Nandan K. Mondal

Oxidative Stress, DNA Damage and Repair in Heart Failure Patients after Implantation of Continuous Flow Left Ventricular Assist Devices

Objective: To study the status of oxidative stress and DNA damage repair in circulating blood leukocytes of heart failure patients supported by continuous flow left ventricular assist devices (LVADs). Materials and methods: Ten HF patients implanted with LVAD as bridge to transplant or destination therapy were enrolled in the study and 10 age and sex matched volunteers were recruited as the study control. Reactive oxygen species (ROS) in blood leukocytes and superoxide dismutase (SOD) in erythrocytes were measured by flow cytometry/immunofluorescence microscopy and spectrophotometry, respectively. ELISA was used to measure oxidized low density lipoproteins (oxLDL) in plasma. Markers of DNA damage (γ-H2AX) and repairs (Mre11, DNA ligase IV, Ku70, and Ku80) were quantified in blood lymphocytes by immunocytochemistry. Results: Levels of ROS and oxLDL were significantly higher in HF patients with LVAD than baseline as well as the control group; moreover, SOD levels were decreased with increasing post-operative periods. All the changes indicated enhanced oxidative stress among LVAD recipients. Significantly higher γ-H2AX foci in lymphocytes confirmed DNA double strand breaks in LVAD recipients. γ-H2AX foci numbers in lymphocytes were positively correlated with the ROS and oxLDL and negatively with SOD levels (p<0.0001). Expressions of DNA ligase IV, Ku70 and Ku80 proteins were highest after one week and Mre11 protein after 3 months of LVAD transplantation; indicated abnormal DNA repair. Conclusions: The study, for the first time shows that, continuous flow LVAD implanted HF patients not only exhibit elevated oxidative stress and DNA damage in blood leukocytes but also have abnormalities in DNA repair pathways.

Platelet glycoprotein Ibα ectodomain shedding and non-surgical bleeding in heart failure patients supported by continuous-flow left ventricular assist devices

BACKGROUND Non-surgical bleeding (NSB) is a major complication among heart failure (HF) patients supported by continuous-flow left ventricular assist devices (CF-LVADs). Understanding the hemostatic defects contributing to NSB after CF-LVAD implantation is crucial for prevention of this adverse event. The aim of this study was to examine the link between platelet glycoprotein Ibα (GPIbα) ectodomain shedding and NSB in CF-LVAD recipients and to identify a potential biomarker of NSB. METHODS Serial blood samples were collected from 35 HF patients supported with CF-LVADs. Platelet function was evaluated by a platelet function analysis device and thromboelastography (TEG). Platelet GPIbα shedding, von Willebrand factor (vWF) antigen and vWF collagen binding capacity were determined using enzyme-linked immunosorbent assays (ELISAs). The structural analysis of vWF was performed by gel electrophoresis. These platelet function measures with vWF parameters of the patients who had NSB between 4 and 32 days after CF-LVAD implantation (bleeder) were analyzed against those without NSB (non-bleeder). Blood samples from 7 healthy individuals were collected to obtain healthy reference values for the laboratory assays. RESULTS Elevated GPIbα shedding was found to be a pre-existing condition in all HF patients prior to CF-LVAD implantation. Post-operative level of GPIbα shedding increased and remained elevated in the bleeder group, whereas a consistent decrease was found in the non-bleeder group. A receiver operating characteristic (ROC) analysis indicated that the level of GPIbα shedding had a predictive power of NSB in patients on CF-LVAD support. CONCLUSIONS Platelet GPIbα ectodomain shedding which attenuates platelet reactivity is associated with NSB. Plasma GPIbα level may potentially be used to refine bleeding risk stratification in CF-LVAD patients.

