Search

With the increasing use of vaping devices that deliver high levels of nicotine (NIC) to the lungs, sporadic lung injury has been observed. Commercial vaping solutions can contain high NIC concentrations of 150 mM or more. With high NIC levels, its metabolic products may induce toxicity. NIC is primarily metabolized to form NIC iminium (NICI) which is further metabolized by aldehyde oxidase (AOX) to cotinine. We determine that NICI in the presence of AOX is a potent trigger of superoxide generation. NICI stimulated superoxide generation from AOX with K m  = 2.7 μM and V max  = 794 nmol/min/mg measured by cytochrome-c reduction. EPR spin-trapping confirmed that NICI in the presence of AOX is a potent source of superoxide. AOX is expressed in the lungs and chronic e-cigarette exposure in mice greatly increased AOX expression. NICI or NIC stimulated superoxide production in the lungs of control mice with an even greater increase after chronic e-cigarette exposure. This superoxide production was quenched by AOX inhibition. Furthermore, e-cigarette-mediated NIC delivery triggered oxidative lung damage that was blocked by AOX inhibition. Thus, NIC metabolism triggers AOX-mediated superoxide generation that can cause lung injury. Therefore, high uncontrolled levels of NIC inhalation, as occur with e-cigarette use, can induce oxidative lung damage., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

While chronic smoking triggers cardiovascular disease, controversy remains regarding its effects on the brain and cognition. We investigated the effects of long-term cigarette smoke (CS) exposure (CSE) on cerebrovascular function, neuronal injury, and cognition in a novel mouse exposure model. Longitudinal studies were performed in CS or air-exposed mice, 2 hours/day, for up to 60 weeks. Hypertension and carotid vascular endothelial dysfunction (VED) occurred by 16 weeks of CSE, followed by reduced carotid artery blood flow, with oxidative stress detected in the carotid artery, and subsequently in the brain of CS-exposed mice with generation of reactive oxygen species (ROS) and secondary protein and DNA oxidation, microglial activation and astrocytosis. Brain small vessels exhibited decreased levels of endothelial NO synthase (eNOS), enlarged perivascular spaces with blood brain barrier (BBB) leak and decreased levels of tight-junction proteins. In the brain, amyloid-β deposition and phosphorylated-tau were detected with increases out to 60 weeks, at which time mice exhibited impaired spatial learning and memory. Thus, long-term CSE initiates a cascade of ROS generation and oxidative damage, eNOS dysfunction with cerebral hypoperfusion, as well as cerebrovascular and BBB damage with intracerebral inflammation, and neuronal degeneration, followed by the onset of impaired cognition and memory., Competing Interests: Declaration of conflicting interestsThe author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Background: We investigated the role of A2B-adenosine receptor in regulating immunosuppressive metabolic stress in the tumor microenvironment. Novel A2B-adenosine receptor antagonist PBF-1129 was tested for antitumor activity in mice and evaluated for safety and immunologic efficacy in a phase I clinical trial of patients with non-small cell lung cancer., Methods: The antitumor efficacy of A2B-adenosine receptor antagonists and their impact on the metabolic and immune tumor microenvironment were evaluated in lung, melanoma, colon, breast, and epidermal growth factor receptor-inducible transgenic cancer models. Employing electron paramagnetic resonance, we assessed changes in tumor microenvironment metabolic parameters, including pO2, pH, and inorganic phosphate, during tumor growth and evaluated the immunologic effects of PBF-1129, including its pharmacokinetics, safety, and toxicity, in patients with non-small cell lung cancer., Results: Levels of metabolic stress correlated with tumor growth, metastasis, and immunosuppression. Tumor interstitial inorganic phosphate emerged as a correlative and cumulative measure of tumor microenvironment stress and immunosuppression. A2B-adenosine receptor inhibition alleviated metabolic stress, downregulated expression of adenosine-generating ectonucleotidases, increased expression of adenosine deaminase, decreased tumor growth and metastasis, increased interferon γ production, and enhanced the efficacy of antitumor therapies following combination regimens in animal models (anti-programmed cell death 1 protein vs anti-programmed cell death 1 protein plus PBF-1129 treatment hazard ratio = 11.74 [95% confidence interval = 3.35 to 41.13], n = 10, P < .001, 2-sided F test). In patients with non-small cell lung cancer, PBF-1129 was well tolerated, with no dose-limiting toxicities; demonstrated pharmacologic efficacy; modulated the adenosine generation system; and improved antitumor immunity., Conclusions: Data identify A2B-adenosine receptor as a valuable therapeutic target to modify metabolic and immune tumor microenvironment to reduce immunosuppression, enhance the efficacy of immunotherapies, and support clinical application of PBF-1129 in combination therapies., (© The Author(s) 2023. Published by Oxford University Press.)

