scholarly journals Neutralization of Inflammasome-Processed Cytokines Reduces Inflammatory Mechanisms and Leukocyte Recruitment in the Vasculature of TNF-α-Stimulated Sickle Cell Disease Mice

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 856-856
Author(s):  
Lidiane S. Torres ◽  
Erica M.F. Gotardo ◽  
Flávia Costa Leonardo ◽  
Pamela L. Brito ◽  
Irmgard Förster ◽  
...  

Abstract The chronic inflammatory state associated with sickle cell disease (SCD) incurs pan-cellular activation and the recruitment of leukocytes to the activated endothelium of blood vessels. The resultant rheological alterations and red cell sickling culminate in acute vaso-occlusive processes. Cytokines IL-1β and IL-18, the bio-active products of the activated inflammasome, are elevated in the plasma of SCD patients (ASH Abstract (2016) 128 (22): 854), and a previous study reported that anti-IL1β therapy alleviated reperfusion injury and flow stasis in NY1DD transgenic sickle mice exposed to hypoxia/reoxygenation (ASH Abstract (2011) 118(21): 848). The aim of this study was to determine whether antibodies that neutralize IL-1β and IL-18 could individually, or synergistically, diminish inflammatory processes and leukocyte recruitment in mice with SCD. Townes mice (5-months old; N=4-7 per group) received an i.p. administration of either saline, 200 µg/mouse anti-murine IL-1β (01BSUR), and/or 250 or 500 µg/mouse anti-murine IL-18 (SK113AE-4), or an IgG1 control antibody (iProt105125; 200 or 500 µg/mouse). At 21h after treatments, vaso-occlusive-like processes were induced in mice by the injection of tumor necrosis factor-α (TNF; 0.5μg, i.p.). At 3h after TNF, the cremaster muscles of anesthetized mice were surgically exposed, and leukocyte TNF recruitment and extravasation in venules of the microcirculation were observed using intravital microscopy. Another set of Townes mice (N=4-12 per group) was submitted to the same procedures, with blood sampling for ELISA at 3h post TNF. Optimal concentrations of antibodies were determined by observing leukocyte recruitment by intravital microscopy (doses; 100, 200 µg/mouse anti-IL-1β [N=2; 3, respectively] and 250, 500 µg/mouse anti-IL-18, [N=2 each]) in TNF-stimulated C57BL/6J mice (data not shown). Figure 1 (A-C) demonstrates the extensive recruitment and extravasation of leukocytes that occurs in the microvasculature of Townes mice at 3h post-TNF (saline group). Pre-treatment of mice with either anti-IL-1β or anti-IL-18 significantly abrogated (P<0.01) the TNF-induced adhesion (Fig. 1B) and extravasation (Fig. 1C) of leukocytes in venules, while only anti-IL-18 significantly reduced leukocyte rolling along the venule endothelium (Fig. 1A; P<0.05). In contrast, the administration of 500 µg/mouse (Fig. 1) or 200 µg/mouse (data not shown) of a non-specific IgG1 did not significantly affect TNF-induced leukocyte recruitment/extravasation (P>0.05). The combined use of the anti-IL-1β (200 µg/mouse) together with an intermediate dose of anti-IL-18 (250 µg/mouse) did not further reduce leukocyte recruitment and extravasation in this model, when compared with the effects of anti-IL-1β alone (Fig. 1A-C; P>0.05). Investigating the effects of these biological agents on inflammatory molecules production in TNF-stimulated Townes mice, we found that the administration of anti-IL-1β (200 µg) reduced IL-6 production, a pleiotropic inflammatory molecule that is upregulated by IL-1β (Fig. 1D), as did the combination of anti-IL-1β plus anti-IL-18 (200 µg and 250 µg/mouse, respectively). Pre-treatment of TNF-stimulated SCD mice with anti-IL-1β plus anti-IL-18 decreased Interferon (IFN)-γ production, a molecule that is upregulated synergistically by IL-18 and IL-12 and that mediates early host immune defenses (Fig. 1E). In contrast, anti-inflammatory IL-10 production was not significantly modulated by the pre-treatment of TNF-stimulated mice with these biological agents (Fig. 1F). As such, IL-1β and IL-18 neutralization significantly reduced inflammatory processes and leukocyte recruitment in the microvasculature of mice with SCD, indicating that biological agents that inhibit the effects of inflammasome-processed cytokines may hold potential for reducing vaso-occlusive processes in patients with this disease. Figure 1 Figure 1. Disclosures Kovarik: Novartis Institutes for Biomedical Research: Current Employment. Costa: Novartis: Consultancy. Conran: Novartis Pharma AG: Research Funding.

