1Department of Gene Therapy, Faculty of Medicine, The National Heart and Lung Institute, Imperial College London, Manresa Road, London SW3 6LR, UK
2Klink für Herz-Thorax-Gefasschirurgie, Universitatsklinik, Magdeburg, Germany
3Genzyme Corporation, Framingham, MA, USA
Received 2 July 2004; revised 25 January 2005; accepted 17 February 2005; online publish-ahead-of-print 8 April 2005.
* Corresponding author. Tel: +44 207 351 8339; fax: +44 207 351 8340. E-mail address: a.heinl-green{at}ic.ac.uk
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Abstract |
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Methods and results Ameroid occluders were placed around the left circumflex coronary artery of 74 pigs. Three weeks later, pigs were randomized to receive (i) adenovirus encoding HIF-1 (Ad2/HIF-1
VP-16 1010 particles); (ii) plasmid DNA encoding HIF-1
(pHIF-1
NF
B 500 µg); (iii) pHIF-1
NF
B 2500 µg; and (iv) adenoviral control (Ad2/CMV-empty vector 1010 particles). Twenty injections (50 µL each) were administered epicardially via re-thoracotomy. Three weeks after gene delivery significant (ANOVA P=0.02) changes in myocardial perfusion during stress were seen in the area adjacent to injections. Post hoc testing (Bonferroni) demonstrated that the AdHIF-1
group was significantly (P=0.02) different from the Ad2/control. There were also significant (ANOVA P=0.02) differences in resting left ventricular (LV) function. Post hoc (Bonferroni) showed that the AdHIF-1
group was significantly different from the Ad2/control (P=0.03). No significant changes in any parameter were seen with plasmid HIF-1
. There were no differences in collateralization or capillary growth.
Conclusion Ad2/HIF-1 increased myocardial perfusion and improved LV function. Plasmid HIF-1
was not associated with improvements in any bioactivity endpoints.
Key Words: Angiogenesis HIF-1 Gene therapy Myocardium
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Introduction |
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Mature blood vessel formation requires the interplay of a number of different cell types3 and sequential expression of a number of growth factors together with their receptors. Perhaps not surprisingly, therefore, co-administration, for example, of vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (FGF2) produces synergistic effects.4 Thus, up-regulation of a single growth factor may be insufficient. Hypoxia-inducible factor-1 (HIF-1) has been described as a master switch gene. HIF-1
is able to induce production of inducible nitric oxide synthase (iNOS), VEGF-A, transforming growth factor beta (TGFß3), insulin-like growth factor (IGF-2) plasminogen activator inhibitor 1, erythropoietin in addition to FLT-1, a VEGF receptor. Thus, HIF-1
acts as an inducible transcription regulatory factor, which in combination with constitutively expressed HIF-1ß, plays a principal role in the cellular response to hypoxia. To allow for constitutive expression of this factor as a transgene, transactivating domains either from the herpes simplex virus (VP16) or from the nuclear factor kappa (NFkB) can be incorporated.5 Angiogenesis may be induced by application of the candidate protein or by gene therapy. The latter is, however, the only option for certain transcription factors, such as HIF-1
, because in its protein form it is very rapidly degraded.6 Gene delivery to the myocardium can be achieved using a number of vectors and delivery routes. Generally, adenoviral-mediated gene transfer produces the highest expression levels, but is associated with dose-related inflammation. Plasmid DNA has also proved effective in some studies and is generally less likely to induce toxicity. For either vector, direct injection into the myocardium via an epicardial approach is most likely to circumvent delivery issues. We set out to establish the efficacy and safety of plasmid and adenoviral HIF-1
in a porcine model of chronic myocardial ischaemia.
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Methods |
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Ameroid placement
A left lateral thoracotomy was performed through the fourth to fifth intercostal space, and an ameroid constrictor (2.02.5 mm inner diameter, Research Instruments, Escondido, CA, USA) was placed as proximally as possible around the left circumflex coronary artery (LCX).
Gene delivery
Screening and entry criteria
Three weeks after ameroid placement, left heart catheterization was performed by standard techniques (Philips Angio Diagnost 2U Optimus M200, The Netherlands). After administration of a heparin bolus (100 U/kg), angiograms of the left and right coronary arteries were obtained. The degree of stenosis and antegrade flow were assessed in the LCX and scored using TIMI criteria.7 In addition, collateralization of the LCX was quantified using a Rentrop score.8 Pigs were randomized if: (i) the LCX was totally occluded, (ii) the LCX had a >95% flow limiting stenosis and a (TIMI2 flow), and (iii) there was evidence of right-to-left (contralateral) collaterals (Rentrop class
1). Animals not fulfilling these criteria were terminated with administration of pentobarbital (16003400 mg).
