scholarly journals A Quantitative Method to Measure Low Levels of ROS in Nonphagocytic Cells by Using a Chemiluminescent Imaging System

2019 ◽  
Vol 2019 ◽  
pp. 1-11 ◽  
Author(s):  
Jun-Sub Kim ◽  
Kyuho Jeong ◽  
James M. Murphy ◽  
Yelitza A. R. Rodriguez ◽  
Ssang-Taek Steve Lim
Keyword(s):  
Real Time ◽  
Intracellular Signaling ◽  
Imaging System ◽  
Low Cost ◽  
Fiber Formation ◽  
Imaging Method ◽  
Actin Stress Fiber ◽  
Dependent Manner ◽  
Protein Tyrosine ◽  
Low Levels

Chemiluminescence (CL) is one of the most useful methods for detecting reactive oxygen species (ROS). Although fluorescence dyes or genetically encoded biosensors have been developed, CL is still used due to its high sensitivity, ease of use, and low cost. While initially established and used to measure high levels of ROS in phagocytic cells, CL assays are not ideal for measuring low levels of ROS. Here, we developed a newly modified CL assay using a chemiluminescent imaging system for measuring low concentrations of ROS in nonphagocytic cells. We found that dissolving luminol in NaOH, rather than DMSO, increased the H2O2-induced CL signal and that the addition of 4-iodophenylboronic acid (4IPBA) further increased CL intensity. Our new system also increased the rate and intensity of the CL signal in phorbol 12-myristate 13-acetate- (PMA-) treated HT-29 colon cancer cells compared to those in luminol only. We were able to quantify ROS levels from both cells and media in parallel using an H2O2standard. A significant benefit to our system is that we can easily measure stimulus-induced ROS formation in a real-time manner and also investigate intracellular signaling pathways from a single sample simultaneously. We found that PMA induced tyrosine phosphorylation of protein tyrosine kinases (PTKs), such as focal adhesion kinase (FAK), protein tyrosine kinase 2 (Pyk2), and Src, and increased actin stress fiber formation in a ROS-dependent manner. Interestingly, treatment with either N-acetyl-L-cysteine (NAC) or diphenyleneiodonium (DPI) reduced the PMA-stimulated phosphorylation of these PTKs, implicating a potential role in cellular ROS signaling. Thus, our newly optimized CL assay using 4IPBA and a chemiluminescent imaging method provides a simple, real-time, and low-cost method for the quantification of low levels of ROS.

RhoA Effector mDia1 Is Required for PI 3-Kinase-Dependent Actin Remodeling and Spreading by Thrombin in Platelets.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 5077-5077
Author(s):  
Xiequn Chen ◽  
Guangxun Gao ◽  
Jishi Wang
Keyword(s):  
Kinase Inhibitors ◽  
Conflicts Of Interest ◽  
Fiber Formation ◽  
Actin Stress Fiber ◽  
Dependent Manner ◽  
Critical Pathway ◽  
Actin Remodeling ◽  
Cytoskeletal Remodeling ◽  
Phosphoinositide 3 Kinase ◽  
Platelet Spreading

Abstract Abstract 5077 The RhoA effector mDia1 is involved in controlling the balance between filamentous and monomeric actin, but its role in modulating thrombin-induced actin remodeling and platelet spreading on fibrinogen matrices remains unclear. In this study, mDia1 was shown to translocate to the platelet cytoskeleton following thrombin stimulation, in a phosphoinositide 3-kinase (PI 3-kinase)-dependent manner. AntimDia1 loading or pretreatment with PI 3-kinase inhibitors essentially abrogated thrombin-elicited actin stress fiber formation, with a corresponding decrease in the proportion of platelets exhibiting a fully spread morphology. We also investigated the mechanisms underlying the effects of mDia1 on thrombin-induced actin remodeling and platelet spreading, and found that these involved PI 3-kinase-mediated induction of mDia1 interaction with RhoA. Collectively, these results suggest that the PI 3-kinase/RhoA/mDia1 axis is a critical pathway for coupling thrombin signaling to actin cytoskeletal remodeling during platelet spreading. Disclosures No relevant conflicts of interest to declare.

