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Ann Thorac Surg 2007;83:946-953
© 2007 The Society of Thoracic Surgeons

Original Articles: Cardiovascular

Transforming Growth Factor-ß1 Mechanisms in Aortic Valve Calcification: Increased Alkaline Phosphatase and Related Events

Jocelyn N. Clark-Greuel, PhDa, Jeanne M. Connolly, MSa, Elizabeth Sorichillo, BSa, Navneet R. Narula, MDb, H. Scott Rapoport, PhDa, Emile R. Mohler, III, MDb, Joseph H. Gorman, III, MDb, Robert C. Gorman, MDb, Robert J. Levy, MDa,*

a Division of Cardiology, The Children’s Hospital of Philadelphia, Philadelphia, Pennsylvania
b University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania

Accepted for publication October 6, 2006.

* Address correspondence to Dr Levy, Children’s Hospital of Philadelphia, Abramson Research Center, Suite 702, 3615 Civic Center Boulevard, Philadelphia, PA 19104-4318 (Email: levyr{at}email.chop.edu).


   Abstract
Top
 Abstract
Introduction
 Material and Methods
 Results
 Comment
 Acknowledgments
 References
 
Background: Aortic valve stenosis is the most frequent indication for valve replacement surgery, and is commonly associated with pathologic calcification. Previous investigations by our group have shown a strong association of transforming growth factor-beta1 (TGF-ß1)-related mechanisms with calcific aortic stenosis in both cell culture and clinical pathology studies.

Methods: In the present investigations we sought to investigate the sequence of events involved in TGF-ß1-initiated aortic valve interstitial cell calcification in cell culture, and to study related gene expression pattern differences comparing calcific aortic stenosis surgical specimens with normal aortic valve leaflets.

Results: Sheep aortic valve interstitial cells (SAVIC) in culture progressively calcified over 14 days after the addition of TGF-ß1 to a significantly greater extent than non-TGF-ß1 controls. The TGF-ß1-induced SAVIC calcification was associated with maximal levels of alkaline phosphatase by 72 hours. Annexin V positive apoptosis was increased in TGF-ß1-treated SAVIC cultures at 14 days compared with controls. Matrix metalloproteinase 9 per gel zymography was detectable only in SAVIC cultures treated with TGF-ß1 from seven days on. Matrix metalloproteinase 2 was present in all SAVIC cultures per gel zymograms, either with or without TGF-ß1, but the active form of matrix metalloproteinase 2 significantly increased over 14 days in response to TGF-ß1. Quantitative gene expression studies (re: RNA levels) of human aortic valve cusps obtained at cardiac surgery demonstrated a number of related trends, including upregulation of the expression of TGF-ß1, alkaline phosphatase, and matrix metalloproteinase 9 in calcified human aortic valves.

Conclusions: Transforming growth factor-ß1 causes SAVIC to calcify due to an early maximal increase in alkaline phosphatase activity with associated apoptotic events and increased matrix metalloproteinase 9. These TGF-ß1-related mechanistic events may be of clinical relevance based upon the gene expression pattern changes observed in calcific aortic stenosis valve cusps.


   Introduction
Top
 Abstract
 Introduction
Material and Methods
 Results
 Comment
 Acknowledgments
 References
 
Dystrophic calcification of the aortic valve is associated with cuspal deterioration and stenosis, often resulting in valve replacement surgery [1, 2]. Cardiovascular calcification in general is a multifaceted disorder that is actively regulated and comparable in many aspects with bone mineralization [3]. Indeed, the expression of extracellular matrix proteins and cytokines associated with physiologic mineralization such as tenascin-C [4, 5], transforming growth factor-beta1 (TGF-ß1) [6], bone morphogenetic protein 2 (BMP2) [4], matrix Gla-protein [7, 8], osteopontin [9–11], and alkaline phosphatase (ALP) [12, 13] has been described in calcific valve disease. In addition, Mohler and colleagues [14] found active bone formation and remodeling in calcified human aortic valves.

