Crosslinked hyaluronan scaffolds as a biologically active carrier for valvular interstitial cells

Hyaluronic acid (HA), a major component of the cardiac jelly during heart morphogenesis, is a polysaccharide that upon modification can be photopolymerized into hydrogels. Previous work in our lab has found that photopolymerizable HA hydrogels are suitable scaffolds for the culture and proliferation of valvular interstitial cells (VICs), the most prevalent cell type in native heart valves. The physical properties of HA gels are easily modified through alteration in material crosslink density or by copolymerizing with other reactive macromolecules. Degradation products of HA gels and the starting macromers significantly increased VIC proliferation when added to cell cultures. With low molecular weight HA (<6700 Da) exhibiting greatest stimulation of VIC proliferation. Low molecular weight HA degradation products added to VIC cultures also resulted in a four-fold increase in total matrix production and a two-fold increase in elastin production over untreated controls. VIC internalization of HA, as shown by cellular uptake of fluorescently labeled HA, likely activates signaling cascades resulting in the biological responses seen here. Lastly, VICs encapsulated within HA hydrogels remained viable, and significant elastin production was observed after 6 weeks of culture. This work shows promise for the creation of a tissue-engineered heart valve utilizing the synergistic relationship between hyaluronic acid and VICs.     

Regulation of valvular interstitial cell calcification by components of the extracellular matrix

Understanding the interactions between extracellular matrix (ECM) components and valvular interstitial cells (VICs) is relevant to both treating heart valve disease and designing heart valve tissue engineering scaffolds, yet the VIC-ECM relationship has not been well characterized. Thus, the aim of this study was to characterize VIC-ECM interactions, paying specific attention to whether ECM composition affected the in vitro calcification of VICs. Our results show that the number and size of calcific nodules formed in VIC cultures, as well as the expression of the mineralization markers alkaline phosphatase (ALP) and CBFa1, were highly dependent upon the composition of the culture surface. VICs cultured on certain ECM components, that is, collagen and fibronectin, were resistant to calcification, even upon treatment with mineralization-inducing growth factors. Meanwhile, cultures of VICs on fibrin, laminin, and heparin coatings had a high number of calcified nodules, although only VICs on fibrin expressed significantly elevated levels of ALP and CBFa1. Nodule composition analysis revealed the presence of multiple types of mineralization. Although apoptotic and necrotic cells were more concentrated in nodules, these nodules did contain a strong majority population of viable cells. Characterizing this ECM-dependence of VIC calcification will help us to identify appropriate biomaterial environments for heart valve tissue engineering as well as elucidate mechanisms of valvular disease.     

The emerging role of valve interstitial cell phenotypes in regulating heart valve pathobiology

The study of the cellular and molecular pathogenesis of heart valve disease is an emerging area of research made possible by the availability of cultures of valve interstitial cells (VICs) and valve endothelial cells (VECs) and by the design and use of in vitro and in vivo experimental systems that model elements of valve biological and pathobiological activity. VICs are the most common cells in the valve and are distinct from other mesenchymal cell types in other organs. We present a conceptual approach to the investigation of VICs by focusing on VIC phenotype-function relationships. Our review suggests that there are five identifiable phenotypes of VICs that define the current understanding of their cellular and molecular functions. These include embryonic progenitor endothelial/mesenchymal cells, quiescent VICs (qVICs), activated VICs (aVICs), progenitor VICs (pVICs), and osteoblastic VICs (obVICs). Although these may exhibit plasticity and may convert from one form to another, compartmentalizing VIC function into distinct phenotypes is useful in bringing clarity to our understanding of VIC pathobiology. We present a conceptual model that is useful in the design and interpretation of studies on the function of an important phenotype in disease, the activated VIC. We hope this review will inspire members of the investigative pathology community to consider valve pathobiology as an exciting new frontier exploring pathogenesis and discovering new therapeutic targets in cardiovascular diseases.     

