Mitral valvular interstitial cell responses to substrate stiffness depend on age and anatomic region
Introduction
Valve disease afflicts a substantial portion of the population: 1–2% of 26- to 84-year-olds are afflicted by mitral valve disorders [1]. Valve disease incurs significant morbidity and mortality, requiring over 100,000 surgeries in the USA each year [2]. In many of these disease states the mechanical properties of these valves are altered, often contributing to the poor valve function requiring surgical intervention. Valvular interstitial cells (VICs) are the dynamic, living component of heart valves responsible for synthesizing and maintaining the valve matrix composition, which in turn determines the valve’s material behavior. VICs and valves are known to be responsive to changes in their mechanical environment [3], [4], [5]. However, the interplay between matrix-driven material properties such as stiffness and the phenotype and synthetic behavior of VICs, particularly in the mitral valve (MV), has largely been overlooked. Recent work has demonstrated age-related significant changes in valve composition [6], [7], [8] and material properties [9]; other studies have shown substantial heterogeneity in material behavior among the different anatomic regions of the MV [10]. Given that different aged VICs and VICs from different regions of the MV reside in valve tissues with distinct stiffnesses [9], it was hypothesized that there may be an age- and valve-region-specific response of VICs to substrate stiffness.
In order to test this hypothesis, separate groups of VICs from three distinct age groups and from two different regions of the MV were cultured on poly(ethylene) glycol (PEG) hydrogels of two different stiffnesses. After 48 h, the resulting VIC expression of cell phenotype and collagen synthesis markers was assessed using immunocytochemistry (ICC).
PEG hydrogels were chosen for this experiment based on their promise as a potential platform for the design of scaffolds for tissue-engineered heart valves. PEG hydrogels are extremely hydrophilic, providing prevention against protein adsorption, a critical step in the immunogenicity and degradation of bioprosthetics [11]. They are also highly permeable, allowing the exchange of nutrients and waste materials [12]. Their stiffness can be regulated by changing the molecular weight and concentration of PEG [13]. However, one of the factors that makes these gels particularly attractive is the ability to customize them by conjugating to the PEG backbone various peptides, including cell ligands and growth factors, as well as incorporating enzyme-degradable sequences allowing tunability of the degradation rate of the hydrogel. This designer biofunctionality makes PEG hydrogels advantageous for the tissue engineering of heart valves. In the present study PEG hydrogels were conjugated with an Arg–Gly–Asp–Ser (RGDS) peptide, enabling VIC attachment to the hydrogel, and methacrylated heparin, which is necessary for normal VIC morphology [14], [15]. These functionalized PEG hydrogels of two different stiffnesses were formulated to keep the concentration of biological cues constant, thus isolating the effect of stiffness on VIC phenotype.
Section snippets
Synthesis of PEG hydrogel components
PEG-diacrylate (PEG-DA) of 3400 Da MW was synthesized from PEG (Sigma–Aldrich, St. Louis, MO) as previously described [13]. 1H nuclear magnetic resonance (NMR) analysis revealed >95% acrylation. Methacrylated heparin was synthesized as described previously [14]. Briefly, a 10 mg ml–1 solution of heparin (Sigma–Aldrich) dissolved in ultrapure water was reacted with 40 molar excess methacrylic anhydride (Sigma–Aldrich). The pH of the solution was adjusted to 7.5 using 4 M NaOH and stirred for 24 h.
Stiffness of different weight–volume fraction functionalized PEG hydrogels
Uniaxial testing revealed that functionalized 5% weight–volume PEG gels had a mean modulus of 34.5 kPa, while functionalized 15% weight–volume PEG gels had a mean modulus of 323.3 kPa (Fig. 3).
SMaA expression of VICs on gels of different stiffnesses
Not all VICs stained positively for SMaA, as expected. Analysis of the fraction of VICs expressing SMaA (SMaA+ VICs) on the different gels revealed a trend towards decreased SMaA+ VICs on the 15% gels compared to the 5% in the 6-week-old MVAC VICs (Fig. 4, p = 0.1), but there was no difference between gels
Discussion
In this study, mitral VICs grown on functionalized PEG hydrogels demonstrated both age- and valve-region-specific (MVAC vs. PML) responses to substrate stiffness. These results underscore the range of unique phenotypes found in valvular cells and provide compelling motivation for further studies of heart valve mechanobiology.
Conclusions
In this study, the response of MV VICs to substrate stiffness has been investigated for the first time. VIC populations taken from different regions of the same MV and VICs of different ages cultured on PEG hydrogels of different stiffnesses demonstrated age- and valve-region-specific responses to substrate stiffness. These findings should be taken into consideration in the design of an age-specific tissue-engineered heart valve and in future investigations of heart valve mechanobiology.
