Xanthine Oxidoreductase Function Contributes to Normal Wound Healing

Chronic, nonhealing wounds result in patient morbidity and disability. Reactive oxygen species (ROS) and nitric oxide (NO) are both required for normal wound repair, and derangements of these result in impaired healing. Xanthine oxidoreductase (XOR) has the unique capacity to produce both ROS and NO. We hypothesize that XOR contributes to normal wound healing. Cutaneous wounds were created in C57Bl6 mice. XOR was inhibited with dietary tungsten or allopurinol. Topical hydrogen peroxide (H₂O₂, 0.15%) or allopurinol (30 μg) was applied to wounds every other day. Wounds were monitored until closure or collected at d 5 to assess XOR expression and activity, cell proliferation and histology. The effects of XOR, nitrite, H₂O₂ and allopurinol on keratinocyte cell (KC) and endothelial cell (EC) behavior were assessed. We identified XOR expression and activity in the skin and wound edges as well as granulation tissue. Cultured human KCs also expressed XOR. Tungsten significantly inhibited XOR activity and impaired healing with reduced ROS production with reduced angiogenesis and KC proliferation. The expression and activity of other tungsten-sensitive enzymes were minimal in the wound tissues. Oral allopurinol did not reduce XOR activity or alter wound healing but topical allopurinol significantly reduced XOR activity and delayed healing. Topical H₂O₂ restored wound healing in tungsten-fed mice. In vitro, nitrite and H₂O₂ both stimulated KC and EC proliferation and EC migration. These studies demonstrate for the first time that XOR is abundant in wounds and participates in normal wound healing through effects on ROS production.     

Xanthine oxidoreductase: a journey from purine metabolism to cardiovascular excitation-contraction coupling

Xanthine oxidoreductase (XOR) is a ubiquitous complex cytosolic molybdoflavoprotein which controls the rate limiting step of purine catabolism by converting xanthine to uric acid. It is known that optimum concentrations of uric acid (UA) and reactive oxygen species (ROS) are necessary for normal functioning of the body. The ability of XOR to perform detoxification reactions, and to synthesize UA and reactive oxygen species (ROS) makes it a versatile intra- and extra-cellular protective "housekeeping enzyme". It is also an important component of the innate immune system. The enzyme is a target of drugs against gout and hyperuricemia and the protein is of major interest as it is associated with ischemia reperfusion (I/R) injury, vascular disorders in diabetes, cardiovascular disorders, adipogenesis, metabolic syndrome, cancer, and many other disease conditions. Xanthine oxidoreductase in conjugation with antibodies has been shown to have an anti-tumor effect due to its ability to produce ROS, which in turn reduces the growth of cancer tissues. Apart from this, XOR in association with nitric oxide synthase also participates in myocardial excitation-contraction coupling. Although XOR was discovered over 100 years ago, its physiological and pathophysiological roles are still not clearly elucidated. In this review, various physiological and pathophysiological functional aspects of XOR and its association with various forms of cancer are discussed in detail.     

Xanthine oxidoreductase: A journey from purine metabolism to cardiovascular excitation-contraction coupling

Xanthine oxidoreductase (XOR) is a ubiquitous complex cytosolic molybdoflavoprotein which controls the rate limiting step of purine catabolism by converting xanthine to uric acid. It is known that optimum concentrations of uric acid (UA) and reactive oxygen species (ROS) are necessary for normal functioning of the body. The ability of XOR to perform detoxification reactions, and to synthesize UA and reactive oxygen species (ROS) makes it a versatile intra- and extra-cellular protective “housekeeping enzyme”. It is also an important component of the innate immune system. The enzyme is a target of drugs against gout and hyperuricemia and the protein is of major interest as it is associated with ischemia reperfusion (I/R) injury, vascular disorders in diabetes, cardiovascular disorders, adipogenesis, metabolic syndrome, cancer, and many other disease conditions. Xanthine oxidoreductase in conjugation with antibodies has been shown to have an anti-tumor effect due to its ability to produce ROS, which in turn reduces the growth of cancer tissues. Apart from this, XOR in association with nitric oxide synthase also participates in myocardial excitation-contraction coupling. Although XOR was discovered over 100 years ago, its physiological and pathophysiological roles are still not clearly elucidated. In this review, various physiological and pathophysiological functional aspects of XOR and its association with various forms of cancer are discussed in detail.     

