Endothelial Leukocyte Adhesion Research Articles

Safety of Anti-Reelin Therapeutic Approaches for Chronic Inflammatory Diseases.

Reelin, a large extracellular glycoprotein, plays critical roles in neuronal development and synaptic plasticity in the central nervous system (CNS). Recent studies have revealed non-neuronal functions of plasma Reelin in inflammation by promoting endothelial-leukocyte adhesion through its canonical pathway in endothelial cells (via ApoER2 acting on NF-κB), as well as in vascular tone regulation and thrombosis. In this study, we have investigated the safety and efficacy of selectively depleting plasma Reelin as a potential therapeutic strategy for chronic inflammatory diseases. We found that Reelin expression remains stable throughout adulthood and that peripheral anti-Reelin antibody treatment with CR-50 efficiently depletes plasma Reelin without affecting its levels or functionality within the CNS. Notably, this approach preserves essential neuronal functions and synaptic plasticity. Furthermore, in mice induced with experimental autoimmune encephalomyelitis (EAE), selective modulation of endothelial responses by anti-Reelin antibodies reduces pathological leukocyte infiltration without completely abolishing diapedesis. Finally, long-term Reelin depletion under metabolic stress induced by a Western diet did not negatively impact the heart, kidney, or liver, suggesting a favorable safety profile. These findings underscore the promising role of peripheral anti-Reelin therapeutic strategies for autoimmune diseases and conditions where endothelial function is compromised, offering a novel approach that may avoid the immunosuppressive side effects associated with conventional anti-inflammatory therapies.

Cyclophilin A is a ligand for RAGE in thrombo-inflammation.

Cyclophilin A (CyPA) induces leucocyte recruitment and platelet activation upon release into the extracellular space. Extracellular CyPA therefore plays a critical role in immuno-inflammatory responses in tissue injury and thrombosis upon platelet activation. To date, CD147 (EMMPRIN) has been described as the primary receptor mediating extracellular effects of CyPA in platelets and leucocytes. The receptor for advanced glycation end products (RAGE) shares inflammatory and prothrombotic properties and has also been found to have similar ligands as CD147. In this study, we investigated the role of RAGE as a previously unknown interaction partner for CyPA. Confocal imaging, proximity ligation, co-immunoprecipitation, and atomic force microscopy were performed and demonstrated an interaction of CyPA with RAGE on the cell surface. Static and dynamic cell adhesion and chemotaxis assays towards extracellular CyPA using human leucocytes and leucocytes from RAGE-deficient Ager-/- mice were conducted. Inhibition of RAGE abrogated CyPA-induced effects on leucocyte adhesion and chemotaxis in vitro. Accordingly, Ager-/- mice showed reduced leucocyte recruitment and endothelial adhesion towards CyPA in vivo. In wild-type mice, we observed a downregulation of RAGE on leucocytes when endogenous extracellular CyPA was reduced. We furthermore evaluated the role of RAGE for platelet activation and thrombus formation upon CyPA stimulation. CyPA-induced activation of platelets was found to be dependent on RAGE, as inhibition of RAGE, as well as platelets from Ager-/- mice showed a diminished activation and thrombus formation upon CyPA stimulation. CyPA-induced signalling through RAGE was found to involve central signalling pathways including the adaptor protein MyD88, intracellular Ca2+ signalling, and NF-κB activation. We propose RAGE as a hitherto unknown receptor for CyPA mediating leucocyte as well as platelet activation. The CyPA-RAGE interaction thus represents a novel mechanism in thrombo-inflammation.

P06.16.A FRACTIONATED IRRADIATION ACTIVATES GLIOMA IMMUNE MICROENVIRONMENT AND BOOSTS ANTIGEN-SPECIFIC T CELL RESPONSE IN MALIGNANT GLIOMAS

