In The Name Of God

Review of
Diabetic Microangiopathic
Complications

Javad Tavakkoly Bazzaz MD, PhD;

Mahsa M. Amoli MD, PhD, Bagher Larijani MD
Endocrinology & Metabolism Research Center (EMRC)
Tehran University of Medical Sciences (TUMS)

Definition Classification of Diabetes

Diabetes mellitus: A chronic disorder Type 1 Diabetes Mellitus
characterized by a deficiency of insulin Type 2 Diabetes Mellitus
secretion and/or insulin effect, which causes Other Specific Types
hyperglycemia, disturbances of carbohydrate,
fat and protein metabolism, and a constellation Gestational Diabetes Mellitus
of chronic complications .

Microvascular Complications Langerhans islet Diabetic Nephropathy

Diabetic retinopathy
background retinopathy
macular edema
proliferative retinopathy a c
Diabetic nephropathy
Diabetic neuropathy
distal symmetrical polyneuropathy
mononeuropathy (peripheral, cranial
nerves) b d
autonomic neuropathy a) Insulitis c) Nodular glomerulosclerosis
b) Amyloid deposition d) NG & hyaline arteriolosclerosis

1
 Diabetes Complications
Diabetic Retinopathy Diabetic foot
The Diabetes Control and
Complications Trial (DCCT)
Randomized, multicenter,
multicenter, prospective
trial including 1441 patients with Type 1
diabetes; 2 treatment groups
Average follow-
follow-up 7 years
> 98% completion of outcome
measures
Standard therapy: mean HbA1c=9.2%
Intensified therapy: mean HbA1c=7.2%
DCCT: Adverse Effects of
Intensified Management
Three-
Three-fold increase in severe
hypoglycemia (events requiring
assistance
Increased weight gain
No signigicant effects on cognitive
function, quality of life, cardiovascular
events
2
Diabetic chronic Complications:
Magnitude of Problem
Diabetic retinopathy: most common
cause of blindness before age 65
Nephropathy: most common cause of
ESRD
Neuropathy: most common cause of
non-
non-traumatic amputations
2-3 fold increase in cardiovascular
disease
DCCT: Effects of Intensified
Therapy on Complications
Reduced risk of retinopathy by 76%
Reduced risk of nephropathy by 54%
Reduced risk of neuropathy by 60%
No glycemic threshold for risk
reduction; any improvement in
BG=reduced risk
Same benefits in adults and
adolescents
Benefits greatest when initiated early
Does the DCCT Apply to
Type 2 Diabetes?
Vascular complications are the same in
Type 1 and Type 2
Epidemiologic studies show same
relationship between BG and
complications in Type 1 and Type 2
Short term clinical trials in Type 2:
reducing blood glucose improves
nerve, kidney function
 Etiology of diabetes complications Intra-Individual Variation of Complications
1-Biochemical factors: Endothelial cells, as a main target
Hyperglycemia, Polyol pathway, AGE, in diabetic chronic complications
alteration in the synthesis of Growth factors
2-Physical factors: Microangiopathic complications:
Rheological, HTN, hypercoagulability, -Retinal endothelial cells DR
3-General factors: -Glomerular endothelial cells DN
Duration of diabetes, age at the onset, -End. cells of endoneurial capillaries DNU
quality of metabolic control, sex, ...
Macrovascular complications:
4-Genetics
4-Genetics Atherosclerosis (cardiovascular com.)

Early increased microvascular pressure and flow

Diabetic Vascular diseases
Endothelial injury response
Microangiopathy Macroangiopathy
Diabetic Retinopathy Atheroscerosis
Diabetic Neuropathy Microvascular response
Diabetic Nephropathy
Myocardial Infarction
- +
Impaired microvascular hyperaemia

Loss of autoregulation
VEGF Pathogenesis of diabetic microangiopathy in IDDM
(Hemodynamic hypothesis)

