RSPO3 impairs barrier function of human vascular endothelial monolayers and synergizes with pro-inflammatory IL-1.

BACKGROUND: Endothelial barrier dysfunction characterized by hyperpermeability of the vascular endothelium is a key factor in the pathogenesis of chronic inflammatory diseases and affects clinical outcomes. In states of chronic inflammation, mediators secreted by activated immune cells or vascular endothelium may affect the barrier function and permeability of the vascular endothelium. The matricellular R-spondin family member RSPO3 is produced by inflammatory-activated human monocytes and vascular endothelial cells, but its effects in the regulation of vascular endothelial barrier function remains elusive. METHODS: The present study investigates the effects of RSPO3 on the barrier function of adult human primary macro- and micro- vascular endothelial monolayers. Tight monolayers of primary endothelial cells from human coronary and pulmonary arteries, and cardiac, brain, and dermal microvascular beds were treated with RSPO3 either alone or in combination with pro-inflammatory mediator IL-1ß. Endothelial barrier function was assessed non-invasively in real-time using Electric Cell-substrate Impedance Sensing. RESULTS: RSPO3 treatment critically affected barrier function by enhancing the permeability of all vascular endothelial monolayers investigated. To confer hyperpermeable phenotype in vascular endothelial monolayers, RSPO3 induced inter-endothelial gap formation by disrupting the ß-catenin and VE-cadherin alignment at adherens junctions. RSPO3 synergistically enhanced the barrier impairing properties of the pro-inflammatory mediator IL-1ß. CONCLUSION: Here, we show that the matricellular protein RSPO3 is a mediator of endothelial hyperpermeability that can act in synergy with the inflammatory mediator IL-1ß. This finding stimulates further studies to delineate the endothelial barrier impairing properties of RSPO3 and its synergistic interaction with IL-1ß in chronic inflammatory diseases.

Glucocorticoid treatment skews human monocyte differentiation into a hemoglobin-clearance phenotype with enhanced heme-iron recycling and antioxidant capacity.

Glucocorticoids are used extensively to treat autoimmune hemolytic anemias. Some beneficial effects of glucocorticoid pulse therapy have also been reported in sickle cell disease and paroxysmal nocturnal hemoglobinuria. Based on established concepts of hemoglobin (Hb) toxicity and physiologic Hb scavenger systems, we evaluated whether glucocorticoids could support an adaptive response to extracellular Hb independently of their immunosuppressive activities. Using global proteome and transcriptome analysis with mass-spectrometry (isobaric tag for relative and absolute quantitation and liquid chromatography-mass spectrometry) and gene-array experiments, we found that glucocorticoid treatment in vitro and in patients on glucocorticoid-pulse therapy polarized monocytes into a M2/alternatively activated phenotype with high Hb-scavenger receptor (CD163) expression and enhanced Hb-clearance and detoxification capability. Monocytes concurrently exposed to the interactive activity of glucocorticoids and extracellular Hb were characterized by high expression of a group of antioxidant enzymes known to be regulated by the conserved oxidative response transcription factor nuclear factor E2-related factor. Further, suppressed transferrin receptor, together with high ferroportin expression, pointed to a shift in iron homeostasis directed toward an increased cellular export of heme-derived iron. Therefore, stimulating Hb-endocytosis by CD163 and enhancing antioxidative homeostasis and iron recycling may be an essential activity of glucocorticoids that helps alleviate the adverse effects of extracellular Hb.

Wingless-related integration site (WNT) signaling is activated during the inflammatory response upon cardiac surgery: A translational study.

