Cell Microparticles Research Articles

Blood cell microparticles as biomarkers of hemostatic abnormalities in patients with implanted cardiac assist devices.

For heart failure patients unable to undergo cardiac transplantation, mechanical circulatory support with left ventricular assist devices can be utilized. These devices improve quality of life and prolong life expectancy, but they are associated with bleeding and thrombotic complications impacting patient survival. Little is known of the relevant mechanisms of these hemostatic issues, hindering identification of a clinically useful biomarker. However, there is suggestive evidence that blood cell-derived microparticles may fulfill this unmet clinical need. Recent publications have shown an association of up regulated microparticle production with implanted left ventricular assist devices and the potential to use this as a biomarker to predict thrombosis (and perhaps other adverse events) with an onset time earlier than currently used clinical indicators.

Alterations in Platelet Activity and Elastic Modulus of Healthy Subjects, Carriers of G20210A Polymorphism in the Prothrombin Gene

Summary Platelet activation is a complex process in which platelet reorganization takes place associated with changes in the cell shape, topology, membrane elasticity and microparticle production. The aim of this study was to investigate the changes/aberrations in the platelet activity, elasticity and morphology in healthy subjects, carriers of A allele of prothrombin G20210A polymorphism. Blood samples from 18 healthy subjects were used for platelet analysis by force-mode atomic force microscopy. Restriction analysis was used to investigate the carriage of G20210A polymorphism in the prothrombin gene. Flow- cytometry was applied to evaluate platelet activation. Young’s modulus of the plasma membranes of platelets derived from healthy subjects, carriers of variant A allele of prothrombin 20210G>A polymorphism (407±69 kPa) is two times higher than the one determined for non­carriers (195.4±48.7 kPa; p<0.05). The background activity of platelets measured as an interrelation of Cd41/Cd61 and CD62 by flow cytometry was also higher in carriers of variant A allele of prothrombin 20210G>A polymorphism (5.0%) than in non-carriers (1.3%). Platelets isolated from healthy carriers of variant A allele of prothrombin 20210G>A polymorphism exhibited a higher level of activity and a higher degree of stiffness at the stage of spreading as compared to platelets from non­carriers.

Circulating Erythrocyte Microparticles and the Biochemical Extent of Myocardial Injury in ST Elevation Myocardial Infarction

Objectives: Red blood cell microparticles (RBCm) have potential adverse vascular effects and they have been shown to be elevated in ST elevation myocardial infarction (STEMI). The purpose of this study is to investigate their relationship with biochemical infarct size. Methods: RBCm were quantified with flow cytometry in blood drawn from 60 STEMI patients after a primary angioplasty. The creatine kinase-myocardial brain fraction (CK-MB) was measured at predefined time points and the area under the curve (AUC) was calculated. Results: RBCm count was correlated with CK-MB AUC (Spearman's ρ = 0.83, p < 0.001). The CK-MB AUC values per RBCm quartile (lower to upper) were: 3,351 (2,452-3,608), 5,005 (4,450-5,424), 5,903 (4,862-10,594), and 8,406 (6,848-12,782) ng × h/ml, respectively. From lower to upper quartiles, the maximal troponin I values were: 42.2 (23.3-49.3), 49.6 (28.8-54.1), 59.2 (41.4-77.3), and 69.1 (48.0-77.5) ng/ml (p = 0.005). In multivariable analysis, RBCm remained a significant predictor of CK-MB AUC (standardized β = 0.63, adjusted p = 0.001). Conclusions: Erythrocyte microparticles appear to be related to the total myocardial damage biomarker output. The exact pathophysiologic routes, if any, for this interaction remain to be identified. However, these results suggest that erythrocytes may be a - thus far virtually ignored - player in the pathogenesis of ischemic injury.

Endothelial Dysfunction in Resuscitated Cardiac Arrest (ENDO-RCA): safety and efficacy of low-dose prostacyclin administration and blood pressure target in addition to standard therapy, as compared to standard therapy alone, in post-cardiac arrest syndrome patients: study protocol for a randomized controlled trial.

