A global assessment of hemostatic function of healthy allogeneic stem cell donors undergoing apheresis by rotational thromboelastometry.

INTRODUCTION: Peripheral blood stem cell (PBSC) collection via apheresis requires the administration of granulocyte colony-stimulating factor (filgrastim) to stem cell donors. Several reports have shown that filgrastim administration and apheresis procedure induce a hypercoagulable state across PBSC collection, which might predispose certain donors to thrombotic complications. METHODS: We evaluated the hemostatic functions of healthy allogeneic stem cell donors by rotational thromboelastometry (ROTEM). Blood samples from healthy donors (n = 30) were collected at defined time points: before filgrastim (baseline), on the day of apheresis before and after the procedure, and 1 week after apheresis. RESULTS: The results indicated that hemostatic changes are temporary since all parameters in both EXTEM and INTEM assays are restored to their initial values 1 week after the apheresis. CONCLUSION: We concluded that stem cell apheresis does not induce a hypercoagulable state in healthy donors. This is the first study evaluating the hemostatic functions of stem cell donors by ROTEM.

Platelet Proteome Reveals Novel Targets for Hypercoagulation in Pseudoexfoliation Syndrome.

Pseudoexfoliation syndrome (PEX) is characterized by the accumulation of abnormal extracellular matrix material in ocular and non-ocular tissues, including blood vessel walls. Clot-forming dysfunction might be responsible for venous thrombosis in PEX. We investigated global coagulation, the proteome, and functions of platelets in PEX patients and aimed to determine prognostic biomarkers for thrombosis risk in PEX. Peripheral blood was collected from PEX and retinal vein occlusion (RVO) patients, and age-sex matched controls. Viscoelastic hemostasis was evaluated by rotational thromboelastometry (ROTEM). Platelet markers (CD41, CD42, CD61, and CD62p) and endothelial markers (P-selectin, E-selectin, and von Willebrand factor) were investigated by flow cytometry and ELISA, respectively. The platelet proteome was analyzed by 2D fluorescence difference gel electrophoresis followed by mass spectrometry. Clot formation time (CFT) is significantly reduced in PEX patients compared to the controls (p < 0.05). P-selectin levels were higher in PEX patients than in controls (p < 0.05); E-selectin and von Willebrand factor remained unchanged. The monitorization of CFT by ROTEM, and soluble P-selectin, may help assess thrombotic risk in PEX patients. Proteomic analysis revealed differential expression of Profilin-1 in platelets. Profilin-1 regulates the stability of actin-cytoskeleton and may contribute to impaired platelet hemostatic functions. Increased P-selectin levels together with impaired coagulation dynamics might be responsible for the thrombotic events in PEX disease.

A preliminary study of phosphodiesterases and adenylyl cyclase signaling pathway on red blood cell deformability of sickle cell patients.

Sickle cell disease (SCD) is an inherited hemoglobinopathy characterized by chronic anemia, intravascular hemolysis, and the occurrence of vaso-occlusive crises due to the mechanical obstruction of the microcirculation by poorly deformable red blood cells (RBCs). RBC deformability is a key factor in the pathogenesis of SCD, and is affected by various factors. In this study, we investigated the effects of adenylyl cyclase (AC) signaling pathway modulation and different phosphodiesterase (PDE) modulatory molecules on the deformability and mechanical stress responses of RBC from SCD patients (HbSS genotype) by applying 5 Pa shear stress with an ektacytometer (LORRCA). We evaluated RBC deformability before and after the application of shear stress. AC stimulation with Forskolin had distinct effects on RBC deformability depending on the application of 5 Pa shear stress. RBC deformability was increased by Forskolin before shear stress application but decreased after 5 Pa shear stress. AC inhibition with SQ22536 and protein kinase A (PKA) inhibition with H89 increased RBC deformability before and after the shear stress application. Non-selective PDE inhibition with Pentoxifylline increased RBC deformability. However, modulation of the different PDE types had distinct effects on RBC deformability, with PDE1 inhibition by Vinpocetine increasing deformability while PDE4 inhibition by Rolipram decreased RBC deformability after the shear stress application. The effects of the drugs varied greatly between patients suggesting some could benefit from one drug while others not. Developing drugs targeting the AC signaling pathway could have clinical applications for SCD, but more researches with larger patient cohorts are needed to identify the differences in the responses of sickle RBCs.

