Physiological Shear Stress Research Articles

TCT-663 Comparison of Conformability and Wall Shear Stress Between Resolute Integrity Zotarolimus-Eluting Stent and the XIENCE Xpedition Everolimus-Eluting Stent in Angulated Vessels: An Interim Analysis of the SHEAR-STENT Randomized Controlled Study

Given its continuous wire molding and sinusoidal design, we hypothesized that Resolute Integrity stent (R-ZES) is more conformable than the XIENCE Xpedition stent (X-EES) and therefore may induce more physiologic wall shear stress (WSS) in angulated coronary arteries. Interim analysis of

Shear stress regulated uptake of liposome-decorated microgels coated with a poly(dopamine) shell.

Advanced multicompartment drug delivery platforms ensure the co-localization of several drugs within the same carrier, thus making it possible to achieve a more effective and safe therapeutic outcome. Herein, we report a novel multicompartment architecture by combining two intrinsically different systems, i.e., polymeric microgels and liposomes, with the aim to achieve different release kinetics for model compounds. We assemble poly(N-isopropylacrylamide-co-acrylic acid) microgels decorated with liposomes which are subsequently coated with a protective poly(dopamine) shell and a poly(ethylene glycol) (PEG) layer. Since any intravenous administered drug delivery vehicle will get in contact with the dynamics of the blood flow, we evaluate the stealth properties of this novel multicompartment carrier towards protein adsorption and cellular uptake by three relevant cell lines (macrophages, endothelial and cancer cells) under physiological shear stress conditions. Our results demonstrate less protein adsorption for the PEGylated carriers and differences in the extent of internalized carriers depending on the presence of a PEG coating, the studied cell line and the intensity of the applied shear stress. Additionally, we demonstrate that, for all three tested cell lines, shear stress results in the activation of different cell entry pathways as compared to static conditions. All in all, we report a thorough study about the effect of shear stress on the cell association/uptake with a novel multicompartment carrier.

Abstract 2029: Notch signaling inhibitor increases E-selectin/ligand interactions and alters cell migration of mesenchymal-like breast cancer cells

Abstract Activation of Notch signaling has been shown to promote cell migration properties and hematogenous metastasis by inducing a dramatic phenotypic change in breast cancer cells via the epithelial-to-mesenchymal transition (EMT). In response to these findings, Notch signaling inhibitors are being explored as therapeutics to prevent EMT in cancers. However, research examining the effects of Notch inhibitors on the activity of sialofucosylated ligands used by circulating tumor cells to adhere to vascular endothelial E-selectin during metastasis is lacking. To advance knowledge in this area, our study investigated the use of an indirect Notch signaling inhibitor (DAPT) on modifying E-selectin ligand activity of breast cancer cell lines by shear flow adhesion assays performed at physiological bone marrow shear stress. DAPT exposure to mesenchymal-like MDA-MB-231 and MDA-MB-468 (induced to the mesenchymal-like state by exogenous epidermal growth factor) breast cancer cells increased E-selectin ligand activity. Furthermore, mesenchymal-like MDA-MB-468 cells after DAPT exposure demonstrated E-selectin ligand activity similar to that of epithelial-like MDA-MB-468 cells. This supports the hypothesis by our group that epithelial-like and mesenchymal-like phenotypes determine activity of functional E-selectin ligands in breast cancer cells, as we have previously shown that epithelial cells display high E-selectin ligand activity while mesenchymal cells display little or no ligand activity. Cell migration ability was assessed using wound closure assays. Mesenchymal-like MDA-MB-468 cells exposed to DAPT exhibited decreased migration compared to mesenchymal-like MDA-MB-468 cells exposed to the DMSO diluent control, whereas mesenchymal-like MDA-MB-231 cells demonstrated no difference in migration compared to DMSO exposed cells. Altogether, our results suggest Notch inhibitors may both increase fluid-shear resistant adhesion and alter migration ability of mesenchymal-like breast cancer cells, and that factors present in the blood might impact the metastatic potential of cancer cells via regulating cellular phenotypes. Citation Format: Christian A. Showalter, Monica M. Burdick. Notch signaling inhibitor increases E-selectin/ligand interactions and alters cell migration of mesenchymal-like breast cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2029.

