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

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.

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Anti-inflammatory therapy ameliorates leukocyte adhesion and microvascular flow abnormalities in transgenic sickle mice

AJP Heart and Circulatory Physiology ◽

10.1152/ajpheart.01150.2003 ◽

2004 ◽

Vol 287 (1) ◽

pp. H293-H301 ◽

Cited By ~ 81

Author(s):

Dhananjay K. Kaul ◽

Xiao-du Liu ◽

Stephana Choong ◽

John D. Belcher ◽

Gregory M. Vercellotti ◽

...

Keyword(s):

Blood Flow ◽

Sickle Cell Disease ◽

Sickle Cell ◽

Leukocyte Adhesion ◽

Endothelial Activation ◽

Leukocyte Recruitment ◽

Microvascular Blood Flow ◽

Cell Disease ◽

Anti Inflammatory ◽

Hypoxia Reoxygenation

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.

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

Blood ◽

10.1182/blood-2010-05-285718 ◽

2011 ◽

Vol 117 (2) ◽

pp. 727-735 ◽

Cited By ~ 73

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.

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Inclacumab, a Fully Human Anti-P-Selectin Antibody, Directly Binds to PSGL-1 Binding Region and Demonstrates Robust and Durable Inhibition of Cell Adhesion

Blood ◽

10.1182/blood-2020-140530 ◽

2020 ◽

Vol 136 (Supplement 1) ◽

pp. 10-11

Author(s):

Xin Geng ◽

Radu Mihaila ◽

Yue Yuan ◽

Steven Strutt ◽

Jörg Benz ◽

...

Keyword(s):

Monoclonal Antibody ◽

Cell Adhesion ◽

Sickle Cell Disease ◽

Healthy Volunteers ◽

Sickle Cell ◽

Cell Disease ◽

Binding Region ◽

Publicly Traded ◽

Blood Samples

Sickle cell-related vaso-occlusive crises (VOCs) are among the primary clinical manifestations of sickle cell disease (SCD) and are associated with many acute and chronic complications that lead to significant morbidity and mortality. VOCs are caused by the adhesion of leukocytes and sickle erythrocytes to the endothelium, which results in vascular obstruction and tissue ischemia. By blocking the P-selectin- PSGL-1 (P-selectin glycoprotein ligand 1) mediated cell adhesion, crizanlizumab, a recently FDA approved humanized IgG2 anti-P-selectin antibody, reduced the frequency of VOCs in SCD patients and established the proof of principle for this approach (Ataga KI et al., N Engl J Med, 2017). Inclacumab is a novel, fully human IgG4 monoclonal antibody that selectively targets P-selectin and has safely demonstrated sustained anti-cell adhesion effects in over 700 participants including healthy volunteers and patients with cardiovascular disease (Schmitt C et al., J Cardiovasc Pharmacol. 2015; Tardif JC et al., J Am Coll Cardiol, 2013; Morrison M et al., Eur J Clin Pharmacol, 2015; Kling D et al., Thromb Res, 2013). A crystal structure of inclacumab and P-selectin reveals that inclacumab directly binds to an epitope in the PSGL-1 binding region on P-selectin and thus competitively inhibits P-selectin and its ligand interaction. In contrast, crizanlizumab binds to a more distant epitope to the PSGL-1 binding site on P-selectin. To further elucidate differences between the two antibodies, we characterized inclacumab and crizanlizumab in a series of in vitro functional assays including ligand binding affinity, competitive ligand binding by surface plasmon resonance (SPR), P-selectin mediated cell-based adhesion assay and cell-cell interaction with human whole blood samples. In vitro, inclacumab binds to human P-selectin with high affinity and potently suppresses the interaction of P-selectin with its main ligand PSGL-1. Both antibodies exhibited similar binding affinities to P-selectin (KD of 9.9 and 9.1 nM for inclacumab and crizanlizumab, respectively) and comparable potencies at preventing a PSGL-1 mimetic peptide from binding P-selectin (IC50 of 1.9 and 2.2 µg/mL for inclacumab and crizanlizumab, respectively) or blocking the adhesion of PSGL-1 expressing cells to an immobilized P-selectin (IC50 = 430 ng/mL for inclacumab and IC50 = 453 ng/mL for crizanlizumab). However, inclacumab demonstrated greater maximal platelet-leukocyte cell adhesion inhibition in response to thrombin receptor activating peptide (TRAP) in blood samples from both healthy volunteers and subjects with SCD in an in vitro efficacy assay (see figure). Inclacumab is differentiated from crizanlizumab as a fully human monoclonal antibody that directly blocks the PSGL-1 binding region of P-selectin and shows greater maximal inhibition of cell-cell interactions in vitro. At doses up to 20 mg/kg Q4W, which previous clinical trials have shown to be safe and well-tolerated, inclacumab has much greater drug exposure than the approved dose of crizanlizumab (5 mg/kg W0/W2/Q4W) (Ataga KI et al., N Engl J Med, 2017; Schmitt C et al., J Cardiovasc Pharmacol. 2015; Tardif JC et al., J Am Coll Cardiol, 2013). A single dose of inclacumab 20 mg/kg demonstrated full PLA inhibition for ≥84 days in healthy volunteers (Morrison M et al., Eur J Clin Pharmacol, 2015; Kling D et al., Thromb Res, 2013). Inclacumab may allow for a substantially longer and therefore more convenient dosing interval as compared with crizanlizumab. In aggregate, these data suggest that inclacumab has the potential to be a best-in-class P-selectin inhibitor to reduce VOCs in sickle cell disease. Clinical studies of inclacumab in patients with SCD are planned for the 1st half of 2021. Disclosures Geng: Global Blood Therapeutics: Current Employment, Current equity holder in publicly-traded company. Mihaila:Global Blood Therapeutics: Current Employment, Current equity holder in publicly-traded company. Yuan:Global Blood Therapeutics: Current Employment, Current equity holder in publicly-traded company. Strutt:Global Blood Therapeutics: Current Employment, Current equity holder in publicly-traded company. Benz:Roche Pharmaceuticals: Current Employment. Tang:Global Blood Therapeutics: Current Employment, Current equity holder in publicly-traded company. Mayer:Global Blood Therapeutics: Current Employment, Current equity holder in publicly-traded company. Oksenberg:Global Blood Therapeutics: Current Employment, Current equity holder in publicly-traded company.