Zinc improved erythrocyte deformability and aggregation in patients with beta-thalassemia: An in vitro study.

BACKGROUND: Thalassemia patients have reduced red cell deformability and decreased plasma zinc levels in their blood. OBJECTIVE: This study aimed to evaluate the effects of zinc (Zn) on the hemorheological parameters and antioxidant enzyme activities in ß-thalassemia major (TM) and healthy volunteers (HV). METHODS: Hemorheological parameters were measured using LORCA (laser-assisted optical rotational cell analyzer) after adjusting the hematocrit to 40%. Zinc sulfate (ZnSO4.7H2O) was used for Zn incubation with a concentration of 0.5µg/dl. Oxidative stress and antioxidant status were determined using commercial kits. RESULTS: Data showed that after Zn incubation, EImax, the area under the EI-osmolarity curve (Area), and Omax decreased in TM. However, no significant difference was observed in the osmotic deformability parameters of HV. The increased elongation index was obtained at different shear stresses for TM and HV, and SS1/2 decreased in both groups. The AMP and aggregation index (AI) decreased in TM, and the required time for half of the maximum aggregation (t1/2) increased in HV. However, Zn did not affect oxidative parameters in both groups. CONCLUSIONS: This study showed that Zn incubation increased deformability and decreased aggregation in thalassemic erythrocytes. It means that Zn supplementation will contribute to microcirculation in thalassemia patients.

Signaling mechanisms in red blood cells: A view through the protein phosphorylation and deformability.

Intracellular signaling mechanisms in red blood cells (RBCs) involve various protein kinases and phosphatases and enable rapid adaptive responses to hypoxia, metabolic requirements, oxidative stress, or shear stress by regulating the physiological properties of the cell. Protein phosphorylation is a ubiquitous mechanism for intracellular signal transduction, volume regulation, and cytoskeletal organization in RBCs. Spectrin-based cytoskeleton connects integral membrane proteins, band 3 and glycophorin C to junctional proteins, ankyrin and Protein 4.1. Phosphorylation leads to a conformational change in the protein structure, weakening the interactions between proteins in the cytoskeletal network that confers a more flexible nature for the RBC membrane. The structural organization of the membrane and the cytoskeleton determines RBC deformability that allows cells to change their ability to deform under shear stress to pass through narrow capillaries. The shear stress sensing mechanisms and oxygenation-deoxygenation transitions regulate cell volume and mechanical properties of the membrane through the activation of ion transporters and specific phosphorylation events mediated by signal transduction. In this review, we summarize the roles of Protein kinase C, cAMP-Protein kinase A, cGMP-nitric oxide, RhoGTPase, and MAP/ERK pathways in the modulation of RBC deformability in both healthy and disease states. We emphasize that targeting signaling elements may be a therapeutic strategy for the treatment of hemoglobinopathies or channelopathies. We expect the present review will provide additional insights into RBC responses to shear stress and hypoxia via signaling mechanisms and shed light on the current and novel treatment options for pathophysiological conditions.

Theories and Molecular Basis of Vascular Aging: A Review of the Literature from VascAgeNet Group on Pathophysiological Mechanisms of Vascular Aging.

Vascular aging, characterized by structural and functional alterations of the vascular wall, is a hallmark of aging and is tightly related to the development of cardiovascular mortality and age-associated vascular pathologies. Over the last years, extensive and ongoing research has highlighted several sophisticated molecular mechanisms that are involved in the pathophysiology of vascular aging. A more thorough understanding of these mechanisms could help to provide a new insight into the complex biology of this non-reversible vascular process and direct future interventions to improve longevity. In this review, we discuss the role of the most important molecular pathways involved in vascular ageing including oxidative stress, vascular inflammation, extracellular matrix metalloproteinases activity, epigenetic regulation, telomere shortening, senescence and autophagy.

Proteomic Analysis of the Role of the Adenylyl Cyclase-cAMP Pathway in Red Blood Cell Mechanical Responses.

