This Article Abstract Full Text (PDF) Correction (v43,pe9) All Versions of this Article: 41/1/156    most recent 01.HYP.0000053552.86367.12v1 Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Amano, K. Articles by Iwasaka, T. Search for Related Content PubMed PubMed Citation Articles by Amano, K. Articles by Iwasaka, T. Related Collections Angiogenesis

(Hypertension. 2003;41:156.)
© 2003 American Heart Association, Inc.

Scientific Contributions

Enhancement of Ischemia-Induced Angiogenesis by eNOS Overexpression

Katsuya Amano; Hiroaki Matsubara; Osamu Iba; Mitsuhiko Okigaki; Soichiro Fujiyama; Takanobu Imada; Hiroyuki Kojima; Yoshihisa Nozawa; Seinosuke Kawashima; Mitsuhiro Yokoyama; Toshiji Iwasaka

From the Department of Medicine II and Cardiovascular Center (K.A., H.M., O.I., M.O., S.F., T. Imada, T. Iwasaka) and Radiology (H.K.), Kansai Medical University, Moriguchi, Osaka, Japan; Pharmacobioregulation Research Laboratory, Taiho Pharmaceutical Co Ltd (Y.N.), Saitama, Japan; and the Division of Cardiovascular and Respiratory Medicine, Kobe University Graduate School of Medicine (S.K., M.Y.), Kobe, Japan.

Correspondence to Hiroaki Matsubara, MD, Department of Medicine II, Kansai Medical University, Moriguchi, Osaka 570-8507, Japan. E-mail matsubah{at}takii.kmu.ac.jp

	Abstract

Top Abstract Introduction Methods Results Discussion References

 
It remains undetermined whether continuous endothelial nitric oxide (NO) overexpression exerts angiogenic action. We surgically induced hindlimb ischemia in transgenic mice overexpressing endothelial NO synthase in the endothelium (eNOS-Tg) and studied neocapillary formation, ischemia-induced vascular endothelial growth factor (VEGF) expression, cGMP accumulation, and Akt/PKB signaling. Laser Doppler imaging revealed a markedly increased recovery of blood perfusion in ischemic limbs of eNOS-Tg mice (44% increase) compared with that in wild-type mice. Angiography showed a marked increase in basal and ischemia-induced collateral vessel formation in eNOS-Tg mice. Basal capillary densities and tissue cGMP levels were increased in eNOS-Tg mice (1.8-fold and 1.6-fold versus wild-type mice, respectively). Ischemia-induced neocapillary formation and cGMP accumulation were markedly increased in eNOS-Tg mice (3.6-fold and 4.1-fold versus preischemia levels, respectively), whereas those in wild-type mice were much less (1.8-fold and 1.5-fold, respectively). Basal and time-dependent VEGF expression in ischemic muscles did not differ between eNOS-Tg and wild-type mice. Basal and VEGF-mediated Akt phosphorylation in aortas was similar between eNOS-Tg and wild-type mice. Aortic basal eNOS expression was increased 3.3-fold, and VEGF-mediated eNOS phosphorylation was markedly induced in aortas of eNOS-Tg compared with preischemia levels (4.2-fold), whereas much smaller changes were observed in wild-type mice (1.8-fold increase). Our study demonstrates that overexpression of eNOS protein causes a marked increase in neocapillary formation in response to tissue ischemia without affecting ischemia-induced VEGF expression or VEGF-mediated Akt phosphorylation.

Key Words: endothelium • ischemia • nitric oxide synthase • nitric oxide • vasculature

	Introduction

Top Abstract Introduction Methods Results Discussion References

 
Nitric oxide (NO), constitutively produced by endothelial nitric oxide synthase (eNOS), plays critical roles in vascular biology, including regulation of vascular tone and blood pressure.¹ We generated transgenic (Tg) mice overexpressing eNOS in the vascular endothelium and reported that the mice exhibited an increase in basal NO production and basal cGMP levels in the vasculature.²

Previous investigations have provided inferential evidence that biological processes modulated by NO might extend to include angiogenesis. Brock et al³ found that vascular endothelial growth factor (VEGF) increased cytosolic Ca²⁺ in human umbilical vein endothelial cells. Ku et al⁴ documented dose-dependent relaxation of isolated canine coronary arteries in response to VEGF that could be abolished by prior endothelial disruption and/or N^G-monomethyl-L-arginine (L-NMMA). In vitro studies demonstrated that VEGF stimulates the release of NO from the normal arterial wall⁵ and promotes the recovery of disturbed endothelium-dependent flow in the rabbit ischemic hindlimb.⁶ Direct in vitro evidence that NO may induce angiogenesis was demonstrated recently by Papapetropoulos et al.^7,8 Ziche et al^9,10 established the first line of evidence that NO can induce angiogenesis in vitro. Murohara et al¹¹ clearly showed NO-mediated angiogenesis in response to tissue ischemia by using NO-deficient mice. However, we have reported that NO overexpression attenuates NO/cGMP-mediated vasoactive actions by reducing soluble guanylate cyclase (sGC) activity and cGMP-dependent protein kinase (PKG) levels.¹² Although NO was shown to downregulate the expression of VEGF gene,^13–16 there is a considerable body of evidence that NO upregulates VEGF gene expression in various cell types.^17–19 Jozkowicz et al¹⁷ reported that NO derived from NO donors or generated by NOS within the cells upregulates the synthesis of VEGF in vascular smooth muscle cells. Esumi et al (Kimura et al¹⁸ and Chin et al¹⁹) showed that NO upregulates VEGF gene transcription through the HIF-1 (hypoxia-inducible factor-1) binding site and HIF-1 ancillary sequence within hypoxia-response element in human glioblastoma and hepatoma cell lines.

