Leukocyte Adhesion Research Articles

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.

RG7774 (Vicasinabin), an orally bioavailable cannabinoid receptor 2 (CB2R) agonist, decreases retinal vascular permeability, leukocyte adhesion, and ocular inflammation in animal models.

Preclinical studies suggest that cannabinoid receptor type 2 (CB2R) activation has a therapeutic effect in animal models on chronic inflammation and vascular permeability, which are key pathological features of diabetic retinopathy (DR). A novel CB2R agonist, triazolopyrimidine RG7774, was generated through lead optimization of a high-throughput screening hit. The aim of this study was to characterize the pharmacology, absorption, distribution, metabolism, elimination, and toxicity (ADMET) profile of RG7774, and to explore its potential for managing the key pathological features associated with retinal disease in rodents. The in vitro pharmacology of RG7774 was investigated for CB2R binding and receptor activation using recombinant human and mouse CB2R expression in Chinese hamster ovary cells, and endogenous CB2R expression in human Jurkat cells, and rat and mouse spleen cells. The ADMET profile was evaluated and the effects of RG7774 on retinal permeability, leukocyte adhesion, and choroidal neovascularization (CNV) were investigated in rodent models of retinal disease. Pharmacokinetic (PK) parameters and the exposure-response relationship were characterized in healthy animals and in animals with laser-induced CNV. RG7774 was found to be a potent (EC50: 2.8nM and Ki: 51.3nM), selective, and full CB2R agonist with no signs of cannabinoid receptor type 1 (CB1R) binding or activation. The ligand showed a favorable ADMET profile and exhibited systemic and ocular exposure after oral delivery. Functional potency in vitro translated from recombinant to endogenous expression systems. In vivo, orally administered RG7774 reduced retinal permeability and leukocyte adhesion in rodents with lipopolysaccharide (LPS)-induced uveitis and streptozotocin (STZ)-induced DR, and reduced lesion areas in rats with laser-induced CNV with an ED50 of 0.32mg/kg. Anatomically, RG7774 reduced the migration of retinal microglia to retinal lesions. RG7774 is a novel, highly selective, and orally bioavailable CB2R agonist, with an acceptable systemic and ocular PK profile, and beneficial effects on retinal vascular permeability, leukocyte adhesion, and ocular inflammation in rodent animal models. Results support the development of RG7774 as a potential treatment for retinal diseases with similar pathophysiologies as addressed by the animal models.

Bioactive Flavonoids in Protecting Against Endothelial Dysfunction and Atherosclerosis.

Atherosclerosis is a common cardiovascular disease closely associated with factors such as hyperlipidaemia and chronic inflammation. Among them, endothelial dysfunction serves as a major predisposing factor. Vascular endothelial dysfunction is manifested by impaired endothelium-dependent vasodilation, enhanced oxidative stress, chronic inflammation, leukocyte adhesion and hyperpermeability, endothelial senescence, and endothelial-mesenchymal transition (EndoMT). Flavonoids are known for their antioxidant activity, eliminating oxidative stress induced by reactive oxygen species (ROS), thereby preventing the oxidation of low-density lipoprotein (LDL) cholesterol, reducing platelet aggregation, alleviating ischemic damage, and improving vascular function. Flavonoids have also been shown to possess anti-inflammatory activity and to protect the cardiovascular system. This review focuses on the protective effects of these naturally-occuring bioactive flavonoids against the initiation and progression of atherosclerosis through their effects on endothelial cells including, but not limited to, their antioxidant, anti-inflammatory, anti-thrombotic, and lipid-lowering properties. However, more clinical evidences are still needed to determine the exact role and optimal dosage of these compounds in the treatment of atherosclerosis.

The crosstalk between neuropilin-1 and tumor necrosis factor-α in endothelial cells.

Tumor necrosis factor-α (TNFα) is a master cytokine which induces expression of chemokines and adhesion molecules, such as intercellular adhesion molecule 1 (ICAM-1) and vascular cell adhesion molecule 1 (VCAM-1), in endothelial cells to initiate the vascular inflammatory response. In this study, we identified neuropilin-1 (NRP1), a co-receptor of several structurally diverse ligands, as a modulator of TNFα-induced inflammatory response of endothelial cells. NRP1 shRNA expression suppressed TNFα-stimulated leukocyte adhesion and expression of ICAM-1 and VCAM-1 in human umbilical vein endothelial cells (HUVECs). Likewise, it reduced TNFα-induced phosphorylation of MAPK p38 but did not significantly affect other TNF-induced signaling pathways, such as the classical NFκB and the AKT pathway. Immunofluorescent staining demonstrated co-localization of NRP1 with the two receptors of TNF, TNFR1 and TNFR2. Co-immunoprecipitation further confirmed that NRP1 was in the same protein complex or membrane compartment as TNFR1 and TNFR2, respectively. Modulation of NRP1 expression, however, neither affected TNFR levels in the cell membrane nor the receptor binding affinities of TNFα. Although a direct interface between NRP1 and TNFα/TNFR1 appeared possible from a protein docking model, a direct interaction was not supported by binding assays in cell-free microplates and cultured cells. Furthermore, TNFα was shown to downregulate NRP1 in a time-dependent manner through TNFR1-NFκB pathway in HUVECs. Taken together, our study reveals a novel reciprocal crosstalk between NRP1 and TNFα in vascular endothelial cells.

