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

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.

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.

Increased Erythrocyte Rigidity Is Sufficient to Cause Endothelial Dysfunction in Sickle Cell Disease

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 818-818 ◽  
Author(s):  
Robert Mannino ◽  
David R Myers ◽  
Yumiko Sakurai ◽  
Russell E. Ware ◽  
Gilda Barabino ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Endothelial Cell ◽  
Endothelial Dysfunction ◽  
Sickle Cell Disease ◽  
Sickle Cell ◽  
Adhesion Molecule ◽  
Endothelial Cell Dysfunction ◽  
Cell Disease ◽  
Cell Dysfunction ◽  
Sickle Erythrocytes

Abstract Abstract 818 Endothelial dysfunction is a major component of sickle cell disease (SCD) pathophysiology. Interestingly, previous cardiovascular research has definitively shown that endothelial cells biologically respond to mechanical forces and aberrations in these forces cause endothelial dysfunction via pro-inflammatory pathways that are also involved in SCD. While endothelial dysfunction in SCD has been well characterized biologically, little research has focused on the direct biophysical effects of SCD blood on endothelium. As endothelial cells are in constant contact with flowing “stiffened” sickle erythrocytes, we propose that the direct mechanical interactions between the physically altered sickle erythrocytes and endothelial cells are an additional cause of endothelial dysfunction in SCD (Figure 1A). Endothelial dysfunction in SCD is thought to be caused by the downstream effects of vaso-occlusion and/or hemolysis. Our laboratory has recently developed and published a description of an in vitro microvasculature model comprised of endothelial cells that are cultured throughout the entire 3D inner surface of a microfluidic system designed for investigating cellular interactions in hematologic diseases (Tsai, et al, JCI, 2012), (Figure 1B-D). This microvasculature-on-a-chip recapitulates an ensemble of physiological processes and biophysical properties, including adhesion molecule expression, blood cell-endothelial cell interactions, cell deformability, cell size/shape, microvascular geometry, hemodynamics, and oxygen levels (Myers et al. JoVE, 2012), all of which may contribute to endothelial dysfunction in SCD. We hypothesize that the mechanical interactions between sickle erythrocytes and endothelial cells alone are sufficientto cause endothelial dysfunction in our microvasculature-on-a-chip. To test our hypothesis, we flowed different suspensions of healthy red blood cells (RBCs), and stiffened RBCs, through our microvasculature on a chip cultured with HUVECs. We suspended fresh human RBCs in media at a low hematocrit recapitulating the anemic conditions typically seen in SCD patients as a control. The experimental conditions used the same solution as the control, but also contained glutaraldehyde-stiffened RBCs, which are of the same stiffness as irreversibly sickled cells (ISCs), at approximately the same concentrations as ISCs in SCD patients. The stiffened RBC suspension was washed multiple times to eliminate all traces of glutaraldehyde and to ensure that any endothelial cell dysfunction in our system was due to mechanical effects between the endothelium and RBCs. After 4 hours of perfusion, the number of occlusions in our microsystem was counted and the cells were fixed and stained for Vascular Cell Adhesion Molecule 1 (VCAM-1). VCAM-1 been shown to be a marker of endothelial cell dysfunction and is a biomarker for severe vasculopathy in SCD (Dworkis, Am J Hematol, 2011). Immunofluorescence staining in our microsystem confirmed that VCAM1 is upregulated (Figure 2) in HUVECs when exposed to flowing stiffened RBCs compared to control RBCs. VCAM-1 upregulation appears to be diffuse throughout the length of the device. After experimentation, endothelial cells in our system can be isolated for further RT-PCR or microarray analysis. As such, ongoing work involves investigating and quantifying the expression of other pro-inflammatory molecules to elucidate the underlying mechanisms of this biomechanical process involving RBCs and endothelial cells. Additional experiments complementary experiments using endothelial cells from other anatomic areas, SCD patient samples, and murine SCD models are also underway. Our data indicates that purely physical interactions between endothelial cells and stiffened RBCs are sufficient to cause some degree of endothelial dysfunction, even in the absence of vaso-occlusion, ischemia, or oxidative stress due to hemolysis. As sickle RBCs and ISCs are constantly circulating in the blood of SCD patients, our results have profound implications for SCD pathophysiology and may help explain why SCD patients develop chronic diffuse vasculopathy over time. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Endothelial TLR4 Expression Mediates Vaso-Occlusive Crisis in Sickle Cell Disease