Blood ◽  
2017 ◽  
Vol 130 (Suppl_1) ◽  
pp. 961-961
Author(s):  
Athos Rodrigues Moraes ◽  
Hanan Chweih ◽  
Nicola Conran ◽  
Kleber Yotsumoto Fertrin ◽  
Fernando Ferreira Costa ◽  
...  

Abstract Introduction: Sickle cell disease (SCD) vaso-occlusion process involves different cell types, such as red blood cells,activated endothelial cells, platelets and leukocytes. Endothelialdysfunction contributes to the vaso-occlusion process and leadsto inflammation. Data suggest that Rho-kinase signaling may regulate numerous aspects of the inflammatory process. Alterations in the Rho-A/Rho-kinase signaling pathway modulate pathophysiological aspects of the sickle cell disease such as priapism, and these enzymes are involved in increased reactive oxygen species generation and altered sickle cell cytoskeletal phosphorylation. In addition, Rho kinase inhibitors were able to reduce endothelial activation and consequent eosinophil adhesion in vitro and reduced allergic inflammation in the lungs of SCD mice. However, the involvement of Rho/Rho-kinase signaling pathways in the mechanism underlying systemic vascular occlusion in SCD remains unclear. This study aimed to determine whether Rho/Rho-kinase pathways are involved in the mechanism of microvascular SCD vaso-occlusion. We investigated the effect of fasudil, a specific inhibitor of Rho-kinase, in the initial steps of the leukocyte transmigration process in a model of allergic inflammation using intravital microscopy of the cremaster muscle in SCD mice. Methods: Experimental groups consisted of male homozygous Tim Townes transgenic sickle cell mice and C57BL/6JuniB control mice. SCD and control mice were actively sensitized with a subcutaneous injection of 100 μg of ovalbumin (OVA) mixed with 1.6 mg Al(OH)3 in 0.9% NaCl (Day zero). On day 7, mice received a second injection of 100 μg of OVA. On day 14, mice were subcutaneously challenged with eotaxin (100 ng/per dose) with or without pre-treatment with intraperitoneal fasudil (10 mg/kg) 1 hour before eotaxin. Four hours later, the animals were surgically prepared for intravital microscopy of the microvasculature of the cremaster muscle. Results: Intravital microscopy showed that eotaxin challenge in OVA-sensitized animals increased rolling and adhesion of leukocytes to the endothelium, and accumulation of leukocytes outside the vessels was observed. However, this increase is significantly higher in SCD mice compared to control animals (Rolling: 23±1.9 and 15±1.2 leukocyte min-1; Adhesion: 16.3±0.9 and 11.7±0.5 leukocyte adhered 100µm-1; Extravasation: 3.88±0.3 and 2.4±0.2 leukocyte perx100x50 µm2, p<0.05, respectively). The leukocyte rolling flow was similarly inhibited by fasudil treatment in control animals by 50% and in SCD mice by 42%. Eotaxin-induced firm adhesion after fasudil was also reduced by 57% in control animals and 63% in SCD mice. Notably, pre-treatment with fasudil caused a greater decrease in leukocyte extravasation in SCD mice (44%) than in control animals (16%), p<0.0001. Conclusion: Our data show that inhibition of Rho-kinase decreased endothelial-leukocyte interaction. These findings suggest that Rho-kinase inhibitors may have therapeutic benefits in the vaso-occlusive process in SCD, limiting the extravasation of leukocytes, and reducing vascular inflammation. Disclosures Conran: Bayer AG: Research Funding. Fertrin: Alexion Pharmaceuticals: Consultancy.


Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 976-976
Author(s):  
Natacha Ralainirina ◽  
Ferron Lynn Sonderegger ◽  
Hong Pei ◽  
Grzegorz Chodaczek ◽  
Joel Linden

Abstract Although sickle cell anemia is initiated by red cell pathology, it is accompanied by an inflammatory immune response involving platelets and white blood cells that contribute to vaso-occlusvie episodes including painful vaso-occlusive crises (pVOC) and acute chest syndrome (ACS). In order to better understand the cellular and molecular bases of vaso-occlusion we are in the process of developing procedures to image microvessels in the lung, liver and spleen of living mice by 2-photon microscopy, a procedure that is based on excitation of a fluorophore by two photons simultaneously. The two-photon technique utilizes infrared light that efficiently penetrates tissues up to 200 microns with low phototoxicity allowing time-lapse imaging. Two-photon intravital microscopy can be used to study the behavior of intravascular cells during vaso-occlusive events. Mice are prepared for lung intravital microscopy by the intraperitoneal injection of a mixture of ketamine and xylazine. Additional anesthesia is added during experimentation. The trachea is opened and the mouse intubated. The chest is opened to allow access to the left lobe of the lung through a window that is a few millimeters in diameter. PBS is applied to keep the lung moist. A custom built suction device is placed on the lung and covered with a cover glass at the same time pressure is exerted to seal the organ and the glass cover together. Throughout the procedure, the mouse is held at a temperature of 37°C. Once surgery is completed, a mixture of antibodies coupled to fluorophores is given by retro-orbital injection. In order to minimize photobleaching we used antibodies conjugated to Alexa Fluor 488, Alexa Fluor 555 or Alexa Fluor 647. We are able to visualize and quantify interactions between red blood cells, white blood cells, and endothelial cells as well as the expression of adhesion molecules on endothelial cells in real time. During pVOC triggered by hypoxia, cell adhesion of neutrophils, lymphocytes and monocytes to the endothelium is observed that is associated with an increase in endothelial expression of ICAM-1 and V-CAM. We label endothelial cells with anti-CD31, lymphocytes with anti-CD45, monocytes with anti-Ly6C and neutrophils with anti-Ly6G. Platelets are labeled with anti-CD41 or anti-CD62P, NK cells with anti-NKp46, and macrophages with anti-F4/80 and anti-CD1d. We are able to quantify cell shape, rolling, adhesion and movement. Our preliminary results demonstrate that it is possible in real time to image the sequence of events occurring during pulmonary vasoocclusion in sickle cell disease. In conclusion, intravital 2-photon microscopy holds great potential for enabling us to better understand inflammatory responses within the blood vessels of living SCD mice. Disclosures: No relevant conflicts of interest to declare.


2004 ◽  
Vol 287 (1) ◽  
pp. H293-H301 ◽  
Author(s):  
Dhananjay K. Kaul ◽  
Xiao-du Liu ◽  
Stephana Choong ◽  
John D. Belcher ◽  
Gregory M. Vercellotti ◽  
...  

In sickle cell disease, inflammatory activation of vascular endothelium and increased leukocyte-endothelium interaction may play an important role in the occurrence of vasoocclusion. In sickle mouse models, inflammatory stimuli (e.g., hypoxia-reoxygenation and cytokines) result in increased leukocyte recruitment and can initiate vasoocclusion, suggesting that anti-inflammatory therapy could be beneficial in management of this disease. We have tested the hypothesis that inhibition of endothelial activation in a transgenic mouse model by anti-inflammatory agents would lead to reduced leukocyte recruitment and improved microvascular blood flow in vivo. In transgenic sickle mice, hypoxia-reoxygenation resulted in greater endothelial oxidant production than in control mice. This exaggerated inflammatory response in transgenic mice, characterized by increased leukocyte recruitment and microvascular flow abnormalities, was significantly attenuated by antioxidants (allopurinol, SOD, and catalase). In contrast, control mice exhibited a muted response to antioxidant treatment. In addition, hypoxia-reoxygenation induced activation of NF-κB in transgenic sickle mice but not in control mice. In transgenic sickle mice, sulfasalazine, an inhibitor of NF-κB activation and endothelial activation, attenuated endothelial oxidant generation, as well as NF-κB activation, accompanied by a marked decrease in leukocyte adhesion and improved microvascular blood flow. Thus targeting oxidant generation and/or NF-κB activation may constitute promising therapeutic approaches in sickle cell disease.


