Isolation of Human Umbilical Vein Endothelial Cells and Their Use in the Study of Neutrophil Transmigration Under Flow Conditions

Ganguly, Anutosh; Zhang, Hong; Sharma, Ritu; Parsons, Sean A.; Patel, Kamala D.

doi:10.3791/4032

Cited by 32 publications

(34 citation statements)

References 17 publications

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“…Despite blocking .85% of FAK protein expression and completely blocking FAK phosphorylation, downregulation of FAK had no effect on GATA6, VCAM-1 or CCL26 expression. Pyk2 is functionally related to FAK, expressed in endothelial cells, and can also be regulated by FRNK (Govindarajan et al, 2000;Schaller, 2010;Vockel and Vestweber, 2013). When we examined the effect of Pyk2 knockdown alone or in conjunction with FAK in this model Fig.…”

Section: Discussionmentioning

confidence: 99%

“…Pyk2 is functionally related to FAK, expressed in endothelial cells and can also be regulated by FRNK (Govindarajan et al, 2000;Fig. 3.…”

Section: Effect Of Gata6 On Il-4-mediated Expression Of Vcam-1 and Ccl26mentioning

confidence: 99%

“…Eosinophils were typically .95% pure, with lymphocytes as the main contaminating cell type. Endothelial cells were isolated from human umbilical cords (Foothills Hospital, Calgary, Alberta, Canada) as previously described, HUVECs were maintained in M199 medium (Invitrogen, Carlsbad, CA) supplemented with 20% human serum (Ganguly et al, 2012). Cells were used at first passage.…”

Section: Cell Isolation and Culturementioning

confidence: 99%

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Focal adhesion kinase-related nonkinase (FRNK) negatively regulates IL-4-mediated inflammation

Sharma

Colarusso

Zhang

et al. 2015

Journal of Cell Science

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Focal adhesion kinase (FAK)-related nonkinase (PTK2 isoform 6 in humans, hereafter referred to as FRNK) is a cytoskeletal regulatory protein that has recently been shown to dampen lung fibrosis, yet its role in inflammation is unknown. Here, we show for the first time that expression of FRNK negatively regulates IL-4-mediated inflammation in a human model of eosinophil recruitment. Mechanistically, FRNK blocks eosinophil accumulation, firm adhesion and transmigration by preventing transcription and protein expression of VCAM-1 and CCL26. IL-4 activates STAT6 to induce VCAM-1 and CCL26 transcription. We now show that IL-4 also increases GATA6 to induce VCAM-1 expression. FRNK blocks IL-4-induced GATA6 transcription but has little effect on GATA6 protein expression and no effect on STAT6 activation. FRNK can block FAK or Pyk2 signaling and we, thus, downregulated these proteins using siRNA to determine whether signaling from either protein is involved in the regulation of VCAM-1 and CCL26. Knockdown of FAK, Pyk2 or both had no effect on VCAM-1 or CCL26 expression, which suggests that FRNK acts independently of FAK and Pyk2 signaling. Finally, we found that IL-4 induces the late expression of endogenous FRNK. In summary, FRNK represents a novel mechanism to negatively regulate IL-4-mediated inflammation.

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Section: Discussionmentioning

confidence: 99%

“…Pyk2 is functionally related to FAK, expressed in endothelial cells and can also be regulated by FRNK (Govindarajan et al, 2000;Fig. 3.…”

Section: Effect Of Gata6 On Il-4-mediated Expression Of Vcam-1 and Ccl26mentioning

confidence: 99%

Section: Cell Isolation and Culturementioning

confidence: 99%

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Focal adhesion kinase-related nonkinase (FRNK) negatively regulates IL-4-mediated inflammation

Sharma

Colarusso

Zhang

et al. 2015

Journal of Cell Science

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“…This can be used to observe the interaction of relevant primary cells with the multilayers in contact with the implant such as epithelial cells or endothelial cells. Either isolated 25 or commercially available cells can be used. This film layer provides a surface where an inner lining for a tubular organ can be developed.…”

Section: Discussionmentioning

confidence: 99%

Multi-Scale Modification of Metallic Implants With Pore Gradients, Polyelectrolytes and Their Indirect Monitoring <em>In vivo</em>