Propose estimates of Endothelial Dysfunction in humans Altered vascular

Mediators Vascular Cells remodelling
Interpretation of altered (Cytokines) (resident & non-resident)
Measurement endothelial function Altered cell and
Growth factors: matrix turnover
Transcapillary escape rate of intraven-
intraven- Permeability to macromolecules TGF-, IGF, VEGF,
ousely injected radiolabeled albumin PDGF, FGF
Impaired enothelium-
enothelium-dependent Production of vasodilators such as NO Altered cell-to-cell &
vasodilation Vasoactive factors: cell-to matrix contacts
NO, ET-1, PG,
Endothelina Synthesis of vasoconstrictors TX, AT-II, ANP
Von Willebrand factora Prothrombotic and procoagulant activity Impaired vascular
tone regulation
sThrombomodulin
a Anticoagulant activity Coagulation factors:
PA, PAI-1, Thrombin,
Tissue-
Tissue-type plasminogen activator, Profibrinolytic activity Tissue factor PKC
Plasminogen activator inhibitor-
inhibitor-1a Polyols Increased vascular
GlcN ROS permeability
sE-selectin,
selectin, sVCAM-
VCAM-1a Permeability to leucocytes; inflammatory
Adhesion molecules:
activations AGEs
Integrins, Cadherins, Lectins,
Cellular fibronectina, type IV collagen Altered extracellular matrix synthesis Ig superfamily (PECAM-1 ?) Pro-aggregant &
fragments pro-coagulant pattern
s= soluble ; a =Increased plasma or serum level.

3
 Causes of growth factors upregulation Hyperglycemia
in Diabetes
GH AT-II
-Direct effect of glucose & glucoseamine
IGF-I TGF-
-AGE formation (PDGF, ET-1, PAI-1)

-Ischemia (due to loss of capillaries) matrix deposition

(synthesis & degradation)
- PKC activation
-Mesangial stretch Glomerulosclerosis

-GFR Nephropathy

ET-1
Diabetes Growth factors
NO
Involved in angiogenesis
Retinal blood VEGF Other factors: Endothelial cell response
flow sensitivity ROI, AGE, NADH/NAD Activation Enzyme Differe- End.
Migration proliferation Differe-
GFs release/ ntiation cell
expression specific

Hypoxia VEGF VEGF Hypoxia VEGF + + + ?

+
TGF-1
TGF- _ _ _ _ + _
ET-1 NO

IGF-
IGF-I + + +
_
Capillay Retinopathy
closure progression TNF-
TNF- + + + +
_ _
RBF
FGF + + + + + _
Neovascularization Insulin + +
_
+, stimulation; _ , inhibition; blank, not tested

TGF-1 and Diabetic Nephropathy Hyperglycemia

Aminoguanidine
LY333531
Oxidants
Glycations
mRNA of
TGF-
1 Vit. E
DAG/PKC ,
ECM components
Metalloproteinases

TGF- C-fos, VEGF ET-1, Caldesmon
(collagens) PLA2
Metalloproteinases ANP, TGF eNOS PAI-I
ICAMs
Type IV collagen; PGE2
Decreased degradation inhibitors PKC
Increased
Fibronectin
of ECM components Na+-K+-
collagen Basemembrane ATPase
deposition Thickening; Permeability; P Fibrinolysis;
Extracellular matrix Angiogenesis; Flow;
expansion; Cell turnover Contractility Adhesion
Cardiomyopathy
Accumulation of O
ECM components
P
Diabetic vascular complications

4
 Hyperglycemia
Polyol Glucose Protein Vasoacive G
pathway autoxidation glycation hormones
Antioxidant NGF NADPH GSSG
+
defence IGF-I AR ROS PKC
IGFI
TGF-
NADH/NAD+ Oxidative VEGF
NADP+ GSH
stress NCV

Glyk
trioses O2--NO axonal +
degeneration Vascular Sorbitol AGE MGO DHAP Gly-3P Gly DAG
DAG permeability
VEGF
ECM NAD + GA-3P
PKC Ca+ + crosslinking SD
ECM GAPDH
NOS acumulaion NADH 1,3 -DPG Glycolysis
TGF-
IGF-I Vascular Proteinuria
dysfunction Fructose F-6P
GFAT
Retinopathy Neuropathy Nephropathy GlcN-6P GlcNAC-6P GlcNAC-1P UDP-GlcNAC

Inter-individual variation of
Hyperglycemia
diabetes complications
(case specifity)
I- Enzymes affecting Enzymes affecting
-Variation of complication prevalence in glucose metabolism vascular risk
different ethnic group, and different families.
-Familial clustering of diabetes complications.
-Follow-up studies. Genetics
-Sib-pair, Mono- and Di-zygotic twin studies.

