Mammalian peroxidasin (PXDN): From physiology to pathology.

Heme-containing peroxidases catalyze the oxidation of a variety of substrates by consuming hydrogen peroxide (H2O2), and play diversified roles in physiology and pathology including innate immunity, the synthesis of thyroid hormone and the extracellular matrix, as well as the pathogenesis of several inflammatory diseases. Peroxidasin (PXDN), also known as Vascular Peroxidase-1 (VPO1), is a newly identified peroxidase and expresses in multiple cells and tissues including cardiovascular system and the lung. Recent studies imply its roles in the innate immunity, cardiovascular physiology and diseases, and extracellular matrix formation. Studies on the role of PXDN in human diseases are entering a new and exciting stage, and this review provides the insights into this emerging field of PXDN.

Recombinant Myeloperoxidase as a New Class of Antimicrobial Agents.

Heme-containing peroxidases are widely distributed in the animal and plant kingdoms and play an important role in host defense by generating potent oxidants. Myeloperoxidase (MPO), the prototype of heme-containing peroxidases, exists in neutrophils and monocytes. MPO has a broad spectrum of microbial killing. The difficulty of producing MPO at a large scale hinders its study and utilization. This study aimed to overexpress recombinant human MPO and characterize its microbicidal activities in vitro and in vivo. A human HEK293 cell line stably expressing recombinant MPO (rMPO) was established as a component of this study. rMPO was overexpressed and purified for studies on its biochemical and enzymatic properties, as well as its microbicidal activities. In this study, rMPO was secreted into culture medium as a monomer. rMPO revealed enzymatic activity similar to that of native MPO. rMPO, like native MPO, was capable of killing a broad spectrum of microorganisms, including Gram-negative and -positive bacteria and fungi, at low nM levels. Interestingly, rMPO could kill antibiotic-resistant bacteria, making it very useful for treatment of nosocomial infections and mixed infections. The administration of rMPO significantly reduced the morbidity and mortality of murine lung infections induced by Pseudomonas aeruginosa or methicillin-resistant Staphylococcus aureus. In animal safety tests, the administration of 100 nM rMPO via tail vein did not result in any sign of toxic effects. Taken together, the data suggest that rMPO purified from a stably expressing human cell line is a new class of antimicrobial agents with the ability to kill a broad spectrum of pathogens, including bacteria and fungi with or without drug resistance. IMPORTANCE Over the past 2 decades, more than 20 new infectious diseases have emerged. Unfortunately, novel antimicrobial therapeutics are discovered at much lower rates. Infections caused by resistant microorganisms often fail to respond to conventional treatment, resulting in prolonged illness, greater risk of death, and high health care costs. Currently, this is best seen with the lack of a cure for coronavirus disease 2019 (COVID-19). To combat such untreatable microorganisms, there is an urgent need to discover new classes of antimicrobial agents. Myeloperoxidase (MPO) plays an important role in host defense. The difficulty of producing MPO on a large scale hinders its study and utilization. We have produced recombinant MPO at a large scale and have characterized its antimicrobial activities. Most importantly, recombinant MPO significantly reduced the morbidity and mortality of murine pneumonia induced by Pseudomonas aeruginosa or methicillin-resistant Staphylococcus aureus. Our data suggest that recombinant MPO from human cells is a new class of antimicrobials with a broad spectrum of activity.

Peroxidasin promotes diabetic vascular endothelial dysfunction induced by advanced glycation end products via NOX2/HOCl/Akt/eNOS pathway.

Reactive oxygen species (ROS) derived from NADPH oxidases (NOX) plays an essential role in advanced glycation end products (AGEs)-induced diabetic vascular endothelial dysfunction. Peroxidasin (PXDN, VPO1) is one member of peroxidases family that catalyzes hydrogen peroxide (H2O2) to hypochlorous acid (HOCl). This present study aimed to elucidate the role of PXDN in promoting vascular endothelial dysfunction induced by AGEs in diabetes mellitus. We found that, compared to non-diabetic (db/m) mice, PXDN expression was notably increased in db/db mice with impaired endothelium-dependent relaxation. Knockdown of PXDN in vivo through tail vein injection of siRNA restored the impaired endothelium-dependent relaxation function of db/db mice which is accompanied with up-regulation of eNOS Ser1177 phosphorylation and NO production. AGEs significantly elevated expression of PXDN and 3-Cl-Tyr, but decreased phosphorylation of Akt and eNOS and NO release in HUVECs. All these effects induced by AGEs were remarkable alleviated by silencing PXDN with small interfering RNAs. In addition, HOCl treatment alone as well as HOCl added with Akt inhibitor MK2206 inhibited phosphorylation of Akt and eNOS, reducing NO production. More importantly,AGEs-induced up-regulation of PXDN and 3-Cl-Tyr with endothelial dysfunction were transformed by NOX2 silencing and H2O2 scavengers. Thus, these results support the conclusion that PXDN promotes AGEs-induced diabetic vascular endothelial dysfunction by attenuating eNOS phosphorylation at Ser1177 via NOX2/HOCl/Akt pathway.

