Reduced Tie2 in Microvascular Endothelial Cells Is Associated with Organ-Specific Adhesion Molecule Expression in Murine Health and Endotoxemia

Endothelial cells (ECs) in the microvasculature in organs are active participants in the pathophysiology of sepsis. Tyrosine protein kinase receptor Tie2 (Tek; Tunica interna Endothelial cell Kinase) is thought to play a role in their inflammatory response, yet data are inconclusive. We investigated acute endotoxemia-induced changes in the expression of Tie2 and inflammation-associated endothelial adhesion molecules E-selectin and VCAM-1 (vascular cell adhesion molecule-1) in kidneys and lungs in inducible, EC-specific Tie2 knockout mice. The extent of Tie2 knockout in healthy mice differed between microvascular beds, with low to absent expression in arterioles in kidneys and in capillaries in lungs. In kidneys, Tie2 mRNA dropped more than 70% upon challenge with lipopolysaccharide (LPS) in both genotypes, with no change in protein. In renal arterioles, tamoxifen-induced Tie2 knockout was associated with higher VCAM-1 protein expression in healthy conditions. This did not increase further upon challenge of mice with LPS, in contrast to the increased expression occurring in control mice. Also, in lungs, Tie2 mRNA levels dropped within 4 h after LPS challenge in both genotypes, while Tie2 protein levels did not change. In alveolar capillaries, where tamoxifen-induced Tie2 knockout did not affect the basal expression of either adhesion molecule, a 4-fold higher E-selectin protein expression was observed after exposure to LPS compared to controls. The here-revealed heterogeneous effects of absence of Tie2 in ECs in kidney and lung microvasculature in health and in response to acute inflammatory activation calls for further in vivo investigations into the role of Tie2 in EC behavior. </p

Requirement of respiratory support in acute bronchiolitis in infants is linked to endothelial and neutrophil activation

BACKGROUND: Evidence shows that activation of pulmonary vascular endothelium and neutrophils are involved in the pathophysiology of acute bronchiolitis. We hypothesized that levels of markers of endothelial activation and leukocyte counts are associated with requirement and duration of respiratory support. METHODS: Thirty-four infants with bronchiolitis and eight controls were included. Nasopharyngeal swabs and blood samples were taken at admission. Serum levels of Angiopoietin (Ang)-1, Ang-2, sP-selectin, sE-selectin, vascular cell adhesion molecule-1 (sVCAM-1), intercellular adhesion molecule-1 (sICAM-1), and leukocyte counts were measured. For univariate analysis, bronchiolitis cases were grouped into two groups, namely those not requiring and those requiring any form of respiratory support. To control for known risk factors for poor outcome (i.e., age, prematurity, and congenital heart disease), and for days post symptom onset, linear regression analysis was performed with duration of any type of respiratory support in days. RESULTS: Ang-2 levels, Ang-2/Ang-1 ratios, sE-selectin levels, immature neutrophil count, and neutrophil/lymphocyte ratio (NLR) were higher in acute bronchiolitis versus controls. Ang-2, and NLR levels were significantly higher, and lymphocyte counts significantly lower, in infants that required respiratory support versus those that did not. Ang-2 levels (β: .32, 95% confidence interval [CI]: 0.19-1.19) and NLR (β: .68, 95% CI: 0.17-1.19) were positive predictors for the duration of respiratory support. CONCLUSIONS: Markers of endothelial and neutrophil activation are associated with respiratory support for acute bronchiolitis. Admission Ang-2 levels and NLR may be promising markers to determine requirement of respiratory support and deserve further study

Serum concentrations of endothelial cell adhesion molecules and their shedding enzymes and early onset sepsis in newborns in Suriname

