Capillary morphogenesis is a complex cellular process that occurs in response to external stimuli. A number of assays have been used to study critical regulators of the process, but those assays are typically limited by the inability to control biochemical gradients and to obtain images on the single cell level. We have recently developed a new microfluidic platform that has the capability to control the biochemical and biomechanical forces within a three dimensional scaffold coupled with accessible image acquisition. Here, the developed platform is used to evaluate and quantify capillary growth and endothelial cell migration from an intact cell monolayer. We also evaluate the endothelial cell response when placed in co-culture with physiologically relevant cell types, including cancer cells and smooth muscle cells. This resulted in the following observations: cancer cells can either attract (MTLn3 cancer cell line) endothelial cells and induce capillary formation or have minimal effect (U87MG cancer cell line) while smooth muscle cells (10T 1/2) suppress endothelial activity. Results presented demonstrate the capabilities of this platform to study cellular morphogenesis both qualitatively and quantitatively while having the advantage of enhanced imaging and internal biological controls. Finally, the platform has numerous applications in the study of angiogenesis, or migration of other cell types including tumor cells, into a three-dimensional scaffold or across an endothelial layer under precisely controlled conditions of mechanical, biochemical and co-culture environments.

Biomechanical forces are emerging as critical regulators of embryogenesis, particularly in the developing cardiovascular system 1,2 . After initiation of the heartbeat in vertebrates, cells lining the ventral aspect of the dorsal aorta, the placental vessels, and the umbilical and vitelline arteries initiate expression of the transcription factor Runx1 (refs 3-5), a master regulator of haematopoiesis, and give rise to haematopoietic cells 4 . It remains unknown whether the biomechanical forces imposed on the vascular wall at this developmental stage act as a determinant of haematopoietic potential 6 . Here, using mouse embryonic stem cells differentiated in vitro, we show that fluid shear stress increases the expression of Runx1 in CD41 + c-Kit + haematopoietic progenitor cells 7 ,concomitantly augmenting their haematopoietic colony-forming potential. Moreover, we find that shear stress increases haematopoietic colony-forming potential and expression of haematopoietic markers in the paraaortic splanchnopleura/aorta-gonads-mesonephros of mouse embryos and that abrogation of nitric oxide, a mediator of shear-stress-induced signalling 8 , compromises haematopoietic potential in vitro and in vivo. Collectively, these data reveal a critical role for biomechanical forces in haematopoietic development.In the mouse, the first haemogenic areas appear in the yolk sac starting at day 7.5 of development (E7.5) 9 . After the establishment of circulation and the onset of vascular flow at day 8.5, additional haemogenic sites appear between day 9 and 10.5 as Runx1 + regions within

Functional endothelialized networks constitute a critical building block for vascularized replacement tissues, organ assist devices, and laboratory tools for in vitro discovery and evaluation of new therapeutic compounds. Progress towards realization of these functional artificial vasculatures has been gated by limitations associated with the mechanical and surface chemical properties of commonly used microfluidic substrate materials and by the geometry of the microchannels produced using conventional fabrication techniques. Here we report on a method for constructing microvascular networks from polystyrene substrates commonly used for tissue culture, built with circular cross-sections and smooth transitions at bifurcations. Silicon master molds are constructed using an electroplating process that results in semi-circular channel cross-sections with smoothly varying radii. These master molds are used to emboss polystyrene sheets which are then joined to form closed bifurcated channel networks with circular cross-sections. The mechanical and surface chemical properties of these polystyrene microvascular network structures enable culture of endothelial cells along the inner lumen. Endothelial cell viability was assessed, documenting nearly confluent monolayers within 3D microfabricated channel networks with rounded cross-sections.