Characterization of valvular interstitial cell function in three dimensional matrix metalloproteinase degradable PEG hydrogels

Valvular interstitial cells (VICs) maintain functional heart valve structure and display transient fibroblast and myofibroblast properties. Most cell characterization studies have been performed on plastic dishes; while insightful, these systems are limited. Thus, a matrix metalloproteinase (MMP) degradable poly(ethylene glycol) (PEG) hydrogel system is proposed in this communication as a useful tool for characterizing VIC function in 3D. When encapsulated, VICs attained spread morphology, and proliferated and migrated as shown through real-time cell microscopy. Additionally, fibronectin derived pendant RGD was incorporated into the system to promote integrin binding. As RGD concentration increased from 0 to 2000 μm, VIC process extension and integrin α_vβ₃ binding increased within two days. By day 10, integrin binding was equalized between conditions. VIC morphology and rate of process extension were also increased through decreasing the hydrogel matrix density presented to the cells. VIC differentiation in response to exogenously delivered transforming growth factor-beta1 (TGF-β1) was also examined within the hydrogel networks. TGF-β1 increased expression of alpha smooth muscle actin (αSMA) and collagen-1 at both the mRNA and protein level by day 2 of culture, indicating myofibroblast differentiation, and was sustained over the course of the study (2 weeks). These studies demonstrate the utility, flexibility, and biological activity of this MMP-degradable system for the characterization of VICs, an important cell population for tissue engineering viable valve replacements and understanding valvular pathobiology.     

Differences in tissue-remodeling potential of aortic and pulmonary heart valve interstitial cells

Heart valve interstitial cells (VICs) appear to have a dynamic and reversible phenotype, an attribute speculated to be necessary for valve tissue remodeling during times of development and repair. Therefore, we hypothesized that the cytoskeletal (CSK) remodeling capability of the aortic and pulmonary VICs (AVICs and PVICs, respectively), which are dominated by smooth muscle alpha-actin, would exhibit unique contractile behaviors when seeded on collagen gels. Using a porcine cell source, we observed that VIC populations did not contract the gels at early time points (2 and 4 hours) as dermal fibroblasts did, but formed a central cluster of cells prior to contraction. After clustering, VICs appeared to radiate out from the center of the gels, whereas fibroblasts did not migrate but contracted the gels locally. VIC gels treated with transforming growth factor beta1 contracted the gels rapidly, revealing similar sensitivity to the cytokine. Moreover, we evaluated the initial mechanical state of the underlying CSK by comparing AVIC and PVIC stiffness with atomic force microscopy. Not only were AVICs significantly stiffer (p < 0.001) than the PVICs, but they also contracted the gels significantly more at 24 and 48 hours (p < 0.001). Taken together, these findings suggest that the AVICs are capable of inducing greater extra cellular matrix contraction, possibly manifesting in a more pronounced ability to remodel valvular tissues. Moreover, significant mechanobiological differences between AVICs and PVICs exist, and may have implications for understanding native valvular tissue remodeling. Elucidating these differences will also define important functional endpoints in the development of tissue engineering approaches for heart valve repair and replacement.     

Dynamic stiffening of poly(ethylene glycol)-based hydrogels to direct valvular interstitial cell phenotype in a three-dimensional environment

Valvular interstitial cells (VICs) are active regulators of valve homeostasis and disease, responsible for secreting and remodeling the valve tissue matrix. As a result of VIC activity, the valve modulus can substantially change during development, injury and repair, and disease progression. While two-dimensional biomaterial substrates have been used to study mechanosensing and its influence on VIC phenotype, less is known about how these cells respond to matrix modulus in a three-dimensional environment. Here, we synthesized MMP-degradable poly(ethylene glycol) (PEG) hydrogels with elastic moduli ranging from 0.24 kPa to 12 kPa and observed that cell morphology was constrained in stiffer gels. To vary gel stiffness without substantially changing cell morphology, cell-laden hydrogels were cultured in the 0.24 kPa gels for 3 days to allow VIC spreading, and then stiffened in situ via a second, photoinitiated thiol-ene polymerization such that the gel modulus increased from 0.24 kPa to 1.2 kPa or 13 kPa. VICs encapsulated within soft gels exhibited αSMA stress fibers (∼40%), a hallmark of the myofibroblast phenotype. Interestingly, in stiffened gels, VICs became deactivated to a quiescent fibroblast phenotype, suggesting that matrix stiffness directs VIC phenotype independent of morphology, but in a manner that depends on the dimensionality of the culture platform. Collectively, these studies present a versatile method for dynamic stiffening of hydrogels and demonstrate the significant effects of matrix modulus on VIC myofibroblast properties in three-dimensional environments.     

