Obesity-driven changes in breast tissue exhibit a pro-angiogenic extracellular matrix signature.

Obesity has reached epidemic proportions in the United States, emerging as a risk factor for the onset of breast cancer and a harbinger of unfavorable outcomes [1], [2], [3]. Despite limited understanding of the precise mechanisms, both obesity and breast cancer are associated with extracellular matrix (ECM) rewiring [4], [5], [6]. Utilizing total breast tissue proteomics, we analyzed normal-weight (18.5 to < 25 kg/m2), overweight (25 to < 30 kg/m2), and obese (≥30 kg/m2) individuals to identify potential ECM modifying proteins for cancer development and acceleration. Obese individuals exhibited substantial ECM alterations, marked by increased basement membrane deposition, angiogenic signatures, and ECM-modifying proteins. Notably, the collagen IV crosslinking enzyme peroxidasin (PXDN) emerged as a potential mediator of the ECM changes in individuals with an elevated body mass index (BMI), strongly correlating with angiogenic and basement membrane signatures. Furthermore, glycan-binding proteins galectin-1 (LGALS1) and galectin-3 (LGALS3), which play crucial roles in matrix interactions and angiogenesis, also strongly correlate with ECM modifications. In breast cancer, elevated PXDN, LGALS1, and LGALS3 correlate with reduced relapse-free and distant-metastatic-free survival. These proteins were significantly associated with mesenchymal stromal cell markers, indicating adipocytes and fibroblasts may be the primary contributors of the obesity-related ECM changes. Our findings unveil a pro-angiogenic ECM signature in obese breast tissue, offering potential targets to inhibit breast cancer development and progression.

Reactive Halogen Species: Role in Living Systems and Current Research Approaches.

Reactive halogen species (RHS) are highly reactive compounds that are normally required for regulation of immune response, inflammatory reactions, enzyme function, etc. At the same time, hyperproduction of highly reactive compounds leads to the development of various socially significant diseases - asthma, pulmonary hypertension, oncological and neurodegenerative diseases, retinopathy, and many others. The main sources of (pseudo)hypohalous acids are enzymes from the family of heme peroxidases - myeloperoxidase, lactoperoxidase, eosinophil peroxidase, and thyroid peroxidase. Main targets of these compounds are proteins and peptides, primarily methionine and cysteine residues. Due to the short lifetime, detection of RHS can be difficult. The most common approach is detection of myeloperoxidase, which is thought to reflect the amount of RHS produced, but these methods are indirect, and the results are often contradictory. The most promising approaches seem to be those that provide direct registration of highly reactive compounds themselves or products of their interaction with components of living cells, such as fluorescent dyes. However, even such methods have a number of limitations and can often be applied mainly for in vitro studies with cell culture. Detection of reactive halogen species in living organisms in real time is a particularly acute issue. The present review is devoted to RHS, their characteristics, chemical properties, peculiarities of interaction with components of living cells, and methods of their detection in living systems. Special attention is paid to the genetically encoded tools, which have been introduced recently and allow avoiding a number of difficulties when working with living systems.

Identification of tyrosine brominated extracellular matrix proteins in normal and fibrotic lung tissues.

Peroxidasin (PXDN) is a secreted heme peroxidase that catalyzes the oxidative crosslinking of collagen IV within the extracellular matrix (ECM) via intermediate hypobromous acid (HOBr) synthesis from hydrogen peroxide and bromide, but recent findings have also suggested alternative ECM protein modifications by PXDN, including incorporation of bromide into tyrosine residues. In this work, we sought to identify the major target proteins for tyrosine bromination by HOBr or by PXDN-mediated oxidation in ECM from mouse teratocarcinoma PFHR9 cells. We detected 61 bromotyrosine (BrY)-containing peptides representing 23 proteins in HOBr-modified ECM from PFHR9 cells, among which laminins displayed the most prominent bromotyrosine incorporation. Moreover, we also found that laminin α1, laminin ß1, and tubulointerstitial nephritis antigen-like (TINAGL1) contained BrY in untreated PFHR9 cells, which depended on PXDN. We extended these analyses to lung tissues from both healthy mice and mice with experimental lung fibrosis, and in lung tissues obtained from human subjects. Analysis of ECM-enriched mouse lung tissue extracts showed that 83 ECM proteins were elevated in bleomycin-induced fibrosis, which included various collagens and laminins, and PXDN. Similarly, mRNA and protein expression of PXDN and laminin α/ß1 were enhanced in fibrotic mouse lung tissues, and also in mouse bone-marrow-derived macrophages or human fibroblasts stimulated with transforming growth factor ß1, a profibrotic growth factor. We identified 11 BrY-containing ECM proteins, including collagen IV α2, collagen VI α1, TINAGL1, and various laminins, in both healthy and mouse fibrotic lung tissues, although the relative extent of tyrosine bromination of laminins was not significantly increased during fibrosis. Finally, we also identified 7 BrY-containing ECM proteins in human lung tissues, again including collagen IV α2, collagen VI α1, and TINAGL1. Altogether, this work demonstrates the presence of several bromotyrosine-modified ECM proteins, likely involving PXDN, even in normal lung tissues, suggesting a potential biological function for these modifications.

