Acute intensive insulin therapy exacerbates diabetic blood-retinal barrier breakdown via hypoxia-inducible factor-1α and VEGF
Vassiliki Poulaki, … , George D. Yancopoulos, Anthony P. Adamis
Vassiliki Poulaki, … , George D. Yancopoulos, Anthony P. Adamis
Published March 15, 2002
Citation Information: J Clin Invest. 2002;109(6):805-815. https://doi.org/10.1172/JCI13776.
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Article Vascular biology

Acute intensive insulin therapy exacerbates diabetic blood-retinal barrier breakdown via hypoxia-inducible factor-1α and VEGF

Abstract

Acute intensive insulin therapy is an independent risk factor for diabetic retinopathy. Here we demonstrate that acute intensive insulin therapy markedly increases VEGF mRNA and protein levels in the retinae of diabetic rats. Retinal nuclear extracts from insulin-treated rats contain higher hypoxia-inducible factor-1α (HIF-1α) levels and demonstrate increased HIF-1α–dependent binding to hypoxia-responsive elements in the VEGF promoter. Blood-retinal barrier breakdown is markedly increased with acute intensive insulin therapy but can be reversed by treating animals with a fusion protein containing a soluble form of the VEGF receptor Flt; a control fusion protein has no such protective effect. The insulin-induced retinal HIF-1α and VEGF increases and the related blood-retinal barrier breakdown are suppressed by inhibitors of p38 mitogen-activated protein kinase (MAPK) and phosphatidylinositol (PI) 3-kinase, but not inhibitors of p42/p44 MAPK or protein kinase C. Taken together, these findings indicate that acute intensive insulin therapy produces a transient worsening of diabetic blood-retinal barrier breakdown via an HIF-1α–mediated increase in retinal VEGF expression. Insulin-induced VEGF expression requires p38 MAPK and PI 3-kinase, whereas hyperglycemia-induced VEGF expression is HIF-1α–independent and requires PKC and p42/p44 MAPK. To our knowledge, these data are the first to identify a specific mechanism for the transient worsening of diabetic retinopathy, specifically blood-retinal barrier breakdown, that follows the institution of intensive insulin therapy.

Authors

Vassiliki Poulaki, Wenying Qin, Antonia M. Joussen, Peter Hurlbut, Stanley J. Wiegand, John Rudge, George D. Yancopoulos, Anthony P. Adamis

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(a) Nuclear HIF-1α expression levels in the retinae of nondiabetic and d...
(a) Nuclear HIF-1α expression levels in the retinae of nondiabetic and diabetic rats treated with control and insulin implants. Following normalization to β-actin, the HIF-1α protein levels in the diabetic animals increased 19.9% compared with the nondiabetic animals (100 ± 0.62 vs. 119.6 ± 4.26, n = 5, P < 0.05). The HIF-1α content increased an additional 96.4% in the diabetic animals treated with the insulin implants (235.04 ± 15.37, n = 5, P < 0.05 vs. control implants). (b) Western blot analysis of HIF-1α retinal nuclear protein levels in nondiabetic and diabetic rats treated with control or insulin implants. Insulin treatment upregulates the HIF-1α nuclear content in the diabetic rats. (c) Electrophoretic mobility shift assay for HIF-1α in retinal nuclear extracts from nondiabetic rats and diabetic rats treated with control or insulin implants. The extracts were incubated with labeled HRE-containing oligonucleotides from the VEGF promoter. Lane 1, free probe; lane 2, nondiabetic rats; lane 3, diabetic rats; lanes 4 and 5, insulin implant–treated rats; lane 6, control implant–treated rats; lane 7, competition with the cold oligonucleotide; lanes 8 and 9, incubation with the HIF-1α antibody (n = 5 retinae per lane for all). The markings on the left indicate the HIF-1α complexes. The marking on the right identifies the supershifted bands following exposure to the HIF-1α antibody.