Role In Vaso-occlusion Research Articles

COVID-19-induced endotheliitis: emerging evidence and possible therapeutic strategies.

The coronavirus disease 2019 (COVID‐19) pandemic, a viral illness caused by the severe acute respiratory syndrome coronavirus‐2 (SARS‐CoV‐2), 1 has produced at the time of this writing nearly 33 million cases of infection, with over a million deaths in 235 countries, 2 causing an unprecedented burden on healthcare systems and a severe global socioeconomic crisis. As the pandemic spreads, knowledge on the disease course, as well as potential risk factors and predictors of severity is increasing daily, and initial data from randomised controlled studies have allowed care providers to refine therapeutic strategies. Nonetheless, mortality is markedly elevated among those presenting with severe disease, long‐term sequelae among survivors are unknown, and vaccine‐based therapies currently remain at early stages of development. Most reported cases are asymptomatic or present with mild symptoms; however, 7–26% of hospitalised patients experience severe disease, often requiring admission to intensive care units (ICUs), with progressive multiple organ dysfunction and high mortality. 3 , 4 , 5 Such differences in clinical outcomes have led physicians to initiate diverse pharmacological therapies at various stages of the disease, generating challenges as to the most appropriate therapeutic choice for COVID‐19. In this context, the use of dexamethasone has significantly reduced mortality rates in critically ill patients requiring supplemental oxygen or mechanical ventilation, 6 and remdesivir has demonstrated clinical benefit in hospitalised patients, but with unknown survival benefit to date 7 ; additional effective treatment options are therefore urgently needed. In an initial attempt to provide a uniform and widely reproducible methodology to guide systematic treatment strategies, a three‐stage classification of COVID‐19 has been proposed. 8 The Stage I or ‘early infection’ occurs at the initial establishment of disease with high viral replication, and commonly presents with a range of complaints that can include mild and often non‐specific influenza‐like signs and symptoms. Stage II is the ‘pulmonary phase’, with preferential viral‐mediated injury of the lung parenchyma and this is characterised by shortness of breath, hypoxia and pulmonary infiltrates with some degree of lung inflammation. Stage III is characterised by an exaggerated host immune‐inflammatory response to the virus, leading to acute respiratory distress syndrome (ARDS) and multi‐organ failure (MOF).

Deletion of TLR4 in Townes SS Mice Prevents Microvascular Stasis in Response to Hemin, LPS and Hypoxia/Reoxygenation: Role of Inflammation

We have previously shown that heme, a damage-associated molecular pattern (DAMP), released from sickle RBCs interacts with the innate immune toll-like receptor 4 (TLR4) on endothelium and blood cells to activate cell signaling. This activates the pro-inflammatory transcription factor NF-κB leading to the production of cytokines and adhesion molecules that promote inflammation, coagulation, and vaso-occlusion (VO). Previous studies in our lab have demonstrated that inhibition of TLR4 in Townes-SS mice with TAK242 reduces microvascular stasis in response to hemin, hypoxia/reoxygenation and LPS. To further delineate the role of TLR4 in sickle cell disease (SCD), 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. These Tlr-/- Townes mice were backcrossed 10 generations to homogenize their genetic background with our Tlr4+/+ Townes colony. Townes-SS Tlr4-/- mice developed less microvascular stasis than Townes-SS Tlr4+/+ mice in response to challenges with heme infusion (4% vs 30% at 1 hr, p<0.05, Fig 1A); LPS infusion (13% vs 36% at 1 hr, p<0.05, Fig 1B); and hypoxia/reoxygenation (3% vs 20% at 1 hr, p<0.05, Fig 1C). We next measured the complete blood counts, serum chemistries, and urine analytes in the Tlr4+/+ and -/- mice. We also quantified mRNA levels via qPCR for liver and kidney cytokines, adhesion molecule, heme oxygenase 1 (HO-1) and coagulation factors in all four genotypes (AA Tlr4+/+; AA Tlr4-/-; SS Tlr4+/+; SS Tlr4-/-). Hemoglobin levels in SS mice were lower than AA mice, but there were no differences between Tlr4+/+ and Tlr4-/- mice. WBC counts were greater in SS than AA mice, but there were no differences between Tlr4+/+ and Tlr4-/- mice. Bilirubin and liver function tests (LFTs) were higher in SS than AA, but there were no differences between Tlr4+/+ and Tlr4-/- mice. In the liver, adhesion molecules and HO-1 were higher in SS than AA, but there were no differences between Tlr4+/+ and Tlr4-/- mice. To define a potential mechanism for decreased microvascular stasis in response to hemin infusion in SS Tlr4-/-, we measured cytokines and adhesion molecule expression after heme challenge. When SS Tlr4+/+ mice were infused with hemin, we found increased expression of pro-inflammatory cytokines and cell adhesion molecules. SS Tlr4-/- mice infused with hemin had reduced expression of cytokines and cell adhesion molecules compared to SS Tlr4+/+ mice; most notably in IL-6 in the liver, adhesion molecules VCAM-1 and ICAM-1 in the liver and kidneys (p<0.05), and P-selectin and von Willebrand factor in the lungs (Fig 2). Our data thus far indicate that TLR4 of the innate immune system plays a critical role in VO and inflammation in challenged SS mice. We speculate that modulation of TLR4 with targeted inhibitors would be beneficial for SCD patients. Disclosures Belcher: Mitobridge, an Astellas Company: Consultancy, Research Funding. Vercellotti:Mitobridge, an Astellas Company: Consultancy, Research Funding.

Disrupting the vicious cycle created by NOX activation in sickle erythrocytes exposed to hypoxia/reoxygenation prevents adhesion and vasoocclusion

In sickle cell disease (SCD), recurrent painful vasoocclusive crisis are likely caused by repeated episodes of hypoxia and reoxygenation. The sickle erythrocyte (SSRBC) adhesion plays an active role in vasoocclusion. However, the effect of prolonged reoxygenation after hypoxic stress on the molecular mechanisms in SSRBCs involved in onset of episodic vasoocclusion remain unclear. Exposure of human SSRBCs to hypoxia followed by 2 h reoxygenation, increased reactive oxygen species (ROS) production. Using specific pharmacological inhibitors, we show that excess ROS production in both reticulocytes and mature SSRBCs is regulated by NADPH oxidases (NOXs), the mitogen-activated protein kinase (ERK1/2), and G-protein coupled-receptor kinase 2 (GRK2). Consequently, SSRBC ROS create an intracellular positive feedback loop with ERK1/2 and GRK2 to mediate SSRBC adhesion to endothelium in vitro, and vasoocclusion in a mouse model of vasoocclusion in vivo. Importantly, reducing ROS levels in SSRBCs with redox-active manganese (Mn) porphyrins, commonly known as mimics of superoxide dismutase (SOD), disrupted the cycle created by ROS by affecting NOX and GRK2 activities and ERK1/2 phosphorylation, thus abrogating RBC-endothelial interactions. Inhibition adhesion assays show that LW (ICAM-4, CD242) blood group glycoprotein and CD44 are the RBC adhesion molecules mediating endothelial binding. Conversely, hypoxia/reoxygenation of normal RBCs failed to activate this feedback loop, and adhesion. These findings provide novel insights into the pathophysiological significance of the deleterious cycle created by NOX-dependent ROS, GRK2 and ERK1/2 within SSRBCs activated by hypoxia/reoxygenation, and involved in SSRBC adhesion and vasoocclusion. Thus, this loop in SSRBCs, which can be disrupted by Mn porphyrins, likely drives the profound SCD vasculopathy, and may point to new therapeutic targets to prevent chronic vasoocclusive events.

