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(Circulation. 2003;108:2250.)
© 2003 American Heart Association, Inc.

Basic Science Reports

Angiogenesis Induced by Endothelial Nitric Oxide Synthase Gene Through Vascular Endothelial Growth Factor Expression in a Rat Hindlimb Ischemia Model

Tsunetatsu Namba, MD; Hiromi Koike; Kazushi Murakami, MD, PhD; Motokuni Aoki, MD, PhD; Hirofumi Makino, MD; Naotaka Hashiya, MD; Toshio Ogihara, MD, PhD; Yasufumi Kaneda, MD, PhD; Masakazu Kohno, MD, PhD; Ryuichi Morishita, MD, PhD

From the Division of Gene Therapy Science (T.N., Y.K.), the Division of Clinical Gene Therapy (H.K., M.A., H.M., N.H., R.M.), and the Department of Geriatric Medicine (K.M., M.A., H.M., N.H., T.O.), Osaka University Medical School, Osaka, and the Second Department of Internal Medicine (T.N., K.M., M.K.), Kagawa Medical University, Kagawa, Japan.

Correspondence to Ryuichi Morishita, MD, PhD, Professor, Division of Clinical Gene Therapy, Osaka University Medical School, 2-2 Yamada-oka, Suita 565-0871, Japan. E-mail morishit{at}cgt.med.osaka-u.ac.jp

Received August 5, 2002; de novo received March 31, 2003; revision received July 9, 2003; accepted July 10, 2003.

	Abstract

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Background— Because the mechanism of the angiogenic property of nitric oxide (NO) was not fully understood in vivo, we focused on the role of vascular endothelial growth factor (VEGF) in angiogenesis induced by endothelial NO synthase (eNOS) gene transfer.

Methods and Results— After intramuscular injection of eNOS DNA into a rat ischemic hindlimb, transfection of eNOS vector resulted in a significant increase in eNOS protein 1 week after transfection. In addition, tissue concentrations of nitrite and nitrate were significantly increased in rats transfected with the eNOS gene up to 2 weeks after transfection. The increase in tissue nitrite and nitrate concentrations was completely inhibited by N^G-nitro-L-arginine methyl ester (L-NAME). In contrast, serum concentrations of nitrite and nitrate and blood pressure were not changed by eNOS gene transfer. Importantly, overexpression of the eNOS gene resulted in a significant increase in peripheral blood flow, whereas L-NAME inhibited the increase in blood flow. Interestingly, basal blood flow was significantly lower in rats treated with L-NAME than in control rats. A significant increase in capillary number was consistently detected in rats transfected with the eNOS gene at 4 weeks after transfection, accompanied by a significant increase in VEGF. Moreover, administration of neutralizing anti-VEGF antibody abolished the increase in blood flow and capillary density induced by eNOS plasmid injection.

Conclusions— Overall, intramuscular injection of bovine eNOS plasmid induced therapeutic angiogenesis in a rat ischemic hindlimb model, a potential therapy for peripheral arterial disease. The stimulation of angiogenesis by NO might be due to upregulation of local VEGF expression.

Key Words: peripheral vascular disease • endothelium • angiogenesis • genes • nitric oxide

	Introduction

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Angiogenesis requires several key processes, including dissolution of matrix, endothelial cell proliferation and migration, and organization into tubes, followed by lumen formation.¹ Because nitric oxide (NO) is an important regulator of endothelial cell growth and angiogenesis, it has been implicated to play a role in all of these processes. Indeed, inhibition of NO synthesis blocks vessel formation in the cornea micropocket assay,² reduces flow in tumor-associated neovasculature,³ and retards closure of excisional wounds.⁴ Interestingly, a number of angiogenic factors, including vascular endothelial growth factor (VEGF), upregulate the expression of endothelial NO synthase (eNOS) and stimulate the release of endothelium-derived NO.^5–7 The release of NO by these factors is believed to play a critical role in their angiogenic actions. In fact, previous studies have demonstrated angiogenesis induced by the eNOS gene with the use of adenoviral vectors.^8,9 In a rabbit corneal model, VEGF-induced angiogenesis was blocked by the NOS inhibitor N^G-nitro-L-arginine methyl ester (L-NAME).¹⁰ The angiogenic response to hindlimb ischemia is impaired in eNOS-deficient mice, an effect that cannot be reversed by VEGF.¹¹ From these findings, NO appears to be a downstream mediator of VEGF-induced endothelial cell proliferation and migration.^10,12,13

On the other hand, NO has been reported to positively regulate the expression of VEGF.^14,15 However, others have suggested that NO downregulates VEGF synthesis.^16,17 Most studies regarding the involvement of NO in VEGF expression have been performed with NO donors such as sodium nitroprusside (SNP). The drawback of this approach is that the influence of the released NO might be masked by the NO-independent actions of donating compounds or their derivatives. A better strategy might be to manipulate the endogenous generation of NO. Therefore, in this study, we examined overexpression of the eNOS gene in a rat hindlimb ischemia model to determine whether the effect of NO on VEGF synthesis could be achieved in an ischemic limb. The results strongly support a role for NO in the regulation of VEGF. Here, we demonstrated that injection of bovine eNOS plasmid induced angiogenesis through VEGF upregulation.

