Interfering with VE-PTP stabilizes endothelial junctions in vivo via Tie-2 in the absence of VE-cadherin

Vascular endothelial (VE)–protein tyrosine phosphatase (PTP) associates with VE-cadherin, thereby supporting its adhesive activity and endothelial junction integrity. VE-PTP also associates with Tie-2, dampening the tyrosine kinase activity of this receptor that can support stabilization of endothelial junctions. Here, we have analyzed how interference with VE-PTP affects the stability of endothelial junctions in vivo. Blocking VE-PTP by antibodies, a specific pharmacological inhibitor (AKB-9778), and gene ablation counteracted vascular leak induction by inflammatory mediators. In addition, leukocyte transmigration through the endothelial barrier was attenuated. Interference with Tie-2 expression in vivo reversed junction-stabilizing effects of AKB-9778 into junction-destabilizing effects. Furthermore, lack of Tie-2 was sufficient to weaken the vessel barrier. Mechanistically, inhibition of VE-PTP stabilized endothelial junctions via Tie-2, which triggered activation of Rap1, which then caused the dissolution of radial stress fibers via Rac1 and suppression of nonmuscle myosin II. Remarkably, VE-cadherin gene ablation did not abolish the junction-stabilizing effect of the VE-PTP inhibitor. Collectively, we conclude that inhibition of VE-PTP stabilizes challenged endothelial junctions in vivo via Tie-2 by a VE-cadherin–independent mechanism. In the absence of Tie-2, however, VE-PTP inhibition destabilizes endothelial barrier integrity in agreement with the VE-cadherin–supportive effect of VE-PTP.The endothelium of the blood vessel wall forms a barrier for blood solutes and for leukocytes. The entry of molecules and cells of the immune system into inflamed tissue is mainly regulated via endothelial junctions. Vascular endothelial (VE)–cadherin is a central component of these junctions, and it is generally considered one of the major adhesive mechanisms that controls the stability and barrier function of the endothelium (Dejana and Vestweber, 2013). Antibodies against VE-cadherin can destabilize endothelial junctions in vitro and in vivo (Breviario et al., 1995; Gotsch et al., 1997; Corada et al., 1999). In agreement with this, enhancing the adhesive function of VE-cadherin by directly fusing it to α-catenin blocks the induction of vascular permeability in the skin of the respective knock-in mice and strongly reduces leukocyte extravasation in various tissues (Schulte et al., 2011).The VE–protein tyrosine phosphatase (PTP) was shown to associate with VE-cadherin and thereby enhance the adhesive function of VE-cadherin (Nawroth et al., 2002; Nottebaum et al., 2008). Permeability-inducing mediators such as VE growth factor (VEGF) and the attachment of leukocytes to endothelial cells (ECs) both stimulate a signaling pathway that triggers the dissociation of VE-PTP from VE-cadherin (Nottebaum et al., 2008; Vockel and Vestweber, 2013). This dissociation is necessary for the induction of vascular permeability and for leukocyte extravasation in vivo. Evidence for this has been based on the analysis of knock-in mice where modified forms of VE-cadherin and VE-PTP, each containing a different binding site for a small molecular weight compound, had been knocked into the VE-cadherin locus (Broermann et al., 2011). In these mice, administration of the appropriate compound inhibited the dissociation of VE-PTP from VE-cadherin and thereby attenuated the induction of vascular permeability and leukocyte extravasation, demonstrating the importance of VE-cadherin–associated VE-PTP for the control of endothelial junction stability in vivo. Moreover, HIF2α was recently reported to enhance endothelial barrier integrity, in part through induced VE-PTP expression (Gong et al., 2015). In line with these findings, tyrosine phosphorylation of VE-cadherin has been demonstrated to be involved in the regulation of EC contacts in vitro (Allingham et al., 2007; Turowski et al., 2008; Monaghan-Benson and Burridge, 2009) and in vivo (Orsenigo et al., 2012; Wessel et al., 2014).VE-PTP also associates with Tie-2 (Fachinger et al., 1999), an endothelial tyrosine kinase receptor that regulates angiogenesis and can support the integrity of endothelial junctions. VE-PTP gene ablation causes embryonic lethality at embryonic day (E) 9.5 (Bäumer et al., 2006; Dominguez et al., 2007). This is caused by a defect in blood vessel remodeling leading to enlarged and fused vessel structures. Antibodies against VE-PTP caused similar defects when incubated with explant cultures of allantois from WT mice, but not if the tissue originated from Tie-2 gene–deficient mice (Winderlich et al., 2009). In addition, these antibodies against the extracellular part of VE-PTP dissociated the phosphatase from Tie-2 and caused phosphorylation of this receptor and signaling (Winderlich et al., 2009).We have recently shown that a specific pharmacological inhibitor of VE-PTP catalytic activity, AKB-9778, activated Tie-2 in the mouse, and this correlated with suppression of ocular neovascularization and blocking of VEGF-induced vascular leak (Shen et al., 2014). Although direct evidence for the relevance of Tie-2 is still missing, these results are in line with studies reporting that the Tie-2 ligand Angiopoietin-1 (Ang1) protects the vasculature against plasma leakage (Gamble et al., 2000; Thurston et al., 2000; Mammoto et al., 2007; Gavard et al., 2008). In a mouse breast cancer model, AKB-9778 normalized tumor vessels and delayed tumor growth (Goel et al., 2013). VE-PTP was also found to associate with VEGF receptor-2 (VEGFR-2; Mellberg et al., 2009), and this interaction was suggested to affect VEGFR-2 activity in endothelial sprouts of mouse embryoid bodies (Hayashi et al., 2013).Here we have analyzed how interference with VE-PTP activity influences endothelial junctions in adult mice and how this relates to the functions of Tie-2 and VE-cadherin. We found that conditional gene ablation of VE-PTP, as well as interference with antibodies or administering the inhibitor AKB-9778 each stabilized challenged endothelial junctions, thereby preventing enhanced permeability and leukocyte extravasation induced by inflammatory mediators. These effects required the expression of Tie-2 because blocking its expression in vivo reversed the effect of the VE-PTP inhibitor on endothelial junction integrity. Thus, VE-PTP inhibition stabilizes challenged endothelial junctions via Tie-2 and destabilizes them in the absence of Tie-2. Remarkably, the junction-stabilizing effect was even observed in mice conditionally gene deficient for VE-cadherin. Inhibition of leak formation was accompanied by activation of Rap1 and cytoskeletal remodeling and reduced radial stress fiber formation. Our results reveal that inhibition of VE-PTP in vivo has opposing effects on endothelial junctions as the result of its different effects on VE-cadherin and on Tie-2. Activation of Tie-2 via inhibition of VE-PTP protects endothelial junctions against inflammation-induced destabilization and overrides the negative effect of VE-PTP inhibition on the adhesive function of VE-cadherin.     