Accordingly, we tested the hypothesis that overexpression of endothelial eNOS modulates angiogenesis in response to ischemia by using eNOS-Tg mice.

	Methods

Top Abstract Introduction Methods Results Discussion References

 
Endothelial NOS
Tg mice and hindlimb ischemia heterozygous male eNOS-Tg mice and their littermate male control mice,² at 14 to 16 weeks of age, were used. To inhibit NO synthase chronically, the mice were provided water containing 1 mg/mL L-NAME for 4 weeks. Unilateral hindlimb ischemia was induced by resecting the left femoral artery and vein. Four weeks after surgery, skeletal muscles were isolated and snap-frozen in liquid nitrogen. All animal protocols were approved by the Institutional Animal Care and Use Committee.

Immunohistochemical Analysis
Four pieces of ischemic tissues from the adductor and semimembranous muscles were obtained. Frozen sections were stained with anti–von Willebrand factor (vWF) serum (DAKO) followed by incubation with TRITC-conjugated secondary antisera.²⁰ Five fields from 2 muscle samples of each animal were randomly selected for capillary counts. To ensure that capillary densities were not overestimated as a consequence of myocyte atrophy or underestimated because of interstitial edema, the capillary/muscle fiber ratio was determined.

Laser Doppler Perfusion Image and Angiography
We measured the ratio of the ischemic (left)/normal (right) limb blood flow using a laser Doppler perfusion image (LPDI) analyzer (Moor Instruments). After scanning blood flow twice, stored images were subjected to computer-assisted quantification of blood flow, and the average flows of ischemic and nonischemic limbs were calculated. To minimize data variables caused by ambient light and temperature, the LPDI index was expressed as the ratio of ischemic (left) to nonischemic (right) limb blood flow. Vessel density was evaluated with a microfocus x-ray television device (Hitex Co Ltd) 28 days after ischemia. Longitudinal laparotomy was performed to introduce a catheter into the abdominal aorta followed by injection of contrast medium (lipiodol). Angiography was performed for 2 seconds after the injection. We quantitatively analyzed collateral vessel numbers as previously reported.²⁰ Briefly, numbers of vessels in the thigh area were counted by using 5-mm² grids by 2 radiologists who were unaware of the group identity of the angiographic film. Interobserver variation was <5%. The procedure for microangiography with the use of monochromatic synchrotron radiography was previously described.²¹

cGMP Assay and Measurement of NOx
The assay for tissue cGMP was performed by cGMP enzyme immunoassay system (Biotrak; Amersham), as previously described.^2,12 The tissues remaining after cGMP measurement were digested by use of a bicinchoninic acid protein assay kit (Pierce). Plasma nitrite and nitrate (NOx) were measured by the Griess method, as previously described.²²

Northern and Western Blotting
Frozen samples from the adductor and semimembranous muscles were homogenized in TRIZOL Reagent (GIBCO BRL). Blots were hybridized with a random-primed ³²P-labeled cDNA probe for VEGF^20,23 and normalized by densities for 28S rRNA as an internal control. Hybridized signals were measured by scanning densitometry, and VEGF mRNA levels were arbitrarily normalized relative to the 28S rRNA levels.

VEGF-mediated phosphorylation of Akt (serine 473) and eNOS (serine 1177) was analyzed by Western blotting, with the use of phosphospecific antibodies (New England Biolabs). Abdominal aortas were excised from eNOS-Tg mice and exposed to VEGF (100 ng/mL) in serum-free DMEM for 10 minutes. Aortas were homogenized in lysis buffer. Lysates were immunoblotted with antiphospho antibodies and detected with an enhanced chemiluminescence kit (Amersham).²⁴

Statistics
Statistical analyses were performed by 1-way ANOVA followed by pairwise contrasts using Dunnett’s test. Data (mean±SEM) were considered significant at a value of P<0.05.

	Results

Top Abstract Introduction Methods Results Discussion References

 
Blood Perfusion Analysis
Progressive recovery of limb perfusion was disclosed in both eNOS-Tg and control wild-type mice after induction of limb ischemia. A greater degree of blood perfusion was observed in the ischemic limbs of eNOS-Tg mice compared with wild-type control mice (44% increase at day 21, P<0.001) (Figures 1A and 1B). Inhibition of NOS activity by L-NAME administration reduced the increased blood perfusion in eNOS-Tg mice toward the control level. Blood flow in L-NAME–treated eNOS-Tg mice or L-NAME-treated wild-type mice tended to be lower than that in wild-type mice, but this difference was not significant (Figure 1B). To test whether L-NAME treatment used in this study was sufficient to abolish endogenous NO production, we measured plasma NOx levels in eNOS-Tg, eNOS-Tg treated with L-NAME, wild-type mice, and wild-type mice treated with L-NAME (n=5, each). We found that plasma NOx levels (µmol/L) were 41±2.2, 12±1.1, 19±1.6, and 11±0.8, respectively. Thus, L-NAME treatment used here reduced NO production in eNOS-Tg mice to the level below baseline, whereas endogenous NO was still generated.

