Gene Ontology Analysis for Drug Targets of the Whole Genome Transcriptome of Human Vascular Endothelial Cells in Response to Proinflammatory IL-1

The innate immune system combats tissue injury and infection by activating the proinflammatory responses involving the humoral complement system, granulocytes, macrophages and vascular endothelial cells (VEC) (Newton and Dixit, 2012; Zhu et al., 2012). Macrophages mediate proinflammatory responses by releasing inflammatory cytokines such as IL-1β. Once secreted, IL-1β paracrinically acts on the VEC and massively change their functions. These perturbations include a change from the anticoagulant phenotype to a procoagulant state, enhanced expression of vasoactive substances, cell adhesion molecules as well as inflammatory mediators including chemoattractants, and endothelial barrier dysfunction causing microvascular leakage (Pober and Sessa, 2007). Although essential for the effective immune defense, uncontrolled or chronic inflammatory response causes tissue damage and loss of organ function (Lon et al., 2012)

LAPORAN KERJA PRAKTEK ANALISIS SISTEM PENDAFTARAN ONLINE SKKTR SERTA PEMELIHARAAN HARDWARE DAN SOFTWARE di DINAS PERMODALAN DAN PERIZINAN KOTA YOGYAKARTA

Barrier function of Wnt5A-treated VEC in the presence or absence of WIF1 and sFRP1. a ECIS assisted measurements (Additional file 4: supplementary methods) showing resistance of HCAEC monolayers grown in 8W10E+ arrays treated with vehicle (black), Wnt5A (green), Wnt5A + WIF1 (yellow) and Wnt5A + sFRP1 (purple). Data shown are the resistance measurements conducted at 4000 Hz and are mean ± SEM of 2 wells from 1 out of three representative experiments. b Barrier function measurements indicating the significance of WIF1’s antagonistic effect on Wnt5A and are mean ± SEM of three independent experiments run with duplicate wells. *P < 0.05 vs non-treated, **P < 0.05 vs Wnt5A. (PDF 2125 kb

Thromboelastography to Monitor Clotting/Bleeding Complications in Patients Treated with the Molecular Adsorbent Recirculating System

Background. The Molecular Adsorbent Recirculating System (MARS) has been shown to clear albumin-bound toxins from patients with liver failure but might cause bleeding complications potentially obscuring survival benefits. We hypothesized that monitoring clotting parameters and bed-side thromboelastography allows to reduce bleeding complications. Methods. Retrospective analysis of 25 MARS sessions during which clotting parameters were monitored by a standardized protocol. Results. During MARS therapy median INR increased significantly from 1.7 to 1.9 platelet count and fibrinogen content decreased significantly from 57 fL−1 to 42 fL−1 and 2.1 g/L to 1.5 g/L. Nine relevant complications occurred: the MARS system clotted 6 times 3 times we observed hemorrhages. Absent thrombocytopenia and elevated plasma fibrinogen predicted clotting of the MARS system (ROC 0.94 and 0.82). Fibrinolysis, detected by thromboelastography, uniquely predicted bleeding events. Conclusion. Bed-side thromboelastography and close monitoring of coagulation parameters can predict and, therefore, help prevent bleeding complications during MARS therapy

Hyperferritinemia without iron overload in patients with bilateral cataracts: a case series

Hepatologists and internists often encounter patients with unexplained high serum ferritin concentration. After exclusion of hereditary hemochromatosis and hemosiderosis, rare disorders like hereditary hyperferritinemia cataract syndrome should be considered in the differential diagnosis. This autosomal dominant syndrome, that typically presents with juvenile bilateral cataracts, was first described in 1995 and has an increasing number of recognized molecular defects within a regulatory region of the L-ferritin gene (FTL). CASE PRESENTATION: Two patients (32 and 49-year-old Caucasian men) from our ambulatory clinic were suspected as having this syndrome and a genetic analysis was performed. In both patients, sequencing of the FTL 5' region showed previously described mutations within the iron responsive element (FTL c.33 C > A and FTL c.32G > C). CONCLUSION: Hereditary hyperferritinemia cataract syndrome should be considered in all patients with unexplained hyperferritinemia without signs of iron overload, particularly those with juvenile bilateral cataracts. Liver biopsy and phlebotomy should be avoided in this disorder

Transcriptional Regulation of Drug Metabolizing CYP Enzymes by Proinflammatory Wnt5A Signaling in Human Coronary Artery Endothelial Cells