Investigation of Some Autosomal Recessive Inherited Diseases (BLAD, DUMPS, CVM, and FXID) in Holstein Cattle

Genetic disorders are often transmitted via autosomal recessive inheritance, which negatively affect health, welfare, and yield traits in farm animals such as cattle. In this study, a total of 80 animals belonging to Holstein Friesian (HF) reared in the dairy farm of Akdeniz University were investigated in terms of Bovine Leukocyte Adhesion Deficieny (BLAD), Deficiency of Uridine Monophosphate Synthase (DUMPS), Factor XI Deficiency (FXID), and Complex Vertebral Malformation (CVM) via three molecular genotyping methods such as Polymerase Chain Reaction (PCR), PCR-Restriction Fragment Length Polymorphism (PCR-RFLP), and Allele-Sprcific PCR (AS-PCR). BLAD and DUMPS were analysed by PCR-RFLP, while PCR and AS-PCR were utilized to investigate FXID and CVM disorders, respectively. Based on fragment patterns on agarose gel electrophoresis, animals were genotyped as normal, mutant, and carrier. In this study, no carrier or mutant animals were detected for BLAD, CVM, DUMPS, and FXID diseases in HF cattle, since these animals were previously imported from disease-free dairy farms located in different provinces of Türkiye. Nevertheless, this population should be periodically checked for autosomal genetic disorders, since inherited diseases may be observed in the next generations due to causative mutations in the related genomic regions.

Leukocyte reaction of peripheral blood in secondary chronic carrageenan inflammation after administration of the thrombin blocker dabigatran etexilate

The experimental study was conducted on 72 adult male WAG laboratory rats weighing 180–200 g. The study was conducted in accordance with the guidelines of Directive 2010/63/EU of the European Parliament and of the Council on the protection of animals used for scientific purposes. Intact rats served as a control for the natural course of inflammation, and rats receiving dabigatran etexilate without further induction of chronic inflammation served as a control for inflammation in the setting of dabigatran etexilate administration. The model of inflammation was carrageenan secondary chronic aseptic inflammation, which was induced by intramuscular injection of 10 mg λ-carrageenan (Sigma, USA) dissolved in 1 ml of isotonic sodium chloride solution into the right thigh. Dabigatran etexilate was administered intragastrically through a gavage tube at a dose of 15 mg/kg/day dissolved in 1 ml of isotonic sodium chloride solution daily throughout the experiment, which lasted 28 days. The results of the study have shown that the use of a thrombin blocker dabigatran etexilate, compared with the natural course of secondary chronic carrageenan inflammation, promotes the involvement of leukocytes on the 14th day, which leads to greater elimination of phlogogen during this period, and a decrease in the number of leukocytes on the 21st and 28th days during the period of chronic inflammation, which indicates a decrease in the need for leukocytes in these periods. The tendency to increase the number of segmented neutrophils by 1.3 times on the 28th day of inflammation after administration of dabigatran etexilate, compared with the natural course of inflammation, indicates a less pronounced emigration of leukocytes to the inflammatory focus due to a decrease in the intensity of chronic inflammation. A decrease in the number of banded neutrophils in the peripheral blood on the 28th day may be associated with a decrease in their emigration to the inflammatory focus. A significant decrease in the number of monocytes by 1.62 times (p<0.01) on the 28th day indicates that the use of the thrombin blocker dabigatran etexilate reduces leukocyte adhesion and reduces the chronicity of inflammation. Keywords: toxic action, biochemical variant, antimicrobials.

Use of Topical Gaseous Nitric Oxide/Plasma Energy in the Treatment of Recalcitrant Wounds.

Nitric oxide (NO) is involved in many biological functions and has been demonstrated to be important in wound healing. When delivered to a wound in its gaseous state, NO stimulates vasodilatation and angiogenesis, inhibits platelet and erythrocyte aggregation, reduces leukocyte adhesion, and is an important anti-inflammatory and antimicrobial agent. Many patients with chronic and hard-to-heal wounds have a deficiency of NO in their tissues ,which may contribute to slow and even arrested healing. However, it has been difficult to use NO for treatment because of its short half-life, which is measured in seconds. A recently developed device provides a way to generate NO and combine it with a stream of plasma energy, which extends its half-life to the point that it can provide a clinical effect. This device creates NO from the ambient air, and no other gases are needed. The combination of atmospheric oxygen and nitrogen at a high temperature generated by an electric arc results in NO and plasma energy (N2 + O2 = 2NO + 181 KJ energy). After generation, the NO/plasma energy-containing gas flow is cooled to 18-20°C, and NO is delivered to the tissues in a "dose" between 800 and 1000 ppm. When NO gas was combined with the plasma energy stream, the NO was found to penetrate intact skin or tissue up to 3cm to treat an underlying problem. Studies have shown that NO/plasma energy therapy promotes healing. This report summarizes current applications of this unique approach in the treatment of chronic, hard-to-heal and infected wounds.

Endothelial Dysfunction and Heart Failure with Preserved Ejection Fraction-An Updated Review of the Literature.