Critical role of C5a in sickle cell disease.

Innate immune complement activation may contribute to sickle cell disease (SCD) pathogenesis. Ischemia-reperfusion physiology is a key component of the inflammatory and vaso-occlusive milieu in SCD and is associated with complement activation. C5a is an anaphylatoxin, a potent pro-inflammatory mediator that can activate leukocytes, platelets, and endothelial cells, all of which play a role in vaso-occlusion. We hypothesize that hypoxia-reoxygenation (H/R) in SCD mice activates complement, promoting inflammation and vaso-occlusion. At baseline and after H/R, sickle Townes-SS mice had increased C3 activation fragments and C5b-9 deposition in kidneys, livers and lungs and alternative pathway Bb fragments in plasma compared to control AA-mice. Activated complement promoted vaso-occlusion (microvascular stasis) in SS-mice; infusion of zymosan-activated, but not heat-inactivated serum, induced substantial vaso-occlusion in the skin venules of SS-mice. Infusion of recombinant C5a induced stasis in SS, but not AA-mice that was blocked by anti-C5a receptor (C5aR) IgG. C5a-mediated stasis was accompanied by inflammatory responses in SS-mice including NF-κB activation and increased expression of TLR4 and adhesion molecules VCAM-1, ICAM-1, and E-selectin in the liver. Anti-C5aR IgG blocked these inflammatory responses. Also, C5a rapidly up-regulated Weibel-Palade body P-selectin and von Willebrand factor on the surface of human umbilical vein endothelial cells in vitro and on vascular endothelium in vivo. In SS-mice, a blocking antibody to P-selectin inhibited C5a-induced stasis. Similarly, an antibody to C5 that blocks murine C5 cleavage or an antibody that blocks C5aR inhibited H/R-induced stasis in SS-mice. These results suggest that inhibition of C5a may be beneficial in SCD.

Complement Activation in a Murine Model of Sickle Cell Disease: Inhibition of Vaso-Occlusion By Blocking C5 Activation

▪IntroductionInnate immune complement activation may contribute to sickle cell disease (SCD) pathogenesis. The alternative complement pathway is abnormally activated in SCD and is additionally activated by phosphatidylserine (PS) on the outer leaflet of SS-red blood cells (SS-RBC). PS on the surface of SS-RBC and activated platelets accelerates the assembly of prothrombinase complexes leading to generation of thrombin, which can cleave circulating C5 protein into two biologically active fragments, C5a and C5b. C5a is an anaphylatoxin, a potent pro-inflammatory mediator, that can activate leukocytes, platelets, and endothelial cells, all of which play a role in vaso-occlusion (VO). Active C5b fragments stimulate the formation of membrane attack complexes (MAC) on SS-RBC that increases their susceptibility to lysis. We hypothesize that complement activation on the surface of SS-RBC may stimulate VO and RBC turnover in SCD.Methods and ResultsWhole blood from Townes-AA, -AS, and -SS mice (n=4) was collected in EDTA and RBC were immunostained with erythroid specific anti-Ter119 (Ly-76) IgG conjugated to PE-Cy7, anti-C5b-9 (MAC) IgG conjugated to Alexa Fluor 647, and anti-C3 (also specific for activation fragments C3b, iC3b, C3d and C3dg) IgG conjugated to PE. The percentages of Ter119-positive RBC that were positive for MAC were 6.3%, 6.5%, and 26.0% for AA-, AS-, and SS-RBCs, respectively (p<0.01 SS vs. AA and AS) suggesting enhanced C5 activation in Townes-SS mice. However, EDTA plasma C5a levels measured by ELISA were not significantly different between Townes-AA, -AS and -SS mice. C3 activation fragments were also found on a subset of MAC positive RBC. We used a mouse model of hypoxia/reoxygenation (H/R)-induced stasis to investigate the role of complement in VO. Townes-SS mice with implanted dorsal skin-fold chambers were infused with 30 µg of either anti-C5 IgG mAb BB5.1 (which blocks murine C5 cleavage ) (n=4), annexin V (n=2), or with isotype control IgG (n=4) 30 minutes prior to H/R (1 hour of hypoxia at 7% O2, followed by reoxygenation for 4 hours in room air). Percent microvascular stasis (% non-flowing venules) in the subcutaneous venules in the dorsal skin fold chamber window was measured using intravital microscopy at 1 and 4 hours post-hypoxia. Percent stasis was significantly lower in the Townes-SS mice receiving anti-C5 IgG or annexin V than the mice receiving the isotype control IgG at 1 and 4 hours post-hypoxia (Figure 1, means ± SD, *p<0.01 anti-C5 IgG or annexin V vs. control IgG). This finding implies that H/R may acutely activate C5 , thus leading to stasis. Initial studies suggest increased plasma C5a levels in Towne-SS mice treated with isotype control IgG compared to mice treated with anti-C5 IgG in response to H/R.ConclusionsThese results demonstrate an increased percentage of SS-RBC expressing MAC on their surface and inhibition of H/R-induced stasis with anti-C5 IgG. However, circulating C5a levels were not different between untreated Townes-AA, -AS, or -SS mice suggesting that C5a is not chronically elevated in SCD. Rather, the generation of C5a may occur locally and in the acute setting. Current studies underway are examining the effects of anti-C5 IgG on MAC deposition and RBC half-lives; the mechanism(s) of complement activation in SCD mice (classical, alternative, tic-over, etc); the ability of recombinant murine C5a to induce stasis; and complement activation markers on RBC and in plasma from SCD patients and controls. [Display omitted] DisclosuresChen:Imara: Research Funding. Belcher:Imara: Research Funding; CSL-Behring: Research Funding. Vercellotti:Imara: Research Funding; CSL-Behring: Research Funding.

Neutrophil-Platelet Aggregation Enables Vaso-Occlusion in Sickle Cell Disease

Introduction: Sickle Cell Disease (SCD) is an autosomal-recessive-genetic disorder that leads to sickling and hemolysis of red blood cells (RBCs). Acute vaso-occlusive pain crisis (VOC) is the predominant pathophysiology faced by SCD patients and the primary reason for emergency medical care. Although neutrophils have been shown to play a role in vaso-occlusion by interacting with sickle RBCs in the cremaster venules of transgenic SCD mice, the cellular, molecular and biophysical mechanisms that promote vaso-occlusion in SS patients is not completely understood. Materials and Methods: Freshly collected heparinized blood from steady-state SS patients and race matched control (AA) subjects was perfused through silicone based microfluidic flow channels with a glass bottom coated a cocktail of recombinant human P-selectin, ICAM-1 and IL-8 at a physiological wall shear stress (6 dyn cm-2). Fluorescent Abs against CD16 and CD49b were added to the blood for in-situ staining of neutrophils and platelets, respectively. Cellular interactions were recorded at a single cell-resolution using quantitative microfluidic fluorescence microscopy (qMFM)1, which allows quantitative assessment of vaso-occlusive events at an unprecedented single cell resolution2. Results: Vaso-occlusion in the microfluidic channel involved neutrophil arrest followed by nucleation of platelets on arrested neutrophils, formation of neutrophil-platelet-aggregates (NPA) and partial occlusion of the microfluidic flow channel. Remarkably, the number of platelet-neutrophil interactions and the lifetime of these interactions were several folds higher in SS patient than control AA blood. Surprisingly, preincubation with 250 ng/ml of bacterial lipopolysaccharide (LPS) led to a significant increase in the number and lifetime of platelet-neutrophil interactions in SS but not AA blood. This enhanced NPA formation in SS patient blood was attenuated to the level observed in AA blood by simultaneous blockage of P-selectin on platelets and Mac-1 on neutrophils as well as pretreatment with a small molecule inhibitor of toll-like-receptor-4 (TLR4) signaling pathway. Conclusion: Our data shows that the vaso-occlusive pathophysiology in SCD involves sequential steps of neutrophil arrest, nucleation of platelets on arrested neutrophils, formation of large NPAs and obstruction of blood flow. Platelet-neutrophil aggregation can be ameliorated by the simultaneous blockage of P-selectin on platelets and Mac-1 on neutrophils. The inflammatory milieu of SS patient blood sets a lower threshold for bacterial endotoxin induced neutrophil-platelet aggregation than control blood. The enhanced platelet-neutrophil aggregation in SS blood is dependent on activation of TLR-4 pathway. Understanding the molecular mechanism of vaso-occlusion will enable the development of therapeutics to prevent VOC in SS patients.

