Stroke dependent mechanisms of endothelial autoimmune PAR2 activation

Protease-activated receptor 2 (PAR2) is broadly expressed on endothelial cells contributing to inflammatory processes and angiogenesis. Autoantibodies against PAR2 can regulate these functions in health and disease. Recently, a collaboration group discovered in a prospective observational cohort study that stroke patients with a higher concentration (highest quartile Q4) of PAR2-IgG presented a poor clinical outcome compared to others (lower quartiles Q1-3). However, the biological functions and molecular mechanism remain unclear. Therefore this study aims to explore the possible function of PAR2-IgG on endothelial-based angiogenesis which might have an essential impact on clinical outcome after stroke. Methods: IgGs were isolated from the sera of stroke patients (Q1-3 and Q4), then Matrigel angiogenesis assay was used to study vascular tube formation by IgGs and the natural PAR2 agonist trypsin in vascular endothelial cells (HMEC-1 and hCMEC/D3, a blood-brain barrier human endothelial cell line). Additionally, qRT-PCR, western blot, and ELISA were used to investigate the angiogenic signaling pathways. The activation of G-protein subunits was examined by the Luciferase reporter assay, based on mutations of wild-type PAR2 and extracellular loops (ECLs). The receptor internalization was studied by Nano-Glo ®HiBiT detection assay. The main inductor of angiogenesis VEGF was examined in detail by promoter assay with regard to the activation of the promoter and potential binding fragments of transcription factors. Results: The PAR2 agonist trypsin was involved in the regulation of mRNA synthesis and protein release of VEGF, IL-6, IL-8, CXCL1 and ANG2 through the ERK1/2 signaling pathway, which promoted vascular tube formation of HMEC-1. Q1-3 PAR2-IgG enhanced angiogenesis via PAR2-ERK1/2-induced angiogenic cytokines and receptor internalization when compared to Q4 PAR2-IgG. Q1-3 PAR2-IgG 10 triggered Gq/11 activation -, but not G12/13 -, and the mutation of ECL1-3 did not alter the activation. As for Q4, the ECL3 mutation increased the Gq/11 activation and ERK1/2 activation. Different truncations of the VEGF promoter assay confirmed the binding sites of transcription factors located in the VEGF promoter sequence -1339 to -839 bp, and the inhibition of AP-1 completely blocked the promoter activation. To summarize, in contrast to the Q4 group, Q1-3 PAR2-IgG induced greater receptor internalization, Gq/11 activation, ERK1/2 activation, and angiogenic gene production, which resulted in increased formation of vascular tubes. Conclusion: The present work elucidated new PAR2 signaling pathways that are induced by PAR2-IgG in human endothelial cells and identified contrary functional consequences on the angiogenesis of PAR2-IgGs from two different stroke patient cohorts classified by different PAR2-IgG titer ranges. Novel therapeutics modulating PAR2-IgGs in stroke patients might thereby already limit stroke progression at an early