IGF-I, IGF-I receptor and IGF-I binding proteins in human retinal endothelial cell cultures from diabetic and nondiabetic donors

P. E. Spoerri, E. A. Ellis, R. W. Tarnuzzer, M. B. Grant

Research output: Contribution to journalArticle

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Abstract

Purpose. To determine the protein and mRNA levels of IGF-I, IGF-I type I receptor and IGF-I binding proteins in cultures of retinal endothelial cells of diabetic and nondiabetic origin. Methods. Human diabetic and nondiabetic eyes were dissected, the retinas were removed, digested and cultured as previously described (Grant, M.B. and Guay, C., 1991, Invest. Opthalmol. Vis. Res. 31:53-63). Human retinal endothelial cell (HREC) cultures of passage one were either processed for electron microscopy and/or mRNA was isolated for RT-PCR analysis. Colloidal gold immunocytochemical localization of IGF-I, IGF-I receptor and six IGF-I binding proteins was carried out on sections on nickel grids of diabetic and nondiabetic HREC cultures. The degree of immunolocalization was determined by morphometric analysis. The message levels for IGF-I, IGF-I receptor, and IGF-I binding proteins were measured using quantitative RT-PCR. Results. Intense immunoreactivity to IGF-I receptor and the five IGF-I stimulatory binding proteins (IGFBP-1, IGFBP-2, IGFBP-3, IGFBP-5, IGFBP-6) was seen in diabetic HREC cultures. The immunoreactivity for these six proteins was significantly less in nondiabetic HREC cultures. In contrast, diabetic HREC cultures showed significantly less immunoreactivity to IGF-I and one IGF-I inhibitory binding protein (IGFBP-4) as compared to nor diabetic HREC cultures. Message levels for IGF-I were approximately 1 copy/cell in diabetic HREC cultures and 20 copies/cell in nondiabetic HREC cultures. Message levels for IGF-I receptor were 200 copies/cell and for IGFBP-1, IGFBP-4 and IGFBP-5 were 200,000-400,000 copies/cell with no significant difference between diabetic and nondiabetic HREC cultures. Conclusions. The changes in protein levels for IGF-I correlates with the observed mRNA levels in diabetic and nondiabetic HREC cultures. However, the levels of IGF-I binding proteins do not correlate with mRNA levels, suggesting a post-transcriptional regulation of the binding proteins in these cultures. The observed increase in the IGF-I binding proteins in diabetic retinal endothelial cells may enhance IGF-I action which is an important cellular event in human diabetic retinopathy.

Original languageEnglish (US)
JournalInvestigative Ophthalmology and Visual Science
Volume37
Issue number3
StatePublished - Feb 15 1996
Externally publishedYes

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Insulin-Like Growth Factor Binding Proteins
IGF Type 1 Receptor
Insulin-Like Growth Factor I
Endothelial Cells
Cell Culture Techniques
Insulin-Like Growth Factor Binding Protein 5
Insulin-Like Growth Factor Binding Protein 4
Insulin-Like Growth Factor Binding Protein 1
Carrier Proteins
Messenger RNA
Insulin-Like Growth Factor Binding Protein 6
Insulin-Like Growth Factor Binding Protein 2
Gold Colloid
Polymerase Chain Reaction
Insulin-Like Growth Factor Binding Protein 3
Proteins
Diabetic Retinopathy
Nickel

ASJC Scopus subject areas

  • Ophthalmology

Cite this

IGF-I, IGF-I receptor and IGF-I binding proteins in human retinal endothelial cell cultures from diabetic and nondiabetic donors. / Spoerri, P. E.; Ellis, E. A.; Tarnuzzer, R. W.; Grant, M. B.

In: Investigative Ophthalmology and Visual Science, Vol. 37, No. 3, 15.02.1996.

Research output: Contribution to journalArticle

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abstract = "Purpose. To determine the protein and mRNA levels of IGF-I, IGF-I type I receptor and IGF-I binding proteins in cultures of retinal endothelial cells of diabetic and nondiabetic origin. Methods. Human diabetic and nondiabetic eyes were dissected, the retinas were removed, digested and cultured as previously described (Grant, M.B. and Guay, C., 1991, Invest. Opthalmol. Vis. Res. 31:53-63). Human retinal endothelial cell (HREC) cultures of passage one were either processed for electron microscopy and/or mRNA was isolated for RT-PCR analysis. Colloidal gold immunocytochemical localization of IGF-I, IGF-I receptor and six IGF-I binding proteins was carried out on sections on nickel grids of diabetic and nondiabetic HREC cultures. The degree of immunolocalization was determined by morphometric analysis. The message levels for IGF-I, IGF-I receptor, and IGF-I binding proteins were measured using quantitative RT-PCR. Results. Intense immunoreactivity to IGF-I receptor and the five IGF-I stimulatory binding proteins (IGFBP-1, IGFBP-2, IGFBP-3, IGFBP-5, IGFBP-6) was seen in diabetic HREC cultures. The immunoreactivity for these six proteins was significantly less in nondiabetic HREC cultures. In contrast, diabetic HREC cultures showed significantly less immunoreactivity to IGF-I and one IGF-I inhibitory binding protein (IGFBP-4) as compared to nor diabetic HREC cultures. Message levels for IGF-I were approximately 1 copy/cell in diabetic HREC cultures and 20 copies/cell in nondiabetic HREC cultures. Message levels for IGF-I receptor were 200 copies/cell and for IGFBP-1, IGFBP-4 and IGFBP-5 were 200,000-400,000 copies/cell with no significant difference between diabetic and nondiabetic HREC cultures. Conclusions. The changes in protein levels for IGF-I correlates with the observed mRNA levels in diabetic and nondiabetic HREC cultures. However, the levels of IGF-I binding proteins do not correlate with mRNA levels, suggesting a post-transcriptional regulation of the binding proteins in these cultures. The observed increase in the IGF-I binding proteins in diabetic retinal endothelial cells may enhance IGF-I action which is an important cellular event in human diabetic retinopathy.",
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T1 - IGF-I, IGF-I receptor and IGF-I binding proteins in human retinal endothelial cell cultures from diabetic and nondiabetic donors

AU - Spoerri, P. E.