BackgroundMorbidity and mortality following initial survival of cardiac arrest remain high despite great efforts to improve resuscitation techniques and post-resuscitation care, in part due to the ischemia-reperfusion injury secondary to the restoration of the blood circulation. Patients resuscitated from cardiac arrest display evidence of endothelial injury and coagulopathy (hypocoagulability, hyperfibrinolysis), which in associated with poor outcome. Recent randomized controlled trials have revealed that treatment with infusion of prostacyclin reduces endothelial damage after major surgery and AMI. Thus, a study is pertinent to investigate if prostacyclin infusion as a therapeutic intervention reduces endothelial damage without compromising, or even improving, the hemostatic competence in resuscitated cardiac arrest patients. Post-cardiac arrest patients frequently have a need for vasopressor therapy (catecholamines) to achieve the guideline-supported blood pressure goals. To evaluate a possible catecholamine interaction with the primary endpoints of this study, included patients will be randomized into two different blood pressure goals within guideline-recommended targets.Methods/designA randomized, placebo-controlled, double-blind investigator-initiated pilot trial in 40 out-of-hospital-cardiac-arrest (OHCA) patients will be conducted. Patients will be randomly assigned to either the active treatment group (48 hours of active study drug (iloprost, 1 ng/kg/min) or to the control group [placebo (saline) infusion]. Target mean blood pressure levels will be allocated 1:1 to 65 mmHg or approximately 75 mmHg, which gives four different permutations, namely: (i) iloprost/65 mHg, (ii) iloprost/75 mmHg, (iii) placebo/65 mmHg, and (iv) placebo/75 mmHg. All randomized patients will be treated in accordance with state-of-the art therapy including targeted temperature management.The primary endpoint of this study is change in biomarkers indicative of endothelial activation and damage, [soluble thrombomodulin (sTM), sE-selectin, syndecan-1, soluble vascular endothelial growth factor (sVEGF), nucleosomes] and sympathoadrenal over activation (epinephrine/norepinephrine) from baseline to 48 hours post-randomization.The secondary endpoints of this trial will include: (1) the hemostatic profile [change in functional hemostatic blood test (thrombelastography (TEG) and whole blood platelet aggregometry (multiplate)) blood cell and endothelial cell-derived microparticles]; (2) feasibility of blood pressure target intervention (target 90 %); (3) interaction of primary endpoints and blood pressure target; (4) levels of neuron-specific enolase at 48 hours post-inclusion according to blood pressure targets.DiscussionThe ENDO-RCA study is a pilot study trial that investigates safety and efficacy of low-dose infusion of prostacyclin administration as compared to standard therapy in post-cardiac arrest syndrome patients.Trial registrationTrial registration at ClinicalTrials.gov (identifier NCT02685618) on 18 February 2016.

Multiple Natural and Experimental Inflammatory Rabbit Lacrimal Gland Phenotypes

PurposeTo investigate lacrimal gland (LG) immunophysiological and immune-mediated inflammatory process (IMIP) phenotype diversity. MethodsEx vivo matured dendritic cells (mDC) were loaded with acinar cell microparticles (MP). Peripheral blood lymphocytes (PBL) were activated in mixed cell reactions with mDC and injected directly into autologous, unilateral LG (1°ATD-LG) of two rabbit cohorts, one naïve, one immunized with a LG lysate membrane fraction (Pi). Autoimmune IgG titers were assayed by ELISA, MCR PBL stimulation indices (SI) by [3H]-thymidine incorporation. Schirmer tests without and with topical anesthetic (STT-I, STT-IA) and rose Bengal (RB) staining tests were performed. H&E and immunohistochemically stained sections were examined. RNA yields and selected transcript abundances were measured. Immune cell number and transcript abundance data were submitted to Principal Component Analysis (PCA). ResultsImmunizing Pi dose influenced SI but not IgG titers. STT scores were decreased, and rose Bengal scores increased, by day 118 after immunization. Previous immunization exacerbated scores in 1°ATD-eyes and exacerbated 1°ATD-LG atrophy. IMIP were evident in 2°ATD-LG as well as 1°ATD-LG. PCA described diverse immunophysiological phenotypes in control LG and diverse IMIP phenotypes in ATD-LG. IgG titers and SI pre-adoptive transfer were significantly associated with certain post-adoptive transfer IMIP phenotype features, and certain LG IMIP features were significantly associated with RB and STT IA scores. ConclusionsThe underlying variability of normal states may contribute to the diversity of experimental IMIP phenotypes. The ability to generate and characterize diverse phenotypes may lead to phenotype-specific diagnostic and therapeutic paradigms.

Effect of endothelin-1 and endothelin receptor blockade on the release of microparticles.

Increased levels of endothelial cell microparticles (EMP) are known to reflect endothelial dysfunction (ED). In diabetes mellitus type 2 (T2DM), the expression of endothelin (ET)-1 is increased. As treatment with an ET-1 antagonist significantly inhibited atherosclerosis in animal models, we sought to investigate whether treatment with ET-1 antagonists affects EMP levels in vitro and in vivo in patients with T2DM. In vitro study: Human umbilical vein endothelial cells (HUVEC) were stimulated with ET-1 alone and ET-1 in combination with a dual ET-A and ET-B endothelin receptor blocker. In vivo study: Patients with T2DM were randomized to treatment with the ET receptor antagonist bosentan or placebo. After 4 weeks, the patients were re-examined and blood samples were obtained. EMP counts in supernatants and plasma samples were determined using flow cytometry. In vitro study: In supernatants of ET-1-stimulated HUVECs, the increased release of EMP was reduced significantly by co-incubation with an ET-1 receptor antagonist (e.g. CD31+/CD42b-EMP decreased from 37·1% ± 2·8 to 31·5% ± 2·8 SEM, P = 0·0078). In vivo study: No changes in EMP levels in blood samples of patients with T2DM were found after 4 weeks of bosentan treatment (n = 36, P = ns). Our in vitro results suggest that ET-1 stimulates the release of EMP from HUVECs via a receptor-dependent mechanism. Co-incubation with an endothelin receptor blocker abolished ET-1-dependent EMP release. However, treatment with bosentan for 4 weeks failed to alter EMP levels in patients with T2DM. Other factors seem to have influenced EMP release in patients with T2DM independent of ET-1 receptor-mediated mechanisms.

