Aortic valve calcification is mediated by a differential response of aortic valve interstitial cells to inflammation

doi:10.1016/j.jss.2014.03.051

Journal of Surgical Research

Volume 190, Issue 1, July 2014, Pages 1-8

Presented at the ninth Annual Meeting of the Academic Surgical Congress, San Diego, California, February 4–7, 2013.

https://doi.org/10.1016/j.jss.2014.03.051 Get rights and content

Abstract

Background

Although calcific aortic stenosis is common, calcification of the other three heart valves is not. The aortic valve interstitial cell (VIC) has been implicated in the pathogenesis of aortic stenosis. Proinflammatory stimulation of aortic VICs induces an osteogenic and inflammatory phenotypic change. We hypothesized that the VICs of the other heart valves do not undergo these changes. Using isolated human VICs from normal aortic, mitral, pulmonary, and tricuspid valves, our purpose was to compare the osteogenic response to proinflammatory stimulation via toll-like receptor 4 (TLR-4).

Materials and methods

Aortic, pulmonic, mitral, and tricuspid (n = 4 for each valve type) VICs were isolated from hearts valves explanted from patients undergoing cardiac transplantation. Cells were cultured and grown to confluence in passage 2–6 before treatment with Lipopolysaccharide (LPS) (100–200 ng/mL) for 24 or 48 h. Cells were characterized by immunofluorescent staining. TLR-4 expression was analyzed (immunoblotting, flow cytometry). Bone morphogenetic protein 2 and intercellular adhesion molecule 1 production were determined (immunoblotting). Monocyte chemoattractant protein 1 levels were determined by enzyme-linked immunosorbent assay. Statistics were by Mann–Whitney U test.

Results

TLR-4 stimulation induced bone morphogenetic protein 2 production only in aortic VICs (P < 0.05). Intra-cellular adhesion molecule 1 production and monocyte chemoattractant protein 1 secretion increased in a similar fashion among TLR-4-stimulated VICs from all four valves.

Conclusions

Proinflammatory stimulation induces an osteogenic phenotype in aortic VICs but not mitral, pulmonic, or tricuspid VICs. We conclude that this differential osteogenic response of aortic VICs contributes to the pathogenesis of calcific aortic stenosis.

Introduction

Calcific aortic stenosis is the third most prevalent cardiovascular disease in the United States, exceeded in prevalence only by hypertension and coronary artery disease [1]. It is the leading indication for heart valve replacement surgery. Although calcification of the aortic valve is common, it is noteworthy that calcification of the other heart valves is not.

The pathogenesis of calcific aortic stenosis is not well understood. It has traditionally been considered a degenerative process, one in which calcium passively accumulates on the aortic valve leaflets. However, evidence is accumulating that aortic stenosis is an active disease process—one in which mechanisms of inflammation play an important role [2], [3].

The valve interstitial cell (VIC) is the principle cell type found in cardiac valve leaflets [4]. In the aortic valve, the aortic VIC has been implicated in the pathogenesis of calcific aortic stenosis [5]. In response to proinflammatory stimulation via activation of toll-like receptor 4 (TLR-4), the aortic VIC has been shown to undergo a phenotypic change from that of a myofibroblast to that of an osteogenic and inflammatory phenotype [6]. In aortic VICs, such osteogenic phenotypic changes are characterized by the production of the important bone-forming protein, bone morphogenetic protein 2 (BMP-2), and an inflammatory phenotype is characterized by the production of intra-cellular adhesion molecule 1 (ICAM-1) [6]. Previous studies have also demonstrated that these inflammatory responses are exaggerated in VICs isolated from stenotic aortic valves [7], [8], further emphasizing their importance to the disease process.

Little is known about the biological behavior of VICs from the mitral, tricuspid, and pulmonary valves. It is interesting to note that these three valves rarely, if ever, calcify. But as the aortic VIC has been implicated in calcification of the aortic valve, we hypothesized that intrinsic differences may exist among the four cardiac valves that help to explain why aortic valves calcify but the mitral, tricuspid, and pulmonary valves rarely, if ever, do. We specifically hypothesized that unlike aortic VICs, the VICs from mitral, tricuspid, and pulmonary valves do not undergo an osteogenic phenotypic change in response to proinflammatory stimulation.

Using isolated human VICs from aortic, mitral, tricuspid, and pulmonary valves, the purposes of this study were to determine the differences in TLR-4–induced expression of an osteogenic phenotype and an inflammatory phenotype. The results of this study demonstrate that TLR-4 stimulation induced an inflammatory response in all four types of VICs. However, TLR-4 stimulation induced an osteogenic phenotype only in aortic VICs.

Section snippets

Materials and methods

This study was approved by the Colorado Multiple Institutional Review Board at the University of Colorado School of Medicine.