Blood ◽  
2001 ◽  
Vol 97 (11) ◽  
pp. 3401-3404 ◽  
Author(s):  
Anthony T. W. Cheung ◽  
Paul Harmatz ◽  
Ted Wun ◽  
Peter C. Y. Chen ◽  
Edward C. Larkin ◽  
...  

The Stroke Prevention Trial has confirmed that utilization of transcranial Doppler ultrasonography (TCD), which examines blood flow in large intracranial vessels, can identify children with sickle cell disease (SCD) who are at high risk of developing a premature stroke. It is not known to what extent the vasculopathy in SCD involves small vessels and whether the abnormalities, if present, correlate with large-vessel vasculopathy. Eighteen children with SCD were examined with TCD to determine middle cerebral artery (MCA) velocity and computer-assisted intravital microscopy (CAIM) to determine bulbar conjunctival vessel velocity during the same visit for vasculopathy correlation. High MCA velocity (≥ 200 cm/sec) was found by TCD in 4 patients who also showed abnormal conjunctival velocity (< 0.2 mm/sec or intermittent trickle flow) by CAIM. Three patients had conditional (≥ 170 cm/sec and < 200 cm/sec) MCA velocity: 2 showed abnormal (trickle) and 1 showed normal conjunctival velocity (1.9 mm/sec). One patient with unmeasurable MCA velocity had abnormal (trickle) conjunctival velocity. Of the remaining 10 patients who had normal MCA velocity, 2 showed abnormal (0.05 mm/sec and 0.1 mm/sec) and 8 showed normal conjunctival velocities (1.1-2.4 mm/sec). The MCA velocities correlated significantly with bulbar conjunctival flow velocities (P ≤ .008, Fisher exact test). A correlation exists between MCA (large-vessel) and conjunctival (small-vessel) flow velocities. CAIM is a noninvasive quantitative technique that might contribute to the identification of SCD patients at high risk of stroke. Small-vessel vasculopathy might be an important pathological indicator and should be further explored in a large-scale study.


Blood ◽  
2002 ◽  
Vol 99 (11) ◽  
pp. 3999-4005 ◽  
Author(s):  
Anthony T. W. Cheung ◽  
Peter C. Y. Chen ◽  
Edward C. Larkin ◽  
Patricia L. Duong ◽  
Sahana Ramanujam ◽  
...  

The conjunctival microcirculation of 18 homozygous sickle cell disease (SCD) patients during steady-state, painful crisis, and postcrisis conditions was recorded on high-resolution videotapes using intravital microscopy. Selected videotape sequences were subsequently coded, frame-captured, studied, and blindly analyzed using computer-assisted image analysis protocols. At steady-state (baseline), all SCD patients exhibited some of the following morphometric abnormalities: abnormal vessel diameter, comma signs, blood sludging, boxcar blood flow phenomenon, distended vessels, damaged vessels, hemosiderin deposits, vessel tortuosity, and microaneurysms. There was a decrease in vascularity (diminished presence of conjunctival vessels) in SCD patients compared with non-SCD controls, giving the bulbar conjunctiva a “blanched” avascular appearance in most but not all SCD patients during steady-state. Averaged steady-state red cell velocity in SCD patients was slower than in non-SCD controls. During painful crisis, a further decrease in vascularity (caused by flow stoppage in small vessels) and a 36.7% ± 5.2% decrease in large vessel (mostly venular) diameter resulted. In addition, the conjunctival red cell velocities either slowed significantly (6.6% ± 13.1%; P < .01) or were reduced to a trickle (unmeasurable) during crisis. The microvascular changes observed during crisis were transient and reverted to steady-state baseline after resolution of crisis. When combined, intravital microscopy and computer-assisted image analysis (computer-assisted intravital microscopy) represent the availability of a noninvasive tool to quantify microvascular abnormalities in vascular diseases, including sickle cell disease. The ability to identify and relocate the same conjunctival vessels for longitudinal studies uniquely underscores the applicability of this quantitative real-time technology.