AU - Ellis, E. A.

AU - Tarnuzzer, R. W.

AU - Grant, M. B.

PY - 1996/2/15

Y1 - 1996/2/15

N2 - Purpose. To determine the protein and mRNA levels of IGF-I, IGF-I type I receptor and IGF-I binding proteins in cultures of retinal endothelial cells of diabetic and nondiabetic origin. Methods. Human diabetic and nondiabetic eyes were dissected, the retinas were removed, digested and cultured as previously described (Grant, M.B. and Guay, C., 1991, Invest. Opthalmol. Vis. Res. 31:53-63). Human retinal endothelial cell (HREC) cultures of passage one were either processed for electron microscopy and/or mRNA was isolated for RT-PCR analysis. Colloidal gold immunocytochemical localization of IGF-I, IGF-I receptor and six IGF-I binding proteins was carried out on sections on nickel grids of diabetic and nondiabetic HREC cultures. The degree of immunolocalization was determined by morphometric analysis. The message levels for IGF-I, IGF-I receptor, and IGF-I binding proteins were measured using quantitative RT-PCR. Results. Intense immunoreactivity to IGF-I receptor and the five IGF-I stimulatory binding proteins (IGFBP-1, IGFBP-2, IGFBP-3, IGFBP-5, IGFBP-6) was seen in diabetic HREC cultures. The immunoreactivity for these six proteins was significantly less in nondiabetic HREC cultures. In contrast, diabetic HREC cultures showed significantly less immunoreactivity to IGF-I and one IGF-I inhibitory binding protein (IGFBP-4) as compared to nor diabetic HREC cultures. Message levels for IGF-I were approximately 1 copy/cell in diabetic HREC cultures and 20 copies/cell in nondiabetic HREC cultures. Message levels for IGF-I receptor were 200 copies/cell and for IGFBP-1, IGFBP-4 and IGFBP-5 were 200,000-400,000 copies/cell with no significant difference between diabetic and nondiabetic HREC cultures. Conclusions. The changes in protein levels for IGF-I correlates with the observed mRNA levels in diabetic and nondiabetic HREC cultures. However, the levels of IGF-I binding proteins do not correlate with mRNA levels, suggesting a post-transcriptional regulation of the binding proteins in these cultures. The observed increase in the IGF-I binding proteins in diabetic retinal endothelial cells may enhance IGF-I action which is an important cellular event in human diabetic retinopathy.

AB - Purpose. To determine the protein and mRNA levels of IGF-I, IGF-I type I receptor and IGF-I binding proteins in cultures of retinal endothelial cells of diabetic and nondiabetic origin. Methods. Human diabetic and nondiabetic eyes were dissected, the retinas were removed, digested and cultured as previously described (Grant, M.B. and Guay, C., 1991, Invest. Opthalmol. Vis. Res. 31:53-63). Human retinal endothelial cell (HREC) cultures of passage one were either processed for electron microscopy and/or mRNA was isolated for RT-PCR analysis. Colloidal gold immunocytochemical localization of IGF-I, IGF-I receptor and six IGF-I binding proteins was carried out on sections on nickel grids of diabetic and nondiabetic HREC cultures. The degree of immunolocalization was determined by morphometric analysis. The message levels for IGF-I, IGF-I receptor, and IGF-I binding proteins were measured using quantitative RT-PCR. Results. Intense immunoreactivity to IGF-I receptor and the five IGF-I stimulatory binding proteins (IGFBP-1, IGFBP-2, IGFBP-3, IGFBP-5, IGFBP-6) was seen in diabetic HREC cultures. The immunoreactivity for these six proteins was significantly less in nondiabetic HREC cultures. In contrast, diabetic HREC cultures showed significantly less immunoreactivity to IGF-I and one IGF-I inhibitory binding protein (IGFBP-4) as compared to nor diabetic HREC cultures. Message levels for IGF-I were approximately 1 copy/cell in diabetic HREC cultures and 20 copies/cell in nondiabetic HREC cultures. Message levels for IGF-I receptor were 200 copies/cell and for IGFBP-1, IGFBP-4 and IGFBP-5 were 200,000-400,000 copies/cell with no significant difference between diabetic and nondiabetic HREC cultures. Conclusions. The changes in protein levels for IGF-I correlates with the observed mRNA levels in diabetic and nondiabetic HREC cultures. However, the levels of IGF-I binding proteins do not correlate with mRNA levels, suggesting a post-transcriptional regulation of the binding proteins in these cultures. The observed increase in the IGF-I binding proteins in diabetic retinal endothelial cells may enhance IGF-I action which is an important cellular event in human diabetic retinopathy.

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