scholarly journals Sodium phenyl butyrate downregulates endothelin-1 expression in cultured human endothelial cells: Relevance to sickle-cell disease

2007 ◽  
Vol 82 (5) ◽  
pp. 357-362 ◽  
Author(s):  
Marie-Hélène Odièvre ◽  
Manuel Brun ◽  
Rajagopal Krishnamoorthy ◽  
Claudine Lapouméroulie ◽  
Jacques Elion
Keyword(s):  
Endothelial Cells ◽  
Sickle Cell Disease ◽  
Sickle Cell ◽  
Cell Disease ◽  
Human Endothelial Cells ◽  
Endothelin 1

Abstract 347: Endothelin-1 Regulates Molecules of the Major Histocompatibility Complex: Role in Sickle Cell Disease

2016 ◽  
Vol 36 (suppl_1) ◽  
Author(s):  
Isamar Alicea ◽  
Daniel Gil de Lamadrid ◽  
Analaura Santiago ◽  
Joshua Cazares ◽  
Shirley Valentin ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Endothelial Cells ◽  
Major Histocompatibility Complex ◽  
Endothelial Cell ◽  
Sickle Cell Disease ◽  
Sickle Cell ◽  
Major Histocompatibility ◽  
Cell Disease ◽  
Endothelin 1 ◽  
Histocompatibility Complex

Sickle Cell Disease (SCD) is characterized, in part, by vascular endothelial cell activation, increased oxidative stress and sickle cell adhesion. Excess levels of the vasoconstrictor, endothelin-1 (ET-1), activate endothelial cells, induce oxidative stress and inflammation in the vascular wall and regulate erythrocyte homeostasis, in part, via activation of protein disulfide isomerase (PDI). We measured circulating ET-1, TGFβ1 and PDI activity in two sickle transgenic knockout mouse models expressing human sickle hemoglobin (Hb), BERK and βS Antilles . We observed significant increases in all three markers (n=6; p<0.01) when compared to either C57BL/J6 mice or knockout mice expressing normal human Hb A. We then intraperitoneally injected BERK mice for 14 days with ET-1 receptor antagonists and observed significant reductions in these circulating markers (n=6; p<0.05). These studies are important as PDI is proposed to control hemostasis, thrombosis and is reported to regulate the major histocompatibility complex (MHC). In addition, activated endothelial cells express PDI and MHC. However, the relationship between ET-1 and MHC in SCD remains unclear. We characterized the role of ET-1 on MHC expression in the endothelial cell line, EA.hy926. We observed dose-dependent increases in the expression of MHC class I (HLA-A2 4.8 ± 2.1 folds p<0.01 n=4), MHC class II (HLA-DR 4.4 ± 1.7 folds p<0.01 n=4) and MHC transcription factor (CIITA 3.5 ± 1.8 folds p<0.05 n=4) in EA.hy926 cells. Increased expression of MHC was significantly blocked by co-incubation of cells with 10μM BQ788, a selective blocker of ET-1 type B receptors. Chromatin immunoprecipitation studies showed that ET-1 incubation produced an increase in Histone H3 acetylation of the promoter in MHC molecules (HLA-A2 4.6 ± 1.4 folds, HLA-DRB 6.0 ± 0.7 folds, p<0.01, n=3) in these cells; an event that was likewise blocked by BQ788. In addition, ET-1 stimulated recruitment of CIITA to the promoter of HLA-A2 (1.9 ± 0.7 folds, p<0.05, n=3) and HLA-DRB (3.0 ± 0.6 folds, p<0.01, n=3). These results implicate ET-1 as a novel regulator of MHC promoter activity and suggest that ET-1 receptor blockade represents an important therapeutic approach to improve both the inflammatory and vascular complications of SCD.

