title: Forskolin modifies retinal vascular development in Mrp4-knockout mice.
abstract: OBJECTIVE
  Multidrug resistance protein 4 (MRP4) effluxes a wide variety of endogenous compounds, including cyclic adenosine monophosphate (cAMP), and is exclusively expressed in vascular endothelial cells (ECs) of the retina. This study aimed to investigate the role of MRP4 in retinal vascular development.
  
  
  METHODS
  The retinal vascular phenotype of Mrp4(-/-) mice was examined by whole-mount immunohistochemistry at P3, P6, and P14. The retinas from P6 pups that received an intraperitoneal injection of either solvent control or forskolin, an inducer of intracellular cAMP formation, at P4 and P5 were analyzed in terms of their vascular formation (vascular length, vascular branching, vascular density, and the number of tip cells), cell proliferation and apoptosis, and vessel stability.
  
  
  RESULTS
  The Mrp4(-/-) mice exhibited no overt abnormalities in the development of the retinal vasculature, but retinal vascular development in the Mrp4(-/-) mice was suppressed in response to forskolin administration. There was a significant decrease in the vascular length, vascular branching, and vascular density, and inhibited tip cell formation at the vascular front. The forskolin-treated Mrp4(-/-) mice showed an increased number of Ki67-positive and cleaved caspase 3-positive ECs, a significant decrease in the amount of pericyte coverage, and a reduced number of empty sleeves. In pups exposed to hyperoxia (75% oxygen) from P7 to P12, the Mrp4(-/-) mice showed a significant increase in the unvascularized retinal area.
  
  
  CONCLUSIONS
  Mrp4(-/-) mice exhibited suppressed retinal vascular development in response to forskolin treatment. Thus, Mrp4 might have protective roles in retinal vascular development by regulating the intracellular cAMP level.
authors.affiliation: Division of Ophthalmology, Department of Surgery, Kobe University Graduate School of Medicine, Kobe, Japan.
authors.forename: Wataru
authors.initials: W
authors.lastname: Matsumiya
authors.forename: Sentaro
authors.initials: S
authors.lastname: Kusuhara
authors.forename: Keiko
authors.initials: K
authors.lastname: Hayashibe
authors.forename: Kazuichi
authors.initials: K
authors.lastname: Maruyama
authors.forename: Hiroyuki
authors.initials: H
authors.lastname: Kusuhara
authors.forename: Mizuki
authors.initials: M
authors.lastname: Tagami
authors.forename: John D
authors.initials: JD
authors.lastname: Schuetz
authors.forename: Akira
authors.initials: A
authors.lastname: Negi
mesh_terms: D000818:Animals; D000831:Animals, Newborn; D017209:Apoptosis; D049109:Cell Proliferation; D005576:Colforsin / Q000494:pharmacology*; D000242:Cyclic AMP / Q000378:metabolism; D004195:Disease Models, Animal; D004730:Endothelium, Vascular / Q000187:drug effects; D006801:Humans; D007150:Immunohistochemistry; D007231:Infant, Newborn; D007274:Injections, Intraperitoneal; D051379:Mice; D008810:Mice, Inbred C57BL; D018345:Mice, Knockout; D018613:Microscopy, Confocal; D027425:ATP-Binding Cassette, Sub-Family C Proteins / Q000502:physiology*; D010100:Oxygen / Q000633:toxicity; D012171:Retinal Vessels / Q000254:growth & development*; D012178:Retinopathy of Prematurity / Q000209:etiology / Q000378:metabolism
chemical_list: D005576:Colforsin; D000242:Cyclic AMP; D027425:ATP-Binding Cassette, Sub-Family C Proteins; D010100:Oxygen; C489540:Abcc4 protein, mouse