NADPH Oxidase 2 Is Critical for Heterotypic Neutrophil-Platelet Interactions during Vascular Inflammation

Abstract Intravital microscopic studies have shown that neutrophil-platelet interactions on activated endothelial cells are the major determinant of vascular occlusion under thromboinflammatory conditions. Heterotypic neutrophil-platelet interactions are mainly mediated by two crucial receptors and counter receptors, P-selectin-P-selectin glycoprotein ligand-1 and glycoprotein Ibalpha-alphaMbeta2 integrin. Although the major receptors and counter receptors are well identified, it remains unclear how heterotypic cell-cell interactions are regulated during vascular disease. Recently, we demonstrated that neutrophil AKT2 is a key regulator for neutrophil-platelet interactions during vascular inflammation. Since it is known that AKT2 is important for NADPH oxidase 2 (NOX2) activity and reactive oxygen species (ROS) generation during neutrophil activation, we investigated whether NOX2 regulates the heterotypic neutrophil-platelet interaction during vascular inflammation. Using real-time fluorescence intravital microscopy, we found that platelet-neutrophil interactions are nearly completely inhibited during TNF-alpha-induced cremaster venular inflammation in NOX2 KO mice, compared with WT mice. Studies with bone marrow chimera further demonstrated that both hematopoietic and endothelial cell NOX2 are crucial for neutrophil-platelet interactions on the inflamed venules. In vitro studies with neutrophils and platelets isolated from WT and NOX2-deficient mice indicated that both neutrophil and platelet NOX2 are required for neutrophil-platelet aggregation under shear conditions. Using ROS scavengers and NOX2 KO cells, we observed that neutrophil NOX2-generated ROS regulate the activation and ligand-binding activity of alphaMbeta2 integrin during neutrophil activation and that platelet NOX2-produced ROS are important for P-selectin exposure upon agonist stimulation and the ligand-binding function of glycoprotein Ibalpha. Interestingly, neutrophil NOX2 was crucial for store-operated calcium entry (SOCE), but not calcium release from the endoplasmic reticulum, whereas platelet NOX2 modulated intracellular calcium release, but not SOCE. We further demonstrated that the differential regulation of platelet and neutrophil NOX2 in calcium signaling correlates with the differences in the phosphorylation of AKT, ERK, and p38MAPK, suggesting the importance of AKT and its downstream molecules for NOX2-mediated calcium signaling. Our results indicate that neutrophil and platelet NOX2-produced ROS play critical roles in regulating the function of surface receptors required for heterotypic neutrophil-platelet interactions during vascular inflammation. Disclosures No relevant conflicts of interest to declare.

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Neutrophil azurophilic granule exocytosis is primed by TNF-α and partially regulated by NADPH oxidase

Innate Immunity ◽

10.1177/1753425916668980 ◽

2016 ◽

Vol 22 (8) ◽

pp. 635-646 ◽

Cited By ~ 18

Author(s):

Renee M Potera ◽

Melissa J Jensen ◽

Brieanna M Hilkin ◽

Gina K South ◽

Jessica S Hook ◽

...

Keyword(s):

Nadph Oxidase ◽

Tissue Damage ◽

Chronic Inflammatory Disease ◽

Host Tissue ◽

Ros Generation ◽

Oxygen Species ◽

Granule Exocytosis ◽

Azurophilic Granule ◽

Tnf Α

Neutrophil (polymorphonuclear leukocyte) activation with release of granule contents plays an important role in the pathogenesis of acute lung injury, prompting clinical trials of inhibitors of neutrophil elastase. Despite mounting evidence for neutrophil-mediated host tissue damage in a variety of disease processes, mechanisms regulating azurophilic granule exocytosis at the plasma membrane, and thus release of elastase and other proteases, are poorly characterized. We hypothesized that azurophilic granule exocytosis would be enhanced under priming conditions similar to those seen during acute inflammatory events and during chronic inflammatory disease, and selected the cytokine TNF-α to model this in vitro. Neutrophils stimulated with TNF-α alone elicited intracellular reactive oxygen species (ROS) generation and mobilization of secretory vesicles, specific, and gelatinase granules. p38 and ERK1/2 MAPK were involved in these components of priming. TNF-α priming alone did not mobilize azurophilic granules to the cell surface, but did markedly increase elastase release into the extracellular space in response to secondary stimulation with N-formyl-Met-Leu-Phe (fMLF). Priming of fMLF-stimulated elastase release was further augmented in the absence of NADPH oxidase-derived ROS. Our findings provide a mechanism for host tissue damage during neutrophil-mediated inflammation and suggest a novel anti-inflammatory role for the NADPH oxidase.