Transforming growth factor-ß1 is a member of the same gene super-family as the bone morphogenic proteins [15] and is a pleiotropic cytokine involved in a variety of signaling pathways [16–19]. Previous findings have demonstrated that TGF-ß1 promotes calcification of aortic smooth muscle cells [20] as well as canine, human, and sheep aortic valve interstitial cells [6, 21]. Our laboratory has shown the presence of TGF-ß1 localized within interstitial cells, inflammatory cells, and calcific deposits in clinical-pathology studies of calcific aortic stenosis cusps; normal human aortic cusps contain little TGF-ß1by immunostaining [6].

Matrix metalloproteinases (MMP) are zinc- and calcium-dependent endopeptidases that are involved in tissue remodeling in both physiological and pathological settings. In addition, MMPs also modify the biological activity of several nonmatrix proteins, including growth factors, cytokines, chemokines, growth factor binding proteins, as well as other proteinases [22–24]. In particular, the gelatinases MMP2 and MMP9 have both been shown to have an increased presence in calcific aortic stenosis valve cusps [25–28]. The MMP9 expression is modulated by several growth factors and inflammatory cytokines such as TGF-ß1 [29], interleukin-1ß[30], and TNF{alpha} [31].

Thus, the present studies investigated the hypothesis that TGF-ß1 initiates a sequence of mechanistic events in the development and progression of calcific aortic stenosis. Thus, the goals of these studies were the following: (1) to characterize the onset and progression of TGF-ß1-induced calcification of sheep aortic valve interstitial cells (SAVIC) in cell culture; (2) to investigate TGF-ß1-related changes in gene expression patterns in calcific aortic stenosis versus normal aortic valve cusps.


   Material and Methods
Top
 Abstract
 Introduction
 Material and Methods
Results
 Comment
 Acknowledgments
 References
 
Cell Culture
Sheep aortic valve interstitial cells were isolated and characterized as previously described [6] from normal, mature female sheep aortic valve cusps (Western Cross, from Thomas Morris, Reisterstown, MD). Cells were maintained with M199 medium containing 10% fetal bovine serum (FBS; Hyclone, Logan, UT) supplemented with penicillin and streptomycin (Invitrogen Life Technologies, Carlsbad, CA). The SAVICs were used in the experiments between passages 3 and 7 as previously published [6]. Cells were cultured on plates coated with bovine dermal type I collagen (Cohesion Technologies Inc, Palo Alto, CA) as previously described [6, 32], in calcification medium (0.5% FBS M199 medium supplemented with 1.5 mM calcium chloride [CaCl2] (Sigma, St Louis, MO) and 10 mM ß-glycerophosphate (Sigma), and treated without or with TGF-ß1 (R&D Systems, Minneapolis, MN) at 10 ng/mL. Every three days the media was replaced.

Cell Culture Staining
At designated time points, cells were fixed with 4% paraformaldehyde and stained for ALP activity using nitroblue tetrazolium/5-bromo-4-chloro-indoly-phosphate-4-toludine (NBT/BCIP; Roche Diagnostics Inc, Indianapolis, IN) according to established methods [6]. Calcium deposits were stained using alizarin red S (Sigma) as described previously [6, 10]. Apoptosis was visualized [6] prior to fixation using an annexin V-fluorescein isothiocyanate (FITC) staining kit (R&D Systems, Minneapolis, MN), with subsequent nuclear counterstaining with 4,6-diamidino-2-phenylindole (DAPI; Vector Laboratories, Burlingame, CA).

Quantitation of Cellular Staining
Multiple series of representative photomicrographs of the SAVIC cultures were taken under the same conditions of luminosity, exposure, and resolution, for each time point and specific stains (as above) using a Leica DC500 digital camera (Leica Microsystems, Wetzlar, Germany) mounted on a Nikon Eclipse TE300 inverted microscope (Nikon, Tokyo, Japan) at 100x magnification. Data were calculated based on at least three individual images analyzed from each of triplicate cultures in order to attain sufficient power. Image backgrounds were masked using minor adaptations of published methods [33, 34], using Adobe Photoshop (ver. 7.0, Adobe, San Jose, CA) prior to intensity analysis of cellular areas of interest using the PC version of NIH Image, Scion Image (ver. 4.0.2, Scion Corporation, Frederick, MD). Similar analysis of positive controls for each stain yielded the following values for comparison: alizarin red staining of pure hydroxyapatite crystals equals 227.6 ± 4.3 intensity units (AU); nitroblue tetrazolium staining of 60 U/mL ALP equals 169.5 ± 0.7 AU; annexin V-FITC staining of hydrogen peroxide-induced apoptotic cells [35] equals 196.1 ± 0.8 AU. These data were used as standard references.

