title: Extracellular HSP70 blocks CD40L-induced apoptosis and tubular formation in endothelial cells.
abstract: BACKGROUND
  Recent studies have shown that CD40, a key player in angiogenesis and tubular formation, is an extracellular receptor of the heat shock protein 70 (HSP70)-peptide complex in endothelial cells. The aim of the present study was to determine the effect of extracellular HSP70 treatment on CD40L-suppressed apoptosis and CD40L-induced tubular formation in human umbilical vein endothelial cells (HUVEC).
  
  
  METHODS
  The apoptotic index of CD40L-stimulated HUVEC with or without recombinant human HSP70 was evaluated using terminal deoxynucleotidyl transferase biotin-dUTP nick end labeling assay analysis. Binding of HSP70-peptide complex to CD40 on HUVEC was determined by double-labeling immunofluorescence methods. To evaluate the biological activity of CD40 engagement pretreated with rhHSP70 (0.5, 1 and 3 ng/mL), the extent of new capillary-like networking structure (tubular formation) formation in HUVEC was counted using an Olympus digital camera. Vascular invasion into MNK-28 cell clusters was assessed by counting the number of tubular structures extending from the HUVEC into growth factor-depleted Matrigel. Scores for CD34, HSP70 and CD40L expression levels in gastric cancer tissues were determined by immunostaining.
  
  
  RESULTS
  CD40L stimulation inhibited vincristine-induced apoptosis of HUVEC in a dose-dependent manner. Extracellular HSP70 treatment significantly blocked the inhibition of apoptosis by CD40L in HUVEC exposed to vincristine. HSP70-peptide complex bound to CD40 on HUVEC. Extracellular HSP70 treatment also significantly reduced CD40L-induced tubular formation in a dose-dependent manner. HSP70 treatment also suppressed invasive tubular formation into MKN-28 cells clusters by CD40L-activated HUVEC. There was a significant relationship between CD40L expression levels and microvessel density; however, the relationship between HSP70 expression level and microvessel density in gastric cancer tissues was not significant.
  
  
  CONCLUSIONS
  Extracellular HSP70 treatment blocks CD40L inhibition of apoptosis and CD40L induction of tubular formation in HUVEC.
authors.affiliation: Division of Gastroenterology, Internal Medicine, Nippon Medical School, Bunkyo-ku, Tokyo, Japan. seiji-f@nms.ac.jp
authors.forename: Seiji
authors.initials: S
authors.lastname: Futagami
authors.forename: Tetsuro
authors.initials: T
authors.lastname: Hiratsuka
authors.forename: Tomotaka
authors.initials: T
authors.lastname: Shindo
authors.forename: Tatsuhiko
authors.initials: T
authors.lastname: Hamamoto
authors.forename: Akane
authors.initials: A
authors.lastname: Horie
authors.forename: Nobue
authors.initials: N
authors.lastname: Ueki
authors.forename: Masafumi
authors.initials: M
authors.lastname: Kusunoki
authors.forename: Katya
authors.initials: K
authors.lastname: Gudis
authors.forename: Kazumasa
authors.initials: K
authors.lastname: Miyake
authors.forename: Taku
authors.initials: T
authors.lastname: Tsukui
authors.forename: Choitsu
authors.initials: C
authors.lastname: Sakamoto
mesh_terms: D018952:Antigens, CD34 / Q000032:analysis; D000972:Antineoplastic Agents, Phytogenic / Q000494:pharmacology; D017209:Apoptosis / Q000187:drug effects; D019013:CD40 Antigens / Q000378:metabolism*; D023201:CD40 Ligand / Q000378:metabolism*; D045744:Cell Line, Tumor; D002465:Cell Movement; D002478:Cells, Cultured; D042783:Endothelial Cells / Q000187:drug effects / Q000378:metabolism* / Q000473:pathology; D018840:HSP70 Heat-Shock Proteins / Q000378:metabolism*; D006801:Humans; D055806:Microvessels / Q000737:chemistry / Q000473:pathology; D018919:Neovascularization, Physiologic; D011994:Recombinant Proteins / Q000378:metabolism; D013274:Stomach Neoplasms / Q000098:blood supply / Q000378:metabolism / Q000473:pathology; D014750:Vincristine / Q000494:pharmacology
chemical_list: D018952:Antigens, CD34; D000972:Antineoplastic Agents, Phytogenic; D019013:CD40 Antigens; D018840:HSP70 Heat-Shock Proteins; D011994:Recombinant Proteins; D023201:CD40 Ligand; D014750:Vincristine