STUDY OF VASCULAR ENDOTHELIAL GROWTH FACTOR (VEGF)
AND BCL-2 PROTEIN LEVELS IN SERUM AND VITREOUS HUMOR
Of PATIENTS WITH PROLIFERATIVE DIABETIC
RETINOPATHY
by
Ola Hussein Aly Elgaddar
ADVISORS
Prof. Dr. Ahmad Mohamad Zaki Professor of Chemical Pathology
Medical Research Institute
Alexandria University
Prof. Dr. Ahmad Magdy Bedda Professor of Ophthalmology
Faculty of Medicine
Alexandria University
Dr. Amel Abd El-Fattah Kamel Assistant Professor of Chemical Pathology
Medical Research Institute
Alexandria University
Dr. Hoda Ali El-Attar Assistant Professor of Chemical Pathology
Medical Research Institute
Alexandria University
INTRODUCTION INTRODUCTION
DIABETIC RETINOPATHY (DR)
Normal vision DR vision
DR is a devastating microvascular complication
of diabetes mellitus.
It is considered the leading cause for adult
blindness.
Its prevalence among diabetic patients in Egypt
is 42%.
Risk factors includes: poor glycemic control,
duration of diabetes, hypertension,
hyperlipidemia and proteinuria.
DR can be classified into:
Early non-proliferative diabetic
retinopathy
Advanced non-proliferative diabetic
retinopathy
Proliferative diabetic retinopathy
Non-proliferative Diabetic Retinopathy
Proliferative Diabetic Retinopathy
Hypoxia occurring early in the course of DR
triggers the release of several growth
factors that promote retinal
neovascularization.
Among these growth factors are:
Insulin-like growth factor-I (IGF-I)
Basic fibroblast growth factor (bFGF)
Hepatocyte growth factor (HGF)
Vascular endothelial growth factor (VEGF)
VASCULAR ENDOTHELIAL
GROWTH FACTOR (VEGF)
VEGF-A is a 34- to 42-kDa dimeric
glycoprotein.
It is a member of the VEGF family that
currently comprises seven members;
VEGF-A (hereafter referred to as VEGF),
VEGF-B, VEGF-C, VEGF-D, VEGF-E,
VEGF-F, and placental growth factor (PlGF)
Monomer
Monomer Dimer
VEGF STRUCTURE
ACTIVITIES OF VEGF
A. Mitogenesis, angiogenesis, and endothelial
cell survival.
B. Stimulatory effect on bone marrow cells and
hematopoiesis.
C. Enhancement of vascular permeability.
REGULATION OF
VEGF PRODUCTION
I) Hypoxia: The main stimulus
II) Growth factors & hormones: PlGF, TNF- α, bFGF, TGF-ß, PDGF,Ang-1, Ang-2,
IGF-1, IL-1 and IL-6
TSH, ACTH, estrogens & progestins
III) Glucose: Hyperglycemia or hypoglycemia???
VEGF RECEPTORS
Receptor tyrosine kinases family (RTKs):
VEGFR-1 (Flt-1)
VEGFR-2 (KDR)
VEGFR-3 (Flt-4)
Co receptors:
Neuropilin-1&-2
VEGF IN PATHOLOGICAL CONDITIONS
A) Solid & hematological tumors:
As in Lung, breast, renal, ovarian and
intracranial tumors as well as in some
lymphomas and leukemias.
In these tumors, VEGF Induces Bcl-2 production
thus increasing tumor cells survival.
B) Intraocular neovascular syndromes:
Like Diabetes mellitus, occlusion of central retinal vein and neonatal prematurity.
Retinal ischemia is the main stimulus for VEGF production in such conditions.
In the eye, VEGF is produced by retinal pericytes, endothelial cells and glial cells.
It leads to ocular neovascularization, hemorrhages and vascular permeability, which results in visual impairment/blindness.
B-Cell lymphocyte/leukemia-2
(Bcl-2)
Bcl-2 family members function as
regulators of apoptosis.
All family members share the presence of
at least one Bcl-2 homology (BH)
domain.
BCL-2 PROTEIN
Bcl-2 is the founding antiapoptotic member of this family of proteins.
It is a 25 KDa protein.
It is a membrane protein that is localized to the outer mitochondrial membrane, endoplasmic reticulum membrane, and nuclear envelope.
Lymphoid germinal center.
Proliferative precursor cells in the bone marrow.