Abstract 187: Breast cancer cells exhibit enhanced proliferation, invasion, PLAU expression and chemoresistance when exposed to fluid shear stress in an innovative 3D bioreactor

Abstract The cells within the mammary tumor microenvironment experience a range of shear stresses from both interstitial fluid flow, as well as, vascular blood flow, and this shear stress is increased as the tumor expands. Many in vitro models recapitulate the 3D microenvironment but fail to include physiological shear stress. To address this issue, a bioreactor capable of applying 3D shear stress to cells encapsulated within a 3D environment was designed, characterized, and utilized to investigate the impact of shear stimulation on breast cancer cell morphology, proliferation, gene expression, and chemoresistance. A shear bioreactor capable of applying variable 3D shear stress to hydrogel constructs was designed and fabricated in house. Shear stress of 4.92 dynes/cm2 was applied for 72 hours to MDA-MB-231, MDA-MB-468, and MCF7 breast cancer cells housed within an interpenetrating agarose-collagen type I hydrogel (IPN). The IPN hydrogel was characterized using SEM imaging and rheometric testing, and the viscoelastic modulus was determined to be 10 kPa, within the range of aggressive tumor microenvironments. Histological stains were evaluated for morphology. Immunohistochemistry staining was used for proliferation and protein evaluation. Changes in gene expression were monitored through qPCR analysis. The chemotherapeutic drug paclitaxel was investigated to observe changes in chemoresistance while under shear stress stimulation. Finally, the PLAU inhibitor amiloride was investigated for impact on cellular response to shear stress stimulus. All cell lines were found to have a significant increase in cellular area and a decrease in circularity when exposed to shear stress. Shear stimulated cells also displayed an increase in proliferation as quantified by Ki67 expression. qRTPCR analysis revealed a greater than 2-fold increase in the expression of PLAU, a common factor in breast cancer progression and metastasis. Preliminary results also show enhanced chemoresistance. Overall, shear stress stimulus was found to enhance the expression of PLAU, contribute to breast cancer proliferation, morphological changes, and chemoresistance. This bioreactor provides an ideal in vitro 3D platform for understanding the influence of shear stress stimulus on a variety of cell types and co-culture investigations. Acknowledgements: This material is based upon work supported by the DOD OCRP Early Career Investigator Award W81XWH-13-1-0134, MIOCA and Rivkin Center for Ovarian Cancer grants to GM. This research was supported by the National Cancer Institute of the National Institutes of Health under award number P30CA046592. CMN is supported by the National Science Foundation Graduate Research Fellowship under Grant No. 1256260. Citation Format: Caymen Novak, Eric Horst, Shreya Raghavan, Geeta Mehta. Breast cancer cells exhibit enhanced proliferation, invasion, PLAU expression and chemoresistance when exposed to fluid shear stress in an innovative 3D bioreactor [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 187.

Collagen-Mimetic Proteins with Tunable Integrin Binding Sites for Vascular Graft Coatings.

Achieving graft endothelialization following implantation continues to be a challenge in the development of "off-the-shelf," small-caliber, arterial prostheses. Coating grafts with biomolecules to support the retention, migration, and differentiation of adherent endothelial precursor cells (EPCs) is a promising approach toward improving graft endothelialization. Designer Collagen Scl2-2 with 1 integrin binding site per strand (DC2-1X) is a Streptococcus pyogenes-derived, collagen-like protein that has previously been evaluated as a graft coating due to its ability to resist platelet aggregation and to promote attachment and migration of "late outgrowth" EPCs (EOCs). However, these prior assessments were performed in the absence of physiological shear. In addition, although DC2-1X coatings supported increased migration rates relative to native collagen coatings, EOC attachment and spreading remained inferior to collagen controls at all DC2-1X concentrations assayed. Thus, the objectives of the present work were the following: (1) to improve EOC attachment on DC2 coatings by modulating the number and spacing of DC2 integrin binding sites (IBS) and (2) to evaluate the retention, migration, and differentiation of adherent EOCs under physiological shear stress. Using single point mutations, three novel DC2 variants were generated containing either two IBS (DC2-2X) or three IBS (DC2-3X1 and DC2-3X2) per strand. After initial evaluation of the potential of each DC2 variant to support increased EOC attachment relative to DC2-1X, DC2-2X and DC2-3X1 coatings were further assessed under physiological shear for their capacity to promote EOC retention, migration, and differentiation relative to DC2-1X and collagen controls. An increase in the number of IBS from 1 to 3 significantly improved EOC retention on DC2 coatings while also supporting increased average migration rates. Moreover, EOCs on DC2-3X1 coatings showed increased gene-level expression of intermediate endothelial cell differentiation markers relative to collagen. Overall, the current results suggest that DC2-3X1 warrants further investigation as a vascular graft coating.

