1
HEPATOCYT GROWTH FACTOR AND INSULIN LIKE GROWTH
FACTOR-1 IN EVOLUTION OF DIABETIC RETINOPATHY
By
Eman El-Masry El-Damarany*, Alaa El-Din Fathy. **
Departments of *Biochemistry, and **Ophthalmology,
El-Minia Faculty of Medicine
ABSTRACT :
Aim::Despite remarkable advance in the diagnosis and treatment of diabetic
retinopathy (DR) and its associated complications, visual disability due to DR thus
remains a serious health and socioeconomic problem. It is important to show the
factors that affect the pathogenesis and progression of the disease in diabetic patient
population. Recent advances in diabetic retinopathy research have reframed our
thinking in regards to the role of growth factors in promoting angiogenesis
particularly the hepatocyte growth factor (HGF). The aim of our study is to present
simple non invasive clue(s) to predict the progression of diabetic retinopathy through
the study of the plasma levels of insulin like growth factor-1(IGF-1) and hepatocyte
growth factor (HGF) at different stages of diabetic retinopathy and to study the
relationship of these parameters with glycemic control as reflected by the level of
glycosylated hemoglobin (HbA1C) and if we can use theses parameters as risk
markers.
Method : The present study was carried out on seventy subjects of both sexes and
different age groups selected from the patients who attended the Ophthalmology
Outpatient Clinic in Minia University Hospital scheduled for cataract surgery where,
fifty of which were diabetic patients (insulin dependent and non- insulin dependent)
and twenty subjects (controls) were normal and healthy. Subjects were divided into 4
groups: Group I included 20 diabetic patients with no retinopathy, Group II included
20 diabetic patients with non-proliferative diabetic retinopathy and Group III included
10 diabetic patients with proliferative diabetic retinopathy. Beside 20 healthy subjects
with senile cataract as a control group (Group IV). Venous blood and aqueous
samples were collected where plasma IGF-1, HGF, glycosylated hemoglobin
(HbA1C), fasting blood glucose (FBG) , serum creatinine and liver function testes
(serum alanine aminotransferase – ALT and serum aspartate aminotransferase -AST)
in addition to aqueous IGF-1 and HGF were determined for each subject in the
studied groups.
Results: There were statistically significant differences in the plasma and aqueous
levels of IGF-1 in all diabetic groups as compared to the control group as well as
when group I is compared to group II and group III (except for aqueous level between
groups I and II). On the other hand, there were statistically significant differences in
the plasma levels of HGF in group II and group III as compared to control group and
in the aqueous level between group III and group IV and in the plasma and aqueous
levels of HGF in group III when compared to group I and group II. There were
statistically significant differences in FBG and HbAIC in all diabetic groups when
compared to the control group and in group III when compared to group I and group
II. Moreover, there was statistically significant difference as regard duration when
group I was compared to group II and group III.
There was significant positive correlation between plasma and aqueous levels of IGF1(in group III), between IGF-1, HbAIC and. FBG (in group III) and IGF-1and duration
(in group II and group III). On the other hand, there was significant positive
2
correlation between plasma and aqueous levels of HGF (in group III) and between
HGF, FBG and HbAIC (in group III). Moreover there was significant positive
correlation between plasma levels of IGF-1and HGF (in group III).
Conclusion: Our data have shown that plasma IGF-1 and HGF levels represent early
markers for diabetic retinopathy and may be correlated with the degree of retinopathy
and thus plasma levels of IGF-1 and HGF may possibly serve as predictors for
diabetic retinopathy.
KEY WORDS:
Diabetic retinopathy
Insulin like growth factor-1
Hepatocyte growth factor
Glycosylated hemoglobin
INTRODUCTION:
Diabetic retinopathy (DR) is the most severe of the several ocular
complications of diabetes. The earliest clinical signs of diabetic retinopathy are microaneurysms, and dot intra retinal hemorrhages. These signs are present in nearly all
persons who have had type -1 diabetes for 20 years and in nearly 80 percent of those
with type- 2 disease of this duration.1 Diabetes mellitus remains a profound health
issue worldwide. The World Health Organization (WHO) estimates that there are
currently 150 million people with diabetes and that this number will double by the
year 2025. More than 90% of the new cases of diabetes are type 2 (2).
It is now quite evident that there is a plethora of growth factors which regulate
the retinal vasculature and are involved in the development and progression of
diabetic retinopathy. However, identifying the role of each growth factor is difficult
since growth factors can act alone or, as appears to be more often the case, interact
with each other. Examples include: one growth factor inducing the synthesis of a
more potent growth factor, synergy between growth factors and commonality in the
downstream transduction cascade. Although many growth factors with potential
angiogenic property have been identified, only five of them have so far been
implicated in DR. They are basic fibroblast growth factor (b-FGF), insulin like growth
factor-1 (IGF-1), vascular endothelial growth factor (VEGF), platelet-derived growth
factor (PDGF) and hepatocyte growth factor (HGF). 3
Insulin like Growth Factor-1 (IGF-1)
The first indication as to the role of IGF-I in diabetic retinopathy came from a
study in which hypo-physectomy led to a reduction in the severity of the diabetic eye
condition4. Subsequently the pituitary factor has been identified as growth hormone
and the mitogenic mediator of growth hormone action is Insulin like growth factor-I
(IGF-I).5 IGF-I was one of the first growth factors to be directly linked with diabetic
retinopathy.6 Initial reports demonstrated an acute increase in serum levels of IGF-I
preceded the onset of proliferative diabetic retinopathy (PDR) in animal models. 7.&8
IGF-I can induce almost all steps of the angiogenesis process including endothelial
cell proliferation, migration and basement membrane degradation9-11.