Mechanism of peroxidasin inactivation in hyperglycemia: Heme damage by reactive oxygen species.

Diabetic complications present a serious health problem. Functional damage to proteins due to post-translational modifications by glycoxidation reactions is a known factor contributing to pathology. Extracellular proteins are especially vulnerable to diabetic damage because robust antioxidant defenses are lacking outside the cell. We investigated glucose-induced inactivation of peroxidasin (PXDN), a heme protein catalyzing sulfilimine crosslinking of collagen IV that reinforce the basement membranes (BM). Experiments using physiological diabetic glucose levels were carried out to exclude several potential mechanisms of PXDN inactivation i.e., direct adduction of glucose, reactive carbonyl damage, steric hindrance, and osmotic stress. Further experiments established that PXDN activity was inhibited via heme degradation by reactive oxygen species. Activity of another extracellular heme protein, myeloperoxidase, was unaffected by glucose because its heme was resistant to glucose-induced oxidative degradation. Our findings point to specific mechanisms which may compromise BM structure and stability in diabetes and suggest potential modes of protection.

Identification of brominated proteins in renal extracellular matrix: Potential interactions with peroxidasin.

Peroxidasin (PXDN) is an extracellular peroxidase, which generates hypobromous acid to form sulfilimine cross-links within collagen IV networks. We have previously demonstrated that mouse and human renal basement membranes (BM) are enriched in bromine due to PXDN-dependent post-translational bromination of protein tyrosine residues. The goal of the present study was identification of specific brominated sites within renal BM. A comprehensive analysis of brominated proteome of mouse glomerular matrix had been performed using liquid chromatography-tandem mass spectrometry. We found that out of over 200 identified proteins, only three were detectably brominated, each containing a single distinct brominated tyrosine site i.e., Tyr-1485 in collagen IV α2 chain, Tyr-292 in TINAGL1 and Tyr-664 in nidogen-2. To explain this highly selective bromination, we proposed that these proteins interact with PXDN within the glomerular matrix. Experiments using purified proteins demonstrated that both TINAGL1 and nidogen-2 can compete with PXDN for binding to collagen IV and that TINAGL1 can directly interact with PXDN. We propose that a protein complex, including PXDN, TINAGL1, nidogen-2 and collagen IV, may exist in renal BM.

Quantitative proteomics identifies tumour matrisome signatures in patients with non-small cell lung cancer.

Introduction: The composition and remodelling of the extracellular matrix (ECM) are important factors in the development and progression of cancers, and the ECM is implicated in promoting tumour growth and restricting anti-tumour therapies through multiple mechanisms. The characterisation of differences in ECM composition between normal and diseased tissues may aid in identifying novel diagnostic markers, prognostic indicators and therapeutic targets for drug development. Methods: Using tissue from non-small cell lung cancer (NSCLC) patients undergoing curative intent surgery, we characterised quantitative tumour-specific ECM proteome signatures by mass spectrometry. Results: We identified 161 matrisome proteins differentially regulated between tumour tissue and nearby non-malignant lung tissue, and we defined a collagen hydroxylation functional protein network that is enriched in the lung tumour microenvironment. We validated two novel putative extracellular markers of NSCLC, the collagen cross-linking enzyme peroxidasin and a disintegrin and metalloproteinase with thrombospondin motifs 16 (ADAMTS16), for discrimination of malignant and non-malignant lung tissue. These proteins were up-regulated in lung tumour samples, and high PXDN and ADAMTS16 gene expression was associated with shorter survival of lung adenocarcinoma and squamous cell carcinoma patients, respectively. Discussion: These data chart extensive remodelling of the lung extracellular niche and reveal tumour matrisome signatures in human NSCLC.

