ABSTRACT
Heparin-binding epidermal growth factor (EGF)-like growth factor (HBEGF) is expressed in the embryo and uterus at the implantation site, stimulating trophoblast invasive activity essential for placentation. The effect of extraembryonic HBEGF deficiency on placental development was investigated by breeding mice heterozygous for the Hbegf null mutation. On gestation day 13.5, the average placental weights of the wild-type (Hbegf+/+) and heterozygous (Hbegf+/-) mice were approximately 76 and 77 mg, respectively, as opposed to reduced average placental weights of approximately 61 mg in homozygous null (Hbgef-/-) females. In contrast, fetal weights were not significantly affected by genotype. HBEGF immunostaining in placental sections was Hbegf gene dosage-dependent, while expression of other EGF family members was comparable in Hbegf+/+ and Hbegf-/- placentas. Histological analysis revealed no apparent differences in trophoblast giant cells, but the spongiotrophoblast region was reduced compared to labyrinth (P < 0.05) in Hbegf null placentas. While no differences in cell apoptosis were noted, proliferation as assessed by nuclear Ki67 staining was elevated in the labyrinth and decreased in the spongiotrophoblast region of Hbegf-/- placentas. Labyrinth morphology appeared disrupted in Hbegf -/- placentas stained with laminin, a marker for capillary basement membrane, and the capillary density was reduced. Immunohistochemical staining revealed reduced vascular endothelial growth factor (VEGF) levels in both spongiotrophoblast and labyrinth (P < 0.01) regions of Hbegf-/- placentas. In vitro, HBEGF supplementation increases the expression of VEGF in a human trophoblast cell line. These findings suggest that trophoblast HBEGF promotes placental capillary formation by inducing VEGF in the developing placenta of mice.
Subject(s)
Extraembryonic Membranes/metabolism , Heparin-binding EGF-like Growth Factor/genetics , Placenta Diseases/genetics , Placentation/genetics , Animals , Cell Line , Extraembryonic Membranes/blood supply , Female , Heparin-binding EGF-like Growth Factor/deficiency , Heparin-binding EGF-like Growth Factor/metabolism , Male , Mice , Mice, Inbred C57BL , Mice, Knockout , Neovascularization, Physiologic/genetics , Placenta/blood supply , Placenta/metabolism , Placenta/pathology , Placenta Diseases/pathology , Placentation/physiology , Pregnancy , Trophoblasts/metabolism , Trophoblasts/pathology , Vascular Endothelial Growth Factor A/metabolismABSTRACT
OBJECTIVE: The incidence of blindness is increasing because of the increase in abnormal ocular neovascularization. Anti-VEGF (vascular endothelial growth factor) therapies have led to good results, although they are not a cure for the blindness. The purpose of this study was to determine what role HB-EGF (heparin-binding epidermal growth factor-like growth factor) plays in ocular angiogenesis. APPROACH AND RESULTS: We examined the role played by HB-EGF in ocular neovascularization in 2 animal models of neovascularization: laser-induced choroidal neovascularization (CNV) and oxygen-induced retinopathy. We also studied human retinal microvascular endothelial cells in culture. Our results showed that the neovascularization was decreased in both the CNV and oxygen-induced retinopathy models in HB-EGF conditional knockout mice compared with that in wild-type mice. Moreover, the expressions of HB-EGF and VEGF were increased after laser-induced CNV and oxygen-induced retinopathy, and their expression sites were located around the neovascular areas. Exposure of human retinal microvascular endothelial cells to HB-EGF and VEGF increased their proliferation and migration, and CRM-197 (cross-reactive material-197), an HB-EGF inhibitor, decreased the HB-EGF-induced and VEGF-induced cell proliferation and migration. VEGF increased the expression of HB-EGF mRNA. VEGF-dependent activation of EGFR (epidermal growth factor receptor)/ERK1/2 (extracellular signal-regulated kinase 1/2) signaling and cell proliferation of endothelial cells required stimulation of the ADAM17 (a disintegrin and metalloprotease) and ADAM12. CRM-197 decreased the grades of the fluorescein angiograms and size of the CNV areas in marmoset monkeys. CONCLUSIONS: These findings suggest that HB-EGF plays an important role in the development of CNV. Therefore, further investigations of HB-EGF are needed as a potential therapeutic target in the treatment of exudative age-related macular degeneration.
Subject(s)
Autocrine Communication , Choroidal Neovascularization/metabolism , Heparin-binding EGF-like Growth Factor/metabolism , Neovascularization, Pathologic , Paracrine Communication , Retinal Neovascularization/metabolism , Retinal Vessels/metabolism , ADAM12 Protein/genetics , ADAM12 Protein/metabolism , ADAM17 Protein/genetics , ADAM17 Protein/metabolism , Angiogenesis Inhibitors/pharmacology , Animals , Autocrine Communication/drug effects , Bacterial Proteins/pharmacology , Callithrix , Cell Movement , Cell Proliferation , Cells, Cultured , Choroidal Neovascularization/genetics , Choroidal Neovascularization/pathology , Choroidal Neovascularization/prevention & control , Disease Models, Animal , Endothelial Cells/metabolism , Endothelial Cells/pathology , Extracellular Signal-Regulated MAP Kinases/metabolism , Heparin-binding EGF-like Growth Factor/deficiency , Heparin-binding EGF-like Growth Factor/genetics , Humans , Mice, Knockout , Paracrine Communication/drug effects , Retinal Neovascularization/genetics , Retinal Neovascularization/pathology , Retinal Neovascularization/prevention & control , Retinal Vessels/drug effects , Retinal Vessels/pathology , Signal Transduction , Vascular Endothelial Growth Factor A/genetics , Vascular Endothelial Growth Factor A/metabolismABSTRACT
BACKGROUND: Structural and functional changes of the hippocampus are correlated with psychiatric disorders and cognitive dysfunctions. Genetic deletion of heparin-binding epidermal growth factor-like growth factor (HB-EGF), which is predominantly expressed in cortex and hippocampus, also causes similar psychiatric and cognitive dysfunctions, accompanying down-regulated NMDA receptor signaling. However, little is known of such dysfunctions in hippocampus-specific Hbegf cKO mice. RESULTS: We successfully developed hippocampus-specific cKO mice by crossbreeding floxed Hbegf and Gng7-Cre knock-in mice, as Gng7 promoter-driven Cre is highly expressed in hippocampal neurons as well as striatal medium spiny neurons. In mice lacking hippocampus Hbegf gene, there was a decreased neurogenesis in the subgranular zone (SGZ) of the dentate gyrus as well as down-regulation of PSD-95/NMDA-receptor-NR1/NR2B subunits and related NMDA receptor signaling. Psychiatric, social-behavioral and cognitive abnormalities were also observed in hippocampal cKO mice. Interestingly, D-cycloserine and nefiracetam, positive allosteric modulators (PAMs) of NMDA receptor reversed the apparent reduction in NMDA receptor signaling and most behavioral abnormalities. Furthermore, decreased SGZ neurogenesis in hippocampal cKO mice was reversed by nefiracetam. CONCLUSIONS: The present study demonstrates that PAMs of NMDA receptor have pharmacotherapeutic potentials to reverse down-regulated NMDA receptor signaling, neuro-socio-cognitive abnormalities and decreased neurogenesis in hippocampal cKO mice.
