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IAS Visiting Fellowship
Award Report
To the International Atherosclerosis Society:
I would like to thank the
Society for supporting my visit to Professor
Mark Cooper’s laboratory
at Baker IDI Heart and Diabetes Institute and
giving me a chance to study the effects of
urotensin II (UII) and urotensin II receptor
antagonist on the development of diabetes asociated
atherosclerosis. My major role in this project
was to handle a series of in vitro experiments
including cell culture, RNA extraction and
Real-time PCR. Below I would like to present
a brief report of the results I have obtained.
In vivo experiments are still being carried
by Dr. Cooper’s research group. As soon
as the work is completed, we anticipate to
publish our results in a prestigious journal.
Once again, I would like to thank the Society
for this scholarship because it has made possible
my visit to this excellent laboratory to improve
my professional skills.
With my best regards,
Murat Olukman, MD
Ege University School of Medicine
Department of Pharmacology and Clinical Pharmacology
Izmir-TURKEY
In addition to metabolic
stimuli such as glucose and classical haemodynamic
pathways such as the
renin-angiotensin system, other vasoconstrictor
hormone pathways may exert direct effects to
promote macrovascular disease in diabetes1. In
our study, we hypothesised that UII via its vascular
receptor GPR14, may exhibit disparate actions
on the various cellular populations (e.g. endothelial
cells & macrophages) in the vasculature to
induce pathological changes such as enhancing
macrophage lipid uptake and endothelial dysfunction
in the diabetic milieu 2,3. Thus, a series of
experiments was performed, both in vitro and
in vivo, including the use of a specific UII
receptor antagonist, to delineate the role of
the UII system in the development of progression
of diabetes associated atherosclerosis. It was
predicted that UII might play a pivotal role
in diabetic macrovascular complications and with
the advent of specific UII antagonists might
provide the rationale to explore interruption
of this system as a new therapeutic strategy
to lower cardiovascular burden in diabetes.
Therefore, the overall aim of our study was
to examine the role of the urotensin II system
in diabetes associated atherosclerosis. Specifically,
- To examine the effect on inhibition of UII
using the novel non-peptidic UII receptor antagonist,
SB-657510 on atherosclerosis in control and
diabetic apoE-/- mice.
- To examine the UII system in endothelial
cells, smooth muscle cells, and macrophages in
the presence of diabetes-associated stimuli including
glucose, TGF
 and
advanced glycation products, and its effects
on processes that contribute
toward atherosclerotic plaque development such
as SMC (smooth muscle cell) proliferation, foam
cell formation, and endothelial dysfunction.
As I mentioned above, my major duty was to participate in the
in vitro experiments of this project. Therefore I first started
with determining the most appropriate cell type for this project.
After analysing human aortic endothelial cells, human umbilical
venous endothelial cells, and human vascular smooth muscle cells
for preprourotensin and UII receptor expression, I decided that
the human aortic endothelial cell was the most appropriate cell
type to assess first. I had to initially determine which of the
various protocols reported in the literature examining the effects
of the duration and dose of glucose concentrations on the gene
expression of adhesion molecules implicated the development of
diabetes associated atherosclerosis should be used. In establishing
my protocol I decided to examine three different groups.
Group I: Cells exposed to 25 mM high glucose concentration for
3 weeks;
Group II: Cells exposed to 25 mM mannitol for 3 weeks;
Group III: Cells exposed to normal glucose concentration for 3
weeks.
At the end of 3 weeks, cells were exposed to 4 different doses
of UII for 24 hours. The next day cells were removed and RNA extraction
was performed. However I could not achieve enough RNA and suspected
that the long duration of high glucose treatment might have induced
apopitosis. Therefore I reduced the duration of high glucose treatment
to 1 week. In each group 2x105 endothelial cells were added into
each well. Four different doses of UII were administered as 10-8,
10-9, 10-10 and 10-11M respectively.
In each group approximately 3 µg RNA was converted to 5 µg
cDNA and used for analysing VCAM-1, ICAM-1, MCP-1, TGF-, TGF-1 receptor, TGF-2
receptor and NFK- B gene expression by real time PCR. Statistical
analysis
of my results showed that urotensin had a considerable effect on
the gene expression of above mentioned molecules which are known
to play an important role on the development of diabetic atherosclerosis.
However none of these findings reached statistical significance.
I postulated that this might have been related to temporal changes
such as an initial up-regulation followed by subsequent down-regulation
in gene expression.
In order to address this postulate, I decided
to limit the duration of high glucose treatment to three days.
In this new protocol,
it appears that UII caused a decrease in NFK-B,
MCP-1, ICAM, and TGF- gene
expression in normal glucose concentrations. Interestingly
in high glucose concentrations urotensin led to the opposite effect
with increased gene expressions of these specific molecules. Therefore
I concluded that UII exhibits dose-dependent effects which are
also influenced by the prevailing glucose concentration.
This surprising finding can be interpreted as follows: Whilst UII
plays a protective role against atherosclerosis under physiological
conditions, it increases the expression of molecules that contribute
to the development of atherosclerosis such as adhesion molecules
and key signaling molecules which take part in intracellular pathways
promoting vascular damage, under high glucose conditions.
Currently there are several UII receptor antagonists which
are not released commercially for clinical use. One of
these antagonists
SB-756510 was provided by the company GlaxoSmithKline for my
project. I used this antagonist at three different doses
for 24 hours. After
analysing the real time PCR results, I saw that at all concentrations
SB-756510 inhibited the enhanced gene expression of certain molecules
induced by UII. This evidence suggests that UII plays a critical
role in the development of diabetic atherosclerosis. Therefore,
it is worth determining in vivo, if controlling UII production
release, or action would prevent or attenuate the development
and progression of diabetic atherosclerosis.
In conclusion this visiting fellowship provided me with an
incredible oppurtunity to improve my skills particularly
with respect to cell
culture techniques, RNA extraction, converting RNA to c-DNA and
state of the art real time PCR methods as a result of my visit
to the Baker IDI Heart and Diabetes Institute. I think these
techniques as well as the knowledge gained in this field
will be very useful
in my home laboratory. Thus, I would like to thank the International
Atherosclerosis Society once again for supporting my visit.
- Watanabe T, Kanome T, Miyazaki A,
Katagiri T.
Human urotensin II as
a link between
hypertension and coronary artery
disease.
Hypertens Res
2006 Jun; 29(6): 375-87.
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