The Role of the Microtubules in Tumor Necrosis Factor-α–Induced Endothelial Cell Permeability

Abstract

Tumor necrosis factor (TNF)-alpha, a major proinflammatory cytokine, triggers endothelial cell activation and barrier dysfunction which are implicated in the pathogenesis of pulmonary edema associated with acute lung injury syndromes. The mechanisms of TNF-alpha-induced vascular permeability are not completely understood. Our initial experiments demonstrated that TNF-alpha-induced decreases in transendothelial electrical resistance across human pulmonary artery endothelial cells are independent of myosin light chain phosphorylation catalyzed by either myosin light chain kinase or Rho kinase. We next assessed the involvement of another cytoskeletal component, the tubulin-based microtubule network, and found TNF-alpha to induce a decrease in stable tubulin content and partial dissolution of peripheral microtubule network as evidenced by anti-acetylated tubulin and anti-beta-tubulin immunofluorescent staining, respectively. Microtubule-stabilizing agents, paclitaxel and epothilone B, significantly attenuated TNF-alpha-induced decreases in transendothelial electrical resistance, inhibited the cytokine-induced increases in actin stress fibers, formation of intercellular gap, and restored the TNF-alpha-compromised vascular endothelial (VE)-cadherin-based cell-cell junctions. Importantly, neither TNF-alpha nor paclitaxel treatment was associated with endothelial cell apoptosis. Inhibition of p38 mitogen-activated protein kinase by SB203580 significantly attenuated TNF-alpha-induced microtubule destabilization, actin rearrangement, and endothelial barrier dysfunction. These results strongly suggest the involvement of microtubule rearrangement in TNF-alpha-induced endothelial cell permeability via p38 mitogen-activated protein kinase activation.

Probing the mechanical properties of TNF-α stimulated endothelial cell with atomic force microscopy

“…The actin filaments are known to form a network under the cell membrane providing mechanical strength and to undergo extensive remodeling and reorganization upon TNF-α stimulation. [34][35][36] The observed increase in filament density could explain the higher stiffness of the stimulated cells as compared with the controls. Also, the level of expression of ICAM-1 was measured through ELISA, demonstrating a statistically significant increase in the surface density for these adhesive molecules (Figure 4), normally overexpressed on the inflamed endothelium.…”

Agonist of growth hormone-releasing hormone reduces pneumolysin-induced pulmonary permeability edema

“…These pores facilitate increased intracellular Ca 2+ levels (9), which in turn activate mechanisms leading to contraction of endothelial cells (10,11). These pathways include both myosin light chain (MLC)-dependent mechanisms and microtubule depolymerization, the latter of which can cause disassembly of adherens junction proteins, such as vascular endothelial (VE)-cadherin (12).…”

Suppression of endotoxin-induced inflammation by taxol

“…The capacity of inhibitors of microtubule polymerisation to increase transendothelial permeability to solutes in vitro has been reported [2]. Conversely, microtubule stabilisation by taxol attenuates solute permeability across the endothelial monolayer and paracellular gap formation induced by tumour necrosis factor (TNF)-a [3] and thrombin [4]. Furthermore, stabilisation of endothelial microtubules with taxol decreases leukocyte transmigration through endothelial monolayers, whereas disassembly of microtubules increases leukocyte migration [5].…”