The Adhesion of Sickle Cell Disease Neutrophils to Endothelial Layers, In Vitro, Is Mediated by the Mac-1, LFA-1 and VLA-4 Integrins.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 2264-2264 ◽  
Author(s):  
Andreia A. Canalli ◽  
Renata F. Proenca ◽  
Sara T.O. Saad ◽  
Nicola Conran ◽  
Fernando F. Costa
Keyword(s):  
Endothelial Cells ◽  
Sickle Cell Disease ◽  
Sickle Cell ◽  
Umbilical Vein ◽  
In Vivo Studies ◽  
Integrin Subunit ◽  
Cell Disease ◽  
Inflammatory Conditions ◽  
Tnf Α

Abstract Leukocytes may have a propagating and, possibly, initiating role in sickle cell disease (SCD) vaso-occlusion. In vivo studies suggest that adherent leukocytes capture sickle erythrocytes in the microcirculation and in vitro studies demonstrate an increased ability of SCD neutrophils (neu) to adhere to fibronectin, endothelial cells and endothelial proteins. Previous studies suggest that the expressions of the major neu integrins, CD11a/CD18 (LFA-1) and CD11b/CD18 (Mac-1) may only be upregulated on the surface of SCD neu following their stimulation, indicating that alterations in integrin function (affinity or avidity) contribute to alter SCD neu adhesion. The objective of this study was to identify the integrins responsible for altered SCD neu adhesion. Neus were isolated from the peripheral blood of healthy controls and SCD individuals in steady state over ficoll-paque gradients. Cell adhesion (2×106cells/ml in RPMI) to cultured human umbilical vein endothelial cells (HUVEC) at confluence was assessed using static adhesion assays (30min, 37°C, 5%CO2). Neus from SCD patients demonstrated a significantly greater adhesion to HUVEC than control neus (20.2±2.8% compared to 11.2±1.0%; n≥7; p<0.03; Mann Whitney test). Subsequently, cells were co-incubated with adhesion molecule-blocking monoclonal antibodies (mAbs) during assays. Control neu adhesion to HUVEC was significantly inhibited by the anti-CD11b mAb (6.7±1.5%;n=6; P<0.05, paired t test), but not by mAbs against CD11a, the VLA-4-integrin subunit, CD49d, or a non-specific negative control mAb (neg control) (data not shown). In contrast, the adhesion of SCD neus to HUVEC was significantly inhibited by both the anti-CD11a and the anti-CD11b mAbs (20.2±2.8% reduced to 11.4±1.2% and 9.1±1.5%; n=9; P<0.01 and P<0.001, respect.). Interestingly, a mAb against CD49d was also found to significantly decrease SCD neu adhesion to HUVEC (10.4±1.1%; n=9; P<0.01), while the neg control mAb did not significantly affect SCD neu adhesion (data not shown). Following the stimulation of HUVEC with TNF-α (10 ng/ml) (3h, 37°C, 5%CO2) to simulate an endothelial layer under inflammatory conditions, the adhesions of control and SCD neus were increased but statistically similar (38.4±2.9% and 34.4±5.0%; n≥4, respect.). Under these conditions anti-CD11a and CD11b mAbs significantly inhibited control neu adhesion to HUVEC (reduced to 28.8±2.9% and 19.6±4.6%; n=4; P<0.01 and P<0.05, respect.). In contrast, SCD neu adhesion to HUVEC was significantly inhibited by mAbs for CD11a (19.5±2.6%; n=6; p<0.01) and CD11b (15.2±2.0%; n=6; p<0.001). The anti-CD49d, but not the neg control mAb, also significantly decreased SCD neu adhesion to TNF-α-stimulated HUVEC (19.5±3.7%; n=6; p<0.05). In conclusion, data indicate that control neu adhesion to endothelial cells appears mainly to be mediated by the Mac-1 (CD11b/18) integrin with a contribution from the LFA-1 integrin (CD11a/18) under inflammatory conditions. In contrast, SCD neu adhesion to endothelium (under both basal and stimulated conditions), at least in vitro, appears to be mediated by the Mac-1 and LFA-1 integrins and, interestingly, by VLA-4 (CD49d/CD29), an integrin found expressed at low levels on neus during certain inflammatory conditions. We speculate that alterations in the affinity/ avidity of these molecules contribute to SCD neu adhesion. Approaches to inhibit the adhesion of all three integrins may be important for preventing leukocyte adhesion to the vascular endothelium and, in turn, vaso-occlusion.

scholarly journals Soluble MD-2 and Heme in Sickle Cell Disease Plasma Promote Pro-Inflammatory Signaling in Endothelial Cells