Association of High Mobility Group Box Chromosomal Protein 1 and Receptor for Advanced Glycation End Products Serum Concentrations With Extraglandular Involvement and Disease Activity in Sjögren's Syndrome

Objective

To assess serum levels of high mobility group box chromosomal protein 1 (HMGB‐1) and the soluble receptor for advanced glycation end products (sRAGE) in patients with Sjögren's syndrome (SS) and explore correlations with disease activity.

Methods

Thirty‐nine patients with SS and 21 healthy controls were included in this cross‐sectional study. Clinical and laboratory values were obtained from all patients. Disease activity was assessed using the European League Against Rheumatism SS Disease Activity Index (ESSDAI). Serum samples were collected and HMGB‐1 and sRAGE levels were measured using enzyme‐linked immunosorbent assay (ELISA), and HMGB‐1 concentrations were semiquantified by Western blotting.

Results

In ELISA, HMGB‐1 serum levels did not differ between healthy controls and patients with SS (P = 0.783). When measured by semiquantitative Western blotting, HMGB‐1 levels were increased in patients with SS compared to healthy controls (P = 0.012). HMGB‐1 serum levels detected by Western blotting were higher in patients with extraglandular manifestations (P = 0.003) and were correlated with ESSDAI disease activity (r = 0.544, P < 0.0001). Furthermore, sRAGE was elevated in the sera of patients with SS (P = 0.003) compared to healthy controls and was also correlated with the ESSDAI (r = 0.545, P = 0.002).

Conclusion

Serum levels of total HMGB‐1 and sRAGE were elevated in patients with SS compared to healthy controls and correlated with disease activity as measured by the ESSDAI. Patients with extraglandular involvement had high serum levels of HMGB‐1.

     

Stress and burnout in chiropractic students of European chiropractic colleges:: A cross-sectional study

ObjectiveHigh levels of stress and burnout are known to negatively impact academic success, quality of life, and well-being of students. The purpose of this study was to investigate the degrees of stress and burnout levels of students from several European chiropractic colleges.MethodsStress and burnout were assessed using the Perceived Stress Scale (PSS-10) and the Maslach Burnout Inventory–Student Survey (MBI-SS). Surveys were delivered electronically in November 2017 to chiropractic students from 4 different chiropractic colleges. Data were analyzed using t test and 1-way ANOVA to determine differences between demographic data. Scores in perceived stress and burnout subscales were compared to the general, chiropractic, and medical student populations.ResultsBoth the MBI-SS and PSS had similar response rates (30%–34%) and demonstrated statistically significant differences between institutions, with C-3 demonstrating the highest levels of exhaustion (p < .001) and the highest levels of perceived stress (p = .012). MBI-SS results show that in the general chiropractic student population, 26.4% presented high emotional exhaustion, 18.2% high cynicism, and 43.8% low academic efficacy. Meanwhile, the PSS score indicated “moderate” levels of stress.ConclusionsEuropean chiropractic students experience higher levels of perceived stress than the general population and they may suffer levels of burnout similar to those of medical students. These results suggest that colleges should monitor stress and burnout levels in their students. This may help to establish student support systems in order to improve students'' quality of life and academic performance, as well as help new graduates transition to their professional lives.     