View larger version (70K): [in this window] [in a new window]   Figure 1. Laser Doppler perfusion images. A, Greater blood perfusion (red to yellow) in ischemic limbs was observed in eNOS-Tg mice in contrast to reduced perfusion (green to blue) in wild-type mice (WT). B, Computer-assisted analyses of LDPI revealed significantly greater blood perfusion values in eNOS-Tg mice than in wild-type mice. Administration of L-NAME (1 mg/mL) in drinking water reduced the increased perfusion in eNOS-Tg mice toward normal levels. Values shown are mean±SEM (n=10) at each time point. *P<0.001 vs wild-type mice.

Angiographic Analysis
In the angiography using contrast medium (lipiodol), collateral vessel numbers were markedly increased in ischemic limbs of eNOS-Tg mice (4.8±0.8-fold at day 28, P<0.001) compared with those in wild-type mice, whereas there was no significant difference in basal vessel numbers before ischemia between eNOS-Tg and wild-type mice (Figure 2). L-NAME treatment of eNOS-Tg mice completely prevented an increase in collateral vessel formation in ischemic limbs (data not shown). Angiography used here is a conventional x-ray imaging, which cannot detect capillaries less than 200 µm in diameter. Recently, a new microangiography was developed that uses monochromatic synchrotron radiography that can record capillaries less than 200 µm in diameter.²¹ When the basal vessel numbers before ischemia were examined by the microangiography, vessel formation was markedly increased in eNOS-Tg mice (2.6±0.2-fold, P<0.001) compared with those in wild-type mice (Figure 2).

View larger version (81K): [in this window] [in a new window]   Figure 2. Angiographic analysis. Representative angiograms for conventional radiographic imaging and microangiography with monochromatic synchrotron radiography. Arrows indicate ligated ends of femoral arteries. Numerous collateral vessels were observed at postoperative day 28 and preischemia (by microangiography) in eNOS-Tg mice compared with those in wild-type mice.

Analysis of Capillary Density
Immunohistochemical staining of endothelial cells with anti-vWF antibody (Figure 3) revealed that basal capillary vessel numbers were significantly increased in eNOS-Tg mice than those in wild-type mice (1.4-fold). Twenty-eight days after the hindlimb ischemia, the vessel numbers in the wild-type mice were increased 1.8-fold compared with preischemic numbers. Neocapillary formation in eNOS-Tg mice was more markedly increased (2.7-fold relative to preischemic numbers). L-NAME treatment of eNOS-Tg mice reduced an increase in capillary formation to the control level (Figure 3).

View larger version (42K): [in this window] [in a new window]   Figure 3. Immunohistochemical analysis. Endothelial cells were stained with anti-vWF antibody followed by incubation with TRITC-conjugated secondary antisera. Five fields from two muscle samples of each animal (n=10) were randomly selected, and capillary density was shown as the capillary/muscle fiber ratio. *P<0.001 vs basal values in each group, P<0.05 vs basal values in wild-type mice. Bars, 50 µm.

Ischemia-Induced VEGF Expression in eNOS-Tg Mice
VEGF mRNA levels were examined by using hindlimb muscles dissected at days 0, 1, 3, 7, 14, and 21. As shown in Figure 4, the basal VEGF mRNA levels were similar between eNOS-Tg and wild-type mice. VEGF mRNA levels were markedly decreased at day 1 and day 3 and reverted to the basal level at day 7 in both wild-type and eNOS-Tg mice. Thereafter, VEGF mRNA levels were gradually increased and showed a peak level approximately at day 14. There were no significant differences in time-dependent induction of VEGF mRNA levels after hindlimb ischemia between eNOS-Tg and wild-type mice.

View larger version (36K): [in this window] [in a new window]   Figure 4. VEGF mRNA expression in ischemic limbs. Skeletal muscles were dissected after hindlimb ischemia and RNA was extracted. Densities of VEGF mRNA signals were measured by densitometry and normalized relative to those of 28S rRNA signals. Results (mean±SEM, n=6) were arbitrarily indicated as values relative to VEGF mRNA levels at day 0. *P<0.001 vs day 0 control.

cGMP Levels in Ischemic Limbs
Basal cGMP levels in skeletal muscles were significantly increased in eNOS-Tg mice (1.6-fold) compared with wild-type mice. After hindlimb ischemia, tissue cGMP levels were significantly increased in eNOS-Tg mice at day 14, day 21, and day 28 (2.4-fold, 3.7-fold, and 4.1-fold, respectively, versus preischemic levels, n=6 each, P<0.001), whereas there was no significant difference at day 7 (1.3-fold). Much smaller changes were observed in the ischemic limbs of wild-type mice (1.6-fold increase at day 28 versus preischemic levels, P<0.05) (Figure 5).

View larger version (15K): [in this window] [in a new window]   Figure 5. cGMP accumulation in ischemic limbs. Skeletal muscles were dissected at days 7, 14, 21, and 28 after hindlimb ischemia and the tissue cGMP levels were measured. Results shown are mean±SEM (n=6 each). *P<0.05, **P<0.001 vs day 0 control.