2021 ◽  
Vol 12 ◽  
Author(s):  
Ping Zhang ◽  
Julia Nguyen ◽  
Fuad Abdulla ◽  
Alexander T. Nelson ◽  
Joan D. Beckman ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Sickle Cell Disease ◽  
Sickle Cell ◽  
Umbilical Vein ◽  
Acute Chest Syndrome ◽  
Cell Disease ◽  
Inflammatory Signaling ◽  
Inflammatory Conditions ◽  
Vis Spectroscopy ◽  
Umbilical Vein Endothelial Cells

Recent evidence indicates that hemolysis in sickle cell disease (SCD) promotes inflammation via innate immune signaling through toll-like receptor 4 (TLR4). Free heme released by hemolyzed red blood cells can bind to myeloid differentiation factor-2 (MD-2) and activate TLR4 pro-inflammatory signaling on endothelium to promote vaso-occlusion and acute chest syndrome in murine models of SCD. MD-2 is co-expressed with TLR4 on cell membranes, but in inflammatory conditions, soluble MD-2 (sMD-2) is elevated in plasma. sMD-2 levels were significantly increased in human and murine sickle (SS) plasma as compared to normal (AA) plasma. Human umbilical vein endothelial cells (HUVEC) and human lung microvascular endothelial cells incubated with human SS plasma had significant increases in pro-inflammatory IL-8, IL-6, and soluble VCAM-1 secretion compared to endothelial cells incubated with AA plasma. The increase in HUVEC IL-8 secretion was blocked by depletion of sMD-2 from SS plasma and enhanced by the addition of sMD-2 to AA plasma. The TLR4 signaling inhibitor, TAK-242, inhibited HUVEC IL-8 secretion in response to SS plasma by 85%. Heme-agarose pull-down assays and UV/Vis spectroscopy demonstrated that heme binds to sMD-2. Hemopexin, a high affinity heme-binding protein, inhibited HUVEC IL-8 secretion induced by SS plasma or SS and AA plasma supplemented with sMD-2. These data suggest that sMD-2 bound to heme might play an important role in pro-inflammatory signaling by endothelium in 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<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<0.000001), and reticulocyte count (r=0.60, p<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.

Activated monocytes in sickle cell disease: potential role in the activation of vascular endothelium and vaso-occlusion

Blood ◽  
2000 ◽  
Vol 96 (7) ◽  
pp. 2451-2459 ◽  
Author(s):  
John D. Belcher ◽  
Paul H. Marker ◽  
Jill P. Weber ◽  
Robert P. Hebbel ◽  
Gregory M. Vercellotti
Keyword(s):  
Endothelial Cells ◽  
Sickle Cell Disease ◽  
Sickle Cell ◽  
Sickle Cell Anemia ◽  
Nuclear Translocation ◽  
Mononuclear Leukocytes ◽  
Cell Contact ◽  
Cell Disease ◽  
Tnf Α ◽  
Activated Monocytes

Sickle cell anemia is characterized by painful vaso-occlusive crises. It is hypothesized that monocytes are activated in sickle cell disease and can enhance vaso-occlusion by activating endothelium. To test this hypothesis, human umbilical vein endothelial cells (HUVEC) and human microvascular endothelial cells (MVEC) with sickle and normal mononuclear leukocytes were incubated, and endothelial activation was measured. Endothelial cells incubated with sickle mononuclear leukocytes were more activated than those incubated with normal mononuclear leukocytes, as judged by the increased endothelial expression of adhesion molecules and tissue factor and the adhesion of polymorphonuclear leukocytes (PMNL). Monocytes, not lymphocytes or platelets, were the mononuclear cells responsible for activating endothelial cells. Sickle monocytes triggered endothelial nuclear factor-kappa B (NF-κB) nuclear translocation. Cell-to-cell contact of monocytes and endothelium enhanced, but was not required for, activation. Antibodies to tumor necrosis factor-alpha (TNF-α) and interleukin-1-beta (IL-1β) blocked activation of the endothelium by monocytes. Peripheral blood monocytes from patients with sickle cell disease had 34% more IL-1β (P = .002) and 139% more TNF-α (P = .002) per cell than normal monocytes. Sixty percent of sickle monocytes expressed the adhesion molecule ligand CD11b on their surfaces compared with only 20% of normal monocytes (P = .002). Serum C-reactive protein, a marker of systemic inflammation, was increased 12-fold in sickle serum than in normal serum (P = .003). These results demonstrate that sickle monocytes are activated and can, in turn, activate endothelial cells. It is speculated that vascular inflammation, marked by activated monocytes and endothelium, plays a significant role in the pathophysiology of vaso-occlusion in sickle cell anemia.

