scholarly journals STAT3 activation by hypoxia in in vitro models of cervix cancer and endothelial cells

Biomédica ◽  
2017 ◽  
Vol 37 (1) ◽  
pp. 119 ◽  
Author(s):  
Óscar Ortega ◽  
Alejandro Ondo-Méndez ◽  
Ruth Garzón
Keyword(s):  
Endothelial Cells ◽  
Western Blot ◽  
In Vitro Models ◽  
Cervix Cancer

Introducción. El microambiente tumoral influye en el comportamiento de las células cancerosas. Especialmente, el estímulo de agentes estresantes, como la hipoxia, se convierte en un factor crítico para la evolución y el tratamiento del cáncer. La reacción celular frente a diversos estímulos se manifiesta en la activación de vías de señalización como la JAK/STAT, una de las más importantes por sus efectos en la diferenciación y proliferación celular.Objetivo. Evaluar el estado de la vía JAK/STAT mediante la expresión o activación de la proteína STAT3 en células de cáncer de cuello uterino (HeLa) y en células endoteliales (EA.hy926) sometidas a hipoxia.Materiales y métodos. Las líneas celulares se sometieron a condiciones de hipoxia física (1 % de O2) o química (100 μM de deferoxamina, DFO) durante dos, seis y 24 horas. Mediante Western blot se determinó el cambio en la expresión y activación de STAT3, y mediante inmunofluorescencia indirecta, su localización subcelular.Resultados. La hipoxia se evidenció por la activación y translocación al núcleo del HIF-1. Ni la hipoxia física ni la química alteraron la expresión de STAT3, pero sí la activación, según se comprobó por su fosforilación y su translocación al núcleo en los dos modelos bajo estudio.Conclusiones. Se evidenció la importancia de la hipoxia como un estímulo que modifica la activación de la proteína STAT3 en las células HeLa y EA.hy926, lo cual la convierte en un elemento importante en el diseño de estrategias terapéuticas contra el cáncer.

scholarly journals Radiobiological Studies of Microvascular Damage through In Vitro Models: A Methodological Perspective

Cancers ◽  
2021 ◽  
Vol 13 (5) ◽  
pp. 1182
Author(s):  
Luca Possenti ◽  
Laura Mecchi ◽  
Andrea Rossoni ◽  
Veronica Sangalli ◽  
Simone Bersini ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
In Vitro Models ◽  
Microvascular Endothelial Cells ◽  
Microvascular Damage ◽  
Essential Information ◽  
Reliable Model ◽  
Critical Issues ◽  
Irradiation Scheme ◽  
In Vitro Effects

Ionizing radiation (IR) is used in radiotherapy as a treatment to destroy cancer. Such treatment also affects other tissues, resulting in the so-called normal tissue complications. Endothelial cells (ECs) composing the microvasculature have essential roles in the microenvironment’s homeostasis (ME). Thus, detrimental effects induced by irradiation on ECs can influence both the tumor and healthy tissue. In-vitro models can be advantageous to study these phenomena. In this systematic review, we analyzed in-vitro models of ECs subjected to IR. We highlighted the critical issues involved in the production, irradiation, and analysis of such radiobiological in-vitro models to study microvascular endothelial cells damage. For each step, we analyzed common methodologies and critical points required to obtain a reliable model. We identified the generation of a 3D environment for model production and the inclusion of heterogeneous cell populations for a reliable ME recapitulation. Additionally, we highlighted how essential information on the irradiation scheme, crucial to correlate better observed in vitro effects to the clinical scenario, are often neglected in the analyzed studies, limiting the translation of achieved results.

scholarly journals Conditioned medium-preconditioned EPCs enhanced the ability in oligovascular repair in cerebral ischemia neonatal rats

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Ning Zhou ◽  
Lei Wang ◽  
Ping Fu ◽  
Zihao Cui ◽  
Yuhang Ge ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Western Blot ◽  
Conditioned Medium ◽  
Cognitive Outcome ◽  
Adult Brain ◽  
Neonatal Rat ◽  
Vascular Density ◽  
Cxcr4 Axis