Cardiac valve interstitial cells secrete fibronectin and form fibrillar adhesions in response to injury.

BACKGROUND: Fibronectin, an extracellular matrix protein, is associated with the general process of tissue repair and is present in heart valves. In order to understand the cellular mechanisms of heart valve repair, we hypothesized that fibronectin is produced and secreted by valvular interstitial cells (VICs), and when up-regulated in VICs involved in active repair, it is associated with prominent fibrillar adhesions composed of tensin and alpha(5)beta(1) integrin. We investigated the interaction of porcine mitral VICs with the underlying fibronectin matrix and the formation and localization of focal and fibrillar adhesion complexes in an in vitro wound model. METHODS: Confluent monolayers of VICs were wounded with a 1-mm-wide cell scraper, maintained in standard media and 10% fetal bovine serum, and fixed at various time points after wounding. Immunohistochemistry was used to localize fibronectin, paxillin, tensin, and alpha(5)beta(1) integrin. F-actin was localized with an Alexa-Fluor-568-labeled phalloidin. Cells were examined with a scanning confocal laser microscope. RESULTS: In response to in vitro mechanical wounding, migrating VICs at the wound edge expressed cytoplasmic fibronectin compared to nonwounded confluent monolayers. Over 24 to 48 h, fibrils were deposited into the subcellular space. Coincident with this, staining for alpha(5)beta(1) appeared, and tensin redistributed from focal adhesions to fibrillar adhesions, which colocalized with alpha(5)beta(1). CONCLUSIONS: Fibronectin in association with fibrillar adhesions is a component of the matrix that may be secreted by migrating VICs to regulate repair at sites of valve injury.     

Investigating the role of substrate stiffness in the persistence of valvular interstitial cell activation

During heart valve remodeling and in many disease states, valvular interstitial cells (VICs) shift to an activated myofibroblast phenotype characterized by enhanced synthetic and contractile activity. Pronounced alpha smooth muscle actin (αSMA)-positive stress fibers, the hallmark of activated myofibroblasts, are also observed in VICs cultured on stiff substrates especially in the presence of transforming growth factor-beta1 (TGF-β1), however, the detailed relationship between stiffness and VIC phenotype has not been explored. The goal of this study was to characterize VIC activation as a function of substrate stiffness over a wide range of stiffness levels including that of diseased valves (stiff), normal valves (compliant), and hydrogels for heart valve tissue engineering (very soft). VICs obtained from porcine aortic valves were cultured on stiff tissue culture plastic to activate them, then, cultured on collagen-coated polyacrylamide substrates of predefined stiffness in a high-throughput culture system to assess the persistence of activation. Metrics extracted from regression analysis demonstrate that relative to a compliant substrate, stiff substrates result in higher cell numbers, more pronounced expression of αSMA-positive stress fibers, and larger spread area which is in qualitative agreement with previous studies. Our data also indicate that VICs require a much lower substrate stiffness level to "deactivate" them than previously thought. The high sensitivity of VICs to substrate stiffness demonstrates the importance of the mechanical properties of materials used for valve repair or for engineering valve tissue.     

