7ad481c566d592d70ae8c3e551d63565 309037.pdf 71e55362de51b23baa4447cf6e600acc859b544f 309037.pdf cfdac34cb8544b755065175aa906c5e9d45ff84791620ca6d8efa48f508dd4a0 309037.pdf Title: NADPH Oxidase 5 (NOX5) Upregulates MMP-10 Production and Cell Migration in Human Endothelial Cells Subject: NADPH oxidases (NOXs) have been described as critical players in vascular remodeling, a mechanism modulated by matrix metalloproteinases. In this study, we describe for the first time the upregulation of MMP-10 through the activation of NOX5 in endothelial cells. In a chronic NOX5 overexpression model in human endothelial cells, MMP-10 production was measured at different levels: extracellular secretion, gene expression (mRNA and protein levels), and promoter activity. Effects on cell migration were quantified using wound healing assays. NOX5 overexpression increased MMP-10 production, favoring cell migration. In fact, NOX5 and MMP-10 silencing prevented this promigratory effect. We showed that NOX5-mediated MMP-10 upregulation involves the redox-sensitive JNK/AP-1 signaling pathway. All these NOX5-dependent effects were enhanced by angiotensin II (Ang II). Interestingly, MMP-10 protein levels were found to be increased in the hearts of NOX5-expressing mice. In conclusion, we described that NOX5-generated ROS may modulate the MMP-10 expression in endothelial cells, which leads to endothelial cell migration and may play a key role in vascular remodeling. Keywords: oxidative stress; NADPH oxidase 5; MMP-10; AP-1; cell migration; endothelial cells Author: Javier Marqués, Elena Ainzúa, Josune Orbe, María Martínez-Azcona, José Martínez-González and Guillermo Zalba Creator: LaTeX with hyperref Producer: pdfTeX-1.40.25 CreationDate: Thu Oct 3 09:29:46 2024 CEST ModDate: Thu Oct 3 09:34:14 2024 CEST Custom Metadata: no Metadata Stream: no Tagged: no UserProperties: no Suspects: no Form: none JavaScript: no Pages: 19 Encrypted: no Page size: 595.276 x 841.89 pts (A4) Page rot: 0 File size: 4769167 bytes Optimized: no PDF version: 1.7 name type encoding emb sub uni object ID ------------------------------------ ----------------- ---------------- --- --- --- --------- OSMCID+VnURWPalladioL-Bold Type 1 Custom yes yes yes 10 0 PPNDQH+URWPalladioL-Bold Type 1 Custom yes yes yes 16 0 MJAFQF+URWPalladioL-Roma Type 1 Custom yes yes yes 22 0 GSCHJN+URWPalladioL-Ital Type 1 Custom yes yes yes 27 0 FJMQIG+VnURWPalladioL Type 1 Custom yes yes yes 32 0 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Silencing Destination: subsection.2.2 Item: Title: NOX5 Acute Overexpression Model Destination: subsection.2.3 Item: Title: NOX5 Chronic Overexpression Model Destination: subsection.2.4 Item: Title: Cell Culture Reagents Destination: subsection.2.5 Item: Title: Wound Healing Assay Destination: subsection.2.6 Item: Title: RNA Isolation, Reverse-Transcription and qPCR Destination: subsection.2.7 Item: Title: gDNA Extraction and Cell Line Genotyping Destination: subsection.2.8 Item: Title: Protein Extraction and Immunodetection Destination: subsection.2.9 Item: Title: Extracellular MMP-10 Detection by ELISA Destination: subsection.2.10 Item: Title: MMP-10 Promoter Activity Assays Destination: subsection.2.11 Item: Title: ROS Production Destination: subsection.2.12 Item: Title: Real-Time Proliferation Measurement Destination: subsection.2.13 Item: Title: In Vivo Studies Destination: subsection.2.14 Item: Title: Statistical Analysis Destination: subsection.2.15 Item: Title: Results Destination: section.3 Children: Item: Title: NOX5 Enhances MMP-10 Production and Cell Migration in Human Endothelial Cells Destination: subsection.3.1 Item: Title: MMP-10 Is Involved in Cell Migration in Human Endothelial Cells Destination: subsection.3.2 Item: Title: NOX5 Chronic Overexpression Enhances MMP-10 Production and the Associated Cell Migration in Human Endothelial Cells Destination: subsection.3.3 Item: Title: Ang II Stimulation of NOX5-Overexpressing Human Endothelial Cells Enhances MMP-10 Production and Cell Migration Destination: subsection.3.4 Item: Title: ERK, p38, and JNK Are Involved in the Ang II/NOX5/MMP-10/Cell Migration Pathway Destination: subsection.3.5 Item: Title: NOX5 Enhances MMP-10 Promoter Activity via JNK and AP-1 Destination: subsection.3.6 Item: Title: Endothelial NOX5 Expression Leads to Cardiac MMP-10 Upregulation at Protein Levels Destination: subsection.3.7 Item: Title: Discussion Destination: section.4 Item: Title: Conclusions Destination: section.5 Item: Title: References Destination: appendix.A. Info: Title: NADPH Oxidase 5 (NOX5) Upregulates MMP-10 Production and Cell Migration in Human Endothelial Cells Author: Javier Marqués, Elena Ainzúa, Josune Orbe, María Martínez-Azcona, José Martínez-González and Guillermo Zalba Subject: NADPH oxidases (NOXs) have been described as critical players in vascular remodeling, a mechanism modulated by matrix metalloproteinases. In this study, we describe for the first time the upregulation of MMP-10 through the activation of NOX5 in endothelial cells. In a chronic NOX5 overexpression model in human endothelial cells, MMP-10 production was measured at different levels: extracellular secretion, gene expression (mRNA and protein levels), and promoter activity. Effects on cell migration were quantified using wound healing assays. NOX5 overexpression increased MMP-10 production, favoring cell migration. In fact, NOX5 and MMP-10 silencing prevented this promigratory effect. We showed that NOX5-mediated MMP-10 upregulation involves the redox-sensitive JNK/AP-1 signaling pathway. All these NOX5-dependent effects were enhanced by angiotensin II (Ang II). Interestingly, MMP-10 protein levels were found to be increased in the hearts of NOX5-expressing mice. In conclusion, we described that NOX5-generated ROS may modulate the MMP-10 expression in endothelial cells, which leads to endothelial cell migration and may play a key role in vascular remodeling. 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