Objective: Cardiac surgery and the use of cardiopulmonary bypass initiate a systemic inflammatory response. Wingless-related integration site (WNT) signaling is part of the innate immunity and has been attributed a major role in the regulation of inflammation. In preclinical research, WNT-5a may sustain an inflammatory response and cause endothelial dysfunction. Our aim was to investigate WNT signaling after cardiac surgery and its association with postoperative inflammation (Clinicaltrials.gov, NCT04058496). Methods: In this prospective, single-center, observational study, 64 consecutive patients for coronary artery bypass grafting (CABG) ± valve surgery were assigned into three groups: off-pump CABG (n = 28), on-pump CABG (n = 16) and combined valve-CABG surgery (n = 20). Blood samples were acquired before surgery, at intensive care unit (ICU) admission and 4, 8, and 48 h thereafter. Plasma concentrations of WNT-5a and its antagonists Secreted frizzled-related protein 1 (sFRP-1), Secreted frizzled-related protein 5 (sFRP-5), and WNT inhibitory factor 1 (WIF-1) were determined by enzyme-linked immunosorbent assay. In addition, plasma concentrations of six inflammatory cytokines were measured by multiplex immunoassay. Parameters were analyzed for evolution of plasma concentration over time, interactions, intergroup differences, and association with clinical outcome parameters. Results: At baseline, WNT-5a, sFRP-1, and WIF-1 were present in a minimal concentration, while sFRP-5 was elevated. A higher baseline value of WNT-5a, sFRP-5, and WIF-1 resulted in higher subsequent values of the respective parameter. At ICU admission, WNT-5a and sFRP-5 reached their maximum and minimum value, respectively. WIF-1 decreased over time and was lowest 8 h after surgery. sFRP-1 changed minimally over time. While WNT-5a returned to the baseline within 48 h, sFRP-5 and WIF-1 did not reach their baseline value at 48 h. Of the investigated WNT system components, only WIF-1 partially reflected the severity of surgery. WNT-5a and WIF-1 had an impact on postoperative fluid balance and noradrenaline requirement. Conclusion: WNT-5a, sFRP-5, and WIF-1 are part of the systemic inflammatory response after cardiac surgery. WNT-5a peaks immediately after cardiac surgery and returns to baseline within 48 h, presumably modulated by its antagonist sFRP-5. Based on this translational study, WNT-5a antagonism may be further investigated to assess potentially beneficial effects in patients with a dysregulated inflammation after cardiac surgery.

Extracellular hemoglobin polarizes the macrophage proteome toward Hb-clearance, enhanced antioxidant capacity and suppressed HLA class 2 expression.

Peripheral blood monocytes and macrophages are the only cell population with a proven hemoglobin (Hb) clearance capacity through the CD163 scavenger receptor pathway. Hb detoxification and related adaptive cellular responses are assumed to be essential processes to maintaining tissue homeostasis and promoting wound healing in injured tissues. Using a dual platform mass spectrometry analysis with MALDI-TOF/TOF and LTQ-Orbitrap instruments combined with isobaric tag for relative and absolute quantitation (iTRAQ), we analyzed how Hb exposure could modulate the macrophage phenotype on a proteome level. We identified and relatively quantified 3691 macrophage proteins, representing the largest human macrophage proteome published to date. The Hb polarized macrophage phenotype was characterized by an induced Hb:Hp-CD163-HO1-ferritin pathway and enhanced antioxidant enzymes while suppression of HLA class 2 was the most prominent effect. Enhanced Hb clearance and antioxidant capacity together with reduced antigen presentation might therefore be essential qualities of Hb polarized macrophages in wounded tissues and hemorrhage or atherosclerotic plaques.

Haptoglobin preserves the CD163 hemoglobin scavenger pathway by shielding hemoglobin from peroxidative modification.

Detoxification and clearance of extracellular hemoglobin (Hb) have been attributed to its removal by the CD163 scavenger receptor pathway. However, even low-level hydrogen peroxide (H(2)O(2)) exposure irreversibly modifies Hb and severely impairs Hb endocytosis by CD163. We show here that when Hb is bound to the high-affinity Hb scavenger protein haptoglobin (Hp), the complex protects Hb from structural modification by preventing alpha-globin cross-links and oxidations of amino acids in critical regions of the beta-globin chain (eg, Trp15, Cys93, and Cys112). As a result of this structural stabilization, H(2)O(2)-exposed Hb-Hp binds to CD163 with the same affinity as nonoxidized complex. Endocytosis and lysosomal translocation of oxidized Hb-Hp by CD163-expressing cells were found to be as efficient as with nonoxidized complex. Hp complex formation did not alter Hb's ability to consume added H(2)O(2) by redox cycling, suggesting that within the complex the oxidative radical burden is shifted to Hp. We provide structural and functional evidence that Hp protects Hb when oxidatively challenged with H(2)O(2) preserving CD163-mediated Hb clearance under oxidative stress conditions. In addition, our data provide in vivo evidence that unbound Hb is oxidatively modified within extravascular compartments consistent with our in vitro findings.

