Cyclic strain induces dual-mode endothelial-mesenchymal transformation of the cardiac valve - PubMed (original) (raw)

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

Kartik Balachandran et al. Proc Natl Acad Sci U S A. 2011.

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.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.

In vitro experimental model. Isotropic (A and B) and anisotropic (C and D) valve endothelial lamellae were engineered via micropatterning of fibronectin and seeding VECs, as shown by phase contrast microscopy. (Scale bar: 100 µm.) Tissue lamellae were engineered on an elastomeric membrane and clamped using aluminum brackets. The silicone membrane can be assembled such that strain can be imposed parallel (physiological) or orthogonal (pathological) to tissue alignment (E). Tissue was cyclically stretched at 1 Hz (F). Plot of one cycle of Lagrange strain that was representative of low (10%) or high (20%) cyclic strain magnitude (G). Schematic of all experimental conditions are shown in

Fig S1

Fig. 2.

Cyclic strain resulted in robust tissue alignment and increased actin OOP and nuclear eccentricity. Representative micrographs of actin and DAPI immunostained valve tissue (A). (Scale bar: 20 µm.) Actin orientation order parameter (B), and nuclear eccentricity (C) were quantified for each of the tissue treatments based on the immunostained tissue samples. All graphs, mean ± SEM; n = 8; *p < 0.05.

Fig. 3.

Cyclic strain resulted in EMT of valve endothelial tissue. Representative micrographs (A) VEC tissue stained for DAPI and α-SMA with α-SMA expression indicative of EMT (blue, nuclei; white, α-SMA). (Scale bar: 50 µm.) Western blotting quantitatively demonstrate EMT via increased α-SMA expression (B) and reduced CD31 expression (C) in samples that were cyclically strained. Quantitative flow cytometry (D) also demonstrated EMT via reduced VE-cadherin-positive cells and increased α-SMA-positive cells. All bar graphs, mean ± SEM; n = 6; *p < 0.05.

Fig. 4.

Valve thin film experiments demonstrating altered tissue function due to cyclic strain-induced EMT. Schematic representation of experimental model (A). Anisotropic VEC issue was engineered on an elastic membrane and stretched to induce EMT. Tissue was released from membrane and attached to polytetrafluoroethylene posts to quantify contractile stress response, based on alteration in radius of curvature of tissue samples. Representative thresholded vTF images of 20% strain samples showing initial configuration, contraction after ET-1, KCl, and final relaxed configuration after HA-1077 (B). (Scale bar: 1 mm.) Temporal change in tissue stress demonstrated that induced contraction due to ET-1 and KCl, and tissue relaxation following HA-1077 was dependent on strain magnitude (C). Induced contractile stress due to ET-1 relative to initial tone in tissues stretched to 0%, 10%, and 20% strain demonstrated strain magnitude-dependent responses (D). Total tissue basal tone relative to initial tone in samples stretched to 0%, 10%, and 20% strain demonstrated strain magnitude-dependent responses (E). All graphs, mean ± SEM; n = 8; *p < 0.05.

Fig. 5.

Dual-mode EMT depending on cyclic strain magnitude. RT-PCR revealed significantly lower VEGF expression in groups that underwent EMT (A). TGF-β1 mRNA (B) and protein (C) expression was significantly higher at low strain (10%) compared to unstretched and high-strain (20%) tissues (n = 4). Culture with TGF-β1 antagonist SB-431542 inhibited EMT at 10% strain but not at 20% strain (D). Representative micrographs demonstrating normal β-catenin expression (E) in 0% and 10% tissue but increased β-catenin staining within cellular cytoplasm (yellow arrows) in tissue stretched orthogonally to high- strain (blue, nuclei; white: β-catenin). (Scale bar: 20 µm.) Western blotting (F) demonstrates significantly increased β-catenin expression for tissue stretched to 20% strain compared to 0% or 10% strain. The β-catenin expression was also significantly increased in tissue stretched orthogonal to axis of alignment (n = 6). There was significantly increased wnt (G) expression at 20% strain and when strain was applied orthogonally to axis of tissue alignment (n = 4). All graphs, mean ± SEM; *p < 0.05.

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Cyclic strain induces dual-mode endothelial-mesenchymal transformation of the cardiac valve - PubMed (original) (raw)