Flow-Dependent Mass Transfer May Trigger Endothelial Signaling Cascades

It is well known that fluid mechanical forces directly impact endothelial signaling pathways. But while this general observation is clear, less apparent are the underlying mechanisms that initiate these critical signaling processes. This is because fluid mechanical forces can offer a direct mechanical input to possible mechanotransducers as well as alter critical mass transport characteristics (i.e., concentration gradients) of a host of chemical stimuli present in the blood stream. However, it has recently been accepted that mechanotransduction (direct mechanical force input), and not mass transfer, is the fundamental mechanism for many hemodynamic force-modulated endothelial signaling pathways and their downstream gene products. This conclusion has been largely based, indirectly, on accepted criteria that correlate signaling behavior and shear rate and shear stress, relative to changes in viscosity. However, in this work, we investigate the negative control for these criteria. Here we computationally and experimentally subject mass-transfer limited systems, independent of mechanotransduction, to the purported criteria. The results showed that the negative control (mass-transfer limited system) produced the same trends that have been used to identify mechanotransduction-dominant systems. Thus, the widely used viscosity-related shear stress and shear rate criteria are insufficient in determining mechanotransduction-dominant systems. Thus, research should continue to consider the importance of mass transfer in triggering signaling cascades

Analytical and numerical analyses of the micromechanics of soft fibrous connective tissues

State of the art research and treatment of biological tissues require accurate and efficient methods for describing their mechanical properties. Indeed, micromechanics motivated approaches provide a systematic method for elevating relevant data from the microscopic level to the macroscopic one. In this work the mechanical responses of hyperelastic tissues with one and two families of collagen fibers are analyzed by application of a new variational estimate accounting for their histology and the behaviors of their constituents. The resulting, close form expressions, are used to determine the overall response of the wall of a healthy human coronary artery. To demonstrate the accuracy of the proposed method these predictions are compared with corresponding 3-D finite element simulations of a periodic unit cell of the tissue with two families of fibers. Throughout, the analytical predictions for the highly nonlinear and anisotropic tissue are in agreement with the numerical simulations

In vitro calibration of a system for measurement of in vivo convective heat transfer coefficient in animals

BACKGROUND: We need a sensor to measure the convective heat transfer coefficient during ablation of the heart or liver. METHODS: We built a minimally invasive instrument to measure the in vivo convective heat transfer coefficient, h in animals, using a Wheatstone-bridge circuit, similar to a hot-wire anemometer circuit. One arm is connected to a steerable catheter sensor whose tip is a 1.9 mm × 3.2 mm thin film resistive temperature detector (RTD) sensor. We used a circulation system to simulate different flow rates at 39°C for in vitro experiments using distilled water, tap water and saline. We heated the sensor approximately 5°C above the fluid temperature. We measured the power consumed by the sensor and the resistance of the sensor during the experiments and analyzed these data to determine the value of the convective heat transfer coefficient at various flow rates. RESULTS: From 0 to 5 L/min, experimental values of h in W/(m(2)·K) were for distilled water 5100 to 13000, for tap water 5500 to 12300, and for saline 5400 to 13600. Theoretical values were 1900 to 10700. CONCLUSION: We believe this system is the smallest, most accurate method of minimally invasive measurement of in vivo h in animals and provides the least disturbance of flow

Proteomic Profiling of Mesenchymal Stem Cell Responses to Mechanical Strain and TGF-β1

Mesenchymal stem cells (MSCs) are a potential source of smooth muscle cells (SMCs) for constructing tissue-engineered vascular grafts. However, the details of how specific combinations of vascular microenvironmental factors regulate MSCs are not well understood. Previous studies have suggested that both mechanical stimulation with uniaxial cyclic strain and chemical stimulation with transforming growth factor-β1 (TGF-β1) can induce smooth muscle markers in MSCs. In this study, we investigated the combined effects of uniaxial cyclic strain and TGF-β1 stimulation on MSCs. By using a proteomic analysis, we found differential regulation of several proteins and genes, such as the up-regulation of TGF-β1-induced protein ig-h3 (BGH3) protein levels by TGF-β1 and up-regulation of calponin 3 protein level by cyclic strain. At the gene expression level, BGH3 was induced by TGF-β1, but calponin 3 was not significantly regulated by mechanical strain or TGF-β1, which was in contrast to the synergistic up-regulation of calponin 1 gene expression by cyclic strain and TGF-β1. Further experiments with cycloheximide treatment suggested that the up-regulation of calponin 3 by cyclic strain was at post-transcriptional level. The results in this study suggest that both mechanical stimulation and TGF-β1 signaling play unique and important roles in the regulation of MSCs at both transcriptional and post-transcriptional levels, and that a precise combination of microenvironmental cues may promote MSC differentiation

