Flow perfusion effects on three-dimensional culture and drug sensitivity of Ewing sarcoma

Three-dimensional tumor models accurately describe different aspects of the tumor microenvironment and are readily available for mechanistic studies of tumor biology and for drug screening. Nevertheless, these systems often overlook biomechanical stimulation, another fundamental driver of tumor progression. To address this issue, we cultured Ewing sarcoma (ES) cells on electrospun poly(ε-caprolactone) 3D scaffolds within a flow perfusion bioreactor. Flow-derived shear stress provided a physiologically relevant mechanical stimulation that significantly promoted insulin-like growth factor-1 (IGF1) production and elicited a superadditive release in the presence of exogenous IGF1. This finding is particularly relevant, given the central role of the IGF1/IGF-1 receptor (IGF-1R) pathway in ES tumorigenesis and as a promising clinical target. Additionally, flow perfusion enhanced in a rate-dependent manner the sensitivity of ES cells to IGF-1R inhibitor dalotuzumab (MK-0646) and showed shear stress-dependent resistance to the IGF-1R blockade. This study demonstrates shear stress-dependent ES cell sensitivity to dalotuzumab, highlighting the importance of biomechanical stimulation on ES-acquired drug resistance to IGF-1R inhibition. Furthermore, flow perfusion increased nutrient supply throughout the scaffold, enriching ES culture over static conditions. Our use of a tissue-engineered model, rather than human tumors or xenografts, enabled precise control of the forces experienced by ES cells, and therefore provided at least one explanation for the remarkable antineoplastic effects observed by some ES tumor patients from IGF-1R targeted therapies, in contrast to the lackluster effect observed in cells grown in conventional monolayer culture.

Download Full-text

Shear stress induces hepatocyte PAI-1 gene expression through cooperative Sp1/Ets-1 activation of transcription

AJP Gastrointestinal and Liver Physiology ◽

10.1152/ajpgi.00467.2005 ◽

2006 ◽

Vol 291 (1) ◽

pp. G26-G34 ◽

Cited By ~ 30

Author(s):

Hideki Nakatsuka ◽

Takaaki Sokabe ◽

Kimiko Yamamoto ◽

Yoshinobu Sato ◽

Katsuyoshi Hatakeyama ◽

...

Keyword(s):

Gene Expression ◽

Shear Stress ◽

Consensus Sequence ◽

Early Gene ◽

Immediate Early ◽

Mrna Levels ◽

Consensus Sequences ◽

Static Conditions ◽

Stress Dependent ◽

Pai 1

Partial hepatectomy causes hemodynamic changes that increase portal blood flow in the remaining lobe, where the expression of immediate-early genes, including plasminogen activator inhibitor-1 (PAI-1), is induced. We hypothesized that a hyperdynamic circulatory state occurring in the remaining lobe induces immediate-early gene expression. In this study, we investigated whether the mechanical force generated by flowing blood, shear stress, induces PAI-1 expression in hepatocytes. When cultured rat hepatocytes were exposed to flow, PAI-1 mRNA levels began to increase within 3 h, peaked at levels significantly higher than the static control levels, and then gradually decreased. The flow-induced PAI-1 expression was shear stress dependent rather than shear rate dependent and accompanied by increased hepatocyte production of PAI-1 protein. Shear stress increased PAI-1 transcription but did not affect PAI-1 mRNA stability. Functional analysis of the 2.1-kb PAI-1 5′-promoter indicated that a 278-bp segment containing transcription factor Sp1 and Ets-1 consensus sequences was critical to the shear stress-dependent increase of PAI-1 transcription. Mutations of both the Sp1 and Ets-1 consensus sequences, but not of either one alone, markedly prevented basal PAI-1 transcription and abolished the response of the PAI-1 promoter to shear stress. EMSA and chromatin immunoprecipitation assays showed binding of Sp1 and Ets-1 to each consensus sequence under static conditions, which increased in response to shear stress. In conclusion, hepatocyte PAI-1 expression is flow sensitive and transcriptionally regulated by shear stress via cooperative interactions between Sp1 and Ets-1.

Download Full-text

NPM1 is a Novel Therapeutic Target and Prognostic Biomarker for Ewing Sarcoma

Frontiers in Genetics ◽

10.3389/fgene.2021.771253 ◽

2021 ◽

Vol 12 ◽

Author(s):

Yangfan Zhou ◽

Yuan Fang ◽

Junjie Zhou ◽

Yulian Liu ◽

Shusheng Wu ◽

...

