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Ann Thorac Surg 2008;86:71-76. doi:10.1016/j.athoracsur.2008.03.008
© 2008 The Society of Thoracic Surgeons
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Original Articles: Adult Cardiac

Lipopolysaccharide Stimulation of Human Aortic Valve Interstitial Cells Activates Inflammation and Osteogenesis

Ashok N. Babu, MD, Xianzhong Meng, MD, PhD, Ning Zou, MD, Xiaoping Yang, PhD, Maorong Wang, MD, Yong Song, MD, Joseph C. Cleveland, MD, Michael Weyant, MD, Anirban Banerjee, PhD, David A. Fullerton, MD*

Division of Cardiothoracic Surgery, University of Colorado at Denver, Denver, Colorado

Accepted for publication March 5, 2008.

* Address correspondence to Dr Fullerton, Cardiothoracic Surgery, University of Colorado at Denver and Health Sciences Center, 12631 East 17th Ave, Room 6602, MSC310, PO Box 6511, Aurora, CO 80045 (Email: david.fullerton{at}uchsc.edu).

Presented at the Poster Session of the Forty-third Annual Meeting of The Society of Thoracic Surgeons, San Diego, CA, Jan 29–31, 2007.


   Abstract
Top
 Abstract
Introduction
 Material and Methods
 Results
 Comment
 Acknowledgments
 References
 
Background: Calcific aortic stenosis may be an inflammatory disease with active bone formation in the valve leaflets rather than a disease of passive calcium deposition. Epidemiologic data demonstrating correlation of poor dental hygiene to atherosclerotic pathologies suggests that circulating bacterial products could be involved in the pathogenesis of aortic valve stenosis. We hypothesized that lipopolysaccharide (LPS) stimulation of human aortic valve interstitial cells (HAVICs) would induce inflammatory and osteogenic gene expression.

Methods: The HAVICs were isolated from normal aortic valves obtained from explanted hearts during transplantation (n = 5) and grown in culture. Cells underwent 4 and 24 hours of LPS stimulation (LPS, 200 ng/mL) or β-glycerol phosphate treatment (BGP) (osteogenic media as positive control). Media was removed for interleukin (IL)-6 and IL-8 immunoassay. Ribonucleic acid was extracted for microarray analysis. Statistics were by analysis of variance with post-hoc analysis (p < 0.05).

Results: The LPS stimulation induced the gene expression of proinflammatory cytokines, chemokines, and adhesion molecules. Protein level confirmation by immunoassay demonstrated 3.4-fold (± 0.35, p < 0.01) and 9.5-fold (± 1.5 p < 0.01) increase over control of IL-6 and IL-8, respectively. The LPS and BGP both induced critical mediators of osteogenesis including bone morphogenetic protein 2 and platelet-derived growth factor alpha.

Conclusions: The LPS stimulation of HAVICs not only induces inflammatory mediators but also induces gene expression of osteogenic factors, similar to that induced by osteogenic media. Bacterial products stimulation, likely by toll-like receptor 4 and the innate immune system, may contribute to the pathogenesis of aortic valve stenosis.


   Introduction
Top
 Abstract
 Introduction
Material and Methods
 Results
 Comment
 Acknowledgments
 References
 
Calcific aortic valve stenosis is the third most common cardiovascular disease in the United States, exceeded only by coronary artery disease and hypertension [1]. It is present in at least 2% to 5% of the elderly [2]. It has traditionally been considered a "degenerative" disease, with passive accumulation of calcium on the valve leaflets. However, recent data suggest that the pathogenesis of calcific aortic stenosis may involve both inflammation and active bone formation in the leaflets. Skeletal-type bone deposition has been identified in the leaflets of explanted stenotic valves, as has the presence of bone-specific proteins such as osteocalcin, osteopontin, and bone sialoprotein [3, 4]. Histologic studies have also identified the infiltration of lymphocytes, monocytes, and mast cells into calcified aortic valve leaflets, suggesting an active inflammatory component to the disease [5]. Despite these histologic findings, the molecular and cellular mechanisms of inflammation and those leading to acquisition of an osteogenic phenotype are unknown.