Abstract 368: Novel Role of Cofilin in Retinal Neovascularization

2016 ◽  
Vol 36 (suppl_1) ◽  
Author(s):  
Raj Kumar ◽  
Jagadish Janjanam ◽  
Nikhlesh K Singh ◽  
Gadiparthi N Rao
Keyword(s):  
Cell Proliferation ◽  
Endothelial Cell ◽  
Cell Formation ◽  
Fiber Formation ◽  
Endothelial Cell Proliferation ◽  
Actin Stress Fiber ◽  
Dependent Manner ◽  
Tip Cell ◽  
Cofilin Phosphorylation ◽  
Retinal Endothelial Cell

Pak1 plays an important role in several cellular processes including cell migration, but its role in pathological angiogenesis is not known. Here we have determined its role in pathological retinal angiogenesis using Oxygen Induced Retinopathy (OIR) model. VEGFA induced Pak1 and its effector cofilin phosphorylation in time-dependent as well as p38β-dependent manner in HRMVECs. Depletion of the levels of any of these molecules inhibited VEGFA-induced HRMVECs F-actin stress fiber formation, migration, proliferation, sprouting, and tube formation. In accordance with these observations, hypoxia induced Pak1 and Cofilin phosphorylation with p38β being downstream to Pak1 and upstream to cofilin. Furthermore, Pak1 deficiency abolished hypoxia-induced p38β and cofilin phosphorylation and abrogated retinal endothelial cell proliferation, tip cell formation and neovascularization. In addition, siRNA-mediated downregulation of p38β or cofilin levels in WT mouse retina also diminished endothelial cell proliferation, tip cell formation and neovascularization. Together, these observations suggest that, while p38β-Pak1-cofilin axis is required for HRMVECs migration, proliferation, sprouting and tubulogenesis, Pak1-p38β-cofilin signaling is essential for hypoxia-induced retinal endothelial cell proliferation, tip cell formation and neovascularization.

scholarly journals Malaysian Tualang Honey Inhibits Hydrogen Peroxide-Induced Endothelial Hyperpermeability

2019 ◽  
Vol 2019 ◽  
pp. 1-10 ◽  
Author(s):  
Kogilavanee Devasvaran ◽  
Jun Jie Tan ◽  
Chin Theng Ng ◽  
Lai Yen Fong ◽  
Yoke Keong Yong
Keyword(s):  
Hydrogen Peroxide ◽  
Calcium Release ◽  
Therapeutic Potential ◽  
Umbilical Vein ◽  
Fiber Formation ◽  
Actin Stress Fiber ◽  
Dependent Manner ◽  
Cortical Actin ◽  
Junction Protein ◽  
Tualang Honey

Malaysian Tualang honey (TH) is a known therapeutic honey extracted from the honeycombs of the Tualang tree (Koompassia excelsa) and has been reported for its antioxidant, anti-inflammatory, antiproliferative, and wound healing properties. However, the possible vascular protective effect of TH against oxidative stress remains unclear. In this study, the effects of TH on hydrogen peroxide- (H2O2-) elicited vascular hyperpermeability in human umbilical vein endothelial cells (HUVECs) and Balb/c mice were evaluated. Our data showed that TH concentrations ranging from 0.01% to 1.00% showed no cytotoxic effect to HUVECs. Induction with 0.5 mM H2O2 was found to increase HUVEC permeability, but the effect was significantly reversed attenuated by TH (p<0.05), of which the permeability with the highest inhibition peaked at 0.1%. In Balb/c mice, TH (0.5 g/kg-1.5 g/kg) significantly (p<0.05) reduced H2O2 (0.3%)-induced albumin-bound Evans blue leak, in a dose-dependent manner. Immunofluorescence staining confirmed that TH reduced actin stress fiber formation while increasing cortical actin formation and colocalization of caveolin-1 and β-catenin in HUVECs. Signaling studies showed that HUVECs pretreated with TH significantly (p<0.05) decreased intracellular calcium release, while sustaining the level of cAMP when challenged with H2O2. These results suggested that TH could inhibit H2O2-induced vascular hyperpermeability in vitro and in vivo by suppression of adherence junction protein redistribution via calcium and cAMP, which could have a therapeutic potential for diseases related to the increase of both oxidant and vascular permeability.

scholarly journals Review of Low-Cost Photoacoustic Sensing and Imaging Based on Laser Diode and Light-Emitting Diode

Sensors ◽  
2018 ◽  
Vol 18 (7) ◽  
pp. 2264 ◽  
Author(s):  
Hongtao Zhong ◽  
Tingyang Duan ◽  
Hengrong Lan ◽  
Meng Zhou ◽  
Fei Gao
Keyword(s):  
Laser Diode ◽  
Imaging System ◽  
Light Emitting Diode ◽  
Low Cost ◽  
Coded Modulation ◽  
Imaging Systems ◽  
Laser Source ◽  
Imaging Method ◽  
Light Emitting ◽  
Laser Sources