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The Effect of Anti-GPIIIa49-66 Antibody on Megakaryocyte Differentiation.

Blood ◽

10.1182/blood.v116.21.1434.1434 ◽

2010 ◽

Vol 116 (21) ◽

pp. 1434-1434

Author(s):

Jianhui Wang ◽

Ruimin Pan ◽

Michael A. Nardi ◽

Zongdong Li

Keyword(s):

Nadph Oxidase ◽

In Vitro Culture ◽

Conflicts Of Interest ◽

Signal Pathways ◽

Mouse Bone Marrow ◽

Colony Forming Units ◽

Sequential Activation ◽

12 Lipoxygenase ◽

Hiv 1

Abstract Abstract 1434 We previously reported that patients with early-onset HIV-1 ITP develop a unique anti-platelet integrin GPIIIa antibody against the GPIIIa49-66 epitope. Anti-GPIIIa49-66 antibody-induced platelet fragmentation requires sequential activation of the platelet 12-lipoxygenase (12-LO) and NADPH oxidase to release reactive oxygen species (ROS). 12-LO is upstream of the NADPH oxidase pathway and 12(S)-HETE, the product of 12-LO, induces the same oxidative platelet fragmentation as anti-GPIIIa49-66. Since the megakaryocyte (MK) is the progenitor cells for platelets and may contain similar signal pathways, we have investigated the effect of anti-GPIIIa49-66 on MK differentiation and, in particular, the potential role of anti-GPIIIa49-66 induced ROS in this process. We first show that polyclonal anti-GPIIIa49-66 antibody isolated from HIV-1 ITP patients inhibits MK proliferation 2.5 fold in in vitro culture of mouse bone marrow Lin-/- cells driven by thrombopoietin (TPO). We also observed a 3 fold decrease in the number of MK colony-forming units in the presence of a human monoclonal anti-GPIIIa49-66 antibody we generated. However, we could not detect ROS release in DCFH-loaded MEG-01 cells treated with anti-GPIIIa49-66 antibody. In addition, 12(S)-HETE does not inhibit the in vitro differentiation of MK cell line L8057 induced by TPO. In fact, we found a dose dependent increasing of the percentage of αIIb integrin positive cells (from 17.1% to 48.7%) in in vitro culture of L8057 treated by various concentration of H2O2 (from 5 to 20μM). Thus, our data suggests that ROS is not involved in the inhibition of MK differentiation induced by anti-GPIIIa49-66, in contrast to the effect that this antibody has on mature platelets. We therefore conclude that the anti-GPIIIa49-66 antibody dysregulates ROS independent β3 integrin signaling to inhibit MK differentiation. Disclosures: No relevant conflicts of interest to declare.

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Leukocyte Adhesion and Activation in Atherothrombosis

Blood ◽

10.1182/blood.v122.21.sci-52.sci-52 ◽

2013 ◽

Vol 122 (21) ◽

pp. SCI-52-SCI-52

Author(s):

Meinrad Gawaz

Keyword(s):

