ABSTRACT
This study is to investigate the optimal treatment option for synchronous bilateral breast cancer (SBBC) by comparing dosimetric and radiobiological parameters of intensity-modulated radiotherapy (IMRT) and volumetric modulated arc therapy (VMAT) plans using single and dual isocenters. Twenty patients with SBBC without lymph node involvement were selected retrospectively. Four treatment plans were generated for each patient using the Eclipse treatment planning system (Varian Medical System, Palo Alto, CA, USA) following two delivery techniques with two isocenter conditions-IMRT using a single isocenter (IMRT_Iso1), VMAT using a single isocenter (VMAT_Iso1), IMRT using dual isocenters (IMRT_Iso2), and VMAT using dual isocenters (VMAT_Iso2). A dose of 42.56 Gy in 16 fractions was prescribed for the planning target volume (PTV). All plans were calculated using the Acuros XB algorithm and a photon optimizer for a 6-MV beam of a Vital Beam linear accelerator. PTV-related dosimetric parameters were analyzed. Further, the homogeneity index, conformity index, and conformation number were computed to evaluate plan quality. Dosimetric parameters were also measured for the organs at risk (OARs). In addition, the equivalent uniform dose corresponding to an equivalent dose related to a reference of 2 Gy per fraction, the tumor control probability, and the normal tissue complication probability were calculated based on the dose-volume histogram to investigate the radiobiological impact on PTV and OARs. IMRT_Iso1 exhibited similar target coverage and a certain degree of dosimetric improvement in OAR sparing compared to the other techniques. It also exhibited some radiobiological improvement, albeit insignificant. Although IMRT_Iso1 significantly increased monitor unit compared to VMAT_Iso1, which is the best option in terms of delivery efficiency, there was only a 22% increase in delivery time. Therefore, in conclusion, IMRT_Iso1, the complete treatment of which can be completed using a single setup, is the most effective method for treating SBBC.
Subject(s)
Breast Neoplasms , Radiotherapy, Intensity-Modulated , Breast Neoplasms/radiotherapy , Female , Humans , Organs at Risk , Radiotherapy Dosage , Radiotherapy Planning, Computer-Assisted/methods , Radiotherapy, Intensity-Modulated/methods , Retrospective StudiesABSTRACT
Peroxidasin (PXDN) has been reported to crosslink the C-terminal non-collagenous domains of collagen IV (Col IV) by forming covalent sulfilimine bond. Here, we explored the physiological role of PXDN and its mechanism of action in endothelial cell survival and growth. Silencing of PXDN using siRNAs decreased cell proliferation without increase of the number of detached cells and decreased cell viability under serum-starved condition with increased fragmented nuclei and caspase 3/7 activity. Conditioned medium (CM) containing wild-type PXDN restored the proliferation of PXDN-depleted cells, but CM containing mutant PXDN with deletion of either N-terminal extracellular matrix (ECM) motifs or peroxidase domain failed to restore PXDN function. Accordingly, anti-PXDN antibody [raised against IgC2 (3-4) subdomain within ECM motifs] and peroxidase inhibitor phloroglucinol prevented the rescue of the PXDN-depleted cells by PXDN-containing CM. PXDN depletion resulted in loss of sulfilimine crosslinks, and decreased dense fibrillar network assembly of not only Col IV, but also fibronectin and laminin like in Col IV knockdown. Exogenous PXDN-containing CM restored ECM assembly as well as proliferation of PXDN-depleted cells. Accordingly, purified recombinant PXDN protein restored the proliferation and ECM assembly, and prevented cell death of the PXDN-depleted cells. PXDN depletion also showed reduced growth factors-induced phosphorylation of FAK and ERK1/2. In addition, siPXDN-transfected cell-derived matrix failed to provide full ECM-mediated activation of FAK and ERK1/2. These results indicate that both the ECM motifs and peroxidase activity are essential for the cellular function of PXDN and that PXDN is crucial for ECM assembly for survival and growth signaling.