VEGF-Mediated Phosphorylation of Akt and eNOS
Abdominal aortas were dissected, and VEGF-mediated phosphorylation of Akt and eNOS was examined. The basal expression and phosphorylation levels of Akt were similar between eNOS-Tg and wild-type mice. Akt phosphorylation in the aortas from eNOS-Tg mice was induced by VEGF to an extent similar to that observed in wild-type mice (Figure 6A).

View larger version (56K): [in this window] [in a new window]   Figure 6. VEGF-mediated phosphorylation of Akt and eNOS. Abdominal aortas were isolated and exposed to VEGF (100 ng/mL) in serum-free medium for 10 minutes. Aortas were homogenized and immunoblotted with antiphospho antibodies for Akt and eNOS. Phosphosignals in filters were stripped and reprobed with anti-Akt or anti-eNOS antibodies. Phospho-Akt or phospho-eNOS densities were measured by densitometry and normalized relative to those of Akt or eNOS signals, respectively. Results are arbitrarily indicated as values relative to signal densities in wild-type mice before VEGF stimulation. Results shown are mean±SEM (n=6); representative data are shown. *P<0.001 vs values in wild-type mice before VEGF stimulation.

The aortic basal eNOS expression in eNOS-Tg mice was increased 3.3-fold compared with that in wild-type mice, and the expression levels were not modified by VEGF stimulation. In contrast, the basal phospho-eNOS levels were increased in eNOS-Tg mice (2.3 fold, P<0.001) compared with that in wild-type mice. VEGF stimulation induced a further increase in phospho-eNOS levels in both eNOS-Tg and wild-type mice (2.5-fold and 1.8-fold, respectively) relative to their basal levels. However, when phospho-eNOS levels were normalized with expression levels of eNOS protein, there was no significant difference between eNOS-Tg and wild-type mice (Figure 6B), suggesting that VEGF-mediated phosphorylation of eNOS is not affected by eNOS overexpression.

	Discussion

Top Abstract Introduction Methods Results Discussion References

 
Our present study demonstrated that continuous endothelial NO production augments collateral vessel formation in tissue ischemia without affecting ischemia-induced VEGF expression or VEGF-mediated phosphorylation of Akt and eNOS proteins. Akt acts downstream of VEGF to confer endothelial survival and ensure proper blood vessel development as an activator of endothelial NO production through its ability to phosphorylate eNOS.²⁵ In the eNOS-Tg mice, Akt phosphorylates increased amounts of eNOS protein, resulting in an enhancement of collateral vessel formation. The role of NO in angiogenesis is controversial. Lau and Ma²⁶ reported that NO inhibits migration of cultured endothelial cells. Pipili-Synetos et al²⁷ reported that NO donors inhibit angiogenesis in the chick chorioallantoic membrane and tube formation. In contrast, Guo et al²⁸ reported that an NO donor stimulated the proliferation of aortic endothelial cells. Ziche et al¹⁰ suggested that NO may play a role in angiogenesis elicited by VEGF but not basic fibroblast growth factor.

Murohara et al¹¹ demonstrated that angiogenesis developing in response to limb ischemia was severely reduced in mice lacking eNOS gene. In the eNOS^-/- mice, ischemia-induced VEGF expression was normal and exogenous administration of VEGF did not improve the impaired angiogenesis, suggesting the impairment downstream of VEGF signaling. We established mice overexpressing eNOS in the vascular endothelium exhibited a NO/cGMP-dependent decrease in blood pressure,² whereas we also found that eNOS overexpression reduced the sGC activity and PKG expression leading to reduction of endothelium-dependent or endothelium-independent activities.¹² This raised the possibility that continuous activation of endogenous NO may impair angiogenesis. Our present study thus extended the work of Murohara et al¹¹ to establish that endogenous NO production enhances angiogenesis in hindlimb ischemia affecting neither ischemia-induced VEGF expression nor VEGF-mediated Akt-eNOS signaling.

Previous studies indicated that NO may function as an endogenous negative regulator of VEGF expression in the vascular wall,¹³ whereas there is a considerable body of evidence that NO upregulates VEGF gene expression in various cell types, including vascular smooth muscle cells.^17–19 Although we considered the possibility that a similar paradigm might lead to affect expression of VEGF in ischemic skeletal muscle of eNOS-Tg mice, we were unable to detect any such difference between control and eNOS-Tg mice, consistent with the finding observed in eNOS^-/- mice.¹¹ The reason for this discrepancy remains enigmatic, but it must be recognized that the two paradigms differ in several important respects. The arterial wall contains smooth muscle, which is (normally) in direct contact with a continuous layer of endothelial cells, and the regulatory activity of NO was demonstrated after shear stress–induced or balloon-induced stretch of the artery wall. In contrast, the ischemic hindlimb consisted of skeletal muscle, lacking such an adjacent organized endothelial monolayer, and the injury (arterial excision) in this case results in profound ischemia, including necrosis, with a corresponding inflammatory cell infiltrate. Precisely how these salient differences may directly contribute to modulated NO-VEGF interaction remains to be elucidated.