Red blood cell (RBC) deformability is modulated by the phosphorylation status of the cytoskeletal proteins that regulate the interactions of integral transmembrane complexes. Proteomic studies have revealed that receptor-related signaling molecules and regulatory proteins involved in signaling cascades are present in RBCs. In this study, we investigated the roles of the cAMP signaling mechanism in modulating shear-induced RBC deformability and examined changes in the phosphorylation of the RBC proteome. We implemented the inhibitors of adenylyl cyclase (SQ22536), protein kinase A (H89), and phosphodiesterase (PDE) (pentoxifylline) to whole blood samples, applied 5 Pa shear stress (SS) for 300 s with a capillary tubing system, and evaluated RBC deformability using a LORRCA MaxSis. The inhibition of signaling molecules significantly deteriorated shear-induced RBC deformability (p < 0.05). Capillary SS slightly increased the phosphorylation of RBC cytoskeletal proteins. Tyrosine phosphorylation was significantly elevated by the modulation of the cAMP/PKA pathway (p < 0.05), while serine phosphorylation significantly decreased as a result of the inhibition of PDE (p < 0.05). AC is the core element of this signaling pathway, and PDE works as a negative feedback mechanism that could have potential roles in SS-induced RBC deformability. The cAMP/PKA pathway could regulate RBC deformability during capillary transit by triggering significant alterations in the phosphorylation state of RBCs.

A Novel Fragmentation Sensitivity Index Determines the Susceptibility of Red Blood Cells to Mechanical Trauma.

Supraphysiological shear stresses (SSs) induce irreversible impairments of red blood cell (RBC) deformability, overstretching of RBC membrane, or fragmentation of RBCs that causes free hemoglobin to be released into plasma, which may lead to anemia. The magnitude and exposure tisme of the SSs are two critical parameters that determine the hemolytic threshold of a healthy RBC. However, impairments in the membrane stability of damaged cells reduce the hemolytic threshold and increase the susceptibility of the cell membrane to supraphysiological SSs, leading to cell fragmentation. The severity of the RBC fragmentation as a response to the mechanical damage and the critical SS levels causing fragmentation are not previously defined. In this study, we investigated the RBC mechanical damage in oxidative stress (OS) and metabolic depletion (MD) models by applying supraphysiological SSs up to 100 Pa by an ektacytometer (LORRCA MaxSis) and then assessed RBC deformability. Next, we examined hemolysis and measured RBC volume and count by Multisizer 3 Coulter Counter to evaluate RBC fragmentation. RBC deformability was significantly impaired in the range of 20-50 Pa in OS compared with healthy controls (p < 0.05). Hemolysis was detected at 90-100 Pa SS levels in MD and all applied SS levels in OS. Supraphysiological SSs increased RBC volume in both the damage models and the control group. The number of fragmented cells increased at 100 Pa SS in the control and MD and at all SS levels in OS, which was accompanied by hemolysis. Fragmentation sensitivity index increased at 50-100 Pa SS in the control, 100 Pa SS in MD, and at all SS levels in OS. Therefore, we propose RBC fragmentation as a novel sensitivity index for damaged RBCs experiencing a mechanical trauma before they undergo fragmentation. Our approach for the assessment of mechanical risk sensitivity by RBC fragmentation could facilitate the close monitoring of shear-mediated RBC response and provide an effective and accurate method for detecting RBC damage in mechanical circulatory assist devices used in routine clinical procedures.

Applications of deep learning to the assessment of red blood cell deformability.

