Group Title: Cardiovascular Diabetology 2005, 4:12
Title: Endothelial cell injury by high glucose and heparanase is prevented by insulin, heparin and basic fibroblast growth factor
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Title: Endothelial cell injury by high glucose and heparanase is prevented by insulin, heparin and basic fibroblast growth factor
Series Title: Cardiovascular Diabetology 2005, 4:12
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Creator: Han J
Mandal AK
Hiebert LM
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Cardiovascular Diabetology ioMed



Original investigation

Endothelial cell injury by high glucose and heparanase is prevented
by insulin, heparin and basic fibroblast growth factor
Juying Han', Anil K Mandal2 and Linda M Hiebert*

Address: 'Department of Veterinary Biomedical Sciences, University of Saskatchewan, Saskatoon, Saskatchewan, S7N 5B4, Canada and
2Department of Medicine, University of Florida, Jacksonville, Florida, 32086, USA
Email: Juying Han juyinghan@yahoo.ca; Anil K Mandal amandal@med-spec.com; Linda M Hiebert* linda.hiebert@usask.ca
* Corresponding author


Published: 09 August 2005
Cardiovascular Diabetology 2005, 4:12 doi: 10.1 186/1475-2840-4-12


Received: 23 June 2005
Accepted: 09 August 2005


This article is available from: http://www.cardiab.com/content/4/1/12
2005 Han et al; licensee BioMed Central Ltd.
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0),
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.



Abstract
Background: Uncontrolled hyperglycemia is the main risk factor in the development of diabetic
vascular complications. The endothelial cells are the first cells targeted by hyperglycemia. The
mechanism of endothelial injury by high glucose is still poorly understood. Heparanase production,
induced by hyperglycemia, and subsequent degradation of heparan sulfate may contribute to
endothelial injury. Little is known about endothelial injury by heparanase and possible means of
preventing this injury.
Objectives: To determine if high glucose as well as heparanase cause endothelial cell injury and if
insulin, heparin and bFGF protect cells from this injury.
Methods: Cultured porcine aortic endothelial cells were treated with high glucose (30 mM) and/
or insulin (I U/ml) and/or heparin (0.5 ug/ml) and /or basic fibroblast growth factor (bFGF) (I ng/
ml) for seven days. Cells were also treated with heparinase I (0.3 U/ml, the in vitro surrogate
heparanase), plus insulin, heparin and bFGF for two days in serum free medium. Endothelial cell
injury was evaluated by determining the number of live cells per culture and lactate dehydrogenase
(LDH) release into medium expressed as percentage of control.
Results: A significant decrease in live cell number and increase in LDH release was found in
endothelial cells treated with high glucose or heparinase I. Insulin and/or heparin and/or bFGF
prevented these changes and thus protected cells from injury by high glucose or heparinase I. The
protective ability of heparin and bFGF alone or in combination was more evident in cells damaged
with heparinase I than high glucose.
Conclusion: Endothelial cells injured by high glucose or heparinase I are protected by a
combination of insulin, heparin and bFGF, although protection by heparin and/or bFGF was variable.


Background
Diabetes mellitus is characterized by hyperglycemia and
vascular complications including microangiopathy and
macroangiopathy [1,2]. The hallmarks of diabetic micro-
angiopathy are retinopathy and nephropathy leading to


blindness and renal failure respectively [3,4]. Macroangi-
opathy in diabetes, includes coronary artery disease,
peripheral vascular disease, and cerebrovascular disease,
and results from an acceleration of atherosclerosis and
increased thrombosis thus increasing the risk of


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Cardiovascular Diabetology 2005, 4:12


myocardial infarction, stroke and ischaemia [5,6]. It is
reported that under better glycemic control fewer patients
develop eye and/or renal complications [7].

Since the initial injury by hyperglycemia occurs in the
blood vessel, endothelial cells (ECs) are considered to be
the first target. Heparan sulfate proteoglycans (HSPGs),
an important EC component, are synthesized by ECs and
incorporated into the plasma membrane and extracellular
matrix (ECM) [8,9]. In the ECM, HSPGs interact with
fibronectin, laminin, collagen and growth factors such as
basic fibroblast growth factor (bFGF) and help maintain
vascular integrity [10,11]. HSPGs with their negative
charged sulfate and carboxylate residues, create a "charge
barrier", which decrease the permeability of anionic
plasma proteins [12]. Thus degradation of HSPGs could
lead to an increase in vascular permeability, decrease in
vascular integrity, and changes in growth factor activity.
Depletion of heparan sulfate (HS) and/or abnormal gly-
cosaminoglycan (GAG) metabolism appears to be a piv-
otal mechanism associated with diabetic EC injury. HSPG
or HS were decreased in the glomerular basement mem-
brane (GBM) of patients with overt diabetic nephropathy
which correlated with the degree of proteinuria [13,14]. A
similar decrease in HS content was observed in the aortic
intima of diabetic patients [15]. Skin basement mem-
brane thickness was significantly reduced in patients with
diabetic nephropathy compared to those without neph-
ropathy. As well, HSPG synthesis was decreased in aorta,
liver and intestinal epithelium of diabetic rats [16-18].
Thus in the diabetic condition, changes in HS metabolism
may occur in any tissue suggesting the link between HS
abnormalities and vascular complications in both large
and small vessels.

Heparanase is an endo-Yf-D-glucuronidase that cleaves HS
at specific interchain sites. Under normal physiological
conditions, heparanase is expressed in platelets, cytotro-
phoblasts, mast cells, neutrophils, macrophages, and the
placenta [19]. Heparanase activity was found in the urine
of some diabetic patients and heparanase protein was
expressed in both the glomerular mesangial and epithelial
cell lysates, but not in intact cells [20]. HSPG degradation
by heparanase upregulation may contribute to EC injury
by hyperglycemia. Thus we wished to determine if hepara-
nase as well as high glucose injured ECs.

