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Hemophilia A is an inherited bleeding disorder caused by defective or deficient coagulation factor VIII (FVIII) activity. Until recently, the only treatment for prevention of bleeding involved IV administration of FVIII. Gene therapy with adeno-associated vectors (AAVs) has shown some efficacy in patients with hemophilia A. However, limitations persist due to AAV-induced cellular stress, immunogenicity, and reduced durability of gene expression. Herein, we examined the efficacy of liver-directed gene transfer in FVIII knock-out mice by AAV8-GFP. Surprisingly, compared with control mice, FVIII knockout (F8TKO) mice showed significant delay in AAV8-GFP transfer in the liver. We found that the delay in liver-directed gene transfer in F8TKO mice was associated with absence of liver sinusoidal endothelial cell (LSEC) fenestration, which led to aberrant expression of several sinusoidal endothelial proteins, causing increased capillarization and decreased permeability of LSECs. This is the first study to link impaired liver-directed gene transfer to liver-endothelium maladaptive structural changes associated with FVIII deficiency in mice.

Aging is the most significant risk factor for the majority of chronic diseases, including liver disease. The cellular, molecular, and pathophysiological mechanisms that promote age-induced hepatovascular changes are unknown due to our inability to visualize changes in liver pathophysiology in live mice over time. We performed quantitative liver intravital microscopy (qLIM) in live C57BL/6J mice to investigate the impact of aging on the hepatovascular system over a 24-mo period. qLIM revealed that age-related hepatic alterations include reduced liver sinusoidal blood flow, increased sinusoidal vessel diameter, and loss of small hepatic vessels. The ductular cell structure deteriorates with age, along with altered expression of hepatic junctional proteins. Furthermore, qLIM imaging revealed increased inflammation in the aged liver, which was linked to increased expression of proinflammatory macrophages, hepatic neutrophils, liver sinusoidal endothelial cells, senescent cells, and procoagulants. Finally, we detected elevated NF-κB pathway activity in aged livers. Overall, these findings emphasize the importance of inflammation in age-related hepatic vasculo-epithelial alterations and highlight the utility of qLIM in studying age-related effects in organ pathophysiology.

Sickle cell disease (SCD) is an autosomal-recessive blood disorder affecting over 100 000 individuals in the United States.1 Mutant sickle hemoglobin polymerizes within red blood cells (RBCs) under deoxygenated conditions resulting in cell rigidity, hemolysis, and vaso-occlusion.2 These events catalyze an inflammatory response, which can lead to acute painful vaso-occlusive episodes.2,3 Acute pain is the predominant cause for seeking medical treatment, resulting in estimated medical care costs of over $1.1 billion dollars annually for those with SCD.4,5 The role of impaired hemorheology in SCD and its contribution toward vaso-occlusion is well documented.6,7 Poor blood rheology including elevated whole-blood viscosity, plasma viscosity, RBC membrane rigidity, and cytosolic viscoelasticity impedes microvascular blood flow by increasing intravascular resistance and decreasing RBC velocity.8,9 Abnormal cellular adhesion markers presented on sickle RBCs due to membrane damage and on prematurely released reticulocytes further contribute to impaired flow and the precipitation of vaso-occlusion.3,10,11 Recently, blood additives known as drag-reducing polymers (DRPs) have been shown to alter blood rheology beneficially and to improve the hemodynamics and outcomes of animals following hemorrhagic shock,12 liver ischemia/reperfusion injury,13 and traumatic brain injury significantly,14 while simultaneously providing increased capillary recruitment, perfusion, and oxygenation.15 However, the effects of DRP are unknown in the context of SCD, where a rheology-based therapy to improve microcirculatory blood flow has the potential to greatly reduce the incidence of vaso-occlusion. In this study, we have used quantitative liver intravital microscopy (qLIM) to show that the administration of nanomolar concentrations of DRP reduces vaso-occlusion within liver sinusoids of transgenic SCD mice following inflammatory stimulus via lipopolysaccharide (LPS).

Rationale: Intraerythrocytic polymerization of Hb S promotes hemolysis and vasoocclusive events in the microvasculature of patients with sickle cell disease (SCD). Although platelet–neutrophil aggr...

Acute lung injury, referred to as the acute chest syndrome, is a major cause of morbidity and mortality in patients with sickle cell disease (SCD), which often occurs in the setting of a vaso-occlusive painful crisis. P-selectin antibody therapy reduces hospitalization of patients with SCD by ∼50%, suggesting that an unknown P-selectin–independent mechanism promotes remaining vaso-occlusive events. In patients with SCD, intraerythrocytic polymerization of mutant hemoglobin promotes ischemia-reperfusion injury and hemolysis, which leads to the development of sterile inflammation. Using intravital microscopy in transgenic, humanized mice with SCD and in vitro studies with blood from patients with SCD, we reveal for the first time that the sterile inflammatory milieu in SCD promotes caspase-4/11–dependent activation of neutrophil–gasdermin D (GSDMD), which triggers P-selectin–independent shedding of neutrophil extracellular traps (NETs) in the liver. Remarkably, these NETs travel intravascularly from liver to lung, where they promote neutrophil-platelet aggregation and the development of acute lung injury. This study introduces a novel paradigm that liver-to-lung embolic translocation of NETs promotes pulmonary vascular vaso-occlusion and identifies a new GSDMD-mediated, P-selectin–independent mechanism of lung injury in SCD.

Understanding the kinetics and spatiotemporal interactions of living cells within the tissue environment requires real-time imaging. The introduction of two-photon microscopy has substantially boosted the power of live intravital imaging, making it possible to obtain information of individual cells in near-physiologic conditions within intact tissues nondestructively. Intravital imaging of the liver has proved useful in understanding its 3D structure, function, and dynamic cellular interactions. Recently we have shown that integrity of the blood-bile barrier in different physiologic and pathophysiologic conditions can be imaged in real time using intravital microscopy. Here we discuss the real-time intravital imaging method to visualize blood-bile barrier integrity in the murine liver. © 2021 Wiley Periodicals LLC. Basic Protocol 1: Live imaging in the mouse liver Support Protocol: Monitoring vital signs of the mouse during live liver imaging Basic Protocol 2: Visualizing blood and bile transport using intravital microscopy.