Heart failure (HF) is a clinical syndrome consisting of typical symptoms and signs due to structural and/or functional abnormalities of the heart, resulting in elevated intracardiac pressures and/or inadequate cardiac output. The vascular system plays a crucial role in the development and progression of HF regardless of ejection fraction, with endothelial dysfunction (ED) as one of the principal features of HF. The main ED manifestations (i.e., impaired endothelium-dependent vasodilation, increased oxidative stress, chronic inflammation, leukocyte adhesion, and endothelial cell senescence) affect the systemic and pulmonary haemodynamic and the renal and coronary circulation. The present review is aimed to discuss the contribution of ED to HF pathophysiology-in particular, HF with preserved ejection fraction-ED role in HF patients, and the possible effects of pharmacological and non-pharmacological approaches. For this purpose, relevant data from a literature search (PubMed, Scopus, EMBASE, and Medline) were reviewed. As a result, ED, assessed via venous occlusion plethysmography or flow-mediated dilation, was shown to be independently associated with poor outcomes in HF patients (e.g., mortality, cardiovascular events, and hospitalization due to worsening HF). In addition, SGLT2 inhibitors, endothelin antagonists, endothelial nitric oxide synthase cofactors, antioxidants, and exercise training were shown to positively modulate ED in HF. Despite the need for future research to better clarify the role of the vascular endothelium in HF, ED represents an interesting and promising potential therapeutic target.

Inhaled nitric oxide suppresses neuroinflammation in experimental ischemic stroke

Ischemic stroke is a major global health issue and characterized by acute vascular dysfunction and subsequent neuroinflammation. However, the relationship between these processes remains elusive. In the current study, we investigated whether alleviating vascular dysfunction by restoring vascular nitric oxide (NO) reduces post-stroke inflammation. Mice were subjected to experimental stroke and received inhaled NO (iNO; 50 ppm) after reperfusion. iNO normalized vascular cyclic guanosine monophosphate (cGMP) levels, reduced the elevated expression of intercellular adhesion molecule-1 (ICAM-1), and returned leukocyte adhesion to baseline levels. Reduction of vascular pathology significantly reduced the inflammatory cytokines interleukin-1β (Il-1β), interleukin-6 (Il-6), and tumor necrosis factor-α (TNF-α), within the brain parenchyma. These findings suggest that vascular dysfunction is responsible for leukocyte adhesion and that these processes drive parenchymal inflammation. Reversing vascular dysfunction may therefore emerge as a novel approach to diminish neuroinflammation after ischemic stroke and possibly other ischemic disorders.

Relationship between Vascular Cell Adhesion Molecule-1 (Vcam-1) Level with Severity Degree of COVID-19 Patients at General Hospital H. Adam Malik Medan

Background: The mechanism and pathogenesis of COVID-19 that distinguishes asymptomatic, mild, moderate, severe to critical symptoms is not yet known with certainty. Endothelial dysfunction and immune thrombosis serve as a profound pathogenic mechanism in COVID-19. Vascular Cell Adhesion Molecule-1 (VCAM-1) is a major regulator of leukocyte adhesion that contributes to the formation of thrombosis. The purpose of the study determine the relationship between VCAM-1 level with the severity degree in COVID-19 patients. Methods: An analytical study with a cross-sectional design was conducted from September – March 2022. This study was followed by 50 patients aged over 18 years with moderate, severe, and critical degrees of COVID-19 admitted to Haji Adam Malik General Hospital Medan by excluding malignancy and pregnancy patients. Serum VCAM-1 levels were measured using Chemwell Analyzer. Results: Among 50 patients, 33 patients (66%) were male and 17 patients (34%) were female. The mean age was 57 years with the youngest 19 years old and the oldest 81 years old. The median level of VCAM-1 was 23,02 ng/mL (10.96 ng/mL - 50.63 ng/mL). The median concentration of VCAM-1 was 37.85 ng/mL (15,59 – 50.63), 36.68 ng/mL (19.58 – 49.71), and 18.83 ng/mL (10.96 – 46.32) for critical, severe, and moderate degree respectively (p = 0.001). The cut-off value of VCAM-1 levels to predict the severity of COVID-19 is 32.01 ng/mL. Conclusions: There is a significant relationship between VCAM-1 levels and the severity of COVID-19.

Vasoinhibin's Apoptotic, Inflammatory, and Fibrinolytic Actions Are in a Motif Different From Its Antiangiogenic HGR Motif.