Subject(s)
Cell Proliferation/drug effects , Cell Survival/drug effects , Endothelial Cells/drug effects , Extracellular Matrix Proteins/metabolism , Extracellular Matrix/metabolism , Imines/pharmacology , Peroxidase/metabolism , Signal Transduction/drug effects , Basement Membrane/drug effects , Basement Membrane/metabolism , Cell Death/drug effects , Cells, Cultured , Collagen Type IV/metabolism , Endothelial Cells/metabolism , Fibronectins/metabolism , Focal Adhesion Kinase 1/metabolism , HEK293 Cells , Human Umbilical Vein Endothelial Cells , Humans , Laminin/metabolism , MAP Kinase Signaling System/drug effects , Peroxidases/metabolism , PeroxidasinABSTRACT
BACKGROUND AND AIMS: Bile acid (BA) dysregulation is related to not only metabolic diseases but also nonalcoholic fatty liver disease (NAFLD). We investigated whether circulating BA levels are altered according to the histological severity of NAFLD independent of metabolic derangements. METHODS: Global metabolic profiling and targeted BA analysis using sera collected from biopsy-proven no-NAFLD (n = 67), nonalcoholic fatty liver (NAFL) (n = 99), and nonalcoholic steatohepatitis (NASH, n = 75) subjects were performed sequentially. Circulating metabolome analysis integrated with the hepatic transcriptome was performed to elucidate the mechanistic basis of altered circulating BA profiles after stratification by obesity (body mass index ≤ 25 kg/m2 ). Circulating BA alterations were also validated in an independent validation cohort (29 no-NAFLD, 70 NAFL and 37 NASH). RESULTS: Global profiling analysis showed that BA was the metabolite significantly altered in NASH compared to NAFL. Targeted BA analysis demonstrated that glyco-/tauro-conjugated primary BAs were commonly increased in nonobese and obese NASH, while unconjugated primary BAs increased only in nonobese NASH. These characteristic primary BA level changes were maintained even after stratification according to diabetes status and were replicated in the independent validation cohort. Compared to nonobese NAFL patients, nonobese NASH patients exhibited upregulated hepatic expression of CYP8B1. CONCLUSIONS: BA metabolism is dysregulated as the histological severity of NAFLD worsens, independent of obesity and diabetes status; dysregulation is more prominent in nonobese NAFLD patients. Metabolome-driven omics approach provides new insight into our understanding of altered BA metabolism associated with individual phenotypes of NAFLD.
Subject(s)
Diabetes Mellitus , Non-alcoholic Fatty Liver Disease , Bile Acids and Salts/metabolism , Humans , Liver/pathology , Non-alcoholic Fatty Liver Disease/complications , Obesity/complications , Obesity/metabolismABSTRACT
A growing body evidence suggests that selenium (Se) deficiency is associated with an increased risk of developing Alzheimer's disease (AD). Se-dependent glutathione peroxidase-1 (GPx-1) of a major antioxidant enzyme, and the most abundant isoform of GPx in the brain. In the present study, we investigated whether GPx-1 is protective against memory impairments induced by beta-amyloid (Aß) (1-42) in mice. As the alteration of protein kinase C (PKC)-mediated ERK activation was recognized in the early stage of AD, we examined whether the GPx-1 gene modulates Aß (1-42)-induced changes in PKC and ERK levels. We observed that Aß (1-42) treatment (400 pmol, i.c.v.) significantly decreased PKC ßII expression in the hippocampus of mice. Aß (1-42)-induced neurotoxic changes [i.e., oxidative stress (i.e., reactive oxygen species, 4-hydroxy-2-noneal, and protein carbonyl), reduced PKC ßII and phospho-ERK expressions, and memory impairment under Y-maze and passive avoidance test] were more pronounced in GPx-1 knockout than in wild type mice. Importantly, exposure to a GPx-1 gene-encoded adenovirus vector (Adv-GPx-1) significantly increased GPx-1 mRNA and GPx activity in the hippocampus of GPx-1 knockout mice. Adv-GPx-1 exposure also significantly blocked the neurotoxic changes induced by Aß (1-42) in GPx-1 knockout mice. Treatment with ERK inhibitor U0126 did not significantly change Adv-GPx-1-mediated attenuation in PKC ßII expression. In contrast, treatment with PKC inhibitor chelerythrine (CHE) reversed Adv-GPx-1-mediated attenuation in ERK phosphorylation, suggesting that PKC ßII-mediated ERK signaling is important for Adv-GPx-1-mediated potentials against Aß (1-42) insult. Our results suggest that treatment with the antioxidant gene GPx-1 rescues Aß (1-42)-induced memory impairment via activating PKC ßII-mediated ERK signaling.
Subject(s)
Glutathione Peroxidase/deficiency , Glutathione Peroxidase/pharmacology , MAP Kinase Signaling System/drug effects , Memory Disorders/enzymology , Memory/drug effects , Protein Kinase C beta/metabolism , Adenoviridae/genetics , Amyloid beta-Peptides , Animals , Gene Expression/drug effects , Genetic Therapy , Glutathione Peroxidase/genetics , Hippocampus/enzymology , Hippocampus/metabolism , Male , Memory Disorders/chemically induced , Memory Disorders/genetics , Memory Disorders/therapy , Mice, Inbred C57BL , Mice, Knockout , Peptide Fragments , Glutathione Peroxidase GPX1ABSTRACT
Cellular antioxidant enzymes play crucial roles in aerobic organisms by eliminating detrimental oxidants and maintaining the intracellular redox homeostasis. Therefore, the function of antioxidant enzymes is inextricably linked to the redox-dependent activities of multiple proteins and signaling pathways. Here, we report that the VEGFR2 RTK has an oxidation-sensitive cysteine residue whose reduced state is preserved specifically by peroxiredoxin II (PrxII) in vascular endothelial cells. In the absence of PrxII, the cellular H(2)O(2) level is markedly increased and the VEGFR2 becomes inactive, no longer responding to VEGF stimulation. Such VEGFR2 inactivation is due to the formation of intramolecular disulfide linkage between Cys1199 and Cys1206 in the C-terminal tail. Interestingly, the PrxII-mediated VEGFR2 protection is achieved by association of two proteins in the caveolae. Furthermore, PrxII deficiency suppresses tumor angiogenesis in vivo. This study thus demonstrates a physiological function of PrxII as the residential antioxidant safeguard specific to the redox-sensitive VEGFR2.