Repair of epithelial defect is complicated by infection and related metabolites. Pyocyanin (PYO) is one such metabolite that is secreted during Pseudomonas aeruginosa infection. Keratinocyte (KC) migration is required for the closure of skin epithelial defects. This work sought to understand PYO-KC interaction and its significance in tissue repair. Stable Isotope Labeling by Amino acids in Cell culture proteomics identified mitochondrial dysfunction as the top pathway responsive to PYO exposure in human KCs. Consistently, functional studies showed mitochondrial stress, depletion of reducing equivalents, and adenosine triphosphate. Strikingly, despite all stated earlier, PYO markedly accelerated KC migration. Investigation of underlying mechanisms revealed, to our knowledge, a previously unreported function of keratin 6A in KCs. Keratin 6A was PYO inducible and accelerated closure of epithelial defect. Acceleration of closure was associated with poor quality healing, including compromised expression of apical junction proteins. This work recognizes keratin 6A for its role in enhancing KC migration under conditions of threat posed by PYO. Qualitatively deficient junctional proteins under conditions of defensive acceleration of KC migration explain why an infected wound close with deficient skin barrier function as previously reported., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Electron paramagnetic resonance (EPR) spectroscopy and imaging coupled with the use of suitable probes is a promising tool for assessment of the tumor microenvironment (TME). Measurement of multiple TME parameters by EPR is very desirable but challenging. Herein, we designed and synthesized a class of negative-charged trityl quinodimethane MTPs as unimolecular triple-function extracellular probes for redox, pH, and oxygen (O 2 ) levels. Using the deuterated analogue, dMTP 5, which has an optimal p K a as well as high sensitivity to bioreduction and O 2 , we reasonably evaluated pH effects on efflux of reducing agents from HepG2 cells and cellular O 2 consumption.

Background: CD38 is a transmembrane glycoprotein that catabolizes nicotinamide adenine dinucleotide (NAD + ) and is the main source for the age-dependent decrease in NAD + levels. Increased CD38 enzymatic activity has been implicated in several neurological diseases. However, its role in the pathogenesis of cerebral small vessel disease (CSVD) remains unknown. We aimed to characterize CD38 expression and enzymatic activity in the brain of spontaneously hypertensive stroke-prone rats (SHRSP), a genetic model for hypertension and human CSVD, in comparison to age-matched normotensive Wistar Kyoto rats (WKY). Materials and Methods: Age-matched male 7- and 24-week-old WKY and SHRSP were studied. CD38 enzymatic activity was determined in the brain homogenate. Immunohistochemistry and Western Blotting (WB) were used to characterize CD38 expression and localize it in the different cell types within the brain. In addition, expression of nitric oxide synthase (NOS) isoforms and the levels of nitric oxide (NO), superoxide, nicotinamide dinucleotide (phosphate) NAD(P)H were measured the brain of in WKY and SHRSP. Results: CD38 expression and enzymatic activity were increased in SHRSP brains compared to age matched WKY starting at 7 weeks of age. CD38 expression was localized to the endothelial cells, astrocytes, and microglia. We also identified increased CD38 expression using WB with age in SHRSP and WKY. CD38 enzymatic activity was also increased in 24-week SHRSP compared to 7-week SHRSP. In association, we identified evidence of oxidative stress, reduced NO level, reduced NAD(P)H level and endothelial NOS expression in SHRSP compared to age matched WKY. NAD(P)H also decreased with age in WKY and SHRSP. Additionally, activation of astrocytes and microglia were present in SHRSP compared to WKY. Conclusions: CD38 is overexpressed, and its enzymatic activity is increased in SHRSP, a genetic model for marked hypertension and human CSVD. Our results suggest a potential role for CD38 enzymatic activation in the pathogenesis of CSVD and points to the need for future mechanistic and pharmacological studies., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Hannawi, Ewees, Moore and Zweier.)

