ALBERT

Description: Leukocytes normally marginate toward the vascular wall in large vessels and within the microvasculature. Reversal of this process, leukocyte demargination, leads to substantial increases in the clinical white blood cell and granulocyte count and is a well-documented effect of glucocorticoid and catecholamine hormones, although the underlying mechanisms remain unclear. Here we show that alterations in granulocyte mechanical properties are the driving force behind glucocorticoid- and catecholamine-induced demargination. First, we found that the proportions of granulocytes from healthy human subjects that traversed and demarginated from microfluidic models of capillary beds and veins, respectively, increased after the subjects ingested glucocorticoids. Also, we show that glucocorticoid and catecholamine exposure reorganizes cellular cortical actin, significantly reducing granulocyte stiffness, as measured with atomic force microscopy. Furthermore, using simple kinetic theory computational modeling, we found that this reduction in stiffness alone is sufficient to cause granulocyte demargination. Taken together, our findings reveal a biomechanical answer to an old hematologic question regarding how glucocorticoids and catecholamines cause leukocyte demargination. In addition, in a broader sense, we have discovered a temporally and energetically efficient mechanism in which the innate immune system can simply alter leukocyte stiffness to fine tune margination/demargination and therefore leukocyte trafficking in general. These observations have broad clinically relevant implications for the inflammatory process overall as well as hematopoietic stem cell mobilization and homing.

Description: Background: Disruption of red blood cell (RBC) volume regulation and water homeostasis is a major component of sickle cell pathophysiology. As such, hydration is a mainstay of prevention and treatment of vaso-occlusive crises (VOC) in patients with sickle cell disease (SCD). However, evidence for guiding clinicians' choice for intravenous (IV) fluid hydration in the acute setting is lacking (Okomo, Coch Data Syst Rev, 2012). Pediatric hematologists typically discourage rapid infusion of hypotonic fluid given the risk of hyponatremia, although such fluids as 5% dextrose with 34meq/L or 77meq/L sodium are often used after stabilizing various acute clinical situations relevant to SCD. Although altered sodium concentration and osmolarity have been shown to affect erythrocyte swelling and rheology, dextrose-containing fluids used clinically such as D5 ¼ and D5 ½ normal saline (NS), have not been studied (Reinhart et al., Mic Res, 2015; Hijiya et al., J Lab Clin Med, 1991). To those ends, we sought to investigate the effect of different clinically relevant IV fluid formulations on normal and sickle RBC stiffness and deformability. Methods: Fresh blood was obtained from healthy volunteers and patients with sickle cell anemia (SS) on hydroxyurea and not transfused for at least 100 days. Sterile, clinical grade fluids stored at room temperature were used for the experiments (Baxter, Figure 1A). Our laboratory has previously published a description of a microfluidic device comprised of multiple parallel microchannels 5μm wide recapitulating the in vivo geometry of capillaries (Rosenbluth et al., Lab Chip, 2008). This microvasculature-on-a-chip enables measurements of single-RBC transit times, which correlate with cell stiffness (Figure 1B,C). For the transit time experiments, a master silicon wafer was used to mold the microfluidic channels out of polydimethylsiloxane (PDMS) silicone. Centrifuged RBCs were washed with phosphate-buffered saline (PBS) and then diluted to 0.5% hematocrit (HCT) in the various fluids prior to flow. Cell suspensions were perfused into a microfluidic device pre-coated with 2% bovine serum albumin (BSA) at an average linear flow rate of 0.50 mm s-1 in the smallest channels with a syringe pump (Harvard Apparatus) and then imaged at 20x at 20 frames per second. Images were recorded for future analysis. For the experiments assessing RBC shape, washed RBCs were diluted with PBS to 0.5% HCT and imaged at 40x in plastic wells at time 0. PBS was removed and 100 μL of each clinical fluid formulation was added to the wells and images were then obtained over time. RBC circularity was calculated using custom-written scripts in Matlab (Figure 1D). Results: RBC transit times in both healthy and sickle blood were affected by osmolarity and the various solute concentrations (Figures 2A,B). Transit times of sickle RBCs in all IV fluid formulations were significantly higher, over 10 times, than that of RBCs from healthy donors. Transit times for both healthy and sickle donors were least in the D5 ¼ NS solution. Of note, sickle RBC transit time was greatest in the NS solution. Sickle RBC circularity also changed with solute concentration and osmolarity with statistical significance (Figure 2C). Cells with the highest change in circularity from baseline were also those exposed to the D5 ¼ NS solution. Conclusion: Our results suggest the stiffness of sickle RBCs is affected by different formulations of clinical IV fluids. Increased transit time of sickle RBCs in NS through our device may in part be explained by the decreased circularity, indicating that RBCs adopt more irregular shapes in this fluid. This, in turn, could lead to increased propensity of microchannel obstruction. Although the exact mechanisms are unclear, this begs the question of whether NS is an appropriate choice for initial fluid resuscitation for VOC and other SCD-related complications as it could exacerbate the already high stiffness and shape irregularity of sickle RBCs, further increasing microvascular occlusion. As these in vitro results have significant clinical implications, ongoing experiments involve investigating how these fluid-dependent effects may alter sickle RBC adhesion in 'endothelialized' microfluidic devices, how different oxygen tensions affect these fluid-mediated effects, potential differences on and off of hydroxyurea, and the underlying mechanisms of this IV fluid formulation-dependent effect. Disclosures No relevant conflicts of interest to declare.

