Talin-Dependent Integrin Activation Regulates VE-Cadherin Localization and Endothelial Cell Barrier Function

Abstract

Rationale: Endothelial barrier function depends on the proper localization and function of the adherens junction protein VE-cadherin. Previous studies have suggested a functional relationship between integrin-mediated adhesion complexes and VE-cadherin yet the underlying molecular links are unclear. Binding of the cytoskeletal adaptor protein talin to the β integrin cytoplasmic domain is a key final step in regulating the affinity of integrins for extracellular ligands (activation) but the role of integrin activation in VE-cadherin mediated endothelial barrier function is unknown. Objective: To test the requirement of talin-dependent activation of β1 integrin in VE-cadherin organization and endothelial cell barrier function. Methods and Results: Endothelial cell-specific deletion of talin in adult mice resulted in impaired stability of intestinal microvascular blood vessels, hemorrhage and death. Talin-deficient endothelium showed altered VE-cadherin organization at endothelial cell-cell junctions in vivo. shRNA-mediated knockdown of talin1 expression in cultured endothelial cells led to increased radial actin stress fibers, increased adherens junction width and increased endothelial monolayer permeability measured by electrical cell-substrate impedance sensing. Restoring β1 integrin activation in talin-deficient cells with a β1 integrin activating antibody normalized both VEcadherin organization and endothelial cell barrier function. In addition, VE-cadherin organization was normalized by re-expression of talin or integrin activating talin head domain but not a talin head domain mutant that is selectively deficient in activating integrins.

Integrin-dependent regulation of the endothelial barrier

“…Interestingly, deletion of talin1 (Tln1), a key modulator of integrin activation, in the ECs of established vessels induced intestinal vascular leak, cell-cell junction disassembly and lethality 16-21 days after Tln1 deletion. 31 Loss of EC Tln1 reduced β1 integrin activation and cell-cell junctions of tln1-depleted ECs displayed significant VE-cadherin disorganization. Deletion of tln1 in vitro resulted in reduced monolayer barrier function measured by electrical cell-substrate impedance sensing (ECIS).…”

“…2,4,9,28,29 Notably, both integrin and cadherin adhesion complexes share important components, including vinculin, that function to link these complexes to the actin cytoskeleton. Integrins themselves have been observed at the EC junctions 30,31 but the significance of this observation is unclear.…”

Integrin Affinity Modulation Critically Regulates Atherogenic Endothelial Activationin vitroandin vivo

“…While talin1 reexpression restores endothelial adherens junction structure, rescuing integrin activation with the talin1 head domain (but not a L325R head domain mutant) only partially reverts this phenotype. These data suggest an important role for both talin1dependent integrin activation and talin1-mediated actin binding in maintaining vascular barrier function 21 . Similarly, β1 integrin expression is crucial for the development of stable, non-leaky vessels 42 .…”

“…Global knockout of talin1 is embryonic lethal at 8.5-9.5 days and associated with vascular defects 20 . Inducible talin1 deletion in endothelial cells results in an unstable intestinal vasculature resulting, in hemorrhage and premature death 21 . However, these effects may be due to talin1's role in maintaining cell adhesion rather than integrin activation.…”

“…A mutation in the talin1 head domain that selectively impairs the ability of talin1 to bind integrin β subunit MPR (L325R) 22,23 prevents integrin activation without affecting talin1's ability to bind the β-integrin subunit and form focal adhesions. Mice expressing only this L325R talin1 mutant in platelets and neutrophils show defects in thrombus formation and neutrophil homing to sites of inflammation, respectively 21,22 . Therefore, we sought to utilize this model to characterize the importance of talin1-dependent integrin activation in endothelial phenotype in the context of atherogenic endothelial activation.…”