	Methods

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Construction of Plasmids
To produce the eNOS expression vector, full-length, bovine, endothelial, constitutive NOS (ecNOS) cDNA (3.7 kb; kindly provided by T. Michel, Harvard Medical School, Cambridge, Mass^18,19) was inserted into a simple eukaryotic expression plasmid that utilizes the chicken ß-actin promoter/cytomegalovirus (CMV) enhancer (pUC-CAGGS; donated by Prof Miyazaki, Osaka University, Osaka, Japan²⁰) The CAGGS expression vector plasmid, which does not contain eNOS cDNA, was used as a control.

In Vivo Gene Transfer by the Direct Injection Approach
Sprague-Dawley rats (400 to 500 g; Charles River Breeding Laboratories, Tokyo, Japan) were anesthetized with an injection of sodium pentobarbital (0.2 mg/g IP).^21,22 Creation of the hindlimb ischemia model was performed as previously described.^21,22 Naked bovine NOS (500 µg/body) or control vector (500 µg/body) was carefully injected directly into the right ischemic limb of rats with a 27-gauge needle (Terumo) 1 week after surgery. The injection volume of plasmid was 400 µL (100 µL x 4 points). To evaluate the effect of NOS inhibition, freshly prepared L-NAME (1 g/L) was given in the drinking water ad libitum.¹⁰ The efficacy of L-NAME administration was evaluated at the end of treatment. Systolic blood pressure was measured by the tail-cuff method. All protocols were approved by the Osaka University Institutional Animal Care and Use Committee.

Measurement of Nitrite and Nitrate Levels
Release of NO from muscles transfected with the eNOS gene was confirmed by measurement of nitrite and nitrate with an automated NO detector–high-performance liquid chromatography system (ENO-20, Eicom). Nitrite and nitrate were separated by a column (NO-PAK packed with polystyrene polymer, 4.6x50 mm; Eicom), and nitrate was reduced to nitrite in a cadmium reduction column (NO-RED, Eicom). Nitrite was mixed with a Griess reagent to form a purple azo dye in a reaction coil. The mobile phase, which was delivered by a pump at a rate of 0.33 mL/min, was 10% methanol containing 0.15 mol/L NaCl-NH₄Cl and 0.5 g/L of tetrasodium EDTA. The Griess reagent, which was 1.25% HCl containing 5 g/L sulfanilamide with 0.25 g/L N-naphthylethylenediamine, was delivered at a rate of 0.1 mL/min. Total NO metabolite levels were calculated as the sum of nitrite and nitrate levels.

Measurement of eNOS Protein and VEGF Protein
Rats were humanely killed 1 week after transfection of naked plasmid into the hindlimb. Protein concentration was measured by the bicinchoninic acid assay method (Pierce Chemical Co). The concentration of eNOS or VEGF protein in blood vessels was determined by Western blotting with an anti-eNOS antibody (1:1000, Cayman Chemical Co) or an anti-VEGF antibody (1:1000, Santa Cruz Biotechnology, Inc). Both antibodies cross-reacted with rat eNOS or VEGF, respectively. Secondary antibody (RPN2132, 1:5000; ECL Plus Western blotting detection reagents, Amersham Life Science) was visualized with the ECL Western blotting detection system (Amersham Life Science). To quantify and compare levels of proteins, the density of each band was measured by densitometry. Finally, amounts of loaded proteins were normalized by Western blotting of tubulin with an anti-tubulin antibody (1:1000, Calbiochem).

Measurement of Blood Flow by Laser Doppler Imaging
Rats were placed on a heating plate at 37°C to minimize temperature variations.^21,22 A laser Doppler imager (LDI, Moor Instruments) uses a 12-mW helium-neon laser beam that sequentially scans a 5x5-cm surface area with extremely high speed to be able to measure blood flow in the ischemic hindlimb. The blood flow 1 mm under the surface can be measured. Low or no perfusion is displayed as dark blue, whereas the highest perfusion interval is displayed as white. These laser images were quantitatively converted into histograms that represented the amount of blood flow on the x axis and the number of pixels on the y axis in the traced area. The average blood flow in each histogram was calculated for evaluation. We confirmed that the blood flow measured by this method correlated well with capillary density (r=0.78, P<0.01).