Insulin and heparin alone, or in combination, prevented
the intercellular gaps formed in ECs cultured in high glu-
cose [21]. Several previous studies have shown that insu-
lin increases nitric oxide (NO) production in cultured ECs
and ensures normal vascular function [22,23]. Heparin
can accumulate in ECs at a greater concentration than in
plasma, increase HS on the EC surface, and prevent ECs
from free radical injury [24-26]. Therefore, we postulated


that insulin and/or heparin would protect ECs from high
glucose or heparanase injury.

bFGF has a high affinity for heparin and HS which is
required for interaction with its receptor (bFGFR). How-
ever, HS in the ECM also limits bFGF release into intersti-
tial spaces [27,28]. The high affinity of bFGF for heparin
and HS, together with the EC proliferation potential of
bFGF [29], may protect ECs. Previous studies have shown
that both heparanase and plasmin degrade HSPG and
decrease the stability of the bFGF/HS/bFGFR complex
resulting in loss of bFGF which was corrected by exoge-
nous heparin [30,31]. Thus, the stabilization of bFGF/HS/
bFGFR complex, by supplying heparin and bFGF, may
protect ECs from injury by high glucose or heparanase.

Therefore, the purposes of our study were to determine if
high glucose or heparanase induced cultured EC injury
and if supplementation of insulin, heparin and bFGF
would protect ECs from this damage.

Methods
Culture of Porcine Aortic Endothelial Cells (PAECs)
PAECs were cultured according to the method of Gotlieb
and Spector [32]. Porcine aortic segments were rinsed in
three changes of calcium- and magnesium-free Dulbecco's
phosphate-buffered saline (CMF-DPBS), while the adven-
titial connective tissue and end pieces were trimmed. One
end of the aorta was clamped with two hemostats (ensur-
ing no leakage from bottom or branch points) and the
lumen was rinsed three times with CMF-DPBS and then
filled with collagenase solution (Type IV, SIGMA, St.
Louis, MO, USA; 1 mg/ml in CMF-DPBS at 37C). After 6
minutes the collagenase solution was removed and the
endothelial surface was gently rinsed with M199 (Gibco-
BRL, Life Technologies, Inc., Grand Island, NY, USA) con-
taining 5% fetal bovine serum (FBS, GibcoBRL), 50 /g/ml
penicillin (SIGMA) and 10 /g/ml streptomycin (SIGMA).
The medium was removed and plated onto 60 mm culture
dishes. The volume was made up to 2 ml medium/dish.
Cells were incubated at 370C with 5% C02/ 95% air in a
humidified environment. ECs were identified by their
morphological appearance of cobblestone-like flattened
cells and the presence ofvon Willebrand Factor (vWF) in
initial cultures. Non-endothelial-like cells, such as
smooth muscle cells and fibroblasts, were destroyed by
mechanical suction before the first passage.

To subculture, confluent 60 mm dishes of PAECs were
washed twice with sterile CMF-DPBS, followed by expo-
sure to a sterile 0.025% trypsin solution for two or three
minutes at room temperature. The cells were then resus-
pended in 6 ml of culture medium and seeded onto three
60 mm dishes (2 ml/dish). Confluent cultures at passage
4, in 35 mm dishes, were used in experiments.


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Reagents
Reagents were first prepared as stock solutions in CMF-
DPBS at the following concentrations: glucose (D-Glu-
cose, BDH Inc. Toronto, Canada) 3M, unfractionated
bovine lung heparin (151 USP U/mg Upjohn Pharmaceu-
ticals, Kalamazoo, MI, USA) 0.1 mg/ml, insulin (Humu-
lin N) 100 U/ml. Stock solutions of heparinase I
(SIGMA) 10 and 1 U/ml and bFGF (SIGMA) 0.1 ng//l
were prepared in M199 without serum.

Cell Treatment
Cell medium was changed just prior to addition of rea-
gents to 1 ml of medium per dish. For high glucose, 10 /l
of 3 M stock solution was added to give a final added con-
centration of 30 mM. For heparin, 5 /l of 0.1 mg/ml of
stock solution was added to give a final concentration 0.5
y/g/ml. For insulin, 10 /l of 100 U/ml stock solution was
added to give a final concentration of 1 U/ml. For bFGF,
10 /l of 0.1 ng//ul stock solution was added to give a final
concentration of 1 ng/ml. Cell medium was changed and
reagents were added fresh every other day for seven days.
Cells were harvested 48 hours after the last addition.

In order to determine the culture conditions and damag-
ing doses of heparinase I in PAECs, heparinase I was
added to cultures at concentrations of 0.01, 0.05, 0.1, 0.3
and 0.5 U/ml in medium, which was produced by adding
10 /l of 1 U/ml and 5, 10, 30 and 50 /ul of 10 U/ml of
heparinase I to 1 ml medium respectively. Cells were cul-
tured either in medium with serum for six or ten days,
where cell medium was changed and fresh heparinase I
was added every other day; or in serum free medium for
two days by adding heparinase I once.

To determine the effect of heparin, insulin and bFGF in
the presence heparinase I, cell medium was changed to
M199 without serum. Then 30 /l of 10 U/ml of hepari-
nase I was added to give a final concentration of0.3 U/ml.
Then heparin, insulin or bFGF was added to medium as
described above. Cells were harvested two days later.

Assessment of Cell Injury
Trypan blue exclusion
Number of viable cells was determined by using trypan
blue dye. The cells were washed with CMF-DPBS,
detached from the culture dishes using 0.5 ml of 0.025%
trypsin for 2-3 minutes and then were suspended in 1 ml
of culture medium. An aliquot of 100 /l of the cell sus-
pension was mixed with 10 /l of 0.4% of trypan blue solu-
tion (SIGMA) for 2-3 minutes. Cells were counted in a
hemocytometer chamber with the light microscope. The
number of live cells, those excluding trypan blue, and
dead cells, those taking up trypan blue, were counted and
calculated per dish. The number of live cells in experimen-


tal cultures was expressed as percent of live cells in control
cultures.

