Cardiovascular Research

Cardiovascular Research 2002 54(3):576-583; doi:10.1016/S0008-6363(02)00259-6
© 2002 by European Society of Cardiology

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Copyright © 2002, European Society of Cardiology

Intramyocardial injection of naked DNA encoding HIF-1/VP16 hybrid to enhance angiogenesis in an acute myocardial infarction model in the rat

Kou-Gi Shyu^a, Mei-Ti Wang^b, Bao-Wei Wang^a, Chih-Chuna Chang^a, Jyh-Gang Leu^b, Peiliang Kuan^a and Hang Chang^c,*

^aDivision of Cardiology, Shin Kong Wu Ho-Su Memorial Hospital, 95 Wen-Chang Road, Taipei 111, Taiwan
^bInstitute of Pharmacology, National Yang Ming University, No. 155, Section 2, Lee-Long Street, Taipei 111, Taiwan
^cDepartment of Emergency Medicine, Shin Kong Wu Ho-Su Memorial Hospital, 95 Wen-Chang Road, Taipei 111, Taiwan

^* Corresponding author. Tel.: +886-2-2833-2211; fax: +886-2-2836-5775 m001043{at}ms.skh.org.tw

Received 19 July 2001; accepted 25 January 2002

	Abstract

Top Abstract 1 Introduction 2 Methods 3 Results 4 Discussion Acknowledgments References

 
Objectives: The therapeutic utility of hypoxia-inducible factor-1 (HIF-1) transcriptional regulatory system for ischemic hindlimb has been demonstrated. It is not yet known whether this transcriptional regulatory system can be used as a therapeutic strategy to enhance collateral vessel formation in myocardial tissues, where acute hypoxia occurs due to inadequate perfusion. We aimed to test the hypothesis that exogenous administration of HIF-1/VP16 could enhance collateral vessel formation in a rat acute myocardial infarction model. Methods: Sprague–Dawley rats received ligation of the proximal left anterior descending coronary artery to induce acute myocardial infarction. Immediately after the ligation, 50 µg total plasmid DNA (control, plasmid encoding human vascular endothelial growth factor (pVEGF₁₆₅), or pHIF-1/VP16) was injected into the infarct area at three locations. Results: Reverse transcription–polymerase chain reaction (RT–PCR) showed the presence of HIF-1 and VEGF mRNA in the myocardium, but not in other organs at days 3 and 7. The infarct size significantly decreased from 37±4% (control) to 24±2% in the VEGF-treated group and 23±2% in the HIF-1/VP16 treated group (P<0.05). Capillary density also significantly increased from 550±75/mm² (control) to 850±75/mm² in the VEGF group and 850±50/mm² in the HIF-1/VP16-treated group (P<0.01). Combined therapy with HIF-1/VP16 and VEGF resulted in higher capillary density (1230±50/mm²) than treatment with either therapy alone. Regional myocardial blood flow was also higher in the treated groups than in the control. Plasma levels of VEGF were also significantly higher in the HIF-1/VP16 and VEGF-treated group than in the control group. Conclusions: The HIF-1/VP16 hybrid transcription factor is able to reduce infarct size and enhance neovascularization in an acute ischemic myocardium. The potency of VEGF and HIF-1/VP16 hybrid as therapeutic angiogenic factors in acute hypoxic myocardium is similar.

KEYWORDS Angiogenesis; Collateral circulation; Gene therapy; Infarction

	1 Introduction

Top Abstract 1 Introduction 2 Methods 3 Results 4 Discussion Acknowledgments References

