Endothelial Intercellular Adhesion Molecule-1 Research Articles

High-Density Lipoprotein Anti-Inflammatory Capacity and Acute Kidney Injury After Cardiac and Vascular Surgery: A Prospective Observational Study.

Acute kidney injury (AKI) predicts death after cardiac and vascular surgery. Higher preoperative high-density lipoprotein (HDL) concentrations are associated with less postoperative AKI. In animals, HDL's anti-inflammatory capacity to suppress endothelial cell adhesion molecule expression reduces kidney damage due to ischemia and hemorrhagic shock. The objective of this study is to evaluate the statistical relationship between HDL anti-inflammatory capacity and AKI after major cardiac and vascular surgery. Prospective observational study. Quaternary medical center. One hundred adults with chronic kidney disease on long-term statin therapy undergoing major elective cardiac and vascular surgery. None. Apolipoprotein B-depleted serum collected at anesthetic induction was incubated with tumor necrosis factor alpha stimulated human endothelial cells. Reverse transcriptase-polymerase chain reaction was used to measure intercellular adhesion molecule-1 (ICAM-1) messenger RNA. Enzyme-linked immunosorbent assay assays were used to measure apolipoprotein A-I and postoperative soluble ICAM-1 concentrations in patient plasma. HDL concentration did not correlate with HDL ICAM-1 suppression capacity (Spearman R = 0.05; p = 0.64). Twelve patients (12%) were found to have dysfunctional, pro-inflammatory HDL. Patients with pro-inflammatory HDL had a higher rate of postoperative AKI than patients with anti-inflammatory HDL (p = 0.046). After adjustment for AKI risk factors, a higher preoperative HDL capacity to suppress endothelial ICAM-1 was independently associated with lower odds of AKI (odds ratio, 0.88; 95% CI, 0.80-0.98; p = 0.016). The association between HDL anti-inflammatory capacity and postoperative AKI was independent of HDL concentration (p = 0.018). Further, a higher long-term statin dose was associated with higher HDL capacity to suppress endothelial ICAM-1 (p = 0.045). Patients with chronic kidney disease undergoing cardiac and vascular surgery who have dysfunctional, pro-inflammatory HDL have a higher risk of postoperative AKI compared with patients with anti-inflammatory HDL. Conversely, a higher HDL anti-inflammatory capacity is associated with a lower risk of postoperative AKI, independent of HDL concentration. Higher long-term statin dose is associated with higher HDL anti-inflammatory capacity.

Bezafibrate mitigates oxidized-low density lipoprotein (ox-LDL)-induced the attachment of monocytes to endothelial cells: An implication in atherosclerosis.

Oxidized forms of low-density lipoproteins (ox-LDL)-associated endothelial dysfunction and subsequent monocyte adhesion play an important role in the development of atherosclerosis (AS). Bezafibrate (BEZ) is a peroxisome proliferator-activated receptor (pan-PPAR) agonist licensed as a hypolipidemic drug. However, the effects of BEZ on endothelial dysfunction are less reported. In this study, we aim to investigate the protective effects of BEZ on ox-LDL-challenged vascular endothelial cells to evaluate its potential value in treating AS. Human aortic endothelial cells (HAECs) and THP-1 cells were used to establish an In Vitro AS model. Cell Counting Kit-8 (CCK-8) assay, Real-time PCR, Western blot analysis, and Enzyme-linked immunosorbent assay (ELISA) were used to test the data. As expected, treatment with BEZ suppressed the expression of vascular endothelial growth factor A (VEGF-A), tissue factor (TF), Interleukin 12 (IL-12), tumor necrosis factor (TNF-α), and monocyte chemoattractant protein-1 (MCP-1). BEZ was also found to inhibit ox-LDL-induced expression of the endothelial adhesion molecules vascular cellular adhesion molecule-1 (VCAM-1) and intercellular adhesion molecule-1 (ICAM-1) in HAECs. Correspondingly, BEZ prevented attachment of THP-1 monocytes to ox-LDL-incubated HAECs. Mechanically, BEZ was found to prevent NF-κB activation by reducing the levels of nuclear NF-κB p65 and inhibiting luciferase activity of NF-κB. Our study revealed the pharmacological function of BEZ in protecting endothelial dysfunction against ox-LDL, which may provide valuable insight for the clinical application of BEZ.

MiR-486-5p protects against rat ischemic kidney injury and prevents the transition to chronic kidney disease and vascular dysfunction.

