Selectin Expression Research Articles

Correlation and clinical significance of placental tissue selectin (E), angiotensin II and its receptors, and oxidized lipid levels in patients with preeclampsia

Background: The purpose was to analyze the levels of placental tissue selectins (E), angiotensin II (AngII) and its receptors (ATRs), and oxidized lipids (malondialdehyde (MDA), 8-isoprostane 2a) in patients with preeclampsia (PE). (8-iso-PGF2a)) correlation and clinical significance. Methods: Select 30 PE pregnant women who were admitted to our hospital from March 2023 to January 2024 as the case group, and select another 30 normal pregnant women who were registered in our hospital during the same period as the health group .The general information of the two groups and placental tissue selectin (E), plasma AngII, ATRs, placental tissue MDA, 8-iso-PGF2a and blood pressure levels (systolic blood pressure (SBP), diastolic blood pressure (DBP)) were compared. Pearson correlation was used to analyze the correlation between the expression of selectin (E), AngII, ATRs, MDA, 8-iso-PGF2a and the levels of SBP and DBP. Linear regression analysis was used to analyze the correlation between the expression of selectin (E), AngII, ATRs, MDA, 8-iso-PGF2a, SBP, DBP and other indicators and the occurrence of PE. ROC curves were drawn to analyze the value of placental tissue selectin (E), AngII, ATRs, MDA, and 8-iso-PGF2a individually and jointly in predicting the risk of PE. Results The expression of placental tissue selectin (E), AngII, ATRs, MDA, 8-iso-PGF2a and the levels of SBP and DBP in the case group were higher than those in the healthy group (P<0.05). Pearson correlation showed that the expression levels of placental tissue selectin (E), AngII, ATRs, MDA, and 8-iso-PGF2a were positively correlated with SBP and DBP (r>0, P<0.05). Linear regression analysis showed that placental tissue selectin (E), AngII, ATRs, MDA, 8-iso-PGF2a expression and SBP, DBP levels were closely related to the occurrence of PE (P<0.05). The results of drawing the ROC curve showed that the AUCs of placental tissue selectin (E), AngII, ATRs, MDA, and 8-iso-PGF2a expression in predicting the occurrence of PE were 0.854, 0.756, 0.745, 0.885, 0.900, and 0.905 respectively. Conclusion Placenta tissue selectin (E), AngII, ATRs, MDA, and 8-iso-PGF2a are highly expressed in pregnant women with PE. The expression of the above indicators is related to maternal blood pressure levels and the occurrence of PE, and their combination can effectively increase predictive value of the risk of PE.

Effect of Arabica coffee and Robusta coffee on aortic tissue P selectin expression and plasma apolipoprotein A-1 (ApoA-1) levels in the early process of atherosclerosis

Effect of Arabica coffee and Robusta coffee on aortic tissue P selectin expression and plasma apolipoprotein A-1 (ApoA-1) levels in the early process of atherosclerosis

Comparison of storage solutions on saphenous vein endothelial function using immunofluorescence. A pilot study.

Our aim was to establish which of the current and novel storage fluids caused the least harm to the endothelium of the saphenous vein. Unmanipulated samples of saphenous vein were obtained. This vein was exposed to four fluids, DuraGraft®, Plasma-Lyte, autologous blood and sodium chloride, and a control medium. The expression of endothelial selectin (E-selectin) and endothelial nitric oxide synthase (eNOS) was measured using confocal microscopy. We identified and quantified platelet endothelial cell adhesion molecules, vascular cell adhesion molecules and endothelial nitric oxide synthase 3 on the endothelium. The results were widely variable. The protein expression of endothelial selectin (p = .17) and endothelial nitric oxide synthase 3 (p = .73) showed no statistically significant differences in levels of preservation when the different solutions were compared. In this study, we could not determine that any of the solutions were superior at preserving saphenous vein endothelium.

Fucosyltransferase VII (FUT7) Activity in Patients with Sickle Cell Disease and Healthy Controls

