Cell signalling and mechanics influence vascular pathophysiology and there is an increasing demand for in vitro model systems that enable examination of signalling between vascular cells under hemodynamic conditions. Current 3D vessel wall constructs do not recapitulate the mechanical conditions of the native tissue nor do they allow examination of cell–cell interactions under relevant hemodynamic conditions. Here, we describe a 3D microfluidic chip model of arterial endothelial and smooth muscle cells where cellular organization, composition and interactions, as well as the mechanical environment of the arterial wall are mimicked. The hemodynamic EC–VSMC-signalling-on-a-chip consists of two parallel polydimethylsiloxane (PDMS) cell culture channels, separated by a flexible, porous PDMS membrane, mimicking the porosity of the internal elastic lamina. The hemodynamic EC–VSMC-signalling-on-a-chip allows co-culturing of human aortic endothelial cells (ECs) and human aortic vascular smooth muscle cells (VSMCs), separated by a porous membrane, which enables EC–VSMC interaction and signalling, crucial for the development and homeostasis of the vessel wall. The device allows real time cell imaging and control of hemodynamic conditions. The culture channels are surrounded on either side by vacuum channels to induce cyclic strain by applying cyclic suction, resulting in mechanical stretching and relaxation of the membrane in the cell culture channels. The blood flow is mimicked by creating a flow of medium at the EC side. Vascular cells remain viable during prolonged culturing, exhibit physiological morphology and organization and make cell–cell contact. During dynamic culturing of the device with a shear stress of 1–1.5 Pa and strain of 5–8%, VSMCs align perpendicular to the given strain in the direction of the flow and EC adopt a cobblestone morphology. To our knowledge, this is the first report on the development of a microfluidic device, which enables a co-culture of interacting ECs and VSMCs under hemodynamic conditions and presents a novel approach to systematically study the biological and mechanical components of the intimal-medial vascular unit.

We present a novel and highly reproducible process to fabricate transferable porous PDMS membranes for PDMS-based Organs-on-Chips (OOCs) using microelectromechanical systems (MEMS) fabrication technologies. Porous PDMS membranes with pore sizes down to 2.0 μm in diameter and a wide porosity range (2–65%) can be fabricated. To overcome issues normally faced when using replica moulding and extend the applicability to most OOCs and improve their scalability and reproducibility, the process includes a sacrificial layer to easily transfer the membranes from a silicon carrier to any PDMS-based OOC. The highly reliable fabrication and transfer method does not need of manual handling to define the pore features (size, distribution), allowing very thin (<10 μm) functional membranes to be transferred at chip level with a high success rate (85%). The viability of cell culturing on the porous membranes was assessed by culturing two different cell types on transferred membranes in two different OOCs. Human umbilical endothelial cells (HUVEC) and MDA-MB-231 (MDA) cells were successfully cultured confirming the viability of cell culturing and the biocompatibility of the membranes. The results demonstrate the potential of controlling the porous membrane features to study cell mechanisms such as transmigrations, monolayer formation, and barrier function. The high control over the membrane characteristics might consequently allow to intentionally trigger or prevent certain cellular responses or mechanisms when studying human physiology and pathology using OOCs.

The intermediate filament (IF) cytoskeleton has been proposed to regulate morphogenic processes by integrating the cell fate signaling machinery with mechanical cues. Signaling between endothelial cells (ECs) and vascular smooth muscle cells (VSMCs) through the Notch pathway regulates arterial remodeling in response to changes in blood flow. Here we show that the IF-protein vimentin regulates Notch signaling strength and arterial remodeling in response to hemodynamic forces. Vimentin is important for Notch transactivation by ECs and vimentin knockout mice (VimKO) display disrupted VSMC differentiation and adverse remodeling in aortic explants and in vivo. Shear stress increases Jagged1 levels and Notch activation in a vimentin-dependent manner. Shear stress induces phosphorylation of vimentin at serine 38 and phosphorylated vimentin interacts with Jagged1 and increases Notch activation potential. Reduced Jagged1-Notch transactivation strength disrupts lateral signal induction through the arterial wall leading to adverse remodeling. Taken together we demonstrate that vimentin forms a central part of a mechanochemical transduction pathway that regulates multilayer communication and structural homeostasis of the arterial wall.

Tissue development and homeostasis are controlled by mechanical cues. Perturbation of the mechanical equilibrium triggers restoration of mechanostasis through changes in cell behavior, while defects in these restorative mechanisms lead to mechanopathologies, for example, osteoporosis, myopathies, fibrosis or cardiovascular disease. Therefore, sensing mechanical cues and integrating them with the biomolecular cell fate machinery is essential for the maintenance of health. The Notch signaling pathway regulates cell and tissue fate in nearly all tissues. Notch activation is directly and indirectly mechanosensitive, and regulation of Notch signaling, and consequently cell fate, is integral to the cellular response to mechanical cues. Fully understanding the dynamic relationship between molecular signaling, tissue mechanics and tissue remodeling is challenging. To address this challenge, engineered microtissues and computational models play an increasingly large role. In this Review, we propose that Notch takes on the role of a ‘mechanostat’, maintaining the mechanical equilibrium of tissues. We discuss the reciprocal role of Notch in the regulation of tissue mechanics, with an emphasis on cardiovascular tissues, and the potential of computational and engineering approaches to unravel the complex dynamic relationship between mechanics and signaling in the maintenance of cell and tissue mechanostasis.

Significance: Notch signaling and hemodynamics are widely known to regulate arterial morphogenesis, remodeling, and homeostasis. Recent studies suggest that Notch signaling and mechanics interact in vascular remodeling, but the impact on vascular homeostasis is still unclear. Here, using a computational–experimental approach, we show that expression of Notch ligands, receptors, and target genes are down-regulated with mechanical strain. Incorporation of these results in a computational model of the arterial wall reveals that this mechanosensitivity leads to a sudden transition from synthetic toward contractile smooth muscle cells at a certain wall thickness, which varies per arterial location and closely agrees with reported anatomical data. This result provides an explanation for how mechanical forces can regulate arterial morphogenesis and homeostasis through Notch signaling.