Download Inhibition of 14q32 microRNAs drastically improves blood flow

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:
1053
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.
1054
Cardiovascular biology
Conclusions: Inhibition of 14q32 miRs with GSOs provides a powerful therapeutic tool in neovascularization after ischemia.
lymphangiogenesis. This observation may be important for the design of
adenosine-based therapies to stimulate cardiac repair.
P5669 | BENCH
Aging and longevity genes p66shc and junD mediate age-related
dysfunction of angiogenic early outgrowth cells: mechanistic
insights into vascular repair
P5671 | BENCH
Controlled delivery of platelet lysate by polymer nanoparticles in
ischemic tissue
F. Paneni, S. Costantino, N. Krankel, F. Cosentino, T.F. Luscher. Department of
Cardiovascular Research, Institute of Physiology, University of Zurich, Zurich,
Switzerland
Purpose: Cardiovascular disease is markedly age-dependent. Early outgrowth
cells (EOCs) are important modulators of the vascular repair process, favouring
myocardial neovascularization. Impairment of EOCs functionality in human aging
is mostly driven by reactive oxygen species (ROS), but the molecular mechanisms
remain largely unknown. We previously reported that transcription factor JunD
and mitochondrial adaptor p66Shc are critically involved ROS-induced vascular
aging. The present study investigates the role of JunD and p66Shc in age-related
EOCs dysfunction.
Methods: EOCs were isolated and cultured from peripheral blood mononuclear
cells of young (24±4 years; n=5) and old (63±5 years; n=6) healthy volunteers enrolled via the blood donation service of the University Hospital Zürich,
Switzerland. Gene silencing of p66Shc was performed with siRNA technology
(Microsynth® ), while JunD overexpression was obtained with a predesigned vector (Origene® ). Scrambled siRNA or empty vector were used as negative controls
for p66Shc and JunD, respectively. Three days after transfection young and old
EOCs were harvested for measurement of O2- levels by ESR spectroscopy, migration assay and real-time PCR. Written informed consent was obtained from all
subjects.
Results: EOCs isolated form old individuals showed higher p66Shc expression
and JunD downregulation as compared with young subjects. p66Shc and JunD
deregulation in old EOCs was associated with increased O2- generation, blunted
migration, upregulation of the NADPH subunit Nox2 as well as reduced expression of the scavenger enzymes manganese superoxide dismutase (MnSOD) and
aldehyde dehydrogenase-2 (ALDH2). Interestingly, either p66Shc knockdown or
JunD overexpression significantly suppressed age-related O2- production, improved EOCs migration and restored the balance between oxidant and antioxidant enzymes.
Conclusions: p66Shc and JunD are critically involved in age-dependent EOCs
dysfunction by altering their redox state. Modulation of such aging and longevity
genes restores normal repair capacities in EOCs from aged individuals and may
be useful as an ex vivo strategy to improve the clinical efficacy of stem cell therapy
in elderly cardiovascular patients.
P5670 | BENCH
Adenosine inhibits the proliferation of lymphatic endothelial cells
B. Lenoir 1 , A. Noel 2 , M. Theresine 1 , S. Blacher 2 , I. Ernens 1 , Y. Devaux 1 ,
D.R. Wagner 3 . 1 Public Research Centre (CRP) - Health, Luxembourg,
Luxembourg; 2 University of Liege, Liege, Belgium; 3 Hospital Centre,
Luxembourg, Luxembourg
Background: The presence of a lymphatic system in the human heart has been
demonstrated several decades ago. After myocardial infarction, lymphatic vessel
density increases, predominantly in the myocardium, but also in the epicardium
and endocardium of both ventricles and atria. The biological relevance of this
increase is poorly known. Recent studies suggest that it may be a protective element in the response to myocardial injury, through efficient removal of cellular
debris produced by cardiomyocyte death. Adenosine has the potential to stimulate cardiac repair, partly through activation of angiogenesis. However, whether
adenosine affects the lymphatic system is unknown.
Methods: Human adult dermal microvascular lymphatic endothelial cells
(HMVEC) were used to study the direct effect of adenosine on the lymphatic
system. Expression of adenosine receptors was investigated using quantitative
PCR. Proliferation of lymphatic cells was measured using CyQUANT and Ki-67,
migration of the cells was assessed using a wound healing assay and a Boyden
chamber assay, and tube formation was evaluated using a tubulogenesis assay
in collagen and spheroids matrix. Cytotoxicity was evaluated using the Live/Dead
viability/cytotoxicity assay. Lymphangiogenesis was characterized ex vivo using
mouse lymphatic ring explants. To identify potential paracrine effects of adenosine
on lymphangiogenesis, the murine cardiomyocyte cell line HL-1 and the human
fibroblast cell line 1226 KI were used.
Results: HMVEC expressed A2A and A2B, but not A1 and A3, adenosine receptors. Adenosine dose-dependently decreased the proliferation of HMVEC (40% for 10 μmol/L adenosine), and inhibited their migration and tube formation.
Adenosine also decreased microvessel outgrowth from lymphatic rings explanted
from mouse lymphatic thoracic duct. A potential cytotoxic effect of adenosine was
ruled out. Adenosine decreased the production of vascular endothelial growth
factor C (VEGFC), the main growth factor of lymphatic endothelial cells, by fibroblasts. Conditioned medium from fibroblasts treated with adenosine inhibited
the proliferation of HMVEC. Adenosine did not alter the production of VEGFC by
cardiomyocytes.