stage and lead to improved clinical outcome.Hintergrund: Protease-aktivierter Rezeptor 2 (PAR2) ist auf Endothelzellen hoch exprimiert und reguliert inflammatorische Prozesse und Angiogenese. Autoantikörper gegen PAR2 können diese Funktionen physiologisch und pathophysiologisch beeinflussen. Vor kurzem entdeckten Kollaborationspartner in einer prospektiven Kohortenstudie, dass Schlaganfallpatienten mit hohen PAR2-Spiegeln (höchstes Quartil Q4) einen schlechteren klinischen Outcome hatten als Patienten mit niedrigeren Spiegeln (Quartile Q1-3). Die molekularen Mechanismen dafür waren allerdings ungeklärt. Es war daher das Ziel dieser Studie, eine mögliche Bedeutung dieser PAR2-IgGs auf endotheliale Angiogenese zu untersuchen, die sich positiv auf den klinischen Outcome auswirken könnten. Methoden: IgGs wurden von Schlaganfallpatienten (Q1-3 und Q4) isoliert und dann der Matrigel Angiogenese Assay benutzt, um Gefäßbildungen von Endothelzellen (HMEC-1 und hCMEC/D3, einer humanen Bluthirnschranken-Endothelzelllinie) durch die IgGs und den natürlichen Agonisten von PAR2, Trypsin, zu untersuchen. Weiterhin dienten qRT-PCR, Westernblots und ELISA zur Erforschung angiogenetischer Signalwege. Luziferase-Reporter-Assays wurden durchgeführt, um die Aktivierung von G-Protein-Untereinheiten mithilfe von Mutationen von Wildtyp-PAR2 und der extrazellulären Schleifen (ECLs) zu untersuchen. Die Internalisierung des Rezeptors wurde visualisiert mit dem Nano-Glo ®HiBiT Detektionsassay. Der Hauptaktivator der Angiogenese, VEGF, wurde detailliert untersucht mithilfe von Promotoraktivierungsstudien und Bindungsfragmente. Ergebnisse: Der PAR2 Agonist Trypsin beeinflusste die mRNA Synthese und Proteinfreisetzung von VEGF, IL-6, IL-8, CXCL1 und ANG2 über den ERK1/2 Signalweg und förderte die Ausbildung vaskulärer Gefäßschläuche in HMEC-1. Im Vergleich zu Q4 PAR2-IgG erhöhte Q1-3 PAR2-IgG Angiogenese durch Freisetzung von 13 angiogentischer Zytokine über PAR2-ERK1/2-Aktivierung und Rezeptorinternalisierung. Q1-3 PAR2-IgG triggerte die Aktivierung von Gq/11, aber nicht von G12/13, und Mutationen von ECL1-3 hatten keinen Einfluss auf die Aktivierung. VEGF Promotorassays bestätigten die Bindungsstelle des Transkriptionsfaktors AP-1 an der VEGF-Promotorsequenz -1339 bis -839 bp. Zusammenfassend induzierte Q1-3 PAR2-IgG im Gegensatz zu Q4 PAR2-IgG eine verstärkte Rezeptorinternalisierung, Gq/11 – und ERK1/2-Aktivierung und Induktion angiogenetischer Gene, was in einer verstärkten Netzwerkbildung resultierte. Schlussfolgerung: Die hier dargestellte Arbeit beschreibt neue Signalwege von PAR2 in humanen Endothelzellen, die durch PAR2-IgG aktiviert werden und identifizierte gegensätzliche Auswirkungen auf Angiogenese durch PAR2-IgG von zwei unterschiedlichen Schlaganfallkohorten mit besonders hohen (Q4) oder niedrig-mäßigen (Q1-3) PAR2-IgG-Spiegeln. Die Modulation von PAR2-IgGs könnte eine neue Therapieform bei Schlaganfall darstellen, um die Progression im Verlauf zu verhindern und zu verbessertem klinischem Outcome zu führen