Membranoproliferative glomerulonephritis complicating Waldenström’s macroglobulinemia

Background

Lymphoproliferative disorders causing paraproteinemia can be associated with various kidney injuries including the deposition of monoclonal immunoglobulins (Ig). A known glomerular manifestation of Waldenström’s macroglobulinemia is characterized by prominent intracapillary hyaline thrombi and lack of conspicuous glomerular proliferation. The present case was special in 2 aspects: 1. the diagnosis of glomerulonephritis was unexpected before renal biopsy, 2. the prominent glomerular proliferation paired with large intracapillary hyaline thrombi is uncommon in Waldenström’s macroglobulinemia-associated glomerulonephritis.

Case presentation

A 73-year-old Caucasian woman with a long-standing history of rheumatoid arthritis and Waldenström’s macroglobulinemia was admitted for acute renal failure (ARF), which initially was presumed to be the consequence of extrarenal causes. Proteinuria and hematuria were only mild. In renal core biopsy, a membranoproliferative glomerulonephritis (MPGN) and prominent intracapillary hyaline monoclonal IgM thrombi were found in addition to acute tubular necrosis. Of note, the patient’s history was positive for purpuric skin changes, suspicious for cryoglobulinemia. However, serological tests for cryoglobulins were repeatedly negative. The ARF resolved before the start of immunomodulatory therapy for Waldenström’s macroglobulinemia.

Conclusion

The presence of MPGN with prominent hyaline thrombi in the context of Waldenström’s macroglobulinemia is uncommon and can be oligosymptomatic. We discuss this case in the context of previous literature and classifications suggested for monoclonal Ig-related renal pathologies.

     

Large-scale generation of natural killer lymphocytes for clinical application

Natural killer (NK) lymphocytes can be used for adoptive immunotherapeutic strategies. Alternatively, they may be employed as adjuvants for stem cell/bone marrow transplantation, either to re-induce remission, or to purge autografts of contaminating malignant cells. We developed a new protocol that enables the generation of NK cells on a clinical scale in a closed system that enables good manufacturing practice (GMP) conformity. Aside from the initial NK cell inoculum, our protocol includes activated feeder cells [irradiated peripheral blood mononuclear cells (PBMC) and no transformed blasts], cytokines [interleukin-2 (IL-2) and IL-15], human serum, and a complex basic media formulation. During the whole expansion period of approximately 14 days, the cells were handled in PTFE (Teflon) bags, whereby fresh medium was added without opening the system. The use of immortalized or virus-transformed feeder cells, as used in many other current research protocols, was completely avoided. A precise controlling of a number of environmental factors was necessary to achieve reproducible results. Increases in NK cell number ranged between 80- and 200-fold. The resulting NK cells were CD56(+), CD3(-), and CD16(+) (75%). They were highly cytotoxic against different malignant target cells and did not produce significant levels of interferon-gamma. Therefore, they belonged to the cytotoxic rather than the immunoregulatory NK subpopulation. No non-specific activation against normal allogenous lymphocytes occurred. This work might permit the realization of future protocols for evaluating the clinical effect of NK lymphocytes in human disease.     

Sensitivity to Escherichia coli Nissle 1917 in mice is dependent on environment and genetic background

Escherichia coli Nissle 1917 (EcN) is a well-characterized probiotic bacterium. Although genomic comparisons of EcN with the uropathogenic E. coli strain CFT073 revealed high degrees of similarity, EcN is generally considered a non-pathogenic organism. However, as recent evidence suggests that EcN is capable of inducing inflammatory responses in host intestinal epithelial cells, we aimed to investigate potential pathogenic properties of EcN in an in vivo model using various germ-free (GF) mouse strains. With the exception of C3H/HeJZtm mice, which carry a defective toll-like receptor (TLR)4-allele, no lesions were obvious in mice of different strains orally inoculated with EcN for 1 week, although organ cultures (blood, lung, mesenteric lymph node, pancreas, spleen, liver and kidney) tested positive to various degrees. C3H/HeJZtm mice inoculated with EcN became clinically ill and the majority died or had to be euthanized. Organs of all gnotobiotic C3H/HeJZtm mice were positive for EcN by culture; major histological findings were moderate to severe pyogranulomatous serositis, typhlitis and pancreatitis. Histological findings were corroborated by highly elevated tumour necrosis factor (TNF) serum levels. Lesions were not detected in specified pathogen free maintained C3H/HeJZtm mice, GF C3H/HeJ mice lacking the interleukin-10 gene, or GF C3H/HeJZtm mice that were inoculated with E. coli K12 strain MG1655 as a control. In addition, mild histological lesions were detected in Ztm:NMRI mice 3 months after oral inoculation with EcN. This study shows that EcN is capable of displaying a virulent phenotype in GF C3H/HeJZtm mice. Whether this phenotype is linked to the bacterium's probiotic nature should be the focus of further studies.     