As part of the cross industry efforts to get aircraft flying again during the April/May 2010 eruption of Eyjafjallajokull RollsRoyce produced a chart that plotted examples of aircraft engine exposure to volcanic ash against the ash concentration the engines had been exposed to. This chart became known as the Rolls-Royce 'Safe-to-Fly' chart, and it was used to guide decisions on how the UK Met Office ash concentration charts for the Eyjafjallajokull eruption could be utilised to help aviators plan their flight paths. Over the period 2011-2013, this paper's authors reassessed the engine data that made up the 'Safe-to-Fly' chart, and in particular the data relating to two key exposure events at high ash concentrations, flight BA009 on 24 June 1982 and flight KLM867 on 15 December 1989. Through a combination of reassessment of the original engineering calculations carried out for these events (i.e. calculations based on evidence from engine hardware) and assessments of relevant volcanological and ash cloud visibility data, it has been concluded that these events are unlikely to have occurred at or near the 2000 mg/m 3 ash concentration arrived at in 2010; based on current evidence, it is more plausible that these events occurred in ash concentrations of around 200 mg/m 3 . As a consequence, a revision to the 'Safe-to-Fly' chart is recommended. In addition, to more easily present the main considerations associated with flight within ash concentrations where the ash would start to become visible, a new chart is proposed that plots duration of exposure against ash concentration. The points plotted on this chart are the revised understanding of the BA009 and KLM867 events, other relevant engine exposure events and speculative regions where flight would be unsafe and where flight would be safe, but engines would be susceptible to long-term damage.

The minimum angles of taper that can in theory be formed to various preparations were established for representative dental preparations and operating distances. These values were compared with the taper of dental preparations formed both under laboratory conditions and in clinical practice, the latter by measurement of taper on the dies of clinically successful crowns and inlays. A possible explanation for the discrepancy noted between recommended degrees of taper and the tapers produced under clinical conditions was considered to be due to the requirement by a dental surgeon to avoid forming undercuts to the line of withdrawal of a cast intracoronal or extracoronal retainer.

. Force-induced focal adhesion translocation: effects of force amplitude and frequency. Am J Physiol Cell Physiol 287: C954 -C962, 2004. First published June 9, 2004 10.1152/ajpcell. 00567.2003.-Vascular endothelial cells rapidly transduce local mechanical forces into biological signals through numerous processes including the activation of focal adhesion sites. To examine the mechanosensing capabilities of these adhesion sites, focal adhesion translocation was monitored over the course of 5 min with GFPpaxillin while applying nN-level magnetic trap shear forces to the cell apex via integrin-linked magnetic beads. A nongraded steady-load threshold for mechanotransduction was established between 0.90 and 1.45 nN. Activation was greatest near the point of forcing (Ͻ7.5 m), indicating that shear forces imposed on the apical cell membrane transmit nonuniformly to the basal cell surface and that focal adhesion sites may function as individual mechanosensors responding to local levels of force. Results from a continuum, viscoelastic finite element model of magnetocytometry that represented experimental focal adhesion attachments provided support for a nonuniform force transmission to basal surface focal adhesion sites. To further understand the role of force transmission on focal adhesion activation and dynamics, sinusoidally varying forces were applied at 0.1, 1.0, 10, and 50 Hz with a 1.45 nN offset and a 2.25 nN maximum. At 10 and 50 Hz, focal adhesion activation did not vary with spatial location, as observed for steady loading, whereas the response was minimized at 1.0 Hz. Furthermore, applying the tyrosine kinase inhibitors genistein and PP2, a specific Src family kinase inhibitor, showed tyrosine kinase signaling has a role in force-induced translocation. These results highlight the mutual importance of force transmission and biochemical signaling in focal adhesion mechanotransduction. mechanotransduction; endothelial cell; paxillin; viscoelastic model MECHANOTRANSDUCTION IS AN essential function of the cell, controlling its growth, proliferation, protein synthesis, and gene expression (8,18). Extensive data exist documenting the cellular responses to external force (15, 41, 44), but less is known about how force affects rapid biological signaling. Although integrins/focal adhesion sites (42), cytoskeleton constituents, G proteins (6), ion channels, intercellular junction proteins, and membrane biomolecules have all been identified as potential mechanosensors (6,16,42,43), we know little about the force level and frequency-dependent thresholds required to initiate mechanotransduction or the role of intracellular force transmission on mechanosensor activation. Biological readouts used to study mechanotransduction range from long-term gene expression and cell morphology changes (8, 26) to rapid variations in intracellular ion concentration and protein activity (8,26,42). Morphological and gene expression comparisons provide a robust marker of mechanotransduction, but the response is slow on the scale of hours, a...