El ácido acetilsalicílico reduce la liberación de micropartículas eritrocitarias, monocitarias y de células del músculo liso vascular en pacientes diabéticos

Introduction and objectivesDiabetes mellitus is associated with an enhanced risk for cardiovascular disease and its prevalence is increasing. Diabetes induces metabolic stress on blood and vascular cells, promoting platelet activation and vascular dysfunction. The level of vascular cell activation can be measured by the number and phenotype of microparticles found in the circulation. The aim of this study was to investigate the effect of a platelet-inhibitory dose of aspirin on the number and type of microparticles shed to the circulation. MethodsForty-three diabetic patients were enrolled in the study and received a daily dose of 100mg of aspirin for 10 days to cover the average platelet life-span in the circulation. Before and after the intervention period, circulating microparticles were characterized and quantified by flow cytometry. ResultsType 1 diabetic patients had about twice the number of tissue factor-positive circulating microparticles (derived both from platelets and monocytes) and endothelial-derived E-selectin positive microparticles than type 2 diabetic patients. Aspirin therapy significantly inhibited platelets since cyclooxygenase 1 derived thromboxane generation levels were reduced by 99%. Microparticles derived from erythrocytes, activated monocytes, and smooth muscle cells were significantly reduced after 10 days of aspirin administration. ConclusionsThese results indicate that: a) vascular and blood cells in type 1 diabetic patients are exposed to more sustained stress shown by their specific microparticle origin and levels; b) aspirin therapy inhibits vascular wall cell activation and microparticle shedding, and c) the effects of aspirin are similar in type 1 and 2 diabetes.Full English text available from: www.revespcardiol.org/en

Microparticle Shedding by Erythrocytes, Monocytes and Vascular Smooth Muscular Cells Is Reduced by Aspirin in Diabetic Patients

Introduction and objectivesDiabetes mellitus is associated with an enhanced risk for cardiovascular disease and its prevalence is increasing. Diabetes induces metabolic stress on blood and vascular cells, promoting platelet activation and vascular dysfunction. The level of vascular cell activation can be measured by the number and phenotype of microparticles found in the circulation. The aim of this study was to investigate the effect of a platelet-inhibitory dose of aspirin on the number and type of microparticles shed to the circulation. MethodsForty-three diabetic patients were enrolled in the study and received a daily dose of 100mg of aspirin for 10 days to cover the average platelet life-span in the circulation. Before and after the intervention period, circulating microparticles were characterized and quantified by flow cytometry. ResultsType 1 diabetic patients had about twice the number of tissue factor-positive circulating microparticles (derived both from platelets and monocytes) and endothelial-derived E-selectin positive microparticles than type 2 diabetic patients. Aspirin therapy significantly inhibited platelets since cyclooxygenase 1 derived thromboxane generation levels were reduced by 99%. Microparticles derived from erythrocytes, activated monocytes, and smooth muscle cells were significantly reduced after 10 days of aspirin administration. ConclusionsThese results indicate that: a) vascular and blood cells in type 1 diabetic patients are exposed to more sustained stress shown by their specific microparticle origin and levels; b) aspirin therapy inhibits vascular wall cell activation and microparticle shedding, and c) the effects of aspirin are similar in type 1 and 2 diabetes.

Low simvastatin concentrations reduce oleic acid-induced steatosis in HepG2 cells: An in vitro model of non-alcoholic fatty liver disease.

Non-alcoholic fatty liver disease (NAFLD) is an inflammatory condition caused by hepatic lipid accumulation that is associated with insulin resistance, diabetes and metabolic syndrome. Although statins should be used with caution in liver diseases, they are increasingly investigated as a possible treatment for NAFLD. The present study recreated an in vitro model of NAFLD using HepG2 cells exposed to oleic acid (OA), which was used to quantify OA-induced lipid accumulation in HepG2 cells treated with various concentrations of simvastatin. In addition, the effect of simvastatin on HepG2 cell morphology and microparticle generation as a marker of cell apoptosis was assessed. OA-induced lipid accumulation was quantified by Oil Red O staining and extraction for optical density determination. Stained lipid droplets were visualized using phase contrast microscopy. Furthermore, HepG2 cell-derived microparticles were counted by flow cytometry subsequent to staining for Annexin V. HepG2 cells treated with 0-1 mM OA showed dose-dependent lipid accumulation. Treatment of HepG2 cells with increasing concentrations of simvastatin followed by treatment with 1 mM OA showed that low simvastatin concentrations (4-10 µM) were able to reduce lipid accumulation by ~40%, whereas high simvastatin concentrations (20 and 30 µM) induced apoptotic changes in cell morphology and increased the production of Annexin V+ microparticles. This suggests that low simvastatin doses may have a role in preventing NAFLD. However, further investigations are required to confirm this action in vivo and to determine the underlying mechanism by which simvastatin reduces hepatic steatosis.

Microparticle Shedding from Neural Progenitor Cells and Vascular Compartment Cells Is Increased in Ischemic Stroke.

