scholarly journals Zyxin Mediates Vascular Repair via Endothelial Migration Promoted by Forskolin in Mice

2021 ◽  
Vol 12 ◽  
Author(s):  
Xuya Kang ◽  
Yanan Deng ◽  
Yang Cao ◽  
Yingqing Huo ◽  
Jincai Luo
Keyword(s):  
Wound Healing ◽  
Cell Migration ◽  
Carotid Artery ◽  
Vascular Injury ◽  
Camp Signaling ◽  
Blue Staining ◽  
Wild Type ◽  
Wound Healing Assay ◽  
Endothelial Repair ◽  
Endothelial Migration

Background and Purpose: Endothelial repair upon vascular injury is critical for the protection of vessel integrity and prevention of the development of vascular disorders, but the underlying mechanisms remain poorly understood. In this study, we investigated the role of zyxin and its associated cyclic adenosine monophosphate (cAMP) signaling in the regulation of re-endothelialization after vascular injury.Experimental Approach: In zyxin-/- and wild-type mice, wire injury of the carotid artery was carried out, followed by Evans blue staining, to evaluate the re-endothelialization. Mice with endothelium-specific zyxin knockout were used to further determine its role. An in vitro wound-healing assay was performed in primary human endothelial cells (ECs) expressing zyxin-specific short-hairpin RNAs (shRNAs) or scrambled controls by measuring cell migration and proliferation. The effects of the cAMP signaling agonist forskolin were assessed.Key Results: The re-endothelialization of the injured carotid artery was impaired in zyxin-deficient mice, whereas the rate of cell proliferation was comparable with that in wild-type controls. Furthermore, endothelium-specific deletion of zyxin led to similar phenotypes. Knockdown of zyxin by shRNAs in primary human ECs significantly reduced cell migration in the wound-healing assay. Notably, forskolin enhanced endothelial migration in a dose-dependent manner, and this was dependent on zyxin through its interaction with vasodilator-stimulated phosphoprotein. In addition, forskolin promoted the re-endothelialization of the injured carotid artery, and this was compromised by zyxin deficiency.Conclusion and Implications: This study reveals zyxin as a new player in endothelial repair, which is promoted by forskolin, after vascular injury. Thus, zyxin-mediated signaling might be a potential treatment target for diseases involving vascular injury.

A Method for Quantitative Analysis of the Kinematics of Fibroblast Migration in a Monolayer Wound Model

2011 ◽  
Author(s):  
Gil Topman ◽  
Orna Sharabani-Yosef ◽  
Amit Gefen
Keyword(s):  
Wound Healing ◽  
Cell Migration ◽  
Wound Healing Assay ◽  
Complete Coverage ◽  
Time Intervals ◽  
Fibroblast Migration ◽  
Wound Model ◽  
Regular Time

A wound healing assay is simple but effective method to study cell migration in vitro. Cell migration in vitro was found to mimic migration in vivo to some extent [1,2]. In wound healing assays, a “wound” is created by either scraping or mechanically crushing cells in a monolayer, thereby forming a denuded area. Cells migrate into the denuded area to complete coverage, and thereby “heal” the wound. Micrographs at regular time intervals are captured during such experiments for analysis of the process of migration.

Wound Healing Assay for Melanoma Cell Migration

2021 ◽  
pp. 65-71
Author(s):  
Juliano T. Freitas ◽  
Ivan Jozic ◽  
Barbara Bedogni
Keyword(s):  
Wound Healing ◽  
Cell Migration ◽  
Melanoma Cell ◽  
Wound Healing Assay

Very Low Molecular Weight Heparins Retain the Capacity to Inhibit Endothelial Cell Migration and Capillary-Like Tube Formation Induced by Tumor Cells.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 3047-3047
Author(s):  
Marina Marchetti ◽  
Alfonso Vignoli ◽  
Elena Cantalino ◽  
Erika Diani ◽  
Gaia Bonacina ◽  
...  
Keyword(s):  
Molecular Weight ◽  
Wound Healing ◽  
Tumor Cells ◽  
Tube Formation ◽  
Low Molecular Weight ◽  
Wound Healing Assay ◽  
Low Molecular Weight Heparins ◽  
Microvascular Endothelium ◽  
Angiogenic Activity ◽  
Endothelial Migration