Abstract Several ongoing clinical trials employ vaccine, immune checkpoint inhibitors and/or adoptive T cell therapy to treat glioblastoma. However, the impact of irradiation as part of the standard of care on the immune microenvironment and response to immunotherapy is ill-defined. In the present study, we analyzed the post-irradiation changes in the GBM immune microenvironment, and further elucidated the impact of fractionated tumor irradiation (FIR) on the efficacy of T cell-mediated immunotherapies in experimental gliomas. FIR boosted the response of orthotopically-implanted GL261 tumors expressing the tumor-associated antigen glycoprotein (gp)100 to a gp100 vaccine. Furthermore, FIR enhanced the infiltration of GL261-gp100 gliomas by adoptively-transferred gp100 T cell receptor-transgenic T cells, which also displayed a more activated and less exhausted phenotype post-irradiation. Single cell transcriptomic studies of tumor-infiltrating CD45+ leukocytes revealed that FIR promotes the expression of leukocyte-endothelial adhesion molecules in T cells, and transcripts encoding proinflammatory M1-like genes in tumor-associated microglia and macrophages. Collectively, our preclinical findings support the use of FIR with to increase response to T cell-based immunotherapies.

Increased Levels of Inflammatory and Endothelial Biomarkers in Blood of Long COVID Patients Point to Thrombotic Endothelialitis.

The prevailing hypotheses for the persistent symptoms of Long COVID have been narrowed down to immune dysregulation and autoantibodies, widespread organ damage, viral persistence, and fibrinaloid microclots (entrapping numerous inflammatory molecules) together with platelet hyperactivation. Here we demonstrate significantly increased concentrations of von Willebrand factor (VWF), platelet factor 4 (PF4), serum amyloid A (SAA), α-2 antiplasmin (α-2AP), endothelial-leukocyte adhesion molecule 1 (E-selectin), and platelet endothelial cell adhesion molecule (PECAM-1) in the soluble part of the blood. It was noteworthy that the mean level of α-2 antiplasmin exceeded the upper limit of the laboratory reference range in Long COVID patients, and the other 5 were significantly elevated in Long COVID patients as compared to the controls. This is alarming if we take into consideration that a significant amount of the total burden of these inflammatory molecules has previously been shown to be entrapped inside fibrinolysis-resistant microclots (thus decreasing the apparent level of the soluble molecules). We conclude that presence of microclotting, together with relatively high levels of six biomarkers known to be key drivers of endothelial and clotting pathology, points to thrombotic endothelialitis as a key pathological process in Long COVID.

Preservation of vascular endothelial glycocalyx and barrier by activation of adenosine A2A receptor (A2AR) improved renal dysfunction in cirrhotic rats

Cirrhosis-related hepatic and renal endothelial dysfunction is characterized by macrophage-endothelium adhesion-mediated inflammation, glycocalyx/barrier damage, and impaired vasodilation. Activation of adenosine A2A receptor (A2AR) protects cirrhotic rats from impairment of hepatic microcirculation post hepatectomy. This study evaluates the effects of A2AR activation on the cirrhosis-related hepatic and renal endothelial dysfunction in biliary cirrhotic rats receiving two weeks of A2AR agonist PSB0777 [bile duct ligated (BDL)+PSB0777] treatment.Endothelial dysfunction in cirrhotic liver, renal vessels, and kidney is characterized by downregulation of the A2AR expressions, decreased vascular endothelial vasodilatory (p-eNOS)/anti-inflammatory (IL-10/IL-10R)/barrier [VE-cadherin (CDH5) and β-catenin (CTNNB1)]/glycocalyx [syndecan-1 (SDC1) and hyaluronan synthase-2 (HAS2)] markers, and increased leukocyte-endothelium adhesion molecules (F4/80, CD68, ICAM-1, and VCAM-1). In BDL rats, PSB0777 treatment improves hepatic and renal endothelial dysfunction, ameliorates portal hypertension, and attenuates renal hypoperfusion by restoring of the vascular endothelial anti-inflammatory, barrier, glycocalyx markers and vasodilatory response as well as inhibiting the leukocyte-endothelium adhesion. In an in vitro study, conditioned medium (CM) of bone marrow-derived macrophage (BMDM) of BDL rats [BMDM-CM (BDL)] induced barrier/glycocalyx damage, which was reversed by the PSB0777 pre-treatment. The A2AR agonist is a potential agent that can simultaneously correct cirrhosis-related hepatic and renal endothelial dysfunction, portal hypertension, renal hypoperfusion, and renal dysfunction.

Chimeric Antigen Receptor (CAR) T-cell Therapy: A New Genetically Engineered Method of Immunotherapy for Cancer.