PXDN reduces autophagic flux in insulin-resistant cardiomyocytes via modulating FoxO1.

Autophagy, a well-observed intracellular lysosomal degradation process, is particularly important to the cell viability in diabetic cardiomyopathy (DCM). Peroxidasin (PXDN) is a heme-containing peroxidase that augments oxidative stress and plays an essential role in cardiovascular diseases, while whether PXDN contributes to the pathogenesis of DCM remains unknown. Here we reported the suppression of cell viability and autophagic flux, as shown by autophagosomes accumulation and increased expression level of LC3-II and p62 in cultured H9C2 and human AC16 cells that treated with 400 µM palmitate acid (PA) for 24 h. Simultaneously, PXDN protein level increased. Moreover, cell death, autophagosomes accumulation as well as increased p62 expression were suppressed by PXDN silence. In addition, knockdown of PXDN reversed PA-induced downregulated forkhead box-1 (FoxO1) and reduced FoxO1 phosphorylation, whereas did not affect AKT phosphorylation. Not consistent with the effects of si-PXDN, double-silence of PXDN and FoxO1 significantly increased cell death, suppressed autophagic flux and declined the level of FoxO1 and PXDN, while the expression of LC3-II was unchanged under PA stimulation. Furthermore, inhibition of FoxO1 in PA-untreated cells induced cell death, inhibited autophagic flux, and inhibited FoxO1 and PXDN expression. Thus, we come to conclusion that PXDN plays a key role in PA-induced cell death by impairing autophagic flux through inhibiting FoxO1, and FoxO1 may also affect the expression of PXDN. These findings may develop better understanding of potential mechanisms regarding autophagy in insulin-resistant cardiomyocytes.

Oxidative Stress in Pulmonary Fibrosis.

Oxidative stress has been linked to various disease states as well as physiological aging. The lungs are uniquely exposed to a highly oxidizing environment and have evolved several mechanisms to attenuate oxidative stress. Idiopathic pulmonary fibrosis (IPF) is a progressive age-related disorder that leads to architectural remodeling, impaired gas exchange, respiratory failure, and death. In this article, we discuss cellular sources of oxidant production, and antioxidant defenses, both enzymatic and nonenzymatic. We outline the current understanding of the pathogenesis of IPF and how oxidative stress contributes to fibrosis. Further, we link oxidative stress to the biology of aging that involves DNA damage responses, loss of proteostasis, and mitochondrial dysfunction. We discuss the recent findings on the role of reactive oxygen species (ROS) in specific fibrotic processes such as macrophage polarization and immunosenescence, alveolar epithelial cell apoptosis and senescence, myofibroblast differentiation and senescence, and alterations in the acellular extracellular matrix. Finally, we provide an overview of the current preclinical studies and clinical trials targeting oxidative stress in fibrosis and potential new strategies for future therapeutic interventions. © 2020 American Physiological Society. Compr Physiol 10:509-547, 2020.

Vascular peroxidase 1 is a novel regulator of cardiac fibrosis after myocardial infarction.

Cardiac fibrosis is the most important mechanism contributing to cardiac remodeling after myocardial infarction (MI). VPO1 is a heme enzyme that uses hydrogen peroxide (H2O2) to produce hypochlorous acid (HOCl). Our previous study has demonstrated that VPO1 regulates myocardial ischemic reperfusion and renal fibrosis. We investigated the role of VPO1 in cardiac fibrosis after MI. The results showed that VPO1 expression was robustly upregulated in the failing human heart with ischemic cardiomyopathy and in a murine model of MI accompanied by severe cardiac fibrosis. Most importantly, knockdown of VPO1 by tail vein injection of VPO1 siRNA significantly reduced cardiac fibrosis and improved cardiac function and survival rate. In VPO1 knockdown mouse model and cardiac fibroblasts cultured with TGF-ß1, VPO1 contributes to cardiac fibroblasts differentiation, migration, collagen I synthesis and proliferation. Mechanistically, the fibrotic effects following MI of VPO1 manifested partially through HOCl formation to activate Smad2/3 and ERK1/2. Thus, we conclude that VPO1 is a crucial regulator of cardiac fibrosis after MI by mediating HOCl/Smad2/3 and ERK1/2 signaling pathways, implying a promising therapeutic target in ischemic cardiomyopathy.