Subject(s)
Antioxidants/metabolism , Aorta/enzymology , Endothelial Cells/enzymology , Endothelium, Vascular/enzymology , Neovascularization, Pathologic/enzymology , Peroxiredoxins , Vascular Endothelial Growth Factor Receptor-2 , Animals , Aorta/cytology , Carcinoma, Lewis Lung/enzymology , Carcinoma, Lewis Lung/pathology , Caveolae/enzymology , Cysteine/chemistry , Cysteine/metabolism , Disulfides/chemistry , Disulfides/metabolism , Endothelial Cells/cytology , Endothelium, Vascular/cytology , Gene Silencing , Humans , Hydrogen Peroxide/metabolism , Mice , Mice, Knockout , Mutagenesis, Site-Directed , Neoplasm Transplantation , Neovascularization, Pathologic/genetics , Oxidation-Reduction , Peroxiredoxins/antagonists & inhibitors , Peroxiredoxins/genetics , Peroxiredoxins/metabolism , Protein Binding , Protein Structure, Tertiary , RNA, Small Interfering , Reactive Oxygen Species/metabolism , Signal Transduction , Vascular Endothelial Growth Factor Receptor-2/genetics , Vascular Endothelial Growth Factor Receptor-2/metabolismABSTRACT
Germ cell tumours (GCTs) are one of the most threatening malignancies in young men and women. Although several reports have suggested the importance of OCT4 in human GCTs, its role has not been clearly investigated on a molecular level. In this study, we revealed GCT-specific direct transcriptional target genes of OCT4. Conditional knockdown of OCT4 in GCT cell lines reduced cell proliferation by affecting both cell cycle and death. Knockdown of OCT4 also reduced stemness of GCTs, as assessed by the expression of other stemness factors, alkaline phosphatase staining, and tumour sphere formation ability. Analysis of whole mRNA expression patterns among GCT cells harbouring endogenous, depleted, and rescued OCT4 revealed 1133 OCT4 target genes in GCT. Combined analysis of both the chromatin binding signature of OCT4 and the genes whose expression levels were changed by OCT4 revealed 258 direct target genes of OCT4 in GCTs. In a similar way, 594 direct target genes in normal embryonic stem cells (ESCs) were identified. Among these two sets of OCT4 direct target genes, 38 genes were common between GCTs and ESCs, most of which were related to regulation of pluripotency, and 220 genes were specific to GCTs, most of which were related to focal adhesion and extracellular matrix organisation. These results provide a molecular basis for how OCT4 regulates GCT stemness and will aid our understanding of the role of OCT4 in other cancers.
Subject(s)
Extracellular Matrix/genetics , Neoplasms, Germ Cell and Embryonal/genetics , Neoplasms, Germ Cell and Embryonal/pathology , Neoplastic Stem Cells/pathology , Octamer Transcription Factor-3/metabolism , Alkaline Phosphatase/analysis , Alkaline Phosphatase/metabolism , Cell Cycle Checkpoints/drug effects , Cell Death/drug effects , Cell Proliferation/drug effects , Cells, Cultured , Doxycycline/pharmacology , Flow Cytometry , Gene Regulatory Networks/genetics , Humans , Neoplastic Stem Cells/metabolism , Octamer Transcription Factor-3/antagonists & inhibitors , Octamer Transcription Factor-3/genetics , Transcription, Genetic/geneticsABSTRACT
BACKGROUND: The aim of this study was to determine the optimal strategy among various arc arrangements in prostate plans of stereotactic body radiotherapy with volumetric modulated arc therapy (SBRT-VMAT). PATIENTS AND METHODS: To investigate how arc arrangements affect dosimetric and biological metrics, SBRT-VMAT plans for eighteen patients were generated with arrangements of single-full arc (1FA), single-partial arc (1PA), double-full arc (2FA), and double-partial arc (2PA). All plans were calculated by the Acuros XB calculation algorithm. Dosimetric and radiobiological metrics for target volumes and organs at risk (OARs) were evaluated from dosevolume histograms. RESULTS: All plans were highly conformal (CI<1.05, CN=0.91) and homogeneous (HI=0.09-0.12) for target volumes. For OARs, there was no difference in the bladder dose, while there was a significant difference in the rectum and both femoral head doses. Plans using 1PA and 2PA showed a strong reduction to the mean rectum dose compared to plans using 1FA and 2FA. Contrastively, the D2% and mean dose in both femoral heads were always lower in plans using 1FA and 2FA. The average tumor control probability and normal tissue complication probability were comparable in plans using all arc arrangements. CONCLUSIONS: The use of 1PA had a more effective delivery time and produced equivalent target coverage with better rectal sparing, although all plans using four arc arrangements showed generally similar for dosimetric and biological metrics. However, the D2% and mean dose in femoral heads increased slightly and remained within the tolerance. Therefore, this study suggests that the use of 1PA is an attractive choice for delivering prostate SBRT-VMAT.
