Malgorzata Wachowska1, Agata Osiak2, Angelika Muchowicz3, Magdalena Gabrysiak3, Antoni Domagała3, Witold W Kilarski4, Jakub Golab5. 1. Department of Immunology, Medical University of Warsaw, Warsaw, Poland; Department of Laboratory Diagnostics and Clinical Immunology of Developmental Age Medical University of Warsaw, Warsaw, Poland. 2. Department of Immunology, Medical University of Warsaw, Warsaw, Poland; Laboratory of Immunology, Institute of Cardiology, Warsaw, Poland. 3. Department of Immunology, Medical University of Warsaw, Warsaw, Poland. 4. Institute of Bioengineering and Swiss Institute for Cancer Research (ISREC), School of Life Sciences, École Polytechnique Fédérale de Lausanne (EPFL), Lausanne, Switzerland; Institute for Molecular Engineering, University of Chicago, USA. 5. Department of Immunology, Medical University of Warsaw, Warsaw, Poland. Electronic address: jakub.golab@wum.edu.pl.
Abstract
BACKGROUND: Photodynamic therapy (PDT) has been shown to induce ablation and functional occlusion of tumor-associated lymphatic vessels. However, direct effects of PDT on lymphatic endothelial cells (LECs) have not been studied so far. The aim of this study was to elucidate molecular mechanisms of cell death induced by PDT in human LECs. METHODS: Verteporfin was used as a photosensitizer to investigate PDT-mediated damage of lymphatic vessels in mice using immunofluorescent staining and stereomicroscopy. In vitro dose-response studies were carried-out with crystal violet staining. Immunofluorescence, flow cytometry, immunoblotting and DNA electrophoresis were used to investigate the mechanisms of cell death in human LECs undergoing PDT. RESULTS: PDT induced an increase in the number of propidium iodide positive lymphatic endothelial cells in the mouse dermis. In in vitro studies dose-dependent cytotoxic effects of PDT towards LECs were observed. Typical hallmarks of apoptotic cell death, including Annexin V binding, loss of mitochondrial membrane potential, caspase activation, cleavage of PARP as well as DNA fragmentation were observed in LECs when PDT was used at high irradiation conditions, causing >80% cell death. At lower light fluencies causing <50% cell death PDT induced autophagy rather than apoptosis, as revealed by conversion of LC3-I to the autophagosomal LC3-II and formation of LC3 puncta. Z-VAD-FMK, a caspase inhibitor, prevented cell death induced by high-dose PDT only, while 3-methyladenine, an autophagy suppressor, inhibited cell death induced by low-dose PDT. CONCLUSIONS: Both apoptosis and autophagy are involved in cell death induced by verteporfin-PDT in LECs.
BACKGROUND: Photodynamic therapy (PDT) has been shown to induce ablation and functional occlusion of tumor-associated lymphatic vessels. However, direct effects of PDT on lymphatic endothelial cells (LECs) have not been studied so far. The aim of this study was to elucidate molecular mechanisms of cell death induced by PDT in human LECs. METHODS:Verteporfin was used as a photosensitizer to investigate PDT-mediated damage of lymphatic vessels in mice using immunofluorescent staining and stereomicroscopy. In vitro dose-response studies were carried-out with crystal violet staining. Immunofluorescence, flow cytometry, immunoblotting and DNA electrophoresis were used to investigate the mechanisms of cell death in human LECs undergoing PDT. RESULTS: PDT induced an increase in the number of propidium iodide positive lymphatic endothelial cells in the mouse dermis. In in vitro studies dose-dependent cytotoxic effects of PDT towards LECs were observed. Typical hallmarks of apoptotic cell death, including Annexin V binding, loss of mitochondrial membrane potential, caspase activation, cleavage of PARP as well as DNA fragmentation were observed in LECs when PDT was used at high irradiation conditions, causing >80% cell death. At lower light fluencies causing <50% cell death PDT induced autophagy rather than apoptosis, as revealed by conversion of LC3-I to the autophagosomal LC3-II and formation of LC3 puncta. Z-VAD-FMK, a caspase inhibitor, prevented cell death induced by high-dose PDT only, while 3-methyladenine, an autophagy suppressor, inhibited cell death induced by low-dose PDT. CONCLUSIONS: Both apoptosis and autophagy are involved in cell death induced by verteporfin-PDT in LECs.
Authors: Antoine Louveau; Jasmin Herz; Maria Nordheim Alme; Andrea Francesca Salvador; Michael Q Dong; Kenneth E Viar; S Grace Herod; James Knopp; Joshua C Setliff; Alexander L Lupi; Sandro Da Mesquita; Elizabeth L Frost; Alban Gaultier; Tajie H Harris; Rui Cao; Song Hu; John R Lukens; Igor Smirnov; Christopher C Overall; Guillermo Oliver; Jonathan Kipnis Journal: Nat Neurosci Date: 2018-09-17 Impact factor: 24.884