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L. Luo, H. Uehara, N. Singh, V. Chiodo, W. Hauswirth, N. Ferrara, J. Ambati, Y. Fu, W. Baehr, B. K. Ambati; Soluble Vascular Endothelial Growth Factor Receptor 1 (sflt-1) is Essential for Subretinal Vascular Zoning. Invest. Ophthalmol. Vis. Sci. 2010;51(13):6385.
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© ARVO (1962-2015); The Authors (2016-present)
The retinal pigment epithelium(RPE)-Bruch’s membrane(BrM)- choroicapillaris(CC) complex is a crucial anatomic structure for subretinal vascular zoning that presents highly vascularized, highly permeable fenestrated CC on its outer basal aspect, whereas the photoreceptor layer is completely avascular. The pathological conditions of RPE- BrM-CC arouse choroidal neovascularization(CNV) which breach the subretinal vascular barrier. The molecular underpinnings of maintaining the normal subretinal vascular zoning have remained obscure. VEGF- A is a potent stimulator of angiogenesis. sFlt-1 specifically binds VEGF and inhibits its activity. We previously showed corneal avascularity is due to sFlt-1. Here we sought to determine whether sFlt-1 in subretinal space is vital for subretinal vascular zoning.
Three independent strategies were used to test if CNV can be induced by suppressing sflt-1. First, a neutralizing antibody against sflt-1 was injected into subretinal space, isotype IgG performed as control. The second strategy was knockdown of sflt-1 using adeno-associated viral deliveryviral delivery of small RNA interference targeted to soluble VEGF receptor 1(AAV.siRNA.sFLT-1) by subretinal injection. PBS, aav.GFP and aav.nonspecific siRNA served as controls. The third strategy was genomic deletion: in FltloxP/loxP mice, subretinal NLS-Cre would reduces sFlt expression and then CNV would be expected. CNV was observed and evaluated by FA and ICG angiography and OCT using the Heidelberg Spectralis. ERG and histology were also used to evaluate functional and anatomic status after sacrifice. RT-PCR , Western blot, IHC and ELISA were performed to confirm sflt knocked down or cre expression and VEGF-A level.
RPE expresses sflt-1 and suppression of this endogenous VEGF-A trap by neutralizing antibodies, RNA interference or Cre-lox-mediated gene ablation in Flt-loxp mice induced CNV(P < 0.05). RPE secretes VEGF toward its basal side where its receptor sflt is located on the apical RPE. Free VEGF-A was elevated in these models. Subretinal pCre injection in VEGF-loxp animals prevented CNV. AAV.siRNA.sFlt-induced CNV more closely resembles human CNV than laser-induced CNV.
sflt-1 is essential for subretinal vascular zoning. Aav.sirna.sflt induced CNV has significant correlates to human CNV that the laser-induced CNV model does not.
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