ABSTRACT
The purpose of this study was to compare the performance of different commercial quality assurance (QA) systems for the pretreatment verification plan of stereotactic body radiotherapy (SBRT) with volumetric arc therapy (VMAT) technique using a flattening-filter-free beam. The verification for 20 pretreatment cancer patients (seven lung, six spine, and seven prostate cancers) were tested using three QA systems (EBT3 film, I'mRT MatriXX array, and MapCHECK). All the SBRT-VMAT plans were optimized in the Eclipse (version 11.0.34) treatment planning system (TPS) using the Acuros XB dose calculation algorithm and were delivered to the Varian TrueBeam® accelerator equipped with a high-definition multileaf collimator. Gamma agreement evaluation was analyzed with the criteria of 2% dose difference and 2 mm distance to agreement (2%/2 mm) or 3%/3 mm. The highest passing rate (99.1% for 3%/3 mm) was observed on the MapCHECK system while the lowest passing rate was obtained on the film. The pretreatment verification results depend on the QA systems, treatment sites, and delivery beam energies. However, the delivery QA results for all QA systems based on the TPS calculation showed a good agreement of more than 90% for both the criteria. It is concluded that the three 2D QA systems have sufficient potential for pretreatment verification of the SBRT-VMAT plan.
Subject(s)
Quality Assurance, Health Care , Radiometry/methods , Radiosurgery , Algorithms , Bone Neoplasms/radiotherapy , Humans , Lung Neoplasms/radiotherapy , Male , Prostatic Neoplasms/radiotherapy , Radiometry/instrumentation , Radiotherapy Dosage , Retrospective StudiesABSTRACT
Redox balance has been suggested as an important determinant of "stemness" in embryonic stem cells (ESCs). In this study, we demonstrate that peroxiredoxin (Prx) plays a pivotal role in maintenance of ESC stemness during neurogenesis through suppression of reactive oxygen species (ROS)-sensitive signaling. During neurogenesis, Prx I and Oct4 are expressed in a mutually dependent manner and their expression is abruptly downregulated by an excess of ROS. Thus, in Prx I(-/-) or Prx II(-/-) ESCs, rapid loss of stemness can occur due to spontaneous ROS overload, leading to their active commitment into neurons; however, stemness is restored by the addition of an antioxidant or an inhibitor of c-Jun N-terminal kinase (JNK). In addition, Prx I and Prx II appear to have a tight association with the mechanism underlying the protection of ESC stemness in developing teratomas. These results suggest that Prx functions as a protector of ESC stemness by opposing ROS/JNK cascades during neurogenesis. Therefore, our findings have important implications for understanding of maintenance of ESC stemness through involvement of antioxidant enzymes and may lead to development of an alternative stem cell-based therapeutic strategy for production of high-quality neurons in large quantity.
Subject(s)
Embryonic Stem Cells/enzymology , MAP Kinase Kinase 4/metabolism , Neurogenesis/physiology , Peroxiredoxins/metabolism , Reactive Oxygen Species/metabolism , Animals , Embryonic Stem Cells/cytology , MAP Kinase Kinase 4/genetics , Mice , Mice, Knockout , Peroxiredoxins/geneticsABSTRACT
Peroxiredoxin (Prx) II is an intracellular antioxidant molecule that eliminates hydrogen peroxide, employing a high substrate-binding affinity. PrxII deficiency increases the levels of intracellular reactive oxygen species in many types of cells, which may increase reactive oxygen species-mediated inflammation. In this study, we investigated the susceptibility of PrxII knockout (KO) mice to experimentally induced colitis and the effects of PrxII on the immune system. Wild-type mice displayed pronounced weight loss, high mortality, and colon shortening after dextran sulfate sodium administration, whereas colonic inflammation was significantly attenuated in PrxII KO mice. Although macrophages were hyperactivated in PrxII KO mice, the amount of IFN-γ and IL-17 produced by CD4(+) T cells was substantially reduced. Foxp3(+) regulatory T (Treg) cells were elevated, and Foxp3 protein expression was increased in the absence of PrxII in vitro and in vivo. Restoration of PrxII into KO cells suppressed the increased Foxp3 expression. Interestingly, endogenous PrxII was inactivated through hyperoxidation during Treg cell development. Furthermore, PrxII deficiency stabilized FoxO1 expression by reducing mouse double minute 2 homolog expression and subsequently activated FoxO1-mediated Foxp3 gene transcription. PrxII overexpression, in contrast, reduced FoxO1 and Foxp3 expression. More interestingly, adoptive transfer of naive CD4(+) T cells from PrxII KO mice into immune-deficient mice attenuated T cell-induced colitis, with a reduction in mouse double minute 2 homolog expression and an increase in FoxO1 and Foxp3 expression. These results suggest that inactivation of PrxII is important for the stability of FoxO1 protein, which subsequently mediates Foxp3(+) Treg cell development, thereby attenuating colonic inflammation.