VPO1 Modulates Vascular Smooth Muscle Cell Phenotypic Switch by Activating Extracellular Signal-regulated Kinase 1/2 (ERK 1/2) in Abdominal Aortic Aneurysms.

Background Hydrogen peroxide (H2O2) is a critical molecular signal in the development of abdominal aortic aneurysm ( AAA ) formation. Vascular peroxidase 1 ( VPO 1) catalyzes the production of hypochlorous acid ( HOC l) from H2O2 and significantly enhances oxidative stress. The switch from a contractile phenotype to a synthetic one in vascular smooth muscle cells ( VSMC s) is driven by reactive oxygen species and is recognized as an early and important event in AAA formation. This study aims to determine if VPO 1 plays a critical role in the development of AAA by regulating VSMC phenotypic switch. Methods and Results VPO 1 is upregulated in human and elastase-induced mouse aneurysmal tissues compared with healthy control tissues. Additionally, KLF 4, a nuclear transcriptional factor, is upregulated in aneurysmatic tissues along with a concomitant downregulation of differentiated smooth muscle cell markers and an increase of synthetic phenotypic markers, indicating VSMC phenotypic switch in these diseased tissues. In cultured VSMC s from rat abdominal aorta, H2O2 treatment significantly increases VPO 1 expression and HOC l levels as well as VSMC phenotypic switch. In support of these findings, depletion of VPO 1 significantly attenuates the effects of H2O2 and HOC l treatment. Furthermore, HOC l treatment promotes VSMC phenotypic switch and ERK 1/2 phosphorylation. Pretreatment with U0126 (a specific inhibitor of ERK 1/2) significantly attenuates HOC l-induced VSMC phenotypic switch. Conclusions Our results demonstrate that VPO 1 modulates VSMC phenotypic switch through the H2O2/ VPO 1/ HOC l/ ERK 1/2 signaling pathway and plays a key role in the development of AAA . Our findings also implicate VPO 1 as a novel signaling node that mediates VSMC phenotypic switch and plays a key role in the development of AAA . Clinical Trial Registration URL : www.chictr.org.cn . Unique identifier: Chi CTR 1800016922.

Peroxidasin contributes to lung host defense by direct binding and killing of gram-negative bacteria.

Innate immune recognition is classically mediated by the interaction of host pattern-recognition receptors and pathogen-associated molecular patterns; this triggers a series of downstream signaling events that facilitate killing and elimination of invading pathogens. In this report, we provide the first evidence that peroxidasin (PXDN; also known as vascular peroxidase-1) directly binds to gram-negative bacteria and mediates bactericidal activity, thus, contributing to lung host defense. PXDN contains five leucine-rich repeats and four immunoglobulin domains, which allows for its interaction with lipopolysaccharide, a membrane component of gram-negative bacteria. Bactericidal activity of PXDN is mediated via its capacity to generate hypohalous acids. Deficiency of PXDN results in a failure to eradicate Pseudomonas aeruginosa and increased mortality in a murine model of Pseudomonas lung infection. These observations indicate that PXDN mediates previously unrecognized host defense functions against gram-negative bacterial pathogens.

Vascular peroxidase 1 mediates hypoxia-induced pulmonary artery smooth muscle cell proliferation, apoptosis resistance and migration.

