AdVEGF-B186 and AdVEGF-D(Delta)N(Delta)C induce angiogenesis and increase perfusion in porcine myocardium

UEF//eRepository

DSpace https://erepo.uef.fi

Rinnakkaistallenteet Terveystieteiden tiedekunta

2016

AdVEGF-B186 and

AdVEGF-D(Delta)N(Delta)C induce

angiogenesis and increase perfusion in porcine myocardium

Nurro Jussi

BMJ

Tieteelliset aikakauslehtiartikkelit

© 2016 BMJ Publishing Group Ltd & British Cardiovascular Society CC BY-NC http://creativecommons.org/licenses/by-nc/4.0/

http://dx.doi.org/10.1136/heartjnl-2016-309373

https://erepo.uef.fi/handle/123456789/26359

Downloaded from University of Eastern Finland's eRepository

AdVEGF-B

and AdVEGF-D

induce angiogenesis and increase perfusion in porcine myocardium

Jussi Nurro, MD,* Paavo J. Halonen, MD,*† Antti Kuivanen, MD,*† Miikka Tarkia, PhD,‡§ Antti Saraste, MD, PhD,‡ Krista Honkonen, MD,* Johanna Lähteenvuo, MD, PhD,* Tuomas T. Rissanen, MD, PhD,*||

Juhani Knuuti, MD, PhD,‡ and Seppo Ylä-Herttuala, MD, PhD*#**

From the *Department of Biotechnology and Molecular Medicine, A. I. Virtanen Institute, University of Eastern Finland, Kuopio, Finland; ‡Turku PET Centre, Turku University Hospital, Turku, Finland;

§Department of Pharmacology, University of Helsinki, Helsinki, Finland; ||Department of Internal Medicine, Central Hospital of North Karelia, Joensuu, Finland; #Science Service Center, Kuopio University Hospital, Kuopio, Finland; and the **Gene Therapy Unit, Kuopio University Hospital, Kuopio, Finland;

†authors with equal contribution

Address for correspondence:

Seppo Ylä-Herttuala, MD, PhD, FESC, Department of Biotechnology and Molecular Medicine, A. I. Virtanen Institute, University of Eastern Finland, P.O. Box 1627 FIN-70211 Kuopio, Finland

phone +358 40 355 2075, seppo.ylaherttuala@uef.fi

Word count: 3366

Key words: Gene transfer, positron emission tomography, therapeutic angiogenesis, preclinical, safety

The Corresponding Author has the right to grant on behalf of all authors and does grant on behalf of all authors, an exclusive licence (or non exclusive for government employees) on a worldwide basis to the BMJ Publishing Group Ltd and its Licensees to permit this article (if accepted) to be published in HEART editions and any other BMJPGL products to exploit all subsidiary rights.

Abstract

Objective

Coronary heart disease (CHD) remains a significant clinical problem and new therapies are needed especially for refractory angina patients for whom the current therapies do not provide sufficient relief.

Whilst angiogenic gene therapy has shown its potential in capillary enlargement, functional improvements are necessary for therapeutic purposes. The aim of this study was to find out if angiogenic gene therapy would increase myocardial perfusion as measured by the positron emission tomography (PET) ¹⁵O- imaging, and whether there would be coronary steal effect to the contralateral side. Furthermore, safety of intramyocardial angiogenic adenoviral gene transfer was evaluated.

Methods

Intramyocardial adenoviral (Ad) VEGF-B186 or AdVEGF-D^ΔNΔC gene transfers were given endovascularly into the porcine posterolateral wall of the left ventricle (n=34). Six days later, PET ¹⁵O-imaging for myocardial perfusion and coronary angiography were performed.

Results

AdVEGF-B186 and AdVEGF-D^ΔNΔC induced angiogenesis and increased total microvascular area 1.8-fold (95

% CI 0.2 to 3.5) and 2.8-fold (95 % CI 1.4 to 4.3), respectively. At rest, perfusion was maintained at normal levels, but at stress, relative perfusion was increased 1.4-fold (95 % CI 1.1 to 1.7) for AdVEGF-B186 and 1.3- fold (95 % CI 1.0 to 1.7) for AdVEGF-D^ΔNΔC, without causing coronary steal effect in the control area. The therapy was well tolerated and did not lead to any significant changes in laboratory safety parameters.