Microvascular engineering in perfusion culture: immunohistochemistry and CLSM findings

BACKGROUND: One of the most challenging problems in tissue engineering is the establishment of vascular supply. A possible approach might be the engineering of microvasculature in vitro and the supply by engineered feeder vessels. METHODS: An in vitro model for a small-diameter vessel was developed and made from adipose tissue stromal cells and human umbilical vein endothelial cells in a tube-like gelatine scaffold. The number of "branches" emerging from the central lumen and the morphology of the central lumen of the vessel equivalent were assessed after 16 days of either pulsatile perfusion culture or culture in rotating containers by evaluation of immunohistochemically stained sections (n = 6 pairs of cultures). Intramural capillary network formation was demonstrated in five experiments with confocal laser scanning microscopy. RESULTS: Perfused specimens showed a round or oval lumen lined by a single layer of endothelial cells, whereas following rotation culture the lumen tended to collapse. Confocal laser scanning microscopy showed more extended network formation in perfused specimens as compared to specimens after rotation culture. Partially highly interconected capillary-like networks were imaged which showed orientation around the central lumen. Perfused specimens exhibited significantly more branches emerging from the central lumen. There were, however, hardly any capillary branches crossing the whole vessel wall. CONCLUSION: Pulsatile perfusion supports the development of vascular networks with physiological appearance. Advances in reactor development, acquisition of functional data and imaging procedures are however necessary in order to attain the ultimate goal of a fully functional engineered supplying vessel

Modulation of ATP/ADP Concentration at the Endothelial Cell Surface by Flow: Effect of Cell Topography

Determining how flow affects the concentration of the adenine nucleotides ATP and ADP at the vascular endothelial cell (EC) surface is essential for understanding flow-induced mobilization of intracellular calcium. Previously, mathematical models were formulated to describe the ATP/ADP concentration at the EC surface; however, all previous models assumed the endothelium to be flat. In the present study we investigate the effect of surface undulations on ATP/ADP concentration at the EC surface. The results demonstrate that under certain geometric and flow conditions, the ATP + ADP concentration at the EC surface is considerably lower for a wavy cell surface than for a flat surface. Because ECs in regions of disturbed arterial flow are expected to have larger undulations than cells in non-disturbed flow zones, our findings suggest that ECs in regions of flow disturbance would exhibit lower ATP + ADP concentrations at their surfaces, which may lead to impaired calcium signaling. If validated experimentally, the present results may contribute to our understanding of endothelial cell dysfunction observed in regions of disturbed flow

Stress analysis in a layered aortic arch model under pulsatile blood flow

BACKGROUND: Many cardiovascular diseases, such as aortic dissection, frequently occur on the aortic arch and fluid-structure interactions play an important role in the cardiovascular system. Mechanical stress is crucial in the functioning of the cardiovascular system; therefore, stress analysis is a useful tool for understanding vascular pathophysiology. The present study is concerned with the stress distribution in a layered aortic arch model with interaction between pulsatile flow and the wall of the blood vessel. METHODS: A three-dimensional (3D) layered aortic arch model was constructed based on the aortic wall structure and arch shape. The complex mechanical interaction between pulsatile blood flow and wall dynamics in the aortic arch model was simulated by means of computational loose coupling fluid-structure interaction analyses. RESULTS: The results showed the variations of mechanical stress along the outer wall of the arch during the cardiac cycle. Variations of circumferential stress are very similar to variations of pressure. Composite stress in the aortic wall plane is high at the ascending portion of the arch and along the top of the arch, and is higher in the media than in the intima and adventitia across the wall thickness. CONCLUSION: Our analysis indicates that circumferential stress in the aortic wall is directly associated with blood pressure, supporting the clinical importance of blood pressure control. High stress in the aortic wall could be a risk factor in aortic dissections. Our numerical layered aortic model may prove useful for biomechanical analyses and for studying the pathogeneses of aortic dissection