Aims: Reactive oxygen species (ROS) play essential roles in the pulmonary vascular remodelling associated with hypoxia-induced pulmonary hypertension (PH). Vascular peroxidase 1 (VPO1) is a newly identified haeme-containing peroxidase that accelerates oxidative stress development in the vasculature. This study aimed to determine the potential role of VPO1 in hypoxia-induced PH-related vascular remodelling. Methods and results: The vascular morphology and VPO1 expression were assessed in the pulmonary arteries of Sprague-Dawley (SD) rats. Nicotinamide adenine dinucleotide phosphate (NADPH) oxidase 4 (NOX4) and VPO1 expression and HOCl production were significantly increased in hypoxic rats, which also exhibited obvious vascular remodelling. Furthermore, a hypoxia-induced PH model was generated by exposing primary rat pulmonary artery smooth muscle cells (PASMCs) to hypoxic conditions (3% O2, 48 h), which significantly increased the expression of NOX4 and VPO1 and the production of HOCl. These hypoxic changes were accompanied by enhanced proliferation, apoptosis resistance, and migration. In PASMCs, hypoxia-induced changes, including effects on the expression of cell cycle regulators (cyclin B1 and cyclin D1), apoptosis-related proteins (bax, bcl-2, and cleaved caspase-3), migration promoters (matrix metalloproteinases 2 and 9), and NF-κB expression, as well as the production of HOCl, were all inhibited by silencing VPO1 with small interfering RNAs. Moreover, treatment with HOCl under hypoxic conditions upregulated NF-κB expression and enhanced proliferation, apoptosis resistance, and migration in PASMCs, whereas BAY 11-7082 (an inhibitor of NF-κB) significantly inhibited these effects. Conclusion: Collectively, these results demonstrate that VPO1 promotes hypoxia-induced proliferation, apoptosis resistance, and migration in PASMCs via the NOX4/VPO1/HOCl/NF-κB signalling pathway.

The role of losartan in preventing vascular remodeling in spontaneously hypertensive rats by inhibition of the H2O2/VPO1/HOCl/MMPs pathway.

Vascular peroxidase 1 (VPO1) has been proved to be associated with vascular endothelial cell apoptosis by producing reactive oxygen species. However, the contribution of VPO1 to the development of vascular remodeling (VR) remains to be fully characterized. We explored the role of VPO1 in VR in spontaneously hypertensive rats (SHRs) and the underlying mechanism of losartan in inhibiting VR. Compared to Wistar-Kyoto (WKY) rats, the SHR showed remodeling of their vascular walls. The level of VPO1 and the hydrogen peroxide (H2O2) concentration were increased in the SHRs. However, the SHRs pretreated with losartan showed significant inhibition of blood pressure and VR and decreased levels of VPO1 and H2O2 compared to the non-treated SHRs. Angiotensin II significantly increased the expressions of MMP-2, MMP-9 and the concentrations of H2O2 and hypochlorous acid (HOCl) in vascular smooth muscle cells (VSMCs). However, only the H2O2 level increased in VSMCs when transfected with VPO1 shRNA. These results support a critical but previously unrecognized role of VPO1 in VR and suggest that therapies to reduce VPO1 may be novel approaches for VR.

Critical role of vascular peroxidase 1 in regulating endothelial nitric oxide synthase.

Vascular peroxidase 1 (VPO1) is a member of the peroxidase family which aggravates oxidative stress by producing hypochlorous acid (HOCl). Our previous study demonstrated that VPO1 plays a critical role in endothelial dysfunction through dimethylarginine dimethylaminohydrolase2 (DDAH2)/asymmetric Dimethylarginine (ADMA) pathway. Hereby we describe the regulatory role of VPO1 on endothelial nitric oxide synthase (eNOS) expression and activity in human umbilical vein endothelial cells (HUVECs). In HUVECs AngiotensinII (100nM) treatment reduced Nitric Oxide (NO) production, decreased eNOS expression and activity, which were reversed by VPO1 siRNA. Knockdown of VPO1 also attenuated ADMA production and eNOS uncoupling while enhancing phosphorylated ser1177 eNOS expression level. Furthermore, HOCl stimulation was shown to directly induce ADMA production and eNOS uncoupling, decrease phosphorylated ser1177 eNOS expression. It also significantly suppressed eNOS expression and activity together with NO production. Therefore, VPO1 plays a vital role in regulating eNOS expression and activity via hydrogen peroxide (H2O2)-VPO1-HOCl pathway.

Involvement of vascular peroxidase 1 in angiotensin II-induced hypertrophy of H9c2 cells.