Metabolites changes of a low-temperature and low-salt fermented Chinese kohlrabi during fermentation based on non-targeted metabolomic analysis

A low-temperature and low-salt industrially fermented Chinese kohlrabi (LSCK) was developed in this study, with the salt usage decreased by approximately 70% compared to the traditional high-salt fermented Chinese kohlrabi (HSCK). The differences in physicochemical properties, metabolites and overall flavors during LSCK fermented for 0, 45 and 90 days (d) were analyzed by gas chromatography-time-of-flight mass spectrometry (GC-TOF-MS), electronic nose (E-nose) and other techniques. The results showed that the total acid content increased significantly from 3.68 to 8.59 g/kg. However, the protein content significantly decreased from 2.52/100 to 0.66 g/100 g. The number of lactic acid bacteria cells increased significantly from 3.69 to 4.46 log10CFU/g. Based on multivariate statistical analysis, 21, 14, and 15 differential metabolites were identified in the three treatment groups A1 (0 and 45 days), A2 (45 and 90 days), and A3 (0 and 90 days) respectively (VIP > 1, p < 0.05, |log2FC| ≥ 1.1). Carbohydrates, sugar alcohols, amino acids and their derivatives were the main differential metabolites in the LSCKs fermented for different periods. Aminoacyl−tRNA biosynthesis and glycine, serine and threonine metabolism pathways significantly correlated with the differential metabolites based on Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis (p < 0.05). Furthermore, the overall odors were significantly different among the LSCKs with different fermentation periods, as detected by E-nose. The present study describes the change trend of metabolites during LSCK fermentation and elucidates important metabolic pathways in LSCK, providing a theoretical basis for the target regulation of functional metabolites in kohlrabi and the optimization of LSCK processing

Autoantibodies from Patients with Scleroderma Renal Crisis Promote PAR-1 Receptor Activation and IL-6 Production in Endothelial Cells

Background. Scleroderma renal crisis (SRC) is a life-threatening complication of systemic sclerosis (SSc). Autoantibodies (Abs) against endothelial cell antigens have been implicated in SSc and SRC. However, their detailed roles remain poorly defined. Pro-inflammatory cytokine interleukin-6 (IL-6) has been found to be increased in SSc, but its role in SRC is unclear. Here, we aimed to determine how the autoantibodies from patients with SSc and SRC affect IL-6 secretion by micro-vascular endothelial cells (HMECs). Methods. Serum IgG fractions were isolated from either SSc patients with SRC (n = 4) or healthy individuals (n = 4) and then each experiment with HMECs was performed with SSc-IgG from a separate patient or separate healthy control. IL-6 expression and release by HMECs was assessed by quantitative reverse transcription and quantitative PCR (RT-qPCR) and immunoassays, respectively. The mechanisms underlying the production of IL-6 were analyzed by transient HMEC transfections with IL-6 promoter constructs, electrophoretic mobility shift assays, Western blots and flow cytometry. Results. Exposure of HMECs to IgG from SSc patients, but not from healthy controls, resulted in a time- and dose-dependent increase in IL-6 secretion, which was associated with increased AKT, p70S6K, and ERK1/2 signalling, as well as increased c-FOS/AP-1 transcriptional activity. All these effects could be reduced by the blockade of the endothelial PAR-1 receptor and/or c-FOS/AP-1silencing. Conclusions. Autoantibodies against PAR-1 found in patients with SSc and SRC induce IL-6 production by endothelial cells through signalling pathways controlled by the AP-1 transcription factor. These observations offer a greater understanding of adverse endothelial cell responses to autoantibodies present in patients with SRC

Angiogenic Role of Mesothelium-Derived Chemokine CXCL1 During Unfavorable Peritoneal Tissue Remodeling in Patients Receiving Peritoneal Dialysis as Renal Replacement Therapy

Peritoneal dialysis (PD) is a valuable 'home treatment' option, even more so during the ongoing Coronavirus pandemic. However, the long-term use of PD is limited by unfavourable tissue remodelling in the peritoneal membrane, which is associated with inflammation-induced angiogenesis. This appears to be driven primarily through vascular endothelial growth factor (VEGF), while the involvement of other angiogenic signaling pathways is still poorly understood. Here, we have identified the crucial contribution of mesothelial cell-derived angiogenic CXC chemokine ligand 1 (CXCL1) to peritoneal angiogenesis in PD. CXCL1 expression and peritoneal microvessel density were analysed in biopsies obtained by the International Peritoneal Biobank (NCT01893710 at www.clinicaltrials.gov), comparing 13 children with end-stage kidney disease before initiating PD to 43 children on chronic PD. The angiogenic potential of mesothelial cell-derived CXCL1 was assessed in vitro by measuring endothelial tube formation of human microvascular endothelial cells (HMECs) treated with conditioned medium from human peritoneal mesothelial cells (HPMCs) stimulated to release CXCL1 by treatment with either recombinant IL-17 or PD effluent. We found that the capillary density in the human peritoneum correlated with local CXCL1 expression. Both CXCL1 expression and microvessel density were higher in PD patients than in the age-matched patients prior to initiation of PD. Exposure of HMECs to recombinant CXCL1 or conditioned medium from IL-17-stimulated HPMCs resulted in increased endothelial tube formation, while selective inhibition of mesothelial CXCL1 production by specific antibodies or through silencing of relevant transcription factors abolished the proangiogenic effect of HPMC-conditioned medium. In conclusion, peritoneal mesothelium-derived CXCL1 promotes endothelial tube formation in vitro and associates with peritoneal microvessel density in uremic patients undergoing PD, thus providing novel targets for therapeutic intervention to prolong PD therapy