Mechanical stress activates xanthine oxidoreductase through MAP kinase-dependent pathways

Xanthine oxidoreductase (XOR) plays a prominent role in acute lung injury because of its ability to generate reactive oxygen species. We investigated the role of XOR in ventilator-induced lung injury (VILI). Male C57BL/6J mice were assigned to spontaneous ventilation (sham) or mechanical ventilation (MV) with low (7 ml/kg) and high tidal volume (20 ml/kg) for 2 h after which lung XOR activity and expression were measured and the effect of the specific XOR inhibitor allopurinol on pulmonary vascular leakage was examined. In separate experiments, rat pulmonary microvascular endothelial cells (RPMECs) were exposed to cyclic stretch (5% and 18% elongation, 20 cycles/min) for 2 h before intracellular XOR activity measurement. Lung XOR activity was significantly increased at 2 h of MV without changes in XOR expression. There was evidence of p38 MAP kinase, ERK1/2, and ERK5 phosphorylation, but no change in JNK phosphorylation. Evans blue dye extravasation and bronchoalveolar lavage protein concentration were significantly increased in response to MV, changes that were significantly attenuated by pretreatment with allopurinol. Cyclic stretch of RPMECs also caused MAP kinase phosphorylation and a 1.7-fold increase in XOR activity, which was completely abrogated by pretreatment of the cells with specific MAP kinase inhibitors. We conclude that XOR enzymatic activity is significantly increased by mechanical stress via activation of p38 MAP kinase and ERK and plays a critical role in the pathogenesis of pulmonary edema associated with VILI.     

A combination of ischemic preconditioning and allopurinol protects against ischemic injury through a nitric oxide-dependent mechanism

This study examined the cytoprotective mechanisms of a combination of ischemic preconditioning (IPC) and allopurinol against liver injury caused by ischemia/reperfusion (I/R). Allopurinol (50 mg/kg) was intraperitoneally administered 18 and 1 h before sustained ischemia. A rat liver was preconditioned by 10 min of ischemia, followed by 10 min of reperfusion, and then subjected to 90 min of ischemia, followed by 5 h of reperfusion. Rats were pretreated with adenosine deaminase (ADA), 3,7-dimethyl-1-[2-propargyl]-xanthine (DMPX), and N-nitro-l-arginine methyl ester (l-NAME) before IPC. Hepatic nitrite and nitrate and eNOS protein expression levels were increased by the combination of IPC and allopurinol. This increase was attenuated by ADA, DMPX, and l-NAME. I/R induced an increase in alanine aminotransferase activity, whereas it decreased the hepatic glutathione level. A combination of IPC and allopurinol attenuated these changes, which were abolished by ADA, DMPX, and l-NAME. The increase in the liver wet weight-to-dry weight ratio after I/R was attenuated by the combination of IPC and allopurinol. In contrast, hepatic bile flow was decreased after I/R, which was attenuated by the combination of IPC and allopurinol. These changes were restored by l-NAME. I/R induced a decrease in the level of mitochondrial dehydrogenase, whereas it increased mitochondrial swelling. A combination of IPC and allopurinol attenuated these changes, which were restored by ADA, DMPX, and l-NAME. Our findings suggest that a combination of IPC and allopurinol reduces post-ischemic hepatic injury by enhancing NO generation.     

