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: Treatment of thrombosis relies on prompt resolution of thrombi to restore blood flow to avoid ischemic injury. However, our understanding of the step-by-step process of how thromboses resolve remains limited due in large part to the lack of sufficient technologies. In addition, as thromboses require days to weeks to resolve, existing in vitro and in vivo systems cannot monitor this process, especially in the microvasculature where thrombi are difficult to visualize. As such, questions such as how do the cellular and biochemical composition of a clot change as it resolves and how do hemodynamics affect this process remain unanswered. This is particularly important in thromboinflammatory conditions such as autoimmune/inflammatory disorders in which patients are chronically at risk for microvascular thromboses but the pathophysiology and therefore optimal therapies remain unclear. Thus, a pressing need exists for an assay that assesses how microvascular thromboses resolve over long time scales, especially under thromboinflammatory conditions. We recently developed a microfluidic system that assesses microvascular events, including endothelial dysfunction and permeability, in response to proinflammatory signals over months (Qiu, Nature Biomed Eng. 2018). Here we leverage this system to monitor not only how microvascular thromboses form but, importantly, how they resolve over long timescales. Moreover, our system also enables the monitoring of how antithrombotic drugs and anticoagulants "work" in the context of existing inflammatory thrombi, which will provide insight into the pathophysiology as well as provide evidence for the use of different therapies. Results and Discussions: Our engineered microvasculature on chip recapitulates the biophysical microenvironment, such as microvessel size, geometry, wall shear stress, and shear gradients (Fig 1A-C), as well as the biological microenvironment, such as perfusion of whole blood, endothelial cells activated with inflammatory mediators, and vascular permeability, to assess how these factors interact during microvascular thrombi formation and resolution over long timescales (Fig 1D). As the entire "lifetime" of a clot is monitored with high spatiotemporal resolution (Fig 2), how the innate immunity, platelets, and coagulation cascade interact during microvascular thrombosis and resolution can be systematically studied. Interestingly, exposure of the microvasculature to TNF-α induces VWF multimers that deposit onto the inflamed endothelium at bifurcations of the smallest vessels, where wall shear stress gradients exist. The deposited VWF multimers then induce platelet aggregation in the bifurcation within minutes and is accompanied with gradual fibrin formation (Fig 1E-H). Neutrophils adhere to the inflamed endothelium at a relatively later stage primarily in areas with lower wall shear stress, aggregating with platelets and incorporating with the growing fibrin mesh. Interestingly, as thrombi start to resolve, platelets are mostly undetectable by 1 day post-thrombosis, while neutrophils and fibrin persist, occluding flow and preventing endothelial barrier function recovery. With this novel system, we are also able to monitor, for the first time, the effects of commonly used anticoagulants such as enoxaparin on clot resolution (Fig 2).When given as a prophylactic under thromboinflammatory conditions, enoxaparin prevents fibrin formation yet does not attenuate platelet-neutrophil aggregates, highlighting the critical role of interaction of platelets and neutrophils in microvascular occlusion. Surprisingly, enoxaparin also decreases endothelial dysfunction and restores barrier function, suggesting that a significant part of enoxaparin's antithrombotic effects may be endothelial in nature. While post-thrombosis overnight perfusion of the thrombolytic tPA expectedly results in complete fibrin degradation and restoration of flow, tPA also surprising induces endothelial barrier function. Conclusions: We have, for the first time, developed a perfusable vascularized thrombus resolution assay that enables the tracking of inflammatory thrombi over weeks and is ideal for studying antithrombotic drugs effects and how they may restore microvascular barrier function. Studies assessing the formation of microvascular emboli in this context are ongoing. Disclosures Lam: Sanguina, LLC: Equity Ownership; Sanguina, LLC: Equity Ownership.

