In conditions of prolonged stress valvular cells can contribute to valve disease progression. In this Review, Wang and colleagues describe how the biophysical and biochemical properties of the extracellular matrix... can regulate valve cell function in the context of calcific valvular diseases. The authors also describe how new cell culture approaches can be employed to better understand the pathophysiology of valve diseases.
Abstract
During every heartbeat, cardiac valves open and close coordinately to control the unidirectional flow of blood. In this dynamically challenging environment, resident valve cells actively maintain homeostasis, but the signalling between cells and their microenvironment is complex. When homeostasis is disrupted and the valve opening obstructed, haemodynamic profiles can be altered and lead to impaired cardiac function. Currently, late stages of cardiac valve diseases are treated surgically, because no drug therapies exist to reverse or halt disease progression. Consequently, investigators have sought to understand the molecular and cellular mechanisms of valvular diseases using in vitro cell culture systems and biomaterial scaffolds that can mimic the extracellular microenvironment. In this Review, we describe how signals in the extracellular matrix regulate valve cell function. We propose that the cellular context is a critical factor when studying the molecular basis of valvular diseases in vitro, and one should consider how the surrounding matrix might influence cell signalling and functional outcomes in the valve. Investigators need to build a systems-level understanding of the complex signalling network involved in valve regulation, to facilitate drug target identification and promote in situ or ex vivo heart valve regeneration.
Key points
- The interaction between valve cells and their microenvironment signals determine the functional output of cardiac valve tissues; cardiac valve diseases might be caused by a disruption of these interactions
- In vitro cell culture studies can help understand cardiac valve disease pathobiology, which is enhanced by culturing cells on biomaterials that mimic the native valve cell matrix environment
- Many biochemical signals regulate pathogenic phenotypes of cells in the valve; however, their effects are dependent on the matrix microenvironment
- Synthetic biomaterials can be used to understand valve cell regulation by biochemical, biophysical, and other matrix-associated signals, especially related to the development of valvular diseases
- Understanding the dynamic interplay between microenvironmental signals and valvular cell phenotypes will enable the identification of new therapies and the design of ex vivo tissue engineered valves
As the heart evolved from a single-chamber to a multiple-chamber structure, cardiac valves arose to control the unidirectional flow of blood during cardiac cycles. For example, aortic valves open in response to higher blood pressure in the left ventricle than in the aorta, and close when the pressure equilibrates. These valves function in a similar manner to valves in water dams or car engines, but cardiac valves are living tissue with the ability to repair and remodel in response to damage. During an average human life span, heart valves open and close approximately 3 billion times,1 withstanding various mechanical stresses, including fluid shear stresses and bending stretch.2, 3
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| Figure 1: Valve cells and their matrix regulate tissue homeostasis and disease progression.
a | Cardiac valves have three extracellular matrix layers: the ventricularis, spongiosa, and fibrosa. In this image, a cross-section of a porcine aortic valve was visualized by second-harmonic generation confocal imaging. Elastin in green, collagen in red. Scale bar: 100 μm. b | Two main cell populations comprise the cardiac valve. VECs, which are CD31+ (green), line the boundaries of the leaflets. VICs reside throughout the valve leaflet. Nuclear staining (DAPI; blue), Scale bar: 100 μm. c | In healthy valves (left), very few VICs and VECs are activated to repair local tissue damage during normal valve function. Collagen fibres are circumferentially aligned, and activated VICs undergo continual turn over into deactivated fibroblasts and other cell fates (such as apoptosis). In diseased valves (right), VICs and VECs are recruited to remodel the tissue, leading to excessive collagen accumulation, a degraded and disarrayed matrix, which in some cases becomes calcified. Valve cells lose the homeostatic equilibrium and remain in an activated state. Abbreviations: VEC, valvular endothelial cell; VIC, valvular interstitial cell.
