Endothelial to mesenchymal transformation is induced by altered extracellular matrix in aortic valve endothelial cells

Abstract

Alterations in shear stress, mechanical deformation, extracellular matrix (ECM) composition and exposure to inflammatory conditions are known to cause endothelial to mesenchymal transformation (EndMT). This change in endothelial phenotype has only recently been linked to adult pathologies such as cancer progression, organ fibrosis, and calcific aortic valve disease; and its function in adult physiology, especially in response to tissue mechanics, has not been rigorously investigated. EndMT is a response to mechanical and biochemical signals that results in the remodeling of underlying tissues. In diseased aortic valves, glycosaminoglycans (GAGs) are present in the collagen-rich valve fibrosa, and are deposited near calcified nodules. In this study, in vitro models of early and late-stage valve disease were developed by incorporating the GAGs chondroitin sulfate (CS), hyaluronic acid, and dermatan sulfate into 3D collagen hydrogels with or without exposure to TGF-β1 to simulate EndMT in response to microenvironmental changes. High levels of CS induced the highest rate of EndMT and led to the most collagen I and GAG production by mesenchymally transformed cells, which indicates a cell phenotype most likely to promote fibrotic disease. Mesenchymal transformation due to altered ECM was found to depend on cell-ECM bond strength and extracellular signal-regulated protein kinases 1/2 signaling. Determining the environmental conditions that induce and promote EndMT, and the subsequent behavior of mesenchymally transformed cells, will advance understanding on the role of endothelial cells in tissue regeneration or disease progression. © 2017 Wiley Periodicals Inc. J Biomed Mater Res Part A: 105A: 2729-2741, 2017.

The role of shear stress and altered tissue properties on endothelial to mesenchymal transformation and tumor-endothelial cell interaction

“…We examined the characteristics of HUVEC by analyzing the protein expression of the pro-EndMT marker a-SMA from the confocal images. Collagen þ GAG samples were compared to their corresponding static collagen-only stiffness controls, as described by Dahal et al, 21 to make it possible to determine if stiffness or GAG content induces EndMT. For the lowest gel concentration, there was a significant difference in GAG containing gels (1.5 mg/ml collagen þ 1 mg/ml HA gels under static and 1.5 mg/ml collagen þ 1 mg/ml CS gels under shear conditions) when compared to 1.5 mg/ml collagen-only stiffness control gels.…”

“…The stiffness of the collagen-only ECM was matched to the collagen þ GAG gels as described previously. 21 Supplementary material Fig. 3 illustrates the types of ECM tissue properties that were studied within the microfluidic device chamber.…”

“…Dahal et al measured the collagen gel mechanical stiffness to provide a comparison between native and pathological tissue stiffness. 21 From the hydrogel stiffness tests, they determined the elastic modulus (Young's modulus) of collagen-only hydrogels and collagen þ GAG hydrogels. A rheometer (AR 1000, TA Instruments, New Castle, DE) was utilized to measure various collagen-only concentrations (1.5, 2.0, and 2.2 mg/ml collagen) and 1.5 mg/ml collagen þGAGs (1, 10, and 20 mg/ml HA or CS) elastic modulus.…”

“…For the lowest concentration (1.5 mg/ml collagen-only control), elastic modulus was determined to be 2.45 6 0.45 kPa and for the highest concentration of 2.2 mg/ml collagen-only control to be 37.28 6 5.12 kPa. 21 Depending on location, composition, and type of tissue, in vivo conditions exhibit a gradient of stiffnesses (e.g., elastic modulus of brain tissue ranges from 0.1 to 1 kPa and muscles ranges from 8 to 17 kPa). 22,23 The stiffness (elastic modulus) of blood vessels ranges from 0.2 to 4.0 kPa.…”

Cell Phenotype Transitions in Cardiovascular Calcification

“…As mentioned above, the layers of the valve are rich with collagens, elastin, proteoglycans, and glycosaminoglycans, and it is well-established that these ECM proteins can initiate and propagate signaling events. For example, the glycosaminoglycans chondroitin sulfate and hyaluronic acid can drive EndMT in healthy adult VECs in a 3D in vitro culture system ( 74 ). The constant movement of the valves exposes the leaflets to both mechanical and shear forces.…”