Conclusion: We have shown for the first time that adenosine down-regulates
M.C. Barsotti 1 , R. Di Stefano 1 , F. Chiellini 2 , A. Lisella 2 , C. Errico 2 , R. Feriani 1 ,
S. Burchielli 3 , A. Balbarini 1 , G. Soldani 4 . 1 Department of Surgery, Medical,
Molecular, and Critical Area Pathology - University of Pisa, Pisa, Italy; 2 BIOlab UdR INSTM - Department of Chemistry and Industrial Chemistry - University of
Pisa, Pisa, Italy; 3 Gabriele Monasterio Foundation-CNR Region Toscana, Pisa,
Italy; 4 Institute of Clinical Physiology (CNR), Massa, Italy
Purpose: Platelet lysate (PL) contains growth factors, cytokines and chemokines
useful for angiogenesis and tissue repair. Polymer-based nanoparticles (NP)
could be used as PL carriers for a controlled release in ischemia. Our aim was to
characterize the in vitro (viability, tissue repair and inflammatory response activation) and in vivo (injection in rat ischemic hindlimb and Laser Doppler Perfusion
Imaging, LDPI) effect of PL-loaded NP.
Methods: Human umbilical vein endothelial cells (HUVEC) were obtained from
healthy donors. Experiments were performed at p3, with a pool from 3 donors.
PL obtained by thrombapheresis from healthy donors was resuspended (6x108
thrombocytes/ml), subjected to 3 freeze-thaw cycles and stored at -80°C. Two
polymers, Poly(Gly-co-HPMA) (fast drug release, e.g. few days) and PLGA (slow
drug release, e.g. several weeks), were used to obtain NP for different application. HUVEC were incubated for 48h at 37°C with PL-loaded NP (corresponding
to 1.5% PL), with 5% FBS and EGF to assess cell viability. ERK1/2 and NFkB
were detected by FACE ELISA, monitoring proteins activated by phosphorylation.
HUVEC were treated with PL-loaded NP for 30 and 60’ (ERK 1/2) or 1, 24 and
48h (NFkB). Unilateral hindlimb ischemia was induced in adult male Wistar rats
(n=24) by surgical excision of the femoral vessels. Blood flow was monitored by
LDPI at different time points, up to 21d. After 7d, intramuscular injections of PL,
PL-loaded fast NP and saline were performed using fibrin for enhancing engraftment; 6 rats received no treatment. Blood flow was monitored by LDPI.
Results: PL-loaded NP did not affect cell viability. PL-loaded fast NP induced
ERK1/2 activation at 60’, unlike PL-loaded slow NP. PL-loaded slow NP activated
NFkB at 48 hours, unlike PL-loaded fast NP. After a short postoperative period
of depressed perfusion (p<0.005), the rats showed moderate hindlimb ischemia
from d7 onwards, with constant decreased perfusion. Rats receiving only fibrin
were comparable to untreated rats. At 7d after intramuscular injection, PL-loaded
fast NP significantly increased blood flow (1.01±0.15 ischemic/contralateral hind
limb perfusion ratio) in the ischemic hindlimb vs. PL (0.78±0.12, p<0.05).
Conclusions: The nanosystems showed cytocompatibility and a differential capability to activate important inflammatory pathways such as ERK 1/2 and NFkB,
involved in angiogenesis and tissue repair. Release of PL from loaded NP was
effective for blood flow enhancement in a model of chronic, moderate hindlimb
ischemia, underlining the advantages of using such drug-controlled release in
regenerative medicine.
P5672 | BENCH
Direct epicardial shock wave therapy improves ventricular function
in a porcine model of ischemic heart disease: evidence for
induction of angiogenesis and stimulation of VEGF receptors
J. Holfeld 1 , D. Zimpfer 2 , K. Albrecht-Schgoer 3 , A. Stojadinovic 4 , P. Paulus 5 ,
A. Thomas 2 , W. Schaden 6 , R. Kirchmair 3 , S. Aharinejad 2 , M. Grimm 1 .
1 Innsbruck Medical University, Department of Cardiac Surgery, Innsbruck,
Austria; 2 Medical University of Vienna, Department of Cardiac Surgery, Vienna,
Austria; 3 Innsbruck Medical University, Department of Internal Medicine I,
Innsbruck, Austria; 4 Walter Reed Army Medical Center, Washington, United
States of America; 5 Johann Wolfgang Goethe-University Hosp., Frankfurt am
Main, Germany; 6 AUVA Trauma Center Meidling, Vienna, Austria
Purpose: Shock waves were shown to induce angiogenesis in ischemic myocardium in rodent models. In the present experiments we aimed to address
safety and efficacy of direct epicardial shock wave therapy in a preclinical large
animal model and to further evaluate mechanism of action of this novel therapy.
Methods: Four weeks after left anterior descending (LAD) artery ligation in pigs,
animals underwent re-thoracotomy with (SW group, n=6) or without (CTR, n=5)
direct epicardial shock waves (300 impulses at 0.38mJ/mm2 ) applied to the infarcted anterior wall. Efficacy endpoints were improvement of LVEF and induction
of angiogenesis 6 weeks after shock wave therapy. Safety endpoints were hemodynamic stability during treatment and myocardial damage.
A receptor tyrosine kinase profiler was performed in human coronary artery endothelial cells to proof receptor activation.
Results: Four weeks after LAD ligation, LVEF decreased in both shock wave
(43±3%, p<0.001) and control (41±4%, p=0.012) group. LVEF markedly improved in shock wave animals 6 weeks after treatment (62±9%, p=0.006), no
improvement was observed in controls (41±4%, p=0.36). Quantitative histology
revealed significant angiogenesis six weeks after treatment as shown by number of arterioles (CTR 2±0.4 arterioles/high power field vs. SW 9±3, p=0.004),
number of capillaries (CTR 9±4 vs. SWT 53±12, p<0.001) and number of vital
cells (CTR 212±32 vs. SWT 310±48, p=0.003). No acute or chronic adverse ef-