Chimeric antigen receptor (CAR T) cell treatment for solid tumours faces significant challenges. CAR T cells are unable to pass the vascular barrier in tumours due to a lack of endothelial leukocyte adhesion molecules. The invasion, activity, and durability of CAR T cells may be hampered by additional immunosuppressive mechanisms present in the solid tumour environment. The use of CAR T cells to attack cancer vascular endothelial metabolic targets from within the blood may simplify the fight against cancer. These are the principles that govern our examination of CAR T cell treatment for tumor cells, with a specific eye toward tumour venous delivery. CAR T cells may also be designed such that they can be readily, safely, and successfully transferred.

JAK-STAT inhibition reduces endothelial prothrombotic activation and leukocyte–endothelial proadhesive interactions

BackgroundVascular activation is characterized by increased proinflammatory, pro thrombotic, and proadhesive signaling. Several chronic and acute conditions, including Bcr-abl-negative myeloproliferative neoplasms (MPNs), graft-vs-host disease, and COVID-19 have been noted to have increased activation of the janus kinase (JAK)-signal transducer and downstream activator of transcription (STAT) pathways. Two notable inhibitors of the JAK-STAT pathway are ruxolitinib (JAK1/2 inhibitor) and fedratinib (JAK2 inhibitor), which are currently used to treat MPN patients. However, in some conditions, it has been noted that JAK inhibitors can increase the risk of thromboembolic complications. ObjectivesWe sought to define the anti-inflammatory and antithrombotic effects of JAK-STAT inhibitors in vascular endothelial cells. MethodsWe assessed endothelial activation in the presence or absence of ruxolitinib or fedratinib by using immunoblots, immunofluorescence, qRT-PCR, and function coagulation assays. Finally, we used endothelialized microfluidics perfused with blood from normal and JAK2V617F+ individuals to evaluate whether ruxolitinib and fedratinib changed cell adhesion. ResultsWe found that both ruxolitinib and fedratinib reduced endothelial cell phospho-STAT1 and STAT3 signaling and attenuated nuclear phospho-NK-κB and phospho-c-Jun localization. JAK-STAT inhibition also limited secretion of proadhesive and procoagulant P-selectin and von Willebrand factor and proinflammatory IL-6. Likewise, we found that JAK-STAT inhibition reduced endothelial tissue factor and urokinase plasminogen activator expression and activity. ConclusionsBy using endothelialized microfluidics perfused with whole blood samples, we demonstrated that endothelial treatment with JAK-STAT inhibitors prevented rolling of both healthy control and JAK2V617F MPN leukocytes. Together, these findings demonstrate that JAK-STAT inhibitors reduce the upregulation of critical prothrombotic pathways and prevent increased leukocyte–endothelial adhesion.

Hemoglobin increases leukocyte adhesion and initiates lung microvascular endothelial activation via Toll-like receptor 4 signaling.

Cell-free hemoglobin is a pathophysiological driver of endothelial injury during sepsis and acute respiratory distress syndrome (ARDS), but the precise mechanisms are not fully understood. We hypothesized that hemoglobin (Hb) increases leukocyte adhesion and endothelial activation in human lung microvascular endothelial cells (HLMVEC). We stimulated primary HLMVEC, or leukocytes isolated from healthy human donors, with Hb (0.5 mg/mL) and found that leukocyte adhesion to lung endothelium in response to Hb is an endothelial-dependent process. Next, we stimulated HLMVEC with Hb over time (1, 3, 6, and 24 h) and found increased transcription and release of inflammatory cytokines (IL-1β, IL-8, and IL-6). In addition, Hb exposure variably upregulated transcription, total protein expression, and cell-surface localization of adhesion molecules E-selectin, P-selectin, intercellular adhesion molecule-1 (ICAM-1), and vascular cell adhesion molecule-1 (VCAM-1). Since VCAM-1 was most upregulated by Hb, we further tested mechanisms for Hb-mediated upregulation of VCAM-1 in HLMVEC. Although upregulation of VCAM-1 was not prevented by hemoglobin scavenger haptoglobin, heme scavenger hemopexin, or inhibition of nod-like receptor protein 3 (NLRP3) signaling, blocking Toll-like receptor 4 (TLR4) with small molecule inhibitor TAK-242 (1 µM) prevented upregulation of VCAM-1 in response to Hb. Consistently, Hb increased nuclear factor-κB (NF-κB) activation and intracellular reactive oxygen species (ROS), which were both prevented by TLR4 inhibition. Together, these data demonstrate that Hb increases leukocyte-endothelial adhesion and activates HLMVEC through TLR4 signaling, indicating a potential mechanism for Hb-mediated pulmonary vascular injury during inflammatory and hemolytic conditions.

