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LC Shaw, AM Timmers, S Caballero, EA Ellis, PE Spoerri, MB Grant; Reduction of the Insulin-like Growth Factor 1 Receptor mRNA by Ribozyme Cleavage Results in a Reduction in Retinal Angiogenesis . Invest. Ophthalmol. Vis. Sci. 2002;43(13):4024.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose:Retinal neovascularization is associated with proliferative diabetic retinopathy (PDR) and retinopathy of prematurity (ROP). Clinical studies suggest that insulin like growth factor-1 (IGF-1) is involved in both diseases and has direct effects on retinal vasculature. To understand the mechanisms by which IGF-1 contributes to normal and abnormal vascular physiology, several processes must be understood in greater detail. This includes how the level of IGF-1 receptor (IGF-1R) expression affects normal and abnormal retinal vascular growth. Our goal was to design ribozymes that selectively reduced the expression of the IGF-1R and to test these ribozymes in in vitro and in vivo angiogenesis models. Methods:We have designed two hammerhead ribozymes that specifically cleave the human IGF-1R mRNA. These ribozymes have been cloned into recombinant Adeno Associated Viral vectors (rAAV). We have used these rAAV constructs to transfect Human Retinal Endothelial Cells (HREC) and used the modified Boyden chamber assay to examine ribozyme effects. We have injected these constructs intravitreally into an oxygen induced ROP mouse model (Smith, et al. 1994) and sectioned the eyes to determine the effect of the ribozymes on neovascularization. For both the transfection and intravitreal injection experiments we have determined the mRNA and protein levels for the IGF-1R. Results:The IGF-1R hammerhead ribozymes reduced migration of HRECs by as much as 90% indicating that IGF-1R levels were reduced in cells treated with the IGF-1R ribozyme constructs. Catalytically inactive versions of the IGF-1R ribozymes that still retain the ability to bind the target mRNA showed antisense effects by reducing migration of HRECs by as much as 60%. The ribozymes reduced the amount of in vivo neovascularization, as measured by the average number endothelial cell nuclei present above the ILM, by 45%. In both HRECs and in the ROP mouse model, both ribozymes reduced the levels of IGF-1R RNA and protein corresponding to the observed growth inhibiting effect. effect. Conclusion:The IGF-1R ribozymes are effective at reducing endothelial cell migration in vitro and endothelial cell proliferation in vivo by reducing the expression of the IGF-1R. Therefore, the use of ribozymes, in general, and the IGF-1R ribozymes, in particular, is an effective method for studying the process of angiogenesis. In addition, these ribozymes may ultimately be effective as a gene therapy tools for the reduction of pathological retinal angiogenesis.
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