917 helper dependent adenoviruses expressing apolipoprotein a i do not alter endothelial cell function in vitro: potential for vascular gene therapy

917 Helper Dependent Adenoviruses Expressing Apolipoprotein A I Do Not Alter Endothelial Cell Function In Vitro Potential for Vascular Gene Therapy Molecular Therapy Volume 17, Supplement 1, May 2009[.]

Molecular Therapy Volume 17, Supplement 1, May 2009

Copyright © The American Society of Gene Therapy S350

CARDIOVASCULAR

randomly divided into fi ve groups: Sham, MI+Saline, MI+MSC,

MI+MSC+SAA, MI+SAA (n=20) For MSC treatment animals,

immediately after LAD ligation, 2.5 millions of BrdU labeled MSCs

in 150 ml of saline was injected into the border zone of the infarcted

heart The control animals were only injected with 150 ml of saline

After closing the chests, animals in SAA treatment groups were

daily administrated with SAA (i.p., 10 mg/kg, in 1 ml of saline for

each rat) until the animals were killed for examination At days 3,

animals were killed and the hearts were harvested for RNA and protein

isolation The expression of MI related cytokines including HSP70,

CTnT, IL-1, IL-6, TNF-a, Connexin 43 and VEGF were analyzed

with Real Time PCR The protein level of HSP70, CTnT, IL-1, IL-6,

TNF-a and Connexin 43 were analyzed by Western Blot At day 28,

animals were examined for heart function by 2D echocardiography

Heart tissues were analyzed for pathologic change, infarction size,

MSCs survival and differentiation Results: Data of Real Time-PCR

shows that at day 3 the expression of IL-1, IL-6, CTnT, HSP70 and

TNF-a were elevated in saline group However, the expression of

these genes was inhibited to about 50-60% in MI+MSC group and

MI+SAA group More interestingly, MI+MSC+SAA group showed

the lowest gene expression which is only 40-50% of that in MI+MSC

group and MI+SAA group, and less than 20% of that in saline group

At day 30, the combination treatment group showed the highest

VEGF expression and normalized CX43 expression Western blot

data showed that for HSP70, Connexin 43, CTnT, IL-1, IL-6, and

TNF-a expression, the difference between saline group and the three

treatment groups are signifi cant (p<0.05) Greater difference was

observed between saline group and the combination treatment group

(p<0.01) The expression of IL-1, IL-6, CTnT, TNF-a and HSP70

was almost normalized in the combination treatment group Saline

group showed severe muscle necrosis, disordered and broken muscle

fi bers Improvement can be seen in treatment groups The combination

treatment group has the smallest infarction size compared to other

groups Heart function was evaluated for LVDd, LVDs, EF and FS

In all the four parameters, the combination treatment group showed

the best improvement Our further mechanistic study revealed that

increased JNK phosphorylation stimulated by H2O2 was abolished

by SAA treatment Conclusion: Administration of SAA combined

with MSCs transplantation can inhibit infl ammatory factors, reduce

myocardial infarction and improve heart functions It could be a

promising approach in treatment of myocardial infarction

for the Long-Term Monitoring of Vascular Gene

Transfer

Ilia Fishbein,1 Michael Chorny,1 Marina Bakay,1 Peter Sobolewski,1

Ivan S Alferiev,1 Robert J Levy.1

1 The Children’s Hospital of Philadelphia, Philadelphia.

Background: An accurate quantifi cation of reporter transgene

expression over time is instrumental to the development of gene

therapies to combat vascular diseases, such as atherosclerosis and

restenosis Due to the high sensitivity and ability to monitor intravital

gene expression for a prolonged time, bioluminescence methods of

reporter detection are gaining an increasing popularity In contrast to

luciferase assays in cell or tissue lysates, bioluminescence imaging

relies on the luciferin uptake by living cells, therefore it exhibits

higher dependence on substrate (luciferin) levels than the standard

luciferase assays However, this aspect of bioluminescence imaging is

often overlooked leading to circumstances when chemoluminescence,

which is taken as the measure of reporter activity, is limited in reality

by luciferin availability Methods: In vitro, primary rat aortic smooth

muscle cells were transduced with AdLuc at MOIs of 300, 2000

and 7500 24 hours post-transduction, the transgene expression was

examined by bioluminescence 5 min after the addition of 50 or 250

µg of luciferin In vivo 5x108 pfu of AdLuc were locally delivered into

the balloon injured rat carotid arteries (n=6) 2 days after gene delivery the rats underwent imaging (IVIS-100) as 30 sec-long series’ spanning 2-35 min following IV injection of 50 mg/kg luciferin (i1) At 13 min post-i1, the second 50 mg/kg luciferin injection (i2) was performed Six hours post-i2, when the luminescence signals went down to background levels, 200 µl of 5 mg/ml luciferin/Pluronic-127/PBS solution was locally applied to the transduced arterial segment (a1) and allowed to gel in situ for 1 min The 30 sec imaging series’ were carried out for 35 min post-a1 At 13 min after a1 an additional 200 µl