Abstract Abstract 3047 Poster Board II-1023 Introduction A beneficial effect of low molecular weight heparins (LMWH) on the survival of cancer patients is suggested by clinical data and has been attributed to a broad range of activities, including the capacity to affect the hemostatic and angiogenic properties of endothelial cells. We have previously observed that unfractionated heparin and LMWHs with a mean molecular weight (MW) of 4.5 to 5.7 KDa can effectively antagonise tumor cell-induced angiogenesis in a microvascular endothelium-based Matrigel assay (Marchetti et al, Thromb Res 2008). Aim Since this anti-angiogenic activity of heparins may depend on the mean MW and the composition of their polysaccharidic chains, this study was designed to evaluate the capacity of ‘second generation’ LMWHs, with a lower MW, to counteract the pro-angiogenic stimulus of tumor cells: i.e. the LMWH Bemiparin, that possesses the lowest MW among commercially available LMWH (i.e. 3.6 KDa), and of its ultra low MW derivative RO-14 (MW=2.5 KDa, Laboratorios Farmaceuticos Rovi S.A., Madrid, Spain). Methods Tumor cell conditioned media (TCM) were obtained from three human cell lines of different tumor types, i.e. H69 (small cell lung cancer), MDA.MB.231 (breast cancer), and NB4 (acute promyelocytic leukemia). Human microvascular endothelial cells (HMEC-1) were incubated for 24h in Matrigel with TCM, or purified proangiogenic factors (VEGF, FGF-2), in the absence or presence of increasing concentrations (0.01-0.1-1 IU/ml) of Bemiparin or RO-14. Tubule formation was photographed under inverted microscopy and quantified by an image analysis software. The same heparins were tested for their capacity to counteract endothelial migration elicited by TCM in the wound healing assay. The levels of the pro-angiogenic factors VEGF, FGF-2 and IL-1beta in TCM were quantified by ELISA. Results All three TCM induced a significant (p<0.05) increase in total tube length (43-61% mean increase) compared to control medium. These increases were dose-dependently inhibited by both Bemiparin and RO-14, with about 75-100% inhibition at the heparin concentration of 1 IU/ml. In the same experimental condition the anti-VEGF antibody inhibited by 67-98% the TCM-induced capillary tube formation. Both heparins significantly inhibited tube formation induced by standard VEGF and FGF-2. In the wound healing assay, Bemiparin and RO-14 prevented the endothelial migration induced by all three different TCM. The evaluation of proangiogenic cytokine content in TCM showed that VEGF was the main product in all of the three cancer cell lines. Conclusions Bemiparin and RO-14 counteract the proangiogenic stimulus of tumor cells and of standard proangiogenic factors on microvascular endothelium. Interestingly, the Ultra-low MWH RO-14 presents an anti-angiogenic activity similar to that of the LMWH bemiparin. This suggests that the anti-angiogenic action can be retained by heparins with shorter polysaccharidic chains, which may be of interest in patients with cancer receiving LMWH for prophylaxis or therapy of thrombosis. Disclosures No relevant conflicts of interest to declare.

scholarly journals A Novel In Vitro Wound Healing Assay to Evaluate Cell Migration

10.3791/56825 ◽  
2018 ◽  
Author(s):  
Floriana Cappiello ◽  
Bruno Casciaro ◽  
Maria Luisa Mangoni
Keyword(s):  
Wound Healing ◽  
Cell Migration ◽  
Wound Healing Assay

scholarly journals Anti-cancer effect of dihydrokaempferol in human malignant melanoma cell is mediated via inhibition of cell migration and invasion and up-regulation of NF-kB/MAPK signalling pathways

2016 ◽  
Vol 11 (4) ◽  
pp. 810
Author(s):  
Ning Zeng ◽  
Hong Qiu ◽  
Min Wu ◽  
Yi Xu ◽  
Hai-Ping Wang ◽  
...  
Keyword(s):  
Wound Healing ◽  
Cell Migration ◽  
Malignant Melanoma ◽  
Signalling Pathways ◽  
Migration And Invasion ◽  
Wound Healing Assay ◽  
Human Malignant Melanoma ◽  
Cell Migration And Invasion ◽  
Mapk Signalling

<p class="Abstract">The purpose of the present research work was to demonstrate the antitumor activity of dihydrokaempferol in SK-Mel-28 human malignant melanoma cells. MTT assay was used to study the cytotoxic effects induced by dihydrokaempferol in these cells. In vitro wound healing assay and invasion assay were used to examine its effects on cell migration and invasion. Fluorescence microscopy using acridine orange/propidium iodide was used to study effects on cell morphology and apoptosis. Western blot assay revealed its effects on NF-kB/mitogen-activated protein kinase (MAPK) protein expression levels. The results indicated that dihydrokaempferol significantly inhibited the growth of these cells and the cytotoxicity pattern was shown to follow the drug dose and incubation times. Dihydrokaempferol led to onset of red fluorescence in these cells indicating that its treatment with different doses leads to induction of apoptosis. Dihydrokaempferol also led to inhibition of cell migration and invasion in a dose-dependent manner. It was also shown to up-regulate NF-kB/MAPK signalling pathways.</p><p class="Abstract"><strong>Video Clip:</strong></p><p class="Abstract"><a href="https://youtube.com/v/g8vkXiPHG4A"><em>In vitro</em> wound healing assay:</a> 4 min 25 sec</p><p> </p>

scholarly journals A microfluidic wound-healing assay for quantifying endothelial cell migration