Background Whole lung tissue transcriptomic profiling studies in chronic obstructive pulmonary disease (COPD) have led to the identification of several genes associated with the severity of airflow limitation and/or the presence of emphysema, however, the cell types driving these gene expression signatures remain unidentified. Methods To determine cell specific transcriptomic changes in severe COPD, we conducted single-cell RNA sequencing (scRNA seq) on n = 29,961 cells from the peripheral lung parenchymal tissue of nonsmoking subjects without underlying lung disease (n = 3) and patients with severe COPD (n = 3). The cell type composition and cell specific gene expression signature was assessed. Gene set enrichment analysis (GSEA) was used to identify the specific cell types contributing to the previously reported transcriptomic signatures. Results T-distributed stochastic neighbor embedding and clustering of scRNA seq data revealed a total of 17 distinct populations. Among them, the populations with more differentially expressed genes in cases vs. controls (log fold change >|0.4| and FDR = 0.05) were: monocytes (n = 1499); macrophages (n = 868) and ciliated epithelial cells (n = 590), respectively. Using GSEA, we found that only ciliated and cytotoxic T cells manifested a trend towards enrichment of the previously reported 127 regional emphysema gene signatures (normalized enrichment score [NES] = 1.28 and = 1.33, FDR = 0.085 and = 0.092 respectively). Among the significantly altered genes present in ciliated epithelial cells of the COPD lungs, QKI and IGFBP5 protein levels were also found to be altered in the COPD lungs. Conclusions scRNA seq is useful for identifying transcriptional changes and possibly individual protein levels that may contribute to the development of emphysema in a cell-type specific manner.

Background: Whole lung tissue transcriptomic profiling studies in chronic obstructive pulmonary disease (COPD) have led to the identification of several genes associated with the severity of airflow limitation and/or the presence of emphysema, however, the cell types driving these gene expression signatures remain unidentified.Methods: To determine cell specific transcriptomic changes in severe COPD, we conducted single-cell RNA sequencing (scRNA seq) on n= 29,961 cells from the peripheral lung parenchymal tissue of nonsmoking subjects without underlying lung disease (n=3) and patients with severe COPD (n=3). The cell type composition and cell specific gene expression signature was assessed. Gene set enrichment analysis (GSEA) was used to identify the specific cell types contributing to the previously reported transcriptomic signatures.Results: T-distributed stochastic neighbor embedding and clustering of scRNA seq data revealed a total of 17 distinct populations. Among them, the populations with more differentially expressed genes in cases vs. controls (log fold change >|0.4| and FDR=0.05) were: monocytes (n=1499); macrophages (n=868) and ciliated epithelial cells (n= 590), respectively. Using GSEA, we found that only ciliated and cytotoxic T cells manifested a trend towards enrichment of the previously reported 127 regional emphysema gene signatures (normalized enrichment score [NES] = 1.28 and =1.33, FDR= 0.085 and =0.092 respectively). Among the significantly altered genes present in ciliated epithelial cells of the COPD lungs, QKI and IGFBP5 protein levels were also found to be altered in the COPD lungs. Conclusions: scRNA seq is useful to identify transcriptional changes and possibly individual protein levels that may contribute to the development of emphysema in a cell-type specific manner.

SummaryYAP1 regulates cell plasticity during liver injury, regeneration and cancer, but its role in liver development is unknown. YAP1 activity was detected in biliary cells and in cells at the hepato-biliary bifurcation in single-cell RNA-sequencing analysis of developing livers. Hepatoblast deletion ofYap1led to no impairment in Notch-driven SOX9+ ductal plate formation, but prevented the formation of the abutting second layer of SOX9+ ductal cells, blocking the formation of a patent intrahepatic biliary tree. Intriguingly, the mice survived for 8 months with severe cholestatic injury and without any hepatocyte-to-biliary transdifferentiation. Ductular reaction in the perihilar region suggested extrahepatic biliary proliferation likely seeking the missing intrahepatic biliary network. Long-term survival of these mice occurred through hepatocyte adaptation via reduced metabolic and synthetic function including altered bile acid metabolism and transport. Overall, we show YAP1 as a key regulator of bile duct development while highlighting a profound adaptive capability of hepatocytes.

Patients with hereditary or acquired hemolytic anemias have a high risk of developing in situ thrombosis of the pulmonary vasculature. While pulmonary thrombosis is a major morbidity associated with hemolytic disorders, the etiological mechanism underlying hemolysis-induced pulmonary thrombosis remains largely unknown. Here, we use intravital lung microscopy in mice to assess the pathogenesis of pulmonary thrombosis following deionized water–induced acute intravascular hemolysis. Acute hemolysis triggered the development of αIIbβ3-dependent platelet-rich thrombi in precapillary pulmonary arterioles, which led to the transient impairment of pulmonary blood flow. The hemolysis-induced pulmonary thrombosis was phenocopied with intravascular ADP- but not thrombin-triggered pulmonary thrombosis. Consistent with a mechanism involving ADP release from hemolyzing erythrocytes, the inhibition of platelet P2Y(12) purinergic receptor signaling attenuated pulmonary thrombosis and rescued blood flow in the pulmonary arterioles of mice following intravascular hemolysis. These findings are the first in vivo studies to our knowledge to suggest that acute intravascular hemolysis promotes ADP-dependent platelet activation, leading to thrombosis in the precapillary pulmonary arterioles, and that thrombin generation most likely does not play a significant role in the pathogenesis of acute hemolysis–triggered pulmonary thrombosis.

β-Catenin, the downstream effector of the Wnt signaling, plays important roles in hepatic development, regeneration, and tumorigenesis. However, its role at hepatocyte adherens junctions (AJ) is relatively poorly understood, chiefly due to spontaneous compensation by γ-catenin. We simultaneously ablated β- and γ-catenin expression in mouse liver by interbreeding β-catenin-γ-catenin double-floxed mice and Alb-Cre transgenic mice. Double knockout mice show failure to thrive, impaired hepatocyte differentiation, cholemia, ductular reaction, progressive cholestasis, inflammation, fibrosis, and tumorigenesis, which was associated with deregulation of tight junctions (TJ) and bile acid transporters, leading to early morbidity and mortality, a phenotype reminiscent of progressive familial intrahepatic cholestasis (PFIC). To address the mechanism, we specifically and temporally eliminated both catenins from hepatocytes using adeno-associated virus 8 carrying Cre-recombinase under the thyroid-binding globulin promoter (AAV8-TBG-Cre). This led to a time-dependent breach of the blood-biliary barrier associated with sequential disruption of AJ and TJ verified by ultrastructural imaging and intravital microscopy, which revealed unique paracellular leaks around individual hepatocytes, allowing mixing of blood and bile and leakage of blood from one sinusoid to another. Molecular analysis identified sequential losses of E-cadherin, occludin, claudin-3, and claudin-5 due to enhanced proteasomal degradation, and of claudin-2, a β-catenin transcriptional target, which was also validated in vitro. Conclusion We report partially redundant function of catenins at AJ in regulating TJ and contributing to the blood-biliary barrier. Furthermore, concomitant hepatic loss of β- and γ-catenin disrupts structural and functional integrity of AJ and TJ via transcriptional and posttranslational mechanisms. Mice with dual catenin loss develop progressive intrahepatic cholestasis, providing a unique model to study diseases such as PFIC. (Hepatology 2018;67:2320-2337).