Solvent extraction optimisation and validation of the HPLC-DAD method for quantification of 5-hydroxyindolin-2-one inProtaetia brevitarsis seulensis larvae extracts

Recent studies on the composition of edible insects have placed more emphasis on the quantity and quality of macro- and micronutrients and less emphasis on their bioactive components. This study aimed to assess the effect of 0-100% ethanol solvent ratio considering 5-hydroxyindolin-2-one (5-HI) content on the extraction efficiency inProtaetia brevitarsis seulensis larvae (PBSL) extract and to investigate the effect of PBSL on the induction of cell adhesion molecules (CAMs) in tumour necrosis factor (TNF)-α-activated human umbilical vein endothelial cells (HUVECs). A reversed-phase high-performance liquid chromatography method coupled to a diode array detector (HPLC-DAD) was validated for the quantification of 5-HI. The HPLC-DAD method was validated for its specificity, linearity, precision, accuracy, limit of detection, and limits of quantitation. The calibration curve of 5-HI showed significant linearity (R2>1). The limits of detection and quantitation were 0.016 μg/ml and 0.048 μg/ml, respectively. The relative standard deviation of the precision and intraday tests was less than 0.5%. In the recovery test, the accuracy ranged from 94.48-105.5% and the relative standard deviation value was less than 0.13%. The method validation parameters indicate the applicability of the HPLC method for quality control of food or pharmaceutical formulations containing PBSL. Comparative analysis of the 5-HI content by extraction solvent revealed that it was the highest at 357.7 μg/ml in 70% ethanol. Additionally, the analysis result of 5-HI content according to the growth conditions was the highest at 439 μg/ml at 31 °C for 30 days. Furthermore, in the condition where PBSL was at 31 °C for 30 days inhibition of the TNF-α-induced upregulation of protein expression of vascular cell adhesion molecule-1 (VCAM-1), intracellular adhesion molecule-1 (ICAM-1), and endothelial-leukocyte adhesion molecule-1 (E-selectin) was observed. Therefore, PBSL may interfere with early atherosclerotic events involving endothelial CAM induction.

Antibody-based in vivo leukocyte label for two-photon brain imaging in mice.

.SignificanceTo study leukocyte-endothelial interactions in a living system, robust and specific leukocyte labeling techniques are needed for in vivo two-photon microscopy of the cerebral microvasculature.AimWe tested fluorophore-conjugated anti-CD45.2 monoclonal antibodies (mAb) to optimize dosing and two-photon imaging parameters for leukocyte labeling in healthy mice and a venous microstroke model.ApproachWe retro-orbitally injected anti-CD45.2 mAb at 0.04, 0.4, and into BALB/c mice and used flow cytometry to analyze antibody saturation. Leukocyte labeling in the cortical microvasculature was examined by two-photon imaging. We also tested the application of CD45.2 mAb in a pathological leukocyte-endothelial adhesion model by photothrombotically occluding cortical penetrating venules.ResultsWe found that of anti-CD45.2 antibody intravenously was sufficient to label 95% of circulating leukocytes. There was no depletion of circulating leukocytes after 24 h at the dosages tested. Labeled leukocytes could be observed as deep as from the cortical surface. The antibody reliably labeled rolling, crawling, and adherent leukocytes in venules around the stroke-affected tissues.ConclusionWe show that the anti-CD45.2 mAb is a robust reagent for acute labeling of leukocytes during in vivo two-photon microscopy of the cortical microvasculature.

P2RX7 stimulates Mac-1/ICAM-1-dependent leukocyte-endothelial Adhesion through promotion of Neurovascular Insult during Septic Encephalopathy (P9-10.005)

P2RX7 stimulates Mac-1/ICAM-1-dependent leukocyte-endothelial Adhesion through promotion of Neurovascular Insult during Septic Encephalopathy (P9-10.005)

Different Contribution of Monocyte- and Platelet-Derived Microvesicles to Endothelial Behavior.