Alkaline phosphatase staining was used as a marker of endothelial cells.^21,22 To analyze the number of vessels in the right ischemic hindlimb, 3 individual sections from the middle of the transfected muscle were analyzed. The number of vessels was counted under a light microscope (magnification, 100x) in a blinded manner. At least 10 individual sections were evaluated in each muscle. The areas in which the number of vessels was quantified were randomly selected in the injected site and around the injected site. The animals were coded so that analysis was performed without any knowledge of which treatment each animal had received. Intraobserver variability was 1.8±0.2%, whereas interobserver variability was 2.1±0.8% (mean±SD). These observers were blinded to other data concerning the rats, as well as to the results of the other observer.

Inhibition of VEGF Activity
In vivo suppression of endogenous VEGF activity was done with a VEGF-specific, neutralizing rabbit polyclonal IgG (cross-reactive to human and murine VEGF; NeoMarkers Co).²³ One day before surgery, the disposable micro-osmotic pump (Alza Co) with either nonimmunized rabbit IgG or anti-VEGF IgG was implanted into the peritoneal cavity. This pump continuously releases these solutions at a rate of 0.5 to 1.0 µL/h for 28 days. Soon after the ischemia-related surgery, additional bolus administration of these antibodies (100 µg) was given via the pineal vein.

Statistical Analysis
All values are expressed as mean±SEM. ANOVA with a subsequent Duncan’s test was used to determine the significance of differences in multiple comparisons, whereas a t test was used for the data in Figure 1. Differences with a probability value <0.05 were considered significant.

View larger version (25K): [in this window] [in a new window]   Figure 1. Top, Western blotting of eNOS protein in muscle of ischemic rats transfected with eNOS or control vector at 7 days after transfection. Bottom, Increase in eNOS protein in muscle of ischemic rats transfected with eNOS vector at 7 days after transfection. Control indicates intramuscular injection of control naked plasmid; eNOS, intramuscular injection of naked plasmid of eNOS gene. Data were expressed as fold increase normalized to tubulin expression. Each group contains 4 animals.

	Results

Top Abstract Introduction Methods Results Discussion References

 
First, we measured eNOS protein in the ischemic hindlimb transfected with eNOS vector by using naked plasmid DNA to confirm successful gene transfer. As expected at 7 days after transfection, eNOS protein was readily detected in the hindlimb transfected with eNOS vector (Figure 1). As shown in Figure 1, a significant increase in eNOS protein could be observed in the hindlimb transfected with eNOS vector compared with control vector (P<0.01). After an increase in eNOS protein, injection of eNOS vector into the ischemic hindlimb resulted in a significant increase in tissue concentration of nitrite and nitrate from 1 to 2 weeks after transfection (Figure 2A, P<0.05). Successful transfection was also confirmed by the observation that L-NAME attenuated the increase in tissue nitrite and nitrate levels (P<0.01). Importantly, serum concentrations of nitrite and nitrate did not differ between eNOS vector and control vector at both 1 and 2 weeks after transfection (Figure 2B). Transfection of the eNOS gene did not alter systemic hemodynamics, because no significant change in systolic blood pressure was observed by eNOS gene transfer compared with control vector (Figure 2C). In contrast, administration of L-NAME increased systolic blood pressure from 1 to 2 weeks after treatment of both eNOS and control vector (Figure 2C, P<0.01).

View larger version (35K): [in this window] [in a new window]   Figure 2. A, Percent change in tissue concentration of nitrite and nitrate in muscle transfected with control or eNOS gene at 7 and 14 days after transfection. B, Percent change in serum concentration of nitrite and nitrate in rats transfected with control or eNOS gene at 7 and 14 days after transfection. C, Effect of L-NAME treatment on systolic blood pressure in rats transfected with control or eNOS gene at 7 and 14 days after transfection. Control indicates rats transfected with control vector; eNOS, rats transfected with eNOS vector; L-NAME, L-NAME treatment. Each group contains 6 to 8 animals. *P<0.01 vs control, #P<0.01 vs eNOS.