Lactate dehydrogenase (LDH) assay
Cell medium (100 /l) from each culture was saved in a
microcentrifuge tube for LDH determination using Sigma
Diagnostic Kit No. 228-UV. A 50 #ul sample was added to
500 #ul of reagent, and LDH was quantified spectrophoto-
metrically by the rate of change in absorbance at 340 nm
at room temperature. The increased absorbance at 340 nm
is the result of reduction of NAD+ to NADH as LDH cata-
lyzes the conversion of lactate to pyruvate, thus the rate of
NADH production is directly proportional to the LDH
activity in the sample.

Statistical Analysis
All data was expressed as mean +/- standard error (SE)
from three dishes/group. A one-way ANOVA was used to
determine significant differences between groups. Values
ofP < 0.05 were considered to be statistically significant.

Results
Effect of Heparin and Insulin on PAECs Injured by High
Glucose
PAECs were exposed to high glucose (30 mM) alone, insu-
lin (1 U/ml) alone, heparin (0.5 /g/ml) alone and glucose
plus heparin plus insulin for seven days. PAECs treated
with high glucose showed a significant decrease in live cell
number and increase in LDH release compared to control
cells. Compared to control cells, there were significant
changes in live cell number and LDH release in insulin
alone treated cells, but not in heparin alone treated cells.
Live cell number was significantly greater in heparin or
insulin alone versus high glucose treated cultures. The
combination of heparin and insulin in the presence of
high glucose significantly increased live cell number and
decreased LDH release compared to cells injured by high
glucose alone (Figure 1).

To determine if insulin and /or heparin protect PAECs
from high glucose injury, PAECs were treated with high
glucose (30 mM), glucose plus insulin (1 U/ml), glucose
plus heparin (0.5 /g/ml), and glucose plus insulin plus
heparin for seven days (Figure 2). A trend towards a
decrease in live cell number and a significant increase in
LDH release were seen in PAECs treated with high glucose
compared to control cultures. A significant increase in live
cell number and decrease in LDH release was seen in
PAECs treated with high glucose and a combination of
insulin and heparin compared to high glucose treatment
alone, similar to results shown in Figure 1. A significant
increase in live cell number and decrease in LDH release
was seen when insulin was added to high glucose injured
cells. High glucose plus heparin treated cultures showed a
trend towards an increase in live cell number, and a


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C G H


G+H+I


C- control
G- glucose (30 mM)
H- heparin (0.5 ug/ml)
I- insulin (1 U/ml)
G+H+I- glucose+heparin+insulin

Figure I
PAECs Injured by High Glucose were Protected by a Combination of Heparin and Insulin. PAECs were exposed
to glucose (30 mM), insulin (I U/ml), heparin (0.5 /g/ml) and glucose plus heparin plus insulin for seven days. Cell medium was
changed and fresh reagents were added every other day. Cells were counted and LDH release in medium was determined 48
hrs after the last addition of reagents. Results are expressed as mean +/- SE of three dishes per group. Significantly different
than a, control; b, glucose; c, glucose+heparin+insulin (P < 0.01) (one-way ANOVA).


significant decrease in LDH release compared to high glu-
cose treatment alone (Figure 2).

Effect of Insulin andlor Heparin on PAECs Injured by High
Glucose in the Presence of bFGF
PAECs were treated with high glucose (30 mM), glucose
plus bFGF (1 ng/ml), glucose plus bFGF plus insulin (1 U/
ml), glucose plus bFGF plus heparin (0.5 /g/ml) and glu-
cose plus bFGF plus insulin plus heparin for seven days. A
significant decrease in live cell number and increase in
LDH release was shown in high glucose treated versus
control cells. When bFGF was present in cell medium, the
combination of insulin and heparin had a protective


effect on high glucose injured cells as shown by a signifi-
cant increase in live cell number and decrease in LDH
release in cells treated with glucose plus bFGF plus insulin
plus heparin versus high glucose alone. In addition, a sig-
nificant increase in live cell number and decrease in LDH
release was shown in cells treated with high glucose plus
insulin plus bFGF versus glucose alone. Heparin with
bFGF or bFGF added to high glucose treated cells showed
a significant increase in live cell number versus high glu-
cose treatment alone. Although LDH release was less in
high glucose plus heparin plus bFGF and high glucose
plus bFGF versus the high glucose alone treated cells, this
difference did not reach significance. The combination of


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200
180
160
140
120
100
80
60
40
20


b
4-


abc
-


nrN IE I


C G G+H G+I G+H+I
C- control
G-glucose (30 mM)
G+H- glucose+heparin (0.5 ug/ml)
G+I- glucose+insulin (1 U/ml)
G+H+I- glucose+heparin+insulin
Figure 2
The Protective Effect of Insulin andlor Heparin on PAECs Injured by High Glucose. PAECs were treated with glu-
cose (30 mM), glucose plus insulin (I U/ml), glucose plus heparin (0.5 /g/ml) and glucose plus insulin plus heparin for seven
days. Cell medium was changed and fresh reagents were added every other day. Cells were counted and LDH release to
medium was determined 48 hrs after the last addition of reagents. Results are expressed as mean +/- SE of three dishes per
group. Significantly different than a, control; b, glucose; c, glucose + heparin; d, glucose + insulin (P < 0.01) (one-way ANOVA).


insulin plus bFGF, and insulin plus heparin plus bFGF
was more protective than bFGF or bFGF plus heparin on
high glucose treated cultures, when numbers of live cells
were considered. The combination of insulin plus heparin
plus bFGF was more protective than bFGF plus heparin
when LDH was considered (Figure 3).

Damaging Effect of Heparinase I on PAECs
PAECs were exposed to different doses of heparinase I
(0.01, 0.05, 0.1 and 0.3 U/ml) for six or ten days in M199
with 5% serum. Cells were harvested 24 hours after the
last addition of heparinase I. There were no significant dif-
ferences in live cell number and LDH release in control


cultures compared to those treated with different doses of
heparinase I. PAECs exposed to heparinase I (0.05, 0.1,
0.3 and 0.5 U/ml) for 48 hours in serum free M199
showed a significant decrease in cell viability and increase
in LDH release compared to the control group. Cell injury
was dose dependent since there was a significant decrease
in cell viability, with heparinase I 0.5 U/ml compared to
0.05 U/ml (data not shown). Doses of 0.3 U/ml hepari-
nase I in serum free media conditions were chosen for the
following experiments.





