Gold nanoparticles disrupt actin organization and pulmonary endothelial barriers.

This study explored the impact of gold nanoparticles on the metabolic activity and morphology of human pulmonary endothelial cell monolayers. We developed a gold nanoparticle library of three different sizes and two surface chemistries that include anionic citrate and the cationic polyelectrolyte poly(allylamine hydrochloride). The nanoparticles were characterized in cell culture medium to assess how their physical properties are altered after exposure to biological fluids. A bovine serum albumin pretreatment protocol was developed to stabilize the nanoparticles in cell culture medium. Results of this study show that an 18 h exposure of human pulmonary artery endothelial cells to the different nanoparticles modestly affects cellular metabolic activity. However, nanoparticle exposure perturbs the cortical actin networks and induces the formation of intercellular gaps. In particular, exposure to the poly(allylamine hydrochloride)-coated particles reduces the area of cell-cell junctions-a change that correlates with increased leakiness of endothelial barriers. The presence of excess polyelectrolyte capping agents in the supernatant of poly(allylamine hydrochloride)-coated nanoparticles significantly impacts endothelial morphology. Pretreatment of the particle supernatant with bovine serum albumin mitigates the negative effects of free or bound polyelectrolytes on endothelial cell monolayers.

VE-PTP stabilizes VE-cadherin junctions and the endothelial barrier via a phosphatase-independent mechanism.

Vascular endothelial (VE) protein tyrosine phosphatase (PTP) is an endothelial-specific phosphatase that stabilizes VE-cadherin junctions. Although studies have focused on the role of VE-PTP in dephosphorylating VE-cadherin in the activated endothelium, little is known of VE-PTP's role in the quiescent endothelial monolayer. Here, we used the photoconvertible fluorescent protein VE-cadherin-Dendra2 to monitor VE-cadherin dynamics at adherens junctions (AJs) in confluent endothelial monolayers. We discovered that VE-PTP stabilizes VE-cadherin junctions by reducing the rate of VE-cadherin internalization independently of its phosphatase activity. VE-PTP serves as an adaptor protein that through binding and inhibiting the RhoGEF GEF-H1 modulates RhoA activity and tension across VE-cadherin junctions. Overexpression of the VE-PTP cytosolic domain mutant interacting with GEF-H1 in VE-PTP-depleted endothelial cells reduced GEF-H1 activity and restored VE-cadherin dynamics at AJs. Thus, VE-PTP stabilizes VE-cadherin junctions and restricts endothelial permeability by inhibiting GEF-H1, thereby limiting RhoA signaling at AJs and reducing the VE-cadherin internalization rate.

Dynamic Imaging Reveals Coordinate Effects of Cyclic Stretch and Substrate Stiffness on Endothelial Integrity.

We describe an equibiaxial cell stretcher and hybrid, elastic membrane platform designed for dynamic imaging of cells on substrates with physiological stiffness undergoing cyclic stretch. Studies enabled by this device revealed that both substrate stiffness and cyclic stretch coordinately protect pulmonary endothelial monolayers against thrombin-induced disruption. The fluorescence imaging possible with the designed hybrid membranes further revealed similarities and differences in actin and cell dynamics during monolayer recovery. The improved live-cell imaging capabilities of this platform, when used in conjunction with fluorescent probes, will have broad applications for investigations of the impact of biochemical stimuli and mechanotransduction mechanisms on mechanically perturbed tissues.

A genetic variant of cortactin linked to acute lung injury impairs lamellipodia dynamics and endothelial wound healing.

Inflammatory mediators released in acute lung injury (ALI) trigger the disruption of interendothelial junctions, leading to loss of vascular barrier function, protein-rich pulmonary edema, and severe hypoxemia. Genetic signatures that predict patient recovery or disease progression are poorly defined, but recent genetic screening of ALI patients has identified an association between lung inflammatory disease and a single nucleotide polymorphism (SNP) in the gene for the actin-binding and barrier-regulatory protein cortactin. This study investigated the impact of this disease-linked cortactin variant on wound healing processes that may contribute to endothelial barrier restoration. A microfabricated platform was used to quantify wound healing in terms of gap closure speed, lamellipodia dynamics, and cell velocity. Overexpression of wild-type cortactin in endothelial cells (ECs) improved directional cell motility and enhanced lamellipodial protrusion length, resulting in enhanced gap closure rates. By contrast, the cortactin SNP impaired wound closure and cell locomotion, consistent with the observed reduction in lamellipodial protrusion length and persistence. Overexpression of the cortactin SNP in lung ECs mitigated the barrier-enhancing activity of sphingosine 1-phosphate. These findings suggest that this common cortactin variant may functionally contribute to ALI predisposition by impeding endothelial wound healing.