 
New blood vessel formation has been recognized as an adaptive response to cellular hypoxia [1]. In the case of local hypoxia due to inadequate perfusion (ischemia), angiogenesis is stimulated by the production of vascular endothelial growth factor (VEGF), a well-known phenomenon in coronary artery disease [2], tumor angiogenesis [3,4], and diabetic neovascularization [5]. Hypoxia also plays a significant role in regulating angiogenesis and vasoformation during mammalian embryonic development [6]. VEGF is a protein that is essential for angiogenesis [7]. Increased expression of VEGF gene in hypoxic cells is mediated in part by increased gene transcription [8–11]. Transcription of genes encoding erythropoietin, VEGF, and glycolytic enzymes is activated in hypoxic cells by a common molecular mechanism [12]. Transcriptional activation of these genes is mediated by the binding of hypoxia-inducible factor 1 (HIF-1) to cis-acting hypoxia-response elements located primarily within 5'-flanking regions of these genes [13]. These observations suggest that activation of HIF-1 may be involved in the regulation of vascular growth and cellular metabolism.

HIF-1, a DNA binding complex first identified as a factor critical for the inducible activity of the erythropoietin 3' enhancer [14], is a key physiological regulator of gene expression that responds to changes in cellular oxygen tension [15]. HIF-1 is a heterodimeric DNA complex composed of two basic helix-loop-helix Per-AHR-ARNT-Sim-proteins (HIF-1 and HIF-1β) [16]. HIF-1 protein levels, which determine the level of HIF-1 DNA-binding and transcriptional activity, increases exponentially as cellular oxygen concentration is reduced [17]. Under hypoxic conditions, both HIF-1 protein level and the activity of the HIF-1 transactivation domains increase [17,18]. Pugh et al. demonstrated that sequences from HIF-1 but not HIF-1β convey hypoxia-inducible activity when fused to the DNA binding domain of heterologous transcription factors [19]. The HIF-1 gene has been shown to be involved in tumor angiogenesis and growth [20–22]. The therapeutic utility of this transcriptional regulatory system for targeting gene expression at hypoxic tumor cells or ischemic hindlimb has been demonstrated [22,23]. However, it is not known whether this transcriptional regulatory system can be used as a therapeutic strategy to enhance collateral vessel formation in myocardial tissues where acute hypoxia occurs due to inadequate perfusion.

The HIF-1/VP16 hybrid is a constitutively active and more potent form of HIF-1 by constructing a hybrid transcription factor consisting of the DNA-binding and dimerization domains from HIF-1 and the transactivation domain from herpes simplex virus VP16 protein [24]. The HIF-1/VP16 hybrid up-regulates exogenous VEGF expression in vitro and enhances angiogenesis in rabbit hindlimb ischemia [23].

Accordingly, we ought to test the hypothesis that exogenous administration of HIF-1/VP16 hybrid could enhance collateral vessel formation in a rat acute myocardial infarction model and to compare the potency of HIF-1/VP16 hybrid to that of VEGF as a therapeutic angiogenic factor.

	2 Methods

Top Abstract 1 Introduction 2 Methods 3 Results 4 Discussion Acknowledgments References

 
2.1 Production of acute myocardial infarction
Male Sprague–Dawley rats weighing 250–350 g were intraperitoneally anesthetized with sodium pentobarbital (45 mg/kg). The rats were intubated and ventilated with a volume-cycled small-animal ventilator. An anterior thoracotomy was performed to open the pericardium. The heart was then rapidly exteriorized, and a 6-0 silk suture was tightened around the proximal left anterior descending coronary artery (before the first branch of diagonal artery). Positive end-expiratory pressure was applied to fully inflate the lungs. The muscle layer and skin were closed separately after plasmid injection, and the animals were allowed to recover. The experimental protocol was approved by the Shin Kong WHS Memorial Hospital committee on animal experiments.

2.2 Plasmids
The HIF-1/VP16 plasmid was generated by Genzyme Corporation (Framingham, MA, USA). cDNA fragments of HIF-1 at amino acid (aa) 390 as well as VP16 C-terminal aa 413–490 were assembled into a simple eukaryotic expression plasmid that uses a CMV promoter to drive HIF-1 expression. Downstream from the HIF-1 cDNA is a BGH polyadenylation sequence. These fragments occur in the pUC19 vector, which includes an SV40-neo gene for neomycin resistance. The plasmid pCMVβ encoding β-galactosidase under control of CMV promoter/enhancer was used for the control transfection experiments. Plasmids were purified with QIAGEN kits. Ethanol precipitation was used to sterilize all plasmids in preparation for myocardial injection, after which, the DNA pellets were reconstituted with sterile PBS containing 5% sucrose, and stored at –20 °C. Prior to injection, DNA concentrations were determined by a spectrophotometer. The plasmid constructs phVEGF₁₆₅ and pCMVβ have been previously described [25].