PXDNL activates the motility of urothelial bladder carcinoma cells through the Wnt/ß-catenin pathway and has a prognostic value.

AIMS: Although aberrant expression of peroxidasin-like (PXDNL) has been associated with carcinogenesis, its potential role in the Urothelial Carcinoma of the Bladder (UCB) remains unknown. The present study aimed to explore the role of PXDNL in UCB carcinogenesis and its potential clinical value. MAIN METHODS: Based on The Cancer Genome Atlas (TCGA) data, bioinformatics was used to explore the potential clinical value of PXDNL. Wound healing and Transwell invasion assays were employed for the purpose of assessing the cell motility, while the Western Blotting experiments were utilized for investigating the protein expression pattern of PXDNL in UCB and investigating the Epithelial-to-Mesenchymal Transition (EMT) and Wnt/ß-catenin pathways for understanding the probable mechanisms involved. KEY FINDS: PXDNL mRNA was overexpressed in UCB tissues and indicated a poor prognosis. High PXDNL mRNA levels were also associated with advanced clinicopathological features and were regarded as independent prognostic factors for UCB. However, PXDNL showed a weak correlation with immune cell infiltration in UCB. In addition, the findings of the study verified that the existing form of the PXDNL protein was 57-kDa and it was upregulated in the UCB cell lines and tissue samples. Furthermore, silencing PXDNL inhibited, while overexpressing PXDNL promoted EMT and motility of UCB cells in vitro. Mechanistic studies showed that PXDNL activated UCB cell motility via the Wnt/ß-catenin pathway. SIGNIFICANCE: The results reveal a novel molecular target that could be further explored for developing preventive, predictive, and individualized treatment strategies for UCB.

Peroxidasin Inhibition by Phloroglucinol and Other Peroxidase Inhibitors.

Human peroxidasin (PXDN) is a ubiquitous peroxidase enzyme expressed in most tissues in the body. PXDN represents an interesting therapeutic target for inhibition, as it plays a role in numerous pathologies, including cardiovascular disease, cancer and fibrosis. Like other peroxidases, PXDN generates hypohalous acids and free radical species, thereby facilitating oxidative modifications of numerous biomolecules. We have studied the inhibition of PXDN halogenation and peroxidase activity by phloroglucinol and 14 other peroxidase inhibitors. Although a number of compounds on their own potently inhibited PXDN halogenation activity, only five were effective in the presence of a peroxidase substrate with IC50 values in the low µM range. Using sequential stopped-flow spectrophotometry, we examined the mechanisms of inhibition for several compounds. Phloroglucinol was the most potent inhibitor with a nanomolar IC50 for purified PXDN and IC50 values of 0.95 µM and 1.6 µM for the inhibition of hypobromous acid (HOBr)-mediated collagen IV cross-linking in a decellularized extracellular matrix and a cell culture model. Other compounds were less effective in these models. Most interestingly, phloroglucinol was identified to irreversibly inhibit PXDN, either by mechanism-based inhibition or tight binding. Our work has highlighted phloroglucinol as a promising lead compound for the design of highly specific PXDN inhibitors and the assays used in this study provide a suitable approach for high-throughput screening of PXDN inhibitors.

Measuring peroxidasin activity in live cells using bromide addition for signal amplification.

Peroxidasin (PXDN) is involved in the crosslinking of collagen IV, a major constituent of basement membranes. Disruption of basement membrane integrity as observed in genetic alterations of collagen IV or PXDN can result in developmental defects and diverse pathologies. Hence, the study of PXDN activity in (patho)physiological contexts is highly relevant. So far, measurements of PXDN activity have been reported from purified proteins, cell lysates and de-cellularized extracellular matrix. Here, for the first time we report the measurement of PXDN activity in live cells using the Amplex Red assay with a signal amplifying modification. We observe that bromide addition enhances the obtained signal, most likely due to formation of HOBr. Abrogation of signal amplification by the HOBr scavenger carnosine supports this hypothesis. Both, pharmacological inhibition as well as complementary genetic approaches confirm that the obtained signal is indeed related to PXDN activity. We validate the modified assay by investigating the effect of Brefeldin A, to inhibit the secretory pathway and thus the access of PXDN to the extracellular Amplex Red dye. Our method opens up new possibilities to investigate the activity of PXDN in (patho)physiological contexts.

Uric Acid Reacts with Peroxidasin, Decreases Collagen IV Crosslink, Impairs Human Endothelial Cell Migration and Adhesion.