Current knowledge on oxidative stress in hepatic ischemia/reperfusion

Abstract

Ischemia/reperfusion (I/R) injury associated with hepatic resections and liver transplantation remains a serious complication in clinical practice, despite several attempts to solve the problem. The redox balance, which is pivotal for normal function and integrity of tissues, is dysregulated during I/R, leading to an accumulation of reactive oxygen species (ROS). Formation of ROS and oxidant stress are the disease mechanisms most commonly invoked in hepatic I/R injury. The present review examines published results regarding possible sources of ROS and their effects in the context of I/R injury. We also review the effect of oxidative stress on marginal livers, which are more vulnerable to I/R-induced oxidative stress. Strategies to improve the viability of marginal livers could reduce the risk of dysfunction after surgery and increase the number of organs suitable for transplantation. The review also considers the therapeutic strategies developed in recent years to reduce the oxidative stress induced by hepatic I/R, and we seek to explain why some of them have not been applied clinically. New antioxidant strategies that have yielded promising results for hepatic I/R injury are discussed.     

Circular RNA YAP1 acts as the sponge of microRNA‐21‐5p to secure HK‐2 cells from ischaemia/reperfusion‐induced injury

Circular RNA YAP1 (circYAP1) was reported to participate in progression of gastric cancer. However, the role of circYAP1 in acute kidney injury (AKI) remains obscure. We attempted to examine the effects of circYAP1 on ischaemia/reperfusion‐stimulated renal injury. AKI model was established by treating HK‐2 cells in ischaemia/reperfusion (I/R) environment. CircYAP1 expression in blood of AKI patients and I/R‐treated HK‐2 cells was evaluated via RT‐qPCR. CCK‐8, flow cytometry, ELISA and ROS assay were executed to test the impact of circYAP1 on cell viability, apoptosis, inflammatory cytokines and ROS generation. Bioinformatic analysis was executed to explore miRNA targets. The relativity between circYAP1 and miR‐21‐5p was verified by RT‐qPCR and luciferase assay. The functions of miR‐21‐5p in I/R‐triggered injury were reassessed. PI3K/AKT/mTOR pathway was detected by Western blot. Down‐regulated circYAP1 was observed in AKI blood samples and I/R‐treated HK‐2 cells. CircYAP1 overexpression expedited cell growth and weakened secretion of inflammatory factors and ROS generation in I/R‐disposed cells. Besides, we found circYAP1 could sponge to miR‐21‐5p. Interestingly, miR‐21‐5p overexpression overturned the repressive effects of circYAP1 on cell injury. Moreover, PI3K/AKT/mTOR pathway was activated by circYAP1 via inhibiting miR‐21‐5p. We demonstrated that circYAP1 activated PI3K/AKT/mTOR pathway and secured HK‐2 cells from I/R injury via sponging miR‐21‐5p.     

Alda-1, an ALDH2 activator,protects against hepatic ischemia/reperfusion injury in rats via inhibition of oxidative stress

Previous studies have proved that activation of aldehyde dehydrogenase two (ALDH2) can attenuate oxidative stress through clearance of cytotoxic aldehydes, and can protect against cardiac, cerebral, and lung ischemia/reperfusion (I/R) injuries. In this study, we investigated the effects of the ALDH2 activator Alda-1 on hepatic I/R injury. Partial warm ischemia was performed in the left and middle hepatic lobes of Sprague-Dawley rats for 1?h, followed by 6?h of reperfusion. Rats received either Alda-1 or vehicle by intravenous injection 30?min before ischemia. Blood and tissue samples of the rats were collected after 6-h reperfusion. Histological injury, proinflammatory cytokines, reactive oxygen species (ROS), cellular apoptosis, ALDH2 expression and activity, 4-hydroxy-trans-2-nonenal (4-HNE) and malondialdehyde (MDA) were measured. BRL-3A hepatocytes were subjected to hypoxia/reoxygenation (H/R). Cell viability, ROS, and mitochondrial membrane potential were determined. Pretreatment with Alda-1 significantly alleviated I/R-induced elevations of alanine aminotransferase and aspartate amino transferase, and significantly blunted the pathological injury of the liver. Moreover, Alda-1 significantly inhibited ROS and proinflammatory cytokines production, 4-HNE and MDA accumulation, and apoptosis. Increased ALDH2 activity was found after Alda-1 administration. No significant changes in ALDH2 expression were observed after I/R. ROS was also higher in H/R cells than in control cells, which was aggravated upon treatment with 4-HNE, and reduced by Alda-1 treatment. Cell viability and mitochondrial membrane potential were inhibited in H/R cells, which was attenuated upon Alda-1 treatment. Activation of ALDH2 by Alda-1 attenuates hepatic I/R injury via clearance of cytotoxic aldehydes.     