Acknowledgements
The authors appreciate the assistance of the members of the Grande-Allen laboratory, as well as members of the West laboratory: Michael Cuchiara, Stephanie Nemir, Ph.D., Melissa McHale, Ph.D., Maude Rowland, Jean Altus, Ph.D. and Jerome Saltarelli, Ph.D. The authors also appreciate the counsel of Scott Baggett, Ph.D. regarding statistics and the assistance of Sean Moran, Ph.D. in NMR analysis. This research was supported in part by a Hertz Foundation Graduate Fellowship (E.H.S.), a NIH Ruth
References (40)
- et al.
Valve proteoglycan content and glycosaminoglycan fine structure are unique to microstructure mechanical load and age: relevance to an age-specific tissue-engineered heart valve
Acta Biomater
(2008) - et al.
Material-based regulation of the myofibroblast phenotype
Biomaterials
(2007) - et al.
New method for quantitative determination of uronic acids
Anal Biochem
(1973) - et al.
Mitral valve stiffening in end-stage heart failure: evidence of an organic contribution to functional mitral regurgitation
J Thorac Cardiovasc Surg
(2005) - et al.
Fibroblast adaptation and stiffness matching to soft elastic substrates
Biophys J
(2007) - et al.
In-vivo dynamic deformation of the mitral valve anterior leaflet
Ann Thorac Surg
(2006) - et al.
Matrix elasticity directs stem cell lineage specification
Cell
(2006) - et al.
Functional diversity of lysyl hydroxylase 2 in collagen synthesis of human dermal fibroblasts
Exp Cell Res
(2006) - et al.
Transforming growth factor-beta regulates in vitro heart valve repair by activated valve interstitial cells
Am J Pathol
(2008) - et al.
The cardiac valve interstitial cell
Int J Biochem Cell Biol
(2003)
Dual structural and functional phenotypes of the porcine aortic valve interstitial population: characteristics of the leaflet myofibroblast
J Surg Res
Activation of valvular interstitial cells is mediated by transforming growth factor-beta1 interactions with matrix molecules
Matrix Biol
Heart disease and stroke statistics – 2007 update: a report from the American Heart Association Statistics Committee and Stroke Statistics Subcommittee
Circulation
Collagen synthesis by mesenchymal stem cells and aortic valve interstitial cells in response to mechanical stretch
Cardiovasc Res
An ex vivo study of the biological properties of porcine aortic valves in response to circumferential cyclic stretch
Ann Biomed Eng
Cyclic pressure and shear stress regulate matrix metalloproteinases and cathepsin activity in porcine aortic valves
J Heart Valve Dis
Age-related changes in collagen synthesis and turnover in porcine heart valves
J Heart Valve Dis
Human semilunar cardiac valve remodeling by activated cells from fetus to adult: implications for postnatal adaptation pathology, and tissue engineering
Circulation
Age-related changes in material behavior of porcine mitral and aortic valves and correlation to matrix composition
Tissue Eng Part A
Cited by (34)
The impact of biological factors, anatomy, and mechanical forces on calcification and fibrosis of cardiac and vascular structures
2022, Debulking in Cardiovascular Interventions and Revascularization Strategies: Between a Rock and the HeartDevelopment and Evaluation of a Tissue-Engineered Fibrin-based Canine Mitral Valve Three-dimensional Cell Culture System
2018, Journal of Comparative PathologyCitation Excerpt :The fibrin hydrogel used in this study is favourable for cell distribution, cell communication and the synthesis and accumulation of ECM within tissue-engineered constructs, but is also affected by cell-mediated contraction and shrinkage (Ye et al., 2000; Jockenhoevel et al., 2001; Flanagan et al., 2007). VICs do have contractile and force generation properties and are also sensitive and responsive to the mechanical properties of the surrounding matrix (Stephens et al., 2010, 2011). Human aortic VICs activate spontaneously when grown on mechanically soft methacrylated gelatine hydrogels, while activated myofibroblasts have heightened contractility and force generation capability, compared with fibroblasts, when grown on collagen gels (Stephens et al., 2010).
A survey of membrane receptor regulation in valvular interstitial cells cultured under mechanical stresses
2017, Experimental Cell ResearchValve interstitial cell shape modulates cell contractility independent of cell phenotype
2016, Journal of BiomechanicsCitation Excerpt :COL1A1, COL3A1 and RhoA gene expression (Supplementary Fig. S4) were not significantly altered as a function of pattern width or osteogenic culture medium. Over the last decade, extensive research has implicated biochemical mediators, the mechanical environment, and the substrate as factors that regulate the VIC phenotype and function (Balachandran et al., 2011, 2009, 2010; Chen et al., 2015; Gould et al., 2013; Hutcheson et al., 2012; Ku et al., 2006; Liu et al., 2013; Quinlan and Billiar, 2012; Stephens et al., 2011; Sucosky et al., 2009; Wang et al., 2013; Yip and Simmons, 2011). In the current study, we used micropatterned VICs with enforced shapes via constraining boundary conditions to study the relationship between cell shape and contractile output.