Association of homocysteine and inflammatory-related molecules in sickle cell anemia

Objective: Investigate the role of homocysteine (Hcy), Th17-related cytokines, and adhesion molecules in the inflammatory state seen in the sickle cell anemia (SCA).Methods: We studied the Hcy, interleukin (IL)-17, and transforming growth factor β (TGF-β) cytokine levels of 62 SCA patients, as well as the expression levels of inflammatory and endothelial activation markers.Results: We found significant associations between Hcy levels and increased expression of IL-17 and TGF-β among SCA patients, and a positive significant correlation between Hcy and soluble vascular cellular adhesion molecules (sVCAM). SCA individuals had raised IL-17 levels when compared with controls.Discussion: These results suggest a possible role of Hyc in the induction of TGF-β and IL-17. Other authors proposed that Hcy may contribute to the initiation and progression of vascular disease by monocyte activation, resulting in the secretion of cytokines that amplify the inflammatory response. The role of Hcy in cytokine production and oxidative stress in the endothelium may explain the increase of sVCAM expression and, the vascular activation currently described among the SCA individuals with the highest Hcy serum levels. The chronic inflammation was observed in hyperhomocysteinemic mice, with an increased expression of VCAM-1 and plasma levels of tumor necrosis factor-alpha, showing an association of this inflammatory molecule and vascular changes.Conclusion: Our findings suggest that the increased levels of IL-17,Hcy and sVCAM contributes contributes to the vascular inflammation and activation presented by SCA patients, which probably have an important role in vaso-occlusion. On the basis of the presented data, IL-17 and Hcy might be considered as important components in the pathogenesis of SCA.

Inhibition Of the Rho/Rho-Kinase Pathway Reduces In Vitro Adhesion Of Sickle Cell Anemia Eosinophils To Human Endothelial Cells

IntroductionSickle cell anemia (SCA) individuals present a chronic inflammatory condition that contributes to the recurrent episodes of vaso-occlusion. This process is a multi-step process that involves different cell types such as red blood cells, reticulocytes, activated endothelial cells, platelets and leukocytes. Endothelial dysfunction contributes to the vaso-occlusion process and leads to inflammation. Leukocytes may have a propagating and, possibly, initiating role in SCA vaso-occlusion. In vivo and in vitro studies using animal models show that neutrophils play a key role in vaso-occlusion; however, eosinophils (EOs) may also participate in this phenomenon in humans. We have previously shown that SCA eosinophils demonstrating greater adhesion to fibronectin. A small GTPase, Rho, and its target molecule, Rho-kinase, play an important role in the cell functions, including contractility, chemotaxis, adhesion, and migration. Y-27632 and NSC23766 are Rho-kinase inhibitors, that reduce leukocyte infiltration in several models of EOs inflammation, such as endotoxic liver and lung injury. The objective of this study was to investigate the effect of Rho kinase pathway on the in vitro adhesion of sickle EOs to activated human endothelial cell layers (umbilical vein endothelial cells, HUVEC). MethodsEosinophils were isolated from peripheral blood of healthy controls (n=5), steady-state SCA patients (SCA, n=7) and SCA patients on HU therapy (SCAHU, n=5), using Percoll gradient separation, followed by immunomagnetic sorting. EO adhesion (1x106 cell/ml in Ham's F12 K) to cultured HUVEC grown to confluence was assessed using static adhesion assays. HUVEC cells were treated, or not, with 100 µM NSC23766 or Y-27632 for 1h before incubation with an inflammatory stimulus (10nM TNF-alpha – 3 h), subsequently Eos were added to the cell layer (1 hour, 37°C, 5%CO2). For adhesion under flow conditions, biochips with microchannels coated with HUVEC were pre treated or not with Y-27632 or NSC23766 per 1 h before the TNF-alphastimulus. Purified eosinophils was added to HUVEC-coated microchannels at a flow rate of 0.5 dynes/cm2. Images of adherent eosinophils were recorded using real-time video microscopy and analysed using Duco Cell software. ResultsEOs from SCA patients demonstrated a significantly greater adhesion to HUVEC than healthy controls (14.42 ± 1.8% vs 9.35 ± 1.7 %; p<0.02); no significant difference was observed between the SCAHU group, compared to the healthy control and SCA group (10.5 ± 1.6%). Pre treatment of HUVEC with TNF-alpha significantly augmented the adhesion of healthy control, SCA, and SCAHU eosinophils to HUVEC (16.1 ± 3.2%, 33.1 ± 5.1%, 17.9 ± 3.5%, P<0.01, respectively). However, this adhesion was significantly higher for SCA EOs compared to healthy control and SCAHU EOs (p<0.05). Interestingly, when the HUVEC was pre-treated with NSC23766 then stimulated with TNF-alpha, adhesion of EOs from SCA and SCAHU was significantly reduced (22.9 ± 5,6%, 17.2 ± 6.1%, respectively, p<0.05). However, when HUVEC was pre-treated with Y-27632 no significant difference was found. In addition, EOs from SCA patients demonstrated greater adhesion to HUVEC under flow conditions when compared to healthy control and SCAHU. EOs when the HUVEC was protect with Rho inhibitors (Y-27632 and NSC23766) before stimulation with TNF-alpha, EOs adhesion under physiological shear stress was diminished in all groups investigated (healthy control, SCA and SCAHU). ConclusionRho kinase inhibitors were able to reduce endothelial activation and consequent eosinophil adhesion, in an in vitro model. Therapy with such Rho-kinase inhibitors could be used in patients with SCA, with beneficial effects on the vascular occlusion process.Financial Support: FAPESP/CNPq/INCTS Disclosures:No relevant conflicts of interest to declare.

