From the Department of Medicine, Teikyo University School of Medicine,
Tokyo (S.T., T.I., M.O., K.E., T.S.); the Department of Physiology, Tokai
University School of Medicine, Isehara (H.M., E.T.); and the Central Research
Laboratory, Hitachi Ltd., Tokyo (K.U.), Japan.
Correspondence to Satoshi Takeshita, MD, Department of Medicine, Teikyo University School of Medicine, 2-11-1 Kaga, Itabashi, Tokyo 173-8605, Japan. E-mail stake{at}blue.ocn.ne.jp
Methods and Results—Ischemia was induced in the hindlimb
of 35 rats by excision of the femoral artery. Immediately thereafter,
400 µg of a plasmid encoding VEGF or ß-galactosidase (control) was
transfected into limb muscles. Four weeks later, synchrotron radiation
microangiography, with a spatial resolution of 30 µm, was
performed to document the reactivity of collateral microvessels.
Administration of the endothelium-dependent vasodilator
acetylcholine failed to induce dilation of collateral microvessels in
control animals. By contrast, profound dilation of collaterals was
observed after acetylcholine in VEGF-treated animals. This response was
evident in vessels with a linear appearance but not in those with an
undulating appearance. The resulting blood flow in the ischemic
limb after administration of acetylcholine in the control animals was
only 64.6±17.0% of that of the contralateral normal limb, whereas
blood flow was augmented to 106.1±8.4% in VEGF-treated animals
(P<0.05).
Conclusions—These results demonstrate in vivo that the use of
VEGF restores impaired vasomotor responses in some types of collateral
microvessels, which may help to provide a basis for understanding the
microcirculation after therapeutic angiogenesis with VEGF.
Recent investigations have established that exogenously administered
angiogenic growth factors can induce the formation of new blood vessels
and enhance collateral blood flow to ischemic
tissues.3 A number of naturally occurring growth
factors could potentially induce or accelerate angiogenesis by
stimulating endothelial cell proliferation and
migration. Among these, the most potent endothelial
mitogen may be vascular endothelial growth factor
(VEGF).4
Previous in vivo studies have suggested that VEGF not only augments the
formation of collateral vessels but also modifies their vasomotor
responses. Bauters et al1 demonstrated
angiographically that administration of VEGF results in improved
endothelium-dependent responses of relatively large
collateral vessels. A more recent study demonstrated in vitro that VEGF
improves preservation of endothelium-dependent
relaxation of collaterals at the microvascular
level.5 Whether impaired responses of collateral
microvessels can be improved by the use of VEGF remains to be confirmed
in vivo.
The purpose of this study was to document in vivo the altered
endothelium-dependent responses of naturally occurring
collateral vessels at the microvascular level and to investigate the
extent to which the function of these microvessels is modified after
VEGF administration.
Direct Transfection of Limb Muscles
Microangiographic Assessment
Quantitative angiographic analysis of collateral vessel
development was performed by directly counting the number of vessels
that crossed a line drawn diagonally across the mid thigh
(perpendicular to the femur).9 This
analysis was performed by a single observer who was blinded to
the treatment regimen.
Measurements of Limb Blood Flow by Use of Colored
Microspheres
Statistical Analyses
Administration of ACh to the normal limb induces relaxation of
microvessels (Figure 1A
Limb Blood Flow Measurement
At least 2 different mechanisms could contribute to the improvement in
the vasodilatory responses of collateral microvessels. First, VEGF
significantly increases blood pressure in ischemic
limbs3 8 ; therefore, the characteristics of flow
and perfusion pressure in arterioles distal to the site of vessel
occlusion should change significantly. Development of an increase in
perfusion pressure may lead to repair of dysfunctional
endothelium in distal vasculatures. The second
possibility is that VEGF leads to a direct improvement in
endothelial function. Asahara et
al10 demonstrated that VEGF accelerates recovery
of endothelium-dependent reactivity of balloon-injured
vessels. Thus, VEGF may modulate qualitative aspects of
endothelial cell function by directly repairing
endothelial cells damaged by protracted
ischemia and thereby restore normal,
endothelium-dependent blood flow.