Uric acid is considered the main substrate for peroxidases in plasma. The oxidation of uric acid by human peroxidases generates urate free radical and urate hydroperoxide, which might affect endothelial function and explain, at least in part, the harmful effects of uric acid on the vascular system. Peroxidasin (PXDN), the most recent heme-peroxidase described in humans, catalyzes the formation of hypobromous acid, which mediates collagen IV crosslinks in the extracellular matrix. This enzyme has gained increasing scientific interest since it is associated with cardiovascular disease, cancer, and renal fibrosis. The main objective here was to investigate whether uric acid would react with PXDN and compromise the function of the enzyme in human endothelial cells. Urate decreased Amplex Red oxidation and brominating activity in the extracellular matrix (ECM) from HEK293/PXDN overexpressing cells and in the secretome of HUVECs. Parallelly, urate was oxidized to 5-hydroxyisourate. It also decreased collagen IV crosslink in isolated ECM from PFHR9 cells. Urate, the PXDN inhibitor phloroglucinol, and the PXDN knockdown impaired migration and adhesion of HUVECs. These results demonstrated that uric acid can affect extracellular matrix formation by competing for PXDN. The oxidation of uric acid by PXDN is likely a relevant mechanism in the endothelial dysfunction related to this metabolite.

Halogenation Activity of Mammalian Heme Peroxidases.

Mammalian heme peroxidases are fascinating due to their unique peculiarity of oxidizing (pseudo)halides under physiologically relevant conditions. These proteins are able either to incorporate oxidized halides into substrates adjacent to the active site or to generate different oxidized (pseudo)halogenated species, which can take part in multiple (pseudo)halogenation and oxidation reactions with cell and tissue constituents. The present article reviews basic biochemical and redox mechanisms of (pseudo)halogenation activity as well as the physiological role of heme peroxidases. Thyroid peroxidase and peroxidasin are key enzymes for thyroid hormone synthesis and the formation of functional cross-links in collagen IV during basement membrane formation. Special attention is directed to the properties, enzymatic mechanisms, and resulting (pseudo)halogenated products of the immunologically relevant proteins such as myeloperoxidase, eosinophil peroxidase, and lactoperoxidase. The potential role of the (pseudo)halogenated products (hypochlorous acid, hypobromous acid, hypothiocyanite, and cyanate) of these three heme peroxidases is further discussed.

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.

Peroxidasin Deficiency Re-programs Macrophages Toward Pro-fibrolysis Function and Promotes Collagen Resolution in Liver.

BACKGROUND & AIMS: During liver fibrosis, tissue repair mechanisms replace necrotic tissue with highly stabilized extracellular matrix proteins. Extracellular matrix stabilization influences the speed of tissue recovery. Here, we studied the expression and function of peroxidasin (PXDN), a peroxidase that uses hydrogen peroxide to cross-link collagen IV during liver fibrosis progression and regression. METHODS: Mouse models of liver fibrosis and cirrhosis patients were analyzed for the expression of PXDN in liver and serum. Pxdn-/- and Pxdn+/+ mice were either treated with carbon tetrachloride for 6 weeks to generate toxin-induced fibrosis or fed with a choline-deficient L-amino acid-defined high-fat diet for 16 weeks to create nonalcoholic fatty liver disease fibrosis. Liver histology, quantitative real-time polymerase chain reaction, collagen content, flowcytometry and immunostaining of immune cells, RNA-sequencing, and liver function tests were analyzed. In vivo imaging of liver reactive oxygen species (ROS) was performed using a redox-active iron complex, Fe-PyC3A. RESULTS: In human and mouse cirrhotic tissue, PXDN is expressed by stellate cells and is secreted into fibrotic areas. In patients with nonalcoholic fatty liver disease, serum levels of PXDN increased significantly. In both mouse models of liver fibrosis, PXDN deficiency resulted in elevated monocyte and pro-fibrolysis macrophage recruitment into fibrotic bands and caused decreased accumulation of cross-linked collagens. In Pxdn-/- mice, collagen fibers were loosely organized, an atypical phenotype that is reversible upon macrophage depletion. Elevated ROS in Pxdn-/- livers was observed, which can result in activation of hypoxic signaling cascades and may affect signaling pathways involved in macrophage polarization such as TNF-a via NF-kB. Fibrosis resolution in Pxdn-/- mice was associated with significant decrease in collagen content and improved liver function. CONCLUSION: PXDN deficiency is associated with increased ROS levels and a hypoxic liver microenvironment that can regulate recruitment and programming of pro-resolution macrophages. Our data implicate the importance of the liver microenvironment in macrophage programming during liver fibrosis and suggest a novel pathway that is involved in the resolution of scar tissue.