Keyword(s):

Ewing Sarcoma ◽

Es Cells ◽

Differentially Expressed Gene ◽

Bioinformatic Analysis ◽

Dependent Manner ◽

Hub Gene ◽

Immune Infiltration ◽

Proliferation And Apoptosis ◽

Immune Related Genes ◽

Dose Dependent

Ewing sarcoma (ES) is a cancer that may originate from stem mesenchymal or neural crest cells and is highly prevalent in children and adolescents. In recent years, targeted therapies against immune-related genes have shown good efficacy in a variety of cancers. However, effective targets for immunotherapy in ES are yet to be developed. In our study, we first identified the immune-associated differential hub gene NPM1 by bioinformatics methods as a differentially expressed gene, and then validated it using real time-PCR and western blotting, and found that this gene is not only closely related to the immune infiltration in ES, but also can affect the proliferation and apoptosis of ES cells, and is closely related to the survival of patients. The results of our bioinformatic analysis showed that NPM1 can be a hub gene in ES and an immunotherapeutic target to reactivate immune infiltration in patients with ES. In addition, treatment with NPM1 promoted apoptosis and inhibited the proliferation of ES cells. The NPM1 inhibitor NSC348884 can induce apoptosis of ES cells in a dose-dependent manner and is expected to be a potential therapeutic agent for ES.

Download Full-text

Shear Stress Modulates the Expression of Thrombospondin-1 and CD36 in Endothelial Cells in vitro and during Shear Stress-Induced Angiogenesis in vivo

International Journal of Immunopathology and Pharmacology ◽

10.1177/205873920601900104 ◽

2006 ◽

Vol 19 (1) ◽

pp. 205873920601900 ◽

Cited By ~ 35

Author(s):

M. Bongrazio ◽

L. DA Silva-Azevedo ◽

E.C. Bergmann ◽

O. Baum ◽

B. Hinz ◽

...

Keyword(s):

Endothelial Cells ◽

Shear Stress ◽

Western Blot ◽

Dependent Manner ◽

Vascular Networks ◽

Thrombospondin 1 ◽

Matrix Bound ◽

Stress Dependent

Binding of thrombospondin-1 (TSP-1) to the CD36 receptor inhibits angiogenesis and induces apoptosis in endothelial cells (EC). Conversely, matrix-bound TSP-1 supports vessel formation. In this study we analyzed the shear stress-dependent expression of TSP-1 and CD36 in endothelial cells in vitro and in vivo to reveal its putative role in the blood flow-induced remodelling of vascular networks. Shear stress was applied to EC using a cone-and-plate apparatus and gene expression was analyzed by RT-PCR, Northern and Western blot. Angiogenesis in skeletal muscles of prazosin-fed (50 mg/1 drinking water; 4 d) mice was assessed by measuring capillary-to-fiber (C/F) ratios. Protein expression in whole muscle homogenates (WMH) or BS-1 lectin-enriched EC fractions (ECF) was analyzed by Western blot. Shear stress down-regulated TSP-1 and CD36 expression in vitro in a force- and time-dependent manner sustained for at least 72 h and reversible by restoration of no-flow conditions. In vivo, shear stress-driven increase of C/F in prazosin-fed mice was associated with reduced expression of TSP-1 and CD36 in ECF, while TSP-1 expression in WMH was increased. Down-regulation of endothelial TSP-1/CD36 by shear stress suggests a mechanism for inhibition of apoptosis in perfused vessels and pruning in the absence of flow. The increase of extra-endothelial (e.g. matrix-bound) TSP-1 could support a splitting type of vessel growth.

Download Full-text

Patient-specific three-dimensional simulation of LDL accumulation in a human left coronary artery in its healthy and atherosclerotic states

AJP Heart and Circulatory Physiology ◽

10.1152/ajpheart.01182.2008 ◽

2009 ◽

Vol 296 (6) ◽

pp. H1969-H1982 ◽

Cited By ~ 66

Author(s):

Ufuk Olgac ◽

Dimos Poulikakos ◽

Stefan C. Saur ◽

Hatem Alkadhi ◽

Vartan Kurtcuoglu

Keyword(s):