Oxidative stress has been implicated in cardiac hypertrophy and heart failure. Vascular peroxidase 1 (VPO1), a peroxidase in the cardiovascular system, uses the hydrogen peroxide (H2O2) derived from co-expressed NADPH oxidases (NOX) to produce hypochlorous acid (HOCl) and catalyze peroxidative reactions. Our previous studies showed that VPO1 contributes to the vascular smooth muscle cell proliferation and endothelial dysfunction in spontaneous hypertensive rats (SHRs); however, the role of VPO1 in cardiomyocytes hypertrophy is still uninvestigated. The present study was therefore undertaken to examine the role of VPO1 in the angiotensin II-induced cardiac hypertrophy, and the underlying mechanism by which VPO1 regulates the redox signaling. As compared to WKY rats, the SHRs exhibited increased myocyte cross sectional area, enhanced Nox2 and VPO1 expression level in cardiac tissue, and an increased Ang II level in plasma. In cultured H9c2 cell line, Ang II increased the hypertrophy-related gene (BNP/ANF) expression and the cellular surface area, which was attenuated by knocking down of VPO1 via VPO1 siRNA or pharmacological inhibition of NOX/VPO1 pathway. Moreover, the enhanced hypochlorous acid (HOCl) production and phosphorylation of ERK1/2 was suppressed by VPO1 knockdown. Furthermore, the protective role of VPO1 siRNA transfection on H9c2 cardiomyocytes hypertrophy was abrogated on the HOCl stimulation, and the phosphorylated ERK1/2 expression level was found also upregulated after HOCl stimulation. In conclusion, these results suggest that the Nox2/VPO1/HOCl/ERK1/2 redox signaling pathway was implicated in the pathogenesis of Ang II-induced cardiac hypertrophy.

Vascular peroxidase 1 up regulation by angiotensin II attenuates nitric oxide production through increasing asymmetrical dimethylarginine in HUVECs.

Asymmetric dimethylarginine (ADMA), the endogenous inhibitor of nitric oxide synthase, contributes to endothelial dysfunction and subsequent cardiovascular events including hypertension. Vascular peroxidase 1 (VPO1) is a novel heme-containing peroxidase that uses hydrogen peroxide (H2O2) generated from co-expressed nicotinamide adenine dinucleotide phosphate (NADPH) oxidase to catalyze peroxidative reactions. Our previous study revealed a clear connection between VPO1 gene expression and endothelial dysfunction in spontaneously hypertensive rats. In the present study, we explored whether VPO1 participates in endothelial dysfunction during hypertension by increasing ADMA production. Spontaneously hypertensive rats displayed impaired endothelium-dependent relaxation, decreased eNOS expression and nitric oxide production, significantly increased VPO1 expression in both plasma and aorta tissue, and an increased ADMA level in plasma. In cultured endothelial cells, angiotensin II increased the ADMA level by inhibiting dimethylarginine dimethylaminohydrolase activity, which was inhibited by knockdown of VPO1 using small hairpin RNA. Moreover, the NADPH oxidase inhibitor and the hydrogen peroxide scavenger attenuated angiotensin II-mediated up-regulation of VPO1 and generation of hypochlorous acid. Furthermore, VPO1-derived hypochlorous acid suppressed recombinant dimethylarginine dimethylaminohydrolase activity and increased ADMA production. VPO1 plays a critical role in ADMA production via H2O2-VPO1-hypochlorous acid pathways, which may contribute to endothelial dysfunction in hypertension.

VPO1 mediates oxidation of LDL and formation of foam cells.

Deposition of oxidized-LDL in vascular walls is essential in the initiation of atherosclerosis. Oxidation of LDL has been attributed to myeloperoxidase as its generation of potent oxidants. However, the exact mechanism of LDL oxidation and foam cell formation in atherosclerosis remains to be elucidated. Vascular peroxidase-1 (VPO1), a newly-identified heme-containing peroxidase, is primarily expressed in cardiovascular systems, and secreted into the circulation. The present study evaluates VPO1-mediated LDL oxidation and its role in atherosclerosis. VPO1 was first demonstrated binding to LDL. VPO1-mediated oxidation of proteins and lipids in LDL was verified by a variety of methods including immunoblot analysis, free tryptophan assay, UV absorbance, and thiobarbituric acid assay. VPO1-oxidized LDL caused accumulation of LDL in monocyte-like cells and promoted formation of foam cells. Administration of inflammation factors, LPS or TNF-α, induced increasing expression of VPO1 in aorta and secretion to plasma. TNF-α also promoted formation and retention of VPO1-oxidized LDL in aortic walls. Our data suggest that VPO1 contributes to oxidation and retention of LDL in vessel walls, and formation foam cells, indicating VPO1 as a novel potential mediator of atherosclerosis.