Peroxidasin Enhances Basal Phenotype and Inhibits Branching Morphogenesis in Breast Epithelial Progenitor Cell Line D492.

The human breast is composed of terminal duct lobular units (TDLUs) that are surrounded by stroma. In the TDLUs, basement membrane separates the stroma from the epithelial compartment, which is divided into an inner layer of luminal epithelial cells and an outer layer of myoepithelial cells. Stem cells and progenitor cells also reside within the epithelium and drive a continuous cycle of gland remodelling that occurs throughout the reproductive period. D492 is an epithelial cell line originally isolated from the stem cell population of the breast and generates both luminal and myoepithelial cells in culture. When D492 cells are embedded into 3D reconstituted basement membrane matrix (3D-rBM) they form branching colonies mimicking the TDLUs of the breast, thereby providing a well-suited in vitro model for studies on branching morphogenesis and breast development. Peroxidasin (PXDN) is a heme-containing peroxidase that crosslinks collagen IV with the formation of sulfilimine bonds. Previous studies indicate that PXDN plays an integral role in basement membrane stabilisation by crosslinking collagen IV and as such contributes to epithelial integrity. Although PXDN has been linked to fibrosis and cancer in some organs there is limited information on its role in development, including in the breast. In this study, we demonstrate expression of PXDN in breast epithelium and stroma and apply the D492 cell line to investigate the role of PXDN in cell differentiation and branching morphogenesis in the human breast. Overexpression of PXDN induced basal phenotype in D492 cells, loss of plasticity and inhibition of epithelial-to-mesenchymal transition as is displayed by complete inhibition of branching morphogenesis in 3D culture. This is supported by results from RNA-sequencing which show significant enrichment in genes involved in epithelial differentiation along with significant negative enrichment of EMT factors. Taken together, we provide evidence for a novel role of PXDN in breast epithelial differentiation and mammary gland development.

Characterization of the Proprotein Convertase-Mediated Processing of Peroxidasin and Peroxidasin-like Protein.

Peroxidasin (PXDN) and peroxidasin-like protein (PXDNL) are members of the peroxidase-cyclooxygenase superfamily. PXDN functions in basement membrane synthesis by forming collagen IV crosslinks, while the function of PXDNL remains practically unknown. In this work, we characterized the post-translational proteolytic processing of PXDN and PXDNL. Using a novel knock-in mouse model, we demonstrate that the proteolytic cleavage of PXDN occurs in vivo. With the help of furin-specific siRNA we also demonstrate that the proprotein-convertase, furin participates in the proteolytic processing of PXDN. Furthermore, we demonstrate that only the proteolytically processed PXDN integrates into the extracellular matrix, highlighting the importance of the proteolysis step in PXDN's collagen IV-crosslinking activity. We also provide multiple lines of evidence for the importance of peroxidase activity in the proteolytic processing of PXDN. Finally, we show that PXDNL does not undergo proteolytic processing, despite containing sequence elements efficiently recognized by proprotein convertases. Collectively, our observations suggest a previously unknown protein quality control during PXDN synthesis and the importance of the peroxidase activity of PXDN in this process.

Peroxidasin protein expression and enzymatic activity in metastatic melanoma cell lines are associated with invasive potential.

Peroxidasin, a heme peroxidase, has been shown to play a role in cancer progression. mRNA expression has been reported to be upregulated in metastatic melanoma cell lines and connected to the invasive phenotype, but little is known about how peroxidasin acts in cancer cells. We have analyzed peroxidasin protein expression and activity in eight metastatic melanoma cell lines using an ELISA developed with an in-house peroxidasin binding protein. RNAseq data analysis confirmed high peroxidasin mRNA expression in the five cell lines classified as invasive and low expression in the three non-invasive cell lines. Protein levels of peroxidasin were higher in the cell lines with an invasive phenotype. Active peroxidasin was secreted to the cell culture medium, where it accumulated over time, and peroxidasin protein levels in the medium were also much higher in invasive than non-invasive cell lines. The only well-established physiological role of peroxidasin is in the formation of a sulfilimine bond, which cross-links collagen IV in basement membranes via catalyzed oxidation of bromide to hypobromous acid. We found that peroxidasin secreted from melanoma cells formed sulfilimine bonds in uncross-linked collagen IV, confirming peroxidasin activity and hypobromous acid formation. Moreover, 3-bromotyrosine, a stable product of hypobromous acid reacting with tyrosine residues, was detected in invasive melanoma cells, substantiating that their expression of peroxidasin generates hypobromous acid, and showing that it does not exclusively react with collagen IV, but also with other biomolecules.