Fucosylation Inhibitor, 2-Fluorofucose, Inhibits NF-κB Activation and Vaso-Occlusion In Transgenic Sickle Mice

Polymorphonuclear cells (PMNs) roll along vessel walls by binding to E- and P-selectins on the endothelial surface. These vascular adhesion molecules are involved in the initial tethering of PMNs on endothelium rather than their firm adhesion. Endothelial cell–expressed P- and E-selectins interact with leukocyte counterreceptors P-selectin glycoprotein ligand-1 (PSGL-1) and E-selectin ligand-1 (ESL-1). These glycoprotein counterreceptors are active only when modified posttranslationally by fucosylated oligosaccharides represented by the sialyl Lewis x tetrasaccharide (sLex) or its structural variants. Because of the important role that fucose plays in protein glycosylation and cellular functions, specific inhibitors of its incorporation could have significant applications in basic research and therapy. One such inhibitor, 2-fluorofucose peracetate, was recently reported to block fucosylation of cellular proteins and to inhibit binding of PMNs to P- and E-selectin. One potential application is the inhibition of leukocyte recruitment in pro-inflammatory disease states such as sickle cell disease (SCD). P-selectin expression on endothelium has been demonstrated to play an important role in vaso-occlusion, a hallmark of SCD. Inhibition of P-selectin binding inhibits vaso-occlusion in transgenic sickle mice. Therefore, we hypothesized that 2-fluorofucose would inhibit tissue inflammation and vaso-occlusion in SCD mice. MethodsNY1DD sickle mice were given plain water or 20 or 100 mM 2-fluorofucose in their drinking water ad libitum. The first cohort of mice (n=8/group) was treated for 7 days. On day 7 the mice were sacrificed with CO2, an EDTA blood sample was collected from the heart and the liver was removed and frozen in liquid nitrogen. Total white blood cell counts and differentials were performed by manual counting using a hemocytometer and Wright-stained blood smears, respectively. Nuclear extracts were prepared from nuclei isolated from liver homogenates. Western blots of the nuclear extracts were immunostained with antibodies to NF-ĸB phospho-p65 a marker of NF-ĸB activation. The second cohort of mice (n=4/group) was also treated for 7 days. On day 4, dorsal skin fold chambers were implanted onto the mice. On day 7, flowing venules in the dorsal skin-fold chamber window were selected and mapped using intravital microscopy. Thereafter, the mice were infused via the tail vein with human stroma-free hemoglobin (0.32 µmols heme/kg), a known inducer of vascular stasis in SCD mice. At 1 and 4 hours after infusion the same venules were re-examined and the percentage of vessels that had become static (no flow) was recorded. A positive control group of NY1DD mice (n=3) with implanted dorsal skin-fold chambers was given water to drink and pretreated with hemin (40 µmols/kg X 3 days, i.p.), a known inhibitor of vascular stasis. ResultsWhite blood cells counts were 16.3±3.2 (K/µL, mean±SD) in SCD mice treated with water. The white counts increased to 22.1±5.2 in SCD mice treated with 20 mM 2-fluorofucose and 34.2±7.2 in SCD mice treated with 100 mM 2-fluorofucose, respectively (p<0.05 for all pairwise comparisons). NF-ĸB in liver and other organs is activated in SCD mice compared to normal C57BL/6 mice. Nuclear NF-ĸB phospho-p65 was partially diminished in mice treated with 20 mM 2-fluorofucose and markedly decreased in mice treated with 100 mM 2-fluorofucose or pretreated with heme (Fig. 1).Infusion of hemoglobin induced ∼30% microvascular stasis at 1 and 4 hours in SCD mice treated with water (Fig. 2). Treatment with 20 mM 2-fluorofucose partially inhibited stasis at 1 and 4 hours (p<0.05 compared to water). Treatment with 100 mM 2-fluorofucose or heme inhibited stasis to 6.7% or less at 1 and 4 hours (p<0.025 compared to water). [Display omitted] [Display omitted] ConclusionsOral administration of 2-fluorofucose has significant anti-inflammatory and anti-vaso-occlusive properties in SCD mice. Disclosures:Belcher:Seattle Genetics, Inc.: Research Funding. Chen:Seattle Genetics, Inc.: Research Funding. Nguyen:Seattle Genetics, Inc.: Research Funding. Okeley:Seattle Genetics, Inc.: Employment. Senter:Seattle Genetics, Inc.: Employment. Benjamin:Seattle Genetics, Inc.: Employment. Vercellotti:Seattle Genetics, Inc.: Research Funding.

Cigarette Smoking Is An Independent Predictor of Chronic Pain In Sickle Cell Patients: Results From the Walk-PHaSST Study

AbstractAbstract 4804 Introduction:Smoking is known to promote vascular inflammation, in-vitro platelet aggregation and expression of endothelial adhesion molecules, processes that contribute to vasculopathy. Inflammation, abnormal platelet activation with thrombus formation and endothelial cell activation also play a role in vaso-occlusion in sickle cell disease (SCD). These overlapping pathobiological mechanisms suggest the possibility of a relationship between smoking and SCD vaso-occlusive pain. While small single center studies have suggested a link between environmental smoke exposure and hospitalization rate for acute chest syndrome and SCD pain (West et al 2003, Cohen et al 2010) there is a paucity of data derived from large multicenter studies about the interplay between smoking and pain phenotype in SCD. Aims:To determine the relationship between patient self reported chronic pain and history of current or former cigarette smoking in the SCD subjects screened in the walk-PHaSST study. Methods:Walk-PHaSST was a multi-center, placebo-controlled, double-blind 16-week trial designed to evaluate the safety and efficacy of sildenafil in patients with tricuspid regurgitant velocity [TRV] ≥2.7m/s and decreased exercise capacity as assessed by the six-minute walk distance (6MWD).We analyzed the data from all subjects screened for the walk-PHaSST trial. In the screening trial, subjects were evaluated by medical history, physical examination, laboratory screening, echocardiography and 6MWD testing.Univariate and stepwise multivariable logistic regression was used for this analysis. P value of <0.05 was considered statistically significant. Results:Of the 720 patients screened, medical history on pain and smoking was obtained in a total of 673 subjects. Of these, 483/673 subjects (72%) had HbSS disease and 137 (20.4%) had HbSC disease. Mean age was 36.6 years (median 36.1 years).A total of 104 (15.5%) were current smokers and 114 (17.4%) reported having smoked in the past and 451 (67.1%) subjects reported no history of any life-time or current smoking. Subjects had smoked for a mean of 11.5 years (median 9 years, range 1–42 years). The mean pack years of smoking were 8.8 (median 5 years, range <1- 60 pack years).In a multivariable logistic regression model, being a ‘current’ cigarette smoker was associated with an increased odds ratio (OR 3.0, 95% CI 1.8–4.9) (See Table 1) of reporting chronic SCD pain as compared to no smoking history when adjusted for 1) self reported acute pain, 2) age, 3) gender, 4) hematocrit 4) marijuana use, 5) SCD genotype and 6) current hydroxyurea treatment.Additionally, in the multivariable model, being a ‘former’ cigarette smoker was associated with a smaller effect size (OR 2.2, 95% CI 1.4–3.5) of reporting chronic SCD pain as compared with no smoking history when adjusted for the above listed variables.On examining by SCD genotypes, the effect size was similar for HbSS genotype (OR 2.79, 95% CI: 1.54–5.0) while the effect size was significantly higher for HbSC genotype (OR 5.6, 95% CI: 1.7–19.0).The increase in self reported pain with number of pack years adjusted for age was not statistically significant although an increase in reported pain was observed with increase in the number of pack years (See Figure 1). Conclusions:These data suggest that being a current or former smoker is independently associated with ‘self reported chronic SCD pain’ after adjusting for potential confounding variables including age of the patient, gender, hematocrit, marijuana use and being on hydroxyurea treatment.However, it is unclear whether smoking might worsen pain, represent a stress relieving behavior by patients to chronic pain or even provide a beneficial adaptation to chronic pain mediated by nicotine, carbon monoxide, nitric oxide, or other substances known to be present in tobacco smoke. These data provide a rationale for further mechanistic studies of the relationship between smoking and chronic pain in patients with SCD.Table 1:Odds Ratio Estimates: Multivariable AnalysisEffectODDS RATIO Point Estimate95% Wald Confidence LimitsSMOKING Current smoker vs None3.01.85.0SMOKING Former smoker vs None2.21.43.5Age (per 10 yrs increment)1.31.11.5Gender: Female vs Male1.30.91.9Marijuana Use1.30.82.1SS vs Sß01.30.27.8SC vs Sß01.70.311.0Sß+ vs Sß02.50.318.3Hematocrit1.01.01.0Current Hydroxyurea Use0.80.51.1 [Display omitted] Disclosures:Hassell: Novartis: Research Funding. Barst: Pfizer: Consultancy, Research Funding. Rosenzweig: Pfizer: Research Funding. Badesch: Pfizer: Honoraria, Research Funding.