Regulation of Endothelin-1-Mediated Reactive Oxygen Species Production by Protein Disulfide Isomerase in Endothelial Cells: Role in Sickle Cell Disease

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 1073-1073
Author(s):  
Patricia Neuman ◽  
Gregory N. Prado ◽  
Philip Asamoah ◽  
Daphne Diaz ◽  
Jose R Romero ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Sickle Cell Disease ◽  
Sickle Cell ◽  
Ros Production ◽  
Cell Disease ◽  
Rt Pcr ◽  
Endothelin 1 ◽  
Endogenous Controls ◽  
Protein Disulfide

Abstract Abstract 1073 Endothelial cell activation and increased levels of endothelin-1 (ET-1) have been proposed as important contributors to vaso-occlusion in the pathophysiology of Sickle Cell Disease (SCD). We have reported a role for ET-1 receptor antagonists in improving hematological complications of two transgenic mouse models of SCD in vivo (Rivera A., 2008, Amer J Physiol). Activation of endothelial cells leads to, among other factors, increased levels of reactive oxygen species (ROS) and protein disulfide isomerase (PDI). PDI mediates redox modifications, catalyzes disulfide interchange reactions in the plasma membrane and is up-regulated under hypoxic conditions as commonly observed in SCD. We tested the hypothesis that ET-1 would regulate PDI activity in endothelial cells. We now report on the detection of PDI and ET-1 receptor B by western blot analyses in membranes from the human endothelial cell line, EA.hy926 (EA). We studied the effects of ET-1 on cellular ROS production by fluorimetry of DCFDA-AM loaded EA cells and observed that 10−7 M ET-1 for 60 mins led to significant increases in cellular ROS production (1720±78 vs 1447±86 Relative Fluorescent Units (RFU), P<0.001, n=6) that were significantly blocked by ET-1 receptor B antagonist, 10−6 M BQ-788 (1720±78 vs 1426±55 RFU, P<0.0007, n=3). We also tested the effects of the specific ET-1 receptor B agonist, 10−7 M BQ-3020 on cellular ROS production and observed similar results (1748±194 vs 1447±86 RFU, P<0.02, n=3). We then studied the effect of a well-described PDI inhibitor, monoclonal antibodies to PDI (RL90), on ET-1 stimulated ROS production and showed that RL90 likewise blocked ET-1 stimulated ROS production in EA cells (P<0.001, n=3), thus suggesting a role for PDI in ET-1-stimulated ROS production. To further explore the role of PDI on ROS production, we employed a molecular approach and show that siRNA against PDI likewise led to reduced ET-1-stimulated ROS production (P<0.0001, n=2) that was associated with significantly reduced PDI mRNA levels in siRNA transfected cells, but not when cells were transfected with scrambled siRNA as determined by quantitative RT-PCR with ABI TaqMan detection probes and GAPDH and β2 microglobulin as endogenous controls. We then optimized conditions to measure PDI activity using fluorescently labeled GSSG conversion to GSH in supernatants of ET-1-stimulated cells. We observed that incubation of EA cells for 60 mins with 10−7 M ET-1 was associated with increased extracellular PDI activity (232±183 to 664±94 (RFU), n=3, P<0.02) that was blunted by PDI siRNA knockdown (1731±147 to 757±141 RFU, n=2) when compared to scrambled siRNA transfected cells. Consistent with these data we observed increases in PDI protein levels in supernatants of EA cells stimulated with ET-1 by ELISA. Moreover, incubation of EA cells for 12 hr with 10−8 M ET-1 led to increases in cell-associated PDI levels by western blot analyses. We then tested the in vivo effects of ET receptor antagonist on plasma PDI activity in the BERK mouse model of SCD. We blocked ET-1 receptors in vivo by treatment with BQ-788 and BQ-123 (360mg/Kg/Day) for 14 days and observed a reduction of plasma PDI activity when compared to vehicle treatment (67.7±3 to 34.3±6, RFU, P<0.03, n=3). We also characterized the effects of ET-1 on PDI expression in EA cells, using quantitative RT-PCR with ABI TaqMan probes and GAPDH and β-actin as endogenous controls and observed that stimulation of EA cells with 10−8 M ET-1 for 4 hr was associated with increased PDI mRNA expression levels that were 1.89 fold greater than vehicle treated cells (n=6, P<0.04). Thus, our results provide evidence to suggest that endothelin-1 receptor blockade leads to improved measures of ROS via regulation of PDI and thus implicate PDI as a novel regulator of Sickle Cell Disease pathophysiology. Disclosures: No relevant conflicts of interest to declare.