Several contributions of circulating microvesicles (MVs) to the endothelial dysfunction have been reported in the past; a head-to-head comparison of platelet- and monocyte–derived MVs has however never been performed. To this aim, we assessed the involvement of these MVs in vessel damage related processes, i.e., oxidative stress, inflammation, and leukocyte-endothelial adhesion. Platelets and monocytes isolated from healthy subjects (HS, n = 15) were stimulated with TRAP-6 and LPS to release MVs that were added to human vascular endothelial cell (hECV) culture to evaluate superoxide anion production, inflammatory markers (IL-6, TNFα, NF-κB mRNA expression), and hECV adhesiveness. The effects of the MVs-induced from HS were compared to those induced by MVs spontaneously released from cells of patients with ST-segment elevation myocardial infarction (STEMI, n = 7). MVs released by HS-activated cells triggered a threefold increase in oxidative burst in a concentration-dependent manner. Only MVs released from monocytes doubled IL-6, TNFα, and NF-κB mRNA expression and monocyte-endothelial adhesion. Interestingly, the effects of the MVs isolated from STEMI-monocytes were not superimposable to previous ones except for adhesion to hECV. Conversely, MVs released from STEMI-platelets sustained both redox state and inflammatory phenotype. These data provide evidence that MVs released from activated and/or pathologic platelets and monocytes differently affect endothelial behavior, highlighting platelet-MVs as causative factors of impaired endothelial function in the acute phase of STEMI.

Grape Pomace Extract Attenuates Inflammatory Response in Intestinal Epithelial and Endothelial Cells: Potential Health-Promoting Properties in Bowel Inflammation.

Inflammatory bowel disease (IBD) implies the chronic inflammation of the gastrointestinal tract, combined with systemic vascular manifestations. In IBD, the incidence of cardiovascular disease appears to be related to an increase of oxidative stress and endothelial dysfunction. Grape pomace contains high levels of anti-oxidant polyphenols that are able to counteract chronic inflammatory symptoms. The aim of this study was to determine whether grape pomace polyphenolic extract (GPE) was able to mitigate the overwhelming inflammatory response in enterocyte-like cells and to improve vascular function. Intestinal epithelial Caco-2 cells, grown in monolayers or in co-culture with endothelial cells (Caco-2/HMEC-1), were treated with different concentrations of GPE (1, 5, 10 µg/mL gallic acid equivalents) for 2 h and then stimulated with lipopolysaccharide (LPS) and tumor necrosis factor (TNF)-α for 16 h. Through multiple assays, the expression of intestinal and endothelial inflammatory mediators, intracellular reactive oxygen species (ROS) levels and NF-κB activation, as well as endothelial-leukocyte adhesion, were evaluated. The results showed that GPE supplementation prevented, in a concentration-dependent manner, the intestinal expression and release of interleukin (IL)-6, monocyte chemoattractant protein (MCP)-1, and matrix metalloproteinases (MMP)-9 and MMP-2. In Caco-2 cells, GPE also suppressed the gene expression of several pro-inflammatory markers, such as IL-1β, TNF-α, macrophage colony-stimulating factor (M-CSF), C-X-C motif ligand (CXCL)-10, intercellular adhesion molecule (ICAM)-1, vascular cell adhesion molecule (VCAM)-1, and cyclooxygenase (COX)-2. The GPE anti-inflammatory effect was mediated by the inhibition of NF-κB activity and reduced intracellular ROS levels. Furthermore, transepithelial GPE suppressed the endothelial expression of IL-6, MCP-1, VCAM-1, and ICAM-1 and the subsequent adhesion of leukocytes to the endothelial cells under pro-inflammatory conditions. In conclusion, our findings suggest grape pomace as a natural source of polyphenols with multiple health-promoting properties that could contribute to the mitigation of gut chronic inflammatory diseases and improve vascular endothelial function.

Quantification of Leukocyte-Endothelial Cell Interaction During In Vitro Models of Thrombosis at Arterial and Venous Shear Rates.