BACKGROUND: Measurement of abnormal Red Blood Cell (RBC) deformability is a main indicator of Sickle Cell Anemia (SCA) and requires standardized quantification methods. Ektacytometry is commonly used to estimate the fraction of Sickled Cells (SCs) by measuring the deformability of RBCs from laser diffraction patterns under varying shear stress. In addition to estimations from model comparisons, use of maximum Elongation Index differences (ΔEImax) at different laser intensity levels was recently proposed for the estimation of SC fractions. OBJECTIVE: Implement a convolutional neural network to accurately estimate rigid-cell fraction and RBC concentration from laser diffraction patterns without using a theoretical model and eliminating the ektacytometer dependency for deformability measurements. METHODS: RBCs were collected from control patients. Rigid-cell fraction experiments were performed using varying concentrations of glutaraldehyde. Serial dilutions were used for varying the concentration of RBC. A convolutional neural network was constructed using Python and TensorFlow. RESULTS AND CONCLUSIONS: Measurements and model predictions show that a linear relationship between ΔEImax and rigid-cell fraction exists only for rigid-cell fractions less than 0.2. The proposed neural network architecture can be used successfully for both RBC concentration and rigid-cell fraction estimations without a need for a theoretical model.

Rotational Thromboelastometry Reveals Distinct Coagulation Profiles for Patients With COVID-19 Depending on Disease Severity.

Identifying a hypercoagulable state in patients with COVID-19 may help identify those at risk for virus-induced thromboembolic events and improve clinical outcomes using personalized therapeutic approaches. Herein, we aimed to perform a global assessment of the patients' hemostatic system with COVID-19 using rotational thromboelastometry (ROTEM) and to describe whether patients with different disease severities present different coagulation profiles. Together with 37 healthy volunteers, a total of 65 patients were included and then classified as having mild, moderate, and severe disease depending on clinical severity. Peripheral blood samples were collected and analyzed using a ROTEM Coagulation Analyzer. Also, complete blood count and coagulation parameters including prothrombin time, activated partial thromboplastin time, fibrinogen levels, and D-dimer levels were measured at admission. EXTEM and INTEM MCF (P < 0.001) values were significantly higher and the EXTEM CFT (P = 0.002) value was significantly lower in patients with COVID-19 when compared with controls. In particular, patients with the severe disease showed a significant decrease in CFT (P < 0.001) and an increase in MCF (P < 0.001) in both INTEM and EXTEM assays compared with patients with the non-severe disease. Correlation analysis revealed significant correlations between ROTEM parameters and other coagulation parameters. There were significant positive correlations between fibrinogen, D-dimer, platelet count, and MCF in both EXTEM and INTEM assays. Our data demonstrate thromboelastographic signs of hypercoagulability in patients with COVID-19, which is more pronounced in patients with increased disease severity. Therefore, ROTEM analysis can classify subsets of patients with COVID-19 at significant thrombotic risk and assist in clinical decisions.

Calcium/protein kinase C signaling mechanisms in shear-induced mechanical responses of red blood cells.

Red blood cell (RBC) deformability has vital importance for microcirculation in the body, as RBCs travel in narrow capillaries under shear stress. Deformability can be defined as a remarkable cell ability to change shape in response to an external force which allows the cell to pass through the narrowest blood capillaries. Previous studies showed that RBC deformability could be regulated by Ca2+/protein kinase C (PKC) signaling mechanisms due to the phosphorylative changes in RBC membrane proteins by kinases and phosphatases. We investigated the roles of Ca2+/PKC signaling pathway on RBC mechanical responses and impaired RBC deformability under continuous shear stress (SS). A protein kinase C inhibitor Chelerythrine, a tyrosine phosphatase inhibitor Calpeptin, and a calcium channel blocker Verapamil were applied into human blood samples in 1 micromolar concentration. Samples with drugs were treated with or without 3 mM Ca2+. A shear stress at 5 Pa level was applied to each sample continuously for 300 s. RBC deformability was measured by a laser-assisted optical rotational cell analyzer (LORRCA) and was calculated as the change in elongation index (EI) of RBC upon a range of shear stress (SS, 0.3-50 Pa). RBC mechanical stress responses were evaluated before and after continuous SS through the parameterization of EI-SS curves. The drug administrations did not produce any significant alterations in RBC mechanical responses when they were applied alone. However, the application of the drugs together with Ca2+ substantially increased RBC deformability compared to calcium alone. Verapamil significantly improved Ca2+-induced impairments of deformability both before and after 5 Pa SS exposure (p < 0.0001). Calpeptin and Chelerythrine significantly ameliorated impaired deformability only after continuous SS (p < 0.05). Shear-induced improvements of deformability were conserved by the drug administrations although shear-induced deformability was impaired when the drugs were applied with calcium. The blocking of Ca2+ channel by Verapamil improved impaired RBC mechanical responses independent of the SS effect. The inhibition of tyrosine phosphatase and protein kinase C by Calpeptin and Chelerythrine, respectively, exhibited ameliorating effects on calcium-impaired deformability with the contribution of shear stress. The modulation of Ca2+/PKC signaling pathway could regulate the mechanical stress responses of RBCs when cells are under continuous SS exposure. Shear-induced improvements in the mechanical properties of RBCs by this signaling mechanism could facilitate RBC flow in the microcirculation of pathophysiological disorders, wherein Ca2+ homeostasis is disturbed and RBC deformability is reduced.