Alterations in hemodynamic shear stress acting on the vascular endothelium are critical for adaptive arterial remodeling. The molecular mechanisms regulating this process, however, remain largely uncharacterized. Here, we sought to define the responses evoked in endothelial cells exposed to shear stress waveforms characteristic of coronary collateral vessels and the subsequent paracrine effects on smooth muscle cells. A lumped parameter model of the human coronary collateral circulation was used to simulate normal and adaptive remodeling coronary collateral shear stress waveforms. These waveforms were then applied to cultured human endothelial cells (EC), and the resulting differences in EC gene expression were assessed by genome-wide transcriptional profiling to identify genes distinctly regulated by collateral flow. Analysis of these transcriptional programs identified several genes to be differentially regulated by collateral flow, including genes important for endothelium-smooth muscle interactions. In particular, the transcription factor KLF2 was up-regulated by the adaptive remodeling coronary collateral waveform, and several of its downstream targets displayed the expected modulation, including the down-regulation of connective tissue growth factor. To assess the effect of endothelial KLF2 expression on smooth muscle cell migration, a three-dimensional microfluidic assay was developed. Using this three-dimensional system, we showed that KLF2-expressing EC co-cultured with SMC significantly reduce SMC migration compared with control EC and that this reduction can be rescued by the addition of exogenous connective tissue growth factor. Collectively, these results demonstrate that collateral flow evokes distinct EC gene expression profiles and functional phenotypes that subsequently influence vascular events important for adaptive remodeling.Coronary collateralization or arteriogenesis is a vascular adaptive remodeling process that occurs in the context of arterial occlusion when preexisting collateral arterioles remodel to form larger diameter bypass arteries. The current arteriogenesis paradigm implicates local hemodynamics, recruitment/activation of monocytes, and structural remodeling of the vascular wall as key steps in collateralization (1). Two primary stages of collateralization have been described based predominantly on observations from femoral artery ligation models that induce collateral remodeling via an instantaneous increase in collateral flow. The initial stage of collateralization is characterized by a wave of monocyte-driven inflammation and rapid vasodilation, an immediate compensatory response to increased collateral flow. In the second phase of collateralization, the initial inflammatory response is resolved, and the newly formed conductance artery wall is stabilized (2). To date, considerable attention has been dedicated to investigating the inflammatory regulation of this process (i.e. MCP-1 and GM-CSF), but despite evidence on the importance of the endothelium and endothelium-derived ...

The regulation of valvular endothelial phenotypes by the hemodynamic environments of the human aortic valve is poorly understood. The nodular lesions of calcific aortic stenosis (CAS) develop predominantly beneath the aortic surface of the valve leaflets in the valvular fibrosa layer. However, the mechanisms of this regional localization remain poorly characterized. In this study, we combine numerical simulation with in vitro experimentation to investigate the hypothesis that the previously documented differences between valve endothelial phenotypes are linked to distinct hemodynamic environments characteristic of these individual anatomical locations. A finite-element model of the aortic valve was created, describing the dynamic motion of the valve cusps and blood in the valve throughout the cardiac cycle. A fluid mesh with high resolution on the fluid boundary was used to allow accurate computation of the wall shear stresses. This model was used to compute two distinct shear stress waveforms, one for the ventricular surface and one for the aortic surface. These waveforms were then applied experimentally to cultured human endothelial cells and the expression of several pathophysiological relevant genes was assessed. Compared to endothelial cells subjected to shear stress waveforms representative of the aortic face, the endothelial cells subjected to the ventricular waveform showed significantly increased expression of the “atheroprotective” transcription factor Kruppel-like factor 2 (KLF2) and the matricellular protein Nephroblastoma overexpressed (NOV), and suppressed expression of chemokine Monocyte-chemotactic protein-1 (MCP-1). Our observations suggest that the difference in shear stress waveforms between the two sides of the aortic valve leaflet may contribute to the documented differential side-specific gene expression, and may be relevant for the development and progression of CAS and the potential role of endothelial mechanotransduction in this disease.