2.3 Intramyocardial gene transfer
After ligation of the left anterior descending coronary artery, 50 µg of total plasmid DNA in 0.1 ml of normal saline were injected intramuscularly at the left anterior free wall by using an insulin syringe with a 30-gauge needle. After the left ventricle was accessed, the needle was advanced along the left ventricular free wall and plasmid DNA was injected over a period of 5 to 10 s at three separate sites. The injected sites were chosen at least 5 mm away from the left ventricular apex. All animals received three intramyocardial injections of plasmid, with the control plasmid being pCMVβ. In the treated animals, pHIF-1/VP16 or phVEGF₁₆₅ was injected. After injection, the chest was closed and the animals were allowed to recover.

2.4 β-Galactosidase gene expression
To localize the areas of injection and reporter gene expressions relative to the infarcts, β-galactosidase activity was assessed by incubating muscles in 5-bromo-chloro-3-indolyl-β-D-galactosidase chromogen (X-Gal, Sigma, St. Louis, MO, USA) overnight at 37 °C after intramyocardial injection of pCMVβ in four additional rats. After staining, the muscles were rinsed in saline, post-fixed in 1% paraformaldehyde, and were then paraffin embedded, sectioned, and counterstained with hematoxylin and eosin. Five sections from each sample were randomly selected, and the numbers of positive and total myocytes in five high-power fields among an area including pericardium were counted manually for each specimen.

2.5 Human VEGF and HIF-1 gene expression in ischemic myocardial muscles
Gene expression was evaluated by detecting mRNA levels using RT–PCR with rats of myocardial infarction that were put to death at days 1, 3, 7, 14 and 28 after the transfection with phVEGF₁₆₅ and HIF-1/VP16 hybrid (n=2 at each time point) or control plasmid (n=2 at 7 days after transfection). In the eight rats killed at 3 and 7 days after transfection, remote tissues (lung, liver, spleen, and aorta) were also retrieved for analysis of human VEGF and HIF-1 mRNA. To ensure specificity and avoid amplification of endogenous rat VEGF and HIF-1, each primer was selected from a region that was not conserved among different species. Sequences of primers used for VEGF were 5'-GAGGGCAGAATCATCACGAAGT-3' (sense) and 5'-TGAGAGATCTGGTTCCCGAAAC-3' (antisense). Sequences of primers used for HIF-1 were 5'-AGAAAAAGATAAGTTCTGAACGTC-3' (sense) and 5'-GAGAAAAAAGCTTCGCTGTGTG-3' (antisense). RT–PCR was performed according to the manufacture's protocol (Access RT–PCR System, Promega, Madison, WI, USA). The size of the PCR product for VEGF and HIF-1 was 531 and 478 bp, respectively. RT–PCR products were analyzed by 2% agarose gel electrophoresis. To detect the endogenous gene response to the ischemic change, real time PCR using a Lightcycler (Roche Diagnostics, Mannheim, Germany) was performed with rat specific primers for VEGF and HIF-1. Sequences of primers used for rat VEGF were 5'-CACCCACGACAGAAGG-3' (sense) and 5'-TCACAGTGAACGCTCC-3' (antisense). Sequences of primers used for rat HIF-1 were 5'-AGTCGGACAGCCTCAC-3' (sense) and 5'-TGCTGCCTTGTATGGGA-3' (antisense). The initial denaturation phase was 10 min at 95 °C followed by an amplification phase as described below: denaturation at 95 °C for 10 s; annealing at 55 °C for 5 s; elongation at 72 °C for 15 s and for 30 cycles. Amplification, fluorescence detection, and post-processing calculations were also performed using the Lightcycler apparatus.