A micropillar-based microfluidic viscometer for Newtonian and non-Newtonian fluids.

In this study, a novel viscosity measurement technique based on measuring the deflection of flexible (poly) dimethylsiloxane (PDMS) micropillars is presented. The experimental results show a nonlinear relationship between fluid viscosity and the deflection of micropillars due to viscoelastic properties of PDMS. A calibration curve, demonstrating this nonlinear relationship, is generated, and used to determine the viscosity of an unknown fluid. Using our method, viscosity measurements for Newtonian fluids (glycerol/water solutions) can be performed within 2-100 cP at shear rates γ = 60.5-398.4 s-1. We also measured viscosity of human whole blood samples (non-Newtonian fluid) yielding 2.7-5.1 cP at shear rates γ = 120-345.1 s-1, which compares well with measurements using conventional rotational viscometers (3.6-5.7 cP). With a sensitivity better than 0.5 cP, this method has the potential to be used as a portable microfluidic viscometer for real-time rheological studies.

Increased Hemoglobin Oxygen Affinity With 5-Hydroxymethylfurfural Supports Cardiac Function During Severe Hypoxia.

Acclimatization to hypoxia or high altitude involves physiological adaptation processes, to influence oxygen (O2) transport and utilization. Several natural products, including aromatic aldehydes and isothiocyanates stabilize the R-state of hemoglobin (Hb), increasing Hb-O2 affinity and Hb-O2 saturation. These products are a counter intuitive therapeutic strategy to increase O2 delivery during hypoxia. 5-Hydroxymethylfurfural (5-HMF) is well known Amadori compound formed during the Maillard reaction (the non-enzymatic browning and caramelization of carbohydrate-containing foods after thermal treatment), with well documented effects in Hb-O2 affinity. This study explores the therapeutic potential of 5-HMF on left ventricular (LV) cardiac function (LVCF) during hypoxia. Anesthetized Golden Syrian hamsters received 5-HMF i.v., at 100 mg/kg and were subjected to stepwise increased hypoxia (15, 10, and 5%) every 30 min. LVCF was assessed using a closed chest method with a miniaturized conductance catheter via continuous LV pressure-volume (PV) measurements. Heart hypoxic areas were studied using pimonidazole staining. 5-HMF improved cardiac indices, including stroke volume (SV), cardiac output (CO), ejection fraction (EF), and stroke work (SW) compared to the vehicle group. At 5% O2, SV, CO, EF, and SW were increased by 53, 42, 33, and 51% with 5-HMF relative to vehicle. Heart chronotropic activity was not statistically changed, suggesting that differences in LV-CF during hypoxia by 5-HMF were driven by volume dependent effects. Analysis of coronary blood flow and cardiac muscle metabolism suggest no direct pharmacological effects from 5-HMF, therefore these results can be attributed to 5-HMF-dependent increase in Hb-O2 affinity. These studies establish that naturally occurring aromatic aldehydes, such as 5-HMF, produce modification of hemoglobin oxygen affinity with promising therapeutic potential to increase O2 delivery during hypoxic hypoxia.

Nitrite may serve as a combination partner and a biomarker for the anti-cancer activity of RRx-001.

