Shear stress magnitude and transforming growth factor-beta 1 regulate endothelial to mesenchymal transformation in a three-dimensional culture microfluidic device† Sara G. Mina, a Wei Wang,‡ b Qingfeng Cao, b Peter Huang, b Bruce T. Murray b and Gretchen J. Mahler * a Normal fibroblasts are present within the extracellular matrix (ECM). They can become activated, leading to increased proliferation and ECM protein secretion such as collagen type I to promote tissue remodeling. These cells are also involved in adult pathologies including cancer metastasis and cardiac and renal fibrosis. One source of activated fibroblasts is endothelial to mesenchymal transformation (EndMT), in which endothelial cells lose their cell–cell and cell–ECM adhesions, gain invasive properties, and become mesenchymal cells. While EndMT is well characterized in developmental biology, the mechanisms and functional role of EndMT in adult physiology and pathology have not been fully investigated. A microfluidic device with an incorporated three-dimensional ECM chamber was developed to study the role of combined steady fluid shear stress magnitudes and transforming growth factor-beta 1 (TGF-b1) on EndMT. Low (1 dyne per cm 2 ) steady shear stress and TGF-b1 exposure induced EndMT in endothelial cells, including upregulation of mesenchymal protein and gene expression markers. Cells exposed to TGF-b1 and high (20 dynes per cm 2 ) steady shear stress did not undergo EndMT, and protein and gene expression of mesenchymal markers was significantly downregulated. Mesenchymally transformed cells under static conditions with and without TGF-b1 showed significantly more collagen production when compared to fluidic conditions. These results confirm that both low shear stress and TGF-b1 induce EndMT in endothelial cells, but this process can be prevented by exposure to physiologically relevant high shear stress. These results also show conditions most likely to cause tissue pathology. Introduction Endothelial to mesenchymal transformation (EndMT) is a physi- ological cellular response involved in embryonic heart valve development. 1 However, recent studies have shown that mesen- chymal transformation can also occur in tissue homeostasis, such as wound healing, and in adult pathologies, including cancer, atherosclerosis, cardiac and renal brosis, and calcic aortic valve disease. 1,2 EndMT begins when vascular or valvular endothelial cells delaminate from their cell monolayer, and lose cell–cell contacts and endothelial markers such as vascular endothelial cadherin (VE-cadherin). These cells gain mesen- chymal or broblast-like markers such as alpha-smooth muscle actin (a-SMA), and acquire mesenchymal cell-like properties. 3–5 Cell transformation from endothelial to mesenchymal phenotype is followed by cell invasion into and remodeling of the extracel- lular matrix (ECM). This transformation has been found to be responsible for generating cancer activated broblasts (CAFs) in transgenic tumor grown mice. 3 These CAFs remodel the ECM and secrete biochemical signaling factors that affect the behavior of cells within the tumor microenvironment leading to metastasis. 6 Two stimuli that have been shown to induce EndMT are changes in hemodynamic forces (such as uid shear stress) 7 and increased exposure of biochemical signals (such as transforming growth factor-b; TGF-b). 3,7 Previous studies have shown the effects of TGF-b 3,7,18,19 and the magnitude of shear ow 7 on EndMT; however, the interplay of these stimuli on EndMT and the role of mesenchymally transformed cells in disease progres- sion or tissue regeneration is still not well dened. Understanding the role of parameters in a physiological environment is more cost effective in microuidic devices than in animals. Microuidic devices allow for control of various shear stress magnitudes and direct biochemical factors exposure. Microuidic devices integrate perfused recirculating cell culture a Department of Biomedical Engineering, Binghamton University, PO Box 6000, Binghamton, NY 13902, USA. E-mail: gmahler@binghamton.edu b Department of Mechanical Engineering, Binghamton University, PO Box 6000, Binghamton, NY 13902, USA † Electronic supplementary information (ESI) available. See DOI: 10.1039/c6ra16607e ‡ Currently at the Department of Mechanical Engineering, Colorado State University, Fort Collins, CO 80523, USA. Cite this: RSC Adv. , 2016, 6, 85457 Received 27th June 2016 Accepted 30th August 2016 DOI: 10.1039/c6ra16607e www.rsc.org/advances This journal is © The Royal Society of Chemistry 2016 RSC Adv., 2016, 6, 85457–85467 | 85457 RSC Advances PAPER Published on 30 August 2016. Downloaded by State University of New York at Binghamton on 12/12/2018 6:20:49 PM. View Article Online View Journal | View Issue