Staphylococcus aureus clumping factor A is a force-sensitive molecular switch that activates bacterial adhesion

Clumping factor A (ClfA), a cell-wall-anchored protein from Staphylococcus aureus, is a virulence factor in various infections and facilitates the colonization of protein-coated biomaterials. ClfA promotes bacterial adhesion to the blood plasma protein fibrinogen (Fg) via molecular forces that have not been studied so far. A unique, yet poorly understood, feature of ClfA is its ability to favor adhesion to Fg at high shear stress. Unraveling the strength and dynamics of the ClfA-Fg interaction would help us better understand how S. aureus colonizes implanted devices and withstands physiological shear stress. By means of single-molecule experiments, we show that ClfA behaves as a force-sensitive molecular switch that potentiates staphylococcal adhesion under mechanical stress. The bond between ClfA and immobilized Fg is weak (∼0.1 nN) at low tensile force, but is dramatically enhanced (∼1.5 nN) by mechanical tension, as observed with catch bonds. Strong bonds, but not weak ones, are inhibited by a peptide mimicking the C-terminal segment of the Fg γ-chain. These results point to a model whereby ClfA interacts with Fg via two distinct binding sites, the adhesive function of which is regulated by mechanical tension. This force-activated mechanism is of biological significance because it explains at the molecular level the ability of ClfA to promote bacterial attachment under high physiological shear stress.

Fabrication of a co-culture micro-bioreactor device for efficient hepatic differentiation of human induced pluripotent stem cells (hiPSCs)

Primary hepatocytes, as the gold standard cell type for in vitro models, lose their characteristic morphology and functions after few days. There is an urgent need to develop physiologically relevant models that recapitulate liver microenvironment to obtain mature hepatocyte from stem cells. We designed and fabricated a micro-bioreactor device mimicking the physiological shear stress and cell–cell interaction in liver sinusoid microenvironment. Induced pluripotent stem cells (iPSCs) were co-cultured with human umbilical vein endothelial cells (HUVECs) in the micro-bioreactor device with continuous perfusion of hepatic differentiation medium (100 μL/h). Simulation results showed that flow field inside our perfusion device was uniform and shear stress was adjusted to physiological condition (<2 dyne/cm2). IPSCs-derived hepatocytes (iPSCs-Heps) that were cultured in micro-bioreactor device showed a higher level of hepatic markers compared to those in static condition. Flow cytometry and immunocytochemistry analysis revealed iPSCs cultured in the device sequentially acquired characteristics of definitive endodermal cells (SOX17 positive), hepatoblasts (AFP positive) and mature hepatocyte (ALB positive). Moreover, the albumin and urea secretion were significantly higher in micro-bioreactor device than those cultured in culture dishes during experiment. Thus, based on our results, we propose our micro-bioreactor as a beneficial device to generate mature hepatocytes for drug screening and basic research.

Role of the mechanosensitive ion channel Piezo1 in Autosomal Dominant Polycystic Kidney Disease (ADPKD)