2.6 Infarct size determination
Two weeks after myocardial infarction, rats were deeply anesthetized with pentobarbital and put to death by rapid excision of the heart. The atria were trimmed from the ventricles, and the right ventricle and left ventricle plus septum were separated and weighed. The tissues were then immersed and fixed in 10% buffered formalin. Each heart was sliced in cross section at four levels spanning from the apex to the base and prepared for routine histology. These sections from each level were stained with 1% triphenyltrtrazolium chloride (TTC) for 20 min. The histological sections of all four slices were projected on a digitalization screen. A planimeter was used to obtain the length of the entire endocardial circumference and that segment of the endocardial circumference made up by the infarcted portion from each of the four slices of the left ventricle. The infarct size, expressed as a percentage of the left ventricle, was calculated by dividing the circumference of the infarct by the total circumference of the left ventricle including the septum. The person who measured the infarct size was unaware of the treatment group.

2.7 Capillary density by immunohistochemistry
At the day of sacrifice (days 14 and 28 after transfection, respectively), the left ventricle was harvested, fixed in methanol, and sliced into 5 µm paraffin sections. To block endogenous peroxide activity and nonspecific binding, sections were incubated with 3% hydrogen peroxide followed by 10% normal horse serum. Specimens were incubated with a monoclonal anti-mouse CD31 antibody at 4 °C overnight. Bound primary antibodies were detected with the avidin–biotin–immunoperoxidase method (Signet). Nonimmune normal rabbit IgG was used to confirm specificity. The number of capillaries was counted in regions with transversely sectioned myocytes in the border zone and in the area of infarction. Twenty sections per heart were evaluated to estimate capillarity by another investigator unaware of the treatment groups. Five fields per section were randomly selected and analyzed at a magnification of 400x. The number of capillaries was assessed from photomicrographs by computerized image analysis.

2.8 Myocardial perfusion detection
At day 28 after transfection, animals were reanesthetized and their chests were opened. Radioactive microspheres (PerkinElmer Life Sciences, Inc., Boston, MA, USA), approximately 700 000 in number, labeled with ¹⁴¹Cr, were injected into the left ventricular cavity. The microspheres were allowed to circulate for 1 min. Then, the animals were euthanized, and the hearts were excised. Tissue specimens from the left ventricle including the septum and right ventricular free wall were excised for measurement of radioactivity. The activities in the left ventricle were expressed per weight of heart tissue as a ratio relative to the activity in the right ventricular free wall.

2.9 Measurement of plasma VEGF
For the first six rats in each group, blood samples were drawn from the left carotid artery at day 3 and from the right femoral artery at day 7 using a 23-gauge needle after coronary ligation. After sampling 0.5 ml of blood, the left carotid and right femoral arteries were ligated. The blood sample was stored at 4 °C for 30 min and then centrifuged at 3000 rev./min for 15 min. Plasma was frozen at –80 °C until assay of VEGF by a mouse VEGF ELISA kit, purchased for R&D Systems. The lower limit of detection of plasma VEGF was 5 pg/ml. The assay was performed in duplicate for each sample.

2.10 Statistical analysis
All results were expressed as mean±S.E.M. Statistical significance was evaluated by analysis of variance followed by Scheffe's procedure. A value of P<0.05 was considered to denote statistical significance.

	3 Results

Top Abstract 1 Introduction 2 Methods 3 Results 4 Discussion Acknowledgments References

 
3.1 Evidence of transfection
To demonstrate the activity of reporter genes, X-Gal staining was performed 4 days after intramyocardial injection. In the myocardium transfected with pCMVβ, successful transfection was evidenced by dark-blue stains that were observed at the area near the infarct size, but not in the infarct area (Fig. 1). This evidence showed that the plasmids were delivered into the peri-infarct zone. The efficiency of gene transfer using β-galactosidase detection was 3.6±0.5%.