Upon injury, stable thrombi formation requires the recruitment of platelets, leukocytes, and various clotting factors, to provide sufficient inhibition of hemostasis. Classical models of thrombosis involve either ex vivo isolation of platelets and subsequent quantification of aggregation through light transmission aggregometry or in vivo murine intravital thrombosis models (laser injury, ferric chloride, or rose Bengal). Flow adhesion models allow for accurate quantification of the contribution of cell-types to thrombi formation. Here, we describe the use of flow chambers to flow human blood over activated endothelial cells to observe leukocyte-endothelial adhesion at arterial and venous shear rates.

Brain capillary obstruction during neurotoxicity in a mouse model of anti-CD19 chimeric antigen receptor T-cell therapy.

Immunotherapy for haematologic malignancies with CD19-directed chimeric antigen receptor T cells has been highly successful at eradicating cancer but is associated with acute neurotoxicity in ∼40% of patients. This neurotoxicity correlates with systemic cytokine release syndrome, endothelial activation and disruption of endothelial integrity, but it remains unclear how these mechanisms interact and how they lead to neurologic dysfunction. We hypothesized that dysfunction of the neurovascular unit is a key step in the development of neurotoxicity. To recapitulate the interaction of the intact immune system with the blood–brain barrier, we first developed an immunocompetent mouse model of chimeric antigen receptor T-cell treatment-associated neurotoxicity. We treated wild-type mice with cyclophosphamide lymphodepletion followed by escalating doses of murine CD19-directed chimeric antigen receptor T cells. Within 3–5 days after chimeric antigen receptor T-cell infusion, these mice developed systemic cytokine release and abnormal behaviour as measured by daily neurologic screening exams and open-field testing. Histologic examination revealed widespread brain haemorrhages, diffuse extravascular immunoglobulin deposition, loss of capillary pericyte coverage and increased prevalence of string capillaries. To measure any associated changes in cerebral microvascular blood flow, we performed in vivo two-photon imaging through thinned-skull cranial windows. Unexpectedly, we found that 11.9% of cortical capillaries were plugged by Day 6 after chimeric antigen receptor T-cell treatment, compared to 1.1% in controls treated with mock transduced T cells. The capillary plugs comprised CD45+ leucocytes, a subset of which were CD3+ T cells. Plugging of this severity is expected to compromise cerebral perfusion. Indeed, we found widely distributed patchy hypoxia by hypoxyprobe immunolabelling. Increased serum levels of soluble ICAM-1 and VCAM-1 support a putative mechanism of increased leucocyte–endothelial adhesion. These data reveal that brain capillary obstruction may cause sufficient microvascular compromise to explain the clinical phenotype of chimeric antigen receptor T-cell neurotoxicity. The translational impact of this finding is strengthened by the fact that our mouse model closely approximates the kinetics and histologic findings of the chimeric antigen receptor T-cell neurotoxicity syndrome seen in human patients. This new link between systemic immune activation and neurovascular unit injury may be amenable to therapeutic intervention.

Anti-inflammatory iron chelator, DIBI, reduces leukocyte-endothelial adhesion and clinical symptoms of LPS-induced interstitial cystitis in mice.

Interstitial cystitis (IC) is a prevalent and debilitating chronic inflammatory disease of the urinary bladder. Currently there are no fully effective therapeutic agents available, in part due to the still obscure pathogenesis of IC. Lipopolysaccharide (LPS) also known as endotoxin from Gram negative bacteria elicits IC in mice and has formed the basis of model systems for investigation. Excess free iron plays an important role in inflammation through generation of reactive oxygen species (ROS). The novel iron chelator DIBI has been shown to sequester excess free iron and dampen excess inflammatory responses to systemic LPS administration and also to Gram negative bacterial infections. The overall objective of this study was to evaluate the effects of DIBI on LPS induced IC in mice. Leukocyte activation, endothelial adhesion and functional capillary density were assessed by intravital microscopy of the bladder microcirculation following a single intravesical LPS administration with or without intravesical DIBI treatment. Clinical IC symptoms were also assessed through behavioral and pain threshold force measurements. Four groups of female BALB/c mice (n = 5-6/group) were randomized in this study: control group, IC group without therapy, IC group with DIBI therapy and control group with DIBI therapy. The groups were examined using intravital microscopy (IVM) of the bladder for leukocyte-endothelial interactions (adherent leukocytes, temporarily interacting leukocytes) and functional capillary density (FCD). A modified behavioral score by Boucher et al. and Von-Frey-Aesthesiometry were used to evaluate key behavioral indices related to pain and visceral pain perception. LPS introduced intravesically induced an early (≤2h) inflammation of the bladder evidenced by leukocyte activation and adhesion to bladder capillary walls. Intravesical DIBI therapy of mice 30min following LPS administration and assessed after 1.5h treatment showed a significant decrease in the number of adherent leukocytes compared to IC animals without DIBI treatment. DIBI treated mice showed a significantly lowered increase in behavioral distress scores compared to IC mice without therapy. Untreated IC mice exhibited a significantly decreased threshold force value for evoked pain response and DIBI treatment improved the threshold pain response. A significant inverse correlation was found for the two pain and suffering evaluation methods results. DIBI reduced inflammatory endothelial leukocyte adhesion and key indices related to pain and suffering over those observed in untreated IC mice. Our findings suggest a potential therapeutic role for DIBI for IC treatment.