Sickle Red Cell–Endothelium Interactions

Periodic recurrence of painful vaso-occlusive crisis is the defining feature of sickle cell disease. Among multiple pathologies associated with this disease, sickle red cell-endothelium interaction has been implicated as a potential initiating mechanism in vaso-occlusive events. This review focuses on various interrelated mechanisms involved in human sickle red cell adhesion. We discuss in vitro and microcirculatory findings on sickle red cell adhesion, its potential role in vaso-occlusion, and the current understanding of receptor-ligand interactions involved in this pathological phenomenon. In addition, we discuss the contribution of other cellular interactions (leukocytes recruitment and leukocyte-red cell interaction) to vaso-occlusion, as observed in transgenic sickle mouse models. Emphasis is given to recently discovered adhesion molecules that play a predominant role in mediating human sickle red cell adhesion. Finally, we analyze various therapeutic approaches for inhibiting sickle red cell adhesion by targeting adhesion molecules and also consider therapeutic strategies that target stimuli involved in endothelial activation and initiation of adhesion.

Acute Lung Inflammation in Sickle Mice Is Mediated by Increase of CXC Chemokines and Matrix Metalloproteinases

Studies in vitro, and in vivo using animal models show that leukocytes play a key role in vasoocclusion and clinical research suggests that high leukocyte counts correlate with mortality, stroke and acute chest syndrome in sickle cell disease (SCD). Lungs are particularly vulnerable to vaso-occlusive events because of their anatomic features in SCD. Transgenic mice expressing exclusively human sickle hemoglobin (SS) are well-established models for the study of vascular inflammation. Previous studies have shown that systemic LPS challenge causes exaggerated inflammation, including increased serum and broncoalveolar lavage (BAL) TNF-α and IL-1 cytokines and sVCAM-1 in sickle mice. The aim of this study was to examine the contribution of acute airway inflammation in SCD using SS mice and the role of chemokines and matrix metalloproteinases (MMPs) in this process. Acute lung inflammation and injury were induced by intranasal administration of lipopolysaccharide (LPS, 50 μl of 250 μg/ml) in control (C57BL/6) and SS mice. The vehicle mice group received a similar volume of sterile PBS. BAL was performed 4 h after LPS challenge. qRT-PCR analysis was used to examine gene expression and ELISA protein production. The intranasal administration of LPS to mice triggered a huge influx of leukocytes (neutrophils, NS) in BAL of control and SS mice compared with the respective vehicle groups, but this influx was greater in SS mice, when compared with control mice (1.4 ± 0.06 vs 0.66 ± 0.12 WBCx106/BAL); p=0.0006, 1.06 ± 0.1 vs 0.40 ±0.12 NSx106/BAL; p=0.004, respectively). At baseline levels, KC and MIP-2 chemokines (functional homologues of human IL-8 in mice) are higher in BAL fluid of SS mice compared to control mice (186.6 ± 14.1 pg/ml vs 14.1 ± 5.8 pg/ml; 41.2 ± 7.9 pg/ml vs 11.4 ± 7.3 pg/ml, p=, respectively). Corresponding with influx of NS, lung lavage levels of KC and MIP-2 were significantly higher in SS BALF compared to control mice (2491 ± 454 pg/ml vs 798.1 vs 98.2 pg/ml; 1726 ± 307 pg/ml vs 887.3 ± 149.5 pg/ml, respectively). Enhanced levels of TNF-α were also observed at baseline and after LPS instillation compared to those of the control mice (20.8 ± 8.8 pg/ml vs 2.5 ± 1.6 pg/ml; 4250 ± 636 pg/ml vs 1585 ± 263 pg/ml, respectively). Instillation of LPS markedly increased KC, TNF-α, MMP-8, MMP-9 and TIMP-1 mRNA levels in the lungs of control and SS mice compared to animals that received PBS instead of LPS (Control, KC: 0.19 ± 0.047 vs 0.01 ± 0.005; TNF-α: 0.30 ± 0.07 vs 0.01 ± 0.002; MMP-8: 0.2 ± 0.06 vs 0.016 ± 0.004; MMP-9: 0.22 ± 0.03 vs 0.08 ± 0.01; TIMP-1: 0.32 ± 0.06 vs 0.09 ± 0.03); (SS, KC: 0.42 ± 0.1 vs 0.039 ± 0.02; TNF-α: 0.23 ± 0.025 vs 0.02 ± 0.007; MMP-8: 0.42 ± 0.06 vs 0.06 ± 0.03; MMP-9: 0.49 ± 0.11 vs 0.11 ± 0.05; TIMP-1: 0.49 ± 0.11 vs 0.09 ± 0.03). However, the LPS-induced KC, MMP-8 and MMP-9 expression was significantly higher in SS mice lung compared than that of the control group (p<0.05). Lung MMP-2, MMP-12 and TIMP-2 gene expressions were similar in the PBS and LPS groups and were not significantly different between SS and control mice. Our results indicate that chemokines and MMPs are critically involved in the recruitment of neutrophils to the lung following LPS challenge, and suggest that these inflammatory mediators may play a role in the development of pulmonary diseases in SCD. The findings from this study provide further support to the claim that a proinflammatory state is present in SCD and have important implications for the pathophysiology of lung injury in SCD.

Hemoglobin Blocks Von Willebrand Factor Proteolysis by ADAMTS-13: A Mechanism Associated with Acquired ADAMTS-13 Deficiency in Patients with Sickle Cell Disease