Strain-specific differences in sensitivity to ischemia-reperfusion lung injury in mice.

Ischemia-reperfusion (I/R) lung injury is characterized by increased pulmonary endothelial permeability and edema, but the genetic basis for this injury is unknown. We utilized an in vivo mouse preparation of unilateral lung I/R to evaluate the genetic determinants of I/R lung injury. An index of pulmonary vascular protein permeability was measured by the ratio of left-to-right lung Evans blue dye of eight inbred mouse strains after 30 min of left lung ischemia and 150 min of reperfusion. The order of strain-specific sensitivity to I/R lung injury was BALB/c < SJL/J < CBA/J < C57BL/6J < 129/J < A/J < C3H/H3J < SWR/J. The reciprocal F1 offspring of the BALB/c and SWR/J progenitor strains had intermediate phenotypes but a differing variance. A similar pattern of right lung Evans blue dye content suggested the presence of contralateral injury because baseline vascular permeability was not different. Lung I/R injury was attenuated by NADPH oxidase inhibition, indicating a role for NADPH oxidase-derived reactive oxygen species (ROS). There was no strain-dependent difference in lung NADPH oxidase expression. Strain-related differences in zymosan-stimulated neutrophil ROS production did not correlate with I/R lung injury in that neutrophil ROS production in SWR/J mice was greater than C57BL/6J but not different from BALB/c mice. These data indicate the presence of a genetic sensitivity to lung I/R injury that involves multiple genes including a maternal-related factor. Although neutrophil-derived ROS production is also modulated by genetic factors, the pattern did not explain the genetic sensitivity to lung I/R injury.     

Therapeutic administration of IL-11 exhibits the postconditioning effects against ischemia-reperfusion injury via STAT3 in the heart

Activation of cardiac STAT3 by IL-6 cytokine family contributes to cardioprotection. Previously, we demonstrated that IL-11, an IL-6 cytokine family, has the therapeutic potential to prevent adverse cardiac remodeling after myocardial infarction; however, it remains to be elucidated whether IL-11 exhibits postconditioning effects. To address the possibility that IL-11 treatment improves clinical outcome of recanalization therapy against acute myocardial infarction, we examined its postconditioning effects on ischemia/reperfusion (I/R) injury. C57BL/6 mice were exposed to ischemia (30 min) and reperfusion (24 h), and IL-11 was intravenously administered at the start of reperfusion. I/R injury mediated the activation of STAT3, which was enhanced by IL-11 administration. IL-11 treatment reduced I/R injury, analyzed by triphenyl tetrazolium chloride staining [PBS, 46.7 ± 14.4%; IL-11 (20 μg/kg), 28.6 ± 7.5% in the ratio of infarct to risk area]. Moreover, echocardiographic and hemodynamic analyses clarified that IL-11 treatment preserved cardiac function after I/R. Terminal deoxynucleotide transferase-mediated dUTP nick-end labeling staining revealed that IL-11 reduced the frequency of apoptotic cardiomyocytes after I/R. Interestingly, IL-11 reduced superoxide production assessed by in situ dihydroethidium fluorescence analysis, accompanied by the increased expression of metallothionein 1 and 2, reactive oxygen species (ROS) scavengers. Importantly, with the use of cardiac-specific STAT3 conditional knockout (STAT3 CKO) mice, it was revealed that cardiac-specific ablation of STAT3 abrogated IL-11-mediated attenuation of I/R injury. Finally, IL-11 failed to suppress the ROS production after I/R in STAT3 CKO mice. IL-11 administration exhibits the postconditioning effects through cardiac STAT3 activation, suggesting that IL-11 has the clinical therapeutic potential to prevent I/R injury in heart.     

