The changes in endothelial cytoskeleton and calcium in vascular barrier breakdown: a response of ever‐growing complexity

The endothelium forms a tightly regulated semi-permeable barrier that dictates the rate of passage of fluid and macromolecules from the vessel lumen towards the surrounding extravascular space. This endothelial barrier is a critical aspect of fluid homeostasis. Many inflammatory stimuli promote the disruption of this barrier, leading to the formation of tissue edema. While this is a necessary aspect of the inflammatory response, when dysregulated, it can lead to tissue damage and organ injury, such as during acute respiratory distress syndrome. Given its importance, it is not surprising that multiple mediators regulate the strength of this barrier, including a large number of inflammatory and edemagenic factors that include cytokines, growth factors, proteases, and small molecules acting systemically or in a paracrine fashion to activate a plethora of intracellular signaling mechanisms. As Hamilton et al. now show in their article in this issue of Pulmonary Circulation, a novel immunophilin is a new player in this complex system of endothelial regulators. The best understood mechanisms of endothelial barrier disruption involve acute responses that lead to short-term opening of the barrier, mediated in large part by the activation of the Src family of kinases and the Rho family of small GTPases. However, many aspects of this response are still a matter of intense research. Some the questions that are still unanswered are: How do these pathways become activated? When is the activation of these pathways required? For which edemagenic mediators and under which pathological conditions? Thrombin, a protease originally identified as the enzyme that mediates the cleavage of fibrin to regulate clotting, promotes in cultured endothelial cells a dramatic increase in permeability that lasts for approximately 2 h via the activation of the protease-activated receptor (PAR) 1, a member of the G protein-coupled receptors (GPCR) superfamily. PAR1 activation promotes RhoA-mediated actin stress fibers formation and the disassembly of the endothelial cell–cell junctions, thus leading to monolayer gap formation barrier breakdown. Multiple GPCRs induce calcium entry via the store-operated calcium entry (SOCE) pathway, causing RhoA and myosin light chain kinase (MLCK) activation to form new actin stress fibers. This way, SOCE promotes an acute endothelial response that includes a sharp decrease in barrier function that is very similar to thrombin. Whether calcium mediates thrombin/PAR1-induced endothelial permeability increases is still a matter of active debate. SOCE is a physiological cellular response to the depletion of inositol 1,4,5-trisphosphate (IP3)-sensitive endoplasmic reticulum calcium contents (Fig. 1). This Ca2þ depletion promotes extracellular calcium entry via the activation of several plasma membrane channels, including the highly selective Ca2þ release-activated Ca2þ (CRAC) channel (mediated by STIM-1/Orai-1) and the less Ca2þ-selective store-operated channel (SOC) mediated by the transient receptor potential (TRP) proteins. Normally, endoplasmic reticulum calcium stores are maintained through the activity of the sarcoplasmic/endoplasmic reticulum calcium ATPase (SERCA), a Ca2þ pump. Thapsigargin, a SERCA inhibitor, prevents Ca2þ pumping into the endoplasmic reticulum, thus effectively inducing the depletion of the calcium stores (Fig. 1). It has been widely used to induce SOCE independently of IP3. Thapsigargin can also induce other responses that are initiated by endoplasmic reticulum Ca2þ depletion, such as the unfolded protein response. In the paper published in this issue, Hamilton et al. used thapsigargin to study a new regulator of SOCE-induced barrier function loss. Using rat pulmonary artery endothelial cells (PAECs) as a model, the authors show that the immunophilin FK506 binding protein (FKBP) 51, a protein that was previously known to bind SOC channel components, negatively regulates an inward Ca2þ current in cells treated with thapsigargin that is compatible with the SOC current. Cells overexpressing FKBP51 not only show reduced SOC current, but also a stabilization of microtubules, reduced formation of actin stress fibers, and a drastically diminished barrier function loss after thapsigargin treatment, mechanistically linking SOCE and FKBP51 to cytoskeletal changes and endothelial barrier disruption. Because of the observed