Patient characteristics

Aortic, pulmonary, mitral, and tricuspid valves were obtained from four male patients who underwent cardiac transplantation for idiopathic dilated cardiomyopathy. Patient ages ranged from 20–49 y. Before transplantation, these patients all had echocardiograms showing no significant valvular disease. All patients also underwent coronary angiography before transplantation, and none had coronary artery disease. None of the patients used tobacco.

VICs from all four valves demonstrated a myofibroblast phenotype

Aortic, pulmonic, mitral, and tricuspid VICs were

Discussion

The results of the present study demonstrate an important difference in the response to TLR-4 stimulation among the VICs from the four types of heart valves. TLR-4 expression was not different among the different types of VICs. In addition, TLR-4–induced production of ICAM-1 and MCP-1 increased in all four types of VICs. However, TLR-4 stimulation induced an osteogenic phenotype as characterized by BMP-2 production in aortic VICs but not VICs from mitral, tricuspid, or pulmonary valves. These

Conclusions

The results of the present study demonstrated that although TLR-4 expression was not different among VICs from all four heart valves, the response to TLR-4 stimulation was different; TLR-4 stimulation induced an osteogenic phenotype in aortic VICs but not in VICs from mitral, tricuspid, or pulmonary valves. Such a differential response to proinflammatory stimulation in human aortic VICs offers mechanistic insight into the observation that the aortic valve commonly calcifies and the other three

Acknowledgment

Author contributions: N.V., N.A.N., M.J.W. and T.B.R. contributed to the conception of the article. N.V., N.A.N., M.J.W., T.B.R., X.M. and D.A.F. contributed to the design of the article. N.V., M.J.W., T.B.R., X.M. and D.A.F. did the analysis. N.V., M.J.W., X.M. and D.A.F. did the interpretation. N.V., N.A.N. and Q.Z. did the data collection. N.V. and D.A.F. wrote the article. X.M. and D.A.F. did the critical revision of the article and obtained funding.

The study was funded by grants from the

References (13)

M. Lindroos et al.
Prevalence of aortic valve abnormalities in the elderly: an echocardiographic study of a random population sample
J Am Coll Cardiol
(1993)
C. Li et al.
The progression of calcific aortic valve disease through injury, cell dysfunction, and disruptive biologic and physical force feedback loops
Cardiovasc Pathol
(2013)
A.C. Liu et al.
The emerging role of valve interstitial cell phenotypes in regulating heart valve pathobiology
Am J Pathol
(2007)
X. Yang et al.
Pro-osteogenic phenotype of human aortic valve interstitial cells is associated with higher levels of Toll-like receptors 2 and 4 and enhanced expression of bone morphogenetic protein 2
J Am Coll Cardiol
(2009)
J. López et al.
Viral and bacterial patterns induce TLR-mediated sustained inflammation and calcification in aortic valve interstitial cells
Int J Cardiol
(2012)
N. Nadlonek et al.
Interleukin-1 beta induces an inflammatory phenotype in human aortic valve interstitial cells through nuclear factor kappa beta
Ann Thorac Surg
(2013)

There are more references available in the full text version of this article.

Cited by (38)