Stretch-Induced Stress Fiber Remodeling and the Activations of JNK and ERK Depend on Mechanical Strain Rate, but Not FAK

BACKGROUND: Cells within tissues are subjected to mechanical forces caused by extracellular matrix deformation. Cells sense and dynamically respond to stretching of the matrix by reorienting their actin stress fibers and by activating intracellular signaling proteins, including focal adhesion kinase (FAK) and the mitogen-activated proteins kinases (MAPKs). Theoretical analyses predict that stress fibers can relax perturbations in tension depending on the rate of matrix strain. Thus, we hypothesized stress fiber organization and MAPK activities are altered to an extent dependent on stretch frequency. PRINCIPAL FINDINGS: Bovine aortic endothelial cells and human osteosarcoma cells expressing GFP-actin were cultured on elastic membranes and subjected to various patterns of stretch. Cyclic stretching resulted in strain rate-dependent increases in stress fiber alignment, cell retraction, and the phosphorylation of the MAPKs JNK, ERK and p38. Transient step changes in strain rate caused proportional transient changes in the levels of JNK and ERK phosphorylations without affecting stress fiber organization. Disrupting stress fiber contractile function with cytochalasin D or Y27632 decreased the levels of JNK and ERK phosphorylation. Previous studies indicate that FAK is required for stretch-induced cell alignment and MAPK activations. However, cyclic uniaxial stretching induced stress fiber alignment and the phosphorylation of JNK, ERK and p38 to comparable levels in FAK-null and FAK-expressing mouse embryonic fibroblasts. CONCLUSIONS: These results indicate that cyclic stretch-induced stress fiber alignment, cell retraction, and MAPK activations occur as a consequence of perturbations in fiber strain. These findings thus shed new light into the roles of stress fiber relaxation and reorganization in maintenance of tensional homeostasis in a dynamic mechanical environment

Endothelial cells enhance the in vivo bone-forming ability of osteogenic cell sheets

Addressing the problem of vascularization is of vital importance when engineering three-dimensional (3D) tissues. Endothelial cells are increasingly used in tissue-engineered constructs to obtain prevascularization and to enhance in vivo neovascularization. Rat bone marrow stromal cells were cultured in thermoresponsive dishes under osteogenic conditions with human umbilical vein endothelial cells (HUVECs) to obtain homotypic or heterotypic cell sheets (CSs). Cells were retrieved as sheets from the dishes after incubation at 20 °C. Monoculture osteogenic CSs were stacked on top of homotypic or heterotypic CSs, and subcutaneously implanted in the dorsal flap of nude mice for 7 days. The implants showed mineralized tissue formation under both conditions. Transplanted osteogenic cells were found at the new tissue site, demonstrating CS bone-inductive effect. Perfused vessels, positive for human CD31, confirmed the contribution of HUVECs for the neovascularization of coculture CS constructs. Furthermore, calcium quantification and expression of osteocalcin and osterix genes were higher for the CS constructs, with HUVECs demonstrating the more robust osteogenic potential of these constructs. This work demonstrates the potential of using endothelial cells, combined with osteogenic CSs, to increase the in vivo vascularization of CS-based 3D constructs for bone tissue engineering purposes.We would like to acknowledge Mariana T Cerqueira for the illustration in Figure 1. This study was supported by Formation of Innovation Center for Fusion of Advanced Technologies in the Special Coordination Funds for Promoting Science and Technology 'Cell Sheet Tissue Engineering Center (CSTEC)' and the Global CUE program, the Multidisciplinary Education and Research Center for Regenerative Medicine (MERCREM), from the Ministry of Education, Culture, Sports, Science and Technology (MEXT), Japan. Financial support to RP Pirraco by the Portuguese Foundation for Science and Technology (FCT) through the PhD Grant SFRH/BD/44893/2008 is also acknowledged