Vasoinhibin, a proteolytic fragment of the hormone prolactin, inhibits blood vessel growth (angiogenesis) and permeability, stimulates the apoptosis and inflammation of endothelial cells, and promotes fibrinolysis. The antiangiogenic and antivasopermeability properties of vasoinhibin were recently traced to the HGR motif located in residues 46 to 48 (H46-G47-R48), allowing the development of potent, orally active, HGR-containing vasoinhibin analogues for therapeutic use against angiogenesis-dependent diseases. However, whether the HGR motif is also responsible for the apoptotic, inflammatory, and fibrinolytic properties of vasoinhibin has not been addressed. Here, we report that HGR-containing analogues are devoid of these properties. Instead, the incubation of human umbilical vein endothelial cells with oligopeptides containing the sequence HNLSSEM, corresponding to residues 30 to 36 of vasoinhibin, induced apoptosis, nuclear translocation of NF-κB, expression of genes encoding leukocyte adhesion molecules (VCAM1 and ICAM1) and proinflammatory cytokines (IL1B, IL6, and TNF), and adhesion of peripheral blood leukocytes. Also, intravenous or intra-articular injection of HNLSSEM-containing oligopeptides induced the expression of Vcam1, Icam1, Il1b, Il6, and Tnf in the lung, liver, kidney, eye, and joints of mice and, like vasoinhibin, these oligopeptides promoted the lysis of plasma fibrin clots by binding to plasminogen activator inhibitor-1 (PAI-1). Moreover, the inhibition of PAI-1, urokinase plasminogen activator receptor, or NF-κB prevented the apoptotic and inflammatory actions. In conclusion, the functional properties of vasoinhibin are segregated into 2 different structural determinants. Because apoptotic, inflammatory, and fibrinolytic actions may be undesirable for antiangiogenic therapy, HGR-containing vasoinhibin analogues stand as selective and safe agents for targeting pathological angiogenesis.

Green Solvent-Based Antifouling Polymer Brushes Demonstrate Excellent Hemocompatibility.

Medical devices are crucial for patient care, yet even the best biomaterials lead to infections and unwanted activation of blood coagulation, potentially being life-threatening. While hydrophilic polymer brushes are the best coatings to mitigate these issues, their reliance on fossil raw materials underscores the urgency of bio-based alternatives. In this work, we introduce polymer brushes of a green solvent-based monomer, prohibiting protein adsorption, bacterial colonization, and blood clot formation at the same level as fossil-based polymer brushes. The polymer brushes are composed of N,N-dimethyl lactamide acrylate (DMLA), can be polymerized in a controlled manner, and show strong hydrophilicity as determined by thermodynamic analysis of the surface tension components. The contact of various challenging protein solutions results in repellency on the poly(DMLA) brushes. Furthermore, the poly(DMLA) brushes completely prevent the adhesion and colonization of Escherichia coli. Remarkably, upon blood contact, the poly(DMLA) brushes successfully prevent the formation of a fibrin network and leukocyte adhesion on the surface. While showcasing excellent antifouling properties similar to those of N-hydroxypropyl methacrylamide (HPMA) polymer brushes as one of the best antifouling coatings, the absence of hydroxyl groups prevents activation of the complement system in blood. We envision the polymer brushes to contribute to the future of hemocompatible coatings.

Roles of Sphingosine Kinase and Sphingosine-1-Phosphate Receptor 2 in Endotoxin-Induced Acute Retinal Inflammation

ABSTRACT Purpose To investigate the roles of sphingosine kinases (SphKs) and sphingosine-1-phosphate receptors (S1PRs) in endotoxin-induced uveitis (EIU) mice. Methods EIU model was induced using an intraperitoneal injection of lipopolysaccharide (LPS). The expression of SphKs and S1PRs in the retina was assessed using quantitative polymerase chain reaction (qPCR) and immunofluorescence. The effects of S1PR antagonists on the expression of inflammatory cytokines in the retina were evaluated using qPCR and western blotting. Effects of leukocyte infiltration of the retinal vessels were evaluated to determine the effects of the S1PR2 antagonist and genetic deletion of S1PR2 on retinal inflammation. Results Retinal SphK1 expression was significantly upregulated in EIU. SphK1 was expressed in the GCL, IPL, and OPL and S1PR2 was expressed in the GCL, INL, and OPL. Positive cells in IPL and OPL of EIU retina were identified as endothelial cells. S1PR2 antagonist and genetic deletion of S1PR2 significantly suppressed the expression of IL-1α, IL-6, TNF-α, and ICAM-1, whereas S1PR1/3 antagonist did not. Use of S1PR2 antagonist and S1PR2 knockout in mice significantly ameliorated leukocyte adhesion induced by LPS. Conclusion SphK1/S1P/S1PR2 signaling was upregulated in EIU and S1PR2 inhibition suppressed inflammatory response. Targeting this signaling pathway has potential for treating retinal inflammatory diseases.

P-selectin-dependent leukocyte adhesion is governed by endolysosomal two-pore channel 2

Upon proinflammatory challenges, endothelial cell surface presentation of the leukocyte receptor P-selectin, together with the stabilizing co-factor CD63, is needed for leukocyte capture and is mediated via demand-driven exocytosis from the Weibel-Palade bodies that fuse with the plasma membrane. We report that neutrophil recruitment to activated endothelium is significantly reduced in mice deficient for the endolysosomal cation channel TPC2 and in human primary endothelial cells with pharmacological TPC2 block. We observe less CD63 signal in whole-mount stainings of proinflammatory-activated cremaster muscles from TPC2 knockout mice. We find that TPC2 is activated and needed to ensure the transfer of CD63 from endolysosomes via Weibel-Palade bodies to the plasma membrane to retain P-selectin on the cell surface of human primary endothelial cells. Our findings establish TPC2 as a key element to leukocyte interaction with the endothelium and a potential pharmacological target in the control of inflammatory leukocyte recruitment.