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Cardiovascular Diabetology 2005, 4:12


C G GF
C- control
G- glucose (30 mM)
GF- glucose+bFGF (1 ng/ml)
GFI- glucose+bFGF+insulin (1 U/ml)
GFH- glucose+bFGF+heparin (0.5 ug/ml)
GFIH- glucose+bFGF+insulin+heparin


GFI GFH GFIH


Figure 3
Insulin andlor Heparin Protected PAECs from High Glucose Injury when bFGF was Present in Cell Medium.
PAECs were treated with glucose (30 mM), glucose plus bFGF (I ng/ml), glucose plus bFGF plus insulin (I U/ml), glucose plus
bFGF plus heparin (0.5 /g/ml) and glucose plus bFGF plus insulin plus heparin for seven days. Cell medium was changed and
fresh reagents were added every other day. Cells were counted and medium LDH was determined 48 hrs after the last addi-
tion of reagents. Results are expressed as mean +/- SE of three dishes per group. Significantly different than a, control; b, glu-
cose; c, glucose+bFGF; d, glucose+bFGF+heparin (P < 0.05) (one-way ANOVA).


Effect of Insulin, Heparin and bFGF on Heparinase I
Induced PAEC Injury
PAECs were treated with heparinase I (0.3 U/ml) and/or
insulin (1 U/ml) and/or heparin (0.5 /g/ml) for 48 hours
in serum free M199. Treatment with heparinase I showed
a significant decrease in live cell number and increase in
LDH release compared to control cells. Addition of insu-
lin or heparin to heparinase I treated cells showed a signif-
icant increase in live cell number and decrease in LDH
release compared to heparinase I treatment alone. Fur-
thermore, the combination of insulin and heparin
showed a significant increase in live cell number com-


pared to all other groups, the LDH levels were also the
lowest in this group (Figure 4).

To determine the protective effect of insulin and/or
heparin on PAECs injured by heparinase I when bFGF was
present in cell medium, PAECs were treated with hepari-
nase I, heparinase I plus bFGF (1 ng/ml), heparinase I plus
insulin plus bFGF, heparinase I plus heparin plus bFGF
and heparinase I plus insulin plus heparin plus bFGF for
48 hours in serum free M199. Heparinase I treated PAECs
showed a significant decrease in live cell number and
increase in LDH release compared control cells. Cells


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Cardiovascular Diabetology 2005, 4:12


n


+


b b


0-I
C Hs Hs+H Hs+l Hs+H+I

C- control
Hs- heparinase I (0.3 U/ml)
Hs+H- heparinase I+heparin (0.5 ug/ml)
Hs+l- heparinase I+insulin (1 U/ml)
Hs+H+I- heparinase I+heparin+insulin


Figure 4
Heparinase I Induced PAECs Injury was Prevented by Insulin andlor Heparin. PAECs were treated with heparinase
I (0.3 U/ml) and/or insulin (I U/ml) and/or heparin (0.5 uig/ml) for 48 hrs in serum free medium 199, then cells were counted
and media LDH was determined. Insulin and/or heparin were added immediately after heparinase I addition. All reagents were
only added once. Results are expressed as mean +/- SE of three dishes per group. Significantly different than a, control; b, hepa-
rinase I; c, heparinase l+heparin; d, heparinase l+insulin (P < 0.005) (one-way ANOVA).


treated with heparinase I and bFGF showed a significant
decrease in LDH release, but not an increase in live cell
number when compared to heparinase I treated cells. A
significant increase in live cell number and decrease in
LDH release was seen in cultures treated with bFGF plus
insulin, bFGF plus heparin and bFGF plus insulin plus
heparin in the presence of heparinase I versus heparinase
I treatment alone. Furthermore, when compared to bFGF,
bFGF plus insulin plus heparin showed a significant
increase in live cell number in the presence of heparinase
I (Figure 5).


Discussion
Vascular complications are the main causes of morbidity
and mortality in diabetes mellitus. ECs play a pivotal role
in the regulation of vascular tone, as well as in the main-
tenance of vascular integrity, blood fluidity and homeos-
tasis. EC injury is the initial step leading to irreversible
structural abnormalities, followed by progressive microv-
ascular occlusion in the eye and kidney as well as intimal
proliferation in large vessels [33-35]. The exact cause of
EC injury is still unclear.

In this study, PAECs were used as an in vitro model to
study human vascular disease associated with EC injury


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140

120

100

80

60

40


400


300


200


100


0


b
-I--


b


abc

-I-


-a- j

- - -- ---T


a
_1_


b


b
i3E


b

I -n


m


C Hs HsF HsFH HsFI HsFIH

C- control
Hs- heparinase I (0.3 U/ml)
HsF- heparinase I+bFGF (1 ng/ml)
HsFH- heparinase I+bFGF+heparin (0.5 ug/ml)
HsFI- heparinase I+bFGF+insulin (1 U/ml)
HsFIH-heparinase I+bFGF+heparin+insulin


Figure 5
The Protective Effect of Insulin andlor Heparin on PAECs Injured by Heparinase I when bFGF was Present in
Cell Medium. PAECs were treated with heparinase I (0.3 U/ml), heparinase I plus bFGF (I ng/ml), heparinase I plus insulin (I
U/ml) plus bFGF, heparinase I plus heparin (0.5 /g/ml) plus bFGF and heparinase I plus insulin plus heparin plus bFGF for 48 hrs
in serum free medium 199. After 48 hrs, cells were counted and media LDH was determined. Insulin, heparin and bFGF were
added immediately after heparinase I addition. Results are expressed as mean +/- SE of three culture dishes per group. Signifi-
cantly different than a, control ; b, heparinase I; c, heparinase l+bFGF (P < 0.05) (one-way ANOVA).