Myocardial Infarction ◽

Progenitor Cells ◽

Myocardial Injury ◽

Conflicts Of Interest ◽

Vascular Inflammation ◽

Dynamic Change ◽

Cyclophilin A ◽

Ppiase Activity ◽

Monocyte Migration

Abstract Platelets interact with a variety of cells, including leukocytes and progenitor cells. Upon activation, platelets release chemokines such as CXCL12/SDF-1 that modulate important functional aspects of vascular inflammation and atherogenesis. Platelet-derived SDF-1 and CyPA regulate chemotaxis, migration, and cell activation in the microenvironment of accumulating platelets. Previously we have shown that platelets are the major source of CXCL12/SDF-1. Platelet-derived SDF-1 provides a strong signal for recruitment of circulating progenitor cells and leukocytes towards vascular and myocardial injury. Patients with severe coronary artery disease have enhanced levels of plasma and platelet-associated SDF-1 that correlates with an enhanced number of circulating progenitor cells and the development of cardiovascular diseases. In patients with both acute myocardial infarction platelet-SDF-1 and an increase in circulating platelet/progenitor cell aggregates, the combination is associated with myocardial recovery and improved prognosis, indicating that platelet-SDF-1 is critical for repair and regeneration of vascular or myocardial injury. Administration of a stable recombinant SDF-1 fusion protein reduces infarct size and preserves myocardial infarction in mice. We showed that platelet-derived SDF-1 is an important survival factor both for platelets (in an autocrine manner) and for surrounding cells. SDF-1 released from activated platelets binds to its receptor CXCR4 and induces internalization of the SDF-1/CXCR4 complex. SDF-1-ligation of CXCR4 augments surface expression of CXCR7; a process coupled via ERK1/2 and cyclophilin A. SDF-1α caused downstream phosphorylation of Erk1/2 and cyclophilin A (CyPA). NIM-811 a CyPA-PPIase-activity inhibitor significantly abolished SDF-1α-driven CXCR7 surface exposure. Moreover, Cypa-/-murine platelets failed to show SDF-1α/CXCR4-mediated CXCR7 translocation. SDF-1α-induced ubiquitination of CXCR7 was essential for its surface translocation and was dependent on Erk1/2 and CyPA-PPIase activity, inhibited by U0126 and NIM-811, respectively. In contrast to wild-type, Cypa-/- murine platelets failed to exhibit a dynamic change in CXCR7 ubiquitination status upon SDF-1α exposure and thus failed in subsequent CXCR7 externalization. SDF-1/CXR4-driven CXCR7 translocation inhibits activation-induced apoptosis of platelets and prolongs platelet survival in vitro and in vivo. Similarly, platelet SDF-1 interacts with monocytes, reduces apoptosis and promotes differentiation of monocytes into macrophages. Inhibition of platelet release and adhesion to endothelial cells reduces monocyte migration and differentiation into macrophages in vitro and attenuates vascular inflammation and atheroprogression in apoE-deficient mice. In conclusion, platelets and platelet interaction with leukocytes are critical in regulation of vascular inflammation and atheroprogression and may offer promising targets for modulating inflammation in disease states. Disclosures: No relevant conflicts of interest to declare.

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High Glucose/Thrombin-Induced Endothelial Inflammation Via Microrna-146a and Nox4 Regulation

Blood ◽

10.1182/blood.v124.21.5952.5952 ◽

2014 ◽

Vol 124 (21) ◽

pp. 5952-5952

Author(s):

Ching-Tien Peng ◽

Wan Yu Lo ◽

Huang Joe Wang

Keyword(s):

High Glucose ◽

Conflicts Of Interest ◽

Mrna Level ◽

In Silico Analysis ◽

Fold Increase ◽

Ros Generation ◽

Aortic Endothelial Cells ◽

Protein Levels ◽

Endothelial Inflammation

Abstract Diabetes is associated with hyperglycemia and increased thrombin generation. It is unknown whether high glucose (HG)/thrombin can modulate the expression of NAPDH oxidase (Nox) subtypes in human aortic endothelial cells (HAECs). Besides, we investigate whether miR-146a is involved in endothelial cell inflammation. We observed that HG (25 mmol/l) exerted a synergistic effect with thrombin (2 U/ml) for induction of Nox4 mRNA level in HAECs. The increased Nox4 mRNA was associated with increased Nox4 protein and ROS production. We also demonstrated that HG/thrombin treatment increased interleukin-8 and interleukin-6 protein levels. Besides, HG/thrombin treatment caused an 11.43-fold increase of THP-1 adhesion to HAECs. In Silico analysis identified homology between miR-146a and the 3’-UTR of the human Nox4 mRNA, suggesting a potential regulation of Nox4 by miR-146a. Furthermore, HG/thrombin treatment decreased miR146a expression to 58% of the control, indicating an impaired feedback restrain of HG/thrombin-induced endothelial inflammation. MiR-146a mimic transfection prevented HG/thrombin-induced upregulation of Nox4 mRNA, Nox4 protein, and ROS generation. In addition, inflammatory phenotypes were attenuated in miR-146a mimic-transfected HAECs. In conclusion, miR-146a is involved in the regulation of endothelial inflammation via modulation of Nox4 in an in-vitro milieu mimicking diabetic atherothrombosis. Disclosures No relevant conflicts of interest to declare.