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-1ß 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.

The wnt pathway: a macrophage effector molecule that triggers inflammation.

Wnt proteins are members of the highly conserved wingless family of proteins responsible for cell differentiation and development and for neoplastic and degenerative processes. Recently, Toll-like receptor-mediated Wnt signaling was found to be associated with innate immunity in Drosophila. Upregulation of Wnt5A in human macrophages upon microbial challenge indicated a similar mechanism. Toll-like receptor-mediated Wnt5A expression is a key process for sustained inflammatory macrophage activation through autocrine and paracrine signaling. Downregulation of Wnt5A expression and subsequent attenuation of inflammatory macrophage responses by activated protein C supports the link between inflammation and coagulation, another highly conserved biologic system. Direct evidence for the relevance of Wnt5A in severe systemic inflammation is provided by the finding of higher Wnt5A levels in patients with sepsis than in healthy individuals. The fact that Wnt5A signaling can be modulated by anti-inflammatory mediators makes this effector molecule an attractive target for therapeutic intervention in inflammatory diseases.

Wnt5A/CaMKII signaling contributes to the inflammatory response of macrophages and is a target for the antiinflammatory action of activated protein C and interleukin-10.

OBJECTIVE: Sepsis is a major cause of death for intensive care patients. High concentrations of inflammatory cytokines are characteristic of severe systemic inflammation and activated monocytes are their predominant cellular source. To identify targets for antiinflammatory intervention, we investigated the response of human macrophages to inflammatory and antiinflammatory mediators. METHODS AND RESULTS: We profiled gene expression in human macrophages exposed to lipopolysaccharide (LPS) and interferon (IFN)-gamma in the presence or absence of recombinant activated protein C (APC) or IL-10 and identified Wnt5A as one of the transcripts most highly induced by LPS/IFN-gamma and suppressed by APC and IL-10. We confirmed regulation of Wnt5A protein in macrophages and detected it in sera and bone marrow macrophages of patients with severe sepsis. We established that a functional Wnt5A/frizzled-5/CaMKII signaling pathway was essential for macrophage inflammatory activation. To prove the essential contribution of Wnt5A we measured inflammatory cytokines after stimulation with Wnt5A, silenced Wnt5A by siRNA, and blocked receptor binding with soluble Frizzled-related peptide-1 (sFRP1). CONCLUSIONS: Wnt5A is critically involved in inflammatory macrophage signaling in sepsis and is a target for antiinflammatory mediators like APC or antagonists like sFRP1.

Heme carrier protein (HCP-1) spatially interacts with the CD163 hemoglobin uptake pathway and is a target of inflammatory macrophage activation.

Macrophages constitute the major cellular compartment for hemoglobin (Hb) degradation and subsequent recycling of heme-iron to erythropoiesis. Dysregulation of macrophage iron and heme metabolism is a major pathophysiologic determinant of anemia of chronic disease. In this study, we show that the heme transporter heme carrier protein 1 (HCP-1) is expressed in human macrophages. Within early endosomes, HCP-1 colocalizes with endocytosed Hb-haptoglobin (Hp) complexes, which are taken up via the CD163 scavenger receptor pathway. Hb-Hp passes the divalent metal transporter 1B/HCP-1-positive endosomal compartment on its route from the cell surface to lysosomes. HCP-1 mRNA and protein expression are down-regulated by stimulation of macrophages with various TLR agonists and IFN-gamma. The profound suppression of HCP-1 expression by inflammatory macrophage activation parallels the regulation of the iron exporter ferroportin. In contrast, dexamethasone enhanced HCP-1 expression significantly. Given the spatial relationship, we propose that the Hb scavenger receptor CD163 and HCP-1 constitute a linked pathway for Hb catabolism and heme-iron recycling in human macrophages.

The reaction of hydrogen peroxide with hemoglobin induces extensive alpha-globin crosslinking and impairs the interaction of hemoglobin with endogenous scavenger pathways.