Background:Sickle cell disease (SCD) is a severe monogenic disorder with a majority of the affected individuals living in Africa. Vaso-occlusion is a significant cause of morbidity and mortality in SCD, and selectin mediated intercellular adhesion is recognized as an important target for therapy. As such Anti-P-selectin agents are in clinical use to reduce the frequency of vaso-occlusive crises (VOCs) in SCD patients. Sialyl lewis x is a crucial ligand common to all members of the selectin family and the final step in the biosynthesis of sialyl lewis X antigen (fucosylation), is catalysed by Fucosyltransferase VII (FUT7). This enzyme is most expressed in leukocytes and endothelial cells. We hypothesized that FUT7 enzyme activity is abnormal in SCD patients and differs by VOC severity. We sought to measure plasma levels of FUT7 in SCD patients and healthy controls, and investigate their association with clinical and laboratory features, such as the frequency of VOCs. Methods:This wasa single centre cross-sectional analytical study at the University of Nigeria Teaching Hospital, Enugu. 38 steady state adult SCD patients and 40 HbAA age- and sex-matched healthy control subjects were enrolled. SCD patients were stratified by clinical severity into: Group A- 19 subjects with mild disease (0-1 crises and no disease complication such as stroke, chronic leg ulcers, nephropathy, or avascular necrosis in the previous year), and Group B- 19 patients with moderate to severe disease (2 or more crises in the previous year, with or without complications). We excluded patients with red cell transfusion within 4 months, on hydroxyurea, or other agents that may affect vaso-occlusive crisis frequency, and co-existing chronic disorders unrelated to SCD, such as asthma and diabetes mellitus. We performed a CBC and plasma FUT7 enzyme concentration measurements (ELISA kit, MyBiosource Inc, USA). Statistical analysis was performed with SPSS version 22.0 for Windows. A 2-tailed P value of <0.05 was used. Results:Our subjects were predominantly female (SCD 52.5%, HbAA 55%), with median age 28 years (range 19-46 and 19-45 for SCD and HbAA, respectively). Median frequency of VOCs in patients was 1 (range 0-5). Avascular necrosis (32.5%) was the most common complication, while stroke, acute chest syndrome, and eye disease were the least common. As expected, SCD patients had lower haemoglobin levels than HbAA (7.9±1.3g/dL vs. 12.6±1.4g/dL, P<0.001). Significantly higher WBC, ANC, and platelet counts were observed in SCD patients compared with HbAA controls (P<0.001). No significant correlations were found between FUT7 and haematocrit (r =0.06), WBC count (r = -0.08), neutrophil count (r = -0.12), and platelet count (r =0.1), with P values of 0.7, 0.6, 0.5, and 0.6, respectively. We found no difference between the median and IQR values of plasma FUT7 of SCD (2.1; 1.8-2.5) and controls (2.2; 1.8-2.8), P = 0.7. FUT7 activity was higher in SCD subjects in Group A (2.2; 1.9-2.9) than in Group B (2.0; 1.2- 2.4), with a trend towards significance P = 0.1. Discussion:In this study, FUT7 activity did not differ between SCD patients and controls. We also did not find correlations between FUT7 and blood cell counts, suggesting that plasma FUT7 activity is not increased at baseline in SCD and that the degrees of anemia or leukocytosis are not associated with changes in FUT7. Nonetheless, a mild decrease in FUT7 levels was found in more severe SCD patients and should be confirmed in a larger study. Our study did not evaluate patients in crises or who received hydroxyurea and/or other SCD-modifying therapies. Since FUT7 catalyses selectin expression, variation in its activity may be more relevant in subgroups of SCD patients on anti-selectin therapies. Future studies should be carried out in such patients to explore FUT7 activity as a marker of acute vaso-occlusion and as a modifier of response to therapy.

Abstract 157: Role Of Systemic Platelet Levels In Venous Thrombosis And Resolution In A Murine Model Of Inferior Vena Cava Constriction

Background: Platelet role in primary clot formation in response to vessel wall injury is well established; however, the role of systemic platelet levels in deep vein thrombosis (DVT) and its sequelae is unclear. Their interaction with the venous endothelium under low shear stress, regulation of thrombosis, and mediation of both innate and adaptive immune responses through selectin and toll-like receptor expression, respectively, warrants further investigation into their role in DVT formation and resolution. In this study, we present a role for systemic platelet levels in regulating thrombus resolution. Methods: Wildtype, male or female CD1 mice were treated with a thrombopoietin antisense oligonucleotide (TPO-ASO) at 10 mg/kg, eltrombopag at 75 mg/ kg, saline (control), or a scrambled TPO-ASO (control) at 8 mg/ kg prior to surgical creation of a venous thrombus by ligation of the infra-renal inferior vena cava (IVC). Peak scaled thrombus weights (i.e., linear density measured using a ratio of the clot and IVC weight scaled to the length of the IVC) were measured to characterize the early resolution of the thrombus through involution and recanalization. Mice were euthanized on postoperative days 3 and 7 to measure the formation and resolution of the thrombus, respectively. Results: Platelet levels were elevated and decreased by over 30% by the day of surgical treatment after TPO-ASO and eltrombopag treatments, respectively. Scaled thrombus weights were significantly different (p = 0.045, Kruskal Wallis) between groups on day 7 after IVC constriction. Post-hoc analysis (Dunn Test with Benjamini-Hochberg correction) revealed that the TPO-ASO group (mean = 25.4 +/- 0.9 mg/cm) had significantly decreased (p = 0.018) scaled thrombus weights compared to the mice treated with eltrombopag (mean = 35.3 +/- 6.5 mg/cm) on day 7. Thrombus formation on day 3 was not statistically different between treatment groups. Conclusion: Achieving a minimum threshold of 30% reduction of systemic platelet levels by TPO-ASO enhanced thrombus resolution, but not thrombus formation, in our stasis model of rodent DVT resolution. These data suggest a potential role for systemic platelet levels in regulating thrombus resolution.