Epidemiologic studies, as well as the histologic association of inflammation with atherosclerosis, have led to the theory that periodontal disease and its associated bacteria may be causative. Similarities in the risk factors for the development of aortic stenosis to those for atherosclerosis led us to consider the potential role of circulating bacterial products in this disease process [6]. Aortic valve interstitial cells constitute the majority of the valve leaflet's interstitium and are known to be important contributors to the pathogenesis of aortic stenosis [7–10]. We sought to link the roles of inflammation and osteogenesis in the pathogenesis of aortic stenosis to these cells. We postulated that in response to bacterial products, the human aortic valve interstitial cells (HAVICs) acquire an osteogenic phenotype, thereby contributing to the pathogenesis of calcific aortic stenosis. Given the central role of toll-like receptors (TLRs), particularly TLR4 (an important lipopolysaccharide [LPS] receptor), in the mediation of many inflammatory conditions including atherosclerosis [11, 12], we specifically hypothesized that LPS stimulation of HAVICs would trigger proinflammatory and pro-osteogenic gene programs and cytokine expression in HAVICs. The results of the present study confirm both of these objectives.


   Material and Methods
Top
 Abstract
 Introduction
 Material and Methods
Results
 Comment
 Acknowledgments
 References
 
Chemicals and Reagents
Medium 199 (M199) and fetal bovine serum were purchased from Cambrex Walkervile Bioscience Inc (Walkervile, MD). Collagenase LPS, and other reagents otherwise mentioned were purchased from Sigma Chemical Co (St. Louis, MO).

Cell Isolation and Culture
Five aortic valves were obtained from explanted hearts of patients undergoing heart transplantation at the University of Colorado Health Sciences Center. There were four men and one woman with ages ranging between 40 and 70 years. Etiology of heart failure was ischemic cardiomyopathy. The study was approved by the Colorado Multiple Institutional Review Board of University of Colorado at Denver and Health Sciences Center. All patients gave informed consent for collection of aortic valve leaflets from the explanted heart.

Isolation of human aortic valve interstitial cells was performed after a previously described method [13]. Briefly, healthy human aortic valves were collected in normal saline after removal from explanted hearts. The valves were characterized as healthy ones by histologic analysis (hematoxylin and eosin stain, data not shown). Valve leaflets from five explanted hearts were rinsed in Earle's balanced salt solution and then digested in 2.5 mg/mL collagenase in M199 at 37°C for 30 minutes.

After removing endothelial cells by vortex, the leaflet was further digested with a milder solution of collagenase medium (0.8 mg/mL) at 37°C for 3 hours. After vortexing and aspirating repeatedly to break up the tissue mass, the cell suspension was spun at 500g for 2 minutes to remove remaining tissue mass. The supernatant was transferred into a fresh tube and spun again at 1,100g, 4°C, for 8 minutes. The cells were resuspended in full medium (M199 with penicillin G, streptomycin, amphotericin B, and 10% fetal bovine serum), plated onto a 75-mm flask, and cultured in a cell culture incubator supplied with 5% carbon dioxide. The purification of cells was characterized by smooth muscle alpha-actin and vimentin fluorescence stain (data not shown). Cells with 95% positive stain were used for further culture. When the flask reached 70% to 90% confluence, cells were subcultured on plates and chamber slides. Cells at passage 3 or 4 were used for experiments. The cells from each patient were maintained as independent cultures.

Treatment of HAVIC
After reaching approximately 90% confluence, HAVIC were treated with media alone (Controls), LPS (E. coli 0111:B4, 200 ng/mL), or 10 mM β-glycerol phosphate (organic phosphate donor used as noninflammatory positive control for osteogenic phenotypic change) for 4 hours or 24 hours. After treatment, the cells were washed with cold phosphate-buffered saline twice and collected with centrifugation at 1,400 rpm at 4°C for subsequent isolation of ribonucleic acid (RNA) for microarray experiment. Cell culture supernatants from 4-hour LPS treatment were collected for the measurement of levels of proinflammatory mediators (interleukin [IL]-6 and IL-8) by enzyme-linked immunosorbent assay (ELISA).