Plasma Proteome Profiling of Sickle Cell Patients Reveals Activation of Inflammatory Pathways during Steady State and Vaso-Occlusive Crisis

Background The pathophysiology of Sickle Cell Disease (SCD) is characterized by chronic intravascular hemolysis and ischemia-reperfusion, resulting in oxidative stress and chronic inflammation. Although inflammation seems to play an important role in the development of tissue damage in SCD, little is known about the various cellular and cytokine pathways involved in the pathophysiology of SCD. This study aims to further elucidate the inflammatory pathways in SCD using targeted plasma protein profiling both in steady state and during vaso-occlusive crisis (VOC). Methods In this prospective study, SCD patients with the genotypes HbSS and HbSβ 0-thalassemia and healthy ethnicity matched controls were included. Plasma levels of 92 proteins involved in various inflammatory pathways were measured in EDTA plasma using Olink Proximity Extension Assay (PEA) technology. In brief, pairs of oligonucleotide-labeled antibody probes were added to 20 μL of plasma, resulting in the binding of these antibody probes to their targeted protein. Subsequently DNA polymerase was added, leading to a proximity-dependent DNA polymerization event, generating a unique PCR target sequence. The final assay read-out is presented in Normalized Protein eXpression (NPX) values, which is an arbitrary unit on a log2-scale with an NPX difference (estimate) of 1 meaning a doubling of protein concentration. RStudio version 4.2.1 was used for data analysis and visualization. Differences between subgroups were tested using an unpaired or paired t-test as appropriate. Results were corrected for multiple testing using the Benjamini-Hochberg method. Results with p < 0.05 were considered significant. Furthermore Over-representation Analysis (ORA) was performed to investigate the association of the differentially expressed proteins with specific biological processes. Results Plasma samples were collected from 43 SCD patients in steady state (median age: 25 years (IQR: 20 - 32)) and 13 healthy ethnicity matched controls (median age: 29 (IQR: 28-39)). From 15 SCD patients plasma samples were collected during both steady state and VOC (median age: 27 (IQR: 19 - 34)). Comparison of protein levels between healthy controls and SCD patients in steady state identified 38 proteins (IL-18, ANGPT2, Gal-9, CD4, HGF, HO-1, CCL4, ARG1, ADA, GZMA, PD-L1, CCL23, LAG3, TNFRSF4, CCL3, CXCL11, CRTAM, IL-10, GZMB, CD27, FGF2, IL-8, IL-6, TNF, PGF, NCR1, TNFSF14, CASP-8, IL-7, FASLG, TNFRSF9, LAP TGF-beta-1, PD-L2, CCL19, CD83, GZMH, MCP-4, CD244, TIE2) that were significantly higher in SCD patients as shown in Figure 1. Furthermore soluble CD70 was significantly lower in SCD patients in steady state compared to healthy controls (estimate: -0.34, p < 0.05). Over-representation Analysis revealed a significant upregulation of proteins involved in the regulation of hematopoiesis, the IL-18 signaling pathway, T-cell activation, leukocyte differentiation and leukocyte adhesion (Figure 2). Pairwise comparison of protein levels between steady state and VOC in SCD patients revealed 6 proteins (TWEAK, VEGFR-2, CD244, PDCD1, GZMA, TNFRSF4) which were significantly lower and 12 proteins (IL-6, TNFSF14, ADA, IL-15, HGF, IL-10, IL-8, Gal-9, CSF-1, CCL20, MMP7, VEGFA) which were significantly higher during VOC as compared to steady state. During VOC, pathways involved in the regulation of cell death (IL-6, TNFSF14, ADA, HGF, IL-10, PDCD1, VEGFA) and tissue development (IL-6, ADA, HGF, IL-10, VEGFA) were significantly enriched. Summary/conclusion The results of these proteomic analyses provide new insights into the pathways that regulate inflammation in SCD, both during steady state and VOC. These findings might contribute to a better understanding of SCD pathophysiology and identifying potential new targets for therapeutic interventions in SCD patients. Our results warrant further in-depth investigation of the different activated pathways.

Effect of Integrin α 4β 1 -VCAM-1 Interactions in JAK2V617F Myeloproliferative Neoplasm Pro-Thrombotic Interactions