Randomization and gene delivery
Pigs meeting the entry criteria were randomized in a blinded fashion to receive one of the following:
Following randomization, a resting transthoracic echocardiogram was performed to assess left ventricular (LV) function. Regional myocardial blood flow measurements were undertaken with stable isotope-labelled microspheres at rest and during stress. A re-thoracotomy was then performed in the sixth to seventh space. The LCX territory was identified and 20 injections (each 50 µL) of randomized substance were injected evenly into a 6.25 cm2 (2.5x2.5 cm2) area with a 29 G needle to a depth of 5 mm. Two 5.0 prolene stitches were applied to the epicardial fat at the transverse boundaries of the injected area to assist with subsequent identification during tissue harvesting.
Evaluation of toxicity and efficacy
Three weeks after gene delivery, repeat echocardiography, angiography, and microsphere studies were performed. Animals were then terminated under anaesthesia (pentobarbital 16003400 mg), the heart excised, and the target area identified using the stitches placed in the epicardial fat. This area was then scored with a blade on the epicardial surface and the heart cut into five transverse sections from apex to base (sections 15). The middle ring, anatomically located at the mid-papillary level, was selected for determination of myocardial blood flow. The fourth ring was used for protein and immunohistochemistry assays.
Vectors
Ad2/HIF-1/VP16 is a recombinant replication-deficient adenovirus in which the virus backbone is E1, E3+, E4 (but retaining E4/ORF6) and contains the pIX gene insertion in the E4 region.9 The deleted E1 region has been replaced with an expression cassette encoding the chimeric cDNA for HIF-1
/VP165 Ad2/CMVEV is similar to Ad2/HIF-1
/VP16 except that it lacks a transgene. A second constitutively active version of HIF-1
contains amino acids 1390 of HIF-1
fused to amino acids 350550 of the human NFkB p65 subunit.
Transthoracic echocardiography
Echocardiography was performed at rest. Standard two-dimensional and M-mode transthoracic images were used (750 Sonotron, 3.25 MHz transthoracic transducer, VINGMED, Horten, Norway). From a right parasternal approach, short axis, mid-papillary views were obtained at rest. Global cardiac function was estimated using fractional LV shortening, calculated as [(diastolic LV diametersystolic LV diameter)/diastolic diameter]x100.
Assessment of myocardial blood flow
A dedicated angiography catheter was advanced into the left atrium (LA). Pressure recordings within the LA and a test injection of contrast dye verified correct positioning. Reference arterial blood was withdrawn for 2 min at a constant speed of 5 mL/min from the femoral access by a blood withdrawal pump (Harvard Apparatus, Bedford, MA, USA). This was commenced 10 s before 4 mL of microspheres (106 per mL 15 µm diameter; Biopal, Worcester, MA, USA) were injected into the LA over a period of 30 s at rest and under stress. Stress was defined by a dobutamine continuous infusion (664 µg/kg) sufficient to increase resting heart rate by 100%. Samarium and gold labelled microspheres were administered at rest and stress prior to gene delivery, whereas lutetium and iridium labelled microspheres were used 3 weeks after gene delivery. On completion of the study, the mid-papillary myocardial ring was subdivided into 15 segments (Figure 1). Tissue samples were washed in Sansaline© (Biopal), weighed, and placed in sodium- and contamination-free tubes (Biopal). Blood and tissues samples were dried in an oven at 70°C for 24 h and then sent to a core laboratory for neutron activation (Biopal). Values are expressed as number of disintegrations per minute (d.p.m.). To calculate the blood flow per unit gram of tissue, we used:
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Histological analysis of non-target organs
Samples from the liver, heart, spleen, lung, and kidneys were placed in 10% formalin and processed as described earlier to assess potential toxicity.