Shear-induced platelet receptor shedding by non-physiological high shear stress with short exposure time: Glycoprotein Ibα and glycoprotein VI ☆

INTRODUCTION The structural integrity of platelet receptors is essential for platelets to function normally in hemostasis and thrombosis in response to physiological and pathological stimuli. The aim of this study was to examine the shedding of two key platelet receptors, glycoprotein (GP) Ibα and GPVI, after exposed to the non-physiological high shear stress environment which commonly exists in blood contacting medical devices and stenotic blood vessels. MATERIALS AND METHODS In this in vitro experiment, we exposed healthy donor blood in our specially designed blood shearing device to three high shear stress levels (150, 225, 300 Pa) in combination with two short exposure time conditions (0.05 and 0.5 sec.). The expression and shedding of platelet GPIbα and GPVI receptors in the sheared blood samples were characterized using flow cytometry. The ability of platelet aggregation induced by ristocetin and collagen related to GPIbα and GPVI in the sheared blood samples, respectively, was evaluated by aggregometry. RESULTS AND CONCLUSIONS Compared to the normal blood, the surface expression of platelet GPIbα and GPVI in the sheared blood significantly decreased with increasing shear stress and exposure time. Moreover, the platelet aggregation induced by ristocetin and collagen reduced remarkably in a similar fashion. In summary non-physiological high shear stresses with short exposure time can induce shedding of platelet GPIbα and GPVI receptors, which may lead platelet dysfunction and influence the coagulation system. This study may provide a mechanistic insight into the platelet dysfunction and associated bleeding complication in patients supported by certain blood contacting medical devices.

Indoor Air Pollution from Biomass Burning Activates Akt in Airway Cells and Peripheral Blood Lymphocytes A Study among Premenopausal Women in Rural India

Biomass burning is a major source of indoor air pollution in rural India. The authors investigated in this study whether cumulative exposures to biomass smoke cause activation of the serine/threonine kinase Akt in airway cells and peripheral blood lymphocytes (PBL). For this, the authors enrolled 87 premenopausal (median age 34 years), nonsmoking women who used to cook with biomass (wood, dung, crop wastes) and 85 age-matched control women who cooked with cleaner fuel liquefied petroleum gas. Immunocytochemical and immunoblotting assays revealed significantly higher levels of phosphorylated forms of Akt protein (p-Aktser473 and p-Aktthr308) in PBL, airway epithelial cells, alveolar macrophages, and neutrophils in sputum of biomass-using women than control. Akt activation in biomass users was associated with marked rise in generation of reactive oxygen species and concomitant depletion of superoxide dismutase. Measurement of particulate matter having a diameter of less than 10 and 2.5 µm in indoor air by real-time aerosol monitor showed 2 to 4 times more particulate pollution in biomass-using households, and Akt activation was positively associated with particulate pollution after controlling potential confounders. The findings suggest that chronic exposure to biomass smoke activates Akt, possibly via generation of oxidative stress.

Systemic Inflammatory Response Syndrome in End-Stage Heart Failure Patients Following Continuous-Flow Left Ventricular Assist Device Implantation: Differences in Plasma Redox Status and Leukocyte Activation.

The role of oxidative stress and leukocyte activation has not been elucidated in developing systemic inflammatory response syndrome (SIRS) in heart failure (HF) patients after continuous-flow left ventricular assist device (CF-LVAD) implantation. The objective of this study was to investigate the change of plasma redox status and leukocyte activation in CF-LVAD implanted HF patients with or without SIRS. We recruited 31 CF-LVAD implanted HF patients (16 SIRS and 15 non-SIRS) and 11 healthy volunteers as the control. Pre- and postimplant blood samples were collected from the HF patients. Plasma levels of oxidized low-density lipoprotein (oxLDL), malondialdehyde (MDA), total antioxidant capacity (TAC), superoxide dismutase (SOD) in erythrocyte, myeloperoxidase (MPO), and polymorphonuclear elastase (PMN-elastase) were measured. The HF patients had a preexisting condition of oxidative stress than healthy controls as evident from the higher oxLDL and MDA levels as well as depleted SOD and TAC. Leukocyte activation in terms of higher plasma MPO and PMN-elastase was also prominent in HF patients than controls. Persistent oxidative stress and reduced antioxidant status were found to be more belligerent in HF patients with SIRS after the implantation of CF-LVAD when compared with non-SIRS patients. Similar to oxidative stress, the activation of blood leukocyte was significantly highlighted in SIRS patients after implantation compared with non-SIRS. We identified that the plasma redox status and leukocyte activation became more prominent in CF-LVAD implanted HF patients who developed SIRS. Our findings suggest that plasma biomarkers of oxidative stress and leukocyte activation may be associated with the development of SIRS after CF-LVAD implant surgery.