Introduction: The JAK2 V617Fmutation, which occurs in up to 95% of polycythemia vera (PV) myeloproliferative neoplasm (MPN) patients, increases thrombosis risk 6-fold. A recent study in PV mice demonstrated that blocking JAK2 V617F+ leukocyte-mediated β1/β2 integrin interactions with anti-VCAM-1 antibodies decreases thrombosis. Interestingly, hydroxyurea (HU), a common PV treatment, reduces leukocyte and red cell α4β1 integrin expression. Overall, JAK2 V617F+ mouse models show inconsistent thrombosis and bleeding phenotype, hindering pre-clinical study reproducibility; therefore, we have developed an in vitro endothelialized microfluidic model to assess MPN-flow behavior. We hypothesized that in our microfluidic model, disrupting α4β1 integrin expression on JAK2 V617F+ leukocytes would reduce pro-adhesive interactions. We tested this hypothesis by assessing JAK2 V617F+ MPN blood flow in untreated and hydroxyurea treated subjects and through addition of a monoclonal antibody against α4β1 integrin, natalizumab. Methods: Primary human umbilical vein endothelial cells (HUVECs) were exposed to venous shear (15 dynes/cm 2) in microfluidics for a 4-day culture period. On day 4, endothelialized microfluidics were treated +/- 10 ng/mL TNFα for 4 hours. Blood samples were obtained from consented JAK2 V617F MPN individuals aged >18 years with ET, PV, or MF as diagnosed by the WHO 2016 Criteria identified in the M Health Fairview Masonic Cancer Center Clinics. Healthy age, race, and sex-matched blood samples served as controls. Clinical data on patients was collected and stored in REDCAP. Platelets and leukocytes were visualized using Calcein AM. Samples were treated +/- 0.25 μg/mL natalizumab for 30 minutes, then perfused through the microfluidic under a microscope to observe blood-endothelial interactions in real-time. Post-experiment, videos were analyzed to quantify velocity and adhesion of platelets and leukocytes for each treatment condition. Results: When perfused over TNF-α activated endothelium, both controls ( JAK2 negative, n=11) and JAK2 V617F MPN (n=20) had a significant reduction in cell velocity. Furthermore, compared to controls, JAK2 V617F MPN individuals exhibited significantly lower cell velocities. In multi-variate analysis, reduction in cell velocity was independent of white blood cell, hemoglobin, hematocrit or platelet counts. Hydroxyurea is a standard therapy in JAK2 V617F MPN and is known to reduce α4β1 expression. Therefore, we performed subgroup analysis in our JAK2 V617F MPN cohort based on treatment. When perfused over TNF-α activated endothelium, individuals with JAK2 V617F MPN (n=9) not on therapy (newly diagnosed) or on phlebotomy/aspirin only had a significant reduction in cell velocity. In the same conditions, HU-treated JAK2 V617F MPN patients (n=9) had no change in cell velocity. There was no difference in complete blood counts between these groups. In a separate subgroup analysis, compared to untreated JAK2 V617F MPN patients (n=9), interferon-treated JAK2 V617F MPN patients (n=5) had a greater reduction in cell velocity. Last, we specifically targeted α4β1 integrin by adding natalizumab to either control (n=7) or untreated JAK2 V617F MPN (n=3) samples. On inactivated endothelium, treatment of either control or JAK2 V617F MPN blood with natalizumab did not significantly change cell velocity. On a TNF-α activated endothelium, treatment of control (n=7) and JAK2 V617F MPN blood (n=3) with natalizumab led to a recovery in cell velocity. Conclusion: Our data suggests that the interaction between integrin α4β1 on JAK2 V617F cell surface and VCAM-1 on activated endothelium is important for initiation of pro-thrombotic interactions. JAK2 V617F MPN individuals treated with HU exhibit decreased adhesion to activated endothelium compared to control; JAK2 V617F MPN individuals treated with interferon exhibit increased adhesion. In the microfluidic system, based on current findings, blockage of integrin α4β1-VCAM-1 interactions using natalizumab is a potential therapeutic strategy. Further mechanistic studies evaluating interferon and cell populations changed by therapies as they pertain to integrin α4β1 are underway.

Smaart Criz: Sickle Cell Mechanisms of Activation, Adhesion, Rheology, and Thrombosis (SMAART) in Response to P-Selectin Inhibition