Glandular epithelium of the breast, thyroid & prostate.
Normally, it is expressed in cells and
tissues characterized by apoptotic turn
over, like:
Pathologically, Bcl-2 is over expressed in
many malignant & non-malignant conditions:
Malignant
• B- Cell lymphoma
• Ovarian cancer
• HCC
• Malignant melanoma
• Breast cancer
• Colorectal cancer
• Lung cancer
• Kaposi sarcoma
Non-malignant
• Epilepsy
• Endometriosis
• Liver cirrhosis
• Multiple sclerosis
1) Blocking the release of cytochrome- c from the
mitochondria to the cytosol.
2) Inhibition of the proapoptotic effects of the Bcl-2
family proteins (e.g., Bax and Bak).
3) Maintenance of sufficient Ca++ in the
sarcoplasmic/endoplasmic reticulum and
mitochondria.
4) Direct antioxidant activity.
The mechanisms by which Bcl-2 suppresses apoptosis
So……
Is there a role for VEGF and Bcl-2
in the pathogenesis of PDR?
Is it possible that the angiogenic effect of VEGF in PDR is via Bcl-2 up regulation?
AIM OF THE WORK
This study aimed at evaluating the
levels of VEGF and Bcl-2 protein
in the serum and vitreous humor
of patients with proliferative
diabetic retinopathy
SUBJECTS
SUBJECTS
40 subjects were included in the present study divided as follow:
Patients group
25 subjects with PDR
Undergoing vitrectomy
Control group
15 non-diabetic subjects
Undergoing vitrectomy
METHODS
To all studied subjects the following was done:
I) Full clinical examination.
II) Laboratory investigations:
Preliminary tests in serum.
(F.B.G, RFTs, lipid profile & aminotransferases activities)
Measurement of glycated hemoglobin (Hb A1C
) by resin chromatography technique.
Estimation of serum and vitreous humor levels of both VEGF & Bcl-2 by ELISA technique.
VITREOUS HUMOR SAMPLING
VITREOUS HUMOR
A clear avascular gel which occupies the posterior compartment of the eye.
It has the following composition:
• Water (99%)
• Network of collagen fibrils
• Large molecules of hyaluronic acid
• Peripheral cells (hyalocytes)
• Inorganic salts, sugar and ascorbic acid
VITRECTOMY
RESULTS
0
20
40
60
80
100
120
140
160
180
mg/ dl
1 2
Controls Patients
F.B.G in the studied groups
VEGF IN THE VITREOUS HUMOR
PatientsControls
Vitre
ou
s V
EG
F (p
g/m
l)
500
400
300
200
100
0
p= 0.001
VEGF IN THE SERUM
PatientsControls
Se
ru
m V
EG
F (p
g/m
l)
500
400
300
200
100
0
BCL-2 IN THE VITREOUS HUMOR
p= 0.003
PatientsControls
Vitre
ou
s B
cl-2
(n
g/m
l)
35
30
25
20
15
10
5
0
-5
p= 0.001
BCL-2 IN THE SERUM
PatientsControls
Se
ru
m B
cl-2
(n
g/m
l)
35
30
25
20
15
10
5
0
CORRELATION BETWEEN SERUM
VEGF AND BCL-2 IN THE PATIENTS
Serum VEGF (pg/ml)
7006005004003002001000
Se
ru
m B
cl-2
(n
g/m
l)
50
40
30
20
10
0
CONCLUSIONS
From the present study the following could be concluded:
1) Levels of VEGF and Bcl-2 were
significantly higher in the vitreous
humor of patients with PDR when
compared to their corresponding levels
in the control group suggesting that both
factors are incriminated in the pathogenesis
of this disease.
2) VEGF and Bcl-2 did not show any
statistical difference in the serum of the
studied groups. This finding may support
the hypothesis that their increased levels
in the vitreous is probably attributed to
intraocular synthesis, in response to local
retinal hypoxia, rather than to serum filtration.
3) A significant positive correlation was
found between serum Bcl-2 and serum
VEGF in the proliferative diabetic
retinopathy patients. This might suggest
that the role of VEGF in inducing
pathological angiogenesis in PDR might be
in part due to up regulation of the anti-
apoptotic protein Bcl-2.
RECOMMENDATIONS
VEGF together with Bcl-2 will hopefully lead
to the discovery of new targets for future
therapy for proliferative diabetic
retinopathy as well as other diseases with a
neovascular component.
THANK YOU
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