One of the hallmark events of sickle cell disease (SCD) is vasoocclusion and episodic pain crisis. Although the mechanism of vascular occlusion is very complicated, processes like thrombosis and thromboembolism have been recognized to play an important role in the development of such clinical manifestation in SCD. Studies have shown that the von Willebrand factor (VWF), especially the ultra-large (UL) multimers play a major role in vasoocclusion, which clearly indicates a possible impairment of the VWF-cleaving metalloproteae ADAMTS-13 in these patients with SCD. In a recent work, indeed we have mentioned that the plasma ADAMTS-13 in patients with SCD having normal antigen level showed 35% less protease activity than the normal. There may be several plasma factors responsible for the acquired deficiency of ADAMTS-13 in SCD. Since, the increasing evidences suggest that the elevated level of extracellular hemoglobin (Hb) in plasma parallely associated with the pathogenesis of SCD, we investigated the effects of extracellular Hb on VWF proteolysis by ADAMTS-13. We observed that purified Hb dose-dependently inhibited the ADAMTS-13 cleavage of recombinant(r) VWF and endothelial ULVWF multimers under static and flow conditions. Hb bound to VWF multimers in a saturation-dependent manner and more potently to the rVWFA2 domain (affinity Kd~24nM), which contains the cleavage site for ADAMTS-13. Hb bound also to the ADAMTS-13 (Kd~65nM), with 2.7 times less affinity than to VWFA2. The bindings were neither calcium-dependent nor affected by haptoglobin. However, it is the Hb-binding to VWF that prevented the substrate from being cleaved by ADAMTS-13. These in vitro findings are consistent with the in vivo observations in patients with SCD. An elevated level of extracellular Hb in plasma was inversely correlated (linear regression, r2 =0.6354) with the low activity of ADAMTS-13 in a cohort of ten adult patients with SCD (mean±SE, Hb 346±138 mg/l; activity 33.3±30%) compared to age and gender-matched normal individuals (n=10; Hb 24±8 mg/l; activity 76.2±16%). The data together suggest that patients with SCD suffer from acquired ADAMTS-13 deficiency, primarily because Hb competitively binds and inhibits the proteolysis of VWF multimers, leading to ULVWF accumulation on vascular endothelium and in circulation. The Hb-VWF interaction may therefore be considered as a therapeutic target for reducing thrombotic and vasoocclusive complications in patients with severe hemolysis such as those with SCD.

Intravenous Immunoglobulins Reverse Acute Vaso-Occlusive Crises in Sickle Cell Mice through Rapid Inhibition of Neutrophil Adhesion.

Previous studies using intravital microscopy in a sickle cell disease (SCD) mouse model suggest that adherent leukocytes (WBCs) play a key role in vaso-occlusion by capturing circulating erythrocytes (RBCs) in venules. Commercial intravenous human gamma globulin (IVIG) given prior to the inflammatory stimuli increased microcirculatory blood flow and improved survival (Blood 2004 103(6):2397–400). To mimic the clinical situation where SCD patients seek medical attention after the onset of symptoms, we have developed an in vivo model in which the therapeutic intervention was administered after in the inflammatory challenge. Berkeley SCD bone marrow was transplanted into irradiated C57BL/6 to generate age- and gender-matched genetically identical cohorts of SCD mice. Fully engrafted male SCD mice were treated with TNF-α and prepared for intravital microscopy examination of the cremaster muscle. Seventy minutes following administration of TNF-α (0.5 μg i.p.), IVIG (800 mg/kg) or an equivalent volume of control PBS or albumin (800 mg/kg) was administered by a programmable syringe pump. Twenty minutes after IVIG, PBS, or albumin exposure, 8–10 venules were recorded over a period of 60 min with each venule recorded continuously for at least 3 min. IVIG significantly increased the number of rolling leukocytes (2–3 fold, p<0.05) compared to control groups treated with albumin or PBS. Moreover, further analyses of leukocyte behavior revealed that IVIG significantly increased rolling velocities, indicating that it alters adhesion pathways involved in slow rolling. In contrast, the number of WBCs adherent to the endothelium was reduced by ∼50% in the IVIG group (p<0.05). The effect of IVIG on leukocyte adhesion was rapid, being statistically significant in the first ∼10 min after completion of IVIG infusion. The number of adherent WBCs increased progressively over time in the PBS group but remained lower throughout the recording period in the IVIG group. To assess the effect of IVIG on sickle RBC adhesion, we quantified the interactions between circulating RBCs and adherent WBCs, and found that IVIG dramatically reduced the number of RBC-WBC interactions by 10–19 fold (p<0.05). These effects resulted in improved microcirculatory blood flow and Kaplan-Meier curves revealed a dramatic improvement in survival of sickle cell mice in the IVIG-treated sickle cell mice compared to control groups. Longer survival correlated positively with blood flow (p=0.003) and negatively with the number of adherent WBCs (p=0.01) and RBC-WBC interactions (p=0.05). In order to exclude the possibility that irradiation and graft-versus-host disease were confounding factors, we treated parental (non-transplanted) sickle mice with IVIG, and found similar results, although statistical significance was only reached for RBC-WBC interactions (p<0.001) due to the small number of mice. In addition, IVIG treatment reduced the number of adherent WBCs recruited in wild-type C57BL/6 mice (p<0.002). Using multichannel digital fluorescence videomicroscopy that we recently developed (Nat Methods. 2007 4:219–22), we found that IVIG affected specifically the recruitment of adherent neutrophils, but did not change the number of adherent monocytes or lymphocytes. Thus, these results suggest that IVIG may improve VOC by inhibiting neutrophil activation, and underscore the encouraging possibility that therapeutic interventions initiated when patients have an established crisis may change the outcome of acute episodes, preventing their progression into life-threatening complications.

A Novel Selectin Antagonist, GMI-1070, Prevents Vaso-Occlusion in Sickle Cell Mice by Inhibiting Leukocyte Adhesion and Activation.

Previous studies using intravital microscopy in a sickle cell disease (SCD) mouse model (Berkeley) suggest that adherent leukocytes (WBCs) play a key role in vaso-occlusion by capturing circulating erythrocytes (RBCs) in venules. In addition, mice deficient in both P-and E-selectins are protected from vaso-occlusion (VOC) induced by surgical trauma and TNF-α stimulation, suggesting that targeting selectins or their ligands represents a potentially useful strategy. Selectins bind to specific sialylated and fucosylated carbohydrate structures presented by glycoprotein or glycolipid ligands. Here, we tested the effect of novel small glycomimetic selectin inhibitors, GMI-1070 and GMI-1077, on leukocyte behavior and sickle cell VOC. Berkeley SCD mouse bone marrow was transplantated into lethally irradiated C57BL/6 animals to generate age- and gender-matched genetically identical cohorts of SCD mice. Fully engrafted male SCD mice were treated with TNF-α and prepared for intravital microscopy examination of the cremaster muscle 90 min later. GMI-1070, GMI-1077 (both 20 mg/kg) or vehicle (PBS) were administered immediately prior to cytokine stimulation (t=0 min), and an additional dose was given at t=70min. Another group of mice was injected with antibodies against P-and E-selectins (PES, 1 mg/kg) as positive control. Several post-capillary and collecting venules were examined between t= 90min and t= 150min. Antibody blockade of endothelial selectins completely ablated leukocyte rolling, whereas GMI-1070 and GMI-1077 significantly increased the rolling flux fractions (PBS: 5.0±1.2 GMI-1070: 10.6±1.3%%; GMI-1077: 9.9±1.0%; p< 0.001). Furthermore GMI-1070 and GMI-1077 significantly reduced the recruitment of adherent leukocytes (914±172 and 1433±119 cells/mm2, respectively) compared to sickle mice injected with PBS control (2400±392 cells/mm2, p< 0.001). Although the reduction in leukocyte adhesion was not as marked as with anti-P and E-selectins (61±25 cells/mm2, p< 0.001), GMI-1070, in particular, dramatically inhibited the capture of sickle RBCs by adherent leukocytes (PBS: 0.9±0.4, GMI-1077: 0.6±0.2, GMI-1070: 0.07±0.05 and PES: 0.01±0.01 RBC interactions/WBC/min, p< 0.05) and markedly improved the blood flow in venules (PBS: 312±24, GMI-1077: 398±41, GMI-1070: 710±68 and PES: 683±75 nL/s, p< 0.001), to levels observed in non-sickle mice. The increased leukocyte rolling fluxes by these glycomimetics suggest that they inhibit E-selectin > P-selectin. Since the hallmark of E-selectin-mediated adhesion is the slow leukocyte rolling, we analyzed leukocyte rolling velocities in the various group and indeed found a 2-fold increase in rolling velocities in sickle mice treated with GMI-1070 compared to PBS control (PBS: 21±1 μm/s, GMI-1070: 38±1 μm/s, p<0.001). Consistent with these results, other studies using a parallel plate flow chamber (0.9 dynes/cm2) revealed that GMI-1070 was much more potent (1000-fold difference) in inhibiting the binding of human PMNs to TNF-α-stimulated (to induce E-selectin) endothelial cells (HUVEC) than with IL-4 and histamine stimulated HUVECs (to induce P-selectin). Further, competitive inhibition assays revealed that the IC50 of GMI-1070, relative to the standard glycyrrhizin, was much lower for E-selectin than P-selectin. These studies suggest that E-selectin-mediated adhesion/signaling may play a more important role than previously appreciated in the pathophysiology of SCD, and suggest that GMI-1070 may be beneficial for the treatment of sickle cell vaso-occlusion.