We recently reported a mouse model of chronic electronic cigarette (e-cig) exposure-induced cardiovascular pathology, where long-term exposure to e-cig vape (ECV) induces cardiac abnormalities, impairment of endothelial function, and systemic hypertension. Here, we delineate the underlying mechanisms of ECV-induced vascular endothelial dysfunction (VED), a central trigger of cardiovascular disease. C57/BL6 male mice were exposed to ECV generated from e-cig liquid containing 0, 6, or 24 mg/mL nicotine for 16 and 60 wk. Time-dependent elevation in blood pressure and systemic vascular resistance were observed, along with an impairment of acetylcholine-induced aortic relaxation in ECV-exposed mice, compared with air-exposed control. Decreased intravascular nitric oxide (NO) levels and increased superoxide generation with elevated 3-nitrotyrosine levels in the aorta of ECV-exposed mice were observed, indicating that ECV-induced superoxide reacts with NO to generate cytotoxic peroxynitrite. Exposure increased NADPH oxidase expression, supporting its role in ECV-induced superoxide generation. Downregulation of endothelial nitric oxide synthase (eNOS) expression and Akt-dependent eNOS phosphorylation occurred in the aorta of ECV-exposed mice, indicating that exposure inhibited de novo NO synthesis. Following ECV exposure, the critical NOS cofactor tetrahydrobiopterin was decreased, with a concomitant loss of its salvage enzyme, dihydrofolate reductase. NADPH oxidase and NOS inhibitors abrogated ECV-induced superoxide generation in the aorta of ECV-exposed mice. Together, our data demonstrate that ECV exposure activates NADPH oxidase and uncouples eNOS, causing a vicious cycle of superoxide generation and vascular oxidant stress that triggers VED and hypertension with predisposition to other cardiovascular disease. NEW & NOTEWORTHY Underlying mechanisms of e-cig-induced vascular endothelial dysfunction are delineated. e-cig exposure activates and increases expression of NADPH oxidase and disrupts activation and coupling of eNOS, leading to a vicious cycle of superoxide generation and peroxynitrite formation, with tetrahydrobiopterin depletion, causing loss of NO that triggers vascular endothelial dysfunction. This process is progressive, increasing with the duration of e-cig exposure, and is more severe in the presence of nicotine, but observed even with nicotine-free vaping.

Ultrasound coupled with activated persulfate can synergistically degrade aqueous organic contaminants. Here, in situ electron paramagnetic resonance spin trapping was used to compare radicals produced by ultrasonically activated persulfate (US-PS) and its individual technologies, ultrasound alone (US) and heat-activated persulfate (PS), with respect to temperature. Radicals were trapped using 5,5-dimethyl-1-pyrroline- N -oxide, DMPO, to form detectable nitroxide adducts. Using initial rates of radical adduct formation, and compared to US and PS, US-PS at 40 and 50 °C resulted in the largest synergistic production of radicals. Radicals generated from US were reasonably consistent from 40 to 70 °C, indicating that temperature had little effect on cavitational bubble collapse over this range. However, synergy indexes calculated from initial rates showed that ultrasonic activation of persulfate at the bubble interface changes with temperature. From these results, we speculate that higher temperatures enhance persulfate uptake into cavitation bubbles via nanodroplet injection. DMPO-OH was the predominant adduct detected for all conditions. However, competition modeling and spin trapping in the presence of nitrobenzene and atrazine probes showed that SO 4 •- predominated. Therefore, the DMPO-OH signal is derived from SO 4 •- trapping with subsequent DMPO-SO 4 - hydrolysis to DMPO-OH. Spin trapping is effective in quantifying total radical adduct formation but limited in measuring primary radical speciation in this case.