Another major finding of this study is that VEGF improved the vasomotor
response of linear but not of tortuous collaterals. We have documented
recently, by SR microangiography, that tortuous collaterals exist in
ischemic but not in normal limbs.7 It is
possible that linear collaterals result from the functional opening
and/or dilation of preexisting vessels, whereas tortuous microvessels
result from remodeling of preexisting vessels or are newly formed and
thus possess functionally different endothelial cells
that have a smaller vasodilatory response to ACh.
We chose ACh to examine endothelium-dependent vasomotor
reactivity because it can be used in human patients. However, ACh has
vasoconstrictive actions in vascular smooth muscle
cells, and in this regard, other agents, such as substance P, may also
be useful for examining endothelium-dependent
reactivity of collaterals in animals.
In summary, the use of VEGF restores the impaired vasomotor responses
of a selected group of microvascular collaterals. These data may
provide new insights regarding collateral microcirculation and
therapeutic angiogenesis.
Received June 19, 1998;
revision received July 29, 1998;
accepted July 30, 1998.
2.
Sellke FW, Quillen JE, Brooks LA, Harrison DG.
Endothelial modulation of the coronary
vasculature in vessels perfused via mature collaterals.
Circulation. 1990;81:1938–1947.
3.
Takeshita S, Zheng LP, Brogi E, Kearney M, Pu LO,
Bunting S, Ferrara N, Symes JF, Isner JM. Therapeutic angiogenesis: a
single intraarterial bolus of vascular
endothelial growth factor augments
revascularization in a rabbit ischemic hind
limb model. J Clin Invest. 1994;93:662–670.
4.
Leung DW, Cachianes G, Kuang WJ, Goeddel DV, Ferrara
N. Vascular endothelial growth factor is a secreted
angiogenic mitogen. Science. 1989;246:1306–1309.
5.
Harada K, Friedman M, Lopez JJ, Wang SY, Li J, Prasad
PV, Pearlman JD, Edelman ER, Sellke FW, Simons M. Vascular
endothelial growth factor administration in chronic
myocardial ischemia. Am J Physiol. 1996;270:H1791–H1802.
6.
Takeshita S, Isshiki T, Sato T. Increased expression
of direct gene transfer into skeletal muscles observed after acute
ischemic injury in rats. Lab Invest. 1996;74:1061–1065.[Medline]
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7.
Takeshita S, Isshiki T, Mori H, Tanaka E, Eto K,
Miyazawa Y, Tanaka A, Shinozaki Y, Hyodo K, Ando M, Kubota M, Tanioka
K, Umetani K, Ochiai M, Sato T, Miyashita H. Use of synchrotron
radiation microangiography to assess development of small collateral
arteries in a rat model of hindlimb ischemia.
Circulation. 1997;95:805–808.
8.
Tsurumi Y, Takeshita S, Chen D, Kearney M, Rossow ST,
Passeri J, Horowitz JR, Symes JF, Isner JM. Direct intramuscular gene
transfer of naked DNA encoding vascular endothelial
growth factor augments collateral development and tissue perfusion.
Circulation. 1996;94:3281–3290.
9.
Takeshita S, Pu L-Q, Stein LA, Sniderman AD, Bunting
S, Ferrara N, Isner JM, Symes JF. Intramuscular administration of
vascular endothelial growth factor induces
dose-dependent collateral artery augmentation in a rabbit model of
chronic limb ischemia. Circulation. 1994;90(suppl
II):II-228–II-234.
10.
Asahara T, Chen D, Tsurumi Y, Kearney M, Rossow S,
Passeri J, Symes JF, Isner JM. Accelerated restitution of
endothelial integrity and
endothelium-dependent function after phVEGF165 gene
transfer. Circulation. 1996;94:3291–3302.