Rationale: Epidemiological evidence suggests that prior exposure to cigarette smoke (CS) or habitual smoking increases the risk of influenza A virus (IAV)-triggered respiratory failure (severe flu). Although emerging evidence supports the role of thrombo-inflammation in the development of CS and IAV-triggered lung injury, the innate immune mechanism that contributes to this morbidity remains poorly understood. Materials and methods: We have developed a two-hit model of CS-induced severe flu in mice. Mice were exposed to four weeks of room air (air) or CS followed by intra-nasal administration of A/PR/8/34 (H1N1) IAV. The body weight was measured every day for two weeks after IAV administration followed by assessment of lung injury at day 7 and 14. Lungs were harvested for histological assessment of lung injury and estimation of viral titer by RT-PCR. Quantitative fluorescence intravital lung microscopy (qFILM) was conducted at 2-, 3- and 4-days post IAV-infection to visualize dynamics of neutrophil and platelet recruitment in the lung of mice IV administered with fluorescent dextran, anti-Ly6G Ab and anti-CD49Ab. Results: Mice exposed to CS+IAV manifested significantly more weight loss, lung injury, lung congestion and alveolar hemorrhage compared to mice administered room-air+IAV. QFILM revealed that severity of lung injury was associated with significantly more entrapment of neutrophil-platelet aggregates within the pulmonary microcirculation and infiltration into the air spaces of CS+IAV than room-air+IAV administered mice. Conclusion: These initial results suggest that CS primes innate immune signaling in neutrophils and platelets to promote their recruitment in the lung following flu. Currently, studies are underway to identify innate immune pathways in neutrophils and platelets that drive this severe thrombo-inflammatory response. Disclosures Sundd: CSL Behring Inc: Research Funding; Bayer: Research Funding; Novartis: Membership on an entity's Board of Directors or advisory committees, Research Funding.

Sickle cell disease (SCD) is caused by a homozygous mutation in the β-globin gene, which leads to erythrocyte sickling, vaso-occlusion, and premature hemolysis . Vaso-occlusion and hemolysis are the two predominant pathophysiologic events in SCD that contribute to chronic organ damage and acute systemic painful vaso-occlusive episode (VOE). Previous studies both in vivo and in vitro have confirmed the role of endothelial and platelet adhesion marker P-selectin in sickle cell vasoocclusive crisis. Moreover, a phase 2 study showed a significant reduction in painful vaso-occlusive episodes among SCD patients receiving the P-selectin-blocking antibody crizanlizumab . However, the long-term effect of crizanlizumab in SCD and associated organ injuries is not known. To understand the roles and regulation of P-selectin dependent vasoocclusion in SCD associated organ injuries, we have introduced the first SCD mice genetically lacking P-selectin in hematopoietic and nonhematopoietic compartments. Using this model (SS-Selp −/−) we have shown that P-selectin deficiency protects SCD mice from lung vaso-occlusion. Here we have used this model to study the liver injury in SCD. Using quantitative liver intravital imaging (qLIM) technique, we show that P-selectin deficiency protects SCD mice from liver vaso-occlusion. However, we found persistent hepatobiliary injury in SS-Selp −/− mice. Mechanistically, we show that blocking P-selectin causes significant enrichment of circulating inflammatory cells however the organ specific recruitment of inflammatory cells was drastically impaired. Remarkably, impairment of organ specific recruitment of inflammatory cells exacerbated cellular senescence and injury in sickle mouse. We found significant enrichment of senescent markers including P21, P16 and phosphor P53 using both western blot and immunohistochemistry. Colocalization analysis confirmed that hepatocytes, cholangiocytes and macrophages were susceptible to senescence. Moreover, we also found impaired iron trafficking in in SS-Selp −/− mice. Work is currently underway to understand how p-selectin loss promotes liver senescence and impaired iron trafficking in the liver. In summary, our results reveal a significant defect in iron homeostasis and exacerbated senescence in the liver of SS-Selp −/− mice suggesting that increased liver senescence might impair iron homeostasis which then contribute to hepatobiliary injury in SCD. Thus, the efficacy of P-selectin inhibition in preventing SCD warrants further studies to determine whether long term inhibition of P-selectin would lead to end stage complications. Disclosures Sundd: Bayer: Research Funding; Novartis: Membership on an entity's Board of Directors or advisory committees, Research Funding; CSL Behring Inc: Research Funding.

Acute systemic painful vaso-occlusive episode (VOE) often serves as an antecedent to acute chest syndrome (ACS), which is a type of acute lung injury and the leading cause of mortality among sickle cell disease (SCD) patients. Based on clinical epidemiology, ACS is often preceded by thrombocytopenia and involves massive thrombosis across pulmonary artery branches in 10-20% of ACS patients. Although, released during hemolysis, adenosine diphosphate (ADP) is known to activate platelets by stimulating their P2Y1 and P2Y12 purinergic receptors, antagonists of P2Y12 have not shown any benefit in ACS therapy, justifying the need for better understanding of purinergic signaling in SCD. Ecto-nucleoside-tri-phosphate-diphosphohydrolase-1 (E-NTPDase1; CD39) maintains ADP homeostasis by degrading excessive ADP. Though CD39 inhibits ADP-dependent platelet activation and vascular thrombosis, its role in ASC is still unidentified. Here, we use intravital lung microscopy in transgenic humanized SCD mice to show that intravascular (IV) administration of ADP triggered pulmonary thrombosis in control mice but failed to trigger pulmonary thrombosis in SCD mice. Identical to intravital findings, IV ADP administration also evoked transient thrombocytopenia in control but not SCD mice, while, IV collagen led to comparable drop in platelet count in both SCD and control mice. In vitro turbidimetric aggregation study yet again demonstrated impaired SCD mouse platelet response to ADP, which was significantly augmented by CD39 inhibitor (sodium metatungstate, POM-1). Indeed, we found significantly higher plasma levels and activity of CD39 in SCD mice compared to control mice using ELISA and malachite green assays, respectively. Hemin, a major host-derived damage associated molecular pattern (DAMP) in SCD, was incubated with Human Lung MicroVascular Endothelial Cell (HMVEC-L) to assess CD39 expression using western blotting. Hemin (10 -100 µM) in a dose dependent manner decreased CD39 levels in HMVEC-L. Our current findings suggest that elevated CD39 plasma levels and activity possibly prevents ADP-mediated platelet aggregation and pulmonary thrombosis in SCD. We demonstrated that SCD milieu promotes loss of endothelial CD39, which may be directly associated with increased CD39 plasma levels and activity. Current study explains why P2Y12 blockers are not effective in SCD therapy and warrant the need for further studies to understand the role of purinergic signaling in pathogenesis of ACS. Disclosures Sundd: Novartis: Membership on an entity's Board of Directors or advisory committees, Research Funding; CSL Behring Inc: Research Funding; Bayer: Research Funding.