since there is a similarity between human and porcine tis-
sue [36]. ECs of both micro and macro vascular origin
present similar pathological features in diabetic complica-
tions. Thus ECs from porcine aorta (macro vessel) injured
by high glucose could mimic EC injury associated with
uncontrolled hyperglycemia in diabetes. High glucose
injury in our model agrees with previous observations of
ECs grown under hyperglycemic conditions showing
decreased proliferation and fibrinolytic potential and
increased programmed cell death [37,38]. It was previ-
ously reported that normal human umbilical vein ECs
showed increased proliferation when cultured in medium


with high glucose (30 mM) for ten to twelve days [39], a
longer period compared to the seven day treatment in our
study. Cell proliferation increased similarly when umbili-
cal ECs were obtained from pregnant diabetic women
[40]. PAECs treated with high glucose for seven days in the
present study may represent early forms of injury and
showed reduced live cell number indicating decreased cell
proliferation. Some variation exists in the response of ECs
to high glucose conditions. Cell conditions such as varia-
tion between animals, subtle differences in medium, CO2
levels, humidity, and other unidentified factors may be
responsible for this variation.


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Depletion and abnormalities in HS and HSPG have been
found in the kidney, skin and aortic intima of diabetic
patients with nephropathy [13-15,41]. The degradation of
HSPG may play a role in EC injury leading to diabetic vas-
cular complications. Heparanase cleaves HS chains at spe-
cific sites and may be responsible for HSPG degradation
contributing to EC injury. Heparanase has been found in
the kidney and urine of diabetic patients [20]. In order to
determine if heparanase as well as high glucose damage
ECs, PAECs were treated with heparinase I.

Several heparanases have been purified and characterized
from platelets, placenta, and Chinese Hamster Ovary
(CHO) cells including connective tissue activating pep-
tide III (CTAP-III), Hpa I, Hpa II and CHO cell hepara-
nases [42]. Heparinase I, from Flacobacterium heparinum
(Cytophagia heparinia), the commercially available hepara-
nase, was chosen for PAEC treatment, and cleaves HS [43].
Heparinase I did not cause injury when PAECs were cul-
tured in M199 with serum for six to ten days, but showed
a dose effect when cultured in serum-free medium for two
days. These findings suggest that a serum constituent
inhibits heparanase activity. A recently discovered cell sur-
face protein, HS/heparin-interacting protein (HIP), was
shown to prevent heparanase access to its substrate HS by
competing with the same binding recognition site as in
the HS chain [44,45]. Thus in our experiments, serum
may contain HIP so that heparanase was active only in
serum free medium.

Our findings showing cell injury with both high glucose
and heparinase I treatment suggest that high glucose may
induce heparanase upregulation which degrades HS caus-
ing cell injury. This injury occurs in the presence of serum
which would contain HIP that may interact with hepara-
nase at the cell surface. This suggests that with high glu-
cose, heparanase may be produced within the cell.
Heparanase activity is optimal between pH 5.0 and 6.5,
with much less activity above pH 7.0 [46]. Glucose (30
mM) added for seven days to ECs lowers the medium pH
(medium color become yellow) and may further stimu-
late heparanase activity.

Exogenous heparin significantly reduces proteinuria in
diabetic patients and animals [47,48]. Heparin promotes
antioxidant and barrier properties of blood vessels, pre-
vents the formation of occlusive vascular thrombi, pro-
tects against proteolytic or oxidative damage, and lowers
blood pressure [26,49,50]. Heparin and HS, similar in
chemical structure, possess common physiological and
biological features important in the vasculature. Heparin
modifies the synthesis and the structure of HSPG [51,52].
In our study, addition of heparin to heparinase I treated
ECs significantly increased live cell number and decreased
LDH release compared to ECs treated with heparinase I


alone suggesting that heparin has the ability to prevent
cell injury by heparanase. A significant decrease in LDH
release and a trend towards an increase in live cell number
(close to control levels) seen in high glucose and heparin
treated cells compared to high glucose alone also indicate
the potential of heparin to protect ECs injured by high
glucose.

HS and heparin have high affinity for bFGF and are part
of the bFGF/bFGFR complex that affects the growth, dif-
ferentiation and migration of many cell types [53]. Thus,
bFGF function is protected by HS synthesis and perturbed
by its degradation. Our results showed a significant
increase in live cell number and a trend towards a decrease
in LDH release both in cells treated with high glucose plus
bFGF and high glucose plus bFGF plus heparin when
compared to high glucose alone indicating some protec-
tive effects of bFGF. However, the live cell number in con-
trols is significantly greater than high glucose plus bFGF
indicating bFGF alone dose not eliminate high glucose
injury. Since high glucose produces many metabolic and
biochemical abnormalities through several cellular path-
ways, normal bFGF function may be altered by its interac-
tion with abnormal metabolites. Previous studies showed
that in hyperglycemia, nonenzymatic glycosylation of
bFGF decreased bFGF activity [54] and could explain our
observations here. Moreover, we observed similar results
when ECs were damaged by heparinase I. The protective
effect of bFGF on heparinase I injury is shown by a signif-
icantly decreased LDH release and a trend towards an
increase in cell number compared to heparinase I injury
alone. This protective ability of bFGF is consistent with
that seen in high glucose plus bFGF treated ECs.

The binding of heparin to bFGF depends on the molecular
mass, degree of sulfation and the disaccharide composi-
tion. Unfractionated bovine lung heparin used here is
highly sulfated and of high molecular weight and has pre-
viously been shown to protect bFGF from tryptic cleavage.
This capacity was reduced by N-desulfation and N-acetyla-
tion of the bovine lung heparin [55]. Previous studies
have also suggested that heparin first needs to bind to the
cell surface to fulfill the role of heparan sulfate in bFGF
receptor interactions [56]. We have observed that bovine
lung heparin binds to the surface of cultured porcine
endothelial cells and thus would be able to interact with
bFGF [57].

