Epithelial Cell Line Derived from Endometriotic Lesion Mimics Macrophage Nervous Mechanism of Pain Generation on Proteome and Metabolome Levels

Endometriosis is a benign disease affecting one in ten women of reproductive age worldwide. Although the pain level is not correlated to the extent of the disease, it is still one of the cardinal symptoms strongly affecting the patients’ quality of life. Yet, a molecular mechanism of this pathology, including the formation of pain, remains to be defined. Recent studies have indicated a close interaction between newly generated nerve cells and macrophages, leading to neurogenic inflammation in the pelvic area. In this context, the responsiveness of an endometriotic cell culture model was characterized upon inflammatory stimulation by employing a multi-omics approach, including proteomics, metabolomics and eicosanoid analysis. Differential proteomic profiling of the 12-Z endometriotic cell line treated with TNFα and IL1β unexpectedly showed that the inflammatory stimulation was able to induce a protein signature associated with neuroangiogenesis, specifically including neuropilins (NRP1/2). Untargeted metabolomic profiling in the same setup further revealed that the endometriotic cells were capable of the autonomous production of 7,8-dihydrobiopterin (BH2), 7,8-dihydroneopterin, normetanephrine and epinephrine. These metabolites are related to the development of neuropathic pain and the former three were found up-regulated upon inflammatory stimulation. Additionally, 12-Z cells were found to secrete the mono-oxygenated oxylipin 16-HETE, a known inhibitor of neutrophil aggregation and adhesion. Thus, inflammatory stimulation of endometriotic 12-Z cells led to specific protein and metabolite expression changes suggesting a direct involvement of these epithelial-like cells in endometriosis pain development.

Differential Leukocyte and Platelet Profiles in Distinct Models of Traumatic Brain Injury

Traumatic brain injury (TBI) affects over 3 million individuals every year in the U.S. There is growing appreciation that TBI can produce systemic modifications, which are in part propagated through blood–brain barrier (BBB) dysfunction and blood–brain cell interactions. As such, platelets and leukocytes contribute to mechanisms of thromboinflammation after TBI. While these mechanisms have been investigated in experimental models of contusion brain injury, less is known regarding acute alterations following mild closed head injury. To investigate the role of platelet dynamics and bioenergetics after TBI, we employed two distinct, well-established models of TBI in mice: the controlled cortical impact (CCI) model of contusion brain injury and the closed head injury (CHI) model of mild diffuse brain injury. Hematology parameters, platelet-neutrophil aggregation, and platelet respirometry were assessed acutely after injury. CCI resulted in an early drop in blood leukocyte counts, while CHI increased blood leukocyte counts early after injury. Platelet-neutrophil aggregation was altered acutely after CCI compared to sham. Furthermore, platelet bioenergetic coupling efficiency was transiently reduced at 6 h and increased at 24 h post-CCI. After CHI, oxidative phosphorylation in intact platelets was reduced at 6 h and increased at 24 h compared to sham. Taken together, these data demonstrate that brain trauma initiates alterations in platelet-leukocyte dynamics and platelet metabolism, which may be time- and injury-dependent, providing evidence that platelets carry a peripheral signature of brain injury. The unique trend of platelet bioenergetics after two distinct types of TBI suggests the potential for utilization in prognosis.

Hairy Platelet-Derived Extracellular Vesicles Promote Lung Vaso-Occlusion in Sickle Cell Disease

Background: Acute chest syndrome (ACS) is a type of acute lung injury and the leading cause of mortality in Sickle Cell Disease (SCD). Current treatments for ACS are primarily supportive, and there is a critical need for rescue therapies. ACS is often a sequela of acute systemic vaso-occlusive crisis and preceded by thrombocytopenia. However, the role of platelets in the pathogenesis of ACS remains largely unknown. Methods: We used our validated model of vaso-occlusive crisis in transgenic, humanized SCD mice, which is triggered by intravenous challenge with nanogram levels of the TLR4 ligand, lipopolysaccharide (LPS). Platelet-neutrophil aggregates and blood flow in the lung microcirculation was visualized in real time in vivo, using multi-photon-excitation microscopy of intact lung in live SCD mice. SCD or control human blood was perfused through microfluidic channels in vitro and neutrophil-platelet aggregation was visualized using fluorescence microscopy. Platelet derived extracellular vesicles were characterized using nanoparticle tracking and biochemical approaches. Results: We have made a novel finding that the arrest of blood flow and injury in the lung is secondary to blockade of pulmonary arterioles by platelet-neutrophil aggregates. Using in vitro microfluidic studies, we confirmed that platelet-neutrophil aggregation is higher in LPS-treated SCD patient blood compared with healthy controls, and this correlates with increased numbers of platelet-derived extracellular vesicles (EVs) that express IL-1β. Our studies also reveal that platelet-neutrophil aggregation in pulmonary arterioles of SCD mice is associated with an increase in peripheral blood levels of platelet-derived EVs containing IL-1β. Remarkably, inhibition of TLR4 or TLR4/NLRP3-inflammasome activated caspase-1, or inhibition of IL-1β signaling, attenuated release of platelet EVs and platelet-neutrophil aggregation in the lung arterioles of SCD mice in vivo and SCD human blood in vitro . Conclusions: TLR4 and NLRP3-inflammasome-mediated caspase-1 activation in platelets during vaso-occlusive crisis leads to release of IL-1β-containing EVs into the circulation. These circulating platelet EVs promote platelet-neutrophil aggregation in pulmonary arterioles, which results in arrest of blood flow in the lung, leading to ACS. Therapeutic inhibition of TLR4/NLRP3-caspase-1 signaling in platelets or IL-1β signaling is a potential therapy for ACS in SCD patients. Acknowledgments: This study was supported by 1R01HL128297-01 (P.S.) and VMI startup funds (P.S.). Disclosures No relevant conflicts of interest to declare.