Hemophilia A is an X-linked recessive bleeding disorder that affects 1 in 5000 males and is caused by procoagulant factor VIII deficiency. Affected people are at danger of spontaneous bleeding into organs, which can be fatal and lead to persistent damage. Current therapy includes intravenous infusion of FVIII protein concentrate which carries the danger of transmitting blood-borne diseases. As a result of recent advancements in liver-directed gene transfer, gene therapy based innovative strategy for treating hemophilia has emerged. In patients with severe hemophilia B, intravenous infusion of an adeno-associated viral (AAV) vector encoding factor IX (FIX) under the control of a liver-directed promoter resulted in expression of FIX for a considerable period of time. In hemophilia-A patients, gene treatment utilizing AAV vectors has demonstrated to be less effective than Hemophilia B due to the size of the F8 coding sequence and the decreased release of FVIII protein. Among other concerns high immunogenicity of FVIII with 25-30% of hemophilia A patients forming inhibitors and overexpression of FVIII in hepatocytes triggering a cellular stress response are significantly challenging. A phase 1 clinical trial is now being conducted to examine the AAV8 induced liver directed gene expression strategy to circumvent these challenges. The Factor VIII null mouse has been effective in understanding the disease pathogenesis as well as the development of liver directed novel gene therapy techniques to treat hemophilia. FVIII is predominantly produced in the liver. Thus, liver directed adenoviral and retroviral vectors have been studied by several groups to understand the gene delivery method in hemophilia. A few of these studies have shown limited effectiveness in hemophilia animal models. Although the coagulation anomaly seen in hemophilia murine model was completely repaired immediately after liver directed adenovirus-mediated treatment, the effect was transient. Additionally, adeno associated virus (AAV8)-FVIII overexpression has been associated with increased cellular stress. In this study we evaluated the stability and efficacy of liver driven gene transfer mechanism in FVIII null mouse using recombinant AAV8 vector. Recombinant AAV8 vector delivered through the systemic circulation successfully transduces to target tissues via passing through the permeable barrier of sinusoidal endothelial cell. The bidirectional passage through sinusoidal endothelial cell is mainly supported by the presence of discontinuous fenestrated endothelium. Remarkably, we found that liver directed gene transfer was significantly delayed in FVIII null mice. Using quantitative liver intravital imaging we found that upon AAV8-GFP administration liver sinusoidal endothelial cells shows increased apoptosis. Moreover, structural analysis of the liver sinusoidal endothelial cells using intravital and electron micrograph imaging showed significant structural functional difference in liver sinusoidal endothelial cells of FVIII KO mouse. Work is currently underway to understand how absence of FVIII can affect the LSECs. In conclusion, detailed molecular characterization of LSEC-mediated liver directed gene transfer in a hemophilia mouse model is critical for understanding the efficacy and stability of gene-based hemophilia treatment. Disclosures Sundd: Bayer: Research Funding; Novartis: Membership on an entity's Board of Directors or advisory committees, Research Funding; CSL Behring Inc: Research Funding.

Sickle cell disease (SCD) is a monogenic disorder, estimated to affect over three million people worldwide(1,2). Acute systemic painful vaso-occlusive episode (VOE) is the primary reason for emergency medical care among SCD patients(3). VOE may also progress to acute chest syndrome (ACS), a type of acute lung injury and one of the primary reasons for mortality among SCD patients(3). Recently, P-selectin monoclonal antibodies were shown to attenuate VOE in SCD patients and lung vaso-occlusion in transgenic humanized SCD mice, highlighting the therapeutic benefit of P-selectin inhibition in SCD(4,5). Here, we use quantitative fluorescence intravital lung microscopy (qFILM) to show that tandem-P-selectin-glycoprotein-ligand-immunoglobulin (TSGL-Ig) fusion molecule containing four P-selectin binding sites, significantly attenuated intravenous (IV) oxy-hemoglobin (oxy-Hb) triggered lung vaso-occlusion in SCD mice. These findings highlight the therapeutic potential of TSGL-Ig to prevent VOE and ACS in SCD.

BACKGROUND AND AIMS Hepatic crisis is an emergent complication affecting patients with sickle cell disease (SCD); however, the molecular mechanism of sickle cell hepatobiliary injury remains poorly understood. Using the knock-in humanized mouse model of SCD and SCD patient blood, we sought to mechanistically characterize SCD-associated hepato-pathophysiology applying our recently developed quantitative liver intravital imaging, RNA sequence analysis, and biochemical approaches. APPROACH AND RESULTS SCD mice manifested sinusoidal ischemia, progressive hepatomegaly, liver injury, hyperbilirubinemia, and increased ductular reaction under basal conditions. Nuclear factor kappa B (NF-κB) activation in the liver of SCD mice inhibited farnesoid X receptor (FXR) signaling and its downstream targets, leading to loss of canalicular bile transport and altered bile acid pool. Intravital imaging revealed impaired bile secretion into the bile canaliculi, which was secondary to loss of canalicular bile transport and bile acid metabolism, leading to intrahepatic bile accumulation in SCD mouse liver. Blocking NF-κB activation rescued FXR signaling and partially ameliorated liver injury and sinusoidal ischemia in SCD mice. CONCLUSIONS These findings identify that NF-κB/FXR-dependent impaired bile secretion promotes intrahepatic bile accumulation, which contributes to hepatobiliary injury of SCD. Improved understanding of these processes could potentially benefit the development of therapies to treat sickle cell hepatic crisis.