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In vitro effect of Allopurinol on sickling rate and uric acid level in sickle cell erythrocyte.

Asian Journal of Medical Sciences ◽

10.3126/ajms.v4i3.7992 ◽

2013 ◽

Vol 4 (3) ◽

pp. 5-7

Author(s):

Johnkennedy Nnodim

Keyword(s):

Uric Acid ◽

Sickle Cell Disease ◽

Sickle Cell ◽

Uric Acid Level ◽

Acid Level ◽

Standard Dose ◽

Cell Disease ◽

Plasma Uric Acid ◽

Vitro Effect

Objective: Allopurinol is a drug used for the prevention of gout. The in vitro effect of allopurinol on human plasma and erythrocyte of sickle cell haemoglobin(HbSS) were investigated on uric acid level and sickling rate respectively. Methods: The determination was carried out on samples from 9-25years attending General Hospital Owerri. The plasma uric acid level and sickling rate were determined prior to and after incubation with 100mg standard dose of allopurinol. Results: The plasma uric acid level was significantly decreased (p<0.05) when compared with the control while sickling rate was significantly increased when compared with the control (p <0.05).The results obtained demonstrated that allopurinol could hasten sickling rate and decrease uric acid level. Conclusion: These observations suggest that allopurinol concentration in the circulation might induce marked increased in erythrocyte sickling in vivo and reduced the antioxidant ability of uric acid level in sickle cell disease. Consequently, slow recovery from a sickling crisis. This may be detrimental in the management of sickle cell disease DOI: http://dx.doi.org/10.3126/ajms.v4i3.7992 Asian Journal of Medical Sciences 4(2013) 5-7

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Anti-Inflammatory Activities of Sulfated Non-Anticoagulant Heparin Derivative Beyond Its Anti-Sickling and Anti-Selectin for the Effective Management of Sickle Cell Disease

Blood ◽

10.1182/blood-2020-141632 ◽

2020 ◽

Vol 136 (Supplement 1) ◽

pp. 27-27

Author(s):

Noureldien Darwish ◽

Osheiza Abdulmalik ◽

Shaker A Mousa

Keyword(s):