BACKGROUND: RRx-001 is an anti-cancer immunotherapeutic that increases the sensitivity of drug resistant tumors via multiple mechanisms which involve binding to hemoglobin and enhancing nitrite reductase activity of deoxyhemoglobin. OBJECTIVE: In the present study, the effect of clinically used doses of RRx-001 on erythrocyte deformability was examined. METHODS: A dose dependent effect of RRx-001 (1-1000 micro molar) on erythrocyte deformability was measured by ektacytometer under hypoxia (n = 8). Low dose RRx-001 (20 micro molar) in the presence of ODQ (1H-[1,2,4]Oxadiazolo[4,3-a]quinoxalin-1-one), L-NAME (L-NG-Nitroarginine methyl ester) or nitrite were examined both in normoxia and hypoxia. Intracellular nitric oxide (NO) levels were measured fluorometrically with DAF-FM-DA. RESULTS: Higher doses of RRx-001 (100, 1000 micro molar) significantly decreased erythrocyte deformability under hypoxia (p < 0.01; p < 0.05, respectively). RRx-001 (20 micro molar), alone or in combination with ODQ or L-NAME, did not change deformability. However, RRx-001 and nitrite caused an increase in deformability (p < 0.01) under hypoxia. RRx-001 induced NO production was more pronounced in the presence of nitrite (p < 0.05). CONCLUSIONS: Co-administration of RRx-001 and nitrite under hypoxic conditions results in a significant increase in erythrocyte deformability that is related to increased NO production. We suggest that measurement of serum nitrite level in RRx-001 treated cancer patients should be routinely undertaken and supplemented if levels are low for maximal activity.

A Short-Term In Vivo Evaluation of the Istanbul Heart Left Ventricular Assist Device in a Pig Model.

OBJECTIVES: A continuous-flow centrifugal blood pump system has been recently developed as an implantable left ventricular assist device for patients with endstage heart failure. The objective of this study was to evaluate the initial in vivo performance of a newly developed left ventricular assist device (iHeart or Istanbul heart; Manufacturing and Automation Research Center, Koc University, Istanbul, Turkey) in an acute setting using a pig model. MATERIALS AND METHODS: Three pigs (77, 83, 92 kg) received implants via a median sternotomy, with animals supported for up to 6 hours. An outflow cannula was anastomosed to the ascending aorta. Anticoagulation was applied by intravenous heparin administration. During the support period, pump performance was evaluated under several flow and operating conditions. All pigs were humanely sacrificied after the experiments, and organs were examined macroscopically and histopathologically. RESULTS: Flow rate ranged between 1.5 and 3.6 L/min with pump speeds of 1500 to 2800 revolutions/min and motor current of 0.6 to 1.3 A. Initial findings confirmed thatthe iHeart ventricular assist device had sufficient hydraulic performance to support the circulation. During the experimental period, plasma free hemoglobin levels were found to be within normalranges.Thrombus formation was not observed inside the pump in all experiments. CONCLUSIONS: The iHeart ventricular assist device demonstrated encouraging hemodynamic performance and good biocompatibility in the pig model for use as an implantable left ventricular assist device. Further acute in vivo studies will evaluate the short-term pump performance prior to chronic studies for long-term evaluation.

Differential effects of adenylyl cyclase-protein kinase A cascade on shear-induced changes of sickle cell deformability.

BACKGROUND: Erythrocyte deformability is impaired in sickle cell disease (SCD). The regulation of cytoskeletal protein organization plays a key role in erythrocyte deformability. The activation of adenylyl cyclase (AC)/cAMP/Protein kinase A (PKA) signaling pathway was associated with increased deformability in healthy erythrocytes, however the role of this pathway in SCD is unknown. OBJECTIVE: We evaluated mechanical responses of sickle red blood cells under physiological levels of shear stress and the possible link between their deformability and AC/cAMP/PKA signaling pathway. METHODS: The shearing of sickle red blood cells at physiological level (5 Pa) and the measurement of deformability were performed by a laser assisted optical rotational cell analyzer (LORRCA). RESULTS: Red blood cell deformability increased of 2.5-6.5% by blocking the activity of phosphodiesterase with Pentoxifylline (10µM) (p < 0.05). The inhibition of AC with SQ22536 (100µM) produced more significant rise in deformability (+4.8-12%, p < 0.01). No significant change was observed by the inhibition of PKA with H89 (10µM). CONCLUSION: Pentoxifylline and SQ22536 increased the deformability of sickle red blood cells under fluid shear stress. Modulation of the AC/cAMP/PKA pathway could have the potential to be an effective therapeutic approach for SCD through shear-induced improvements of RBC deformability.