Background: Early onset sepsis (EOS) is defined as onset of sepsis within 72 hours after birth. Leucocyte-endothelial interactions play a pivotal part in EOS pathophysiology. Endothelial cell adhesion molecules (CAMs) orchestrate these interactions and their soluble isoforms (sCAMs) are released into the vasculature by enzymes called sheddases. Purpose: This study was undertaken to explore further the pathophysiology of EOS and to investigate the potential of sCAM and their sheddases as potential biomarkers for EOS. Methods: Stored serum aliquots were used from 71 Surinamese newborns suspected of EOS and 20 healthy newborns from an earlier study. Serum had been collected within 72 hours after birth and six (8.6%) newborns had a positive blood culture with gram-negative pathogens. Concentrations of sCAMs sP-selectin, sE-selectin, soluble vascular cell adhesion molecule-1, intercellular adhesion molecule-1 and platelet and endothelial cell adhesion molecule-1, sheddases matrix metalloproteinase-9 (MMP-9) and neutrophil elastase (NE) and sheddase antagonist tissue-inhibitor of metalloproteinases-1 (TIMP-1) were measured simultaneously with Luminex and ELISA. Results: MMP-9 and TIMP-1 levels were measured in serum of n=91 newborns and sCAMs and NE levels in serum of n=80 newborns, respectively. We found no differences in median concentrations of sCAMs, MMP-9 and TIMP-1 or NE between blood culture positive EOS, blood culture negative EOS and control groups at start of antibiotic treatment. Conclusions: Our data indicate that serum concentrations of sCAMs and their sheddases have no clinical utility as biomarkers for EOS. Trial registration number: NCT02486783. Results

Pattern of tamoxifen-induced Tie2 deletion in endothelial cells in mature blood vessels using endo SCL-Cre-ERT transgenic mice

Tyrosine-protein kinase receptor Tie2, also known as Tunica interna Endothelial cell Kinase or TEK plays a prominent role in endothelial responses to angiogenic and inflammatory stimuli. Here we generated a novel inducible Tie2 knockout mouse model, which targets mature (micro)vascular endothelium, enabling the study of the organ-specific contribution of Tie2 to these responses. Mice with floxed Tie2 exon 9 alleles (Tie2floxed/floxed) were crossed with end-SCL-Cre-ERT transgenic mice, generating offspring in which Tie2 exon 9 is deleted in the endothelial compartment upon tamoxifen-induced activation of Cre-recombinase (Tie2ΔE9). Successful deletion of Tie2 exon 9 in kidney, lung, heart, aorta, and liver, was accompanied by a heterogeneous, organ-dependent reduction in Tie2 mRNA and protein expression. Microvascular compartment-specific reduction in Tie2 mRNA and protein occurred in arterioles of all studied organs, in renal glomeruli, and in lung capillaries. In kidney, lung, and heart, reduced Tie2 expression was accompanied by a reduction in Tie1 mRNA expression. The heterogeneous, organ- and microvascular compartment-dependent knockout pattern of Tie2 in the Tie2floxed/floxed;end-SCL-Cre-ERT mouse model suggests that future studies using similar knockout strategies should include a meticulous analysis of the knockout extent of the gene of interest, prior to studying its role in pathological conditions, so that proper conclusions can be drawn

Pharmacological inhibition of protein tyrosine kinases axl and fyn reduces TNF-α-induced endothelial inflammatory activation in vitro

Major surgery induces systemic inflammation leading to pro-inflammatory activation of endothelial cells. Endothelial inflammation is one of the drivers of postoperative organ damage, including acute kidney injury Tumour Necrosis Factor alpha (TNF-α) is an important component of surgery-induced pro-inflammatory activation of endothelial cells. Kinases, the backbone of signalling cascades, can be targeted by pharmacological inhibition. This is a promising treatment option to interfere with excessive endothelial inflammation. In this study, we identified activated kinases as potential therapeutic targets. These targets were pharmacologically inhibited to reduce TNF-α-induced pro-inflammatory signalling in endothelial cells. Kinome profiling using PamChip arrays identified 64 protein tyrosine kinases and 88 serine-threonine kinases, the activity of which was determined at various timepoints (5–240 min) following stimulation with 10 ng/ml TNF-α in Human umbilical vein endothelial cells in vitro. The PTKs Axl and Fyn were selected based on high kinase activity profiles. Co-localisation experiments with the endothelial-specific protein CD31 showed Axl expression in endothelial cells of glomeruli and Fyn in arterioles and glomeruli of both control and TNF-α-exposed mice. Pharmacological inhibition with Axl inhibitor BMS-777607 and Fyn inhibitor PP2 significantly reduced TNF-α-induced pro-inflammatory activation of E-selectin, VCAM-1, ICAM-1, IL-6 and IL-8 at mRNA and VCAM-1, ICAM-1, and IL-6 at protein level in HUVEC in vitro. Upon pharmacological inhibition with each inhibitor, leukocyte adhesion to HUVEC was also significantly reduced, however to a minor extent. In conclusion, pre-treatment of endothelial cells with kinase inhibitors BMS-777607 and PP2 reduces TNF-α-induced endothelial inflammation in vitro