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AGER1 regulates endothelial cell NADPH oxidase-dependent oxidant stress via PKC-δ: implications for vascular disease

AJP Cell Physiology ◽

10.1152/ajpcell.00463.2009 ◽

2010 ◽

Vol 298 (3) ◽

pp. C624-C634 ◽

Cited By ~ 50

Author(s):

Weijing Cai ◽

Massimo Torreggiani ◽

Li Zhu ◽

Xue Chen ◽

John Cijiang He ◽

...

Keyword(s):

Nadph Oxidase ◽

Vascular Disease ◽

Nuclear Translocation ◽

Oxidant Stress ◽

Growth Factor Receptor ◽

Ros Generation ◽

In Vivo Models ◽

Carboxymethyl Lysine

Advanced glycated end-product receptor 1 (AGER1) protects against vascular disease promoted by oxidants, such as advanced glycated end products (AGEs), via inhibition of reactive oxygen species (ROS). However, the specific AGEs, sources, and pathways involved remain undefined. The mechanism of cellular NADPH oxidase (NOX)-dependent ROS generation by defined AGEs, Nε-carboxymethyl-lysine- and methylglyoxal (MG)-modified BSA, was assessed in AGER1 overexpressing (AGER1+ EC) or knockdown (sh-mRNA-AGER1+ EC) human aortic endothelial (EC) and ECV304 cells, and aortic segments from old (18 mo) C57BL6-F2 mice, propagated on low-AGE diet (LAGE), or LAGE supplemented with MG (LAGE+MG). Wild-type EC and sh-mRNA-AGER1+ EC, but not AGER1+ EC, had high NOX p47 phox and gp91 phox activity, superoxide anions, and NF-κB p65 nuclear translocation in response to MG and Nε-carboxymethyl-lysine. These events involved epidermal growth factor receptor-dependent PKC-δ redox-sensitive Tyr-311 and Tyr-332 phosphorylation and were suppressed in AGER1+ ECs and enhanced in sh-mRNA-AGER1+ ECs. Aortic ROS, PKC-δ Tyr-311, and Tyr-332 phosphorylation, NOX expression, and nuclear p65 in older LAGE+MG mice were significantly increased above that in age-matched LAGE mice, which had higher levels of AGER1. In conclusion, circulating AGEs induce NADPH-dependent ROS generation in vascular aging in both in vitro and in vivo models. Furthermore, AGER1 provides protection against AGE-induced ROS generation via NADPH.

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Plant Extract of Limonium gmelinii Attenuates Oxidative Responses in Neurons, Astrocytes, and Cerebral Endothelial Cells In Vitro and Improves Motor Functions of Rats after Middle Cerebral Artery Occlusion

Antioxidants ◽

10.3390/antiox10111814 ◽

2021 ◽

Vol 10 (11) ◽

pp. 1814

Author(s):

Tulendy Nurkenov ◽

Andrey Tsoy ◽

Farkhad Olzhayev ◽

Elvira Abzhanova ◽

Anel Turgambayeva ◽

...

Keyword(s):

Oxidative Stress ◽

Nadph Oxidase ◽

Middle Cerebral Artery ◽

Artery Occlusion ◽

Ros Generation ◽

Tnf Α ◽

Cerebral Artery Occlusion ◽

Nadph Oxidase Activation

There are numerous publications demonstrating that plant polyphenols can reduce oxidative stress and inflammatory processes in the brain. In the present study we have investigated the neuroprotective effect of plant extract isolated from the roots of L. gmelinii since it contains a rich source of polyphenols and other biologically active compounds. We have applied an oxidative and inflammatory model induced by NMDA, H2O2, and TNF-α in human primary neurons and astrocytes, and mouse cerebral endothelial cell (CECs) line in vitro. The levels of ROS generation, NADPH oxidase activation, P-selectin expression, and activity of ERK1/2 were evaluated by quantitative immunofluorescence analysis, confocal microscopy, and MAPK assay. In vivo, sensorimotor functions in rats with middle cerebral artery occlusion (MCAO) were assessed. In neurons NMDA induced overproduction of ROS, in astrocytes TNF-α initiated ROS generation, NADPH oxidase activation, and phosphorylation of ERK1/2. In CECs, the exposure by TNF-α induced oxidative stress and triggered the accumulation of P-selectin on the surface of the cells. In turn, pre-treatment of the cells with the extract of L. gmelinii suppressed oxidative stress in all cell types and pro-inflammatory responses in astrocytes and CECs. In vivo, the treatment with L. gmelinii extract improved motor activity in rats with MCAO.