Immunohistochemistry
Three 5 µm sections, separated by 50 µm intervals, were immunostained using mouse anti-human Von Willebrand Factor (Dako M0616) and anti-human CD31 (Dako M0823) monoclonal antibodies for identification of vascular endothelial cells. The EnVisionTM plus horseradish peroxidase kit (HRP) was used to enhance the immuno signal.11 HRP was detected using diaminobenzidine-positive labelling of endothelial cells demonstrated by a dark brown pigment. The number of capillaries was assessed using a squared eyepiece graticule to avoid repeat counts (x200 magnification). The data were expressed as number of capillaries per field.
Western blot protein estimation
Epicardial and endocardial surfaces were removed from the target zone segment, so that only the mid-myocardial area was analysed. All samples for protein analysis were snap frozen and stored in liquid nitrogen until analysis by standard techniques.12 As a positive control, 100 µg of Escherichia coli-derived recombinant human VEGF (Autogen Bioclear, Wiltshire, UK) was applied.
Statistics
For sample size consideration, we used a previously published study16 to calculate that a size of n=8 could detect an angiogenic effect. Samples were coded and all data analysed in a blinded fashion, using SPSS version 10. All data are presented as mean (SEM). Group means were compared with ANOVA and post hoc Bonferroni correction; all tests were two-sided. The null hypothesis was rejected at P<0.05.
Results
Seventy-four animals underwent thoracotomy for ameroid placement, of which 37 were available for gene delivery following exclusion criteria and perioperative losses. Table 1 shows that there were no significant differences among any of the four groups for any parameters following randomization.
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Inflammation within the myocardium
There were no significant differences in inflammatory foci between any group, either in the target area ANOVA (P=0.6) or in the septum (P=0.8) (Figure 2).
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Efficacy
Perfusion
The relative (per cent) change in perfusion from gene delivery to final studies is presented for convenience. Absolute differences were also calculated, which gave comparable results. The administration of dobutamine (stress) resulted in four-fold increase in flow as expected. The diastolic blood pressure as the main variable for coronary perfusion was comparable among groups during rest and stress (data not shown).
At rest, no significant (ANOVA) changes in myocardial perfusion were detected in the target area (P=0.3), adjacent (P=0.3), left anterior descending artery (LAD) (P=0.2), and septum (P=0.2). However, during stress, significant differences among groups were seen for the adjacent area (ANOVA P=0.02). Post hoc testing (Bonferroni) demonstrated that the AdHIF-1 group was significantly (P=0.02) different from the Ad2/control group. The improvement in flow was 54% (SEM 22%) in the AdHIF-1
group compared with a reduction of 14% (SEM 11%) in the Ad2/control. This corresponded to an absolute change in myocardial blood flow of 0.87 mL/g/min (SEM 0.42 mL/g/min) and 0.48 mL/g/min (SEM 0.35 mL/g/min), respectively. The differences in other myocardial territories were not statistically significant [ANOVA target area (P=0.07), LAD (P=0.08), and septum (P=0.1)]. No differences were seen with either plasmid dose in any of the four territories studied (Bonferroni, P=1.0) for all territories (Figure 3).
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There was a significant (ANOVA P=0.02) difference in fractional shortening 3 weeks after gene delivery. Post hoc (Bonferroni) testing demonstrated that the AdHIF-1 group was significantly (P=0.03) different from the Ad2/control. The improvement in the AdHIF-1
group was 7% (SEM 5%) compared with a reduction of 6% (SEM 3%) in the Ad2/control (Figure 4). The latter showed a reduction in shortening, compatible with the known disease progression in this model.
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Haemodynamics
There were no detectable differences among groups, at rest or during stress, for heart rate, arterial pressure, or left atrial pressure (data not shown).
VEGF protein
The half-life of HIF-1 is too short to assess protein level; we therefore investigated a down-stream protein VEGF. No VEGF protein could be detected 3 weeks after gene delivery by western blotting in any group. However, a single band at an appropriate site was visible after samples were spiked with 100 µg of VEGF protein.
Discussion
In this study, animals that received Ad2/HIF-1 demonstrated increased perfusion during pharmacological stress and improved global LV function compared with an Ad2/control. There were no detectable differences in safety or toxicity among groups.
