(128e) In Vitro Generation of Red Blood Cell Extracellular Vesicles (REVs) and Functional Evaluation of REV-Mediated Acute Endothelial Activation in Individual Patients with Genetic Hemoglobin Disorders

Extracellular vesicles (EVs), composed of a lipid bilayer, comprised of transmembrane proteins, enclose intracellular remnants, including cytosolic proteins, RNA, and micro-RNA (miRNA). EVs can serve as a vehicles for cellular communication, in near and remote proximity, and can reflect the parent cell’s activation state. Genetic hemoglobin disorders including sickle cell disease (SCD) are inherited hemoglobinopathy, afflicting an estimated 300,000 to 400,000 newborns/year worldwide. The mutation of a single amino acid in the adult beta chain results in sickle hemoglobin (HbS). Upon deoxygenation, HbS polymerizes in red blood cells (RBCs), induces RBC sickling, and provokes sickling a complex pathophysiology of acute and chronic organ damage. SCD RBCs show reduced deformability, increased fragility, increased adhesion to vascular wall, and are prone to intravascular hemolysis, especially within microcapillaries, where cells experience low oxygen content and high shear stress. During intravascular hemolysis, SCD RBC release EVs that express surface phosphatidylserine (PS), contain heme and microRNAs. The RBC-derived EVs (REVs) are capable of promoting blood coagulation and a pro-inflammatory/pro-adhesive endothelial phenotype. The activation of endothelial cells triggers abnormal RBC adhesion, which can promote vaso-occlusion causing acute and chronic organ damage for patients with SCD. Despite the significant potential role of REV as candidate biomarkers, REV-induced RBC adhesion are often evaluated using mouse models, and thus do not reflect the well-known clinical heterogeneity and the abnormal RBC adhesion amongst patients with SCD.

Materials and Methods

Here, we have developed two in vitro microfluidic platforms, the EV-µCapChip and EV-BioChip. EV-µCapChip was used to activate SCD RBCs for REV generation under physiological oxygen content and shear stress in human microcapillaries. SCD-EV-BioChip was used to assess RBC adhesion as a biomarker for REV-mediated lung microvascular endothelial dysfunction. The EV-µCapChip contains microchannels for accurately control gas content and apply shear stress on SCD RBCs at physiologically and clinically-relevant levels for REV generation. SCD RBCs were exposed to: 1) shear stress; 2) hypoxia condition; and 3) shear stress under hypoxia condition, for REV generation. The generated REVs were characterized by Nanoparticle Tracking Analysis (NTA) measure size distribution and concentration. The EV-BioChip contains microfluidic channels lined with human pulmonary microvascular endothelial cell (HPMEC) that are maintained under precise shear stress at physiologically and clinically-relevant levels. HPMECs were incubated with REVs generated in vitro in individual patients with SCD and healthy donors. RBC adhesion on REV-treated HPMECs was quantified in 14 patients with SCD (HbSS) using EV-BioChip assay.

Results and Discussion

Three SCD RBC activation methods (shear stress, hypoxia, shear + hypoxia) generated REVs of similar size (Fig. 1A). Application of shear stress under hypoxia condition generated 10 times more REVs than the other two approaches (Fig. 1B). These diverse REV properties demonstrate the importance of exposing purified RBCs to physiological/pathological relevant conditions for generating autologous REVs that can potentially recapitulate the signatures of endogenous REVs from SCD patients during disease activity. Within 2 hours, von Willebrand factor (vWF) expression was significantly increased on HPMECs incubated with SCD (SS) REVs (Fig. 2A) comparing to HPMECs incubated with healthy donor (AA) REVs (Fig. 2B&C, p = 0.012, Mann-Whitney). In patient-specific testing, SCD RBC adhesion profiles are associated with clinical outcomes of the patients with SCD. The results demonstrated that the SCD RBC adhesion profiles are associated with patient hemolytic biomarkers (Fig. 2A&B), white blood cell counts (Fig. 2C), ferritin levels (Fig. 2D), and clinical record of deep vein thrombosis (Fig. 2E). Altogether, these evidence suggest that subjects with higher RBC adhesion are at elevated disease status, and the developed microfluidic assay is capable of identifying those patients who may need acute clinical care for disease management.

Conclusions

Here, we present two in vitro microfluidic platforms for in vitro generation REVs and for in vitro evaluating REV-mediated endothelial activation using functional biomarker of RBC adhesion under physiologically conditions. The results demonstrate strong association between the outputs from in vitro microfluidic assay and the outputs from patient clinical records. We envision that the strategies presented here have potential to enable diagnosis, prognosis and assist in clinical decision making for individual patients with CD.

Figure. 1: Characterization of REVs generated using multiple in vitro approaches: REVs generated by exposing SCD RBCs to shear stress (light gray), hypoxia condition (dark gray) and shear stress under hypoxia condition (red) (A) REVs generated using different methods have similar size distribution (B) Higher concentration of REVs are generated from SCD RBCs using shear stress under hypoxia condition, comparing to exposing SCD RBCs to hypoxia or shear stress only.

Figure. 2: RBC adhesion to REV-activated HPMECs correlates with subject clinical phenotype including hemolytic and inflammatory biomarkers. (A): A subpopulation (Group 1, N = 6) with distinct hemolysis markers of lactate dehydrogenase (LDH) levels and absolute reticulocyte count (ARCs) comparing to the rest (Group 2, N = 9) via k-means clustering analysis. RBCs from subjects in Group 2, with significantly higher LDH levels and ARCs, have greater adhesion to REV-activated HPMECs compared to the RBCs from subjects in Group 1 (B, p = 0.016, One-way ANOVA). The gray and green shaded areas indicate normal ranges for ARC and LDH, respectively. (C): Subjects in Group 2 with higher LDH and ARC and enhanced RBC adhesion have significantly higher WBC counts, than subjects in Group 1 (p = 0.034, one-tailed Mann-Whitney). Shadowed area: normal range of WBC counts. (D): Subjects in Group 2 with higher LDH and ARC and enhanced RBC adhesion have higher ferritin levels, although not statistically significant, than subjects in Group 1 (p = 0.071, one-tailed Mann-Whitney) Shadowed area: normal range of ferritin level (2 to 1000 µg/L) [47, 48]. Subjects with deep vein thrombosis (DVT) had significantly higher RBC adhesion to REV activated HPMECs. Six out of six subjects in Group 1 patient with lower RBC adhesion, and three out of six subjects in Group 2 did not have DVT. Four out of eight subjects in group 2 with higher RBC adhesion had DVT. DVT status of two out of eight subjects in Group 2 were not available (1 patient diagnosed as ‘unclear’, 1 patient record not accessible).