Acute kidney injury (AKI) increases the risk for progressive chronic kidney disease (CKD). MicroRNA (miR)-486-5p protects against kidney ischemia-reperfusion (IR) injury in mice, although its long-term effects on the vasculature and development of CKD are unknown. We studied whether miR-486-5p would prevent the AKI to CKD transition in rat, and affect vascular function. Adult male rats were subjected to bilateral kidney IR followed by i.v. injection of liposomal-packaged miR-486-5p (0.5 mg/kg). Kidney function and histologic injury were assessed after 24 h and 10 weeks. Kidney endothelial protein levels were measured by immunoblot and immunofluorescence, and mesenteric artery reactivity was determined by wire myography. In rats with IR, miR-486-5p blocked kidney endothelial cell increases in intercellular adhesion molecule-1 (ICAM-1), reduced neutrophil infiltration and histologic injury, and normalized plasma creatinine (P<0.001). However, miR-486-5p attenuated IR-induced kidney endothelial nitric oxide synthase (eNOS) expression (P<0.05). At 10 weeks, kidneys from rats with IR alone had decreased peritubular capillary density and increased interstitial collagen deposition (P<0.0001), and mesenteric arteries showed impaired endothelium-dependent vasorelaxation (P<0.001). These changes were inhibited by miR-486-5p. Delayed miR-486-5p administration (96 h, 3 weeks after IR) had no impact on kidney fibrosis, capillary density, or endothelial function. In rats, administration of miR-486-5p early after kidney IR prevents injury, and protects against CKD development and systemic endothelial dysfunction. These protective effects are associated with inhibition of endothelial ICAM-1 and occur despite reduction in eNOS. miR-486-5p holds promise for the prevention of ischemic AKI and its complications.

Neutrophil extracellular traps extrusion from neutrophils stably adhered to ICAM-1 by lipoteichoic acid stimulation

The effect of neutrophil extracellular traps (NETs) on promoting intravascular microthrombi formation and exacerbating the severity of sepsis in patients has gained extensive attention. However, in sepsis, the mechanisms and key signaling molecules mediating NET formation during direct interactions of endothelial cells and neutrophils still need further explored. Herein, we utilized lipoteichoic acid (LTA), a component shared by Gram-positive bacteria, to induce NET extrusion from neutrophils firmly adhered to the glass slides coated with intercellular adhesion molecule-1(ICAM-1). We also used Sytox green to label NET-DNA and Flou-4 AM as the intracellular Ca 2+ signaling indicator to observe the NET formation and fluctuation of Ca 2+ signaling. Our results illustrated that LTA was able to induce NET release from neutrophils firmly attached to ICAM-1-coated glass slides, and the process was time-dependent. In addition, our study indicated that LTA-induced NET release by neutrophils stably adhered to ICAM-1 depended on Ca 2+ signaling but not intracellular reactive oxygen species (ROS). This study reveals NET formation mediated by direct interactions between endothelial ICAM-1 and neutrophils under LTA stimulation and key signaling molecules involved, providing the theoretical basis for medicine development and clinical treatment for related diseases.

Biochanin A inhibits endothelial dysfunction induced by IL‑6‑stimulated endothelial microparticles in Perthes disease via the NFκB pathway.

Endothelial dysfunction caused by the stimulation of endothelial microparticles (EMPs) by the inflammatory factor IL-6 is one of the pathogenic pathways associated with Perthes disease. The natural active product biochanin A (BCA) has an anti-inflammatory effect; however, whether it can alleviate endothelial dysfunction in Perthes disease is not known. The present in vitro experiments on human umbilical vein endothelial cells showed that 0-100 pg/ml IL-6-EMPs could induce endothelial dysfunction in a concentration-dependent manner, and the results of the Cell Counting Kit 8 assay revealed that, at concentrations of <20 µM, BCA had no cytotoxic effect. Reverse transcription-quantitative PCR demonstrated that BCA reduced the expression levels of the endothelial dysfunction indexes E-selectin and intercellular cell adhesion molecule-1 (ICAM-1) in a concentration-dependent manner. Immunofluorescence and western blotting illustrated that BCA increased the expression levels of zonula occludens-1 and decreased those of ICAM-1. Mechanistic studies showed that BCA inhibited activation of the NFκB pathway. In vivo experiments demonstrated that IL-6 was significantly increased in the rat model of ischemic necrosis of the femoral head, whereas BCA inhibited IL-6 production. Therefore, in Perthes disease, BCA may inhibit the NFκB pathway to suppress IL-6-EMP-induced endothelial dysfunction, and could thus be regarded as a potential treatment for Perthes disease.

Exosomes From IgE-Stimulated Mast Cells Aggravate Asthma-Mediated Atherosclerosis Through circRNA CDR1as-Mediated Endothelial Cell Dysfunction in Mice.

IgE has been known for mediating endothelial cell dysfunction and mast cell (MC) activation to fuel asthma-aggravated high-fat diet-induced atherosclerosis. However, it remains unclear for the mechanism of asthma-mediated atherosclerosis, especially the potential involvement of IgE in the exacerbation of asthma-mediated atherosclerosis with a standard laboratory diet, and the cross talk between endothelial cells and MCs. Asthma-mediated atherosclerosis mice models under a standard laboratory diet and FcεR1 knock-out mice were used to determine the role of IgE-FcεR1 signaling in asthma-mediated atherosclerosis, which was assessed by Oil Red O staining and immunohistochemistry. Various in vitro assays including nanoparticle tracking analysis and transmission electron microscopy were used to evaluate exosome characteristics. Immunofluorescence and fluorescent in situ hybridization approaches were used to evaluate the effect and mechanism of MC-secreted exosomes encapsulated circular RNA CDR1as (cerebellar degeneration-related 1 antisense) on endothelial cells in vivo and in vitro. Finally, cohort studies examined the plasma CDR1as levels in patients with atherosclerosis with or without allergies. Asthma mice with a standard laboratory diet showed increased atherosclerotic lesions and inflammatory infiltration depending on IgE-FcεR1 signal. FcεR1 knockout mice and blockage of IgE-FcεR1 signaling with IgE monoclonal antibody, omalizumab, all significantly alleviated asthma-mediated atherosclerosis and vascular inflammatory remodeling. Anti-inflammation with dexamethasone and stabilization of MC with cromolyn partially alleviated atherosclerotic lesions and mitigated the inflammatory infiltration in arteries. Mechanistically, IgE stimulation upregulates MC CDR1as expression in exosomes and upregulates the endothelial cell adhesive factors VCAM-1 (vascular cell adhesion molecule-1) and ICAM-1 (intercellular adhesion molecule-1) via the CDR1as-FUS (fused in sarcoma)-phos-p65 axis. Knockdown of CDR1as in vivo significantly decreased the endothelial adhesion function and mitigated asthma-mediated atherosclerosis. Furthermore, a cohort study indicated higher plasma CDR1as levels in patients with atherosclerosis with allergies than in patients with atherosclerosis and healthy controls. Exosomes from IgE-stimulated MCs aggravated atherosclerosis through circular RNA CDR1as-mediated endothelial dysfunction, providing a novel insight into asthma-mediated atherosclerosis and potential diagnostic and therapeutic targets.