Epidemiological evidence suggests that prior exposure to cigarette smoke (CS) or habitual smoking increases the risk of influenza A virus (IAV)-triggered respiratory failure. Although emerging evidence supports the role of thrombo-inflammation in the development of CS and IAV-triggered lung injury, the innate immune mechanism remains poorly understood. We have developed a two-hit model of CS-induced severe flu in mice. Mice were exposed to four weeks of room air (air) or CS followed by intra-nasal administration of A/PR/8/34 IAV. The body weight was measured every day for two weeks after IAV administration followed by assessment of lung injury at day 7. Lungs were harvested for histological assessment and viral titration by qPCR. Quantitative fluorescence intravital lung microscopy (qFILM) was conducted at 2-, 3- and 4-days post IAV-infection to visualize dynamics of neutrophil and platelet recruitment in the lung of mice IV administered with fluorescent dextran, anti-Ly6G Ab and anti-CD49Abs. Mice exposed to CS+IAV manifested significantly more weight loss, lung injury, lung congestion, hemorrhage and hypoxemia compared to mice administered IAV only. QFILM revealed that severity of lung injury was associated with significantly larger area with impaired blood flow and more vascular leakage secondary to vascular occlusion by platelet-rich neutrophil-platelet aggregates in the lung of CS+IAV than IAV administered mice. These initial results suggest that CS primes innate immune signaling in neutrophils and platelets to promote their recruitment in the lung following flu, leading to severe acute lung injury. Currently, studies are underway to identify innate immune pathways that drive hyper thrombo-inflammatory response.

SUMMARY Yes-associated protein 1 (YAP1) regulates cell plasticity during liver injury, regeneration, and cancer, but its role in liver development is unknown. We detect YAP1 activity in biliary cells and in cells at the hepatobiliary bifur-cation in single-cell RNA sequencing analysis of developing livers. Deletion of Yap1 in hepatoblasts does not impair Notch-driven SOX9+ ductal plate formation but does prevent the formation of the abutting second layer of SOX9+ ductal cells, blocking the formation of a patent intrahepatic biliary tree. Intriguingly, these mice survive for 8 months with severe cholestatic injury and without hepatocyte-to-biliary transdifferentiation. Ductular reaction in the perihilar region suggests extrahepatic biliary proliferation, likely seeking the missing intrahepatic biliary network. Long-term survival of these mice occurs through hepatocyte adaptation via reduced metabolic and synthetic function, including altered bile acid metabolism and transport. Overall, we show YAP1 as a key regulator of bile duct development while highlighting a profound adaptive capability of hepatocytes., Graphical Abstract, In brief Molina et al. demonstrate that loss of Yap1 in developing livers leads to absence of intrahepatic bile ducts, preventing bile secretion from liver to small intestines. Yap1 knockout mice are small and jaundiced and exhibit adaptation via altering bile acid transport and metabolism, but they eventually die around 8 months of age.

Hepatic crisis is an emergent complication affecting sickle cell disease (SCD) patients, however, the molecular mechanism of sickle cell hepatobiliary crisis remains poorly understood. We have used intravital imaging to show that sinusoidal ischemia occurs in the liver of transgenic-humanized SCD mice under basal condition. SCD mice manifested progressive hepatomegaly, liver injury and hyperbilirubinemia. RNA-sequence analysis identified dysregulation of genes encoding proteins responsible for fibrosis, bile acid synthesis, and bile transport in the SCD mice liver. Live injury was the result of NF-κB-dependent inhibition of FXR signaling and its downstream targets in hepatocytes, leading to loss of canalicular bile transport and bile metabolism. Blocking NF-κB activation rescued FXR-signaling, ameliorated liver injury and resolved sinusoidal ischemia in SCD mice. Moreover, administration of FXR agonist (GW4064) also ameliorated liver injury seen in SCD mice. These findings are the first to identify that FXR-dependent impaired bile secretion promotes intrahepatic bile accumulation, which contributes to hepatobiliary injury of SCD. Improved understanding of these pathways could potentially benefit the development of new therapies to treat sickle cell hepatic crisis. Disclosures No relevant conflicts of interest to declare.

Pulmonary thrombosis is a major complication associated with high morbidity. Despite advances in diagnosis and treatment, the pathophysiology of pulmonary thrombosis remains incompletely understood. New clinical evidence suggests that in situ platelet activation resulting in enhanced procoagulant activity may promote pulmonary thrombosis. Improved understanding of the etiological mechanism would enable the development of new therapies for pulmonary thrombosis. Collagen and thromboplastin (TF) were administered intravascularly (IV) to C57BL/6 (WT) mice and the pulmonary microcirculation was visualized using quantitative fluorescence intravital fluorescence lung microscopy (qFILM). Fluorochrome-conjugated anti-mouse CD49b Ab and dextran was administered IV for in vivo staining of circulating platelets and visualization of blood vessels, respectively. Pulmonary thrombosis was defined as occlusion of blood vessels with platelet aggregates leading to pulmonary ischemia. Additionally, quantitative microfluidic fluorescence microscopy (qMFM) was used to study the effect of platelet αIIbβ3 inhibition on platelet procoagulant activity in human blood under vascular mimetic flow conditions. Collagen and TF triggered dose-dependent pulmonary thrombosis in mice in vivo, which involved development of platelet-rich thrombi in the pulmonary arteriolar bottlenecks (junction of pulmonary arteriole and capillaries), resulting in a transient ischemia in the arteriole and the down-stream capillary tree. The pulmonary arteriole thrombosis triggered by IV collagen or TF was protracted, lethal and completely abrogated following IV administration of αIIbβ3 receptor inhibitor (eptifibatide). Inhibition of platelet αIIbβ3 also significantly reduced platelet procoagulant activity, fibrin formation and thrombus formation in human blood flowing through microfluidic channels ex vivo. Our current findings suggest that αIIbβ3-dependent platelet procoagulant activity promotes pulmonary thrombosis. Both our models have potential application in investigating the molecular determinants of pulmonary thrombosis in diverse pulmonary disorders as well as evaluating efficacy of new antithrombotic drugs. Disclosures No relevant conflicts of interest to declare.

