted microscope with phase contrast. Two independent observers counted all sets of cells on consecutive days. Cells which had detached were not included in calculations. Cell counts were then normalized to the number of cells in the initial inoculum and a growth fold induction was calculated by expressing the number of cells counted/initial count on day 1.

2.5. Statistics
Differences between groups were tested by Student's t-test. A P-value of less than 0.05 was considered significant. All analyses were performed on the Statistica software package, version 5.5 (Tulsa, OK).


   3. Results
Top
 Abstract
 1. Introduction
 2. Materials and methods
 3. Results
4. Discussion
 References
 
3.1. Growth rate
In general, PVEC grew more rapidly than PAEC. The maximal fold induction on day 5 for PVEC on type IV collagen is 12, whereas PAEC on the same substrate is 2.3 (P<0.05). On day 5, at the conclusion of the experiment, PVEC grew more rapidly than PAEC on all substrates except lysine, for which the fold induction was 3.1 for both cell types. Lysine appears to be growth stimulatory for PAEC versus growth inhibitory for PVEC. PVEC grew most rapidly on gelatin, collagen types I and IV as well as fibronectin. PAEC preferred laminin, lysine, and to a lesser extent, gelatin. When the growth rates are plotted, most cell types had a linear lag phase, after which there was an exponential growth phase (Fig. 1) . This is consistent with other cell types in culture and may be due to the induction of autocrine and paracrine growth factor loops. For most subsequent experiments, we chose to culture PVEC on gelatin due to the ease of dish pretreatment and favorable growth kinetics. PAEC were grown on standard tissue culture dishes due to the relative ineffectiveness of specialty coatings in promoting growth.



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  		Fig. 1. Graphical representation of growth curves for valve endothelial cells (top) and aortic endothelial cells (bottom). The abscissa represents the day on which cells were counted, and the ordinate the fold induction, which has been normalized to the initial count. Note that the fold increase axis (ordinate) is on a different scale for both cell types, with a higher growth induction for valve endothelial cells.

 
3.2. Microarray analysis
Of 847 genes analyzed, 69 were transcriptionally active in PAEC and 89 on valve endothelial cells. This corresponds to (69/847) 8.1% of tested genes active on PAEC and (89/847) 10.5% on PVEC. Of the activated genes, 55 were common to both cell types, which corresponds to (55/69) 79.7% commonly activated on PAEC and, (55/89) 61.8% on PVEC. The common genes are presented in Table 1. Normalized counts are listed, referenced to ß-actin. The Unigene sequence is listed in Table 1. Table 2 lists sequences unique to PAEC, and Table 3 lists sequences unique to PVEC. All counts are normalized across the three tables to ß-actin, which has been arbitrarily set to 6000 activity units. Overall, PVEC appeared to be more transcriptionally active, with 75% (41/55) of genes common to both cell types more active in PVEC than PAEC, which was consistent with proliferation indices. Individual genes are highlighted and further explored in Section 4.


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  		Table 1. Microarray sequences of RNA common to aortic and valve endothelial cells

 

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  		Table 2. Microarray sequences unique to aortic endothelial cells

 

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  		Table 3. Microarray sequences unique to valve endothelial cells

 

   4. Discussion
Top
 Abstract
 1. Introduction
 2. Materials and methods
 3. Results
 4. Discussion
References
 
In this report we describe a methodology to establish primary porcine valve endothelial cell cultures. We expand upon our previous report [1] and establish the growth kinetics on various substrates for both aortic and valve endothelial cells. Additionally, we describe a transcriptional snapshot of the both valve and aortic endothelial cells, and compare and contrast some of the activated transcripts. It is hoped that this information will be useful in the culture of these cells for various methodologies, including the study of valve immunology, the clonal expansion of valve endothelial cells for the repopulation of tissue engineered valves, as well as other basic science investigations of this unique cell type.

We demonstrate that valve endothelial cells have a higher proliferative rate than aortic endothelial cells. Valve endothelial cells grew more rapidly, especially when placed on gelatin, types I- and IV-collagen and fibronectin. This is consistent with previous reports that the extracellular matrix stimulates proliferation of cells through kinases [18], such as the focal adhesion kinase (FAK). Differences in the growth of valvular versus aortic endothelial cells may be due to developmental cues, since there is a paucity of vessels in valvular tissue, the endothelium may not efficiently recognize components such as laminin. Interestingly, Johnson and Fass [16] had reported in the initial description of the culture of valve endothelial cell that these cells had a paucity of fibronectin production in culture. We demonstrate that the various matrix components have differential effects on valve versus aortic endothelial cells, and this is likely due to differing effects of cell-matrix interactions as well as potential differences in the handling and presentation of various growth factors found in serum or attached to heparin [19] (which is an additive in the culture medium).