© 1998 American Heart Association, Inc.
Brief Rapid Communications
Endothelium-Dependent Relaxation of Collateral Microvessels After Intramuscular Gene Transfer of Vascular Endothelial Growth Factor in a Rat Model of Hindlimb Ischemia
Abstract
Top
Abstract
Introduction
Methods
Results
Discussion
References
Background—Recent investigations
have demonstrated the ability of vascular endothelial
growth factor (VEGF) to augment the development of collateral arteries
in vivo. In vitro studies have suggested that the use of VEGF also
improves the endothelium-dependent relaxation of
collaterals at the microvascular level. The purpose of this study was
to determine in vivo the extent to which vasomotor responses of
collateral microvessels are altered after VEGF treatment.
Key Words: angiogenesis • collateral circulation • endothelium • microcirculation
Introduction
Top
Abstract
Introduction
Methods
Results
Discussion
References
Collateral vessels respond to numerous vasoactive agents
and play an active role in regulation of blood flow to ischemic
tissues. However, the vasomotor responses of collaterals, whether these
collaterals are large conduit vessels or small arterioles, differ
markedly from those of innate vessels. Specifically, vasomotor
responses of relatively large collateral vessels to
endothelium-dependent agents such as acetylcholine
(ACh) have been shown angiographically to be impaired in
ischemic limbs in vivo.1 In vitro studies
also have shown that endothelium-dependent relaxation
of coronary collaterals is depressed markedly at the
microvascular level.2
Methods
Top
Abstract
Introduction
Methods
Results
Discussion
References
Plasmids
The plasmid phVEGF165 used in this study
contains the cDNA for recombinant human VEGF165
under the transcriptional control of the cytomegalovirus
promoter/enhancer (a gift from Genentech Inc). The biological activity
of VEGF165 secreted from the cells transfected
with phVEGF165 was confirmed
previously.4 A promoter-matched reporter plasmid
pCMVß (Clontech) encoding ß-galactosidase was used in control
transfection experiments.
The endothelium-dependent microvessel reactivity
of collaterals was investigated in a model of rat limb
ischemia.6 7 Male Wistar rats weighing
300 to 350 g (Charles River Japan) were anesthetized with
an injection of sodium pentobarbital 40 mg/kg IP, after which the left
femoral artery was surgically excised to induce limb ischemia.
Rats were then transfected with either VEGF (n=17) or ß-galactosidase
(n=18) plasmid. The plasmid (400 µg) was dissolved in saline (in a
final volume of 1.0 mL) and injected directly into the limb muscles
with a syringe with a 27-gauge needle.6 8 For
each animal, DNA was injected into 5 different sites in the 3 major
thigh muscles: the adductor (2 sites), quadriceps (2 sites), and
semimembranous (1 site). After 5 injections (80 µg · 0.2
mL-1 · site-1)
were completed, the incision was closed in layers.
Four weeks after transfection, 10 VEGF-treated and 12 control
animals were reanesthetized, and angiographic assessment was
performed with synchrotron radiation (SR) microangiography with a
spatial resolution of 30 µm.7 After
obtaining a baseline angiogram, we allowed a 10-minute interval to
reestablish basal hindlimb blood flow. ACh (Sigma) prepared in saline
(final volume of 0.4 mL) was then infused over 2 minutes (0.2 µg
· kg-1 · min-1),
after which a second angiogram was recorded. Preliminary studies
have shown that the dose of ACh used here significantly dilates normal
limb arteries (Figure 1A
and 1B) and
increases limb blood flow to 199.3±31.5% (n=5) without an effect on
the systemic blood pressure.
View larger version (113K):
[in a new window]
Figure 1. Microangiography of normal and ischemic
rat limbs. Microangiography of a normal limb (A) before and (B) after
ACh, a nontreated ischemic limb (C) before and (D) after ACh,
and a VEGF-treated ischemic limb (E) before and (F) after ACh.