1987 ◽  
Vol 80 (1) ◽  
pp. 117-127 ◽  
Author(s):  
H H Lipowsky ◽  
N U Sheikh ◽  
D M Katz

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 2475-2475 ◽  
Author(s):  
Camila B. Almeida ◽  
Fernanda Gonçalves Pereira ◽  
Irene Lorand Metze ◽  
Sara T.O. Saad ◽  
Fernando F. Costa ◽  
...  

Abstract Increased white cell counts are associated with augmented mortality and morbidity in sickle cell disease (SCD) and leukocytes play a central role in the vaso-occlusive process of sickle cell disease by adhering to the vascular endothelium and participating in mechanisms of oxidative stress and inflammation. The relevance of neutrophil death to the pathogenesis of inflammatory disease is now recognized, since alterations in the apoptotic processes of leukocytes may affect their cellular function and inflammatory processes. As such, this study investigated the effect of serum from control individuals (CON), SCD patients in steady state (SCD) and SCD patients on hydroxyurea therapy (SCDHU; 20–30 mg/kg/day HU) on the apoptotic processes of neutrophils (Neu). Neu were separated from whole blood of CON individuals using a ficoll-paque gradient; serum was also separated and filtered through 0.22μm ultra-filters. Pools of serum were prepared by mixing serum from 10 individuals from each subject group. Neutrophils (4×106 cells/ml) were cultured in the presence of pooled filtered serum (10% v/v) for 16h (37°C, 5%CO2) in DMEM. Apoptosis was evaluated by detection of annexin V binding, and viability was determined by MTS assay. CON Neu cultured in the presence of pooled SCD serum demonstrated a significantly higher percentage of apoptotic cells (67.6 ± 4.3%; n=4) compared to Neu cultured with CON serum (52.3 ± 3.6%; n=4; P<0.01 comp. SCD, paired t-test) or SCDHU serum (55.4 ± 4.2%; n=4; P<0.01 comp. SCD). In contrast, Caspase-3 activity was not significantly different in CON Neu following 16h of culture in the presence of CON and SCD serum (2.46±0.13 and 2.50±0.19-fold increased caspase-3 activity at 16h compared to basal activity, respect., n>5; P>0.05); Neu cultured with SCDHU serum demonstrated a higher caspase-3 activity (4.23±0.70-fold increased activity, n=8, P>0.05 Wilcox. matched pairs), but this increase was not statistically different to that of Neu cultured with CON serum. Viability did not differ significantly following 16h-culture of CON Neu with CON, SCD, or SCDHU serum (0.70±0.05; 0.80±0.06; 0.86±0.08 OD490nm, respect., n>9, P>0.05); furthermore co-incubation of the Neu with serums in the presence of the programmed necrosis inhibitor, Necrostatin (10μM), had no significant effect on cell viability (0.80±0.05; 0.83±0.05; 0.86±0.05 OD490nm, for CON, SCD and SCDHU respect., n>9, P>0.05), indicating that SCD serum does not appear to induce a process of necroptosis in Neu. Neutrophil death in SCD, like other inflammatory diseases, appears to be regulated by a complex network of both intracellular death and survival signaling pathways that may be modulated by extracellular stimuli. We demonstrate, under the conditions described herein, that filtered SCD serum can induce an apoptotic process in non-SCD neutrophils that does not appear to be mediated by alterations in caspase-3 activity. High levels of circulating TNF-α, a cytokine that has been previously found elevated in the serum of our population of SCD patients, are known to elicit neutrophil death via a caspase-independent mechanism; likewise, intracellular reactive oxygen species (ROS) production also induces caspase-independent neutrophil apoptosis. Interestingly, activity levels of superoxide dismutase (SOD), were found in significantly lower levels in SCD serum compared to CON serum (data not shown), suggesting that SCD serum may have a greater capacity to induce ROS production in neutrophils. Notably, serum from SCDHU individuals did not stimulate increased apoptosis in control neutrophils, indicating a reduction in the serum factor(s) that elicit apoptosis in these cells. As such, Neu may be subject to both anti-and pro-apoptotic stimuli in the circulation of SCD individuals. Since alterations in Neu apoptotic processes may have significant effects on Neu function and number, in turn contributing to inflammatory processes and cellular damage at sites of inflammation, studies to understand the complex balance of these mechanisms in SCD are necessary.


Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 1982-1982
Author(s):  
Lanetta B Jordan ◽  
Francis Vekeman ◽  
Anirban Sengupta ◽  
Mitra Corral ◽  
Amy Guo ◽  
...  

Abstract Abstract 1982 Poster Board I-1004 Background: Patients with sickle-cell disease (SCD) receiving chronic transfusions of red blood cells are at risk of developing serious adverse effects. Iron chelating therapies (ICTs) help eliminate iron surplus by binding with plasma iron to form a non-toxic conjugate that can be safely excreted from the body. Two iron chelating agents are currently available in the US: deferoxamine (DFO) is an injectable formulation and deferasirox (Exjade®) is an oral suspension. This study compared the frequency of hospitalizations, persistence, and compliance of SCD patients from Medicaid programs treated with DFO versus deferasirox. Methods: An analysis of patients' electronic claims records from the Florida (1998-2007), Missouri (1993-2008), and New Jersey (1996-2008) Medicaid programs was conducted. Patients with continuous enrollment for at least six months prior to ICT initiation and ≥1 SCD diagnosis were included in the analysis. Patients were divided into four cohorts: patients treated with DFO (any DFO group) and patients treated with deferasirox (any deferasirox group); the latter was further divided into patients initiated on DFO and then switched to deferasirox (deferasirox switchers), and patients treated with deferasirox only (deferasirox only group). Frequency of hospitalization as well as length of stay pre- and post-ICT treatment initiation were assessed. Persistence was defined as time to drug discontinuation with at least one Rx gap, using Kaplan-Meier approach. Compliance was estimated using a medication possession ratio (MPR), calculated as the total days of supply divided by the number of days between the first and last dispensing plus the days of supply of the last dispensing. Results: A total of 217 (mean age=19.4), 275 (20.1), 105 (19.4), and 166 (20.4) patients were included in the any DFO, any deferasirox, deferasirox switchers, and deferasirox only groups, respectively. Exposure period, defined as the time from the date of the first dispensing to the end of the days supply of the last dispensing, was approximately two times longer in the DFO group than in the deferasirox groups (days, DFO: 783, any deferasirox: 353, deferasirox switchers: 416, deferasirox only groups: 317). After ICT initiation, both DFO and deferasirox groups experienced significant reduction in the frequency of hospitalizations relative to pre-treatment (DFO: from 0.92 to 0.64 hospitalizations per patient per month, P=.0010; any deferasirox: from 1.22 to 0.4, P<.0001). Median length of hospitalization stay remained similar after ICT initiation relative to pre-treatment in both the DFO group (pre- and post-ICT initiation: 3 days) and the any deferasirox group (from 3 days pre-ICT to 4 days post-ICT initiation). The Kaplan-Meier rates of medication persistence during the first year of ICT were significantly greater for all three deferasirox cohorts compared to the DFO cohort (see Table 1). Patients treated with deferasirox were significantly more compliant compared to the DFO group (see Table 2). Conclusions: Based on a Medicaid population, deferasirox patients were more compliant and persistent to their treatment than those treated with DFO. Frequency of hospitalizations was significantly reduced in the observation period for all ICT treated patients, although the length of hospitalization stay remained similar. Prospective studies are warranted to validate these results. Disclosures: Jordan: Analysis Group, Inc.: Consultancy. Vekeman:Analysis Group, Inc.: Employment; Novartis Pharmaceuticals Corporation: Research Funding. Sengupta:Analysis Group, Inc.: Employment; Novartis Pharmaceuticals Corporation: Research Funding. Corral:Novartis Pharmaceuticals Corporation: Employment. Guo:Novartis Pharmaceuticals Corporation: Employment. Duh:Novartis Pharmaceuticals Corporation: Research Funding; Analysis Group, Inc.: Employment.


