title: Inhibition of development of laser-induced choroidal neovascularization with suppression of infiltration of macrophages in Smad3-null mice.
abstract: We evaluated the effects of the loss of Smad3 on the development of experimental argon laser-induced choroidal neovascularization (CNV) in mice. An in vitro angiogenesis model was also used to examine the role of transforming growth factor-β1 (TGFβ1)/Smad3 signaling in vessel-like tube formation by human umbilical vein endothelial cells (HUVECs). CNV was induced in eyes of 8-12-week-old B6.129-background Smad3-deficient (KO) mice (n=47) and wild-type (WT) mice (n=47) by argon laser irradiation. Results showed that the size of the CNV induced was significantly smaller in KO mice as compared with WT mice at day 14 as revealed by high-resolution angiography with fluorescein isothiocyanate-dextran. Immunohistochemistry and real-time reverse transcription-polymerase chain reaction of RNA extracted from laser-irradiated choroidal tissues were conducted on specimens at specific timepoints. Invasion of macrophages (F4/80+), but not neutrophils (myeloperoxidase+), and appearance of myofibroblasts (α-smooth muscle actin+) were suppressed in laser-irradiated KO tissues. mRNA expression of inflammation-related factors, that is, vascular endothelial growth factor (VEGF), macrophage-chemoattractant protein-1 (MCP-1), interleukin-6 (IL-6) and TGFβ1 in choroidal tissues was suppressed by the loss of Smad3. We then examined the effects of adding a Smad3 inhibitor, SIS3, or an ALK5 inhibitor, SB431542, on tube formation promoted by TGFβ1 or VEGF in HUVECs cocultured with fibroblast feeder. Further addition of SIS3 or SB431542 augmented vessel-like tube formation by HUVECs in the presence of TGFβ1 or VEGF. In conclusion, lack of Smad3 attenuated the growth of laser-induced CNV with suppression of inflammation by macrophages in mice. Because blocking TGFβ1/Smad3 signal stimulated the activity of angiogenesis of HUVECs in vitro, the reduction of CNV in vivo in KO mice is attributed to a decrease in growth factor levels in the tissue by the loss of Smad3.
authors.affiliation: Department of Ophthalmology, Wakayama Medical University School of Medicine, Wakayama, Japan.
authors.forename: Hiroki
authors.initials: H
authors.lastname: Iwanishi
authors.affiliation: Department of Ophthalmology, Wakayama Medical University School of Medicine, Wakayama, Japan.
authors.forename: Norihito
authors.initials: N
authors.lastname: Fujita
authors.affiliation: Department of Ophthalmology, Wakayama Medical University School of Medicine, Wakayama, Japan.
authors.forename: Katsuo
authors.initials: K
authors.lastname: Tomoyose
authors.affiliation: Department of Ophthalmology, Wakayama Medical University School of Medicine, Wakayama, Japan.
authors.forename: Yuka
authors.initials: Y
authors.lastname: Okada
authors.affiliation: Department of Ophthalmology, Wakayama Medical University School of Medicine, Wakayama, Japan.
authors.forename: Osamu
authors.initials: O
authors.lastname: Yamanaka
authors.affiliation: Laboratory of Cancer Biology and Genetics, National Cancer Institute, National Institutes of Health, Bethesda, MD, USA.
authors.forename: Kathleen C
authors.initials: KC
authors.lastname: Flanders
authors.affiliation: Department of Ophthalmology, Wakayama Medical University School of Medicine, Wakayama, Japan.
authors.forename: Shizuya
authors.initials: S
authors.lastname: Saika
mesh_terms: D000818:Animals; D020256:Choroidal Neovascularization / Q000209:etiology / Q000473:pathology / Q000517:prevention & control*; D004195:Disease Models, Animal; D061307:Human Umbilical Vein Endothelial Cells; D006801:Humans; D018836:Inflammation Mediators / Q000378:metabolism; D007546:Isoquinolines / Q000494:pharmacology; D054020:Lasers, Gas / Q000009:adverse effects; D008264:Macrophages / Q000473:pathology*; D008297:Male; D051379:Mice; D057507:Mice, 129 Strain; D008810:Mice, Inbred C57BL; D018345:Mice, Knockout; D011725:Pyridines / Q000494:pharmacology; D011758:Pyrroles / Q000494:pharmacology; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D051900:Smad3 Protein / Q000037:antagonists & inhibitors / Q000172:deficiency* / Q000235:genetics; D053773:Transforming Growth Factor beta1
chemical_list: C508504:6,7-dimethyl-2-(2E)-3-(1-methyl-2-phenyl-1H-pyrrolo(2,3-b)pyridin-3-yl-prop-2-enoyl)-1,2,3,4-tetrahydroisoquinoline hydrochloride; D018836:Inflammation Mediators; D007546:Isoquinolines; D011725:Pyridines; D011758:Pyrroles; D012333:RNA, Messenger; C497790:SMAD3 protein, human; D051900:Smad3 Protein; C497791:Smad3 protein, mouse; C508960:TGFB1 protein, human; D053773:Transforming Growth Factor beta1