When heparin was added to ECs treated with heparinase I
and bFGF, live cell number increased and LDH release
decreased significantly compared to heparinase I treat-
ment alone and showed a more pronounced increase in
live cells and decrease in LDH than addition of bFGF
alone suggesting that bFGF and heparin bind together to
prevent HSPG from degradation by heparinase I. These


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Cardiovascular Diabetology 2005, 4:12


findings cause us to speculate that heparin may exert its
protective effect in two steps: firstly, heparin increases EC
synthesis of HS; secondly, newly synthesized HS with
exogenous bFGF and heparin form the bFGF/HS or
heparin/ bFGFR complex which allows bFGF to play its
physiological role in cell growth, differentiation, prolifer-
ation. As well, in the case of heparanase injury, heparin in
the medium may compete with HS for heparanase and
thus may prevent the degradation of HS.

Insulin not only stimulates cells to utilize glucose, but
also promotes DNA synthesis and cell growth. The latter
effect was supported in this study when ECs, treated with
insulin alone, significantly increased live cell number
compared to controls (Figure 1). Insulin protection of
high glucose or heparinase I treated ECs was shown in all
treatment combinations including insulin alone, insulin
plus heparin, insulin plus bFGF and insulin plus heparin
plus bFGF. The mechanism by which insulin protects ECs
from high glucose injury is not entirely understood. Other
vasoprotective actions of insulin are its ability to increase
NO production [58], act as an antioxidant and prevent
atherosclerosis by reducing oxygen consumption [59].
Our present study suggests that the combination of insu-
lin, heparin, and bFGF may have additive effects with sig-
nificantly increased live cell number and decreased LDH
compared to high glucose or heparinase I alone. With
high glucose injury the three combined treatments were
more effective than bFGF and bFGF plus heparin and sug-
gested increased effectiveness compared to insulin plus
bFGF when medium LDH levels were considered. With
heparinase I injury combined treatments were signifi-
cantly more effective than bFGF with a tendency towards
increased effectiveness with heparin or insulin plus bFGF
when live cell number was considered.

Conclusion
This study demonstrates that both high glucose and hepa-
rinase I cause EC injury and suggests a link between hyper-
glycemia and heparanase induction in diabetic
complications. Exogenous heparinase I damages ECs only
in serum free conditions. The mechanism of EC injury by
high glucose is a complicated process during which a vari-
ety of metabolic abnormalities occur, and the induction
of heparanase may be one of them. The protective effect of
heparin and bFGF alone or in combination was more evi-
dent in heparinase I versus high glucose injury indicating
the limited damage induced by heparinase I and the com-
plexity of glucose-induced cell injury. Cell injury by hepa-
rinase I further confirms that degradation of HSPG on the
EC surface or the ECM contributes to the diabetic vascu-
lopathy consistent with previous observations, both in
vivo and in vitro. Our findings are the first to show the pro-
tective effects of heparin and/or insulin and/or bFGF on
cells injured by high glucose or heparinase I. Interestingly,


we found that the protective effects of bFGF in the pres-
ence of heparin or insulin in cell medium were more evi-
dent when cells were treated with heparinase I compared
to high glucose. Regardless of the interaction between
heparin, insulin and bFGF, this study demonstrated that
these three compounds in combination protect cells from
high glucose or heparanase injury. These findings provide
the basis for further studies in the understanding and
treatment of diabetic vascular complications.

List of abbreviations
bFGF-basic fibroblast growth factor

bFGFR-basic fibroblast growth factor receptor

CHO-Chinese Hamster Ovary

CMF-DPBS-calcium- and magnesium-free Dulbecco's
phosphate-buffered saline

CTAP-III-connective tissue activating peptide III

ECM-extracellular matrix

EC(s)-endothelial cell(s)

GAG-glycosaminoglycan

GBM-glomerular basement membrane

HIP-HS/heparin-interacting proteinHS- heparan sulfate

HSPG(s)-Heparan sulfate proteoglycan(s)

LDH-lactate dehydrogenase

NO-nitric oxide

PAEC(s)-Porcine Aortic Endothelial Cell(s)

SE-standard error

vWF-von Willebrand Factor

Competing interests
The authors) declare that they have no competing
interests.

Authors' contributions
JH participated in the design of the study carried out the
experiments and drafted the manuscript. AM conceived
the study and participated in the design. LH conceived the
study, participated in the design and co-ordination and
contributed to the writing of the manuscript.



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Cardiovascular Diabetology 2005, 4:12


Acknowledgements
This study was supported by a grant from the Canadian Diabetes Associa-
tion in honor of Mildred I. Wright and from private sources. We are
indebted to Tilly Ping and Dr. Adrienne Woytowich for technical
assistance.