The insulin-like growth factors IGFs (Somatomedins) are group of peptide
growth factors which collectively include the two distinct peptides responsible for the
3
growth promoting effects of growth hormone; Insulin like growth factor-1 (IGF-1)
and insulin like growth factor-2 (IGF-2)12.
IGF-1 (somatomedin- C) 13 is a 70 amino acid, straight chain, basic peptide
that is homologous to human proinsulin. The other somatomedin, IGF-2, is a 67
amino acid neutral peptide that is homologous to IGF-1 (12). The IGFs, act by binding
to cell surface receptors, IGF-IR and IGF-IIR. IGF-IR binds IGF-I with higher affinity
than IGF-II and insulin. IGF-IIR, on the other hand, preferentially binds IGF-II.
Several circulating proteins, insulin like growth factor binding proteins (IGFBPs), are
responsible for the bioavailability and half-life of the IGFs and these binding proteins,
like the IGFs, are synthesized primarily in the liver. IGFs and their binding proteins
are also produced locally by most tissues, where they act in an autocrine or paracrine
manner.14
None of the cellular sources of IGF-1 can store preformed IGF-115. The
various sources of IGF-1 production, the apparent lack of any known forms of
intracellular storage, and the existence of both local and endocrine effects suggest
that, unlike insulin, IGF-1 is more like a cytokine than a hormone16. Insulin-like
growth factor-1 can affect target cells by acting on a specific type I receptor, the
insulin receptor, and on the IGF type II receptor17. Several studies in vitro have shown
that IGF-I and IGFBPs are subject to regulation by hypoxia.18–20
IGF-I directly participates in the pathophysiology of diabetic retinopathy by
increasing vascular endothelial growth factor (VEGF) expression via a phosphatidylinositol- 3-kinase/Akt dependent mechanism.21 IGF-I can also increase
transforming growth factor- ß (TGF - ß) bioavailability via increased plasminogen
activator (PA) activity. This suggests that IGF-I might be important in directing
angiogenesis by itself and also by regulating various other growth factors. 22
Hepatocyte Growth Factor (HGF)
HGF is a mesenchyme derived pleiotropic growth factor that is secreted as a
single chain, biologically inactive, glycoprotein precursor which is converted into its
active form by proteolytic digestion.23.24 Mature HGF is a heterodimer consisting of
an -chain (62 kDa) and a ß-chain (32–34 kDa) attached by a disulphide bond.23 The
-chain contains a heparin binding domain and sulphated polysaccharides such as
heparin and heparan sulphate can enhance the potency of HGF.25 It was initially
isolated from serum of hepatectomized rats26. It is a most potent mitogenic factor for a
number of cell types, including hepatocytes, myeloid precursor cells, and various
epithelial and endothelial cells.27-29
HGF also promotes epithelial and endothelial cell motility in addition to
regulating tube morphogenesis and tube branching.30 The HGF receptor has been
identified as the protein product of the c-met proto-oncogene which encodes a transmembrane tyrosine kinase and it has been demonstrated that large and microvessel
derived endothelial cells express the c-met receptor and respond to HGF.31 Therefore,
it seems logical that HGF might induce the proliferation of some intraocular
endothelial cells during the angiogenic response that occurs in PDR. HGF binds to the
c-Met receptor and initiates signaling via activation of both protein kinase- C (PKC)
and phosphate-dylinositol- 3-kinase (PI-3K), inducing mitogen-activated protein
kinase (MAPK) phosphorylation that is critical for migration and growth. In addition
4
to promoting cell growth and offering protection against apoptosis (HGF strongly
induces bcl-x expression and, thus, inhibits apoptosis), HGF regulates cell
dissociation, migration into extracellular matrices, and branching morphogenesis 31-33.
While this factor plays an important role in development and tissue
homeostasis there is considerable evidence that it may also be an important regulator
of angiogenesis.34.35 Firstly, the mitogenic action of HGF on human endothelial cells
is the most potent among growth factors, including VEGF. Secondly, HGF acts
directly on vascular endothelial cells, which possess the c-met receptor, to promote
stimulation of cell migration, proliferation, protease production, tissue invasion, and
organization into capillary-like tubes, all essential facets of angiogenesis. Thirdly, the
angiogenic activity of HGF can be blocked by specific neutralizing antibodies.