Zymography
Conditioned medium was collected and concentrated (10x) in Centricon-30 filters (Millipore, Billerica, MA). Cell-associated MMPs were not assessed because SAVICs could only be dissociated from the type I collagen substrate with collagenase, thus precluding zymography. Equal amounts of concentrated conditioned medium were loaded on 10% polyacrylamide-sodium dodecyl sulfate precast zymogram gels containing 0.1% gelatin (Bio-Rad Laboratories, Hercules, CA). After electrophoresis, gels were renatured, developed, and stained with Coomassie Blue. Human MMP2 and MMP9 zymography standards (Chemicon International Inc, Temecula, CA) were used to determine the approximate size of bands indicating proteolytic activity. Control zymograms of 0.5% FBS containing culture media showed trace levels of pro-MMP2 and no active MMP2, thus indicating negligible levels of MMP2 due to the serum-starved conditions used. The protein concentration was determined with the Bradford protein assay (Bio-Rad Laboratories, Hercules, CA). Densitometric quantitation of MMP activity was performed using the GelPlot2 function of Scion Image on tif images of zymograms developed from three to four individual experiments. The results of individual experiments were normalized to the day three control result for that zymogram in order to allow comparisons of fold-changes between experiments.

Isolation of Human Aortic Valve RNA
Human aortic valves were collected and immediately examined by a cardiovascular pathologist (N.N.) at the time of valve replacement surgery under an approved Institutional Review Board exemption issued by the Hospital of the University of Pennsylvania. The five calcified cases included three males (age 56 to 66 years) and two females (57 to 78 years). Normal human heart valves for RNA extraction were obtained from explanted hearts without cardiac valve disease at the time of transplant surgery, and the six samples included four males (34 to 59 years) and two females (44 to 61 years). After surgical pathology evaluation, representative samples of the valve cusps were immediately placed in RNALater (Ambion Inc, Austin, TX) and stored in a –80°C freezer. Frozen valve cusps were pulverized in liquid nitrogen using a liquid nitrogen mill (Mikro-Dismembranator, B. Braun Biotech Inc, Allentown, PA). Pulverized samples were extracted with Trizol (Invitrogen Life Technologies). Individual sample RNA integrity was documented with Agilent RNA 6000 lab chips run on an Agilent 2100 Bioanalyzer (Agilent Technologies, Palo Alto, CA).

Quantitative Real-Time Polymerase Chain Reaction (qRT-PCR) Analyses of Human Heart Valve RNA
The RNA samples obtained as described above were converted to single strand cDNA for the RNA of each individual valve cusp using the high capacity DNA archive kit (Applied Biosystems, Foster City, CA). Taqman universal master mix (Applied Biosystems) was added to cDNA for real-time PCR analyses using Applied Biosystems custom low density arrays (LDA) for real-time PCR quantitation using the Taqman system (Applied Biosystems) to assess selected genes in a customized LDA per real-time PCR known to be associated with pathologic calcification (see Table 1). Five hundred nanograms of RNA was converted to cDNA for each card. The PCR primers for each of the selected genes were preloaded into each LDA well by Applied Biosystems. Primer sequences were proprietary (Applied Biosystems) based on GenBank data. The LDA real-time PCR runs were carried out in quadruplicate, normalized per glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and expressed as relative fold change comparing statistical data based on individual normal valve runs with each of the calcified valves.


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  		Table 1 Gene Expression Patterns in Calcified Valves Compared to Noncalcified per Quantitative Real-Time Polymerase Chain Reaction a
 
The formula used to calculate individual expression levels in calcified valves relative to GAPDH was the following:_____________________________


Formula

_____________________________

The individual quadruplicate data (y) for the RNA of each normal valve leaflet was used to calculate a mean set of data for comparisons with the individual calcified valve data sets. Thus, the normal valve qRT-PCR levels (y) were used to calculate a mean relative to GAPDH (termed y-b), and each quadruplicate data set for each individual gene of interest for every calcified valve was then calculated by subtracting the normal valve leaflet mean value, Mean(y-b), from the mean values of each individual calcified leaflet, thus calculating (x-a)-Mean(y-b). This value was then used to express fold change (relative quantity [RQ]), calculated as 2–(x-a)-Mean(y-b) relative to the mean normal valve cusp data with an assumed value of one for the purposes of this comparison.