Improving vasculoprotective effects of MSCs in coronary microvessels - benefits of 3D culture, sub-populations and heparin.

Opening occluded coronary arteries in patients with myocardial infarction (MI) damages the delicate coronary microvessels through a process called myocardial ischaemia-reperfusion injury. Although mesenchymal stromal cells (MSCs) have the potential to limit this injury, clinical success remains limited. This may be due to (i) poor MSC homing to the heart (ii) infused MSCs, even if derived from the same site, being a heterogeneous population with varying therapeutic efficacy and (iii) conventional 2D culture of MSCs decreasing their homing and beneficial properties. This study investigated whether 3D culture of two distinctly different bone marrow (BM)-derived MSC sub-populations could improve their homing and coronary vasculoprotective efficacy. Intravital imaging of the anaesthetised mouse beating heart was used to investigate the trafficking and microvascular protective effects of two clonally-derived BM-derived MSC lines, namely CD317neg MSCs-Y201 and CD317pos MSCs-Y202, cultured using conventional monolayer and 3D hanging drop methods. 3D culture consistently improved the adhesive behaviour of MSCs-Y201 to various substrates in vitro. However, it was their differential ability to reduce neutrophil events within the coronary capillaries and improve ventricular perfusion in vivo that was most remarkable. Moreover, dual therapy combined with heparin further improved the vasculoprotection afforded by 3D cultured MSCs-Y201 by also modifying platelet as well as neutrophil recruitment, which subsequently led to the greatest salvage of viable myocardium. Therapeutic benefit could mechanistically be explained by reductions in coronary endothelial oxidative stress and intercellular adhesion molecule-1 (ICAM-1)/vascular cell adhesion molecule-1 (VCAM-1) expression. However, since this was noted by both 2D and 3D cultured MSCs-Y201, therapeutic benefit is likely explained by the fact that 3D cultured MSCs-Y201 were the most potent sub-population at reducing serum levels of several pro-inflammatory cytokines. This novel study highlights the importance of not only 3D culture, but also of a specific CD317neg MSC sub-population, as being critical to realising their full coronary vasculoprotective potential in the injured heart. Since the smallest coronary blood vessels are increasingly recognised as a primary target of reperfusion injury, therapeutic interventions must be able to protect these delicate structures from inflammatory cells and maintain perfusion in the heart. We propose that relatively feasible technical modifications in a specific BM-derived MSC sub-population could achieve this.

LFA-1/ICAM-1 Adhesion Pathway Mediates the Homeostatic Migration of Lymphocytes from Peripheral Tissues into Lymph Nodes through Lymphatic Vessels.

Lymphocyte function-associated antigen-1 (LFA-1) and its endothelial ligand intercellular adhesion molecule-1 (ICAM-1) are important for the migration of lymphocytes from blood vessels into lymph nodes. However, it is largely unknown whether these molecules mediate the homeostatic migration of lymphocytes from peripheral tissues into lymph nodes through lymphatic vessels. In this study, we find that, in naive mice, ICAM-1 is expressed on the sinus endothelia of lymph nodes, but not on the lymphatic vessels of peripheral tissues. In in vivo lymphocyte migration assays, memory CD4+ T cells migrated to lymph nodes from peripheral tissues much more efficiently than from blood vessels, as compared to naive CD4+ T cells. Moreover, ICAM-1 deficiency in host mice significantly inhibited the migration of adoptively transferred wild-type donor lymphocytes from peripheral tissues, but not from blood vessels, into lymph nodes. The migration of LFA-1-deficient donor lymphocytes from peripheral tissues into the lymph nodes of wild-type host mice was also significantly reduced as compared to wild-type donor lymphocytes. Furthermore, the number of memory T cells in lymph nodes was significantly reduced in the absence of ICAM-1 or LFA-1. Thus, our study extends the functions of the LFA-1/ICAM-1 adhesion pathway, indicating its novel role in controlling the homeostatic migration of lymphocytes from peripheral tissues into lymph nodes and maintaining memory T cellularity in lymph nodes.