Haematologica ◽  
2020 ◽  
Vol 105 (10) ◽  
pp. 2380-2390 ◽  
Author(s):  
Nicola Conran ◽  
Erich V. De Paula

Sickle cell disease (SCD) is an inherited hemoglobinopathy that is caused by the presence of abnormal hemoglobin S (HbS) in red blood cells, leading to alterations in red cell properties and shape, as the result of HbS dexoygenation and subsequent polymerization. SCD pathophysiology is characterized by chronic inflammatory processes, triggered by hemolytic and vaso-occlusive events, which lead to the varied complications, organ damage and elevated mortality seen in individuals with the disease. In association with activation of the endothelium and leukocytes, hemostatic alterations and thrombotic events are well-documented in SCD. Here we discuss the role for inflammatory pathways in modulating coagulation and inducing platelet activation in SCD, due to tissue factor activation, adhesion molecule expression, inflammatory mediator production and the induction of innate immune responses, amongst other mechanisms. Thromboinflammatory pathways may play a significant role in some of the major complications of SCD, such as stroke, venous thromboembolism and possibly acute chest syndrome, besides exacerbating the chronic inflammation and cellular interactions that trigger vaso-occlusion, ischemia-reperfusion processes, and eventually organ damage.


Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 3383-3383
Author(s):  
Jing Li ◽  
Andrew Barazia ◽  
Kyungho Kim ◽  
Jaehyung Cho

Abstract We recently demonstrated that the basal levels of AKT phosphorylation were significantly increased in neutrophils and platelets isolated from sickle cell disease (SCD) patients compared with those cells from healthy donors and that specific inhibition of AKT2 impaired neutrophil-endothelial cell and neutrophil-platelet interactions in venules of Berkeley (SCD) mice (Li et al. J Clin Invest 2014). Although hydroxyurea (HU), an inducer of fetal hemoglobin, is the main therapy for treatment of SCD, it is unclear whether the short-term treatment with HU has immediate benefits on acute vaso-occlusive events in SCD. Using real-time fluorescence intravital microscopy, we demonstrated that co-administration of HU (100 microg/g body weight, iv injection) and Akti XII (3 microg/g body weight, iv injection), an AKT2 inhibitor, efficiently reduced neutrophil adhesion and platelet-neutrophil aggregation in cremaster muscle venules of TNF-α-challenged Berkeley mice. Importantly, compared with HU or Akti XII treatment alone, treatment with both agents significantly improved blood flow rates and survival in the mice. Further, similar results were obtained in Berkeley mice challenged with hypoxia (8% oxygen for 3 hours) and subsequent reoxygenation (room air for 3 hours). As determined by histochemistry of cremaster muscle sections following intravital microscopy of Berkeley mice, the expression of endothelial E-selectin and intercellular adhesion molecule 1 (ICAM-1) was significantly decreased by treatment with either HU or Akti XII. Leukocyte transmigration in the lung section of Berkeley mice was also inhibited by either agent, and the inhibitory effect was potentiated by co-administration of HU and Akti XII. Using the plasma and isolated cells of Berkeley mice, we found that the level of plasma nitric oxide species (NOx) was significantly elevated by treatment with HU but not Akti XII and that AKT2 phosphorylation levels in activated neutrophils and platelets were reduced by treatment with Akti XII but not HU. Taken together, these results suggest that short-term treatment of Berkeley mice with either HU or Akti XII inhibits inflammatory conditions: treatment with HU significantly increases plasma NOx levels, treatment with Akti XII decreases AKT2 phosphorylation in neutrophils and platelets without affecting plasma NOx levels, and administration of either agent reduces the surface expression of ICAM-1 and E-selectin on activated endothelial cells. Thus, our results provide evidence that co-administration of HU and a specific AKT2 inhibitor has immediate benefits on acute vaso-occlusive events and survival in SCD mice exceeding those seen for single therapy. Disclosures No relevant conflicts of interest to declare.


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