Disruption of intracellular calcium likely underlies the upregulation of cyclic AMP (cAMP) signaling and the proliferative and secretory phenotype of the cystic epithelium in ADPKD. However, the mechanisms by which polycystins reduce cytoplasmic calcium remain elusive. Impaired flow‐induced calcium entry through ciliary polycystin complex has been previously proposed as a pathogenic mechanism, but newer evidence suggests the involvement of polycystins in modulation of other mechanosensitive ion channels such as Piezo1, the newly‐identified stretch‐activated non‐selective cation channel. We hypothesized that polycystins, through their physical and functional interaction with Piezo1, modulate various cellular responses to mechanical stimulation.Piezo1 was expressed in both wild type mouse inner medullary collecting duct cells (mIMCD3) cells and PKD2 null cells (~3 fold higher than wild type). Wild type mIMCD3 cells were stably transfected with genetically encoded biosensors, jRGeco1a and Flamindo2, to study spatiotemporal dynamics of calcium and cAMP during live cell imaging, respectively. Piezo1 activation by Yoda1 increased cytoplasmic calcium and simultaneously lowered forskolin‐induced cAMP (Figure 1A). Additionally, exerting physiological fluid shear stress (FSS) (ibidi pump system) led to reduction in cAMP (Figure 1B). Both Yoda1 and FSS lead to an increase in ATP release in mIMCD3 cells (Figure 1C). Furthermore, Yoda1 significantly reduced forskolin‐induced in vitro cystogenesis by 68% in WT and by 48% in PKD2 null mIMCD3 cells (Figure 1D).These results suggest that Piezo1 is involved in renal cystogenesis by controlling FSS‐induced calcium entry. Further studies are required to determine in vivo effect of Piezo1 on renal cystogenesis.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Hemodynamic shear stress induces protective autophagy in HeLa cells through lipid raft-mediated mechanotransduction.

During metastatic dissemination, cancer cells experience shear stresses in narrow confinements of in vivo vasculature. Such stresses are currently known to influence a gamut of cellular processes. While a host of cells emanating from a primary tumor perish in circulation due to shear, some cells manage to migrate to distant niches and form secondary tumors. Current research focuses on how cancer cells avert such mechanical stresses and adapt themselves in order to survive. This study deals with the autophagic response of cervical cancer cells HeLa and its subline HeLa 229, exposed to physiological shear stresses in vitro, and evaluates its role as a pro-survival mechanism. It also delineates the probable mechanotransduction pathway that is involved in eliciting the stress-adaptive response in cervical cancer cells. Our results show that shear stress of physiological regime elicits protective autophagy in cervical cancer cells as an immediate response and inhibiting the same, leads to early onset of apoptosis. An effort to study the underlying mechanotransduction revealed that autophagy induction by shear stress requires intact lipid rafts which serve as signalling platforms to trigger phosphorylation of p38 mitogen activated protein kinases, leading to autophagy. This study thus gives novel insights into the mechanobiology of cervical cancer and hints at promising therapeutic targets in metastasis, the major cause of cancer mortality.

Biomechanics of the Circulating Tumor Cell Microenvironment.

Circulating tumor cells (CTCs) exist in a microenvironment quite different from the solid tumor tissue microenvironment. They are detached from matrix and exposed to the immune system and hemodynamic forces leading to the conclusion that life as a CTC is "nasty, brutish, and short." While there is much evidence to support this assertion, the mechanisms underlying this are much less clear. In this chapter we will specifically focus on biomechanical influences on CTCs in the circulation and examine in detail the question of whether CTCs are mechanically fragile, a commonly held idea that is lacking in direct evidence. We will review multiple lines of evidence indicating, perhaps counterintuitively, that viable cancer cells are mechanically robust in the face of exposures to physiologic shear stresses that would be encountered by CTCs during their passage through the circulation. Finally, we present emerging evidence that malignant epithelial cells, as opposed to their benign counterparts, possess specific mechanisms that enable them to endure these mechanical stresses.

Effector and Regulatory T Cells Roll at High Shear Stress by Inducible Tether and Sling Formation

The adaptive immune response involves Tcell differentiation and migration to sites of inflammation. Tcell trafficking is initiated by rolling on inflamed endothelium. Tethers and slings, discovered in neutrophils, facilitate cell rolling at high shear stress. Here, we demonstrate that the ability to form tethers and slings during rolling is highly inducible in T helper 1 (Th1), Th17, and regulatory T (Treg) cells but less in Th2 cells. Invivo, endogenous Treg cells rolled stably in cremaster venules at physiological shear stress. Quantitative dynamic footprinting nanoscopy of Th1, Th17, and Treg cells uncovered the formation of multiple tethers per cell. Human Th1 cells also showed tethers and slings. RNA sequencing (RNA-seq) revealed the induction of cell migration and cytoskeletal genes in sling-forming cells. We conclude that differentiated CD4 Tcells stabilize rolling by inducible tether and sling formation. These phenotypic changes approximate the adhesion phenotype of neutrophils and support CD4 Tcell access to sites of inflammation.