Shear Stress ◽

Coronary Artery ◽

Left Coronary Artery ◽

Three Dimensional ◽

Arterial Blood Flow ◽

Patient Specific ◽

Computed Tomography Images ◽

Arterial Blood ◽

Diseased State ◽

Stress Dependent

We calculate low-density lipoprotein (LDL) transport from blood into arterial walls in a three-dimensional, patient-specific model of a human left coronary artery. The in vivo anatomy data are obtained from computed tomography images of a patient with coronary artery disease. Models of the artery anatomy in its healthy and diseased states are derived after segmentation of the vessel lumen, with and without the detected plaque, respectively. Spatial shear stress distribution at the endothelium is determined through the reconstruction of the arterial blood flow field using computational fluid dynamics. The arterial endothelium is represented by a shear stress-dependent, three-pore model, taking into account blood plasma and LDL passage through normal junctions, leaky junctions, and the vesicular pathway. Intraluminal pressures of 70 and 120 mmHg are employed as the normal and hypertensive operating pressures, respectively. By applying our model to both the healthy and diseased states, we show that the location of the plaque in the diseased state corresponds to one of the two sites with predicted high-LDL concentration in the healthy state. We further show that, in the diseased state, the site with high-LDL concentration has shifted distal to the plaque, which is in agreement with the clinical observation that plaques generally grow in the downstream direction. We also demonstrate that hypertension leads to increased number of regions with high-LDL concentration, elucidating one of the ways in which hypertension may promote atherosclerosis.

Download Full-text

Abstract 927: The Role Of Peroxiredoxins As Mechanosensitive Antioxidants In Endothelial Cells

Circulation ◽

10.1161/circ.116.suppl_16.ii_182-c ◽

2007 ◽

Vol 116 (suppl_16) ◽

Author(s):

Amy Mowbray ◽

Sang Won Kang ◽

Sue Goo Rhee ◽

Hanjoong Jo

Keyword(s):

Endothelial Cells ◽

Shear Stress ◽

Subcellular Localization ◽

Molecular Mechanisms ◽

Immunofluorescent Staining ◽

Dependent Manner ◽

Laminar Shear Stress ◽

Arterial Tree ◽

Aortic Endothelial Cells ◽

Stress Dependent

Atherosclerosis is an inflammatory disease occurring primarily in curved or branching regions of the arterial tree where disturbed flow patterns, such as oscillation, exist. We have previously shown that oscillatory shear stress (OS) increases reactive oxygen species (ROS) levels in endothelial cells, while laminar shear stress (LS) reduces ROS compared to static controls. OS stimulation of ROS has been shown to occur in an NADPH oxidase-dependent manner. However, the mechanism by which LS removes ROS remains unclear. Peroxiredoxins (PRX) are a family of antioxidant proteins that have been linked to the prevention of oxidative stress and inflammation, but their role in atherosclerosis is unknown. Here, we hypothesize that shear stress regulates ROS levels in endothelial cells by controlling antioxidant peroxiredoxins. To test this hypothesis, bovine aortic endothelial cells (BAEC) were subjected to static, laminar, and oscillatory fluid flow conditions via cone-and-plate viscometer. Western blot analysis and immunofluorescent staining were used to evaluate the expression and subcellular localization of six known mammalian peroxiredoxins (PRX I-VI). Immunoblots indicated that BAEC express all six isoforms of peroxiredoxin proteins and that LS upregulated PRX I levels significantly compared to static controls and OS. Immunofluorescence also showed a distinct subcellular localization of each PRX: PRX I, II, IV, V and VI in the cytoplasm, PRX I, IV and V in the Golgi, PRX III in the mitochondria, and PRX I in the nucleus. These results indicate that peroxiredoxins are mechanosensitive antioxidants, removing ROS in a subcellular-specific manner. Based on these data, we suggest that peroxiredoxin antioxidants are likely involved in the molecular mechanisms that control shear stress-dependent atherosclerotic plaque development.

Download Full-text

Mobility and invasiveness of metastatic esophageal cancer are potentiated by shear stress in a ROCK- and Ras-dependent manner

AJP Cell Physiology ◽

10.1152/ajpcell.00626.2005 ◽

2006 ◽

Vol 291 (4) ◽

pp. C668-C677 ◽

Cited By ~ 37

Author(s):

Karen Lawler ◽

Eilis Foran ◽

Gerald O'Sullivan ◽

Aideen Long ◽

Dermot Kenny

Keyword(s):