Sickle cell disease (SCD) is an autosomal-recessive-genetic disorder that affects millions of people worldwide. Although hepatic crisis affects 10-40% of hospitalized SCD patients and can progress to fatal liver failure, the current treatment is primarily supportive and the molecular pathophysiology remains largely unknown. We found that transgenic, humanized SCD mice developed liver injury with age, manifested by increased inflammation, necrosis and hepatic iron accumulation. The presence of iron particles in SCD liver was confirmed by transmission electron micrograph (TEM) analysis and prussian blue staining which revealed increased iron accumulation in the central and midzonal region of the SCD liver tissue. An increase in aggregates of iron pigment reminiscent of hemosiderin-laden macrophages was also observed in SCD liver tissue. Interestingly, the SCD mice also showed significant enrichment of both hepatic (p=0.02) and serum iron (p=0.04) compared to control AS mice. We determined the expression level of genes commonly involved in iron homeostasis by RT-PCR. Interestingly, a significantly lower expression level of hepcidin transcripts was observed in the hepatocytes of SCD mice compared to control mice (AS) (p=0.01). In order to define the pathways controllinghepcidintranscription in SCD, we performed an RNA-seq analysis in the liver of SCD mice. Remarkably, our data showed significant misexpression of BMP signaling pathways. Further analysis revealed a significant misexpression in BMP2 and 6 levels in the liver of SCD mice by western blot. Reduced levels of hepcidin were also confirmed in serum samples from SCD patients compared to controls. Work is currently underway to understand how BMP2/6 might hypothetically regulate hepcidin expression in the liver. In summary, our results reveal a significant defect in iron homeostasis in the liver of SCD mice, suggesting that impaired iron homeostasis may contribute to hepatobiliary injury in SCD independent of blood transfusions. Our study also highlights the importance of hepcidin as potential therapeutic target in regulation of hepatic injury in SCD. Disclosures No relevant conflicts of interest to declare.

Intraerythrocytic polymerization of hemoglobin S promotes hemolysis and vaso-occlusive events in the microvasculature of sickle cell disease (SCD) patients. Although platelet-neutrophil aggregates-dependent vaso-occlusion is known to occur in the lung and contribute to lung injury, the etiological mechanisms that trigger acute chest syndrome are largely unknown. Here, we use intravital imaging of the lung in transgenic humanized SCD mice and in vitro imaging of SCD patient blood flowing through a microfluidic system to identify the innate-immune mechanism that promotes platelet-neutrophil aggregate-dependent lung vaso-occlusion and injury in SCD. Platelet-inflammasome activation led to generation of IL-1β and caspase-1 carrying platelet extracellular vesicles (EVs) that bind to neutrophils and promote platelet-neutrophil aggregation in lung arterioles of SCD mice in vivo and SCD human blood in microfluidics in vitro. The inflammasome activation, platelet EV generation and platelet-neutrophil aggregation were enhanced by the presence of lipopolysaccharide at a nanogram dose in SCD but not control human blood. Inhibition of the inflammasome effector caspase-1 or IL-1β pathway attenuated platelet EV generation, prevented platelet-neutrophil aggregation, and restored microvascular blood flow in lung arterioles of SCD mice in vivo and SCD human blood in microfluidics in vitro. These results are the first to identify that platelet-inflammasome dependent shedding of IL-1β and caspase-1 carrying platelet EVs promote lung vaso-occlusion in SCD. The current findings also highlight the therapeutic potential of targeting the platelet-inflammasome dependent innate immune pathway to prevent acute chest syndrome in SCD.

Background and aims The Wnt/β-catenin signaling pathway has a well-described role in liver pathobiology. Its suppression was recently shown to decrease bile acid (BA) synthesis, thus preventing the development of cholestatic liver injury and fibrosis after bile duct ligation (BDL). Approach and results To generalize these observations, we suppressed β-catenin in Mdr2 knockout (KO) mice, which develop sclerosing cholangitis due to regurgitation of BA from leaky ducts. When β-catenin was knocked down (KD) in KO for 2 weeks, hepatic and biliary injury were exacerbated in comparison to KO given placebo, as shown by serum biochemistry, ductular reaction, inflammation, and fibrosis. Simultaneously, KO/KD livers displayed increased oxidative stress and senescence and an impaired regenerative response. Although the total liver BA levels were similar between KO/KD and KO, there was significant dysregulation of BA transporters and BA detoxification/synthesis enzymes in KO/KD compared with KO alone. Multiphoton intravital microscopy revealed a mixing of blood and bile in the sinusoids, and validated the presence of increased serum BA in KO/KD mice. Although hepatocyte junctions were intact, KO/KD livers had significant canalicular defects, which resulted from loss of hepatocyte polarity. Thus, in contrast to the protective effect of β-catenin KD in BDL model, β-catenin KD in Mdr2 KO aggravated rather than alleviated injury by interfering with expression of BA transporters, hepatocyte polarity, canalicular structure, and the regenerative response. Conclusions The resulting imbalance between ongoing injury and restitution led to worsening of the Mdr2 KO phenotype, suggesting caution in targeting β-catenin globally for all cholestatic conditions.

Background & Aims Liver fibrosis, hepatocellular necrosis, inflammation, and proliferation of liver progenitor cells are features of chronic liver injury. Mouse models have been used to study the end-stage pathophysiology of chronic liver injury. However, little is known about differences in the mechanisms of liver injury among different mouse models because of our inability to visualize the progression of liver injury in vivo in mice. We developed a method to visualize bile transport and blood-bile barrier (BBlB) integrity in live mice. Methods C57BL/6 mice were fed a choline-deficient, ethionine-supplemented (CDE) diet or a diet containing 0.1% 3,5-diethoxycarbonyl-1, 4-dihydrocollidine (DDC) for up to 4 weeks to induce chronic liver injury. We used quantitative liver intravital microscopy (qLIM) for real-time assessment of bile transport and BBlB integrity in the intact livers of the live mice fed the CDE, DDC, or chow (control) diets. Liver tissues were collected from mice and analyzed by histology, immunohistochemistry, real-time polymerase chain reaction, and immunoblots. Results Mice with liver injury induced by a CDE or a DDC diet had breaches in the BBlB and impaired bile secretion, observed by qLIM compared with control mice. Impaired bile secretion was associated with reduced expression of several tight-junction proteins (claudins 3, 5, and 7) and bile transporters (NTCP, OATP1, BSEP, ABCG5, and ABCG8). A prolonged (2-week) CDE, but not DDC, diet led to re-expression of tight junction proteins and bile transporters, concomitant with the reestablishment of BBlB integrity and bile secretion. Conclusions We used qLIM to study chronic liver injury, induced by a choline-deficient or DDC diet, in mice. Progression of chronic liver injury was accompanied by loss of bile transporters and tight junction proteins.