Enzymatic cross-linking of collagens in organ fibrosis - resolution and assessment.

Introduction: Enzymatic cross-linking of the collagens within the extracellular matrix (ECM) catalyzed by enzymes such as lysyl oxidase (LOX) and lysyl oxidase like-enzymes 1-4 (LOXL), transglutaminase 2 (TG2), and peroxidasin (PXDN) contribute to fibrosis progression through extensive collagen cross-linking. Studies in recent years have begun elucidating the important role of collagen cross-linking in perpetuating progression of organ fibrosis independently of inflammation through an increasingly stiff and noncompliant ECM. Therefore, collagen cross-linking and the cross-linking enzymes have become new targets in anti-fibrotic therapy as well as targets of novel biomarkers to properly assess resolution of the fibrotic ECM.Areas covered: The enzymatic actions of enzymes catalyzing collagen cross-linking and their relevance in organ fibrosis. Potential biomarkers specifically quantifying proteolytic fragments of collagen cross-linking is discussed based on Pubmed search done in November 2020 as well as the authors knowledge.Expert opinion: Current methods for the assessment of fibrosis involve the use of invasive and/or cumbersome and expensive methods such as tissue biopsies. Thus, an unmet need exists for the development and validation of minimally invasive biomarkers of proteolytic fragments of cross-linked collagens. These biomarkers may aid in the development and proper assessment of fibrosis resolution in coming years.

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.

Inhibition of Myeloperoxidase.

Myeloperoxidase participates in innate immune defense mechanism through formation of microbicidal reactive oxidants and diffusible radical species. A unique activity is its ability to use chloride as a cosubstrate with hydrogen peroxide to generate chlorinating oxidants such as hypochlorous acid, a potent antimicrobial agent. However, chronic MPO activation can lead to indiscriminate protein modification causing tissue damage, and has been associated with chronic inflammatory diseases, atherosclerosis, and acute cardiovascular events. This has attracted considerable interest in the development of therapeutically useful MPO inhibitors. Today, based on the profound knowledge of structure and function of MPO and its biochemical and biophysical differences with the other homologous human peroxidases, various rational and high-throughput screening attempts were performed in developing specific irreversible and reversible inhibitors. The most prominent candidates as well as MPO inhibitors already studied in clinical trials are introduced and discussed.

Peroxidasin mediates bromination of tyrosine residues in the extracellular matrix.

Peroxidasin is a heme peroxidase that oxidizes bromide to hypobromous acid (HOBr), a powerful oxidant that promotes the formation of the sulfilimine crosslink in collagen IV in basement membranes. We investigated whether HOBr released by peroxidasin leads to other oxidative modifications of proteins, particularly bromination of tyrosine residues, in peroxidasin-expressing PFHR9 cells. Using stable isotope dilution LC-MS/MS, we detected the formation of 3-bromotyrosine, a specific biomarker of HOBr-mediated protein modification. The level of 3-bromotyrosine in extracellular matrix proteins from normally cultured cells was 1.1 mmol/mol tyrosine and decreased significantly in the presence of the peroxidasin inhibitor, phloroglucinol. A negligible amount of 3-bromotyrosine was detected in peroxidasin-knockout cells. 3-Bromotyrosine formed both during cell growth in culture and in the isolated decellularized extracellular matrix when embedded peroxidasin was supplied with hydrogen peroxide and bromide. The level of 3-bromotyrosine was significantly higher in extracellular matrix than intracellular proteins, although a low amount was detected intracellularly. 3-Bromotyrosine levels increased with higher bromide concentrations and decreased in the presence of physiological concentrations of thiocyanate and urate. However, these peroxidase substrates showed moderate to minimal inhibition of collagen IV crosslinking. Our findings provide evidence that peroxidasin promotes the formation of 3-bromotyrosine in proteins. They show that HOBr produced by peroxidasin is selective for, but not limited to, the crosslinking of collagen IV. Based on our findings, the use of 3-bromotyrosine as a specific biomarker of oxidative damage by HOBr warrants further investigation in clinical conditions linked to high peroxidasin expression.