PurposeIschemic stroke has shown to induce platelet and endothelial microparticle shedding, but whether stroke induces microparticle shedding from additional blood and vascular compartment cells is unclear. Neural precursor cells have been shown to replace dying neurons at sites of brain injury; however, if neural precursor cell activation is associated to microparticle shedding, and whether this activation is maintained at long term and associates to stroke type and severity remains unknown. We analyzed neural precursor cells and blood and vascular compartment cells microparticle shedding after an acute ischemic stroke.MethodsForty-four patients were included in the study within the first 48h after the onset of stroke. The cerebral lesion size was evaluated at 3–7 days of the stroke. Circulating microparticles from neural precursor cells and blood and vascular compartment cells (platelets, endothelial cells, erythrocytes, leukocytes, lymphocytes, monocytes and smooth muscle cells) were analyzed by flow cytometry at the onset of stroke and at 7 and 90 days. Forty-four age-matched high cardiovascular risk subjects without documented vascular disease were used as controls.ResultsCompared to high cardiovascular risk controls, patients showed higher number of neural precursor cell- and all blood and vascular compartment cell-derived microparticles at the onset of stroke, and after 7 and 90 days. At 90 days, neural precursor cell-derived microparticles decreased and smooth muscle cell-derived microparticles increased compared to levels at the onset of stroke, but only in those patients with the highest stroke-induced cerebral lesions.ConclusionsStroke increases blood and vascular compartment cell and neural precursor cell microparticle shedding, an effect that is chronically maintained up to 90 days after the ischemic event. These results show that stroke induces a generalized blood and vascular cell activation and the initiation of neuronal cell repair process after stroke. Larger cerebral lesions associate with deeper vessel injury affecting vascular smooth muscle cells.

Utility of the Red Blood Cell-Derived Microparticles as a Marker of Periprocedural Adverse Effects amongst Patients with Acute ST-Segment Elevation Myocardial Infarction

The short commentary is depicted the role of the circulating number of red blood cell (RBC) microparticles (MPs) as predictive biomarker in acute myocardial infarction patients after primary percutaneous coronary intervention (PCI). The commonly used cardiac biomarkers (i.e., troponins, creatine kinase-myocardial band isoenzymes, myoglobin, heart-type fatty acid-binding protein, copeptin and B-type natriuretic peptide) have now exhibited broad spectrum limitations regarding short-term and long-term-term mortality rate. Recent clinical studies have shown that the number of RBC-MPs has increased in acute myocardial infarction as compared to healthy volunteers and patients with unstable angina, associated with the extent of myocardial damage and have potential adverse vascular and thrombotic effects. It has been suggested that the number of RBC-MPs might be better predictor compared to other cardiac biomarkers in scintigraphically measured infarct size, periprocedural left ventricular ejection fraction and survival rate.

Elevated Red Cell Microparticles (RMP) in Hematologic and Thrombotic Disorders

Introduction. Circulating cell-derived microparticlesa (MP) from platelets (PMP), leukocytes (LMP), and endothelia (EMP) have been well-documented for their roles in hemostasis, thrombosis and inflammation but the clinical significance of RMP is less well understood. The purpose of this study is to study the relation of elevated RMP to selected hematologic and thrombotic disorders.Methods. This is a retrospective study on RMP profiles for patients referred to University of Miami Hospital and Clinics for hematologic consultation over the last 8 years. The patient population includes 51 hemolytic anemia (HA), 459 thrombocytopenia (TP), 26 myeloproliferative disorder (MPD), 413 hypercoagulable state (HCS), and 446 thrombotic disorders (TBS). Some patients were analyzed in more than one of the above disorders. Levels of RMP were measured by flow cytometry with PE-conjugated anti-CD235a labeling as previously described [Thromb. & Haemost., 2013;110:751-60]. Levels of RMP above mean +2SD of controls (> 2,000 counts/mL) are designated as "elevated RMP".Results. (I) Prevalence of elevated RMP in patient populartion: Elevated RMP were found in 31 of 51 HA (60.8%), 138 of 459 TP (30.1%), 20 of 26 MPD (78.1%), 175 of 413 HCS (42.4%), and 251 of 446 TBS (56.3%). (II) Association of elevated RMP with thrombosis: Of 31 HA patients with elevated RMP, 11 were positive for TBS, and the remaining 20 were negative. Levels of RMP (mean ±SD) for TBS(+) and TBS(-) were 5,824 ±3713 and 3,265 ±1,048, counts /mL, respectively (p<0.01). Of 138 TP patients with elevated RMP, 31 were TBS(+) and 107 were TBS(-). Levels of RMP for TBS(+) and TBS(-) were 4,698 ±3,208 and 3,012 ±1,503, counts/mL, respectively (p <0.001). (III) RMP in HCS: Of 175 HCS with elevated RMP, 116 were TBS(+) and 59 were TBS(-). The levels of RMP for TBS(+) and TBS(-) were 4,062 ±3,285 and 2,987 ±1,454, counts/mL, respectively (p <0.02). Among these 116 TBS(+) patients, 22 did not receive antithrombotic treatment at the time of assay (HCS-1), 84 received treatment at time of assay (HCS-2), and 10 developed recurrent TBS despite therapy (HCS-3). Levels of RMP for HCS-1, HCS-2, and HCS-3 were 5,091 ±3,804, 3,973 ±3,044, and 6,191 ±3,763, respectively. Of these, HCS-2 differed significantly from HCS-3, p < 0.05. (IV) TBS with vs. without risk factors: Of 251 TBS with elevated RMP, 226 had known hematological or coagulation disorders, or risk factors for TBS (such as HA, TP, MPD, thrombocytosis, cancers, atrial fibrillation, APS, lupus, or markers for HCS). The remaining 25 without risk factors nevertheless had TBS and elevated RMP, suggesting RMP may be a useful biomarker for thrombotic risk.Discussion. (i) The cause of RMP elevation in HA is reasonably attributed to red cell destruction. The cause in TP/ITP is likely due to products of platelet destruction or leukocyte activation. The cause in MPD could result from clearance of the high burden of red cells and platelets. (ii) Patients who were TBS(+) had higher RMP than TBS(-), seen in HCS, HA, TP, raising the question of whether high RMP is a cause or consequence of TBS. To answer this will require further study. (iii) The finding of exceptionally high RMP levels in recurrent TBS vs. non-recurrent TBS (HCS-3 vs. HC-2) indicates that RMP levels reflect severity of TBS. (iv) Finally, these data indicate that RMP may be a useful biomarker of thrombotic risk, particularly because some TBS patients had elevated RMP, yet tested negative by all conventional markers of HCS workups. DisclosuresNo relevant conflicts of interest to declare.

