Microdevice array-based identification of distinct mechanobiological response profiles in layer-specific valve interstitial cells

Abstract

Aortic valve homeostasis is mediated by valvular interstitial cells (VICs) found in spatially distinct and mechanically dynamic layers of the valve leaflet. Disease progression is associated with the pathological differentiation of VICs to myofibroblasts, but the mechanobiological response profiles of cells specific to different layers in the leaflet remains undefined. Conventional mechanically dynamic macroscale culture technologies require a large number of cells per set of environmental conditions. However, large scale expansion of primary VICs in vitro does not maintain in vivo phenotypes, and hence conventional macroscale techniques are not well-suited to systematically probe response of these cell types to combinatorially manipulated mechanobiological cues. To address this issue, we developed a microfabricated composite material screening array to determine the combined effects of dynamic substrate stretch, soluble cues and matrix proteins on small populations of primary cells. We applied this system to study VICs isolated from distinct layers of the valve leaflet and determined that (1) mechanical stability and cellular adhesion to the engineered composite materials were significantly improved as compared to conventional stretching technologies; (2) VICs demonstrate layer-specific mechanobiological profiles; and (3) mechanical stimulation, matrix proteins and soluble cues produce integrated and distinct responses in layer-specific VIC populations. Strikingly, myofibroblast differentiation was most significantly influenced by cell origin, despite the presence of potent mechanobiological cues such as applied strain and TGF-β1. These results demonstrate that spatially-distinct VIC subpopulations respond differentially to microenvironmental cues, with implications for valve tissue engineering and pathobiology. The developed platform enables rapid identification of biological phenomena arising from systematically manipulating the cellular microenvironment, and may be of utility in screening mechanosensitive cell cultures with applications in drug screening, tissue engineering and fundamental cell biology.

Hemodynamic and Cellular Response Feedback in Calcific Aortic Valve Disease

“…44,45,106,108 These findings have potentially provocative implications linking pathological stretch with the deterioration in aortic valve tissue properties and function that occurs with disease. Indeed, Thayer et al 45 found that subjecting aortic valve leaflets to a combination of pathological stretch and pressure loads ex vivo caused a significant increase in the thickness of the fibrosa and spongiosa layers ( Figure 4C).…”

“…However, when both tension and TGF-β1 were applied to the valve leaflets simultaneously, αSMA expression increased synergistically. Moraes et al 108 also observed interaction between stretching and TGF-β1 treatment in the activation of isolated VICs but found that synergism depended on the layer from which the VICs were isolated (ie, fibrosa versus ventricularis) and the ECM protein on which they were cultured. The link between mechanical stimulation and TGF-β1 signaling was further supported by subsequent research that implicated cell-mediated mechanical activation of latent TGF-β1.…”

“…However, VICs were grown in TCPS under static conditions in these studies; improved insight into the role of ECM proteins in regulating VIC phenotype will require culture substrates with physiologically relevant elasticity and dynamic strain culture conditions. To that end, Moraes et al 108 stretched VICs on collagen-I and fibronectin using a microscale cell-stretching platform and measured myofibroblast differentiation. They found that myofibroblast differentiation by VICs in response to strain was indeed dependent on the ECM protein, with collagen being less myofibrogenic than fibronectin, particularly for VICs isolated from the fibrosa layer of the leaflet.…”

Emerging Trends in Heart Valve Engineering: Part I. Solutions for Future

“…In the context of VIC biology, TGF-b1 and cyclic strain synergistically act to enhance VIC myofibroblast differentiation and collagen synthesis. 66,69,96 VIC response to cyclic strain is ECM protein-dependent, 69 similar to the MSC response to ECM elasticity. 26 To develop a means of delivering customized combinations of microenvironmental cues to the cells within a TEHV, there is growing interest in the use of scaffolds based on synthetic hydrogel materials.…”

“…Both can elicit responses associated not only with valve disease, but also development, repair, and regeneration, including increased matrix synthesis and remodeling, 6-8 and VIC activation to the secretory myofibroblast phenotype. 6,66,69 All in all, there is significant interaction between various types of microenvironmental cues, which profoundly impact cell responses. In the context of VIC biology, TGF-b1 and cyclic strain synergistically act to enhance VIC myofibroblast differentiation and collagen synthesis.…”