Statistical Analysis
Data are reported as mean ± standard error, of at least triplicate experiments. One-way analysis of variance was used to compare differences between groups, followed by post hoc Dunn or Holm-Sidak analyses. Comparisons between normal and calcified valve mRNA levels for individual genes were made using the Student t test or the Mann-Whitney rank sum test as appropriate. All statistical analyses were accomplished using SigmaStat (ver. 3.0, SPSS, Inc., Chicago, IL) analysis software. A p value of 0.05 or less was considered significant, and values of p less than 0.001 were only expressed as such.


   Results
Top
 Abstract
 Introduction
 Material and Methods
 Results
Comment
 Acknowledgments
 References
 
TGF-ß1-Mediated SAVIC Calcification
The SAVICs cultivated on type I collagen with TGF-ß1 added formed cellular condensations that progressed to multicellular nodule formation, as previously published [6], by three days (Fig 1). Quantitative alizarin red staining confirmed that these nodules were calcifying, and time points assessed at 7 and 14 days confirmed the progressive nature of the calcification process (p < 0.05). By comparison, SAVICs grown on type I collagen without TGF-ß1 formed fewer nodules and had significantly less calcification (p < 0.001 at any time point), that did not increase over the 14 day time course of these studies (Fig 1).


Figure 1
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  		Fig 1. Progression of sheep aortic valve interstitial cell (SAVIC) calcification without and with transforming growth factor-beta 1 (TGF-ß1) treatment. Panels A, B, and C show quantitation of alizarin red staining of SAVIC at days 3, 7, and 14, respectively, of treatment without (black) or with (white) 10 ng/mL TGF-ß1. Data are expressed as relative intensity in arbitrary units (AU) (see Methods) and are derived from four representative fields analyzed from each of three cell culture experiments. Representative alizarin red stained culture results (calcifications stained red, magnification 100x) are shown above each quantitative panel (calcification increases with TGF-ß1 treatment, p = 0.049; *p < 0.001 vs without TGF-ß1 at the same time point).

 
Alkaline phosphatase histochemistry was used as a mechanistic measure of biomineralization activity in these studies (Fig 2). The TGF-ß1 stimulated an intense early increase in ALP activity in SAVIC studies that reached what proved to be maximal levels by only 72 hours, with no significant change in the ALP amounts at the 7 and 14 day time points. The SAVICs cultivated with TGF-ß1 had sixfold to eightfold greater levels of ALP than did non-TGF-ß1 cultures at all of the time points studied (p < 0.001). Annexin V was used to assess apoptotic activity (Fig 3), that did not become prominently greater in TGF-ß1 SAVIC cultures until seven days, and was only greater in TGF-ß1cultures versus controls at 14 days (p = 0.009).


Figure 2
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  		Fig 2. Sheep aortic valve interstitial cells (SAVIC) alkaline phosphatase (ALP) activity without and with transforming growth factor-beta 1 (TGF-ß1) treatment over time. Panels A, B, and C demonstrate the quantitation of ALP staining of SAVIC at days 3, 7, and 14, respectively, of treatment without (black) or with (white) 10 ng/mL TGF-ß1. The ALP-stained culture results (nitroblue tetrazolium positive, blue-black, magnification 100x) are shown as inserts above each quantitative panel, which are derived from five representative fields from each of three cell culture experiments. Data are shown as arbitrary units (AU). The ALP activity increases with TGF-ß1 treatment, p < 0.001; *p < 0.001 vs without TGF-ß1 at the same time point.

 

Figure 3
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  		Fig 3. Apoptosis of sheep aortic valve interstitial cells (SAVIC) without and with transforming growth factor-beta 1 (TGF-ß1) treatment. Panels A, B, and C show annexin V staining quantitated for SAVIC at days 3, 7, and 14, respectively, of treatment without (black) or with (white) 10 ng/mL TGF-ß1. Results expressed as arbitrary units (AU). Representative annexin V (green, fluorescein isothiocyanate) fluorescent micrographs from SAVIC culture results with 4,6-diamidino-2-phenylindole (blue) stained nuclei (magnification 100x) are shown as insets above each quantitative panel (apoptosis increases with TGF-ß1 treatment, p = 0.009, which is derived from four representative fields from each of three cell culture experiments.

