Effects of shear stress pattern and magnitude on mesenchymal transformation and invasion of aortic valve endothelial cells - PubMed (original) (raw)

. 2014 Nov;111(11):2326-37.

doi: 10.1002/bit.25291. Epub 2014 Aug 5.

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Effects of shear stress pattern and magnitude on mesenchymal transformation and invasion of aortic valve endothelial cells

Gretchen J Mahler et al. Biotechnol Bioeng. 2014 Nov.

Abstract

Understanding the role of mechanical forces on cell behavior is critical for tissue engineering, regenerative medicine, and disease initiation studies. Current hemodynamic bioreactors are largely limited to 2D substrates or the application of general flow conditions at a tissue level, which eliminates the investigation of some essential physiological and pathological responses. One example is the mesenchymal transformation of endothelial cells in response to shear stress. Endothelial to mesenchymal transformation (EndMT) is a valve morphogenic mechanism associated with aortic valve disease initiation. The aortic valve experiences oscillatory shear on the disease-susceptible fibrosa, and the role of hemodynamics on adult EndMT is unknown. The goal of this work was to develop and characterize a microfluidic bioreactor that applies physiologically relevant laminar or oscillatory shear stresses to endothelial cells and permits the quantitative analysis of 3D cell-extracellular matrix (ECM) interactions. In this study, porcine aortic valve endothelial cells were seeded onto 3D collagen I gels and exposed to different magnitudes of steady or oscillatory shear stress for 48 h. Cells elongated and aligned perpendicular to laminar, but not oscillatory shear. Low steady shear stress (2 dyne/cm(2) ) and oscillatory shear stress upregulated EndMT (ACTA2, Snail, TGFB1) and inflammation (ICAM1, NFKB1) related gene expression, EndMT-related (αSMA) protein expression, and matrix invasion when compared with static controls or cells exposed to high steady shear (10 and 20 dyne/cm(2) ). Our system enables direct testing of the role of shear stress on endothelial cell mesenchymal transformation in a dynamic, 3D environment and shows that hemodynamics regulate EndMT in adult valve endothelial cells.

Keywords: 3D culture; endothelial to mesenchymal transformation; inflammation; side specific.

© 2014 Wiley Periodicals, Inc.

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Figures

Fig. 1

Fig. 1

(A) Schematic of the shear stress bioreactor. (B) The bioreactor set-up for steady shear experiments, (C) the 3D parallel plate bioreactor, and (D) the bioreactor arranged with a syringe pump for oscillatory shear experiments.

Fig. 1

Fig. 1

(A) Schematic of the shear stress bioreactor. (B) The bioreactor set-up for steady shear experiments, (C) the 3D parallel plate bioreactor, and (D) the bioreactor arranged with a syringe pump for oscillatory shear experiments.

Fig. 2

Fig. 2

Simulated flow streamlines for the 2 (A), 10 (B), and 20 (C) dyne/cm2 chambers under steady shear conditions with a graph of corresponding simulated wall shear stress values at nine different sites. Dotted lines represent approximate gel location.

Fig. 3

Fig. 3

Porcine aortic valve endothelial cell (PAVEC) invasion into the collagen matrix after a 48 hour exposure to 2, 10, or 20 dyne/cm2 steady or oscillatory shear. Error bars show ±SEM, n = 6. Bars that do not share any letters are significantly different according to a one-way ANOVA with Tukey’s post-test (p ≤ 0.05).

Fig. 4

Fig. 4

Porcine aortic valve endothelial cells (PAVEC) following 48 hours of exposure to 2 (A), 10 (B), or 20 (C) dyne/cm2 steady or 2 (D), 10 (E), or 20 (F) dyne/cm2 oscillatory shear. Cells are stained for F-actin (green) and DNA (blue), and arrows indicate the fluid flow direction.

Fig. 5

Fig. 5

Image analysis of cells following 48 hours steady (SS) or oscillatory (OSC) shear exposure. (A) Cell circularity, (B) F-actin alignment for steady shear. Error bars show ±SEM, n = at least 50 cells from 10 representative confocal images. Bars that do not share any letters are significantly different according to a one-way ANOVA with Tukey’s post-test (p ≤ 0.05).

Fig 6

Fig 6

Porcine aortic valve endothelial cells (PAVEC) following 48 hours of exposure to 2 (A), 10 (B), or 20 (C) dyne/cm2 steady or 2 (D), 10 (E), or 20 (F) dyne/cm2 oscillatory shear. Panel G shows porcine aortic valve interstitial cells seeded onto a 3D collagen I gel and grown for 48 hours in static conditions, and panel H is PAVEC seeded onto a 3D collagen I gel and grown for 48 hours in static conditions. Cells are stained for PECAM-1 (red), αSMA (green), and DNA (blue), arrows indicate the fluid flow direction.

Fig. 7

Fig. 7

Protein quantification of cells following 48 hours steady (SS) or oscillatory (OSC) shear exposure. (A) αSMA expression per cell in steady sheared cells, and (B) αSMA per cell expression in oscillatory sheared cells. Error bars show ±SEM, n = at least 50 cells from 10 representative confocal images. Bars that do not share any letters are significantly different according to a one-way ANOVA with Tukey’s post-test (p ≤ 0.05).

Fig. 8

Fig. 8

Porcine aortic valve endothelial cells (PAVEC) EndMT-related (A, C) and inflammatory (B, D) gene expression following 48 hours of exposure to steady (A, B; SS) or oscillatory (C, D; OSC) shear stress. Error bars show ±SEM, n = 6. Bars that do not share any letters are significantly different according to a one-way ANOVA with Tukey’s post-test (p ≤ 0.05).

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