Download Cardiovascular biology 1053

Cardiovascular biology
P5665 | BENCH
Static, but not cyclic tensional forces on the heart increase
anti-angiogenic sFlt-1 expression in endothelial cells and inhibit
VEGFR-2 activation
K. Ablasser 1 , R. Gasser 1 , P. Del Nido 2 , B. Pieske 1 , I. Friehs 2 . 1 Medical
University of Graz, Department of Cardiology, Graz, Austria; 2 Boston Children’s
Hospital, Boston, United States of America
Purpose: The left ventricle (LV) is subject to increased mechanical forces in a
variety of diseases that cause chronic increased systolic or diastolic pressures.
These are often associated with endothelial dysfunction and/or reduced numbers
of capillaries. Further mechanical forces are known to modulate endothelial gene
expression. Therefore, we hypothesized, that increase in static tensional forces
on capillary endothelial cells of the LV result in up-regulation of soluble VEGF
receptor 1 (sFlt-1) and thus leading to an imbalance of pro- and anti-angiogenic
stimuli in the heart.
Methods: A rabbit Langendorff heart with retrograde constant pressure perfusion was used as model. As perfusate oxygenated Dulbecco’s modified Eagle’s
medium was used to keep the heart viable as long as possible without deterioration of its function or sings of ischemia. For the tensional force 5g per g of
heart weight was applied to the apex of the beating LV either as static load or
every 30 seconds (cyclic). For a set of experiments cardiac contractions were
completely arrested using butanedione monoxime (BDM). Quantitative RT-PCR,
immunoblots and co-immunoprecipitation was used to quantify mRNA and proteins of sFlt-1, vascular endothelial growth factor (VEGF) and VEGF receptor-2
(VEGFR-2). Immunohistochemistry was performed for colocalized of proteins.
Results: Increased static tensional force on the LV leads to significant increase
in mRNA (3,8 fold, p < 0.05) and protein expression of sFlt-1 (1,4 fold, p < 0.05),
which can be detected as early as after 30min and remains increased for the
duration of the experiments (up to 6h). Cyclic tensional forces, however, do not
affect sFlt-1 mRNA or protein levels. VEGF levels are unaffected by both static
and cycle forces on the beating heart. Complete cardiac arrest without affecting
the endothelium by BDM, an ATPase inhibitor of skeletal myosin 2, does not affect
sFlt-1 or VEGF expression. Increased sFlt-1 due to increased static force on the
LV binds more VEGF, shown by co-immunoprecipitation of sFlt-1 and VEGF, and
reduced VEGFR-2 phosphorylation. Endothelial cells of the heart were colocalized by immunohistochemistry as source of sFlt-1 release.
Conclusions: Static, but not cyclic tensional forces on the LV result in increased
release of anti-angiogenic sFlt-1 from capillary endothelial cells. While expression
levels of VEGF remain unchanged, sFlt-1 binds VEGF and reduces activation
of the signalling Receptor VEGFR-2. Thus, chronic increased forces in the LV
contribute to the anti-angiogenic setting and reduced capillary density as seen in
many heart diseases.
P5666 | BENCH
Intracoronary injection of encapsulated antagomir-92a promotes
angiogenesis and prevents adverse infarct remodeling in a pig
model of myocardial infarction
N. Bellera 1 , I. Barba 1 , A. Rodriguez-Sinovas 1 , M.T. Gonzalez-Alujas 1 ,
J. Perez-Rodon 1 , M. Esteves 1 , C. Fonseca 1 , B. Garcia Del Blanco 1 , A. Perez 2 ,
D. Garcia-Dorado 1 . 1 Universitary Hospital Vall d’Hebron, Cardiology Department,
Barcelona, Spain; 2 Pierre Fabre Iberica S.A., Cerdanyola del Valles, Spain
Background: Intravenous antagomir-92a based-therapy enhanced neoangiogenesis and improved left ventricular contractility in a mouse model of acute myocardial infarction (MI), but its effect on postMI remodeling is unknown and clinical
translation limited by the need of repeated administration and potential systemic
adverse effects (AE). We investigated whether a single intracoronary administration of antagomir-92a encapsulated in microspheres (Antag92aME) could prevent
deleterious myocardial remodeling one month (1mo) after MI.
Methods and results: We developed poly-d,-lactide-co-glycolide 9 μm ME with
7-10% loads of 3 mg Antag92a. In a first phase, ME were injected in the LAD coronary artery of healthy pigs. Repetitive injections to a total dose of 240 mg were
used (n=3) to rule out persistent effects in myocardial contraction (intramyocardial piezoelectric crystals), LAD flow (Doppler probe) or necrosis (histology), and
fluorescence labeled ME injection (n=4) showed no ME content in myocardium
outside the LAD territory or in lung, spleen or liver (fluorescence microscopy).
Finally, miR-92a expression was quantified by RT-PCR in 3 pigs euthanized at
1, 3 and 10 days after Antag92aME injection demonstrating local and sustained
miR-92a inhibition (> 8x and >4x fold at 1 and 10 days). In a second phase, MI
was induced inflating a 2.5/12 mm balloon (49 min) in LAD of 27 closed-chest
minipigs, randomly to blind receive Antag92aME, placeboME or saline administration, 5 minutes after reperfusion. Intravascular echocardiography was performed during ischemia, reperfusion and repeated 1mo later immediately before
the animals were euthanized and the hearts excised, ex-vivo MRI was performed
(maximum necrotic wall thickness (Tmax), largest diameter (D) between remodeled and contralateral wall (DR) and its largest perpendicular D between unaffected walls (DN) were measured in short-axis transverse ventricular slices)
and ventricle histologic sections were obtained. Mortality was 23% (1, 2, 3 died
in Antag92aME, placeboME and saline). Antag92aME induced vessel growth
(161.57±58.71 vessels/cm2 , 68.49±23.56, 73.91±24.97; p=0.001) reduced regional wall motion dysfunction (28.6% and 76.9% of dyskinesia in treated vs
non-treated p=0.03) and prevented adverse remodeling 1mo after injury (Tmax:
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9.01±0.6, 5.61±0.5, 6.07±0.9 p=0.006, DR/DN: 1.29±0.1, 2.02±0.2, 1.93±0.2
p=0.03).