of the luciferin/Pluronic formulation was applied (a2) Results: In cell

culture the higher luciferin dose resulted in 3.1-4.2-fold more intense signal across all examined MOIs In vivo, after the IV administration

of luciferin a 50% drop of luminescence signal intensity was observed within 8-13 min post-i1 in all rats There was a 48.3%±10.2% increase

of peak signal intensity post-i2 in comparison with post-i1, confi rming that the circulating levels of luciferin were indeed a limiting factor

of bioluminescence signal intensity under this imaging protocol The signal was signifi cantly more stable after local perivascular application of luciferin/Pluronic formulation (less than a 10% drop of the signal in the fi rst 13 min post-a1 in 5 of 6 animals) Moreover, the signal recorded after local administration of luciferin was uniformly higher than after IV administration (2.16±0.32 fold) and had almost not changed upon re-administration (0.6%±7.5% increase of peak

signal intensity post-a2 in comparison with post-a1) Conclusion:

Bioluminescence intensity at the peak levels of luciferase expression

is limited by the substrate availability both in vitro and in vivo Local perivascular administration of luciferin in Pluronic gel provides high and stable substrate levels and thus is advantageous over systemic luciferin administration for the purposes of vascular imaging of luciferase gene delivery and transgene expression

Expressing Apolipoprotein A-I Do Not Alter Endothelial Cell Function In Vitro: Potential for Vascular Gene Therapy

Rowan Flynn,1 Joshua Buckler,1 David A Dichek.1

1 Department of Medicine, Division of Cardiology, University of Washington, Seattle, WA.

Objective: Transduction of endothelial and other cell types

in vitro with adenoviral vectors (Ad) can increase expression of proinflammatory transcription factors, cytokines, and adhesion molecules and can alter cell migration and apoptosis Blood vessel health is largely dependent upon preservation of functional, intact endothelium Therefore, a clinically useful gene transfer vector must not generate proinfl ammatory endothelium or interfere with normal endothelial physiology because this would likely worsen vascular disease We previously reported that helper-dependent (HD)-Ad has promise for vascular gene therapy, with several advantages over fi rst-generation (FG)-Ad Here, we investigate whether HD-Ad or FG-Ad have proinfl ammatory effects on endothelium or otherwise impair normal endothelial functions We also tested whether Ad-mediated expression of apolipoprotein (apo) A-I, a potential therapeutic gene which has been shown to reduce infl ammation in vivo, would affect

the endothelial phenotype Methods: Bovine aortic endothelial

cells (BAoEC) were mock-transduced or exposed to FG-AdNull, HD-AdNull (both AdNull vectors contain an empty expression cassette), FG-AdApoAI, or HD-AdApoAI (both AdApoAI vectors express rabbit apoA-I) HD-Ad reagents were from Merck ApoA-I expression was detected by western blot and quantitative RT-PCR Cell proliferation, apoptosis, and expression of the adhesion molecules ICAM-1 and VCAM-1 were measured 24, 48, and 72 hours after transduction by MTT assay, annexin binding with fl ow cytometry, and quantitative RT-PCR, respectively Migration was examined 24 hours after transduction, using the monolayer wound-healing assay