Contrast Enhanced Ultrasound Molecular Imaging of Spontaneous Chronic Inflammatory Bowel Disease in an Interleukin-2 Receptor α-/- Transgenic Mouse Model Using Targeted Microbubbles.

Inflammatory bowel disease (IBD) is a lifelong inflammatory disorder with relapsing–remission cycles, which is currently diagnosed by clinical symptoms and signs, along with laboratory and imaging findings. However, such clinical findings are not parallel to the disease activity of IBD and are difficult to use in treatment monitoring. Therefore, non-invasive quantitative imaging tools are required for the multiple follow-up exams of IBD patients in order to monitor the disease activity and determine treatment regimens. In this study, we evaluated a dual P- and E-selectin-targeted microbubble (MBSelectin) in an interleukin-2 receptor α deficient (IL-2Rα−/−) spontaneous chronic IBD mouse model for assessing long-term anti-inflammatory effects with ultrasound molecular imaging (USMI). We used IL-2Rα−/− (male and female on a C57BL/6 genetic background; n = 39) and C57BL/6 wild-type (negative control; n = 6) mice for the study. USMI of the proximal, middle, and distal colon was performed with MBSelectin using a small animal scanner (Vevo 2100) up to six times in each IL-2Rα−/− mouse between 6–30 weeks of age. USMI signals were compared between IL-2Rα−/− vs. wild-type mice, and sexes in three colonic locations. Imaged colon segments were analyzed ex vivo for inflammatory changes on H&E-stained sections and for selectin expression by immunofluorescence staining. We successfully detected spontaneous chronic colitis in IL-2Rα−/− mice between 6–30 weeks (onset at 6–14 weeks) compared to wild-type mice. Both male and female IL-2Rα−/− mice were equally (p = 0.996) affected with the disease, and there was no significant (p > 0.05) difference in USMI signals of colitis between the proximal, middle, and distal colon. We observed the fluctuating USMI signals in IL-2Rα−/− mice between 6–30 weeks, which might suggest a resemblance of the remission-flare pattern of human IBD. The ex vivo H&E and immunostaining further confirmed the inflammatory changes, and the high expression of P- and E-selectin in the colon. The results of this study highlight the IL-2Rα−/− mice as a chronic colitis model and are suitable for the long-term assessment of treatment response using a dual P- and E-selectin-targeted USMI.

Cold Atmospheric Plasma Promotes the Immunoreactivity of Granulocytes In Vitro.

Cold atmospheric plasma (CAP) reduces bacteria and interacts with tissues and cells, thus improving wound healing. The CAP-related induction of neutrophils was recently described in stained sections of wound tissue in mice. Consequently, this study aimed to examine the functionality of human polymorphonuclear cells (PMN)/granulocytes through either a plasma-treated solution (PTS) or the direct CAP treatment with different plasma modes and treatment durations. PTS analysis yielded mode-dependent differences in the production of reactive oxygen species (ROS) and reactive nitrogen species (RNS) after CAP treatment. Live-cell imaging did not show any chemo-attractive or NETosis-inducing effect on PMNs treated with PTS. The time to maximum ROS production (TmaxROS) in PMNs was reduced by PTS and direct CAP treatment. PMNs directly treated with CAP showed an altered cell migration dependent on the treatment duration as well as decreased TmaxROS without inducing apoptosis. Additionally, flow cytometry showed enhanced integrin and selectin expression, as a marker of activation, on PMN surfaces. In conclusion, the modification of PMN immunoreactivity may be a main supporting mechanism for CAP-induced improvement in wound healing.

Cadherins, Selectins, and Integrins in CAM-DR in Leukemia.