It was described as a scatter factor (SF) because it induced the dissociation of
colonies of epithelial cells. 34 This growth factor is produced mainly in the liver, but it
has been found in several tissues such as lung, skin, spleen, brain, bone marrow,
kidney, placenta, and even in intraocular structures such as the cornea, the lens, and
the retina36-38. Interestingly, it has been shown that corneal cells, the retinal pigment
epithelium, and epiretinal membranes in proliferative vitreoretinopathy express the cmet receptor.39 Its mitogenic activity is the most potent compared with that of basic
fibroblast growth factor (b-FGF), VEGF, interleukin-1 and 6 (IL-1 and 6).40
HGF and its receptor levels have been shown to significantly increase in the
vitreous of diabetic patients compared to normal control groups. HGF can also induce
VEGF production by a variety of cells and tissues. Since VEGF does not appear to
mediate these initial HGF effects it suggests that HGF acts as a co-factor promoting
retinal neovascularization 41&.42.
Local production of HGF could play a crucial part in the pathogenesis of
diabetic retinopathy. The potential sites of HGF production could be the retinal
pigment epithelial cells,28 epiretinal membranes,21 and macrophages.43. HGF-induced
endothelial cell growth is augmented by b-FGF, a factor that appears to potentiate
retinal neovascularization but does not initiate it.44&.45 it has been demonstrated that
administration of heparin in patients with coronary disease caused significant
increases in plasma HGF.46 The serum collected after heparin administration had
more prominent angiogenic properties than the serum collected before heparin
administration, thus suggesting that HGF could play a significant part in the
angiogenic effect of heparin. On the other hand, heparin sulphate glycosaminoglicans
protect other growth factors such as b-FGF from proteolytic degradation by
extracellular proteinases.47
Hepatocyte growth factor (HGF) increases paracellular permeability and
decreases transendothelial cell resistance, by decreasing occludin tight junction
protein content. These characteristics are considered important attributes of factors
involved in mediating ischemic retinopathies48.
SUBJECTS AND METHODS:
The present study was carried out on seventy subjects of both sexes and
different age groups selected from those patients attended the Ophthalmology
Outpatient Clinic in El-Minia University Hospital scheduled for cataract surgery
5
where, fifty of them were diabetic patients and twenty subjects (controls) were normal
and healthy. Subjects were divided into 4 groups:
Group I: Included 20 diabetic patients without retinopathy (2 insulin dependant
diabetes mellitus -IDDM and18 non-insulin dependant diabetes mellitus - NIDDM).
Group II: Included 20 diabetic patients with non-proliferative diabetic retinopathy
NPDR (5 IDDM and 15 NIDDM).
Group III: Included 10 diabetic patients with proliferative diabetic retinopathy PDR
(2 IDDM and 8 NIDDM).
Group IV: Included 20 healthy subjects with senile cataract admitted to the
Ophthalmology Department of El-Minia University Hospital to do cataract surgery as
a control.
Subjects with serum creatinine level more than 1.5 mg/dl were excluded from
the study to eliminate substantial renal failure as a variable. Subjects with serum
alanine aminotransferase (ALT) over 32 IU/l in women and over 40 IU/l in men and
serum aspartate aminotransferase (AST) over 30 IU/l in women and over 37 IU/l in
men were excluded from the study to avoid hepatic disorder as a variable. In addition,
patients with hypertension, ischemic cardiovascular, ocular (vascular disorders rather
than diabetic retinopathy, glaucoma and previous laser treatment (pan retinal
photocoagulation-PRP) for DR), and malignancy were excluded from the study. The
diabetic patients were considered controlled if the glycosylated hemoglobin (HbA1C)
level was ≤ 7% and uncontrolled if it was more than 7%. All patients were subjected
to the following:
Medical Examination: including history, duration of the disease, type of treatment and
the presence of any diabetic complication.
Laboratory Investigations: fasting blood glucose FBG), glycosylated hemoglobin
(HbAIC), serum creatinine, liver function tests (serum alanine aminotransferase ALT
and aspartate aminotransferase AST), plasma and aqueous IGF-1 and HGF.
Ophthalmic Examination
- Slit lamp examination.
- Direct and indirect ophthalmoscopy with dilated pupils by tropicamide (Mydriacyl)
1% eye drops.
- Fundus photography (including fluorescien angiography if needed).
On the other hand, clinical and laboratory investigations were performed for each
subject of the control group to exclude the presence of diabetes mellitus or any
associated disease.
Sampling
All patients and controls were fasting for twelve hours and then ten-ml of blood were
drawn by venous puncture and the blood samples were divided into:
Tube I (5ml blood) without anticoagulant to get serum for immediate estimation of
fasting blood sugar, liver function tests and creatinine where it is left to clot at room
temperature to separate sera after centrifuging for 10 min at 3000 rpm.
6
Tube II (2ml blood) is placed in clean dry tube containing EDTA for
estimation of glycosylated hemoglobin in whole blood, where it is kept at 2-8˚c(stable
for one week).