Sickle cell disease (SCD) pathophysiology involves uncontrolled inflammation, dysregulated coagulation, and adherence of erythrocytes, leukocytes, and platelets to endothelium, all contributing to disrupted rheology and ultimately vaso-occlusion. Crizanlizumab (Novartis), an FDA-approved humanized monoclonal antibody, binds to p-Selectin and blocks endothelial interactions with blood cells. The phase 2 SUSTAIN trial demonstrated a reduction in the median rate of pain crisis, as well as prolonged time to first and second crisis ( Ataga 2017). Not all participants benefited, however, nor have all treated patients achieved pain reduction in the post-marketing period. Paradoxical infusion-related pain has also been reported ( Kanter 2022). Baseline differences between responders, non-responders, and those suffering infusion-related pain remain uncharacterized. In January 2023, Novartis issued a preliminary update on the ongoing global crizanlizumab phase 3 STAND trial which indicated no statistically significant pain reduction with crizanlizumab therapy compared to placebo. A better understanding of patient suitability for the various disease modifying therapies for SCD would however be beneficial. Our center is a pilot site for a lab conducting an assay measuring flow adhesion of whole blood to P-selectin (FA-WB-Psel) utilizing a protein coated microfluidic chamber by Functional Fluidics. Previously published data identified a steady state reference range for FA-WB-Psel in non-SCD as well as SCD patients ( Pittman 2021). The SMAART Criz study aims to characterize downstream biological effects of crizanlizumab (FA-WB-Psel) and identify biomarkers with the potential to predict treatment response, with the goal of facilitating more individualized prescribing. This is an interim analysis of an investigator-initiated, prospective, IRB-approved, 18-week longitudinal study at the Children's Hospital of Michigan. Patients desiring crizanlizumab treatment were offered enrollment. Following informed consent, samples were obtained for FA-WB-Psel before and after each crizanlizumab dose. Flow adhesion of whole blood to vascular cell adhesion molecule-1 (FA-WB-VCAM) was assessed as a control substrate. Thromboelastography (TEG) and whole blood platelet aggregometry were performed to evaluate platelet function at baseline, week 6, and week 18. Patient reported response to treatment was used to measure response to therapy. As of August 2023, 7 patients have completed the study with a median age of 20 years (range 16-25 years; 4 female). Genotypes are HbSS (5), HbSC (1), and HbS-G-Philadelphia (1). Four of 7 patients self-reported clinical response to therapy (Table 1). Treatment was discontinued after 1st dose due to treatment-related acute pain in 2 participants, and after the 4th dose due to patient-reported worsening pain trend (Figure 1). Clinical responders had a median baseline FA-WB-Psel adhesion index of 86 cells/mm 2 (n=4, mean 106, range of 56-193). Clinical responders experienced a 65% reduction in FA-WB-Psel from baseline (mean 86) to the mean pre-infusion level calculated using visit 2 and 3 levels (p=0.05, later visits affected by dropout). Non-responders had a median baseline FA-WB-PSel of 49 cells/mm 2 (n=3, mean 46, range 13-75) with widely variable FA-WB-PSel responses (Fig. 1). The two patients with acute treatment-induced pain had the lowest baseline values at 13 and 49 cells/mm 2. No significant trends were observed in adhesion to a control substrate using FA-WB-VCAM. There was an overall decrease in both arachidonic acid (AA) and adenosine diphosphate (ADP) platelet aggregation over time (AA: 23.5 Ohms to 11.3 Ohms, p=0.254; ADP: 23.4 Ohms to 10.6 Ohms, p=0.036). Early indications from SMAART Criz are that administration of crizanlizumab decreases FA-WB-Psel immediately after infusion with continued decreases over time. Interim analysis suggests elevated baseline FA-WB-Psel may predict individuals with SCD likely to benefit from crizanlizumab, while normal FA-WB-Psel levels at baseline may predict non-response or even adverse reactions. This cohort showed an overall decrease in both AA and ADP aggregation, indicating reduced platelet aggregation following crizanlizumab treatment, a potential downstream biological change contributing to treatment effect (Table 1). Further investigation is warranted. Enrollment and analyses are ongoing.

Caspase-1 As a Therapeutic Target in Intravascular Hemolysis-Induced Microvascular Inflammation and Occlusive Processes

Hemolytic anemias, such as sickle cell disease (SCD), are a group of acute and acquired conditions characterized by the accelerated destruction of red blood cells (RBC). Intravascular hemolysis (IVH), or RBC lysis within the bloodstream, incurs the release of damage-associated molecular patterns (DAMPs), trigging cell activation and immune responses. Caspase-1 (Casp-1), a protease enzyme, plays a critical role during inflammatory processes by initiating programed cell death and promoting the cleavage of pro-interleukin (IL)-1β into its active form, IL-1β, through inflammasome assembly. Here, we aimed to elucidate the complex mechanisms involved in IVH-induced inflammation, leukocyte-endothelium interactions, and microvascular dynamics. Acute IVH was induced in C57BL/6 mice (HEM) by the injection of sterile water (i.v., 150 µl), and control mice received i.v. saline (CON) (n=3-8 per group). Intravital microscopy was performed at 15 min post IVH induction on the cremaster muscle of mice to quantify venular leukocyte (WBC) rolling and adhesion, and non-invasive laser Doppler flowmetry (LDF) measured blood perfusion in the murine pelvis microvasculature. Blood samples were collected at 1-hour post-IVH for ELISA and flow cytometry analyses. Immortalized human endothelial cells (HUVEC; n=7 per group) were stimulated with heme or S100A8 for 3 hours. HEM mice demonstrated significantly increased plasma heme (P<0.0001) and cell-free hemoglobin (P<0.001), in association with significantly decreased plasma haptoglobin (P<0.05), compared to CON mice, thereby confirming the occurrence of IVH. Furthermore, HEM mice exhibited significantly increased plasma concentrations of the S100A8 alarmin (P<0.001) and IL1-β (P<0.001), as well as augmentations (P<0.001) in plasma TNF-ɑ and IL-6 levels. Flow cytometry was used to measure active Casp-1 in neutrophils and monocytes of mice using the FAM-FLICA probe. FLICA staining was significantly higher in the neutrophils (P<0.05) and monocytes (P<0.05) of HEM mice, compared to CON mice, implying Casp-1 activation in these cell subsets. WBC-endothelium interactions are a hallmark of vaso-occlusion in SCD. HEM mice exhibited elevated venular WBC rolling and adhesion to the endothelium, compared to CON mice (P<0.001), as previously shown (Almeida et al., 2015; PMID: 26019278). In addition, IVH significantly reduced the velocity of microvascular blood flow in mice (P<0.001), and impaired perfusion (P<0.001). To address the role of IVH-induced Casp-1 activation in microvascular dynamics, mice received i.p. administrations of the Casp-1 inhibitor, YVAD, at 1 hour prior to induction of IVH, or not, and were subjected to intravital microscopy or LDF. Interestingly, the inhibition of Casp-1 significantly abrogated IVH-induced WBC rolling (P<0.01) and adhesion to vessel walls (P<0.05) in HEM mice and significantly improved microvascular blood perfusion and flow velocity (P<0.05), suggesting a role for Casp-1 activation in the WBC-endothelium and occlusive interactions observed (See Figure). We then evaluated how heme and S100A8 may induce endothelial cell activation, as these DAMPs were found elevated in the plasma of HEM mice. As expected, compared to unstimulated HUVEC, heme induced endothelial cell activation in vitro, as shown by higher expressions of ICAM-1 (P<0.05), VCAM-1 (P<0.01) and E-selectin (P<0.01), and increased Casp-1 activity (P<0.01). In turn, S100A8 alone significantly elevated ICAM-1 (P<0.05) and E-selectin (P<0.01) expressions on HUVEC, as well as IL-1β release (P<0.001), however no significant increase in Casp-1 activity was observed. HUVEC, when pre-treated with YVAD, displayed a significant reduction in S100A8-induced E-selectin expression (P<0.05), but neither ICAM-1 nor VCAM-1 expression were significantly decreased. In contrast, no changes in the expressions of these adhesion molecules were observed in heme-stimulated cells after YVAD treatment. Taken together, these findings suggest that targeting caspase-1 activation and its associated pathways may represent a potential therapeutic intervention to mitigate the adverse inflammatory and microvascular effects of IVH, a disease mechanism that occurs in severe hemolytic anemias such as SCD.