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Increased Inflammatory Properties of Neutrophils in Patients with Venous Thromboembolism and the Effect of Simvastatin on Abrogating Inflamed Neutrophils Adhesiveness

Blood ◽

10.1182/blood.v128.22.3681.3681 ◽

2016 ◽

Vol 128 (22) ◽

pp. 3681-3681

Author(s):

Kiara Cristina Senger Zapponi ◽

Fernanda Andrade Orsi ◽

Ingrid Rafaela de Brito ◽

Anna Virginia Calazans Romano ◽

José Luiz Rosenberis Cunha Junior ◽

...

Keyword(s):

Venous Thromboembolism ◽

Conflicts Of Interest ◽

Serum Levels ◽

Neutrophil Activation ◽

Ros Generation ◽

Inflammatory Condition ◽

Chronic Inflammatory Condition ◽

Cell Death Detection Elisaplus ◽

Hs Crp ◽

Adhesive Molecules

Abstract Neutrophils have a complex migrating process out of the vascular lumen, that consists of chemoattraction and rolling, followed by firm attachment and migration to extravascular tissues. In these sites, neutrophils are capable of promoting phagocytosis, secreting proteases, generating reactive oxygen species (ROS), and probably releasing neutrophil extracellular traps (NETs). Furthermore, neutrophil activation also induces major tissue injury associated with acute and chronic inflammatory disorders, such as the venous thromboembolism (VTE). Recently, animal models and clinical studies in acute VTE have explored the participation of neutrophils in the pathophysiology of VTE. However, VTE has been associated with a chronic inflammatory condition, and it remains unclear whether the activation of neutrophils is persistent after the acute phase of the disease. Furthermore, there are clinical evidences supporting that simvastatin may prevent VTE, since the drug has pleiotropic anti-inflammatory effects. The aim of this study is to evaluate the occurrence of neutrophil activation in patients with VTE compared to health controls and determines the effect of simvastatin in these adhesive properties of the neutrophils. Neutrophils were separated from blood collected over Ficoll-Paque densities. Neutrophils activation was determined by the expression of activated adhesive molecules (LFA-1/CD11a and MAC-1/CD11b) and ROS generation, detected by flow cytometry. Chemotaxis assays (chemoTx, Neuro Probe, Inc) and serum nucleosome, a marker of NETs, were quantified by optical density (OD) (Cell Death Detection ELISAPLUS Kit, Roche). Serum high sensitive CRP (hs-CRP) levels were measured in BN ProSpec System (Siemens) by nephelometry. For CD11a and CD11b integrins expression, cells were evaluated under basal conditions and after TNFα inflammatory stimulus, pretreated, or not, with simvastatin. For the migration assays, neutrophils were treated, or not, with IL-8, a neutrophil chemotactic factor. The results were displayed as mean and standard deviation (±SD). The study group consisted of thirty-seven patients with personal history of VTE, the median time since VTE occurred was 25 months (range 13 - 42 months), the event was spontaneous in 51.35% of the cases and 23 patients presented proximal VTE. Thirty-seven controls, matched with patients according to age, gender and ethnicity, were also included. The mean fluorescence intensity (MFI) of CD11a was higher in VTE patient neutrophils, both in basal conditions (30.84 ± 6.82 vs. 38.72 ± 22.75, P= 0.04) and after TNFα stimulus (34.09 ± 9.64 vs. 45.65 ± 33.06, P= 0.01). Higher MFI of CD11b was observed in patient neutrophils, compared with controls, only after TNFα stimulus (149.10 ± 52.74 vs. 200.0 ± 100.5, P= 0.02), and the stimulus was reverted by pre-treatment with simvastatin (200.8 ± 100.50 vs. 174.60 ± 80.63, P= 0.001). The amount of ROS (MFI) was similar in patients and controls (908.30 ± 423.7 vs. 844.0 ± 312.0, P= 0.83). Neutrophils from VTE patients also presented increased basal chemotaxis (17.55% ± 9.79 vs. 12.64% ± 4.78, P=0.02) and IL-8-stimulated chemotaxis (63.48% ± 29.73 vs. 49.88% ± 19.48%, P=0.06). Serum levels of nucleosomes were similar in patients and controls (1.05 ± 0.81 vs. 0.88 ± 0.62, P= 0.64), however higher levels of circulating nucleosomes were observed in patients with severe post-thrombotic syndrome (PTS), compared to patients with non-severe PTS, without PTS and controls (1.49 ± 0.81 vs. 1.06 ± 0.64 vs. 0.64 ± 0.60 vs. 0.88 ± 0.62, P=0.04). Furthermore, serum levels of hs-CRP were significantly higher in VTE patients when compared with controls (0.59 mg/dl ± 0.58 vs. 0.17 mg/dl ± 0.12, P=0.00). We demonstrated that patients with VTE presented patterns of neutrophil activation long time after the acute thrombotic episode. In particular, the stimuli for neutrophil adhesion and chemotaxis were higher in patients, as detected by the increased activation of adhesive molecules and cell migration. Furthermore, we observed that simvastatin may abrogate the expression of CD11b in inflamed neutrophils. Neutrophil activities associated with ROS generation and the releases of nucleosomes were not increased in these patients. The results may support the hypothesis that increased neutrophils activation is part of the chronic inflammatory condition associated with VTE and may be downregulated by the effects of simvastatin. Disclosures No relevant conflicts of interest to declare.