II-Environmental: Hyperglycemia, Diabetes Complications

e x ia ss
a Hyperglycaemia b Retinal Ischemia os po tre s s
GF F-
uc tS re
Gl Hy n St VE TN
ida ea
r
Ox Sh
A1 A2 A3 A
(AGEs) (Tissue RAS) (GFR) (VEGF)
PECAM-1
Ph
os Do
B1 B2 B3 B1 B2 B3 ph
ory wn M
(Renal AGE (Renal AGTII)
Deposition)
(Mesangial
Stretch)
(Angiogenesis) (Vascular
lat reg ono
permeability)
ion u la /di
ti o me
n ris
a tio
n
C
(TGF-) C
(DR)
Signaling Cascade Adhesion Cascade
Cyt & Chem. Production Trans-endothelial migration of Mon.
D T Cell activ. & Proliferation Endothelial migration
(DN) Angiogenesis Platelet Anti-agregation
The contributing pattern of the player as a convergent(a) or divergent(b) Chemotaxy
mediator in disease development

5
 A NOVEL POLYMORPHISM IN PECAM-1 GENE PROMOTER REGION:
PECAM-1 (CD31) ASSOCIATION STUDY WITH T1DM AND ITS MICROANGIOPATHIC COMPLICATIONS
Tavakkoly Bazzaz J*, Larijani B*, Boulton AJM**, Pravica V*** & Hutchinson IV****
*Endocrinology & Metabolism Research Center (EMRC), Tehran University of Medical Sciences, Tehran 14114, IRAN

Adhesion Molecule Signalling Molecule

**The Manchester Diabetes Centre, Manchester Royal Infirmary, Manchester M13 9WL, UK
***School of Biological Sciences, University of Manchester, Manchester M13 9PT, UK
****National Institute of Transplantation, Los Angeles, USA
INTRODUCTION Table 1. The Frequency Distribution of PECAM- 1 Gene Polymorphisms (-265* C/T) in IDDM
se ia ss s Patients
Platelet endothelial cell adhesion molecule-1 (PECAM-1, or CD31) is a 130-kDa co re es F
ox F -

Endothelial Cells (106 Copies/Cell)

St tr G
G lu H yp nt rS VE TN
100 60 Diabetic Retinopathy
+ 55.3 60
Diabetic Nephropathy
56.4
54.5 60
Diabetic
+ Neuropathy
56.6

member of the Ig superfamily. As a transmembrane glycoproteine PECAM-1 is da Control +

Shear Stress
52
ea 51.6
O xi
_ _ _
Sh
IDDM 50 50 Diabetic
80 Neuropathy 50

65
expressed at high levels at the borders between endothelial cells and on the 40 40 40

P E C A M -1
60
50.4 28.7
30 27 28.3
30 30

%
surface of circulating platelets, monocytes, neutrophils, and particular T cell

%
%
25

%
23 23.2 21.7 21.7
40 20.2 19.7
27.6 20 17.6 20 17.2 20
22 20
subsets. Further to its main role in the adhesion cascade leading to Ph
os D ow
20 15
10 10 10

ph
transendothelial migration of leukocytes, PECAM-1 is also a downstream M

Platelets (5000 Copies/Platelet)

0 0 0 0
o ry nr on
la tio eg o/ di
TT CC CT TT CC CT TT CC CT TT CC CT

ul

Hypoxia signaling molecule for diverse stimuli. PECAM-1 is able to interact with itself
and other non-PECAM-1 molecules, homophilic and heterophilic interactions
respectively. Diverse stimuli like high concentration of glucose, shear stress,
n at
io
n
m
er
is
at
io
n
Table 2. The Expression of PECAM-1 Molecule (%) in Controls Among its Polymorphic
Genotypes
53.66
S ig n a ling C a sc a d e Ad h e s io n C a s c a d e 48.58 55.5 55.05
57.00
hypoxia, ROS and VEGF, which are present in diabetic context, can activate Cy t & Ch em . P rod uc tio n
60
50
8
70
60
43.17
Tra ns -en doth elial m igration o f M on.

