Individuals with sickle cell disease (SCD) have persistently elevated thrombin generation that results in a state of systemic hypercoagulability. Antithrombin‐III (ATIII), an endogenous serine protease inhibitor, inhibits several enzymes in the coagulation cascade, including thrombin. Here, we utilize a biomimetic microfluidic device to model the morphology and adhesive properties of endothelial cells (ECs) activated by thrombin and examine the efficacy of ATIII in mitigating the adhesion of SCD patient‐derived red blood cells (RBCs) and EC retraction. Microfluidic devices were fabricated, seeded with ECs, and incubated under physiological shear stress. Cells were then activated with thrombin with or without an ATIII pretreatment. Blood samples from subjects with normal haemoglobin (HbAA) and subjects with homozygous SCD (HbSS) were used to examine RBC adhesion to ECs. Endothelial cell surface adhesion molecule expression and confluency in response to thrombin and ATIII treatments were also evaluated. We found that ATIII pretreatment of ECs reduced HbSS RBC adhesion to thrombin‐activated endothelium. Furthermore, ATIII mitigated cellular contraction and reduced surface expression of von Willebrand factor and vascular cell adhesion molecule‐1 (VCAM‐1) mediated by thrombin. Our findings suggest that, by attenuating thrombin‐mediated EC damage and RBC adhesion to endothelium, ATIII may alleviate the thromboinflammatory manifestations of SCD.
In sickle cell disease (SCD), ‘disease severity’ associates with increased RBC adhesion to quiescent endothelium, but the impact on activated endothelium is not known. Increased concentrations of free heme result from intravascular hemolysis in SCD. Heme is essential for aerobic metabolism and plays an important role in numerous biological processes. Excess free heme induces reactive oxygen species generation and endothelial activation, which are associated with cardiovascular disorders including atherosclerosis, hypertension, and thrombosis. Here, we utilized an endothelialized microfluidic platform (Endothelium‐on‐a‐chip) to assess adhesion of sickle hemoglobin‐containing red blood cells (HbS RBCs), from adults with homozygous SCD, to heme‐activated human endothelial cells (EC)
- NSF-PAR ID:
- 10236486
- Publisher / Repository:
- Wiley Blackwell (John Wiley & Sons)
- Date Published:
- Journal Name:
- American Journal of Hematology
- Volume:
- 93
- Issue:
- 8
- ISSN:
- 0361-8609
- Page Range / eLocation ID:
- p. 1050-1060
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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Abstract Sickle cell disease (SCD) is a recessive genetic blood disorder exhibiting abnormal blood rheology. Polymerization of sickle hemoglobin, due to a point mutation in the
β‐globin gene of hemoglobin, results in aberrantly adhesive and stiff red blood cells (RBCs). Hemolysis, abnormal RBC adhesion, and abnormal blood rheology together impair endothelial health in people with SCD, which leads to cumulative systemic complications. Here, we describe a microfluidic assay combined with a micro particle image velocimetry technique for the integrated in vitro assessment of whole blood viscosity (WBV) and RBC adhesion. We examined WBV and RBC adhesion to laminin (LN) in microscale flow in whole blood samples from 53 individuals with no hemoglobinopathies (HbAA, N = 10), hemoglobin SC disease (HbSC, N = 14), or homozygous SCD (HbSS, N = 29) with mean WBV of 4.50 cP, 4.08 cP, and 3.73 cP, respectively. We found that WBV correlated with RBC count and hematocrit in subjects with HbSC or HbSS. There was a significant inverse association between WBV and RBC adhesion under both normoxic and physiologically hypoxic (SpO2of 83%) tests, in which lower WBV associated with higher RBC adhesion to LN in subjects with HbSS. Low WBV has been found by others to associate with endothelial activation. Altered WBV and abnormal RBC adhesion may synergistically contribute to the endothelial damage and cumulative pathophysiology of SCD. These findings suggest that WBV and RBC adhesion may serve as clinically relevant biomarkers and endpoints in assessing emerging targeted and curative therapies in SCD. -