Conclusions

Both AdVEGF-B186 and AdVEGF-D^ΔNΔC gene transfers induced efficient angiogenesis in the myocardium resulting in an increased myocardial perfusion measured by PET. Importantly, local perfusion increase did not induce any coronary steal effect. As such, both of the treatments seem suitable candidates for the induction of therapeutic angiogenesis for the treatment of ischemic heart disease.

Key Messages

What is already known about this subject?

Vascular endothelial growth factors (VEGFs) have been studied extensively in the past years as their ability to produce angiogenesis in myocardium is hypothesized to become an alternative treatment option for ischemic diseases. By using a gene therapy approach angiogenesis can be induced in the myocardium by various different VEGFs.

What does this study add?

The newly formed microvasculature in the myocardium could be aberrant and unable to provide the much needed perfusion in the tissue. In this study we have measured the myocardial perfusion after VEGF gene therapy by using positron emission tomography ¹⁵O-radiowater perfusion imaging, which is considered the “gold standard” for perfusion measurement. We have shown that adenoviral gene therapy of different

VEGFs can be used to increase myocardial perfusion at stress conditions. In addition, coronary steal effect was not observed in the non-treated area of the myocardium.

How might this impact on clinical practice?

We have shown that using adenoviral VEGF gene therapy is safe and a significant perfusion increase can be achieved in the preclinical setting. This will further encourage conducting clinical trials by using adenoviral VEGF gene therapy possibly leading to a new treatment option for ischemic patients.

Main text

INTRODUCTION

Even though death rates from cardiovascular diseases have declined in the United States[1] and EU,[2]

cardiovascular diseases still remain a major cause of morbidity and mortality in the western world.

Coronary heart disease (CHD) is accountable for nearly half of these deaths.[1, 2] Thus, new therapies are needed for those patients with chronic stable CHD with severe symptoms, advanced disease and possibly left ventricular dysfunction but for whom conventional treatments including pharmacological therapy, lifestyle change, angioplasty, stenting and revascularization are insufficient or unavailable. These patients are often referred to as refractory angina patients.[3]

Therapeutic angiogenesis to improve blood supply to ischemic myocardium is a potential treatment option shown efficacy in preclinical settings.[4-7] Many recent studies show that neovascularization is possible in myocardium using gene transfer of different vascular endothelial growth factors (VEGFs).[4, 5, 7-12] However, recent clinical trials have failed to show a significant functional improvement in patients.[13, 14] One explanation might be that in the preclinical setting there have not been appropriate studies with adequate focus on the functional effects of VEGF gene therapy on large animal myocardium.

Furthermore, past studies have shown that VEGF family members in high concentration may cause harmful tissue edema and aberrant angiogenesis.[4, 7] Newly formed vessels might not always be functional and increased angiogenesis does not always mean increased myocardial perfusion. Also, particularly during stress conditions, the heart should be performing at peak efficiency with maximal myocardial perfusion.

For determining myocardial perfusion there are various methods, each with their own limitations: single- photon emission computed tomography, contrast echocardiography, cardiac magnetic resonance imaging, computed tomography angiography and positron emission tomography (PET).[15] However, PET

imaging is regarded the “gold standard” for absolute myocardial perfusion measurement as it provides a

quantitative perfusion value in mL/min/kg. It is crucial that the therapy produces functional vasculature and increases perfusion, leading to functional improvement.

Furthermore, the coronary steal effect has to be taken into account especially in CHD patients. Coronary steal effect usually refers to a phenomenon where global small vessel dilation increases perfusion in an area already well perfused leading to a decrease in perfusion in another area with borderline perfusion and limited coronary flow reserve due to a coronary stenosis.[16] Angiogenic gene therapy might lead to a similar situation even in a healthy heart, where increased blood flow in the treated area decreases the absolute flow in intact areas as according to a path of least resistance. If this phenomenon is observed in a healthy heart it might indicate severe adverse effects in similarly treated CHD patients.

