title: Investigation of cell death mechanisms in human lymphatic endothelial cells undergoing photodynamic therapy.
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.
authors: 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.
authors: Malgorzata
authors: M
authors: Wachowska
authors: Department of Immunology, Medical University of Warsaw, Warsaw, Poland; Laboratory of Immunology, Institute of Cardiology, Warsaw, Poland.
authors: Agata
authors: A
authors: Osiak
authors: Department of Immunology, Medical University of Warsaw, Warsaw, Poland.
authors: Angelika
authors: A
authors: Muchowicz
authors: Department of Immunology, Medical University of Warsaw, Warsaw, Poland.
authors: Magdalena
authors: M
authors: Gabrysiak
authors: Department of Immunology, Medical University of Warsaw, Warsaw, Poland.
authors: Antoni
authors: A
authors: Domagała
authors: 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.
authors: Witold W
authors: WW
authors: Kilarski
authors: Department of Immunology, Medical University of Warsaw, Warsaw, Poland. Electronic address: jakub.golab@wum.edu.pl.
authors: Jakub
authors: J
authors: Golab
mesh_terms: D000818:Animals; D015153:Blotting, Western; D016923:Cell Death / Q000187:drug effects; D045744:Cell Line, Tumor; D004305:Dose-Response Relationship, Drug; D042783:Endothelial Cells / Q000187:drug effects*; D005455:Fluorescent Antibody Technique; D006801:Humans; D008196:Lymph / Q000166:cytology*; D051379:Mice; D010778:Photochemotherapy; D017319:Photosensitizing Agents / Q000494:pharmacology / Q000627:therapeutic use; D011166:Porphyrins / Q000494:pharmacology* / Q000627:therapeutic use; D000077362:Verteporfin
chemical_list: D017319:Photosensitizing Agents; D011166:Porphyrins; D000077362:Verteporfin
keywords: Apoptosis; Autophagy; Lymphatic endothelial cells; Photodynamic therapy