References
I. Ruderman NB, Williamson JR, Brownlee M: Glucose and diabetic
vascular disease. FASEBJ 1992, 6:2905-2914.
2. Lee TS, Saltsman KA, Ohashi H, King GL: Activation of protein
kinase C by elevation of glucose concentration: proposal for
a mechanism in the development of diabetic vascular
complications. Proc Natl Acad Sci U S A 1989, 86:5141-5145.
3. Kamata K, Miyata N, Abiru T, Kasuya Y: Functional changes in
vascular smooth muscle and endothelium of arteries during
diabetes mellitus. Life Sci 1992, 50:1379-1387.
4. Aiello LP, Avery RL, Arrigg PG, Keyt BA, Jampel HD, Shah ST, Pas-
quale LR, Thieme H, Iwamoto MA, Park JE,.: Vascular endothelial
growth factor in ocular fluid of patients with diabetic retin-
opathy and other retinal disorders 2. N Engl ] Med 1994,
331:1480-1487.
5. Mogensen CE, Schmitz A, Christensen CK: Comparative renal
pathophysiology relevant to IDDM and NIDDM patients 5.
Diabetes Metab Rev 1988, 4:453-483.
6. Jarrett RJ, McCartney P, Keen H: The Bedford survey: ten year
mortality rates in newly diagnosed diabetics, borderline dia-
betics and normoglycaemic controls and risk indices for cor-
onary heart disease in borderline diabetics. Diabetologio 1982,
22:79-84.
7. Pirart J: [Diabetes mellitus and its degenerative complica-
tions: a prospective study of 4,400 patients observed
between I947 and I973 (3rd and last part) (author's transl)].
Diabete Metab 1977, 3:245-256.
8. Muir H: Structure and function of proteoglycans of cartilage
and cell-matrix interactions. Soc Gen Physiol Ser 1977, 32:87-99.
9. Hook M, Kjellen L, Johansson S: Cell-surface
glycosaminoglycans. Annu Rev Biochem 1984, 53:847-869.
10. Kraemer PM: Heparan sulfates of cultured cells. I. Membrane-
associated and cell-sap species in Chinese hamster cells. Bio-
chemistry 1971, 10:1437-1445.
II. Hedman K, Johansson S, Vartio T, Kjellen L, Vaheri A, Hook M:
Structure of the pericellular matrix: association of heparan
and chondroitin sulfates with fibronectin-procollagen fibers.
Cell 1982, 28:663-671.
12. Hardebo JE, Kahrstrom J: Endothelial negative surface charge
areas and blood-brain barrier function. Acto Physiol Scand 1985,
125:495-499.
13. van den Bj, van den Heuvel LP, Bakker MA, Veerkamp JH, Assmann
KJ, Berden JH: A monoclonal antibody against GBM heparan
sulfate induces an acute selective proteinuria in rats. Kidney
Int 1992,41:115-123.
14. van den Bj, van den Heuvel LP, Bakker MA, Veerkamp JH, Assmann
KJ, Weening JJ, Berden JH: Distribution of GBM heparan sulfate
proteoglycan core protein and side chains in human glomer-
ular diseases. Kidney Int 1993, 43:454-463.
15. Wasty F, Alavi MZ, Moore S: Distribution of glycosaminoglycans
in the intima of human aortas: changes in atherosclerosis
and diabetes mellitus. Diabetologia 1993, 36:316-322.
16. Brown DM, Klein DJ, Michael AF, Oegema TR: 35S-gly-
cosaminoglycan and 35S-glycopeptide metabolism by dia-
betic glomeruli and aorta. Diabetes 1982, 31:418-425.
17. Kjellen L, Bielefeld D, Hook M: Reduced sulfation of liver
heparan sulfate in experimentally diabetic rats. Diabetes 1983,
32:337-342.
18. Levy P, Picard J, Bruel A: Evidence for diabetes-induced altera-
tions in the sulfation of heparin sulfate intestinal epithelial
cells. Life Sci 1984, 35:2613-2620.
19. Freeman C, Browne AM, Parish CR: Evidence that platelet and
tumour heparanases are similar enzymes. Biochem 1999, 342
(Pt 2):36 1-368.
20. Katz A, Van Dijk DJ, Aingorn H, Erman A, Davies M, Darmon D, Hur-
vitz H, Vlodavsky I: Involvement of human heparanase in the
pathogenesis of diabetic nephropathy. Isr Med Assoc j 2002,
4:996-1002.


21. Mandal AK, Puchalski JT, Lemley-Gillespie S, Taylor CA, Kohno M:
Effect of insulin and heparin on glucose-induced vascular
damage in cell culture 15. Kidney Int 2000, 57:2492-2501.
22. Zeng G, Quon MJ: Insulin-stimulated production of nitric oxide
is inhibited by wortmannin. Direct measurement in vascular
endothelial cells 8. j Clin Invest 1996, 98:894-898.
23. Vallance P, Collier J, Moncada S: Effects of endothelium-derived
nitric oxide on peripheral arteriolar tone in man 20. Lancet
1989, 2:997-1000.
24. Hiebert LM, Wice SM, McDuffie NM, Jaques LB: The heparin tar-
get organ--the endothelium. Studies in a rat model. QJ Med
1993, 86:341-348.
25. Nader HB, Toma L, Pinhal MA, Buonassisi V, Colburn P, Dietrich CP:
Effect of heparin and dextran sulfate on the synthesis and
structure of heparan sulfate from cultured endothelial cells.
Semin Thromb Hemost 1991, 17 Suppl 1:47-56.
26. Hiebert LM, Liu JM: Heparin protects cultured arterial
endothelial cells from damage by toxic oxygen metabolites.
Atherosclerosis 1990, 83:47-5 1.
27. Moscatelli D: High and low affinity binding sites for basic
fibroblast growth factor on cultured cells: absence of a role
for low affinity binding in the stimulation of plasminogen
activator production by bovine capillary endothelial cells. j
Cell Physiol 1987, 131:123-130.
28. Flaumenhaft R, Moscatelli D, Rifkin DB: Heparin and heparan sul-
fate increase the radius of diffusion and action of basic fibrob-
last growth factor. j Cell Biol 1990, I 1:1651-1659.
29. Broadley KN, Aquino AM, Woodward SC, Buckley-Sturrock A, Sato
Y, Rifkin DB, Davidson JM: Monospecific antibodies implicate
basic fibroblast growth factor in normal wound repair. Lab
Invest 1989, 61:571-575.
30. Yayon A, Klagsbrun M, Esko JD, Leder P, Ornitz DM: Cell surface,
heparin-like molecules are required for binding of basic
fibroblast growth factor to its high affinity receptor. Cell 1991,
64:841-848.
31. Nissen NN, Shankar R, Gamelli RL, Singh A, DiPietro LA: Heparin
and heparan sulphate protect basic fibroblast growth factor
from non-enzymic glycosylation. Biochem j 1999, 338 ( Pt
3):637-642.
32. Gotlieb Al, Spector W: Migration into an in vitro experimental
wound: a comparison of porcine aortic endothelial and
smooth muscle cells and the effect of culture irradiation. Am
j Pathol 1981, 103:271-282.
33. Bresnick GH, Davis MD, Myers FL, de Venecia G: Clinicopathologic
correlations in diabetic retinopathy. II. Clinical and histo-
logic appearances of retinal capillary microaneurysms. Arch
Ophthalmol 1977, 95:1215-1220.
34. Steffes MW, Osterby R, Chavers B, Mauer SM: Mesangial expan-
sion as a central mechanism for loss of kidney function in dia-
betic patients. Diabetes 1989, 38:1077-1081.
35. Baumgartner-Parzer SM, Wagner L, Pettermann M, Grillari J, Gessl A,
Waldhausl W: High-glucose--triggered apoptosis in cultured
endothelial cells 2. Diabetes 1995, 44:1323-1327.
36. Lee KT: Swine as animal models in cardiovascular research.
In Swine in biomedical research Edited by: Tumbleson ME. New York,
Plenum; 1986:1481-1496.
37. Lorenzi M: Glucose toxicity in the vascular complications of
diabetes: the cellular perspective. Diabetes Metab Rev 1992,
8:85-103.
38. Cagliero E, Roth T, Roy S, Lorenzi M: Characteristics and mech-
anisms of high-glucose-induced overexpression of basement
membrane components in cultured human endothelial cells.
Diabetes 1991, 40:102-110.
39. Sank A, Wei D, Reid J, Ertl D, Nimni M, Weaver F, Yellin A, Tuan TL:
Human endothelial cells are defective in diabetic vascular
disease. j Surg Res 1994, 57:647-653.
40. Tamsma jT, van den BJ, Bruijn JA, Assmann KJ, Weeningjj, Berden jH,
Wieslander J, Schrama E, Hermans J, Veerkamp JH: Expression of
glomerular extracellular matrix components in human dia-
betic nephropathy: decrease of heparan sulphate in the
glomerular basement membrane. Diabetologia 1994,
37:313-320.
41. Bame KJ: Heparanases: endoglycosidases that degrade
heparan sulfate proteoglycans 2. Glycobiology 2001, I 1:91 R-98R.