Isolation of RNA
The RNA was isolated using RNeasy mini columns (Qiagen, Valencia, CA) per manufacturer's protocol. The RNA content was determined by spectrophotometry at 260 nm. The integrity of the RNA preparation was determined using an Agilent bioanalyzer (model 2100; Agilent Technologies, Palo Alto, CA). Four hundred to 800 ng of RNA was extracted from each specimen, so 400 ng of each sample was used to create complementary (c)DNA.

Custom Microarray Analysis
The microarray analysis was performed in three individual HAVIC cultures using CustomArray 4 x 2K microarrays from CombiMatrix Corp (Mukilteo, WA). These microarrays are manufactured using in situ oligonucleotide synthesis on microelectrodes embedded into a semiconductor chip. The microarray probe set was designed by CombiMatrix from a list of 650 genes related to osteogenesis, cytokines, inflammatory signaling, TLR signaling, extracellular matrix molecules, growth factors, and transforming growth factor beta (TGF-β) signaling pathway. Three different probes were designed for each gene of interest.

Microarray Hybridization and Data Acquisition-Analysis-Interpretation
Total RNA was transcribed to cDNA, then transcribed to cRNA and biotinylated using the MessageAmp II-Biotin Enhanced (#1791; Ambion, Austin, TX). The cRNA content was determined by spectrophotometry at 260 nm. The integrity of the cRNA preparation was determined again. The 50 µL labeled cRNA was added to 150 µL prehybridization solution which contains 6X subacute sclerosing panencephalitis (SSPE), 0.05% Tween-20, 20 mM ethylenediaminetetraacetic acid (EDTA), 5X Denhardt's solution, 100 ng/µL denatured salmon sperm DNA and 0.05% sodium dodecyl sulfate (SDS) and prehybridized at 45°C for 30 minutes. Each cRNA was hybridized to the chip mixed in hybridization solution of 30 µL containing 6X SSPE, 0.05% Tween-20, 20 mM EDTA, 25% formamide, 50 ng/µL cRNA, 100 ng/µL salmon sperm DNA and 0.04% SDS at 45°C overnight. The chips were scanned using a Hewlett Packard Gene Array Scanner and the images were saved as tagged image file format. The data were extracted from the image using the CombiMatrix Microarray Imager Software. Gene array data wereanalyzed using both the Affymetrix Microarray Suite 5.0 and GeneSpring GX 7.3 Expression Analysis (Agilent Technologies) software analysis programs. Genes with greater than twofold change versus control were analyzed for significance at a 95% confidence limit.

Cytokine Assay
Cytokine assay was performed in all five individual HAVIC cultures. Cell culture medium was collected after 24 hours of treatment with LPS or control media and cytokine levels were determined using immunoassay as described previously [14, 15].

Statistical Analysis
Data are presented as mean ± standard error (SE). Analysis of variance with a post hoc Bonferroni-Dunn test was performed to analyze differences between groups. Statistical significance was accepted within 95% confidence limit.


   Results
Top
 Abstract
 Introduction
 Material and Methods
 Results
Comment
 Acknowledgments
 References
 
LPS and Phosphate Donor Induce Significant Changes in Gene Expression
The HAVICs grown to 90% confluence were treated with control media, LPS, or β-glycerol phosphate (organic phosphate donor which is a component of "osteogenic media" known to induce osteoblast activity without inflammation [16]) for 4 hours and 24 hours. The RNA expression was evaluated in three individual cultures by a custom microarray, and gene expression was considered altered if fold change from control was greater than 2 or less than 0.5. At 4 hours and 24 hours, LPS affected the expression of 79 and 16 genes, respectively. At 4 hours and 24 hours, β-glycerol phosphate affected the expression of 37 and 122 genes, respectively. At 4 hours and 24 hours, 10 genes and 2 genes were significantly altered by both treatments, respectively (Fig 1). Unique genes altered at 4 hours or 24 hours were 82 by LPS, 137 by β-glycerol phosphate, and 27 by both treatments (Fig 2).


