Activation of valvular interstitial cells is mediated by transforming growth factor-β1 interactions with matrix molecules
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.
Section snippets
VIC isolation and passage number dictate α-SMA expression
Myofibroblasts exist along a continuum between fibroblasts and smooth muscle cells, having characteristics of both cell types. Identification of myofibroblasts is commonly performed through immunocytochemical labeling of expressed cytoskeletal proteins, of which α-SMA is the most prevalent muscle-specific contractile protein. Tissue-culture polystyrene surfaces were first explored to establish a baseline scenario for VIC behavior on a synthetic extracellular environment that is non-specifically
Materials
Recombinant human transforming growth factor-β1 was purchased from PeproTech Inc., Rocky Hill, NJ. Pan-specific TFG-β neutralizing antibody was used at a concentration of 50 μg/ml, and was purchased from R and D Systems, Minneapolis, MN. Mouse monoclonal Anti-β-Tubulin I (clone SAP.4G5), SIGMAScreen™ Collagen I Plates, and heparin (isolated from porcine intestinal mucosa) were purchased from Sigma, Saint Louis, MO. Human Fibronectin BIOCOAT® Cellware was purchased from Becton Dickinson Labware,
Acknowledgments
Funding for this project was provided by the Howard Hughes Medical Institute and a grant from the American Heart Association (0355488Z), as well as fellowships to MCC from the Department of Education Graduate Assistance in Areas of National Need (GAANN) and NIH Leadership Training in Pharmaceutical Biotechnology, and support to JTL from the Undergraduate Research Opportunities Program at the University of Colorado.
References (41)
- et al.
Cell composition of the human pulmonary valve: a comparative study with the aortic valve—the VESALIO* project
Ann. Thor. Surg.
(2000) - et al.
Improved quantitation and discrimination of sulfated glycosaminoglycans by use of dimethylmethylene blue
Biochim. Biophys. Acta
(1986) - et al.
Progression of aortic valve stenosis: TGF-beta1 is present in calcified aortic valve cusps and promotes aortic valve interstitial cell calcification via apoptosis
Ann. Thorac. Surg.
(2003) - et al.
Porcine cardiac valvular subendothelial cells in culture: cell isolation and growth characteristics
J. Mol. Cell. Cardiol.
(1987) - et al.
Transforming growth factor-beta1 stimulates collagen matrix remodeling through increased adhesive and contractive potential by human renal fibroblasts
Biochim. Biophys. Acta
(2004) - et al.
The interaction of the transforming growth factor-betas with heparin/heparan sulfate is isoform-specific
J. Biol. Chem.
(1997) - et al.
Assay-based quantitative analysis of PC12 cell differentiation
J. Neurosci. Methods
(2002) - et al.
Altered response of vascular smooth muscle cells to exogenous biochemical stimulation in two-and three-dimensional culture
Exp. Cell Res.
(2003) - et al.
Low-molecular-weight heparin prevents high glucose- and phorbol ester-induced TGF-beta 1 gene activation
Kidney Int.
(2001) - et al.
Type-I collagen promotes modulation of cultured rabbit arterial smooth-muscle cells from a contractile to a synthetic phenotype
Exp. Cell Res.
(1993)
Image processing with ImageJ
Biophoton. Int.
Heparin treatment reduces glomerular injury in rats with adriamycin-induced nephropathy but does not modify tubulointerstitial damage or the renal production of transforming growth factor-beta
Nephron
Role of transforming growth factor {beta} in human disease
N. Engl. J. Med.
Extracellular matrix and integrin signalling: the shape of things to come
Biochem. J.
Heparin–protein interactions
Angew. Chem. Int. Ed. Engl.
Heparin stimulates production of bFGF and TGF-beta 1 by human normal, keloid, and fetal dermal fibroblasts
Med. Sci. Monit.
Binding and internalization of heparin by vascular smooth muscle cells
J. Cell. Physiol.
Heparin induces alpha-smooth muscle actin expression in cultured fibroblasts and in granulation tissue myofibroblasts
Lan. Invest. Dec.
Transforming growth-factor beta-modulates the expression of collagenase and metalloproteinase inhibitor
EMBO J.
Cited by (106)
Generating robust human valvular interstitial cell cultures: Protocol and considerations
2022, Journal of Molecular and Cellular CardiologyCitation Excerpt :They also respond in a myofibroblastic pattern to TGF-β stimulation—specifically, they form calcific nodules, show upregulation of ACTA2 and ECM-related genes COL1A1, COL3A1, and ELN. This finding is consistent with the literature [3,68,69]. We also showed that FGF-2 stimulation promotes a quiescent phenotype.
Valve Strain Quantitation in Normal Mitral Valves and Mitral Prolapse With Variable Degrees of Regurgitation
2021, JACC: Cardiovascular ImagingCitation Excerpt :This is plausible with what is known about MVP pathology. In mitral valves with myxomatous degeneration, transforming growth factor-β mediates endothelial-mesenchymal transdifferentiation of endothelial cells into myofibroblasts, promoting increased matrix production along with collagen and elastin degradation within the valvular fibrosa (28–30). The end result is a valve tissue that is thickened, weakened, and more prone to excessive deformation (31), a characteristic captured by leaflet thickness measurements and strain calculations.
Scaffold-free high throughput generation of quiescent valvular microtissues
2017, Journal of Molecular and Cellular CardiologyCitation Excerpt :This study, as well as others, have shown that the cultivation of VIC onto 2D rigid polystyrene substrates, leads to an activated VIC phenotype which is shown by the expression of α-SMA in primary cultures [18,19]. To date, there is still some discrepancy whether or not α-SMA levels increase [18], decrease [28] or remain relatively unchanged when passage number increases [29]. Recently, Xu et al. showed that confluent VIC monolayers express lower α-SMA levels compared to sub-confluent cultures [30].
7.25 Cardiac patch with cells: Biological or synthetic
2017, Comprehensive Biomaterials IIInterleukin 18 promotes myofibroblast activation of valvular interstitial cells
2016, International Journal of Cardiology