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The Antioxidant from Ethanolic Extract of Rosa cymosa Fruits Activates Phosphatase and Tensin Homolog In Vitro and In Vivo: A New Insight on Its Antileukemic Effect

International Journal of Molecular Sciences ◽

10.3390/ijms20081935 ◽

2019 ◽

Vol 20 (8) ◽

pp. 1935 ◽

Cited By ~ 1

Author(s):

Kuan-Chih Wang ◽

Yi-Chang Liu ◽

Mohamed El-Shazly ◽

Shou-Ping Shih ◽

Ying-Chi Du ◽

...

Keyword(s):

Calcium Release ◽

Herbal Remedy ◽

Lymphoblastic Leukemia ◽

Ethanolic Extract ◽

Control Group ◽

Ros Generation ◽

Phosphatase And Tensin Homolog ◽

Tensin Homolog

Rosa cymosa Tratt is a Chinese herbal remedy that is used in the treatment of diarrhea, burns, rheumatoid arthritis, and hemorrhage. Despite its use in Asian folk medicine, there are limited reports on the biological activity of R. cymosa fruits. This study focused on the investigation of the antitumor effect of the antioxidative ethanolic extract of R. cymosa fruits (RCE) along with its underlying mechanism of action. RCE showed a potent cytotoxic effect against Sup-T1 and Molt-4 lymphoblastic leukemia cells. In the xenograft animal model, the tumor size was significantly reduced to about 59.42% in the RCE-treated group in comparison with the control group. The use of RCE (37.5, 75, or 150 μg/mL) triggered apoptosis by 26.52–83.49%, disrupted mitochondrial membrane potential (MMP) by 10.44–58.60%, and promoted calcium release by 1.29-, 1.44-, and 1.71-fold compared with the control group. The extract induced redox oxygen species (ROS) generation through the elimination of Nrf2/Keap1/P62-mediated oxidative stress response. The loss of phosphatase and tensin homolog (PTEN) activation by RCE impaired PI3K/Akt/Foxo and Jak/Stat activation pathways, which contributed to tumorigenesis. These multiple targets of R. cymosa against hematologic cancer cells suggested its potential application as an antileukemic dietary supplement.

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Shear and Integrin Outside-In Signaling Activates NADPH-Oxidase 2 to Promote Platelet Activation

Arteriosclerosis Thrombosis and Vascular Biology ◽

10.1161/atvbaha.120.315773 ◽

2021 ◽

Author(s):

Zheng Xu ◽

Ying Liang ◽

M. Keegan Delaney ◽

Yaping Zhang ◽

Kyungho Kim ◽

...