Shear Stress ◽

Leading Edge ◽

Basement Membranes ◽

Dependent Manner ◽

Shear Forces ◽

Membrane Blebbing ◽

Metastatic Cells ◽

Bleb Formation ◽

Static Conditions

To metastasize, tumor cells must adopt different morphological responses to resist shear forces encountered in circulating blood and invade through basement membranes. The Rho and Ras GTPases play a critical role in regulating this dynamic behavior. Recently, we demonstrated shear-induced activation of adherent esophageal metastatic cells, characterized by formation of dynamic membrane blebs. Although membrane blebbing has only recently been characterized as a rounded mode of cellular invasion promoted through Rho kinase (ROCK), the role of shear forces in modulating membrane blebbing activity is unknown. To further characterize membrane blebbing in esophageal metastatic cells (OC-1 cell line), we investigated the role of shear in cytoskeletal remodeling and signaling through ROCK and Ras. Our results show that actin and tubulin colocalize to the cortical ring of the OC-1 cell under static conditions. However, under shear, actin acquires a punctuate distribution and tubulin localizes to the leading edge of the OC-1 cell. We show for the first time that dynamic bleb formation is induced by shear alone independent of integrin-mediated adhesion ( P < 0.001, compared with OC-1 cells). Y-27632, a specific inhibitor of ROCK, causes a significant reduction in shear-induced bleb formation and inhibits integrin αvβ3-Ras colocalization at the leading edge of the cell. Direct measurement of Ras activation shows that the level of GTP-bound Ras is elevated in sheared OC-1 cells and that the shear-induced increase in Ras activity is inhibited by Y-27632. Finally, we show that shear stress significantly increases OC-1 cell invasion ( P < 0.007), an effect negated by the presence of Y-27632. Together our findings suggest a novel physiological role for ROCK and Ras in metastatic cell behavior.

Download Full-text

Assessment with clinical data of a coupled bio-hemodynamics numerical model to predict leukocyte adhesion in coronary arteries

Scientific Reports ◽

10.1038/s41598-021-92084-4 ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Umberto Ciri ◽

Ruth L. Bennett ◽

Rita Bhui ◽

David S. Molony ◽

Habib Samady ◽

...

Keyword(s):

Shear Stress ◽

Numerical Model ◽

Wall Shear Stress ◽

Coronary Arteries ◽

Clinical Data ◽

Leukocyte Adhesion ◽

Three Dimensional ◽

High Adhesion ◽

Wall Shear ◽

Stress Dependent

AbstractNumerical simulations of coupled hemodynamics and leukocyte transport and adhesion inside coronary arteries have been performed. Realistic artery geometries have been obtained for a set of four patients from intravascular ultrasound and angiography images. The numerical model computes unsteady three-dimensional blood hemodynamics and leukocyte concentration in the blood. Wall-shear stress dependent leukocyte adhesion is also computed through agent-based modeling rules, fully coupled to the hemodynamics and leukocyte transport. Numerical results have a good correlation with clinical data. Regions where high adhesion is predicted by the simulations coincide to a good approximation with artery segments presenting plaque increase, as documented by clinical data from baseline and six-month follow-up exam of the same artery. In addition, it is observed that the artery geometry and, in particular, the tortuosity of the centerline are a primary factor in determining the spatial distribution of wall-shear stress, and of the resulting leukocyte adhesion patterns. Although further work is required to overcome the limitations of the present model and ultimately quantify plaque growth in the simulations, these results are encouraging towards establishing a predictive methodology for atherosclerosis progress.

Download Full-text

Colonization of distant organs by tumor cells generating circulating homotypic clusters adaptive to fluid shear stress

Scientific Reports ◽

10.1038/s41598-021-85743-z ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Manabu Maeshiro ◽

Satoru Shinriki ◽

Rin Liu ◽

Yutaka Nakachi ◽

Yoshihiro Komohara ◽

...

Keyword(s):

Shear Stress ◽

Tumor Cells ◽

Fluid Shear Stress ◽

Dna Barcode ◽

Dependent Manner ◽

Fluid Shear ◽

Cortical Actin ◽

Metastatic Recurrence ◽

Static Conditions ◽

E Cadherin

AbstractOnce disseminated tumor cells (DTCs) arrive at a metastatic organ, they remain there, latent, and become seeds of metastasis. However, the clonal composition of DTCs in a latent state remains unclear. Here, we applied high-resolution DNA barcode tracking to a mouse model that recapitulated the metastatic dormancy of head and neck squamous cell carcinoma (HNSCC). We found that clones abundantly circulated peripheral blood dominated DTCs. Through analyses of multiple barcoded clonal lines, we identified specific subclonal population that preferentially generated homotypic circulating tumor cell (CTC) clusters and dominated DTCs. Despite no notable features under static conditions, this population significantly generated stable cell aggregates that were resistant to anoikis under fluid shear stress (FSS) conditions in an E-cadherin-dependent manner. Our data from various cancer cell lines indicated that the ability of aggregate-constituting cells to regulate cortical actin-myosin dynamics governed the aggregates’ stability in FSS. The CTC cluster-originating cells were characterized by the expression of a subset of E-cadherin binding factors enriched with actin cytoskeleton regulators. Furthermore, this expression signature was associated with locoregional and metastatic recurrence in HNSCC patients. These results reveal a biological selection of tumor cells capable of generating FSS-adaptive CTC clusters, which leads to distant colonization.