scholarly journals Mycobacterium aviumComplex Infection in a Patient with Sickle Cell Disease and Severe Iron Overload

2014 ◽  
Vol 2014 ◽  
pp. 1-5 ◽  
Author(s):  
Kamal Shemisa ◽  
Nasima Jafferjee ◽  
David Thomas ◽  
Gretta Jacobs ◽  
Howard J. Meyerson
Keyword(s):  
Sickle Cell Disease ◽  
Iron Overload ◽  
Sickle Cell ◽  
Unknown Origin ◽  
Mycobacterial Infection ◽  
In Vivo Studies ◽  
Recurrent Pain ◽  
Cell Disease

A 34-year-old female with sickle cell anemia (hemoglobin SS disease) and severe iron overload presented to our institution with the subacute presentation of recurrent pain crisis, fever of unknown origin, pancytopenia, and weight loss. A CT scan demonstrated both lung and liver nodules concerning for granulomatous disease. Subsequent biopsies of the liver and bone marrow confirmed the presence of noncaseating granulomas and blood cultures isolatedMycobacterium aviumcomplex MAC. Disseminated MAC is considered an opportunistic infection typically diagnosed in the immunocompromised and rarely in immunocompetent patients. An appreciable number of mycobacterial infection cases have been reported in sickle cell disease patients without immune dysfunction. It has been reported that iron overload is known to increase the risk for mycobacterial infection in vitro and in vivo studies. While iron overload is primarily known to cause end organ dysfunction, the clinical relationship with sickle cell disease and disseminated MAC infection has not been reported. Clinical iron overload is a common condition diagnosed in the sub-Saharan African population. High dietary iron, genetic defects in iron trafficking, as well as hemoglobinopathy are believed to be the etiologies for iron overload in this region. Patients with iron overload in this region were 17-fold more likely to die fromMycobacterium tuberculosis. Both experimental and clinical evidence suggest a possible link to iron overload and mycobacterial infections; however larger observational studies are necessary to determine true causality.

Simvastatin Reduces the in Vitro Adhesion of Sickle Cell Disease Neutrophils to Endothelial Layers

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 2489-2489 ◽  
Author(s):  
Andreia A Canalli ◽  
Renata P. Ferreira ◽  
Sara T.O. Saad ◽  
Nicola Conran ◽  
Fernando F. Costa
Keyword(s):  
Cell Adhesion ◽  
Sickle Cell Disease ◽  
Sickle Cell ◽  
Leukocyte Adhesion ◽  
Cell Disease ◽  
Tnf Α ◽  
Cell Layers ◽  
Anti Inflammatory ◽  
Vitro Effect

Abstract Leukocytes may have a propagating and, possibly, initiating role in sickle cell disease (SCD) vaso-occlusion. Endothelial dysfunction contributes to the vaso-occlusion process and leads to inflammation, leukocyte and red cell adhesion. Markers of neutrophil activation are also increased in SCD, in association with increased levels of circulating cytokines and increased leukocyte adhesion. In animal models, vaso-occlusion causes hypoxia/reperfusion, leading to vascular endothelium damage and an inflammatory response. We postulate that anti-inflammatory agents may reduce the participation of activated endothelium in the vaso-occlusive process. Statins are commonly used to treat arteriosclerosis and have anti-inflammatory effects that include a regulatory action on endothelial function, reduced oxidative stress and inflammation. The objective of this study was to investigate the in vitro effect of simvastatin on the adhesion of sickle neutrophils to activated endothelial cell layers (HUVEC). Neutrophils (Neu) were isolated from the peripheral blood of healthy controls (ConNeu) and SCD (SCDNeu) individuals in steady state over ficoll-paque gradients. Cell adhesion (2×106 cell/ml in Ham’s F12 K) to cultured human umbilical vein endothelial cells (HUVEC) grown to confluence was assessed using static adhesion assays. HUVEC cells were treated with or without 1 μg/ml simvastatin for 6 hours in the absence or presence of a 10nM TNF-α activating stimulus (3 hours) before allowing adhesion of Neu to the cell layers (30 min, 37°C, 5%CO2). Neu from SCD patients demonstrated a significantly greater adhesion to HUVEC than ConNeu (20.5 ± 1.9% compared to 13.8 ± 1.7 %; n=15; p<0.02; Mann Whitney test). Subsequently, Neu from patients and controls were allowed to adhere to endothelial layers previously treated with simvastatin; adhesion was not significantly different to the adhesion of Neu to nonsimvastatin treated HUVEC (16.7 ± 3.2% for ConNeu; n=8, p>0.05 and 19.8 ±2.7% for SCDNeu; n=11, p>0.05, paired t test). Pre-treatment of HUVEC with the cytokine TNF-α increased the adhesion of SCD and Con Neu to HUVEC (40.9 ± 5.4%; 28.9 ± 5.0%, respect, N>8, P<0.01 compared to adhesion to non-activated HUVEC). Interestingly, when the endothelium layer was protected with simvastatin and then stimulated with TNF-α, SCDNeu adhesion was significantly diminished (reduced to 31.3% ± 3.6%; n=11, p<0.005 comp. to adhesion to non-simvastatin-treated HUVEC); in contrast, no difference in the adhesion of ConNeu to HUVEC treated with TNF-α and simvastatin was observed (31.9 ± 5.8%, n=8, p>0.05 for ConNeu). In conclusion, data indicate that under in vitro inflammatory conditions, simvastatin appears to protect endothelium layers and reduces SCD leukocyte adhesion. We speculate that statins may have anti-inflammatory properties and, as such, may be useful for diminishing endothelial activation and, in turn, preventing the adhesion of leukocytes adhesion to the vascular wall in SCD, a mechanism that is essential to the vaso-occlusive process.