Tube III (3ml blood) is placed in clean dry tube containing EDTA to get
EDTA plasma for estimation of IGF-1 and HGF, after centrifuging for 10 min at 3000
rpm. The plasma samples are stored at -70 ˚c until assay.
Aqueous Collection
At the beginning of cataract surgery,(to avoid breakdown of the blood -aqueous
barrier because of surgical manipulation) the sample of undiluted aqueous humor
(100–200-μl) was manually aspirated from the central papillary area using a
disposable tuberculin syringe by limbal paracentesis with a great caution in order not
to touch the corneal endothelium, the lens or the iris ,transferred immediately to a
sterile tube, kept in ice box in the operating theater and then transferred to be stored at
−70°C until assay .
 The concentration of HGF in the aqueous and plasma samples was measured by a
double-antibody sandwich enzyme-linked immunosrbent assay (ELISA) designed
to measure HGF in body fluids or cell culture supernates (R&D Systems,
Minneapolis. MN, USA).Briefly, the standard HGF or the test sample was added
to microtiter ELISA plates coated with monoclonal antibody to HGF then after 2
hours of incubation at room temperature, the samples were aspirated and each well
was washed well with buffer solution 4 times. A polyclonal antibody to HGF
conjugated to horseradish peroxide was added to each well and incubated for 2
hours (for plasma samples) and for 1.75 hours (for aqueous samples).The reagents
were removed and washed 4 times then a freshly prepared substrate containing
hydrogen peroxide and tetramethylbenzidine (TMB) was added and incubated for
30 minutes. Color develops in proportion to the amount of HGF bound in the
initial step. The color development is stopped and the intensity of the color is
measured. The test is specific and the minimum detectable dose of HGF is less
than 40 pg/ml.
 The concentration of IGF-1 in the aqueous and plasma samples was measured by
a double-antibody sandwich enzyme-linked immunosrbent assay (ELISA)
designed to measure IGF- 1 in body fluids or cell culture supernates (R&D
Systems, Minneapolis. MN, USA) .Briefly; a monoclonal antibody specific for
IGF-I has been pre-coated onto a microplate. Standards and pretreated samples are
pipetted into the wells and any IGF-I present is bound by the immobilized
antibody. After washing away any unbound substances, an enzyme-linked
polyclonal antibody specific for IGF-I is added to the wells. Following a wash to
remove any unbound antibody-enzyme reagent, a substrate solution is added to the
wells and color develops in proportion to the amount of IGF-I bound in the initial
step. The Stop Solution changes the color from blue to yellow, and the intensity of
the color is measured at 450nm. The test is specific and the minimum detectable
dose of IGF-1 ranged from 0.007 - 0.056 ng/ml.
 Estimation of blood glucose level was done by Enzymatic colourimetric test,
GOD-PAP method according to Trender, using kits from Quimica Clinica
Aplicada (QCA), Spain (49).
 Estimation of serum creatinine was done by Jaffe reaction using kits from
Quimica Clinica Aplicada (QCA), Spain.
 Estimation of HbA1C was carried out using kits from BioSystems S.A. Spain.
7

Estimation of liver function tests (ALT and AST) in serum was carried out using
kits from bioMerieux, France.
STATISTICAL ANALYSIS:
Data entry and analysis were all done with IBM compatible computer using
software called SPSS (statistical package for social science) for windows version 13.
Quantitative data were presented by mean and standard deviation (mean ±
SD).Correlation, Student t test and one way ANOVA test were done. The probability
of ≤ 0.05 used as a cut off points for all significant tests.
RESULTS:
Table 1 (A and B) shows the demographic and biochemical data of the studied
groups, where the results were expressed as meanSD. In addition, it shows
comparison of the statistical significance (P- value) of all parameters in the studied
groups, where there was a significant increase in the plasma and aqueous levels of
IGF-1 in all diabetic groups as compared to the control group as well as when group I
is compared to group II and group III (except for aqueous level between group I and
group II). On the other hand, there was a significant increase in the plasma levels of
HGF in group II and group III as compared to control group and in the aqueous level
between group III and group IV and there was a significant increase in the plasma
and aqueous levels of HGF in group III when compared to group I and group II.
There were statistically significant differences in FBG and HbAIC in all diabetic
groups when compared to the control group and in group III when compared to group
I and group II. Moreover, there was statistically significant difference as regard
duration when group I was compared to group II and group III.
Table 2 shows comparison of the statistical correlation coefficient (r) of the
plasma and aqueous levels of IGF-1 and HGF among the studied groups where there
was significant positive correlation between plasma and aqueous levels of IGF-1(in
group III), between IGF-1, HbAIC and. FBG (in group III) and IGF-1and duration (in
group II and group III). On the other hand, there was significant positive correlation
between plasma and aqueous levels of HGF (in group III) and between HGF, FBG
and HbAIC (in group III). Moreover there was significant positive correlation between
plasma levels of IGF-1and HGF (in group III).