Stiffness and adhesivity control aortic valve interstitial cell behavior within hyaluronic acid based hydrogels

Bioactive and biodegradable hydrogels that mimic the extracellular matrix and regulate valve interstitial cells (VIC) behavior are of great interest as three-dimensional (3-D) model systems for understanding mechanisms of valvular heart disease pathogenesis in vitro and the basis for regenerative templates for tissue engineering. However, the role of stiffness and adhesivity of hydrogels in VIC behavior remains poorly understood. This study reports the synthesis of methacrylated hyaluronic acid (Me-HA) and oxidized and methacrylated hyaluronic acid, and the subsequent development of hybrid hydrogels based on modified HA and methacrylated gelatin (Me-Gel) for VIC encapsulation. The mechanical stiffness and swelling ratio of the hydrogels were tunable with the molecular weight of the HA and the concentration/composition of the precursor solution. The encapsulated VIC in pure HA hydrogels with lower mechanical stiffness showed a more spreading morphology compared to their stiffer counterparts and dramatically up-regulated alpha smooth muscle actin expression, indicating more activated myofibroblast properties. The addition of Me-Gel in Me-HA facilitated cell spreading, proliferation and VIC migration from encapsulated spheroids and better maintained the VIC fibroblastic phenotype. The VIC phenotype transition during migration from encapsulated spheroids in both Me-HA and Me-HA/Me-Gel hydrogel matrixes was also observed. These findings are important for the rational design of hydrogels for controlling the VIC morphology, and for regulating the VIC phenotype and function. The Me-HA/Me-Gel hybrid hydrogels accommodated with VIC are promising as valve tissue engineering scaffolds and 3-D models for understanding valvular pathobiology.     

Versican and CD44 in in vitro valvular interstitial cell injury and repair

BackgroundVersican is one of the key components of the extracellular matrix (ECM) that is expressed during injury, inflammatory, and repair processes. The current study evaluated the relationship between versican and the membrane receptor CD44 during in vitro valvular interstitial cell (VIC) injury and repair.MethodsSubconfluent, confluent, and wounded cultures of human VICs were fixed and immunostained to detect versican and the membrane receptor CD44. To examine the relationship between versican and CD44, a blocking antibody to CD44 was added to cultured VICs, and in vitro wound repair along with pericellular versican organization and stress fiber formation were examined.ResultsImmunohistochemistry demonstrated that versican is prominent intracellularly, as well as extracellularly, in actively proliferating VICs. In contrast, versican was only localized to fibrils in the extracellular space in between cells in confluent (quiescent) cultures. Following wounding, versican expression was up-regulated, and it was secreted as ECM at the trailing edge of migrating cells. The staining for CD44 was similarly localized to the trailing edge of migrating VICs in wounded cultures. Treatment of VICs with a CD44-blocking antibody disrupted the organization of versican in the pericellular matrix and inhibited stress fiber formation in these cells. Functionally, blocking CD44 significantly inhibited VIC-mediated contraction of type I collagen gels (35.7%±0.7% vs. 23.3%±1.4% of initial gel area, P<.01).ConclusionsVersican is a key component of the provisional wound repair ECM that is expressed following injury to VICs. The receptor CD44 plays an important role in organizing the provisional ECM.SummaryOur data suggests VICs synthesize and secrete versican following injury. These cells also up-regulate CD44, a receptor that binds versican. Blocking CD44 disrupted the organization of versican and inhibited stress fiber formation. Functionally, blocking CD44 inhibited cell-mediated contraction of a collagen matrix. Collectively, these data suggest versican expression and organization are important to valve cell injury and repair.     

A collagen-glycosaminoglycan co-culture model for heart valve tissue engineering applications

In order to develop efficient design strategies for a tissue-engineered heart valve, in vivo and in vitro models of valvular structure and cellular function require extensive characterisation. Collagen and glycosaminoglycans (GAGs) provide unique functional characteristics to the heart valve structure. In the current study, type I collagen-GAG hydrogels were investigated as biomaterials for the creation of mitral valve tissue. Porcine mitral valve interstitial cells (VICs) and endothelial cells (VECs) were isolated and co-cultured for 4 weeks in hydrogel constructs composed of type I collagen. The metabolic activity and tissue organisation of mitral valve tissue constructs was evaluated in the presence and absence of chondroitin sulphate (CS) GAG, and comparisons were made with normal mitral valve tissue. Both collagen and collagen-CS mitral valve constructs contracted to form tissue-like structures in vitro. Biochemical assay demonstrated that over 75% of CS was retained within collagen-CS constructs. Morphological examination demonstrated enhanced VEC surface coverage in collagen-CS constructs compared to collagen constructs. Ultrastructural analysis revealed basement membrane synthesis and cell junction formation by construct VECs, with an increased matrix porosity observed in collagen-CS constructs. Immunohistochemical analyses demonstrated enhanced extracellular matrix production in collagen-CS constructs, including expression of elastin and laminin by VICs. Both native valve and collagen-CS construct VECs also expressed the vasoactive molecule, eNOS, which was absent from collagen construct VECs. The present study demonstrates that collagen gels can be used as matrices for the in vitro synthesis of tissue structures resembling mitral valve tissue. Addition of CS resulting in a more porous model was shown to positively influence the bioactivity of seeded valve cells and tissue remodelling. Collagen-GAG matrices may hold promise for a potential use in heart valve tissue engineering and improved understanding of heart valve biology.     