High levels of endothelial ICAM-1 prohibit natalizumab mediated abrogation of CD4+ T cell arrest on the inflamed BBB under flow in vitro

IntroductionThe humanized anti-α4 integrin blocking antibody natalizumab (NTZ) is an effective treatment for relapsing–remitting multiple sclerosis (RRMS) that is associated with the risk of progressive multifocal leukoencephalopathy (PML). While extended interval dosing (EID) of NTZ reduces the risk for PML, the minimal dose of NTZ required to maintain its therapeutic efficacy remains unknown.ObjectiveHere we aimed to identify the minimal NTZ concentration required to inhibit the arrest of human effector/memory CD4+ T cell subsets or of PBMCs to the blood–brain barrier (BBB) under physiological flow in vitro.ResultsMaking use of three different human in vitro BBB models and in vitro live-cell imaging we observed that NTZ mediated inhibition of α4-integrins failed to abrogate T cell arrest to the inflamed BBB under physiological flow. Complete inhibition of shear resistant T cell arrest required additional inhibition of β2-integrins, which correlated with a strong upregulation of endothelial intercellular adhesion molecule (ICAM)-1 on the respective BBB models investigated. Indeed, NTZ mediated inhibition of shear resistant T cell arrest to combinations of immobilized recombinant vascular cell adhesion molecule (VCAM)-1 and ICAM-1 was abrogated in the presence of tenfold higher molar concentrations of ICAM-1 over VCAM-1. Also, monovalent NTZ was less potent than bivalent NTZ in inhibiting T cell arrest to VCAM-1 under physiological flow. In accordance with our previous observations ICAM-1 but not VCAM-1 mediated T cell crawling against the direction of flow.ConclusionTaken together, our in vitro observations show that high levels of endothelial ICAM-1 abrogate NTZ mediated inhibition of T cell interaction with the BBB. EID of NTZ in MS patients may thus require consideration of the inflammatory status of the BBB as high levels of ICAM-1 may provide an alternative molecular cue allowing for pathogenic T cell entry into the CNS in the presence of NTZ.

Neutrophil-Membrane-Coated Biomineralized Metal-Organic Framework Nanoparticles for Atherosclerosis Treatment by Targeting Gene Silencing.

Antisense oligonucleotides (ASOs) are promising tools for gene silencing and have been exploited as therapeutics for human disease. However, delivery of therapeutic ASOs to diseased tissues or cells and subsequent escape from the endosomes and release of ASO in the cytosol remain a challenge. Here, we reported a neutrophil-membrane-coated zeolitic imidazolate framework-8 (ZIF-8) nanodelivery platform (AM@ZIF@NM) for the targeted transportation of ASOs against microRNA-155 (anti-miRNA-155) to the endothelial cells in atherosclerotic lesions. Neutrophil membrane could improve plaque endothelial cells targeting through the interaction between neutrophil membrane protein CD18 and endothelial cell membrane protein intercellular adhesion molecule-1 (ICAM-1). The ZIF-8 "core" provided high loading capacity and efficient endolysosomal escaping ability. Delivery of anti-miR-155 effectively downregulated miR-155 expression and also saved the expression of its target gene BCL6. Moreover, RELA expression and the expression of its downstream target genes CCL2 and ICAM-1 were correspondingly reduced. Consequently, this anti-miR-155 nanotherapy can inhibit the inflammation of atherosclerotic lesions and alleviate atherosclerosis. Our study shows that the designed biomimetic nanodelivery system has great application prospects in the treatment of other chronic diseases.

Mechano-Redox Control of Macrophage-1 Antigen De-Adhesion From ICAM-1 (Intercellular Adhesion Molecule 1) by Protein Disulfide Isomerase Promotes Directional Movement Under Flow.

Neutrophil migration is critical to the initiation and resolution of inflammation. Macrophage-1 antigen (Mac-1; CD11b/CD18, αMβ2) is a leukocyte integrin essential for firm adhesion to endothelial ICAM-1 (intercellular adhesion molecule 1) and migration of neutrophils in the shear forces of the circulation. PDI (protein disulfide isomerase) has been reported to influence neutrophil adhesion and migration. We aimed to elucidate the molecular mechanism of PDI control of Mac-1 affinity for ICAM-1 during neutrophil migration under fluid shear. Neutrophils isolated from whole blood were perfused over microfluidic chips coated with ICAM-1. Colocalization of Mac-1 and PDI on neutrophils was visualized by fluorescently labeled antibodies and confocal microscopy. The redox state of Mac-1 disulfide bonds was mapped by differential cysteine alkylation and mass spectrometry. Wild-type or disulfide mutant Mac-1 was expressed recombinantly in Baby Hamster Kidney cells to measure ligand affinity. Mac-1 conformations were measured by conformation-specific antibodies and molecular dynamics simulations. Neutrophils crawling on immobilized ICAM-1 were measured in presence of oxidized or reduced PDI, and the effect of PDI inhibition using isoquercetin on neutrophil crawling on inflamed endothelial cells was examined. Migration indices in the X- and Y-direction were determined and the crawling speed was calculated. PDI colocalized with high-affinity Mac-1 at the trailing edge of stimulated neutrophils when crawling on ICAM-1 under fluid shear. PDI cleaved 2 allosteric disulfide bonds, C169-C176 and C224-C264, in the βI domain of the β2 subunit, and cleavage of the C224-C264 disulfide bond selectively controls Mac-1 disengagement from ICAM-1 under fluid shear. Molecular dynamics simulations and conformation-specific antibodies reveal that cleavage of the C224-C264 bond induces conformational change and mechanical stress in the βI domain. This allosterically alters the exposure of an αI domain epitope associated with a shift of Mac-1 to a lower-affinity state. These molecular events promote neutrophil motility in the direction of flow at high shear stress. Inhibition of PDI by isoquercetin reduces neutrophil migration in the direction of flow on endothelial cells during inflammation. Shear-dependent PDI cleavage of the neutrophil Mac-1 C224-C264 disulfide bond triggers Mac-1 de-adherence from ICAM-1 at the trailing edge of the cell and enables directional movement of neutrophils during inflammation.

