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)
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
- NSF-PAR ID:
- 10453161
- Publisher / Repository:
- Wiley Blackwell (John Wiley & Sons)
- Date Published:
- Journal Name:
- American Journal of Hematology
- Volume:
- 95
- Issue:
- 11
- ISSN:
- 0361-8609
- Page Range / eLocation ID:
- p. 1246-1256
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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Abstract in vitro . Confluent EC monolayers in microchannels were treated with pathophysiologically relevant levels of heme in order to simulate the highly hemolytic intravascular milieu seen in SCD. RBC adhesion to heme‐activated ECs varied from subject to subject, and was associated with plasma markers of hemolysis (LDH) and reticulocytosis, thereby linking those RBCs that are most likely to adhere with those that are most likely to hemolyze. These results re‐emphasize the critical contribution made by heterogeneous adhesive HbS RBCs to the pathophysiology of SCD. We found that adhesion of HbS RBCs to heme‐activated ECs varied amongst individuals in the study population, and associated with biomarkers of hemolysis and inflammation, age, and a recent history of transfusion. Importantly, the microfluidic approach described herein holds promise as a clinically feasible Endothelium‐on‐a‐chip platform with which to study complex heterocellular adhesive interactions in SCD. -
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Summary 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.
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Abstract Objectives We present a standardized in vitro microfluidic assay and Occlusion Index (OI) for the assessment of red blood cell (RBC)–mediated microcapillary occlusion and its clinical associations in sickle cell disease (SCD).
Methods Red blood cell mediated microcapillary occlusion represented by OI and its clinical associations were assessed for seven subjects with hemoglobin‐SC disease (HbSC), 18 subjects with homozygous SCD (HbSS), and five control individuals (HbAA).
Results We identified two sub‐populations with HbSS based on the OI distribution. HbSS subjects with relatively higher OIs had significantly lower hemoglobin levels, lower fetal hemoglobin (HbF) levels, and lower mean corpuscular volume (MCV), but significantly higher serum lactate dehydrogenase levels and absolute reticulocyte counts, compared to subjects with HbSS and lower OIs. HbSS subjects who had relatively higher OIs were more likely to have had a concomitant diagnosis of intrapulmonary shunting (IPS). Further, lower OI associated with hydroxyurea (HU) responsiveness in subjects with HbSS, as evidenced by significantly elevated HbF levels and MCV.
Conclusions We demonstrated that RBC‐mediated microcapillary occlusion and OI associated with subject clinical phenotype and HU responsiveness in SCD. The presented standardized microfluidic assay may be useful for evaluating clinical phenotype and assessing therapeutic outcomes in SCD, including emerging targeted and curative treatments that aim to improve RBC deformability and microcirculatory health.
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Abstract Objectives The advancement of microfluidic technology has facilitated the simulation of physiological conditions of the microcirculation, such as oxygen tension, fluid flow, and shear stress in these devices. Here, we present a micro‐gas exchanger integrated with microfluidics to study
RBC adhesion under hypoxic flow conditions mimicking postcapillary venules.Methods We simulated a range of physiological conditions and explored
RBC adhesion to endothelial or subendothelial components (FN orLN ). Blood samples were injected into microchannels at normoxic or hypoxic physiological flow conditions. Quantitative evaluation ofRBC adhesion was performed on 35 subjects with homozygousSCD .Results Significant heterogeneity in
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RBC adhesion in a cost‐efficient and patient‐specific manner. We identified a unique patient population in whichRBC s showed enhanced adhesion in hypoxia in vitro. Clinical correlates suggest a more severe clinical phenotype in this subgroup. -
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