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Publications (2)18.98 Total impact

  • [Show abstract] [Hide abstract] ABSTRACT: Epac1 and its effector Rap1 are important mediators of cAMP induced tightening of endothelial junctions and consequential increased barrier function. We have investigated the involvement of Rap1 signalling in basal, unstimulated, barrier function of a confluent monolayer of HUVEC using real time Electric Cell-substrate Impedance Sensing. Depletion of Rap1, but not Epac1, results in a strong decrease in barrier function. This decrease is also observed when cells are depleted of the cAMP independent Rap exchange factors PDZ-GEF1 and 2, showing that PDZ-GEFs are responsible for Rap1 activity in control of basal barrier function. Monolayers of cells depleted of PDZ-GEF or Rap1 show an irregular, zipper-like organization of VE-cadherin and live imaging of VE-cadherin-GFP reveals enhanced junction motility upon depletion of PDZ-GEF or Rap1. Importantly, activation of Epac1 increases the formation of cortical actin bundles at the cell-cell junctions, inhibits junction motility and restores barrier function of PDZ-GEFs depleted, but not Rap1 depleted cells. We conclude that PDZ-GEF activates Rap1 under resting conditions to stabilize cell-cell junctions and maintain basal integrity. Activation of Rap1 by cAMP/Epac1 induces junctional actin to further tighten cell-cell contacts.
    No preview · Article · Aug 2011 · Cellular Signalling
  • [Show abstract] [Hide abstract] ABSTRACT: Resection of primary colorectal cancer is associated with enhanced risk of development of liver metastases. It was previously demonstrated that surgery initiated an early inflammatory response resulting in elevated tumour cell adhesion in the liver. Because reactive oxygen species (ROS) are shown to be produced and released during surgery, the effects of ROS on the liver vascular lining and tumour cell adhesion were investigated. Human endothelial cell monolayers (human umbilical vein endothelial cells (HUVECs) and human microvascular endothelial cells of the lung (HMEC-1s)) were exposed to ROS production, after which electrical impedance, cellular integrity and tumour cell adhesion were investigated. Furthermore, surgery-induced tumour cell adhesion as well as the role of ROS and liver macrophages (Kupffer cells) in this process were studied in vivo. Production of ROS decreased cellular impedance of endothelial monolayers dramatically. Moreover, formation of intercellular gaps in endothelial monolayers was observed, exposing subendothelial extracellular matrix (ECM) on which colon carcinoma cells adhered via integrin molecules. Endothelial damage was, however, prevented in the presence of ROS-scavenging enzymes. Additionally, surgery induced downregulation of both rat and human liver tight junction molecules. Treatment of rats with the ROS scavenger edaravone prevented surgery-induced tumour cell adhesion and downregulation of tight junction proteins in the liver. Interestingly, depletion of Kupffer cells prior to surgery significantly reduced the numbers of adhered tumour cells and prevented disruption of expression of tight junction proteins. In this study it is shown that surgery-induced ROS production by macrophages damages the vascular lining by downregulating tight junction proteins. This leads to exposure of ECM, to which circulating tumour cells bind. In light of this, perioperative therapeutic intervention, preventing surgery-induced inflammatory reactions, may reduce the risk of developing liver metastases, thereby improving the clinical outcome of patients with colorectal cancer.
    No preview · Article · Jul 2011 · Gut