Galectin-1 mediates interactions between polymorphonuclear leukocytes and vascular endothelial cells, and promotes their extravasation during lipopolysaccharide-induced acute lung injury.

The lung airway epithelial surface is heavily covered with sialic acids as the terminal carbohydrate on most cell surface glycoconjugates and can be removed by microbial neuraminidases or endogenous sialidases. By desialylating the lung epithelial surface, neuraminidase acts as an important virulence factor in many mucosal pathogens, such as influenza and S. pneumoniae. Desialylation exposes the subterminal galactosyl moieties - the binding glycotopes for galectins, a family of carbohydrate-recognition proteins playing important roles in various aspects of immune responses. Galectin-1 and galectin-3 have been extensively studied in their roles related to host immune responses, but some questions about their role(s) in leukocyte recruitment during lung bacterial infection remain unanswered. In this study, we found that both galectin-1 and galectin-3 bind to polymorphonuclear leukocytes (PMNs) and enhance the interaction of endothelial intercellular adhesion molecule-1 (ICAM-1) with PMNs, which is further increased by PMN desialylation. In addition, we observed that in vitro galectin-1 mediates the binding of PMNs, particularly desialylated PMNs, onto the endothelial cells. Finally, in a murine model for LPS-mediated acute lung injury, we observed that galectin-1 modulates PMN infiltration to the lung without altering the expression of chemoattractant cytokines. We conclude that galectins, particularly galectin-1, may function as adhesion molecules that mediate PMN-endothelial cell interactions, and modulate PMN infiltration during acute lung injury.

Notch Pathway Driven Delayed Dedifferentiation of Liver Sinusoidal Endothelium Impacts Liver-Directed Gene Transfer in Hemophilia-a Mice

Background: Hemophilia A is an X-linked recessive bleeding disorder that affects 1 in 5000 males and is caused by procoagulant factor VIII deficiency. Affected people are at danger of spontaneous bleeding into organs, which can be fatal and lead to persistent damage. Current therapy includes intravenous infusion of FVIII protein concentrate which is costly, inconvenient, and does not fully prevent bleeding. As a result of recent advancements in liver-directed gene transfer, gene therapy based innovative strategies for treating hemophilia have emerged. The Factor VIII null mouse (F8TKO mice) has been effective in understanding the disease pathogenesis as well as the development of liver directed novel gene therapy techniques to treat hemophilia. FVIII is predominantly produced in the liver sinusoidal endothelial cells (LSECs). Thus, liver directed adenoviral and retroviral vectors have been studied by several groups to understand the gene delivery method in hemophilia. Methods: In this study we evaluated the stability and efficacy of liver driven gene transfer mechanism in F8TKO mouse using recombinant adeno associated virus (AAV)8 vector. AAV8-TBG-GFP was administered through IP and IV, and GFP expression was analyzed in the liver 15- and 30-days post injection. Results: Immunofluorescence of GFP at day 15 and day 30 post injection revealed significantly fewer GFP+ cells (p=0.04) in the liver of F8TKO compared with control mice post AAV8-GFP injection. This was further validated by ELISA assays and western blot for GFP. Moreover, Caspase-3 staining revealed significant enrichment (p=0.05) in the liver of F8TKO mice compared with control mice after AAV8-GFP administration at both the 15- and 30-day time points suggestive of increased LSEC apoptosis post AAV8-GFP injection. To further confirm apoptosis of LSECs, we next used liver intravital imaging to visualize LSECs in F8TKO mice at baseline and upon AAV8-GFP administration. TXR-dextran and AF488-anti-CD31 antibody were IV administered to visualize hepatic blood flow and LSECs, respectively. At baseline, both control and F8TKO mice showed normal blood flow and enriched expression of AF488-anti-CD31. However, there were fewer AF488-anti-CD31+endothelial cells in F8TKO than control mice at day 15 post-AAV8-GFP treatment (p=0.01), suggestive of apoptosis of LSECs in F8TKO mice post-AAV8-GFP administration. Scanning electron microscopy revealed dramatic reduction (p=0.01) in endothelial fenestration in the liver of F8TKO as compared with control mouse liver. The absence of fenestrae has been associated with LSEC capillarization, which promotes basement membrane formation while decreasing permeability. Remarkably, we found significant upregulation of LSEC markers associated with capillarization, including CD31, vascular endothelial growth factor (VEGF), ICAM1 (Intercellular Adhesion Molecule-1), stabilin2, ID1 (Inhibitor of DNA Binding-1), Gata4, and Ehd3 (EH Domain Containing-3) in F8TKO mice by qRT-PCR) and western blot. Mechanistically we show significant upregulation of Notch pathway components (Notch 1, 3, and Jagged 2) by qRT-PCR and western in the liver of F8TKO mice. IHC analysis showed significant upregulation of notch 3 early in development in F8TKO mice leading to pseudocapillarization of LSECs. Moreover, we also show that notch activation regulates nitric oxide/soluble guanylyl cyclase signaling by upregulating downstream targets (NOS-1, 2, 3, PDE1a, b, c and 10B) in LSECs which directly controls the development of LSEC fenestration. Conclusion: In conclusion, we demonstrate molecular characterization of LSEC-mediated liver-directed gene transfer in a null hemophilia A mouse model, which is critical for understanding the efficacy and stability of AAV-FVIII gene-based hemophilia treatment. Work is currently underway to understand how absence of FVIII can affect the LSECs mediated hepatic gene transfer in Hemophilia-A mice and how modulation of Notch and nitric oxide/sGC signaling can manipulate LSEC fenestration and improve the efficiency of liver directed gene transfer.