Vrana

Dupret‐Bories

Chaubaroux

et al. 2013

JoVE

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Metallic implants, especially titanium implants, are widely used in clinical applications. Tissue in-growth and integration to these implants in the tissues are important parameters for successful clinical outcomes. In order to improve tissue integration, porous metallic implants have being developed. Open porosity of metallic foams is very advantageous, since the pore areas can be functionalized without compromising the mechanical properties of the whole structure. Here we describe such modifications using porous titanium implants based on titanium microbeads. By using inherent physical properties such as hydrophobicity of titanium, it is possible to obtain hydrophobic pore gradients within microbead based metallic implants and at the same time to have a basement membrane mimic based on hydrophilic, natural polymers. 3D pore gradients are formed by synthetic polymers such as Poly-L-lactic acid (PLLA) by freeze-extraction method. 2D nanofibrillar surfaces are formed by using collagen/alginate followed by a crosslinking step with a natural crosslinker (genipin). This nanofibrillar film was built up by layer by layer (LbL) deposition method of the two oppositely charged molecules, collagen and alginate. Finally, an implant where different areas can accommodate different cell types, as this is necessary for many multicellular tissues, can be obtained. By, this way cellular movement in different directions by different cell types can be controlled. Such a system is described for the specific case of trachea regeneration, but it can be modified for other target organs. Analysis of cell migration and the possible methods for creating different pore gradients are elaborated. The next step in the analysis of such implants is their characterization after implantation. However, histological analysis of metallic implants is a long and cumbersome process, thus for monitoring host reaction to metallic implants in vivo an alternative method based on monitoring CGA and different blood proteins is also described. These methods can be used for developing in vitro custom-made migration and colonization tests and also be used for analysis of functionalized metallic implants in vivo without histology. Video LinkThe video component of this article can be found at

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“…The vessel wall is modeled by either coating of biomolecules or growth of cells on one surface of the flow chamber 3 . Particles [4][5][6][7] or cells [8][9][10][11][12][13][14][15][16] are then flowed in at desired range of flow rates to quantify the number of adhering particles under various shear rates.…”

Section: Introductionmentioning

confidence: 99%

Generation of Shear Adhesion Map Using SynVivo Synthetic Microvascular Networks

Prabhakarpandian

Pant

2014

JoVE

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Cell/particle adhesion assays are critical to understanding the biochemical interactions involved in disease pathophysiology and have important applications in the quest for the development of novel therapeutics. Assays using static conditions fail to capture the dependence of adhesion on shear, limiting their correlation with in vivo environment. Parallel plate flow chambers that quantify adhesion under physiological fluid flow need multiple experiments for the generation of a shear adhesion map. In addition, they do not represent the in vivo scale and morphology and require large volumes (~ml) of reagents for experiments. In this study, we demonstrate the generation of shear adhesion map from a single experiment using a microvascular network based microfluidic device, SynVivo-SMN. This device recreates the complex in vivo vasculature including geometric scale, morphological elements, flow features and cellular interactions in an in vitro format, thereby providing a biologically realistic environment for basic and applied research in cellular behavior, drug delivery, and drug discovery. The assay was demonstrated by studying the interaction of the 2 µm biotin-coated particles with avidin-coated surfaces of the microchip. The entire range of shear observed in the microvasculature is obtained in a single assay enabling adhesion vs. shear map for the particles under physiological conditions. Video LinkThe video component of this article can be found at

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Isolation of Human Umbilical Vein Endothelial Cells and Their Use in the Study of Neutrophil Transmigration Under Flow Conditions

Cited by 32 publications

References 17 publications

Focal adhesion kinase-related nonkinase (FRNK) negatively regulates IL-4-mediated inflammation

Focal adhesion kinase-related nonkinase (FRNK) negatively regulates IL-4-mediated inflammation

Multi-Scale Modification of Metallic Implants With Pore Gradients, Polyelectrolytes and Their Indirect Monitoring <em>In vivo</em>

Generation of Shear Adhesion Map Using SynVivo Synthetic Microvascular Networks

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