We showed that VEGF mRNA levels were decreased immediately after limb ischemia and then gradually increased. At present, we cannot explain why VEGF mRNA levels were temporally decreased after ischemia. To prepare ischemic limbs, we ligated the femoral artery and extensively stripped the branch arteries. Given a marked decrease in VEGF mRNA levels at day 1 and day 3, this operation may suddenly cause severe necrosis of skeletal muscle cells rather than tissue ischemia. A decrease in VEGF expression in the early phase after hindlimb ischemia was also observed by Li et al.²⁹ Necrotic skeletal muscle cells lose the ability of VEGF synthesis. Blood flow was gradually restored by the expansion of preexisting collaterals (arteriogenesis), and only surviving skeletal muscle cells can synthesize VEGF in response to hypoxia. Thus, it may be possible to consider that restoration of blood flow in early phase after ligation of femoral artery is due to expansion of preexisting collaterals (arteriogenesis), which produces the discrepancy between VEGF expression and restoration of blood flow.

Hypotension in eNOS-Tg mice may contribute to the enhanced angiogenic process. We previously reported that arterial pressure in eNOS-Tg mice (82±2 mm Hg) was reversed by L-NAME treatment (102±3 mm Hg) toward the normal level (101±2 mm Hg).² Thus, as inhibition of NO synthesis by L-NAME normalized both hypotension and enhanced angiogenic process in eNOS-Tg mice, we could not define the influence of blood pressure itself to the angiogenic process. Native angiogenic response to ischemia was reported to be impaired in spontaneous hypertensive rats (SHR).^30,31 In contrast, Murohara et al¹¹ reported that an elevation in blood pressure in eNOS^-/- mice did not affect the native vessel numbers in hindlimb and that blood pressure per se probably does not affect angiogenesis in vivo. Coronary capillary angiogenesis was reported to develop uninhibitedly in SHR³² or hypertension induced by constriction of the left renal artery.³³ Further studies will be needed to define the effect of blood pressure on the angiogenic process in response to tissue ischemia.

Perspectives
This study demonstrated that continuous endothelial NO production augments collateral vessel formation in tissue ischemia without affecting ischemia-induced VEGF expression or VEGF-mediated Akt activation. In the eNOS-Tg mice, basal eNOS expression is increased in vascular endothelium and Akt phosphorylates the increased amounts of basal eNOS protein, resulting in an enhancement of collateral vessel formation. The role of NO was reported in ischemia-induced coronary collateral formation by Matsunaga et al.³⁴ Thus, the findings observed in two gene-engineered models of hindlimb ischemia that used eNOS-Tg (this study) and eNOS^-/-11 mice established the pivotal role of endothelial NO production in the angiogenic process, suggesting that supplemental treatment leading to production of endogenous NO is effective as a compensatory strategy for angiogenic gene or cell therapies.³⁵

	Acknowledgments

 
This study was supported in part by Grants-in-Aid from the Ministry of Education, Science, and Culture and from the Ministry of Health Labor and Welfare, Japan.

Received June 10, 2002; first decision July 1, 2002; accepted October 25, 2002.

	References

Top Abstract Introduction Methods Results Discussion References

 
1. Moncada S, Palmer RMJ, Higgs EA. Nitric oxide: physiology, pathophysiology, and pharmacology. Pharmacol Rev. 1991; 43: 109–142.[Medline] [Order article via Infotrieve]

2. Ohashi Y, Kawashima S, Hirata K, Yamashita T, Ishida T, Inoue N, Sakoda T, Kurihara H, Yazaki Y, Yokoyama M. Hypotension and reduced nitric oxide-elicited vasorelaxation in transgenic mice overexpressing endothelial nitric oxide synthase. J Clin Invest. 1998; 102: 2061–2071.[Medline] [Order article via Infotrieve]

3. Brock TA, Dvorak HF, Senger DR. Tumor-secreted vascular permeability factor increases cytosolic Ca²⁺ and von Willebrand factor release in human endothelial cells. Am J Pathol. 1991; 138: 213–221.[Abstract]

4. Ku DD, Zaleski JK, Liu S, Brock TA. Vascular endothelial growth factor induces EDRF-dependent relaxation in coronary arteries. Am J Physiol. 1993; 265: H586–H592.[Medline] [Order article via Infotrieve]

5. van der Zee R, Murohara T, Luo Z, Zollmann F, Passeri J, Lekutat C, Isner JM. Vascular endothelial growth factor (VEGF)/vascular permeability factor (VPF) augments nitric oxide release from quiescent rabbit and human vascular endothelium. Circulation. 1997; 95: 1030–1037.[Abstract/Free Full Text]

6. Bauters C, Asahara T, Zheng LP, Takeshita S, Bunting S, Ferrara N, Symes JF, Isner JM. Recovery of disturbed endothelium-dependent flow in the collateral-perfused rabbit ischemic hindlimb after administration of vascular endothelial growth factor. Circulation. 1995; 91: 2802–2809.[Abstract/Free Full Text]

7. Papapetropoulos A, Desai KM, Rudic RD, Mayer B, Zhang R, Ruiz-Torres MP, Garcia-Cardena G, Madri JA, Sessa WC. Nitric oxide synthase inhibitors attenuate transforming-growth-factor-ß1–stimulated capillary organization in vitro. Am J Pathol. 1997; 150: 1835–1844.[Abstract]

