RESUMO
BACKGROUND/AIMS: Hypoxia induces angiogenesis while hyperoxia promotes vasoregression in the retina. We investigated herein the effect of prolonged hyperoxia on retinal angiogenesis and the underlying mechanism in an oxygen-induced retinopathy (OIR) model. METHODS: Vascular morphology was quantified in whole-mount retina from the mice subjected to the conventional OIR model (c-OIR) or the OIR model with prolonged hyperoxia (p-OIR). Expressions of genes related to angiogenesis were determined by real-time PCR. RESULTS: p-OIR retinas showed few intraretinal neovascular tufts at the border of avascular zones, lacking preretinal neovascularization, whereas c-OIR retinas had numerous preretinal neovascularizations. p-OIR retinas demonstrated outgrowth of capillaries in the deep layers despite persistent hyperoxia and possess a larger avascular zone compared with the c-OIR retinas. The capillaries in the p-OIR retinas were well-formed in contrast to those in the c-OIR retinas. p-OIR retinas expressed significantly higher TNFα (â¼4 fold) than c-OIR retinas. The expression of vascular endothelial growth factor, Erythropoietin, Angiopoietin 1 and 2 remained unchanged. CONCLUSION: Our data demonstrate that TNFα transcription is increased in hyperoxia-promoted retinal angiogenesis, implicating it, in association with low VEGF levels, as a possible proponent in retinal angiogenesis under hyperoxia.
Assuntos
Hiperóxia , Neovascularização Retiniana/genética , Transcrição Gênica , Fator de Necrose Tumoral alfa/genética , Regulação para Cima , Angiopoietina-1/genética , Angiopoietina-1/metabolismo , Angiopoietina-2/genética , Angiopoietina-2/metabolismo , Animais , Eritropoetina/genética , Eritropoetina/metabolismo , Imunofluorescência , Regulação da Expressão Gênica , Hipóxia , Camundongos Endogâmicos C57BL , Neovascularização Retiniana/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fator de Necrose Tumoral alfa/metabolismo , Fator A de Crescimento do Endotélio Vascular/genética , Fator A de Crescimento do Endotélio Vascular/metabolismoRESUMO
OBJECTIVE: Nucleoside diphosphate kinase B (NDPKB) participates in the activation of heterotrimeric and monomeric G proteins, which are pivotal mediators in angiogenic signaling. The role of NDPKB in angiogenesis has to date not been defined. Therefore, we analyzed the contribution of NDPKB to angiogenesis and its underlying mechanisms in well-characterized in vivo and in vitro models. APPROACH AND RESULTS: Zebrafish embryos were depleted of NDPKB by morpholino-mediated knockdown. These larvae displayed severe malformations specifically in vessels formed by angiogenesis. NDPKB-deficient (NDPKB(-/-)) mice were subjected to oxygen-induced retinopathy. In this model, the number of preretinal neovascularizations in NDPKB(-/-) mice was strongly reduced in comparison with wild-type littermates. In accordance, a delayed blood flow recovery was detected in the NDPKB(-/-) mice after hindlimb ligation. In in vitro studies, a small interfering RNA-mediated knockdown of NDPKB was performed in human umbilical endothelial cells. NDPKB depletion impaired vascular endothelial growth factor (VEGF)-induced sprouting and hampered the VEGF-induced spatial redistributions of the VEGF receptor type 2 and VE-cadherin at the plasma membrane. Concomitantly, NDPKB depletion increased the permeability of the human umbilical endothelial cell monolayer. CONCLUSIONS: This is the first report to show that NDPKB is required for VEGF-induced angiogenesis and contributes to the correct localization of VEGF receptor type 2 and VE-cadherin at the endothelial adherens junctions. Therefore, our data identify NDPKB as a novel molecular target to modulate VEGF-dependent angiogenesis.