Results: Cell proliferation was comparable in all transduced and

Molecular Therapy Volume 17, Supplement 1, May 2009

CARDIOVASCULAR

mock-transduced cells at 24 and 48 hours At 72 hours, a 20% decrease

in proliferation was observed in cells transduced with either of the

FG-Ad vectors versus mock-transduced cells (P < 0.01) No signifi cant

differences in cell migration, apoptosis or VCAM-1 expression were

detected amongst the groups at any of the time points examined An

approximately 80% increase in ICAM-1 expression was detected

in FG-AdApoAI transduced cells versus mock-transduced cells at

72 hours (P < 0.001), although no increase in ICAM-1 expression

was observed at earlier time points Conclusion: Transduction of

endothelial cells in vitro with HD-Ad does not upregulate expression

of important vascular cell adhesion molecules and has no effect on

critical cellular functions including proliferation, migration, and

survival Expression of apoA-I by HD-Ad does not impair critical

endothelial functions and has neither proinfl ammatory nor

anti-infl ammatory effects HD-Ad is a promising vector for vascular gene

therapy, and might be used in vivo to enhance cholesterol transport

out of the artery wall through expression of apoA-I

Peptide Delivery Leads to Reduced Heart Size in

Normal Rats

Jason M Tonne,1 Martin L Fernando,2 Gerald E Harders,2

Stephen J Russell,1 John C Burnett,2 Yasuhiro Ikeda.1

1 Molecluar Medicne, Mayo Clinic, Rochester, MN;

2 Cardiovascular Diseases, Mayo Clinic, Rochester, MN.

B-type natriuretic peptide (BNP) is a cardiac hormone with

potent natriuretic, renin-angiotensin-aldosterone suppressing,

vasodilating, anti-fi brotic and anti-hypertrophic activities The use

of BNP and BNP-derived designer peptides as therapeutic agents

is highly attractive based upon their selectivity and potency in

human disease which may go beyond small molecules and other less

specifi c pharmacological agents Indeed, synthetic BNP is used as a

therapeutic tool to treat acute congestive heart failure However, use

of BNP for long-term therapy has been limited by their short in vivo

half-life and by their inherent protein structures that prevent their

oral administration We reasoned that delivery of BNP-encoding

DNA sequence would allow long-term BNP delivery in vivo In this

study, we employed adeno-associated virus serotype 9 (AAV9)-based

vectors, which demonstrated effi cient long-term cardiac transduction,

to achieve continuous BNP over-expression Normal Wister rats

were intravenously administered with AAV9 vectors carrying rat

pre-proBNP or GFP sequences At 5 weeks post infection, high

levels of GFP expression were evident in the heart sections of

AAV9-GFP-infected rats, while AAV9-BNP-injected rats showed elevated

plasma BNP concentrations and reduced heart weights No statistical

signifi cance was observed in the blood pressures of the

AAV9-BNP-infected rats The lack of statistical signifi cance could be, in part, due

to the intact counter regulatory systems in normal rats which may

have blunted the hypotensive effects of BNP Nevertheless, our results

demonstrated the feasibility of AAV9 vector-mediated gene delivery

for long-lasting BNP therapy We will use this system to examine the

infl uence of BNP expression on hypertension and cardiac remodeling

in spontaneously hypertensive rats

Doxorubicin-Induced Cardiomyopathy Using a

Vigilant Vector

Marcio C Bajgelman,1 Juliana S Nakamuta,1 Christian Merkel,1

Samantha V Omae,1 Newton A Lopes,1 Jose E Krieger,1 Bryan E

Strauss.1

1 Laboratory of Genetics and Molecular Cardiology, Heart Institute

(InCor), University of São Paulo, Sao Paulo, SP, Brazil.

Doxorubicin is an anthracyclin widely used in cancer therapy Even

though its effi cacy is well known, the drug has an inherent effect of

cardiac toxicity, which results in functional damage to the heart Swain

et al., 2003, reported that 26% of patients who had undergone cancer treatment using anthracycline were affected by cardiomyopathy One of the most characteristic intracellular alterations, related to the administration of doxorubicin, is the activation of p53 The p53 protein acts as a potent transcription factor and is involved in controlling the cell cycle and apoptosis Thus the activation of p53 may provide an opportunity to induce expression of a therapeutic transgene encoded by a p53-responsive gene transfer vector and thus induce a cardioprotective effect In this work, we constructed a recombinant AAV vector which has an expression cassette containing the luciferase (luc) reporter gene driven by a chimerical promoter which is responsive to p53 (Bajgelman et al., 2008) We performed

in vitro assays in a cell line containing temperature sensitive p53 and demonstrated that the vector has a high specifi city for p53 and robust expression of the reporter, showing levels of expression of

up to 1,000 fold greater than that observed at the non-permissive temperature (Figure 1) Moreover, when using primary cultures of cardiomyocytes, we observed a dose-dependent relationship between doxorubicin concentration and luc expression (Figure 2) We have chosen VEGF as the cardioprotective gene because of its potential to improve cardiac function by arteriogenesis (Crotogini et al., 2003) as well as induce mitosis and hyperplasia of cardiomyocytes (Laguens

et al, 2004) The cDNA of the human VEGF(165) gene was cloned

in the p53-responsive AAV vector In vitro assays using the p53 responsive AAV vector encoding VEGF were performed in primary cultures of cardiomyocytes isolated from newborn rats showed that the expression of the therapeutic gene was modulated depending

on the dose of chemotherapy (Figure 2) Different in vivo routes

of viral administration, such as intracoronary, intrapericardial and intramyocardial, were compared Intramyocardial injection showed the highest rate of viral incorporation, as observed about 4 weeks after in vivo transduction Given the stability, long-term expression and induction of the expression cassette mediated by chemotherapy, our new recombinant adeno-associated vector may prove to be useful as a tool for providing cardioprotection on demand, avoiding