8. Papapetropoulos A, Garcia-Cardena G, Madri JA, Sessa WC. Nitric oxide production contributes to the angiogenic properties of vascular endothelial growth factor in human endothelial cells. J Clin Invest. 1997; 100: 3131–3139.[Medline] [Order article via Infotrieve]

9. Ziche M, Morbidelli L, Masini E, Amerini S, Granger HJ, Maggi CA, Geppetti P, Ledda F. Nitric oxide mediates angiogenesis in vivo and endothelial cell growth and migration in vitro promoted by substance P. J Clin Invest. 1994; 94: 2036–2044.[Medline] [Order article via Infotrieve]

10. Ziche M, Morbidelli L, Choudhuri R, Zhang H-T, Donnini S, Granger HJ, Bicknell R. Nitric oxide synthase lies downstream from vascular endothelial growth factor-induced but not fibroblast growth factor-induced angiogenesis. J Clin Invest. 1997; 99: 2625–2634.[Medline] [Order article via Infotrieve]

11. Murohara T, Asahara T, Silver M, Bauters C, Masuda H, Kalka C, Kearney M, Chen D, Chen D, Symes JF, Fishman MC, Huang PL, Isner JM. Nitric oxide synthase modulates angiogenesis in response to tissue ischemia. J Clin Invest. 1998; 101: 2567–2578.[Medline] [Order article via Infotrieve]

12. Yamashita T, Kawashima S, Ohashi Y, Ozaki M, Rikitake Y, Inoue N, Hirata K, Akita H, Yokoyama M. Mechanisms of reduced nitric oxide/cGMP-mediated vasorelaxation in transgenic mice overexpressing endothelial nitric oxide synthase. Hypertension. 2000; 36: 97–102.[Abstract/Free Full Text]

13. Tsurumi Y, Murohara T, Krasinski K, Chen D, Witzenbichler B, Kearney M, Couffinhal T, Isner JM. Reciprocal relation between VEGF and NO in the regulation of endothelial integrity. Nature Med. 1997; 3: 879–886.[CrossRef][Medline] [Order article via Infotrieve]

14. Tuder RM, Flook BE, Voelkel NF. Increased gene expression for VEGF and the VEGF receptors KDR/Flk and Flt in lungs exposed to acute or to chronic hypoxia: modulation of gene expression by nitric oxide. J Clin Invest. 1995; 95: 1798–1807.[Medline] [Order article via Infotrieve]

15. Sogawa K, Numayama-Tsuruta K, Ema M, Abe M, Abe H, Fujii-Kuriyama Y. Inhibition of hypoxia-inducible factor 1 activity by nitric oxide donors in hypoxia. Proc Natl Acad Sci U S A. 1998; 95: 7368–7373.[Abstract/Free Full Text]

16. Huang LE, Willmore WG, Gu J, Goldberg MA, Bunn HF. Inhibition of hypoxia-inducible factor 1 activation by carbon monoxide and nitric oxide: implications for oxygen sensing and signaling. J Biol Chem. 1999; 274: 9038–9044.[Abstract/Free Full Text]

17. Jozkowicz A, Cooke JP, Guevara I, Huk I, Funovics P, Pachinger O, Weidinger F, Dulak J. Genetic augmentation of nitric oxide synthase increases the vascular generation of VEGF. Cardiovasc Res. 2001; 51: 773–783.[Abstract/Free Full Text]

18. Kimura H, Weisz A, Kurashima Y, Hashimoto K, Ogura T, D’Acquisto F, Addeo R, Makuuchi M, Esumi H. Hypoxia response element of the human vascular endothelial growth factor gene mediates transcriptional regulation by nitric oxide: control of hypoxia-inducible factor-1 activity by nitric oxide. Blood. 2000; 95: 189–197.[Abstract/Free Full Text]

19. Chin K, Kurashima Y, Ogura T, Tajiri H, Yoshida S, Esumi H. Induction of vascular endothelial growth factor by nitric oxide in human glioblastoma and hepatocellular carcinoma cells. Oncogene. 1997; 15: 437–442.[CrossRef][Medline] [Order article via Infotrieve]

20. Kamihata H, Matsubara H, Nishiue T, Fujiyama S, Nozawa Y, Iwasaka T. Implantation of autologous bone marrow cells into ischemic myocardium enhances collateral perfusion and regional function via side-supply of angioblasts, angiogenic ligands and cytokines. Circulation. 2001; 104: 1046–1052.[Abstract/Free Full Text]

21. Mori H, Hyodo K, Tobita K, Chujo M, Shinozaki Y, Sugishita Y, Ando M. Visualization of penetrating transmural arteries in situ by monochromatic synchrotron radiation. Circulation. 1994; 89: 863–871.[Abstract/Free Full Text]

22. Yamashita T, Kawashima S, Ozaki M, Namiki M, Kobayashi A, Semo T, Matsuda Y, Inoue N, Hirata K, Akita H, Umetani K, Tanaka E, Mori H, Yokoyama M. Role of endogenous nitrite oxide generation in the regulation of vascular tone and reactivity in small vessels as investigated in transgenic mice using synchrotron radiation microangiography. Nitric Oxide. 2001; 5: 494–503.[Medline] [Order article via Infotrieve]

