Molecular mechanisms of endothelial hyperpermeability: implications in inflammation

Abstract

Endothelial hyperpermeability is a significant problem in vascular inflammation associated with trauma, ischaemia-reperfusion injury, sepsis, adult respiratory distress syndrome, diabetes, thrombosis and cancer. An important mechanism underlying this process is increased paracellular leakage of plasma fluid and protein. Inflammatory stimuli such as histamine, thrombin, vascular endothelial growth factor and activated neutrophils can cause dissociation of cell-cell junctions between endothelial cells as well as cytoskeleton contraction, leading to a widened intercellular space that facilitates transendothelial flux. Such structural changes initiate with agonist-receptor binding, followed by activation of intracellular signalling molecules including calcium, protein kinase C, tyrosine kinases, myosin light chain kinase, and small Rho-GTPases; these kinases and GTPases then phosphorylate or alter the conformation of different subcellular components that control cell-cell adhesion, resulting in paracellular hypermeability. Targeting key signalling molecules that mediate endothelial-junction-cytoskeleton dissociation demonstrates a therapeutic potential to improve vascular barrier function during inflammatory injury.

Figure 1. Schematic diagram of microvascular endothelial barrier structure

The barrier is formed by endothelial cells that connect to each other through the junctional adhesive molecule vascular endothelial (VE)-cadherin, which binds to another VE-cadherin molecule from an adjacent cell and connects to the actin cytoskeleton via a family of catenins (α, β, γ and p120). This endothelial lining is tethered to the extracellular matrix through focal adhesions mediated by transmembrane integrins composed of α and β subunits, focal adhesion kinase (FAK), and cytoskeleton-linking proteins including paxillin and vinculin. The integrity of this barrier is maintained by VE-cadherin-mediated cell–cell adhesions and focal-adhesion-supported cell–matrix attachment. A dynamic interaction among these structural elements controls the opening and closing of the paracellular pathways for fluid, proteins and cells to move across the endothelium. In particular, the Ca²⁺/calmodulin (CaM)-dependent myosin light chain kinase (MLCK) catalyses phosphorylation of myosin light chains (small red circles), triggering binding of the myosin heavy chain motor domains to actin and their cross-bridge movement. This reaction promotes cytoskeleton contraction and cell retraction. In parallel, phosphorylation of VE-cadherin and/or catenins may cause the junction complex to dissociate from its cytoskeletal anchor, leading to weakened cell–cell adhesion. The cytoskeletal and junctional responses act in concert causing paracellular hyperpermeability. These structural changes are caused by signalling reactions depicted in Figure 2.

Figure 2. Signal transduction in endothelial hyperpermeability

(Legend; see previous page for figure) Multiple cascades of intracellular signalling reactions are initiated when an inflammatory agonist binds to its respective receptor expressed on the endothelial surface [e.g. thrombin binds the protease-activated receptor 1 (PAR-1), histamine binds its receptor H₁, and vascular endothelial growth factor (VEGF) binds its receptor VEGFR-2 (KDR)]. Occupancy of G-protein-coupled receptors activates RhoA and its effector kinase ROCK (left), or it triggers phospholipase (PLC)-catalysed protein kinase C (PKC) activation and elevated intracellular calcium, which stimulates nitric oxide production and cGMP-dependent protein kinase (PKG) activation (right). Agonist binding of receptor tyrosine kinase also activates the mitogen-activated protein (MAP) kinase cascades characterised by phosphorylation of extracellular-signal-regulated kinases (ERK1/2) (middle). The three pathways (Rho GTPases, MAP kinases and protein kinases) interact with each other, causing changes in the endothelial barrier structure. Abbreviations: cGMP, cyclic guanosine monophosphate; CRAF, Raf-1; DAG, diacylglycerol; ER, endoplasmic reticulum; GC, guanylate cyclase; GDP, guanosine diphosphate; GEF-H1, guanine-nucleotide-exchange factor H1; GRB2, growth factor receptor-bound protein 2; GTP, guanosine triphosphate; JNK, c-Jun N-terminal kinase; JNKK, c-Jun N-terminal kinase kinase; l-Arg, l-arginine; NO, nitric oxide; NOS, nitric oxide synthase; p115RhoGEF, 115 kDa guanine-nucleotide-exchange factor; p190RhoGAP, p190 Rho GTPase-activating protein; Ras, ras gene product; Rho-GDI, GDP dissociation inhibitor (GDI); Rho-GDl, Rho GDP-dissociation inhibitor 1; ROCK, Rho kinase; RTK, receptor tyrosine kinase; SAPK, stress-activated protein kinase; SEK, stress-activated protein kinase/ERK kinase; Sos, Son of sevenless; Src, src gene product.