Expanded Hemodialysis Therapy Ameliorates Uremia-Induced Systemic Microinflammation and Endothelial Dysfunction by Modulating VEGF, TNF-α and AP-1 Signaling

Systemic chronic microinflammation and altered cytokine signaling, with adjunct cardiovascular disease (CVD), endothelial maladaptation and dysfunction is common in dialysis patients suffering from end-stage renal disease and associated with increased morbidity and mortality. New hemodialysis filters might offer improvements. We here studied the impact of novel improved molecular cut-off hemodialysis filters on systemic microinflammation, uremia and endothelial dysfunction. Human endothelial cells (ECs) were incubated with uremic serum obtained from patients treated with two different hemodialysis regimens in the Permeability Enhancement to Reduce Chronic Inflammation (PERCI-II) crossover clinical trial, comparing High-Flux (HF) and Medium Cut-Off (MCO) membranes, and then assessed for their vascular endothelial growth factor (VEGF) production and angiogenesis. Compared to HF membranes, dialysis with MCO membranes lead to a reduction in proinflammatory mediators and reduced endothelial VEGF production and angiogenesis. Cytokine multiplex screening identified tumor necrosis factor (TNF) superfamily members as promising targets. The influence of TNF-alpha and its soluble receptors (sTNF-R1 and sTNF-R2) on endothelial VEGF promoter activation, protein release, and the involved signaling pathways was analyzed, revealing that this detrimental signaling was indeed induced by TNF-alpha and mediated by AP-1/c-FOS signaling. In conclusion, uremic toxins, in particular TNF-signaling, promote endothelial maladaptation, VEGF expression and aberrant angiogenesis, which can be positively modulated by dialysis with novel MCO membranes

Control of neutrophil influx during peritonitis by transcriptional cross‐regulation of chemokine CXCL1 by IL‐17 and IFN‐γ

Neutrophil infiltration is a hallmark of peritoneal inflammation, but mechanisms regulating neutrophil recruitment in patients with peritoneal dialysis (PD)-related peritonitis are not fully defined. We examined 104 samples of PD effluent collected during acute peritonitis for correspondence between a broad range of soluble parameters and neutrophil counts. We observed an association between peritoneal IL-17 and neutrophil levels. This relationship was evident in effluent samples with low but not high IFN-γ levels, suggesting a differential effect of IFN-γ concentration on neutrophil infiltration. Surprisingly, there was no association of neutrophil numbers with the level of CXCL1, a key IL-17-induced neutrophil chemoattractant. We investigated therefore the production of CXCL1 by human peritoneal mesothelial cells (HPMCs) under in vitro conditions mimicking clinical peritonitis. Stimulation of HPMCs with IL-17 increased CXCL1 production through induction of transcription factor SP1 and activation of the SP1-binding region of the CXCL1 promoter. These effects were amplified by TNFα. In contrast, IFN-γ dose-dependently suppressed IL-17-induced SP1 activation and CXCL1 production through a transcriptional mechanism involving STAT1. The SP1-mediated induction of CXCL1 was also observed in HPMCs exposed to PD effluent collected during peritonitis and containing IL-17 and TNFα, but not IFN-γ. Supplementation of the effluent with IFN-γ led to a dose-dependent activation of STAT1 and a resultant inhibition of SP1-induced CXCL1 expression. Transmesothelial migration of neutrophils in vitro increased upon stimulation of HPMCs with IL-17 and was reduced by IFN-γ. In addition, HPMCs were capable of binding CXCL1 at their apical cell surface. These observations indicate that changes in relative peritoneal concentrations of IL-17 and IFN-γ can differently engage SP1–STAT1, impacting on mesothelial cell transcription of CXCL1, whose release and binding to HPMC surface may determine optimal neutrophil recruitment and retention during peritonitis