Abstract 345: Syzygium Jambos: A Potential Therapeutic Approach to Treat the Vascular and Hematological Complications of Sickle Cell Disease

2016 ◽  
Vol 36 (suppl_1) ◽  
Author(s):  
Analaura Santiago-Perez ◽  
Yaritza Inostroza-Nieves ◽  
Daniel Gil de la Madrid ◽  
Isamar Alicea ◽  
Christopher Vega ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Sickle Cell Disease ◽  
Sickle Cell ◽  
Therapeutic Approach ◽  
Thrombus Formation ◽  
Critical Role ◽  
Antioxidant Properties ◽  
Cell Disease ◽  
Syzygium Jambos ◽  
Promising Therapeutic Approach

Protein disulfide isomerase (PDI) is an oxidoreductase that mediates thiol/disulfide interchange reactions and has been reported to play a critical role in thrombus formation following vascular injury. PDI has also been shown to regulate leukocyte adherence to the endothelium and nitric oxide delivery. We recently reported that PDI is present at high levels and regulates erythrocyte homeostasis and Gardos Channel activity in humans with Sickle Cell Disease (SCD). Thus, PDI inhibition has been proposed as a promising therapeutic approach to ameliorate both the vascular and hematological complications of SCD. Syzygium jambos (S. jambos) is purported to have anti-inflammatory and antioxidant properties. However, the regulation of PDI activity by S. jambos has not been studied. We studied in vitro PDI activity in the presence of the S. jambos aqueous leaf extract using a PDI insulin turbidity assay. We observed significant reductions in PDI activity at 25 μg/mL (66.0 ± 9.7%, p<0.01, n=3), 50 μg/mL (83.3 ± 6.0%, p<0.01, n=3), and 100 μg/mL (91.6 ± 11.5%, p<0.01, n=3). S. jambos extract showed a dose-dependent anti-PDI activity with an IC50 of 14.40 μg/mL. We then tested the effects of S. jambos on endothelin-1 (ET-1)-stimulated PDI activity in human endothelial cells. Using a fluorescence based PDI activity assay, we observed that ET-1 increased PDI activity (1.7 ± 0.7 folds, n=3) that was dose-dependently blocked by S. jambos extract. In addition, we observed that ET-1 stimulated ex vivo human polymorphic nucleated (PMN) leukocyte migration toward the endothelial cells that was likewise dose-dependently blocked by S. jambos extract. (p<0.01, n=3). We also quantified the levels of reactive oxygen species (ROS) production in ET-1 treated endothelial cells. ET-1 stimulation significantly increased ROS levels [3 fold] when compared to vehicle treatment (p<0.05, n=3). S. jambos extract reduced ET-1 stimulated ROS to baseline levels (p<0.05, n=3). Our results suggest that S. Jambos may represent a novel pharmacological approach to treat complications of SCD.