View larger version (137K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 1 Expression of β-galactosidase in the ischemic myocardium. Positive expression is identified as dark-blue staining of myocytes (arrow).

 
3.2 Human HIF-1/VP16 and VEGF gene expression in myocardium
To confirm HIF-1/VP16 and VEGF gene expression in the transfected rat myocardium, we analyzed the gene expression of transfected myocardium by detecting mRNA using RT–PCR. HIF-1 and VEGF mRNA were detected in the myocardium at days 1, 3, 7 and 14 after gene transfection (Fig. 2). The mRNA of HIF-1 and VEGF could not be detected at day 28 after transfection. Neither HIF-1 nor VEGF mRNA was detected in distal tissues at days 3 and 7. Rat myocardium injected with pCMVβ plasmid was consistently negative for HIF-1 and VEGF mRNA. To detect the endogenous gene response to the ischemic change, real time PCR using a Lightcycler (Roche Diagnostics, Mannheim, Germany) was performed with rat specific primers for VEGF and HIF-1. The mRNA of endogenous VEGF at day 7 after infarction in the pHIF-1/VP16, phVEGF₁₆₅, and pCMVβ-treated groups increased 2.4-, 4.2-, and 2.1-fold, respectively as compared to the normal rat without infarction. The mRNA of endogenous HIF-1 at 7 days after infarction in the pHIF-1/VP16, phVEGF₁₆₅, and pCMVβ-treated groups increased 4.3-, 3.8-, and 2.8-fold, respectively as compared to the rat without infarction.

View larger version (38K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 2 Expression of human HIF-1 and VEGF mRNA by RT–PCR in ischemic myocardium. (A) Human HIF-1 and VEGF mRNA expression at different periods of time. M, Promega 100-bp DNA ladder; H+ and V+, positive control for HIF-1 and VEGF, respectively; H1, H3, H7, H14, and H28, ischemic myocardium transfected with pHIF-1/VP16 at days 3, 7, 14, and 28; V1, V3, V7, V14 and V28, ischemic myocardium transfected with phVEGF165 at days 1, 3, 7, 14 and 28; Corresponding β-actin expression is indicated below. (B) No expression of human HIF-1 and VEGF mRNA at extramyocardial tissues. M, Promega 100-bp DNA ladder; +, positive control; 1, 4, 7, 10, and 13, brain, lung, liver, kidney and limb muscle, respectively, from rats transfected with pHIF-1/VP16 at day 7; 2, 5, 8, 11, and 14, brain, lung, liver, kidney and limb muscle, respectively, from rats treated with phVEGF165; 3, 6, 9, 12, and 15, same tissue as mentioned above from rats treated with pCMVβ. Corresponding β-actin expression is indicated below.

 
3.3 Infarct size
Since the infarct size of the rats transfected with pCMVβ was similar to that of rats transfected with saline only (data not shown), we used pCMVβ-treated rats as the control. A total of 44 rats were studied for the determination of infarct size with surgical mortality rates after coronary ligation and intramyocardial injection at 31% in each group. Measurement of the infarct size of the left ventricle was performed in 31 hearts (four pHIF-1/VP16 plus phVEGF₁₆₅-treated hearts, nine pHIF-1/VP16-treated hearts, nine phVEGF₁₆₅-treated hearts and nine controls) at day 14 after transfection. Infarct size was significantly smaller in the pHIF-1/VP16-treated and phVEGF₁₆₅-treated groups than in the control group (23±2 and 24±2% vs. 37±4%, P<0.01 and P<0.05, respectively) as shown in Fig. 3. The infarct size was similar between the HIF-1/VP16-treated and VEGF-treated groups. The infarction ratio (20±3%) was further reduced by the combination of pHIF-1/VP16 and phVEGF₁₆₅. The infarct size was also significantly smaller in the pHIF-1/VP16 and phVEGF₁₆₅-treated groups than in the control at day 7 after transfection (data not shown).

