Novel Role of the IGF-1 Receptor in Endothelial Function and Repair: Studies in Endothelium-Targeted IGF-1 Receptor Transgenic Mice

Diabetes, Sep 2012

We recently demonstrated that reducing IGF-1 receptor (IGF-1R) numbers in the endothelium enhances nitric oxide (NO) bioavailability and endothelial cell insulin sensitivity. In the present report, we aimed to examine the effect of increasing IGF-1R on endothelial cell function and repair. To examine the effect of increasing IGF-1R in the endothelium, we generated mice overexpressing human IGF-1R in the endothelium (human IGF-1R endothelium-overexpressing mice [hIGFREO]) under direction of the Tie2 promoter enhancer. hIGFREO aorta had reduced basal NO bioavailability (percent constriction to NG-monomethyl-l-arginine [mean (SEM) wild type 106% (30%); hIGFREO 48% (10%)]; P < 0.05). Endothelial cells from hIGFREO had reduced insulin-stimulated endothelial NO synthase activation (mean [SEM] wild type 170% [25%], hIGFREO 58% [3%]; P = 0.04) and insulin-stimulated NO release (mean [SEM] wild type 4,500 AU [1,000], hIGFREO 1,500 AU [700]; P < 0.05). hIGFREO mice had enhanced endothelium regeneration after denuding arterial injury (mean [SEM] percent recovered area, wild type 57% [2%], hIGFREO 47% [5%]; P < 0.05) and enhanced endothelial cell migration in vitro. The IGF-1R, although reducing NO bioavailability, enhances in situ endothelium regeneration. Manipulating IGF-1R in the endothelium may be a useful strategy to treat disorders of vascular growth and repair.

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Novel Role of the IGF-1 Receptor in Endothelial Function and Repair: Studies in Endothelium-Targeted IGF-1 Receptor Transgenic Mice

Helen Imrie Hema Viswambharan Piruthivi Sukumar Afroze Abbas Richard M. Cubbon Nadira Yuldasheva Matthew Gage Jessica Smith Stacey Galloway Anna Skromna Sheik Taqweer Rashid T. Simon Futers Shouhong Xuan V. Kate Gatenby Peter J. Grant Keith M. Channon David J. Beech Stephen B. Wheatcroft Mark T. Kearney - We recently demonstrated that reducing IGF-1 receptor (IGF-1R) numbers in the endothelium enhances nitric oxide (NO) bioavailability and endothelial cell insulin sensitivity. In the present report, we aimed to examine the effect of increasing IGF-1R on endothelial cell function and repair. To examine the effect of increasing IGF-1R in the endothelium, we generated mice overexpressing human IGF-1R in the endothelium (human IGF-1R endothelium-overexpressing mice [hIGFREO]) under direction of the Tie2 promoter enhancer. hIGFREO aorta had reduced basal NO bioavailability (percent constriction to NG-monomethyl-Larginine [mean (SEM) wild type 106% (30%); hIGFREO 48% (10%)]; P , 0.05). Endothelial cells from hIGFREO had reduced insulin-stimulated endothelial NO synthase activation (mean [SEM] wild type 170% [25%], hIGFREO 58% [3%]; P = 0.04) and insulin-stimulated NO release (mean [SEM] wild type 4,500 AU [1,000], hIGFREO 1,500 AU [700]; P , 0.05). hIGFREO mice had enhanced endothelium regeneration after denuding arterial injury (mean [SEM] percent recovered area, wild type 57% [2%], hIGFREO 47% [5%]; P , 0.05) and enhanced endothelial cell migration in vitro. The IGF-1R, although reducing NO bioavailability, enhances in situ endothelium regeneration. Manipulating IGF-1R in the endothelium may be a useful strategy to treat disorders of vascular growth and repair. Diabetes 61:23592368, 2012 I nsulin-resistant type 2 diabetes characterized by perturbation of the insulin/IGF-1 system is a multisystem disorder of nutrient homeostasis, cell growth, and tissue repair (1). As a result, type 2 diabetes is a major risk factor for the development of a range of disorders of human health, including occlusive coronary artery disease (2), peripheral vascular disease (3), stroke (4), chronic vascular ulcers (5), proliferative retinopathy (6), and nephropathy (7). A key hallmark of these pathologies is See accompanying commentary, p. 2225. endothelial cell dysfunction characterized by a reduction in bioavailability of the signaling radical nitric oxide (NO). In the endothelium, insulin binding to its tyrosine kinase receptor stimulates release of NO (8). Insulin resistance at a whole-body level (9,10) and specific to the endothelium (11) leads to reduced bioavailability of NO, indicative of a critical role for insulin in regulating NO bioavailability. The insulin receptor (IR) and IGF-1 receptor (IGF-1R) are structurally similarboth composed of two extracellular a and two transmembrane b subunits linked by disulfide bonds (12). As a result, IGF-1R and IR can heterodimerize to form insulin-resistant hybrid receptors composed of one IGF-1R-ab complex and one IR-ab subunit complex (13,14). We recently demonstrated that reducing IGF-1R (by reducing the number of hybrid receptors) enhances insulin sensitivity and NO bioavailability in the endothelium (15). To examine the effect of increasing IGF-1R specifically in the endothelium on NO bioavailability, endothelial repair, and metabolic homeostasis, we generated a transgenic mouse with targeted overexpression of the human IGF-1R in the endothelium (hIGFREO). RESEARCH DESIGN AND METHODS Generation of hIGFREO mice. To overcome the limitations of random and multiple copy insertion sites seen in standard transgenics, we used the hypoxanthine phosphoribosyl transferase (Hprt) targeting system (Genoway; see ref. 16) to generate genetically modified embryonic stem (ES) cells. This approach uses homologous recombination to target a single copy of a transgene (in this case, the human IGF-1r), driven by a promoter (in this case, the Tie2 promoter), into the Hprt locus on the X chromosome. The model was developed with E15Tg2a (E14) cells derived from the strain 129P2/OiaHsd (12901a). In E14 cells, 35 kb of the Hprt gene encompassing the 59 untranslated region up to intron 2 is deleted. The Hprt gene encodes a constitutively expressed housekeeping enzyme involved in the synthesis of purines from the degradation products of nucleotide bases (salvage pathway). Cells normally synthesize purines by the salvage and de novo pathways. In Hprt-deleted cell lines, only the de novo pathway is functional, enabling the cells to grow in classical medium. However, in the presence of the aminopterin drug, the de novo pathway is blocked. As a result, Hprt-deleted cells die in hypoxanthine-aminopterin-thymidine (HAT) media (containing HAT substrates). The targeted insertion of a transgenic cassette in E14 ES cells with a functional Hprt gene rescues these cells, which can then be selected using HAT media to identify ES cells showing the correct targeting event. ES cells with the correct insertion can be se (...truncated)


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Helen Imrie, Hema Viswambharan, Piruthivi Sukumar, Afroze Abbas, Richard M. Cubbon, Nadira Yuldasheva, Matthew Gage, Jessica Smith, Stacey Galloway, Anna Skromna, Sheik Taqweer Rashid, T. Simon Futers, Shouhong Xuan, V. Kate Gatenby, Peter J. Grant, Keith M. Channon, David J. Beech, Stephen B. Wheatcroft, Mark T. Kearney. Novel Role of the IGF-1 Receptor in Endothelial Function and Repair: Studies in Endothelium-Targeted IGF-1 Receptor Transgenic Mice, Diabetes, 2012, pp. 2359-2368, 61/9, DOI: 10.2337/db11-1494