Figure 1
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  		Fig 1. Venn diagrams demonstrating extent of gene expression alteration resulting from treatment with lipopolysaccharide (LPS) and β-glycerol phosphate (BGP) at 4 hours and 24 hours. Altered genes were defined as those that were twofold increased or twofold decreased compared with controls.

 

Figure 2
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  		Fig 2. Interleukin (IL)-6 and IL-8 protein induced by lipopolysaccharide (LPS). The LPS stimulation induced significant increases in IL-6 and IL-8 protein levels at 24 hours.

 
LPS Stimulation Induces Proinflammatory Genes
We sought to determine whether exposure of HAVIC to LPS would stimulate a proinflammatory gene program. The LPS stimulation significantly increased the gene expression of several proinflammatory cytokines, chemokines, adhesion molecules, and those involved in nuclear factor-kappa B signaling (Table 1).


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  		Table 1 Inflammatory Genes Encoding Cytokines, Chemokines, Adhesion Molecules and Nuclear Factor-Kappa B (NF-{kappa}B) Signaling Molecules Were Induced by Lipopolysaccharide (LPS) Stimulation. Data Are Presented as Mean ± Standard Deviation
 
LPS and Phosphate Donor Induce Osteogenic Genes
The main goal of this study was to determine whether a physiologic inflammatory stimulus could induce an osteogenic gene program in aortic valve interstitial cells. β-glycerol phosphate (a noninflammatory organic phosphate donor) was used as a positive control. Four genes which are critical to osteogenesis were significantly increased in expression after treatment (Table 2).


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  		Table 2 Genes Associated With Osteogenesis Were Induced by Both β-Glycerol Phosphate (BGP) and Lipopolysaccharide (LPS) Stimulation. Data Are Presented as Mean ± Standard Deviation
 
Proinflammatory Cytokines Are Induced at the Protein Level
To confirm microarray data, levels of proinflammatory cytokines IL-6 and IL-8 in the supernatant were measured in the five individual HAVIC cultures. At 24 hours after LPS stimulation, IL-6 was increased 3.4-fold (± 0.35, p < 0.01) and IL-8 was increased 9.5-fold (± 1.5, p < 0.01) over control (Fig 2).


   Comment
Top
 Abstract
 Introduction
 Material and Methods
 Results
 Comment
Acknowledgments
 References
 
The results of the present study demonstrate that stimulation of cells implicated in the pathogenesis of aortic stenosis by bacterial products can lead to increased expression of proinflammatory genes and proteins and the expression of some prototypical osteogenic genes, similar in effect to a phosphate donor commonly used to stimulate osteoblast differentiation.

The hypothesis that calcific aortic stenosis is an active process incited by inflammation of unknown etiology has only been set forth over the past 15 years. Observational studies that identified similar risk factors for the development of aortic stenosis, as for atherosclerosis, suggested a similar process was at play [6]. Histologic studies revealing infiltration of inflammatory cells [4, 17], lipid deposition [18], and skeletal-type bone with expression of osteoblast proteins [3] strengthened this hypothesis. Acceptance that aortic valve calcification is an active biologic process as opposed to passive calcium deposition opens the door to potential therapies to prevent or reverse the disease. However, for these therapies to be developed, the molecular and cellular mechanisms leading to eventual calcification must be understood.

In this study we utilized LPS as an inflammatory stimulant for the human aortic valve interstitial cells. Lipopolysaccharide is a relevant stimulus because two groups of patients who are at greater risk for vascular disease have been shown to have higher circulating levels of LPS; obese diabetics [19] as well as those with periodontal disease. The physiologically relevant dose for in vitro studies has been the subject of much debate and remains controversial. Circulating serum levels of LPS under normal conditions are significantly lower than the 200 ng/mL used in this study. However, the actual tissue level concentrations in humans under various pathologic states are unknown and the concentration we have used is in line with many other in vitro studies.