ICAM-1 on the luminal surface of endothelial cells is induced to a greater extent in mouse retina than in other tissues in diabetes.

Induction of intercellular adhesion molecule-1 (ICAM-1) has been implicated in the development of macrovascular and microvascular diseases such as diabetic retinopathy. Lesions of diabetic retinopathy are unique to the retina but the reason for this is unclear, as all tissues are exposed to the same hyperglycaemic insult. We tested whether diabetes induces ICAM-1 on the luminal surface of endothelial cells to a greater extent in the retina than in other tissues and the role of vision itself in that induction. Experimental diabetes was induced in C57Bl/6J, P23H opsin mutant and Gnat1-/- × Gnat2-/- double knockout mice using streptozotocin. The relative abundance of ICAM-1 on the luminal surface of endothelial cells in retina and other tissues was determined by conjugating anti-ICAM-1 antibodies to fluorescent microspheres (2μm), injecting them intravenously and allowing them to circulate for 30min. After transcardial perfusion, quantification of microspheres adherent to the endothelium in tissues throughout the body was carried out by fluorescent microscopy or flow cytometry. Mice injected with lipopolysaccharide (LPS) were used as positive controls. The difference in leucostasis between retinal and non-retinal vasculature was evaluated. Diabetes significantly increased ICAM-1-mediated adherence of microspheres to retinal microvessels by almost threefold, independent of sex. In contrast, diabetes had a much smaller effect on endothelial ICAM-1 in other tissues, and more tissues showed a significant induction of endothelial ICAM-1 with LPS than with diabetes. The diabetes-induced increase in endothelial ICAM-1 in retinal vasculature was inhibited by blocking phototransduction in photoreceptor cells. Diabetes significantly increased leucostasis in the retina by threefold compared with a non-ocular tissue (cremaster). The diabetes-induced upregulation of ICAM-1 on the luminal surface of the vascular endothelium varies considerably among tissues and is highest in the retina. Induction of ICAM-1 on retinal vascular endothelial cells in diabetes is influenced by vision-related processes in photoreceptor cells. The unique presence of photoreceptors in the retina might contribute to the greater susceptibility of this tissue to vascular disease in diabetes.

The Effect of Diammonium Glycyrrhizinate Lipid Ligand on Nonalcoholic Fatty Liver Disease Induced Leukocyte Infiltration in Rat Model

Objective: To investigate the effects of diammonium glycyrrhizinate lipid ligand (DGLL) on nonalcoholic fatty liver disease (NAFLD)-induced leukocyte infiltration in rat and underlying mechanisms. Methods: Sixty SD rats were randomly distributed into six groups, control group, NAFLD model group, DGLL (30, 60, 120 mg/kg) treatment group and positive control (biphenyl biester tablets (BDP), 200 mg/kg) group. The expression and activity of myeloperoxidase (MPO) in liver tissues were measured by immunohistochemistry and ELISA, respectively. The expressions of leukocyte adhesion molecules in whole blood were determined by flow cytometry. The intercellular adhesion molecule-1 (ICAM-1) expressions in liver tissues was detected by western blotting. Results: Compared with NAFLD model groups, the expression and activity of MPO in liver tissues of DGLL (30, 60, 120 mg/kg) treatment group were all significantly ameliorated by the administration of DGLL (P &lt; 0.05). Meanwhile, the up-regulation of adhesion molecules expressed on monocytes and neutrophils in whole blood, as well as endothelial ICAM-1 expression in NAFLD model groups were eliminated by DGLL treatment (P &lt; 0.05). Conclusion: DGLL ameliorated leukocyte activation and infiltration in rat liver with NAFLD, which might be related to suppression of adhesion molecules expression on leukocyte and endothelium.