SCARF-1 promotes adhesion of CD4+ T cells to human hepatic sinusoidal endothelium under conditions of shear stress

Liver-resident cells are constantly exposed to gut-derived antigens via portal blood and, as a consequence, they express a unique repertoire of scavenger receptors. Whilst there is increasing evidence that the gut contributes to chronic inflammatory liver disease, the role of scavenger receptors in regulating liver inflammation remains limited. Here, we describe for the first time the expression of scavenger receptor class F, member 1 (SCARF-1) on hepatic sinusoidal endothelial cells (HSEC). We report that SCARF-1 shows a highly localised expression pattern and co-localised with endothelial markers on sinusoidal endothelium. Analysis of chronically inflamed liver tissue demonstrated accumulation of SCARF-1 at sites of CD4+ T cell aggregation. We then studied the regulation and functional role of SCARF-1 in HSEC and showed that SCARF-1 expression by HSEC is regulated by proinflammatory cytokines and bacterial lipopolysaccharide (LPS). Furthermore, SCARF-1 expression by HSEC, induced by proinflammatory and gut-derived factors acts as a novel adhesion molecule, present in adhesive cup structures, that specifically supports CD4+ T cells under conditions of physiological shear stress. In conclusion, we show that SCARF-1 contributes to lymphocyte subset adhesion to primary human HSEC and could play an important role in regulating the inflammatory response during chronic liver disease.

C-Abl tyrosine kinase regulates neutrophil crawling behavior under fluid shear stress via Rac/PAK/LIMK/cofilin signaling axis.

The excessive recruitment and improper activation of polymorphonuclear neutrophils (PMNs) often induces serious injury of host tissues, leading to inflammatory disorders. Therefore, to understand the molecular mechanism on neutrophil recruitment possesses essential pathological and physiological importance. In this study, we found that physiological shear stress induces c-Abl kinase activation in neutrophils, and c-Abl kinase inhibitor impaired neutrophil crawling behavior on ICAM-1. We further identified Vav1 was a downstream effector phosphorylated at Y174 and Y267. Once activated, c-Abl kinase regulated the activity of Vav1, which further affected Rac1/PAK1/LIMK1/cofilin signaling pathway. Here, we demonstrate a novel signaling function and critical role of c-Abl kinase during neutrophil crawling under physiological shear by regulating Vav1. These findings provide a promising treatment strategy for inflammation-related disease by inactivation of c-Abl kinase to restrict neutrophil recruitment.

The collective influence of 1, 25-dihydroxyvitamin D3 with physiological fluid shear stress on osteoblasts

1, 25-dihydroxyvitamin D3 (1, 25 (OH)2 D3) and mechanical stimuli in physiological environment contributes greatly to osteoporosis pathogenesis. Wide investigations have been conducted on how 1, 25-dihydroxyvitamin D3 and mechanical stimuli separately impact osteoblasts. This study reports the collective influences of 1, 25-dihydroxyvitamin D3 and flow shear stress (FSS) on biological functions of osteoblasts. 1, 25 (OH)2 D3 were prepared in various kinds of concentrations (0, 1, 10, 100 nmmol/L), while physiological fluid shear stress (12 dynes/cm2) was produced by using a parallel-plate fluid flow system. 1, 25 (OH)2 D3 affects the responses of ROBs to FSS, including the inhibition of NO release and cell proliferation as well as the promotion of PGE2 release and cell differentiation. These findings provide a possible mechanism by which 1, 25(OH)2 D3 influences osteoblasts’ responses to FSS, thus most probably providing guidance for the selection of 1, 25(OH)2 D3 concentration and mechanical loading in order to produce functional bone tissues in vitro.