Toll-like receptor 4 (TLR4) is a critical component of the innate immune response; its presence is critical to the cellular inflammatory response to LPS from gram negative bacteria [20]. It is likely that TLR4 mediates the proinflammatory and osteogenic effects of LPS on HAVICs. Its function has been shown to be important to the development of atherosclerotic plaque in animal models [11, 12]; hence, a likely involvement in the pathogenesis of aortic stenosis. We have demonstrated increased gene expression of cytokines, chemokines, and adhesion molecules in HAVICs in response to LPS stimulation. Expression of IL-8, chemokine (C-X-C) ligand 1, and chemokine (CC) ligand 5 (CCL5) were all significantly induced by LPS stimulation, predominantly after 4 hours. These mediators are potent chemokines responsible for attracting neutrophils, monocytes, and lymphocytes to the tissue as well as for inducing angiogenesis. Inflammatory cell infiltrate with neoangiogenesis is known to be associated with aortic valve stenosis [5]. It is certainly possible that, in response to activation by circulating LPS, the HAVIC may release these chemoattractants to recruit effector cells and subsequently begin the early stages of valvular inflammation. Effector cell recruitment also requires cell adhesion molecules, classically thought to be expressed by the endothelium. However, this study demonstrates that HAVICs had significantly greater gene expression of the adhesion molecules, intercellular adhesion molecule 1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1), and Selectin-E after LPS stimulation, suggesting they may play a role in leukocyte adhesion in the aortic valve. Interestingly, tumor necrosis factor alpha (TNF-{alpha}) was not upregulated in this model, though others have demonstrated an important role for this cytokine in vascular as well as aortic valve calcification in cell culture experiments [8]. Perhaps TNF-{alpha} in aortic valve calcification is produced by a cell type other than HAVIC and acts in a paracrine fashion.

Interleukin-6 and IL1-β are proinflammatory cytokines with multipotent effects on the adaptive immune system and inflammation [21]. In a previous study, IL1-β expression was shown to be associated with extracellular matrix remodeling in calcific aortic stenosis [7]. In this study, HAVICs expressed both cytokines in response to LPS stimulation at 4 and 24 hours.

Treatment of HAVICs with β-glycerol phosphate was utilized in this study as a positive control of osteogenic phenotypic change. Treatment of cells with this "osteogenic media" has been demonstrated to induce osteoblast phenotypic change in vitro in multiple cell types [16]. Interestingly, several genes induced by this treatment were also induced by LPS stimulation. Most notably, bone morphogenetic protein-2 (BMP2) gene expression was significantly increased after 4 hours of treatment. The BMP2 is known to be a critical signaling molecule in bone formation and recent studies have demonstrated its importance in aortic stenosis; BMP2 was found to be highly expressed in stenotic valves but not in normal specimens [22]. Additionally, treatment of HAVIC with exogenous BMP2 was demonstrated to induce osteogenic phenotypic change in vitro [10]. The production of BMP2 by HAVIC exposed to LPS demonstrates that inflammatory stimulus to the aortic valve could lead to osteogenic changes. Platelet derived growth factor alpha (PDGF-{alpha}) and fibroblast growth factor 2 (FGF2) were significantly induced by both β-glycerol phosphate and LPS. The PDGF is highly expressed in forming bone, perhaps because of its role as a potent mitogen for mesenchymal cells [23], and has been shown to be highly expressed in animal model lesions of aortic stenosis [24]. The FGF2 appears to play a modulatory role with respect to BMP2 induction of osteogenesis. At low concentrations, it promotes bone formation whereas at higher concentrations it may inhibit the effects of BMP-2 [25].

In summary, stimulation of human aortic valve interstitial cells by a physiologically relevant bacterial product triggers proinflammatory and proosteogenic gene and protein expression, likely by effects on the innate immune system. These findings offer mechanistic insight into the pathogenesis of calcific aortic stenosis.


   Acknowledgments
Top
 Abstract
 Introduction
 Material and Methods
 Results
 Comment
 Acknowledgments
References
 
This study was supported in part by the National Institutes of Health (grant T32GM008315) and the University of Colorado Denver. We would also like to acknowledge Lihua Ao, BS, for assistance in development of ELISA protocol.


   References
Top
 Abstract
 Introduction
 Material and Methods
 Results
 Comment
 Acknowledgments
 References
 

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