Description: Background: Originally described as a monogenic hemoglobin disorder resulting in increased red blood cell (RBC) stiffness leading to vaso-occlusion, sickle cell disease (SCD) is now known to be a vasculopathic disease with some semblance to cardiovascular disease in which the endothelium is inflamed. While adhesive RBC-endothelial interactions, inflammatory cytokines, and hemolysis all contribute to SCD vasculopathy, whether the increased stiffness of sickle RBCs directly contributes to endothelial inflammation is unknown. Endothelial cells are now known to mechanotransduce shear forces into biological signals. Pathological alteration of such forces leads to proinflammatory endothelial cell signaling including upregulation of VCAM-1 and E-selectin, which contribute to atherosclerotic plaques leading to myocardial infarction and stroke (Abe, ATVB, 2014). In addition, under normal homeostatic conditions, RBCs do not come into contact with the endothelium due to a cell-free layer created by the Fåhræus-Lindqvist effect. Studies including our own have shown in silico that increasing RBC stiffness diminishes or eliminates the cell-free layer, allowing stiff RBCs to contact the vessel wall (Kumar, Phys Rev E, 2011). This is particularly pertinent in SCD, as all patients have a small population (1-10%) of sickle RBCs that are permanently stiff and misshapen. We therefore hypothesize that purely physical interactions - akin to "scratches" or collisions - between endothelial cells and stiff SCD RBCs breaking through the cell-free layer are sufficient to cause endothelial inflammation in the absence of adhesion or vaso-occlusion (Fig. 1A). Methods: We performed computational direct numerical simulations using the boundary integral method for a binary suspension of flexible biconcave discs and stiff curved prolate spheres modeling healthy RBCs and ISCs, respectively. Experimentally, we leveraged our microfluidic microvasculature models of human umbilical vein endothelial cells cultured throughout each microchannel (Fig. 2). RBCs from SCD patients were "spiked" into normal RBC suspensions to comprise 5 and 10% of the overall population (a representation of ISCs in vivo), suspended in media to 25% hematocrit mimicking conditions seen in SCD patients, and perfused into the microfluidics for 4 hours. Samples of 100% normal RBCs or SCD RBCs were run in parallel. To isolate the stiffness effects of sickle RBCs without confounding hemolytic and adhesive effects, parallel experiments were conducted using nystatin-treated normal RBCs to create artificially stiffened RBC subpopulations, defined by elevated mean corpuscular hemoglobin concentrations (MCHCs), at the same proportion of the overall RBC population (0, 5, 10 and 100%). The endothelialized models were then fixed, permeabilized, and immunostained with antibodies against VCAM-1 and E-selectin. Mean fluorescence intensity was measured to quantify endothelial inflammation. Results: In silico, we observed that ISCs strongly marginate towards the vessel walls due to their stiffness and "pointy" shape, and heterogeneous suspensions with small fractions of stiff, pointy cells (5 and 10%) caused the highest degree of margination (Fig. 1B). Experimentally, endothelium exposed to 5, 10, and 100% SCD RBCs exhibited increased VCAM-1 and E-selectin expression over normal RBCs, and the degree of expression increased with higher percentages of SCD RBCs. While endothelial cells exposed to nystatin-stiffened RBCs also showed increased VCAM-1 and E-selectin expression, those exposed to a lower percentage of stiff cells (5 and 10%) exhibited higher expression than the homogenously stiff (100%) condition (Fig. 3), which is consistent with our computer simulations. Conclusions: Here we demonstrate that purely non-adhesive, physical interactions between endothelial cells and SCD RBCs are sufficient to cause endothelial inflammation. Furthermore, heterogeneous RBC populations, comprised of a small minority of stiff cells, cause more inflammation than uniformly stiff RBCs. Studies elucidating the underlying mechanisms, using different endothelial cell types, and analyzing the effect of vessel curvature are ongoing. Our results introduce a new paradigm for understanding SCD pathophysiology and may help explain how chronic diffuse vasculopathy develops, which could lead to more biophysically-based therapeutic strategies. Disclosures Carden: GBT: Honoraria; NIH: Research Funding. Mannino:Sanguina, LLC: Employment, Equity Ownership. Lam:Sanguina, LLC: Equity Ownership.