The role of mitochondrial reactive oxygen species,NO and H2S in ischaemia/reperfusion injury and cardioprotection

Redox signalling in mitochondria plays an important role in myocardial ischaemia/reperfusion (I/R) injury and in cardioprotection. Reactive oxygen and nitrogen species (ROS/RNS) modify cellular structures and functions by means of covalent changes in proteins including among others S‐nitros(yl)ation by nitric oxide (NO) and its derivatives, and S‐sulphydration by hydrogen sulphide (H₂S). Many enzymes are involved in the mitochondrial formation and handling of ROS, NO and H₂S under physiological and pathological conditions. In particular, the balance between formation and removal of reactive species is impaired during I/R favouring their accumulation. Therefore, various interventions aimed at decreasing mitochondrial ROS accumulation have been developed and have shown cardioprotective effects in experimental settings. However, ROS, NO and H₂S play also a role in endogenous cardioprotection, as in the case of ischaemic pre‐conditioning, so that preventing their increase might hamper self‐defence mechanisms. The aim of the present review was to provide a critical analysis of formation and role of reactive species, NO and H₂S in mitochondria, with a special emphasis on mechanisms of injury and protection that determine the fate of hearts subjected to I/R. The elucidation of the signalling pathways of ROS, NO and H₂S is likely to reveal novel molecular targets for cardioprotection that could be modulated by pharmacological agents to prevent I/R injury.     

Further evidence for the role of free radicals in the limb teratogenicity of L-NAME

BACKGROUND: L-NAME (N(G)-nitro-(L)-arginine methyl ester), a nitric oxide synthase inhibitor, causes severe limb reduction malformations when gravid rats are treated intraperitoneally on gd-17. Hemorrhages, appearing within hours of L-NAME administration, and defects at term can be significantly reduced by co-treatment with PBN (alpha-phenyl-N-t-butylnitrone), a spin trap antioxidant. We have proposed that limb defects result from ischemia-reperfusion injury. We examine the role of xanthine oxidase and ROS formation in the limb effects of L-NAME. METHODS: Gravidas were treated with L-NAME (50 mg/kg) in the presence or absence of allopurinol, a xanthine oxidase inhibitor. Spatial patterns of limb hemorrhage were determined promptly and at term as was digit length at the latter interval. Xanthine oxidase activities were assayed in control and treated limbs with and without allopurinol co-treatment. RESULTS: Allopurinol significantly reduced hemorrhage severity in a dose-responsive fashion when fetuses were examined at term. Higher doses of allopurinol significantly preserved digit length. Xanthine oxidase activities in fetal limb were significantly increased by L-NAME treatment whereas co-treatment with allopurinol restored activities to near-control levels. CONCLUSIONS: These findings support the role of excess reactive oxygen species (ROS) formation in L-NAME-induced limb reduction. We propose that nitric oxide (NO) depletion by L-NAME interferes with vascular integrity, and causes vasoconstriction. Resultant hypoxia stimulates superoxide formation and nitric oxide formation catalyzed by the inducible isoform of nitric oxide synthase. The reduction products of superoxide or the products of its reaction with nitric oxide oxidize or nitrate endothelial components resulting in limb reduction defects.     