VEGF family members have different receptor binding profiles: VEGF-B is a VEGF receptor 1 (VEGFR-1) agonist while VEGF-D is a VEGFR-2 and VEGFR-3 agonist.[17] It has been previously shown that adenoviral gene transfer with either of these VEGFs induces angiogenesis,[5, 6] with VEGF-D even increasing perfusion, as measured by contrast echocardiography and microspheres.[5] Both VEGF-B186 and VEGF- D^ΔNΔC are very soluble and diffusible factors in tissues since they do not bind effectively to heparan sulphates and they also bind to neuropilin co-receptors.[17] As the angiogenic pathways are not yet fully understood, it is important to evaluate angiogenesis produced by VEGFR-1 and VEGFR-2 pathways. Either or both of these potent VEGFs could be useful for the induction of therapeutic vascular growth in the human heart.[7]

The aim of the present study was to find out if angiogenic gene therapy would increase myocardial perfusion as measured by the “gold standard” PET ¹⁵O-imaging, and whether there would be any coronary steal effect on the contralateral side. Furthermore, AdVEGF-B186 and AdVEGF-D^ΔNΔC gene transfers were compared and the safety of intramyocardial angiogenic Ad gene transfer was evaluated.

METHODS

Study overview

In this study the effects of AdVEGF-B186 and AdVEGF-D^ΔNΔC gene transfers were evaluated in healthy porcine myocardium. All porcine experiments (n = 34) were performed using female domestic pigs initially weighing from 25 to 40 kg and were approved by the Animal Experiment Board in Finland. The catheterization and imaging of the pigs were done as previously described[18] in a catheterization laboratory dedicated for the animal use.

Table 1. Study groups

Group Day 0 Day 6

Number of animals

AdCMV AdVEGF-B AdVEGF-D AdLacZ

1

Gene transfer, angiography, stroke volume, cardiac output

Angiography, LVEF, stroke

volume, cardiac output,

vascular permeability,

histology, Western Blot

4 4 7 -

2 Gene transfer PET perfusion 5 5 5 -

3 Gene transfer histology - - - 4

LVEF = left ventricular ejection fraction, PET = positron emission tomography

Study groups are described in Table 1. A separate group was used for the PET perfusion imaging due to scanner access. After cardiac catheterization and imaging intramyocardial gene transfers of AdVEGF-B186, AdVEGF-D^ΔNΔC or Ad control cytomegalovirus (CMV) promoter or AdLacZ encoding beta-galactosidase (β- gal) were made using the protocol described below. The assessment of AdVEGF-B186 and AdVEGF-D^ΔNΔC gene transfers on angiogenesis and myocardial perfusion in healthy myocardium was done at the time of the peak effect of Ad gene transfer, at six days’ time point. At the same timepoint, AdLacZ was used to evaluate Ad transduction efficiency.

Medication and anesthesia

Pigs received daily 200 mg amiodarone and 2.5 mg bisoprolol p.o. to prevent fatal ventricular arrhythmias, from one week before operation until the end of the follow up.[18] One day before the operation pigs received a loading dose of 300 mg clopidogrel and 300 mg acetylsalicylic acid p.o. to prevent acute thrombosis. Immediately before the operation, pigs received 100 mg lidocain i.v. 10 and 2.5 mL MgSo4 i.v.

to prevent ventricular arrhythmias, 500 mg cefuroxime i.m. for infection prophylaxis and 30 mg enoxaparin i.v. for thrombosis prevention.

Pigs were sedated with i.m. injection of 1.5 mL atropine and 6 mL azaperone. Propofol anesthesia was introduced intravenously at 15 mg · kg^-1 · h^-1 in combination with analgetic phentanyl infusion at 10 µg · kg^-1 · h^-1. After placing the femoral sheath, 1.25 mg of sublingual dinitrate was given to induce coronary vasodilatation.

Gene transfer

At the beginning of the experiment (day 0), MyoStar® intramyocardial injection catheter system (Biosense Webster, a Johnson & Johnson company, Diamond Bar, CA, USA) was introduced to the left ventricle via femoral sheat. Under fluoroscopic guidance, using perpendicular views antreroposterior and right anterior oblique 90°, 3 x 10¹⁰ plaque forming units of Ad vectors encoding either VEGF-B186, VEGF-D^ΔNΔC,

the control CMV or transduction marker β-gal were delivered by 15 injections of 0.2 mL each in the lateral and posterior wall of the left ventricle. This Ad dose has been shown to induce potent myocardial gene transfer in our previous experiments[5].

Ejection fraction analysis, cardiac output and stroke volume

Left ventricle ejection fraction (LVEF) was measured using the angio workstation ventricle analysis program (Innova® 3100^IQ; GE Healthcare, USA). Cardiac output was measured with a percutaneous thermodilution method using a Swan-Ganz Standard Thermodilution Pulmonary Artery Catheter (Edwards Lifesciences, Irvine, California). The stroke volumes were calculated using the data from ejection fraction measurements.