Cardioprotective Effect of Phase 3 Clinical Anticancer Agent, RRx-001, in Doxorubicin-Induced Acute Cardiotoxicity in Mice.

Anthracycline chemotherapy (e.g., doxorubicin or DOX) is associated with a cumulative dose-dependent cardiac dysfunction that may lead to congestive heart failure, which limits both its use and usefulness in the clinic. The cardiotoxicity may manifest acutely and/or months or years after treatment with doxorubicin has ended. Experimental and human data have demonstrated that angiotensin-converting enzyme/angiotensin-receptor antagonists mediate a cardioprotective effect against anthracycline toxicity. In this study, with the angiotensin receptor blocker, candesartan, as a positive control, we evaluated whether pretreatment with the hypoxic nitric oxide generating anticancer agent, RRx-001, could reduce acute DOX-induced cardiotoxicity. A total of 24 BALB/c mice were randomized for prophylactic treatment with vehicle, RRx-001, candesartan, or no-intervention control. Within each of the three intervention arms, mice received treatment with DOX. Murine pressure-volume analysis was performed with microconductance catheters to characterize the degree of cardiovascular dysfunction within each group. The following hemodynamic parameters were monitored: left ventricular systolic pressure (LVSP), heart rate, and maximal rate of increase of left ventricular pressure (±d P/d tmax). Five days after doxorubicin injection, untreated (with RRx-001) mice displayed significantly impaired systolic (LVSP, -27%; d P/d tmax, -25%; left ventricular developed pressure (LVDP), +33%; P < 0.05) and global (stroke volume (SV), -52%; ejection fraction (EF), -20%; stroke work (SW), -62.5%; heart rate (HR), -18%; cardiac output (CO), -57%; mean blood arterial pressure (MAP), -30%; systemic vascular resistance (SVR), +20%; P < 0.05) LV functions when compared with the untreated (with RRx-001) group. In contrast, RRx-001-treated mice showed improved variables of systolic (LVSP, +27%; d P/d tmax, +25%; LVDP, -33%; P < 0.05) and global (SV, +52%; EF, +20%; SW, +62.5%; HR, +18%; CO, +57%; MAP, +30%; SVR, -20%; P < 0.05) LV functions compared with untreated doxorubicin mice. Similar to the positive control, candesartan, the cardiotoxic effects of DOX in mice were partially attenuated by the prophylactic administration of RRx-001. These results suggest that RRx-001 as a multifunctional anticancer agent, which sensitizes cancer cells to the cytotoxic effects of chemotherapy and radiation, may also have beneficial cardioprotective effects.

Image-Based Flow Cytometry and Angle-Resolved Light Scattering to Define the Sickling Process.

Red blood cells (RBCs) from sickle cell patients exposed to a low oxygen tension reveal highly heterogeneous cell morphologies due to the polymerization of sickle hemoglobin (HbS). We show that angle-resolved light scattering approach with the use of image-based flow cytometry provides reliable quantitative data to define the change in morphology of large populations of RBCs from sickle cell patients when the cells are exposed for different times to low oxygen. We characterize the RBC morphological profile by means of a set of morphological and physical parameters, which includes cell shape, size, and orientation. These parameters define the cell as discocyte, sickle, elongated, as well as irregularly or abnormal RBC shaped cells, including echinocytes, holly-leaf, and granular structures. In contrast to microscopy, quick assessment of large numbers of cells provides statistically relevant information of the dynamic process of RBC sickling in time. The use of this approach facilitates the understanding of the processes that define the propensity of sickle blood samples to change their shape, and the ensuing vaso-occlusive events in the circulation of the patients. Moreover, it assists in the evaluation of treatments that include the use of anti-sickling agents, gene therapy-based hemoglobin modifications, as well as other approaches to improve the quality of life of sickle cell patients. © 2019 International Society for Advancement of Cytometry.