Cytoglobin (Cygb) has been identified as the major nitric oxide (NO) metabolizing protein in vascular smooth muscle cells (VSMCs) and is crucial for the regulation of vascular tone. In the presence of its requisite cytochrome B5a (B5)/B5 reductase-isoform-3 (B5R) reducing system, Cygb controls NO metabolism through the oxygen-dependent process of NO dioxygenation. Tobacco cigarette smoking (TCS) induces vascular dysfunction; however, the role of Cygb in the pathophysiology of TCS-induced cardiovascular disease has not been previously investigated. While TCS impairs NO biosynthesis, its effect on NO metabolism remains unclear. Therefore, we performed studies in aortic VSMCs with tobacco smoke extract (TSE) exposure to investigate the effects of cigarette smoke constituents on the rates of NO decay, with focus on the alterations that occur in the process of Cygb-mediated NO metabolism. TSE greatly enhanced the rates of NO metabolism by VSMCs. An initial increase in superoxide-mediated NO degradation was seen at 4 h of exposure. This was followed by much larger progressive increases at 24 and 48 h, accompanied by parallel increases in the expression of Cygb and B5/B5R. siRNA-mediated Cygb knockdown greatly decreased these TSE-induced elevations in NO decay rates. Therefore, upregulation of the levels of Cygb and its reducing system accounted for the large increase in NO metabolism rate seen after 24 h of TSE exposure. Thus, increased Cygb-mediated NO degradation would contribute to TCS-induced vascular dysfunction and partial inhibition of Cygb expression or its NO dioxygenase function could be a promising therapeutic target to prevent secondary cardiovascular disease., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Cytoglobin (Cygb) was discovered as a novel type of globin that is expressed in mammals; however, its functions remain uncertain. While Cygb protects against oxidant stress, the basis for this is unclear, and the effect of Cygb on superoxide metabolism is unknown. From dose-dependent studies of the effect of Cygb on superoxide catabolism, we identify that Cygb has potent superoxide dismutase (SOD) function. Initial assays using cytochrome c showed that Cygb exhibits a high rate of superoxide dismutation on the order of 10 8 M -1 ⋅ s -1 Spin-trapping studies also demonstrated that the rate of Cygb-mediated superoxide dismutation (1.6 × 10 8 M -1 ⋅ s -1 ) was only ∼10-fold less than Cu,Zn-SOD. Stopped-flow experiments confirmed that Cygb rapidly dismutates superoxide with rates within an order of magnitude of Cu,Zn-SOD or Mn-SOD. The SOD function of Cygb was inhibited by cyanide and CO that coordinate to Fe 3+ -Cygb and Fe 2+ -Cygb, respectively, suggesting that dismutation involves iron redox cycling, and this was confirmed by spectrophotometric titrations. In control smooth-muscle cells and cells with siRNA-mediated Cygb knockdown subjected to extracellular superoxide stress from xanthine/xanthine oxidase or intracellular superoxide stress triggered by the uncoupler, menadione, Cygb had a prominent role in superoxide metabolism and protected against superoxide-mediated death. Similar experiments in vessels showed higher levels of superoxide in Cygb -/- mice than wild type. Thus, Cygb has potent SOD function and can rapidly dismutate superoxide in cells, conferring protection against oxidant injury. In view of its ubiquitous cellular expression at micromolar concentrations in smooth-muscle and other cells, Cygb can play an important role in cellular superoxide metabolism., Competing Interests: The authors declare no competing interest.

A serious consequence of myocardial ischemia-reperfusion injury (I/R) is oxidative damage, which causes mitochondrial dysfunction. The cascading ROS can propagate and potentially induce heme bleaching and protein cysteine sulfonation (PrSO 3 H) of the mitochondrial electron transport chain. Herein we studied the mechanism of I/R-mediated irreversible oxidative injury of complex III in mitochondria from rat hearts subjected to 30-min of ischemia and 24-h of reperfusion in vivo. In the I/R region, the catalytic activity of complex III was significantly impaired. Spectroscopic analysis indicated that I/R mediated the destruction of hemes b and c + c 1 in the mitochondria, supporting I/R-mediated complex III impairment. However, no significant impairment of complex III activity and heme damage were observed in mitochondria from the risk region of rat hearts subjected only to 30-min ischemia, despite a decreased state 3 respiration. In the I/R mitochondria, carbamidomethylated C 122 /C 125 of cytochrome c 1 via alkylating complex III with a down regulation of HCCS was exclusively detected, supporting I/R-mediated thioether defect of heme c 1 . LC-MS/MS analysis showed that I/R mitochondria had intensely increased complex III PrSO 3 H levels at the C 236 ligand of the [2Fe2S] cluster of the Rieske iron‑sulfur protein (uqcrfs1), thus impairing the electron transport activity. MS analysis also indicated increased PrSO 3 H of the hinge protein at C 65 and of cytochrome c 1 at C 140 and C 220 , which are confined in the intermembrane space. MS analysis also showed that I/R extensively enhanced the PrSO 3 H of the core 1 (uqcrc1) and core 2 (uqcrc2) subunits in the matrix compartment, thus supporting the conclusion that complex III releases ROS to both sides of the inner membrane during reperfusion. Analysis of ischemic mitochondria indicated a modest reduction from the basal level of complex III PrSO 3 H detected in the mitochondria of sham control hearts, suggesting that the physiologic hyperoxygenation and ROS overproduction during reperfusion mediated the enhancement of complex III PrSO 3 H. In conclusion, reperfusion-mediated heme damage with increased PrSO 3 H controls oxidative injury to complex III and aggravates mitochondrial dysfunction in the post-ischemic heart., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Introduction: Spontaneously hypertensive stroke-prone rats (SHRSP) are used to model clinically relevant aspects of human cerebral small vessel disease (CSVD). To decipher and understand the underlying disease dynamics, assessment of the temporal progression of CSVD histopathological and neuroimaging correlates is essential. Materials and Methods: Eighty age-matched male SHRSP and control Wistar Kyoto rats (WKY) were randomly divided into four groups that were aged until 7, 16, 24 and 32 weeks. Sensorimotor testing was performed weekly. Brain MRI was acquired at each study time point followed by histological analyses of the brain. Results: Compared to WKY controls, the SHRSP showed significantly higher prevalence of small subcortical hyperintensities on T2w imaging that progressed in size and frequency with aging. Volumetric analysis revealed smaller intracranial and white matter volumes on brain MRI in SHRSP compared to age-matched WKY. Diffusion tensor imaging (DTI) showed significantly higher mean diffusivity in the corpus callosum and external capsule in WKY compared to SHRSP. The SHRSP displayed signs of motor restlessness compared to WKY represented by hyperactivity in sensorimotor testing at the beginning of the experiment which decreased with age. Distinct pathological hallmarks of CSVD, such as enlarged perivascular spaces, microbleeds/red blood cell extravasation, hemosiderin deposits, and lipohyalinosis/vascular wall thickening progressively accumulated with age in SHRSP. Conclusions: Four stages of CSVD severity in SHRSP are described at the study time points. In addition, we find that quantitative analyses of brain MRI enable identification of in vivo markers of CSVD that can serve as endpoints for interventional testing in therapeutic studies., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Hannawi, Caceres, Ewees, Powell, Bratasz, Schwab, Rink and Zweier.)