Summary Although commensal flora is involved in the regulation of immunity, the interplay between cytokine signaling and microbiota in atherosclerosis remains unknown. We found that interleukin (IL)-23 and its downstream target IL-22 restricted atherosclerosis by repressing pro-atherogenic microbiota. Inactivation of IL-23-IL-22 signaling led to deterioration of the intestinal barrier, dysbiosis, and expansion of pathogenic bacteria with distinct biosynthetic and metabolic properties, causing systemic increase in pro-atherogenic metabolites such as lipopolysaccharide (LPS) and trimethylamine N-oxide (TMAO). Augmented disease in the absence of the IL-23-IL-22 pathway was mediated in part by pro-atherogenic osteopontin, controlled by microbial metabolites. Microbiota transfer from IL-23-deficient mice accelerated atherosclerosis, whereas microbial depletion or IL-22 supplementation reduced inflammation and ameliorated disease. Our work uncovers the IL-23-IL-22 signaling as a regulator of atherosclerosis that restrains expansion of pro-atherogenic microbiota and argues for informed use of cytokine blockers to avoid cardiovascular side effects driven by microbiota and inflammation.

In patients with sickle cell disease (SCD), the polymerization of intraerythrocytic hemoglobin S promotes downstream vaso-occlusive events in the microvasculature. While vaso-occlusion is known to occur in the lung, often in the context of systemic vaso-occlusive crisis and the acute chest syndrome, the pathophysiological mechanisms that incite lung injury are unknown. We used intravital microscopy of the lung in transgenic humanized SCD mice to monitor acute vaso-occlusive events following an acute dose of systemic lipopolysaccharide sufficient to trigger events in SCD but not control mice. We observed cellular microembolism of precapillary pulmonary arteriolar bottlenecks by neutrophil-platelet aggregates. Blood from SCD patients was next studied under flow in an in vitro microfluidic system. Similar to the pulmonary circulation, circulating platelets nucleated around arrested neutrophils, translating to a greater number and duration of neutrophil-platelet interactions compared with normal human blood. Inhibition of platelet P-selectin with function-blocking antibody attenuated the neutrophil-platelet interactions in SCD patient blood in vitro and resolved pulmonary arteriole microembolism in SCD mice in vivo. These results establish the relevance of neutrophil-platelet aggregate formation in lung arterioles in promoting lung vaso-occlusion in SCD and highlight the therapeutic potential of targeting platelet adhesion molecules to prevent acute chest syndrome.

We love our pets, and when they are separated from us, it is like our whole world comes crashing down on us. We cannot afford to leave any stone unturned in the process of finding them.

Introduction: Acute chest syndrome (ACS) is a type of acute lung injury and among the primary reasons for mortality and morbidity among Sickle Cell Disease (SCD) patients. Although epidemiologic evidence suggests that vaso-occlusion in the lung may serve as an antecedent to ACS, the cellular, molecular and biophysical mechanism of ACS is incompletely understood. Our recent findings revealed that the lung vaso-occlusion is enabled by the entrapment of embolic neutrophil-platelet aggregates in the pulmonary arterioles of transgenic humanized SCD mice. Recent evidence also suggests a role for neutrophil extracellular traps (NETs) in ACS. NETs are web-like structures of decondensed nuclear DNA decorated with citrullinated-histones (H3-cit) and neutrophil granule proteins. Interestingly, circulating nucleosomes and NETs fragments are elevated in SCD patient blood and the levels correlate with onset of ACS, however, the molecular mechanism that promotes generation of circulating NETs and the role of circulating NETs in promoting ACS remains poorly understood. Materials and Methods: Townes knock-in humanized SS (hα/hα:βS/βS) and AS (hα/hα:βA/βS) mice were used as SCD and control mice, respectively. SS and AS mice were intravenously (IV) administered 10 µmole/kg Oxy-Hb followed by Sytox orange, FITC-dextran or fluorescent anti-mouse mAbs against Ly6G, CD49b, H3cit, and neutrophil elastase for in vivo visualization of extracellular DNA, blood vessels, neutrophils, platelets and NETs, respectively. Pulmonary microcirculation was monitored using multi-photon-excitation enabled quantitative fluorescence intravital lung microscopy (qFILM). Results and Discussion: IV Oxy-Hb triggered the occlusion of pulmonary arterioles by neutrophil-platelet aggregates leading to loss of pulmonary blood flow in SCD but not control mice. Surprisingly, pulmonary vaso-occlusion in SCD mice was accompanied by the arrival of circulating cell free DNA (CFD) and NETs fragments into the pulmonary circulation. The cell free DNA (CFD) and NETs fragments entered the lung through the arterial circulation suggesting that they originated outside of lung. These cell free DNA (CFD) and NETs fragments contributed to lung vaso-occlusion and injury by promoting neutrophil-platelet aggregation in the lung arterioles. Conclusion: These findings reveal for the first time that circulating cell free DNA (CFD) and NETs fragments originating outside of lung contribute to pathogenesis of ACS. Currently, experiments are underway to identify the innate immune pathways that promote circulating NETs dependent lung injury in SCD. Disclosures Gladwin: Globin Solutions, Inc: Patents & Royalties: Provisional patents for the use of recombinant neuroglobin and heme-based molecules as antidotes for CO poisoning; United Therapeutics: Patents & Royalties: Co-inventor on an NIH government patent for the use of nitrite salts in cardiovascular diseases ; Bayer Pharmaceuticals: Other: Co-investigator.