Development of the proepicardium in Xenopus laevis

The proepicardium (PE) is an embryonic progenitor cell population, which provides the epicardium, the majority of the cardiac interstitium, the coronary vasculature and possibly some cardiomyocytes. Recent studies have documented (1) the presence of bilaterally paired PE anlagen in several vertebrates, and (2) species-specific differences in the fate of the left and right PE anlagen. Here, we document PE development in Xenopus laevis (stages 37-46). The PE appears at stage 41 in the form of a cone-shaped accumulation of mesothelial cells covering the pericardial surface of the right horn of the sinus venosus. No such structure appears on the left sinus horn. At the end of stage 41, the tip of the PE establishes a firm contact with the developing ventricle. A secondary tissue bridge is established facilitating the transfer of PE cells to the heart. During stages 41-46, this tissue bridge is visible in vivo through the transparent body wall. Corresponding to the morphological data, the PE marker gene Tbx18 is expressed only on the right sinus horn suggesting a right-sided origin of the PE. Left-right lineage tracing has confirmed this idea. These results show that Xenopus PE development proceeds in a bilaterally asymmetric pattern as previously observed in chicks. We speculate that asymmetric PE development is controlled by signals from left-right signaling pathways and that the PE is an indicator for right-sidedness in Xenopus embryos. Xenopus might be a good model to uncover the role of left-right signaling pathways in the control of asymmetric PE development.     

A study on polysialic acid as a biomaterial for cell culture applications

Polysialic acid (PSA) was investigated for its applicability as coating material for mammalian cell cultivation. PSA is involved in post-translational modification of the vertebrate neural cell adhesion molecule (NCAM). It is biocompatible and degradation-controlled. Thus, it becomes interesting for use as a coating and scaffold material for tissue engineering applications, especially for peripheral nerve regeneration. As a preliminary study of the use of PSA as scaffold material it was tested in its soluble form as coating material. The cytotoxicity was investigated and compared to another polysaccharide beta-glucan, to widely used coating substances (collagen I, poly-L-lysine, hyaluronic acid) and uncoated tissue culture plastic material. The interactions between the modified cell culture surface and the cells were investigated using a model liver cell line Hep-G2 and a neurobiological cell line PC-12. The PSA coating itself was analyzed by immunoanalysis. Viability of the cells was investigated by the MTT assay. The number and distribution of adhered cells were studied by cell nuclei staining. Furthermore, the differentiation status of the PC-12 cells was monitored, as well as glucose and lactate levels in the cell culture medium from the Hep-G2 cells. Comparable viability and similar numbers of attached cells were observed. Growth in cell clusters was observed for PSA, beta-glucan, and hyaluronic acid coated materials. In general, the results indicate that PSA is comparable to other well-established coating materials (e.g. collagen I, hyaluronic acid, and poly-L-lysine). Moreover, as a key substance in vertebrate development it offers interesting features for nerve regeneration, especially as an insoluble, modified scaffold material.     

Altered cortical inhibitory function in children with spastic diplegia: a TMS study

Periventricular leukomalacia (PVL) is the most frequent cause of spastic diplegia. The movement disorder is attributed to damage to the corticospinal tract, but there is increasing evidence of additional cortical dysfunction associated with PVL. Aim of the present study was to evaluate the integrity of the corticospinal tract and cortical inhibitory function using transcranial magnetic stimulation. Fifteen children with bilateral PVL and spastic diplegia and twenty-two healthy children underwent single-pulse stimulations to the right tibial anterior muscle. We compared central motor conduction time and amplitudes of motor evoked potentials as markers for corticospinal integrity and the postexcitatory silent period (SP), representing cortical inhibitory interneurons. The patients’ parameters of corticospinal tract function did not differ significantly from those in the control children. In contrast, the SP was significantly shortened in children with PVL (mean 25.6 ± 6.9 ms; controls: mean 47.6 ± 23.2 ms, P = 0.018). This suggests cortical involvement with reduced cortical inhibitory function in PVL. This could be due to impaired functioning of the cortical interneurons themselves, or to decreased input from activating fibres, e.g. thalamocortical or cortico-cortical connections.