Subject(s)
Colitis/immunology , Forkhead Transcription Factors/metabolism , Peroxiredoxins/metabolism , T-Lymphocytes, Regulatory/immunology , Adoptive Transfer , Animals , CD4-Positive T-Lymphocytes/immunology , CD4-Positive T-Lymphocytes/metabolism , Colitis/genetics , Dextran Sulfate , Forkhead Box Protein O1 , Interferon-gamma/metabolism , Interleukin-17/metabolism , Macrophage Activation/immunology , Macrophages/immunology , Mice , Mice, Inbred C57BL , Mice, Knockout , Peroxiredoxins/genetics , Proto-Oncogene Proteins c-mdm2/biosynthesis , Proto-Oncogene Proteins c-mdm2/metabolism , Reactive Oxygen Species/metabolism , T-Lymphocytes, Regulatory/metabolismABSTRACT
The purpose of this study is to investigate the feasibility of using a flattening filter-free (FFF) beam with an endorectal balloon for stereotactic ablative body radiotherapy (SABR) of clinically localized prostate cancer. We assessed plans of SABR with volumetric-modulated arc therapy (VMAT) that used a flattening filter (FF) beam and an FFF beam and compared the verification results of dosimetric quality assurance for all pretreatment plans. A total of 20 patients with prostate cancer were enrolled in the study. SABR plans using VMAT with two full arcs were optimized in the Eclipse treatment planning system. All plans prescribed 42.7 Gy in 7 fractions of 6.1 Gy each. Four SABR plans were computed for each patient: two with FF beams and two with FFF beams of 6 and 10 MV. For all plans, the cumulative dose-volume histograms (DVHs) for the target volumes and organs at risk (OARs) were recorded and compared. Pretreatment quality assurance (QA) was performed using the I'mRT MatriXX system and radiochromic EBT3 film to verify treatment delivery, and gamma analysis was used to quantify the agreement between calculations and measurements. In addition, total monitor units (MUs) and delivery time were investigated as technical parameters of delivery. All four plans achieved adequate dose conformity to the target volumes and had comparable dosimetric data. The DVHs of all four plans for each patient were very similar. All plans were highly conformal with CI < 1.05 and CN > 0.90, and the doses were homogeneous (HI = 0.08-0.15). Sparing for the bladder and rectum was slightly better with the 10 MV FF and FFF plans than with the 6 MV FF and FFF plans, but the difference was negligible. However, there was no significant difference in sparing for the other OARs. The mean agreement with the 3%/3 mm criterion was higher than 97% for verifying all plans. For the 2%/2 mm criterion, the corresponding agreement values were more than 90%, which showed that the plans were acceptable. The mean MUs and delivery time used were 1701 ± 101 and 3.02 ± 0.17 min for 6 MV FF, 1870 ± 116 and 2.01 ± 0.01 min for 6 MV FFF, 1471 ± 86 and 2.68 ± 0.14 min for 10 MV FF, and 1619 ± 101 and 2.00 ± 0.00 min for 10MV FFF, respectively. In the current study, the dose distributions of the prostate SABR plans using 6 and 10 MV FFF beams were similar to those using 6 and 10 MV FF beams. However, this study confirmed that SABR treatment using an FFF beam had an advantage with respect to delivery time. In addition, all pretreatment plans were verified as acceptable and their results were comparable. Therefore, the results of this study suggest that the use of an FFF beam for prostate SABR is a feasible and efficient technique, if carefully applied.
Subject(s)
Prostatic Neoplasms/radiotherapy , Radiosurgery/methods , Radiotherapy Planning, Computer-Assisted/methods , Radiotherapy, Intensity-Modulated/methods , Feasibility Studies , Humans , Male , Quality Assurance, Health Care , Radiotherapy Dosage , Radiotherapy Planning, Computer-Assisted/standards , Radiotherapy Planning, Computer-Assisted/statistics & numerical data , X-Ray FilmABSTRACT
BACKGROUND: Typical 2-Cys peroxiredoxin (Prx) is inactivated by overoxidation of the peroxidatic cysteine residue under oxidative stress. However, the significance in the context of vascular disease is unknown. METHODS AND RESULTS: Immunohistochemical analyses revealed that 2-Cys Prxs, particularly Prx type II, are heavily overoxidized in balloon-injured rodent carotid vessels and in human atherosclerotic lesions. Consistent with this observation, the selective depletion of Prx II exacerbated neointimal hyperplasia in injured carotid vessels. We also found that the epipolythiodioxopiperazine class of fungal metabolites exhibited an enzyme-like activity mimicking 2-Cys Prx peroxidase and manifestly eliminated the intracellular H2O2 in the vascular cells. Functionally, the epipolythiodioxopiperazines reciprocally regulated the platelet-derived growth factor receptor-ß- and vascular endothelial growth factor receptor-mediated signaling in these vascular cells by replacing Prx II. As a consequence, the epipolythiodioxopiperazines inhibited the proliferative and migratory activities of smooth muscle cells but promoted those of endothelial cells in vitro. Moreover, administration of the epipolythiodioxopiperazines to the injured carotid vessels resulted in a successful recovery by inhibiting neointimal hyperplasia without causing cytotoxicity and simultaneously inducing reendothelialization. CONCLUSIONS: This study reveals for the first time the involvement of the 2-Cys Prx overoxidation and thus the therapeutic use of their activity mimetic in vascular injuries like stenting.