scholarly journals Inhibition of cell adhesion by anti–P-selectin aptamer: a new potential therapeutic agent for sickle cell disease

Blood ◽  
2011 ◽  
Vol 117 (2) ◽  
pp. 727-735 ◽  
Author(s):  
Diana R. Gutsaeva ◽  
James B. Parkerson ◽  
Shobha D. Yerigenahally ◽  
Jeffrey C. Kurz ◽  
Robert G. Schaub ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Cell Adhesion ◽  
Sickle Cell Disease ◽  
Sickle Cell ◽  
Therapeutic Agent ◽  
Critical Role ◽  
High Specificity ◽  
Cell Disease ◽  
Adhesive Interactions

Abstract Adhesive interactions between circulating sickle red blood cells (RBCs), leukocytes, and endothelial cells are major pathophysiologic events in sickle cell disease (SCD). To develop new therapeutics that efficiently inhibit adhesive interactions, we generated an anti–P-selectin aptamer and examined its effects on cell adhesion using knockout-transgenic SCD model mice. Aptamers, single-stranded oligonucleotides that bind molecular targets with high affinity and specificity, are emerging as new therapeutics for cardiovascular and hematologic disorders. In vitro studies found that the anti–P-selectin aptamer exhibits high specificity to mouse P-selectin but not other selectins. SCD mice were injected with the anti–P-selectin aptamer, and cell adhesion was observed under hypoxia. The anti–P-selectin aptamer inhibited the adhesion of sickle RBCs and leukocytes to endothelial cells by 90% and 80%, respectively. The anti–P-selectin aptamer also increased microvascular flow velocities and reduced the leukocyte rolling flux. SCD mice treated with the anti–P-selectin aptamer demonstrated a reduced mortality rate associated with the experimental procedures compared with control mice. These results demonstrate that anti–P-selectin aptamer efficiently inhibits the adhesion of both sickle RBCs and leukocytes to endothelial cells in SCD model mice, suggesting a critical role for P-selectin in cell adhesion. Anti–P-selectin aptamer may be useful as a novel therapeutic agent for SCD.

Effect of Quercetin on Lipopolysaccharide and Hydroxyurea Induced Lactate Dehydrogenase Release from Immortalize Mouse Aortic Endothelial Cells

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 4846-4846
Author(s):  
Zachary Monroe Kiser ◽  
Jacqueline Monroe Hibbert ◽  
Alexander Quarshie
Keyword(s):  
Endothelial Cells ◽  
Lactate Dehydrogenase ◽  
Sickle Cell Disease ◽  
Sickle Cell ◽  
Cell Damage ◽  
Vascular Endothelial ◽  
Cell Disease ◽  
Aortic Endothelial Cells ◽  
Dietary Flavonoid