Evaluation of Patients with Vaccine Allergies Prior to mRNA-Based COVID-19 Vaccination

During the initial rollout of coronavirus disease 2019 (COVID-19) vaccination in Singapore, the Ministry of Health (MOH) issued a recommendation that patients with a history of any previous vaccine allergy be referred to an allergist for further review of their suitability to proceed with mRNA-based COVID-19 vaccines. Patients fulfilling the above criterion were divided into three groups: immediate reaction (Group A), delayed reaction (Group B) and no/irrelevant reaction (Group C). They were subjected to either a skin prick test (SPT) and intradermal test (IDT) with polyethylene glycol (PEG) or polysorbate-containing products; direct injection with the Pfizer BNT162b2 vaccine in the allergy clinic; or injection at community vaccination centres, respectively. Groups A and B were also invited to complete a questionnaire survey on post-vaccination reactions, and blood sampling pre-vaccination and 1 h after the first dose of the BNT162b2 vaccine to measure immunoglobulin (Ig) G, IgM and IgE antibodies to the Pfizer BNT162b2 vaccine via ELISA assays immobilised with the BNT162b2 vaccine, as well as levels of allergic cytokines interleukin (IL)-4 and IL-33, complement C5a and the endothelial activation marker intercellular adhesion molecule-1 (ICAM-1). Groups A and B comprised 62 (20.5%) patients each. In Group A, two subjects (3.2%) with equivocal IDT results tolerated both doses of the BNT162b2 vaccine without major allergic reactions. The remaining 60 (96.8%) in Group A and 62 (100%) in Group B completed both doses of BNT162b2 vaccination without major adverse reactions. Among the 99 who completed the questionnaire survey, 13 (13%) patients reported mild allergic reactions after the first dose of the vaccine. Immunoglobulin (Ig) G and M antibodies, but not IgE antibodies to the Pfizer BNT162b2 vaccine were detected in 67 subjects prior to vaccination. The presence of anti-Pfizer BNT162b2 IgG and IgM prior to vaccination did not result in major allergic reactions nor increases in Th2-related cytokines (IL-4, IL-33), complement activation products (C5a) or endothelial activation (ICAM-1). The majority of those with suspected reactions to non-COVID-19 polysorbate-containing vaccines tolerated the BNT162b2 vaccine. Excipient skin tests for PEG and polysorbate prior to vaccination are unnecessary.

IL-1β increased secretion may contribute to NGF-dependent pulmonary arterial remodelling and inflammation

Pulmonary Hypertension (PH) is a severe disease leading to right heart failure and death. We have previously shown that blockade of the nerve growth factor NGF may be of therapeutic interest in PH, since NGF contributes to pulmonary arterial (PA) hyperreactivity, remodelling and inflammation. In this study, we have started to explore the possible links between NGF, inflammation and PA remodelling. More precisely, we have investigated whether interleukin-1β (IL-1β), whose expression is upregulated by NGF, may participate in PA remodelling and inflammation induced by NGF. In vitro, control human PA smooth muscle (hPASMC) or endothelials cells (hPAEC) in primary culture were treated with exogenous IL-1β (10 ng/ml or 100 ng/ml, 24 h). Cell proliferation (cell counting, ki67 staining), cell migration (Transwell assays), cell apoptosis (caspase 3/7 activity assay) and interleukin-6 (IL-6) secretion (ELISA) were then assessed. In hPAEC, IL-1β effects on endothelial nitric oxide synthase (eNOS) and intercellular adhesion molecule-1 (ICAM-1) expression were also investigated (Western Blotting). In vivo, PH was induced in the rat by chronic hypoxia (0.5 atm, 28 days). IL-1β seric and PA levels were assessed (ELISA), and expression of the inflammasome components NLRP3, caspase-1 and pro-IL-1β were evaluated in lung homogenates (Western Blotting). In vitro, in hPASMC, IL-1β significantly increased cell proliferation, cell migration, cell resistance to apoptosis and IL-6 secretion. In hPAEC, IL-1β significantly increased cell proliferation, IL-6 secretion and ICAM-1 expression, and significantly decreased eNOS expression. In vivo, IL-1β seric and PA levels, as well as NLRP3, caspase-1 and pro-IL-1β expression in lung homogenates were significantly increased of chronic hypoxic rats compared to controls. In conclusion, our in vitro results suggest that IL-1β may stimulate both PA smooth muscle and endothelial cells to contribute to PA inflammation, altered reactivity and remodelling. Our in vivo results show inflammasome increased activity and increased IL-1β levels in rat hypoxic PH. Since NGF contributes to IL-1β increased levels in PH, IL-1β may therefore play a role in NGF-dependent PA alterations in this disease.

The NEDD8-activating enzyme inhibitor MLN4924 reduces ischemic brain injury in mice