Downregulation of drug metabolizing enzymes and transporters by proinflammatory mediators in hepatocytes, enterocytes and renal tubular epithelium is an established mechanism affecting pharmacokinetics. Emerging evidences indicate that vascular endothelial cell expression of drug metabolizing enzymes and transporters may regulate pharmacokinetic pathways in heart to modulate local drug bioavailability and toxicity. However, whether inflammation regulates pharmacokinetic pathways in human cardiac vascular endothelial cells remains largely unknown. The lipid modified protein Wnt5A is emerging as a critical mediator of proinflammatory responses and disease severity in sepsis, hypertension and COVID-19. In the present study, we employed transcriptome profiling and gene ontology analyses to investigate the regulation of expression of drug metabolizing enzymes and transporters by Wnt5A in human coronary artery endothelial cells. Our study shows for the first time that Wnt5A induces the gene expression of CYP1A1 and CYP1B1 enzymes involved in phase I metabolism of a broad spectrum of drugs including chloroquine (the controversial drug for COVID-19) that is known to cause toxicity in myocardium. Further, the upregulation of CYP1A1 and CYP1B1 expression is preserved even during inflammatory crosstalk between Wnt5A and the prototypic proinflammatory IL-1β in human coronary artery endothelial cells. These findings stimulate further studies to test the critical roles of vascular endothelial cell CYP1A1 and CYP1B1, and the potential of vascular-targeted therapy with CYP1A1/CYP1B1 inhibitors in modulating myocardial pharmacokinetics in Wnt5A-associated inflammatory and cardiovascular diseases

Wnt5A/Ryk signaling critically affects barrier function in human vascular endothelial cells

Satisfactory therapeutic strategies for septic shock are still missing. Previously we found elevated levels of Wnt5A in patients with severe sepsis and septic shock. Wnt5A is released by activated macrophages but knowledge of its effects in the vascular system remains scant. Here we investigate the response of human coronary artery endothelial cells (HCAEC) to Wnt5A. We used a genome-wide differential expression approach to define novel targets regulated by Wnt5A. Gene ontology analysis of expression profiles revealed clusters of genes involved in actin cytoskeleton remodeling as the predominant targets of Wnt5A. Wnt5A targeted Rho-associated protein serine/threonine kinase (ROCK), leading to phosphorylation of LIM kinase-2 (LIMK2) and inactivation of the actin depolymerization factor cofilin-1 (CFL1). Functional experiments recording cytoskeletal rearrangements in living cells showed that Wnt5A enhanced stress fiber formation as a consequence of reduced actin depolymerization. The antagonist Wnt inhibitory factor 1 (WIF1) that specifically interferes with the WIF domain of Ryk receptors prevented actin polymerization. Wnt5A disrupted β-catenin and VE-cadherin adherens junctions forming inter-endothelial gaps. Functional experiments targeting the endothelial monolayer integrity and live recording of trans-endothelial resistance revealed enhanced permeability of Wnt5A-treated HCAEC. Ryk silencing completely prevented Wnt5A-induced endothelial hyperpermeability. Wnt5A decreased wound healing capacity of HCAEC monolayers; this was restored by the ROCK inhibitor Y-27632. Here we show that Wnt5A acts on the vascular endothelium causing enhanced permeability through Ryk interaction and downstream ROCK/LIMK2/CFL1 signaling. Wnt5A/Ryk signaling might provide novel therapeutic strategies to prevent capillary leakage in systemic inflammation and septic shock

WIF1 prevents Wnt5A mediated LIMK/CFL phosphorylation and adherens junction disruption in human vascular endothelial cells

Abstract Background Wnt5A is released by activated macrophages and elevated levels have been detected in sepsis patients with severe systemic inflammation. However, the signalling and functional effects of Wnt5A in the vascular endothelial cells (VEC) remained unclear. Recently, we showed that Wnt5A affects barrier function in human VEC through Ryk interaction. Wnt5A/Ryk signalling activates LIMK to inactivate the actin depolymerisation factor CFL by phosphorylation, promotes actin polymerisation and disrupts endothelial adherens junctions. Findings Here, we investigate the antagonistic effect of the Ryk specific secreted Wnt antagonist Wnt inhibitory factor (WIF)-1 on Wnt5A-mediated activation/inactivation of LIMK/CFL, and adherens junction disruption in human VEC. In human coronary artery endothelial cells (HCAEC), treatment with Wnt5A enhanced the phosphorylation of LIMK and CFL that was significantly prevented by WIF1. The presence of WIF1 suppressed Wnt5A-mediated disruption of β-catenin and VE-cadherin adherens junctions in HCAEC, thereby preventing barrier dysfunction caused by Wnt5A. Conclusion We conclude that WIF1 or molecules with similar properties could be potent tools for the prevention of vascular leakage due to Wnt5A-mediated actin cytoskeleton remodeling in diseases associated with systemic inflammation