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162 Hypertonie 2011 - Poster Hypertonie 2011 - Poster 163
short period of HFD causes vascular inflamma-
tion mediated by TNF-α/TNFR1 and increases
vasoconstriction through the upregulation of
the Rho kinase pathway.
Methods and Results: C57BL/6 (WT), tumor
necrosis factor receptor 1 knockout (TNFR1-/- )
and severe combined immune deficiency (SCID)
mice (6-8/group) were fed either a normal diet
or HFD for 2 weeks. All groups on HFD were
characterized by elevated cholesterol, increase
in body weight, visceral fat and adipocyte size,
without systemic inflammation. In myograph
experiments small mesenteric arteries from
WT on HFD presented increased constriction
responses induced by noradrenaline and extracellular
calcium. They had a significant decrease
in constriction after incubation with Rho kinase
inhibiton. TNF-α expression and Rho kinase
activity were increased in mesenteric arteries
of WT on HFD. Increase in vasoconstriction was
prevented by pravastatin, anti-TNF-α antibody
and deficiency of TNFR1, independently of cholesterol
and adiposity. Furthermore, TNFR1-/ mice on HFD had less Rho kinase activity compared
to WT on HFD. SCID mice did not show
protection against the effect of HFD.
Conclusion: Brief high fat intake for 2 weeks
increases constriction through vascular TNF-α/
TNFR1/Rho kinase pathway. This mechanism
is independent of systemic inflammation, high
cholesterol, adiposity and lymphocytes. These
findings provide innovative insights into mechanisms
involved in the development of cardiovascular
diseases related to high fat intake.
PS 31
HMGA1 and PPARgamma SUMOylation
Are Required for PPARgamma-Mediated
Transrepression in the Vasculature
Bloch M. 1 , Prock A. 2 , Paonessa F. 3 , Benz V. 1 ,
Bähr I. 1 , Herbst L. 1 , Witt H. 1 , Kappert K. 1 ,
Spranger J. 4 , Stawowy P. 5 , Unger T. 1 , Fusco A. 6 ,
Sedding D. 2 , Brunetti A. 3 , Foryst-Ludwig A. 1 ,
Kintscher U. 1
1Center for Cardiovascular Research, Berlin,
Germany, 2Department of Cardiology, Gießen,
Germany, 3Instituto di Endocrinologia ed Oncologia
Sperimentale del CNR ‘Gaetano Salvatore’,
Napoli, Italy, 4Department of Endocrinology,
Berlin, Germany, 5German Heart Institute
Berlin, Berlin, Germany, 6Department of Clinical
and Experimental Medicine ‘G. Salvatore’,
Catanzaro, Germany
We focused on the mechanism by which
ligand-activated Peroxisome proliferator activated
receptor gamma (PPARgamma) transrepresses
transcriptional activation of the matrix
metalloprotease-9 (MMP-9) gene - a crucial
mediator for vascular injury - in human aortic
smooth muscle cells (hVSMCs); in order to develop
new therapeutic interventions based on
PPARgamma´s vascular protective actions.
PPARgamma-mediated transrepression of
MMP-9 in hVSMCs dependent on the presence
of the high mobility group A1 (HMGA1) protein,
identified by OligoArray expression analysis.
Using siRNA directed against HMGA1 in VSMCs
completely prevented MMP-9 inhibition by
glitazone-activated PPARgamma and HMGA1
overexpression resulted in strongly pioglitazone-induced
MMP-9 repression surporting
the importance of HMGA1.
The Role of PPARgamma SUMOylation was
determined by transactivation assays. Transrepression
of MMP-9 by PPARgamma, and the
regulation by HMGA1 required PPARgamma SU-
MOylation at K367. Furthermore the involvement
of SUMOylation was studied by co-immunoprecipitation
of HMGA1 and the SUMO E2-ligase
(Ubc9). We show that after ligand stimulation
PPARgamma forms a complex with HMGA1-
Ubc9 which likely facilitates its SUMOylation.
ChIP experiments demonstrate that after
PPARgamma-ligand stimulation, HMGA1 and
PPARgamma were recruited to the MMP-9
promoter. ChIP assays using siRNA directed
against HMGA1 show a complete loss of PPARgamma
binding to the MMP-9 promoter.
Consistent with these findings, HMGA1´s relevance
for vascular PPARgamma signalling was
underlined by the complete absence of vascular
protection through PPARgamma-ligand
stimulation (pioglitazone) in HMGA1-/- mice after
arterial wire-injury.
To summarize, our data suggest that liganddependent
formation of HMGA1-Ubc9-PPARgamma
complexes facilitates PPARgamma
SUMOylation which mediate MMP-9 transrepression
by ligand-activated PPARgamma.
PS 32
Heparin Strongly Induces Soluble Fms-Like
Tyrosine Kinase 1 (sFlt1) Release in vivo and
in vitro
Searle J. 1 , Möckel M. 1 , Gwosc S. 1 , Datwyler
S.A. 2 , Qadri F. 3 , Holert F. 4 , Muller R. 5 , Vollert
J.O. 1 , Slagman A. 1 , Mueller C. 4 , Müller D.N. 3 ,
Dechend R. 3 , Herse F. 3
1Department of Cardiology, Campus Virchow
Klinikum, Charité – Universitätsmedizin, Berlin,
Germany, 2Abbott Laboratories, Abbott Park,
United States, 3Experimental and Clinical Research
Center, a joint cooperation between the
Charité Medical Faculty and the Max-Delbrueck
Center for Molecular Medicine, Berlin, Germany,
4Department of Laboratory Medicine and Pathobiochemistry,
Berlin, Germany, 5James Cook
University, School of Public Health and Tropical
Medicine, Townsville, Australia
Background: Soluble fms-like tyrosine kinase
1 (sFlt1) is involved in preeclampsia and coronary
artery disease, which share endothelial
dysfunction in common. Since sFlt1 has a major
heparin-binding site, we aimed to prove that
sFlt1, which is “stored” by heparan sulphate
proteoglycans on the cell surface and/or in the
extracellular matrix, is released upon heparin
administration due to a competitive mechanism.
Methods: We measured sFlt1 in serial plasma
samples taken at 4 time points, before and after
heparin administration from 135 patients
undergoing elective coronary angiography
(CA). We also tested our hypothesis in umbilical
veins, villous explants, cell culture (HUVEC),
and an animal model.
Results: sFlt1 levels in patients (253.6 pg/ml at
admission) increased significantly after heparin
administration (13,440 pg/ml) by a factor of
53-fold (p< 0.001) and returned to baseline
within 6-10 h. Levels further increased after
additional doses of heparin. Not only sFlt1,
but also sFlt1/PLGF- and sFlt1/VEGF-ratios
were significantly elevated (43-fold, 85-fold respectively,
compared to admission, p< 0.0001).
Patients’ plasma sFlt1 were processed for
Western blot that revealed a ~100 kDa isoform.
Heparin also significantly induced the release
of sFlt1 into media by cultured HUVEC (1.4
fold), umbilical veins (2.4 fold) and villous explants
(1.7 fold) compared to vehicle treatment