Pharmacokinetics of Red Cell-Derived Microparticles (RMP) in Rabbits and Rats: Effects of Administration Regimen and Labels for Assay

Background: Circulating cell-derived microparticles (MP) are currently of interest in the fields of hemostasis, thrombosis, and inflammation. Administration of MP has been proposed for use as a hemostatic agent. Efficacy of red cell microparticles (RMP) in reducing blood loss has been documented in rat and rabbit bleeding models [Thromb. & Haemost., 2013;110:751-60]. One of the challenges in developing MP as a hemostatic agent is uncertainty of half-life (T1/2) in circulation, reportedly ranging from several minutes to hours. The purpose of this study was to assess the pharmacokinetics of RMP in rabbits vs. rats, and comparing alternative dosing regimens and assay markers. Although this work was aimed at optimizing the dosage regimen of RMP as a hemostatic agent, it has wider interest because very few studies have addressed the question of MP half-life in circulation.Methods. (i) HumanRMP were produced by high-pressure extrusion [Thromb. & Haemost., 2013; 110:751-60]. The resulting product was washed twice with isotonic saline, lyophilized, and stored at -80°C. (ii) Pharmacokinetics of RMP were measured using either bolus infusion of RMP (3x109 counts/kg) during 1 min., or a combination of bolus (1/3 of total RMP) followed by continuous infusion (2/3 of total RMP) for 30 min to the anesthetized rabbits or rats, via cannulated jugular vein. Blood samples (0.4 mL each) were collected from the cannulated carotid artery at intervals: 5 min pre-injection, and at 1, 2, 3, 5, 7.5, 10, 15, 20, 25, 30, 45, and 60 min post-injection. A sample size of 8 animals was used for each infusion regimen. Levels of RMP were assayed by flow cytometry with 2 separated labels: anti-CD235a-PE (anti-glycophorin) and annexin V-FITC (AnV). The former is specific for human RMP and does not label rabbit or rat RMP. The latter is not species-sensitive, and labels both human and host MP. (iii) The procoagulant activity (PCA) of RMP was assayed by thromboelastogram (TEG) ex vivo.Results: (1) BolusInfusion of RMP (3 x109 MP/kg) resulted in a rapid rise of RMP levels peaking at 1.0 min post-infusion, followed by slower decline to baseline by 10 - 15 min. The half-life (T1/2) of clearance determined by anti-CD235a was 6.2 ±0.7 and was 4.2 ±0.5 min for rabbits and rats, respectively. In contrast, the T1/2 measured by AnV was 4.4 ±1.0 and 2.6 ±0.4 min, respectively. These results indicate that these two MP phenotypes were cleared by different mechanisms, i.e., one or more mechanisms predominates in clearing CD235a(+) MP vs. AnV(+) MP. The RMP enriched in phosphatidylserine (AnV+) are cleared faster than those expressing CD235a. (2) Two-step administration was bolus (1/3 total dosage) followed by continuous infusion (2/3 total dosage), and resulted in a smaller initial spike followed by rapid decline to ≈25-30%, then recovering to a nearly steady-state level of 0.3 -0.6 x107 MP/mL after 20 min. The RMP levels returned to baseline within 15 min after cessation of infusion. The area under the curve (AUC) of the PK curve for bolus infusion was about 40 -60% less than that of two-step administration of the same dose. (4) Procoagulant Activity (PCA): For bolus infusion, the ex vivo TEG data revealed some correlation between circulating RMP and PCA at the early phase (1 to 6 min). At later phase (>10 min), although RMP had dropped to almost baseline, the PCA remained active considerably longer than presence of RMP (T1/2= 9.3±1.2 min for PCA). However, bolus followed by continuous infusion resulted in maintaining stable PCA over the course of infusion.Conclusion/Discussion. (i) The clearance rate of RMP in rats is faster than that in rabbits. (ii) When T1/2 is determined by the two different markers, we found significant differences in half-life, indicating differential of mechanisms of clearance. (iii) The TEG data show that the PCA of RMP persists after detectable RMP have entirely cleared. We interpret this to mean that RMP continue to exert PCA by being bound to blood cells, even after free circulating RMP have cleared. (iv) Continuous infusion is expected to perform better than bolus infusion in controlling excessive bleeding in clinical patients. DisclosuresNo relevant conflicts of interest to declare.

