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.

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.

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.

BackgroundValvular interstitial cells (VICs) in the healthy aortic valve leaflet exhibit a quiescent phenotype, with <5% of VICs exhibiting an activated phenotype. Yet, in vitro culture of VICs on tissue culture polystyrene surfaces in standard growth medium results in rapid transformation to an activated phenotype in >90% of cells. The inability to preserve a healthy VIC phenotype during in vitro studies has hampered the elucidation of mechanisms involved in calcific aortic valve disease. This study describes the generation of quiescent populations of porcine VICs in 2‐dimensional in vitro culture and their utility in studying valve pathobiology.Methods and ResultsWithin 4 days of isolation from fresh porcine hearts, VICs cultured in standard growth conditions were predominantly myofibroblastic (activated VICs). This myofibroblastic phenotype was partially reversed within 4 days, and fully reversed within 9 days, following application of a combination of a fibroblast media formulation with culture on collagen coatings. Specifically, culture in this combination significantly reduced several markers of VIC activation, including proliferation, apoptosis, α‐smooth muscle actin expression, and matrix production, relative to standard growth conditions. Moreover, VICs raised in a fibroblast media formulation with culture on collagen coatings exhibited dramatically increased sensitivity to treatment with transforming growth factor β1, a known pathological stimulus, compared with VICs raised in either standard culture or medium with a fibroblast media formulation.ConclusionsThe approach using a fibroblast media formulation with culture on collagen coatings generates quiescent VICs that more accurately mimic a healthy VIC population and thus has the potential to transform the study of the mechanisms of VIC activation and dysfunction involved in the early stages of calcific aortic valve disease.

In situ vascular tissue engineering aims to regenerate vessels “at the target site” using synthetic scaffolds that are capable of inducing endogenous regeneration. Critical to the success of this approach is a fine balance between functional neo-tissue formation and scaffold degradation. Circulating immune cells are important regulators of this process as they drive the host response to the scaffold and they play a central role in scaffold resorption. Despite the progress made with synthetic scaffolds, little is known about the host response and neo-tissue development during and after scaffold resorption. In this study, we designed a fast-degrading biodegradable supramolecular scaffold for arterial applications and evaluated this development in vivo. Bisurea-modified polycaprolactone (PCL2000-U4U) was electrospun in tubular scaffolds and shielded by non-degradable expanded polytetrafluoroethylene in order to restrict transmural and transanastomotic cell ingrowth. In addition, this shield prevented graft failure, permitting the study of neo-tissue and host response development after degradation. Scaffolds were implanted in 60 healthy male Lewis rats as an interposition graft into the abdominal aorta and explanted at different time points up to 56 days after implantation to monitor sequential cell infiltration, differentiation, and tissue formation in the scaffold. Endogenous tissue formation started with an acute immune response, followed by a dominant presence of pro-inflammatory macrophages during the first 28 days. Next, a shift towards tissue-producing cells was observed, with a striking increase in α-Smooth Muscle Actin-positive cells and extracellular matrix by day 56. At that time, the scaffold was resorbed and immune markers were low. These results suggest that neo-tissue formation was still in progress, while the host response became quiescent, favoring a regenerative tissue outcome. Future studies should confirm long-term tissue homeostasis, but require the strengthening of the supramolecular scaffold if a non-shielded model will be used.

Isolated calcified particles from human aortae are not innocent bystanders but induce a phenotypical and pathological change of VECs and VICs characteristic of activated and pathological cells. Therapy tailored to reduce these calcified particles should be investigated.

Background: Mediastinal ionizing radiotherapy is associated with an increased risk of valvular disease, which demonstrates pathological hallmarks similar to calcific aortic valve disease (CAVD). Despite advances in radiotherapy techniques, the prevalence of comorbidities such as radiation-associated valvular disease is still increasing due to improved survival of patients receiving radiotherapy. However, the mechanisms of radiation-associated valvular disease are largely unknown. CAVD is considered to be an actively regulated disease process, mainly controlled by valvular interstitial cells (VICs). We hypothesize that radiation exposure catalyzes the calcific response of VICs and, therefore, contributes to the development of radiation-associated valvular disease.Methods and Results: To delineate the relationship between radiation and VIC behavior (morphology, calcification, and matrix turnover), two different in vitro models were established: (1) VICs were cultured two-dimensional (2D) on coverslips in control medium (CM) or osteogenic medium (OM) and irradiated with 0, 2, 4, 8, or 16 Gray (Gy); and (2) three-dimensional (3D) hydrogel system was designed, loaded with VICs and exposed to 0, 4, or 16 Gy of radiation. In both models, a dose-dependent decrease in cell viability and proliferation was observed in CM and OM. Radiation exposure caused myofibroblast-like morphological changes and differentiation of VICs, as characterized by decreased αSMA expression. Calcification, as defined by increased alkaline phosphatase activity, was mostly present in the 2D irradiated VICs exposed to 4 Gy, while after exposure to higher doses VICs acquired a unique giant fibroblast-like cell morphology. Finally, matrix turnover was significantly affected by radiation exposure in the 3D irradiated VICs, as shown by decreased collagen staining and increased MMP-2 and MMP-9 activity.Conclusions: The presented work demonstrates that radiation exposure enhances the calcific response in VICs, a hallmark of CAVD. In addition, high radiation exposure induces differentiation of VICs into a terminally differentiated giant-cell fibroblast. Further studies are essential to elucidate the underlying mechanisms of these radiation-induced valvular changes.