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Summary Many hypotheses have been proposed to explain how a glutamate to valine substitution in sickle haemoglobin (HbS) can cause sickle cell disease (SCD). We propose and document a new mechanism in which elevated tyrosine phosphorylation of Band 3 initiates sequelae that cause vaso‐occlusion and the symptoms of SCD. In this mechanism, denaturation of HbS and release of heme generate intracellular oxidants which cause inhibition of erythrocyte tyrosine phosphatases, thus permitting constitutive tyrosine phosphorylation of Band 3. This phosphorylation in turn induces dissociation of the spectrin‐actin cytoskeleton from the membrane, leading to membrane weakening, discharge of membrane‐derived microparticles (which initiate the coagulation cascade) and release of cell‐free HbS (which consumes nitric oxide) and activates the endothelium to express adhesion receptors). These processes promote vaso‐occlusive events which cause SCD. We further show that inhibitors of Syk tyrosine kinase block Band 3 tyrosine phosphorylation, prevent release of cell‐free Hb, inhibit discharge of membrane‐derived microparticles, increase sickle cell deformability, reduce sickle cell adhesion to human endothelial cells, and enhance sickle cell flow through microcapillaries. In view of reports that imatinib (a Syk inhibitor) successfully treats symptoms of sickle cell disease, we suggest that Syk tyrosine kinase inhibitors warrant repurposing as potential treatments for SCD.
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Abstract Objective Vaso‐occlusive crisis (VOC) is a complex process that occurs in patients with sickle cell disease (SCD) and is often associated with pain and urgent hospitalization. A major instigator of VOC is microvascular obstruction by pathologically stiffened sickle red blood cells (RBCs), and thus, therapy relies heavily on optimizing intravenous fluid (IVF) hydration to increase RBC deformability. However, no evidence‐based guidelines regarding the choice of IVF currently exist. We therefore analyzed alterations in biomechanical properties of sickle RBCs isolated from patients with homozygous SCD (hemoglobin SS) after exposure to different osmolarities of clinical IVF formulations.
Methods Atomic force microscopy (AFM) was used to assess stiffness of RBCs after exposure to different IVFs. A microfluidic model of the human capillary system was used to assess transit time (TT) and propensity to occlusion after exposure to the different IVF formulations.
Results Sickle RBCs exposed to normal saline (NS) had increased stiffness, TTs, and propensity to microchannel occlusion compared to other osmolarities.
Conclusion NS, an IVF formulation often used to treat patients with SCD during VOC, may induce localized microvascular obstruction due to alterations of sickle RBC biomechanical properties.
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Abstract Sickle cell disease (SCD) is the most prevalent inherited blood disorder in the world. But the clinical manifestations of the disease are highly variable. In particular, it is currently difficult to predict the adverse outcomes within patients with SCD, such as, vasculopathy, thrombosis, and stroke. Therefore, for most effective and timely interventions, a predictive analytic strategy is desirable. In this study, we evaluate the endothelial and prothrombotic characteristics of blood outgrowth endothelial cells (BOECs) generated from blood samples of SCD patients with known differences in clinical severity of the disease. We present a method to evaluate patient‐specific vaso‐occlusive risk by combining novel RNA‐seq and organ‐on‐chip approaches. Through differential gene expression (DGE) and pathway analysis we find that BOECs from SCD patients exhibit an activated state through cell adhesion molecule (CAM) and cytokine signaling pathways among many others. In agreement with clinical symptoms of patients, DGE analyses reveal that patient with severe SCD had a greater extent of endothelial activation compared to patient with milder symptoms. This difference is confirmed by performing qRT‐PCR of endothelial adhesion markers like E‐selectin, P‐selectin, tissue factor, and Von Willebrand factor. Finally, the differential regulation of the proinflammatory phenotype is confirmed through platelet adhesion readouts in our BOEC vessel‐chip. Taken together, we hypothesize that these easily blood‐derived endothelial cells evaluated through RNA‐seq and organ‐on‐chips may serve as a biotechnique to predict vaso‐occlusive episodes in SCD patients and will ultimately allow better therapeutic interventions.