Three Months of Human Haptoglobin Treatment Decreases Iron Deposition in the Kidneys of Townes Sickle Mice

Introduction: Haptoglobin (Hp), the scavenger for hemoglobin, and hemopexin (Hx), the scavenger for heme, are depleted in most patients with sickle cell disease due to chronic hemolysis. There is mounting evidence of the crucial role of free hemoglobin and/or free heme in mediating inflammatory and oxidative damage in sickle cell disease, including vaso-occlusion and acute chest syndrome. Purified Hp has been used in Japan for a variety of hemolytic conditions and has been proposed as a potential treatment for sickle cell disease. Although infusions of Hp or Hx have been shown to ameliorate vaso-occlusion, acute lung injury, and heme toxicity in sickle cell mouse models, no prior studies have examined the utility of chronic Hp treatment for amelioration of organ damage. We therefore studied the effect of 3 months of chronic Hp treatment in the Townes sickle mouse model.Methods: Male and female Townes mice (Stock number 013071, The Jackson Laboratory) were used for all experiments, starting at 1 or 3 months of age. SS genotype was confirmed by PCR and HPLC. Organ damage in the spleen, liver, and kidneys as previously described was confirmed. Human Hp solution was a kind gift from Bio Product Laboratory (BPL, Hertfordshire, UK). Hp or equivalent volume PBS control was administered intraperitoneally (IP) in the first cohort of 5 mice and then subcutaneously (SC) in the next two cohorts of 7 and 12 mice on a 48-72 hr dosing schedule of Monday, Wednesday, Friday for a period of 3 months. At the end of 3 months treatment, mice were evaluated by the following studies (with concurrent blinding to treatment group for most studies): plasma Hp (ELISA), plasma heme (QuantiChrom heme assay), urine osmolality (osmometer), urine albumin (ELISA), CBC (Advia 120), WBC differential (Advia 120 and manual count), red blood cell ektacyometry (ektacyometer), organ mass (percent of body weight), and organ histology.Results: Mouse Hp levels in SS Townes mice were confirmed to be markedly low compared to Townes AA mice (mean ± SD: SS 2 ± 1 versus AA 39 ± 4 ug/mL). Dose-finding experiments determined that a dose of 200-400 mg/kg IP or SC in SS mice resulted in a 24 hr peak concentration that was 5-14X supraphysiologic, variably physiologic at 48 hr, and absent or almost absent at 72 hr. Chronic dosing at the 400 mg/kg IP in SA mice showed no CBC or organ toxicity. Three successive cohorts of SS mice were treated with Hp (or equivalent volume of PBS): 200 mg/kg IP in 3-month old mice, 400 mg/kg SC in 3-month old mice, and 400 mg/kg SC in 1-month old mice. At the 400 mg/kg dosing levels, there was a significant decrease in iron deposition in the kidneys of both 4-month and 6-month old mice (treatment started at 1-month and 3 months, respectively) (Table 1). There was also a trend towards decreased liver infarction in 6-month old mice (Table 2).Discussion: Functional binding of the administered human Hp to the human Hb of the Townes mice likely occurred, as evidenced by the decrease in iron deposition in the kidneys, suggesting that formation of the complex prevents filtration of Hb into the kidneys. Surprisingly, kidney function as measured by urine osmolality or albumin excretion was not improved, which may be explained by continued heme-laden red cell microparticle filtration (Camus SM, Blood 2015). Encouragingly, however, a trend towards decreased liver infarction in older mice was observed. The less-than-expected effect of Hp on mouse disease severity may also be explained by: 1) continuous physiologic Hp concentrations not being maintained with the dosing frequency while continued hemolysis releases Hb every minute of the day, and 2) CD163-mediated uptake in mice seems to only account for a part of the Hb clearance as opposed to in humans (Etzerodt A, Antioxid Redox Signal 2013). Despite the limitations of the SCD mouse model, the current study suggests haptogobin infusions could be beneficial in SCD patients.Acknowledgment: The authors are grateful to Sandra Suzuka for performing the HPLC.Table 1Table 1. Kidney iron deposition (scale 1-10)Treatment group6-month old SS4-month old SS400 mg/kg Hp4.0 ± 1.41 ± 1.1PBS9.3 ± 0.65 ± 2.9p-value0.0020.02Table 2Liver infarction (scale 1-10)Treatment group6-month old SS4-month old SS400 mg/kg Hp2.6 ± 2.03.7 ± 2.8PBS6.3 ± 2.43.8 ± 2.3p-value0.070.91 DisclosuresBelcher:Biogen Idec: Research Funding; Seattle Genetics: Research Funding; CSL Behring: Research Funding. Vercellotti:CSL Behring: Research Funding; Seattle Genetics: Research Funding; Cydan: Research Funding; Biogen Idec: Research Funding.