View larger version (26K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 3 Effect of intramyocardial pHIF-1/VP16 and phVEGF165 hybrid on infarction ratio. The number of rats used in this analysis was nine in each group. The infarction ratio represents infarction area divided by total left ventricular area.

 
3.4 Capillary density
Given that CD31 antibody specifically identifies capillaries, we used CD31 antibody to detect capillaries at 14 and 28 days after transfection. Capillary densities observed in the myocardium of the pHIF-1/VP16-treated group (850±50/mm²) and the phVEGF₁₆₅-treated group (850±75/mm²) were significantly higher (P<0.01) than that of the control (550±75/mm²) at day 28 after transfection, as shown in Fig. 4. The capillary density in the group of combined pHIF-1/VP16 and phVEGF₁₆₅ was significantly higher than that of either pHIF-1/VP16-treated or phVEGF₁₆₅-treated group. The capillary density was similar between the HIF-1/VP16-treated and VEGF-treated groups. The capillary density of the control group (pCMVβ) was significantly higher than that of the rat without infarction (data not shown). The capillary densities at day 14 after transfection were also significantly higher (P<0.05) in the pHIF-11a/VP16-treated group (650±50/mm²) and the phVEGF₁₆₅-treated group (625±50/mm²) than in the control (400±75/mm²). Grossly and microscopically, no angioma formation was found in any treated-animals or controls. The increased capillary density was mainly limited to the area around the infarct area (border zone).

View larger version (80K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 4 Effect of intramyocardial pHIF-1/VP16 and phVEGF165 on capillary density. The number of rats was also nine in each group. Photomicrography shows representative immunohistochemical CD31 staining of ischemic myocardium harvested at day 28. Dots (arrow) indicate capillaries.

 
3.5 Measurement of regional blood flow
Radioactive microspheres were used to measure relative blood flow in four hearts in each group. The right ventricular free wall, which was noninfarcted in this model, served as the reference region. The weight of the left ventricle and the right ventricular free wall in each heart was about 1.0 and 0.2 g, respectively. In the rat without infarction, the radioactivity of the left ventricle was 2-fold that of the right ventricular free wall. As shown in Fig. 5, the regional blood flow ratio was significantly higher in the treated rats than in the control group. There was no significant difference in relative blood flow among the three treated groups.

View larger version (19K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 5 Effect of intramyocardial pHIF-1/VP16 and phVEGF165 on regional myocardial blood flow. The radioactivities in the left ventricle were expressed per weight of heart tissue as a ratio relative to the activity in the right ventricular free wall. *P<0.05 vs. control.

 
3.6 HIF-1/VP16 hybrid increases plasma VEGF level
As shown in Fig. 6A and B, the plasma VEGF level in the HIF-1/VP16-treated group and the VEGF-treated group was significantly higher than that of the control group at days 3 and 7 after coronary ligation. There was no statistical difference in VEGF levels between the HIF-1/VP16-treated group and the VEGF-treated group.

View larger version (18K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 6 Plasma VEGF levels measured by enzyme immunoassay. (A) VEGF levels at day 3 after transfection. (B) VEGF levels at day 7 after transfection.

 

	4 Discussion

Top Abstract 1 Introduction 2 Methods 3 Results 4 Discussion Acknowledgments References

