Abstract
purpose. Clinical studies have demonstrated that intensive insulin therapy
causes a transient worsening of retinopathy. The mechanisms underlying
the initial insulin-induced deterioration of retinal status in patients
with diabetes remain unknown. Vascular endothelial growth factor (VEGF)
is known to be operative in the pathogenesis of diabetic retinopathy.
The current study was conducted to characterize the effect of insulin
on retinal VEGF gene expression in vitro and in vivo.
methods. The effect of insulin on VEGF expression in vivo was examined by in
situ hybridization studies of rat retinal VEGF transcripts. To examine
the mechanisms by which insulin regulates VEGF expression, human
retinal pigment epithelial (RPE) cells were exposed to insulin, and
VEGF mRNA levels were quantified with RNase protection assays (RPAs).
Conditioned media from insulin-treated RPE cells were assayed for VEGF
protein and capillary endothelial cell proliferation. The capacity of
insulin to stimulate the VEGF promoter linked to a luciferase reporter
gene was characterized in transient transfection assays.
results. Insulin increased VEGF mRNA levels in the ganglion, inner nuclear, and
RPE cell layers. In vitro, insulin increased VEGF mRNA levels in human
RPE cells and enhanced VEGF promoter activity without affecting
transcript stability. Insulin treatment also increased VEGF protein
levels in conditioned RPE cell media in a dose-dependent manner with a
median effective concentration of 5 nM. The insulin-conditioned RPE
cell media stimulated capillary endothelial cell proliferation, an
effect that was completely blocked by anti-VEGF neutralizing antibody.
conclusions. Insulin increases VEGF mRNA and secreted protein levels in RPE cells
through enhanced transcription of the VEGF gene. Intensive insulin
therapy may cause a transient worsening of retinopathy in patients with
diabetes through increased retinal VEGF gene
expression.
Clinical trials
1 2 3 4 5 6 7 8 9 10 have investigated whether
intensive insulin therapy decreases the frequency and severity of
diabetic retinopathy. A consistent finding has been that intensive
insulin therapy causes a transient worsening of retinopathy in the
first 2 years. Continued intensive therapy, however, leads to a marked
reduction in the risk of progression beyond the third
year.
1 10 11
The effect of insulin therapy on the progression of retinopathy in type
2 diabetes has also been examined.
12 13 14 15 Short-term
studies of 1 to 3 years have shown a marked increase in the risk of
retinopathy progression with insulin therapy when compared with oral
hypoglycemic therapy. The risk of progression was related to the degree
of glycemic control by insulin.
13 15 Although
hyperglycemia was identified as a risk factor for the progression of
retinopathy in all patients, change of treatment from oral drugs to
insulin was associated with a twofold increased risk of retinopathy
progression and a threefold increased risk of visual
loss.
15 Progression was seen at all levels of
retinopathy.
13 15 As in type 1 diabetes, long-term
intensive treatment with insulin (more than 6 years) reduced the risk
for the development and progression of retinopathy in patients with
type 2 diabetes.
16 17 The mechanisms underlying the
initial deterioration of retinopathy in diabetes by intensive insulin
therapy remain unknown.
Vascular endothelial growth factor (VEGF) refers to a family of
angiogenic and permeability-enhancing peptides derived from
alternatively spliced mRNAs.
18 19 The isoforms differ in
their affinity for heparin. Smaller isoforms (e.g.,
VEGF
121 and VEGF
165) are
diffusible and can be found in conditioned media and biologic fluids.
By comparison, larger isoforms (e.g., VEGF
189 and
VEGF
206) are bound to heparin-like molecules in
extracellular matrix and on the surface of cell membranes. VEGF is an
endothelial-selective mitogen that binds to high-affinity receptors on
retinal endothelial cells.
20 In addition to stimulating
neovascularization, VEGF, also known as vascular permeability factor,
increases vascular leakage 50,000 times more potently than does
histamine.
18
Recent evidence has identified VEGF as a major mediator of retinal
ischemia-associated neovascularization (for review, see
Ref. 21) . VEGF
has also been causally linked to many of the other changes observed in
diabetic retinopathy, including retinal edema, ischemia, hemorrhage,
and microaneurysm formation.
22 23 24 25 26 Intraocular VEGF levels
are increased in patients with diabetes
27 28 29 and
correlate with the development of
neovascularization.
27 28 30 31 32 Further, the specific
inhibition of VEGF bioactivity prevents neovascularization in animal
models.
22 23 Retinal VEGF mRNA and protein levels are also
increased in rats with background diabetic retinopathy and correlate
with the breakdown of the blood–retinal barrier.
31 32 Injection of VEGF into normal nonhuman primate eyes induces retinal
edema, hemorrhage, intraretinal microvascular abnormalities (IRMA),
ischemia, microaneurysms, and intraretinal
neovascularization.
24 25 Thus, it appears that VEGF may
participate in the pathogenesis of both background and proliferative
diabetic retinopathy. Because the initial worsening of retinopathy
correlates with increased insulin doses, we hypothesized that insulin
indirectly worsens diabetic retinopathy through increases in VEGF gene
expression.