2021 ◽  
Vol 11 ◽  
Author(s):  
Joan D. Beckman ◽  
Fuad Abdullah ◽  
Chunsheng Chen ◽  
Rachel Kirchner ◽  
Dormarie Rivera-Rodriguez ◽  
...  
Keyword(s):  
Sickle Cell Disease ◽  
Adhesion Molecules ◽  
Sickle Cell ◽  
Adhesion Molecule ◽  
Tlr4 Expression ◽  
Cell Disease ◽  
Skin Fold ◽  
Normal Human ◽  
Complete Blood Counts ◽  
Von Willebrand

Heme, released from red blood cells in sickle cell disease (SCD), interacts with toll-like receptor 4 (TLR4) to activate NF-κB leading to the production of cytokines and adhesion molecules which promote inflammation, pain, and vaso-occlusion. In SCD, TLR4 inhibition has been shown to modulate heme-induced microvascular stasis and lung injury. We sought to delineate the role of endothelial verses hematopoietic TLR4 in SCD by developing a TLR4 null transgenic sickle mouse. We bred a global Tlr4-/- deficiency state into Townes-AA mice expressing normal human adult hemoglobin A and Townes-SS mice expressing sickle hemoglobin S. SS-Tlr4-/- had similar complete blood counts and serum chemistries as SS-Tlr4+/+ mice. However, SS-Tlr4-/- mice developed significantly less microvascular stasis in dorsal skin fold chambers than SS-Tlr4+/+ mice in response to challenges with heme, lipopolysaccharide (LPS), and hypoxia/reoxygenation (H/R). To define a potential mechanism for decreased microvascular stasis in SS-Tlr4-/- mice, we measured pro-inflammatory NF-κB and adhesion molecules in livers post-heme challenge. Compared to heme-challenged SS-Tlr4+/+ livers, SS-Tlr4-/- livers had lower adhesion molecule and cytokine mRNAs, NF-κB phospho-p65, and adhesion molecule protein expression. Furthermore, lung P-selectin and von Willebrand factor immunostaining was reduced. Next, to establish if endothelial or hematopoietic cell TLR4 signaling is critical to vaso-occlusive physiology, we created chimeric mice by transplanting SS-Tlr4-/- or SS-Tlr4+/+ bone marrow into AA-Tlr4-/- or AA-Tlr4+/+ recipients. Hemin-stimulated microvascular stasis was significantly decreased when the recipient was AA-Tlr4-/-. These data demonstrate that endothelial, but not hematopoietic, TLR4 expression is necessary to initiate vaso-occlusive physiology in SS mice.

scholarly journals Heme triggers TLR4 signaling leading to endothelial cell activation and vaso-occlusion in murine sickle cell disease

Blood ◽  
2014 ◽  
Vol 123 (3) ◽  
pp. 377-390 ◽  
Author(s):  
John D. Belcher ◽  
Chunsheng Chen ◽  
Julia Nguyen ◽  
Liming Milbauer ◽  
Fuad Abdulla ◽  
...  
Keyword(s):  
Endothelial Cell ◽  
Sickle Cell Disease ◽  
Sickle Cell ◽  
Adhesion Molecule ◽  
Cell Activation ◽  
Adhesion Molecule Expression ◽  
Cell Disease ◽  
Endothelial Cell Activation ◽  
Tlr4 Signaling ◽  
Key Points

Key Points Heme, released from hemoglobin, elicits vaso-occlusion in transgenic sickle mice via endothelial TLR4 signaling. Heme/TLR4 signaling activates NF-κB and triggers vaso-occlusion through Weibel-Palade body degranulation and adhesion molecule expression.

Role of Adhesion Molecules and Vascular Endothelium in the Pathogenesis of Sickle Cell Disease

Hematology ◽  
2007 ◽  
Vol 2007 (1) ◽  
pp. 84-90 ◽  
Author(s):  
Marilyn J. Telen
Keyword(s):  
Sickle Cell Disease ◽  
Red Blood Cells ◽  
Adhesion Molecules ◽  
Sickle Cell ◽  
Vascular Endothelium ◽  
Blood Cells ◽  
Cell Disease ◽  
Adhesive Interactions ◽  
Lines Of Evidence

AbstractA number of lines of evidence now support the hypothesis that vaso-occlusion and several of the sequelae of sickle cell disease (SCD) arise, at least in part, from adhesive interactions of sickle red blood cells, leukocytes, and the endothelium. Both experimental and genetic evidence provide support for the importance of these interactions. It is likely that future therapies for SCD might target one or more of these interactions.

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 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.

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