Vascular permeability

The animals were euthanized after angiography six days after the gene transfer at the time of the maximal effect of Ad gene transfer. Myocardial permeability i.e. plasma protein extravasation was assessed using the modified Miles permeability assay as previously described.[5] Also, echocardiography was used to evaluate pericardial effusion volume at the study end. Short axis view of the heart was acquired under the diaphragm using 3V2c cardiac transducer (Siemens Sequoia 512).

Western blotting

Proteins from the myocardial samples were detected with commercially available VEGF-B (AF751, R&D Systems) and VEGF-D (MAB 286, R&D Systems) primary antibodies followed by horseradish peroxidase–

conjugated secondary antibodies: donkey anti-goat IgG (SC-2020, Santa Cruz Biotechnology) and donkey anti-mouse IgG (HAF018, R&D Systems), respectively.

PET imaging

A subgroup of animals were imaged at the peak effect of Ad gene transfer six days after the treatment using PET scanner for regional myocardial perfusion with ¹⁵O-radiowater. The relative perfusion presented

in the graphs is the ratio of absolute perfusion in the treated posterolateral area and control anteroseptal area of the left ventricle.

PET studies were performed with ECAT EXACT HR+ scanner (Siemens-CTI, Knoxville, TN, USA). Myocardial perfusion was evaluated by PET ¹⁵O-radiolabeled water (805 ± 87 MBq) both at rest and during adenosine stress (200 μg·kg−1·min−1 iv infusion) six days after the gene transfer as previously described.[19] The acquisition frames were as follows: 14 × 5 s, 3 × 10 s, 3 × 20 s and 4 × 30 s (total duration 4 min 40 sec).

The acquired PET data were reconstructed in 2D mode with an iterative reconstruction algorithm OSEM using 6 iteration and 16 subsets in the reconstruction. The transaxial field of view (35 cm ) was reconstructed in a 128 × 128 matrix, yielding a pixel size of 2.57 × 2.57 mm. The measurements were corrected for scatter, random counts, and dead time. The device produces 63 axial planes with a slice thickness of 2.43 mm.

Regional myocardial perfusion (in ml·g−1·min−1) was measured using Carimas 2 software (Turku PET Centre, Turku, Finland; http://www.turkupetcentre.fi/carimas) as previously described.[19]

Histochemistry

Tissue samples were fixed with 4 % paraformaldehyde and embedded in paraffin. Blood vessel endothelium was labeled with lectin (Biotinylated Griffonia (Bandeiraea) Simplicifolia Lectin I; Vector Laboratories, Burlingame, CA, USA) and detected with green fluorochrome for vessel analysis or 3,3'- Diaminobenzidine (DAB) for imaging. The average total microvascular area (%) was evaluated from maximal transduction from five microscopic images using AnalySIS software (Olympus Soft Imaging Solutions, Münster, Germany). The β-gal positive nuclei were labeled using a α-β-galactosidase primary antibody (Promega, Z378B) and detected using biotinylated α-mouse secondary antibody (Vectastain, BA- 2000), HRP-conjugated ABC system (Vectastain, PK-6100) and DAB (Invitrogen, 002020).

Blood markers

Blood samples were collected at the time of gene transfer (day 0) and at the time of peak Ad expression (day 6) to find out if AdVEGF-B186 or AdVEGF-D^ΔNΔC would increase alkaline phosphatase (ALP) or lactate dehydrogenase (LDH) levels as a sign of cardiac of liver injury.

Statistics

Results are expressed separately for each individual animal in dot plots, line indicating the average value for the group. Statistical significance is evaluated by an ordinary one-way ANOVA, or a two-way ANOVA when appropriate, followed by Dunnett’s post-hoc test. The AdCMV group was used as a reference group in comparisons of the group differences. A value of P < 0.05 was considered statistically significant.

Computations were performed with Prism 6 for MAC OS X, Version 6.0c (GraphPad Software, Inc., La Jolla, California, USA).