Abstract 11761: Association of Red Blood Cell Microparticles With Biomarker-Defined Infarct Size in Acute Myocardial Infarction

Background: Red blood cell microparticles (RBCm) have drawn research attention as to their potential prothrombotic and vasoconstrictive effects in experimental settings. Our group has previously reported elevated RBCm counts in patients with ST-elevation myocardial infarction (STEMI) treated with primary percutaneous intervention (PCI). Methods: Circulating microparticles were quantified with flow cytometry in blood samples from consecutive STEMI patients after primary PCI. Creatine kinase myocardial-brain fraction (CK-MB) was measured every 6 hours during the first 3 days and every 12 hours thereafter until discharge. The area under the CK-MB concentration curve (CKMB-AUC), calculated with Simpson’s summing rule, was used as a biochemical measure of infarction size. Results: 51 STEMI patients (age 59.8±8.8 years) were enrolled. RBCm concentration was 33740±21169 /μl. The median values [25th-75th percentile] of CKMB-AUC according to RBCm quartile were (from the lower to the upper quartile) 3351 [2550-4060] ng.h.ml-1, 4883 [4179-5348] ng.h.ml-1, 6932 [5464-13229] ng.h.ml-1, and 8784 [7216-12605] ng.h.ml-1, respectively (Kruskal-Wallis p&lt;0.001) (see Figure). In a multivariable regression analysis, RBCm quartile remained associated with CKMB-AUC, after adjustment for total RBC count, age, serum creatinine, presence of diabetes and Killip class on admission (adjusted p&lt;0.001). Conclusions: Erythrocyte microparticles appear to be related to total myocardial damage biomarker output, which is a parameter known to reliably reflect infarct size in patients with STEMI. This finding may be explained by rheologic and vasoactive properties of RBCm (close interaction of the heavily oxidative heme with the endothelium, depletion of NO etc.), which may act deleteriously on coronary vasculature, resulting in increased myocardial damage.

A novel micro/nano fabrication process based on the combined use of dielectrophoresis, electroosmotic flow, and electrodeposition for surface patterning

In this work, a novel application of electrokinetic forces is presented. Employing a gold interdigitated electrode array (IDEA), dielectrophoresis (a force acting on polarizable material) and electroosmosis (a force acting on ionic fluids) were utilized as microfabrication tools. Through electroosmotic flow, particles were dragged toward dielectrophoretic trapping zones, where they were held. Then polypyrrole, an electroconductive material with good mechanical and electrical properties, wide electrochemical window, and ease of fabrication, was electrodeposited onto the gold IDEA surface, permanently entrapping the microparticles within the structure. The process was tested employing organic (polystyrene), inorganic (silicon), and biologic (yeast cells) microparticles, all of which were successfully trapped. Computational models were developed to predict the electrokinetic response of the microparticles and compared to experimental observations. It was demonstrated that this process can be used to produce hierarchical structures through sequential repetition of entrapment cycles. Additionally, it is compatible with the Carbon-MEMS process. The fabrication process presented in this paper opens a wide range of applications that include: energy storage devices, scaffolds for cell culture, biomedical devices, electrochemical sensors, electrokinetic devices, among many others.

Lectin-Based Characterization of Vascular Cell Microparticle Glycocalyx.

Microparticles (MPs) are released constitutively and from activated cells. MPs play significant roles in vascular homeostasis, injury, and as biomarkers. The unique glycocalyx on the membrane of cells has frequently been exploited to identify specific cell types, however the glycocalyx of the MPs has yet to be defined. Thus, we sought to determine whether MPs, released both constitutively and during injury, from vascular cells have a glycocalyx matching those of the parental cell type to provide information on MP origin. For these studies we used rat pulmonary microvascular and artery endothelium, pulmonary smooth muscle, and aortic endothelial cells. MPs were collected from healthy or cigarette smoke injured cells and analyzed with a panel of lectins for specific glycocalyx linkages. Intriguingly, we determined that the MPs released either constitutively or stimulated by CSE injury did not express the same glycocalyx of the parent cells. Further, the glycocalyx was not unique to any of the specific cell types studied. These data suggest that MPs from both normal and healthy vascular cells do not share the parental cell glycocalyx makeup.

