c3e82de8a3c63a655fb63ee46e568e23 293370.pdf f478e2baea8cb7b58ccf37cfa786c9016bab02f4 293370.pdf d9a129b81b072f6b38c92adf8bdc1b24eda944cbfaeba3a9e10903877e2350e5 293370.pdf Title: Lysyl Oxidase in Ectopic Cardiovascular Calcification: Role of Oxidative Stress Subject: Lysyl oxidase (LOX)-mediated extracellular matrix crosslinking modulates calcification in atherosclerosis and aortic valve disease; however, this enzyme also induces oxidative stress. We addressed the contribution of LOX-dependent oxidative stress to cardiovascular calcification. LOX is upregulated in human-calcified atherosclerotic lesions and atheromas from atherosclerosis-challenged LOX transgenic mice (TgLOXVSMC) and colocalized with a marker of oxidative stress (8-oxo-deoxyguanosine) in vascular smooth muscle cells (VSMCs). Similarly, in calcific aortic valves, high LOX expression was detected in valvular interstitial cells (VICs) positive for 8-oxo-deoxyguanosine, while LOX and LOXL2 expression correlated with osteogenic markers (SPP1 and RUNX2) and NOX2. In human VICs, mito-TEMPO and TEMPOL attenuated the increase in superoxide anion levels and the mineralization induced by osteogenic media (OM). Likewise, in OM-exposed VICs, -aminopropionitrile (a LOX inhibitor) ameliorated both oxidative stress and calcification. Gain- and loss-of-function approaches in VICs demonstrated that while LOX silencing negatively modulates oxidative stress and calcification induced by OM, lentiviral LOX overexpression exacerbated oxidative stress and VIC calcification, effects that were prevented by mito-TEMPO, TEMPOL, and -aminopropionitrile. Our data indicate that LOX-induced oxidative stress participates in the procalcifying effects of LOX activity in ectopic cardiovascular calcification, and highlight the multifaceted role played by LOX isoenzymes in cardiovascular diseases. Keywords: lysyl oxidase; oxidative stress; cardiovascular calcification Author: Carme Ballester-Servera, Judith Alonso, Laia Cañes, Paula Vázquez-Sufuentes, Ana B. García-Redondo, Cristina Rodríguez and José Martínez-González Creator: LaTeX with hyperref Producer: pdfTeX-1.40.25 CreationDate: Fri Apr 26 11:31:35 2024 CEST ModDate: Fri Apr 26 11:35:32 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: 8082955 bytes Optimized: no PDF version: 1.7 name type encoding emb sub uni object ID ------------------------------------ ----------------- ---------------- --- --- --- --------- IOJCNK+VnURWPalladioL-Bold Type 1 Custom yes yes yes 10 0 VVSYYB+EURM10 Type 1 Builtin yes yes yes 16 0 VCPETD+URWPalladioL-Bold Type 1 Custom yes yes yes 21 0 FYYNDW+URWPalladioL-Roma Type 1 Custom yes yes yes 27 0 KYYHSW+URWPalladioL-Ital Type 1 Custom yes yes yes 32 0 YWQWLZ+VnURWPalladioL Type 1 Custom yes yes yes 37 0 KUDXZB+TimesNewRomanPS-BoldItalicMT Type 1C WinAnsi yes yes no 53 0 YFFKLH+CMSY10 Type 1 Builtin yes 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314 0 MCOAEM+PalatinoLinotype TrueType WinAnsi yes yes no 320 0 MCOAEO+PalatinoLinotype,Italic TrueType WinAnsi yes yes no 323 0 Jhove (Rel. 1.28.0, 2023-05-18) Date: 2024-06-06 02:43:48 CEST RepresentationInformation: 293370.pdf ReportingModule: PDF-hul, Rel. 1.12.4 (2023-03-16) LastModified: 2024-06-05 11:17:41 CEST Size: 8082955 Format: PDF Version: 1.7 Status: Well-Formed and valid SignatureMatches: PDF-hul MIMEtype: application/pdf PDFMetadata: Objects: 590 FreeObjects: 1 IncrementalUpdates: 0 DocumentCatalog: PageLayout: SinglePage PageMode: UseNone Outlines: Item: Title: Introduction Destination: section.1 Item: Title: Materials and Methods Destination: section.2 Children: Item: Title: Human Samples Destination: subsection.2.1 Item: Title: VIC Isolation and Osteogenic Induction Destination: subsection.2.2 Item: Title: Immunocytochemistry Destination: subsection.2.3 Item: Title: Analysis of Calcium Deposition in VICs Destination: subsection.2.4 Item: Title: Lentiviral Overexpression in VICs Destination: subsection.2.5 Item: Title: LOX Silencing in VICs Destination: subsection.2.6 Item: Title: Real-Time PCR Destination: subsection.2.7 Item: Title: Animal Handling Destination: subsection.2.8 Item: Title: Histology and Immunohistochemistry Destination: subsection.2.9 Item: Title: ROS Detection Destination: subsection.2.10 Item: Title: Statistical Analysis Destination: subsection.2.11 Item: Title: Results Destination: section.3 Children: Item: Title: LOX Colocalized with 8-oxo-dG in Human-Calcified Atherosclerotic Lesions Destination: subsection.3.1 Item: Title: LOX Transgenesis Enhances Oxidative Stress in Atherosclerotic Lesions Destination: subsection.3.2 Item: Title: LOX and Oxidative Stress in Calcified Human Aortic Valves Destination: subsection.3.3 Item: Title: Antioxidant Treatment Prevents the Calcification of VIC Cultures Induced by High-Phosphate Media Destination: subsection.3.4 Item: Title: LOX Increases Oxidative Stress and Mineralization of VICs Destination: subsection.3.5 Item: Title: Discussion Destination: section.4 Item: Title: Conclusions Destination: section.5 Item: Title: References Destination: appendix.A. Info: Title: Lysyl Oxidase in Ectopic Cardiovascular Calcification: Role of Oxidative Stress Author: Carme Ballester-Servera, Judith Alonso, Laia Cañes, Paula Vázquez-Sufuentes, Ana B. García-Redondo, Cristina Rodríguez and José Martínez-González Subject: Lysyl oxidase (LOX)-mediated extracellular matrix crosslinking modulates calcification in atherosclerosis and aortic valve disease; however, this enzyme also induces oxidative stress. We addressed the contribution of LOX-dependent oxidative stress to cardiovascular calcification. LOX is upregulated in human-calcified atherosclerotic lesions and atheromas from atherosclerosis-challenged LOX transgenic mice (TgLOXVSMC) and colocalized with a marker of oxidative stress (8-oxo-deoxyguanosine) in vascular smooth muscle cells (VSMCs). Similarly, in calcific aortic valves, high LOX expression was detected in valvular interstitial cells (VICs) positive for 8-oxo-deoxyguanosine, while LOX and LOXL2 expression correlated with osteogenic markers (SPP1 and RUNX2) and NOX2. In human VICs, mito-TEMPO and TEMPOL attenuated the increase in superoxide anion levels and the mineralization induced by osteogenic media (OM). Likewise, in OM-exposed VICs, -aminopropionitrile (a LOX inhibitor) ameliorated both oxidative stress and calcification. Gain- and loss-of-function approaches in VICs demonstrated that while LOX silencing negatively modulates oxidative stress and calcification induced by OM, lentiviral LOX overexpression exacerbated oxidative stress and VIC calcification, effects that were prevented by mito-TEMPO, TEMPOL, and -aminopropionitrile. Our data indicate that LOX-induced oxidative stress participates in the procalcifying effects of LOX activity in ectopic cardiovascular calcification, and highlight the multifaceted role played by LOX isoenzymes in cardiovascular diseases. 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