Subject(s)
Biomimetic Materials/therapeutic use , Carotid Artery Injuries/drug therapy , Carotid Artery Injuries/metabolism , Cell Proliferation , Endothelium, Vascular/pathology , Peroxiredoxins/metabolism , Piperazines/therapeutic use , Animals , Biomimetic Materials/pharmacology , Carotid Artery Injuries/pathology , Cell Proliferation/drug effects , Coronary Artery Disease/metabolism , Coronary Artery Disease/pathology , Disease Models, Animal , Endothelium, Vascular/drug effects , Humans , Hydrogen Peroxide/metabolism , Hyperplasia/metabolism , Hyperplasia/pathology , In Vitro Techniques , Male , Mice , Mice, Knockout , Oxidation-Reduction , Piperazines/pharmacology , Rats , Rats, Sprague-Dawley , Reactive Oxygen Species/metabolism , Receptor, Platelet-Derived Growth Factor beta/metabolism , Vascular Endothelial Growth Factor Receptor-2/metabolismABSTRACT
RATIONALE: Peroxiredoxin 2 (Prdx2), a thiol-specific peroxidase, has been reported to regulate proinflammatory responses, vascular remodeling, and global oxidative stress. OBJECTIVE: Although Prdx2 has been proposed to retard atherosclerosis development, no direct evidence and mechanisms have been reported. METHODS AND RESULTS: We show that Prdx2 is highly expressed in endothelial and immune cells in atherosclerotic lesions and blocked the increase of endogenous H(2)O(2) by atherogenic stimulation. Deficiency of Prdx2 in apolipoprotein E-deficient (ApoE(-/-)) mice accelerated plaque formation with enhanced activation of p65, c-Jun, JNKs, and p38 mitogen-activated protein kinase; and these proatherogenic effects of Prdx2 deficiency were rescued by administration of the antioxidant ebselen. In bone marrow transplantation experiments, we found that Prdx2 has a major role in inhibiting atherogenic responses in both vascular and immune cells. Prdx2 deficiency resulted in increased expression of vascular adhesion molecule-1, intercellular adhesion molecule-1, and monocyte chemotactic protein-1, which led to increased immune cell adhesion and infiltration into the aortic intima. Compared with deficiency of glutathione peroxidase 1 or catalase, Prdx2 deficiency showed a severe predisposition to develop atherosclerosis. CONCLUSIONS: Prdx2 is a specific peroxidase that inhibits atherogenic responses in vascular and inflammatory cells, and specific activation of Prdx2 may be an effective means of antiatherogenic therapy.
Subject(s)
Aorta/enzymology , Apolipoproteins E/deficiency , Atherosclerosis/enzymology , Peroxiredoxins/deficiency , Animals , Antioxidants/pharmacology , Aorta/drug effects , Aorta/immunology , Aorta/pathology , Apolipoproteins E/genetics , Atherosclerosis/genetics , Atherosclerosis/immunology , Atherosclerosis/pathology , Atherosclerosis/prevention & control , Azoles/pharmacology , Bone Marrow Cells/enzymology , Bone Marrow Transplantation , Catalase/genetics , Catalase/metabolism , Chemokine CCL2/metabolism , Disease Models, Animal , Endothelial Cells/enzymology , Glutathione Peroxidase/deficiency , Glutathione Peroxidase/genetics , Hydrogen Peroxide/metabolism , Intercellular Adhesion Molecule-1/metabolism , Isoindoles , JNK Mitogen-Activated Protein Kinases/metabolism , Mice , Mice, Inbred C57BL , Mice, Knockout , Organoselenium Compounds/pharmacology , Peroxiredoxins/genetics , Severity of Illness Index , Signal Transduction , Time Factors , Transcription Factor RelA/metabolism , Vascular Cell Adhesion Molecule-1/metabolism , p38 Mitogen-Activated Protein Kinases/metabolism , Glutathione Peroxidase GPX1ABSTRACT
Vascular endothelial growth factor receptor-2 (VEGFR2) plays a key role in maintaining vascular endothelial homeostasis. Here, we show that blood flows determine activation and inactivation of VEGFR2 through selective cysteine modifications. VEGFR2 activation is regulated by reversible oxidation at Cys1206 residue. H2O2-mediated VEGFR2 oxidation is induced by oscillatory flow in vascular endothelial cells through the induction of NADPH oxidase-4 expression. In contrast, laminar flow induces the expression of endothelial nitric oxide synthase and results in the S-nitrosylation of VEGFR2 at Cys1206, which counteracts the oxidative inactivation. The shear stress model study reveals that disturbed blood flow operated by partial ligation in the carotid arteries induces endothelial damage and intimal hyperplasia in control mice but not in knock-in mice harboring the oxidation-resistant mutant (C1206S) of VEGFR2. Thus, our findings reveal that flow-dependent redox regulation of the VEGFR2 kinase is critical for the structural and functional integrity of the arterial endothelium.