Description: Introduction: Sickle Cell Disease (SCD) is an inherited monogenic hemoglobin disorder characterized by decreased red blood cells (RBCs) deformability. While RBCs are directly affected by this mutation, the interaction of these cells in the milieu of other components including white blood cells (WBCs), platelets, and soluble factors in whole blood are also thought to contribute to microvascular occlusion in SCD pathophysiology. Several studies have suggested that platelet activation is increased in SCD, but how platelets affect microvascular occlusion is unknown. As cellular interactions are affected by different flow conditions, we leveraged our previous "endothelialized" microfluidic technology (Tsai et al, JCI, 2012) to develop a novel multi-shear microfludic device to investigate blood cell-endothelial cell interactions in 3 different shear rates spanning 3 orders of magnitude ranging from venous to arteriolar shear conditions found in vivo (Figure 1). As platelets are shear-sensitive, this device is conducive to studying platelet interactions in SCD. In addition, we utilized our multi-shear endothelialized microfluidic device for drug discovery, elucidating the mechanism of action of Purified Poloxamer 188 (MST-188). MST-188 is a non-ionic, block copolymer surfactant that has been studied in nearly 400 patients with SCD and is currently being investigated in EPIC (Evaluation of Purified Poloxamer 188 In Crisis), a Phase III trial. MST-188 is composed of a single chain of hydrophobic polyoxypropylene flanked by two hydrophilic polyoxyethylene chains. It is hypothesized to improve microvascular blood flow by reducing viscosity, particularly under low shear conditions, and reducing adhesive frictional forces (Ballas et al 2004). We utilized our endothlelialized multi-shear microfluidic technology to observe cellular interactions in SCD patient samples treated with MST-188. Methods: Whole blood samples were collected from Pediatric patients with HgbSS SCD, including patients on hydroxyurea (HU) via venipuncture in citrate collecting tubes. Samples were recalcified and perfused through a confluently endothelialized multi-shear microfluidic device for 20 minutes. Time-lapse epiflourescence videomicroscopy was obtained to observe cellular interactions under different physiologic flow conditions. Results: Platelet Aggregation in SCD: Using whole blood samples from SCD patients, we observed that platelet aggregation is markedly increased in Hgb SS patients not on HU compared to samples from control and Hgb SS patients on HU (Figure 2). This effect occurs for all shear rates. Attenuation of phosphotadylserine (PS) exposure by MST-188: When a cell undergoes apoptosis, PS "flips" from the intra- to extracellular surface acting as a signal for macrophage engulfment. In order to identify target cell populations a thin smear whole blood from a patient with HgbSS not on HU (Figure 3A). Samples were fluorescently tagged with anti-CD41 to identify platelets and Annexin V to identify the presence of PS (Figure 3B). Patients with HgbSS not on HU have relatively increased fluorescence that is attenuated with treatment with MST-188 (Figure 3C). Conclusion and Future Directions: We have successfully demonstrated a correlation with increased platelet aggregation in endothelialized microfluidic channels in patients with SCD compared to normal controls. The platelets of SCD patients have an increased propensity to aggregate in an abnormal non-shear dependent fashion which correlated directly with fluorescence. This phenomenon appears to be attenuated in patients with SCD on HU in all shear rates. We have also demonstrated that MST-188 attenuates PS exposure mostly found on irreversibly sickled cells. We believe this data and investigational platform to be a good springboard to unravel the utility of targeting platelet specific therapies to augment the course of VOC. This platform can also be used to continue to determine mechanism of action of MST-188 in disease processes, including SCD where inflammation and increased cellular turnover plays a critical role in pathology. Experiments investigating platelet activation markers, co-localization of other cell types including ISCs, reticulocytes and WBC subpopulations with platelet aggregates, as well as characterizing our microfluidic model under de-oxygenated conditions are currently ongoing. Disclosures No relevant conflicts of interest to declare.

Description: Key Points The multivariate mechanism of FeCl3-induced thrombosis is rooted in colloidal chemistry, mass transfer, and biological clotting. FeCl3-induced thrombosis is mediated by charge-based binding of proteins (cell surface bound and soluble) to the Fe3+ ion.

Description: Background: Recent clinical trials have demonstrated the efficacy and safety of gene therapy utilizing HIV-derived lentiviral vectors (LVs) for blood disorders. However, the LV requirements and clinical ex vivo cell transduction protocols used in these studies exposes the limitations of the technology and beckons the need for improved LV manufacturing and clinical transduction efficiency. Many methods have been devised to enhance efficiency, although none have circumvented the exorbitant amounts of virus required to achieve therapeutic HSC transduction. Furthermore, prolonged ex vivo cell culture is necessary to achieve sufficient transduction despite exposure to toxic byproducts of LV production. To that end, we developed a novel, scalable microfluidic for clinical LV transduction that leverages mass transfer principles to significantly reduce the amount of LV required to achieve therapeutic levels of gene transfer and transduction time by more efficiently exposing cells to virus. Results: Jurkats were transduced with a GFP-encoding clinical LV in microfluidics with surface areas (SAs) comparable to the bottom surface of 96 and 6-well plates. Microfluidic transductions were compared to well plate transductions with matched SA, cell numbers, viral particles, and incubation times. After LV incubation, cells were removed from the microfluidics and well plates, spun down, re-suspended with fresh media, and cultured for at least 72 hours at 37°C and 5% CO2. Cells were assessed for GFP expression with flow cytometry. Preliminary mouse studies utilized Sca+ cells isolated from CD45.1 donor mice via positive selection. The cells were transduced in the scaled up microfluidic with a bioengineered coagulation factor VIII (fVIII) transgene encoding LV and transplanted into host hemophilia A mice after myeloablative conditioning. Two weeks post-transplantation, blood samples were taken from the recipient animals and assayed for donor cell engraftment by flow cytometry and