Environmental stiffness restores mechanical homeostasis in vimentin-depleted cells.

Recent experimental evidence indicates a role for the intermediate filament vimentin in regulating cellular mechanical homeostasis, but its precise contribution remains to be discovered. Mechanical homeostasis requires a balanced bi-directional interplay between the cell's microenvironment and the cellular morphological and mechanical state-this balance being regulated via processes of mechanotransduction and mechanoresponse, commonly referred to as mechanoreciprocity. Here, we systematically analyze vimentin-expressing and vimentin-depleted cells in a swatch of in vitro cellular microenvironments varying in stiffness and/or ECM density. We find that vimentin-expressing cells maintain mechanical homeostasis by adapting cellular morphology and mechanics to micromechanical changes in the microenvironment. However, vimentin-depleted cells lose this mechanoresponse ability on short timescales, only to reacquire it on longer time scales. Indeed, we find that the morphology and mechanics of vimentin-depleted cell in stiffened microenvironmental conditions can get restored to the homeostatic levels of vimentin-expressing cells. Additionally, we observed vimentin-depleted cells increasing collagen matrix synthesis and its crosslinking, a phenomenon which is known to increase matrix stiffness, and which we now hypothesize to be a cellular compensation mechanism for the loss of vimentin. Taken together, our findings provide further insight in the regulating role of intermediate filament vimentin in mediating mechanoreciprocity and mechanical homeostasis.

Radiation Induces Valvular Interstitial Cell Calcific Response in an in vitro Model of Calcific Aortic Valve Disease.

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.

Vimentin regulates Notch signaling strength and arterial remodeling in response to hemodynamic stress.

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.

Host Response and Neo-Tissue Development during Resorption of a Fast Degrading Supramolecular Electrospun Arterial Scaffold.

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.

A biomimetic microfluidic model to study signalling between endothelial and vascular smooth muscle cells under hemodynamic conditions.

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.

Robust Generation of Quiescent Porcine Valvular Interstitial Cell Cultures.

BACKGROUND: Valvular 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 RESULTS: Within 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 ß1, a known pathological stimulus, compared with VICs raised in either standard culture or medium with a fibroblast media formulation. CONCLUSIONS: The 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.

Aortic calcified particles modulate valvular endothelial and interstitial cells.

BACKGROUND: Normal and calcified human valve cusps, coronary arteries, and aortae harbor spherical calcium phosphate microparticles of identical composition and crystallinity, and their role remains unknown. OBJECTIVE: The objective was to examine the direct effects of isolated calcified particles on human valvular cells. METHOD AND RESULTS: Calcified particles were isolated from healthy and diseased aortae, characterized, quantitated, and applied to valvular endothelial cells (VECs) and interstitial cells (VICs). Cell differentiation, viability, and proliferation were analyzed. Particles were heterogeneous, differing in size and shape, and were crystallized as calcium phosphate. Diseased donors had significantly more calcified particles compared to healthy donors (P<.05), but there were no differences between the composition of the particles from healthy and diseased donors. VECs treated with calcified particles showed a significant decrease in CD31 and VE-cadherin and an increase in von Willebrand factor expression, P<.05. There were significantly increased α-SMA and osteopontin in treated VICs (P<.05), significantly decreased VEC and VIC viability (P<.05), and significantly increased number of terminal deoxynucleotidyl transferase dUTP nick end labeling-positive VECs (P<.05) indicating apoptosis when treated with the calcified particles. CONCLUSIONS: 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.