Subject(s)
Endothelial Cells , Hydrogen Peroxide , Animals , Mice , Endothelial Cells/metabolism , Endothelium, Vascular/metabolism , Hydrogen Peroxide/metabolism , Oxidation-Reduction , Signal Transduction , Vascular Endothelial Growth Factor A/metabolism , Vascular Endothelial Growth Factor Receptor-2/metabolismABSTRACT
Dry age-related macular degeneration (AMD) is a type of disease that causes visual impairment due to changes in the macula located in the center of the retina. The accumulation of drusen under the retina is also a characteristic of dry AMD. In this study, we identified a compound (JS-017) that can potentially degrade N-retinylidene-N-retinylethanolamine (A2E), one of the components of lipofuscin, using fluorescence-based screening, which measures A2E degradation in human retinal pigment epithelial cells. JS-017 effectively degraded A2E in ARPE-19 cells and consequently suppressed the activation of the NF-κB signaling pathway and expression of inflammatory and apoptosis genes induced by blue light (BL). Mechanistically, JS-017 induced LC3-II formation and improved autophagic flux in ARPE-19 cells. Additionally, the A2E degradation activity of JS-017 was found to be decreased in autophagy-related 5 protein-depleted ARPE-19 cells, suggesting that autophagy was required for A2E degradation mediated by JS-017. Finally, JS-017 exhibited an improvement in BL-induced retinal damage measured through fundus examination in an in vivo retinal degeneration mouse model. The thickness of the outer nuclear layer and inner/external segments, which was decreased upon exposure to BL irradiation, was also restored upon JS-017 treatment. Altogether, we demonstrated that JS-017 protected human retinal pigment epithelium (RPE) cells from A2E and BL-induced damage by degrading A2E via the activation of autophagy. The results suggest the feasibility of a novel A2E-degrading small molecule as a therapeutic agent for retinal degenerative diseases.
Subject(s)
Light , Retina , Humans , Mice , Animals , Retinal Pigment Epithelium/metabolism , Retinal Pigment Epithelium/radiation effects , Cell Line , Autophagy/physiologyABSTRACT
Endothelial dysfunction and inflammatory immune response trigger dedifferentiation of vascular smooth muscle cells (SMCs) from contractile to synthetic phenotype and initiate arterial occlusion. However, the complex vascular remodeling process playing roles in arterial occlusion initiation is largely unknown. We performed bulk sequencing of small and messenger RNAs in a rodent arterial injury model. Bioinformatic data analyses reveal that six miRNAs are overexpressed in injured rat carotids as well as synthetic-type human vascular SMCs. In vitro cell-based assays show that four miRNAs (miR-130b-5p, miR-132-3p, miR-370-3p, and miR-410-3p) distinctly regulate the proliferation of and monocyte adhesion to the vascular SMCs. Individual inhibition of the four selected miRNAs strongly prevents the neointimal hyperplasia in the injured rat carotid arteries. Mechanistically, miR-132-3p and miR-370-3p direct the cell cycle progression, triggering SMC proliferation. Gene ontology analysis of mRNA sequencing data consistently reveal that the miRNA targets include gene clusters that direct proliferation, differentiation, and inflammation. Notably, bone morphogenic protein (BMP)-7 is a prominent target gene of miR-370-3p, and it regulates vascular SMC proliferation in cellular and animal models. Overall, this study first reports that the miR-370-3p/BMP-7 axis determines the vascular SMC phenotype in both rodent and human systems.
Subject(s)
MicroRNAs , Muscle, Smooth, Vascular , Animals , Humans , Rats , Bone Morphogenetic Protein 7/metabolism , Cell Differentiation/genetics , Cell Proliferation/genetics , Cells, Cultured , MicroRNAs/genetics , MicroRNAs/metabolism , Muscle, Smooth, Vascular/metabolism , Myocytes, Smooth Muscle/metabolism , PhenotypeABSTRACT
The 2-Cys peroxiredoxins (Prx) belong to a family of antioxidant enzymes that detoxify reactive oxygen and nitrogen species and are distributed throughout the intracellular and extracellular compartments. However, the presence and role of 2-Cys Prxs in the nucleus have not been studied. This study demonstrates that the PrxII located in the nucleus protects cancer cells from DNA damage-induced cell death. Although the two cytosolic 2-Cys Prxs, PrxI and PrxII, were found in the nucleus, only PrxII knockdown selectively and markedly increased cell death in the cancer cells treated with DNA-damaging agents. The increased death was completely reverted by the nuclearly targeted expression of PrxII in an activity-independent manner. Furthermore, the antioxidant butylated hydroxyanisole did not influence the etoposide-induced cell death. Mechanistically, the knockdown of Prx II expression impaired the DNA repair process by reducing the activation of the JNK/c-Jun pathway. These results suggest that PrxII is likely to be attributed to a tumor survival factor positively regulating JNK-dependent DNA repair with its inhibition possibly sensitizing cancer cells to chemotherapeutic agents.