23. Maruyama K, Mori Y, Murasawa S, Matsubara H, Iwasaka T, Inada M. Interleukin-1 upregulates cardiac expression of vascular endothelial growth factor and its receptor KDR/flk-1 via activation of protein tyrosine kinases. J Mol Cell Cardiol. 1999; 31: 607–617.[CrossRef][Medline] [Order article via Infotrieve]

24. Murasawa S, Matsubara H, Mori Y, Nozawa Y, Iwasaka T. Angiotensin II AT1 receptor-induced extracellular signal-regulated protein kinase activation is mediated by Ca2+/calmodulin-dependent transactivation of epidermal growth factor receptor. Circ Res. 1998; 82: 1338–1348.[Abstract/Free Full Text]

25. Cooke JP, Losordo DW. Nitric oxide and angiogenesis. Circulation. 2002; 105: 2133–2135.[Free Full Text]

26. Lau Y-T, Ma W-C. Nitric oxide inhibits migration of cultured endothelial cells. Biochem Biophys Res Commun. 1996; 221: 670–674.[CrossRef][Medline] [Order article via Infotrieve]

27. Pipili-Synetos E, Sakkoula E, Maragoudakis ME. Nitric oxide is involved in the regulation of angiogenesis. Br J Pharmacol. 1993; 108: 855–857.[Medline] [Order article via Infotrieve]

28. Guo J-P, Panday MM, Consigny PM, Lefer AM. Mechanisms of vascular preservation by a novel NO donor following rat carotid artery intimal injury. Am J Physiol. 1995; 269: H1122–H1131.[Medline] [Order article via Infotrieve]

29. Li TS, Hamano K, Suzuki K, Ito H, Zempo N, Matsuzaki M. Improved angiogenic potency by implantation of ex vivo hypoxia prestimulated bone marrow cells in rats. Am J Physiol. 2002; 283: H468–H473.

30. Emanueli C, Salis MB, Stacca T, Gaspa L, Chao J, Chao L, Piana A, Madeddu P. Rescue of impaired angiogenesis in spontaneously hypertensive rats by intramuscular human tissue kallikrein gene transfer. Hypertension. 2001; 38: 136–141.[Abstract/Free Full Text]

31. Takeshita S, Tomiyama H, Yokoyama N, Kawamura Y, Furukawa T, Ishigai Y, Shibano T, Isshiki T, Sato T. Angiotensin-converting enzyme inhibition improves defective angiogenesis in the ischemic limb of spontaneously hypertensive rats. Cardiovasc Res. 2001; 52: 314–320.[Abstract/Free Full Text]

32. Tomanek RJ, Searls JC, Lachenbruch PA. Quantitative changes in the capillary bed during developing peak and stabilized cardiac hypertrophy in the spontaneously hypertensive rat. Circ Res. 1982; 51: 295–304.[Free Full Text]

33. Anversa P, Loud AV, Giacomelli F, Wiener J. Absolute morphometric study of myocardial hypertrophy in experimental hypertension, II: ultrastructure of myocytes and interstitium. Lab Invest. 1978; 38: 597–609.[Medline] [Order article via Infotrieve]

34. Matsunaga T, Warltier DC, Weihrauch DW, Moniz M, Tessmer J, Chilian WM. Ischemia-induced coronary collateral growth is dependent on vascular endothelial growth factor and nitric oxide. Circulation. 2000; 102: 3098–3103.[Abstract/Free Full Text]

35. Tateishi-Yuyama E, Matsubara H, Murohara T, Ikeda U, Shintani S, Masaki H, Amano K, Kishimoto Y, Yoshimoto K, Akashi H, Shimada K, Iwasaka T, Imaizumi T. Therapeutic angiogenesis for patients with limb ischemia by autologous transplantation of bone marrow cells: a pilot study and a randomised controlled trial. Lancet. 2002; 360: 427–435.[CrossRef][Medline] [Order article via Infotrieve]

This article has been cited by other articles:

				T. Fujii, M. Onimaru, Y. Yonemitsu, H. Kuwano, and K. Sueishi Statins restore ischemic limb blood flow in diabetic microangiopathy via eNOS/NO upregulation but not via PDGF-BB expression Am J Physiol Heart Circ Physiol, June 1, 2008; 294(6): H2785 - H2791. [Abstract] [Full Text] [PDF]

				B. Mees, S. Wagner, E. Ninci, S. Tribulova, S. Martin, R. van Haperen, S. Kostin, M. Heil, R. de Crom, and W. Schaper Endothelial Nitric Oxide Synthase Activity Is Essential for Vasodilation During Blood Flow Recovery but not for Arteriogenesis Arterioscler. Thromb. Vasc. Biol., September 1, 2007; 27(9): 1926 - 1933. [Abstract] [Full Text] [PDF]

				A. Senthilkumar, R. D. Smith, J. Khitha, N. Arora, S. Veerareddy, W. Langston, J. H. Chidlow Jr, S. C. Barlow, X. Teng, R. P. Patel, et al. Sildenafil Promotes Ischemia-Induced Angiogenesis Through a PKG-Dependent Pathway Arterioscler. Thromb. Vasc. Biol., September 1, 2007; 27(9): 1947 - 1954. [Abstract] [Full Text] [PDF]