NADPH Oxidase 1 Is Associated with Altered Host Survival and T Cell Phenotypes after Influenza A Virus Infection in Mice.

The role of the reactive oxygen species-producing NADPH oxidase family of enzymes in the pathology of influenza A virus infection remains enigmatic. Previous reports implicated NADPH oxidase 2 in influenza A virus-induced inflammation. In contrast, NADPH oxidase 1 (Nox1) was reported to decrease inflammation in mice within 7 days post-influenza A virus infection. However, the effect of NADPH oxidase 1 on lethality and adaptive immunity after influenza A virus challenge has not been explored. Here we report improved survival and decreased morbidity in mice with catalytically inactive NADPH oxidase 1 (Nox1*/Y) compared with controls after challenge with A/PR/8/34 influenza A virus. While changes in lung inflammation were not obvious between Nox1*/Y and control mice, we observed alterations in the T cell response to influenza A virus by day 15 post-infection, including increased interleukin-7 receptor-expressing virus-specific CD8+ T cells in lungs and draining lymph nodes of Nox1*/Y, and increased cytokine-producing T cells in lungs and spleen. Furthermore, a greater percentage of conventional and interstitial dendritic cells from Nox1*/Y draining lymph nodes expressed the co-stimulatory ligand CD40 within 6 days post-infection. Results indicate that NADPH oxidase 1 modulates the innate and adaptive cellular immune response to influenza virus infection, while also playing a role in host survival. Results suggest that NADPH oxidase 1 inhibitors may be beneficial as adjunct therapeutics during acute influenza infection.

The role of vascular peroxidase 1 in ox-LDL-induced vascular smooth muscle cell calcification.

Reactive oxygen species (ROS)-induced osteogenic differentiation of vascular smooth muscle cells (VSMCs) is associated with the pathogenesis of vascular calcification. Vascular peroxidase 1 (VPO1), a peroxidase in the cardiovascular system, utilizes the hydrogen peroxide (H2O2) produced by co-expressed NADPH oxidases to produce hypochlorous acid (HOCl) and catalyze peroxidative reactions. The aim of this study was to determine whether VPO1 plays a role in the osteogenic differentiation of VSMCs in the setting of the vascular calcification induced by oxidized low-density lipoprotein (ox-LDL). In cultured primary rat VSMCs, we observed that the expression of VPO1 was significantly increased in combination with increases in calcification, as demonstrated via increased mineralization, as well as increased alkaline phosphatase (ALP) activity and up-regulated runt-related transcription factor 2 (Runx2) expression in ox-LDL-treated cells. Ox-LDL-induced VSMC calcification and Runx2 expression were both inhibited by knockdown of VPO1 using a small interfering RNA or by an NADPH oxidase inhibitor. Moreover, the knockdown of VPO1 in VSMCs suppressed the production of HOCl and the phosphorylation of AKT, ERK and P38 MAPK. Furthermore, HOCl treatment facilitated the phosphorylation of AKT, ERK1/2 and P38 MAPK and the expression of Runx2, whereas LY294002 (a specific inhibitor of PI3K), U0126 (a specific inhibitor of ERK1/2) and SB203580 (a specific inhibitor of P38 MAPK) significantly attenuated the HOCl-induced up-regulation of Runx2. Collectively, these results demonstrated that VPO1 promotes ox-LDL-induced VSMC calcification via the VPO1/HOCl/PI3K/AKT, ERK1/2, and P38 MAPK/Runx2 signaling pathways.

Myeloperoxidase is increased in human cerebral aneurysms and increases formation and rupture of cerebral aneurysms in mice.