Inhibitory Effect of Punicalagin on Inflammatory and Angiogenic Activation of Human Umbilical Vein Endothelial Cells.

Punicalagin, a major ellagitannin isolated from pomegranate, is proved to have various pharmacological activities with an undefined therapy mechanism. The objective of this research was to demonstrate the effect of punicalagin on anti-inflammatory and angiogenic activation in human umbilical vein endothelial cells (HUVECs) and their potential mechanisms. Endothelial-leukocyte adhesion assay was applied to evaluate primary cultures of HUVECs activation following tumor necrosis factor alpha (TNF-α) treatment. The endothelial cell proliferation, migration, permeability and tube formation were assessed by EdU assay, wound migration assay, trans-endothelial electrical resistances (TEER) assay, and capillary-like tube formation assay, respectively. In addition, the expression of relevant proteins was assessed using Western blot analysis. We confirmed that punicalagin could reduce the adhesion of human monocyte cells to HUVECs in vitro and in vivo. Further, punicalagin decreased the expression of mRNA and proteins of ICAM-1 and VCAM-1 in HUVECs. Moreover, punicalagin inhibited permeability, proliferation, migration, and tube formation in VEGF-induced HUVECs, suppressed IKK-mediated activation of NF-κB signaling in TNF-α-induced endothelial cells, and inhibited vascular endothelial growth factor receptor 2 (VEGFR2) activation and downstream p-PAK1. Our findings indicated that punicalagin might have a protective effect on HUVECs activation, which suggested that punicalagin functions through an endothelial mediated mechanism for treating various disorders such as, cancer, rheumatoid arthritis, and cardiovascular disease.

Contribution of Vascular Cell Adhesion Molecule to Hemodynamics in Sickle Cell Disease