The interaction between leukemia cells and the bone microenvironment is known to provide drug resistance in leukemia cells. This phenomenon, called cell adhesion-mediated drug resistance (CAM-DR), has been demonstrated in many subsets of leukemia including B- and T-acute lymphoblastic leukemia (B- and T-ALL) and acute myeloid leukemia (AML). Cell adhesion molecules (CAMs) are surface molecules that allow cell–cell or cell–extracellular matrix (ECM) adhesion. CAMs not only recognize ligands for binding but also initiate the intracellular signaling pathways that are associated with cell proliferation, survival, and drug resistance upon binding to their ligands. Cadherins, selectins, and integrins are well-known cell adhesion molecules that allow binding to neighboring cells, ECM proteins, and soluble factors. The expression of cadherin, selectin, and integrin correlates with the increased drug resistance of leukemia cells. This paper will review the role of cadherins, selectins, and integrins in CAM-DR and the results of clinical trials targeting these molecules.

Participation of adhesion molecules in changing cell interactions during metastasis development

The review summarizes current information about the role and significance of adhesion molecules in the process of tumor metastasis. For example, different tumors is shown that changes in the expression of adhesion molecules leads to disruption of the regulatory interactions in the processes of proliferation, cytodifferentiation and migration of tumor cells, enables the cascade of pathological processes leading to the colonization of other tumor cells remote from the tumor bodies. The phenomenology of tumor development and metastasis is shown. The participation of the main groups of adhesion molecules (cadherins, integrins, selectins, immunoglobulins and white blood cell homing receptors) at the stages of metastasis is considered. A violation of cadherin expression is accompanied by a decrease in the expression of integrins involved in the formation of hemidesmosomes, while a cascade of reactions develops that contributes to the development of an aggressive malignant phenotype, cells acquire an increased ability to migrate and invade, resulting in the appearance of a tumor in secondary foci. The role of immunoglobulins in the development of metastatic process has been studied in less detail than in cadherins and integrins. It was found that immunoglobulins are involved in the formation of resistance of tumor cells to proapoptotic signals. Immunoglobulins increase the expression of metalloproteinase genes involved in the degradation of the extracellular matrix, which is a factor in the initiation of metastasis. A correlation between increased selectin expression and increased metastasis is shown. An increase in the expression level of white blood cell homing receptors leads to an increase in the invasive potential of tumors. The results of the study of cell adhesion proteins serve as a fundamental basis for the development of methods of antitumor therapy. Over the past decades, a number of immunohistochemical protein detection reactions have become one of the diagnostic methods of cancer clinics. However, the role of cell adhesion molecules in ensuring the metastasis process, as well as their significance in the prognosis of the development of the tumor process and antitumor therapy, needs to be further studied.

Exosome EpCAM promotes the metastasis of glioma by targeting the CD44 signaling molecule on the surface of glioma cells.

Glioma, the most common primary tumor in the central nervous system, originates from glial cells and has a poor prognosis. This experimental laboratory study was designed to explore the role of epithelial cell adhesion molecule (EpCAM) in the metastasis of glioma. Serum samples were collected from patients with non-metastatic or metastatic glioma (n = 20 per group), and healthy volunteers (n = 8). Exosomes were isolated from the serum and the morphological characteristics were observed under a scanning electron microscope (SEM). The expression of CD81 and CD63 was measured to identify exosomes. Glioma tissue and the adjacent normal tissue samples were obtained from patients with non-metastatic or metastatic glioma (n = 12 per group). Meanwhile, 4 normal brain tissue samples were collected. The expression of CD44, hyaluronan-mediated motility receptor (HMMR), and matrix metalloproteinase-9 (MMP-9) was determined in each group using immunohistochemistry. The protein expression of CD44, HMMR, matrix metalloproteinase-2 (MMP-2), MMP-9, and selectin E (SELE) was measured with western blotting. Exosomes were present in the serum, and the proteins CD81 and CD63 were expressed in all 3 groups. CD44 was highly expressed in the non-metastasis and metastasis groups. The expression of HMMR and MMP-9 in the Adj-metastasis and Adj-non-metastasis groups was high, while in the other groups, the levels were low. The expression of CD44 in the metastasis and non-metastasis groups was significantly higher than that of the negative control (NC) group, and the expression in the metastasis group was higher than that of the non-metastasis group. The MMP-2 and MMP-9 were not found in either the metastasis or non-metastasis group. The protein expression of HMMR and SELE was high in all groups. Exosome EpCAM promoted the metastasis of glioma by targeting CD44.

