Author Correction: Heparanase-2 protects from LPS-mediated endothelial injury by inhibiting TLR4 signalling

The Acknowledgements section in the original version of this Article was incomplete. “We are grateful to Prof. Israel Vlodavsky (Technion, Haifa, Israel) for giving us 1c7 antibody to HPSE2. We are grateful to Prof. Fridrich Luft for critical editing the manuscript. Grants from German Federal Ministry of Education and Research (BMBF) Nr. 031A577A and 031A577B funded this research. This work was also supported by a grant for the German Research Council to H.H. Ha1388/17-1.” now reads: “We are grateful to Prof. Israel Vlodavsky (Technion, Haifa, Israel) for giving us 1c7 antibody to HPSE2. We are grateful to Prof. Fridrich Luft for critical editing the manuscript. Grants from German Federal Ministry of Education and Research (BMBF) Nr. 031A577A and 031A577B funded this research. This work was also supported by a grant for the German Research Council to H.H. Ha1388/17-1. This work was also supported by a grant from the Else Kröner-Fresenius-Stiftung (EKFS): Grant 2017_A96.” The original Article has been corrected

Heparanase-2 protects from LPS-mediated endothelial injury by inhibiting TLR4 signalling

The endothelial glycocalyx and its regulated shedding are important to vascular health. Endo-β-D-glucuronidase heparanase-1 (HPSE1) is the only enzyme that can shed heparan sulfate. However, the mechanisms are not well understood. We show that HPSE1 activity aggravated Toll-like receptor 4 (TLR4)-mediated response of endothelial cells to LPS. On the contrary, overexpression of its endogenous inhibitor, heparanase-2 (HPSE2) was protective. The microfluidic chip flow model confirmed that HPSE2 prevented heparan sulfate shedding by HPSE1. Furthermore, heparan sulfate did not interfere with cluster of differentiation-14 (CD14)-dependent LPS binding, but instead reduced the presentation of the LPS to TLR4. HPSE2 reduced LPS-mediated TLR4 activation, subsequent cell signalling, and cytokine expression. HPSE2-overexpressing endothelial cells remained protected against LPS-mediated loss of cell-cell contacts. In vivo, expression of HPSE2 in plasma and kidney medullary capillaries was decreased in mouse sepsis model. We next applied purified HPSE2 in mice and observed decreases in TNFα and IL-6 plasma concentrations after intravenous LPS injections. Our data demonstrate the important role of heparan sulfate and the glycocalyx in endothelial cell activation and suggest a protective role of HPSE2 in microvascular inflammation. HPSE2 offers new options for protection against HPSE1-mediated endothelial damage and preventing microvascular disease. © 2019, The Author(s)

In peripartum cardiomyopathy plasminogen activator inhibitor-1 is a potential new biomarker with controversial roles

Aims Peripartum cardiomyopathy (PPCM) is a life-threatening heart disease occurring in previously heart-healthy women. A common pathomechanism in PPCM involves the angiostatic 16 kDa-prolactin (16 kDa-PRL) fragment, which via NF-kappa B-mediated up-regulation of microRNA-(miR)-146a induces vascular damage and heart failure. We analyse whether the plasminogen activator inhibitor-1 (PAI-1) is involved in the pathophysiology of PPCM. Methods and results In healthy age-matched postpartum women (PP-Ctrl, n = 53, left ventricular ejection fraction, LVEF > 55%), PAI-1 plasma levels were within the normal range (21 +/- 10 ng/mL), but significantly elevated (64 +/- 38 ng/mL, P <0.01) in postpartum PPCM patients at baseline (BL, n = 64, mean LVEF: 23 +/- 8%). At 6-month follow-up (n = 23), PAI-1 levels decreased (36 +/- 14 ng/mL, P <0.01 vs. BL) and LVEF (49 +/- 11%) improved. Increased N-terminal pro-brain natriuretic peptide and Troponin T did not correlate with PAI-1. C-reactive protein, interleukin (IL)-6 and IL-1 beta did not differ between PPCM patients and PP-Ctrl. MiR-146a was 3.6-fold (P <0.001) higher in BL-PPCM plasma compared with PP-Ctrl and correlated positively with PAI-1. In BL-PPCM serum, 16 kDa-PRL coprecipitated with PAI-1, which was associated with higher (P <0.05) uPAR-mediated NF-kappa B activation in endothelial cells compared with PP-Ctrl serum. Cardiac biopsies and dermal fibroblasts from PPCM patients displayed higher PAI-1 mRNA levels (P <0.05) than healthy controls. In PPCM mice (due to a cardiomyocyte-specific-knockout for STAT3, CKO), cardiac PAI-1 expression was higher than in postpartum wild-type controls, whereas a systemic PAI-1-knockout in CKO mice accelerated peripartum cardiac fibrosis, inflammation, heart failure, and mortality. Conclusion In PPCM patients, circulating and cardiac PAI-1 expression are up-regulated. While circulating PAI-1 may add 16 kDa-PRL to induce vascular impairment via the uPAR/NF-kappa B/miR-146a pathway, experimental data suggest that cardiac PAI-1 expression seems to protect the PPCM heart from fibrosis. Thus, measuring circulating PAI-1 and miR-146a, together with an uPAR/NF-kappa B-activity assay could be developed into a specific diagnostic marker assay for PPCM, but unrestricted reduction of PAI-1 for therapy may not be advised