Paradoxical Effect of Nonphysiological Shear Stress on Platelets and von Willebrand Factor

Blood can become hypercoagulable by shear-induced platelet activation and generation of microparticles. It has been reported that nonphysiological shear stress (NPSS) could induce shedding of platelet receptor glycoprotein (GP) Ibα, which may result in an opposite effect to hemostasis. The aim of this study was to investigate the influence of the NPSS on platelets and von Willebrand factor (vWF). Human blood was exposed to two levels of NPSS (25 Pa, 125 Pa) with an exposure time of 0.5 s, generated by using a novel blood-shearing device. Platelet activation (P-selectin expression, GPIIb/IIIa activation and generation of microparticles) and shedding of three platelet receptors (GPIbα, GPVI, GPIIb/IIIa) in sheared blood were quantified using flow cytometry. Aggregation capacity of sheared blood induced by ristocetin and collagen was evaluated using an aggregometer. Shear-induced vWF damage was characterized with Western blotting. Consistent with the published data, the NPSS caused significantly more platelets to become activated with increasing NPSS level. Meanwhile, the NPSS induced the shedding of platelet receptors. The loss of the platelet receptors increased with increasing NPSS level. The aggregation capacity of sheared blood induced by ristocetin and collagen decreased. There was a loss of high molecular weight multimers (HMWMs) of vWF in sheared blood. These results suggest that the NPSS induced a paradoxical effect. More activated platelets increase the risk of thrombosis, while the reduction in platelet receptors and the loss of HMWM-vWF increased the propensity of bleeding. The finding might provide a new perspective to understand thrombosis and acquired bleeding disorder in patients supported with blood contacting medical devices.

Quantification of Shear‐Induced Platelet Activation: High Shear Stresses for Short Exposure Time

Thrombosis and thromboembolism are the life-threatening clinical complications for patients supported or treated with prosthetic cardiovascular devices. The high mechanical shear stress within these devices is believed to be the major contributing factor to cause platelet activation (PA) and function alteration, leading to thrombotic events. There have been limited quantitative data on how the high mechanical shear stress causes platelet activation. In this study, shear-induced PA in the ranges of well-defined shear stress and exposure time relevant to cardiovascular devices was quantitatively characterized for human blood using two novel flow-through Couette-type blood shearing devices. Four markers of platelet activation-surface P-selectin (CD62p), platelet-derived microparticles (PMPs), platelet-monocyte aggregation (PMA), and soluble P-selectin-were measured by flow cytometry and enzyme-linked immunosorbent assay (ELISA), respectively. The results indicated that PA induced by high shear stresses with short exposure time could be reliably detected with surface P-selectin, and, to a lesser extent, PMPs rather than soluble P-selectin. It was also verified that PMA can be a highly sensitive indirect marker of platelet activation. The quantitative relationship between percentage of activated platelets indicated by surface P-selectin expression and shear stress/exposure time follows well the power law functional form. The coefficients of the power law models of PA based on surface P-selectin expression were derived.

Intraplatelet reactive oxygen species, mitochondrial damage and platelet apoptosis augment non-surgical bleeding in heart failure patients supported by continuous-flow left ventricular assist device