After an increase in local NO concentration, injection of eNOS vector into the ischemic hindlimb resulted in a significant increase in blood flow from 2 weeks after transfection (Figure 3, P<0.05), whereas no significant change in blood flow was observed at 1 week after transfection. The laser Doppler imaging score of hindlimbs transfected with control vector was not different from that in nontransfected rats. The increase in blood flow by eNOS gene transfection was completely abolished by L-NAME (P<0.01). Interestingly, basal blood flow was significantly diminished by L-NAME administration at 1 and 2 weeks after surgery (P<0.01). The basal level of NO might modulate the recovery of blood flow after the surgical operation. Moreover, transfection of the eNOS vector significantly increased capillary density around the injection site in the ischemic hindlimb compared with the hindlimb transfected with the control vector at 4 weeks after transfection (Figure 4, P<0.01). Finally, to elucidate the mechanisms of NO-induced angiogenesis, we measured VEGF protein. As shown in Figure 5, rat VEGF164 protein could be detected in hindlimbs by Western blotting. Interestingly, transfection of the eNOS gene resulted in a significant increase in VEGF protein at 1 week after transfection (P<0.01). In contrast, L-NAME administration completely abolished the increase in VEGF protein induced by eNOS gene, whereas basal VEGF protein was not further decreased by L-NAME treatment. More important, angiogenesis induced by eNOS vector was completely abolished by neutralizing anti-VEGF antibody (Figure 6, P<0.01). As shown in Figure 6, the increase in blood flow by eNOS transfection was completely abolished by the neutralizing anti-VEGF antibody at 2 weeks after transfection. Similarly, the increase in capillary density by the eNOS vector was also abolished by an anti-VEGF antibody at 4 weeks after transfection (Figure 6B, P<0.01).

View larger version (52K): [in this window] [in a new window]   Figure 3. A, Typical image of blood flow analyzed by laser Doppler imaging at 2 weeks after transfection. Intramuscular injection of plasmid was performed in right ischemic hindlimb. Panels show color-coded images representing blood flow distribution. Low or no perfusion is displayed as dark blue, whereas highest perfusion is displayed as white. Arrows indicate transfected hindlimb. B, Effect of intramuscular injection of eNOS plasmid in ischemic limb of rats: quantitative analysis of blood flow in ischemic hindlimb at 7 and 14 days after transfection. Normal indicates blood flow in normal, nonischemic hindlimb; control, blood flow in ischemic hindlimb transfected with control vector; eNOS, blood flow in ischemic hindlimb transfected with eNOS vector; and L-NAME, L-NAME treatment. Each group contains 7 or 8 animals. *P<0.01 vs control (ischemia), #P<0.01 vs eNOS (ischemia).

View larger version (47K): [in this window] [in a new window]   Figure 4. Effect of transfection of eNOS vector on vascular formation in ischemic hindlimb of rats at 4 weeks after transfection. Top, Representative cross sections (x200). Bottom, Effect of transfection of eNOS vector on number of vessels. Control indicates muscle from rats transfected with control vector; eNOS, muscle from rats transfected with eNOS vector. Each group contains 7 or 8 animals.

View larger version (23K): [in this window] [in a new window]   Figure 5. Top, Western blotting of VEGF protein in muscle of rat ischemic hindlimb transfected with eNOS or control vector at 7 days after transfection. Bottom, Increase in VEGF164 protein in muscle of ischemic hindlimb transfected with eNOS vector at 7 days after transfection. Control indicates intramuscular injection of control naked plasmid; eNOS, intramuscular injection of naked plasmid of eNOS gene; and L-NAME, L-NAME treatment. Data were expressed as fold increase normalized to tubulin expression. Each group contains 4 animals. *P<0.01 vs control.

View larger version (59K): [in this window] [in a new window]   Figure 6. A, Effect of administration of neutralizing anti-VEGF antibody on blood flow induced by eNOS plasmid injection in ischemic hindlimb at 14 days after transfection. Control indicates blood flow in ischemic hindlimb transfected with control vector; eNOS, blood flow in ischemic hindlimb transfected with eNOS vector; eNOS+anti-VEGF, anti-VEGF antibody treatment. Each group contains 7 or 8 animals. *P<0.01 vs control, #P<0.01 vs eNOS. Top, Representative cross-sections. Bottom, Effect of transfection of eNOS vector on percent change in blood flow compared with normal limbs. B, Effect of administration of neutralizing anti-VEGF antibody on vascular formation in ischemic hindlimb of rats at 4 weeks after transfection. Top, Representative cross-sections (x200). Bottom, Effect of transfection of eNOS vector on number of vessels.