Description: Introduction: Sickle cell disease (SCD) is a genetic blood disorder in which red blood cell (RBC) stiffness is abnormally increased. In addition, chronic endothelial dysfunction, or vasculopathy is another aspect of SCD that involves RBC-endothelial cell interactions, although the underlying mechanisms remain poorly understood. Recent experimental work shows that stiffer RBCs marginate towards the blood vessel walls under physiologic flow conditions due to cell-cell collisions. However, little research has focused on the mechanical interactions between flowing stiff RBCs and endothelium in SCD that are not in the context of vascular occlusion in deoxygenated conditions. We propose that stiff, sickled RBCs in SCD patients constantly interact with the endothelium due to this stiffness-mediated margination, and that this interaction constitutes a purely mechanical cause of endothelial cell dysfunction. Furthermore, we hypothesize that the blood vessel geometry, which controls blood flow patterns and shear stress cellular, will mediate this mechanically-based endothelial dysfunction and may be an important aspect in the development of this vasculopathy. However, an adequate experimental model to test this hypothesis does not exist. To that end, we developed a simple "do-it-yourself" (DIY) perfusable vasculature model that incorporates a confluent endothelial cell monolayer along the channel lumen and recapitulates complex vascular geometries such as curvature. Materials and Methods: To fabricate the DIY endothelialized vasculature model, a strand of 500um diameter PMMA optical fiber was cast and cured in PDMS. The optical fiber was removed, leaving behind a channel that was then cultured with human aortic endothelial cells (HAECs). Bends were introduced into the fibers to create curved geometries. To test the effect of stiff RBCs on the endothelium, suspensions of RBCs from SCD patients were infused into these endothelialized devices, and assessed for endothelial dysfunction via immunostaining for VCAM-1 and E-selectin, known markers of endothelial inflammation. These were then compared to devices infused with control RBCs. To decouple the potential biological causes of endothelial dysfunction in SCD (e.g., adhesion, hemolytic byproducts) from purely physical causes, normal RBCs were dehydrated with nystatin concentrations known to increase the RBC stiffness to similar levels of SCD. Results and Discussion: These DIY vasculature models recapitulate in vivo microvasculature and can be cultured with human aortic endothelial cells (HAECs). (Fig 1. A, B). Simulations show an acute and localized shear rate variability at the site of curvature (Fig 1C). HAECs exposed to SCD RBCs and nystatin-stiffened RBCs perfused at flow rates of 100µL/min exhibited increased VCAM-1 and E-selectin upregulation in the curved regions of the vessel with little effect upstream or downstream of that region (Fig. 2). More specifically, SCD patients show increased endothelial inflammation along the outside wall of the bend (Fig 2). This is an interesting result as the regions of high wall shear stress associated with endothelial dysfunction occur along the outside wall of curved vessels. We speculate that the endothelial inflammation occurring in our system is related to increased collisions with the stiff SCD RBCs that occurs when the stiff RBCs marginate preferentially to the outer wall due to the inertial effects created by fluid flow around a bend. HAECs exposed to RBCs artificially stiffened with nystatin, however, showed increased diffuse VCAM-1 and E-selectin expression throughout the entire region of the curvature compared to healthy and SCD RBCs, potentially due to higher degrees of RBC margination compared to SCD (Fig 2). Overall, these results indicate that the mechanical interactions between stiff RBCs and the endothelium, as well as vascular geometry, plays a role in SCD vasculopathy. Additionally, studies investigating systematically quantifying the effect of varying degrees of vessel curvature on endothelial dysfunction. Conclusion: These results provide new explanations for the complex causes of endothelial dysfunction in SCD by relating the mechanical properties of RBCs as well as the vessel geometry to endothelial cell inflammation. Particularly, these studies have profound implications for understanding stroke in SCD, due to the tortuosity of the cerebral vasculature. Disclosures No relevant conflicts of interest to declare.

