Cyclic strain induces dual-mode endothelial-mesenchymal transformation of the cardiac valve

Abstract

Endothelial-mesenchymal transformation (EMT) is a critical event for the embryonic morphogenesis of cardiac valves. Inducers of EMT during valvulogenesis include VEGF, TGF-β1, and wnt/β-catenin (where wnt refers to the wingless-type mammary tumor virus integration site family of proteins), that are regulated in a spatiotemporal manner. EMT has also been observed in diseased, strain-overloaded valve leaflets, suggesting a regulatory role for mechanical strain. Although the preponderance of studies have focused on the role of soluble mitogens, we asked if the valve tissue microenvironment contributed to EMT. To recapitulate these microenvironments in a controlled, in vitro environment, we engineered 2D valve endothelium from sheep valve endothelial cells, using microcontact printing to mimic the regions of isotropy and anisotropy of the leaflet, and applied cyclic mechanical strain in an attempt to induce EMT. We measured EMT in response to both low (10%) and high strain (20%), where low-strain EMT occurred via increased TGF-β1 signaling and high strain via increased wnt/β-catenin signaling, suggesting dual strain-dependent routes to distinguish EMT in healthy versus diseased valve tissue. The effect was also directionally dependent, where cyclic strain applied orthogonal to axis of the engineered valve endothelium alignment resulted in severe disruption of cell microarchitecture and greater EMT. Once transformed, these tissues exhibited increased contractility in the presence of endothelin-1 and larger basal mechanical tone in a unique assay developed to measure the contractile tone of the engineered valve tissues. This finding is important, because it implies that the functional properties of the valve are sensitive to EMT. Our results suggest that cyclic mechanical strain regulates EMT in a strain magnitude and directionally dependent manner.tight junctions | cytokines | activated myofibroblast C ardiac valves are sophisticated structures that function in a complex mechanical environment, opening and closing more than 3 billion times during the average human lifetime (1). Initially considered passive flaps of tissue, it is now acknowledged that valves contain a highly heterogeneous population of endothelial (VEC) and interstitial (VIC) cells. The VICs exist as synthetic, myofibroblast, or smooth muscle-like phenotypes (2, 3) and alter their tone in response to vasoactive mediators (4-7). The VECs line the surface of the valve leaflet and are unique in their ability to undergo endothelial-mesenchymal transformation (EMT), a process that is crucial for valvulogenesis (8, 9). Recent clinical evidence of EMT has been observed in pathologies such as ischemic cardiomyopathy and concomitant mitral regurgitation and is correlated with increased leaflet mechanical strains (10, 11). These pathological strains can be oriented obliquely to cell and tissue orientation (12, 13), suggesting the possible interaction between mechanical forces and tissue architecture in regulating EMT.Prior work has focused on the regulation of ...

Recapitulating maladaptive, multiscale remodeling of failing myocardium on a chip

“…We fabricated stretchable muscular thin film (MTF) substrates by selectively coating silicone membranes with a temperature-sensitive polymer and a thin layer of polydimethylsiloxane (PDMS; Fig. 1A) (23)(24)(25), building on previous efforts of Zhuang et al (26) and Yamada et al (27). Substrates micropatterned with fibronectin (FN) in a "brick wall" pattern ( Fig.…”

“…1A) (28). Substrates were uniaxially and cyclically stretched at 10% strain and a frequency of 3 Hz by using a custom-built multiwell system (Movie S1) (23,26). After conditioning the tissues, arrays of MTFs were cut from the PDMS layer so that, after the temperature-sensitive layer released, MTFs freely deflected away from the silicone membrane with each paced contraction while remaining fixed at one edge ( Fig.…”

“…Cardiac myocytes were isolated by enzymatically digesting ventricles from 2-d-old Sprague-Dawley rats by using previously published protocols (24,48,57). A custom bioreactor based on a previous design (26) was built to apply cyclic strain to tissues cultured on silicone membranes using a linear motor (LinMot) to stretch as many as 10 samples simultaneously in an incubator (23).…”

“…Membranes treated in a UV-ozone cleaner (Jelight) were micropatterned with PDMS stamps coated with 50 μg/mL FN (BD Biosciences) or coated uniformly with 50 μg/mL FN. To fabricate stretchable MTFs (23,24), silicone membranes were temporarily cured to glass platforms with 200 proof ethanol to maintain baseline tension and provide rigid surfaces for spin-coating. Two pieces of tape were applied across the length or width of the membrane separated by 8 mm.…”

“…Elastic silicone membranes (Specialty Manufacturing) were clamped into stainless steel brackets (23,26) and affixed with 25-mmdiameter rings of silicon tubing. Membranes treated in a UV-ozone cleaner (Jelight) were micropatterned with PDMS stamps coated with 50 μg/mL FN (BD Biosciences) or coated uniformly with 50 μg/mL FN.…”