plasma fVIII activity by chromogenic assay. The high SA:volume ratio of the microfluidic enhances transduction by physically bringing cells and virus into closer proximity and enabling high concentrations of virus to be used without increasing the amount of virus set by the minimum volume requirements of LV transduction platforms (Fig. 1A). The polystyrene bottom of the microfluidic allows for Retronectin coating that immobilizes non-adherent cells on the bottom surface. LV can then be perfused at low concentrations to maintain a constant supply of fresh virus to the cells, increase convective mixing, and to minimize cell exposure to the toxic byproducts of LV production (Fig. 1B). These microfluidics have been scaled up to accommodate 106 cells, with potential to scale up to 107-108 cells (Fig. 1C). Cells transduced in the microfluidics showed 2-6 fold increases in GFP expression over well plates utilizing the same amount of cells, virus, and incubation times (Fig. 2A). The kinetics of LV transduction in the microfluidics also are faster, as seen by the steeper transduction curve. Five hours of incubation in the microfluidic yielded comparable transduction to 24 hours in the 6-well plate (Fig. 2B). Improvements in transduction also were observed by perfusing virus despite using lower virus concentrations (Fig. 2C). Finally, hemophilia A mice transplanted with donor CD45.1 Sca+ cells transduced in the microfluidic have engrafted (Fig. 3A) and produce fVIII (Fig. 3B) after two weeks with similar profiles to control cells transduced in a 6-well plate despite using half the amount of virus and shorter incubation times. Conclusions and ongoing efforts: We describe a novel microfluidic that significantly reduces the amount of virus and ex vivo processing time required for therapeutic levels of transduction in clinical gene therapy. This device is versatile in its compatibility with current transduction strategies such as Retronectin and polybrene in addition to offering new approaches to boosting gene transfer efficiency. Furthermore, we have shown that the device has clinical potential by successfully scaling up cell numbers and transplanting mice with microfluidic transduced cells, of which there is an ongoing effort to monitor fVIII production and determine virus copy number. Future work will involve optimization with transduction-enhancing compounds, further scaling, and continued in vivo experiments. Disclosures Spencer: Expression Therapeutics: Equity Ownership. Doering:Bayer Healthcare: Consultancy, Honoraria, Research Funding; Expression Therapeutics: Equity Ownership.

Description: Background: Endothelial activation and dysfunction play critical roles in vaso-occlusive crises and vasculopathy in sickle cell disease (SCD). However, it remains unclear how the myriad of cellular and biomolecular interactions that occur in SCD directly affect endothelial cell activation and injury, due largely in part to the lack of robust in vitro models for studying these complex biophysical processes. To this end, we engineered the first perfusable hydrogel-based microfluidic device comprised of "endothelialized" channels at the microvascular sizescale. Unlike typical microfluidic devices, which are silicone(PDMS)-based, this device is comprised of a collagen-based hydrogel that is not only more physiologic but also enables real-time monitoring of the endothelium permeability while allowing the user to tightly control hemodynamic conditions and the cellular and molecular components of the perfusate. Interestingly, in this system, upon removal of injurious stimuli, the endothelial cells are able to "self-heal" after injury and fully establish their barrier function. Using this system, we first tested whether interaction of SCD patient RBCs with endothelium under flow can directly cause endothelial permeability. We then studied how shear stress promotes endothelial cell activation and injury caused by free hemin, a byproduct of hemolysis in SCD. Results and Discussions: After seeding into the hydrogel-based device, human endothelial cells formed a monolayer that covered the entire inner surface of the microchannels and can be maintained for 〉1 month under flow conditions. Establishing that cultured endothelial cells are functional in this system, the cells appropriately formed continuous adherens junctions under flow as indicated by VE-cadherin staining (Figure 1A) and also deposited their own subendothelial extracellular matrices, including collagen IV (Figure 1B) and laminin (Figure 1C). Finally, perfusion of fluorescently-tagged albumin (BSA) sufficient endothelial barrier function of our system as all fluorescence signal was contained within the "vascular" space (Figure 1D and 1E). RBCs isolated from SCD patients were perfused into the endothelialized channels for 4 hours. Strikingly, the direct interaction of the perfused SCD RBCs with the engineered endothelium, in and of itself, was sufficient to induce endothelial permeability (Figure 2A), a phenomenon that did not occur with perfusion of control RBCs isolated from healthy volunteers. Interestingly, impermeability was reestablished as the endothelium "healed" 1 day post-interaction with patient RBCs (Figure 2B). We then perfused hemin in the channels under two different flow rates and the flow velocity profile, wall shear stress, shear rate, and pressure were characterized using COMSOL (Figure 3A and 3B). Interestingly, 1 hour of hemin exposure (10 µM) at higher shear stress compared to lower shear stress not only caused increased endothelial permeability and loss of endothelial cells at 1 day post-treatment, but also dampened the "healing" of injured endothelial cells and reestablishment of impermeability after removal of hemin from the system (Figure 3C and D). Conclusions and on-going work: Our physiologic hydrogel-based microvasculature-on-a-chip system enables investigation of how cell/molecular interactions directly affect endothelial permeability in real time and represents a milestone in the use of microfluidic devices for SCD research. In addition, the "self-healing" capability mimics the in vivo microvasculature and is a unique capability of this system as compared to current in vitro models. With this novel system, we determined that RBC-endothelial interactions under physiologic flow are sufficient to induce endothelial barrier dysfunction. We also demonstrated the additive role of hemodynamics in promoting hemin-induced endothelial activation and dysfunction. Ongoing work investigating the mechanisms of our observations will unveil insight into SCD pathogenesis and our system can be applied to other vascular diseases as well. Disclosures No relevant conflicts of interest to declare.