Description: Background: Studying individual red blood cells (RBCs) is critical to understanding hematologic diseases, as pathology often originates at the single-cell level. Measuring biophysical properties, such as deformability, of RBCs is also clinically important as RBC deformability is pathologically altered in numerous disease states including sickle cell disease (SCD), beta-thalassemia major (β-thal), and the "storage lesion" that occurs over time in stored samples of otherwise normal RBCs1-3. While ektacytometry-based systems measure RBC deformability, these assays do not have single-cell resolution4. As evidenced by flow cytometry, single cell measurements are important to detect pathologic cellular subpopulations in hematologic diseases. Recently, microfluidic devices that model the microvascular environment have enabled single RBC deformability measurements, but truly high-throughput measurements have remained elusive due to technical issues such as image processing inaccuracy or aberrant signaling when reconstructing RBC velocity profiles. Methods: To that end, here we introduce a novel integration of a microfluidic device coupled with our UMUTracker, an innovative MATLAB-based automated particle tracking program that is run on a standard desktop computer5. This innovative pairing of the two technologies results in high-throughput velocity tracking of single RBCs through a microfluidic model of a capillary bed that translates to a single deformability index (sDI) for each RBC (Fig 1). To demonstrate the sDI heterogeneity of RBCs across different conditions, data was obtained from healthy volunteers, SCD and β-thal patients, and stored packed RBCs. Results: sDI distribution curves were obtained for healthy RBCs and glutaraldehyde-stiffened RBCs (as positive controls) in which the latter resulted in an expected "left shift" and decrease in the mean sDI (Fig 2). Interestingly, while the mean sDI for SCD RBCs was also expectedly decreased, the sDI distribution was clearly non-normal, indicating the existence of heterogeneous RBC subpopulations with different deformabilities. These heterogeneous sDI distributions were observed in multiple SCD patients, including those on hydroxyurea (HU) and not, although patients on HU exhibited RBCs subpopulations with relatively higher sDIs (Fig 3). While stored RBC samples showed an expected drop in mean sDI over time, our system detected marked shifts in peak sDI with high temporal resolution over only 4 day intervals (Fig 4). Finally, each time a β-thal patient was transfused, the peak sDI shifted to the right and then gradually decreased over the course of the transfusion cycle, while the mean sDI exhibited minimal change over time (Fig 5). Conclusions: Our novel combined microfluidic/portable image analysis system demonstrates the high-throughput capability to detect distinct RBC subpopulations, at the single cell level, of different deformabilities in SCD, β-thal, and aging stored RBCs. This heterogeneity indicates that, in these disease states, RBC deformability cannot be fully characterized with mean or bulk biophysical measurements such as those obtained with ektacytometry. Ongoing studies will determine how changes in sDI profiles are associated with clinical events and different therapies as well as the biological significance of these RBC subpopulations with varied deformabilities and the underlying mechanisms for these differences. References: García-Roa M, del Carmen Vicente-Ayuso M, Bobes AM, et al. Red blood cell storage time and transfusion: current practice, concerns and future perspectives. Blood Transfusion. 2017;15(3):222-231. Li X, Dao M, Lykotrafitis G, Karniadakis GE. Biomechanics and biorheology of red blood cells in sickle cell anemia. Journal of biomechanics. 2017;50:34-41. Mangalani M, Lokeshwar MR, Banerjee R, Nageswari K, Puniyani RR. Hemorheological changes in blood transfusion-treated beta thalassemia major patients. Clin Hemorheol Microcirc. 1998;18(2-3):99-102. Rabai M, Detterich JA, Wenby RB, et al. Deformability analysis of sickle blood using ektacytometry. Biorheology. 2014;51(0):159-170. Zhang H, Stangner T, Wiklund K, Rodriguez A, Andersson M. UmUTracker: A versatile MATLAB program for automated particle tracking of 2D light microscopy or 3D digital holography data. Computer Physics Communications. 2017;219:390-399. Disclosures No relevant conflicts of interest to declare.

Description: Key Points Intravenous fluids are used when treating VOE, but guidelines are lacking, and how IVF tonicity affects sickle red cell biomechanics is unknown. Modifying extracellular fluid tonicity alters deformability, adhesivity, and occlusion risk for sRBCs in microfluidic vascular models.