Cell-free hemoglobin (Hb) enhances the oxidation-related toxicity associated with inflammation, ischemia, and hemolytic disorders. Hb is highly vulnerable to oxidative damage, and irreversible structural changes involving iron/heme oxidation, heme-adduct products, and amino acid oxidation have been reported. Specific structural features of Hb, such as unconstrained alpha-chains and molecular size, determine the efficiency of interactions between the endogenous Hb scavengers haptoglobin (Hp) and CD163. Using HPLC, mass spectrometry, and Western blotting, we show that H(2)O(2)-mediated Hb oxidation results in the formation of covalently stabilized globin multimers, with prominent intramolecular crosslinking between alpha-globin chains. These structural alterations are associated with reduced Hp binding, reduced CD163 interaction, and severely impaired endocytosis of oxidized Hb by the Hp-CD163 pathway. As a result, when exposed to oxidized Hb, CD163-positive HEK293 cells and human macrophages do not increase hemeoxygenase-1 (HO-1) expression, the physiological anti-oxidative macrophage response to Hb exposure. Failed Hb clearance, inadequate HO-1 expression, and the subsequent accumulation of oxidatively damaged Hb species might thus contribute to pathologies related to oxidative stress.

Constitutive endocytosis of CD163 mediates hemoglobin-heme uptake and determines the noninflammatory and protective transcriptional response of macrophages to hemoglobin.

Heme toxicity contributes to the pathogenesis of chronic inflammatory diseases, atherosclerosis, and hemolysis associated vasculopathy. Macrophage clearance of cell free hemoglobin (Hb) is thus an essential homeostatic function of these cells. We examined the transcriptional response of human PBMC derived macrophages to Hb by gene array analysis. The observed noninflammatory macrophage response was characterized by induction of an antioxidative and antiinflammatory gene expression pattern with most prominent induction of the inducible heme oxygenase (HO-1). The metabolically active Hb-CD163-HO-1 pathway resulted in synthesis of ferritin-1 of the antioxidative and antiinflammatory end products linked to heme breakdown by HO-1. This response was mediated by the Hb scavenger receptor CD163 and heme and was not related to Hb mediated depletion of reduced glutathione. In contrast to other cellular responses induced by CD163, there was no role of protein phosphorylation dependent CD163 signaling in the protective macrophage response to Hb. Instead, CD163 acted as an Hb transporter, which undergoes constitutive and ligand independent internalization and recycling between the cell surface and early endosomes. The expression of CD163 and HO-1 in macrophages of neovascularized atherosclerotic lesions suggests that the pathway described herein is active in vivo. Noninflammatory Hb clearance and intimately linked HO-1 expression may provide the long sought-after explanation for the antiinflammatory activity associated with CD163-positive macrophages.

CD163-expressing monocytes constitute an endotoxin-sensitive Hb clearance compartment within the vascular system.

Hemoglobin (Hb) is released into the circulation during intravascular hemolysis and exerts toxic effects through oxidative damage and NO scavenging. According to the traditional concept of Hb clearance, free Hb is bound to the plasma protein haptoglobin (Hp), and the Hb-Hp complexes are cleared by liver and spleen macrophages via the Hb scavenger receptor CD163. Using a novel whole blood assay, we demonstrate that clearance of Hb-Hp is also mediated by CD14(high)/CD64(high) peripheral blood monocytes, which express CD163. Hb-Hp uptake by these cells is Ca(2+)-dependent and is abrogated by the addition of CD163-blocking antibodies. Accordingly, LPS treatment reduces monocyte surface CD163 and impairs Hb-Hp uptake. Monocytes likely mediate Hp-Hb uptake in vivo, as a high expression of the heme breakdown enzyme heme oxygenase-1 was observed in CD163(+) monocytes but not in other leukocyte populations obtained from healthy blood donors. We propose that CD163-mediated Hb-Hp uptake by peripheral blood monocytes constitutes an Hb-Hp clearance pathway, which acts at the site of intravascular hemolysis to reduce Hb-Hp circulation time and toxicity. Disruption of monocyte Hb-Hp clearance may increase Hb-Hp toxicity and contribute to the pathogenesis of systemic inflammatory diseases associated with reduced monocyte CD163 expression.

Heart rate elevations during early sepsis predict death in fluid-resuscitated rats with fecal peritonitis.