Synergistic protective effect of ischemic preconditioning and allopurinol on ischemia/reperfusion injury in rat liver

This study examined the effects of ischemic preconditioning (IPC), allopurinol (Allo) or a combination of both on the extent of mitochondrial injury caused by hepatic ischemia/reperfusion (I/R). I/R increased the serum aminotransferase activity and the level of mitochondrial lipid peroxidation, whereas it decreased the mitochondrial glutathione level. Either IPC or Allo alone attenuated these changes with Allo+IPC having a synergistic effect. Allo increased the serum nitrite and nitrate level after brief ischemia. The significant peroxide production observed after 10 min of reperfusion after sustained ischemia was markedly attenuated by Allo+IPC. The mitochondria isolated after I/R were swollen, which was reduced by Allo+IPC. At the end of ischemia, the hepatic ATP level was lower and there was significant xanthine accumulation, which was attenuated by Allo+IPC. These results suggest that IPC and Allo act synergistically to protect cells against mitochondrial injury and preserve the hepatic energy metabolism during hepatic I/R.     

Exercise-induced peptide EIP-22 protect myocardial from ischaemia/reperfusion injury via activating JAK2/STAT3 signalling pathway

Recent studies have revealed that exercise has myocardial protective effects, but the exact mechanism remains unclear. Studies have increasingly found that peptides play a protective role in myocardial ischaemia-reperfusion (I/R) injury. However, little is known about the role of exercise-induced peptides in myocardial I/R injury. To elucidate the effect of exercise-induced peptide EIP-22 in myocardial I/R injury, we first determined the effect of EIP-22 on hypoxia/reperfusion (H/R)- or H₂O₂-induced injury via assessing cell viability and lactate dehydrogenase (LDH) level. In addition, reactive oxygen species (ROS) accumulation and mitochondrial membrane potential (MMP) was assessed by fluorescence microscope. Meanwhile, Western blot and TUNEL methods were used to detect apoptosis level. Then, we conducted mice I/R injury model and verified the effect of EIP-22 by measuring cardiac function, evaluating heart pathology and detecting serum LDH, CK-MB and cTnI level. Finally, the main signalling pathway was analysed by RNA-seq. In vitro, EIP-22 treatment significantly improved cells viabilities and MMP and attenuated the LDH, ROS and apoptosis level. In vivo, EIP-22 distinctly improved cardiac function, ameliorated myocardial infarction area and fibrosis and decreased serum LDH, CK-MB and cTnI level. Mechanistically, JAK/STAT signalling pathway was focussed by RNA-seq and we confirmed that EIP-22 up-regulated the expression of p-JAK2 and p-STAT3. Moreover, AG490, a selective inhibitor of JAK2/STAT3, eliminated the protective roles of EIP-22. The results uncovered that exercise-induced peptide EIP-22 protected cardiomyocytes from myocardial I/R injury via activating JAK2/STAT3 signalling pathway and might be a new candidate molecule for the treatment of myocardial I/R injury.     

Involvement of GPX4 in irisin's protection against ischemia reperfusion‐induced acute kidney injury

Ischemia reperfusion (I/R)‐induced acute kidney injury (AKI) is a common and serious condition. Irisin, an exercise‐induced hormone, improves mitochondrial function and reduces reactive oxygen species (ROS) production. Glutathione peroxidase 4 (GPX4) is a key regulator of ferroptosis and its inactivation aggravates renal I/R injury by inducing ROS production. However, the effect of irisin on GPX4 and I/R‐induced AKI is still unknown. To study this, male adult mice were subjected to renal I/R by occluding bilateral renal hilum for 30 min, which was followed by 24 hr reperfusion. Our results showed serum irisin levels were decreased in renal I/R mice. Irisin (250 μg/kg) treatment alleviated renal injury, downregulated inflammatory response, improved mitochondrial function, and reduced ER stress and oxidative stress after renal I/R, which were associated with upregulation of GPX4. Treated with RSL3 (a GPX4 inhibitor) abolished irisin's protective effect. Thus, irisin attenuates I/R‐induced AKI through upregulating GPX4.     