Sickle Cell Vaso-Occlusion Is Triggered by E-Selectin Ligand Signaling and Propagated by the Leukocyte Integrin Mac-1.

Vasoocclusion (VOC) is the leading cause of morbidity and mortality in patients with sickle cell disease (SCD). Intravital microscopy studies in a murine model of SCD have revealed that capture of sickle red blood cells (RBC) by intravascular adherent leukocytes (WBC) plays an important role in VOC, and that deficiency in both P-and E-selectins protect from VOC. Here, we have investigated the cellular and molecular mechanisms leading to sickle RBCs interactions with adherent WBCs. Intravital microscopy analyses of the individual role of P- or E-selectin revealed, unexpectedly, a profound reduction in RBC-WBC interactions in Berkeley sickle mice lacking E-selectin (Sele−/−; >97% reduction), whereas the protection was only partial in the absence of endothelial P-selectin. Since E-selectin is expressed exclusively on the endothelium, and its deficiency does not alter WBC recruitment, we hypothesized that E-selectin might provide activation signals to neutrophils that allow them to capture RBCs. During our studies, we observed that RBC-WBC interactions are not exclusive of sickle animals but are also present in wild-type B6 mice treated with TNF-α, suggesting that this phenomenon accompanies a physiological inflammatory response. We found that RBC-WBC interactions in B6 mice occur at a lower frequency than in sickle mice and that these interactions are also reduced in Sele−/− mice (60% reduction; p<0.05). We thus reasoned that B6 mice might provide a convenient model to gain molecular insight into RBC-WBC interactions in vivo. Since PSGL-1, CD44 and ESL-1 harbor the entire E-selectin ligand activity on neutrophils in vivo (Hidalgo et al., Immunity 2007), we investigated which of these glycoproteins mediates the signals allowing RBC capture. High speed digital multichannel fluorescence intravital microscopy analyses revealed that RBC-WBC interactions were only markedly reduced in the absence of ESL-1 (63% reduction, p<0.001), but not in the absence of PSGL-1 or CD44. Further detailed image analyses mapped RBC captures at the leading edge of adherent neutrophils, an area where chemokine receptors and integrins may accumulate. Since selectin-mediated signaling is known to activate β2 integrins, we tested the role of Mac-1, whose expression and affinity are elevated in neutrophils from SCD patients. We found that RBC-WBC interactions were virtually absent in mice deficient in Mac-1 (97% reduction; p<0.0001). To determine whether E-selectin/ESL-1-mediated signaling promoted Mac-1 activation, we developed an assay to assess Mac-1 activity in real time on adherent WBCs in vivo. Albumin-coated fluorobeads bound to subsets of adherent leukocytes in TNF-α-stimulated venules of B6 mice. These interactions were Mac-1-dependent since they were ablated in Itgam−/− mice. Absence of E-selectin or ESL-1, but not P-selectin, PSGL-1 or CD44, significantly reduced Mac-1 activity (by 36% and 52%, respectively; p<0.05). In contrast, Mac-1 activation on adherent leukocytes was dramatically increased in inflamed venules of sickle mice (2.5-fold; p<0.001) and was restricted to a subset of adherent neutrophils. Preliminary experiments in which Mac-1 function is blocked in sickle animals with a monoclonal antibody revealed a reduction of RBC-WBC interactions (by 57%) compared to an isotype control antibody. Our results indicate that the binding of E-selectin to neutrophil ESL-1 promotes Mac-1 activation, which in turn mediates the capture of sickle RBCs. These findings provide attractive therapeutic targets to alleviate this devastating disease.

Inhaled Carbon Monoxide: An Anti-Inflammatory Modulator in Transgenic Sickle Mice.

Inflammation plays a prominent role in vaso-occlusion and organ pathology in sickle cell disease (SCD). We have shown that heme oxygenase-1 (HO-1) plays a critical role in metabolizing the excess heme generated in this hemolytic disease, thereby modulating hypoxia/reoxygenation-induced vaso-occlusion in murine models of SCD. The products of HO-1 activity, carbon monoxide (CO), Fe2+ (ultimately incorporated into ferritin), and biliverdin/bilirubin have demonstrable anti-oxidant and anti-inflammatory effects. We have observed that brief (1 h/d × 3 d) inhalation of CO in SCD mice modulates vaso-occlusion (JCI , 116: 808–816; 2006). We hypothesize that prolonged treatment with inhaled CO will significantly decrease the inflammatory phenotype of SCD mice. Starting at three weeks of age, C57BL-6J and heterozygous BERK mice (mβ/hβS) were treated with inhaled CO at 0, 25, or 250 ppm (1 h/d × 3 d/wk × 10 wks). Over the duration of the ten week study no animals died as result of the CO treatments. Upon completion of the ten week treatment period animals were euthanized, blood was removed by cardiac puncture, and organs were harvested and homogenized. Carboxyhemoglobin levels immediately after 1h of CO treatment, (0, 25, and 250 ppm) were <1%,<1%, and 12%, respectively, but decreased to normal 24 h later. Untreated heterozygous BERK mice had a higher mean white blood cell count (WBC)(14,400/μL) than C57BL-6J mice (9,469/ μL, p<0.005). Treatment for 10 weeks with either 25 or 250 ppm CO significantly decreased heterozygous BERK WBC (8827 and 9222/μl, p<0.02), but had no effect on C57BL-6J WBC. The decrement in heterozygous BERK WBC was primarily due to a significant decrease in neutrophils (p<0.05). There was no significant difference in either the mean hematocrits or reticulocyte counts in CO-treated mice. Since redox-active iron (Fe2+) promotes oxidative stress and inflammation, we measured redox-active iron with Ferene-S in liver homogenates. In untreated heterozygous BERK mice, redox-active iron was two-fold greater than C57BL-6J mice (p<0.05) and treatment with 25 or 250 ppm CO significantly (p<0.01, p<0.02 respectively) decreased the redox-active iron (Fe2+) to levels comparable to untreated C57BL-6J. Since there is red cell congestion in heterozygous BERK livers, it was not surprising that the heme-content of untreated heterozygous BERK mice was three-fold (p<0.05) higher than untreated C57BL-6J. Treatment with 25 or 250 ppm CO significantly (p<0.05) decreased the heme-content of the livers to levels comparable to untreated C57BL-6J. The cytoprotective proteins HO-1 and ferritin heavy-chain were significantly increased in the livers of untreated heterozygous BERK mice compared to C57BL-6J (p<0.02). However, CO treatments had no significant effects on expression of these proteins on Western blots. We conclude that inhaled CO treatments decrease WBC, neutrophils, as well as liver redox-active iron and heme content in heterozygous BERK mice. Further studies are evaluating the effects of CO on inflammation, vaso-occlusion, and organ pathology in the homozygous BERK mouse model. We speculate that inhaled CO treatments may be a potential therapy for patients with SCD by acting as a modulator of oxidative stress and inflammation.