Abstract Background Oligovascular niche mediates interactions between cerebral endothelial cells and oligodendrocyte precursor cells (OPCs). Disruption of OPC-endothelium trophic coupling may aggravate the progress of cerebral white matter injury (WMI) because endothelial cells could not provide sufficient support under diseased conditions. Endothelial progenitor cells (EPCs) have been reported to ameliorate WMI in the adult brain by boosting oligovascular remodeling. It is necessary to clarify the role of the conditioned medium from hypoxic endothelial cells preconditioned EPCs (EC-pEPCs) in WMI since EPCs usually were recruited and play important roles under blood-brain barrier disruption. Here, we investigated the effects of EC-pEPCs on oligovascular remodeling in a neonatal rat model of WMI. Methods In vitro, OPC apoptosis induced by the conditioned medium from oxygen-glucose deprivation-injured brain microvascular endothelial cells (OGD-EC-CM) was analyzed by TUNEL and FACS. The effects of EPCs on EC damage and the expression of cytomokine C-X-C motif ligand 12 (CXCL12) were examined by western blot and FACS. The effect of the CM from EC-pEPCs against OPC apoptosis was also verified by western blot and silencing RNA. In vivo, P3 rat pups were subjected to right common carotid artery ligation and hypoxia and treated with EPCs or EC-pEPCs at P7, and then angiogenesis and myelination together with cognitive outcome were evaluated at the 6th week. Results In vitro, EPCs enhanced endothelial function and decreased OPC apoptosis. Meanwhile, it was confirmed that OGD-EC-CM induced an increase of CXCL12 in EPCs, and CXCL12-CXCR4 axis is a key signaling since CXCR4 knockdown alleviated the anti-apoptosis effect of EPCs on OPCs. In vivo, the number of EPCs and CXCL12 protein level markedly increased in the WMI rats. Compared to the EPCs, EC-pEPCs significantly decreased OPC apoptosis, increased vascular density and myelination in the corpus callosum, and improved learning and memory deficits in the neonatal rat WMI model. Conclusions EC-pEPCs more effectively promote oligovascular remodeling and myelination via CXCL12-CXCR4 axis in the neonatal rat WMI model.

scholarly journals Characterization of human fetal brain endothelial cells reveals barrier properties suitable for in vitro modeling of the BBB with syngenic co-cultures

2017 ◽  
Vol 38 (5) ◽  
pp. 888-903 ◽  
Author(s):  
Allison M Andrews ◽  
Evan M Lutton ◽  
Lee A Cannella ◽  
Nancy Reichenbach ◽  
Roshanak Razmpour ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Neurovascular Unit ◽  
Fetal Brain ◽  
Barrier Properties ◽  
Endothelial Activation ◽  
Fetal Tissue ◽  
In Vitro Models ◽  
Adult Brain ◽  
Alternative Source

Endothelial cells (ECs) form the basis of the blood–brain barrier (BBB), a physical barrier that selectively restricts transport into the brain. In vitro models can provide significant insight into BBB physiology, mechanisms of human disease pathology, toxicology, and drug delivery. Given the limited availability of primary human adult brain microvascular ECs ( aBMVECs), human fetal tissue offers a plausible alternative source for multiple donors and the opportunity to build syngenic tri-cultures from the same host. Previous efforts to culture fetal brain microvascular ECs ( fBMVECs) have not been successful in establishing mature barrier properties. Using optimal gestational age for isolation and flow cytometry cell sorting, we show for the first time that fBMVECs demonstrate mature barrier properties. fBMVECs exhibited similar functional phenotypes when compared to aBMVECs for barrier integrity, endothelial activation, and gene/protein expression of tight junction proteins and transporters. Importantly, we show that tissue used to culture fBMVECs can also be used to generate a syngenic co-culture, creating a microfluidic BBB on a chip. The findings presented provide a means to overcome previous challenges that limited successful barrier formation by fBMVECs. Furthermore, the source is advantageous for autologous reconstitution of the neurovascular unit for next generation in vitro BBB modeling.