 
Matrix Metalloproteinase-Gel Zymography
In the gelatinase studies, gel zymogram electrophoreses of secreted MMPs per conditioned media samples obtained every 72 hours (Fig 4) revealed that MMP2 bands on the gel zymograms were present with or without TGF-ß1addition to the SAVIC cultures. However, the active form of MMP2 significantly increased (threefold) with the addition of TGF-ß1 over the 14 day time course studied (p = 0.01). In contrast, MMP9 activity was not detectable at 72 hours regardless of TGF-ß1 administration, when ALP activity first strongly increased and initial calcification was documented (Figs 2; 3), but was induced by TGF-ß1 (p = 0.008) and could be detected by seven days. Thus, at seven days MMP9 activity was strongly present in the proenzyme form on zymograms (Fig 4), and remained undetectable in the non-TGF-ß1treated cultures. The MMP9 levels increased over 13-fold (p < 0.001) by 14 days in the TGF-ß1 cultures while remaining undetectable in controls. However, while pro-MMP9 secretion was significantly higher with TGF-ß1 than without at these time points (p < 0.03), densitometry did not reveal quantitatively significant differences between the amounts of MMP9 activity detected at seven versus 14 days.


Figure 4
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  		Fig 4. Matrix metalloproteinases (MMP9 and MMP2) activity in sheep aortic valve interstitial cells (SAVIC) cultures without and with transforming growth factor-beta 1 (TGF-ß1) treatment. Gel zymogram bands demonstrate MMP activity in SAVIC cultures at 3, 7, and 14 days, respectively, of treatment without (black) or with (white) 10 ng/mL TGF-ß1. Representative zymograms of conditioned medium from culture are shown as insets above each quantitative panel. Data are shown as optical density (OD) of scans from three individual cell culture experiments. Pro-MMP9 secretion is virtually absent without TGF-ß1 treatment, and increased over 13-fold with TGF-ß1 treatment (p = 0.008; *p = 0.029 and *p = 0.021 vs without TGF-ß1 at 7 and 14 days, respectively). Active MMP2 secretion likewise increased over time with TGF-ß1 treatment (p = 0.01), but only threefold.

 
Gene Expression Patterns in Calcific Aortic Stenosis: TGF-ß1-Related Markers
The qRT-PCR investigations of human aortic valve cusps from calcific aortic stenosis cases and noncalcified cusps obtained from transplant recipients demonstrated trends that reflected mechanistic similarities with the SAVIC cell culture results (Table 1). The TGF-ß1 expression was increased in calcified cusps versus noncalcified indicating the localized production of TGF-ß1. The ALP expression tended to be increased in calcified valves, although not in all cases. The MMP9 was upregulated 5- to 36-fold or more in all calcified valve cusps studied per qRT-PCR (Table 1, p = 0.004). However, changes in MMP2 expression were variable and in general did not demonstrate a trend of upregulation of MMP2 expression (Table 1). The expression of the tissue inhibitors of metalloproteinases (TIMPs) were in general increased in calcified valves versus normals, with TIMP1 demonstrating the greatest levels of increased expression. Caspase expression was not consistently upregulated in calcified cusps versus normals.


   Comment
Top
 Abstract
 Introduction
 Material and Methods
 Results
 Comment
Acknowledgments
 References
 
The novel findings of these studies included: (1) demonstrating that TGF-ß1-induced SAVIC calcification is associated with early and near maximal ALP activity; (2) other mechanistic events occurring after TGF-ß1 administration, both increased MMP9 activity and apoptosis, demonstrated maximal levels after the onset of calcification; and (3) RT-PCR results comparing a representative sampling of both calcified aortic valves and normal human aortic cusps revealed an overall gene expression pattern consistent with TGF-ß1-related mechanisms being involved in the pathogenesis of calcific aortic stenosis. Thus, the results of our investigations provide important mechanistic information linking a sequence of events, including increased ALP activity, apoptosis, and extracellular matrix remodeling activity involving gelatinases to TGF-ß1-induced aortic valve calcification.