Effect of Hydroxyurea Treatment on In Vitro Adhesion of Sickle Erythrocytes to Sickle Leukocyte Subpopulations.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 362-362
Author(s):  
Eileen M. Finnegan ◽  
Aslihan Turhan ◽  
Jennifer Gaines ◽  
David E. Golan ◽  
Gilda Barabino
Keyword(s):  
T Cells ◽  
Endothelial Cells ◽  
T Cell ◽  
Sickle Cell Disease ◽  
Adhesion Molecules ◽  
Sickle Cell ◽  
Cell Disease ◽  
Tnf Α ◽  
Adhesive Interactions ◽  
Cell Cell

Abstract Microvascular vaso-occlusion in sickle cell disease is thought to involve adhesive interactions among erythrocytes (RBCs), leukocytes and vascular endothelial cells. Recent studies have demonstrated the presence of a significant inflammatory response in sickle cell disease, including changes in the cell surface adhesion molecules that mediate cell-cell interactions in the microvasculature. In this study, we used a parallel-plate flow chamber assay to determine the subpopulations of leukocytes that are involved in sickle leukocyte-RBC interactions. We also studied the effect of treatment with hydroxyurea (HU) on these adhesive interactions. Populations of monocytes, neutrophils (PMNs) and T cells were isolated by negative selection from the peripheral blood of untreated patients with sickle cell disease (SS), sickle patients receiving HU (SS-HU), and healthy control subjects (AA). Adhesive interactions involving these leukocyte subpopulations, human umbilical vein endothelial cells (HUVECs) pretreated with tumor necrosis factor-α (TNF-α ), and autologous RBCs were measured under a shear stress of 1 dyne/cm2. Compared to the corresponding cell populations from AA individuals, PMNs, monocytes, and T cells from SS individuals were significantly more adherent to TNF-α-treated HUVECs (774±59 vs. 502±27 cells/mm2, p=0.001; 533±66 vs. 348±36 cells/mm2, p=0.024; and 470±75 vs. 227±26 cells/mm2, p=0.009, respectively). HU therapy significantly decreased the adhesion of SS PMNs to HUVECs (774±59 cells/mm2 for SS vs. 604±36 for SS-HU, p=0.025). Compared to adherent AA leukocytes, adherent SS leukocytes exhibited greater participation in adhesive interactions with autologous RBCs (41±3% for SS vs. 27±3% for AA, p=0.002), and HU treatment decreased the fraction of leukocytes that captured autologous RBCs to the control level (29±3% for SS-HU, p=0.006 vs. SS). Compared to adherent PMNs from SS individuals, adherent PMNs from SS-HU individuals showed significantly reduced participation in the capture of RBCs (53±6% for SS vs. 35±5% for SS-HU, p=0.021). Although adherent T cells from SS individuals participated significantly more in RBC capture than adherent T cells from AA individuals (28±5% for SS vs. 10±2% for AA, p=0.007), HU therapy did not have a significant effect on this parameter (21±5% for SS-HU, p=0.373). Compared to AA leukocytes, SS leukocytes captured more RBCs per participating adherent leukocyte (2.8±0.2 vs. 1.9±0.1 RBCs/cell, p=0.001). HU therapy reduced the number of RBCs captured per PMN but not the number captured per T cell. Compared to AA T cells, SS T cells captured adherent RBCs for a significantly longer period of time (51±9 vs. 26±6 seconds, p=0.035). Our data suggest that sickle neutrophils, monocytes and T cells may all be involved in adhesive interactions with sickle RBCs. PMN-RBC and monocyte-RBC interactions appear to be more numerous than T cell-RBC interactions, although T cell-RBC interactions may be stronger. HU therapy appears to target PMN-RBC and monocyte-RBC interactions preferentially. Future studies will focus on the role of particular adhesion molecules in mediating these interactions and on the potential for therapeutic interventions targeting cell-cell adhesion.

Heme Induces Endothelial Tissue Factor Expression: Potential Role in Hemostatic Activation in Patients with Intravascular Hemolysis Including Sickle Cell Disease.