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The material composition and structure of cardiac valves confer their robustness and durability. In humans, cardiac valves are made of thin (~500 μm) pliable cusps, and only mitral and tricuspid valves have supporting chordae tendineae and papillary muscles.4 A close examination of the tissue architecture of an aortic valve reveals three distinct layers of extracellular matrix (ECM), rich in collagens, proteoglycans, or elastin (Figure 1a).4 These ECM proteins impart unique macroscopic mechanical properties to valves, enabling them to withstand tension when closed and flexure when open. For example, the elastin fibres on the flow side of the valves (known as ventricularis) are radially aligned and elastic, which extend when the valves open and recoil when valves close.5 Proteoglycans in the middle layer, or spongiosa, function as a cushion for absorbing tension and friction between the top and the bottom layers.4 Finally, the fibrosa layer contains circumferentially oriented collagen fibres, which confer stiffness and strength to the valves.4
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| Figure 2: Dynamic cellular phenotypes in cardiac valve diseases.
a | Mineralized spherical particles (red) have been observed in human calcified valves, but not in normal valves. Scale bars 2 μm. b | Cells in the valve adopt diseased phenotypes (myofibroblast and osteoblast-like cells) in response to environmental signals. Activated myofibroblasts can revert back to quiescence, senescence, or undergo apoptosis. Osteoblast-like cells might also undergo apoptosis. (1) Myofibroblasts are characterized by ACTA2-positive stress fibres (green). Scale bar 50 μm. (2) VICs can be induced to mineralize, indicated by Alizarin Red S staining. Scale bar: 50 μm. Abbreviations: ACTA2, actin, aortic smooth muscle; VEC, valvular endothelial cell; VIC, valvular interstitial cell. Permission for panel a obtained from NPG © et al. Nano-analytical electron microscopy reveals fundamental insights into human cardiovascular tissue calcification.
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| Figure 3: Matrix signals regulate valve cell phenotypes.
a | Plastic culture plates have high stiffness and biochemical signals must be introduced as soluble factors in the media. The stiffness of biomaterial-based cultures can mimic native tissues, and enables cells to grow in 3D space, with biochemical signals presented throughout the matrix. b | Adhesive signals (yellow in schematic) are tethered to an inert matrix to study specific cellular phenotype effects. For example, an elastin-derived peptide (VGVAPG, green bars) or a collagen-derived peptide (P15, blue bars) covalently linked to PEG hydrogels has distinct effects on ACTA2 levels. c | Elasticity can elicit intracellular cytoskeletal changes. In a modulus gradient of 7–32 kPa, >80% of PAVICs activated to myofibroblasts near the stiffer region and <10 1="" 3="" abbreviations:="" acta2="" actin="" after="" aortic="" became="" blue="" culture="" day="" days="" differences="" end="" i="" line="" muscle="" myofibroblasts="" near="" not="" observed="" of="" on="" red="" smooth="" softer="" the="" these="" were="">E
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, Young's modulus; PAVIC, porcine aortic valve interstitial cell; PEG, poly(ethylene glycol). Permission for panel b obtained from et al. Small peptide functionalized thiol-ene hydrogels as culture substrates for understanding valvular interstitial cell activation and de novo tissue deposition. Acta Biomaterialia, 8, 3201–3209 (2012). Permission for panel c obtained from et al. In situ elasticity modulation with dynamic substrates to direct cell phenotype.
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| Figure 4: Valve cells and the ECM microenvironment are dynamic and mutually regulated.
Valvular interstitial cells interact with the ECM directly through integrins, with biochemical signals via receptors, and with neighbouring cells via cadherins. In healthy tissue (left), the cells and ECM are in a homeostatic state. When microenvironmental signals of valvular disease are present (right), cells can be activated, and biological outputs are altered. Secretion of ECM proteins, matrix-remodelling enzymes, and biochemical signals is increased; more contractile stress fibres and cell-matrix adhesions form; apoptosis increases where effective matrix interactions are lost. These cellular functions alter the microenvironment and valve tissue structure in diseased valves. Abbreviations: ACTA2, actin, aortic smooth muscle; ECM, extracellular matrix; MMP, matrix metalloproteinase.
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