Cell Adhesion ◽

Sickle Cell Disease ◽

Red Blood Cells ◽

Sickle Cell ◽

Blood Cells ◽

Bleeding Complications ◽

Dependent Manner ◽

Cell Disease ◽

Anti Inflammatory ◽

Heparin Derivative

Sickle cell disease (SCD) is an autosomal recessive genetic disease caused by the inheritance of a single point mutation, resulting in abnormal sickle hemoglobin (HbS). During hypoxia or dehydration, HbS polymerizes to form insoluble aggregates and induces sickling of red blood cells (RBCs). RBC sickling increases the adhesiveness of RBCs to alter the rheological properties of the blood and trigger inflammatory responses, leading to hemolysis, vasoocclusive crisis, pulmonary complications, plethora, and other pathological sequelae. Glycosaminoglycans, such as low molecular weight heparin (LMWH), have been suggested as treatments to relieve coagulation complications in SCD because of their ability to decrease thrombin generation and sickle cell adhesion. However, they are associated with bleeding complications after repeated dosing. An alternative, sulfated non-anticoagulant LMWH derivative (S-NACH) was previously reported to have none to low systemic anticoagulant activity and no bleeding side effects, and it interfered with P-selectin-dependent binding of sickle cells to endothelial cells, with concomitant decrease in the levels of adhesion biomarkers in SCD mice (1,2). S-NACH has been further engineered to possess an aldehyde moiety, which confers anti-sickling properties primarily due to specific interactions with HbS to increase its affinity for oxygen. Our in vitro sickling assay under hypoxic conditions using S-NACH at 0.5 - 2 mM demonstrated that S-NACH significantly reduced the sickling of SS cells and in a concentration-dependent manner, with comparable to that of 1 mM GBT440 (Figure 1 A-C). A similar concentration dependent effect on increasing HbS affinity for oxygen using oxygen equilibrium study was documented. In an vivo animal model using Townes' SCD animals plasma levels of pro-inflammatory cytokines IL-1β, IL-6, IFN-γ, MCP-1, TNF-α, M-CSF, and VEGF were increased in SCD untreated samples in contrast to a significant decrease (*P< 0.001) in S-NACH-treated animals, at both 2 and 6 h. In addition, S-NACH was able to increase the decreased levels of the endogenous anti-inflammatory IL-10 (Figure 1 D). The above set of novel findings about S-NACH established it to be effective for the management of SCD via the modulation of thromboinflammatory pathways, involved in thromboembolism and end organ damage, beside anti-sickling, anti-selectin and without causing any bleeding risk. Reference 1. Alshaiban A, Muralidharan-Chari V, Nepo A, Mousa SA. Modulation of Sickle Red Blood Cell Adhesion and its Associated Changes in Biomarkers by Sulfated Non-anticoagulant Heparin Derivative. Clin Appl Thromb Hemost. 2016 ;22(3):230-8. 2. Mousa SA: Compositions and method for anti-sickling of red blood cells in sickle cell disease. US Patent 9,822,190, November 2017. Disclosures Mousa: Vascular Vision Pharma Co.: Patents & Royalties.

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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 ◽

10.1182/blood.v110.11.2264.2264 ◽

2007 ◽

Vol 110 (11) ◽

pp. 2264-2264 ◽

Cited By ~ 1

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.

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Use of anti-inflammatory analgesics in sickle-cell disease

Journal of Clinical Pharmacy and Therapeutics ◽

10.1111/jcpt.12592 ◽

2017 ◽

Vol 42 (5) ◽

pp. 656-660 ◽

Cited By ~ 5

Author(s):

J. Han ◽

S. L. Saraf ◽

J. P. Lash ◽

V. R. Gordeuk

Keyword(s):

Sickle Cell Disease ◽

Sickle Cell ◽

Cell Disease ◽

Anti Inflammatory

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Hypoxia-induced acute lung injury in murine models of sickle cell disease

AJP Lung Cellular and Molecular Physiology ◽

10.1152/ajplung.00288.2002 ◽

2004 ◽

Vol 286 (4) ◽

pp. L705-L714 ◽

Cited By ~ 57

Author(s):

Kirkwood A. Pritchard ◽

Jingsong Ou ◽

Zhijun Ou ◽

Yang Shi ◽

James P. Franciosi ◽

...