Subject(s)
Cell Nucleus/enzymology , DNA Damage , DNA Repair , MAP Kinase Kinase 4/metabolism , Neoplasms/enzymology , Peroxiredoxins/metabolism , Antineoplastic Agents, Phytogenic/pharmacology , Antioxidants/metabolism , Cell Death/drug effects , Cell Death/genetics , Cell Nucleus/genetics , Cell Survival/drug effects , Cell Survival/genetics , Etoposide/pharmacology , Gene Expression Regulation, Enzymologic/drug effects , Gene Expression Regulation, Enzymologic/genetics , Gene Expression Regulation, Neoplastic/drug effects , Gene Expression Regulation, Neoplastic/genetics , Gene Knockdown Techniques , HeLa Cells , Humans , MAP Kinase Kinase 4/genetics , Neoplasms/genetics , Peroxiredoxins/genetics , Proto-Oncogene Proteins c-jun/genetics , Proto-Oncogene Proteins c-jun/metabolismABSTRACT
Erythroid cells are highly prone to oxidative damage generated during erythropoiesis and thus are well equipped with antioxidant defense systems. However, their roles have been poorly characterized. Here, we investigated the role of peroxiredoxin II in mouse erythropoiesis. Loss of Prx II significantly increased apoptosis and cell cycle arrest leading to abnormal erythropoiesis at 3 weeks of age when erythropoietin levels were almost same between wild type and Prx II(-/-). In Prx II(-/-) bone marrow cells, DNA tail length as an indicator of the oxidative damage was greatly increased and mRNAs of the molecules associated with DNA damage and repair and transcription regulators of antioxidant enzymes were also significantly increased. In addition, N-Acetyl-L-Cysteine treatment significantly decreased immature erythroblasts and apoptotic cells increased in Prx II(-/-) BMCs. These results strongly demonstrate that Prx II plays an essential role in maintaining normal erythropoiesis by protecting DNA damage.
Subject(s)
DNA Damage , Erythroblasts/physiology , Erythropoiesis/physiology , Peroxiredoxins/physiology , Reactive Oxygen Species/metabolism , Animals , Apoptosis/drug effects , Apoptosis/genetics , Apoptosis/physiology , Bone Marrow Cells/cytology , Bone Marrow Cells/physiology , Cysteine/pharmacology , DNA Repair , Erythroblasts/cytology , Erythroblasts/drug effects , Erythropoiesis/genetics , Mice , Mice, Knockout , Peroxiredoxins/genetics , RNA, Messenger/metabolismABSTRACT
Type 2 granular corneal dystrophy (GCD2) is caused by point mutation R124H in the transforming growth factor-ß-induced gene (TGFBI) and is characterized by age-dependent progression of corneal deposits. Mitochondrial features in heterozygous GCD2 and normal corneal tissues was evaluated using electron microscopy. Primary corneal fibroblasts of homozygous and normal corneas were cultured to passage 4 or 8. Keratocytes of normal corneal tissue are narrow, and details of their intracellular organelles are difficult to distinguish. Keratocytes of heterozygous GCD2 tissues exhibited many degenerative mitochondria. MitoTracker and cytochrome c staining demonstrated increased mitochondrial activity in mutated cells at early passages. Decreases in depolarized mitochondria, cellular proliferation, and expression of complexes I to V and increases in apoptotic change were observed in late-passage mutant fibroblasts. PGC-1α, ANT-1, p-Akt, and p-mTOR but not NF-κB expression demonstrated a passage-dependent decrease in all cells. Increased passage- or mutation-related intracellular reactive oxygen species and delayed proliferation of methanethiosulfonate (MTS) were recovered using application of antioxidant butylated hydroxyanisole. Mitochondrial features and function were altered in mutated GCD2 keratocytes, in particular in older cells. Alteration of mitochondrial function is critical for understanding the pathogenesis of GCD2.
Subject(s)
Corneal Dystrophies, Hereditary/genetics , Mitochondria/metabolism , Animals , Cytochromes c/metabolism , Disease Progression , Fibroblasts/metabolism , Heterozygote , Homozygote , Keratinocytes/cytology , Membrane Potentials , Mice , Microscopy, Electron, Transmission/methods , Mitochondrial Membranes/metabolism , Mutation , Point Mutation , Reactive Oxygen Species , Transforming Growth Factor beta1/geneticsABSTRACT
Hydrogen peroxide (H2O2) accumulates transiently in various cell types stimulated with peptide growth factors and participates in receptor signaling by oxidizing the essential cysteine residues of protein tyrosine phosphatases and the lipid phosphatase PTEN. The reversible inactivation of these phosphatases by H2O2 is likely required to prevent futile cycles of phosphorylation-dephosphorylation of proteins and phosphoinositides. The accumulation of H2O2 is possible even in the presence of large amounts of the antioxidant enzymes peroxiredoxin I and II in the cytosol, probably because of a built-in mechanism of peroxiredoxin inactivation that is mediated by H2O2 and reversed by an ATP-dependent reduction reaction catalyzed by sulfiredoxin.