BACKGROUND: In sepsis, early outcome prediction would allow investigation of both adaptive mechanisms underlying survival and maladaptive mechanisms resulting in death. The aim of this study was to test whether early changes in heart rate monitored by telemetry could predict outcome in a long-term rat model of fecal peritonitis. METHODS: Male Wistar rats (n = 24) were instrumented with a central venous line for administration of fluids, antibiotics and analgesics. A telemetry transmitter continuously collected electrocardiogram signals. Sepsis was induced by intraperitoneal injection of fecal slurry, and the animals were observed for 48 h. Additional animals underwent arterial cannulation at baseline (n = 9), 4 h (n = 16), or 24 h (n = 6) for physiology and laboratory measurements. RESULTS: 48-h mortality was 33% (8/24), with all deaths occurring between 4 and 22 h. Septic animals were characterized by lethargy, fever, tachycardia, positive blood cultures, and elevated cytokine (IL-1, IL-6, TNF alpha) levels. An increase in heart rate ≥ 50 bpm during the first 4 h of sepsis predicted death with sensitivity and specificity of 88% (p = 0.001). CONCLUSIONS: In this long-term rat sepsis model, prognostication could be made early by telemetry-monitored changes in heart rate. This model enables the study of underlying mechanisms and the assessment of any differential effects of novel therapies in predicted survivors or non-survivors.

Functional expression of the CD163 scavenger receptor on acute myeloid leukemia cells of monocytic lineage.

The hemoglobin-haptoglobin (Hb-Hp) scavenger receptor CD163 is a monocyte/macrophage-restricted surface antigen, whose expression is strongly up-regulated by glucocorticoids. We have previously shown that CD163 is expressed by acute myeloid leukemia (AML) cells of monocytic lineage. Herein, we expand this finding by demonstrating constitutive and glucocorticoid-enhanced CD163 expression on French-American-British M4/M5 AML cells, and leukemic blasts of other AML subtypes and normal hematopoietic progenitor cells do not express CD163. We provide evidence that the functional characteristics of CD163 are preserved on malignant cells by showing the capability of types M4/M5 blast cells to internalize Hb-Hp by a CD163-mediated mechanism. Together, our results identify CD163 as a potential target for therapeutic intervention. It is important that CD163 does not appear to be released from leukemic blasts under noninflammatory conditions, thus reducing the probability of off-target side-effects as a result of competitive binding of potential therapeutic ligands to nonmembrane-bound CD163.

Inflammatory Wnt5A signalling pathways affecting barrier function of human vascular endothelial cells.

Wnt5A is a chemokine secreted by inflammatory-activated human macrophages that sustains their inflammatory response in an autocrine manner. High levels of Wnt5A are found in sera of patients with sepsis and septic shock. Here, we comment on recently reported Wnt5A signalling pathways in human vascular endothelial cells (VEC). In human VEC, Wnt5A regulates cytoskeleton remodelling and barrier function through Ryk receptor and Rho-associated protein serine/threonine kinase, targeting LIMK2 and CFL1 involved in actin polymerisation. Wnt5A/Ryk signalling in VEC can be antagonised by the naturally occurring Wnt inhibitory factor (WIF)-1 (WIF1). Therapeutic targeting of this mechanism may reduce vascular leakage and edema in severe systemic inflammation and therefore should be subject of further investigations.

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

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 ß-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.

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 ß-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.

IL-4 Causes Hyperpermeability of Vascular Endothelial Cells through Wnt5A Signaling.