Computational analysis of integrated biosensing and shear flow in a microfluidic vascular model

Fluid flow and flow-induced shear stress are critical components of the vascular microenvironment commonly studied using microfluidic cell culture models. Microfluidic vascular models mimicking the physiological microenvironment also offer great potential for incorporating on-chip biomolecular detection. In spite of this potential, however, there are few examples of such functionality. Detection of biomolecules released by cells under flow-induced shear stress is a significant challenge due to severe sample dilution caused by the fluid flow used to generate the shear stress, frequently to the extent where the analyte is no longer detectable. In this work, we developed a computational model of a vascular microfluidic cell culture model that integrates physiological shear flow and on-chip monitoring of cell-secreted factors. Applicable to multilayer device configurations, the computational model was applied to a bilayer configuration, which has been used in numerous cell culture applications including vascular models. Guidelines were established that allow cells to be subjected to a wide range of physiological shear stress while ensuring optimal rapid transport of analyte to the biosensor surface and minimized biosensor response times. These guidelines therefore enable the development of microfluidic vascular models that integrate cell-secreted factor detection while addressing flow constraints imposed by physiological shear stress. Ultimately, this work will result in the addition of valuable functionality to microfluidic cell culture models that further fulfill their potential as labs-on-chips.

Investigation on the effects of variable shear stress on monocyte cell morphology

Microchannels fabricated by soft lithography have been favourable in mechanobiological applications for cell culture platforms, especially shear stress studies. Normally, physiological shear stress levels for veins are between 10 and 70 dynes/cm2 (1–7 Pa) and arteries between 1 and 6 dynes/cm2 (0.1–0.7 Pa). In this work, a circular system has been set up by microfluidic pump to mimic physiological environment of monocyte cells for monitoring structural changes at various levels of shear stresses. Shear stresses at 4, 15 and 45 Pa, low, medium and high stresses, respectively, simulated by CFD have been applied on THP-1 cells under different flow rates for 0.5 and 3 h. These cells, which passed through microchannels under these experimental conditions were analysed for their F-actin content by staining with phalloidin and determination of mean fluorescence intensity by fluorescence microscopy and flow cytometry. An increase in THP-1 monocytic cell actin polymerisation is significant in the case of 15 Pa, and slight in the case of 45 Pa shear stress conditions for 0.5 h. This may reflect the structural adaptation of monocytes during a pathological condition such as atherosclerosis.

Chitosan-based hydrogels for developing a small-diameter vascular graft: in vitro and in vivo evaluation

Aims. Vascular grafts made of synthetic polymers perform poorly in small-diameter applications (cardiac and peripheral bypass). Chitosan is a biocompatible natural polymer that can provide a novel biological scaffold for tissue engineering development. The goal of this study was to demonstrate the biocompatibility of a novel chitosan preparation in vitro and in vivo, and to assess its potential as a scaffold for vascular applications. Methods and results. A series of experiments of increasing complexity, ranging from in vitro biocompatibility and hemocompatibility tests to in vivo studies in small and large animals (rats and sheep), was performed to provide a comprehensive analysis of chitosan hydrogels’ biological properties. In vitro studies established that: (i) chitosan supported human endothelial progenitor cells adhesion, proliferation and resistance to physiological shear stress; (ii) chitosan did not activate platelets, the complement system, or the intrinsic coagulation pathway. In vivo results showed: (iii) no resorption of chitosan and no chronic inflammation at 60 days in a rat heterotopic implantation model (magnetic resonance imaging and histology); (iv) no flow obstruction (Doppler ultrasound) and no thrombus formation (histology and scanning electron microscopy) at 2 h after a carotid arteriotomy repair with chitosan patches in sheep. Finally, two chitosan tubes were implanted as carotid interposition grafts for 3 days in sheep showing that chitosan was strong enough to be sutured, to withstand arterial pressure, and no flow obstruction was observed through this short period. Conclusion. Chitosan-based hydrogels displayed promising in vitro biocompatibility and hemocompatibility properties as well as in vivo short-term performance.

Biodegradability and platelets adhesion assessment of magnesium-based alloys using a microfluidic system.