 
It has been reported that a deletion mutant of HIF-1 truncated at aa 390 exhibits severely reduced transactivation activity but retains a high level of DNA binding that is equivalent in hypoxic and non-hypoxic cells [26]. This result suggests that both the activation domain and the protein region responsible for conferring destabilization with normoxia are located between aa 390 and aa 826. By deleting this region of HIF-1 and replacing it with transactivation domain of herpes simplex virus VP16, the HIF-1/VP16 hybrid up-regulates exogenous VEGF expression in vitro and enhances angiogenesis in rabbit hindlimb ischemia [23]. In this study, we provide the first demonstration that intramyocardial administration of HIF-1/VP16 hybrid can enhance angiogenesis in acute myocardial infarction. The evidence of revascularization in response to administration of HIF-1/VP16 was observed at anatomical and histological levels. Necropsy examination documented a reduction in infarct size and an increase in vascularity at the capillary level that exceeded the negative control for both pHIF-1/VP16 and phVEGF₁₆₅. The physiological study also demonstrated that regional myocardial perfusion and plasma level of VEGF, evidence for functional gene expression, were higher in the treated group than in the control. However, the reduction in infarct size, the increase in capillary density, regional myocardial blood flow, and plasma levels of VEGF in pHIF-1/VP16 and phVEGF₁₆₅ were similar. Therefore, the efficacy of HIF-1/VP16 hybrid and phVEGF₁₆₅ as therapeutic agents in enhancing angiogenesis in acute myocardial infarction is similar. Combined therapy with HIF-1/VP16 and VEGF induced more capillary formation than either therapy alone, although the infarct size could not be further reduced by the combined therapy.

Caution should be taken when interpreting our data. Since the results of histochemical staining with TTC were not validated by direct histologic examination of the affected tissues, we could not exclude the possibility that tissue staining properties were altered by therapy without prevention of tissue necrosis. Because capillary density would likely differ importantly in infarcted and noninfarcted regions, the amount of each tissue type within samples might significantly influence study outcome. Measurements of myocardial perfusion reflect blood flow to the entire left ventricle without distinction between flow to infarct and flow to surviving myocardium. The larger, better-perfused surviving portion of the myocardium is likely to dominate flow results. The observed flow increases in hearts treated with HIF-1/VP16 or phVEGF₁₆₅ might be due to a larger portion of surviving myocardium—or to myocardial hypertrophy, or to a rise in myocardial oxygen demand—rather than to a rise in capillary density.

Expressions of human HIF-1/VP16 and VEGF transgenes were detected by RT–PCR in the myocardium. No gene expression was detected in remote tissues, including lung, liver, kidney and brain. Thus, intramyocardial administrations of phVEGF₁₆₅ and HIF-1/VP16 result in gene expression limited to the target site. Schwarz et al. demonstrated that angioma formation in the infracted tissue was found after intramyocardial injection of 500 µg of phVEGF₁₆₅ in a rat model of myocardial infarction [27]. We did not find any angioma formation in HIF-1/VP16 and VEGF-treated rats in this study: angioma formation may be dose-related. In this study, 50 µg of plasmid was used. A recent study also demonstrated that high levels of VEGF expression caused angioma formation in muscle tissue, but low VEGF levels did not cause angioma formation [28]. Thus, intramyocardial injection of HIF-1/VP16 and phVEGF₁₆₅ may be feasible in patients with acute myocardial infarction or with recent infarcts if a smaller dosage is administered.

Administration of HIF-1/VP16 hybrid via gene therapy may prove to be an effective treatment for ischemia associated with vascular disease. In this application, HIF-1/VP16 may up-regulate a variety of genes, including VEGF. Clinical benefits may be achieved, not only as a result of stimulation of angiogenesis, but also through additional HIF-1-mediated local adaptations to low oxygen tension such as vasodilatation, protection from oxidant stress, and a transition to anaerobic metabolism [29]. This study demonstrated that intramyocardial injection of plasmid DNA encoding HIF-1/VP16 was sufficient to enhance revascularization in a rat model of acute myocardial infarction, although no attempt was made in this study to determine the corresponding extent of functional improvement. This strategy provides an alternative method for therapeutic angiogenesis by exploiting a transcriptional regulatory system. These findings may have important practical implications for the treatment of patients with severe myocardial ischemia.

Time for primary review 22 days.

	Acknowledgments

Top Abstract 1 Introduction 2 Methods 3 Results 4 Discussion Acknowledgments References

 
This study was supported by grant from the National Science Council, Taiwan and Research Committee of Shin Kong Wu Ho-Su Memorial Hospital, Taipei, Taiwan.

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Top Abstract 1 Introduction 2 Methods 3 Results 4 Discussion Acknowledgments References

 

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