Introduction: Sickle cell disease (SCD) is an autosomal-recessive-genetic disorder, which leads to red blood cell sickling, hemolysis and vaso-occlusion. Acute systemic painful vaso-occlusive crisis (VOC) is the predominant pathophysiology requiring emergency medical care by SCD patients. 10-20% of SCD patients hospitalized with VOC tend to develop acute chest syndrome (ACS), a type of lung injury within next few days, suggesting a role for pulmonary vaso-occlusion in ACS. This epidemiology also provides a window for therapeutic intervention provided treatments to prevent vaso-occlusion are available. Earlier, we have shown that VOC involves entrapment of large neutrophil-platelet aggregates in lung arterioles of SCD mice, which is inhibited following intravenous administration of P-selectin function blocking antibody. Recently, a tandem-P-selectin-glycoprotein-ligand-immunoglobulin (TSGL-Ig) with two P-selectin binding sites in tandem, has been shown to prevent P-selectin-dependent liver injury in mice. Here, we test the ability of TSGL-Ig in attenuating P-selectin dependent lung vaso-occlusion in SCD mice. Materials and Methods: Townes knock-in humanized SS (hα/hα:βS/βS) mice were used as SCD mice. SCD mice were intravenously (IV) challenged with 10 µmole/kg Oxy-hemoglobin (Oxy-Hb) without or with 100 µg of TSGL-Ig to trigger lung vaso-occlusion. Fluorescent anti-mouse mAbs against CD49b and Ly6G, and FITC dextran were IV administered for in vivo staining of platelets, neutrophils, and visualizing lung microvasculature, respectively. Presence or absence of lung vaso-occlusion was assessed using multi-photon excitation enabled quantitative fluorescence intravital lung microscopy (qFILM). Results and Discussion: IV administration of Oxy-Hb led to occlusion of pulmonary arterioles by large neutrophil-platelet aggregates. Remarkably, IV administration of TSGL-Ig significantly attenuated lung vaso-occlusion in SCD mice by reducing the number as well as size of neutrophil-platelet aggregates in the pulmonary arterioles of SCD mice. Conclusion: Systemic challenge with Oxy-Hb promotes lung vaso-occlusion in SCD mice. TSGL-Ig significantly reduced oxy-Hb induced lung vaso-occlusion in SCD mice, highlighting the potential of TSGL-Ig to prevent ACS. Disclosures No relevant conflicts of interest to declare.

Background: Vaso-occlusive crisis (VOC) is the primary reason for emergency medical care by sickle cell disease (SCD) patients. SCD patients hospitalized with VOC often develop acute chest syndrome (ACS), a form of acute lung injury, suggesting a role for pulmonary vaso-occlusion in the onset of ACS. However, the cellular, molecular and biophysical mechanism of pulmonary vaso-occlusion is unknown. Methods: SCD transgenic or non-sickle control mice were intravenously (IV) challenged with 2 to 3 ng of bacterial lipopolysaccharide (LPS). Fluorescent anti-mouse Ly-6G and CD49b mAbs were administered IV for in vivo staining of circulating neutrophils and platelets, respectively. Multiphoton excitation enabled quantitative fluorescence intravital lung microscopy (qFILM) was used to determine the molecular mechanism of pulmonary vaso-occlusion in live mice. Function-blocking anti-mouse P-selectin mAb (Fab fragments) was administered IV to assess the role of platelet P-selectin in promoting pulmonary vaso-occlusion. Results: A nanogram dose of IV LPS selectively triggered pulmonary vaso-occlusion in SCD but not control mice. Remarkably, pulmonary vaso-occlusion involved occlusion of the pre-capillary pulmonary arteriole bottle-necks (junction of an arteriole and capillaries) by large neutrophil-platelet embolic aggregates. IV administration of Fab fragments of function blocking anti-P-selectin mAb led to the resolution of pulmonary vaso-occlusion, which was primarily mediated by the attenuation of large neutrophil-platelet aggregates into smaller aggregates that are not stopped by the arteriolar bottle-necks. Conclusion: These results establish the relevance of neutrophil-platelet aggregation in pulmonary arterioles in promoting pulmonary vaso-occlusion in SCD and also highlight the therapeutic potential of inhibiting platelet P-selectin to prevent ACS in SCD patients hospitalized with VOC. Acknowledgments: This study was supported by 1R01HL128297-01 (P.S.), AHA 11SDG7340005 (P.S.), VMI startup funds (P.S.). M.F.B. was supported by NIH-NHLBI training grant T32HL110849 and NIH-NHLBI F32 NRSA 1F32HL131216-01. Disclosures No relevant conflicts of interest to declare.

Introduction: Sickle cell disease (SCD) is an autosomal recessive genetic disorder that affects ~100,000 Americans and millions of people worldwide. The acute chest syndrome (ACS), a form of acute lung injury, is a major cause of morbidity among SCD patients. The current treatment for ACS is primarily supportive and the molecular mechanism remains largely unknown. SCD patients hospitalized with vaso-occlusive pain crisis (VOC) often develop ACS within the ensuing days, suggesting a role for pulmonary vaso-occlusion in the onset of ACS. However, the cellular and molecular mechanism and the anatomical site of pulmonary vaso-occlusion are still elusive. Materials and Methods: Intravenous (IV) bacterial lipopolysaccharide (LPS) was used to induce VOC in SCD mice. Intravital multiphoton excitation (MPE) fluorescence microscopy was used to study the blood cell trafficking within the pulmonary microcirculation of live SCD or control mice. Fluorochrome-conjugated anti-mouse Ly-6G, Ter-119, and CD49b antibodies were administered IV for in vivo staining of circulating neutrophils, red blood cells and platelets, respectively. Cellular trafficking was recorded at baseline and 2 hours after IV challenge with LPS. Image sequences were analyzed to identify vaso-occlusion, which was defined as cellular aggregation and stasis of blood flow within the pulmonary blood vessels. Results and Discussion: Preliminary data using MPE imaging in transgenic SCD mice revealed that vaso-occlusion was absent at baseline in unchallenged SCD mice and the cellular trafficking within the pulmonary microcirculation was comparable in SCD and control mice. Doses of IV LPS (0.01-5 mg/kg of body weight), which were innocuous to control mice were found to be lethal to SCD mice. Remarkably, MPE imaging of the lung microcirculation revealed that IV LPS led to microembolism of the pre-capillary pulmonary arterioles by platelet-neutrophil aggregates in SCD but not control mice. The microembolism involved either entrapment of circulating platelet-neutrophil aggregates or in situ aggregation through sequential steps of neutrophil arrest on the arteriolar endothelium, followed by platelet nucleation on arrested neutrophils and microthrombus formation. Conclusions: Initial findings demonstrate that pulmonary vaso-occlusion in SCD mice involves microembolism of the pre-capillary pulmonary arterioles by platelet-neutrophil aggregates. Future studies will determine the molecular interactions responsible for pulmonary arteriolar microembolism. Acknowledgments: This study is supported by the 11SDG7340005 from the American Heart Association (P.S.) and the VMI startup account (P.S.). M.F.B is supported by the NIH NHLBI VMI T32 training grant T32HL110849. Disclosures No relevant conflicts of interest to declare.