STUDY OF VASCULAR ENDOTHELIAL
GROWTH FACTOR (VEGF)
AND BCL-2 PROTEIN LEVELS
IN SERUM AND VITREOUS HUMOR
Of PATIENTS WITH
P...
ADVISORS
Prof. Dr. Ahmad Mohamad Zaki
Professor of Chemical Pathology
Medical Research Institute
Alexandria University

Pr...
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
blindnes...
DR can be classified into:

Early non-proliferative diabetic
retinopathy

Advanced non-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
neovasc...
VASCULAR ENDOTHELIAL
GROWTH FACTOR (VEGF)
VEGF-A is a 34- to 42-kDa dimeric
glycoprotein.
It is a member of the VEGF famil...
VEGF STRUCTURE
Monomer

Dimer

Monomer
ACTIVITIES OF VEGF
A. Mitogenesis, angiogenesis, and endothelial
cell survival.
B. Stimulatory effect on bone marrow cells...
REGULATION OF
VEGF PRODUCTION
I) Hypoxia:
The main stimulus

II) Growth factors & hormones:
TNF- α, bFGF, TGF-ß, PDGF,Ang-...
VEGF RECEPTORS


Receptor tyrosine kinases family (RTKs):
VEGFR-1 (Flt-1)
VEGFR-2 (KDR)
VEGFR-3 (Flt-4)



Co receptors:...
VEGF IN PATHOLOGICAL
CONDITIONS
A) Solid & hematological tumors:
As in Lung, breast, renal, ovarian and
intracranial tumor...
B) Intraocular neovascular syndromes:
Like Diabetes mellitus, occlusion of central retinal
vein and neonatal prematurity.
...
B-Cell lymphocyte/leukemia-2
(Bcl-2)
Bcl-2 family members function as
regulators of apoptosis.
All family members share th...
BCL-2 PROTEIN


Bcl-2 is the founding antiapoptotic member
of this family of proteins.



It is a 25 KDa protein.



It...
Normally, it is expressed in cells and
tissues characterized by apoptotic turn
over, like:


Lymphoid germinal center.

P...
Pathologically, Bcl-2 is over expressed in
many malignant & non-malignant conditions:
Malignant
• B- Cell lymphoma
• Ovari...
The mechanisms by which Bcl-2
suppresses apoptosis
1) Blocking the release of cytochrome- c from the
mitochondria to the c...
So……


Is there a role for VEGF and Bcl-2
in the pathogenesis of PDR?



Is it possible that the angiogenic
effect of VE...
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 pro...
SUBJECTS
SUBJECTS
40 subjects were included in the
present study divided as follow:
Patients group


25 subjects with PDR

Undergoing vitre...
METHODS
To all studied subjects the following
was done:
I) Full clinical examination.
II) Laboratory investigations:
 Preliminary...
VITREOUS HUMOR SAMPLING
VITREOUS HUMOR


A clear avascular gel which occupies the
posterior compartment of the eye.



It has the following comp...
VITRECTOMY
RESULTS
F.B.G in the studied groups

180

160

140

120

100

mg/ dl
80

60

40

20

0
1

2

Controls

Patients
VEGF IN THE VITREOUS HUMOR
500

p= 0.001
400

300

200

100

0

Controls

Patients
VEGF IN THE SERUM
500

400

300

200

100

0

Controls

Patients
BCL-2 IN THE VITREOUS HUMOR
35

30

p= 0.003
p= 0.001

25

20

15

10

5

0
-5

Controls

Patients
BCL-2 IN THE SERUM
35

30

25

20

15

10

5

0

Controls

Patients
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/...
CONCLUSIONS
From the present study the following
could be concluded:
1) Levels of

VEGF and Bcl-2 were
significantly higher in the vit...
2) VEGF

and Bcl-2 did not show any
statistical difference in the serum of the
studied groups. This finding may support
th...
3) A significant positive correlation was
found between serum Bcl-2 and serum
VEGF in the proliferative diabetic
retinopat...
RECOMMENDATIONS
VEGF together with Bcl-2 will hopefully lead
to the discovery of new targets for future
therapy
for
proliferative diabetic...
THANK YOU
Study of vascular endothelial growth factor (vegf) and bcl 2 protein levels  in serum and vitreous humor  of patients with...
Study of vascular endothelial growth factor (vegf) and bcl 2 protein levels  in serum and vitreous humor  of patients with...
Study of vascular endothelial growth factor (vegf) and bcl 2 protein levels  in serum and vitreous humor  of patients with...
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Study of vascular endothelial growth factor (vegf) and bcl 2 protein levels in serum and vitreous humor of patients with proliferative diabetic retinopathy

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MSc Thesis defense
Ola Elgaddar, 26 - 11 - 2006

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Study of vascular endothelial growth factor (vegf) and bcl 2 protein levels in serum and vitreous humor of patients with proliferative diabetic retinopathy

  1. 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. 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. 3. INTRODUCTION INTRODUCTION
  4. 4. DIABETIC RETINOPATHY (DR) Normal vision DR vision
  5. 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. 6. DR can be classified into: Early non-proliferative diabetic retinopathy Advanced non-proliferative diabetic retinopathy Proliferative diabetic retinopathy
  7. 7. Non-proliferative Diabetic Retinopathy Proliferative Diabetic Retinopathy
  8. 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. 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. 10. VEGF STRUCTURE Monomer Dimer Monomer
  11. 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. 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. 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. 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. 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. 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.
  17. 17. 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.
  18. 18. 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. 
  19. 19. 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
  20. 20. 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.
  21. 21. 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?
  22. 22. AIM OF THE WORK
  23. 23. 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
  24. 24. SUBJECTS SUBJECTS
  25. 25. 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
  26. 26. METHODS
  27. 27. 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. 
  28. 28. VITREOUS HUMOR SAMPLING
  29. 29. 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 • • • • •
  30. 30. VITRECTOMY
  31. 31. RESULTS
  32. 32. F.B.G in the studied groups 180 160 140 120 100 mg/ dl 80 60 40 20 0 1 2 Controls Patients
  33. 33. VEGF IN THE VITREOUS HUMOR 500 p= 0.001 400 300 200 100 0 Controls Patients
  34. 34. VEGF IN THE SERUM 500 400 300 200 100 0 Controls Patients
  35. 35. BCL-2 IN THE VITREOUS HUMOR 35 30 p= 0.003 p= 0.001 25 20 15 10 5 0 -5 Controls Patients
  36. 36. BCL-2 IN THE SERUM 35 30 25 20 15 10 5 0 Controls Patients
  37. 37. 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
  38. 38. CONCLUSIONS
  39. 39. 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.
  40. 40. 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.
  41. 41. 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.
  42. 42. RECOMMENDATIONS
  43. 43. 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.
  44. 44. THANK YOU

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