Microvascular leakage due to endothelial barrier dysfunction is a prominent feature of T helper 2 (Th2) cytokine mediated allergic inflammation. Interleukin-4 (IL-4) is a potent Th2 cytokine, known to impair the barrier function of endothelial cells. However, the effectors mediating IL-4 induced cytoskeleton remodeling and consequent endothelial barrier dysfunction remain poorly defined. Here we have used whole genome transcriptome profiling and gene ontology analyses to identify the genes and processes regulated by IL-4 signaling in human coronary artery endothelial cells (HCAEC). The study revealed Wnt5A as an effector that can mediate actin cytoskeleton remodeling in IL-4 activated HCAEC through the regulation of LIM kinase (LIMK) and Cofilin (CFL). Following IL-4 treatment, LIMK and CFL were phosphorylated, thereby indicating the possibility of actin stress fiber formation. Imaging of actin showed the formation of stress fibers in IL-4 treated live HCAEC. Stress fiber formation was notably decreased in the presence of Wnt inhibitory factor 1 (WIF1). Non-invasive impedance measurements demonstrated that IL-4 increased the permeability and impaired the barrier function of HCAEC monolayers. Silencing Wnt5A significantly reduced permeability and improved the barrier function of HCAEC monolayers upon IL-4 treatment. Our study identifies Wnt5A as a novel marker of IL-4 activated vascular endothelium and demonstrates a critical role for Wnt5A in mediating IL-4 induced endothelial barrier dysfunction. Wnt5A could be a potential therapeutic target for reducing microvascular leakage and edema formation in Th2 driven inflammatory diseases.

Gene expression profiling of inflamed human endothelial cells and influence of activated protein C.

BACKGROUND: During systemic inflammation, activation of vascular endothelium by proinflammatory cytokines leads to hypotension, microvascular thrombosis, and organ damage. Recent data suggest a link between coagulation and inflammation through the activated protein C (APC) pathway. We studied gene expression profiles in human coronary artery endothelial cells (HCAECs) exposed to proinflammatory stimuli and the influence of APC on expression of candidate genes regulated by these stimuli. METHODS AND RESULTS: HCAECs were stimulated with interleukin-1beta, interferon-gamma, and tumor necrosis factor-alpha. In gene expression profiling, 400 of 8400 genes were regulated >2-fold. Verification of selected candidate genes was achieved by measuring expression of mRNA species by real-time polymerase chain reaction, cytokine secretion by ELISA, and metabolites of tetrahydrobiopterin (BH4) biosynthesis by high-performance liquid chromatography. BH4 synthesis, interleukin-6, interleukin-8, monocyte chemotactic protein-1 (MCP-1), and intercellular adhesion molecule-1 (ICAM-1) were downregulated by APC at the transcriptional and protein level. Endothelial nitric oxide synthase, endothelial adhesion molecule, and vascular cell adhesion molecule-1 were not affected by APC. Activities of transcription factors c-Fos, FosB, and c-Rel were inhibited by APC in inflamed HCAECs. CONCLUSIONS: Our study revealed a novel antiinflammatory mechanism of APC-dependent gene regulation in HCAECs since c-Fos-dependent induction of MCP-1 and ICAM-1 was suppressed. APC downregulates expression and activity of genes related to inflammation, most pronounced under intermediate or mild inflammatory conditions.

Functional tetrahydrobiopterin synthesis in human platelets.

BACKGROUND: Previous studies have provided evidence for the importance of platelet-derived nitric oxide (NO) for the regulation of hemostasis. Tetrahydrobiopterin (BH4) is an essential cofactor and regulator of NO synthase activity in the vasculature; however, it is as yet unknown whether platelets dispose over a functional BH4 synthesis. METHODS AND RESULTS: We quantified mRNA expression of genes involved in BH4 synthesis, measured enzymatic activities, and determined intraplatelet levels of pteridines in platelets from healthy volunteers and from patients treated for prolonged periods of time with glucocorticoids. Freshly isolated platelets from healthy volunteers show functional BH4 synthesis, as evidenced by the presence of mRNA species and enzymatic activity of GTP cyclohydrolase I (GTPCH), 6-pyruvoyl tetrahydropterin synthase, and sepiapterin reductase. Biopterin was the major intraplatelet pteridine, whereas no neopterin was found. mRNA expression and enzymatic activity of GTPCH were undetectably low in platelets that had been stored for 5 days, and no pteridines were found in these platelets. Freshly isolated platelets from patients treated with glucocorticoids had decreased mRNA expression and activity of GTPCH compared with platelets from healthy volunteers. CONCLUSIONS: Human platelets dispose over a functional de novo BH4 synthesis. Furthermore, our results indicate the potential of external factors, eg, prolonged storage or glucocorticoid therapy, to significantly affect BH4 synthesis within platelets. Together, these findings offer new insights into the biology and pathobiology of platelet function in humans.