Blood-brain barrier (BBB) breakdown and inflammation occurring at the BBB have a key, mainly a deleterious role in the pathophysiology of ischemic stroke. Neddylation is a ubiquitylation-like pathway that is critical in various cellular functions by conjugating neuronal precursor cell-expressed developmentally down-regulated protein 8 (NEDD8) to target proteins. However, the roles of neddylation pathway in ischemic stroke remain elusive. Here, we report that NEDD8 conjugation increased during acute phase after ischemic stroke and was present in intravascular and intraparenchymal neutrophils. Inhibition of neddylation by MLN4924, also known as pevonedistat, inactivated cullin-RING E3 ligase (CRL), and reduced brain infarction and improved functional outcomes. MLN4924 treatment induced the accumulation of the CRL substrate neurofibromatosis 1 (NF1). By using virus-mediated NF1 silencing, we show that NF1 knockdown abolished MLN4924-dependent inhibition of neutrophil trafficking. These effects were mediated through activation of endothelial P-selectin and intercellular adhesion molecule-1 (ICAM-1), and blocking antibodies against P-selectin or anti-ICAM-1 antibodies reversed NF1 silencing-induced increase in neutrophil infiltration in MLN4924-treated mice. Furthermore, we found that NF1 silencing blocked MLN4924-afforded BBB protection and neuroprotection through activation of protein kinase C δ (PKCδ), myristoylated alanine-rich C-kinase substrate (MARCKS), and myosin light chain (MLC) in cerebral microvessels after ischemic stroke, and treatment of mice with the PKCδ inhibitor rottlerin reduced this increased BBB permeability. Our study demonstrated that increased neddylation promoted neutrophil trafficking and thus exacerbated injury of the BBB and stroke outcomes. We suggest that the neddylation inhibition may be beneficial in ischemic stroke.

Deep Sea Water-Dissolved Organic Matter Intake Improves Hyperlipidemia and Inhibits Thrombus Formation and Vascular Inflammation in High-Fat Diet Hamsters.

Non-alcoholic fatty liver disease (NAFLD) is a chronic liver disease caused by oxidative stress, inflammation and lipid deposition within liver cells, and is subsequently contributing to cardiovascular diseases such as atherosclerosis. Deep sea water (DSW) is characterized by its clearance and abundant nutrients with antioxidant and anti-inflammatory activity to confer therapeutic potential. We aimed to explore the therapeutic capability of our prepared multi-filtration DSW-dissolved organic matter (DSW-DOM) on high-fat diet-induced hyperlipidemia and endothelial dysfunction in hamsters. A high-fat/high-cholesterol diet led to increased oxidative stress, including blood reactive oxygen species (ROS), plasma malondialdehyde (MDA) and hepatic CYP2E1 expression; an increased hyperlipidemic profile and SREBP 1-mediated fatty liver; promoted NFκB p65-mediated hepatic inflammation; triggered PARP-mediated hepatic apoptosis; and enhanced endothelial intercellular adhesion molecule-1 (ICAM-1) and von Willebrand factor (VWF)-mediated atherosclerosis associated with the depressed hepatic antioxidant Paraoxonase 1 (PON1) expression. The DSW-DOM-enriched 1295 fraction, with strong H2O2 scavenging activity, efficiently reduced several oxidative stress parameters, the lipid profile, inflammation, and apoptosis, possibly through the PON1-mediated antioxidant capability. Furthermore, DSW-DOM treatment significantly decreased the endothelial ICAM-1 and VWF expression, subsequently leading to the elongation of time to occlusion of FeCl3-induced arterial thrombosis and to the inhibition of FeCl3-induced fluorescent platelet adhesion to mesentery arterioles in the high-fat diet. Based on the above results, our data suggest that DSW-DOM intake via antioxidant defense mechanisms confers protective effects against high-fat diet-enhanced, oxidative stress-mediated hyperlipidemia, and endothelial dysfunction evoked atherosclerosis by downregulating oxidative injury, lipogenesis, inflammation and apoptosis.

Abstract 12458: Protein L-isoaspartyl O-methyltransferase Attenuates Endothelial Activation Through Inhibitory Interaction With TRAF6 and ICAM1

Introduction: Endothelial activation provokes expression of cell surface adhesion molecules to facilitate circulating leukocyte diapedesis, which demands rigorous surveillance to prevent excessive inflammation response and host damage. As a repair enzyme, protein L-isoaspartyl O-methyltransferase (PIMT) methylates and converts isoaspartyl (isoAsp) residuals back to conventional form to avoid protein damage and aging. However, the role of PIMT in vascular homeostasis remains to be delineated. Methods and Results: Murine acute lung injury (ALI) model was deployed to investigate the significance of PIMT in endothelial activation and vascular inflammation. Results showed that mice harboring heterozygous deletion of PIMT displayed exacerbated pulmonary vascular inflammation. Ectopic overexpression of PIMT protected endothelial cells against endotoxin stimulated endothelial activation, as characterized by attenuated expression of inflammatory cytokines, chemokines, cell adhesion molecules and monocytic adherence to activated endothelial cells. Mechanistically, we found that PIMT inhibits lipopolysaccharide (LPS)-induced endothelial NF-κB transactivation. Tumor necrosis factor receptor-associated factor 6 (TRAF6) was identified as a dynamic binding partner and substrate of PIMT in regulating NF-κB signaling. PIMT catalyzed methylation at an asparagine derived isoAsp residual of TRAF6 directly prevented its oligomerization and auto-ubiquitination. Strikingly, we also found that PIMT specifically impeded N-glycosylation at multiple sites of endothelial intercellular adhesion molecule 1 (ICAM1). The hypo-glycosylated ICAM1 elicited impaired interaction with cell skeletal and reduced stability, which further diminished endothelial activation. Conclusions: PIMT negatively modulates endothelial activation through restricting activation of TRAF6 and inhibiting N-glycosylation of adhesion molecules. Our study provides a novel insight into the role of protein O-methylation in regulating endothelial inflammation and suggest that targeting PIMT may have potential therapeutic applications in treating inflammatory vascular disorders.