Assuntos
Antígenos CD/metabolismo , Caderinas/metabolismo , Células Endoteliais/enzimologia , Músculo Esquelético/irrigação sanguínea , Nucleosídeo NM23 Difosfato Quinases/metabolismo , Neovascularização Fisiológica , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/metabolismo , Proteínas de Peixe-Zebra/metabolismo , Peixe-Zebra/metabolismo , Animais , Células Cultivadas , Modelos Animais de Doenças , Regulação da Expressão Gênica no Desenvolvimento , Técnicas de Silenciamento de Genes , Membro Posterior , Células Endoteliais da Veia Umbilical Humana/enzimologia , Humanos , Isquemia/enzimologia , Isquemia/genética , Isquemia/fisiopatologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Nucleosídeo NM23 Difosfato Quinases/deficiência , Nucleosídeo NM23 Difosfato Quinases/genética , Interferência de RNA , Recuperação de Função Fisiológica , Fluxo Sanguíneo Regional , Neovascularização Retiniana/enzimologia , Neovascularização Retiniana/genética , Neovascularização Retiniana/fisiopatologia , Transdução de Sinais , Fatores de Tempo , Transfecção , Peixe-Zebra/embriologia , Peixe-Zebra/genética , Proteínas de Peixe-Zebra/deficiência , Proteínas de Peixe-Zebra/genéticaRESUMO
As the ex vivo lifetime of nasal ciliary cells is limited, these cells have to be transferred to a culture medium for analysis with vital cytology immediately. Although the ciliary beat frequency (CBF) is likely to change over time, sufficient data regarding changes in the ex vivo CBF has not been published to date. In the present study, nasal epithelial cells were harvested from the mucosa of the inferior turbinate of 19 healthy volunteers with a cytology brush. Beating cilia were visualized with phase-contrast microscopy. Over a 12-h timeframe, 2 s epochs of video were captured every 5 min from the identical group of cells using a high-speed digital camera with a sampling rate of 100 fps. Temperature was maintained at about 22 degrees C and controlled by an infrared pyrometer. The CBF rapidly increased by 47 +/- 53% during the first 3 h of measurement. A relative plateau followed this increase from 3 to 9 h. After 9 h, CBF reduced linearly. After 12 h, the mean frequency reduced to 20 +/- 69% of baseline. However, there was considerable variance between the samples. The initial increase in CBF has not been reported previously. This interval seems to be unsuitable for meaningful measurements. Measurements of CBF are most reliable during the plateau phase between 3 and 9 h. After 9 h, there is clearly a significant decrease in CBF, together with a considerable interindividual variance.
Assuntos
Depuração Mucociliar/fisiologia , Mucosa Nasal/fisiologia , Adulto , Idoso , Feminino , Humanos , Técnicas In Vitro , Masculino , Microscopia de Contraste de Fase , Microscopia de Vídeo , Pessoa de Meia-Idade , Valores de Referência , Temperatura , Fatores de TempoRESUMO
Nucleoside diphosphate kinase B (NDPK-B) is an enzyme required for nucleoside triphosphate homeostasis, which has been shown to interact with caveolin-1 (Cav-1). In endothelial cells (ECs), NDPK-B contributes to the regulation of angiogenesis and adherens junction (AJ) integrity. We therefore investigated whether an interaction of NDPK-B with Cav-1 in ECs is required for this regulation and the involvement of VEGF signaling herein. We report that simultaneous depletion of NDPK-B/Cav-1 in HUVECs synergistically impaired sprouting angiogenesis. NDPK-B depletion alone impaired caveolae formation, VEGF-induced phosphorylation of c-Src/Cav-1 but not of ERK1/2/AKT/eNOS. In vivo, Cav-1-/- mice showed impaired retinal vascularization at postnatal-day five, whereas NDPK-B-/- mice did not. Primary mouse brain ECs (MBMECs) from NDPK-B-/- mice showed no change in caveolae content and transendothelial-electrical resistance upon VEGF stimulation. Interestingly, NDPK-B-/- MBMECs displayed an accumulation of intracellular vesicles and increased Cav-1 levels. Dextran tracer analysis showed increased vascular permeability in the brain of NDPK-B-/- mice compared to wild type. In conclusion, our data indicate that NDPK-B is required for the correct localization of Cav-1 at the plasma membrane and the formation of caveolae. The genetic ablation of NDPK-B could partially be compensated by an increased Cav-1 content, which restored caveolae formation and some endothelial functions.