Monocytes this molecule through its phosphorylation. And with regard to the crucial role 50
T C ell a ctiv. & P rolife ration En do th elial m igra tio n 40
40

VEGF
An giog en es is P la telet A nti-a gre ga tion 30
of PECAM-1 in modulation of migration both of endothelial cells and C he m o ta xy
20
30
20
10
leuckocytes, vascular permeability, and angiogenesis, which are failing in 0 CC CT TT
10
0

IDDM, diabetic retinopathy and nephropathy, the potential role of PECAM-1
in diabetes and its complications has been analysed through its gene
Neutrophils TNF-
polymorphisms in this study. Cross-linking of CD31 itself have demonstrated
its role as a downstream signaling molecule, leading primarily to T cell
proliferation, IL-2R expression, TCR signaling and production of several
cytokines and chemokines. The gene for human PECAM-1 is located on the
long arm of chromosome 17, where is similar region for other adhesion
M molecules, like ICAM-2.
In this study, the genetic susceptibility to IDDM and its chronic complications
were assessed in 205 IDDM Caucasian individuals by seeking differences in the
distribution of the PECAM-1 SNPs in control and diabetic populations with
regard to investigate the association of PECAM-1 alleles with the occurrence of
T cells diabetic complications. In particular, the functional impact of these
polymorphisms on PECAM-1 molecule expression have been assessed by flow
cytometry as quantative approach.
MATERIALS & METHODS
RESULTS
We have discovered polymorphism in the promoter region of
PECAM-1 gene at position 265* CT (Gene Bank Ac. no.
AJ313330). (Fig. 1) While the PECAM-1 molecule is expressing at
high levels in constitute, its stimulation by LPS and PHA result in its
down-regulation until 48 hours. There was no any association on its
polymorphism distribution with its cellular expression, or with
IDDM and its chronic microangiopathic complications. ( Table 1 &
2)
Fig 1. The SSCP gel and sequencing of PECAM-1 Gene Polymorphism (
265* CT)
TT CC
%
GG GC CC
DISCUSSION & CONCLUSION
As the role of genetic factors on the development of both IDDM and its
late complications have been established, We considered PECAM-1 as a
candidate gene, because of a) its wide distribution among different cell
types, those have per se competent role in development of both IDDM and
its complications, and b) its diverse functions as a potential mediator for
these genetically influenced endpoints. While the data of our case-control
association study was showing PECAM-1 is not involved in the
development of these features at least through its gene polymorphism
based regulations, the Flow Cytometry was set up to investigate whether
these polymorphisms are functional, which may predict its level of
expression. Though these polymorphisms did not show any association
with the expressed phenotype in diabetics, but the down-regulated
TC
PECAM-1 following stimulations may suggests its reciprocal role in

Trans-endothelial Migration
compare with other ECAMs, as an anti-inflammatory molecule.
SSCP was developed to find putative polymorphisms in the promoter region of In terms of diabetes complications, it seems that involvement of PECAM-
PECAM-1 gene in over 50 of lab control DNA samples. The PCR products of 1 is due to non-inflammatory based or metabolic factors (i.e.
this region were run on ACRYL/BIS gel (30%). (Fig. 1) To determining the Hyperglycemia) rather than inflammation rised, and the studies of
nucleotide changes supposed by SSCP, DNA sequencing was performed by the PECAM-1 under glucose stimulation should be helpful. Also, the
Fig 2. The Flow cytometry of PECAM-1 in Negative and Positive Control (Non-

TCR Signalling ABI prismTM BigDyeTM terminator cycle sequencing system. activated) qualitative approaches for PECAM-1, like the examination of
A cohort of 205 British Caucasian IDDM patients, who were diagnosed at least phosphorylation state and the capacity/treshold of phosphorylative
3 years prior to this study, was selected. They were evaluated for retinopathy, response among different individuals may provide more insights to
nephropathy and neuropathy, or were designated as complication free. These evaluate the function of PECAM-1 with regard to its genetic background.

T Cell Proliferation
were overlapping, so that most patients had more than one manifestation of
complications. DNA was extracted from peripheral blood, and genotyped for
the two polymorphisms of PECAM-1 gene at the positions -265 (TC), and REFERENCES
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Cytokines & Chemokines Production

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treated 3 times for each samples at : fresh non-activated and activated states Newman PJ. The biology of PECAM-1. J Clin Invest. 1997;100(11 Suppl):S25-9.
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