Endothelium-targeted delivery of dexamethasone by anti-VCAM-1 SAINT-O-Somes in mouse endotoxemia

Microvascular endothelial cells play a pivotal role in the pathogenesis of sepsis-induced inflammatory responses and multiple organ failure. Therefore, they represent an important target for pharmacological intervention in the treatment of sepsis. Glucocorticosteroids were widely used in the treatment of sepsis but vast evidence to support their systemic use is lacking. The limited effects of glucocorticoids in the treatment of sepsis may be explained by differential effects of drug initiated NF-κB inhibition in different cell types and insufficient drug delivery in target cells. The current study aimed therefore to investigate the effects of an endothelial targeted delivery of dexamethasone in a mouse model of endotoxemia induced by two consecutive i.p. injections of lipopolysaccharide (LPS). To achieve endothelial cell specific delivery of dexamethasone, we modified SAINT-O-Somes, a new generation of liposomes that contain the cationic amphiphile SAINT-C18 (1-methyl-4-(cis-9-dioleyl) methyl-pyridinium chloride, with antibodies against vascular cell adhesion molecule-1 (VCAM-1). In LPS challenged mice, the systemic administration of free dexamethasone had negligible effects on the microvascular inflammatory endothelial responses. Dexamethasone-loaded anti-VCAM-1 SAINT-O-Somes specifically localized at VCAM-1 expressing endothelial cells in the microvasculature of inflamed organs. This was associated with a marginal attenuation of the expression of a few pro-inflammatory genes in kidney and liver, while no effects in the lung were observed. This study reveals that, although local accumulation of the targeted drug was achieved, endothelial targeted dexamethasone containing anti-VCAM-1 SAINT-O-Somes exhibited marginal effects on inflammatory endothelial cell activation in a model of endotoxemia. Studies with more potent drugs encapsulated into anti-VCAM-1 SAINT-O-Somes will in the future reveal whether this delivery system can be further developed for efficacious endothelial directed delivery of drugs in the treatment of sepsis

Organ-Specific Differences in Endothelial Permeability-Regulating Molecular Responses in Mouse and Human Sepsis

In patients with sepsis-induced multi-organ dysfunction syndrome, diverging patterns of oedema formation and loss of function in organs such as lung and kidney suggest that endothelial permeability-regulating molecular responses are differentially regulated. This potential differential regulation has been insufficiently studied at the level of components of adherens and tight junctions. We hypothesized that such a regulation by endothelial cells in sepsis takes place in an organ-specific manner. We addressed our hypothesis by studying by quantitative real time polymerase chain reaction the expression of a predefined subset of EC permeability-related molecules (occludin, claudin-5, PV-1, CD-31, endomucin, Angiopoietin-1, Angiopoietin-2, Tie2, VEGFA, VEGFR1, VEGFR2, and VE-cadherin) in kidney and lung after systemic lipopolysacharide injection in mice, and in kidneys of patients who died of sepsis. We showed that baseline endothelial expression of permeability-related molecules differs in mouse kidney and lung. Moreover, we showed differential regulation of these molecules after lipopolysacharide injection in the two mouse organs. In lung we found a decrease in expression levels of molecules of the adherence and tight junctions complex and related signaling systems, compatible with increased permeability. In contrast, in kidney we found expression patterns of these molecules compatible with decreased permeability. Finally, we partially corroborated our findings in mouse kidney in human kidneys from septic patients. These findings may help to understand the clinical difference in the extent of oedema formation in kidney and lung in sepsis-associated organ failure

The renal angiopoietin/Tie2 system in lethal human sepsis

Sepsis-induced multi-organ dysfunction syndrome (MODS) still has a high mortality. Improvements await a better understanding of the pathophysiological mechanisms. The angiopoietin (Ang)1/2 and Tie2 (tyrosine kinase with immunoglobulin and epidermal growth factor homology domains 2) ligand/receptor system is an important regulator of endothelial cell responses to severe insults. Plasma Ang2 levels are prognostic in sepsis, but data on Ang/Tie responses in organs in humans are lacking.. We hypothesized that, in kidneys of patients who died of sepsis with acute kidney injury (AKI), the Ang/Tie signaling system is changed in such a way that microvessels become destabilized