Biomimetic-modified bioprosthetic heart valves with controlled release of glycyrrhizin acid mediated by the inflammatory microenvironment for anti-thrombotic, anti-inflammatory, and anti-calcification
2023, Chemical Engineering Journal
Artificial heart valve replacement is optimum for treating severe valvular heart disease (VHD). Bioprosthetic heart valves (BHVs) have received widespread attention from patients and doctors due to their good hydrodynamics, hemocompatibility, and minimally invasive implantation. However, the inflammatory microenvironment caused by cytotoxicity of residual aldehyde groups, the immunogenicity of heterogeneous tissue, and implantation damage, have led to problems such as endothelialization difficulties, leaflet thrombosis, and calcification of BHVs, limiting service life and applicability to the population of BHVs. In this study, we introduced chondroitin sulfate (CS) and p-aminophenyl boronic acid-glycyrrhizin acid (PBA-GA) into glutaraldehyde (Glut) cross-linked BHV material, thereby preparing a bionic-modified BHV material (CS-GA-PP), which can remould the inflammatory microenvironment at the injured site. CS can form an extracellular matrix (ECM)-like structure, reducing the immunogenicity and thrombosis of BHVs, at the same time, GA can be released under the inflammatory microenvironment, exerting an anti-inflammatory effect, reducing inflammation and calcification. CS-GA-PP maintained the great structural stability and mechanical properties brought about by cross-linking with Glut. In addition, the strategy significantly improved the anti-thrombotic, anti-inflammatory, endothelialization, and anti-calcification properties of the BHV material. It is expected to provide a new idea and a solution for the intelligent biomedical design of Glut cross-linked BHV material.
Generating robust human valvular interstitial cell cultures: Protocol and considerations
2022, Journal of Molecular and Cellular Cardiology
Citation Excerpt :
Human VICs (hVICs) are the most representative subject for CAVD research. There is a substantial amount of literature involving the isolation of hVICs from patients with and without CAVD [36–52]. And while there is great heterogeneity in methods of hVIC isolation chosen by researchers, all seem to involve obtaining fresh donor tissue from either healthy or diseased patients and subjecting the tissue to a short enzymatic digestion to remove endothelial cells, followed by a longer enzymatic digestions to release VICs.
Research in heart valve biology is a growing field that has yet to elucidate the fundamentals of valve disease. Human valvular interstitial cells (hVICs) are the best option for studying the cellular mechanisms behind valvular pathologies. However, there is a wide range of isolation procedures for these cells published in the literature. To what extent various isolation methods, patient pathologies, and seeding densities influence the behaviour of hVICs remains unclear. Here, we present an optimised method of hVIC isolation from diseased human valves donated at the time of surgery. We show that two rounds of 1000 U/mL collagenase digestion for not >2 h results in a phenotypically stable cell culture with a near complete absence of endothelial cell contamination. We also suggest that cells should be seeded at 10,000 cells/cm² for experimentation. We found that patient pathology does not affect the success of the isolation procedure, and that instead, successful cultures are predicted by ensuring >500 mg valve tissue as starting material.
Wnt Signaling Mediates Pro-Fibrogenic Activity in Human Aortic Valve Interstitial Cells
2021, Annals of Thoracic Surgery
Citation Excerpt :
These AVICs were also found to have undergone osteoblastic phenotypic changes and exhibited elevated expression of bone-forming proteins, such as Runt-related transcription factor-2 and bone morphogenetic protein-2. A large body of prior work demonstrated a key role for TLR4 in mediating inflammatory and osteoblastic phenotypic changes in human AVICs.3,4,19,20 In light of the findings in the present study, Wnt signaling may play a role in TLR4-dependent osteogenic and inflammatory activity in AVICs as well.
Proinflammatory activation of toll-like receptor-4 (TLR4) drives phenotypic changes in aortic valve interstitial cells (AVICs) and produces a fibrogenic phenotype that mediates valvular fibrosis and contributes to aortic stenosis. Prior work identified upregulated Wnt signaling in AVICs taken from valves affected by aortic stenosis. Our purpose was to determine the contribution of Wnt signaling to TLR4-dependent fibrogenic activity in isolated human AVICs.
Human AVICs were isolated from hearts explanted for cardiac transplantation (N = 4). To test whether Wnt signaling contributed to TLR4-dependent fibrogenic activity, AVICs were treated with Wnt inhibitor (Dkk1) prior to TLR4 activation (LPS) and fibrogenic markers assessed. To determine the mediator of TLR4-to-Wnt signaling, expression of the key Wnt ligand, Wnt3a, was assessed after TLR4 activation and neutralizing antibodies confirmed the identity of the mediator. Fibrogenic activity was assessed after AVICs were treated with recombinant Wnt3a. Statistics were by analysis of variance (P < .05).
TLR4 activation upregulated in vitro collagen deposition, type IV collagen and MMP2 expression, and Dkk1 inhibited these responses (P < .05). Expression of Wnt3a was upregulated after TLR4 activation (P < .05). Anti-Wnt3a neutralizing antibodies abrogated TLR4-dependent type IV collagen and MMP2 expression (P < .05). Wnt3a upregulated type IV collagen and MMP2 expression independent of TLR4 activation (P < .05).