BACKGROUND AND PURPOSE: Cerebral aneurysm (CA) affects 3% of the population and is associated with hemodynamic stress and inflammation. Myeloperoxidase, a major oxidative enzyme associated with inflammation, is increased in patients with CA, but whether myeloperoxidase contributes to CA is not known. We tested the hypotheses that myeloperoxidase is increased within human CA and is critical for formation and rupture of CA in mice. METHODS: Blood was drawn from the lumen of CAs and femoral arteries of 25 patients who underwent endovascular coiling of CA, and plasma myeloperoxidase concentrations were measured with ELISA. Effects of endogenous myeloperoxidase on CA formation and rupture were studied in myeloperoxidase knockout mice and wild-type (WT) mice using an angiotensin II-elastase induction model of CA. In addition, effects of myeloperoxidase on inflammatory gene expression in endothelial cells were analyzed. RESULTS: Plasma concentrations of myeloperoxidase were 2.7-fold higher within CA than in femoral arterial blood in patients with CA. myeloperoxidase-positive cells were increased in aneurysm tissue compared with superficial temporal artery of patients with CA. Incidence of aneurysms and subarachnoid hemorrhage was significantly lower in myeloperoxidase knockout than in WT mice. In cerebral arteries, proinflammatory molecules, including tumor necrosis factor-α, cyclooxygenase-2 (COX2), chemokine (C-X-C motif) ligand 1 (CXCL1), chemokine (C motif) ligand (XCL1), matrix metalloproteinase (MMP) 8, cluster of differentiation 68 (CD68), and matrix metalloproteinase 13, and leukocytes were increased, and α-smooth muscle actin was decreased, in WT but not in myeloperoxidase knockout mice after induction of CA. Myeloperoxidase per se increased expression of vascular cell adhesion molecule-1 and intercellular adhesion molecule-1 in endothelial cells. CONCLUSIONS: These findings suggest that myeloperoxidase may contribute importantly to formation and rupture of CA.

Negative regulation of NADPH oxidase 4 by hydrogen peroxide-inducible clone 5 (Hic-5) protein.

Hydrogen peroxide-inducible clone 5 (Hic-5) is a focal adhesion adaptor protein induced by the profibrotic cytokine TGF-ß1. We have demonstrated previously that TGF-ß1 induces myofibroblast differentiation and lung fibrosis by activation of the reactive oxygen species-generating enzyme NADPH oxidase 4 (Nox4). Here we investigated a potential role for Hic-5 in regulating Nox4, myofibroblast differentiation, and senescence. In normal human diploid fibroblasts, TGF-ß1 induces Hic-5 expression in a delayed manner relative to the induction of Nox4 and myofibroblast differentiation. Hic-5 silencing induced constitutive Nox4 expression and enhanced TGF-ß1-inducible Nox4 levels. The induction of constitutive Nox4 protein in Hic-5-silenced cells was independent of transcription and translation and controlled by the ubiquitin-proteasomal system. Hic-5 associates with the ubiquitin ligase Cbl-c and the ubiquitin-binding protein heat shock protein 27 (HSP27). The interaction of these proteins is required for the ubiquitination of Nox4 and for maintaining low basal levels of this reactive oxygen species-generating enzyme. Our model suggests that TGF-ß1-induced Hic-5 functions as a negative feedback mechanism to limit myofibroblast differentiation and senescence by promoting the ubiquitin-proteasomal system-mediated degradation of Nox4. Together, these studies indicate that endogenous Hic-5 suppresses senescence and profibrotic activities of myofibroblasts by down-regulating Nox4 protein expression. Additionally, these are the first studies, to our knowledge, to demonstrate posttranslational regulation of Nox4.

Meta-analysis of myeloperoxidase gene polymorphism and coronary artery disease susceptibility.

OBJECTIVE: To assess the association between myeloperoxidase (MPO) gene polymorphism and coronary artery disease (CAD). METHODS: Several databases were used to retrieve relevant literature up to March 2013 by keywords. A Meta-analysis was performed by Stata12.0 software to estimate the pooled odds ratio (OR) and the 95% confidence interval (CI). Heterogeneity among studies was tested and sensitivity analysis was applied. Publication bias was examined using Begg's funnel plot and Egger's linear regression test. RESULTS: A total of 17 studies were included in this Meta-analysis. For MPO -463 G/A polymorphism, the pooled OR of A allele vs G allele was 0.58 [95% CI (0.47-0.72)] and the pooled OR of genotypes AA+AG vs GG was 0.58 [95% CI (0.46-0.72)]. In subgroup analysis of study population, AA and AG genotypes were significantly associated with CAD in Asians but not in Europeans. The MPO -463 G/A polymorphism in the stable angina pectoris subgroup was evaluated in 3 studies and the pooled OR of A allele vs G allele and genotypes AA+AG vs GG for proven CAD was 0.45 [95% CI (0.15-1.37)] and 0.57 [95% CI (0.19- 1.65)]. For MPO -129 A/G gene polymorphism, the pooled OR of genotype GG vs AA+AG was 0.91 [95% CI (0.74-1.10)]. CONCLUSION: A allele of MPO -463 G/A gene is associated with decreased risk of CAD except in the Europeans. There is no association between MPO -129 A/G gene polymorphisms and CAD risk.