Neutrophil-Platelet Aggregation Enables Vaso-Occlusion in Sickle Cell Disease

Introduction: Sickle Cell Disease (SCD) is an autosomal-recessive-genetic disorder that leads to sickling and hemolysis of red blood cells (RBCs). Acute vaso-occlusive pain crisis (VOC) is the predominant pathophysiology faced by SCD patients and the primary reason for emergency medical care. Although neutrophils have been shown to play a role in vaso-occlusion by interacting with sickle RBCs in the cremaster venules of transgenic SCD mice, the cellular, molecular and biophysical mechanisms that promote vaso-occlusion in SS patients is not completely understood. Materials and Methods: Freshly collected heparinized blood from steady-state SS patients and race matched control (AA) subjects was perfused through silicone based microfluidic flow channels with a glass bottom coated a cocktail of recombinant human P-selectin, ICAM-1 and IL-8 at a physiological wall shear stress (6 dyn cm-2). Fluorescent Abs against CD16 and CD49b were added to the blood for in-situ staining of neutrophils and platelets, respectively. Cellular interactions were recorded at a single cell-resolution using quantitative microfluidic fluorescence microscopy (qMFM)1, which allows quantitative assessment of vaso-occlusive events at an unprecedented single cell resolution2. Results: Vaso-occlusion in the microfluidic channel involved neutrophil arrest followed by nucleation of platelets on arrested neutrophils, formation of neutrophil-platelet-aggregates (NPA) and partial occlusion of the microfluidic flow channel. Remarkably, the number of platelet-neutrophil interactions and the lifetime of these interactions were several folds higher in SS patient than control AA blood. Surprisingly, preincubation with 250 ng/ml of bacterial lipopolysaccharide (LPS) led to a significant increase in the number and lifetime of platelet-neutrophil interactions in SS but not AA blood. This enhanced NPA formation in SS patient blood was attenuated to the level observed in AA blood by simultaneous blockage of P-selectin on platelets and Mac-1 on neutrophils as well as pretreatment with a small molecule inhibitor of toll-like-receptor-4 (TLR4) signaling pathway. Conclusion: Our data shows that the vaso-occlusive pathophysiology in SCD involves sequential steps of neutrophil arrest, nucleation of platelets on arrested neutrophils, formation of large NPAs and obstruction of blood flow. Platelet-neutrophil aggregation can be ameliorated by the simultaneous blockage of P-selectin on platelets and Mac-1 on neutrophils. The inflammatory milieu of SS patient blood sets a lower threshold for bacterial endotoxin induced neutrophil-platelet aggregation than control blood. The enhanced platelet-neutrophil aggregation in SS blood is dependent on activation of TLR-4 pathway. Understanding the molecular mechanism of vaso-occlusion will enable the development of therapeutics to prevent VOC in SS patients. References: 1 Jimenez MA, Tutuncuoglu E, Barge S, Novelli EM, Sundd P. Quantitative microfluidic fluorescence microscopy to study vaso-occlusion in sickle cell disease. Haematologica. 2015;100(10):e390-e393. doi:10.3324/haematol.2015.126631. 2 Sundd, P. et al. Quantitative dynamic footprinting microscopy reveals mechanisms of neutrophil rolling. Nat Methods7, 821-824, doi:10.1038/nmeth.1508 (2010). Disclosures Kato: Mast Therapeutics: Consultancy; Bayer: Research Funding.