Tissue expression of endothelial selectin in patients with atopic dermatitis

Objective To investigate the role of endothelial selectin (E-selectin) in atopic dermatitis (AD) through evaluation of the immunohistochemical expressions in lesional skin biopsies. Background AD is a chronic, pruritic inflammatory skin disease that occurs most frequently in children. In AD, the inflammatory cascade is initiated by the release of proinflammatory cytokines from atopic keratinocytes. Patients and methods A total of 50 patients with AD and 10 healthy control participants were included in this case–control study. From all participants, skin biopsies were taken, and immunohistochemical staining by E-selectin monoclonal antibodies was done. Results There were significant elevations in E-selectin cellular localization (P = 0.01) as well as dermal inflammatory infiltrates in AD cases than controls. Conclusion E-selectin participates in AD pathogenesis.

The impact of intramedullary nailing on the characteristics of the pulmonary neutrophil pool in rodents.

Dysregulation of polymorphonuclear neutrophil (PMN) biology is associated with the development of inflammatory complications after trauma, such as acute respiratory distress syndrome (ARDS). It has been demonstrated that intramedullary nailing is both associated with altered pulmonary neutrophil deposition and the occurrence of ARDS. This standardized study aimed to characterize the long-term remote neutrophil response in the lungs in case of a femur fracture and intramedullary nailing. A standardized rat model including intramedullary nailing and a femur fracture was utilized. Groups were terminated after observation times of three, seven and 14 days. Neutrophils were isolated from lung parenchyma and broncho-alveolar lavage fluid (BALF) and analyzed by flow cytometry. Absolute neutrophil numbers as well as membrane expression levels of CD11b, CD62L, and CD11a were compared. Pulmonary neutrophil numbers were increased 3 days after intervention. Membrane expression levels of CD11b (P < 0.01), CD62L (P < 0.01), and CD11a (P = 0.06) on parenchymal PMNs increased as well after 3 days. Thereafter, values restored gradually to physiological levels. Furthermore, neutrophil activation status patterns between parenchymal and BALF neutrophil pools did not correlate. The current study demonstrates that IMN and a femur fracture are associated with transient increased pulmonary PMN deposition, as well as a specific pattern of activation characterized by temporary increased selectin and integrin receptor expression on pulmonary neutrophils. This phenomenon might play an important role in the pathomechanism of ARDS after IMN. Moreover, we found striking differences between parenchymal and BALF-neutrophil populations, demonstrating the limited readout potential of BALF analysis to investigate the entire pulmonary neutrophil pool.

Role of Galectin-9 in Atopic Dermatitis - Is It Mediated Through E Selectin? A Clinical and Immunohistochemical Study.

BackgroundAtopic dermatitis (AD) is a recognized T helper (Th)2, allergic, skin disease. Galectin-9 (gal-9) is a member of galectin family. It alters T-cell balance resulting in Th2 polarization. These Th2 cells yield various cytokines that may influence E selectin expression. Therefore, we hypothesized that gal-9 may have an active role in AD and this role could be mediated through E selectin.ObjectiveTo assess this hypothesis, immunohistochemical expression of gal-9 and E selectin was investigated in skin lesions, from atopic dermatitis patients, and compared.MethodsTwenty-two atopic dermatitis patients and ten controls were included in this case-control study. SCORAD score was used to evaluate atopic dermatitis severity. Biopsies from skin lesions of AD patients and matched sites of controls were taken and stained immunohistochemically by gal-9 and E selectin polyclonal antibodies.ResultsCompared to controls, atopic dermatitis patients exhibited a significant increased gal-9 H score, percent of expression, cellular localization (P˂0.001) and intensity (P=0.04) as well as dermal cellular infiltrate (P˂0.001). Also, there were significant elevations in E selectin H score (P=0.002), percent of expression (P=0.001) and cellular localization (P<0.001) as well as dermal inflammatory infiltrates in AD cases than controls. In AD, 20 cases showed co expression of both gal-9 and E selectin in the epidermis with insignificant correlation between their H scores.Study LimitationsThis study only included a small number of studied subjects.ConclusionGalectin-9 and E selectin participates independently in atopic dermatitis pathogenesis, that may help in development of new therapeutic agents in atopic dermatitis management program.

L-selectin expression is regulated by CXCL8-induced reactive oxygen species produced during human neutrophil rolling.

Neutrophils destroy invading microorganisms by phagocytosis by bringing them into contact with bactericidal substances, among which ROS are the most important. However, ROS also function as important physiological regulators of cellular signaling pathways. Here, we addressed the involvement of oxygen derivatives in the regulation of human neutrophil rolling, an essential component of the inflammatory response. Flow experiments using dihydroethidium-preloaded human neutrophils showed that these cells initiate an early production of intracellular ROS during the rolling phase of the adhesion cascade, a phenomenon that required cell rolling, and the interaction of the chemokine receptor CXCR2 with their ligand CXCL8. Flow cytometry experiments demonstrated that L-selectin shedding in neutrophils is triggered by ROS through an autocrine-paracrine mechanism. Preincubation of neutrophils with the NADPH oxidase complex inhibitor diphenyleniodonium chloride significantly increased the number of rolling neutrophils on endothelial cells. Interestingly, the same effect was observed when CXCL8 signaling was interfered using either a blocking monoclonal antibody or an inhibitor of its receptor. These findings indicate that, in response to CXCL8, neutrophils initiate ROS production during the rolling phase of the inflammatory response. This very early ROS production might participate in the modulation of the inflammatory response by inducing L-selectin shedding in neutrophils.