RESULTS

AdVEGF-B186 and AdVEGF-D^ΔNΔC induce robust angiogenesis

Both treatment gene transfers had an angiogenic effect in the pig myocardium six days a fter the gene transfer (Figure 1, B and C) when compared to the control group (Figure 1, A). Total microvascular area was increased due to increased capillary number and increases in the capillary diameter. AdVEGF- D^ΔNΔC treatment tended to be the most effective in increasing the total microvascular area reaching statistical significance (Figure 1, D), but AdVEGF-B186 treatment also increased this area in the treated posterolateral wall. The increase in total microvascular area was 1.8-fold (95 % CI 0.2 to 3.5) for AdVEGF-B186 and 2.8- fold (95 % CI 1.4 to 4.3) for AdVEGF- D^ΔNΔC when compared to the control (Figure 1, D).

AdVEGF-B186 and AdVEGF-D^ΔNΔC increase myocardial perfusion

An increase in myocardial perfusion in the treated area of the myocardium was observed (Figure 2, D and E) in both treatment groups six days after the gene transfer as measured by ¹⁵O-radiowater PET perfusion

imaging. At rest, increased myocardial perfusion in the treatment groups was localized to the gene transfer area in the targeted posterolateral wall of the left ventricle (Figure 2, B and C) whereas in the control group there was no regional difference in the myocardial perfusion (Figure 2, A). This positive effect of both AdVEGF-B186 and AdVEGF-D^ΔNΔC on the myocardial perfusion was even more pronounced during stress reaching statistical significance (Figure 2, E). At stress, the increase in relative perfusion was 1.4-fold (95 % CI 1.1 to 1.7) for AdVEGF-B186 and 1.3-fold (95 % CI 1.0 to 1.7) for AdVEGF-D^ΔNΔC when comparing to the control (Figure 2, E).

Increased perfusion did not induce coronary steal effect

The absolute perfusion in the untreated myocardium during adenosine-induced stress was not decreased in comparison to the rest values in any of the treated animals by evaluating the bull’s-eye perfusion maps.

This combined with the increase in perfusion in the treated area during stress indicates that no coronary steal effect was present in the treated animals.

Vascular permeability did not cause complications

The extravasated Evans Blue dye was visible six days after the gene transfer in the treatment groups but not in the control group. Also, AdVEGF-B186 and AdVEGF- D^ΔNΔC treated animals had some pericardial effusion at the time of the peak effect of Ad gene transfer (Figure 1, E to G). However, there were no statistical differences in the permeability (data not shown) and no complications occurred due to pericardial effusion.

Treatment had no effect on LVEF, stroke volume or cardiac output

The treatments had no effect on the LVEF, stroke volume or cardiac output measurements as no differences were found between the treatment and control groups (data not shown).

Gene expression in safety tissues was insignificant

Lung, liver, spleen kidney and ovary samples were collected from AdLacZ treated animals. In these tissues there were only a few β-gal transduced cells (Figure 1, K to O). On the contrary, in myocardium the transduced area begun a few millimeters from the endocardium and extended triangularly through the myocardium to the epicardial side (Figure 1, H to J).

Intramyocardial adenoviral gene transfer results in protein production without inflammation

In the Western blotting the VEGF-B186 and VEGF-D^ΔNΔC transduced myocardial samples contained VEGF- B186 and VEGF-D^ΔNΔC protein, respectively, whereas there were no detectable amounts of these proteins in the control CMV samples (Figure 3). Also, VEGF-B186 samples had no detectable amount of VEGF-D^ΔNΔC protein and vice versa. Furthermore, there was no observable neutrophil infiltration in the histological samples of the myocardium at six days timepoint in the control group or in either of the treatment groups.

ALP and LDH levels were not increased in comparison to the control treatment

There were no differences between the groups in ALP levels six days after the gene transfer (Figure 4). By comparing the change in LDH levels the increase at this time period was smaller in the treatment groups than in the control group.

DISCUSSION

Six days after the treatment, the increased capillary area caused by the intramyocardial AdVEGF-B186 and AdVEGF-D^ΔNΔC gene transfers also increased perfusion as measured by PET ¹⁵O-perfusion. It was also important that the angiogenic gene therapy did not lead to steal effect at stress, which has been a concern after the VEGF gene transfers. The increased capillary area and hence the larger blood flow could theoretically cause a decrease in perfusion in the non-treated areas of the myocardium especially during myocardial stress. Also, the perfusion was not increased at rest when compared to the control group. The heart has strong autoregulation, which seems to prevent unnecessary increase in perfusion at rest. This

means that the vessels are recruitable as needed during stress and that the perfusion increase is physiological and hence relevant.