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

2006 ◽  
Vol 19 (1) ◽  
pp. 205873920601900 ◽  
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.

scholarly journals Endothelialized silicone aneurysm models for in vitro evaluation of flow diverters

2020 ◽  
pp. neurintsurg-2020-016859
Author(s):  
Alyssa McCulloch ◽  
Ashley Turcott ◽  
Gabriella Graham ◽  
Sergey Frenklakh ◽  
Kristen O'Halloran Cardinal
Keyword(s):  
Endothelial Cells ◽  
Endothelial Cell ◽  
Umbilical Vein ◽  
Flow Diverter ◽  
In Vitro Models ◽  
Cell Coverage ◽  
Platelet Endothelial Cell Adhesion ◽  
Umbilical Vein Endothelial Cells ◽  
Over Time

ObjectiveThe goal of this work was to endothelialize silicone aneurysm tubes for use as in vitro models for evaluating endothelial cell interactions with neurovascular devices. The first objective was to establish consistent and confluent endothelial cell linings and to evaluate the silicone vessels over time. The second objective was to use these silicone vessels for flow diverter implantation and assessment.MethodsSilicone aneurysm tubes were coated with fibronectin and placed into individual bioreactor systems. Human umbilical vein endothelial cells were deposited within tubes to create silicone vessels, then cultivated on a peristaltic pump and harvested at 2, 5, 7, or 10 days to evaluate the endothelial cell lining. A subset of silicone aneurysm vessels was used for flow diverter implantation, and evaluated for cell coverage over device struts at 3 or 7 days after deployment.ResultsSilicone vessels maintained confluent, PECAM-1 (platelet endothelial cell adhesion molecule 1) positive endothelial cell linings over time. These vessels facilitated and withstood flow diverter implantation, with robust cell linings disclosed after device deployment. Additionally, the endothelial cells responded to implanted devices through coverage of the flow diverter struts with increased cell coverage over the aneurysm seen at 7 days after deployment as compared with 3 days.ConclusionsSilicone aneurysm models can be endothelialized and successfully maintained in vitro over time. Furthermore, these silicone vessels can be used for flow diverter implantation and assessment.

Inflammatory Response of Endothelial Cells to Wall Shear Stress in Three Dimensional Stenotic Models

2009 ◽  
Author(s):  
Leonie Rouleau ◽  
Joanna Rossi ◽  
Jean-Claude Tardif ◽  
Rosaire Mongrain ◽  
Richard L. Leask
Keyword(s):  
Endothelial Cells ◽  
Shear Stress ◽  
Wall Shear Stress ◽  
Three Dimensional ◽  
Realistic Model ◽  
In Vitro Models ◽  
Steady Flows ◽  
Wall Shear

Endothelial cells (ECs) are believed to respond differentially to hemodynamic forces in the vascular tree. Once atherosclerotic plaque has formed in a vessel, the obstruction creates complex spatial gradients in wall shear stress (WSS). In vitro models have used mostly unrealistic and simplified geometries, which cannot reproduce accurately physiological conditions. The objective of this study was to expose ECs to the complex WSS pattern created by an asymmetric stenosis. Endothelial cells were grown and exposed for different times to physiological steady flows in straight dynamic controls and in idealized asymmetric stenosis models. Cell morphology was noticeably different in the regions with spatial WSS gradients, being more randomly oriented and of cobblestone shape. Inflammatory molecule expression was also altered by exposure to shear and endothelial nitric oxide synthase (eNOS) was upregulated by its presence. A regional response in terms of inflammation was observed through confocal microscopy. This work provides a more realistic model to study endothelial cell response to spatial and temporal WSS gradients that are present in vivo and is an important advancement towards a better understanding of the mechanisms involved in coronary artery disease.

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