Page 11 of 12
(page number not for citation purposes)


http://www. cardiab.com/content/4/l/122








Cardiovascular Diabetology 2005, 4:12


42. Desai UR, Wang HM, Linhardt RJ: Substrate specificity of the
heparin lyases from Flavobacterium heparinum. Arch Biochem
Biophys 1993, 306:461-468.
43. Liu S, Zhou F, Hook M, Carson DD: A heparin-binding synthetic
peptide of heparin/heparan sulfate-interacting protein mod-
ulates blood coagulation activities. Proc NatlAcad SciU SA 1997,
94:1739-1744.
44. Marchetti D, Liu S, Spohn WC, Carson DD: Heparanase and a syn-
thetic peptide of heparan sulfate-interacting protein recog-
nize common sites on cell surface and extracellular matrix
heparan sulfate. j Biol Chem 1997, 272:15891-15897.
45. Ihrcke NS, Parker W, Reissner Kj, Platt JL: Regulation of platelet
heparanase during inflammation: role ofpH and proteinases.
j Cell Physiol 1998, 175:255-267.
46. Tamsma JT, van der Woude Fj, Lemkes HH: Effect of sulphated
glycosaminoglycans on albuminuria in patients with overt
diabetic (type I) nephropathy. Nephrol Dial Transplant 1996,
11:182-185.
47. Gambaro G, Cavazzana AO, Luzi P, Piccoli A, Borsatti A, Crepaldi G,
Marchi E, Venturini AP, Baggio B: Glycosaminoglycans prevent
morphological renal alterations and albuminuria in diabetic
rats. Kidney Int 1992, 42:285-291.
48. Fairman RP, Sessler CN, Bierman M, Glauser FL: Protamine sulfate
causes pulmonary hypertension and edema in isolated rat
lungs. j Appl Physiol 1987, 62:1363-1367.
49. Mandal AK, Lyden TW, Fazel A, Saklayen MG, Mehrotra B, Mehling B,
Taylor CA, Yokokawa K, Colvin RV: Heparin-induced endothelial
cell cytoskeletal reorganization: a potential mechanism for
vascular relaxation. Kidney Int 1995, 48:1508-1516.
50. Colburn P, Buonassisi V: Anti-clotting activity of endothelial cell
cultures and heparan sulfate proteoglycans. Biochem Biophys
Res Commun 1982, 104:220-227.
51. Colburn P, Buonassisi V, Dietrich CP, Nader HB: N-glycansulfated
fibronectin: one of the several sulfated glycoproteins synthe-
sized by endothelial cells in culture. Biochem Biophys Res
Commun 1987, 147:920-926.
52. Folkman J, Klagsbrun M: Angiogenic factors. Science 1987,
235:442-447.
53. Giardino I, Edelstein D, Brownlee M: Nonenzymatic glycosylation
in vitro and in bovine endothelial cells alters basic fibroblast
growth factor activity. A model for intracellular glycosyla-
tion in diabetes. j Clin Invest 1994, 94:110-117.
54. Coltrini D, Rusnati M, Zoppetti G, Oreste P, Grazioli G, Naggi A,
Presta M: Different effects of mucosal, bovine lung and chem-
ically modified heparin on selected biological properties of
basic fibroblast growth factor I. Biochem j 1994, 303 ( Pt
2):583-590.
55. Fannon M, Forsten KE, Nugent MA: Potentiation and inhibition of
bFGF binding by heparin: a model for regulation of cellular
response 3. Biochemistry 2000, 39:1434-1445.
56. Hiebert LM, McDuffie NM: The internalization and release of
heparins by cultured endothelial cells: the process is cell
source, heparin source, time and concentration dependent
3. Artery 1990, 17:107-I 18.
57. Shen W, Xu X, Ochoa M, Zhao G, Wolin MS, Hintze TH: Role of
nitric oxide in the regulation of oxygen consumption in con-
scious dogs. Circ Res 1994, 75:1086-1095.



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