Keyword(s):

Nadph Oxidase ◽

Platelet Activation ◽

Nicotinamide Adenine Dinucleotide Phosphate ◽

Regulatory Subunit ◽

Ros Generation ◽

Platelet Response ◽

Nadph Oxidase 2 ◽

Phosphoinositide 3 Kinase ◽

Platelet Spreading

Objective: Despite the importance of reactive oxygen species (ROS) and NOX (nicotinamide adenine dinucleotide phosphate [NADPH] oxidase) 2 in platelet activation and in vivo thrombosis, it is unclear how ROS and NOX2 play a role in platelet activation and why NOX2 deficiencies in humans and mice do not affect hemostasis. Outside-in signaling of integrin α IIb β 3 mediates platelet response to shear stress, secondary platelet activation, and thrombus expansion and is critical to thrombosis but dispensable for hemostasis. We studied the mechanisms of platelet ROS generation, ROS-mediated platelet response, and the role of ROS in integrin α IIb β 3 outside-in signaling. Approach and Results: ROS generation in activated platelets was low and slow without shear but was robust under shear. Shear-enhanced ROS generation and activation of p47phox, an important regulatory subunit of NOX2, were diminished by the integrin antagonist integrilin or β 3 knockout, and by Gα 13 knockout or blocking the Gα 13 -β 3 interaction. Resting platelets spreading on integrin ligand fibrinogen also Gα 13 -dependently stimulated ROS generation and p47phox activation. Hence, Gα 13 -mediated outside-in signaling induces NOX2 activation and ROS generation which is greatly enhanced by shear. Outside-in NOX2 activation requires Src, phosphoinositide 3-kinase and Akt downstream of Gα 13 . Importantly, NOX2-knockout platelets showed defective ROS generation, reduced platelet spreading without shear, and reduced platelet adhesion and thrombus volume on collagen and VWF (von Willibrand factor) under shear, whereas ROS inhibition diminished activation of tyrosine kinase Syk. Conclusions: Outside-in signaling activates the mainly NOX2-mediated ROS generation, which mediates Syk-dependent secondary platelet activation, adhesion, and thrombosis with minimal effect on hemostasis.

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Neuroprotective Ability of Apocynin Loaded Nanoparticles (APO-NPs) as NADPH Oxidase (NOX)-Mediated ROS Modulator for Hydrogen Peroxide-Induced Oxidative Neuronal Injuries

Molecules ◽

10.3390/molecules26165011 ◽

2021 ◽

Vol 26 (16) ◽

pp. 5011

Author(s):

Manisha Singh ◽

Shriya Agarwal ◽

Raj Kumar Tiwari ◽

Silpi Chanda ◽

Kuldeep Singh ◽

...

Keyword(s):

Nadph Oxidase ◽

Therapeutic Efficacy ◽

Polymeric Nanoparticles ◽

In Vitro Release ◽

Ros Generation ◽

Linear Diffusion ◽

Zero Order Kinetics ◽

Diffusion Pattern ◽

The Stability

Apocynin (APO) is a known multi-enzymatic complexed compound, employed as a viable NADPH oxidase (NOX) inhibitor, extensively used in both traditional and modern-day therapeutic strategies to combat neuronal disorders. However, its therapeutic efficacy is limited by lower solubility and lesser bioavailability; thus, a suitable nanocarrier system to overcome such limitations is needed. The present study is designed to fabricate APO-loaded polymeric nanoparticles (APO-NPs) to enhance its therapeutic efficacy and sustainability in the biological system. The optimized APO NPs in the study exhibited 103.6 ± 6.8 nm and −13.7 ± 0.43 mV of particle size and zeta potential, respectively, along with further confirmation by TEM. In addition, the antioxidant (AO) abilities quantified by DPPH and nitric oxide scavenging assays exhibited comparatively higher AO potential of APO-NPs than APO alone. An in-vitro release profile displayed a linear diffusion pattern of zero order kinetics for APO from the NPs, followed by its cytotoxicity evaluation on the PC12 cell line, which revealed minimal toxicity with higher cell viability, even after treatment with a stress inducer (H2O2). The stability of APO-NPs after six months showed minimal AO decline in comparison to APO only, indicating that the designed nano-formulation enhanced therapeutic efficacy for modulating NOX-mediated ROS generation.

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