Platelet Nucleation on Arrested Neutrophils Drives Vaso-Occlusion in Sickle Cell Disease

Introduction: Sickle Cell Disease (SCD) is an autosomal recessive genetic disorder that leads to sickling and hemolysis of RBCs under hypoxic conditions. As a result of chronic hemolysis, SCD is associated with a hyper-inflammatory and hyper-coagulation state, which accounts for enhanced adhesion of leukocytes, platelets, RBCs and vascular endothelial cells leading to vaso-occlusion. Acute vaso-occlusive pain crisis (VOC) is the primary reason for emergency medical care by SCD patients. Although neutrophils have been shown to play a role in the on-set of vaso-occlusion by interacting with sickle RBCs and platelets in cremaster venules of transgenic SCD mice, the cellular, molecular and biophysical mechanisms that promote vaso-occlusion in SCD patients is not completely understood. Materials and Methods: Freshly collected heparinized blood from steady-state SCD (SS) patients and race matched control subjects was perfused through polydimethylsiloxane (PDMS) based microfluidic flow channels (30 µm x 500 µm) with a glass bottom coated with either human microvascular endothelial cells or a cocktail of recombinant human P-selectin, ICAM-1 and IL-8 at a physiological shear stress (6 dyn cm-2). Fluorescent Abs against CD16 and CD49b were added to the blood for in-situ staining of neutrophils and platelets, respectively. Cellular interactions were recorded using quantitative microfluidic fluorescence microscopy (qMFM)1, which is a combination of quantitative dynamic footprinting1 and epifluorescence microscopy. Results and Discussion: Neutrophils in SS blood were observed to roll, arrest and then capture freely flowing platelets leading to the formation of vaso-occlusive aggregates. RBCs were observed getting trapped within the platelet-neutrophil aggregates. The number of platelet-neutrophil interactions, lifetime of these interactions and the extent of platelet-neutrophil aggregation were several folds higher in SS than control subject blood. Bacterial lipopolysaccharide (LPS; 500 ng/ml) pretreatment led to enhanced platelet-neutrophil aggregations in SS but not control blood. The enhanced platelet-neutrophil aggregations in SS blood (+/-LPS) was attenuated to the level observed in control blood by simultaneous blockage of P-selectin on platelets and Mac-1 on neutrophils with functional blocking Abs. Conclusion: Our data demonstrates that the vaso-occlusive pathophysiology in SCD involves sequential steps of neutrophil arrest, nucleation of platelets on arrested neutrophils, formation of platelet-neutrophil aggregates and trapping of RBCs in these aggregates. The inflammatory milieu of SS patient blood sets a lower threshold for bacterial endotoxin induced platelet-neutrophil aggregation than control blood. Vaso-occlusion can be ameliorated in SS blood by simultaneous inhibition of platelet P-selectin and neutrophil Mac-1. Understanding the molecular mechanism of vaso-occlusion will enable the development of therapies that can prevent VOC in SS patients. References: 1. Jimenez MA, Tutuncuoglu E, Barge S, Novelli EM, Sundd P. Quantitative microfluidic fluorescence microscopy to study vaso-occlusion in Sickle Cell Disease. Haematologica, 2015. 2 Sundd, P. et al. Quantitative dynamic footprinting microscopy reveals mechanisms of neutrophil rolling. Nat Methods 7, 821-824, doi:10.1038/nmeth.1508 (2010). Disclosures No relevant conflicts of interest to declare.

PRAVASTATIN IN CORRECTION OF VESSEL WALL ANTIPLATELET CONTROL OVER THE BLOOD CELLS IN PATIENTS WITH ARTERIAL HYPERTENSION AND DYSLIPIDEMIA

Aim. To evaluate in patients with arterial hypertension (AH) and dyslipidemia (DL) a grade of correction influence of pravastatin on antiaggregation activity of vessel wall for erythrocytes, platelets and leukocytes.Material and methods. Totally 47 patients observed with 1-2 levels of AH and the risk 3, with DL IIb type, middle age. Controls were 26 healthy people of the same age. To correct DL all patients were prescribed pravastatin 20 mg before night sleep with already being taken enalapril 10 mg BID. Chemistry, blood count and statistics used. Evaluation of clinical and laboratory parameters was performed in the beginning of treatment, in 4, 12 and 52 weeks.Results. An enforced in AH with DLP erythrocyte, platelet neutrophil aggregation was linked with a control decrease over it by vessel wall and as result of lipid metabolism changes, peroxide oxydation of plasma lipids, lowering of NO generation and of prostacycline as well. After 52-week use of pravastatine in AH with DLP there was significantly better in lipid profile and there was weakening of peroxidation of lipids in plasma, that followed by significant positive dynamics of antiplatelet properties of vessel wall.Conclusion. In AH patients with DLP there is weakening of antiplatlet control by vessels over blood cells, which becomes slightly improved after 52-week pravastatine treatment.