Xanthine oxidoreductase is a critical mediator of cigarette smoke-induced endothelial cell DNA damage and apoptosis

Cigarette smoke (CS) exposure is unquestionably the most frequent cause of emphysema in the United States. Accelerated pulmonary endothelial cell (EC) apoptosis is an early determinant of lung destruction in emphysema. One of the pathogenic causes of emphysema is an alveolar oxidant and antioxidant imbalance. The enzyme xanthine oxidoreductase (XOR) has been shown to be a source of reactive oxygen species (ROS) in a multitude of diseases (S. Sakao et al., FASEB J. 21, 3640–3652; 2007). The contribution of XOR to CS-induced apoptosis is not well defined. Here we demonstrate that C57/bl6 mice exposed to CS have increased pulmonary XOR activity and protein levels compared to filtered-air-exposed controls. In addition, we demonstrate that primary pulmonary human lung microvascular endothelial cells exposed to cigarette smoke extract undergo increased rates of caspase-dependent apoptosis that are reliant on XOR activity, ROS production, and p53 function/expression. We also demonstrate that exogenous XOR is sufficient to increase p53 expression and induce apoptosis, suggesting that XOR is an upstream mediator of p53 in CS-induced EC apoptosis. Furthermore, we show that XOR activation results in DNA double-strand breaks that activate the enzyme ataxia telangiectasia mutated, which phosphorylates histone H2AX and upregulates p53. In conclusion, CS increases XOR expression, and the enzyme is both sufficient and necessary for p53 induction and CS-induced EC apoptosis.     

Protection of NAD(P)H:quinone oxidoreductase 1 against renal ischemia/reperfusion injury in mice

Ischemia/reperfusion (I/R) is the most common cause of acute renal injury. I/R-induced reactive oxygen species (ROS) are thought to be a major factor in the development of acute renal injury by promoting the initial tubular damage. NAD(P)H:quinone oxidoreductase 1 (NQO1) is a well-known antioxidant protein that regulates ROS generation. The purpose of this study was to investigate whether NQO1 modulates the renal I/R injury (IRI) associated with NADPH oxidase (NOX)-derived ROS production in an animal model. We analyzed renal function, oxidative stress, and tubular apoptosis after IRI. NQO1^−/− mice showed increased blood urea nitrogen and creatinine levels, tubular damage, oxidative stress, and apoptosis. In the kidneys of NQO1^−/− mice, the cellular NADPH/NADP⁺ ratio was significantly higher and NOX activity was markedly higher than in those of NQO1^+/+ mice. The activation of NQO1 by β-lapachone (βL) significantly improved renal dysfunction and reduced tubular cell damage, oxidative stress, and apoptosis by renal I/R. Moreover, the βL treatment significantly lowered the cellular NADPH/NADP⁺ ratio and dramatically reduced NOX activity in the kidneys after IRI. From these results, it was concluded that NQO1 has a protective role against renal injury induced by I/R and that this effect appears to be mediated by decreased NOX activity via cellular NADPH/NADP⁺ modulation. These results provide convincing evidence that NQO1 activation might be beneficial for ameliorating renal injury induced by I/R.     

NADPH oxidase 4 promotes cardiac microvascular angiogenesis after hypoxia/reoxygenation in vitro

Microvascular endothelial cell dysfunction plays a key role in myocardial ischemia/reperfusion (I/R) injury, wherein reactive oxygen species (ROS)-dependent signaling is intensively involved. However, the roles of the various ROS sources remain unclear. This study sought to investigate the role of NADPH oxidase 4 (Nox4) in the cardiac microvascular endothelium in response to I/R injury. Adult rat cardiac microvascular endothelial cells (CMECs) were isolated and subjected to hypoxia/reoxygenation (H/R). Our results showed that Nox4 was highly expressed in CMECs, was significantly increased at both mRNA and protein levels after H/R injury, and contributed to H/R-stimulated increase in Nox activity and ROS generation. Downregulation of Nox4 by small interfering RNA transfection did not affect cell viability or ROS production under normoxia, but exacerbated H/R injury as evidenced by increased apoptosis and inhibited cell survival, migration, and angiogenesis after H/R. Nox4 inhibition also increased prolyl hydroxylase 2 (PHD2) expression and blocked H/R-induced increases in HIF-1α and VEGF expression. Pretreatment with DMOG, a specific competitive PHD inhibitor, upregulated HIF-1α and VEGF expression and significantly reversed Nox4 knockdown-induced injury. However, Nox2 was scarcely expressed and played a minimal role in CMEC survival and angiogenesis after H/R, though a modest upregulation of Nox2 was observed. In conclusion, this study demonstrated a previously unrecognized protective role of Nox4, a ROS-generating enzyme and the major Nox isoform in CMECs, against H/R injury by inhibiting apoptosis and promoting migration and angiogenesis via a PHD2-dependent upregulation of HIF-1/VEGF proangiogenic signaling.     

