Marcello Rattazzi1, Maristella Donato2, Elisa Bertacco3, Renato Millioni4, Cinzia Franchin5, Cinzia Mortarino6, Elisabetta Faggin3, Chiara Nardin7, Riccardo Scarpa7, Francesco Cinetto7, Carlo Agostini7, Nicola Ferri2, Paolo Pauletto8, Giorgio Arrigoni5. 1. Department of Medicine, University of Padova, Italy; Medicina Interna I^, Cà Foncello Hospital, Treviso, Italy. Electronic address: marcello.rattazzi@unipd.it. 2. Department of Pharmaceutical and Pharmacological Sciences, University of Padova, Italy. 3. Department of Medicine, University of Padova, Italy. 4. Department of Medicine, University of Padova, Italy; Proteomics Center, University of Padova and Azienda Ospedaliera di Padova, Italy. 5. Department of Biomedical Sciences, University of Padova, Italy; Proteomics Center, University of Padova and Azienda Ospedaliera di Padova, Italy. 6. Department of Statistical Sciences, University of Padova, Italy. 7. Department of Medicine, University of Padova, Italy; Medicina Interna I^, Cà Foncello Hospital, Treviso, Italy. 8. ORAS Rehabilitation Hospital, Motta di Livenza, Treviso, Italy.
Abstract
BACKGROUND AND AIMS: Reduced bioavailability of nitric oxide (NO) has been implicated in the pathogenesis of calcific aortic stenosis. Herein, we investigated the effects of l-Arginine, the main precursor of NO, on the osteogenic differentiation of aortic interstitial valve cells (VICs). METHODS: We isolated a clonal population of bovine VICs that expresses osteogenic markers and induces calcification of collagen matrix after stimulation with endotoxin (LPS 500 ng/mL). VICs were treated in vitro with different combinations of LPS ± l-Arginine (50 or 100 mM) and cell extracts were collected to perform proteomic (iTRAQ) and gene expression (RT-PCR) analysis. RESULTS: l-Arginine prevents the over-expression of alkaline phosphatase (ALP, p < 0.001) and reduces matrix calcification (p < 0.05) in VICs treated with LPS. l-Arginine also reduces the over-expression of inflammatory molecules induced by LPS (TNF-alpha, IL-6 and IL-1beta, p < 0.001). The proteomic analysis allowed to identify 49 proteins with an altered expression profile after stimulation with LPS and significantly modified by l-Arginine. These include proteins involved in the redox homeostasis of the cells (i.e. Xanthine Oxidase, Catalase, Aldehyde Oxidase), remodeling of the extracellular matrix (i.e. ADAMTSL4, Basigin, COL3A1) and cellular signaling (i.e. Fibrillin-1, Legumain, S100A13). The RT-PCR analysis confirmed the modifications of Fibrillin-1, ADAMTSL4, Basigin and Xanthine Oxidase, whose expression levels increase after stimulation with LPS and are reduced by l-Arginine (p < 0.05). CONCLUSIONS: l-Arginine prevents osteogenic differentiation of VICs and reduces matrix calcification. This effect is achieved through the modulation of proteins involved in the cellular redox system, remodeling of extracellular matrix and inflammatory activation of VICs.
BACKGROUND AND AIMS: Reduced bioavailability of nitric oxide (NO) has been implicated in the pathogenesis of calcific aortic stenosis. Herein, we investigated the effects of l-Arginine, the main precursor of NO, on the osteogenic differentiation of aortic interstitial valve cells (VICs). METHODS: We isolated a clonal population of bovineVICs that expresses osteogenic markers and induces calcification of collagen matrix after stimulation with endotoxin (LPS 500 ng/mL). VICs were treated in vitro with different combinations of LPS ± l-Arginine (50 or 100 mM) and cell extracts were collected to perform proteomic (iTRAQ) and gene expression (RT-PCR) analysis. RESULTS:l-Arginine prevents the over-expression of alkaline phosphatase (ALP, p < 0.001) and reduces matrix calcification (p < 0.05) in VICs treated with LPS. l-Arginine also reduces the over-expression of inflammatory molecules induced by LPS (TNF-alpha, IL-6 and IL-1beta, p < 0.001). The proteomic analysis allowed to identify 49 proteins with an altered expression profile after stimulation with LPS and significantly modified by l-Arginine. These include proteins involved in the redox homeostasis of the cells (i.e. Xanthine Oxidase, Catalase, Aldehyde Oxidase), remodeling of the extracellular matrix (i.e. ADAMTSL4, Basigin, COL3A1) and cellular signaling (i.e. Fibrillin-1, Legumain, S100A13). The RT-PCR analysis confirmed the modifications of Fibrillin-1, ADAMTSL4, Basigin and Xanthine Oxidase, whose expression levels increase after stimulation with LPS and are reduced by l-Arginine (p < 0.05). CONCLUSIONS:l-Arginine prevents osteogenic differentiation of VICs and reduces matrix calcification. This effect is achieved through the modulation of proteins involved in the cellular redox system, remodeling of extracellular matrix and inflammatory activation of VICs.
Authors: Huitzilihuitl Saucedo-Orozco; Jesus Vargas-Barron; Ricardo Márquez-Velazco; Julio Iván Farjat-Pasos; Karla Susana Martinez-Zavala; Valentin Jiménez-Rojas; Sergio Andres Criales-Vera; Jose Antonio Arias-Godínez; Giovanni Fuentevilla-Alvarez; Veronica Guarner-Lans; Israel Perez-Torres; Gabriela Melendez-Ramirez; Tomas Efrain Sanchez Perez; Maria Elena Soto Journal: Open Heart Date: 2022-08
Authors: Grzegorz J Lis; Andrzej Dubrowski; Maciej Lis; Bernard Solewski; Karolina Witkowska; Veronika Aleksandrovych; Ewa Jasek-Gajda; Mateusz K Hołda; Krzysztof Gil; Jan A Litwin Journal: Int J Mol Sci Date: 2020-08-31 Impact factor: 5.923