Conclusions: Early intracoronary administration of Antag92aME in a pig model
of reperfused MI prevents ventricular remodeling with no local or distant AE
emerging as a promising therapeutic approach to translate to patients that suffer a large MI.
P5667 | BENCH
Adenosine promotes angiogenesis through up-regulation of
thrombospondin-1
I. Ernens 1 , M. Bousquenaud 1 , B. Lenoir 1 , Y. Devaux 1 , D.R. Wagner 2 . 1 Public
Research Centre (CRP) - Health, Luxembourg, Luxembourg; 2 Hospital Centre,
Luxembourg, Luxembourg
Background: Increase of blood capillary density at the interface between normal
and ischemic tissue after acute Myocardial Infarction (MI) reduces infarct size and
improves cardiac function. Cardiac injury triggers the production of adenosine and
the matricellular protein Thrombospondin-1 (TSP-1). Adenosine is thought to be
involved in cardiac repair and is known to stimulate angiogenesis. However, the
role of TSP-1 in angiogenesis is still debated, since both anti- and pro-angiogenic
activities have been reported. We hypothesized that adenosine controls angiogenesis through modulation of TSP-1 production.
Methods: Primary human macrophages obtained by differentiation of peripheral
blood monocytes from healthy volunteers were treated with adenosine (0.1-50
μmol/L). Angiogenesis was evaluated ex vivo using rat aortic ring explants and in
vivo using matrigel plugs implanted in mice. Rats were subjected to permanent
coronary artery ligation and supplemented with adenosine (2 mg/kg/day for 2
months) to study the effect of adenosine on TSP-1 and angiogenesis after MI.
Results: Adenosine dose-dependently increased the production of TSP-1 by
macrophages, reaching a 5-fold increase at 10 μmol/L (n=5, P<0.001). Use of
agonists and antagonist of adenosine receptors, coupled to RNA interference experiments, implicated the A2A and A2B receptors in the effect of adenosine on
TSP-1. This effect was reproduced by cholera toxin (Gs protein activator) and
forskolin (adenylate cyclase activator), and blocked by the PKA inhibitor H89,
demonstrating the involvement the cAMP/PKA pathway. Conditioned medium
from adenosine-treated macrophages enhanced microvessel outgrowth from aortic ring explants and induced vessel formation in matrigel plugs. Addition of TSP-1
neutralizing antibodies to conditioned medium blocked angiogenesis in both models. Chronic administration of adenosine to rats after MI maintained long term expression of TSP-1 in the infarct border zone and enhanced vascularization and
regional systolic fonction.
Conclusion: Adenosine up-regulates TSP-1 production by macrophages, resulting in stimulation of angiogenesis. The mechanism involves A2-type adenosine
receptors and is mediated through the cAMP/PKA pathway. This may have important implications for therapeutic strategies aiming at improving the revascularization of the ischemic heart.
P5668 | BENCH
Inhibition of 14q32 microRNAs drastically improves blood flow
recovery after ischemia
A.Y. Nossent, S.M.J. Welten, A.J.N.M. Bastiaansen, R.C.M. De Jong, R.M. De
Vries, H.A.B. Peters, P.H.A. Quax. Leiden University Medical Center, Department
of Vascular Surgery, Leiden, Netherlands
Purpose and methods: Arteriogenesis is a multifactorial process in which preexisting arterioles remodel into mature collateral arteries, restoring blood flow
after ischemia. Clinical trials designed to stimulate arteriogenesis have been unsuccessful, mainly because only single pathways involved in arteriogenesis were
targeted. As microRNAs (miRs) regulate expression of several hundred target
genes, we set out to identify miRs that target genes in all pathways of arteriogenesis. We performed a reverse target prediction on a first set of 127 genes involved
in arteriogenesis and on a second set of 90 additional genes that we found upregulated in adductor muscles of C57Bl/6 mice subjected to hind limb ischemia (HLI)
via single femoral artery ligation, a model for effective arteriogenesis. In both analyses we found enrichment of binding sites for miRs in a 14q32 miR gene cluster.
MicroArray analyses showed that 14q32 miRs were down-regulated drastically in
adductor muscles of C57Bl/6 mice after HLI.
Results: Novel miR inhibitors, Gene Silencing Oligonucleotides (GSOs, kindly
provided by Idera Pharmaceuticals), were used to inhibit four 14q32 miRs, miR487b, miR-494, miR-329 and miR-495 in vivo (1mg/mouse), 1 day prior to induction of HLI via double ligation of the left femoral artery. Blood flow recovery was
followed by Laser Doppler Perfusion Imaging (LDPI). All 4 GSOs clearly improved
blood flow recovery after HLI. GSOs targeting miR-495 and miR-329 showed robust effects. Mice treated with GSO-495 or GSO-329 showed increased perfusion already after 3 days (30% perfusion vs. 15% in control) and those treated
with GSO-329 showed a remarkable full recovery of perfusion after 7 days (vs.
60% in control). In concurrence with the LDPI data, mice treated with GSOs displayed larger collateral arteries in the adductor muscle and more cappilaries in
the soleus muscle of the ischemic paw, compared to controls. GSOs gave stable knockdown of their target miRs in the target tissue up to 7 days, and gave
a more specific knockdown than classic antagomiRs in primary human arterial
cells.