It has been shown that increased vascular permeability, tissue edema and pericardial effusion occur as side effects of gene transfer using VEGFR-2 ligands.[5, 20] However, in this study with a dose of 3 x 10¹⁰ plaque forming units these negative effects did not cause any complications. While the permeability was modestly increased in both of the treatment groups it did not reach statistical significance or cause any adverse effects in the animals, even though there was some pericardial effusion at the time of the peak effect of Ad gene transfers.

AdVEGF-D^ΔNΔC increased the total microvascular area of the treated myocardium more than AdVEGF-B186

while robust angiogenesis was still induced by both of the treatments resulting in increased perfusion in the treatment area. It could hence be argued that also the smaller increase in microvascular area caused by the AdVEGF-B186 at this dose is enough for a significant increase in perfusion, at least in a healthy myocardium. This is a major finding as AdVEGF-B186 can produce the same kind of perfusion increase with a smaller increase in microvascular area, thus reducing the risk of excessive and nonfunctional aberrant angiogenesis.

Blood samples taken did not indicate acute heart or liver toxicity as a result of AdVEGF-B186 or AdVEGF- D^ΔNΔC treatment, as the levels of ALP and LDH six days after the gene transfers remained comparable to the control group. However, the variation in the baseline values was high and it remains unclear if the control AdCMV group would show injury on the basis of the increase in LDH levels. Furthermore, these measures of toxicity are of course not very sensitive and should be considered only to give a rough impression of the toxicity. Also, the lack of neutrophil infiltration in the myocardium after the gene transfer indicates that the adenovirus used does not induce an inflammatory response. Therefore, these

results indicate that Ad gene transfers with the therapeutic genes used is safe as no major adverse effects were observed.

Even though there were increased microvascular area and increased perfusion, no differences in the functional parameters, such as ejection fraction or cardiac output were observed. Even though the treatment may have a local effect on the myocardial contraction, it may not be enough to cause a significant improvement in overall myocardial function in this setting. On the other hand, an increased perfusion in a healthy heart might not be expected to result in an improvement in normal function as it is likely that the contracting myocytes already have all the blood they need to function optimally during stress conditions.

Intramyocardial injection resulted in a triangular transduction pattern starting from the needle tip a few millimeters from the endocardial side and continuing through the myocardium into the epicardial side of the myocardium where most of the transduced myocytes were found. Furthermore, only a few of the cells in the safety tissue samples were transduced indicating that the Ad intramyocardial injection is a feasible local delivery method for gene therapy. Even though the transduction efficacy is just around 50 % beyond the needle track, with multiple injections and soluble therapeutic agents real perfusion effects can be achieved. Unlike in some recent trials we conclude that local gene delivery is still the best delivery method for cardiac gene transfer as it provides strong effects with minimal safety issues as seen in this study.

By keeping the clinical world in mind, this study provides some much needed support for planning further clinical trials. Adenoviruses are a very useful tool for proof of concept studies as the expression time is short and controllable without further need for regulation of the expression. Adenoviral gene therapy can also be targeted to the specific treatment area in the myocardium after using an electrophysiological mapping tool, such as the NOGA device, resulting in robust transgene expression in the desired area. This

delivery method eliminates the uncertainties of vector finding the target area still present in other methods, such as with intracoronary infusion of AAV-vectors.

CONCLUSIONS

Both AdVEGF-B186 and AdVEGF-D^ΔNΔC gene transfers induced efficient angiogenesis in the myocardium resulting in an increased myocardial perfusion measured by PET ¹⁵O-perfusion in a healthy heart. Another major finding is that the local increase in perfusion did not induce coronary steal effect. On the basis of this study, both of the treatments seem suitable candidates for the treatment of CHD in future preclinical and clinical studies.

Acknowledgements

The authors acknowledge the contribution of Minna Törrönen, Heikki Karhunen, Olli-Pekka Hätinen, Teemu Karjalainen and Ina-Mari Laine for the data collection, Tuula Tolvanen for the PET imaging and Tiina Koponen and Sari Järveläinen for the adenoviral production.

Affiliations

None.