Mast Cells Contribute to Brain Microvascular Permeability in Sickle Cell Disease

Mast cells are in close proximity to the vasculature and cause endothelial activation, plasma extravasation, and vascular dysfunction (Gupta & Harvima, Immunol Rev 2018). Vascular dysfunction in sickle cell disease (SCD) is accompanied by increased expression of P-selectin. Treatment with Crizanlizumab, an antibody against P-selectin, led to significantly less sickle cell-related pain crises (Ataga et al., NEJM 2017) highlighting the role of endothelial P-selectin in vasoocclusive crises (VOC). Earlier studies demonstrated that mast cell activation with morphine or ischemia/reperfusion stimulates endothelial E- and/or P-selectin expression. However, it is unknown how mast cells stimulate endothelial selectin expression in SCD. Endothelial dysfunction contributes significantly to the pathobiology of SCD including VOC and may play a critical role in increased blood-brain barrier (BBB) permeability, which may contribute to stroke, another major comorbidity of SCD. One of the known triggers of endothelial dysfunction, inflammation and oxidative stress is endoplasmic reticulum (ER) stress. We hypothesize that in a sickle microenvironment, mediators derived from activated mast cells stimulate endothelial P-selectin expression via ER stress leading to increased BBB permeability. We examined the ability of mast cells to stimulate P-selectin expression and BBB permeability via ER stress in a sickle microenvironment.We isolated MCs from HbAA-BERK and HbSS-BERK, control and sickle mice, respectively; incubated them in vitro and collected mast cell conditioned media (MCCM) from HbAA MCs and HbSS MCs. Normal mouse brain microvascular endothelial cells (mBMECs) were treated with unconditioned MCCM, HbAA MCCM, or HbSS MCCM to examine the effect of mast cell activation on endothelium. We observed increased mast cell activity in HbSS mice evinced by significantlyhigher plasma and skin histamine levels, compared to HbAA mice (p< 0.02 for both). Mast cells from HbSS mouse skin showed significantly increased expression of histamine compared to HbAA skin mast cells (p< 0.04). mBMECs incubated with HbAA and HBSS MCCM exhibited about 3- and 6-fold fold increases in P-selectin expression, compared to unconditioned culture medium, respectively (p< 0.0001 for both). Therefore, mast cells in culture release substances that stimulate P-selectin expression which is further increased by mast cells from sickle (HbSS) microenvironment. Preincubation of mBMEC with 5 microM salubrinal, an inhibitor of dephosphorylation of elongation initiation factor-a, which reduces ER stress, significantly inhibited HbSS MCCM-induced P-selectin expression on mBMEC (p< 0.0001) to the level induced by HbAA-MCCM. In contrast, salubrinal did not inhibit HbAA-MCCM-induced P-selectin expression on mBMEC, suggesting that in a sickle microenvironment mast cells contribute to P-selectin expression via ER stress.We next examined mast cell activity on endothelial permeability in vitro on mBMEC monolayers and in vivo in the brain of HbSS mice. mBMECs incubated with HbSS MCCM showed a significant increase in Evans blue leakage compared to unconditioned or HbAA MCCM (p<0.0001 for both), which was inhibited by preincubation of mBMEC with 5 microM salubrinal prior to incubation with HbSS MCCM (p< 0.0001). In vivo female HbSS mice showed a significantly increased leakage in the brain of FITC-dextran injected through tail vein compared to HbAA mice (p< 0.01). HbSS mice treated with 1 mg/kg salubrinal demonstrated inhibition of FITC-dextran leakage in the brain compared to vehicle (p< 0.05). Thus, ER stress contributes to increased BBB permeability in HbSS mice. We observed activated degranulating mast cells in the brain parenchyma of HbSS mice. In HbAA mice, quiescent mast cells were confined to the meninges of the brain but not seen in the parenchyma. Together, these data suggest that mast cell activation contributes to BBB permeability in a sickle microenvironment via ER stress-mediated P-selectin expression. In turn, this mast cell-initiated activity in the brain may underlie the pathobiology of stroke in SCD. Inhibitors of mast cells and P-selectin have been tested clinically leading to reduced VOC in SCD without known adverse events. Therefore, mast cell activation-induced P-selectin via ER stress may serve as a treatable target for reducing the risk of stroke in SCD. DisclosuresGupta:Novartis: Honoraria; Tau tona: Consultancy.