Previous clinical-pathology studies of calcific human aortic stenosis cusps by our group and others have also provided evidence that a number of the individual mechanistic components of interest in the present study are of importance in the pathogenesis of calcific aortic valve disease. A prior clinical-pathology study by our group demonstrated increased TGF-ß1 immunostaining in calcific aortic stenosis cusps versus normal valve cusps with associated increased immunostaining for TGF-ß1-latent binding protein, and TGF-ß receptors I and II [6]. MMP9 [19, 20, 32] and MMP2 [19, 20, 32] were also demonstrated in several studies to be present in calcified human aortic valve cusps to a greater extent than in noncalcified cusps. Similarly, the endogenous inhibitors of the MMPs, the TIMPs are also increased in calcified compared with noncalcified valves [19, 20, 32]. Furthermore, clinical-pathology studies by our group [20] showed that increased MMP2 is associated with the increased presence of tenascin-C, an extracellular matrix protein associated with biomineralization and bone development. Taken together, the present RT-PCR results and prior studies cited above indicate that a complex pathophysiology likely results in calcific aortic stenosis in association with TGF-ß1-related mechanisms.

The SAVIC- TGF-ß1 cell culture system used in the present studies is an accelerated model with maximally increased ALP activity after only 72 hours. By comparison, human aortic valve calcifications develop and progress slowly, typically over decades. However, the parallels between the SAVIC results and the present qRT-PCR data coupled with previous clinical pathology observations strongly support the view that the mechanistic insights from the current SAVIC results may be of relevance. Although there are no comparable cell culture results by others concerning TGF-ß1 increasing MMP levels in heart valve cells in culture, prior clinical-pathology studies by our group [20] and others [25–27] have shown increased MMP2 and MMP9 in calcific aortic stenosis; other investigations have also shown comparable patterns of immunostaining in calcific aortic stenosis cusps for TGF-ß1 and related proteins [6]. Furthermore, data demonstrating TGF-ß1 associated apoptosis (re: annexin results; Fig 3) in SAVIC cultures are also consistent with the increased expression levels of several of the caspases noted in the RT-PCR studies of the calcific aortic stenosis cusp versus normals (Table 1). The source of TGF-ß1 in vivo may be from either inflammatory cells or valvular interstitial cell expression [6]; for example, serotonin-induced stimulation of valve interstitial cell TGF-ß1 expression [36]. Drawing distinctions concerning the source of TGF-ß1 were beyond the scope of the present study; however, our results underscore the strong potential importance of TGFß1 in pathologic calcification of aortic valves.

Conclusions
The results of the present studies support the view that TGF-ß1 has a mechanistic role in the initiation and progression of calcific aortic stenosis. Model studies of initial events associated with TGF-ß1-induced aortic valve interstitial cell calcification indicate that increased ALP plays a crucial role in this process, and this may be comparable to increased ALP activity noted in ultrastructural studies of matrix vesicle biomineralization in bone and aortic valve calcification [37]. Furthermore, RT-PCR studies of gene expression pattern differences between calcific aortic stenosis and normal aortic valve cusps further support this interpretation of the experimental results, demonstrating that calcific aortic cusps have increased ALP and TGF-ß1 expression in association with upregulation of MMP9. Thus, these findings overall support the view that a future therapeutic direction for pharmacotherapy of aortic valve disease could be based upon targeting critical TGF-ß1 mechanisms involved in this important disorder.


   Acknowledgments
Top
 Abstract
 Introduction
 Material and Methods
 Results
 Comment
 Acknowledgments
References
 
The authors thank Jennifer LeBold (Children’s Hospital of Philadelphia) for her assistance in the preparation of the manuscript. This research was supported in part by grants from the National Institutes of Health, P50-HL74731, RO1-HL64388, T32-HL07954, KO8 HL03974, a grant from St. Jude Medical, Inc, and funding provided by both the William J. Rashkind Endowment and the Ethel Brown Foerderer Fund of the Children’s Hospital of Philadelphia.


   References
Top
 Abstract
 Introduction
 Material and Methods
 Results
 Comment
 Acknowledgments
 References
 

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