PP.37.17

Objective: This study try to assess the endothelial function in vivo using flow mediated dilatation (FMD) and several biomarkers of endothelium formation/restoration and damage in patients with obstructive sleep apnoea (OSA) syndrome at baseline and after three months with CPAP therapy. Design and method: Observational study, before and after CPAP therapy. We studied 30 patients with apnoea/hypopnoea index > 15/h that were compared with themselves after three months of CPAP therapy. Flow mediated dilatation (FMD) was assessed non-invasively by in vivo using the Laser-Doppler flowmetry. Circulating cell free DNA (cf-DNA) and microparticles (MPs) were measured as markers of endothelial damage and the vascular endothelial growth factor (VEGF) was determined as a marker of endothelial restoration process. Results: After three month with CPAP, FMD significantly increased (1072.26 ± 483.21 vs. 1604.38 ± 915.69 PU, p < 0.005), and cf-DNA and MPs significantly decreased (respectively: 187.93 ± 115.81 vs. 121.28 ± 78.98 pg/ml, p < 0.01 and 69.60 ± 62.60 vs. 39.82 ± 22.14 U/μL, p < 0.05). Finally, VEGF increased (585.02 ± 246.06 vs. 641.11 ± 212.69 pg/ml, p < 0.05). These changes were higher in patients with more severe disease. There was a relationship between markers of damage (r = -0.53, p < 0.005) but not among markers of damage and restoration, thus suggesting that both types of markers should be measured together. Conclusions: CPAP therapy improves FMD. This improvement may be related to an increase of endothelial restoration process and a decrease of endothelial damage.

Abstract 443: Red Blood Cell Microparticles Mediate Transfer of Functional miR-451 to Endothelial Cells in vitro

Microparticles (MPs) circulate in human blood and can transfer functional miRNA to recipient cells. Red blood cell derived MPs (RMPs) accumulate during storage of human erythrocyte transfusion units, but it is not known whether RMPs can deliver functional miRNAs to endothelial cells. We hypothesized that the RMPs could transfer functional miR-451 to human arterial endothelial cells (HAECs), and that RMP-mediated miRNA transfer could affect clinical outcomes after stored RBC transfusion. miR-451, an erythrocyte specific miRNA, was confirmed to be abundant in RMPs and absent in HAECs, making it a useful miRNA to assess for transfer. RMPs were isolated from a 42 day-old transfusion unit and confirmed by both flow cytometry and electron microscopy. HAECs were incubated for 2 hours with calcein-labeled RMPs derived from 2 ml of blood product. After incubation cells were washed with phosphate buffered saline (PBS) and fixed for microscopy or harvested for RNA. Confocal microscopy confirmed the uptake of RMPs by treated cells. miRNA-451 levels increased ~900-fold in HAECs after treatment with RMPs (fold change = 927 ± 245, p &lt; 1.0 x 10-4). To assess whether transferred miR-451 was functional, we measured mRNA levels of two targets of miR-451 in recipient HAECs, macrophage migration inhibitory factor (MIF) and sphingosine-1-phosphate receptor 2 (S1PR2). RMP treatment decreased expression of MIF (–2.8 fold ± 0.08, p = 0.047) and S1PR2 (–2.9 fold ± 0.06, p = 0.007) as compared to untreated controls, suggesting the transferred miR-451 is functional. Further studies aimed at confirming miR-451 dependent changes in MIF and S1PR2 are underway. This data supports the concept that RMPs, in particular those that accumulate during RBC storage, can function as signaling molecules via MP-mediated miRNA transfer. MP-mediated miRNA transfer represents a novel mechanism by which stored blood products may exert previously unrecognized effects upon transfusion.

Macrophages engulf endothelial cell membrane particles preceding pupillary membrane capillary regression

Programmed capillary regression and remodeling are essential developmental processes. However, the cellular and molecular mechanisms that regulate vessel regression are only the beginning to be understood. Here, using in vivo, dynamic, confocal imaging of mouse transgenic reporters as well as static confocal and electron microscopy, we studied the embryonic development and postnatal regression of the transient mouse pupillary membrane (PM) vasculature. This approach allowed us to directly observe the precise temporal sequence of cellular events preceding and during the elimination of the PM from the mouse eye. Imaging of Tcf/Lef-H2B::GFP Wnt-reporter mice uncovered that, unlike the hyaloid vasculature of the posterior eye, a PM endothelial cell (EC) Wnt/β-catenin response is unlikely to be part of the regression mechanism. Live imaging of EC and macrophage dynamics revealed highly active Csf1r-GFP+ macrophages making direct contact with the Flk1-myr::mCherry+ vessel surface and with membrane protrusions or filopodia extending from the ECs. Flk1-myr::mCherry+ EC membrane particles were observed on and around ECs as well as within macrophages. Electron microscopy studies confirmed that they were in phagosomes within macrophages, indicating that the macrophages engulfed the membrane particles. Interestingly, EC plasma membrane uptake by PM macrophages did not correlate with apoptosis and was found shortly after vessel formation at mid-gestation stages in the embryo; long before vessel regression begins during postnatal development. Additionally, genetic ablation of macrophages showed that EC membrane particles were still shed in the absence of macrophages suggesting that macrophages do not induce the formation or release of EC microparticles. These studies have uncovered a novel event during programmed capillary regression in which resident macrophages scavenge endothelial cell microparticles released from the PM vessels. This finding suggests that there may be an initial disruption in vessel homeostasis embryonically as the PM forms that may underlie its ultimate regression postnatally.