8
9
10
Table (3): Sensitivity and specificity 0f the HGF levels and IGF-1 levels in the studied groups
Item
Cut off value
P*
A*
Sensitivity %
P*
A*
Specificity %
P*
A*
HGF
2.85
0.64
68%
60%
IGF-1
285
127
66%
83%
PPV%
P*
A*
NPV%
P*
A*
77.7%
44%
40%
69%
61%
73.3% 100% 100%
*A= aqueous *P= Plasma
100%
100%
73%
69%
DISCUSSION:
The aim of our study is to present
simple non invasive clue to predict the
progression of diabetic retinopathy and if
we can use theses parameters as risk
markers. Many reports have studied the
concentrations of many growth factors
and cytokines in the vitreous of diabetic
patients but little studies handled the
concentration of these parameters in
serum of diabetic patients. Because the
breakdown of the blood retinal barrier
that occurs during the progression of
diabetic retinopathy particularly in PDR
facilitates the extra capillary leakage of
serum proteins and their passage from
the blood stream to the vitreous fluid,
thus enabling the serum to have an effect
on intra vitreous protein levels and hence
on aqueous levels, therefore, serum
concentrations must be considered for
the accurate interpretation of the results
obtained in vitreous fluid or even in
aqueous.
On the other hand; high
intraocular levels of a particular protein
in diabetic patients do not necessarily
mean its intraocular production.
There are few reports handled the
relation between serum HGF and
diabetic retinopathy. Nishimura et al.,50
found high serum levels of HGF only in
diabetic subjects with PDR who had not
undergone photocoagulation, but they
did not observe differences among
diabetic subjects with background
retinopathy, preproliferative retinopathy,
and PDR who had undergone
photocoagulation.Kulseng
et
al.,51
reported that type 1 diabetic patients
have increased serum HGF levels, but
they could not find significant
differences in serum HGF between
patients with or without PDR.
Katsura et al.,52 found that levels
of HGF in vitreous fluid of PDR patients
were significantly higher than in nondiabetic patients, but they did not show
serum HGF data. Nishimura et al.,53
confirmed these results, showing again
higher levels of HGF in vitreous fluid of
PDR compared with controls. In
addition, these authors compared
vitreous HGF concentrations with their
previously published results obtained in
serum. The mean vitreous HGF
concentrations seen in this study were
27-fold higher than the reported mean
serum HGF levels in PDR subjects.
Cantón et al.,54 studied, serum
and vitreous HGF concentrations in
samples obtained simultaneously (at the
time of vitreoretinal surgery), in the
same group of patients., they showed
increased levels of HGF in serum and
vitreous fluid of PDR patients than in
non-diabetic patients and the mean of
vitreous HGF levels was 25-fold higher
than serum concentrations. In addition,
any relation between serum and vitreous
HGF concentrations was not detected.
Shinoda et al.,55 reported that
aqueous HGF levels increase with
progression of diabetic retinopathy,
being greatest overall in patients with
active PDR but they failed to
demonstrate a correlation between
aqueous HGF levels and serum levels of
HGF
which
remained
constant
11
irrespective of the stage of diabetic
retinopathy.
Cai, W., et al.,29 concluded that
the failure to show a correlation between
serum HGF and the stage of retinopathy
is compelling evidence that the HGF
identified in the aqueous is derived from
intraocular cells which is consistent with
previous studies which demonstrate that
HGF is produced by corneal cells and
the retinal pigment epithelium, and that
these cells express the c-met receptor.29.37
The results of our study however,
revealed increased plasma and aqueous
concentration of HGF in both patients
with NPDR and in patients with PDR
than in diabetic patients without
retinopathy or in the control group.
Positive significant correlation was
found between serum and aqueous levels
of HGF in group III. Moreover positive
significant correlation was found
between serum and aqueous levels in
group III as regard FBS and HbAIC.
Positive significant correlation was also
found between serum HGF and serum
IGF-1 in group III.
The significant correlation of
serum and aqueous levels of HGF with
serum concentrations of HbA1c or
fasting glucose in diabetic subjects
indicates that diabetic control have an
influence on serum HGF and hence on
aqueous
concentrations.
The
insignificant correlation between serum
and aqueous HGF and duration of
diabetes does not deny the involvement
of
systemic
microvascular
complications, including PDR, in serum
HGF concentrations in diabetic subjects,
although further studies are needed to
clarify this point.
HGF appears to play a reparative
role in response to cellular or tissue
injury .it inhibits apoptosis of human
keratocytes induced by ultraviolet rays
and induces synthesis of glutathione37-.39.
It reduces necrotic changes because of
hypoxia in vascular endothelium. The
serum HGF is higher in severe
hypertensive patients. The up-regulation
in various diseases is related to its
involvement in the regenerative events
after organ damage .these findings
support the hypothesis that HGF
function is related to injury and tissue
healing processes29-.40.
On this basis, two hypotheses
may explain the increased concentrations
of serum HGF in patients with diabetic
retinopathy especially the PDR subjects.