Funding

Funding provided by the Academy of Finland, Helsinki, European Research Council Advanced grant, ADVance (EU grant agreement ref. 290002), the Sigrid Juselius Foundation, Helsinki, Finnish Cultural Foundation - North Savo Regional fund, Kuopio, Finnish Cultural Foundation, Helsinki, Maud Kuistila Foundation, Helsinki, Ida Montin Foundation, Helsinki, Finnish Foundation for Cardiovascular Research,

Helsinki, Antti and Tyyne Soininen Foundation, Kuopio, The Finnish Medical Foundation, Helsinki, and Kuopio University Hospital, Kuopio.

Reference list

1. Go AS, Mozaffarian D, Roger VL, et al. Executive Summary: Heart Disease and Stroke Statistics--2013 Update: A Report From the American Heart Association. Circulation 2013;127(1):143-52.

2. Nichols M, Townsend N, Scarborough P, et al. Cardiovascular disease in Europe 2014: epidemiological update. Eur Heart J 2014;35(42):2950-9.

3. Henry TD, Satran D, Jolicoeur EM. Treatment of refractory angina in patients not suitable for revascularization. Nat Rev Cardiol 2014;11(2):78-95.

4. Rissanen TT, Yla-Herttuala S. Current status of cardiovascular gene therapy. Mol Ther 2007;15(7):1233- 47.

5. Rutanen J, Rissanen TT, Markkanen JE, et al. Adenoviral catheter-mediated intramyocardial gene transfer using the mature form of vascular endothelial growth factor-D induces transmural angiogenesis in porcine heart. Circulation 2004;109(8):1029-35.

6. Lahteenvuo JE, Lahteenvuo MT, Kivela A, et al. Vascular endothelial growth factor-B induces myocardium-specific angiogenesis and arteriogenesis via vascular endothelial growth factor receptor-1- and neuropilin receptor-1-dependent mechanisms. Circulation 2009;119(6):845-56.

7. Yla-Herttuala S, Rissanen TT, Vajanto I, et al. Vascular endothelial growth factors: biology and current status of clinical applications in cardiovascular medicine. J Am Coll Cardiol 2007;49(10):1015-26.

8. Roy H, Bhardwaj S, Babu M, et al. VEGF-DdeltaNdeltaC mediated angiogenesis in skeletal muscles of diabetic WHHL rabbits. Eur J Clin Invest 2010;40(5):422-32.

9. Heikura T, Nieminen T, Roschier MM, et al. Baculovirus-mediated vascular endothelial growth factor- D(DeltaNDeltaC) gene transfer induces angiogenesis in rabbit skeletal muscle. J Gene Med 2012;14(1):35-43.

10. Karvinen H, Pasanen E, Rissanen TT, et al. Long-term VEGF-A expression promotes aberrant angiogenesis and fibrosis in skeletal muscle. Gene Ther 2011;18(12):1166-72.

11. Korpisalo P, Hytonen JP, Laitinen JT, et al. Capillary enlargement, not sprouting angiogenesis, determines beneficial therapeutic effects and side effects of angiogenic gene therapy. Eur Heart J 2011;32(13):1664-72.

12. Siddiqui AJ, Blomberg P, Wardell E, et al. Combination of angiopoietin-1 and vascular endothelial growth factor gene therapy enhances arteriogenesis in the ischemic myocardium. Biochem Biophys Res Commun 2003;310(3):1002-9.

13. Kastrup J, Jorgensen E, Ruck A, et al. Direct intramyocardial plasmid vascular endothelial growth factor- A165 gene therapy in patients with stable severe angina pectoris A randomized double-blind placebo-controlled study: the Euroinject One trial. J Am Coll Cardiol 2005;45(7):982-8.

14. Stewart DJ, Kutryk MJ, Fitchett D, et al. VEGF gene therapy fails to improve perfusion of ischemic myocardium in patients with advanced coronary disease: results of the NORTHERN trial. Mol Ther 2009;17(6):1109-15.

15. Salerno M, Beller GA. Noninvasive assessment of myocardial perfusion. Circ Cardiovasc Imaging 2009;2(5):412-24.

16. Rowe GG. Inequalities of myocardial perfusion in coronary artery disease ("coronary steal").

Circulation 1970;42(2):193-4.

17. Nieminen T, Toivanen PI, Rintanen N, et al. The impact of the receptor binding profiles of the vascular endothelial growth factors on their angiogenic features. Biochim Biophys Acta 2014;1840(1):454- 63.

