MSc Thesis defense Ola Elgaddar, 26 - 11 - 2006

1. 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

2. 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

3. INTRODUCTION
INTRODUCTION

4. DIABETIC RETINOPATHY (DR)
Normal vision
DR vision

5. 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.

6. DR can be classified into:
Early non-proliferative diabetic
retinopathy
Advanced non-proliferative diabetic
retinopathy
Proliferative diabetic retinopathy

7. Non-proliferative
Diabetic Retinopathy
Proliferative
Diabetic Retinopathy

8. 
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)





9. 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)

10. VEGF STRUCTURE
Monomer
Dimer
Monomer

11. ACTIVITIES OF VEGF
A. Mitogenesis, angiogenesis, and endothelial
cell survival.
B. Stimulatory effect on bone marrow cells and
hematopoiesis.
C. Enhancement of vascular permeability.

12. REGULATION OF
VEGF PRODUCTION
I) Hypoxia:
The main stimulus
II) Growth factors & hormones:
TNF- α, bFGF, TGF-ß, PDGF,Ang-1, Ang-2,
IGF-1, IL-1 and IL-6
 TSH, ACTH, estrogens & progestins
 PlGF,
III) Glucose:
Hyperglycemia or hypoglycemia???

13. VEGF RECEPTORS

Receptor tyrosine kinases family (RTKs):
VEGFR-1 (Flt-1)
VEGFR-2 (KDR)
VEGFR-3 (Flt-4)

Co receptors:
Neuropilin-1&-2

14. 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.

15. 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.

16. 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.

19. 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.

20. Normally, it is expressed in cells and
tissues characterized by apoptotic turn
over, like:

Lymphoid germinal center.
Proliferative precursor cells in the bone
marrow.

Glandular epithelium of the breast, thyroid
& prostate.


21. 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

22. The mechanisms by which Bcl-2
suppresses apoptosis
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.

23. 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?

24. AIM OF THE WORK

25. 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

26. SUBJECTS
SUBJECTS

27. 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

28. METHODS

29. 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.


30. VITREOUS HUMOR SAMPLING

31. 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
•
•
•
•
•

32. VITRECTOMY

34. RESULTS

35. F.B.G in the studied groups
180
160
140
120
100
mg/ dl
80
60
40
20
0
1
2
Controls
Patients

36. VEGF IN THE VITREOUS HUMOR
500
p= 0.001
400
300
200
100
0
Controls
Patients

37. VEGF IN THE SERUM
500
400
300
200
100
0
Controls
Patients

38. BCL-2 IN THE VITREOUS HUMOR
35
30
p= 0.003
p= 0.001
25
20
15
10
5
0
-5
Controls
Patients

39. BCL-2 IN THE SERUM
35
30
25
20
15
10
5
0
Controls
Patients

40. CORRELATION BETWEEN SERUM
VEGF AND BCL-2 IN THE PATIENTS
50
40
30
20
10
0
0
100
200
300
400
500
Serum VEGF (pg/ml)
600
700

41. CONCLUSIONS

42. 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.

43. 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.

44. 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 antiapoptotic protein Bcl-2.

45. RECOMMENDATIONS

46. 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.

47. THANK YOU