Rapid modification of the glycocalyx caused by ischemia-reperfusion is inhibited by adenosine A2A receptor activation

Ischemia-reperfusion (I/R) has been shown to cause microvascular dysfunction and to alter the appearance of the glycocalyx in electron micrographs. We hypothesized that I/R injury might alter the structure and/or permeability of the glycocalyx. Prior work had shown a role for adenosine in protection from I/R injury, and, therefore, we also explored the idea that activation of the adenosine A(2A) receptor would attenuate I/R glycocalyx injury. Here, we report that I/R causes a rapid and dramatic decrease in the ability of the glycocalyx to exclude FITC-Dextran 70 (Dex70). Over a reperfusion period of 45 min, the glycocalyx dye exclusion zone for Dex70 decreased by one-half in capillaries and postcapillary venules, whereas the red blood cell exclusion zone was very slightly reduced in capillaries only. Pretreatment with the A(2A) agonist ATL-146e significantly inhibited the changes in both vessel types. The modifications of the glycocalyx appear to be an early step in the inflammatory cascade typically associated with reperfusion injury, and adenosine A(2A) receptor activation may play a role in protection from this injury.     

Elevated semicarbazide-sensitive amine oxidase (SSAO) activity in lung with ischemia-reperfusion injury: protective effect of ischemic preconditioning plus SSAO inhibition

Ischemic preconditioning (IP) has been shown to protect the lung against ischemia-reperfusion (I/R) injury. Although the production of reactive oxygen species (ROS) has been postulated to play a crucial role in I/R injury, the sources of these radicals in I/R and the mechanisms of protection in IP remain unknown. Since it was postulated that deamination of endogenous and exogenous amines by semicarbazide-sensitive amine oxidase (SSAO) in tissue damage leads to the overproduction of hydrogen peroxide (H2O2), we investigated the possible contribution of tissue SSAO to excess ROS generation and lipid peroxidation during I/R and IP of the lung. Male Wistar rats were randomized into 6 groups: control lungs were subjected to 30 min of perfusion in absence and presence of SSAO inhibitor, whereas the lungs of the I/R group were subjected to 2 h of cold ischemia following the 30 min of perfusion in absence and presence of SSAO inhibitor. IP was performed by two cycles of 5 min ischemia followed by 5 min of reperfusion prior to 2 h of hypothermic ischemia in absence and presence of SSAO inhibitor. Lipid peroxidation, reduced (GSH) and oxidized (GSSG) glutathione levels, antioxidant enzyme activities, SSAO activity, and H2O2 release were determined in tissue samples of the study groups. Lipid peroxidation, glutathione disulfide (GSSG) content, SSAO activity and H2O2 release were increased in the I/R group, whereas GSH content, GSH/GSSG ratio and antioxidant enzyme activities were decreased. SSAO activity, H2O2 release, GSSG content and lipid peroxidation were markedly decreased in the IP group, whereas GSH content, GSH/GSSG ratio and antioxidant enzyme activities were significantly increased. SSAO activity was found to be positively correlated with H2O2 production in all study groups. Increased lipid peroxidation, SSAO activity, GSSG and H2O2 contents as well as decreased GSH and antioxidant enzyme levels in I/R returned to their basal levels when IP and SSAO inhibition were applied together. The present study suggests that application of IP and SSAO inhibition together may be more effective than IP alone against I/R injury in the lung.