title: Matrigel-based sprouting endothelial cell culture system from mouse corpus cavernosum is potentially useful for the study of endothelial and erectile dysfunction related to high-glucose exposure.
abstract: BACKGROUND
  A proper cavernous endothelial cell culture system would be advantageous for the study of the pathophysiologic mechanisms involved in endothelial dysfunction and erectile dysfunction (ED).
  
  
  OBJECTIVE
  To establish a nonenzymatic technique, which we termed the "Matrigel-based sprouting endothelial cell culture system," for the isolation of mouse cavernous endothelial cells (MCECs) and an in vitro model that mimics in vivo situation for diabetes-induced ED.
  
  
  METHODS
  For primary MCEC culture, mouse cavernous tissue was implanted into Matrigel and sprouting cells from the tissue were subcultivated. To establish an in vitro model for diabetes-induced ED, the primary cultured MCECs were exposed to a normal-glucose (5 mmoL) or a high-glucose (30 mmoL) condition for 48 hours.
  
  
  METHODS
  The purity of isolated cells was determined by fluorescence-activated cell sorting analysis. MCECs incubated under the normal- or the high-glucose condition were used for Western blot, cyclic guanosine monophosphate (cGMP) quantification, and in vitro angiogenesis assay.
  
  
  RESULTS
  We could consistently isolate high-purity MCECs (about 97%) with the Matrigel-based sprouting endothelial cell culture system. MCECs were subcultured up to the fifth passage and no significant changes were noted in endothelial cell morphology or purity. The phosphorylation of Akt and eNOS and the cGMP concentration were significantly lower in MCECs exposed to high glucose than in those exposed to normal glucose. MCECs exposed to the normal-glucose condition formed well-organized capillary-like structures, whereas derangements in tube formation were noted in MCECs exposed to high glucose. The protein expression of transforming growth factor-β1 (TGF-β1) and phospho-Smad2 was significantly increased by exposure to high glucose.
  
  
  CONCLUSIONS
  The Matrigel-based sprouting endothelial cell culture system is a simple, technically feasible, and reproducible technique for isolating pure cavernous endothelial cells in mice. An in vitro model for diabetic ED will be a valuable tool for evaluating the angiogenic potential of novel endogenous or synthetic modulators.
authors.affiliation: Laboratory of Regenerative Sexual Medicine, Department of Urology, Inha University School of Medicine, Incheon, Korea.
authors.forename: Guo Nan
authors.initials: GN
authors.lastname: Yin
authors.forename: Ji-Kan
authors.initials: JK
authors.lastname: Ryu
authors.forename: Mi-Hye
authors.initials: MH
authors.lastname: Kwon
authors.forename: Sun Hwa
authors.initials: SH
authors.lastname: Shin
authors.forename: Hai-Rong
authors.initials: HR
authors.lastname: Jin
authors.forename: Kang-Moon
authors.initials: KM
authors.lastname: Song
authors.forename: Min Ji
authors.initials: MJ
authors.lastname: Choi
authors.forename: Dong-Yeon
authors.initials: DY
authors.lastname: Kang
authors.forename: Woo Jean
authors.initials: WJ
authors.lastname: Kim
authors.forename: Jun-Kyu
authors.initials: JK
authors.lastname: Suh
mesh_terms: D000818:Animals; D017209:Apoptosis / Q000187:drug effects; D002478:Cells, Cultured; D003470:Culture Media; D004195:Disease Models, Animal; D042783:Endothelial Cells / Q000166:cytology / Q000187:drug effects / Q000502:physiology; D007172:Erectile Dysfunction / Q000139:chemically induced / Q000503:physiopathology*; D005947:Glucose / Q000494:pharmacology*; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D018919:Neovascularization, Physiologic / Q000187:drug effects; D010413:Penis / Q000166:cytology* / Q000187:drug effects; D013481:Superoxides / Q000378:metabolism
chemical_list: D003470:Culture Media; D013481:Superoxides; D005947:Glucose