One is that HGF production may be
enhanced in extra ocular organs such as
liver, kidney, lung, or spleen to promote
neovascularization in the retina. After
70% partial hepatectomy in rats, HGF
messenger ribonucleic acid levels in
kidney and spleen increase 3 to 5-fold,
and HGF messenger ribonucleic acid in
spleen is increased after the onset of
renal injury caused by unilateral
nephrectomy. HGF produced in the
uninjured organs may be involved in
regeneration of liver or kidney through
an endocrine mechanism56.-57. The same
endocrinal mechanism may play a role in
the increased concentrations of serum
HGF in patients with diabetic
retinopathy. Another possibility is that
HGF production may have been
enhanced in the diabetic eyes.
Our study revealed significant
positive correlation between serum and
aqueous levels of HGF in group III i.e.,
these levels increase with the stage of
diabetic retinopathy. This result may
confirm the hypotheses of enhanced
production in extra ocular organs on one
side in addition to enhanced intraocular
production in the diabetic eyes on the
other side as a reparative mechanism in
response to diabetic tissue injury.
12
Nakamura et al.,58 reported that
HGF is more efficacious in stimulating
the growth of vascular endothelial cells
than other growth factors such as VEGF,
b-FGF, and IL-6. They also reported that
HGF acts in an additive manner with bFGF, but not with VEGF.
Our
results
support
the
description of Katsura et al.,52 that HGF
also plays an important part in
neovascularization in PDR and that the
roles and induction mechanism may
differ from those of other growth factors.
However, further investigations are
necessary to specify the mechanism.
The exact mechanism by which
IGF-1exerts its action on the retina is
unclear. Possibly, IGF-1 promotes
angiogenesis by stimulating endothelial
cell (EC) migration and preventing cell
death. IGF-1 is also involved in
inflammation-linked angiogenesis 59. On
the other hand, IGF-1 acts as a mediator
to other growth factors that may be
involved, IGF-I potently increases
VEGF expression in retinal pigment
epithelial (RPE) cells in vitro and has an
additive effect with hypoxia in vivo. (21)
Hypoxia also increases retinal IGF-1
production. Thus, increased serum IGF-1
would enhance its local effects by
adding to its local concentrations, and/or
enhance hypoxia induced VEGF activity,
thereby accelerating DR60.
The hypothesis that serum IGF-1
can accelerate diabetic retinopathy may
also work in physiological conditions
with elevation of serum IGF-1, like
puberty and pregnancy, both of which
carry an increased risk of progression of
diabetic retinopathy.61&.62 It further
explains
why
pituitary
ablation
(substantially reducing growth hormone
and IGF-1) acutely improved visual
acuity in some cases of diabetic
retinopathy, and invariably stopped
proliferative diabetic retinopathy. Of
note, proliferative diabetic retinopathy is
rare in dwarfs who are deficient in
growth
hormone
and
IGF-I.63
Importantly,
recombinant
IGF-I
exacerbated diabetic retinopathy, when
administered to diabetic patients in an
attempt to suppress growth hormone
secretion and reverse insulin resistance
64.
Vitreous IGF-I levels correlate
with the presence and severity of
ischemia-associated diabetic retinal
neovascularization; intravitreal IGF-I
injection dose-dependently causes retinal
neovascularization and microangiopathy;
whereas reduction of serum IGF-I levels
inhibits retinal neovascularization in an
ischemic murine model. It has been
proposed that leakage across the blood–
retina barrier and high serum levels of
IGF might be the major source for
vitreous IGF levels65-67.
The results of our study revealed
increased
plasma
and
aqueous
concentration of IGF-1 in patients with
NPDR and in patients with PDR than in
diabetic patients without retinopathy or in
the control group. Positive significant
correlation was found between plasma
and aqueous levels of IGF-1 in group III.
Moreover positive significant correlation
was found between plasma and aqueous
levels of IGF-1 as regard FBS, HbAIC in
group III and duration in group II and
group III. Positive significant correlation
was found between plasma HGF and
plasma IGF-1 in group III.
The results of our study were
also supported by those of Merimee et
al., 68 who found that the serum level of
IGF-1 in diabetic patients with PDR was
twice of that in diabetic patients without
retinopathy, patients with less severe
retinopathy or normal control subjects,
and Dills et al.,69 who reported a
significant increase in IGF-1 level in
diabetic patients with retinopathy in
13
comparison to its level in diabetic
patients without DR.
The significant correlation of
plasma and aqueous levels of IGF-1 with
serum concentrations of HbA1c or fasting
glucose in diabetic subjects indicates that
diabetic control have an influence on
plasma IGF-1and hence on aqueous
concentrations. On the other hand, the
significant correlation between plasma
and aqueous IGF-1 and duration of
diabetes may reflect the involvement of
systemic microvascular complications,
including PDR, in plasma IGF-1
concentrations and hence its aqueous
concentrations in diabetic subjects,
although further studies are needed to
clarify this point.
The observation that there was
significant positive correlation between
plasma HGF and plasma IGF-1 in group
III suggests that the two growth factors
work in an additive fashion systemically
with
each
other
and
operate
independently intraocularly of one
another. To what extent both factors is
under the control of other intraocular
factors has yet to be determined,
although there is evidence that fibroblast
growth factor works in an additive
fashion with HGF but not VEGF when
stimulating endothelial cell function 44 .