	Discussion

Top Abstract Introduction Methods Results Discussion References

 
Although there is a growing body of evidence that NO has angiogenic effects, in part mediated by VEGF, there is not unanimity of opinion in this regard. Indeed, there are some reports of an inhibitory effect of NO donors on the generation of VEGF. Specifically, the NO donor SNP inhibits VEGF synthesis in vascular smooth muscle cells¹⁷ and in retinal epithelial cells.¹⁶ In contrast, other NO donors, such as 3-morpholinosyndnonimine (SIN-1), S-nitroso-N-acetylpenicillamine (SNAP), diethylene triamine (DETA), and S-nitrosoglutathione (GSNO), have been reported to stimulate VEGF synthesis in a variety of cultured cells.^24–27 Because NO chemistry is highly redox sensitive, these discrepancies might be due to subtle differences in the cellular environment in which the assays were performed.²⁷ Furthermore, the incongruent effects of SNP might be due to the fact that, apart from donating NO, SNP fragments into ferrocyanide, ferricyanide, iron ions, and cyanide, each of which might influence a variety of biologic functions.²⁸ We found that overexpression of the eNOS gene increased VEGF production, whereas L-NAME administration attenuated it, and administration of neutralizing anti-VEGF antibody abolished the increase in blood flow and capillary density induced by eNOS gene transfer. Thus, it is supposed that NO might be an important modulator of VEGF synthesis under physiologic conditions, eg, in the vessel wall, and in pathologic situations, eg, in inflammatory diseases. On the other hand, various lines of evidence have revealed that VEGF upregulates the eNOS gene and increases NO level.^23–27,29 It is probable that a paracrine loop might exist between endothelial cells, producing NO, and vascular smooth muscle cells, generating VEGF.

In addition, the present study raises the possibility of a new strategy, therapeutic angiogenesis with the eNOS gene, as gene therapy for the treatment of patients with critical limb ischemia. In patients with critical limb ischemia, amputation, despite its associated morbidity, mortality, and functional implications, is often recommended as a solution to the disabling symptoms. Consequently, the need for alternative treatment strategies in patients with critical limb ischemia is compelling. A novel therapeutic strategy with angiogenic growth factors to expedite and/or augment collateral artery development has recently entered the realm of treatment of ischemic diseases. Gene therapy with VEGF gene has been reported for the treatment of critical limb ischemia and myocardial ischemia.^30–33 As expected, we have demonstrated direct, in vivo evidence of therapeutic angiogenesis induced by eNOS gene transfer in an ischemia model. Notably, a single intramuscular injection of eNOS plasmid was sufficient to induce therapeutic angiogenesis in a rat hindlimb ischemia model. In rats treated with L-NAME, measurement of the Doppler flow ratio between the ischemic and normal limb indicated that restoration of perfusion in the ischemic hindlimb was significantly impaired by endogenous NO blockade. Probably, endogenous NO might be related to the natural recovery of blood flow after ischemia. In contrast, other NOS isoforms, such as inducible NOS (iNOS), might not have a similar angiogenic function, because eNOS but not iNOS plays a predominant role in VEGF-induced angiogenesis and vascular permeability in knockout mice.³⁴ In addition, other VEGF isoforms such as VEGF120 might have different actions on NO release. Thus, selective modulation of eNOS activity is a promising strategy for altering angiogenesis in vivo. One might assume that a higher transfection efficiency of angiogenic growth factors might increase the clinical utility of these treatments. Currently, researchers are using adenoviral gene transfer in clinical trials instead of naked plasmid DNA. However, the potential toxicity of adenovirus, such as strong immunogenicity, is well known. From this viewpoint, we have focused on a plasmid DNA–based gene transfer method, because it is apparently the safest method. Because clinical trials with VEGF to treat peripheral arterial disease seem to be promising, the use of NOS is considered as a safer gene therapy because of its low toxicity.

Overall, the present studies suggest a novel therapeutic strategy that might reduce the symptoms of critical limb ischemia, utilizing the angiogenic properties of the eNOS gene transfer in a rat model. Stimulation of neovascularization induced by NO might be due to induction of VEGF, although enhancement of ischemia-induced angiogenesis by eNOS overexpression was reported not to be dependent on VEGF in transgenic mice.³⁵ Continuous expression of NO might work, as angiogenic factors use other pathways. In addition, stimulation of new vessel formation by eNOS is likely to create new therapeutic options in angiogenesis-dependent conditions such as wound healing, ischemic heart disease, myocardial infarction, and peripheral arterial disease.

	Acknowledgments

 
This work was partially supported by a grant-in-aid from the Organization for Pharmaceutical Safety and Research, a grant-in-aid from the Ministry of Public Health and Welfare, a grant-in-aid from Japan Promotion of Science, and Funds of the Ministry of Education, Culture, Sports, Science and Technology from the Japanese government.

	Footnotes

 
Dr Morishita is a board member and stockholder of AnGes MG, Inc, which has developed gene therapy drugs.

	References

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