Diameter of reference wire (arrowheads) was 130 µm. Imaging
field of each angiogram was 20x20 mm.
Colored microspheres were used to determine the regional
perfusion of the limb muscles at 4 weeks after
transfection.8 Briefly, 7 VEGF-treated and 6
control animals were reanesthetized. A polyethylene catheter
was then introduced through the carotid artery into the aortic arch.
After ACh was infused intra-arterially,
6x105 Dye-Trak microspheres (15
µm in diameter, Triton Technology) were injected. Animals were
killed, and muscle samples were obtained from the medial thigh of both
limbs. Samples were digested with potassium hydroxide, and
microspheres were reclaimed with a vacuum filter. The dye from
the microspheres was extracted with dimethyl formamide. These
dye samples were then analyzed with a spectrophotometer. On the
basis of the optical density (OD) measurements, the percent limb flow
(flow in the ischemic limb expressed as a percentage of that in
the contralateral normal limb) was calculated from the following
equation: (OD of the ischemic limb/OD of the normal
limb)x(tissue weight of the normal limb/tissue weight of the
ischemic limb)x100 (%).
Results are expressed as mean±SEM. Statistical significance was
evaluated by unpaired Student's t test. A value of
P<0.05 was considered statistically significant.
Results
Top
Abstract
Introduction
Methods
Results
Discussion
References
Angiographic Assessment of Collateral Microvessels
Quantitative analysis of collateral vessels showed that
the number of collateral vessels in the VEGF-treated group was
significantly higher than in the controls (18.1±1.3 versus 10.7±1.3,
P<0.01). 
and 1B, arrows). This response was blunted in
collaterals of any size in control animals (Figure 1C and 1D, arrows).
In contrast, VEGF-treated animals showed improved vasodilatory
responses regardless of the vessel size (Figure 1E and 1F).
Importantly, this vasodilatory effect was observed only in the arteries
with a relatively linear appearance (arrows) but not in those with a
corkscrew appearance (open arrows).
At 4 weeks after transfection, blood flow to the ischemic
limbs after ACh in the control animals was reduced to 64.6±17.0% of
that of the normally perfused limb. By contrast, ischemic limb
blood flow in VEGF-transfected animals was completely restored to the
level in their normal limbs (106.1±8.4%, P<0.05 versus
controls) (Figure 2).
View larger version (11K):
[in a new window]
Figure 2. Limb blood flow after gene transfer. Blood flow to
ischemic limb was expressed as % flow of contralateral normal
limb (*P<0.05).
Discussion
Top
Abstract
Introduction
Methods
Results
Discussion
References
SR microangiography demonstrated that
endothelium-dependent relaxation of collateral
microvessels improved markedly in response to VEGF. Although VEGF has
been suggested to induce endothelium-derived relaxing
factor–dependent vasorelaxation, the contribution of this effect on
collateral dilation was not significant in this study because
expression of the transfected VEGF gene was limited to <3
weeks.8 The resultant limb blood flow measured by
colored microspheres also increased significantly in the
VEGF-treated animals. Preliminary studies performed in our laboratory
also showed that 4 weeks after induction of ischemia, blood
flow to the ischemic limb in VEGF-treated animals had already
returned to the level of the normal limb before ACh administration,
which was 
50% of that seen after ACh. Thus, 50% of the limb
flow after ACh administration in VEGF-treated animals seemed to be due
to ACh-induced dilation of collateral vessels.
Acknowledgments
This work was supported in part by a Grant-in-Aid for Scientific
Research from the Ministry of Education, Science, and Culture
(09670755) and by grants from the Terumo Life Science Foundation and
the Japan Society for the Promotion of Science.
References
Top
Abstract
Introduction
Methods
Results
Discussion
References
1.
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.
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