18. Rissanen TT, Nurro J, Halonen PJ, et al. The bottleneck stent model for chronic myocardial ischemia and heart failure in pigs. Am J Physiol Heart Circ Physiol 2013;305(9):H1297-308.

19. Tarkia M, Saraste A, Saanijoki T, et al. Evaluation of 68Ga-labeled tracers for PET imaging of myocardial perfusion in pigs. Nucl Med Biol 2012;39(5):715-23.

20. Rissanen TT, Korpisalo P, Markkanen JE, et al. Blood flow remodels growing vasculature during vascular endothelial growth factor gene therapy and determines between capillary arterialization and sprouting angiogenesis. Circulation 2005;112(25):3937-46.

Figure titles and legends

Figure 1. Intramyocardial injections of AdVEGF-B186 and AdVEGF-D^ΔNΔC induce myocardium specific angiogenesis with negligible transduction elsewhere. (A) to (C) Lectin staining for endothelial cells (brown), magnification 200x. Scale bars are 100 μm. The increase in total microvascular area was 1.8-fold for AdVEGF-B186 (B) and 2.8-fold for AdVEGF- D^ΔNΔC (C) when compared to the control (A) reaching statistical significance with AdVEGF-D^ΔNΔC (D). (E) to (G) Increase in permeability induces minor pericardial effusion at the time of peak effect, as seen in subdiaphragm echo, without causing any adverse effects.

White arrows indicate pericardial effusion. Effusion volume was higher for AdVEGF- D^ΔNΔC treated animals (G). RV = right ventricle; LV = left ventricle. (H) to (O) β-galactosidase nuclear staining (brown) for AdLacZ transduced cells. (H) Myocardial section from endocardium to epicardium, magnification 10x, scale bar is 1 mm. Transduced cell area, indicated by black arrows, begins a few millimeters from the endocardium, which is the length of the injection needle. It spans from there to the epicardium forming a triangular area of transduction. The larger box indicates the section seen in (I) and the smaller box the section seen in (J).

(I) Magnification 40x, scale bar is 1 mm. Transduction efficacy is greatest at a straight line from the needle track to the epicardium whilst disperse transduced cells are seen few millimeters away from this line. (J) Magnification 100x, scale bar is 100 μm. Transducted cells cover at most 50 % of the cells near the needle

tract. However, soluble end products can mediate the effects further. (K) to (O) Lung, liver, spleen, kidney, ovary, respectively. Magnification 200x, scale bars are 100 μm. A few transduced β-gal positive cells were found in the lung (K), spleen (M) and ovary (O).

Figure 2. AdVEGF-B186 and AdVEGF-D^ΔNΔC increase perfusion in healthy pig myocardium. Gene transfers were made to the posterolateral wall of the left ventricle, which corresponds to the lower right sector of the perfusion pictures, indicated by ellipse labeled gt. Color scale is absolute; darkest blue is 0 ml min^-1 g^-

1, green is 1,5 ml min^-1 g^-1 and deepest red is 3,0 ml min^-1 g^-1 or over. (A) Perfusion maximum is not localized in the gene therapy area in the control group indicating no effect of the control treatment. (B) and (C) The peak perfusion of both AdVEGF-B186 and AdVEGF-D^ΔNΔC is localized in the gene transfer area and the changes are even more pronounced at stress. (D) and (E) Relative perfusion was at rest 12 % and at stress 40 % higher for AdVEGF-B186 and at rest 13 % and at stress 34 % higher for AdVEGF-D^ΔNΔC than for AdCMV.

ctrl = control anteroseptal area; gt = posterolateral gene transfer area.

Figure 3. Both AdVEGF-B186 and AdVEGF-D^ΔNΔC treated myocardium produce transduced proteins as determined by Western Blot analysis. The top band represents the VEGF-B monomer and the lower band VEGF-D monomer. Two animals from each group are represented. L = ladder; C = AdCMV; B = AdVEGF-B186; D = AdVEGF-D^ΔNΔC; PB = positive control for VEGF-B186; PD = positive control for VEGF-D^ΔNΔC.

Figure 4. ALP and LDH levels were not increased in comparison to the control treatment. There were no significant differences in ALP levels between the groups. In comparison to the control group, the increase in LDH levels was smaller in the treatment groups than in the control group. ALP = alkaline phosphatase; LDH = lactate dehydrogenase.

Figure 1:

Figure 2:

Figure 3:

Figure 4: