Bosutinib prevents vascular leakage by reducing focal adhesion turnover and reinforcing junctional integrity.

Endothelial barrier dysfunction leads to edema and vascular leak, causing high morbidity and mortality. Previously, Abl kinase inhibition has been shown to protect against vascular leak. Using the distinct inhibitory profiles of clinically available Abl kinase inhibitors, we aimed to provide a mechanistic basis for novel treatment strategies against vascular leakage syndromes. We found that the inhibitor bosutinib most potently protected against inflammation-induced endothelial barrier disruption. In vivo, bosutinib prevented lipopolysaccharide (LPS)-induced alveolar protein extravasation in an acute lung injury mice model. Mechanistically, mitogen-activated protein 4 kinase 4 (MAP4K4) was identified as important novel mediator of endothelial permeability, which signaled via ezrin, radixin and moesin proteins to increase turnover of integrin-based focal adhesions. The combined inhibition of MAP4K4 and Abl-related gene (Arg, also known as ABL2) by bosutinib preserved adherens junction integrity and reduced turnover of focal adhesions, which synergistically act to stabilize the endothelial barrier during inflammation. We conclude that MAP4K4 is an important regulator of endothelial barrier integrity, increasing focal adhesion turnover and disruption of cell-cell junctions during inflammation. Because it inhibits both Arg and MAP4K4, use of the clinically available drug bosutinib might form a viable strategy against vascular leakage syndromes.

A pulmonary endothelial amplification loop aggravates ex-vivo transfusion-related acute lung injury via increased toll-like receptor 4 and intra-cellular adhesion molecule-1 expression.

BACKGROUND: Transfusion-Related Acute Lung Injury (TRALI) is a life-threatening complication of blood transfusions characterized by pulmonary endothelial cell damage and edema, with a high incidence in critically ill patients. The pathophysiology of TRALI is unresolved, but can generally be hypothesized to follow a 2-hit model in which the first hit is elicited by the underlying clinical condition of the patient (e.g., inflammation, which can be reflected by LPS in experimental models), and the second hit is delivered by the blood transfusion product (e.g., HLA class I antibodies). Here, we report a synergistic role for LPS and HLA class I antibody binding to pulmonary endothelium resulting in enhanced inflammatory responses. MATERIALS AND METHODS: Pulmonary endothelial cells were treated with PBS or low-dose LPS, exclusively or in combination with anti-HLA class I. Endothelial surface expression of HLA class I, TLR4, and inflammatory marker ICAM-1 were measured, and trans-endothelial migration (TEM) of neutrophils was investigated. RESULTS: LPS treatment of pulmonary endothelium enhanced HLA class I antibody binding, and combined LPS and HLA class I antibody binding enhanced TLR4 (LPS receptor) and ICAM-1 expression on the endothelial cell surface. Low-dose LPS and HLA antibody together also increased neutrophil TEM under physiological flow by on average 5-fold. CONCLUSION: We conclude that LPS and anti-HLA class I antibody have the ability to activate the pulmonary endothelium into a spiral of increasing inflammation, opening the opportunity to potentially block TLR4 to prevent or reduce the severity of TRALI in vivo.

Double-Hit-Induced Leukocyte Extravasation Driven by Endothelial Adherens Junction Destabilization.

During inflammation, endothelial cells are bombarded with cytokines and other stimuli from surrounding cells. Leukocyte extravasation and vascular leakage are both prominent but believed to be uncoupled as they occur in separate spatiotemporal patterns. In this study, we investigated a "double-hit" approach on primary human endothelial cells primed with LPS followed by histamine. Using neutrophil transendothelial migration (TEM) under physiological flow assays, we found that an LPS-primed endothelium synergistically enhanced neutrophil TEM when additionally treated with histamine, whereas the effects on neutrophil TEM of the individual stimuli were moderate to undetectable. Interestingly, the double-hit-induced TEM increase was not due to decreased endothelial barrier, increased adhesion molecule expression, or Weibel-Palade body release. Instead, we found that it was directly correlated with junctional remodeling. Compounds that increased junctional "linearity" (i.e., stability) counteracted the double-hit effect on neutrophil TEM. We conclude that a compound, in this case histamine (which has a short primary effect on vascular permeability), can have severe secondary effects on neutrophil TEM in combination with an inflammatory stimulus. This effect is due to synergic modifications of the endothelial cytoskeleton and junctional remodeling. Therefore, we hypothesize that junctional linearity is a better and more predictive readout than endothelial resistance for compounds aiming to attenuate inflammation.

Dynamin-2 is a novel NOS1ß interacting protein and negative regulator in the collecting duct.

Nitric oxide synthase 1 (NOS1)-derived nitric oxide (NO) production in collecting ducts is critical for maintaining fluid-electrolyte balance. Rat collecting ducts express both the full-length NOS1α and its truncated variant NOS1ß, while NOS1ß predominates in mouse collecting ducts. We reported that dynamin-2 (DNM2), a protein involved in excising vesicles from the plasma membrane, and NOS1α form a protein-protein interaction that promotes NO production in rat collecting ducts. NOS1ß was found to be highly expressed in human renal cortical/medullary samples; hence, we tested the hypothesis that DNM2 is a positive regulator of NOS1ß-derived NO production. COS7 and mouse inner medullary collecting duct-3 (mIMCD3) cells were transfected with NOS1ß and/or DNM2. Coimmunoprecipitation experiments show that NOS1ß and DNM2 formed a protein-protein interaction. DNM2 overexpression decreased nitrite production (index of NO) in both COS7 and mIMCD-3 cells by 50-75%. mIMCD-3 cells treated with a panel of dynamin inhibitors or DNM2 siRNA displayed increased nitrite production. To elucidate the physiological significance of IMCD DNM2/NOS1ß regulation in vivo, flox control and CDNOS1 knockout mice were placed on a high-salt diet, and freshly isolated IMCDs were treated acutely with a dynamin inhibitor. Dynamin inhibition increased nitrite production by IMCDs from flox mice. This response was blunted (but not abolished) in collecting duct-specific NOS1 knockout mice, suggesting that DNM2 also negatively regulates NOS3 in the mouse IMCD. We conclude that DNM2 is a novel negative regulator of NO production in mouse collecting ducts. We propose that DNM2 acts as a "break" to prevent excess or potentially toxic NO levels under high-salt conditions.

Endothelial cells of pulmonary origin display unique sensitivity to the bacterial endotoxin lipopolysaccharide.

Acute respiratory distress syndrome (ARDS) is a major clinical problem without available therapies. Known risks for ARDS include severe sepsis, SARS-CoV-2, gram-negative bacteria, trauma, pancreatitis, and blood transfusion. During ARDS, blood fluids and inflammatory cells enter the alveoli, preventing oxygen exchange from air into blood vessels. Reduced pulmonary endothelial barrier function, resulting in leakage of plasma from blood vessels, is one of the major determinants in ARDS. It is, however, unknown why systemic inflammation particularly targets the pulmonary endothelium, as endothelial cells (ECs) line all vessels in the vascular system of the body. In this study, we examined ECs of pulmonary, umbilical, renal, pancreatic, and cardiac origin for upregulation of adhesion molecules, ability to facilitate neutrophil (PMN) trans-endothelial migration (TEM) and for endothelial barrier function, in response to the gram-negative bacterial endotoxin LPS. Interestingly, we found that upon LPS stimulation, pulmonary ECs showed increased levels of adhesion molecules, facilitated more PMN-TEM and significantly perturbed the endothelial barrier, compared to other types of ECs. These observations could partly be explained by a higher expression of the adhesion molecule ICAM-1 on the pulmonary endothelial surface compared to other ECs. Moreover, we identified an increased expression of Cadherin-13 in pulmonary ECs, for which we demonstrated that it aids PMN-TEM in pulmonary ECs stimulated with LPS. We conclude that pulmonary ECs are uniquely sensitive to LPS, and intrinsically different, compared to ECs from other vascular beds. This may add to our understanding of the development of ARDS upon systemic inflammation.

ADAM10-Mediated Cleavage of ICAM-1 Is Involved in Neutrophil Transendothelial Migration.

To efficiently cross the endothelial barrier during inflammation, neutrophils first firmly adhere to the endothelial surface using the endothelial adhesion molecule ICAM-1. Upon actual transmigration, the release from ICAM-1 is required. While Integrin LFA1/Mac1 de-activation is one described mechanism that leads to this, direct cleavage of ICAM-1 from the endothelium represents a second option. We found that a disintegrin and metalloprotease 10 (ADAM10) cleaves the extracellular domain of ICAM-1 from the endothelial surface. Silencing or inhibiting endothelial ADAM10 impaired the efficiency of neutrophils to cross the endothelium, suggesting that neutrophils use endothelial ADAM10 to dissociate from ICAM-1. Indeed, when measuring transmigration kinetics, neutrophils took almost twice as much time to finish the diapedesis step when ADAM10 was silenced. Importantly, we found increased levels of ICAM-1 on the transmigrating neutrophils when crossing an endothelial monolayer where such increased levels were not detected when neutrophils crossed bare filters. Using ICAM-1-GFP-expressing endothelial cells, we show that ICAM-1 presence on the neutrophils can also occur by membrane transfer from the endothelium to the neutrophil. Based on these findings, we conclude that endothelial ADAM10 contributes in part to neutrophil transendothelial migration by cleaving ICAM-1, thereby supporting the release of neutrophils from the endothelium during the final diapedesis step.