Magnesium (Mg)-based stents are extensively explored to alleviate atherosclerosis due to their biodegradability and relative hemocompatibility. To ensure the quality, safety and cost-efficacy of bioresorbable scaffolds and full utilization of the material tunability afforded by alloying, it is critical to access degradability and thrombosis potential of Mg-based alloys using improved in vitro models that mimic as closely as possible the in vivo microenvironment. In this study, we investigated biodegradation and initial thrombogenic behavior of Mg-based alloys at the interface between Mg alloys’ surface and simulated physiological environment using a microfluidic system. The degradation properties of Mg-based alloys WE43, AZ31, ZWEK-L, and ZWEK-C were evaluated in complete culture medium and their thrombosis potentials in platelet rich plasma, respectively. The results show that 1) physiological shear stress increased the corrosion rate and decreased platelets adhesion rate as compared to static immersion; 2) secondary phases and impurities in material composition induced galvanic corrosion, resulting in higher corrosion resistance and platelet adhesion rate; 3) Mg-based alloys with higher corrosion rate showed higher platelets adhesion rate. We conclude that a microfluidic-based in vitro system allows evaluation of biodegradation behaviors and platelets responses of Mg-based alloys under specific shear stress, and degradability is related to platelets adhesion.

OP-19 - Localized hydrogen peroxide metabolism and cardiovascular signal transduction

The molecular events underlying the transition from “physiological” to “pathological” reactive oxygen species (ROS) in the vascular endothelium are poorly understood. We constructed differentially-targeted HyPer2 biosensors for H2O2 to identify the intracellular organelles and pathways responsible for dynamic regulation of endothelial H2O2 metabolism. We discovered that laminar (physiological) shear stress robustly increases H2O2 in the endothelial cell nucleus much more than in the cytosol. By contrast, oscillatory (pathological) shear increases H2O2 in the cytosol significantly more than in the nucleus. We generated H2O2 within specific subcellular locales in cultured endothelial cells using recombinant constructs expressing a D-amino acid oxidase (DAAO) that robustly generates H2O2 upon the addition of D-alanine to the cells. Using a series of differentially targeted DAAO constructs, we discovered H2O2 generation in distinct subcellular compartments differentially modulates endothelial transcription. Generation of H2O2 by DAAO in the endothelial cell nucleus enhanced transcription of Nrf2-modulated genes, whereas generation of H2O2 by DAAO targeted to the cytosol activated NF-kB-regulated genes. These findings have important implications for our understanding of flow-responsive genes in the normal blood vessel as well as in disease states associated with disordered blood flow and oxidative stress.

Evidence for shear-mediated Ca2+ entry through mechanosensitive cation channels in human platelets and a megakaryocytic cell line

The role of mechanosensitive (MS) Ca2+-permeable ion channels in platelets is unclear, despite the importance of shear stress in platelet function and life-threatening thrombus formation. We therefore sought to investigate the expression and functional relevance of MS channels in human platelets. The effect of shear stress on Ca2+ entry in human platelets and Meg-01 megakaryocytic cells loaded with Fluo-3 was examined by confocal microscopy. Cells were attached to glass coverslips within flow chambers that allowed applications of physiological and pathological shear stress. Arterial shear (1002.6 s−1) induced a sustained increase in [Ca2+]i in Meg-01 cells and enhanced the frequency of repetitive Ca2+ transients by 80% in platelets. These Ca2+ increases were abrogated by the MS channel inhibitor Grammostola spatulata mechanotoxin 4 (GsMTx-4) or by chelation of extracellular Ca2+. Thrombus formation was studied on collagen-coated surfaces using DiOC6-stained platelets. In addition, [Ca2+]i and functional responses of washed platelet suspensions were studied with Fura-2 and light transmission aggregometry, respectively. Thrombus size was reduced 50% by GsMTx-4, independently of P2X1 receptors. In contrast, GsMTx-4 had no effect on collagen-induced aggregation or on Ca2+ influx via TRPC6 or Orai1 channels and caused only a minor inhibition of P2X1-dependent Ca2+ entry. The Piezo1 agonist, Yoda1, potentiated shear-dependent platelet Ca2+ transients by 170%. Piezo1 mRNA transcripts and protein were detected with quantitative RT-PCR and Western blotting, respectively, in both platelets and Meg-01 cells. We conclude that platelets and Meg-01 cells express the MS cation channel Piezo1, which may contribute to Ca2+ entry and thrombus formation under arterial shear.