Expanded Hemodialysis ameliorates uremia-induced impairment of vasculoprotective KLF2 and concomitant proinflammatory priming of endothelial cells through an ERK/AP1/cFOS-dependent mechanism

AimsExpanded hemodialysis (HDx) therapy with improved molecular cut-off dialyzers exerts beneficial effects on lowering uremia-associated chronic systemic microinflammation, a driver of endothelial dysfunction and cardiovascular disease (CVD) in hemodialysis (HD) patients with end-stage renal disease (ESRD). However, studies on the underlying molecular mechanisms are still at an early stage. Here, we identify the (endothelial) transcription factor Krüppel-like factor 2 (KLF2) and its associated molecular signalling pathways as key targets and regulators of uremia-induced endothelial micro-inflammation in the HD/ESRD setting, which is crucial for vascular homeostasis and controlling detrimental vascular inflammation.Methods and resultsFirst, we found that human microvascular endothelial cells (HMECs) and other typical endothelial and kidney model cell lines (e.g. HUVECs, HREC, and HEK) exposed to uremic serum from patients treated with two different hemodialysis regimens in the Permeability Enhancement to Reduce Chronic Inflammation II (PERCI-II) crossover clinical trial - comparing High-Flux (HF) and Medium Cut-Off (MCO) membranes - exhibited strongly reduced expression of vasculoprotective KLF2 with HF dialyzers, while dialysis with MCO dialyzers led to the maintenance and restoration of physiological KLF2 levels in HMECs. Mechanistic follow-up revealed that the strong downmodulation of KLF2 in HMECs exposed to uremic serum was mediated by a dominant engagement of detrimental ERK instead of beneficial AKT signalling, with subsequent AP1-/c-FOS binding in the KLF2 promoter region, followed by the detrimental triggering of pleiotropic inflammatory mediators, while the introduction of a KLF2 overexpression plasmid could restore physiological KLF2 levels and downmodulate the detrimental vascular inflammation in a mechanistic rescue approach.ConclusionUremia downmodulates vasculoprotective KLF2 in endothelium, leading to detrimental vascular inflammation, while MCO dialysis with the novel improved HDx therapy approach can maintain physiological levels of vasculoprotective KLF2

Autoantibodies from patients with kidney allograft vasculopathy stimulate a proinflammatory switch in endothelial cells and monocytes mediated via GPCR-directed PAR1-TNF-α signaling