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 1902-1902
Author(s):  
Yamaja Setty ◽  
Suhita Gayen Betal ◽  
Jie Zhang ◽  
Nigel S Key ◽  
Marie Stuart
Keyword(s):  
Flow Cytometry ◽  
Endothelial Cells ◽  
Sickle Cell Disease ◽  
Sickle Cell ◽  
Whole Blood ◽  
Dependent Manner ◽  
Cell Disease ◽  
Intravascular Hemolysis ◽  
Tnf Α ◽  
Endothelial Tissue

Abstract Plasma levels of heme in the 20 to 600 μM range are found in clinical conditions associated with intravascular hemolysis including paroxysmal nocturnal hemoglobinuria and sickle cell disease, conditions also associated with a thrombotic tendency. Objectives: To investigate whether heme, an inflammatory mediator and a product of intravascular hemolysis in patients with hemolytic anemia including sickle cell disease (SCD), could modulate hemostasis by an effect on endothelial tissue factor (TF) expression. Additionally, in SCD patient-related studies, we assessed whether any association existed between whole blood TF activity (WBTF) and levels of surrogate markers of intra-vascular hemolysis including lactate dehydrogenase (LDH) and reticulocyte counts. Methods: Following incubation of human endothelial cells (from umbilical vein and/or lung microvasculature) with heme (1 to 100 μM) for various times (30 minutes to 8 hours), levels of TF protein were assessed using ELISA, flow cytometry and/or Western blotting; and TF mRNA by a semi-quantitative RT-PCR. An assay for TF functional activity was performed using a chromogenic tenase activity kit where specificity of TF activity was tested in antibody-blocking experiments. Three TF-specific antibodies including a rabbit polyclonal and two mouse monoclonal (clones hTF-1 and TF9-10H10) antibodies were used in assays involving TF protein analysis. All experiments were performed in media containing polymyxin B to neutralize any potential endotoxin contamination. In patient-related studies, 81 subjects with SCD (1 to 21 years) were evaluated for levels of WBTF, LDH, and reticulocyte counts and data analyzed for potential relationships. Results: Heme induced TF protein expression on the surface of both macro- and micro-vascular endothelial cells in a concentration-dependent manner with 12- to 50-fold induction noted (ELISA assays) between 1 and 100 μM heme (P&lt;0.05, n=3 to 6). Complementary flow cytometry studies showed that the heme-mediated endothelial TF expression was quantitatively similar to that induced by the cytokine TNF-α. Heme also up-regulated endothelial expression of TF mRNA (8- to 26-fold, peak expression at 2 hours postagonist treatment), protein (20- to 39-fold, peak expression at 4 hours) and procoagulant activity (5- to 13-fold, peak activity at 4 hours post-agonist treatment) in a time-dependent manner. Time-course of heme-mediated TF antigen expression paralleled induction of procoagulant activity with antibody blocking studies demonstrating specificity for TF protein. Potential involvement of endogenously released cytokines including IL-1α and TNF-α in mediating the heme effect was next explored. We found that the latter cytokines are not involved, since antibodies against IL-1α and TNF-α, and an IL-1- receptor antagonist failed to block heme-induced endothelial TF expression. Inhibition of heme-induced TF mRNA expression by sulfasalazine and curcumin suggested that the transcription factor NFκB was involved in mediating heme-induced effect. In patient-related studies, whole blood TF levels in SCD correlated positively with both LDH (r=0.72, p&lt;0.000001), and reticulocyte count (r=0.60, p&lt;0.000001). Conclusions: Our findings demonstrate that heme induces TF expression in endothelial cells, and that the observed effects occurred at patho-physiologically relevant heme concentrations. Our results suggest that heme-induced endothelial TF expression may provide a pathophysiologic link between the intravascular hemolytic milieu and the hemostatic perturbations previously noted in patients with hemolytic anemia including sickle cell disease.

