The role of endothelial cell vimentin in the regulation of transendothelial migration of leukocytes

Project: Research project

Project Details

Description

During inflammation, the body's response to tissue damage of virtually any kind, leukocytes are recruited via the bloodstream to the site of injury, where they bind to the walls of local post-capillary venules and then extravasate into their target tissues. This process is called transendothelial migration (TEM). While many molecules on the surface of the leukocytes and endothelial cells (ECs) and within the ECs are involved in this process, the final common mechanism required for TEM appears to be the movement of the lateral border recycling compartment (LBRC), an interconnected reticulum of peri-junctional membrane, to the site of leukocyte/endothelial cell interaction. We focus on EC vimentin, an intermediate filament protein enriched in purified LBRC fractions. Our preliminary data suggest that vimentin has a structural and/or functional role in the normal functioning of the LBRC. The goal is to deduce how EC vimentin functions in the big picture of TEM, and in particular, how it interacts with the LBRC.

The specific aims are to define the role of endothelial cell vimentin on TEM in vitro and in vivo. In vitro studies will provide mechanistic data that are more difficult to obtain in vivo. We will use shRNA transfection to knock down vimentin in EC or Withaferin A to depolymerize it and study the effect on TEM in a well-validated assay. Based on preliminary data, I expect to see a substantial block in TEM. I will rescue vimentin expression using an shRNA-resistant cDNA and expect to see TEM return to control levels, if the shRNA has no off-target effects. Next I will study the effect of EC vimentin knockdown on the LBRC structure and location in the EC using immuno-electron microscopy. I will conduct targeted recycling assays in order to visualize if the LBRC recycling is impaired in the EC with vimentin KD. These studies will tell us whether vimentin is critical for the structure or the function (or both) of the LBRC. For in vivo studies, I will use a tamoxifen-inducible endothelial-specific vimentin KO mouse to compare to non-tamoxifen-treated littermate controls. In the croton oil dermatitis model, I will use confocal microscopy to visualize the stage in TEM at which neutrophil TEM is arrested. In the inflamed mouse cremaster muscle, I will study neutrophil extravasation in real-time by spinning disc confocal intravital microscopy to determine how vimentin in ECs affects the kinetic parameters of TEM.
StatusActive
Effective start/end date4/1/213/31/23

Funding

  • American Heart Association (834372)

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