Vaso-occlusive events (VOEs) and pain crises are common clinical features of sickle cell disease (SCD) that result from sickle-shaped erythrocytes and leukocytes blocking blood flow, particularly in small vessels (Kato 2018). Activated endothelium also plays a role in the pathogenesis of VOEs in SCD. For example, VCAM-1 is expressed on blood vessels after activation by chemical and/or mechanical stimulation, which results in cytokine release. Studies have shown that patients with SCD have higher steady-state serum levels of soluble VCAM-1 compared to controls and that these levels are elevated during VOEs (White 2020). Furthermore, overexpression of these adhesion molecules on the endothelium results in prolonged adherence of white blood cells (WBCs), which has been shown to contribute to the development of VOEs and possibly cerebral vasculopathy. These findings raise the need to explore further the role of aberrant WBC- and/or RBC-endothelial interaction, mediated via VCAM-1, in the pathophysiology of cerebral microvascular hemodynamics and vasculopathy leading to cerebral microinfarcts in SCD. Therefore, we hypothesized that sickle cell mice will show greater cerebral cortical expression of VCAM-1 compared with age-matched controls and that this deposition will be associated with significant evidence of abnormal cerebral microvascular hemodynamic abnormalities.To examine the relationship between abnormalities in cerebral microvascular hemodynamics and VCAM-1 deposition in the cerebral microvasculature, we utilized a humanized sickle cell (with HbSS) and corresponding control (with HbAA). After cranial-window procedures, cortical capillaries, precapillary arterioles, and post-capillary venules were imaged using two-photon microscopy at two time points. In addition, this experiment included pre-and post-transfusion groups as we intend to study the impact of blood transfusion on hemodynamics. Using custom-written but well-validated MATLAB scripts, we analyzed line scans to identify the number and duration of rolling or adherent WBCs and RBCs, the RBC velocity in cerebral microvasculature, and the frequency and magnitude (mL/sec) of cerebral microvascular blood flow reversals. Rolling WBCs were defined as lasting two seconds or more, and adherent RBCs were defined as lasting 0.5 seconds or more. To quantify the expression of VCAM-1, we used immunohistochemistry to stain 50-micron sections of brain tissue for VCAM-1, Lectin to localize the vasculature, and Neun to localize neuronal nuclei. Images were analyzed using Phenochart and ImageJ software to examine the deposition of VCAM-1 throughout the brain tissue.As shown in Figure 1, at the first time point (baseline), we observed a significantly higher maximum RBC velocity (p<0.001) in the sickle cell mice compared to controls (figure 1a). We also found that there was significantly higher expression of VCAM-1 (p<0.001) (figure 1b) as well as significantly more leukocyte rolling (p<0.001) (figure 1c) in the sickle cell mice compared to controls. Additionally, we noted that the sickle cell mice have a significantly higher frequency of blood flow reversals (p<0.01) (figure 1d) as well as higher magnitude of microvascular blood flow reversals (p<0.001) (figure 1e) compared to controls. Interestingly, the sickle cell mice have a slightly lower average or mean capillary blood flow velocity compared to control (figure 1f), but this was not statistically significant (p=0.079). Since the mean capillary velocity is obtained as a smoothened difference between the forward flow and reversals, this decrease was surprising given the significant differences in frequency and magnitude of microvascular blood flow reversal in the sickle cell mice compared to controls (figs 1d and 1e).In conclusion, we see that the high velocity of blood flow might be a mechanical force, among other factors contributing to cerebral microvascular VCAM-1 expression in sickle cell mice. This might be responsible for the increased leukocyte-endothelial interactions and adhesion, ultimately leading to higher frequency and magnitude of cerebral microvascular blood flow reversal. Taken together, this may contribute to the observed slightly lower mean or effective microvascular forward blood flow. These pathophysiological changes might contribute to the reported higher rate of cerebral microinfarct and silent infarct in sickle cell disease. [Display omitted] DisclosuresArcher: Global Blood Therapeutics: Consultancy, Research Funding; Forma Therapeutics: Research Funding. Hyacinth: Novartis: Consultancy; Acuta Capital: Consultancy.

Interleukin 1α (IL-1α) Is a Central Regulator of Leukocyte-Endothelial Adhesion in Myocardial Infarction and in CKD

Interleukin 1α (IL-1α) Is a Central Regulator of Leukocyte-Endothelial Adhesion in Myocardial Infarction and in CKD

Brain Microvascular Endothelial Cell-Derived HMGB1 Facilitates Monocyte Adhesion and Transmigration to Promote JEV Neuroinvasion.

Infection with Japanese encephalitis virus (JEV) induces high morbidity and mortality, including potentially permanent neurological sequelae. However, the mechanisms by which viruses cross the blood-brain barrier (BBB) and invade into the central nervous system (CNS) remain unclear. Here, we show that extracellular HMGB1 facilitates immune cell transmigration. Furthermore, the migration of immune cells into the CNS dramatically increases during JEV infection which may enhance viral clearance, but paradoxically expedite the onset of Japanese encephalitis (JE). In this study, brain microvascular endothelial cells (BMECs) were utilized for the detection of HMGB1 release, and leucocyte, adhesion, and the integrity of the BBB in vitro. Genetically modified JEV-expressing EGFP (EGFP-JEV) and the BBB model were established to trace JEV-infected immune cell transmigration, which mimics the process of viral neuroinfection. We find that JEV causes HMGB1 release from BMECs while increasing adhesion molecules. Recombinant HMGB1 enhances leukocyte-endothelium adhesion, facilitating JEV-infected monocyte transmigration across endothelia. Thus, JEV successfully utilizes infected monocytes to spread into the brain, expanding inside of the brain, and leading to the acceleration of JE onset, which was facilitated by HMGB1. HMGB1-promoted monocyte transmigration may represent the mechanism of JEV neuroinvasion, revealing potential therapeutic targets.