It should be noted that, the
aqueous levels of either HGF or IGF-1 in
our study and the studies of others are
lower than the vitreous level reported by
others which may be due to the result of
anreroposterior gradients in the eye or
the more rapid clearance of these factors
from the anterior chamber.
On the other hand, although the
aqueous level of either HGF or IGF-1 in
our study was lower than the
corresponding serum level with a
positive correlation which indicates that
serum diffusion is the main mechanism,
but in the same time it does not deny the
local production where serum diffusion
can be considered as a trigger for the
local production.
CONCLUSION:
Our data have shown that plasma
IGF-1 and HGF levels represent early
markers for diabetic retinopathy and may
be correlated with the degree of
retinopathy and thus plasma levels of
IGF-1 and HGF may possibly serve as
predictors for diabetic retinopathy.
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‫‪65. Grant M, Russell B, Fitzgerald‬‬
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‫عامل النمو الكبدي و عامل النمو المشابه لألنسولين في نشوء وتطور االعتالل‬
‫الشبكي السكري‬
‫إيمان المصري الدمراني* ‪ -‬عالء الدين فتحي**‬
‫*قسم الكيمياء الحيوية الطبية ‪ ** .‬قسم طب وجراحة العين‬
‫كلية طب المنيا‬
‫الغرض من البحث ‪:‬‬
‫بالرغم من التقدم الملحوظ في تشخيص و عالج االعتالل الشبكي السكري والمضاعفات‬
‫المصاحبة له تظل اإلعاقة البصرية بسببه مشكلة صحية واجتماعية و اقتصادية ‪ ,‬لذلك فانه من‬
‫األهمية بمكان أن نستعرض العوامل التي تؤثر على اإلحداث المرضى و تقدم هذه الحالة في‬
‫مرضى البوال السكري‬
‫وقد عدلت األبحاث الخاصة باالعتالل الشبكي السكري طريقة التفكير في دور عوامل النمو في‬
‫تحفيز عملية تكون األوعية الدموية ‪.‬وعوامل النمو في تحفيز عملية تكون األوعية الدموية‬
‫كثيرة ولكن يبقي هناك خمسة من هذه العوامل هي األكثر تورطا في مثل هذا اإلحداث‬
‫المرضي‪ .‬ويعتبر عامل النمو المشابه لألنسولين ( العامل رقم‪ )1‬و عامل النمو الكبدي من بين‬
‫هذه العوامل الخمسة المؤثرة‪.‬‬
‫والهدف من هذه الدراسة هو تقديم طريقة بسيطة وسهلة للتنبؤ بتقدم حاالت االعتالل الشبكي‬
‫السكري من خالل دراسة مستوىات البالزما لعامل النمو المشابه لألنسولين وعامل النمو الكبدي‬
‫في مراحل مختلفة من حاالت االعتالل الشبكي السكري ودراسة العالقة بين هذه العوامل‬
‫وضبط مستوي السكر في الدم انعكاسا من مستوى الهيموجلوبين السكري وعما إذا ما كان‬
‫بإمكاننا استخدام هذه العوامل كدالالت مبكرة على الخطورة‪.‬‬
‫‪18‬‬
‫الطرق المستخدمة‪:‬‬
‫تم إجراء هذا البحث على سبعين حالة من الجنسين و من مختلف األعمار من المرضى‬
‫المترديين على العيادة الخارجية لقسم طب وجراحة العين بمستشفيات جامعة المنيا والذين تم‬
‫التخطيط لهم إلجراء عملية مياه بيضاء‪ ,‬حيث أن خمسين من هؤالء المرضى من مرضى‬
‫البوال السكري(بنوعيه) وعشرة من المتطوعين األصحاء‪.‬‬
‫وقد قسمت الحاالت إلى أربع مجموعات‪:‬‬
‫المجموعة األولى‪ :‬اشتملت علي عشرين من مرضى البوال السكري بدون االعتالل الشبكي‬
‫السكري ‪.‬‬
‫المجموعة الثانية‪ :‬اشتملت علي عشرين من مرضى البوال السكري المصابين باالعتالل‬
‫الشبكي السكري الغير تشعبي‪.‬‬
‫المجموعة الثالثة‪ :‬اشتملت علي عشرة من مرضى البوال السكري المصابين باالعتالل الشبكي‬
‫السكري التشعبي‪.‬‬
‫المجموعة الرابعة‪ :‬اشتملت علي عشرين من المتطوعين األصحاء المصابين بالمياه البيضاء‬
‫فقط‪..‬‬