Plasmid HIF-1 (500 µg) has been shown to improve perfusion in a rabbit ischaemic hindlimb model.5 More recently, plasmid HIF-1
has been shown to reduce infarct size and increase neovascularization when injected epicardially at the time of LAD ligation in the rat.14 Although no animal model faithfully mimics myocardial disease in man, ameroid-induced ischaemia in the pig is widely considered the gold standard by which to demonstrate efficacy of novel angiogenic agents. We used strict inclusion criteria prior to treatment randomization to try to reduce variability of the outcome measures. Thus, microvessels from chronically ischaemic myocardium show increased reactivity to both FGF-2 and VEGF, while up-regulation of growth factor receptors has also been demonstrated in this tissue.15 Despite our criteria, the Ad2/HIF-1
group showed a non-significant increase in the level of baseline ischaemia in the LCX territory compared with the other groups during stress. It is therefore possible that this may have biased the data in favour of a treatment-induced difference. We also assessed whether the potential increase in inflammation associated with an adenoviral vector could have mediated the improvements in perfusion. However, this was not supported by the histological assessment of myocardial inflammation, which demonstrated no significant difference between Ad2/HIF-1
, plasmid HIF-1
, or an adenoviral control. It is difficult to assess whether baseline data from the two animals that died differed from the surviving population. Although we cannot exclude the possibility that this may have biased the results, any effect is likely to have been minimal.
One of the key findings was an improvement in perfusion, apparently in the absence of new capillaries or larger vessels. We have also seen these effects in a mouse myocardial infarction model. Clearly, one explanation is a lack of sensitivity of our immunohistological assays, although these were based on previously validated techniques. Another potential explanation is improvement in either endothelial or LV function. Growth factor-mediated improvements in endothelial function have been documented.16 Thus, microvessels from chronically ischaemic myocardium have a greater sensitivity to both VEGF and FGF-2 vasodilation15 thought to predominantly arise from nitric oxide (NO) release. HIF-1 may stabilize NO or nitric oxide synthase (NOS) or counteract effects of other substances detrimental to vascular function. In keeping with this, pre-treatment of animals with an NOS inhibitor almost completely suppressed VEGF-induced increases in blood flow.17 In a subsequent study (data not shown), we have demonstrated that nitro-L-arginine methyl ester, an NOS inhibitor, does not effect the improvements in perfusion seen during stress in those pigs treated with Ad2/HIF-1
. A further possible explanation is an improvement in LV function. Studies of FGF-2 on acute ischaemia reperfusion have reported significant salvage of myocardium,18 which could have resulted either from recruitment of pre-existing collaterals or from myocyte protection by the growth factor.
Although others have reported that HIF-1 increases levels of VEGF protein,5,14 we were unable to detect this in vivo 21 days after gene delivery. The most likely explanation is the well-documented time course of adenoviral-mediated expression, which peaks over the first week, and is markedly reduced by the end of 3 weeks.19 Alternatively, additional factors including the time delay between removal of the heart and isolation of the target region may have resulted in VEGF protein degradation or the levels of VEGF protein induced by HIF-1
may have been below the detection sensitivity of the assay.
Vincent et al.5 have demonstrated efficacy with a plasmid HIF-1 in the peripheral hindlimb model. In our study, neither dose of pHIF-1
showed an effect in any of the bioreactivity endpoints. This may reflect differences in gene transfer efficiency in the two organs. Thus, plasmid DNA is well recognized for its efficacy in skeletal muscle, although our data and those of others clearly indicate the superiority of adenoviral-mediated gene transfer in the myocardium. At least at the titre studied this advantage in transfection efficiency was not countered by an increase in inflammatory or other toxic effects.
In summary, plasmid pHIF-1 did not produce any significant changes in bioactivity. Ad2/HIF-1
significantly increased myocardial flow during stress in addition to significantly improving LV function at rest compared with the control group. However, these improvements could not be correlated with histological or angiographic evaluation of vascularity.
Supplementary material
Supplementary material is available at European Heart Journal online.
Acknowledgements
The study was supported by the British Heart Foundation (SP/2001/001), The German Cardiac Society (PWR), and a Wellcome Trust Senior Clinical Fellowship (EWFWA). We thank Tracy Higgins, Antja Mittag, Jens-Uwe Rätzel, Dirk Mahnkopf, Michael Richter, Udo Kellner, and Geoff Akita for their technical support; Mark Post, Roger Laham, Kaori Sato, and Jane Davies for their advice; and Genzyme Corporation, Framingham, MA, USA for the provision of all HIF-1 constructs. This research was performed at The Institute of Medical Technology Mageburg, Germany, and The National Heart and Lung Institute London, UK. There are no conflicts of interest to disclose Karen Vincent is an employee of Genzyme Corporation, USA.
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Footnotes |
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References |
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