Human Pulmonary Endothelial Cell Monolayers Research Articles

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.

Dasatinib increases endothelial permeability leading to pleural effusion

Pleural effusion is a frequent side effect of dasatinib, a second generation BCR-ABL tyrosine kinase inhibitor used in the treatment of chronic myelogenous leukaemia. However, the underlying mechanisms remain unknown. We hypothesized that dasatinib alters endothelial integrity, resulting in increased pulmonary vascular endothelial permeability and pleural effusion. To test this, we established the first animal model of dasatinib-related pleural effusion, by treating rats with a daily regimen of high doses of dasatinib (10 mg/kg/day for 8 weeks). Pleural ultrasonography revealed that rats chronically treated with dasatinib developed pleural effusion after 5 weeks. Consistent with these in vivo observations, dasatinib led to a rapid and reversible increase in paracellular permeability of human pulmonary endothelial cell monolayers as reflected by increased macromolecule passage, loss of VE-cadherin and ZO-1 from cell-cell junctions, and development of actin stress fibers. These results were replicated using HUVECs and confirmed by decreased endothelial resistance. Interestingly, we demonstrated that this increased endothelial permeability is a reactive oxygen species (ROS) dependent mechanism in vitro and in vivo using a co-treatment with an anti-oxidant agent, N-acetyl cysteine (NAC). This study shows that dasatinib alters pulmonary endothelial permeability in a ROS-dependent manner in vitro and in vivo leading to pleural effusion.

Dasatinib increases endothelial permeability leading to pleural effusion.

Pleural effusion is a frequent side-effect of dasatinib, a second-generation tyrosine kinase inhibitor used in the treatment of chronic myelogenous leukaemia. However, the underlying mechanisms remain unknown. We hypothesised that dasatinib alters endothelial integrity, resulting in increased pulmonary vascular endothelial permeability and pleural effusion.To test this, we established the first animal model of dasatinib-related pleural effusion, by treating rats with a daily regimen of high doses of dasatinib (10 mg·kg-1·day-1 for 8 weeks).Pleural ultrasonography revealed that rats chronically treated with dasatinib developed pleural effusion after 5 weeks. Consistent with these in vivo observations, dasatinib led to a rapid and reversible increase in paracellular permeability of human pulmonary endothelial cell monolayers as reflected by increased macromolecule passage, loss of vascular endothelial cadherin and zonula occludens-1 from cell-cell junctions, and the development of actin stress fibres. These results were replicated using human umbilical vein endothelial cells and confirmed by decreased endothelial resistance. Interestingly, we demonstrated that this increased endothelial permeability is a reactive oxygen species (ROS)-dependent mechanism in vitro and in vivo using a cotreatment with an antioxidant agent, N-acetylcysteine.This study shows that dasatinib alters pulmonary endothelial permeability in a ROS-dependent manner in vitro and in vivo leading to pleural effusion.

GSK-3Beta-Dependent Activation of GEF-H1/ROCK Signaling Promotes LPS-Induced Lung Vascular Endothelial Barrier Dysfunction and Acute Lung Injury.

The bacterial endotoxin or lipopolysaccharide (LPS) leads to the extensive vascular endothelial cells (EC) injury under septic conditions. Guanine nucleotide exchange factor-H1 (GEF-H1)/ROCK signaling not only involved in LPS-induced overexpression of pro-inflammatory mediator in ECs but also implicated in LPS-induced endothelial hyper-permeability. However, the mechanisms behind LPS-induced GEF-H1/ROCK signaling activation in the progress of EC injury remain incompletely understood. GEF-H1 localized on microtubules (MT) and is suppressed in its MT-bound state. MT disassembly promotes GEF-H1 release from MT and stimulates downstream ROCK-specific GEF activity. Since glycogen synthase kinase (GSK-3beta) participates in regulating MT dynamics under pathologic conditions, we examined the pivotal roles for GSK-3beta in modulating LPS-induced activation of GEF-H1/ROCK, increase of vascular endothelial permeability and severity of acute lung injury (ALI). In this study, we found that LPS induced human pulmonary endothelial cell (HPMEC) monolayers disruption accompanied by increase in GSK-3beta activity, activation of GEF-H1/ROCK signaling and decrease in beta-catenin and ZO-1 expression. Inhibition of GSK-3beta reduced HPMEC monolayers hyper-permeability and GEF-H1/ROCK activity in response to LPS. GSK-3beta/GEF-H1/ROCK signaling is implicated in regulating the expression of beta-catenin and ZO-1. In vivo, GSK-3beta inhibition attenuated LPS-induced activation of GEF-H1/ROCK pathway, lung edema and subsequent ALI. These findings present a new mechanism of GSK-3beta-dependent exacerbation of lung micro-vascular hyper-permeability and escalation of ALI via activation of GEF-H1/ROCK signaling and disruption of intracellular junctional proteins under septic condition.

Vascular Endothelial Growth Factor May Contribute to Increased Vascular Permeability in Acute Respiratory Distress Syndrome

The development of noncardiogenic pulmonary edema is a characteristic feature of acute respiratory distress syndrome (ARDS). We hypothesized that vascular endothelial growth factor (VEGF) would play an important role in this process. Plasma VEGF was measured in 40 patients with ARDS, 28 at-risk patients, 14 normal control subjects, and 9 ventilated control subjects. Cultured peripheral blood mononuclear cells (PBM) supernatant VEGF was measured in 21 patients with ARDS and 12 at-risk patients, respectively. The functional importance of VEGF as a mediator of endothelial permeability was assessed by measuring albumin flux across human pulmonary endothelial cell monolayers. Plasma VEGF was significantly elevated in patients with ARDS compared with at-risk patients, normal control subjects, and ventilated control subjects (p = 0.01, p = 0.0001, and p = 0.002, respectively). PBM from patients with ARDS produced significantly more VEGF in vitro than at-risk patients (p = 0.05). Albumin flux across human pulmonary endothelial cell monolayers was significantly increased following the addition of plasma from patients with ARDS compared with plasma from normal control subjects (p = 0.008). When VEGF activity in plasma was neutralized by the addition of a soluble VEGF inhibitor, the albumin flux induced by ARDS plasma was reduced by 48%. We conclude that VEGF makes a significant contribution to the endothelial cell permeability-inducing activity in plasma from patients with ARDS, and may play an important role in the development of noncardiogenic pulmonary edema in ARDS.