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
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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)