‫هذا وقد خضعت جميع الحاالت لآلتي‪:‬‬
‫‪ ‬فحص طبي ‪ :‬وشمل قصة المرض ومدته ونوع العالج ووجود أي منن مضناعفات منرض‬
‫البوال السكري‪.‬‬
‫‪ ‬فحص عيني ‪ :‬وشمل توسيع حدقة العين وفحص قاع العين ‪.‬‬
‫‪ ‬فحوصات معملية ‪ :‬وشـــملت‬
‫‪ .1‬تحليل سكر صائم بالدم‪.‬‬
‫‪ .2‬قياس مستوى الهيموجلوبين السكري‬
‫‪ .3‬قياس نسبة الكرياتينين بالدم ‪.‬‬
‫‪ .4‬قياس وظائف الكبد )‪(AST and ALT‬‬
‫‪ .5‬قياس مستوى عامل النمو المشنابه لألنسنولين فني البالزمنا والسنائل المنائي للعنين باسنتخدام‬
‫اختبار إليزا ‪.‬‬
‫‪ .6‬قياس مستوى عامل النمو الكبدي في البالزما والسائل المائي للعين باستخدام اختبار إليزا‬
‫النتائج‪:‬‬
‫أوضحت نتائج دراستنا ما يلي‪:‬‬
‫‪ ‬تزداد مستويات السكر الصائم و الهيموجلوبين السكري زيادة ذات داللة إحصائية في جمينع‬
‫مرضننى البننوال السننكري إذا مننا قورنننت بمجموعننة األصننحاء وكننذلك تننزداد هننذه المسننتويات‬
‫زينننادة ذات داللنننة إحصنننائية كبينننرة بنننين مرضنننى البنننوال السنننكري منننن ذوى المضننناعفات‬
‫ومرضى البوال السكري بدون مضاعفات‪.‬‬
‫‪ ‬تزداد مستويات عامل النمو المشابه لألنسولين زيادة ذات داللة إحصائية في جميع مرضنى‬
‫البوال السكري في البالزما والسائل المائي إذا ما قورنت بمجموعة األصنحاء وكنذلك تنزداد‬
‫مسننتويات عامننل النمننو المشننابه لألنسننولين زيننادة ذات داللننة إحصننائية كبيننرة بننين مرضننى‬
‫البوال السكري منن ذوى المضناعفات ومرضنى البنوال السنكري بندون مضناعفات( منا عندا‬
‫مستواه في السائل المائي بين المجموعة األولى و الثانية) ‪.‬‬
‫‪ ‬تزداد مسنتويات عامنل النمنو الكبندي زينادة ذات داللنة إحصنائية فني جمينع مرضنى البنوال‬
‫السننكري فنني البالزمننا والسننائل المننائي إذا مننا قورنننت بمجموعننة األصننحاء وكننذلك تننزداد‬
‫مستويات عامل النمو الكبدي زيادة ذات داللة إحصائية كبيرة بين مرضنى البنوال السنكري‬
‫من ذوى المضاعفات ومرضى البوال السكري بدون مضاعفات‪.‬‬
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‫‪ ‬وجنند اخننتالف ذو داللننة إحصننائية مننن حيننث فتننرة حنندوث المننرض عننند مقارنننة المجموعننة‬
‫األولى بالمجموعة الثانية و الثالثة‪.‬‬
‫‪ ‬وجد ارتباط ايجابي ذو داللة إحصائية كبيرة بين مستوى عامل النمو المشابه لألنسولين في‬
‫البالزمننا و السننائل المننائي فنني المجموعننة الثالثننة وكننذلك ارتبنناط ايجننابي ذو داللننة إحصننائية‬
‫كبينننرة بنننين مسنننتوى عامنننل النمنننو المشنننابه لألنسنننولين فننني البالزمنننا و السنننائل المنننائي و‬
‫الهيموجلوبين السكري و مستوى السنكر الصنائم فني المجموعنة الثالثنة و وجند هنذا التوافن‬
‫أيضننا بننين مسننتوى عامننل النمننو المشننابه لألنسننولين و فتننرة حنندوث المننرض فنني المجموعننة‬
‫الثانية و الثالثة‪.‬‬
‫‪ ‬وجد ارتباط ايجابي ذو داللة إحصائية كبيرة بين مستوى عامل النمو الكبدي فني البالزمنا و‬
‫السائل المائي في المجموعة الثالثة و أيضا بين مستوى عامل النمو الكبدي و مستوى السكر‬
‫الصائم و الهيموجلوبين السكري في المجموعة الثالثة‪.‬‬
‫‪ ‬وجد ارتباط ايجابي ذو داللة إحصائية كبيرة بين مسنتوى عامنل النمنو المشنابه لألنسنولين و‬
‫عامل النمو الكبدي في المجموعة الثالثة‪.‬‬
‫االستنتاج‪:‬‬
‫أوضننحت نتننائا دراسننتنا أن مسننتوى عامننل النمننو المشننابه لألنسننولين و عامننل النمننو الكبنندي فنني‬
‫البالزمننا يمننثالن دالئننل مبكننرة لالعننتالل الشننبكي السننكري وأن هننذه المسننتويات مننن الممكننن أن‬
‫تتالزم مع درجة االعتالل الشبكي و من الممكن أن تخدم هنذه القياسنات كندالئل مبكنرة لحندوث‬
‫االعتالل الشبكي السكري‪.‬‬