From Experiments to Simulation: Shear-Induced Responses of Red Blood Cells to Different Oxygen Saturation Levels.

Red blood cells (RBC) carry and deliver oxygen (O2) to peripheral tissues through different microcirculatory regions where they are exposed to various levels of shear stress (SS). O2 affinity of hemoglobin (Hb) decreases as the blood enters the microcirculation. This phenomenon determines Hb interactions with RBC membrane proteins that can further regulate the structure of cytoskeleton and affect the mechanical properties of cells. The goal of this study is to evaluate shear-induced RBC deformability and simulate RBC dynamics in blood flow under oxygenated and deoxygenated conditions. Venous blood samples from healthy donors were oxygenated with ambient air or deoxygenated with 100% nitrogen gas for 10 min and immediately applied into an ektacytometer (LORRCA). RBC deformability was measured before and after the application of continuous 5 Pa SS for 300 s by LORRCA and recorded as elongation index (EI) values. A computational model was generated for the simulation of blood flow in a real carotid artery section. EI distribution throughout the artery and its relationships with velocity, pressure, wall SS and viscosity were determined by computational tools. RBC deformability significantly increased in deoxygenation compared to oxygenated state both before and after 5 Pa SS implementation (p < 0.0001). However, EI values after continuous SS were not significant at higher SS levels (>5.15 Pa) in deoxygenated condition. Simulation results revealed that the velocity gradient dominates the generation of SS and the shear thinning effect of blood has a minor effect on it. Distribution of EI was calculated during oxygenation/deoxygenation which is 5-10 times higher around the vessel wall compared to the center of the lumen for sections of the pulsatile flow profile. The extent of RBC deformability increases as RBCs approach to the vessel wall in a real 3D artery model and this increment is higher for deoxygenated condition compared to the oxygenated state. Hypoxia significantly increases shear-induced RBC deformability. RBCs could regulate their own mechanical properties in blood flow by increasing their deformability in hypoxic conditions. Computational tools can be applied for defining hypoxia-mediated RBC deformability changes to monitor blood flow in hypoxic tissues.

Numerical Model for the Determination of Erythrocyte Mechanical Properties and Wall Shear Stress in vivo From Intravital Microscopy.

The mechanical properties and deformability of Red Blood Cells (RBCs) are important determinants of blood rheology and microvascular hemodynamics. The objective of this study is to quantify the mechanical properties and wall shear stress experienced by the RBC membrane during capillary plug flow in vivo utilizing high speed video recording from intravital microscopy, biomechanical modeling, and computational methods. Capillaries were imaged in the rat cremaster muscle pre- and post-RBC transfusion of stored RBCs for 2-weeks. RBC membrane contours were extracted utilizing image processing and parametrized. RBC parameterizations were used to determine updated deformation gradient and Lagrangian Green strain tensors for each point along the parametrization and for each frame during plug flow. The updated Lagrangian Green strain and Displacement Gradient tensors were numerically fit to the Navier-Lame equations along the parameterized boundary to determined Lame's constants. Mechanical properties and wall shear stress were determined before and transfusion, were grouped in three populations of erythrocytes: native cells (NC) or circulating cells before transfusion, and two distinct population of cells after transfusion with stored cells (SC1 and SC2). The distinction, between the heterogeneous populations of cells present after the transfusion, SC1 and SC2, was obtained through principle component analysis (PCA) of the mechanical properties along the membrane. Cells with the first two principle components within 3 standard deviations of the mean, were labeled as SC1, and those with the first two principle components greater than 3 standard deviations from the mean were labeled as SC2. The calculated shear modulus average was 1.1±0.2, 0.90±0.15, and 12 ± 8 MPa for NC, SC1, and SC2, respectively. The calculated young's modulus average was 3.3±0.6, 2.6±0.4, and 32±20 MPa for NC, SC1, and SC2, respectively. o our knowledge, the methods presented here are the first estimation of the erythrocyte mechanical properties and shear stress in vivo during capillary plug flow. In summary, the methods introduced in this study may provide a new avenue of investigation of erythrocyte mechanics in the context of hematologic conditions that adversely affect erythrocyte mechanical properties.