				C. Kupatt, R. Hinkel, M.-L. von Bruhl, T. Pohl, J. Horstkotte, P. Raake, C. El Aouni, E. Thein, S. Dimmeler, O. Feron, et al. Endothelial Nitric Oxide Synthase Overexpression Provides a Functionally Relevant Angiogenic Switch in Hibernating Pig Myocardium J. Am. Coll. Cardiol., April 10, 2007; 49(14): 1575 - 1584. [Abstract] [Full Text] [PDF]

				B. Gigante, G. Morlino, M. T. Gentile, M. G. Persico, and S. De Falco Plgf-/-eNos-/- mice show defective angiogenesis associated with increased oxidative stress in response to tissue ischemia FASEB J, May 1, 2006; 20(7): 970 - 972. [Abstract] [Full Text] [PDF]

				T. Imada, T. Tatsumi, Y. Mori, T. Nishiue, M. Yoshida, H. Masaki, M. Okigaki, H. Kojima, Y. Nozawa, Y. Nishiwaki, et al. Targeted Delivery of Bone Marrow Mononuclear Cells by Ultrasound Destruction of Microbubbles Induces Both Angiogenesis and Arteriogenesis Response Arterioscler. Thromb. Vasc. Biol., October 1, 2005; 25(10): 2128 - 2134. [Abstract] [Full Text] [PDF]

				Y. L. Tang, Q. Zhao, X. Qin, L. Shen, L. Cheng, J. Ge, and M. I. Phillips Paracrine Action Enhances the Effects of Autologous Mesenchymal Stem Cell Transplantation on Vascular Regeneration in Rat Model of Myocardial Infarction Ann. Thorac. Surg., July 1, 2005; 80(1): 229 - 237. [Abstract] [Full Text] [PDF]

				H. Irie, T. Tatsumi, M. Takamiya, K. Zen, T. Takahashi, A. Azuma, K. Tateishi, T. Nomura, H. Hayashi, N. Nakajima, et al. Carbon Dioxide-Rich Water Bathing Enhances Collateral Blood Flow in Ischemic Hindlimb via Mobilization of Endothelial Progenitor Cells and Activation of NO-cGMP System Circulation, March 29, 2005; 111(12): 1523 - 1529. [Abstract] [Full Text] [PDF]

				J. Jacobi, K. Sydow, G. von Degenfeld, Y. Zhang, H. Dayoub, B. Wang, A. J. Patterson, M. Kimoto, H. M. Blau, and J. P. Cooke Overexpression of Dimethylarginine Dimethylaminohydrolase Reduces Tissue Asymmetric Dimethylarginine Levels and Enhances Angiogenesis Circulation, March 22, 2005; 111(11): 1431 - 1438. [Abstract] [Full Text] [PDF]

				K. Minamino, Y. Adachi, M. Okigaki, H. Ito, Y. Togawa, K. Fujitha, M. Tomita, Y. Suzuki, Y. Zhang, M. Iwasaki, et al. Macrophage Colony-Stimulating Factor (M-CSF), As Well As Granulocyte Colony-Stimulating Factor (G-CSF), Accelerates Neovascularization Stem Cells, March 1, 2005; 23(3): 347 - 354. [Abstract] [Full Text] [PDF]

				S. M. Guthrie, L. M. Curtis, R. N. Mames, G. G. Simon, M. B. Grant, and E. W. Scott The nitric oxide pathway modulates hemangioblast activity of adult hematopoietic stem cells Blood, March 1, 2005; 105(5): 1916 - 1922. [Abstract] [Full Text] [PDF]

				O. Baum, L. Da Silva-Azevedo, G. Willerding, A. Wockel, G. Planitzer, R. Gossrau, A. R. Pries, and A. Zakrzewicz Endothelial NOS is main mediator for shear stress-dependent angiogenesis in skeletal muscle after prazosin administration Am J Physiol Heart Circ Physiol, November 1, 2004; 287(5): H2300 - H2308. [Abstract] [Full Text] [PDF]

				B. M. Prior, H. T. Yang, and R. L. Terjung What makes vessels grow with exercise training? J Appl Physiol, September 1, 2004; 97(3): 1119 - 1128. [Abstract] [Full Text] [PDF]

				C. L. Duvall, W. Robert Taylor, D. Weiss, and R. E. Guldberg Quantitative microcomputed tomography analysis of collateral vessel development after ischemic injury Am J Physiol Heart Circ Physiol, July 1, 2004; 287(1): H302 - H310. [Abstract] [Full Text] [PDF]

				T. Namba, H. Koike, K. Murakami, M. Aoki, H. Makino, N. Hashiya, T. Ogihara, Y. Kaneda, M. Kohno, and R. Morishita Angiogenesis Induced by Endothelial Nitric Oxide Synthase Gene Through Vascular Endothelial Growth Factor Expression in a Rat Hindlimb Ischemia Model Circulation, November 4, 2003; 108(18): 2250 - 2257. [Abstract] [Full Text] [PDF]

This Article Abstract Full Text (PDF) Correction (v43,pe9) All Versions of this Article: 41/1/156    most recent 01.HYP.0000053552.86367.12v1 Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Amano, K. Articles by Iwasaka, T. Search for Related Content PubMed PubMed Citation Articles by Amano, K. Articles by Iwasaka, T. Related Collections Angiogenesis