Download Full-text

Acute limb heating improves macro- and microvascular dilator function in the leg of aged humans

AJP Heart and Circulatory Physiology ◽

10.1152/ajpheart.00519.2016 ◽

2017 ◽

Vol 312 (1) ◽

pp. H89-H97 ◽

Cited By ~ 30

Author(s):

Steven A. Romero ◽

Daniel Gagnon ◽

Amy N. Adams ◽

Matthew N. Cramer ◽

Ken Kouda ◽

...

Keyword(s):

Shear Stress ◽

Young Adults ◽

Lower Limb ◽

Local Heating ◽

Reactive Hyperemia ◽

Arterial Occlusion ◽

Lower Limbs ◽

Dependent Manner ◽

Arterial Tree ◽

Stress Dependent

Local heating of an extremity increases blood flow and vascular shear stress throughout the arterial tree. Local heating acutely improves macrovascular dilator function in the upper limbs of young healthy adults through a shear stress-dependent mechanism but has no such effect in the lower limbs of this age group. The effect of acute limb heating on dilator function within the atherosclerotic prone vasculature of the lower limbs of aged adults is unknown. Therefore, the purpose of this study was to test the hypothesis that acute lower limb heating improves macro- and microvascular dilator function within the leg vasculature of aged adults. Nine young and nine aged adults immersed their lower limbs at a depth of ~33 cm into a heated (~42°C) circulated water bath for 45 min. Before and 30 min after heating, macro (flow-mediated dilation)- and microvascular (reactive hyperemia) dilator functions were assessed in the lower limb, following 5 min of arterial occlusion, via Doppler ultrasound. Compared with preheat, macrovascular dilator function was unchanged following heating in young adults ( P = 0.6) but was improved in aged adults ( P = 0.04). Similarly, microvascular dilator function, as assessed by peak reactive hyperemia, was unchanged following heating in young adults ( P = 0.1) but was improved in aged adults ( P < 0.01). Taken together, these data suggest that acute lower limb heating improves both macro- and microvascular dilator function in an age dependent manner. NEW & NOTEWORTHY We demonstrate that lower limb heating acutely improves macro- and microvascular dilator function within the atherosclerotic prone vasculature of the leg in aged adults. These findings provide evidence for a potential therapeutic use of chronic lower limb heating to improve vascular health in primary aging and various disease conditions.

Download Full-text

Mechanism and functional impact of CD40 ligand-induced von Willebrand factor release from endothelial cells

Thrombosis and Haemostasis ◽

10.1160/th14-04-0336 ◽

2015 ◽

Vol 113 (05) ◽

pp. 1095-1108 ◽

Cited By ~ 10

Author(s):

Kerstin Möller ◽

Oliver Adolph ◽

Jennifer Grünow ◽

Julia Elrod ◽

Miruna Popa ◽

...

Keyword(s):

Endothelial Cells ◽

Shear Stress ◽

Von Willebrand Factor ◽

Cd40 Ligand ◽

Dependent Manner ◽

Vascular Remodelling ◽

String Formation ◽

Stress Dependent ◽

Von Willebrand ◽

Willebrand Factor

SummaryCo-stimulation via CD154 binding to CD40, pivotal for both innate and adaptive immunity, may also link haemostasis to vascular remodelling. Here we demonstrate that human platelet-bound or recombinant soluble CD154 (sCD154) elicit the release from and tethering of ultra-large (UL) von Willebrand factor (vWF) multimers to the surface of human cultured endothelial cells (ECs) exposed to shear stress. This CD40-mediated ULVWF multimer release from the Weibel-Palade bodies was triggered by consecutive activation of TRAF6, the tyrosine kinase c-Src and phospholipase Cγ1 followed by inositol-1,4,5 tris-phosphate-mediated calcium mobilisation. Subsequent exposure to human washed platelets caused ULVWF multimer-platelet string formation on the EC surface in a shear stress-dependent manner. Platelets tethered to these ULVWF multimers exhibited P-selectin on their surface and captured labelled monocytes from the superfusate. When exposed to shear stress and sCD154, native ECs from wild-type but not CD40 or vWF-deficient mice revealed a comparable release of ULVWF multimers to which murine washed platelets rapidly adhered, turning P-selectin-positive and subsequently capturing monocytes from the perfusate. This novel CD154-provoked ULVWF multimerplatelet string formation at normal to fast flow may contribute to vascular remodelling processes requiring the perivascular or intravascular accumulation of pro-inflammatory macrophages such as arteriogenesis or atherosclerosis.

Download Full-text