2010 ◽  
Vol 298 (2) ◽  
pp. H719-H725 ◽  
Author(s):  
Andries D. van der Meer ◽  
Kim Vermeul ◽  
André A. Poot ◽  
Jan Feijen ◽  
István Vermes
Keyword(s):  
Wound Healing ◽  
Shear Stress ◽  
Cell Migration ◽  
Endothelial Cell ◽  
Migration Rate ◽  
Microfluidic Channel ◽  
Endothelial Cell Migration ◽  
Wound Healing Assay ◽  
Gradient Generation ◽  
Endothelial Growth Factor

Endothelial migration is an important process in the formation of blood vessels and the repair of damaged tissue. To study this process in the laboratory, versatile and reliable migration assays are essential. The purpose of this study was to investigate whether the microfluidic version of the conventional wound-healing assay is a useful research tool for vascular science. Endothelial cells were seeded in a 500-μm-wide microfluidic channel. After overnight incubation, cells had formed a viable and confluent monolayer. Then, a wound was generated in this monolayer by flushing the channel with three parallel fluid streams, of which the middle one contained the protease trypsin. By analyzing the closing of the wound over time, endothelial cell migration could be measured. Although the migration rate was two times lower in the microfluidic assay than in the conventional assay, an identical 1.5-times increase in migration rate was found in both assays when vascular endothelial growth factor (VEGF165) was added. In the microfluidic wound-healing assay, a stable gradient of VEGF165 could be generated at the wound edge. This led to a two-times increase in migration rate compared with the untreated control. Finally, when a shear stress of 1.3 Pa was applied to the wound, the migration rate increased 1.8 times. In conclusion, the microfluidic assay is a solid alternative for the conventional wound-healing assay when endothelial cell migration is measured. Moreover, it offers unique advantages, such as gradient generation and application of shear stress.

scholarly journals An on-chip wound healing assay fabricated by xurography for evaluation of dermal fibroblast cell migration and wound closure

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Ghazal Shabestani Monfared ◽  
Peter Ertl ◽  
Mario Rothbauer
Keyword(s):  
Wound Healing ◽  
Cell Migration ◽  
Wound Closure ◽  
Dermal Fibroblast ◽  
Lab On A Chip ◽  
Fibroblast Cell ◽  
Serum Starvation ◽  
Circular Membrane ◽  
Wound Healing Assay ◽  
On Chip

Abstract Dermal fibroblast cell migration is a key process in a physiological wound healing. Therefore, the analysis of cell migration is crucial for wound healing research. In this study, lab-on-a-chip technology was used to investigate the effects of basic fibroblast growth factor (bFGF), mitomycin C (MMC), MEK1/2 inhibitor (U0126) and fetal calf serum (FCS) on human dermal fibroblast cell migration. The microdevice was fabricated consisting of microchannels, pneumatic lines and pneumatically-activated actuators by xurographic rapid prototyping. In contrast to current approaches in in vitro wound healing such as scratch assays and silicone inserts in wellplate format, which show high variability and poor reproducibility, the current system aims to automate the wounding procedure at high precision and reproducibility using lab-on-a-chip. Traumatic wounding was simulated on-chip on fibroblast cell monolayers by applying air pressure on the flexible circular membrane actuator. Wound closure was monitored using light microscopy and cell migration was evaluated using image analysis. The pneumatically controlled system generates highly reproducible wound sizes compared to the conventional wound healing assay. As proof-of-principle study wound healing was investigated in the presence of several stimulatory and inhibitory substances and culture including bFGF, MMC, U0126 MEK1/2 inhibitor as well as serum starvation to demonstrate the broad applicability of the proposed miniaturized culture microsystem.

scholarly journals Research Techniques Made Simple: Analysis of Collective Cell Migration Using the Wound Healing Assay

2017 ◽  
Vol 137 (2) ◽  
pp. e11-e16 ◽  
Author(s):  
Ayman Grada ◽  
Marta Otero-Vinas ◽  
Francisco Prieto-Castrillo ◽  
Zaidal Obagi ◽  
Vincent Falanga
Keyword(s):  
Wound Healing ◽  
Cell Migration ◽  
Collective Cell Migration ◽  
Wound Healing Assay ◽  
Simple Analysis ◽  
Research Techniques
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