Involvement of Angiopoietin 2 and vascular endothelial growth factor in uveitis.

Angiopoietin (Ang) 2 is released from vascular endothelial cells by the stimulation of vascular endothelial growth factor (VEGF)A. Ang2 increases the expression of leukocyte adhesion molecules on endothelial cells via nuclear factor κB. The aim of this study was to evaluate the effects of Ang2 and VEGFA on ocular autoimmune inflammation. We measured the concentrations of Ang2 and VEGFA in vitreous samples among patients with uveitis. Vitreous samples were collected from 16 patients with idiopathic uveitis (uveitis group) and 16 patients with non-inflammatory eye disease (control group). Experimental autoimmune uveoretinitis (EAU) was induced in B10.BR mice with a human interphotoreceptor retinoid-binding protein-derived peptide. The retinochoroidal tissues of the EAU mice were removed, and the mRNA levels of Ang2 and VEGFA were examined. EAU mice treated with anti-Ang2, anti-VEGFA, a combination of anti-Ang2 and anti-VEGFA, anti-Ang2/VEGFA bispecific, or IgG control antibodies were clinically and histopathologically evaluated. The protein levels of Ang2 and VEGFA were significantly higher in the vitreous samples of patients with uveitis than in controls (P<0.05). The retinochoroidal mRNA levels of Ang2 and VEGFA were significantly upregulated in EAU mice compared to controls (n = 6, P<0.05). Although there was no significant difference, treatment with anti-VEGFA antibody reduced the clinical and histopathological scores. However, treatment with anti-Ang2 antibody reduced the clinical and histopathological scores (n = 18-20, P<0.05). Furthermore, these scores were further decreased when treated by inhibiting both Ang2 and VEGFA. Based on these results, VEGFA and Ang2 were shown to be upregulated locally in the eye of both uveitis patients and models of uveitis. Dual inhibition of Ang2 and VEGFA is suggested to be a new therapeutic strategy for uveitis.

Non-Invasive Evaluation of Retinal Vascular Alterations in a Mouse Model of Optic Neuritis Using Laser Speckle Flowgraphy and Optical Coherence Tomography Angiography.

Optic neuritis, a characteristic feature of multiple sclerosis (MS), involves the inflammation of the optic nerve and the degeneration of retinal ganglion cells (RGCs). Although previous studies suggest that retinal blood flow alterations occur during optic neuritis, the precise location, the degree of impairment, and the underlying mechanisms remain unclear. In this study, we utilized two emerging non-invasive imaging techniques, laser speckle flowgraphy (LSFG) and optical coherence tomography angiography (OCTA), to investigate retinal vascular changes in a mouse model of MS, known as experimental autoimmune encephalomyelitis (EAE). We associated these changes with leukostasis, RGC injury, and the overall progression of EAE. LSFG imaging revealed a progressive reduction in retinal blood flow velocity and increased vascular resistance near the optic nerve head in the EAE model, indicating impaired ocular blood flow. OCTA imaging demonstrated significant decreases in vessel density, number of junctions, and total vessel length in the intermediate and deep capillary plexus of the EAE mice. Furthermore, our analysis of leukostasis revealed a significant increase in adherent leukocytes in the retinal vasculature of the EAE mice, suggesting the occurrence of vascular inflammation in the early development of EAE pathology. The abovechanges preceded or were accompanied by the characteristic hallmarks of optic neuritis, such as RGC loss and reduced visual acuity. Overall, our study sheds light on the intricate relationship between retinal vascular alterations and the progression of optic neuritis as well as MS clinical score. It also highlights the potential for the development of image-based biomarkers for the diagnosis and monitoring of optic neuritis as well as MS, particularly in response to emerging treatments.