Photoacoustic tomography (PAT), a promising medical imaging method that combines optical and ultrasound techniques, has been developing for decades mostly in preclinical application. A recent trend is to utilize the economical laser source to develop a low-cost sensing and imaging system, which aims at an affordable solution in clinical application. These low-cost laser sources have different modulation modes such as pulsed modulation, continuous modulation and coded modulation to generate different profiles of PA signals in photoacoustic (PA) imaging. In this paper, we review the recent development of the photoacoustic sensing and imaging based on the economical laser sources such as laser diode (LD) and light-emitting diode (LED) in different kinds of modulation types, and discuss several representative methods to improve the performance of such imaging systems based on low-cost laser sources. Finally, some perspectives regarding the future development of portable PAT systems are discussed, followed by the conclusion.

scholarly journals Competitive Real-Time Near Infrared (NIR) Vein Finder Imaging Device to Improve Peripheral Subcutaneous Vein Selection in Venipuncture for Clinical Laboratory Testing

Micromachines ◽  
2021 ◽  
Vol 12 (4) ◽  
pp. 373
Author(s):  
Mark D. Francisco ◽  
Wen-Fan Chen ◽  
Cheng-Tang Pan ◽  
Ming-Cheng Lin ◽  
Zhi-Hong Wen ◽  
...  
Keyword(s):  
Real Time ◽  
Near Infrared ◽  
Imaging System ◽  
Low Cost ◽  
Oxide Semiconductor ◽  
Assessment Process ◽  
Source Image ◽  
Imaging Device ◽  
Dorsal Hand ◽  
Vein Imaging

In this study, near-infrared (NIR) technology was utilized to develop a low-cost real-time near infrared (NIR) guiding device for cannulation. A portable device that can be used by medical practitioners and also by students for their skills development training in performing cannulation. Methods. First, is the development of a reflectance type optical vein finder using three (3) light emitting diode (LED) lights with 960 nm wavelength, complementary metal-oxide-semiconductor-infrared (CMOS-IR) sensor camera with 1920 × 1080 UXGA (1080P), IR filter set for the given wavelength, and an open-source image processing software. Second, is the actual in-vitro human testing in two sites: the arm and dorsal hand of 242 subjects. The following parameters were included, such as gender, age, mass index (BMI), and skin tone. In order to maximize the assessment process towards the device, the researchers included the arm circumference. This augmented subcutaneous vein imaging study using the develop vein finder device compared the difference in the captured vein images through visual and digital imaging approaches. The human testing was performed in accordance with the ethical standards of the Trinity University of Asia—Institutional Ethics Review Committee (TUA—IERC). Results. The NIR imaging system of the developed vein finder in this study showed its capability as an efficient guiding device through real-time vein pattern recognition, for both sites. Improved captured vein images were observed, having 100% visibility of vein patterns on the dorsal hand site. Fourteen (5.79%) out of 242 subjects reported non-visible peripheral subcutaneous veins in the arm sites. Conclusions. The developed vein finder device with the NIR technology and reflected light principle with low-energy consumption was efficient for real-time peripheral subcutaneous vein imaging without the application of a tourniquet. This might be utilized as a guiding device in locating the vein for the purpose of cannulation, at a very low cost as compared to the commercially available vein finders. Moreover, it may be used as an instructional device for student training in performing cannulation.

scholarly journals Essential Role of Rho-Associated Kinase in ABO Immune Complex-Mediated Endothelial Barrier Disruption

Biomedicines ◽  
2021 ◽  
Vol 9 (12) ◽  
pp. 1851
Author(s):  
Hannah L. McRae ◽  
Michelle Warren Millar ◽  
Spencer A. Slavin ◽  
Neil Blumberg ◽  
Arshad Rahman ◽  
...  
Keyword(s):  
Cell Surface ◽  
Stress Fiber ◽  
Fiber Formation ◽  
Normal Donor ◽  
Actin Stress Fiber ◽  
Dependent Manner ◽  
Barrier Permeability ◽  
Phalloidin Staining ◽  
Barrier Disruption ◽  
Stress Fiber Formation

ABO immune complexes (ABO-IC) formed by ABO-incompatible antigen-antibody interaction are associated with hemolysis and platelet destruction in patients transfused with ABO-nonidentical blood products. However, the effects of ABO-IC on endothelial cells (EC) are unclear. ABO-IC were formed in vitro from normal donor-derived plasma and serum. Human pulmonary artery EC (HPAEC) were cultured and treated with media, ABO-identical and –non-identical plasma, and ABO-IC. EC barrier integrity was evaluated using transendothelial electrical resistance (TEER), scanning electron microscopy (SEM), vascular endothelial (VE)-cadherin and phalloidin staining, and Rho-associated Kinase (ROCK) inhibitor treatment. TEER revealed significant/irreversible barrier disruption within 1–2 h of exposure to ABO non-identical plasma and ABO-IC; this occurred independently of EC ABO type. Treatment with ABO-IC resulted in decreased VE-cadherin staining and increased phalloidin staining in a time-dependent manner, suggesting that the resultant increased EC barrier permeability is secondary to actin stress fiber formation and loss of cell surface VE-cadherin. Inhibition of ROCK was effective in protecting against IC-induced barrier disruption even two hours after ABO-IC exposure. ABO-IC causes increased EC barrier permeability by decreasing cell surface VE-cadherin and promoting stress fiber formation, which is preventable by inhibiting ROCK activation to protect against EC contraction and gap formation.