Abstract Non-surgical bleeding (NSB) is the most common clinical complication among heart failure (HF) patients supported by continuous-flow left ventricular assist devices (CF-LVADs). Understanding the role of platelet functionality contributing to NSB after CF-LVAD implantation is crucial for prevention and management of this adverse event. The aim of this study was to examine the role of intraplatelet reactive oxygen species (ROS) and platelet damage on the incidence of bleeding events after CF-LVAD implantation in HF patients. We recruited 25 HF patients implanted with CF-LVADs and 11 healthy volunteers as the control. Intraplatelet ROS generation, platelet mitochondrial damage and platelet apoptosis were quantified by flow cytometry. Among 25 patients, 8 patients developed non-surgical bleeding within one month after CF-LVAD implantation. Intraplatelet ROS, depolarized and apoptotic platelet were found to be pre-existing conditions in all baseline samples of the 25 HF patients when compared to the healthy volunteers. There was no significant difference in the levels of ROS between the non-bleeder and the bleeder groups prior to CF-LVAD implantation, although we noticed 2-fold and 1.5-fold rise in depolarized and apoptotic platelets, respectively, in the bleeder group compared to those in the non-bleeder group. Post implant levels of intraplatelet ROS, depolarized and apoptotic platelets increased and remained elevated in the bleeder group, whereas periodic decreases were noticed in the non-bleeder group, suggesting the potential role of platelet damage on bleeding incidence. ROS generation after CF-LVAD implantation positively associated with platelet apoptosis (ρ = 0.4263, p = 0.0023) and depolarized platelets (ρ = 0.4774, p = 0.0002), especially the latter. In conclusion, elevated intraplatelet ROS and platelet damage may be linked to the NSB among HF patients supported by CF-LVAD. These results provide mechanistic insights into the bleeding complication in patients with CF-LVAD support.

Comparison of intraplatelet reactive oxygen species, mitochondrial damage, and platelet apoptosis after implantation of three continuous flow left ventricular assist devices: HeartMate II, Jarvik 2000, and HeartWare.

Differences in device design may have an effect on platelet damage and associated clinical complications. We aimed to compare device-specific platelet functionality in 26 heart failure patients supported with three continuous-flow left ventricular assist devices: HeartMate II (n = 8), Jarvik 2000 (n = 9), and HeartWare (n = 9). Intraplatelet reactive oxygen species (ROS) generation, mitochondrial damage, and platelet apoptosis were compared between device types before and after the implantation at every week up to 1 month. Overall, the baseline characteristics, demographics, routine laboratory values were comparable between the three device groups. Intraplatelet ROS, mitochondrial damage, and platelet apoptosis significantly elevated in the HeartWare group in comparison with the other two device groups after implantation. The major bleeding, infections, systemic inflammatory response syndrome, and right ventricular failure were found to be more common among the HeartWare group than others. Intraplatelet ROS and platelet damage levels were returned to baseline in both the HeartMate II and the Jarvik groups, whereas in HeartWare group they remained elevated. The patients with the Jarvik and the HeartMate II experienced less clinical complications and the platelet functionality is not compromised by these devices. Data from this study suggests that the continuous-flow left ventricular assist devices design may exert different effects on platelet function.

Infection, Oxidative Stress, and Changes in Circulating Regulatory T Cells of Heart Failure Patients Supported by Continuous-flow Ventricular Assist Devices

The objective of this study was to investigate the changes in oxidative stress (OS) and circulating regulatory T cells (Tregs) of the immune system in patients supported by continuous-flow ventricular assist device (CF-VAD) with or without infection. We recruited 16 CF-VAD patients (5 with infection and 11 without infection) and 7 healthy volunteers. Generation of reactive oxygen species (ROS) from lymphocytes, superoxide dismutase (SOD) in erythrocyte, total antioxidant capacity (TAC), and oxidized low-density lipoprotein (oxLDL) in plasma were measured. Circulating Tregs were evaluated by flow cytometry. Heart failure (HF) patients had elevated OS than healthy volunteers as evident from higher lymphocyte ROS, elevated oxLDL, as well as depleted SOD and TAC levels. At baseline, HF patients had decreased percentage of Tregs (5.12 ± 1.5% vs. 8.14 ± 3.01%, p < 0.01) when compared with healthy volunteers. Postimplant patients with infection illustrated 35% and 44% rise in ROS and oxLDL, respectively, 31% decrease in TAC, and marked rise in percentage of Tregs (14.27 ± 3.17% vs. 9.38 ± 3.41%, p < 0.01) when compared with the patients without infection. Elevated OS and rise in Tregs were more prominent in CF-VAD patients with infection. In conclusion, OS and compromised immune system may be important indicators of systemic response of the body to CF-VAD among HF patients with infection.