The role of valvular endothelial cell paracrine signaling and matrix elasticity on valvular interstitial cell activation

The effects of valvular endothelial cell (VlvEC) paracrine signaling on VIC phenotype and nodule formation were tested using a co-culture platform with physiologically relevant matrix elasticities and diffusion distance. 100 μm thin poly(ethylene glycol) (PEG) hydrogels of 3–27 kPa Young's moduli were fabricated in transwell inserts. VICs were cultured on the gels, as VIC phenotype is known to change significantly within this range, while VlvECs lined the underside of the membrane. Co-culture with VlvECs significantly reduced VIC activation to the myofibroblast phenotype on all gels with the largest percent decrease on the 3 kPa gels (∼70%), while stiffer gels resulted in approximately 20–30% decrease. Additionally, VlvECs significantly reduced αSMA protein expression (∼2 fold lower) on both 3 and 27 kPa gels, as well as the number (∼2 fold lower) of nodules formed on the 27 kPa gels. Effects of VlvECs were prevented when nitric oxide (NO) release was inhibited with l-NAME, suggesting that VlvEC produced NO inhibits VIC activation. Withdrawal of l-NAME after 3, 5, and 7 days with restoration of VlvEC NO production for 2 additional days led to a partial reversal of VIC activation (∼25% decrease). A potential mechanism by which VlvEC produced NO reduced VIC activation was studied by inhibiting initial and mid-stage cGMP pathway molecules. Inhibition of soluble guanylyl cyclase (sGC) with ODQ or protein kinase G (PKG) with RBrcGMP or stimulation of Rho kinase (ROCK) with LPA, abolished VlvEC effects on VIC activation. This work contributes substantially to the understanding of the valve endothelium's role in preventing VIC functions associated with aortic valve stenosis initiation and progression.     

Designing scaffolds for valvular interstitial cells: cell adhesion and function on naturally derived materials

Valvular interstitial cells (VICs) possess many properties that make them attractive for use in the construction of a tissue-engineered valve; however, we have found that the surfaces to which VICs will adhere and spread are limited. For example, VICs adhere and spread on collagen and laminin-coated surfaces, but display altered morphology and do not proliferate. Interestingly, fibronectin (FN) was one adhesion protein that facilitated VIC adhesion and proliferation. Yet VICs did not spread on surfaces modified with RGD, a ubiquitous cell-adhesive peptide, nor with other FN-specific peptide sequences such as EILDV and PHSRN. Hyaluronic acid (HA) is a highly elastic polysaccharide that is involved in natural valve morphogenesis and possesses binding interactions with FN. Hyaluronic acid was modified to form photopolymerizable hydrogels, and VICs were found to spread and proliferate on HA-based gels, forming a confluent monolayer on the gels within 4 days. Modified HA retained its ability to specifically bind FN, allowing for the formation of gels containing both HA and FN. Valvular interstital cells cultured on HA surfaces displayed significantly increased production of extracellular matrix proteins, indicating that HA-based scaffolds may provide useful biological cues to stimulate heart valve tissue formation.     