Digoxin and Platelet Activation in Patients With Atrial Fibrillation: In Vivo and In Vitro Study.

BackgroundDigoxin use was shown to be associated with an increased risk of cardiovascular events in atrial fibrillation (AF). We hypothesized that digoxin may affect cardiovascular risk by increasing platelet activation.Methods and ResultsPost hoc analysis of a prospective study of anticoagulated patients with AF. Patients were divided into 2 groups balanced for age, sex, and cardiovascular risk factors: digoxin users (n=132) and nonusers (n=388). Urinary excretion of 11‐dehydro‐thromboxane B2 (TxB2), a marker of platelet activation, and serum digoxin concentration (SDC) were measured. In vitro experiments were performed on platelets from healthy subjects and AF patients, which were incubated with scalar doses of digoxin (0.6–2.4 ng/mL) with or without prestimulation with a sub‐threshold of collagen. Median 11‐dehydro‐TxB2 was 105.0 (interquartile range, 60.0–190.0) ng/mg creatinine, and median SDC was 0.65 (interquartile range, 0.40–1.00) ng/mL. Urinary 11‐dehydro‐TxB2 and SDC were correlated (r s=0.350, P<0.001). Patients in the upper tertile of SDC showed higher 11‐dehydro‐TxB2 compared with non–digoxin users (P=0.019). In vitro study showed an increased basal platelet activation in patients with AF compared with healthy subjects. Digoxin (2.4 ng/mL) induced calcium mobilization, PAC‐1 (procaspase‐activating compound 1) and platelet aggregation in AF patients but not in healthy subjects. After pretreatment with a sub‐threshold of collagen, digoxin dose‐dependent induced calcium mobilization, arachidonic acid release, TxB2 biosynthesis, PAC‐1 and soluble platelet selectin expression, and platelet aggregation, which were inhibited by antibody against digoxin.ConclusionsWe found a significant in vivo correlation between SDC and platelet activation. Supratherapeutic SDC increased in vitro platelet aggregation via calcium‐related phospholipase A2 phosphorylation. Our findings may have clinical implications for AF patients treated with digoxin.

Forensic age determination of human inflicted porcine bruises inflicted within 10 h prior to slaughter by application of gene expression signatures

Prediction-models based on gene expression profiles from experimental bruises are capable of determining the age of bruises with a precision of ±2 h. However, these models have not yet been applied on tissue from pigs in forensic cases requested by the police. We applied two prediction-models, based on mRNA expression of 13 (prediction-model no. 1) and 4 genes (prediction-model no. 2) involved in inflammation, on forensic cases of porcine bruises in order to determine if gene expression profiles can be used for age determination in forensic cases.Subcutaneous fat tissue from bruises notified to the police was sampled: 1) within 6 h after slaughter (group no. I, n = 142), and 2) after freezing the skin for up to 1 year (group no. II, n = 40). qPCR of genes involved in inflammation was performed to predict the bruise age after partial least squares analysis.mRNA expression data were obtained for 52.8% and 7.5% bruises in group nos. I and II, respectively. Prediction-model no. 2, based on the mRNA expression of Selectin E, Selectin P, Interleukin 6 and Nuclear Factor Kappa Beta Subunit1, was most suitable for predicting the age of bruises within 8 h prior to slaughter.In conclusion, mRNA expression profiles can assist in estimating the age of bruises. However, when applying gene expression signatures in forensic cases the age estimate should be interpreted together with histological manifestations. Subcutaneous tissue must be stabilized hours after the bruises are detected in order to obtain mRNA of a sufficient quality.

US Molecular Imaging of Acute Ileitis: Anti-Inflammatory Treatment Response Monitored with Targeted Microbubbles in a Preclinical Model.

