title: Thromboxane promotes smooth muscle phenotype commitment but not remodeling of hypoxic neonatal pulmonary artery.
abstract: BACKGROUND
  Persistent pulmonary hypertension of the newborn (PPHN) is characterized by vasoconstriction and pulmonary vascular remodeling. Remodeling is believed to be a response to physical or chemical stimuli including pro-mitotic inflammatory mediators such as thromboxane. Our objective was to examine the effects of hypoxia and thromboxane signaling ex vivo and in vitro on phenotype commitment, cell cycle entry, and proliferation of PPHN and control neonatal pulmonary artery (PA) myocytes in tissue culture.
  
  
  METHODS
  To examine concurrent effects of hypoxia and thromboxane on myocyte growth, serum-fed first-passage newborn porcine PA myocytes were randomized into normoxic (21 % O2) or hypoxic (10 % O2) culture for 3 days, with daily addition of thromboxane mimetic U46619 (10(-9) to 10(-5) M) or diluent. Cell survival was detected by MTT assay. To determine the effect of chronic thromboxane exposure (versus whole serum) on activation of arterial remodeling, PPHN was induced in newborn piglets by a 3-day hypoxic exposure (FiO2 0.10); controls were 3 day-old normoxic and day 0 piglets. Third-generation PA were segmented and cultured for 3 days in physiologic buffer, Ham's F-12 media (in the presence or absence of 10 % fetal calf serum), or media with 10(-6) M U46619. DNA synthesis was measured by (3)H-thymidine uptake, protein synthesis by (3)H-leucine uptake, and proliferation by immunostaining for Ki67. Cell cycle entry was studied by laser scanning cytometry of nuclei in arterial tunica media after propidium iodide staining. Phenotype commitment was determined by immunostaining tunica media for myosin heavy chain and desmin, quantified by laser scanning cytometry.
  
  
  RESULTS
  Contractile and synthetic myocyte subpopulations had differing responses to thromboxane challenge. U46619 decreased proliferation of synthetic and contractile myocytes. PPHN arteries exhibited decreased protein synthesis under all culture conditions. Serum-supplemented PA treated with U46619 had decreased G1/G0 phase myocytes and an increase in S and G2/M. When serum-deprived, PPHN PA incubated with U46619 showed arrested cell cycle entry (increased G0/G1, decreased S and G2/M) and increased abundance of contractile phenotype markers.
  
  
  CONCLUSIONS
  We conclude that thromboxane does not initiate phenotypic dedifferentiation and proliferative activation in PPHN PA. Exposure to thromboxane triggers cell cycle exit and myocyte commitment to contractile phenotype.
authors.affiliation: Department of Pediatrics, University of Manitoba, 715 McDermot Avenue, Winnipeg, MB R3E 3P4 Canada.
authors.forename: Fabiana
authors.initials: F
authors.lastname: Postolow
authors.affiliation: Department of Physiology, University of Manitoba, 715 McDermot Avenue, Winnipeg, MB R3E 3P4 Canada ; Biology of Breathing Group, Manitoba Institute of Child Health, 715 McDermot Avenue, Winnipeg, MB R3E 3P4 Canada.
authors.forename: Jena
authors.initials: J
authors.lastname: Fediuk
authors.affiliation: Biology of Breathing Group, Manitoba Institute of Child Health, 715 McDermot Avenue, Winnipeg, MB R3E 3P4 Canada.
authors.forename: Nora
authors.initials: N
authors.lastname: Nolette
authors.affiliation: Biology of Breathing Group, Manitoba Institute of Child Health, 715 McDermot Avenue, Winnipeg, MB R3E 3P4 Canada.
authors.forename: Martha
authors.initials: M
authors.lastname: Hinton
authors.affiliation: Department of Pediatrics, University of Manitoba, 715 McDermot Avenue, Winnipeg, MB R3E 3P4 Canada ; Department of Physiology, University of Manitoba, 715 McDermot Avenue, Winnipeg, MB R3E 3P4 Canada ; Biology of Breathing Group, Manitoba Institute of Child Health, 715 McDermot Avenue, Winnipeg, MB R3E 3P4 Canada ; Section of Neonatology, WS012 Women's Hospital, 735 Notre Dame Ave, Winnipeg, MB R3E 0L8 Canada.
authors.forename: Shyamala
authors.initials: S
authors.lastname: Dakshinamurti