Small heat shock proteins (sHsps) protect the heart from chemotherapeutics-induced heart failure by inhibiting p53-dependent apoptosis. However, mechanism of such protection has not been elucidated yet. Here we test a hypothesis that serine phosphorylation of sHsps is essential to inhibit the doxorubicin-induced and p53-dependent apoptotic pathway. Three transgenic mice (TG) lines with cardiomyocyte-specific overexpression of human heat shock protein 27 (hHsp27), namely, wild-type [myosin heavy chain (MHC)-hHsp27], S82A single mutant [MHC-mut-hHsp27( S82A )], and trimutant [MHC-mut-hHsp27( S15A/S78A/S82A )] were generated. TG mice were treated with Dox (6 mg/kg body wt; once in a week; 4 wk) along with age-matched nontransgenic (non-TG) controls. The Dox-treated MHC-hHsp27 mice showed improved survival and cardiac function (both MRI and echocardiography) in terms of contractility [ejection fraction (%EF)] and left ventricular inner diameter (LVID) compared with the Dox-treated non-TG mice. However, both MHC-mut-hHsp27( S82A ) and MHC-mut-hHsp27( S15A/S78A/S82A ) mutants overexpressing TG mice did not show such a cardioprotection. Furthermore, transactivation of p53 was found to be attenuated only in Dox-treated MHC-hHsp27 mice-derived cardiomyocytes in vitro, as low p53 was detected in the nuclei, not in mutant hHsp27 overexpressing cardiomyocytes. Similarly, only in MHC-hHsp27 overexpressing cardiomyocytes, low Bax, higher mechanistic target of rapamycin (mTOR) phosphorylation, and low apoptotic poly(ADP-ribose) polymerase-1 (PARP-1) cleavage (89 kDa fragment) were detected. Pharmacological inhibition of p53 was more effective in mutant TG mice compared with MHC-hHsp27 mice. We conclude that phosphorylation of overexpressed Hsp27 at S82 and its association with p53 are essential for the cardioprotective effect of overexpressed Hsp27 against Dox-induced dilated cardiomyopathy. Only phosphorylated Hsp27 protects the heart by inhibiting p53 transactivation. NEW & NOTEWORTHY Requirement of serine phosphorylation in Hsp27 for cardioprotective effect against Dox is tested in various mutants overexpressing mice. Cardioprotective effect was found to be compromised in Hsp27 serine mutants overexpressed mice compared with wild-type overexpressing mice. These results indicate that cancer patients, who carry these mutations, may have higher risk of aggravated cardiomyopathy on treated with cardiotoxic chemotherapeutics such as doxorubicin.