Purpose To evaluate whether dual-selectin-targeted US molecular imaging allows longitudinal monitoring of anti-inflammatory treatment effects in an acute terminal ileitis model in swine. Materials and Methods The Institutional Animal Care and Use Committee approved all animal studies. Fourteen swine with chemically induced acute terminal ileitis (day 0) were randomized into the following groups: (a) an anti-inflammatory treatment group (n = 8; meloxicam, 0.25 mg per kilogram of body weight; prednisone, 0.5 mg/kg) and (b) a control group (n = 6; saline). US molecular imaging was performed with a clinical US machine after intravenous injection of clinically translatable dual P- and E-selectin-targeted microbubbles (5 × 108/kg). Three inflamed bowel segments per swine were imaged at baseline, as well as on days 1, 3, and 6 after treatment initiation. At day 6, bowel segments were analyzed ex vivo for selectin expression levels by using quantitative immunofluorescence. Results After induction of inflammation, US molecular imaging signal increased at day 1 in both animal groups (P < .001). At day 3, signal in the treatment group decreased (P < .001 vs day 1), while signal in control animals did not significantly change (P = .18 vs day 1) and was higher (P = .001) compared with that in the treatment group. At day 6, signal in the treatment group further decreased and remained lower (P = .02) compared with that in the control group. Immunofluorescence confirmed significant (P ≤ .04) downregulation of both P- and E-selectin expression levels in treated versus control bowel segments. Conclusion Dual-selectin-targeted US molecular imaging allows longitudinal monitoring of anti-inflammatory treatment effects in a large-animal model of acute ileitis. This supports further clinical development of this quantitative and radiation-free technique for monitoring inflammatory bowel disease. © RSNA, 2018 Online supplemental material is available for this article.

Intravascular hemolysis activates complement via cell-free heme and heme-loaded microvesicles.

In hemolytic diseases, such as sickle cell disease (SCD), intravascular hemolysis results in the release of hemoglobin, heme, and heme-loaded membrane microvesicles in the bloodstream. Intravascular hemolysis is thus associated with inflammation and organ injury. Complement system can be activated by heme in vitro. We investigated the mechanisms by which hemolysis and red blood cell (RBC) degradation products trigger complement activation in vivo. In kidney biopsies of SCD nephropathy patients and a mouse model with SCD, we detected tissue deposits of complement C3 and C5b-9. Moreover, drug-induced intravascular hemolysis or injection of heme or hemoglobin in mice triggered C3 deposition, primarily in kidneys. Renal injury markers (Kim-1, NGAL) were attenuated in C3-/- hemolytic mice. RBC degradation products, such as heme-loaded microvesicles and heme, induced alternative and terminal complement pathway activation in sera and on endothelial surfaces, in contrast to hemoglobin. Heme triggered rapid P selectin, C3aR, and C5aR expression and downregulated CD46 on endothelial cells. Importantly, complement deposition was attenuated in vivo and in vitro by heme scavenger hemopexin. In conclusion, we demonstrate that intravascular hemolysis triggers complement activation in vivo, encouraging further studies on its role in SCD nephropathy. Conversely, heme inhibition using hemopexin may provide a novel therapeutic opportunity to limit complement activation in hemolytic diseases.

Thrombotic Regulation From the Endothelial Cell Perspectives.

Thrombosis, the localized clotting of blood that affects arterial or venous circulation, is one of the leading causes of death worldwide.1 Currently used antithrombotic drugs have increased bleeding risk.2,3 Exploring new antithrombotic strategies that preserve physiological hemostasis is under intensive investigation. Classical triad of causative factors leading to thrombosis includes alterations in blood content, alterations in blood flow, and alterations in the vessel wall. Endothelium, the inner most single layer of cells lining the blood vessels, provides a surface for thrombosis formation and critically regulates blood fluidity and homeostasis. As barrier, endothelium separates blood clotting factors from exposure to subendothelial prothrombotic extracellular matrix components. Endothelium also secretes or expresses vasoactive factors that modulate platelet reactivity, coagulation, fibrinolysis, and vascular contractility, all of which contribute to thrombotic formation. Such factors include nitric oxide, prostacyclin, Von Willebrand factor (VWF), thrombomodulin, endothelin, etc. Accumulating evidences show that endothelial cells (ECs) play a pivotal role in modulating thrombosis, highlighting ECs as a potential target for thrombosis control. In this Recent Highlights, we reviewed recent publications in Arteriosclerosis, Thrombosis, and Vascular Biology and other leading journals that are focused on the mechanisms of endothelial regulation of thrombosis and discussed their merits in therapeutic discovery. Arterial thrombosis is commonly initiated by vascular endothelial erosions and ruptures of an atherosclerotic plaque3,4 while venous thrombosis mainly stems from blood stasis.5 Despite these differences, platelet adhesion/activation, and fibrin deposition as the result of coagulation constitute the fundamental processes of thrombus formation.6,7 Platelet activation occurs when they interact with activated ECs (with increased VWF release and selectin expression) via glycoprotein Ib-V-IX complex binding to VWF, or when they expose to subendothelial extracellular matrix components, such as collagen (via glycoprotein VI receptor), in the cases of endothelial injury or plaques …