Small-molecule cyclic αVβ3 antagonists inhibit sickle red cell adhesion to vascular endothelium and vasoocclusion

Abnormal adhesion of sickle red blood cells (SS RBCs) to vascular endothelium may play an important role in vasoocclusion in sickle cell disease. Accruing evidence shows that endothelial alpha V beta 3-integrin has an important role in SS RBC adhesion because of its ability to bind several adhesive proteins implicated in this interaction. In the present studies, we tested therapeutic efficacy of small-molecule cyclic pentapeptides for their ability to block alpha V beta 3-mediated SS RBC adhesion by using two well-established assay systems, i.e., cultured human umbilical vein endothelial cells (HUVEC) and artificially perfused mesocecum vasculature of the rat under flow conditions. We tested the efficacy of two RGD-containing cyclic pentapeptides, i.e., cRGDFV (EMD 66203) and cRGDF-ACHA (alpha-amino cyclohexyl carboxylic acid) (EMD 270179), based on their known ability to bind alpha V beta 3. An inactive peptide, EMD 135981 (cR beta-ADFV) was used as control. Cyclization and the introduction of D-Phe (F) results in a marked increase in the ability of cyclic peptides to selectively bind alpha V beta 3 receptors. In the mesocecum vasculature, both EMD 66203 and EMD 270179 ameliorated platelet-activating factor-induced enhanced SS RBC adhesion, postcapillary blockage, and significantly improved hemodynamic behavior. Infusion of a fluorescent derivative of EMD 66203 resulted in colocalization of the antagonist with vascular endothelium. Also, pretreatment of HUVEC with either alpha V beta 3 antagonist resulted in a significant decrease in SS RBC adhesion. Because of their metabolic stability, the use of these cyclic alpha V beta 3 antagonists may constitute a novel therapeutic strategy to block SS RBC adhesion and associated vasoocclusion under flow conditions.

The Effect of Sickle Red Blood Cell Adhesion on Gene Expression in a PAF-Pre-Treated Microcirculatory Bed.

The expression profile of a number of genes in the rat mesocecum linked to sickle (SS) red blood cell (RBC) adhesion was examined by microarray analysis. The abnormal adherence of SS RBC to the vascular endothelium has been proposed to play an important role in vaso-occlusion in a double capacity; first, by actual physical blockage of the post capillary venules and, second, by inducing the expression of pleiotropic genes that could lead to further modification of the SS phenotype. The goals of these experiments were to identify these pleiotropic genes expressed upon adhesion of SS RBC, to understand how they contribute to the SS phenotype and, to identify potential targets for therapy. The rat mesocecum preparation was prepared as described (Kaul et al, Blood 95:368, 2000) and platelet activating factor (PAF) was used to induce endothelial adhesion receptors to accentuate human SS RBC binding. Rat cecum/ mesocecum RNA was prepared by Qiagen RNeasy mini-kit and fluorescent labeled cDNA hybridized to 32K oligonucleotide microarrays followed by analysis using Gene Pix Pro 4.1. The effect of SS RBC perfusion on PAF-treated tissues vs untreated tissues was compared to PAF-only tissues vs untreated tissues using hierarchical clustering. SS RBC adhesion was analyzed as a function of venular diameter and was significantly different (p<0.00001) in the PAF treated tissues as revealed by a significantly higher Y-intercept (work quoted above). Of the 533 transcripts that were up-regulated and 353 transcripts that were down-regulated more than 2 fold, 68 were up-regulated and 32 were down-regulated more than 4-fold; of this subset, 91 transcripts were compared by Cluster analysis. Many of the up-regulated genes detected in the PAF treated SS RBC adherent tissues were associated with inflammation such as phospholipase A2, group IIA; glutathione peroxidase 2; sialyltransferase 4C; and integrin beta 4. Interestingly, a number of proteasome subunits and ubiquitin D were also highly up-regulated; these genes were only present in the SS RBC perfused PAF treated mesocecum and could then be considered potential targets for specific therapies e.g. proteasome inhibitors. Recent studies also indicate that the ubiquitin system controls NF-kappaB pathways that, in turn, control integrin expression that can increase cell adhesion. The genes that were down-regulated included apolipoprotein B; cytochrome P450 Cyp4b1; cyclin G associated kinase; and metallothionein 2 (MT2). The down-regulation of MT2 is interesting because it is known to be highly regulated in response to the plasma zinc concentration; due to the chronic inflammatory/oxidative condition present in SS disease, zinc may be depleted and feedback negatively on MT2 synthesis. SS RBC adhesion however, not just PAF treatment, is necessary for MT2 downregulation and may, paradoxically, increase zinc availability since MT expression and zinc depletion are inversely correlated. Overall, the results suggest that SS RBC adhesion regulates a number of mesocecum genes involved in the inflammatory response, regulation of oxidative damage, increased intracellular protein degradation, decreased vesicular transport and regulation of zinc ion concentration. These pleiotropic genes are candidates for epistatic (modifier) genes if found to be polymorphic in different individuals and ethnicities. Enhancing or interfering with these specific genes or metabolic pathways may open up new therapeutic strategies for SS disease.

Critical role of endothelial cell activation in hypoxia-induced vasoocclusion in transgenic sickle mice

Activation of vascular endothelium plays an essential role in vasoocclusion in sickle cell disease. The anti-inflammatory agents dexamethasone and adhesion molecule-blocking antibodies were used to inhibit endothelial cell activation and hypoxia-induced vasoocclusion. Transgenic sickle mice, expressing human alpha-, beta(S)-, and beta(S-Antilles)-globins, had an activated vascular endothelium in their liver, lungs, and skin, as exhibited by increased activation of NF-kappaB compared with normal mice. NF-kappaB activation increased further in the liver and skin after sickle mice were exposed to hypoxia. Sickle mice had decreases in red blood cell (RBC) velocities and developed vasoocclusions in subcutaneous venules in response to hypoxia. Dexamethasone pretreatment prevented decreases in RBC velocities and inhibited vasoocclusions and leukocyte-endothelium interactions in venules after hypoxia. Dexamethasone treatment inhibited NF-kappaB, VCAM-1, and ICAM-1 expression in the liver, lungs, and skin of sickle mice after hypoxia-reoxygenation. VCAM-1 or ICAM-1 blockade with monoclonal antibodies mimicked dexamethasone by inhibiting vasoocclusion and leukocyte adhesion in sickle mice, demonstrating that endothelial cell activation and VCAM-1 and ICAM-1 expression are necessary for hypoxia-induced vasoocclusion in sickle mice. VCAM-1, ICAM-1, and vasoocclusion increased significantly 3 days after dexamethasone discontinuation, possibly explaining rebounds in vasoocclusive crises observed after withdrawal of glucocorticosteroids in sickle patients. We conclude that anti-inflammatory treatments that inhibit endothelial cell activation and adhesion molecule expression can inhibit vasoocclusion in sickle cell disease. Rebounds in vasoocclusive crises after dexamethasone withdrawal are caused by rebounds in endothelial cell activation.