Hydroxyurea Therapy Increases Expression of BCAM/LU and Adhesion to Laminin in Children with Sickle Cell Disease

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 4806-4806
Author(s):  
Clarissa E Johnson ◽  
Marilyn J. Telen
Keyword(s):  
Extracellular Matrix ◽  
Endothelial Cells ◽  
Sickle Cell Disease ◽  
Adhesion Molecules ◽  
Sickle Cell ◽  
Adhesion Molecule ◽  
Extracellular Matrix Protein ◽  
Published Data ◽  
Adhesion Molecule Expression ◽  
Cell Disease

Abstract Vaso-occlusion is the major cause of morbidity and mortality in sickle cell disease. The tendency of red blood cells (RBCs) to adhere to extracellular matrix molecules and the vascular endothelium is believed to be a significant contributor to the vaso-occlusive process. Some published studies have shown that hydroxyurea decreases sickle (SS) RBC adhesion to some ligands, although the mechanism by which this occurs is not completely understood. SS RBCs demonstrate increased expression of several adhesion molecules, especially BCAM/LU, and also conserve functional signaling pathways that are associated with upregulation of adhesion. BCAM/LU mediates adhesion to the extracellular matrix protein laminin. We hypothesized that patients responsive to hydroxyurea (HU) therapy would exhibit reduced adhesion to laminin as well as a decrease in adhesion molecule expression. Our subjects included patients with Hb SS between the ages of 5 to 18. They were divided into three groups: children not receiving HU therapy (n = 3); children receiving HU therapy for over 6 months (n = 5), and children initially not receiving HU but who were initiating therapy at the time of study enrollment (n = 5). Adhesion to laminin was examined using a graduated height flow chamber to quantitate the adhesion of SS RBCs. Expression of adhesion molecules was analyzed by western blot and densitometry, using monoclonal antibody to BCAM/LU. We found that HU therapy was associated with significantly increased expression of BCAM/LU (HU: 145.8 ± 14.0 SEM; no HU: 60.8 ± 11.0 SEM densitometry units, p = .0014). This somewhat unexpected finding confirms results published earlier this year by Odievre et al. (2008). Adhesion to laminin was also increased for patients on HU (HU: 9.3 ± 5.9; no HU: .3 ± .3, p=.2), although this increase was not significant, given the variability in adhesion seen among patients and the small number of subjects. Nevertheless, the increase in adhesion corresponded to the increase in BCAM/LU expression. In contrast, adhesion to endothelial cells was decreased, although not significantly, in patients on HU (HU: 38.1 ± 38; no HU: 127.2 ± 122.5, p=.6). Our findings thus confirm earlier published data showing that HU increases the expression of BCAM/LU measured by flow cytometry and further shows that this increased expression is associated with increased adhesion to laminin but not to endothelial cells. Potential mechanisms by which HU affects adhesion molecule expression and activity merit further investigation, as does the physiologic role of these alterations. Comparison of results from patient-matched pre-treatment and post-treatment samples should also help define the effects of HU. Figure Figure

Platelets From Sickle Cell Disease Individuals Induce Endothelial Activation, Demonstrating ICAM-1 and E-Selectin Adhesion Molecule Expression, Inflammatory Cytokine Production and Activation of NFκB Transcription Factor Gene Expression.

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 2114-2114
Author(s):  
Renata Proença-Ferreira ◽  
Ana Flavia Brugnerotto ◽  
Vanessa Tonin Garrido ◽  
Marilene de Fatima Reis Ribeiro ◽  
Fabiola Traina ◽  
...  
Keyword(s):  
Gene Expression ◽  
Endothelial Cells ◽  
Transcription Factor ◽  
Sickle Cell Disease ◽  
Sickle Cell ◽  
Adhesion Molecule ◽  
Direct Contact ◽  
Endothelial Activation ◽  
Cell Disease ◽  
Gene Encoding