This study found that TLR4-dependent fibrogenic activity was mediated through Wnt signaling. The mediator of profibrogenic TLR4-to-Wnt signaling was a key Wnt ligand, Wnt3a. The abrogation of TLR4-induced fibrogenic activity in human AVICs by Wnt blockade illustrates a potential therapeutic role for Wnt inhibition in treatment and/or prevention of aortic stenosis.
Tumor necrosis factor alpha and interleukin 1 beta suppress myofibroblast activation via nuclear factor kappa B signaling in 3D-cultured mitral valve interstitial cells
2021, Acta Biomaterialia
Citation Excerpt :
We observed downregulation of VIC activation markers (αSMA, SM22α, and contractility), pro-fibrotic signals (TGF-β), and ECM proteins (collagen I and III) by TNF-α and IL-1β, collectively suggesting that these pro-inflammatory cytokines inhibit VIC-mediated fibrotic ECM remodeling. Furthermore, we chose to examine fibrotic rather than osteogenic remodeling in this study because mitral valve calcification, unlike aortic valve calcification, is relatively rare [33]. Nevertheless, it is important to note that the balance between mitral valve homeostasis and fibrosis in vivo is influenced by other cell types, including valvular endothelial cells and macrophages.
Mitral valve disease is a major cause of cardiovascular morbidity throughout the world. Many different mitral valve pathologies feature fibrotic remodeling, often accompanied by an inflammatory state. Mitral valve fibrosis is mediated by valvular interstitial cells (VICs), which reside in the valve leaflets and often differentiate into myofibroblast-like cells during disease conditions. In this study, we investigated the effects of tumor necrosis factor alpha (TNF-α) and interleukin 1 beta (IL-1β) on mitral VICs, since these pro-inflammatory cytokines have been shown to exert pleiotropic effects on various cell types in other fibrotic disorders. Using biomimetic three-dimensional culture systems, we demonstrated that TNF-α and IL-1β suppress myofibroblast differentiation in mitral VICs, as evidenced by gene and protein expression of alpha smooth muscle actin and smooth muscle 22 alpha. Addition of TNF-α and IL-1β also inhibited mitral VIC-mediated contraction of collagen gels. Furthermore, inhibition of NF-κB, which is downstream of TNF-α and IL-1β, reversed these effects. These results reveal targetable pathways for potential development of pharmaceutical treatments for alleviating fibrosis during mitral valve disease.
Mitral valve disease is a common cardiovascular condition that is often accompanied by fibrotic tissue remodeling. Valvular interstitial cells (VICs), the fibroblast-like cells that reside in heart valve leaflets, are thought to drive fibrosis during valve disease by differentiating into activated myofibroblasts. However, the signaling pathways that regulate this process in the mitral valve are not fully understood. In the present study, we cultured mitral VICs in collagen and poly(ethylene glycol) scaffolds designed to mimic the heart valve microenvironment and treated the cell-seeded scaffolds with cytokines. Using these 3D culture models, we found that the pro-inflammatory cytokines TNF-α and IL-1β downregulate myofibroblast and fibrosis markers in mitral VICs via the canonical NF-κB signaling pathway.
Commentary: Closing in on aortic stenosis
2021, Journal of Thoracic and Cardiovascular Surgery
Simvastatin down-regulates osteogenic response in cultured human aortic valve interstitial cells
2021, Journal of Thoracic and Cardiovascular Surgery
Aortic valve interstitial cells have been implicated in the pathogenesis of aortic stenosis. In response to proinflammatory stimuli, aortic valve interstitial cells undergo an osteogenic phenotypic change. The purpose of this study was to determine whether the anti-inflammatory effects of statins prevent osteogenic activity in cultured aortic valve interstitial cells.
Human aortic valve interstitial cells were isolated from hearts explanted for cardiac transplantation. To test whether simvastatin down-regulates TLR4-induced osteogenic response, aortic valve interstitial cells were treated with simvastatin with and without TLR4 agonist lipopolysaccharide (LPS), and osteogenic markers were measured. Simvastatin's influence on in vitro calcium deposition was assessed by alizarin red staining. Knockdown of postreceptor signaling proteins (MyD88 and TRIF) was performed to determine which of 2 TLR4-associated pathways mediates the osteogenic response. Expression levels of TLR4-induced nuclear factor kappa light chain enhancer of activated B cells (NF-κB) and TLR4 expression were assessed after treatment with simvastatin. Statistical testing was done by analysis of variance (P < .05).
Simvastatin decreased LPS-induced ALP and Runx2 expression and inhibited in vitro calcium deposition in aortic valve interstitial cells. Knockdown of MyD88 and TRIF attenuated the osteogenic response. Simvastatin attenuated TLR4-dependent NF-κB signaling and down-regulated TLR4 levels.
Simvastatin prevented TLR4-induced osteogenic phenotypic changes in isolated aortic valve interstitial cells via down-regulation of TLR4 and inhibition of NF-κB signaling. These data offer mechanistic insight into a possible therapeutic role for simvastatin in the prevention of aortic stenosis.

View all citing articles on Scopus

View full text

Association for Academic SurgeryAortic valve calcification is mediated by a differential response of aortic valve interstitial cells to inflammation

Abstract

Background

Materials and methods

Results

Conclusions

Introduction

Section snippets

Materials and methods

Patient characteristics

VICs from all four valves demonstrated a myofibroblast phenotype

Discussion

Conclusions

Acknowledgment

J Am Coll Cardiol

Cardiovasc Pathol

Am J Pathol

J Am Coll Cardiol

Int J Cardiol

Ann Thorac Surg

Association for Academic Surgery
Aortic valve calcification is mediated by a differential response of aortic valve interstitial cells to inflammation