Keyword(s):

Sickle Cell Disease ◽

Lung Injury ◽

Sickle Cell ◽

Globin Gene ◽

Heat Shock Protein 90 ◽

Cell Disease ◽

Vascular Congestion ◽

Nitrite Nitrate ◽

Endothelial Nitric Oxide

Vaso-occlusive events are the major source of morbidity and mortality in sickle cell disease (SCD); however, the pathogenic mechanisms driving these events remain unclear. Using hypoxia to induce pulmonary injury, we investigated mechanisms by which sickle hemoglobin increases susceptibility to lung injury in a murine model of SCD, where mice either exclusively express the human α/sickle β-globin (hαβS) transgene (SCD mice) or are heterozygous for the normal murine β-globin gene and express the hαβStransgene (mβ+/-, hαβS+/-; heterozygote SCD mice). Under normoxia, lungs from the SCD mice contained higher levels of xanthine oxidase (XO), nitrotyrosine, and cGMP than controls (C57BL/6 mice). Hypoxia increased XO and nitrotyrosine and decreased cGMP content in the lungs of all mice. After hypoxia, vascular congestion was increased in lungs with a greater content of XO and nitrotyrosine. Under normoxia, the association of heat shock protein 90 (HSP90) with endothelial nitric oxide synthase (eNOS) in lungs of SCD and heterozygote SCD mice was decreased compared with the levels of association in lungs of controls. Hypoxia further decreased association of HSP90 with eNOS in lungs of SCD and heterozygote SCD mice, but not in the control lungs. Pretreatment of rat pulmonary microvascular endothelial cells in vitro with xanthine/XO decreased A-23187-stimulated nitrite + nitrate production and HSP90 interactions with eNOS. These data support the hypotheses that hypoxia increases XO release from ischemic tissues and that the local increase in XO-induced oxidative stress can then inhibit HSP90 interactions with eNOS, decreasing ·NO generation and predisposing the lung to vaso-occlusion.

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Endothelial Barrier Integrity Is Disrupted In Vitro by Heme and by Serum From Sickle Cell Disease Patients

Frontiers in Immunology ◽

10.3389/fimmu.2020.535147 ◽

2020 ◽

Vol 11 ◽

Author(s):

Vanessa Araujo Gomes Santaterra ◽

Maiara Marx Luz Fiusa ◽

Bidossessi Wilfried Hounkpe ◽

Francine Chenou ◽

Wouitchekpo Vincent Tonasse ◽

...

Keyword(s):

Sickle Cell Disease ◽

Healthy Volunteers ◽

Sickle Cell ◽

Endothelial Barrier ◽

Acute Chest Syndrome ◽

Cell Disease ◽

Experimental Conditions ◽

Cell Monolayers ◽

Junction Protein

Free extracellular heme has been shown to activate several compartments of innate immunity, acting as a danger-associated molecular pattern (DAMP) in hemolytic diseases. Although localized endothelial barrier (EB) disruption is an important part of inflammation that allows circulating leukocytes to reach inflamed tissues, non-localized/deregulated disruption of the EB can lead to widespread microvascular hyperpermeability and secondary tissue damage. In mouse models of sickle cell disease (SCD), EB disruption has been associated with the development of a form of acute lung injury that closely resembles acute chest syndrome (ACS), and that can be elicited by acute heme infusion. Here we explored the effect of heme on EB integrity using human endothelial cell monolayers, in experimental conditions that include elements that more closely resemble in vivo conditions. EB integrity was assessed by electric cell-substrate impedance sensing in the presence of varying concentrations of heme and sera from SCD patients or healthy volunteers. Heme caused a dose-dependent decrease of the electrical resistance of cell monolayers, consistent with EB disruption, which was confirmed by staining of junction protein VE-cadherin. In addition, sera from SCD patients, but not from healthy volunteers, were also capable to induce EB disruption. Interestingly, these effects were not associated with total heme levels in serum. However, when heme was added to sera from SCD patients, but not from healthy volunteers, EB disruption could be elicited, and this effect was associated with hemopexin serum levels. Together our in vitro studies provide additional support to the concept of heme as a DAMP in hemolytic conditions.

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