title: Tumor-induced loss of mural Connexin 43 gap junction activity promotes endothelial proliferation.
abstract: BACKGROUND
  Proper functional association between mural cells and endothelial cells (EC) causes EC of blood vessels to become quiescent. Mural cells on tumor vessels exhibit decreased attachment to EC, which allows vessels to be unstable and proliferative. The mechanisms by which tumors prevent proper association between mural cells and EC are not well understood. Since gap junctions (GJ) play an important role in cell-cell contact and communication, we investigated whether loss of GJ plays a role in tumor-induced mural cell dissociation.
  
  
  METHODS
  Mural cell regulation of endothelial proliferation was assessed by direct co-culture assays of fluorescently labeled cells quantified by flow cytometry or plate reader. Gap junction function was assessed by parachute assay. Connexin 43 (Cx43) protein in mural cells exposed to conditioned media from cancer cells was assessed by Western and confocal microscopy; mRNA levels were assessed by quantitative real-time PCR. Expression vectors or siRNA were utilized to overexpress or knock down Cx43. Tumor growth and angiogenesis was assessed in mouse hosts deficient for Cx43.
  
  
  RESULTS
  Using parachute dye transfer assay, we demonstrate that media conditioned by MDA-MB-231 breast cancer cells diminishes GJ communication between mural cells (vascular smooth muscle cells, vSMC) and EC. Both protein and mRNA of the GJ component Connexin 43 (Cx43) are downregulated in mural cells by tumor-conditioned media; media from non-tumorigenic MCF10A cells had no effect. Loss of GJ communication by Cx43 siRNA knockdown, treatment with blocking peptide, or exposure to tumor-conditioned media diminishes the ability of mural cells to inhibit EC proliferation in co-culture assays, while overexpression of Cx43 in vSMC restores GJ and endothelial inhibition. Breast tumor cells implanted into mice heterozygous for Cx43 show no changes in tumor growth, but exhibit significantly increased tumor vascularization determined by CD31 staining, along with decreased mural cell support detected by NG2 staining.
  
  
  CONCLUSIONS
  Our data indicate that i) functional Cx43 is required for mural cell-induced endothelial quiescence, and ii) downregulation of Cx43 GJ by tumors frees endothelium to respond to angiogenic cues. These data define a novel and important role for maintained Cx43 function in regulation of vessel quiescence, and suggest its loss may contribute to pathological tumor angiogenesis.
authors.affiliation: Department of Radiation Oncology, Wake Forest School of Medicine, Medical Center Boulevard, Winston-Salem, NC, 27157, USA. mayur.choudhary@duke.edu.|Department of Cancer Biology, Wake Forest School of Medicine, Medical Center Boulevard, Winston-Salem, NC, 27157, USA. mayur.choudhary@duke.edu.|Current address: Duke Eye Center, 2351 Erwin Road, AERI Room 4000, Durham, NC, 27705, USA. mayur.choudhary@duke.edu.
authors.forename: Mayur
authors.initials: M
authors.lastname: Choudhary
authors.affiliation: Department of Radiation Oncology, Wake Forest School of Medicine, Medical Center Boulevard, Winston-Salem, NC, 27157, USA. cmcmahan@wakehealth.edu.
authors.forename: Christine
authors.initials: C
authors.lastname: Naczki
authors.affiliation: Department of Radiation Oncology, Wake Forest School of Medicine, Medical Center Boulevard, Winston-Salem, NC, 27157, USA. wechen@wakehealth.edu.
authors.forename: Wenhong
authors.initials: W
authors.lastname: Chen
authors.affiliation: Department of Cancer Biology, Wake Forest School of Medicine, Medical Center Boulevard, Winston-Salem, NC, 27157, USA. kdbarlow@gtcc.edu.
authors.forename: Keith D
authors.initials: KD
authors.lastname: Barlow
authors.affiliation: Department of Biostatistical Sciences, Wake Forest School of Medicine, Medical Center Boulevard, Winston-Salem, NC, 27157, USA. dcase@wakehealth.edu.|Wake Forest Comprehensive Cancer Center, Wake Forest School of Medicine, Medical Center Boulevard, Winston-Salem, NC, 27157, USA. dcase@wakehealth.edu.
authors.forename: L Douglas
authors.initials: LD
authors.lastname: Case
authors.affiliation: Department of Radiation Oncology, Wake Forest School of Medicine, Medical Center Boulevard, Winston-Salem, NC, 27157, USA. lmetheny@wakehealth.edu.|Department of Cancer Biology, Wake Forest School of Medicine, Medical Center Boulevard, Winston-Salem, NC, 27157, USA. lmetheny@wakehealth.edu.|Wake Forest Comprehensive Cancer Center, Wake Forest School of Medicine, Medical Center Boulevard, Winston-Salem, NC, 27157, USA. lmetheny@wakehealth.edu.
authors.forename: Linda J
authors.initials: LJ
authors.lastname: Metheny-Barlow
mesh_terms: D000818:Animals; D001943:Breast Neoplasms / Q000098:blood supply / Q000378:metabolism*; D002450:Cell Communication; D045744:Cell Line, Tumor; D049109:Cell Proliferation; D018031:Connexin 43 / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D017077:Culture Media, Conditioned; D042783:Endothelial Cells / Q000502:physiology*; D004730:Endothelium, Vascular / Q000503:physiopathology*; D017629:Gap Junctions / Q000378:metabolism*; D006801:Humans; D051379:Mice; D032389:Myocytes, Smooth Muscle / Q000378:metabolism*; D009389:Neovascularization, Pathologic / Q000503:physiopathology; D012333:RNA, Messenger / Q000378:metabolism
chemical_list: D018031:Connexin 43; D017077:Culture Media, Conditioned; C500216:GJA1 protein, human; D012333:RNA, Messenger