Non-HLA-directed regulatory autoantibodies (RABs) are known to target G-protein coupled receptors (GPCRs) and thereby contribute to kidney transplant vasculopathy and failure. However, the detailed underlying signaling mechanisms in human microvascular endothelial cells (HMECs) and immune cells need to be clarified in more detail. In this study, we compared the immune stimulatory effects and concomitant intracellular and extracellular signaling mechanisms of immunoglobulin G (IgG)-fractions from kidney transplant patients with allograft vasculopathy (KTx-IgG), to that from patients without vasculopathy, or matched healthy controls (Con-IgG). We found that KTx-IgG from patients with vasculopathy, but not KTx-IgG from patients without vasculopathy or Con-IgG, elicits HMEC activation and subsequent upregulation and secretion of tumor necrosis factor alpha (TNF-α) from HMECs, which was amplified in the presence of the protease-activated thrombin receptor 1 (PAR1) activator thrombin, but could be omitted by selectively blocking the PAR1 receptor. The amount and activity of the TNF-α secreted by HMECs stimulated with KTx-IgG from patients with vasculopathy was sufficient to induce subsequent THP-1 monocytic cell activation. Furthermore, AP-1/c-FOS, was identified as crucial transcription factor complex controlling the KTx-IgG-induced endothelial TNF-α synthesis, and mircoRNA-let-7f-5p as a regulatory element in modulating the underlying signaling cascade. In conclusion, exposure of HMECs to KTx-IgG from patients with allograft vasculopathy, but not KTx-IgG from patients without vasculopathy or healthy Con-IgG, triggers signaling through the PAR1-AP-1/c-FOS-miRNA-let7-axis, to control TNF-α gene transcription and TNF-α-induced monocyte activation. These observations offer a greater mechanistic understanding of endothelial cells and subsequent immune cell activation in the clinical setting of transplant vasculopathy that can eventually lead to transplant failure, irrespective of alloantigen-directed responses

Research on obstacle climbing gait structure design and gait control of hexapod wall climbing robot based on STM32F103 core controller

The hexapod wall climbing robots have the advantages of traversing complex wall surfaces. To traverse complex environments autonomously, it must possess the capability to select gait parameters and paths appropriate for the wall surface. Path planning and gait optimization is a fundamental issue in the aspect of stable, energy efficient robot navigation in complex environments with static and dynamic obstacles. Traditional statistical models have been developed to get the optimal path and gait parameters but the result obtained was very poor. Metaheuristic algorithms are gaining importance in robotic gait planning. In this paper, we proposed robust two stage gait planning approach for predicting collision-free, distance-minimal, smooth navigation path and ensuring stable, energy efficient gait patterns for robots using hybrid metaheuristic algorithms. In the first stage, optimal climbing path for robot is predicted using Tri-objective Grey Wolf Path Optimization (TGWPO) based on obstacle and target detection. In the second stage, the gait parameters adaptive to the constructed climbing path are optimized using Adaptive multi-objective Particle swarm optimization (AMPSO). The hexapod wall climbing robot is designed with STM32F103 as core controller modeled with optimal path planner (using TDWPO) and gait optimizer module (using AMPSO). STM32F103 controller commands and controls the robot to climb on wall with optimized gait parameters according to the optimal path. We analyzed the efficacy of the proposed two stage gait planning approach using TDWPO-AMPSO for hexapod wall climbing robots with existing gait planning approaches in terms of path length, climbing time, gait stability, obstacle avoidance, and energy efficiency. The result analysis showed that the suggested gait planning approach is efficient over conventional strategies for climbing robots

Performance-Based Design of a Main Exhibition Hall and Its Ecological Connectivity with Surroundings

This study aims to verify which the performance-based design was successfully applied in the construction of the main exhibition hall of the first Hebei Garden Expo, China. Based on the idea of “equality and connectivity” among humans, buildings, and the environment, the exhibition hall was designed using digital shape generation technology, and a special double-skin structure was selected. A bright exterior glass curtain wall separated from the cell-shaped reinforced concrete (RC) structure, not only made the exhibition hall fit into the scenery so well, but also presented an adhesion area with functional links to the RC structure. Joints of the steel tubular truss and the glass curtain wall were optimally designed by means of finite element simulations. During the service of more than a decade, extension of space, function of interactivity, and energy conservation required for a green infrastructure were achieved, highlighting the ecological connectivity of the exhibition hall with the surrounding landscape. The exhibition hall performed synergy of structure and architecture functions and it is still a landmark building in Shijiazhuang, showing an excellent application achievement of the performance-based design