Urokinase Receptor Counteracts Vascular Smooth Muscle Cell Functional Changes Induced by Surface Topography

© Ivyspring International Publisher. This is an open-access article distributed under the terms of the Creative Commons Licens

Loss of urokinase receptor sensitizes cells to DNA damage and delays DNA repair.

DNA damage induced by numerous exogenous or endogenous factors may have irreversible consequences on the cell leading to cell cycle arrest, senescence and cell death. The DNA damage response (DDR) is powerful signaling machinery triggered in response to DNA damage, to provide DNA damage recognition, signaling and repair. Most anticancer drugs induce DNA damage, and DNA repair in turn attenuates therapeutic efficiency of those drugs. Approaches delaying DNA repair are often used to increase efficiency of treatment. Recent data show that ubiquitin-proteasome system is essential for signaling and repair of DNA damage. However, mechanisms providing regulation of proteasome intracellular localization, activity, and recruitment to DNA damage sites are elusive. Even less investigated are the roles of extranuclear signaling proteins in these processes. In this study, we report the involvement of the serine protease urokinase-type plasminogen activator receptor (uPAR) in DDR-associated regulation of proteasome. We show that in vascular smooth muscle cells (VSMC) uPAR activates DNA single strand break repair signaling pathway. We provide evidence that uPAR is essential for functional assembly of the 26S proteasome. We further demonstrate that uPAR mediates DNA damage-induced phosphorylation, nuclear import, and recruitment of the regulatory subunit PSMD6 to proteasome. We found that deficiency of uPAR and PSMD6 delays DNA repair and leads to decreased cell survival. These data may offer new therapeutic approaches for diseases such as cancer, cardiovascular and neurodegenerative disorders

H₂O₂-induced PSMD6 nuclear import is impaired in the absence of uPAR.

<p>A. SiCo and uPARsi -nucleofected human VSMC were treated with 100 µM H₂O₂ for 1 h at 37°C. Then cells were fixed and stained for PSMD6 (Alexa 488) DraQ5 was used as nuclear stain. B. Human VSMC were treated as in A and subcellular fractionation was performed. PSMD6 content in nuclear fraction was assessed by western blotting. Histon H3 was used as loading control. Scale bar 100 µm. C. H₂O₂ -induced nuclear import of PSMD6 was quantified from 3 independent experiments.</p

uPAR is essential for MMS-induced DNA SSB signaling and DNA repair.

<p>A. Growth arrested SiCo and uPARsi -nucleofected human VSMC were treated with 1.2 mM MMS for indicated time points. Phosphorylation of Chk1 and Chk2 kinases was detected by western blotting. B. HEK 293 cells were infected with control lentivirus or uPAR-FLAG-expressing virus, and stimulated with 1.2 mM MMS for indicated time points. Phosphorylation of Chk1 and Chk2 kinases was detected by western blotting. C. WT and uPAR−/− mouse VSMC were treated with MMS for 20 min on ice to induce DNA damage. After H₂O₂ removal VSMC were allowed to repair DNA for 4 hrs. Comet tails were quantified as described in the Materials and Methods. D. WT and uPAR−/− mouse VSMC were treated with different concentrations of MMS for 20 min to induce DNA damage. The number of viable cells was calculated 24 hrs after DNA damage using 5(6)CFDA as described in Material and methods.</p

Recruitment of 19S subunits to the proteasome complex calculated as the ratio of normalized intensity of peptide peak to the total input cell extract.

<p>Recruitment of 19S subunits to the proteasome complex calculated as the ratio of normalized intensity of peptide peak to the total input cell extract.</p