Activation of valvular interstitial cells is mediated by transforming growth factor-β1 interactions with matrix molecules

Abstract

Strategies for the tissue-engineering of living cardiac valve replacements are limited by a lack of appropriate scaffold materials that both permit cell viability and actively contribute to the growth of functional tissues. Components of the extracellular matrix can localize and modify growth factor signals, and by doing so impart instructional stimuli for direction of cell phenotype. Fibronectin, collagen I, and heparin were explored as affinity matrices for sequestering and presenting soluble signaling molecules to control differentiation of valvular interstitial cells (VICs) to myofibroblasts. VIC differentiation is commonly characterized by expression of stress fibers containing alpha smooth muscle actin (α-SMA), and transforming growth factor-β1 (TGF-β1) is a central mediator of this transition. Both fibronectin and heparin, which are known to possess TGF-β1 binding interactions, were found to increase VIC α-SMA expression (120% and 258% of expression in controls), while VICs cultured on collagen I-modified substrates had diminished α-SMA expression (66% of control). Heparin treatment significantly stimulated VIC production of TGF-β1 at all concentrations tested (50 to 400 μg/ml). Heparin-modified substrates were found to alter cell morphology through increased adsorption of serum proteins, specifically TGF-β1. In sum, heparin produced α-SMA-positive myofibroblasts through both the de novo production of TGF-β1, and its localization in the pericellular environment. The addition of heparin to fibronectin-modified substrates led to a synergistic increase in VIC α-SMA expression, produced by the reciprocal binding of fibronectin, heparin, cell-produced TGF-β1. The characterization of molecules, both soluble and insoluble, that control VIC activation will be important for the development of tailored 3D culture environments for tissue-engineering applications.

Introduction

The extracellular matrix (ECM) is a hydrated network comprised of fibrillar collagen, elastin and fibronectin glycoproteins, and swollen proteoglycan and glycosaminoglycan connective tissue. Similar to the activities of growth factors and cytokines, the ECM has important intrinsic signaling capacities that govern cell fate processes such as differentiation, migration, survival, and cell growth. Although much research is focused on the regulation of growth factor production and mechanisms of signal transduction, matrix interactions can also modulate growth factor signaling by varying bioactivity, fixing spatial gradients, and altering degradation/activation pathways. The regulation of cell phenotype, an intricate balance of signaling derived from both soluble cytokine signals and insoluble ECM macromolecules, is explored here by the collateral action of matrix glycosaminoglycans to modulate growth factor function and density. Heparin, a highly sulfated glycosaminoglycan, exerts a wide range of biological activities through interactions with innumerable enzymes, growth factors, and ECM proteins (Linhardt, 2003). Although heparin contains a high degree of structural variability, a defined pattern of displayed carboxyl and sulfate groups mediates many specific protein interactions (Capila and Linhardt, 2002). Utilizing these protein interactions is especially attractive in the context of tissue engineering, where physiological processes such as cell–matrix, cell–cytokine, and matrix–cytokine interactions ultimately converge to establish cell phenotype. The study of cell behavior in simplified and controlled microenvironments is a prerequisite to development of complex multi-component tissue-engineered products (Lutolf and Hubbell, 2005).