scholarly journals Disruption of the actin cytoskeleton regulates cytokine-induced iNOS expression

2001 ◽  
Vol 281 (3) ◽  
pp. C932-C940 ◽  
Author(s):  
Chenbo Zeng ◽  
Aubrey R. Morrison
Keyword(s):  
Nitric Oxide ◽  
Serum Response Factor ◽  
Mesangial Cells ◽  
Inos Expression ◽  
Fiber Formation ◽  
Dominant Negative ◽  
Dominant Negative Mutant ◽  
Actin Stress Fiber ◽  
Dependent Manner ◽  
Glomerular Mesangial Cells

Interleukin-1β (IL-1β) induces the inducible nitric oxide synthase (iNOS), resulting in the release of nitric oxide (NO) from glomerular mesangial cells. In this study, we demonstrated that disruption of F-actin formation by sequestration of G-actin with the toxin latrunculin B (LatB) dramatically potentiated IL-1β-induced iNOS protein expression in a dose-dependent manner. LatB by itself had little or no effect on iNOS expression. Staining of F-actin with nitrobenzoxadiazole (NBD)-phallacidin demonstrated that LatB significantly impaired F-actin stress fiber formation. Jasplakinolide (Jasp), which binds to and stabilizes F-actin, suppressed iNOS expression enhanced by LatB. These data strongly suggest that actin cytoskeletal dynamics regulates IL-1β-induced iNOS expression. We demonstrated that LatB decreases serum response factor (SRF) activity as determined by reporter gene assays, whereas Jasp increases SRF activity. The negative correlation between SRF activity and iNOS expression suggests a negative regulatory role for SRF in iNOS expression. Overexpression of a dominant negative mutant of SRF increases the IL-1β-induced iNOS expression, providing direct evidence that SRF inhibits iNOS expression.

Thrombin enhances the barrier function of rat microvascular endothelium in a PAR-1-dependent manner

2008 ◽  
Vol 294 (2) ◽  
pp. L266-L275 ◽  
Author(s):  
B. Troyanovsky ◽  
D. F. Alvarez ◽  
J. A. King ◽  
K. L. Schaphorst
Keyword(s):  
Endothelial Cells ◽  
Pulmonary Artery ◽  
Barrier Function ◽  
Fiber Formation ◽  
Stress Fibers ◽  
Actin Stress Fiber ◽  
Dependent Manner ◽  
Gap Formation ◽  
Vascular Effects ◽  
Microvascular Endothelium

Thrombin is a multifunctional coagulation protease with pro- and anti-inflammatory vascular effects. We questioned whether thrombin may have segmentally differentiated effects on pulmonary endothelium. In cultured rat endothelial cells, rat thrombin (10 U/ml) recapitulated the previously reported decrease in transmonolayer electrical resistance (TER), F-actin stress fiber formation, paracellular gap formation, and increased permeability. In contrast, in rat pulmonary microvascular endothelial cells (PMVEC), isolated on the basis of Griffonia simplicifolia lectin recognition, thrombin increased TER, induced fewer stress fibers, and decreased permeability. To assess for differential proteinase-activated receptor (PAR) expression as a basis for the different responses, PAR family expression was analyzed. Both pulmonary artery endothelial cells and PMVEC expressed PAR-1 and PAR-2; however, only PMVEC expressed PAR-3, as shown by both RT-PCR and Western analysis. PAR-1 activating peptides (PAR-APs: SFLLRN-NH2and TFLLRN-NH2) were used to confirm a role for the PAR-1 receptor. PAR-APs (25–250 μM) also increased TER, formed fewer stress fibers, and did not induce paracellular gaps in PMVEC in contrast to that shown in pulmonary artery endothelial cells. These results were confirmed in isolated perfused rat lung preparations. PAR-APs (100 μg/ml) induced a 60% increase in the filtration coefficient over baseline. However, by transmission electron microscopy, perivascular fluid cuffs were seen only along conduit veins and arteries without evidence of intra-alveolar edema. We conclude that thrombin exerts a segmentally differentiated effect on endothelial barrier function in vitro, which corresponds to a pattern of predominant perivascular fluid cuff formation in situ. This may indicate a distinct role for thrombin in the microcirculation.

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