Small peptide functionalized thiol-ene hydrogels as culture substrates for understanding valvular interstitial cell activation and de novo tissue deposition

A thiol-ene polymerization platform was used to synthesize peptide functionalized poly(ethylene glycol) hydrogels, which were initially characterized and compared to theoretical predictions of Young's modulus via a theoretical crosslinking density equation presented herein. After thorough characterization, this material system's utility for answering specific biological hypotheses was demonstrated with the culture and observation of aortic valvular interstitial cells (VICs). Specifically, these materials were used to better understand the role of substrate elasticity and biochemical functionality on VIC α-smooth muscle (αSMA) expression and secretory properties (i.e. de novo extracellular matrix (ECM)). The Young's moduli of the hydrogels varied from 28kPa (activating, 90% myofibroblasts) to 4kPa (non-activating, 15% myofibroblast), and the biochemical functionality was tailored by incorporating three small adhesive peptide sequences, RGDS, VGVAPG and P15. To promote VIC adhesion, a basal [RGDS] of 0.8mM was used in all formulations, while the [VGVAPG] or [P15] were varied to be lower than, equal to or higher than 0.8mM. The substrates with 1.2mM VGVAPG and all gels with P15 led to significantly higher αSMA expression for both stiff and soft substrates, as compared to 0.8mM RGDS alone. Importantly, all gel conditions αSMA expression were significantly lower than tissue culture poly(styrene) (TCPS; ～4- to 10-fold difference). The ECM produced decreased significantly as the total integrin-binding peptide concentration increased, but was significantly higher than that produced on TCPS. This easily tailored material system provides a useful culture platform to improve the fundamental understanding of VIC biology through isolating specific biological cues and observing VIC function.     

Transforming growth factor-β regulates the growth of valve interstitial cells in vitro

Although valve interstitial cell (VIC) growth is an essential feature of injured and diseased valves, the regulation of VIC growth is poorly understood. Transforming growth factor (TGF)-β promotes VIC proliferation in early-stage wound repair; thus, herein, we tested the hypothesis that TGF-β regulates VIC proliferation under normal nonwound conditions using low-density porcine VIC monolayers. Cell numbers were counted during a 10-day period, whereas proliferation and apoptosis were quantified by bromodeoxyuridine staining and TUNEL, respectively. The extent of retinoblastoma protein phosphorylation and expression of cyclin D1, CDK 4, and p27 were compared using Western blot analysis. Adhesion was quantified using a trypsin adhesion assay, and morphological change was demonstrated by immunofluorescence localization of α-smooth muscle actin and vinculin. TGF-β-treated VICs were rhomboid; significantly decreased in number, proliferation, and retinoblastoma protein phosphorylation; and concomitantly had decreased expression of cyclin D1/CDK4 and increased expression of p27. TGF-β-treated VICs adhered better to substratum and had more vinculin plaques and α-smooth muscle actin stress fibers than did controls. Thus, the regulation of VIC growth by TGF-β is context dependent. TGF-β prevents excessive heart valve growth under normal physiological conditions while it promotes cell proliferation in the early stages of repair, when increased VICs are required.     

Engineering of a bio-functionalized hybrid off-the-shelf heart valve

Currently available heart valve replacements are limited in long-term performance or fail due to leaflet thickening, lack of growth or remodeling potential. In order to address these issues, it is necessary to mimic multiple factors of the native valvular extracellular matrix (ECM) such as architecture, mechanical behavior and biochemical signals. Here, we successfully generated an electrospun PEGdma–PLA scaffold adapted to the structure and mechanical properties of native valve leaflets. Valvular interstitial cells (VICs) and valvular endothelial cells (VECs) were seeded on the scaffold and when cultured under physiological conditions in a bioreactor, the construct performed like a native leaflet. Atomic force microscopy (AFM) was employed to obtain detailed mechanical information from the leaflets, which enabled the first layer-specific measurement of the Young's modulus. Interestingly, spongiosa stiffness was much lower compared to the fibrosa and ventricularis. Moreover, investigations into human fetal heart valve development identified collagen type I and versican as important structural proteins. As a proof of principle, these proteins were introduced to the scaffold, demonstrating the ability to bio-functionalize the hybrid valve based on natures' blueprint.