Abstract Background: A higher than normal basal inflammatory state is characteristic of sickle cell disease (SCD). Hydroxyurea (HU) is the only FDA approved drug for SCD. However, HU is a chemotherapy drug and is therefore naturally cytotoxic, often inducing apoptosis. Chronic inflammation in sickle cell patients is invariably associated with injury to the vascular endothelium. Quercetin is a dietary flavonoid found ubiquitously in plants and foods that have anti-oxidative and anti-inflammatory characteristics. Hypothesis: The dietary flavonoid quercetin will decrease cytotoxic effects of Lipopolysaccharide and HU induced cell damage to vascular endothelial cells. Aims: 1. to develop an in vitro inflammatory model simulating chronic baseline inflammation observed in sickle cell disease. 2. to test the effect of the physiological dose of hydroxyurea (HU), on the inflammatory model. 3. to examine the role of quercetin (QCT), a dietary flavonoid with anti-oxidative and anti-inflammatory characteristics, for reducing the inflammation. Methods: Lipopolysaccharide (LPS) was used to induce inflammation in immortalized mouse aortic endothelial cells (iMAECs), providing an in vitro model of inflamed endothelial cells. The cells were exposed to LPS throughout the entire experiment. Interventions included treating the LPS exposed cells with QCT, HU, or QCT + HU over 50 hours. The 50-hour period included 24 hours of varying treatments, followed by two hours of hypoxic exposure and then 24 hours under normal aerobic exposure. Untreated cells (controls) provided a comparison. Data analyses included comparisons of control cells with the inflammatory model, and pairwise comparisons between the inflammatory model and the different treatments. In this experiment lactate dehydrogenase (LDH) was measured by colorimetric assay, as an indication of cell damage. Results: At the end of the experiment, the LDH level for the inflammatory model was significantly higher than LDH for the control cells (P = 0.0005) fig 1. Treatment with 30 micromoles QCT gleaned a trend toward reduced LDH compared with the inflammatory model (p = 0.1) fig 2. LDH was significantly higher after treatment with 100 micromoles HU compared with the inflammatory model (p = 0.0005) Fig 3. However, LDH was significantly reduced after treatment with a combination of 30 micromoles QCT/100 micromoles HU, compared with 100 micromoles HU alone (p = 0.0008) fig 3. Conclusions: These results suggest that quercetin may be effective against vascular endothelial cell damage for iMAECs in vitro. It also shows promise in preventing HU-induced cytotoxicity, which was a surprising finding from these results. This latter finding is interesting, and should be given more consideration, since HU is the only FDA-approved drug for treating sickle cell patients, and its use is rapidly increasing. Figure 1 Figure 1. Figure 2 Figure 2. Figure 3 Figure 3. Disclosures No relevant conflicts of interest to declare.

Activated monocytes in sickle cell disease: potential role in the activation of vascular endothelium and vaso-occlusion

Blood ◽  
2000 ◽  
Vol 96 (7) ◽  
pp. 2451-2459 ◽  
Author(s):  
John D. Belcher ◽  
Paul H. Marker ◽  
Jill P. Weber ◽  
Robert P. Hebbel ◽  
Gregory M. Vercellotti
Keyword(s):  
Endothelial Cells ◽  
Sickle Cell Disease ◽  
Sickle Cell ◽  
Sickle Cell Anemia ◽  
Nuclear Translocation ◽  
Mononuclear Leukocytes ◽  
Cell Contact ◽  
Cell Disease ◽  
Tnf Α ◽  
Activated Monocytes

Abstract Sickle cell anemia is characterized by painful vaso-occlusive crises. It is hypothesized that monocytes are activated in sickle cell disease and can enhance vaso-occlusion by activating endothelium. To test this hypothesis, human umbilical vein endothelial cells (HUVEC) and human microvascular endothelial cells (MVEC) with sickle and normal mononuclear leukocytes were incubated, and endothelial activation was measured. Endothelial cells incubated with sickle mononuclear leukocytes were more activated than those incubated with normal mononuclear leukocytes, as judged by the increased endothelial expression of adhesion molecules and tissue factor and the adhesion of polymorphonuclear leukocytes (PMNL). Monocytes, not lymphocytes or platelets, were the mononuclear cells responsible for activating endothelial cells. Sickle monocytes triggered endothelial nuclear factor-kappa B (NF-κB) nuclear translocation. Cell-to-cell contact of monocytes and endothelium enhanced, but was not required for, activation. Antibodies to tumor necrosis factor-alpha (TNF-α) and interleukin-1-beta (IL-1β) blocked activation of the endothelium by monocytes. Peripheral blood monocytes from patients with sickle cell disease had 34% more IL-1β (P = .002) and 139% more TNF-α (P = .002) per cell than normal monocytes. Sixty percent of sickle monocytes expressed the adhesion molecule ligand CD11b on their surfaces compared with only 20% of normal monocytes (P = .002). Serum C-reactive protein, a marker of systemic inflammation, was increased 12-fold in sickle serum than in normal serum (P = .003). These results demonstrate that sickle monocytes are activated and can, in turn, activate endothelial cells. It is speculated that vascular inflammation, marked by activated monocytes and endothelium, plays a significant role in the pathophysiology of vaso-occlusion in sickle cell anemia.

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