The transcriptional analysis of these cells reveals a more robust activation of certain transcription factors associated with proliferation, such as jun D, as well as protein kinase C, in valve endothelial cells versus aortic endothelial cells. The jun D proto-oncogene is induced 6.96-fold (13 029/1872) in PVEC versus PAEC. This is consistent with the approximately four fold increase in cell numbers on day 5 between PVEC versus PAEC. Microarray analysis essentially measures steady-state mRNA levels, which is a combination of nascent RNA production (transcription) and mRNA degradation (half-life). To determine definitively the mechanism, it would be necessary to perform nuclear run-on analysis as well as half-life determinations by using an agent such as actinomycin D [20]. Nonetheless, we have shown that valve endothelial cells proliferate more rapidly than aortic endothelial cells, and this is borne out by microarray analysis. A possible explanation of the rapid proliferation of valve endothelial cells is the induction of certain potential proliferative autocrine loops, such as the one identified between fibroblast growth factor (FGF)-3 and FGF-3R. Valve endothelial cells have a higher induction of both agents, which are known to be mitogenic. Further investigations into this phenomenon, perhaps with neutralizing antibodies to FGF or its receptor, may illuminate the contribution of this autocrine or paracrine loop to the proliferation of endothelial cells.

Certain endothelial ‘signature’ genes were differentially activated on these cell types. Vascular cell adhesion molecule (VCAM)-1 and vascular endothelial cell growth factor (VEGF)-B were activated on aortic endothelial cells. Platelet endothelial cell adhesion molecule (PECAM) and platelet-derived growth factor receptor (PDGF-R) were active on valve endothelial cells. Common to both cell types was ICAM-3. The ephrins and their receptors are a large class of tyrosine kinases which have been shown to function in development as arterial and venous ‘switches’ [21]. These have displayed a cell type specific expression. EphB3 receptor is common to both types, while EphB6 is present on aortic endothelial cells. Ephrin A4 ligand, as well as EphB1 receptor, is unique to valve endothelial cells. If translationally relevant, these receptors may determine the switch between valve and aortic lineages, especially since the Ephrins and their receptors have previously been demonstrated as an arterio-venous switch in the mouse [21].

It has recently been postulated that vascular and valvular calcification may be an actively regulated process with similarities to endochondral bone formation [22,23]. Both aortic and valve endothelial cells have certain transcripts activated which may be relevant to calcification. The proteins translated from these transcripts may be able to contribute to hydroxyapatite formation in matrix-competent environments. Osteonectin, osteogenic protein 1 (Bone morphogenetic protein 7) and growth differentiation factor 2 (Bone morphogenetic protein 9) were activated in both cell types. If these are activated in native human aortic valve, they may provide a rationale for the osteoid which has been reported in calcific aortic stenosis [24] and may represent molecular targets for inhibitors of valvular calcification.

We have previously described the presence of the {alpha}Gal epitope on the surface of the valve endothelium [1], and other receptors, such as endoglycan, the PDGF-R, may present antigenic and potentially immunogenic surfaces on valve endothelium [3]. It is unknown whether there is surface expression of these epitopes on fresh homografts or cryopreserved allografts. In the future design of bioengineered valve surfaces, it may be possible to design a scaffolding for an autologous endothelium. Differences in proliferative responses to substrates may be relevant for creating a durable endothelial surface, and a knowledge of the expression of potential immunogenic and antigenic surfaces may be critical for minimizing any inflammatory response.

In summary, transcriptional profiling of PAEC versus PVEC may be useful in the future design of engineered valve surfaces. Furthermore, the differences in growth rate on various cell surfaces may also assist in the rationale design of the next generation of valves and demonstrates the ability to culture selectively various endothelia. Finally, the primary culture of valve endothelial cells, as we have presented, is a model system for the study of the biology of the unique valve endothelium.


   Acknowledgments
 
R.S.F. is a recipient of an individual National Research Service Award from the National Institutes of Health, 1F32 HL67539-01 from the National Heart, Blood and Lung Institute.


   Footnotes
 
Presented at the 16th Annual Meeting of the European Association for Cardio-thoracic Surgery, Monte Carlo, Monaco, September 22–25, 2002.


   References
Top
 Abstract
 1. Introduction
 2. Materials and methods
 3. Results
 4. Discussion
 References
 

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