Abstract Abstract 2114 Introduction: Sickle cell disease (SCD) pathophysiology is associated with a hypercoagulable state that may contribute to the initiation and propagation of vaso-occlusion. Increased platelet activation has been described in SCD and SCD platelets may present augmented adhesion to the vascular endothelium, potentially contributing to vaso-occlusion. Aim: This study investigated whether platelets (PLTs) from SCD individuals are able to activate endothelial cells per se. Methods: Human umbilical vein endothelial cells (HUVEC) were cultured (1×106cells/well) on 6-well plates (37°C, 5% CO2). Subsequently, HUVEC were co-cultured in direct contact, or not, with washed PLTs (1×108PLTs/well) from healthy control individuals (CON, n=23) or steady-state SCD patients (SCD, n=47; 26 of which were on hydroxyurea therapy; 20mg/Kg/day) for 4h, 37°C, 5%CO2. After incubation, PLTs were removed; supernatants were reserved for cytokine quantification by ELISA, and HUVEC were analyzed by flow cytometry for CD62E (E-Selectin) and CD54 (ICAM-1) surface expression; gene expressions of ICAM1 and NFKBIA were analyzed by qPCR. Results: Basal ICAM-1 expression on the surface of HUVEC (39.6±3.2%, n=15) was significantly increased following their incubation in direct contact with SCD PLTs (46.1±3.1%, n=26, p<0.05, Wilcoxon test), but not CON PLTs (41.3±4.7%, n=12). E-selectin expression was also low level on the surface of HUVEC (0.9±0.2%, n=17), and was slightly but significantly increased following incubation of cells with SCD PLTs (6.1±2.2%, n=26, p<0.001), but not CON PLTs (3.6±1.5%, n=12, p>0.05). Repetition of these assays, but with the placement of transwell inserts in culture plates to separate the PLTs from HUVEC resulted in a 45% decrease in ICAM-1 expression (p<0.05), and 85% decrease in E-selectin (p<0.05) expression on the surface of HUVEC, following their incubation with SCD PLTs. HUVEC produce and release interleukin-8 (IL-8); basal IL-8 production by HUVEC (1×106cells/well) was 1.160±0.187ng/mL (n=21); this production was augmented in the presence of SCD PLTs (1.280±0.149ng/mL, n=42, p<0.01), but not by CON PLTs (1.127±0.157ng/mL, n=23). The influence of PLT IL-8 production on these values was negligible, as shown by data (not shown) demonstrating that PLT IL-8 production is low level and does not differ between CON and SCD PLTs. IL-1β is produced and released by PLTs (CON, 3.4±1.4ρg/mL, n=12; SCD, 6.5±1.5 ρg/mL, n=25, p>0.05), but this production was further increased when PLTs were co-cultured with HUVEC: SCD PLTs (10.3±4.2ρg/mL, n=47; p<0.01) and CON PLTs (5.8±2.4ρg/mL, n=25; p>0.05), compared to HUVEC alone (1.27±0.4ρg/mL, n=24). Gene expression of ICAM1 by HUVEC increased 6.3-fold in the presence of SCD PLTs (n=25, p<0.01), compared to basal expression (n=11), but was not altered in the presence of CON PLTs (n=11, p>0.05). The expression of the gene encoding the NFkB transcription factor, NKBIA, increased 3.4-fold in HUVEC following incubation with SCD PLTs (n=25, p<0.05), compared to basal NFKBIA expression (n=12); however NFKBIA expression in HUVEC was not significantly altered by CON PLTs (n=10, p>0.05). Conclusions: Results indicate that the contact of platelets, or products released from platelets, from patients with SCD may activate endothelial cells, in vitro, increasing adhesion molecule and IL-8 production, associated with an augmented expression of the gene encoding NFkB. Platelets produce IL-1β in greater quantities in the presence of endothelial cells, possibly contributing to endothelial cell activation; however the fact that transwell inserts significantly reduced SCD PLT-mediated endothelial activation indicates that the direct contact of PLTs (possibly via adhesion) is required for this activation. Data indicate that platelets adhered to vessel walls may play an important role in endothelial activation and, therefore, vaso-occlusive mechanisms in SCD. Disclosures: No relevant conflicts of interest to declare.

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