Valvular interstitial cells (VICs), the most prevalent cell type of the cardiac valve leaflet, provide a particularly interesting example of phenotypic plasticity that is regulated by both environmental matrix stimuli and soluble signals (Schmittgraff et al., 1994). Activated VICs, or myofibroblasts, are collagen-producing cells that also express contractile proteins commonly found in vascular smooth muscle cells, most notably alpha smooth muscle actin (α-SMA) (Messier et al., 1994). In healthy valves VICs express low levels of α-SMA (Messier et al., 1994, Della Rocca et al., 2000), while valves undergoing remodeling contain a prominent population of activated α-SMA-positive VICs (Rabkin-Aikawa et al., 2004). Myofibroblastic VICs are normally lost by apoptosis when wound healing has ceased; however, myofibroblasts are found to persist in pathological settings, particularly in fibrotic diseases where the normal wound healing responses have failed to terminate (Schmittgraff et al., 1994). Among factors reported to induce α-SMA expression in VICs, transforming growth factor-β1 (TGF-β1) is considered the most efficient and acts via upregulation of both ECM proteins and inhibitors of metalloproteinases (Edwards et al., 1987), as reviewed by (Blobe et al., 2000). When VIC isolates from porcine aortic leaflets are cultured with exogenously added TGF-β1, there is a dose-dependent increase in α-SMA expression, as well as an increase in VIC contractility and fibronectin remodeling (Kondo et al., 2004, Walker et al., 2004). The bioavailability of TGF-β1 is tightly regulated by secretion with a precursor sequence as well as through binding to a larger latent TGF-β-binding protein (LTBP). This work is focused on understanding the role of matrix components in the regulation of TGF-β1, however there are other important molecules not investigated here, such as thrombospondin-1 and plasmin, which are involved in release of TFG-β from inactive complexes. Disassociation of TGF-β1 from inactive complexes by proteolysis or acidic conditions allows the fledged TGF-β1 to bind and signal cell-surface receptors. An additional level of control is provided by the extracellular matrix, which furnishes not only architectural support for the valve but also a supply of matrix-associated growth factors and signals such as TGF-β1 (Taipale et al., 1996).

There is convincing evidence that TGF-β1 contains heparin-binding motifs, comprised of basic residues poised to interact non-covalently with polyanions (McCaffrey et al., 1992, Lyon et al., 1997). Disassociation of the TGF-β1/α2-macroglobulin inactive complex by heparin has been shown to increase the concentration of active TGF-β1, as well as increase specific cell-surface binding of TGF-β1 in smooth muscle cells by increasing the availability of TGF-β1 to its receptor (McCaffrey et al., 1989). Reports are conflicting as to the effect of heparin on the de novo production of TGF-β1 (Baroni et al., 2000, Weigert et al., 2001). In normal, keloid, and dermal fibroblasts the production of TGF-β1 is significantly increased by the addition of heparin to in vitro cultures (Carroll and Koch, 2003). Heparin has been shown to induce α-SMA expression of proliferating fibroblast cultures in vitro and when given subcutaneously in vivo (Desmouliere et al., 1992), but the effects of heparin on valvular interstitial cell activation were unknown prior to this study. We hypothesized that heparin could be used to control the molecular interactions between TGF-β1 and VICs by increasing the stability of the TGF-β1 signal in the pericellular microenvironment. Specifically, this interaction between heparin and TGF-β1 could be exploited to minimize degradative and diffusional loss of TGF-β1.

Furthermore, myofibroblast precursor cells express and actively remodel fibronectin (FN), which is a major component of the insoluble extracellular matrix as well as a soluble constituent of plasma. Alternative splice variants of FN are necessary for induction of the myofibroblast phenotype by TGF-β1 (Serini et al., 1998). FN contains two heparin-binding domains, a collagen-binding domain, and two major fibrin-binding sites, as well as integrin-recognition sequences RGD, LDV, and REDV (Pankov and Yamada, 2002). FN provides a unique linker between cells and their surrounding matrix by binding to both ECM components and cell-surface FN integrin receptors. TGF-β1 is a fibronectin-associated growth factor and FN has been suggested to bind active TGF-β1 directly, as well as through interactions with LTBP (Fava and McClure, 1987, Taipale et al., 1996, Taipale and Keski-Oja, 1997). In this study, VIC monolayer cultures on fibronectin-modified surfaces were explored to manipulate the bioavailability of TGF-β1, either by intrinsic interactions of FN with TGF-β1 or through binding interactions with exogenously added heparin. Modification of substrate chemistry with TGF-β1-binding ECM components offers the potential to alter availability and presentation of TGF-β1 delivered to cell-surface receptors. The ultimate goal of our work is to translate an understanding of phenotype transitions, and the growth factor/matrix interactions that influence these transitions, into the rational design of 3D cell-instructive microenvironments that dynamically promote or suppress selected cell functions. A basic understanding of how cell-material and cell-growth factor interactions combine to alter cellular functions will eventually allow the rational tailoring of tissue-engineered constructs to mimic or replace natural tissue.